MICROORGANISMS AND METHODS FOR THE FERMENTATION OF CANNABINOIDS

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII file, created on Apr. 10, 2020, is named 35066-002WO_SL.txt and is 1,684,311 bytes in size.

BACKGROUND OF THE DISCLOSURE

Cannabis sativa (marijuana, hemp; Cannabaceae) is a medicinal and psychoactive herbal drug. Its unique effects are believed to be caused by cannabinoids, which include Δ⁹-tetrahydrocannabinol (THC) and more than 80 related metabolites. Medical marijuana and cannabinoid drugs are increasingly used to treat a range of diseases and conditions such as multiple sclerosis and chronic pain.

Currently, the production of cannabinoids for pharmaceutical or other use is through the extraction of cannabinoids from plants, for example Cannabis sativa, or by chemical synthesis.

There are several drawbacks of the natural production and extraction of cannabinoids from plants. It is often difficult to reproduce identical cannabinoid profiles in plants using an extraction process. In addition, extraction from Cannabis sativa produces a mixture of cannabinoids, which can be difficult to purify to provide a single compound needed for pharmaceutical applications.

The chemical synthesis of various cannabinoids is a costly process compared to extraction, but it provides the final product as single pure product, which is often required for pharmaceutical use.

The microbial fermentation-based production of cannabigerolic acid (“CBGA”) or cannabinoids can be more economical, more robust, scalable, and can provide specific cannabinoid products for simplified isolation and purification versus current routes.

There are some known microbial fermentation processes. For example, WO 2016/010827 A1 and WO 2011/017798 A1 describe several processes. However, attempts at reproducing the methods disclosed therein, were unsuccessful: CBGA was not produced.

The methods described in WO 2019/071000 A1, incorporated herein by reference, were successful at producing CGBA, however higher titers of CBGA and various cannabinoids are desired. The inventors have discovered ways to produce cannabinoids as described herein.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications herein are incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.

SUMMARY

This application discloses microorganisms that are capable of producing CBGA and cannabinoids (e.g., THC), in an efficient manner, as well as methods of increasing the efficiency of CBGA and cannabinoid, cannabinoid intermediate, or cannabinoid precursor synthesis (“cannabinoid,” “cannabinoid intermediate,” and “cannabinoid precursor” used interchangibly herein). The products that can be made by the processes and microorganism described herein can include, but are not limited to CBGA, Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), and cannabichromevarinic acid (CBCVA), as described in WO 2019/071000 (herein incorporated by reference) and as described herein. In some cases, a combination of mutants, deletions and yeast strains may be used as described or in various combinations.

In an embodiment a genetically modified microorganism comprises at least three polynucleotides that encode for: a) an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 27; b) an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 32; or c) combinations thereof. In an embodiment, the genetically modified microorganism of claim 1 comprises a polynucleotide that encodes for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 27. In an embodiment, the genetically modified microorganism comprises a polynucleotide that encodes for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 32.

In an embodiment, the genetically modified microorganism comprises at least two polynucleotides that encode for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 32.

In an embodiment, the at least three polynucleotides encode for proteins having prenyltransferase activity.

In an embodiment, the microorganism comprises at least one polynucleotide that encodes a F96W mutant of Saccharomyces cerevisiae ERG20. In an embodiment, the microorganism comprises at least one polynucleotide that encodes an N127W mutant of Saccharomyces cerevisiae ERG20. In an embodiment, at least one of the microorganism's engodenous genes is disrupted; preferably wherein the engodenous genes is deleted. In an embodiment, the microorganism further comprises the polynucleotide sequence of the Saccharomyces cerevisiae GAL1/GAL10 promoter. In an embodiment, the GAL1/GAL10 promoter is inserted into the microorganism's native LPP1 locus. In an embodiment, the microorganism's native LPP1 open reading frame is deleted. In an embodiment, the microorganism further comprises at least one polynucleotide that encodes for an amino acid sequence that is substantially identical to a truncated amino acid sequence of the Saccharomyces cerevisiae HMG1, wherein the first 530 amino acids of the HMG1 are truncated.

In an embodiment, the genetically modified microorganism further comprises at least one polynucleotide encoding at least one polypeptide with acyl activating activity; polyketide synthase activity; olivetol synthase activity; tetraketide synthase activity; olivetolic acid cyclase activity; THCA synthase activity; CBDA synthase activity; CBCA synthase activity; HMG-Co reductase activity; farnesyl pyrophosphate synthetase activity; or any combination thereof. In an embodiment, the genetically modified microorganism further comprises at least one polynucleotide encoding an acyl activating enzyme (AAE1); a polyketide synthase (PKS), such as a tetraketide synthase (TKS); an olivetolic acid cyclase (OAC); a THCA synthase (THCAS); a CBDA synthase (CBDAS); a CBCA synthase (CBCAS); a HMG-Co reductase (HMG1); a farnesyl pyrophosphate synthetase (ERG20); or any combination thereof; preferably wherein the AAE1 is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 14; preferably wherein the TKS is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 41; preferably wherein the OAC is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 8; preferably wherein the THCAS is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 10 or SEQ ID NO: 120; preferably wherein the THCAS is a T446A mutant of SEQ ID NO: 120, a T446V mutant of SEQ ID NO: 120, or a T446I mutant of SEQ ID NO: 120, or a combination thereof; preferably wherein the polynucleotide encodes a THCAS signal sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 121 to SEQ ID NO: 138; preferably wherein the CBDAS is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 12; preferably wherein the CBCAS is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 18; preferably wherein the HMG1 is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 20 or SEQ ID NO: 22; preferably wherein the ERG20 is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 24.

In an embodiment, the genetically modified microorganism comprises at least one polynucleotide encoding an enzyme that is capable of converting olivetolic acid to cannabigerolic acid (“CBGA”). In an embodiment, the genetically modified microorganism further comprises at least one polynucleotide encoding an enzyme that is capable of converting butyric acid to cannabigerolic acid (“CBGVA”).

In an embodiment, the genetically modified microorganism further comprises a polynucleotide that encodes for an amino acid sequence that is substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 5.

In an embodiment, the genetically modified microorganism further comprises a polynucleotide that is substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 6.

In an embodiment, the genetically modified microorganism comprises at least two polynucleotides encoding a protein with AAE1 activity. In an embodiment, the genetically modified microorganism comprises at least three polynucleotides encoding a protein with AAE1 activity. In an embodiment, genetically modified microorganism comprises at least two polynucleotides encoding a protein with TKS activity. In an embodiment, the genetically modified microorganism comprises at least three polynucleotides encoding a protein with TKS activity. In an embodiment, the genetically modified microorganism comprises at least two polynucleotides encoding a protein with OAC activity. In an embodiment, the genetically modified microorganism comprises at least three polynucleotides encoding a protein with OAC activity. In an embodiment, the genetically modified microorganism comprises at least three polynucleotides encoding a protein with AAE1 activity; at least three polynucleotides encoding a protein with TKS activity; and at least three polynucleotides encoding a protein with OAC activity.

In an embodiment, the genetically modified microorganism further comprises one or more polynucleotides encoding proteins with Hydroxymethylglutaryl-CoA synthase activity; Hydroxymethylglutaryl-CoA reductase activity; tHMG1 activity; Acetyl-CoA C-acetyltransferase activity; or any combination thereof. In an embodiment, the genetically modified microorganism further comprises one or more polynucleotides encoding a Hydroxymethylglutaryl-CoA synthase (ERG13); a Hydroxymethylglutaryl-CoA reductase (HMG1); a tHMG1; a Acetyl-CoA C-acetyltransferase (ERG10); or any combination thereof. In an embodiment, the genetically modified microorganism further comprises a polynucleotide encoding an ERG13; a polynucleotide encoding a HGM1 and a polynucleotide encoding an amino acid sequence that is substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical SEQ ID NO: 32. In an embodiment, the genetically modified microorganism further comprises a polynucleotide encoding a tHMG1; a polynucleotide encoding an ERG10 and a polynucleotide encoding an EGR13. In an embodiment, the genetically modified microorganism further comprises a polynucleotide encoding a tHMG1; a polynucleotide encoding an ERG13 and a polynucleotide encoding an AAE1. In an embodiment, the genetically modified microorganism further comprises a polynucleotide encoding an enzyme with CBDA synthase activity, a polynucleotide encoding an enzyme with CBCA synthase, a polynucleotide encoding an enzyme with CBCA and CBDA synthase activity, or a combination thereof, preferably wherein the the enzyme with CBDA synthase activity is substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO; 43 or SEQ ID NO: 153 to SEQ ID NO: 287; preferably wherein the polynucleotide encodes a CBDA synthase signal sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 44 to SEQ ID NO: 73 or SEQ ID NO: 104 to SEQ ID NO: 110, preferably wherein the enzyme with CBCA synthase activity is substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 288 to SEQ ID NO: 297 or SEQ ID NO: 305 to SEQ ID NO: 318.

In an embodiment, the genetically modified microorganism further comprises the bCBGA1854 plasmid of SEQ ID No.: 435.

In an embodiment, the genetically modified microorganism further comprises a polynucleotiode encoding a protein with PKS activity, a polynucleotiode encoding a protein with OAC activity, and a polynucleotiode encoding a protein with AAE1 activity.

In an embodiment, the genetically modified microorganism further comprises a polynucleotide encoding a PIR3-CBDA of SEQ ID NO: 302.

In an embodiment, the genetically modified microorganism further comprises a signal peptide corresponding to 0253/asn053-2.

In an embodiment, the genetically modified microorganism's engodenous VPS10 gene is disrupted; preferably wherein the sequence of the disrupted gene is SEQ ID NO: 300. In an embodiment, the coding sequence of the microorganism's engodenous VPS10 gene is deleted

In an embodiment, the microorganism is capable of producing cannabigerolic acid. In an embodiment, the microorganism is capable of producing a cannabinoid. In an embodiment, the cannabinoid is selected from Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), or any combination thereof.

In an embodiment, the genetically modified microorganism comprises one or more endogenous genes is from a pathway that controls beta oxidation of long chain fatty acids. In an embodiment, the at least one endogenous gene is FOX1, FAA1, FAA4, FAT1, PXA1, PXA2, and/or PEX11. In an embodiment, the at least one endogenous gene is FOX1. In an embodiment, the one or more gene is disrupted using a CRISPR/Cas system.

In an embodiment, the genetically modified microorganism is a bacterium or a yeast. In an embodiment, said microorganism is a yeast. In an embodiment, said yeast is from the genus Saccharomyces. In an embodiment, wherein said yeast is from the species Saccharomyces cerevisiae.

In an embodiment, the genetically modified microorganism comprises at least two polynucleotides that encode for amino acid sequences that are substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 27, SEQ ID NO: 32, or combinations thereof.

In an embodiment, the genetically modified microorganism comprises

genetically modified microorganism comprises at least three polynucleotides that encode for a protein with acyl activating activity, at least three polynucleotides that encode for a protein with polyketide synthase activity, at least three polynucleotides that encode for a protein with olivetolic acid cyclase activity.

In an embodiment, the genetically modified microorganism comprises a polynucleotide that encodes for a Saccharomyces cerevisiae TKS with a mutation at Ala125. In an embodiment, the mutation is Ala125Ser.

In an embodiment, the organism comprises a polynucleotide sequence encoding at least one amino acid sequence substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 153 to SEQ ID NO: 287; preferably wherein the polynucleotide further encodes a CBDA synthase signal sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 44 to SEQ ID NO: 73 or SEQ ID NO: 104 to SEQ ID NO: 110.

In an embodiment, the genetically modified organism comprises a polynucleotide sequence encoding at least one amino acid sequence substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 288 to SEQ ID NO: 297 or SEQ ID NO: 305 to SEQ ID NO: 318.

In an embodiment the modified organism comprises a polypeptide comprising an amino acid sequence substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 27.

In an embodiment the modified organism comprises a polynucleotide sequence encoding at least one amino acid sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID: No. 120; preferably wherein the THCAS is a T446A mutant of SEQ ID NO: 120; a T446V mutant of SEQ ID NO: 120; or a T446I mutant of SEQ ID NO: 120, or a combination thereof.

In an embodiment, the genetically modified microorganism comprises at least one polynucleotide encoding for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 320 to SEQ ID NO: 379, a K239A+I240V+L241A combination mutant of SEQ ID NO: 320; a N242D mutant of SEQ ID NO: 320; a N24Q mutant of SEQ ID NO: 320; a G244L+H245K mutant of SEQ ID NO: 320; a K249R mutant of SEQ ID NO: 320; a C264S mutant of SEQ ID NO: 320; a F272I mutant of SEQ ID NO: 320; a R275P mutant of SEQ ID NO: 320; a R275K mutant of SEQ ID NO: 320; a M283I mutant of SEQ ID NO: 320; a M283C+W284F mutant of SEQ ID NO: 320; a F287L mutant of SEQ ID NO: 320; a S295C mutant of SEQ ID NO: 320; a F298G mutant of SEQ ID NO: 320; a F309I mutant of SEQ ID NO: 320; a I314V mutant of SEQ ID NO: 320; a S323A mutant of SEQ ID NO: 320; a S323T mutant of SEQ ID NO: 320; a M326I mutant of SEQ ID NO: 320, a E329Q mutant of SEQ ID NO: 320; a I333L mutant of SEQ ID NO: 320; a L343F mutant of SEQ ID NO: 320; a K348G mutant of SEQ ID NO: 320; a K350N mutant of SEQ ID NO: 320; a L354F mutant of SEQ ID NO: 320; a L354V+F355Y+V356I mutant of SEQ ID NO: 320; a F357Y mutant of SEQ ID NO: 320; a I360C mutant of SEQ ID NO: 320, a F361L mutant of SEQ ID NO: 320; a I363L mutant of SEQ ID NO: 320; a I374L mutant of SEQ ID NO: 320; a Q378K mutant of SEQ ID NO: 320; a T382A mutant of SEQ ID NO: 320; a S398V mutant of SEQ ID NO: 320, a S398V mutant of SEQ ID NO: 320; a T402S mutant of SEQ ID NO: 320; a S417T mutant of SEQ ID NO: 320; a A421L mutant of SEQ ID NO: 320; a M426F mutant of SEQ ID NO: 320, a M428L mutant of SEQ ID NO: 320; a V447+V450I mutant of SEQ ID NO: 320; a S448T mutant of SEQ ID NO: 320; a V450L mutant of SEQ ID NO: 320; a T460S+F461W+V462L mutant of SEQ ID NO: 320, a V473A mutant of SEQ ID NO: 320; a S476L mutant of SEQ ID NO: 320; a W481M mutant of SEQ ID NO: 320; a V484A mutant of SEQ ID NO: 320; a V484L+I489V+I491V mutant of SEQ ID NO: 320, a N488S mutant of SEQ ID NO: 320; a I489V mutant of SEQ ID NO: 320; a S493A mutant of SEQ ID NO: 320; a A495I mutant of SEQ ID NO: 320; a F499S mutant of SEQ ID NO: 320, a C500S mutant of SEQ ID NO: 320; a F503Y mutant of SEQ ID NO: 320; a L510K mutant of SEQ ID NO: 320, a Q520S mutant of SEQ ID NO: 320; a I525L mutant of SEQ ID NO: 320; a L527I mutant of SEQ ID NO: 320; or combinations thereof. In an embodiment, a polynucleotide encoding can encode at least one amino acid sequence disclosed herein. In an embodiment, a vector can comprise a polynucleotide disclosed herein. And in an embodiment, a polypeptide can comprise an amino acid sequence disclosed herein.

In an embodiment, the genetically modified microorganism may comprise at least one polynucleotide encoding for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 153 to SEQ ID NO: 287, or combinations thereof; preferably wherein the at least one polynucleotide further encodes a CBDA synthase signal sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 44 to SEQ ID NO: 73 or SEQ ID NO: 104 to SEQ ID NO: 110. In an embodiment, a polynucleotide encoding can encode at least one amino acid sequence disclosed herein. In an embodiment, a vector can comprise a polynucleotide disclosed herein. And in an embodiment, a polypeptide can comprise an amino acid sequence disclosed herein.

In an embodiment, the genetically modified microorganism may comprise at least one polynucleotide encoding for an amino acid sequence that is substantially identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from THCAS is a T446A mutant of SEQ ID NO: 120; a T446V mutant of SEQ ID NO: 120; or a T446I mutant of SEQ ID NO: 120, or combinations thereof; preferably wherein the at least one polynucleotide encodes a THCAS signal sequence substantially identical to, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to a sequence chosen from SEQ ID NO: 121 to SEQ ID NO: 138. In an embodiement, a polynucleotide encoding can encode at least one amino acid sequence disclosed herein. In an embodiment, a vector can comprise a polynucleotide disclosed herein. And in an embodiment, a polypeptide can comprise an amino acid sequence disclosed herein.

In an embodiment, a method of producing a cannabinoid comprises (a) contacting a carbon substrate with a genetically modified microorganism disclosed herein (b) growing said genetically modified microorganism to produce a cannabinoid; and optionally (c) isolating the cannabinoid from the genetically modified organism. In an embodiment, the carbon substrate is a sugar, alcohol, and/or fatty acid. In an embodiment, the carbon substrate is selected from hexanoic acid, glucose, fructose, xylose, sucrose, dextrins, starch, xylan, cellulose, hemicellulose, arabinose, glycerol, ethanol, butanol, methanol, or any combination thereof. In an embodiment, the carbon substrate is hexanoic acid. In an embodiment, the cannabinoid is converted to Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), or any combination thereof. In an embodiment the conversion is completed outside the microorganism. In an embodiment, the conversion is a non-enzymatic conversion. In an embodiment, the conversion is an enzymatic conversion.

Further embodiments disclose a use of a cannabinoid produced by any one of the disclosed methods for the manufacture of a medicament for the treatment of a disease or a symptom of a disease. In an embodiment, the disease or the symptom of a disease is anorexia, multiple sclerosis, neurodegenerative disorders, epilepsy, glaucoma, osteoporosis, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), asthma, cardiovascular disorders, cancer, obesity, metabolic syndrome-related disorders, depression, anxiety, insomnia, emesis, pain, or inflammation.

Further embodiments disclose a medicament comprising a cannabinoid made by any one of the disclosed methods and a pharmaceutically acceptable excipient. Further embodiments disclose a method of treating a disease or a symptom of a disease comprising administering a subject in need thereof the cannabinoid made by any one of the disclosed methods. In an embodiment, the disease or a symptom of a disease is anorexia, multiple sclerosis, neurodegenerative disorders, epilepsy, glaucoma, osteoporosis, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), asthma, cardiovascular disorders, cancer, obesity, metabolic syndrome-related disorders, depression, anxiety, insomnia, emesis, pain, or inflammation. Further embodiments disclose a method of treating a disease or a symptom of a disease comprising administering a subject in need thereof the disclosed medicament. Further embodiments disclose a use of a cannabinoid produced by any one of the disclosed microorganisms or methods for the manufacture of a medicament for recreational use. In an embodiment, the medicament or cannabinoid is delivered through inhalation, intravenously, oral, or topical application. In an embodiment, the delivery is inhalation and completed through a vaporizer. In an embodiment, the delivery is intravenous and the medicament is delivered through a saline solution. In an embodiment, the delivery is oral and the medicament is delivered with food. In an embodiment, the delivery is oral and the medicament is delivered through drink. In an embodiment, the delivery is topical and the medicament is delivered through a patch. In an embodiment, the delivery is topical and the medicament is delivered through a lotion.

Disclosed herein is a genetically modified microorganism comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. The polynucleotide can encode an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

Disclosed herein is also a genetically modified microorganism comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26. The polynucleotide can encode an amino acid sequence that is at least 60% identical to SEQ ID NO: 27.

Disclosed herein is also a genetically modified microorganism comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31. The polynucleotide can encode an amino acid sequence that is at least 60% identical to SEQ ID NO: 32.

Disclosed herein is also a genetically modified microorganism comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. The polynucleotide can encode an amino acid sequence that is at least 60% identical to SEQ ID NO: 38.

In some cases, the polynucleotide can encode for an enzyme that is capable of converting olivetolic acid to cannabigerolic acid. In other cases, the polynucleotide can encode for a protein having prenyltransferase activity.

In some cases, the genetically modified microorganism can further comprise one or more nucleic acids encoding for acyl activating enzyme (AAE1); polyketide synthase (PKS); tetraketide synthase (TKS) (also referred to as olivetol synthase (OS)); olivetolic acid cyclase (OAC); THCA synthase (THCAS); CBDA synthase (CBDAS); CBCA synthase (CBCAS); HMG-Co reductase (HMG1); farnesyl pyrophosphate synthetase (ERG20); or any combination thereof. For example, if the microorganism comprises an AAE1, the AAE1 can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 14. If the microorganism comprises a PKS, the PKS can be encoded by a polynucleotide sequence that is substantially identical, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. If the microorganism comprises an OAC, the OAC can be encoded by a polynucleotide sequence that is substantially identical, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. If the microorganism comprises a THCAS, the THCAS can be encoded by a polynucleotide sequence that is substantially identical, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. If the microorganism comprises a CBDAS, the CBDAS can be encoded by a polynucleotide sequence that is substantially identical, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. If the microorganism comprises a CBCAS, the CBCAS can be encoded by a polynucleotide sequence that is substantially identical, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. If the microorganism comprises a HMG1, the HMG1 can be encoded by a polynucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or 22. If the microorganism comprises an ERG20, the ERG20 can be encoded by a polynucleotide sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24.

Disclosed herein is a method of making CBGA comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), and iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1; and (b) growing the genetically modified microorganism to make CBGA.

Disclosed herein is a method of making CBGA comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), and iv) a prenyltransferase that comprises an amino acid sequence that is at least 60% identical to SEQ ID NO: 27; and (b) growing the genetically modified microorganism to make CBGA.

Disclosed herein is a method of making CBGA comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), and iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32; and (b) growing the genetically modified microorganism to make CBGA.

Disclosed herein is a method of making CBGA comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), and iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38; and (b) growing the genetically modified microorganism to make CBGA.

The methods can also further comprise isolating the CBGA from (b). The method can also further comprise converting CBGA into CBG, Δ⁹-tetrahydrocannabinolic acid; THC; cannabidiolic acid; CBD; cannabichromenic acid; CBC; Δ⁹-tetrahydrocannabivarinic acid;

THCVA; cannabidivarinic acid; CBDVA; cannabichromevarinic acid; CBCVA; other cannabinoid; or any combination thereof. This CBGA conversion can be completed outside the microorganism. In some cases, the conversion is a non-enzymatic conversion. In other cases, the conversion is an enzymatic conversion.

Also disclosed herein is a method of making a cannabinoid comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and (v) a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof; and (b) growing the genetically modified microorganism to make a cannabinoid.

Also disclosed herein is a method of making a cannabinoid comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27, and (v) a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof; and (b) growing the genetically modified microorganism to make a cannabinoid.

Also disclosed herein is a method of making a cannabinoid comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, and (v) a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof; and (b) growing the genetically modified microorganism to make a cannabinoid.

Also disclosed herein is a method of making a cannabinoid comprising (a) contacting a carbon substrate with a genetically modified microorganism, where the genetically modified microorganism comprises one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) a olivetolic acid cyclase (OAC), iv) a prenyltransferase that comprises an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38, and (v) a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof; and (b) growing the genetically modified microorganism to make a cannabinoid.

The methods can further comprise isolating the cannabinoid from (b).

The carbon substrate used in the methods can be a sugar, alcohol, and/or fatty acid. For example, the sugar, alcohol or fatty acid can include without limitation hexanoic acid, butyric acid, glucose, fructose, xylose, sucrose, dextrins, starch, xylan, cellulose, hemicellulose, arabinose, glycerol, ethanol, butanol, methanol, or any combination thereof. In some cases, the carbon substrate is hexanoic acid. In other cases, the carbon substrate is butyric acid.

The methods can use the same or similar genetically modified microorganism described throughout. For example, if the microorganism comprises an AAE1, the AAE1 can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. If the microorganism comprises a PKS, the PKS can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. If the microorganism comprises an OAC, the OAC can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.

The methods can use a microorganism that can further comprise one or more nucleic acids encoding for THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); or any combination thereof. If the microorganism comprises a THCAS, the THCAS can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. If the microorganism comprises a CBDAS, the CBDAS can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. If the microorganism comprises a CBCAS, the CBCAS can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18. If the microorganism comprises an HMG1, the HMG1 can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or 22. If the microorganism comprises an ERG20, the ERG20 can be encoded by a polynucleotide sequence that is substantially identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. One or more of these enzymes can be present outside of a microorganism.

The methods can use a microorganism that can further comprise one or more genes that are disrupted. For example, the one or more genes that are disrupted can be from a pathway that controls beta oxidation of long chain fatty acids. In some cases, the one or more genes can be endogenous to the microorganism. In some cases, the one or more genes can comprise FOX1, FAA1, FAA4, FAT1, PXA1, PXA2, and/or PEX11.

Disclosed herein is a vector comprising a polynucleotide that is at least 60% identical or at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 and a promoter suitable for expression in a yeast host. Also disclosed herein is a vector comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36 and a promoter suitable for expression in a yeast host. Also disclosed herein is a vector comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31 and a promoter suitable for expression in a yeast host. Also disclosed herein is a vector comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37 and a promoter suitable for expression in a yeast host.

Also disclosed herein is an isolated polynucleotide comprising a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. Also disclosed herein is an isolated polynucleotide comprising a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26.

Also disclosed herein is an isolated polynucleotide comprising a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31.

Also disclosed herein is an isolated polynucleotide comprising a nucleotide sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.

Further disclosed herein is a method of making a genetically modified microorganism capable of synthesizing CBGA comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing CBGA comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing CBGA comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing CBGA comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing cannabinoid comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing cannabinoid comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing cannabinoid comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

Also disclosed herein is a method of making a genetically modified microorganism capable of synthesizing cannabinoid comprising (a) contacting a microorganism with a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37; and (b) growing the microorganism so that the polynucleotide is inserted into the microorganism.

In some cases, the microorganism can translate the polynucleotide into an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some cases, the microorganism can translate the polynucleotide into an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27. In some cases, the microorganism can translate the polynucleotide into an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some cases, the microorganism can translate the polynucleotide into an amino acid sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38. The polynucleotide can encode for a protein having prenyltransferase activity.

In some cases, the microorganism can be a bacterium or a yeast. If a yeast, the yeast can be from the genus Saccharomyces.

The microorganism can also comprise one or more additional polynucleotides that encodes for acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); HMG-Co reductase (HMG1); farnesyl pyrophosphate synthetase (ERG20); or any combination thereof.

In some cases, the method can comprise a genetically modified microorganism that comprises a polynucleotide encoding for an acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); and a prenyltransferase that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

The methods can result in a cannabinoid, where the cannabinoid is Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA) or any combination thereof.

Further disclosed is the use of a cannabinoid made by any one of the microorganisms or methods disclosed throughout for the manufacture of a medicament for recreational use. In some cases, the recreational use is delivered through inhalation, intravenously, oral, or topical. In some cases, the delivery is inhalation and completed through a vaporizer. In some cases, the delivery is intravenous and completed through a saline solution. In some cases, the delivery is oral and completed through food. In some cases, the delivery is oral and completed through drink. In some cases, the delivery is topical and completed through a patch. In some cases, the delivery is topical and completed through a lotion.

Further disclosed herein is a genetically modified microorganism that is capable of making a CBGA, which comprising a disruption of an endogenous gene that is FOX1. Further disclosed herein is a genetically modified microorganism that is capable of making a CBGA or CBGVA, which comprises a disruption of an endogenous gene that is VPS10. Further disclosed herein is a genetically modified microorganism of claims wherein FOX1 and VPS10 genes are deleted.

Further disclosed herein is a genetically modified microorganism comprising a polynucleotide that is at least 60% identical to the sequences depicted in FIG. 6A or 6B or 7.

Further disclosed is a genetically modified microorganism comprising a polynucleotide that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence depicted in Table 1.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 shows the synthesis pathway from hexanoyl-CoA to CBGA. From CBGA, various cannabinoids can be made including but not limited to THC, CBD, CBC, and CBG.

FIG. 2 shows a representative chromatogram of one sample compared to a CBGA standard. This indicates that our strains produce CBGA, since our sample and the CBGA standard overlap.

FIG. 3 shows a representative MRM chromatogram of a THCA containing sample produced by the microorganism described throughout.

FIG. 4 shows a representative UV chromatogram of a THCA containing sample produced by the microorganism described throughout.

FIG. 5 shows the ability of two different yeast strains to produce CBGA, olivetolic acid, and olivetol. yCBGA_0373 strain with a knocked out FOX1 gene produced more CBGA, olivetolic acid, and olivetol compared to its parental yCBGA_0326 strain with wild type FOX1 gene. Error bars show standard deviation of the four replicates measured.

FIG. 6 depicts improved yields on various steps of the high throughput process for production of CBDA.

FIG. 7 depicts a yield of 700 mg/L CBGA using the yCBGA0513 strain in stirred tank fermenter in 3 days from 1 g/L HXA feed.

FIG. 8 depicts the ATG26 locus of the yCBGA0513 strain and the corresponding locuses in the yCBGA0520 strain, the yCBGA0523 strain, and the yCBGA0526 strain which replaced the ATG26 locus. All promoters are labeled beginning with “p.” All coding regions are labeled beginning with “cds.” All terminators are labeled beginning with “t.” The scale ruler (or graphic bar scale) at the top of the figure is in base pairs. Each increment denotes 500 base pairs.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following description and examples illustrate embodiments of the invention in detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

The cannabinoid biosynthetic pathway starts with acyl activating enzyme (AAE1) (also known hexanoyl-CoA synthetase) which converts hexanoic acid to hexanoyl-CoA, which is used as a substrate for a reaction involving two enzymes, polyketide synthase (PKS) and olivetolic acid cyclase (OAC), to form olivetolic acid. Olivetolic acid is then geranylated by a prenyltransferase enzyme (PT) to form cannabigerolic acid (CBGA), a branch-point intermediate that is converted by oxidocyclase enzymes to Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA). These compounds undergo nonenzymatic decarboxylation to their neutral forms, THC and cannabidiol (CBD) and cannabichromene (CBC), respectively. CBGA is a key pathway intermediate that is an important compound for the preparation of both known, commercialized cannabinoids and compounds in development. In some cases, butyric acid is used as a substrate for cannabinoid biosynthesis.

Described herein are genetically modified microorganisms, enzymes, polynucleotides, and methods to more efficiently produce CBGA or cannabinoids, including, THCA, CBDA, CBCA, THC, CBC and CBD.

Definitions

The term “about” in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. For example, the amount “about 10” includes 10 and any amounts from 9 to 11. For example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. In some cases, the numerical disclosed throughout can be “about” that numerical value even without specifically mentioning the term “about.”

The term “genetic modification” or “genetically modified” and their grammatical equivalents as used herein can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within a microorganism's genome. For example, genetic modification can refer to alterations, additions, and/or deletion of nucleic acid (e.g., whole genes or fragments of genes).

The term “disrupting” and its grammatical equivalents as used herein can refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Disrupting a gene can be partially reducing or completely suppressing expression (e.g., mRNA and/or protein expression) of the gene. Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, or any other means to inhibit functionality or expression of a gene or protein.

The term “gene editing” and its grammatical equivalents as used herein can refer to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. For example, gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).

The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”

The term “sugar” and its grammatical equivalents as used herein can include, but are not limited to (i) simple carbohydrates, such as monosaccharides (e.g., glucose fructose, galactose, ribose); disaccharides (e.g., maltose, sucrose, lactose); oligosaccharides (e.g., raffinose, stachyose); or (ii) complex carbohydrates, such as starch (e.g., long chains of glucose, amylose, amylopectin); glycogen; fiber (e.g., cellulose, hemicellulose, pectin, gum, mucilage).

The term “alcohol” and its grammatical equivalents as used herein can include, but are not limited to any organic compound in which the hydroxyl functional group (—OH) is bound to a saturated carbon atom. For example, the term alcohol can include i) monohydric alcohols (e.g., methanol, ethanol, isopropyl alcohol, butanol, pentanol, cetyl alcohol); ii) polyhydric alcohols (e.g., ethylene glycol, propylene glycol, glycerol, erythritol, threitol, xylitol, mannitol, sorbitol, volemitol); iii) unsaturated aliphatic alcohols (e.g., allyl alcohol, geraniol, propargyl alcohol); or iv) alicyclic alcohols (e.g., inositol, menthol).

The term “fatty acid” and its grammatical equivalents as used herein can include, but are not limited to, a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Some examples of unsaturated fatty acids include but are not limited to myristoleic acid, sapienic acid; linoelaidic acid; α-linolenic acid; stearidonic acid; eicosapentaenoic acid; docosahexaenoic acid; linoleic acid; γ-linolenic acid; dihomo-γ-linolenic acid; arachidonic acid; docosatetraenoic acid; palmitoleic acid; vaccenic acid; paullinic acid; oleic acid; elaidic acid; gondoic acid; erucic acid; nervonic acid; and mead acid. Some examples of saturated fatty acids include but are not limited to propionic acid, butyric acid, valeric acid, hexanoic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, and octatriacontanoic acid.

The term “substantially pure” and its grammatical equivalents as used herein can mean that a particular substance does not contain a majority of another substance. For example, “substantially pure CBGA” can mean at least 90% CBGA. In some instances, “substantially pure CBGA” can mean at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% CBGA. For example, substantially pure CBGA can mean at least 70% CBGA. In some cases, substantially pure CBGA can mean at least 75% CBGA. In some cases, substantially pure CBGA can mean at least 80% CBGA. In some cases, substantially pure CBGA can mean at least 85% CBGA. In some cases, substantially pure CBGA can mean at least 90% CBGA. In some cases, substantially pure CBGA can mean at least 91% CBGA. In some cases, substantially pure CBGA can mean at least 92% CBGA. In some cases, substantially pure CBGA can mean at least 93% CBGA. In some cases, substantially pure CBGA can mean at least 94% CBGA. In some cases, substantially pure CBGA can mean at least 95% CBGA. In some cases, substantially pure CBGA can mean at least 96% CBGA. In some cases, substantially pure CBGA can mean at least 97% CBGA. In some cases, substantially pure CBGA can mean at least 98% CBGA. In some cases, substantially pure CBGA can mean at least 99% CBGA. In some cases, substantially pure CBGA can mean at least 99.9% CBGA. In some cases, substantially pure CBGA can mean at least 99.99% CBGA. In some cases, substantially pure CBGA can mean at least 99.999% CBGA. In some cases, substantially pure CBGA can mean at least 99.9999% CBGA.

The term “heterologous” and its grammatical equivalents as used herein can mean “derived from a different species.” For example, a “heterologous gene” can mean a gene that is from a different species. In some instances, as “a yeast comprising a heterologous gene” can mean that the yeast contains a gene that is not from the same yeast. The gene can be from a different microorganism such as bacterium or from a different species such as a different yeast species.

The term “substantially identical” and its grammatical equivalents in reference to another sequence as used herein can mean at least 50% identical. In some instances, the term substantially identical refers to a sequence that is 55% identical. In some instances, the term substantially identical refers to a sequence that is 60% identical. In some instances, the term substantially identical refers to a sequence that is 65% identical. In some instances, the term substantially identical refers to a sequence that is 70% identical. In some instances, the term substantially identical refers to a sequence that is 75% identical. In some instances, the term substantially identical refers to a sequence that is 80% identical. In other instances, the term substantially identical refers to a sequence that is 81% identical. In other instances, the term substantially identical refers to a sequence that is 82% identical. In other instances, the term substantially identical refers to a sequence that is 83% identical. In other instances, the term substantially identical refers to a sequence that is 84% identical. In other instances, the term substantially identical refers to a sequence that is 85% identical. In other instances, the term substantially identical refers to a sequence that is 86% identical. In other instances, the term substantially identical refers to a sequence that is 87% identical. In other instances, the term substantially identical refers to a sequence that is 88% identical. In other instances, the term substantially identical refers to a sequence that is 89% identical. In some instances, the term substantially identical refers to a sequence that is 90% identical. In some instances, the term substantially identical refers to a sequence that is 91% identical. In some instances, the term substantially identical refers to a sequence that is 92% identical. In some instances, the term substantially identical refers to a sequence that is 93% identical. In some instances, the term substantially identical refers to a sequence that is 94% identical. In some instances, the term substantially identical refers to a sequence that is 95% identical. In some instances, the term substantially identical refers to a sequence that is 96% identical. In some instances, the term substantially identical refers to a sequence that is 97% identical. In some instances, the term substantially identical refers to a sequence that is 98% identical. In some instances, the term substantially identical refers to a sequence that is 99% identical. In order to determine the percentage of identity between two sequences, the two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids/nucleotides is determined between the two sequences. For example, methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that can be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.

The term “polyketide synthase”, “PKS”, “tetraketide synthase”, “olivetol synthase”, “OLS”, “OS” and their grammatical equivalents can be interchangeably used, as they refer to the same enzyme.

General

A cannabinoid is one of a class of diverse chemical compounds that acts on cannabinoid receptors. Cannabinoids can alter neurotransmitter release in the brain. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals), the phytocannabinoids (found in cannabis and some other plants), and synthetic cannabinoids (manufactured artificially). The most notable cannabinoid is the phytocannabinoid tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis. Cannabidiol (CBD) is another major constituent of the plant. There are at least 113 different cannabinoids isolated from cannabis, exhibiting varied effects.

Cannabinoids can be useful in treating the side effects of cancer and cancer treatment. For example, one of the severe side effects of chemotherapy is loss of appetite. Marinol (containing delta-9-THC API) has been used to effectively treat this side effect. Other medical uses of cannabinoids include but are not limited to anti-inflammatory activity, blocking cell growth, preventing the growth of blood vessels that supply tumors, antiviral activity, and relieving muscle spasms caused by multiple sclerosis.

Disclosed herein are microorganisms and methods of making CBGA or cannabinoids.

Microrganisms Used in the Synthesis of Cannabinoids
Cell-Types

The cells that can be used include but are not limited to plant or animal cells, fungus, yeast, algae, or bacterium. The cells can be prokaryotes or in some cases can be eukaryotes. For example, the cell can be a Saccharomyces cerevisiae, Yarrowia lipolytica, or Escherichia coli, or any other cell disclosed throughout.

In certain cases, the cells are not naturally capable of producing CBGA or cannabinoids (e.g., THC, CBD, CBC, THCVA, CBDVA, CBCVA). In some cases, the cells are able to produce CBGA or cannabinoids but at a low level. By implementation of the methods described herein, the cells can be modified such that the level of CBGA or cannabinoids in the cells is higher relative to the level of CBGA or the same cannabinoid produced in the unmodified cells.

In some cases, the modified cell is capable of producing a substrate capable of being converted into a CBGA or a cannabinoid, however, the cells is not capable of naturally producing a cannabinoid. The genetically modified microorganisms in some cases are unable to produce a substrate capable of being converted into a CBGA or a cannabinoid (for example, hexanoic acid), and the substrate capable of being converted into a CBGA or a cannabinoid is provided to the cells as part of the cell's growth medium. In this case, the genetically modified microorganism can process the substrate into a desired product such as CBGA, THC, CBD, or CBC.

The cell can naturally comprise one or more enzyme capable of catalyzing one or more of the reactions: Hexanoyl-CoA to Olivetolic Acid; Olivetolic Acid to CBGA; CBGA to THCA; CBGA to CBDA; CBGA to CBCA; THCA to THC; CBDA to CBD; or CBCA to CBC.

The cell can naturally comprise one or more enzyme capable of catalyzing one or more of the reactions from a substrate such as butyric acid: CBGVA to THCVA; CBGVA to CBDVA; CBGVA to CBCVA; THCVA to THCV; CBDVA to CBDV; or CBCVA to CBCV.

In some cases, the modified cell is capable of producing a substrate capable of being converted into a CBGVA or a cannabinoid, however, the cells is not capable of naturally producing a cannabinoid. The genetically modified microorganisms in some cases are unable to produce a substrate capable of being converted into a CBGVA or a cannabinoid (for example, butyric acid), and the substrate capable of being converted into a CBGVA or a cannabinoid is provided to the cells as part of the cell's growth medium. In this case, the genetically modified microorganism can process the substrate into a desired product such as THCVA, CBDVA, or CBCVA.

Enzymes

The cells disclosed can be genetically modified with one or more enzymes that are capable of producing CBGA or CBGVA or a cannabinoid, and other pathway intermediates such as olivetolic acid. The cells disclosed can also be genetically modified with one or more enzymes that are capable of assisting in or enhancing the ability of the cell to produce CBGA or CBGVA or a cannabinoid, and other pathway intermediate (as disclosed throughout).

The cell can be modified to include an enzyme that can perform any one of the following reactions: hexanoic acid to hexanoyl-CoA, hexanoyl-CoA to olivetolic Acid; olivetolic Acid to CBGA; CBGA to THCA; CBGA to CBDA; CBGA to CBCA; THCA to THC; CBDA to CBD; or CBCA to CBC. For example, the cell can be modified with one or more of the following enzymes: polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); or any combination thereof. Additional enzymes that can be included include but are not limited to HMG-CoA reductase, ERG20 reductase, or both. These enzymes can either be endogenous to the cell or heterologous. However, in some cases, even if the enzyme is endogenous, it can be made to be overexpressed. The heterologous enzymes can also be overexpressed.

In some cases, two or more consecutive enzymes in the pathway from a carbon substrate (e.g., sugar) to any of the cannabinoids described throughout (e.g., THCA, CBDA, CBCA, THC, CBD, CBC, CBGVA, THCVA, CBDVA, CBCVA) can be used. In some cases, three or more consecutive enzymes in the pathway can be used. In some cases, four or more consecutive enzymes in the pathway can be used. In some cases, five or more consecutive enzymes in the pathway can be used. In some cases, six or more consecutive enzymes in the pathway can be used. In some cases, seven or more consecutive enzymes in the pathway can be used. In some cases, eight or more consecutive enzymes in the pathway can be used. In some cases, nine or more consecutive enzymes in the pathway can be used. In some cases, ten or more consecutive enzymes in the pathway can be used.

In some cases, when an acyl activating enzyme (AAE1) is desired, the AAE1 can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 50% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 55% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 60% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 65% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 70% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 75% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 81% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 82% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 83% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 84% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 85% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 86% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 87% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 88% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 89% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 90% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 91% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 92% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 93% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 94% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 95% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 96% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 97% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 98% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is at least 99% identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by an amino acid sequence that is identical to SEQ ID NO: 13. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell.

In some cases when a polyketide synthase (PKS) is desired, the PKS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 50% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 55% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 60% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 65% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 70% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 75% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 80% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 81% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 82% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 83% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 84% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 85% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 86% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 87% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 88% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 89% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 91% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 92% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 93% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 94% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 96% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 97% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 98% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is at least 99% identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by an amino acid sequence that is identical to SEQ ID NO: 5. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell.

In some cases when an olivetolic acid cyclase (OAC) is desired, the OAC can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 50% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 55% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 60% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 65% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 70% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 75% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 80% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 81% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 82% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 83% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 84% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 85% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 86% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 87% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 88% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 89% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 91% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 92% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 93% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 94% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 96% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 97% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 98% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is at least 99% identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by an amino acid sequence that is identical to SEQ ID NO: 7. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell.

In some cases when a prenyltransferase (PT) is desired, the PT can be encoded by an amino acid sequence that is substantially identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 50% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 55% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 60% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 65% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 70% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 75% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 80% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 81% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 82% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 83% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 84% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 85% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 86% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 87% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 88% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 89% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 90% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 91% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 92% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 93% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 94% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 95% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 96% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 97% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 98% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be at least 99% identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence encoding a prenyltransferase can be identical to any one of SEQ ID NOs: 1, 27, 32, 38 or 320-379. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell.

Additionally, other enzymes can be used to make different products. These enzymes can include a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof.

In some cases, when a THCA synthase (THCAS) is desired, the THCAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 50% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 55% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 60% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 65% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 70% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 75% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 80% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 81% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 82% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 83% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 84% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 85% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 86% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 87% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 88% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 89% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 90% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 91% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 92% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 93% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 94% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 95% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 96% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 97% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 98% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be at least 99% identical to SEQ ID NO: 9. In some cases, the amino acid sequence encoding a THCAS can be identical to SEQ ID NO: 9. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell. The use of a THCAS, in some cases, can result in the enzymatic synthesis of Δ9-tetrahydrocannabinol (THC) and the accumulation of THC within the cell or culture medium.

In some cases, when a CBDA synthase (CBDAS) is desired, the CBDAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 50% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 55% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 60% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 65% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 70% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 75% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 80% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 81% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 82% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 83% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 84% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 85% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 86% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 87% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 88% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 89% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 90% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 91% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 92% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 93% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 94% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 95% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 96% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 97% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 98% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be at least 99% identical to SEQ ID NO: 11. In some cases, the amino acid sequence encoding a CBDAS can be identical to SEQ ID NO: 11. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell. The use of a CBDAS in some cases can result in the enzymatic synthesis of cannabidiol (CBD) and the accumulation of CBD within the cell or culture medium.

In some cases, when a CBCA synthase (CBCAS) is desired, the CDCS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 50% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 55% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 60% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 65% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 70% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 75% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 80% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 81% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 82% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 83% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 84% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 85% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 86% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 87% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 88% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 89% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 90% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 91% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 92% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 93% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 94% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 95% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 96% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 97% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 98% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be at least 99% identical to SEQ ID NO: 17. In some cases, the amino acid sequence encoding a CBCAS can be identical to SEQ ID NO: 17. In some cases, the amino acid sequence can be optimized to correspond to amino acid usage within a specific host organism/cell. The use of a CBCAS in some cases can result in the enzymatic synthesis of cannabichromene (CBC) and the accumulation of CBC within the cell or culture medium.

The various combinations of enzymes can be used to make a desired product such as olivetolic acid; CBGA; THCA; CBDA; CBCA; THC; CBD; CBC, or any combination thereof.

The enzymes disclosed throughout can be from a plant. For example, the enzymes can be from a plant that is from the genus Cannabis. More specifically, Cannabis plants that can be used include, but are not limited to Cannabis sativa, Cannabis indica, and Cannabis ruderalis. Other plants that can be used can be from the genus Echinacea, Acmella (e.g., Acmella oleracea), Helichrysum (e.g., Helichrysum umbraculigerum), Radula (e.g., Radula marginata), Theobroma (e.g., Theobroma cacao), and/or Piper (e.g., Piper nigrum).

Additional enzymes can be added in order to improve the production of CBGA or cannabinoids. For example, a gene encoding an HMG-CoA reductase, such as HMG1, can be used to increase cannabinoid titers. In some instances, the titer of CBGA can be increased by expressing HMG1. Additionally, HMG1 can be in different forms. For example, a truncated form of HMG1 can be used to increase cannabinoid titers. Other enzymes such as Farnesyl pyrophosphate synthetase, which is encoded by the gene ERG20 can be used to increase cannabinoid/CBGA titers. Additionally, ERG20 can be in different forms, such as mutant forms.

In cases where a HMG-CoA reductase (HMG1) is desired, the HMG1 can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 19 or 21. For example, the HMG1 can be encoded by an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 50% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 60% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 65% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 70% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 75% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 80% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 81% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 82% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 83% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 84% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 85% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 86% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 87% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 88% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 89% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 90% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 91% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 92% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 93% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 94% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 95% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 96% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 97% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 98% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be at least 99% identical to SEQ ID NO: 19 or 21. In some cases, the HMG1 can be identical to SEQ ID NO: 19 or 21. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a farnesyl pyrophosphate synthetase (ERG20) is used, the ERG20 can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 23. For example, the ERG20 can be encoded by an amino acid sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 23. In some cases, the ERG20 can be at least 50% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 60% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 65% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 70% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 75% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 80% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 81% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 82% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 83% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 84% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 85% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 86% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 87% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 88% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 89% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 90% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 91% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 92% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 93% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 94% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 95% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 96% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 97% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 98% identical to SEQ ID NO: 23. In some cases, the ERG20 can be at least 99% identical to SEQ ID NO: 23. In some cases, the ERG20 can be identical to SEQ ID NO: 23. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In some cases, the enzymes described herein can be a fragment thereof. The fragment can still retain its respective biological activity. For example, a fragment of the prenyltransferase can be used as long as the activity of the fragment retains its biological activity.

The enzymes or fragments thereof described throughout can also be in some cases can be fused or linked together. Any fragment linker can be used to link the two or more of the enzymes or fragments thereof together. In some cases, the linker can be any random array of amino acid sequences. In some cases, linkers such as the T2A linker (SEQ ID NO: 15 (amino acid) or 16 (nucleic acid)) can be used.

The fused or linked enzymes can be two or more of any of the enzymes described throughout. For example, the disclosed prenyltransferase can be linked with a CBDA synthase. The resulting fused or linked enzyme can produce increased cannabidiol titers compared to separate enzymes that are not linked or fused. Additionally, other enzymes such as prenyltransferase and THCA synthase can be fused or linked. The resulting fused or linked enzyme can produce increased THC titers compared to separate enzymes that are not linked or fused. Enzymes that can catalyze the product of another enzyme can be fused or linked. For example AAE1 can be fused or linked to PKS. In some cases, OAC can be fused or linked to PKS. This can in some cases, increase the speed of two or more enzymatic conversions due to the proximity of the enzymatic substrates/products.

Vectors

Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host can typically, but not always, comprise a replication system (i.e. vector) recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and can but not necessarily, also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (such as expression vectors) can include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, nucleotide sequences homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides can also be included where appropriate, for example from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

The vectors can be constructed using standard methods (see, e.g., Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. 1989; and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, 1995).

The manipulation of polynucleotides that encode the enzymes disclosed herein is typically carried out in recombinant vectors. Numerous vectors are publicly available, including bacterial plasmids, bacteriophage, artificial chromosomes, episomal vectors and gene expression vectors, which can all be employed. A vector can be selected to accommodate a polynucleotide encoding a protein of a desired size. Following recombinant modification of a selected vector, a suitable host cell (e.g., the microorganisms described herein) is transfected or transformed with the vector. Each vector contains various functional components, which generally include a cloning site, an origin of replication and at least one selectable marker gene. A vector can additionally possess one or more of the following elements: an enhancer, promoter, and transcription termination and/or other signal sequences. Such sequence elements can be optimized for the selected host species. Such sequence elements can be positioned in the vicinity of the cloning site, such that they are operatively linked to the gene encoding a preselected enzyme.

Vectors, including cloning and expression vectors, can contain nucleic acid sequences that enable the vector to replicate in one or more selected microorganisms. For example, the sequence can be one that enables the vector to replicate independently of the host chromosomal DNA and can include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV40, adenovirus) are useful for cloning vectors.

A cloning or expression vector can contain a selection gene (also referred to as a selectable marker). This gene encodes a protein necessary for the survival or growth of transformed microorganisms in a selective culture medium. Microorganisms not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromycin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

The replication of vectors can be performed in E. coli. An E. coli-selectable marker, for example, the β-lactamase gene that confers resistance to the antibiotic ampicillin, can be of use. These selectable markers can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.

Some exemplary vectors that can be used in the methods and microorganisms/cells are SEQ ID NO: 3 and 4. SEQ ID NO: 3 is also called the RUNM000898_511.1 vector, which comprises a Saccharomyces cerevisiae 2μ replication origin, a URA3 gene as an auxotrophic marker and the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter. SEQ ID NO: 4 is the bCBGA0098 vector that comprises a Saccharomyces cerevisiae 2μ replication origin, a LEU2 gene as an auxotrophic marker, and the AAE1 and PT genes under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 25 is a bCBGA0306 is a vector that comprises the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the PT gene under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 34 is the RUNM001233_51.1 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and the THCA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 35 is the RUNM001210_96.1 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker, the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the STE5 promoter.

SEQ ID NO: 36 is the bCBGA0409 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker, the THCA synthase and PT genes under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 29 is the bCBGA0385 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the GFP-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 30 is the bCBGA0305 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the TRP1 gene as an auxotrophic marker and the AAE1 gene under the regulation of the bidirectional GAL1/GAL10 promoter.

SEQ ID NO: 33 is the bCBGA0559 vector comprising the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the ERG20mut-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter.

Promoters

Vectors can contain a promoter that is recognized by the host microorganism. The promoter can be operably linked to a coding sequence of interest. Such a promoter can be inducible or constitutive. Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.

Different promoters can be used to drive the expression of the genes. For example, if temporary gene expression (i.e., non-constitutively expressed) is desired, expression can be driven by inducible promoters.

In some cases, some of the genes disclosed can be expressed temporarily. In other words, the genes are not constitutively expressed. The expression of the genes can be driven by inducible or repressible promoters. For example, the inducible or repressible promoters that can be used include but are not limited to: (a) sugars such as arabinose and lactose (or non metabolizable analogs, e.g., isopropyl β-D-1-thiogalactopyranoside (IPTG)); (b) metals such as lanthanum, copper, calcium; (c) temperature; (d) Nitrogen-source; (e) oxygen; (f) cell state (growth or stationary); (g) metabolites such as phosphate; (h) CRISPRi; (i) jun; (j) fos, (k) metallothionein and/or (l) heat shock.

Constitutively expressed promoters can also be used in the vector systems herein. For example, the expression of some of the genes disclosed throughout can be controlled by constitutively active promoters. For examples, the promoters that can be used include but are not limited to p.Bba.J23111, J23111, and J23100.

Promoters suitable for use with prokaryotic hosts can, for example, include but are not limited to the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Dalgarno sequence operably linked to the coding sequence.

Generally, a strong promoter can be employed to provide for high level transcription and expression of the desired product.

One or more promoters of a transcription unit can be an inducible promoter. For example, a GFP can be expressed from a constitutive promoter while an inducible promoter drives transcription of a gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.

Some vectors can contain prokaryotic sequences that facilitate the propagation of the vector in bacteria. Thus, the vectors can have other components such as an origin of replication (e.g., a nucleic acid sequence that enables the vector to replicate in one or more selected microorganisms), antibiotic resistance genes for selection in bacteria, and/or an amber stop codon which can permit translation to read through the codon. Additional selectable gene(s) can also be incorporated. Generally, in cloning vectors the origin of replication is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences can include the ColEl origin of replication in bacteria or other known sequences.

Genes

The genetically modified microorganisms can comprise a nucleic acid sequence encoding for one or more enzymes that are capable of catalyzing one or more of the following reactions: hexanoic acid to hexanoyl-CoA; hexanoyl-CoA to olivetolic Acid; olivetolic Acid to CBGA; CBGA to THCA; CBGA to CBDA; CBGA to CBCA; THCA to THC; CBDA to CBD; or CBCA to CBC. For example, the genetically modified microorganism can comprise a nucleic acid sequence encoding for one or more of the following enzymes: acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); or any combination thereof. The nucleic acid sequence in some cases can be within a vector. In some cases, the nucleic acid sequences do not need to be within a vector but rather integrated into the microorganism's genome or isolated. In some cases, the isolated nucleic acids can be inserted into the genome of the cell/microorganism used. In some cases, the isolated nucleic acid is inserted into the genome at a specific locus, where the isolated nucleic acid can be expressed in sufficient amounts.

In some cases, two or more genes encoding for consecutive enzymes in the pathway from a carbon substrate (e.g., sugar) to any of the cannabinoids described throughout (e.g., THCA, CBDA, CBCA, THC, CBD, or CBC) can be used. In some cases, three or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, four or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, five or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, six or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, seven or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, eight or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, nine or more genes encoding for consecutive enzymes in the pathway can be used. In some cases, ten or more genes encoding for consecutive enzymes in the pathway can be used.

In some cases, when an acyl activating enzyme (AAE1) is desired, the AAE1 can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 14. For example, the AAE1 can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 50% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 60% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 65% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 70% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 75% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 80% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 81% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 82% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 83% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 84% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 85% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 86% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 87% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 88% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 89% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 90% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 91% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 92% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 93% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 94% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 95% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 96% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 97% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 98% identical to SEQ ID NO: 14. In some cases, the AAE1 can be at least 99% identical to SEQ ID NO: 14. In some cases, the AAE1 can be identical to SEQ ID NO: 14. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a polyketide synthase (PKS) is used, the PKS can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 6. For example, the PKS can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 50% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 60% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 65% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 70% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 75% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 80% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 81% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 82% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 83% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 84% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 85% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 86% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 87% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 88% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 89% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 90% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 91% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 92% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 93% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 94% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 95% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 96% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 97% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 98% identical to SEQ ID NO: 6. In some cases, the PKS can be at least 99% identical to SEQ ID NO: 6. In some cases, the PKS can be identical to SEQ ID NO: 6. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where an olivetolic acid cyclase (OAC) is used, the OAC can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 8. For example, the OAC can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 50% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 60% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 65% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 70% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 75% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 80% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 81% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 82% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 83% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 84% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 85% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 86% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 87% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 88% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 89% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 90% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 91% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 92% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 93% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 94% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 95% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 96% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 97% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 98% identical to SEQ ID NO: 8. In some cases, the OAC can be at least 99% identical to SEQ ID NO: 8. In some cases, the OAC can be identical to SEQ ID NO: 8. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a prenyltransferase (PT) is used, the PT can be encoded by a nucleic acid sequence that is substantially identical to any one of SEQ ID NOs: 2, 26, 31, or 37. For example, the PT can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 50% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 60% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 65% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 70% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 75% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 80% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 81% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 82% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 83% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 84% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 85% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 86% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 87% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 88% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 89% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 90% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 91% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 92% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 93% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 94% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 95% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 96% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 97% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 98% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be at least 99% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the PT can be identical to any one of SEQ ID NOs: 2, 26, 31, or 37. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a THCA synthase (THCAS) is used, the THCAS can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 10. For example, the THCAS can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 50% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 60% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 65% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 70% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 75% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 80% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 81% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 82% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 83% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 84% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 85% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 86% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 87% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 88% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 89% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 90% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 91% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 92% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 93% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 94% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 95% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 96% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 97% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 98% identical to SEQ ID NO: 10. In some cases, the THCAS can be at least 99% identical to SEQ ID NO: 10. In some cases, the THCAS can be identical to SEQ ID NO: 10. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a CBDA synthase (CBDAS) is used, the CBDAS can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 12. For example, the CBDAS can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 50% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 60% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 65% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 70% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 75% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 80% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 81% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 82% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 83% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 84% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 85% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 86% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 87% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 88% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 89% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 90% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 91% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 92% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 93% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 94% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 95% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 96% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 97% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 98% identical to SEQ ID NO: 12. In some cases, the CBDAS can be at least 99% identical to SEQ ID NO: 12. In some cases, the CBDAS can be identical to SEQ ID NO: 12. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a CBCA synthase (CBCAS) is used, the CBCAS can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 18. For example, the CBCAS can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 50% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 60% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 65% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 70% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 75% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 80% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 81% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 82% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 83% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 84% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 85% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 86% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 87% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 88% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 89% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 90% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 91% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 92% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 93% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 94% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 95% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 96% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 97% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 98% identical to SEQ ID NO: 18. In some cases, the CBCAS can be at least 99% identical to SEQ ID NO: 18. In some cases, the CBCAS can be identical to SEQ ID NO: 18. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

The genetically modified microorganism can also further comprise one or more nucleic acids encoding for enzymes (in some cases heterologous enzymes), including but not limited to HMG1, ERG20, and/or isoforms and mutants thereof.

In cases where a HMG-CoA reductase (HMG1) is used, the HMG1 can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 20 or 22. For example, the HMG1 can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 50% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 60% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 65% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 70% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 75% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 80% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 81% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 82% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 83% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 84% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 85% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 86% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 87% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 88% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 89% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 90% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 91% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 92% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 93% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 94% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 95% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 96% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 97% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 98% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be at least 99% identical to SEQ ID NO: 20 or 22. In some cases, the HMG1 can be identical to SEQ ID NO: 20 or 22. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

In cases where a farnesyl pyrophosphate synthetase (ERG20) is used, the ERG20 can be encoded by a nucleic acid sequence that is substantially identical to SEQ ID NO: 24. For example, the ERG20 can be encoded by a polynucleotide that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 50% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 60% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 65% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 70% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 75% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 80% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 81% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 82% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 83% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 84% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 85% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 86% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 87% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 88% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 89% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 90% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 91% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 92% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 93% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 94% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 95% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 96% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 97% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 98% identical to SEQ ID NO: 24. In some cases, the ERG20 can be at least 99% identical to SEQ ID NO: 24. In some cases, the ERG20 can be identical to SEQ ID NO: 24. Further, codon optimized polynucleotides (for a particular host cell/organism) for the above referenced sequences can be used herein.

Modifying Endogenous Gene Expression

The genetically modified microorganisms disclosed herein can have their endogenous genes regulated. This can be useful, for example, when there is negative feedback to the expression of a desired polypeptide, such as any of the enzymes described throughout including but not limited to acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); HMG-CoA reductase (HMG1); farnesyl pyrophosphate synthetase (ERG20); or any combination thereof. Modifying one or more negative regulator can lead to increased expression of a desired polypeptide, and in some cases, increase the production level of the cannabinoids.

Modifying the expression of endogenous genes can be achieved in a variety of ways. For example, antisense or RNA interference approaches can be used to down-regulate expression of the polynucleotides of the present disclosure, e.g., as a further mechanism for modulating cellular phenotype. That is, antisense sequences of the polynucleotides of the present disclosure, or subsequences thereof, can be used to block expression of naturally occurring homologous polynucleotide sequences. In particular, constructs comprising a desired polypeptide coding sequence, including fragments thereof, in antisense orientation, or combinations of sense and antisense orientation, can be used to decrease or effectively eliminate the expression of the desired polypeptide in a cell or plant and obtain an improvement in shelf life as is described herein. Accordingly, this can be used to “knock-out” the desired polypeptide or homologous sequences thereof. A variety of sense and antisense technologies, e.g., as set forth in Lichtenstein and Nellen (Antisense Technology: A Practical Approach IRL Press at Oxford University, Oxford, England, 1997), can be used. Sense or antisense polynucleotide can be introduced into a cell, where they are transcribed. Such polynucleotides can include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.

Other methods for a reducing or eliminating expression (i.e., a “knock-out” or “knockdown”) of a desired polypeptide in a transgenic cell or plant can be done by introduction of a construct which expresses an antisense of the desired polypeptide coding strand or fragment thereof. For antisense suppression, the desired polypeptide cDNA or fragment thereof is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. Further, the introduced sequence need not always correspond to the full length cDNA or gene, and need not be identical to the cDNA or gene found in the cell or plant to be transformed.

Additionally, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced polynucleotide sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, in some embodiments, the introduced antisense polynucleotide sequence in the vector is at least 10, 20, 30, 40, 50, 100 or more nucleotides in length in certain embodiments. Transcription of an antisense construct as described results in the production of RNA molecules that comprise a sequence that is the reverse complement of the mRNA molecules transcribed from the endogenous gene to be repressed.

Other methods for a reducing or eliminating expression can be done by introduction of a construct that expresses siRNA that targets a desired polypeptide (e.g., CBGA synthesis polypeptide). In certain embodiments, siRNAs are short (20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5′ ends and hydroxylated 3′ ends with two overhanging nucleotides.

Other methods for a reducing or eliminating expression can be done by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens or a selection marker cassette or any other non-sense DNA fragments. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in the CBGA synthesis polypeptide (or other desired polypeptide) gene. Plants containing one or more transgene insertion events at the desired gene can be crossed to generate homozygous plant for the mutation, as described in Koncz et al., (Methods in Arabidopsis Research; World Scientific, 1992).

Suppression of gene expression can also be achieved using a ribozyme. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508. Synthetic ribozyme sequences including antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that hybridize to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

A cell or plant gene can also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A cellular or plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

In addition, silencing approach using short hairpin RNA (shRNA) system, and complementary mature CRISPR RNA (crRNA) by CRISPR/Cas system, and virus inducing gene silencing (VIGS) system can also be used to make down regulated or knockout of synthase mutants. Dominant negative approaches can also be used to make down regulated or knockout of desired polypeptides.

The RNA-guided endonuclease can be derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. The CRISPR/Cas system can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cask), Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966.

In general, CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.

The CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein. The CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzyme activity, and/or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR/Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the function of the fusion protein. The CRISPR/Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.

One method to silence a desired gene (or a CBGA synthesis polypeptide gene) is virus induced gene silencing (known to the art as VIGS). In general, in plants infected with unmodified viruses, the viral genome is targeted. However, when viral vectors have been modified to carry inserts derived from host genes (e.g. portions of sequences encoding a desired polypeptide such as CBGA synthesis polypeptide), the process is additionally targeted against the corresponding mRNAs. Thus disclosed is a method of producing a plant expressing reduced levels of a desired gene (such as CBGA synthesis polypeptide) or other desired gene(s), the method comprising (a) providing a plant expressing a desired gene (e.g., a CBGA synthesis polypeptide); and (b) reducing expression of the desired gene in the plant using virus induced gene silencing.

In some cases, one or more genes can be disrupted. In some cases, the one or more genes can be from the pathway that controls beta oxidation of long chain fatty acids. For example, in some cases, the one or more genes that can be disrupted can be any one of FOX1, FAA1, FAA4, FAT1, PXA1, PXA2, and/or PEX11. Any of the methods described throughout, can be used to disrupt one or more of the genes.

In some cases, the one or more genes that can be disrupted can comprise FOX1. For example, a sequence that is substantially identical to SEQ ID NO: 39 can be targeted for disruption. Any of the methods described throughout, can be used to disrupt the FOX1 gene, for example, but use of the CRISPR/Cas system or the use of RNAi technology. As few as a single nucleotide needs to be altered to have a disruptive effect to FOX1 or other genes that are targeted for disruption.

Isolated Polynucleic Acids

The genes described throughout can be in the form of an isolated polynucleic acid. In other words, the genes can be in forms that do not exist in nature, isolated from a chromosome. The isolated polynucleic acids can comprise a nucleic acid sequence of one or more genes encoding a: (i) acyl activating enzyme (AAE1); (ii) polyketide synthase (PKS); (iii) olivetolic acid cyclase (OAC); (iv) prenyltransferase (PT); (v) THCA synthase (THCAS); (vi) CBDA synthase (CBDAS); and/or (vii) CBCA synthase (CBCAS). For example, the isolated polynucleic acid can comprise a PKS gene. The isolated polynucleic acid can comprise an OAC gene. The isolated polynucleic acid can comprise a PT gene. The isolated polynucleic acid can comprise a THCAS gene. The isolated polynucleic acid can comprise a CBDAS gene. The isolated polynucleic acid can comprise a CBCAS gene. The isolated polynucleic acid can comprise an AAE1 gene.

In some cases, the isolated polynucleic acid can encode an acyl activating enzyme (AAE1). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 14. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 14.

In some cases, the isolated polynucleic acid can encode a polyketide synthase (PKS). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 6. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 6.

In some cases, the isolated polynucleic acid can encode an olivetolic acid cyclase (OAC). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 8. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 8.

In some cases, the isolated polynucleic acid can encode a prenyltransferase (PT). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to any one of SEQ ID NOs: 2, 26, 31, or 37. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to any one of SEQ ID NOs: 2, 26, 31, or 37.

In some cases, the isolated polynucleic acid can encode a THCA synthase (THCAS). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 10. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 10.

In some cases, the isolated polynucleic acid can encode a CBDA synthase (CBDAS). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 12. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 12.

In some cases, the isolated polynucleic acid can encode a CBCA synthase (CBCAS). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 18. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 18.

In some cases, the isolated polynucleic acid can encode a HMG-CoA reductase (HMG1). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 50% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 20 or 22. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is identical to SEQ ID NO: 20 or 22.

In some cases, the isolated polynucleic acid can encode a farnesyl pyrophosphate synthetase (ERG20). For example, the isolated polynucleic acid can comprise a nucleotide sequence that is substantially identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 50% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 60% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 65% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 70% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 75% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 80% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 81% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 82% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 83% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 84% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 85% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 86% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 87% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 88% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 89% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 90% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 91% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 92% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 93% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 94% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 95% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 96% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 97% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 98% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can comprise a nucleotide sequence that is at least 99% identical to SEQ ID NO: 24. In some cases, the isolated polynucleic acid can be identical to SEQ ID NO: 24.

Methods of Making Genetically Modified Microorganisms

Disclosed herein is a method of making a genetically modified microorganism capable of converting a carbon substrate into CBGA. Also disclosed herein is a method of making a genetically modified microorganism capable of converting a carbon substrate into a cannabinoid.

In some cases, the microorganism can be made by contacting the microorganism with one or more polynucleotides. The polynucleotides can be a vector. The polynucleotides can also comprise one or more genes encoding for an enzymes.

In some cases, the microorganism can be grown so that the polynucleotides are inserted into the microorganism. In some cases, the insertion can be done any method, e.g., transfections, transformation, etc. The insertion of the microorganism can be by plasmid or in some cases the insertion can lead to a stable integration of the plasmid into the chromosome of the microorganism.

The genes encoding for an enzymes can include (i) acyl activating enzyme (AAE1); (ii) polyketide synthase (PKS); (iii) olivetolic acid cyclase (OAC); (iv) prenyltransferase (PT); (v) THCA synthase (THCAS); (vi) CBDA synthase (CBDAS); and/or (vii) CBCA synthase (CBCAS). In some further cases, the genes encoding for an enzyme can include (viii) a HMG-Co reductase (HMG1) and/or (ix) a farnesyl pyrophosphate synthetase (ERG20).

In some cases, the microorganism can be contacted with a polynucleotide that encodes for a prenyltransferase (PT). In some cases, the PT can be encoded by a nucleotide sequence that is substantially identical to SEQ ID NO: 2. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 1. In some cases, the PT can be encoded by a nucleotide sequence that is substantially identical to SEQ ID NO: 26. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 27. In some cases, the PT can be encoded by a nucleotide sequence that is substantially identical to SEQ ID NO: 31. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 32. In some cases, the PT can be encoded by a nucleotide sequence that is substantially identical to SEQ ID NO: 37. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 38.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for an AAE1. In some cases, the AAE1 is encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 14. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 13.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for a PKS. In some cases, the PKS encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 6. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 5.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for an OAC. In some cases, the OAC encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 8. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 7.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for a THCAS. In some cases, the THCAS encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 10. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 9.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for a CBDAS. In some cases, the CBDAS encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 12. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 11.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for a CBCAS. In some cases, the CBCAS encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 18. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 17.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for an HMG-Co reductase (HMG1). In some cases, the HMG1 encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 20 or 22. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 19 or 21.

In some cases, the microorganism can also be contacted with a polynucleotide that encodes for a farnesyl pyrophosphate synthetase (ERG20). In some cases, the ERG20 encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 24. In some cases, the polynucleotide can be translated into an amino acid sequence that is substantially identical to SEQ ID NO: 23.

The microorganism can be any type of microorganism that is disclosed throughout. For example, the microorganism can be a bacterium or a yeast.

The cannabinoid that can be made can be one or more of the following: cannabinoid is Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), or any combination thereof.

Exemplary Genetically Modified Microorganisms

Disclosed herein is a genetically modified microorganism capable of converting a carbon substrate into CBGA, CBGVA or a cannabinoid.

The genetically modified microorganism can comprise a heterologous polynucleotide encoding an acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); and/or prenyltransferase (PT). In some cases, two or more polynucleotides encoding AAE1, PKS, OAC, and/or PT can be present within the genetically modified microorganism. In some cases, three of the polynucleotides encoding AAE1, PKS, OAC, and/or PT can be present within the genetically modified microorganism. In some cases, all four of the polynucleotides encoding AAE1, PKS, OAC, and PT can be present within the genetically modified microorganism.

Additionally, the genetically modified microorganism can further comprise polynucleotides encoding for a THCA synthase (THCAS); a CBDA synthase (CBDAS), a CBCA synthase (CBCAS), an HMG-Co reductase (HMG1) and/or a farnesyl pyrophosphate synthetase (ERG20). In some cases, the polynucleotides can be heterologous. In some cases, two or more polynucleotides encoding THCAS, CBDAS, CBCAS, HMG1, and/or ERG20 can be present within the genetically modified microorganism. In some cases, three or more of the polynucleotides encoding THCAS, CBDAS, CBCAS, HMG1, and/or ERG20 can be present within the genetically modified microorganism.

Should an AAE1 be present within the genetically modified microorganism, the AAE1 can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 13. In some cases, the AAE1 can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 14.

Should a PKS be present within the genetically modified microorganism, the PKS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 5. In some cases, the PKS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 6.

Should an OAC be present within the genetically modified microorganism, the OAC can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 7. In some cases, the OAC can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 8.

Should a PT be present within the genetically modified microorganism, the PT can be encoded by an amino acid sequence that is substantially identical to any one of SEQ ID NOs: 1, 27, 32, or 38. In some cases, the PT can be encoded by a polynucleotide sequence that is substantially identical to any one of SEQ ID NOs: 2, 26, 31, or 37.

Should a THCAS be present within the genetically modified microorganism, the THCAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 9. In some cases, the THCAS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 10.

Should a CBDAS be present within the genetically modified microorganism, the CBDAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 11. In some cases, the CBDAS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 12.

Should a CBCAS be present within the genetically modified microorganism, the CBCAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 17. In some cases, the CBCAS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 18.

Should a HMG1 be present within the genetically modified microorganism, the CBCAS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 19 or 21. In some cases, the CBCAS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 20 or 22.

Should an ERG20 be present within the genetically modified microorganism, the ERG20 can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO: 23. In some cases, the ERG20 can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 24.

Should a TKS (OS) be present within the genetically modified microorganism, the TKS can be encoded by an amino acid sequence that is substantially identical to SEQ ID NO:41. In some cases, the TKS can be encoded by a polynucleotide sequence that is substantially identical to SEQ ID NO: 40.

In certain cases, the genetically modified microorganism can be a yeast or bacterium. Should the genetically modified microorganism be a yeast, the yeast can be from the genus Saccharomyces. More specifically, the yeast can be from the species Saccharomyces cerevisiae. Should the genetically modified microorganism be a bacterium, the bacterium can be from the genus Escherichia, e.g., Escherichia coli.

The genetically modified microorganism can use hexanoic acid and/or butyric acid. In some cases, the genetically modified microorganism can use sugar as a substrate. In some cases, the genetically modified microorganism can make a CBGA, CBGVA, THCA, CBDA, CBCA or a cannabinoid. If a cannabinoid is made, in some cases, the cannabinoid can be 49-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), and/or cannabichromevarinic acid (CBCVA).

Fermentation Methods and Processes

In general, the microorganisms disclosed herein should be placed in fermentation conditions that are appropriate to convert a carbon source (such as a sugar, alcohol, and/or fatty acid) to CBGA, CBGVA or a cannabinoid (e.g., THC, CBD, CBC, THCVA, CBDVA, CBCVA). Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition.

In some cases, non-genetically modified microorganisms can be used to increase CBGA or cannabinoid production. For example, cells taken from organisms that naturally produce cannabinoids can be used. These cells can be isolated and once isolated they can be used in a fermentation process.

Fermentation Conditions

The fermentation conditions described herein are applicable to any and all methods disclosed throughout the application.

pH can have a profound effect on overall CBGA, CBGVA or cannabinoid production. Therefore, pH adjustments should be made in some cases.

In some cases, the pH during fermentation can vary from 4 to 10. In some instances, the pH can be from 5 to 9; 6 to 8; 6.1 to 7.9; 6.2 to 7.8; 6.3 to 7.7; 6.4 to 7.6; or 6.5 to 7.5. For example, the pH can be from 6.6 to 7.4. In some instances, the pH can be from 5 to 9. In some instances, the pH can be from 6 to 8. In some instances, the pH can be from 6.1 to 7.9. In some instances, the pH can be from 6.2 to 7.8. In some instances, the pH can be from 6.3 to 7.7. In some instances, the pH can be from 6.4 to 7.6. In some instances, the pH can be from 5.5 to 7.5. In some instances, the pH can be from 6.5 to 7.5. In some instances the pH used for the fermentation can be greater than 6. In some instances the pH used for the fermentation can be lower than 10.

Temperature

Temperature can also be adjusted based on cell, microorganism, or enzyme sensitivity. For example, the temperature used during fermentation, can from 27° C. to 45° C. In other instances, the temperature of the fermentation can be from 27° C. to 45° C.; 28° C. to 44° C.; 29° C. to 43° C.; 30° C. to 42° C.; 31° C. to 41° C.; 32° C. to 40° C. For example, the temperature can be from 36° C. to 39° C. (e.g., 36° C., 37° C., 38° C., or 39° C. In some instances, the temperature can be from 27° C. to 45° C. In some instances, the temperature can be from 28° C. to 44° C. In some instances, the temperature can be from 29° C. to 43° C. In some instances, the temperature can be from 30° C. to 42° C. In some instances, the temperature can be from 31° C. to 41° C. In some instances, the temperature can be from 32° C. to 40° C.

Gases

Availability of oxygen and other gases such as gaseous CO₂can affect yield and fermentation rate. For example, when considering oxygen availability, the percent of dissolved oxygen (DO) within the fermentation media can be from 1% to 40%. In certain instances, the DO concentration can be from 1.5% to 35%; 2% to 30%; 2.5% to 25%; 3% to 20%; 4% to 19%; 5% to 18%; 6% to 17%; 7% to 16%; 8% to 15%; 9% to 14%; 10% to 13%; or 11% to 12%. For example, in some cases the DO concentration can be from 2% to 30%. In other cases, the DO can be from 3% to 20%. In some instances, the DO can be from 4% to 10%. In some cases, the DO can be from 1.5% to 35%. In some cases, the DO can be from 2.5% to 25%. In some cases, the DO can be from 4% to 19%. In some cases, the DO can be from 5% to 18%. In some cases, the DO can be from 6% to 17%. In some cases, the DO can be from 7% to 16%. In some cases, the DO can be from 8% to 15%. In some cases, the DO can be from 9% to 14%. In some cases, the DO can be from 10% to 13%. In some cases, the DO can be from 11% to 12%.

For example, when considering atmospheric CO₂, the percent of atmospheric CO₂within an incubator can be from 0% to 10%. In some cases, atmospheric CO₂can help to control the pH within cell culture medium. pH contain within cell culture media is dependent on a balance of dissolved CO₂and bicarbonate (HCO₃). Changes in atmospheric CO₂can alter the pH of the medium. In certain instances, the atmospheric CO₂can be from 0% to 10%; 0.01% to 9%; 0.05% to 8%; 0.1% to 7%; 0.5% to 6%; 1% to 5%; 2% to 4%; 3% to 6%; 4% to 7%; 2% to 6%; or 5% to 10%. For example, in some cases the atmospheric CO₂can be from 0% to 10%. In other cases, the atmospheric CO₂can be from 0.01% to 9%. In some instances, the atmospheric CO₂can be from 0.05% to 8%. In some cases, the atmospheric CO₂can be from 0.1% to 7%. In some cases, the atmospheric CO₂can be from 0.5% to 6%. In some cases, the atmospheric CO₂can be from 1% to 5%. In some cases, the atmospheric CO₂can be from 2% to 4%. In some cases, the atmospheric CO₂can be from 3% to 6%. In some cases, the atmospheric CO₂can be from 4% to 7%. In some cases, the atmospheric CO₂can be from 2% to 6%. In some cases, the atmospheric CO₂can be from 5% to 10%.

Bioreactors

Fermentation reactions can be carried out in any suitable bioreactor. In some embodiments of the invention, the bioreactor can comprise a first, growth reactor in which the microorganisms or cells are cultured, and a second, fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product (for example, CBGA or cannabinoids) is produced.

Media

The medium used to ferment CBGA, CBGVA or cannabinoid with the microorganisms described throughout can include hexanoic acid and/or butyric acid. For example, in some cases, the media can comprise a combination of hexanoic acid, yeast extract, peptone, and glucose. In other cases, the media can comprise a combination of butyric acid, yeast extract, peptone, and glucose. In certain cases, the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid or butyric acid. In some cases, hexanoic acid or butyric acid can be used in an amount of 1 mg/L to 1 g/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 10 mg/to 900 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 25 mg/to 800 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 50 mg/to 700 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 75 mg/to 600 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 100 mg/to 500 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 125 mg/to 400 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 150 mg/to 300 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 175 mg/to 250 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 50 mg/to 250 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 75 mg/to 200 mg/L. In some cases, hexanoic acid or butyric acid can be used in an amount of 90 mg/to 150 mg/L. Henanoic and butyric acid can be used in similar concentrations.

In some cases, hexanoic acid can be used in an amount of 1 g/L to produce 700 mg/L of CBGA or cannabinoids with the microorganism described throughout. In some cases, hexanoic acid can be used in an amount of at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 5.5 g/L, at least about 6 g/L, at least about 6.5 g/L, at least about 7 g/L, at least about 7.5 g/L, at least about 8 g/L, at least about 8.5 g/L, at least about 9 g/L, at least about 9.5 g/L, at least about 10 g/L, at least about 11 g/L, at least about 12 g/L, at least about 13 g/L, at least about 14 g/L, at least about 15 g/L, at least about 16 g/L, at least about 17 g/L, at least about 18 g/L, at least about 19 g/L, at least about 20 g/L, between about 0.1 g/L and about 20 g/L, between about 0.5 g/L and about 15 g/L, between about 1 g/L and about 10 g/L, between about 1 g/L and about 9 g/L, between about 1 g/L and about 8 g/L, between about 1 g/L and about 7 g/L, between about 2 g/L and about 6 g/L, between about 2 g/L and about 5 g/L, or between about 3 g/L and about 4 g/L. In some cases, butyric acid can be used in an amount of 1 g/L to produce 700 mg/L of CBGVA or cannabinoids with the microorganism described throughout. In some cases, butric acid can be used in an amount of at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about 5.5 g/L, at least about 6 g/L, at least about 6.5 g/L, at least about 7 g/L, at least about 7.5 g/L, at least about 8 g/L, at least about 8.5 g/L, at least about 9 g/L, at least about 9.5 g/L, at least about 10 g/L, at least about 11 g/L, at least about 12 g/L, at least about 13 g/L, at least about 14 g/L, at least about 15 g/L, at least about 16 g/L, at least about 17 g/L, at least about 18 g/L, at least about 19 g/L, at least about 20 g/L, between about 0.1 g/L and about 20 g/L, between about 0.5 g/L and about 15 g/L, between about 1 g/L and about 10 g/L, between about 1 g/L and about 9 g/L, between about 1 g/L and about 8 g/L, between about 1 g/L and about 7 g/L, between about 2 g/L and about 6 g/L, between about 2 g/L and about 5 g/L, or between about 3 g/L and about 4 g/L. In certain instances, the microorganism described throughout is fermented in a stirred tank fermentor.

In other cases, olivetolic acid can be used to ferment CBGA or cannabinoids with the microorganism described throughout. For example, in some cases, the media can comprise a combination of olivetolic acid, yeast extract, peptone, and glucose. In certain cases, the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 40 mg/L hexanoic acid. In some cases, olivetolic acid can be used in an amount of 1 mg/to 1 g/L. In some cases, olivetolic acid can be used in an amount of 5 mg/to 900 mg/L. In some cases, olivetolic acid can be used in an amount of 10 mg/to 800 mg/L. In some cases, olivetolic acid can be used in an amount of 15 mg/to 700 mg/L. In some cases, olivetolic acid can be used in an amount of 20 mg/to 600 mg/L. In some cases, olivetolic acid can be used in an amount of 25 mg/to 500 mg/L. In some cases, olivetolic acid can be used in an amount of 30 mg/to 400 mg/L. In some cases, olivetolic acid can be used in an amount of 35 mg/to 300 mg/L. In some cases, olivetolic acid can be used in an amount of 40 mg/to 200 mg/L. In some cases, olivetolic acid can be used in an amount of 50 mg/to 150 mg/L. In some cases, olivetolic acid can be used in an amount of 10 mg/to 100 mg/L. In some cases, olivetolic acid can be used in an amount of 20 mg/to 75 mg/L. In some cases, olivetolic acid can be used in an amount of 30 mg/to 50 mg/L.

Product Recovery

The fermentation of the microorganisms disclosed herein can produce a fermentation broth comprising a desired product (e.g., CBGA, CBGVA, THCA, CBDA or CBCA or cannabinoid) and/or one or more by-products as well as the cells/microorganisms (e.g., a genetically modified microorganism), in a nutrient medium.

In certain embodiments the CBGA, THCA, CBDA or CBCA produced in the fermentation reaction is converted to a cannabinoid, such as THC, CBD, and/or CDC. This conversion can happen directly from the fermentation broth. However, in other embodiments, the CBGA, THCA, CBDA or CBCA can be first recovered from the fermentation broth before conversion to a cannabinoid such as THC, CBD, and/or CDC. In certain embodiments the CBGVA produced in the fermentation reaction is converted to a cannabinoid, such as THCC, CBDV, and/or CDCV. This conversion can happen directly from the fermentation broth. However, in other embodiments, the CBGVA can be first recovered from the fermentation broth before conversion to a cannabinoid such as THCV, CBDV, and/or CDCV.

In some cases, the CBGA, CBGVA, THCA, CBDA or CBCA can be continuously removed from a portion of broth and recovered as purified the CBGA. In particular embodiments, the recovery of the CBGA, CBGVA, THCA, CBDA or CBCA includes passing the removed portion of the broth containing the CBGA, CBGVA, THCA, CBDA or CBCA through a separation unit to separate the microorganisms (e.g., genetically modified microorganism) from the broth, to produce a cell-free CBGA, CBGVA, THCA, CBDA or CBCA containing permeate, and returning the microorganisms to the bioreactor. Additional nutrients can be added to the media to replenish its nutrients before it is returned to the bioreactor. The cell-free CBGA, CBGVA, THCA, CBDA or CBCA permeate can then be stored or be used for subsequent conversion to cannabinoids (or other desired product).

Also, if the pH of the broth was adjusted during recovery of CBGA CBGVA, THCA, CBDA or CBCA the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.

Subsequent purification steps can involve treating the post-fermentation CBGA, CBGVA, THCA, CBDA or CBCA product using methods known in the art to recover individual product species of interest to high purity.

In one example, CBGA, CBGVA, THCA, CBDA or CBCA extracted in an organic phase can be transferred to an aqueous solution. In some cases, the organic solvent can be evaporated by heat and/or vacuum, and the resulting powder can be dissolved in an aqueous solution of suitable pH. The aqueous phase can then be removed by decantation, centrifugation, or another method. For example, when the organic solvent is ethyl acetate, the resulting powder from evaporation is dissolved in a water:acetonitrile mixture (50:50 ratio).

The same methods as described above can be applied to cannabinoids, should they be produced.

CBGA, CBGVA or Cannabinoid Production Levels

The microorganisms and the methods herein can produce CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids at surprisingly high efficiency, more so than other known CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids fermentation processes. For example, the microorganisms and the methods disclosed herein can convert a carbon substrate (such as sugar, alcohol, and/or fatty acid) at a rate of greater than 0.01%.

The genetic modifications to the cells described throughout can be made to produce CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids over what would have been made without any genetic modifications. For example, compared to a non-genetically altered cell, the genetically modified microorganisms described throughout can produce CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids greater than 1.1 times (compared to the production levels of a non-genetically modified microorganism or non-genetically altered cell).

In some cases, the cannabinoid can be THC, CBD, CBC, or any combination thereof. In other cases, the cannabinoid can be THCV, CBDV, CBCV, or any combination thereof.

Methods of Making CBGA, THCA, CBDA or CBCA or cannabinoids

The genetically modified cells or microorganisms described throughout can be used to make CBGA, THCA, CBDA, CBCA and/or cannabinoids, e.g., THC, CBD, and CBC. A substrate capable of being converted into a CBGA or a cannabinoid can be brought in contact with one or more of the following enzymes: acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), HMG-Co reductase (HMG1), and/or farnesyl pyrophosphate synthetase (ERG20).

The CBGA, THCA, CBDA, CBCA or cannabinoids (e.g., THC, CBD, CBC) produced can be recovered and isolated from the modified cells. The CBGA, THCA, CBDA, CBCA or cannabinoids in some cases can be secreted into the media of a cell culture, in which the CBGA, THCA, CBDA, CBCA or cannabinoids is extracted directly from the media. In some cases, the CBGA, THCA, CBDA, CBCA or cannabinoids can be within the cell itself, and the cells will need to be lysed in order to recover the respective CBGA, THCA, CBDA, CBCA or cannabinoids. In some instances, both cases can be true, where some CBGA, THCA, CBDA, CBCA or cannabinoids are secreted and some remains within the cells. In this case, either method or both methods can be used.

The genetically modified cells or microorganisms described throughout can be used to make CBGVA and/or cannabinoids, e.g., THCV, CBDV, and CBCV. A substrate capable of being converted into a CBGVA or a cannabinoid can be brought in contact with one or more of the following enzymes: acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), HMG-Co reductase (HMG1), and/or farnesyl pyrophosphate synthetase (ERG20).

The CBGVA or cannabinoids (e.g., THCV, CBDV, CBCV) produced can be recovered and isolated from the modified cells. The CBGVA or cannabinoids in some cases can be secreted into the media of a cell culture, in which the CBGVA or cannabinoids is extracted directly from the media. In some cases, the CBGVA or cannabinoids can be within the cell itself, and the cells will need to be lysed in order to recover the respective CBGVA or cannabinoids. In some instances, both cases can be true, where some CBGVA or cannabinoids are secreted and some remains within the cells. In this case, either method or both methods can be used.

Accordingly, disclosed herein is a method of making CBGA, CBGVA, THCA, CBDA, CBCA or a cannabinoid comprising (a) contacting the genetically modified microorganism with a medium comprising a carbon source, and (b) growing the genetically modified microorganism to produce the CBGA or cannabinoid. The genetically modified microorganism can comprise any microorganism disclosed throughout. For example, the microorganism can be a genetically modified microorganism capable of converting a carbon substrate into CBGA, CBGVA, THCA, CBDA, CBCA or a cannabinoid, the genetically modified microorganism comprising a heterologous nucleic acid encoding one or more of the enzymes disclosed throughout (e.g., microorganism can comprise a nucleic acid sequence encoding for one or more of the following enzymes: acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); HMG-Co reductase (HMG1); farnesyl pyrophosphate synthetase (ERG20); or any combination thereof).

The carbon source can be any carbon source that can be used by the microorganism. In some cases, the carbon source can be a sugar, alcohol, and/or fatty acid. For example, the sugar, alcohol or fatty acid can include without limitation hexanoic acid, butyric acid, glucose, fructose, xylose, sucrose, dextrins, starch, xylan, cellulose, hemicellulose, arabinose, glycerol, ethanol, butanol, methanol, or any combination thereof. In some cases, the carbon source can be hexanoic acid. In some cases, the carbon source can be olivetolic acid. In other cases, the carbon source can be a mixture of one or more different types of carbon sources.

The cannabinoid produced by the methods disclosed throughout can be any cannabinoid including but not limited to Δ⁹-tetrahydrocannabinolic acid (THCA), cannabigerolic acid (CBGA); cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA) or any combination thereof.

In some cases, the medium does not contain any cells. In other words, this reaction is performed in the media in vitro. In some cases, the reaction does not occur within a cell. For example, the conversion of hexanoic acid to hexanoyl-CoA can occur outside of a cell. In some cases, the conversion of hexanoyl-CoA to olivetolic acid can occur outside of a cell. In some cases, the conversion of olivetolic acid to CBGA can occur outside of a cell. In some cases, the conversion of CBGA to Δ9-tetrahydrocannabinolic acid can occur outside of a cell. In some cases, the conversion of Δ9-tetrahydrocannabinolic acid to Δ9-tetrahydrocannabinol can occur outside of a cell. In some cases, the conversion of CBGA to cannabidiolic acid can occur outside of a cell. In some cases, the conversion of cannabidiolic acid to cannabidiol can occur outside of a cell. In some cases, the conversion of CBGA to cannabichromenic acid can occur outside of a cell. In some cases, the conversion of cannabichromenic acid to cannabichromene can occur outside of a cell.

In some cases, the cannabinoid, such as CBGA, CBGVA, THCA, CBDA, or CBCA can be converted outside of a cell. For example, once CBGA, CBGVA, THCA, CBDA, or CBCA is produced, it can be either isolated (from the cell or the cell media or both). Once isolated it can be converted, enzymatically or non-enzymatically into other a different product, such as another type of cannabinoid. In some cases, the CBGA, CBGVA, THCA, CBDA, or CBCA is just secreted into the media by the microorganism that synthesized it, and then the CBGA, CBGVA, THCA, CBDA, or CBCA is directly converted enzymatically or non-enzymatically into other a different product, such as another type of cannabinoid.

In some cases, this reaction is contained within a cell that is within cell culture media. In other words, the reaction is performed in vivo. For example, the conversion of hexanoic acid to hexanoyl-CoA can occur within a cell. In some cases, the conversion of hexanoyl-CoA to olivetolic acid can occur within a cell. In some cases, the conversion of olivetolic acid to CBGA can occur within a cell. In some cases, the conversion of CBGA to Δ9-tetrahydrocannabinolic acid can occur within a cell. In some cases, the conversion of Δ9-tetrahydrocannabinolic acid to Δ9-tetrahydrocannabinol can occur within a cell. In some cases, the conversion of CBGA to cannabidiolic acid can occur within a cell. In some cases, the conversion of cannabidiolic acid to cannabidiol can occur within a cell. In some cases, the conversion of CBGA to cannabichromenic acid can occur within a cell. In some cases, the conversion of cannabichromenic acid to cannabichromene can occur within a cell.

In some cases, there is a combination of the two. Some reactions along the pathway can occur within a cell, whereas some of the reactions along the pathway occur outside of a cell.

In some cases, the medium is cell culture media. In other instances, the medium is water or other liquid in which the cells (for in vivo reactions) can survive (such as saline buffered water). In other instances, the medium is water or other liquid in which the enzymes (for in vitro reactions) are active.

The CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids produced herein can be useful inter alia in the manufacture of pharmaceutical compositions. Thus, disclosed herein is a method of making a pharmaceutical composition by using the products disclosed herein. In some cases, the CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids are mixed with excipients to produce pharmaceutical compositions.

Upon completion of the methods or reactions described throughout, the amount of a particular cannabinoid, e.g., THCA, CBDA, CBCA, THC, CBD, CBC, THCVA, CBDVA, CBCVA, THCV, CBDV or CBCV, present in the reaction mixture can be about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween. In some instances, the reaction mixture comprises CBGA in a weight percentage of about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween, of total cannabinoids in the reaction mixture. In other instances, the reaction mixture comprises CBGVA in a weight percentage of about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween, of total cannabinoids in the reaction mixture. In other instances, the reaction mixture comprises THCA in a weight percentage of about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween, of total cannabinoids in the reaction mixture. In other instances, the reaction mixture comprises CBDA in a weight percentage of about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween, of total cannabinoids in the reaction mixture. In other instances, the reaction mixture comprises CBCA in a weight percentage of about 0.001% (w/w) to about 99% (w/w), about 0.01% (w/w) to about 25% (w/w), about 0.025% (w/w) to about 20% (w/w), about 0.05% (w/w) to about 15% (w/w), about 0.075% (w/w) to about 12% (w/w), about 0.1% (w/w) to about 10% (w/w), about 0.25% (w/w) to about 5% (w/w), about 0.5% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 1% (w/w), about 0.1% (w/w) to about 0.5% (w/w), about 0% (w/w) to about 1% (w/w), and any range therebetween, of total cannabinoids in the reaction mixture.

Exemplary Uses of the CBGA or Cannabinoids

Preparations of CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids obtained can be used for any and all uses. The CBGA, CBGVA, THCA, CBDA, CBCA or cannabinoids can be isolated and sold as purified products. Or these purified products (e.g., CBGA) can be a feedstock to make additional cannabinoids.

The cannabinoids made can be used to manufacture medicinal compounds.

Accordingly, in one aspect, disclosed is a use of CBGA, THCA, CBDA, or CBCA as a feedstock compound in the manufacture of a cannabinoid. In another aspect, disclosed is a use of a cannabinoid in the manufacture of a medicinal composition.

Pharmaceutical Compositions and Routes of Administration

The cannabinoids (e.g., THC, CBD, CDC, THCV, CBDV, and/or CDCV) can include pharmaceutically acceptable derivatives or prodrugs thereof. A “pharmaceutically acceptable derivative” can mean means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative thereof.

Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

The pharmaceutical preparation can be a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

Exemplary Uses of the Cannabinoids

Preparations of cannabinoids (e.g., CBGA, THCA, CBDA, CBCA, THC, CBD, CBC, CBGVA, THCVA, CBDVA, CBCVA, THCV, CBDV, CBCV) obtained can be used for any and all uses. The cannabinoids can be isolated and sold as purified products. Or these purified products can be a feedstock to make additional types of cannabinoids. For example, purified CBGA can be used as a feedstock to make other cannabinoids such as THCA, CBDA, CBCA, THC, CBD, and CBC. In another example, purified CB GVA can be used as a feedstock to make other cannabinoids such as THCVA, CBDVA, CBCVA, THCV, CBDV, and CBCV.

The cannabinoids made in the processes described throughout can be used to manufacture medicinal compounds. Accordingly, in one aspect, disclosed is a use of cannabinoids as a feedstock compound in the manufacture of a medicinal compound. For example, the cannabinoids can be subsequently processed to prepare a pharmaceutical formulation.

Pharmaceutical Compositions and Routes of Administration

The cannabinoids also include pharmaceutically acceptable derivatives thereof. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative thereof.

One particular delivery system can be through the pulmonary system. In some cases, the cannabinoid can be for into a liquid and vaporized so that it can be inhaled. See e.g., U.S. Pat. No. 9,326,967. Vaporization of cannabinoids and delivery through the pulmonary system can result in quick absorption through the circulatory system and can provide extremely fast systemic effects. Further, vaporization can mimic one of the preferred ways in which natural cannabinoids are inhaled.

Additional delivery system that can work by intravenous injections. See e.g., WO2013009928A1. Similar to vaporization, this intravenous injection can provide extremely fast systemic effects.

Oral delivery systems, such as, delivery through the gastrointestinal tract, can be used to deliver the cannabinoids. For example, the oral delivery system can be in the form of a pharmaceutical dosage unit, food, drink, or anything that can be delivered through the gastrointestinal tract.

Treatment of Disease and Symptoms of Disease

The cannabinoids can be used to treat disease, in particular to treat disease of people that are in need thereof. This includes treating one or more symptoms of the diseases. For example, the cannabinoids can be used to treat one of more of the following diseases: anorexia, multiple sclerosis, neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, Tourette's syndrome, and Alzheimer's disease, epilepsy, glaucoma, osteoporosis, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), asthma, cardiovascular disorders, cancer, obesity, or metabolic syndrome-related disorders.

The cannabinoids can be used to treat one or more symptoms of disease, such as depression, anxiety, insomnia, emesis, pain, or inflammation.

Some of the diseases or symptom of disease can be exclusive to humans, but other diseases or symptom of disease can be shared in more than one animal, such as in all mammals.

Recreational Uses

The cannabinoids produced by the microorganism and methods described throughout can be used for recreational use. For example, the cannabinoids, such as Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), or any combination thereof, can be used for non-medical uses.

In some cases, the cannabinoid can be formed into a liquid and vaporized so that it can be inhaled. See e.g., U.S. Pat. No. 9,326,967. Vaporization of cannabinoids and delivery through the pulmonary system can be used and can be preferred by some recreational users. For example, recreational users who do not like the smell of burning cannabis or those that are afraid of the effects of inhaling burning substances, can use this method. Further, since this method is not invasive and can be used almost anywhere, recreational users can prefer this method.

In some cases, the cannabinoid can be formed into something that can be injected, e.g., injected intravenously. This method can be used in order to deliver substances quickly and efficiently within the blood stream. For example, this liquid can be injected into the saline solution (colloquially known as “IV”) used in hospitals to keep patients hydrated. Further, intravenous injections can be used by recreational users for immediate effects. In some cases, the intravenous cannabinoid injections can be used to treat other drug addictions, such as heroin addiction.

In additional cases, the cannabinoids produced from the microorganisms and methods described throughout can be used as an additive to food or drink. For example, the cannabinoids can be used, for example, in baked goods, such as brownies or cakes. Additionally, the cannabinoids can be added to a beverage such as water, soda, beer, liquor, etc.

Other recreational ways to use the cannabinoids include but are not limited to patches; (similar to nicotine patches); topically (such as in lotions); sprays (breath freshener), or tinctures (mouth drops).

The disclosure is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

EXAMPLES
Example 1—Plasmid Construction

A prenyltransferase of interest was identified. The amino acid sequence (SEQ ID NO: 1) was used by Genscript to design and synthesize the yeast codon optimized sequence coding for the prenyltransferase and used in the experiments.

Plasmids were constructed using the GeneArt Seamless Cloning and Assembly from Thermo Fisher Scientific. The RUNM000898_511.1 vector (SEQ ID NO: 3) contained the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter. The bCBGA0098 vector (SEQ ID NO: 4) contained the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the AAE1 and PT genes under the regulation of the bidirectional GAL1/GAL10 promoter. The bCBGA0306 vector (SEQ ID NO: 25) contained the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the PT gene under the regulation of the bidirectional GAL1/GAL10 promoter.

Example 2—yCBGA0172 Strain Construction

The parental strain for all examples was the Saccharomyces cerevisiae CEN.PK2-1C strain. Its genotype is: MATA, ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8^C; SUC2.

A mutant ERG20 allele was integrated into the GAL80 locus of the host. First, a plasmid was constructed carrying an ERG20 allele with two mutations: F96W and N127W. Second, the ERG20 allele together with the adjacent HygMX cassette was amplified in a PCR reaction and flanking sequences of the chromosomal GAL80 coding sequence were incorporated during the PCR reaction using oligonucleotides with 5′ extensions. Third, this DNA fragment was transformed into the host strain by electroporation. Finally, the strain with integrated mutant ERG20 sequence at the GAL80 locus were identified by its hygromycin B resistance and referred to as yCBGA0172.

Plasmids RUNM000898_511.1 (SEQ ID NO: 3) and bCBGA0098 (SEQ ID NO: 4) were transformed into the yCBGA0172 strain by electroporation. Transformants were selected by their leucine and uracil prototrophy on SD/MSG minimal medium (20 g/L glucose, 1.7 g/L yeast nitrogen base w/o ammonium sulphate and amino acids, 1 g/L monosodium glutamic acid, 20 g/L agar when solid medium is to be used) supplemented with histidine and tryptophan.

In another example plasmid bCBGA0306 (SEQ ID NO: 25) and the VVN4655922 plasmid were transformed into the yCBGA0172 strain by electroporation. The plasmid VVN4655922 encodes for the Saccharomyces cerevisiae HMG1 gene truncated of the first 530 amino acids and has the Saccharomyces cerevisiae TRP1 gene as an auxotrophic selection marker. Transformants were selected by their leucine and tryptophan prototrophies on SD/MSG minimal medium supplemented with histidine and uracil.

Example 3—Growth

Transformant colonies were picked and inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl Synthetic Defined (SD)/MSG liquid medium supplemented with histidine and tryptophan. These inoculums were grown overnight at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter.

After the overnight growth the samples were centrifuged, the supernatant discarded and cells transformed with plasmids RUNM000898_511.1 and bCBGA0098 were re-suspended in 400 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium. In case of cultures transformed with plasmids bCBGA0306 and VVN4655922 the pelleted cells were re-suspended in 400 μl YPD-OLA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 40 mg/L olivetolic acid) medium.

Then samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 16 μl 50% glucose was added to the samples. Samples grown in YPD-OLA medium were supplemented additionally with 20 μl of 800 mg/L olivetolic acid solution too.

Finally, samples were grown for additional 32 hours and were analyzed for CBGA titers.

Example 4—Sample Processing and Analytics

The samples were processed by adding 400 μl acetonitrile, then shaken for 5 minutes at 30° C. at 300 rpm with 50 mm throw. The samples were then centrifuged at 400 rpm for 5 minutes. 200 μl of supernatant were transferred into a new 96 well plate.

The new 96 well plate were transferred to a Waters Acquity UPLC (Binary pump)-TQD MS and set with the following parameters:

- Instrument: Waters Acquity UPLC (Binary pump)-TQD MS
- Stationary phase: Agilent Eclipse Plus C18 RRHD 1.8 mm, 2.1×50 mm
- Mobile phase A: water 0.1% FA
- Mobil phase B: acetonitrile 0.1% FA
- Gradient info (Table 1):

TABLE 1

Time [min]
% A
% B

0
55
45

0.5
45
55

0.6
30
70

2.0
30
70

2.1
0
100

2.2
0
100

2.3
55
45

- Flow: 0.4 mL/min
- Column temp: 35° C.
- Detection: Acquity TQD, MRM Mode (361.2»219.1; 361.2»149.0; 361.2»237.1; 361.2»343.2)

Using the strains and methods described above CBGA was produced at 200 μg/L concentration when YPD-HXA medium was used and at 15 mg/L concentration when YPD-OLA medium was used. A representative chromatogram of the sample and the CBGA standard can be seen in FIG. 2.

Example 5—the yCBGA0189, yCBGA0197 and yCBGA0201 Strains: Testing Additional Prenyl Transferases

A modified sequence with higher prenyl transferase activity was constructed and referred to as GFP-dPT (polynucleotide sequence: SEQ ID NO: 26; amino acid sequence: SEQ ID NO: 27). The GFP-dPT gene is a fusion of two polynucleotide sequences: a gene of a modified fluorescent protein yEVenus (SEQ ID NO: 28) from the plasmid pKT90 and a truncated version of SEQ ID NO: 2 missing its first 246 nucleotides.

Plasmids were constructed using the GeneArt Seamless Cloning and Assembly from Thermo Fisher Scientific. The bCBGA0385 vector (SEQ ID NO: 29) contained the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the GFP-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter. The bCBGA0305 vector (SEQ ID NO: 30) contained the Saccharomyces cerevisiae 2μ replication origin, the TRP1 gene as an auxotrophic marker and the AAE1 gene under the regulation of the bidirectional GAL1/GAL10 promoter.

For testing the GFP-dPT activity a new parental strain was constructed: a polynucleotide fragment of the RUNM000898_511.1 vector coding for OAC, PKS and URA3 genes was transformed into the strain yCBGA0172 by electroporation. The strain with OAC, PKS and URA3 genes inserted was identified by its uracil prototrophy on SD/MSG minimal medium supplemented with histidine, tryptophan and leucine and referred to as yCBGA0189.

In an another experiment a polynucleotide fragment coding for the truncated version of HMG1 gene lacking its first 530 amino acids and a KanMX cassette was transformed into the strain yCBGA0172 by electroporation. The strain with truncated HMG1 gene inserted was identified by its G418 resistance and referred to as yCBGA0197.

The strain yCBGA0197 was transformed with a vector coding for the Saccharomyces cerevisiae HO gene and URA3 gene. The plasmid was cured and a clone with MAT alpha mating type was identified using standard laboratory methods. Finally, this MAT alpha clone was mated with yCBGA0189 and the isolated diploid strain is referred to as yCBGA0201.

Plasmids bCBGA0305 and bCBGA0385 were transformed into the yCBGA0201 strain by electroporation. Transformants were selected by their leucine and tryptophan prototrophies on SD/MSG minimal medium supplemented with histidine.

In another example, plasmid bCBGA0385 was transformed into the yCBGA0197 strain by electroporation. Transformants were selected by their leucine prototrophy on SD/MSG minimal medium supplemented with histidine, uracil and tryptophan.

Transformant colonies were picked and inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl SD/MSG liquid medium supplemented with histidine in case of strains containing both bCBGA0305 and bCBGA3085 and with histidine, uracil and tryptophan in case of strains containing bCBGA0385 plasmid alone. These inoculums were grown overnight at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter.

After the overnight growth the samples were centrifuged, the supernatant discarded and cells re-suspended in 400 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium or 400 μl YPD-800LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 80 mg/L olivetolic acid) medium. In case of cultures transformed with only bCBGA0385 plasmid the YPD-800LA medium was used.

The samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 16 μl 50% glucose was added to the samples.

Finally, samples were grown for additional 32 hours and were analyzed for CBGA titers as described in “Example 4—Sample processing and analytics”.

Using the strains and methods described above CBGA was produced at 11 mg/L concentration when YPD-HXA medium was used and at 50 mg/L concentration when YPD-800LA medium was used.

Example 6—Mutant Prenyl Transferase

Another modified sequence with increased prenyl transferase activity was constructed and referred to as ERG20mut-dPT (polynucleotide sequence: SEQ ID NO: 31; amino acid sequence: SEQ ID NO: 32). The ERG20mut-dPT gene is a fusion of two polynucleotide sequences: ERG20 gene with F96W and N127W mutations and a truncated version of SEQ ID NO: 2 missing its first 246 nucleotides.

Plasmid was constructed using the GeneArt Seamless Cloning and Assembly from Thermo Fisher Scientific. The bCBGA0559 vector (SEQ ID NO: 33) contained the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker and the ERG20mut-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter.

bCBGA0559 was transformed into the yCBGA0197 strain by electroporation. Transformants were selected by their leucine prototrophy on SD/MSG minimal medium supplemented with histidine, uracil and tryptophan.

Transformant colonies were picked and inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl SD/MSG liquid medium supplemented with histidine, uracil and tryptophan. These inoculums were grown overnight at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter.

After the overnight growth the samples were centrifuged, the supernatant discarded and cells re-suspended in 400 μl YPD-1200LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 120 mg/L olivetolic acid) medium.

Then samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 16 μl 50% glucose was added to the samples.

Finally, samples were grown for additional 32 hours and were analyzed for CBGA titers as described in Example 4—Sample processing and analytics.

Using the strains and methods described above CBGA was produced at 90 mg/L concentration.

Example 7—the yCBGA0237, yCBGA0253 and yCBGA0254 Strains: ERG20 Promoter Truncation

yCBGA0237 strain was constructed by deleting the HygMX and KanMX cassettes from the yCBGA0197 strain and inserting the GFP-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter into the YJL144W locus by replacing the native YJL144W open reading frame (ORF). Two more strains were constructed by deleting different fragments from the promoter of the native ERG20 allele of the yCBGA0237 strain. The yCBGA0253 strain contains a 133 nucleotide long deletion between 143 and 275 nucleotides upstream of the translational start site. The yCBGA0254 strain contains a 422 nucleotide long deletion between 69 and 490 nucleotides upstream of the translational start site.

Strains yCBGA0237, yCBGA0253 and yCBGA0254 were inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl SD/MSG liquid medium supplemented with histidine, leucine, uracil and tryptophan. These inoculums were grown overnight at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter.

After the overnight growth 40 μl of the samples were transferred into 360 μl YPD-1200LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 120 mg/L olivetolic acid) medium.

Then samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 16 μl 50% glucose was added to the samples.

Finally, samples were grown for additional 32 hours and were analyzed for CBGA titers as described in Example 4—Sample processing and analytics.

Using the strains and methods described above, yCBGA0237 strain produced 53 mg/L CBGA and yCBGA0253 and yCBGA0254 reached 96 and 78 mg/L CBGA concentration, respectively.

Example 8—Olivetolic Acid Conversion to THCA

A plasmid was constructed using the GeneArt Seamless Cloning and Assembly from Thermo Fisher Scientific. The RUNM001233_51.1 vector (SEQ ID NO: 34) contained the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and the THCA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter.

For testing the olivetolic acid conversion into THCA the RUNM001233_51.1 and bCBGA0385 vectors were transformed into the strain yCBGA0197 by electroporation. Transformants were selected by their leucine and uracil prototrophies on SD/MSG minimal medium supplemented with histidine and tryptophan.

After the 48 hours growth period the samples were centrifuged, the supernatant discarded and cells re-suspended in 400 μl YPD-1200LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 120 mg/L olivetolic acid) medium.

Then samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 16 μl 50% glucose was added to the samples.

Finally, samples were grown for additional 32 hours and were analyzed for THCA and CBGA titers as described below.

Using the strains and methods described above CBGA was produced at 2 mg/L concentration while THCA was produced at 84 mg/L concentration.

Example 9—The yCBGA0269 Strain: Hexanoic Acid Conversion to THCA

Plasmids were constructed using the GeneArt Seamless Cloning and Assembly from Thermo Fisher Scientific. The RUNM001210_96.1 vector (SEQ ID NO: 35) contained the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker, the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the STE5 promoter. The bCBGA0409 vector (SEQ ID NO: 36) contained the Saccharomyces cerevisiae 2μ replication origin, the LEU2 gene as an auxotrophic marker, the THCA synthase and PT genes under the regulation of the bidirectional GAL1/GAL10 promoter.

The yCBGA0251 strain was constructed by inserting the ERG20mut-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter into the YMR145C locus by replacing the native YMR145C ORF of the yCBGA0237 strain. The yCBGA0269 strain was constructed by deleting a 133 nucleotide long fragment between 143 and 275 nucleotides upstream of the translational start site of the native ERG20 allele of the yCBGA0251 strain. Plasmids RUNM001210_96.1 and bCBGA0409 were transformed into the yCBGA0269 strain by electroporation. Transformants were selected by their uracil and leucine prototrophy on SD/MSG minimal medium supplemented with histidine and tryptophan.

Transformant colonies were picked and inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl SC-URA-LEU (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without uracil and leucine, 22 g/L glucose, buffered to pH 6.0). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium. Then samples were grown for 16 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 40 μg hexanoic acid dissolved in 8 μl ethanol was added to the samples. Finally, samples were grown for additional 32 hours and were analyzed for THCA and CBGA titers as described below.

Using the strains and methods described above CBGA was produced at 34 mg/L concentration while THCA was produced at 23 mg/L concentration.

Example 10—Additional Sample Processing

The samples from examples 8 and 9 were processed by dilution with acetonitrile:water mixture (the composition of the mixture depends on the dilution factor, to reach 50% acetonitrile content for further processing), then shaken for 5 minutes at 30° C. at 300 rpm with 50 mm throw. The samples were then centrifuged at 400 rpm for 5 minutes. 200 μl of supernatant were transferred into a new 96 well plate.

The new 96 well plate were transferred to a Waters Acquity UPLC (Binary pump)-TQD MS and set with the following parameters:

- Instrument: Waters Acquity UPLC (Binary pump)-TQD MS
- Stationary phase: Agilent Eclipse Plus C18 RRHD 1.8 mm, 2 1×50 mm
- Mobile phase A: water 0.1% FA
- Mobil phase B: acetonitrile 0.1% FA
- Gradient info (Table 2):

TABLE 2

Time [min]
A [%]
B [%]

0
55
45

0.5
45
55

0.6
30
70

2.0
30
70

2.1
5
95

3.1
5
95

3.2
55
45

5.5
55
45

- Flow: 0.4 mL/min
- Column temp: 35° C.
- Detection: Acquity TQD, MRM Mode (361.2>>219.1; 361.2>>149.0; 361.2>>237.1; 361.2>>343.2); UV at 280 nm wavelength.

Representative chromatograms of a THCA containing sample can be seen in FIG. 3 (MRM chromatogram) and FIG. 4 (UV chromatogram).

Example 11—the yCBGA0314 Strain: Strains with Increased Prenyl Transferase Copy Number

Increased Prenyl Transferase copy number and promoter truncation improves prenyl transferase activity. The yCBGA0314 strain was constructed and tested for the effect of PT copy number on CBGA production. The yCBGA0314 strain was constructed by inserting the ERG20mut-dPT gene under the regulation of the bidirectional GAL1/GAL10 promoter into the LPP1 locus by replacing the native LPP1 ORF of the yCBGA0269 strain. The yCBGA0314 strain and several other previously described strains are listed in Table 3 below along with their respective gene insertions/mutations:

TABLE 3

Mutant

ERG20

integration;

truncated
GFP-dPT
ERG20mut-

HMG1
copy
dPT copy
Promoter

Strain ID
integration
number
number
truncation

yCBGA0197
+
0
0
0

yCBGA0237
+
1
0
0

yCBGA0251
+
1
1
0

yCBGA0269
+
1
1
+

yCBGA0314
+
1
2
+

Transformant colonies were grown using the following protocol: Transformant colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC Medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, uracil, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 24 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 8 μl of 12000 mg/L OLA dissolved in EtOH was added to the samples. Finally, the samples were grown for additional 24 hours and were analyzed for cannabinoids.

The yCBGA0314 strain produced 450 mg/L CBGA using 480 mg/L olivetolic acid as substrate.

Example 12—Preventing Hexanoic Acid Degradation and Increasing CBGA Titers

Cannabinoid production can be limited by the availability of hexanoic acid. We tested to see if the availability of hexanoic acid can be increased by knocking out several genes of the beta-oxidation pathway. In particular, we tested to see if hexanoic acid degradation could be prevented or minimized. Deletion of FAA1, FAA4, FAT1, PXA1, PXA2 and PEX11 had no obvious effect on hexanoic acid titers: after 24 hours of growth in YPD-HXA medium the hexanoic acid concentration was dropped below 5% of the original level (no heterologous cannabinoid pathway genes present). In other words, hexanoic degradation was not affected. A wild type control strain performed similarly, resulting in less than 5% final hexanoic acid concentration. However, deletion of the FOX1 gene (a.k.a. PDX1, systematic name YGL205W) increased hexanoic acid titers. The knockout of FOX1 eliminated HXA degradation almost completely: 95% of the original hexanoic concentration was still present at the end of the 24 hour long growth period.

Yeast strains having the ability to make CBGA were also knocked out for the FOX1 gene. yCBGA0326 strain was constructed by first, inserting the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the HXK1 promoter into the YGL202W locus replacing the native YGL202W ORF, second, inserting the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the HXK1 promoter into the DPP1 locus replacing the native DPP1 ORF, third, inserting the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter into the BTS1 locus replacing the native BTS1 ORF. yCBGA0373 strain was constructed by deleting the FOX1 gene, more specifically the nucleotide fragment between −73 and 3243 relative to the translational start site.

Strains yCBGA0326 and yCBGA0373 were inoculated into separate wells of a 96-well deep well plate. Each well contained 400 μl Synthetic Complete (SC) liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without uracil and leucine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine and uracil). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-50HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 50 mg/L hexanoic acid) medium. Then samples were grown for 24 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter, then 20 μg hexanoic acid dissolved in 8 μl ethanol was added to the samples. Finally, samples were grown for additional 24 hours and four replicates of both strains were analyzed for CBGA titers.

Using the methods described above, CBGA, olivetol, and olivetol acid titers were measured. As seen in FIG. 5, yCBGA0326 produced 14.3 mg/L CBGA and 28.4 mg/L olivetol while the strain yCBGA0373 deleted for the FOX1 gene produced 39.7 mg/L CBGA, 19.6 mg/L olivetolic acid and 66.1 mg/L olivetol.

Example 13—Strain with Increased AAE1—TKS—OAC Copy Number

Increasing the copy number of genes required for olivetolic acid production increased the corresponding CBGA titer. The yCBGA0268 strain was constructed by replacing the native YGL202W ORF of the yCBGA0251 strain by inserting the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the HXK1 promoter into the YGL202W locus. The yCBGA0268 strain and several other previously described strains are listed below along with their respective gene insertions/mutations. The Table 4 illustrates how copy number of the olivetolic acid biosynthetic genes increases the final CBGA titer.

TABLE 4

Combined

CBGA

GFP-dPT

Copy
Copy

titer

and
Copy
num-
num-
Dele-
(mg/L);

ERG20mut-
number
ber
ber
tion
hexanoic

dPT copy
of
of
of
of
acid as

Strain ID
number
CsAAE1
PKS
OAC
FOX1
substrate

yCBGA0251
2
0
0
0
0
0

yCBGA0268
2
1
1
1
0
14

yCBGA0326
2
1
3
3
0
31

yCBGA0373
2
1
3
3
+
63

Example 14—the yCBGA0513 Strain: Strain with Increased Prenyl Transferase Copy Number+Increased AAE1-TKS-OAC Copy Number

The yCBGA0513 strain, a strain containing multiple copies of prenyl transferase and genes required for olivetolic acid production, was constructed. The strain produced increased levels of CBGA using hexanoic acid as its substrate. For the strain construction, the parental strain was the yCBGA0314 strain which contained 3 copies of GFP-dPT and/or ERGOmut-dPT genes in total among other host modifications as described above. Three copies of the PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and three copies of the AAE1 gene under the regulation of the STE5 promoter were inserted into the YDR508C, YRL020C and FOX1 loci replacing the native ORFs of the yCBGA0314 strain. A large number of isolates from this transformation were screened and an isolate with high CBGA productivity and the best reproducibility was identified. Next, to complement the auxotrophic mutations of this strain, URA3, HISS LEU2 and TRP1 genes on a single fragment were inserted 3′ of the genomic ARG2 locus, resulting in the final yCBGA0513 strain.

Colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, uracil, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium. Then samples were grown for 24 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 40 μg hexanoic acid dissolved in 8 μl ethanol was added to the samples. Finally, the samples were grown for additional 24 hours and were analyzed for cannabinoids.

The yCBGA0513 strain was able to produce 140 mg/L CBGA in a standard high throughput screen. 1) The high prenyl transferase activity and optimized GPP productivity, as described for the parental yBGA0314 strain, 2) the replacement of the FOX1 gene that minimizes the degradation of hexanoic acid, and 3) and/or the integration of more than 3 copies of the genes required for olivetolic acid synthesis likely contribute to the high CBGA productivity.

To further test the potential of the yCBGA0513 strain, it was challenged in a modified screening procedure. yCBGA0513 strain was inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, uracil, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium. Then samples were grown for 96 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 40 μg hexanoic acid dissolved in 8 μl ethanol was added to the samples at two time-points: at 24 and 48 hour. At the end of the 96 hour growth period, the samples were analyzed for cannabinoids. The yCBGA0513 strain produced 300 mg/L CBGA using hexanoic acid as substrate.

Example 15—the yCBGA0520, yCBGA0523 and yCBGA0526 Strains: Strains with Increased Prenyl Transferase Copy Number+Genes Required for Olivetolic Acid Production

Several additional strains containing multiple copies of prenyl transferase and genes required for olivetolic acid production were constructed utilizing yCBGA0513 as the parent strain. The yCBGA0520, yCBGA0523 and yCBGA0526 strains are able to produce high levels of CBGA using hexanoic acid as substrate. The yCBGA0520 strain was constructed by inserting the ERG13 gene (encoding Hydroxymethylglutaryl-CoA synthase) under the regulation of the ADH2 promoter, the HMG1 gene (encoding Hydroxymethylglutaryl-CoA reductase) under the regulation of the TEF1 promoter, and the ERG20mut-dPT gene with a TPI1 terminator under the regulation of the HXK1 promoter into the ATG26 locus by replacing the native ATG26 ORF of the yCBGA0513 strain. The yCBGA_0523 strain was constructed by inserting the tHMG1 gene under the regulation of the ADH2 promoter, the ERG10 gene (encoding Acetyl-CoA C-acetyltransferase) under the regulation of the TEF1 promoter, and the ERG13 gene under the regulation of the HXK1 promoter into the ATG26 locus by replacing the native ATG26 locus of the yCBGA0513 strain. The yCBGA_0526 strain was constructed by inserting the tHMG1 gene under the regulation of the ADH2 promoter, the ERG13 gene under the regulation of the TEF1 promoter, and the AAE1 gene with a TEF1 terminator under the regulation of the HXK1 promoter into the ATG26 locus by replacing the native ATG26 ORF of the yCBGA0513 strain. tHMG1 stands for the Saccharomyces cerevisiae HMG1 gene truncated of the first 530 amino acids. ERG10, ERG13, HMG1 and tHMG1 gene cassettes contain their native terminator sequences. ERG20mut-dPT and AAE1 were described earlier. The ATG26 locus of the yCBGA0513 strain and the corresponding locuses in the yCBGA0520 strain, the yCBGA0523 strain, and the yCBGA0526 strain which replaced the ATG26 locus are disclosed in FIG. 13.

The strains and their gene insertions are listed in Table 5 below:

TABLE 5

Target
Pro-

Termi-
Pro-

Termi-
Pro-

Termi-

Strain
locus
moter1
ORF1
nator1
moter2
ORF2
nator2
moter3
ORF3
nator3

yCBGA_
ATG
ADH
ERG13
TEF1
HMG1
HXK
ERG20mut-
TPI1

052

dpt

0

yCBGA_

tHMG1

ERG1

ERG13

052

0

3

yCBGA_

ERG1

AAE1
TEF

052

3

1

6

tHMG1 stands for the Saccharomyces cerevisiae HMG1 gene truncated of the first 530 amino acids. ERG10, ERG13, HMG1 and tHMG1 gene cassettes contain their native terminator sequences. ERG20mut-dPT and AAE1 were described earlier.

All 3 strains outperformed the yCBGA0513 strain in the following modified high throughput screen: Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl Synthetic Complete Medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, uracil, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-HXA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid) medium. Then samples were grown for 72 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 80 μg hexanoic acid dissolved in 8 μl ethanol was added to the samples at two time-points: at 24 and 48 hours. At the end of the 72 hour growth period, the samples were analyzed for cannabinoids. The strains and their final olivetolic acid, olivetol and CBGA titers are listed in Table 6 below:

TABLE 6

Olivetolic

acid
Olivetol
CBGA

Strain
mg/L
mg/L
mg/L

yCBGA0513
94
185
199

yCBGA0520
74
215
313

yCBGA0523
56
204
311

yCBGA0526
79
215
305

Example 16—TKS (OS) Enzyme Evolution

To improve the performance of TKS, an enzyme evolution experiment was conducted. The wild type amino acid and nucleotide sequences of Saccharomyces cerevisiae TKS are included Table 7 below:

TABLE 7

SEQ

Description

ID

of sequence
Sequence
NO:

WT TKS aa
MNHLRAEGPASVLAIGTANPENILIQDEFPD
40

YYFRVTKSEHMTQLKEKFRKICDKSMIRKRN

CFLNEEHLKQNPRLVEHEMQTLDARQDMLVV

EVPKLGKDACAKAIKEWGQPKSKITHLIFTS

ASTTDMPGADYHCAKLLGLSPSVKRVMMYQL

GCYGGGTVLRIAKDIAENNKGARVLAVCCDI

MACLFRGPSDSDLELLVGQAIFGDGAAAVIV

GAEPDESVGERPIFELVSTGQTILPNSEGTI

GGHIREAGLIFDLHKDVPMLISNNIEKCLIE

AFTPIGISDWNSIFWITHPGGKAILDKVEEK

LDLKKEKFVDSRHVLSEHGNMSSSTVLFVMD

ELRKRSLEEGKSTTGDGFEWGVLFGFGPGLT

VERVVVRSVPIKY

WT TKS nt
ATGAATCATTTGAGAGCTGAAGGTCCAGCAT
41

CAGTTTTGGCTATTGGTACTGCAAACCCAGA

AAACATCTTGATCCAAGATGAATTTCCAGAT

TATTACTTCAGAGTTACTAAGTCAGAACATA

TGACACAATTGAAGGAAAAGTTTAGAAAGAT

CTGTGATAAGTCTATGATTAGAAAAAGAAAT

TGTTTCTTGAACGAAGAACATTTGAAGCAAA

ACCCAAGATTAGTTGAACATGAAATGCAAAC

ATTGGATGCTAGACAAGATATGTTGGTTGTT

GAAGTTCCAAAGTTGGGTAAAGATGCATGTG

CTAAAGCAATTAAAGAATGGGGTCAACCAAA

GTCTAAGATCACTCATTTGATTTTTACATCA

GCATCTACTACAGATATGCCAGGTGCTGATT

ACCATTGTGCAAAGTTGTTGGGTTTGTCACC

ATCTGTTAAGAGAGTTATGATGTACCAATTA

GGTTGTTACGGTGGTGGTACTGTTTTGAGAA

TCGCTAAGGATATCGCAGAAAACAATAAGGG

TGCTAGAGTCTTGGCAGTTTGTTGTGATATC

ATGGCTTGTTTGTTTAGAGGTCCATCAGATT

CTGATTTGGAATTGTTAGTTGGTCAAGCTAT

TTTTGGTGACGGTGCTGCAGCTGTTATTGTT

GGTGCAGAACCAGATGAATCAGTTGGTGAAA

GACCAATCTTCGAATTAGTTTCAACTGGTCA

AACAATTTTGCCAAATTCTGAAGGTACAATT

GGTGGTCATATCAGAGAAGCTGGTTTGATCT

TCGATTTGCATAAAGATGTTCCAATGTTGAT

CTCTAACAACATCGAAAAGTGTTTGATCGAA

GCTTTTACTCCAATCGGTATCTCAGATTGGA

ACTCTATTTTCTGGATTACACATCCAGGTGG

TAAAGCAATCTTGGATAAGGTTGAAGAAAAA

TTGGATTTGAAGAAAGAAAAATTTGTTGATT

CAAGACATGTTTTGTCTGAACATGGTAACAT

GTCTTCATCTACTGTTTTGTTCGTTATGGAT

GAATTGAGAAAGAGATCATTAGAAGAGGGTA

AATCTACTACAGGTGACGGTTTTGAATGGGG

TGTTTTATTTGGTTTTGGTCCAGGTTTGACA

GTTGAAAGAGTTGTTGTTAGATCTGTTCCAA

TTAAATACTAA

Each position in TKS listed in Table 8 below was mutagenized and 20-100 yeast colonies carrying randomly changed amino acids for each marked positions were screened for elevated performance.

TABLE 8

Position in TKS
Amino acid

48
Lys

51
Lys

52
Ile

55
Lys

56
Ser

125
Ala

126
Ser

130
Met

156
Gly

157
Cys

185
Asp

186
Ile

187
Met

189
Cys

190
Leu

200
Glu

203
Val

207
Ile

208
Phe

209
Gly

210
Asp

248
Ile

250
Gly

257
Leu

259
Phe

261
Leu

263
Lys

264
Asp

265
Val

266
Pro

297
His

299
Gly

300
Gly

301
Lys

302
Ala

303
Ile

330
Asn

332
Ser

366
Gly

367
Phe

368
Gly

369
Pro

370
Gly

The amino acid position A1a125, coding for Alanine in the wild type sequence was found to be able to elevate the TKS activity when the Alanine was swapped for different amino acids. When this amino acid position of TKS contained Serine, the corresponding olivetolic acid production was 50 mg/L in contrast with the 37 mg/L measured in case of the wild type TKS gene.

The TKS gene was inserted into a plasmid containing the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and the TKS gene under the regulation of the GAL10 promoter. The plasmid ID of the control plasmid with wild type TKS sequence was bCBGA1024. The ID of the plasmid with the Alanine to Serine mutation at position 125 is 0827-01-A1-1 (FIG. 6A).

The yCBGA0368 strain was used for screening these mutant plasmids. yCBGA0368 was constructed by inserting the AAE1 gene under the regulation of the STE5 promoter and the OAC gene under the regulation of the GAL1 promoter into the YKL140W locus by replacing the native YMR145C ORF of the yCBGA0215 strain (FIG. 6B). The yCBGA0215 strain was constructed by deleting the HygMX and KanMX cassettes from the yCBGA0197 strain.

Example 17—Enhanced CBDA Synthase Secretion

The secretion of CBDAs was optimized to be able produce large amounts of CBDA in a single process using yeast cells, the wild type sequences for the Saccharomyces cerevisiae CBDA amino acid and nucleotide sequences are included in Table 9:

TABLE 9

SEQ

Description

ID

of sequence
Sequence
NO:

WT CBDA
ATGAAATGTTCTACATTTTCATTTTGGTTTG
42

synthase nt
TTTGTAAGATCATTTTCTTTTTCTTTTCTTT

TAATATTCAAACTTCAATCGCTAACCCAAGA

GAAAATTTCTTGAAGTGTTTCTCTCAATACA

TTCCAAATAATGCAACAAATTTGAAATTGGT

TTATACTCAAAATAATCCATTATACATGTCT

GTTTTAAATTCTACAATTCATAATTTGAGAT

TTTCTTCAGATACTACACCAAAACCATTGGT

TATTGTTACACCATCTCATGTTTCACATATC

CAAGGTACTATCTTGTGTTCTAAGAAAGTTG

GTTTGCAAATTAGAACTAGATCAGGTGGTCA

TGATTCAGAAGGCATGTCTTACATCTCACAA

GTTCCATTCGTTATCGTTGATTTGAGAAACA

TGAGATCAATTAAAATTGATGTTCATTCACA

AACAGCTTGGGTTGAAGCTGGTGCAACTTTG

GGTGAAGTTTACTACTGGGTTAACGAAAAGA

ATGAATCTTTATCATTGGCTGCTGGTTACTG

TCCAACAGTTTGTGCTGGTGGTCATTTTGGT

GGTGGTGGTTATGGTCCATTAATGAGATCCT

ATGGTTTGGCTGCTGATAACATCATCGATGC

ACATTTGGTTAACGTTCATGGTAAAGTTTTG

GATAGAAAGTCTATGGGTGAAGATTTGTTTT

GGGCTTTGAGAGGTGGTGGTGCTGAATCATT

TGGTATCATCGTTGCTTGGAAGATCAGATTG

GTTGCAGTTCCAAAATCTACTATGTTCTCAG

TTAAGAAAATTATGGAAATCCATGAATTAGT

TAAATTGGTTAATAAGTGGCAAAATATTGCT

TATAAATACGATAAAGATTTGTTATTGATGA

CTCATTTTATTACAAGAAATATTACTGATAA

CCAAGGTAAAAATAAGACAGCTATCCATACT

TACTTTTCTTCAGTTTTCTTGGGTGGTGTTG

ATTCTTTGGTTGATTTGATGAATAAGTCTTT

TCCAGAATTAGGTATTAAGAAAACTGATTGT

AGACAATTGTCTTGGATCGATACTATCATTT

TCTATTCAGGTGTTGTTAACTACGATACAGA

TAACTTCAATAAGGAAATTTTATTGGATAGA

TCAGCTGGTCAAAATGGTGCTTTTAAAATTA

AATTGGATTACGTTAAGAAACCAATTCCAGA

ATCAGTTTTCGTTCAAATTTTAGAAAAATTG

TATGAAGAAGATATTGGTGCTGGCATGTACG

CATTGTATCCATACGGTGGTATCATGGATGA

AATTTCTGAATCAGCTATTCCATTTCCACAT

AGAGCAGGTATTTTATACGAATTGTGGTACA

TTTGTTCTTGGGAAAAGCAAGAAGATAACGA

AAAACATTTGAACTGGATTAGAAACATCTAT

AACTTCATGACTCCATACGTTTCACAAAACC

CAAGATTGGCTTATTTGAACTACAGAGATTT

GGATATCGGTATTAATGATCCTAAAAATCCA

AACAACTATACACAAGCAAGAATTTGGGGTG

AAAAGTACTTCGGTAAAAATTTCGATAGATT

GGTTAAGGTTAAAACTTTGGTTGATCCAAAT

AATTTCTTTAGAAATGAACAATCTATTCCAC

CATTGCCAAGACATAGACATTGA

WT CBDA
MKCSTFSFWFVCKIIFFFFSFNIQTSIANPR
43

synthase aa
ENFLKCFSQYIPNNATNLKLVYTQNNPLYMS

VLNSTIHNLRFSSDTTPKPLVIVTPSHVSHI

QGTILCSKKVGLQIRTRSGGHDSEGMSYISQ

VPFVIVDLRNMRSIKIDVHSQTAWVEAGATL

GEVYYWVNEKNESLSLAAGYCPTVCAGGHFG

GGGYGPLMRSYGLAADNIIDAHLVNVHGKVL

DRKSMGEDLFWALRGGGAESFGIIVAWKIRL

VAVPKSTMFSVKKIMEIHELVKLVNKWQNIA

YKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDC

RQLSWIDTIIFYSGVVNYDTDNFNKEILLDR

SAGQNGAFKIKLDYVKKPIPESVFVQILEKL

YEEDIGAGMYALYPYGGIMDEISESAIPFPH

RAGILYELWYICSWEKQEDNEKHLNWIRNIY

NFMTPYVSQNPRLAYLNYRDLDIGINDPKNP

NNYTQARIWGEKYFGKNFDRLVKVKTLVDPN

NFFRNEQSIPPLPRHRH

A series of plasmids were constructed containing the Saccharomyces cerevisiae replication origin, the URA3 gene as an auxotrophic marker and the CBDA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter. In each plasmid, the predicted plant secretion signal corresponding to the first 21 amino acid of the CBDA synthase was replaced with different yeast secretion signals, see table below.

This set of plasmids were separately transformed into yCBGA0314 strains and their CBDA productivity was assayed in a high throughput screening process optimized for CBDA synthase activity. The nucleotide sequences and protein sequence of the tested signal sequences are included in Tables 10-11 below, along with the plasmid IDs, the corresponding amino acid sequences, the ORF names and CBDA titer.

TABLE 10

Plasmid

Signal Sequence
SEQ ID

ID
ORF name
Amino Acid Sequence
NO:
Titer

bCBGA1827
OST1_signal-and-
MRQVWFSWIVGLFLCFFNVSSAAPVNTT
44
13

alpha_only_pro_
TEDETAQIPAEAVIGYLDLEGDFDVAVL

signal-d21_CBDAs
PFSNSTNNGLLFINTTIASIAAKEEGVS

LDKREAEA

bCBGA1829
alpha_prepro_
MRFPSIFTAVLFAASSALAAPVNTTTED
45
7

signal-d21_CBDAs
ETAQIPAEAVIGYLDLEGDFDVAVLPFS

NSTNNGLLFINTTIASIAAKEEGVSLDK

REAEA

bCBGA1835
AMYI_signal-
MQRPFLLAYLVLSLLFNSALG
46
19

d21_CBDAs

bCBGA1836
BGL2_signal-
MRFSTTLATAATALFFTASQVSA
47
11

d21_CBDAs

bCBGA1837
OST1_signal-
MRQVWFSWIVGLFLCFFNVSSA
48
41

d21_CBDAs

bCBGA1838
SUC2_signal-
MLLQAFLFLLAGFAAKISA
49
11

d21_CBDAs

bCBGA1839
PHO5_signal-and-
MFKSVVYSILAASLANAAPVNTTTEDET
50
44

alpha_only_pro_
AQIPAEAVIGYLDLEGDFDVAVLPFSNS

signal-d21_CBDAs
TNNGLLFINTTIASIAAKEEGVSLDKRE

AEA

bCBGA1840
AGA2_signal-and-
MQLLRCFSIFSVIASVLAAPVNTTTEDE
51
10

alpha_only_pro_
TAQIPAEAVIGYLDLEGDFDVAVLPFSN

signal-d21_CBDAs
STNNGLLFINTTIASIAAKEEGVSLDKR

EAEA

bCBGA1842
AMYI_signal-and-
MQRPFLLAYLVLSLLFNSALGAPVNTTT
52
11

alpha_only_pro_
EDETAQIPAEAVIGYLDLEGDFDVAVLP

signal-d21_CBDAs
FSNSTNNGLLFINTTIASIAAKEEGVSL

DKREAEA

bCBGA1843
BGL2_signal-and-
MRFSTTLATAATALFFTASQVSAAPVNT
53
9

alpha_only_pro_
TTEDETAQIPAEAVIGYLDLEGDFDVAV

signal-d21_CBDAs
LPFSNSTNNGLLFINTTIASIAAKEEGV

SLDKREAEA

bCBGA1845
SUC2_signal-and-
MLLQAFLFLLAGFAAKISAAPVNTTTED
54
9

alpha_only_pro_
ETAQIPAEAVIGYLDLEGDFDVAVLPFS

signal-d21_CBDAs
NSTNNGLLFINTTIASIAAKEEGVSLDK

REAEA

bCBGA1846
HSP150_delta_
MQYKKTLVASALAATTLAAYAPSEPWST
55
102

fragment-d21_
LTPTATYSGGVTDYASTFGIAVQPISTT

CBDAs
SSASSAATTASSKAKRAASQIGDGQVQA

ATTTASVSTKSTAAAVSQIGDGQIQATT

KTTAAAVSQIGDGQIQATTKTTSAKTTA

AAVSQISDGQIQATTTTLAPKSTAAAVS

QIGDGQVQATTTTLAPKSTAAAVSQIGD

GQVQATTKTTAAAVSQIGDGQVQATTKT

TAAAVSQIGDGQVQATTKTTAAAVSQIG

DGQVQATTKTTAAAVSQITDGQVQATTK

TTQAASQVSDGQVQATTATSASAAATST

DPVDAVSCKTSGT

bCBGA1847
alpha_pre_
MRFPSIFTAVLFAASSALAAYAPSEPWS
56
94

signal-and-
TLTPTATYSGGVTDYASTFGIAVQPIST

HSP150_delta_
TSSASSAATTASSKAKRAASQIGDGQVQ

fragment_wo_
AATTTASVSTKSTAAAVSQIGDGQIQAT

signal-d21_CBDAs
TKTTAAAVSQIGDGQVQATTKTTSAKTT

AAAVSQISDGQIQATTTTLAPKSTAAAV

SQIGDGQVQATTTTLAPKSTAAAVSQIG

DGQVQATTKTTAAAVSQIGDGQVQATTK

TTAAAVSQIGDGQVQATTKTTAAAVSQI

GDGQVQATTKTTAAAVSQITDGQVQATT

KTTQAASQVSDGQVQATTATSASAAATS

TDPVDAVSCKTSGT

bCBGA1851
HSP150 secretion
MQYKKTLVASALAATTLA
57
60

signal-d21_CBDAs

bCBGA1852
HSP150_delta_
MQYKKTLVASALAATTLAAYAPSEPWST
58
126

fragment-and-
LTPTATYSGGVTDYASTFGIAVQPISTT

LEKREAEA-
SSASSAATTASSKAKRAASQIGDGQVQA

d21_CBDAs
ATTTASVSTKSTAAAVSQIGDGQIQATT

KTTAAAVSQIGDGQIQATTKTTSAKTTA

AAVSQISDGQIQATTTTLAPKSTAAAVS

QIGDGQVQATTTTLAPKSTAAAVSQIGD

GQVQATTKTTAAAVSQIGDGQVQATTKT

TAAAVSQIGDGQVQATTKTTAAAVSQIG

DGQVQATTKTTAAAVSQITDGQVQATTK

TTQAASQVSDGQVQATTATSASAAATST

DPVDAVSCKTSGTLEKREAEA

bCBGA1860
YDR055W_N term
MQLHSLIASTALLITSALA
59
12

19 AA-d21_CBDAs

bCBGA1861
YGR279C_N term
MRLSNLIASASLLSAATLA
60
44

19 AA-d21_CBDAs

bCBGA1862
YGR279C_N term
MRLSNLIASASLLSAATLAAPANHEHKD
61
24

31 AA-d21_CBDAs
KRA

bCBGA1863
YLR300W_N term
MLSLKTLLCTLLTVSSVLA
62
20

19 AA-d21_CBDAs

bCBGA1864
YLR300W_N term
MLSLKTLLCTLLTVSSVLATPVPA
63
54

24 AA-d21_CBDAs

bCBGA1865
YLR300W_N term
MLSLKTLLCTLLTVSSVLATPVPARDPS
64
58

30 AA-d21_CBDAs
SI

bCBGA1873
YAP_TA57-
MKLKTVRSAVLSSLFASQVLGQTTAQTN
65
50

yEVenus-spacer-
SGGLDVVGLISMAMSKGEELFTGVVPIL

d21_CBDAs
VELDGDVNGHKFSVSGEGEGDATYGKLT

LKLICTTGKLPVPWPTLVTTLGYGLQCF

ARYPDHMKQHDFFKSAMPEGYVQERTIF

FKDDGNYKTRAEVKFEGDTLVNRIELKG

IDFKEDGNILGHKLEYNYNSHNVYITAD

KQKNGIKANFKIRHNIEDGGVQLADHYQ

QNTPIGDGPVLLPDNHYLSYQSALSKDP

NEKRDHMVLLEFVTAAGITHGMDELYKE

EGEPK

bCBGA1874
YAP_TA57-Kex2-
MKLKTVRSAVLSSLFASQVLGQTTAQTN
66
51

yEVenus-spacer-
SGGLDVVGLISMAKRKRMSKGEELFTGV

d21_CBDAs
VPILVELDGDVNGHKFSVSGEGEGDATY

GKLTLKLICTTGKLPVPWPTLVTTLGYG

LQCFARYPDHMKQHDFFKSAMPEGYVQE

RTIFFKDDGNYKTRAEVKFEGDTLVNRI

ELKGIDFKEDGNILGHKLEYNYNSHNVY

ITADKQKNGIKANFKIRHNIEDGGVQLA

DHYQQNTPIGDGPVLLPDNHYLSYQSAL

SKDPNEKRDHMVLLEFVTAAGITHGMDE

LYKEEGEPK

bCBGA1877
YAP_TA57_Kex2_
MKLKTVRSAVLSSLFASQVLGQTTAQTN
67
49

spacer.d21_
SGGLDVVGLISMAKREEGEPK

CBDAs_del_KR_

motif

bCBGA1878
YAP_TA57_Kex2_
MKLKTVRSAVLSSLFASQVLGQTTAQTN
68
50

spacer.d21_
SGGLDVVGLISMAEEGEPK

CBDAs_del_KEX2_

motif

bCBGA1882
MEL1m3 signal-
MRAFLFLTACISLPGVFGVEEGEPK
69
64

d21_CBDAs

bCBGA1884
INU1A signal-
MKLAYSLLLPLAGVSASVINYKRMAMVS
70
61

noSpacer-

d21_CBDAs

bCBGA1885
INU1 signal-
MKFAYSLLLPLAGVSASVINYKRMAMVS
71
60

noSpacer-

d21_CBDAs

bCBGA1886
MEL1m3 signal-
MRAFLFLTACISLPGVFGV
72
25

noSpacer-

d21_CBDAs

bCBGA1887
N/A
MRAFLFLTACISLPGVFG
73
NA

TABLE 11

SEQ

Plasmid
Signal Sequence
ID

ID
Nucleotide Sequence
NO:

bCBGA1827
ATGAGGCAGGTTTGGTTCTCTTGGATTGTGGGATTGTT
74

CCTATGTTTTTTCAACGTGTCTTCTGCTGCTCCAGTCA

ACACTACAACAGAAGATGAAACGGCACAAATTCCGGC

TGAAGCTGTCATCGGTTACTTAGATTTAGAAGGGGAT

TTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAA

TAACGGGTTATTGTTTATAAATACTACTATTGCCAGCA

TTGCTGCTAAAGAAGAAGGGGTATCTTTGGATAAAAG

AGAGGCTGAAGCT

bCBGA1829
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGC
75

AGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACA

ACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTG

TCATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTT

GCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTT

ATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTA

AAGAAGAAGGGGTATCTTTGGATAAAAGAGAGGCTG

AAGCT

bCBGA1835
ATGCAAAGACCATTTCTACTCGCTTATTTGGTCCTTTC
76

GCTTCTATTTAACTCAGCTTTGGGT

bCBGA1836
ATGCGTTTCTCTACTACACTCGCTACTGCAGCTACTGC
77

GCTATTTTTCACAGCCTCCCAAGTTTCAGCT

bCBGA1837
ATGAGGCAGGTTTGGTTCTCTTGGATTGTGGGATTGTT
78

CCTATGTTTTTTCAACGTGTCTTCTGCT

bCBGA1838
ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTT
79

TGCAGCCAAAATATCTGCA

bCBGA1839
ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTC
80

TTTGGCCAATGCAGCTCCAGTCAACACTACAACAGAAG

ATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGG

TTACTTAGATTTAGAAGGGGATTTCGATGTTGCTGTTT

TGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTT

ATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAG

AAGGGGTATCTTTGGATAAAAGAGAGGCTGAAGCT

bCBGA1840
ATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTAT
81

TGCTTCAGTTTTAGCAGCTCCAGTCAACACTACAACAG

AAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCAT

CGGTTACTTAGATTTAGAAGGGGATTTCGATGTTGCTG

TTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG

TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGA

AGAAGGGGTATCTTTGGATAAAAGAGAGGCTGAAGCT

bCBGA1842
ATGCAAAGACCATTTCTACTCGCTTATTTGGTCCTTTC
82

GCTTCTATTTAACTCAGCTTTGGGTGCTCCAGTCAACA

CTACAACAGAAGATGAAACGGCACAAATTCCGGCTGA

AGCTGTCATCGGTTACTTAGATTTAGAAGGGGATTTCG

ATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAAC

GGGTTATTGTTTATAAATACTACTATTGCCAGCATTGC

TGCTAAAGAAGAAGGGGTATCTTTGGATAAAAGAGAG

GCTGAAGCT

bCBGA1843
ATGCGTTTCTCTACTACACTCGCTACTGCAGCTACTGC
83

GCTATTTTTCACAGCCTCCCAAGTTTCAGCTGCTCCAG

TCAACACTACAACAGAAGATGAAACGGCACAAATTCC

GGCTGAAGCTGTCATCGGTTACTTAGATTTAGAAGGG

GATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCAC

AAATAACGGGTTATTGTTTATAAATACTACTATTGCCA

GCATTGCTGCTAAAGAAGAAGGGGTATCTTTGGATAA

AAGAGAGGCTGAAGCT

bCBGA1845
ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTT
84

TGCAGCCAAAATATCTGCAGCTCCAGTCAACACTACAA

CAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGT

CATCGGTTACTTAGATTTAGAAGGGGATTTCGATGTTG

CTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAA

AGAAGAAGGGGTATCTTTGGATAAAAGAGAGGCTGA

AGCT

bCBGA1846
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGC
85

CGCTACTACATTGGCCGCCTATGCTCCATCTGAGCCTT

GGTCCACTTTGACTCCAACAGCCACTTACAGCGGTGG

TGTTACCGACTACGCTTCCACCTTCGGTATTGCCGTTC

AACCAATCTCCACTACATCCAGCGCATCATCTGCAGC

CACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTCC

CAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACTA

CTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGTT

TCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTA

AGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGT

CAAATTCAAGCTACCACCAAGACTACCTCTGCTAAGA

CTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAA

ATCCAAGCTACCACCACTACTTTAGCCCCAAAGAGCA

CCGCTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTT

CAAGCTACCACCACTACTTTAGCCCCAAAGAGCACCG

CTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAA

GCTACTACTAAGACTACCGCTGCTGCTGTCTCTCAAAT

TGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACT

GCTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCA

AGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAA

TCGGTGATGGTCAAGTTCAAGCAACTACCAAAACCAC

TGCCGCAGCTGTTTCCCAAATTACTGACGGTCAAGTTC

AAGCCACTACAAAAACCACTCAAGCAGCCAGCCAAGT

AAGCGATGGCCAAGTCCAAGCTACTACTGCTACTTCC

GCTTCTGCAGCCGCTACCTCCACTGACCCAGTCGATGC

TGTCTCCTGTAAGACTTCTGGTACC

bCBGA1847
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGC
86

AGCATCCTCCGCATTAGCTGCCTATGCTCCATCTGAGC

CTTGGTCCACTTTGACTCCAACAGCCACTTACAGCGGT

GGTGTTACCGACTACGCTTCCACCTTCGGTATTGCCGT

TCAACCAATCTCCACTACATCCAGCGCATCATCTGCAG

CCACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTC

CCAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACT

ACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGT

TTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTA

AGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGT

CAAGTTCAAGCTACCACCAAGACTACCTCTGCTAAGA

CTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAA

ATCCAAGCTACCACCACTACTTTAGCCCCAAAGAGCA

CCGCTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTT

CAAGCTACCACCACTACTTTAGCCCCAAAGAGCACCG

CTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTCCAA

GCTACTACTAAGACTACCGCTGCTGCTGTCTCTCAAAT

TGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACT

GCTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCA

AGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAA

TCGGTGATGGTCAAGTTCAAGCAACTACCAAAACCAC

TGCCGCAGCTGTTTCCCAAATTACTGACGGTCAAGTTC

AAGCCACTACAAAAACCACTCAAGCAGCCAGCCAAGT

AAGCGATGGCCAAGTCCAAGCTACTACTGCTACTTCC

GCTTCTGCAGCCGCTACCTCCACTGACCCAGTCGATGC

TGTCTCCTGTAAGACTTCTGGTACC

bCBGA1851
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGC
87

CGCTACTACATTGGCC

bCBGA1852
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGC
88

CGCTACTACATTGGCCGCCTATGCTCCATCTGAGCCTT

GGTCCACTTTGACTCCAACAGCCACTTACAGCGGTGG

TGTTACCGACTACGCTTCCACCTTCGGTATTGCCGTTC

AACCAATCTCCACTACATCCAGCGCATCATCTGCAGC

CACCACAGCCTCATCTAAGGCCAAGAGAGCTGCTTCC

CAAATTGGTGATGGTCAAGTCCAAGCTGCTACCACTA

CTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCCGTT

TCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTA

AGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGT

CAAATTCAAGCTACCACCAAGACTACCTCTGCTAAGA

CTACCGCCGCTGCCGTTTCTCAAATCAGTGATGGTCAA

ATCCAAGCTACCACCACTACTTTAGCCCCAAAGAGCA

CCGCTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTT

CAAGCTACCACCACTACTTTAGCCCCAAAGAGCACCG

CTGCTGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAA

GCTACTACTAAGACTACCGCTGCTGCTGTCTCTCAAAT

TGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACT

GCTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCA

AGCTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAA

TCGGTGATGGTCAAGTTCAAGCAACTACCAAAACCAC

TGCCGCAGCTGTTTCCCAAATTACTGACGGTCAAGTTC

AAGCCACTACAAAAACCACTCAAGCAGCCAGCCAAGT

AAGCGATGGCCAAGTCCAAGCTACTACTGCTACTTCC

GCTTCTGCAGCCGCTACCTCCACTGACCCAGTCGATGC

TGTCTCCTGTAAGACTTCTGGTACCTTGGAGAAAAGA

GAGGCTGAAGCA

bCBGA1860
ATGCAATTACATTCACTTATCGCTTCAACTGCGCTCTT
89

AATAACGTCAGCTTTGGCT

bCBGA1861
ATGCGTCTCTCTAACCTAATTGCTTCTGCCTCTCTTTT
90

ATCTGCTGCTACTCTTGCT

bCBGA1862
ATGCGTCTCTCTAACCTAATTGCTTCTGCCTCTCTTTT
91

ATCTGCTGCTACTCTTGCTGCTCCCGCTAACCACGAAC

ACAAGGACAAGCGTGCT

bCBGA1863
ATGCTTTCGCTTAAAACGTTACTGTGTACGTTGTTGAC
92

TGTGTCATCAGTACTCGCT

bCBGA1864
ATGCTTTCGCTTAAAACGTTACTGTGTACGTTGTTGAC
93

TGTGTCATCAGTACTCGCTACCCCAGTCCCTGCA

bCBGA1865
ATGCTTTCGCTTAAAACGTTACTGTGTACGTTGTTGAC
94

TGTGTCATCAGTACTCGCTACCCCAGTCCCTGCAAGAG

ACCCTTCTTCCATT

bCBGA1873
ATGAAACTGAAAACTGTAAGATCTGCGGTCCTTTCGT
95

CACTCTTTGCATCGCAGGTTCTCGGTCAAACCACTGCC

CAGACTAATAGTGGCGGACTTGACGTGGTGGGGTTAA

TTTCTATGGCGATGTCTAAAGGTGAAGAATTATTCACT

GGTGTTGTCCCAATTTTGGTTGAATTAGATGGTGATGT

TAATGGTCACAAATTTTCTGTCTCCGGTGAAGGTGAA

GGTGATGCTACTTACGGTAAATTGACCTTAAAATTGAT

TTGTACTACTGGTAAATTGCCAGTTCCATGGCCAACCT

TAGTCACTACTTTAGGTTATGGTTTGCAATGTTTTGCT

AGATACCCAGATCATATGAAACAACATGACTTTTTCA

AGTCTGCCATGCCAGAAGGTTATGTTCAAGAAAGAAC

TATTTTTTTCAAAGATGACGGTAACTACAAGACCAGA

GCTGAAGTCAAGTTTGAAGGTGATACCTTAGTTAATA

GAATCGAATTAAAAGGTATTGATTTTAAAGAAGATGG

TAACATTTTAGGTCACAAATTGGAATACAACTATAAC

TCTCACAATGTTTACATCACTGCTGACAAACAAAAGA

ATGGTATCAAAGCTAACTTCAAAATTAGACACAACAT

TGAAGATGGTGGTGTTCAATTAGCTGACCATTATCAA

CAAAATACTCCAATTGGTGATGGTCCAGTCTTGTTACC

AGACAACCATTACTTATCCTATCAATCTGCCTTATCCA

AAGATCCAAACGAAAAGAGAGACCACATGGTCTTGTT

AGAATTTGTTACTGCTGCTGGTATTACCCATGGTATGG

ATGAATTGTACAAAGAAGAAGGTGAACCAAAA

bCBGA1874
ATGAAACTGAAAACTGTAAGATCTGCGGTCCTTTCGT
96

CACTCTTTGCATCGCAGGTTCTCGGTCAAACCACTGCC

CAGACTAATAGTGGCGGACTTGACGTGGTGGGGTTAA

TTTCTATGGCGAAGAGGAAAAGAATGTCTAAAGGTGA

AGAATTATTCACTGGTGTTGTCCCAATTTTGGTTGAAT

TAGATGGTGATGTTAATGGTCACAAATTTTCTGTCTCC

GGTGAAGGTGAAGGTGATGCTACTTACGGTAAATTGA

CCTTAAAATTGATTTGTACTACTGGTAAATTGCCAGTT

CCATGGCCAACCTTAGTCACTACTTTAGGTTATGGTTT

GCAATGTTTTGCTAGATACCCAGATCATATGAAACAA

CATGACTTTTTCAAGTCTGCCATGCCAGAAGGTTATGT

TCAAGAAAGAACTATTTTTTTCAAAGATGACGGTAAC

TACAAGACCAGAGCTGAAGTCAAGTTTGAAGGTGATA

CCTTAGTTAATAGAATCGAATTAAAAGGTATTGATTTT

AAAGAAGATGGTAACATTTTAGGTCACAAATTGGAAT

ACAACTATAACTCTCACAATGTTTACATCACTGCTGAC

AAACAAAAGAATGGTATCAAAGCTAACTTCAAAATTA

GACACAACATTGAAGATGGTGGTGTTCAATTAGCTGA

CCATTATCAACAAAATACTCCAATTGGTGATGGTCCA

GTCTTGTTACCAGACAACCATTACTTATCCTATCAATC

TGCCTTATCCAAAGATCCAAACGAAAAGAGAGACCAC

ATGGTCTTGTTAGAATTTGTTACTGCTGCTGGTATTAC

CCATGGTATGGATGAATTGTACAAAGAAGAAGGTGAA

CCAAAA

bCBGA1877
ATGAAACTGAAAACTGTAAGATCTGCGGTCCTTTCGT
97

CACTCTTTGCATCGCAGGTTCTCGGTCAAACCACTGCC

CAGACTAATAGTGGCGGACTTGACGTGGTGGGGTTAA

TTTCTATGGCGAAAAGAGAAGAAGGTGAACCAAAA

bCBGA1878
ATGAAACTGAAAACTGTAAGATCTGCGGTCCTTTCGT
98

CACTCTTTGCATCGCAGGTTCTCGGTCAAACCACTGCC

CAGACTAATAGTGGCGGACTTGACGTGGTGGGGTTAA

TTTCTATGGCGGAAGAAGGTGAACCAAAA

bCBGA1882
ATGAGAGCTTTCTTGTTTCTCACCGCATGCATCAGTTT
99

GCCAGGCGTTTTTGGGGTGGAAGAAGGTGAACCAAAA

bCBGA1884
ATGAAGTTAGCATACTCCCTCTTGCTTCCATTGGCAGG
100

AGTCAGTGCTTCAGTTATCAATTACAAGAGAATGGCA

ATGGTATCA

bCBGA1885
ATGAAGTTCGCATACTCCCTCTTGCTTCCATTGGCAGG
101

AGTCAGTGCTTCAGTTATCAATTACAAGAGAATGGCA

ATGGTATCA

bCBGA1886
ATGAGAGCTTTCTTGTTTCTCACCGCATGCATCAGTTT
102

GCCAGGCGTTTTTGGGGTG

bCBGA1887
ATGAGAGCTTTCTTGTTTCTCACCGCATGCATCAGTTT
103

GCCAGGCGTTTTTGGG

A high throughput screening process of the CBDAs synthase activity was conducted as follows: Colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH is added to the samples. Finally, samples were grown for additional 42 hours and are analyzed for cannabinoids. The CBDA titer in the above described screen ranged from 7 to 285 mg/L. For details, see the Table 12 below. (The titers are also included in Table 10 above.)

TABLE 12

CBDA

plasmid ID
ORF name
(mg/L)

bCBGA1829
alpha_prepro_signal-d21_CBDAs
7

bCBGA1843
BGL2_signal-and-alpha_only_pro_signal-
9

d21_CBDAs

bCBGA1845
SUC2_signal-and-alpha_only_pro_signal-
9

d21_CBDAs

bCBGA1840
AGA2_signal-and-alpha_only_pro_signal-
10

d21_CBDAs

bCBGA1836
BGL2_signal-d21_CBDAs
11

bCBGA1842
AMYL_signal-and-alpha_only_pro_signal-
11

d21_CBDAs

bCBGA1838
SUC2_signal-d21_CBDAs
11

bCBGA1860
YDR055W_Nterm 19 AA-d21_CBDAs
12

bCBGA1827
OST1_signal-and-alpha_only_pro_signal-
13

d21_CBDAs

bCBGA1835
AMYI_signal-d21_CBDAs
19

bCBGA1863
YLR300W_Nterm 19 AA-d21_CBDAs
20

bCBGA1862
YGR279C_Nterm 31 AA-d21_CBDAs
24

bCBGA1886
MEL1m3 signal-noSpacer-d21_CBDAs
25

bCBGA1826
OST1_signal-d21_CBDAs
27

bCBGA1837
OST1_signal-d21_CBDAs
41

bCBGA1861
YGR279C_Nterm 19 AA-d21_CBDAs
44

bCBGA1839
PHO5_signal-and-alpha_only_pro_signal-
44

d21_CBDAs

bCBGA1877
YAP_TA57_Kex2_spacer.d21_CBDAs_del_KR_motif
49

bCBGA1873
YAP_TA57-yEVenus-spacer-d21_CBDAs
50

bCBGA1878
YAP_TA57_Kex2_spacer.d21_CBDAs_del_KEX2_motif
50

bCBGA1874
YAP_TA57-Kex2-yEVenus-spacer-d21_CBDAs
51

bCBGA1864
YLR300W_Nterm 24 AA-d21_CBDAs
54

bCBGA1865
YLR300W_Nterm 30 AA-d21_CBDAs
58

bCBGA1851
HSP150 secretion signal-d21_CBDAs
60

bCBGA1885
INU1 signal-noSpacer-d21_CBDAs
60

bCBGA1884
INU1A signal-noSpacer-d21_CBDAs
61

bCBGA1882
MEL1m3 signal-d21_CBDAs
64

bCBGA1847
alpha_pre_signal-and-
94

HSP150_delta_fragment_wo_signal-d21_CBDAs

bCBGA1875
YAP_TA57_Kex2_spacer-yEVenus-d21_CBDAs
101

bCBGA1846
HSP150_delta_fragment-d21_CBDAs
102

bCBGA1853
alpha_pre_signal-and-
114

HSP150_delta_fragment_wo_signal-and-

LEKREAEA-d21_CBDAs

RUNM001233_67.1
YAP_TA57_Kex2_spacer.d21_CBDAs
124

bCBGA1852
HSP150_delta_fragment-and-LEKREAEA-
126

d21_CBDAs

bCBGA1831
YAP_TA57_Kex2_spacer-d21_CBDAs
133

bCBGA1832
PHO5_signal-d21_CBDAs
135

bCBGA1850
PIR3 secretion signal-d21_CBDAs
138

bCBGA1883
K28 viral signal-noSpacer-d21_CBDAs
149

bCBGA1849
alpha_pre_signal-and-
151

PIR3_delta_fragment_wo_singal-d21_CBDAs

bCBGA1880
INU1A signal-d21_CBDAs
181

bCBGA1881
INU1-d21_CBDAs
188

bCBGA1855
alpha_pre_signal-and-
193

PIR3_delta_fragment_wo_signal-and-LEKREAEA-

d21_CBDAs

bCBGA1879
K28 viral signal-d21_CBDAs
219

bCBGA1848
PIR3_delta_fragment-d21_CBDAs
285

bCB GA1854
PIR3_delta_fragment-and-LEKREAEA-d21_CBDAs
285

Proteomic analysis using the methods described in Example 4 confirmed that the most active sample had an elevated CBDA synthase concentration in the culture supernatant, most probably due to more effective CBDA synthase secretion. CBDA synthase supernatant concentration was over 15-fold more in strains transformed with the bCBGA1854 plasmid (SEQ ID No: 435), than strains transformed with RUNM001233_67.1. (The corresponding CBDA titers are 285 mg/L and 124 mg/L, respectively.) That is, CBDA productivity and secretion can be increased by improving CBDA synthase secretion.

The supernatant of the culture transformed with the bCBGA1854 plasmid was mixed with 100 mg/L CBGA, and after 48 hours of incubation, 30 mg/L CBDA was detected, proving that there is active CBDA synthase in the supernatant of the yeast culture.

Example 18—Enhanced CBDA Synthase Secretion II

The secretion of CBDAs was further optimized. A second series of plasmids were constructed containing the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and the CBDA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter. The first 21, or in some cases 28, amino acids of the CBDA synthase in each plasmid was replaced with different yeast secretion signals. The secretion signals included various combinations of the K28 viral secretion signal, the N-terminal 233 amino acid of the PIR3 protein, denoted as PIR3 delta fragment (with or without its predicted native secretion signal), a Kex2p cleavage site and a short spacer motif. The nucleotide sequences and protein sequences of the tested signal sequences are included in Tables 13-14 below, along with the plasmid IDs, the corresponding amino acid sequences, the ORF names and CBDA titer.

TABLE 13

Signal Sequence
SEQ ID

Plasmid ID
ORF name
Amino Acid Sequence
NO:
Titer

0253/asn001-1
K28_signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
104
373

PIR3_delta_
NLKYARGAYAPKDPWSTLTPSATYKGG

fragment_wo_
ITDYSSSFGIAIEAVATSASSVASSKAKR

signal-Spacer-
AASQIGDGQVQAATTTAAVSKKSTAAA

d28_CBDAs_
VSQITDGQVQAAKSTAAAVSQITDGQV

Onofri
QAAKSTAAAVSQITDGQVQAAKSTAAA

VSQITDGQVQAAKSTAAAASQISDGQVQ

ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NS

0253/asn005-1
K28_signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
105
377

PIR3_delta_
NLKYARGAYAPKDPWSTLTPSATYKGG

fragment_wo_
ITDYSSSFGIAIEAVATSASSVASSKAKR

signal-
AASQIGDGQVQAATTTAAVSKKSTAAA

LEKREAEA-
VSQITDGQVQAAKSTAAAVSQITDGQV

Spacer-d28_
QAAKSTAAAVSQITDGQVQAAKSTAAA

CBDAs_Onofri
VSQITDGQVQAAKSTAAAASQISDGQVQ

ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NSTLEKREAE

0253/asn037-3
K28_signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
106
366

PIR3_delta_
NLKYARGAYAPKDPWSTLTPSATYKGG

fragment_wo_
ITDYSSSFGIAIEAVATSASSVASSKAKR

signal-
AASQIGDGQVQAATTTAAVSKKSTAAA

LEKREAEA-
VSQITDGQVQAAKSTAAAVSQITDGQV

d21_CBDAs_
QAAKSTAAAVSQITDGQVQAAKSTAAA

Onofri
VSQITDGQVQAAKSTAAAASQISDGQVQ

ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NSTLEKREAEA

0253/asn049-1
K28_signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
107
374

PIR3_delta_
NLKYARGAYAPKDPWSTLTPSATYKGG

fragment_wo_
ITDYSSSFGIAIEAVATSASSVASSKAKR

signal-
AASQIGDGQVQAATTTAAVSKKSTAAA

Spacer-d21_
VSQITDGQVQAAKSTAAAVSQITDGQV

CBDAs_Onofri
QAAKSTAAAVSQITDGQVQAAKSTAAA

VSQITDGQVQAAKSTAAAASQISDGQVQ

ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NSTEEGEPK

0253/asn052-3
PIR3_delta_
MQYKKPLVVSALAATSLAAYAPKDPWS
108
298

fragment-
TLTPSATYKGGITDYSSSFGIAIEAVATSA

Spacer-
SSVASSKAKRAASQIGDGQVQAATTTA

d21_CBDAs_
AVSKKSTAAAVSQITDGQVQAAKSTAA

Onofri
AVSQITDGQVQAAKSTAAAVSQITDGQ

VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDGQVQATTSTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNSTEEGEPK

0253/asn053-2
K28_signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
109
381

PIR3_delta_
NLKYARGAYAPKDPWSTLTPSATYKGG

fragment_
ITDYSSSFGIAIEAVATSASSVASSKAKR

wo_signal-
AASQIGDGQVQAATTTAAVSKKSTAAA

LEKREAEA-
VSQITDGQVQAAKSTAAAVSQITDGQV

Spacer-
QAAKSTAAAVSQITDGQVQAAKSTAAA

d21_CBDAs_
VSQITDGQVQAAKSTAAAASQISDGQVQ

Onofri
ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NSTLEKREAEAEEGEPK

0253/asn056-1
PIR3_delta_
MESVSSLFNIFSTIMVNYKSLVLALLSVS
110
298

fragment-
NLKYARGAYAPKDPWSTLTPSATYKGG

LEKREAEA-
ITDYSSSFGIAIEAVATSASSVASSKAKR

Spacer-
AASQIGDGQVQAATTTAAVSKKSTAAA

d21_CBDAs_
VSQITDGQVQAAKSTAAAVSQITDGQV

Onofri
QAAKSTAAAVSQITDGQVQAAKSTAAA

VSQITDGQVQAAKSTAAAASQISDGQVQ

ATTSTKAAASQITDGQIQASKTTSGASQ

VSDGQVQATAEVKDANDPVDVVSCNN

NS

TABLE 14

Plasmid ID
Signal Sequence Nucleotide Sequence
SEQ ID NO:

0253/asn001-1
ATGGAATCTGTTTCTTCTTTGTTCAACATCTTCTCTAC
111

TATCATGGTTAACTACAAGTCTTTGGTTTTGGCTTTG

TTGTCTGTTTCTAACTTGAAGTACGCTAGAGGTGCCT

ATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCAT

CAGCTACTTACAAGGGTGGTATAACAGATTACTCTT

CGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCA

GTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAG

CCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTG

CCACTACTACTGCTGCTGTTTCTAAGAAATCCACCGC

TGCTGCTGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATAA

CTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCG

CTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAG

CTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAAC

TGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGC

CGCTGCCTCTCAGATTTCTGACGGCCAAGTTCAGGC

CACTACCTCTACTAAGGCTGCTGCATCCCAAATTAC

AGATGGGCAGATACAAGCATCTAAAACTACCAGTGG

CGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTAC

TGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGT

TGTTTCCTGTAATAACAATAGT

0253/asn005-1
ATGGAATCTGTTTCTTCTTTGTTCAACATCTTCTCTAC
112

TATCATGGTTAACTACAAGTCTTTGGTTTTGGCTTTG

TTGTCTGTTTCTAACTTGAAGTACGCTAGAGGTGCCT

ATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCAT

CAGCTACTTACAAGGGTGGTATAACAGATTACTCTT

CGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCA

GTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAG

CCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTG

CCACTACTACTGCTGCTGTTTCTAAGAAATCCACCGC

TGCTGCTGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATAA

CTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCG

CTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAG

CTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAAC

TGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGC

CGCTGCCTCTCAGATTTCTGACGGCCAAGTTCAGGC

CACTACCTCTACTAAGGCTGCTGCATCCCAAATTAC

AGATGGGCAGATACAAGCATCTAAAACTACCAGTGG

CGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTAC

TGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGT

TGTTTCCTGTAATAACAATAGTACCTTGGAGAAAAG

AGAGGCTGAA

0253/asn037-3
ATGGAATCTGTTTCTTCTTTGTTCAACATCTTCTCTAC
113

TATCATGGTTAACTACAAGTCTTTGGTTTTGGCTTTG

TTGTCTGTTTCTAACTTGAAGTACGCTAGAGGTGCCT

ATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCAT

CAGCTACTTACAAGGGTGGTATAACAGATTACTCTT

CGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCA

GTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAG

CCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTG

CCACTACTACTGCTGCTGTTTCTAAGAAATCCACCGC

TGCTGCTGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATAA

CTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCG

CTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAG

CTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAAC

TGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGC

CGCTGCCTCTCAGATTTCTGACGGCCAAGTTCAGGC

CACTACCTCTACTAAGGCTGCTGCATCCCAAATTAC

AGATGGGCAGATACAAGCATCTAAAACTACCAGTGG

CGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTAC

TGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGT

TGTTTCCTGTAATAACAATAGTACCTTGGAGAAAAG

AGAGGCTGAAGCA

0253/asn049-1
ATGGAATCTGTTTCTTCTTTGTTCAACATCTTCTCTAC
114

TATCATGGTTAACTACAAGTCTTTGGTTTTGGCTTTG

TTGTCTGTTTCTAACTTGAAGTACGCTAGAGGTGCCT

ATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCAT

CAGCTACTTACAAGGGTGGTATAACAGATTACTCTT

CGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCA

GTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAG

CCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTG

CCACTACTACTGCTGCTGTTTCTAAGAAATCCACCGC

TGCTGCTGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATAA

CTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCG

CTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAG

CTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAAC

TGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGC

CGCTGCCTCTCAGATTTCTGACGGCCAAGTTCAGGC

CACTACCTCTACTAAGGCTGCTGCATCCCAAATTAC

AGATGGGCAGATACAAGCATCTAAAACTACCAGTGG

CGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTAC

TGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGT

TGTTTCCTGTAATAACAATAGTACCGAAGAAGGTGA

ACCAAAA

0253/asn052-3
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTA
115

GCTGCTACATCTTTAGCTGCCTATGCTCCAAAGGACC

CGTGGTCCACTTTAACTCCATCAGCTACTTACAAGG

GTGGTATAACAGATTACTCTTCGAGTTTCGGTATTGC

TATTGAAGCCGTGGCTACCAGTGCTTCCTCCGTCGCC

TCATCTAAAGCAAAGAGAGCCGCCTCTCAGATAGGT

GATGGTCAAGTACAGGCTGCCACTACTACTGCTGCT

GTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAA

TAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTG

CCGCTGCTGTTTCCCAAATAACTGACGGTCAAGTTC

AAGCTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAAT

AACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGC

CGCTGCCGTTTCTCAAATAACTGATGGTCAAGTTCA

AGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGATT

TCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAG

GCTGCTGCATCCCAAATTACAGATGGGCAGATACAA

GCATCTAAAACTACCAGTGGCGCTAGTCAAGTAAGT

GATGGCCAAGTCCAGGCTACTGCTGAAGTGAAAGAC

GCTAACGATCCAGTCGATGTTGTTTCCTGTAATAACA

ATAGTACCGAAGAAGGTGAACCAAAA

0253/asn053-2
ATGGAATCTGTTTCTTCTTTGTTCAACATCTTCTCTAC
116

TATCATGGTTAACTACAAGTCTTTGGTTTTGGCTTTG

TTGTCTGTTTCTAACTTGAAGTACGCTAGAGGTGCCT

ATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCAT

CAGCTACTTACAAGGGTGGTATAACAGATTACTCTT

CGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCA

GTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAG

CCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTG

CCACTACTACTGCTGCTGTTTCTAAGAAATCCACCGC

TGCTGCTGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATAA

CTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCG

CTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAG

CTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAAC

TGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGC

CGCTGCCTCTCAGATTTCTGACGGCCAAGTTCAGGC

CACTACCTCTACTAAGGCTGCTGCATCCCAAATTAC

AGATGGGCAGATACAAGCATCTAAAACTACCAGTGG

CGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTAC

TGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGT

TGTTTCCTGTAATAACAATAGTACCTTGGAGAAAAG

AGAGGCTGAAGCAGAAGAAGGTGAACCAAAA

0253/asn056-1
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTA
117

GCTGCTACATCTTTAGCTGCCTATGCTCCAAAGGACC

CGTGGTCCACTTTAACTCCATCAGCTACTTACAAGG

GTGGTATAACAGATTACTCTTCGAGTTTCGGTATTGC

TATTGAAGCCGTGGCTACCAGTGCTTCCTCCGTCGCC

TCATCTAAAGCAAAGAGAGCCGCCTCTCAGATAGGT

GATGGTCAAGTACAGGCTGCCACTACTACTGCTGCT

GTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAA

TAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTG

CCGCTGCTGTTTCCCAAATAACTGACGGTCAAGTTC

AAGCTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAAT

AACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGC

CGCTGCCGTTTCTCAAATAACTGATGGTCAAGTTCA

AGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGATT

TCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAG

GCTGCTGCATCCCAAATTACAGATGGGCAGATACAA

GCATCTAAAACTACCAGTGGCGCTAGTCAAGTAAGT

GATGGCCAAGTCCAGGCTACTGCTGAAGTGAAAGAC

GCTAACGATCCAGTCGATGTTGTTTCCTGTAATAACA

ATAGTACCTTGGAGAAAAGAGAGGCTGAAGCAGAA

GAAGGTGAACCAAAA

This set of plasmids were each individually transformed into the yCBGA0314 strain and their CBDA productivity was assayed in a high throughput screening process optimized for CBDAs synthase activity as described in Example 17 above. The CBDA titer in the above described screen reached up to 381 mg/L. Samples titers for several transformed strains are included in Table 15 below. (The titers are also included in Table 13 above.)

TABLE 15

CBDA

Plasmid ID
ORF name
(mg/L)

0253/asn053-2
K28_signal-PIR3_delta_fragment_wo_signal-
381

LEKREAEA-Spacer-d2l_CB DAs_Onofri

0253/asn005-1
K28_signal-PIR3_delta_fragment_wo_signal-
377

LEKREAEA-Spacer-d28_CBDAs_Onofri

0253/asn049-1
K28_signal-PIR3_delta_fragment_wo_signal-
374

Spacer-d21_CBDAs_Onofri

0253/asn001-1
K28_signal-PIR3_delta_fragment_wo_signal-
373

Spacer-d28_CBDAs_Onofri

0253/asn037-3
K28_signal-PIR3_delta_fragment_wo_signal-
366

LEKREAEA-d21_CBDAs_Onofri

0253/asn052-3
PIR3_delta_fragment-Spacer-
298

d21_CBDAs_Onofri

0253/asn056-1
PIR3_delta_fragment-LEKREAEA-Spacer-
298

d21_CBDAs_Onofri

Example 19—the yCBGA0508 and yCBGA0509 Strains: VPS10 Deletion

CBDA productivity was further improved by deletions in the VPS10 gene of the host. Two deletions were made: yCBGA0508 strain was constructed by deleting the whole coding sequence of the VPS locus of the yCBGA0314 strain. yCBGA0509 strain was made by deleting the majority of the 5′ half of the coding sequence of the VPS locus of the yCBGA0314 strain. (FIG. 7).

The bCBGA1854 plasmid was transformed into the strains yCBGA0314, yCBGA0508 and yCBGA0509 and their CBDA producing capacity was assayed in an improved CBDA screening process.

Improved high throughput screening process of the CBDAs synthase activity.

Colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-pH6-2400LA (10 g/L yeast extract (Ohly), 20 g/L soy peptone (Organotechnie), 20 g/L glucose, buffered to pH6 with sodium phosphate, 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH is added to the samples. Finally, samples were grown for additional 48 hours and are analyzed for cannabinoids.

In this screen, the following CBDA titers were detected: see Table 16 below. Both types of deletion in the VPS10 gene had a positive effect on CBDA productivity.

TABLE 16

Total olivetolic
Parental

CBDA

acid (mg/L)
strain
Plasmid
titer (mg/L)

480
yCBGA0314
bCBGA1854
232

480
yCBGA0508
bCBGA1854
296

480
yCBGA0509
bCBGA1854
286

Example 20—the yCBGA0499 Strain: HXA-to-CBDA Pathway

CBDA production from a hexanoic acid substrate was tested using the yCBGA0499 strain, which contains the bCBAG1854 plasmid described in Example 19 above.

For the strain construction the parental strain was the yCBGA0314 strain. The PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the STE5 promoter were inserted into the YDR508C, YRL020C and FOX1 loci replacing the native ORFs of the yCBGA0314 strain. The PKS and OAC genes (under the regulation of the bidirectional GAL1/GAL10 promoter) and the AAE1 gene (under the regulation of the STE5 promoter) were inserted into the YDR508C locus. A second copy of the PKS and OAC genes (under the regulation of the sa promoter) and the AAE1 gene (under the regulation of the STE5 promoter) were inserted into the YRL020C locus. A third copy of the PKS and OAC genes (under the regulation of the bidirectional GAL1/GAL10 promoter) and the AAE1 gene (under the regulation of the STE5 promoter) were inserted into the FOX1 locus. A large number of isolates from this transformation were screened and an isolate with high CBGA productivity was identified as yCBGA0499.

The yCBGA0499 with bCBGA1854 plasmid was assayed for CBDA productivity using the following high throughput screening process using hexanoic acid for CBDA production. Colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl Synthetic Complete liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-pH6-HXA (10 g/L yeast extract (Ohly), 20 g/L soy peptone (Organotechnie), 20 g/L glucose, buffered to pH6 with sodium phosphate, 100 mg/L hexanoic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 40 μg hexanoic acid dissolved in 8 μl ethanol is added to the samples. Finally, the samples were grown for additional 48 hours and were analyzed for cannabinoids.

Using this screening method, the yCBGA0499 strain produced 51 mg/L CBDA in one single biological process using hexanoic acid as substrate.

Example 21—the yCBGA0519 Strain: HXA-to-CBDA Pathway II

To produce a stable prototrophic strain for CBDA production, the yCBGA0519 strain was constructed by inserting a modified CBDA synthase gene, denoted as PIR3-CBDAs, into the yCBGA0513 strain:

For the strain construction, the parental strain was the prototrophic CBGA producer yCBGA0513 strain. The gene coding for the PIR3-CBDAs under the regulation of the bidirectional GAL1/GAL10 promoter was integrated into the YKL140W locus by replacing the native YKL140W ORF of the yCBGA0513 strain. The amino acid sequence of PIR3-CBDAs consists of the N-terminal 233 amino acid of the PIR3 protein, the LEKREAEA peptide motif (SEQ ID NO: 118) as a Kex2p cleavage site, and the CBDA synthase coding sequence lacking its first 21 amino acids. The nucleotide sequence of PIR3-CBDAs is SEQ ID NO: 301. The amino acid sequence of PIR3-CBDAs is SEQ ID NO: 302.

The yCBGA0519 strain was assayed for CBDA productivity using a modified high throughput screening process using hexanoic acid for CBDA production described in Example 20 above. The yCBGA0519 strain produced 144 mg/L CBDA in one single biological process using hexanoic acid as substrate.

Example 22—Enhanced THCA Synthase Secretion

The secretion of THCAs was optimized to be able produce large amount of THCA in a single process using yeast cells. The wild type sequences for the Saccharomyces cerevisiae THCA synthase amino acid and nucleotide sequences are included in Table 17:

TABLE 17

SEO

Description

ID

of sequence
Sequence
NO:

WT THCA
ATGAACTGCTCCGCATTCTCTTTCTGGTTCGTCTGTAAAATAA
119

synthase nt
TCTTCTTCTTCTTGTCCTTCAACATCCAAATCTCCATCGCAAA

TCCACAAGAAAACTTTTTGAAGTGTTTCTCCGAATACATCCC

AAACAACCCTGCTAACCCAAAGTTTATATATACTCAACATGA

TCAATTGTACATGTCCGTTTTGAACAGTACCATCCAAAATTTG

AGATTCACTTCTGACACTACACCAAAACCTTTAGTCATTGTTA

CACCTTCCAATGTTAGTCACATTCAAGCTTCTATATTGTGCTC

TAAGAAAGTAGGTTTGCAAATCAGAACTAGATCAGGTGGTCA

TGATGCAGAAGGCATGTCTTACATCTCACAAGTTCCATTCGTT

GTAGTCGATTTGAGAAATATGCATTCCATAAAGATCGACGTT

CACAGTCAAACAGCATGGGTAGAAGCAGGTGCCACCTTGGG

TGAAGTTTACTACTGGATCAACGAAAAGAATGAAAACTTTTC

TTTCCCTGGTGGTTACTGTCCAACAGTAGGTGTCGGTGGTCAC

TTTTCTGGTGGTGGTTATGGTGCATTGATGAGAAACTACGGTT

TAGCTGCAGATAATATTATAGACGCCCATTTGGTTAACGTAG

ATGGTAAAGTTTTGGACAGAAAGTCTATGGGTGAAGATTTGT

TTTGGGCCATAAGAGGTGGTGGTGGTGAAAATTTCGGTATCA

TTGCCGCTTGGAAAATTAAGTTAGTCGCTGTTCCTTCCAAAA

GTACTATTTTCTCTGTCAAAAAGAACATGGAAATCCACGGTT

TGGTTAAGTTGTTTAATAAGTGGCAAAACATCGCTTACAAGT

ACGATAAGGACTTGGTTTTGATGACCCATTTCATCACTAAAA

ATATTACAGATAACCATGGTAAAAATAAGACCACTGTTCACG

GTTATTTTTCTTCAATTTTCCATGGTGGTGTAGATTCTTTGGTT

GATTTGATGAATAAGTCATTCCCAGAATTGGGTATTAAAAAG

ACAGATTGCAAGGAATTTTCTTGGATAGACACAACCATCTTC

TATTCAGGTGTTGTAAACTTCAACACCGCTAACTTCAAAAAG

GAAATCTTGTTGGATAGATCCGCTGGTAAAAAGACCGCTTTT

TCTATTAAATTGGACTACGTTAAGAAACCAATCCCTGAAACT

GCAATGGTCAAGATATTGGAAAAGTTGTACGAAGAAGATGT

AGGTGTCGGCATGTACGTTTTGTATCCATACGGTGGTATTATG

GAAGAAATATCTGAATCAGCCATACCATTTCCTCACAGAGCT

GGTATCATGTATGAATTATGGTACACAGCCTCATGGGAAAAG

CAAGAAGATAACGAAAAGCATATCAACTGGGTCAGATCCGT

TTACAACTTCACTACACCTTACGTTAGTCAAAACCCAAGATT

GGCATATTTGAACTACAGAGATTTGGACTTAGGTAAAACTAA

CCCTGAATCTCCAAATAACTATACACAAGCAAGAATTTGGGG

TGAAAAGTACTTTGGTAAAAATTTCAACAGATTAGTTAAAGT

AAAGACTAAAGCCGACCCTAACAACTTTTTCAGAAACGAACA

ATCCATCCCACCTTTGCCACCTCACCACCACTAA

WT THCA
MNCSAFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPA
120

synthase aa
NPKFIYTQHDQLYMSVLNSTIQNLRFTSDTTPKPLVIVTPSNVSHI

QASILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHS

IKIDVHSQTAWVEAGATLGEVYYWINEKNENFSFPGGYCPTVG

VGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSM

GEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIH

GLVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVH

GYFSSIFHGGVDSLVDLMNKSFPELGIKKTDCKEFSWIDTTIFYS

GVVNFNTANFKKEILLDRSAGKKTAFSIKLDYVKKPIPETAMVK

ILEKLYEEDVGVGMYVLYPYGGIMEEISESAIPFPHRAGIMYEL

WYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYR

DLDLGKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPN

NFFRNEQSIPPLPPHHH

A series of plasmids were constructed containing the Saccharomyces cerevisiae replication origin, the URA3 gene as an auxotrophic marker and the THCA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter. In each plasmid various yeast secretion signals were used, in some cases the first 28 amino acids of the THCA synthase protein predicted to be a plant secretion signal was removed. The nucleotide sequences and protein sequences of the tested signal sequences are included in Tables 18-19 below, along with the plasmid IDs, the corresponding amino acid sequences, the ORF names and THCA titer.

TABLE 18

Signal Sequence Amino
SEQ ID

Plasmid ID
ORF name
Acid Sequence
NO:
Titer

0279/asn004-1
PH05_signal-
MFKSVVYSILAASLANA
121
36

THCAs

0279/asn006-2
HSP150_delta_
MQYKKTLVASALAATTLAAYAPSEPWS
122
13

fragment-THCAs
TLTPTATYSGGVTDYASTFGIAVQPISTT

SSASSAATTASSKAKRAASQIGDGQVQA

ATTTASVSTKSTAAAVSQIGDGQIQATT

KTTAAAVSQIGDGQIQATTKTTSANTTA

AAVSQISDGQIQATTTTLAPKSTAAAVS

QIGDGQVQATTTTLAPKSTAAAVSQIGD

GQVQATTKTTAAAVSQIGDGQVQATTK

TTAAAVSQIGDGQVQATTKTTAAAVSQI

GDGQVQATTKTTAAAVSQITDGQVQAT

TKTTQAASQVSDGQVQATTATSASAAA

TSTDPVDAVSCKTSGT

0279/asn009-3
PIR3_delta_frag-
MQYKKPLVVSALAATSLAAYAPKDPWS
123
501

ment-d28_THCAs
TLTPSATYKGGITDYSSSFGIAIEAVATSA

SSVASSKAKRAAYQIGDGQVQAATTTA

AVSKKSTAAAVSQITDGQVQAAKSTAA

AVSQITDGQVQAAKSTAAAVSQITDGQ

VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDDQVQATTYTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNST

0279/asn010-1
PIR3_delta_frag-
MQYKKPLVVSALAATSLAAYAPKDPWS
124
4

ment-THCAs
TLTPSATYKGGITDYSSSFGIAIEAVATSA

SSVASSKAKRAASQIGDGQVQAATTTA

AVSKKSTAAAVSQITDGQVQAAKSTAA

AVSQITDGQVQAAKSTAAAVSQITDGQ

VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDGQVQATTSTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNST

0279/asn012-3
alpha_pre_signal-
MRFPSIFTAVLFAASSALAAYAPKDPWS
125
10

and-
TLTPSATYKGGITDYSSSFGIAIEAVATSA

PIR3_delta_frag-
SSVASSKAKRAASQIGDGQVQAATTTA

ment_wo_singal-
AVSKKSTAAAVSQITDGQVQAAKSTAA

THCAs
AVSQITDGQVQAAKSTAAAVSQITDGQ

VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDGQVQATTSTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNST

0279/asn014-2
PIR3 secretion
MQYKKPLVVSALAATSLA
126
122

signal-THCAs

0279/asn015-2
HSP150_delta_
MQYKKTLVASALAATTLAAYAPSEPWS
127
500

fragment-and-
TLTPTATYSGGVTDYASTFGIAVQQISTT

LEKREAEA-
SSASSAATTASSKAKRAASQIGDGQVQA

d28_THCAs
ATTTASVSTKSTAAAVSQIGDGQIQATT

KTTAAAVSQIGDGQIQATTKTTSAKTTA

AAVSQISDGQIQATTTTLAPKSTAAAVS

QIGDGQVQATTTTLAPKSTAAAVSQIGD

GQVQATTKTTAAAVSQIGDGQVQATTK

TTAAAVSQIGDGQVQATTKTTAAAVSQI

GDGQVQATTKTTAAAVSQITDGQVQAT

TKTTQAASQVSDGQVQATTATSASAAA

TSTDPVDAVSCKTSGTLEKREAEA

0279/asn016-3
HSP150_delta_
MQYKKTLVASALAATTLAAYAPSEPWS
128
9

fragment-and-
TLTPTATYSGGVTDYASTFGIAVQPISTT

LEKREAEA-
SSASSAATTASSKAKRAASQIGDGQVQA

THCAs
ATTTASVSTKSTAAAVSQIGDGQIQATT

KTTAAAVSQICDGQIQATTKTTSAKTTA

AAVSQISDGQIQATTTTLAPKSTAAAVS

QIGDGQVQATTTTLAPKSTAAAVSQIGD

GQVQATTKTTAAAVSQIGDGQVQATTK

TTAAAVSQIGDGQVQATTKTTAAAVSQI

GDGQVQATTKTTAAAVSQITDGQVQAT

TKTTQAASQVSDGQVQATTATSASAAA

TSTDPVDAVSCKTSGTLEKREAEA

0279/asn018-1
alpha_pre_signal-
MRFPSIFTAVLFAASSALAAYAPSEPWST
129
7

and-
LTPTATYSGGVTDYASTFGIAVQPISTTS

HSP150_delta_frag-
SASSAATTASSKAKRAASQIGDGQVQAA

ment_wo_signal-
TTTASVSTKSTAAAVSQIGDGQIQATTK

and-LEKREAEA-
TTAAAVSQIGDGQIQATTKTTSAKTTAA

THCAs
AVSQISDGQIQATTTTLAPKSTAAAVSQI

GDGQVQATTTTLAPKSTAAAVSQIGDG

QVQATTKTTAAAVSQIGDGQVQATTKT

TAAAVSQIGDGQVQATTKTTAAAVSQIG

DGQVQATTKTTAAAVSQITDGQVQATT

KTTQAASQVSDGQVQATTATSASAAAT

STDPVDAVSCKTSGTLEKREAEA

0279/asn019-2
PIR3_delta_
MQYKKPLVVSALAATSLAAYAPKDPWS
130
491

fragment-and-
TLTPSATYKGGITDYSSSFGIAIEAVATSA

LEKREAEA-
SSVASSKAKRAASQIGDGQVQAATTTA

d28_THCAs
AVSKKSTAAAVSQITDGQVQAAKSTAA

AVSQITDGQVQAAKSTAAAVSQITDGQ

VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDGQVQATTSTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNSTLEKREAEA

0279/asn020-3
PIR3_delta_
MQYKKPLVVSALAATSLAAYAPKDPWS
131
37

fragment-and-
TLTPSATYKGGITDYSSSFGIAIEAVATSA

LEKREAEA-
SSVASSKAKRAASQIGDGQVQAATTTA

THCAs
AVSKKSTAAAVSQITDGQVQAAKSTAA

AVSQITDGQVQAAKSTSAAVSQITDGQV

QAAKSTAAAVSQITDGQVQAAKSTAAA

ASQISDGQVQATTSTKAAASQITDGQIQ

ASKTTSGASQVSDGQVQATAEVKDAND

PVDVVSCNNNSTLEKREAEAMNCSAFSF

WFVCKIIFFFLSFNIQISIA

0279/asn021-2
alpha_pre_signal-
MRFPSIFTAVLFAASSALAAYAPKDPWS
132
512

and-
TLTPSATYKGGITDYSSSFGIAIEAVATSA

PIR3_delta_frag-
SSVASSKAKRAASQIGDGQVQAATTTA

ment_wo_signal-
AVSKKSTAAAVSQITDGQVQAAKSTAA

and-LEKREAEA-
AVSQITDGQVQAAKSTAAAVSQITDGQ

d28_THCAs
VQAAKSTAAAVSQITDGQVQAAKSTAA

AASQISDGQVQATTSTKAAASQITDGQI

QASKTTSGASQVSDGQVQATAEVKDAN

DPVDVVSCNNNSTLEKREAEA

0279/asn024-3
YAP_TA57_Kex2
MKLKTVRSAVLSSLFASQVLGQTTAQT
133
233

_spacer-yEVenus-
NSGGLDVVGLISMAKRKREEGEPKMSK

THCAs
GEELFTGVVPILVELDGDVNGHKFSVSG

EGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLQCFARYPDHMKQHDFFKS

AMPEGYVQERTIFFKDDGNYKTRAEVK

FEGDTLVNRIELKGIDFKEDGNILGHKLE

YNYNSHNVYITADKQKNGIKANFKIRHN

IEDGGVQLADHYQQNTPIGDGPVLLPDN

HYLSYQSALSKDPNEKRDHMVLLEFVT

AAGITHGMDELYK

0279/asn026-2
K28 viral signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
134
26

THCAs
NLKYARGEEGEPK

0279/asn028-1
INU1A signal-
MKLAYSLLLPLAGVSASVINYKRMAMV
135
56

THCAs
SEEGEPK

0279/asn030-1
INU1-THCAs
MKFAYSLLLPLAGVSASVINYKRMAMV
136
58

SEEGEPK

0279/asn032-2
K28 viral signal-
MESVSSLFNIFSTIMVNYKSLVLALLSVS
137
111

noSpacer-THCAs
NLKYARG

0279/asn034-4
YAP_TA57_Kex2
MKLKTVRSAVLSSLFASQVLGQTTAQT
138
76

_spacer-THCAs
NSGGLDVVGLISMAKRKREEGEPK

TABLE 19

SEQ

ID

Plasmid ID
Signal Sequence Nucleotide Sequence
NO:

0279/asn004-1
ATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGC
139

CAATGCA

0279/asn006-2
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCT
140

ACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCCACTT

TGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACG

CTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACATC

CAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAA

GAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGC

TACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCC

GTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTAAG

ACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAATTC

AAGCTACCACCAAGACTACCTCTGCTAATACTACCGCCGCTG

CCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCA

CTACTTTAGCCCCAAAGAGCACCGCTGCTGCCGTTTCTCAAA

TCGGTGATGGTCAAGTTCAAGCTACCACCACTACTTTAGCCC

CAAAGAGCACCGCTGCTGCCGTTTCTCAAATCGGTGATGGTC

AAGTTCAAGCTACTACTAAGACTACCGCTGCTGCTGTCTCTC

AAATTGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACTG

CTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAAGCTA

CTACCAAGACTACCGCTGCTGCTGTCTCTCAAATCGGTGATG

GTCAAGTTCAAGCAACTACCAAAACCACTGCCGCAGCTGTTT

CCCAAATTACTGACGGTCAAGTTCAAGCCACTACAAAAACCA

CTCAAGCAGCCAGCCAAGTAAGCGATGGCCAAGTCCAAGCT

ACTACTGCTACTTCCGCTTCTGCAGCCGCTACCTCCACTGACC

CAGTCGATGCTGTCTCCTGTAAGACTTCTGGTACC

0279/asn009-3
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCT
141

ACATCTTTAGCTGCCTATGCTCCAAAGGACCCGTGGTCCACTT

TAACTCCATCAGCTACTTACAAGGGTGGTATAACAGATTACT

CTTCGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCAGTGC

TTCCTCCGTCGCCTCATCTAAAGCAAAGAGAGCCGCCTATCA

GATAGGTGATGGTCAAGTACAGGCTGCCACTACTACTGCTGC

TGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATAACT

GACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCTGTT

TCCCAAATAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACT

GCCGCTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAGCT

GCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAACTGATGGTC

AAGTTCAAGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGA

TTTCTGACGACCAAGTTCAGGCCACTACCTATACTAAGGCTG

CTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTAAAA

CTACCAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCCAGG

CTACTGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGTTG

TTTCCTGTAATAACAATAGTACC

0279/asn010-1
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCT
142

ACATCTTTAGCTGCCTATGCTCCAAAGGACCCGTGGTCCACTT

TAACTCCATCAGCTACTTACAAGGGTGGTATAACAGATTACT

CTTCGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCAGTGC

TTCCTCCGTCGCCTCATCTAAAGCAAAGAGAGCCGCCTCTCA

GATAGGTGATGGTCAAGTACAGGCTGCCACTACTACTGCTGC

TGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATAACT

GACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCTGTT

TCCCAAATAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACT

GCCGCTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAGCT

GCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAACTGATGGTC

AAGTTCAAGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGA

TTTCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAGGCTG

CTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTAAAA

CTACCAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCCAGG

CTACTGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGTTG

TTTCCTGTAATAACAATAGTACC

0279/asn012-3
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCAT
143

CCTCCGCATTAGCTGCCTATGCTCCAAAGGACCCGTGGTCCA

CTTTAACTCCATCAGCTACTTACAAGGGTGGTATAACAGATT

ACTCTTCGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCAG

TGCTTCCTCCGTCGCCTCATCTAAAGCAAAGAGAGCCGCCTC

TCAGATAGGTGATGGTCAAGTACAGGCTGCCACTACTACTGC

TGCTGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATA

ACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCT

GTTTCCCAAATAACTGACGGTCAAGTTCAAGCTGCTAAGTCT

ACTGCCGCTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAA

GCTGCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAACTGATG

GTCAAGTTCAAGCTGCCAAGTCTACTGCTGCCGCTGCCTCTC

AGATTTCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAGG

CTGCTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTA

AAACTACCAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCC

AGGCTACTGCTGAAGTGAAAGACGCTAACGATCCAGTCGATG

TTGTTTCCTGTAATAACAATAGTACC

0279/asn014-2
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCT
144

ACATCTTTAGCT

0279/asn015-2
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCT
145

ACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCAACTT

TGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACG

CTTCCACCTTCGGTATTGCCGTTCAACAAATCTCCACTACATC

CAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAA

GAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGC

TACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCC

GTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTAAG

ACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAATTC

AAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCGCTG

CCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCA

CTACTTTAGCCCCAAAGAGCACCGCTGCTGCCGTTTCTCAAA

TCGGTGATGGTCAAGTTCAAGCTACCACCACTACTTTAGCCC

CAAAGAGCACCGCTGCTGCCGTTTCTCAAATCGGTGATGGTC

AAGTTCAAGCTACTACTAAGACTACCGCTGCTGCTGTCTCTC

AAATTGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACTG

CTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAAGCTA

CTACCAAGACTACCGCTGCTGCTGTCTCTCAAATCGGTGATG

GTCAAGTTCAAGCAACTACCAAAACCACTGCCGCAGCTGTTT

CCCAAATTACTGACGGTCAAGTTCAAGCCACTACAAAAACCA

CTCAAGCAGCCAGCCAAGTAAGCGATGGCCAAGTCCAAGCT

ACTACTGCTACTTCCGCTTCTGCAGCCGCTACCTCCACTGACC

CAGTCGATGCTGTCTCCTGTAAGACTTCTGGTACCTTGGAGA

AAAGAGAGGCTGAAGCA

0279/asn016-3
ATGCAATACAAAAAGACTTTGGTTGCCTCTGCTTTGGCCGCT
146

ACTACATTGGCCGCCTATGCTCCATCTGAGCCTTGGTCCACTT

TGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTACG

CTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACATC

CAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCCAA

GAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCTGC

TACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCGCC

GTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTAAG

ACTACCGCTGCTGCTGTCTCTCAAATTTGTGATGGTCAAATTC

AAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCGCTG

CCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCACCA

CTACTTTAGCCCCAAAGAGCACCGCTGCTGCCGTTTCTCAAA

TCGGTGATGGTCAAGTTCAAGCTACCACCACTACTTTAGCCC

CAAAGAGCACCGCTGCTGCCGTTTCTCAAATCGGTGATGGTC

AAGTTCAAGCTACTACTAAGACTACCGCTGCTGCTGTCTCTC

AAATTGGTGATGGTCAAGTTCAAGCTACCACCAAGACTACTG

CTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAAGCTA

CTACCAAGACTACCGCTGCTGCTGTCTCTCAAATCGGTGATG

GTCAAGTTCAAGCAACTACCAAAACCACTGCCGCAGCTGTTT

CCCAAATTACTGACGGTCAAGTTCAAGCCACTACAAAAACCA

CTCAAGCAGCCAGCCAAGTAAGCGATGGCCAAGTCCAAGCT

ACTACTGCTACTTCCGCTTCTGCAGCCGCTACCTCCACTGACC

CAGTCGATGCTGTCTCCTGTAAGACTTCTGGTACCTTGGAGA

AAAGAGAGGCTGAAGCA

0279/asn018-1
ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCAT
147

CCTCCGCATTAGCTGCCTATGCTCCATCTGAGCCTTGGTCCAC

TTTGACTCCAACAGCCACTTACAGCGGTGGTGTTACCGACTA

CGCTTCCACCTTCGGTATTGCCGTTCAACCAATCTCCACTACA

TCCAGCGCATCATCTGCAGCCACCACAGCCTCATCTAAGGCC

AAGAGAGCTGCTTCCCAAATTGGTGATGGTCAAGTCCAAGCT

GCTACCACTACTGCTTCTGTCTCTACCAAGAGTACCGCTGCCG

CCGTTTCTCAGATCGGTGATGGTCAAATCCAAGCTACTACTA

AGACTACCGCTGCTGCTGTCTCTCAAATTGGTGATGGTCAAA

TTCAAGCTACCACCAAGACTACCTCTGCTAAGACTACCGCCG

CTGCCGTTTCTCAAATCAGTGATGGTCAAATCCAAGCTACCA

CCACTACTTTAGCCCCAAAGAGCACCGCTGCTGCCGTTTCTC

AAATCGGTGATGGTCAAGTTCAAGCTACCACCACTACTTTAG

CCCCAAAGAGCACCGCTGCTGCCGTTTCTCAAATCGGTGATG

GTCAAGTTCAAGCTACTACTAAGACTACCGCTGCTGCTGTCT

CTCAAATTGGTGATGGTCAAGTTCAAGCTACCACCAAGACTA

CTGCTGCCGCCGTTTCTCAAATCGGTGATGGTCAAGTTCAAG

CTACTACCAAGACTACCGCTGCTGCTGTCTCTCAAATCGGTG

ATGGTCAAGTTCAAGCAACTACCAAAACCACTGCCGCAGCTG

TTTCCCAAATTACTGACGGTCAAGTTCAAGCCACTACAAAAA

CCACTCAAGCAGCCAGCCAAGTAAGCGATGGCCAAGTCCAA

GCTACTACTGCTACTTCCGCTTCTGCAGCCGCTACCTCCACTG

ACCCAGTCGATGCTGTCTCCTGTAAGACTTCTGGTACCTTGGA

GAAAAGAGAGGCTGAAGCA

0279/asn019-2
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCT
148

ACATCTTTAGCTGCCTATGCTCCAAAGGACCCGTGGTCCACTT

TAACTCCATCAGCTACTTACAAGGGTGGTATAACAGATTACT

CTTCGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCAGTGC

TTCCTCCGTCGCCTCATCTAAAGCAAAGAGAGCCGCCTCTCA

GATAGGTGATGGTCAAGTACAGGCTGCCACTACTACTGCTGC

TGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATAACT

GACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCTGTT

TCCCAAATAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACT

GCCGCTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAGCT

GCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAACTGATGGTC

AAGTTCAAGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGA

TTTCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAGGCTG

CTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTAAAA

CTACCAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCCAGG

CTACTGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGTTG

TTTCCTGTAATAACAATAGTACCTTGGAGAAAAGAGAGGCTG

AAGCA

0279/asn020-3
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCT
149

ACATCTTTAGCTGCCTATGCTCCAAAGGACCCGTGGTCCACTT

TAACTCCATCAGCTACTTACAAGGGTGGTATAACAGATTACT

CTTCGAGTTTCGGTATTGCTATTGAAGCCGTGGCTACCAGTGC

TTCCTCCGTCGCCTCATCTAAAGCAAAGAGAGCCGCCTCTCA

GATAGGTGATGGTCAAGTACAGGCTGCCACTACTACTGCTGC

TGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATAACT

GACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCTGTT

TCCCAAATAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACT

TCCGCTGCCGTTTCTCAAATAACTGACGGTCAAGTTCAAGCT

GCTAAGTCTACTGCCGCTGCCGTTTCTCAAATAACTGATGGTC

AAGTTCAAGCTGCCAAGTCTACTGCTGCCGCTGCCTCTCAGA

TTTCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAGGCTG

CTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTAAAA

CTACCAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCCAGG

CTACTGCTGAAGTGAAAGACGCTAACGATCCAGTCGATGTTG

TTTCCTGTAATAACAATAGTACCTTGGAGAAAAGAGAGGCTG

AAGCAATGAACTGCTCCGCATTCTCTTTCTGGTTCGTCTGTAA

AATAATCTTCTTCTTCTTGTCCTTCAACATCCAAATCTCCATC

GCA

This set of plasmids was transformed into the yCBGA0314 strain and their CBDA productivity was assayed in the following high throughput screening process optimized for the CBDAs synthase activity. Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH is added to the samples. Finally, samples were grown for additional 42 hours and are analyzed for cannabinoids.

The THCA titer in the above described screen ranged from 4 to 512 mg/L. Sample titers for several transformed strains are included in Table 20 below. (The titers are also included in Table 18 above.)

TABLE 20

THCA

plasmid ID
ORF name
(mg/L)

0279/asn021-
alpha_pre_signal-and-
512

2
PIR3_delta_fragment_wo_signal-and-

LEKREAEA-d28_THCAs

0279/asn009-
PIR3_delta_fragment-d28_THCAs
501

3

0279/asn015-
HSP150_delta_fragment-and-LEKREAEA-
500

2
d28_THCAs

0279/asn011-
alpha_pre_signal-and-
500

2
PIR3_delta_fragment_wo_singal-d28_THCAs

0279/asn009-
PIR3_delta_fragment-d28_THCAs
498

1

0279/asn019-
PIR3_delta_fragment-and-LEKREAEA-
491

2
d28_THCAs

0279/asn031-
K28 viral signal-noSpacer-d28_THCAs
485

4

0279/asn025-
K28 viral signal-d28_THCAs
479

3

0279/asn003-
PHO5_signal-d28_THCAs
473

2

0279/asn013-
PIR3 secretion signal-d28_THCAs
459

1

0279/asn029-
INU1-d28_THCAs
440

2

0279/asn027-
INU1A signal-d28_THCAs
430

1

0279/asn024-
YAP_TA57_Kex2_spacer-yEVenus-THCAs
233

3

0279/asn033-
YAP_TA57_Kex2_spacer-d28_THCAs
212

4

0279/asn001-
YAP_TA57_Kex2_spacer-d28_THCAs
209

3

0279/asn023-
YAP_TA57_Kex2_spacer-yEVenus-d28_THCAs
166

2

0279/asn014-
PIR3 secretion signal-THCAs
122

2

0279/asn032-
K28 viral signal-noSpacer-THCAs
111

2

0279/asn002-
YAP_TA57_Kex2_spacer-THCAs
80

2

0279/asn034-
YAP_TA57_Kex2_spacer-THCAs
76

4

0279/asn030-
INU1-THCAs
58

1

0279/asn028-
INU1A signal-THCAs
56

1

0279/asn020-
PIR3_delta_fragment-and-LEKREAEA-THCAs
37

3

0279/asn004-
PHO5_signal-THCAs
36

1

0279/asn026-
K28 viral signal-THCAs
26

2

0279/asn006-
HSP150_delta_fragment-THCAs
13

2

0279/asn012-
alpha_pre_signal-and-
10

3
PIR3_delta_fragment_wo_singal-THCAs

0279/asn012-
alpha_pre_signal-and-
10

1
PIR3_delta_fragment_wo_singal-THCAs

0279/asn016-
HSP150_delta_fragment-and-LEKREAEA-THCAs
9

3

0279/asn018-
alpha_pre_signal-and-
7

1
HSP150_delta_fragment_wo_signal-and-

LEKREAEA-THCAs

0279/asn010-
PIR3_delta_fragment-THCAs
4

1

Example 23—THCA Synthase Mutagenesis

One amino acid position was mutagenized in the THCA synthase. The mutant genes were screened in yeast. Clones with increased THCA titer were identified.

The parental plasmid for the mutagenesis was the RUNM001233_63.1 plasmid, containing the Saccharomyces cerevisiae 2μ replication origin, the URA3 gene as an auxotrophic marker and a modified THCA synthase gene under the regulation of the bidirectional GAL1/GAL10 promoter. The nucleotide sequence of RUNM001233_63.1 is SEQ ID NO: 303. The modified THCA synthase gene contained a chimeric secretion signal and the THCA synthase gene lacking its predicted 28 amino acid long plant secretion signal.

The mutagenized position is the 469^thamino acid position of this modified THCA synthase protein, corresponding to the 446^thamino acid position of the unmodified THCA synthase protein. This position contains threonine in the natural THCA synthase. We have constructed large number of plasmids where the above-mentioned codon was randomly mutated allowing the incorporation of codons of any of the 20 amino acids.

The RUNM001233_63.1 plasmid and large number of its mutagenized variants were transformed into the yCBGA0517 strain and screened for THCA production. For the strain construction the parental strain was the yCBGA0314 strain. The PKS and OAC genes under the regulation of the bidirectional GAL1/GAL10 promoter and the AAE1 gene under the regulation of the STE5 promoter were inserted into the YDR508C, YRL020C and FOX1 loci replacing the native ORFs of the yCBGA0314 strain. A large number of isolates from this transformation was screened and few of them with high CBGA productivity and best reproducibility were identified. One of these isolates was prototrophic for uracil. Next, its URA3 gene was swapped with HIS3 gene resulting in the strain yCBGA0517, prototrophic for histidine and auxotrophic for uracil, leucine and tryptophan.

Colonies were screened using the following high throughput screening process of the THCA synthase activity: Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl Synthetic Complete liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan and histidine). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter. After the 48 hours growth period 40 μl samples of these cultures are inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples are grown for 48 hours at 30° C. and shaken at 300 rpm with a 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH is added to the samples. Finally, samples were grown for additional 42 hours and are analyzed for cannabinoids.

The plasmid sequence of the best producer clones was determined. In all cases, the plasmids contained mutations only in the region of the mutagenized codon.

When Alanine, Valine or Isoleucine is coded for in the above-mentioned position of the modified THCA synthase gene, increased THCA titer was observed, as summarized in the Table 21 below:

TABLE 21

Amino acid

Original
Position in
Position in
present in
THCA

amino
unmodified
modified
the marked
titer

acid
THCAs
THCAs
position
(mg/L)

Threonine
446
469
Threonine
289

Threonine
446
469
Alanine
477

Threonine
446
469
Valine
565

Threonine
446
469
Isoleucine
445

Example 24—CBDA Synthase Mutagenesis

Eighty-four (84) amino acid positions in the native Cannabis sativa CBDA synthase gene were mutagenized. The mutant genes were screened in yeast. Clones were identified with increased CBDA titer.

The parental plasmid for the mutagenesis was the 0285/asn080-2 plasmid, containing the Saccharomyces cerevisiae 2μ replication origin, the Hygromycin B resistance gene as a dominant marker and one of the modified CBDA synthase genes disclosed in this example under the regulation of the bidirectional GAL1/GAL10 promoter. The modified CBDA synthase gene contained the PIR3 delta fragment as secretion signal and the CBDA synthase gene lacking its predicted 21 amino acid long plant secretion signal. The modified CBDA coding sequences used for the 0285/asn080-2 parental plasmid are included in Table 22 below.

TABLE 22

CBDAs
ATGCAATATAAAAAGCCATTAGTCGTCTCCGCTTTAGCTGCTACATCTTT

DNA
AGCTGCCTATGCTCCAAAGGACCCGTGGTCCACTTTAACTCCATCAGCTA

sequence
CTTACAAGGGTGGTATAACAGATTACTCTTCGAGTTTCGGTATTGCTATT

including
GAAGCCGTGGCTACCAGTGCTTCCTCCGTCGCCTCATCTAAAGCAAAGA

the signal
GAGCCGCCTCTCAGATAGGTGATGGTCAAGTACAGGCTGCCACTACTAC

sequence
TGCTGCTGTTTCTAAGAAATCCACCGCTGCTGCTGTTTCTCAAATAACTG

ACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCTGTTTCCCAAATA

ACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCCGTTTCTCA

AATAACTGACGGTCAAGTTCAAGCTGCTAAGTCTACTGCCGCTGCCGTTT

CTCAAATAACTGATGGTCAAGTTCAAGCTGCCAAGTCTACTGCTGCCGC

TGCCTCTCAGATTTCTGACGGCCAAGTTCAGGCCACTACCTCTACTAAGG

CTGCTGCATCCCAAATTACAGATGGGCAGATACAAGCATCTAAAACTAC

CAGTGGCGCTAGTCAAGTAAGTGATGGCCAAGTCCAGGCTACTGCTGAA

GTGAAAGACGCTAACGATCCAGTCGATGTTGTTTCCTGTAATAACAATA

GTACCAATATTCAAACTTCAATCGCTAACCCAAGAGAAAATTTCTTGAA

GTGTTTCTCTCAATACATTCCAAATAATGCAACAAATTTGAAATTGGTTT

ATACTCAAAATAATCCATTATACATGTCTGTTTTAAATTCTACAATTCAT

AATTTGAGATTTTCTTCAGATACTACACCAAAACCATTGGTTATTGTTAC

ACCATCTCATGTTTCACATATCCAAGGTACTATCTTGTGTTCTAAGAAAG

TTGGTTTGCAAATTAGAACTAGATCAGGTGGTCATGATTCAGAAGGCAT

GTCTTACATCTCACAAGTTCCATTCGTTATCGTTGATTTGAGAAACATGA

GATCAATTAAAATTGATGTTCATTCACAAACAGCTTGGGTTGAAGCTGG

TGCAACTTTGGGTGAAGTTTACTACTGGGTTAACGAAAAGAATGAATCT

TTATCATTGGCTGCTGGTTACTGTCCAACAGTTTGTGCTGGTGGTCATTT

TGGTGGTGGTGGTTATGGTCCATTAATGAGATCCTATGGTTTGGCTGCTG

ATAACATCATCGATGCACATTTGGTTAACGTTCATGGTAAAGTTTTGGAT

AGAAAGTCTATGGGTGAAGATTTGTTTTGGGCTTTGAGAGGTGGTGGTG

CTGAATCATTTGGTATCATCGTTGCTTGGAAGATCAGATTGGTTGCAGTT

CCAAAATCTACTATGTTCTCAGTTAAGAAAATTATGGAAATCCATGAAT

TAGTTAAATTGGTTAATAAGTGGCAAAATATTGCTTATAAATACGATAA

AGATTTGTTATTGATGACTCATTTTATTACAAGAAATATTACTGATAACC

AAGGTAAAAATAAGACAGCTATCCATACTTACTTTTCTTCAGTTTTCTTG

GGTGGTGTTGATTCTTTGGTTGATTTGATGAATAAGTCTTTTCCAGAATT

AGGTATTAAGAAAACTGATTGTAGACAATTGTCTTGGATCGATACTATC

ATTTTCTATTCAGGTGTTGTTAACTACGATACAGATAACTTCAATAAGGA

AATTTTATTGGATAGATCAGCTGGTCAAAATGGTGCTTTTAAAATTAAAT

TGGATTACGTTAAGAAACCAATTCCAGAATCAGTTTTCGTTCAAATTTTA

GAAAAATTGTATGAAGAAGATATTGGTGCTGGCATGTACGCATTGTATC

CATACGGTGGTATCATGGATGAAATTTCTGAATCAGCTATTCCATTTCCA

CATAGAGCAGGTATTTTATACGAATTGTGGTACATTTGTTCTTGGGAAAA

GCAAGAAGATAACGAAAAACATTTGAACTGGATTAGAAACATCTATAA

CTTCATGACTCCATACGTTTCACAAAACCCAAGATTGGCTTATTTGAACT

ACAGAGATTTGGATATCGGTATTAATGATCCTAAAAATCCAAACAACTA

TACACAAGCAAGAATTTGGGGTGAAAAGTACTTCGGTAAAAATTTCGAT

AGATTGGTTAAGGTTAAAACTTTGGTTGATCCAAATAATTTCTTTAGAAA

TGAACAATCTATTCCACCATTGCCAAGACATAGACATTGA

(SEQ ID NO: 150)

CBDAs
MQYKKPLVVSALAATSLAAYAPKDPWSTLTPSATYKGGITDYSSSFGIAIEA

amino
VATSASSVASSKAKRAASQIGDGQVQAATTTAAVSKKSTAAAVSQITDGQV

acid
QAAKSTAAAVSQITDGQVQAAKSTAAAVSQITDGQVQAAKSTAAAVSQIT

sequence
DGQVQAAKSTAAAASQISDGQVQATTSTKAAASQITDGQIQASKTTSGASQ

including
VSDGQVQATAEVKDANDPVDVVSCNNNSTNIQTSIANPRENFLKCFSQYIP

the signal
NNATNLKLVYTQNNPLYMSVLNSTIHNLRFSSDTTPKPLVIVTPSHVSHIQG

sequence
TILCSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTA

WVEAGATLGEVYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRS

YGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKI

RLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNI

TDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWID

TIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILE

KLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQED

NEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

(SEQ ID NO: 151)

CBDAs
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFS

amino
SDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPF

acid
VIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNESLSLAAGYCP

sequence
TVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGKVLDRKSMGEDL

without
FWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQN

the signal
IAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMN

sequence
KSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAF

KIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPH

RAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYR

DLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQS

IPPLPRHRH

(SEQ ID NO: 152)

The eighty-four (84) amino acid positions encoded by the wild-type CBDA synthase gene were selected for mutagenesis based on the fact that they contained amino acid differences with THCA synthase when aligned sequentially. The wild-type CBDA synthase specific amino acid was replaced at each of these positions for the corresponding amino acid found in the THCA synthase protein. 0285/asn080-2 plasmids containing the mutants were transformed into the yCBGA0513 strain and screened for CBDA production.

Several double combinations of these mutations were also introduced into the 0285/asn080-2 plasmid, and along with their corresponding single mutant plasmid counterparts, were transformed into the yCBGA0523 strain and screened for CBDA production.

The CBDA mutant (and wild type) coding sequences used for the 0285/asn080-2 parental plasmid are included in Table 22 in Example 24.

CBDA synthase activity in the strains was screened using the following high throughput screening process. Colonies were inoculated into wells of a 96-well deep well plate. Each well contained 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan, histidine and Hygromycin B). These inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter. After a 48 hour growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH is added to the samples. Finally, samples were grown for additional 42 hours and were analyzed for cannabinoids.

The CBDA titer in the above described screen ranged from 10.5 to 408.6 mg/L. Sample titers for the transformed strains, along with each strain's respective amino acid mutations and amino acid sequences, are included in Tables 23-26 below. (All mutation sites refer to the mutation site on wild-type CBDA's coding sequence without the wild-type signal sequence.)

TABLE 23

yCBGA_0513 mutant strains

mutation site

on CBDAs

coding

sequence

Average

without the
native
new
titer

signal
amino
amino
of CBDA

Constructs
sequence
acid
acid
mg/L

PIR3_CBDAs_S449N
216
S
N
408.6

PIR3_CBDAs_G307A
74
G
A
407

PIR3_CBDAs_H425D
192
H
D
381.1

PIR3_CBDAs_P464PS
231
P
PS
378.7

PIR3_CBDAs_N269D
36
N
D
376.9

PIR3_CBDAs_V454A
221
V
A
376.6

PIR3_CBDAs_M468I
235
M
I
367.5

PIR3_CBDAs_I700L
467
I
L
357

PIR3_CBDAs_Y571F
338
Y
F
352

PIR3_CBDAs_L442I
209
L
I
350.9

PIR3_CBDAs_S328A
95
S
A
343.8

PIR3_CBDAs_I620V
387
I
V
341.6

PIR3_CBDAs_I676V
443
I
V
340.3

PIR3_CBDA5_Q252E
19
Q
E
325.6

PIR3_CBDAs_C392G
159
C
G
323.9

PIR3_CBDAs_R459K
226
R
K
320.8

PIR3_CBDAs_I341V
108
I
V
319.6

PIR3_CBDAs_A626V
393
A
V
319.2

PIR3_CBDAs_L261P
28
L
P
318.2

PIR3_CBDAs_G590T
357
G
T
317.5

PIR3_CBDAs_C658A
425
C
A
310.5

PIR3_CBDAs_V264I
31
V
I
310.3

PIR3_CBDAs_A622V
389
A
V
309.2

PIR3_CBDAs_N675S
442
N
S
308.7

PIR3_CBDAs_R243Q
10
R
Q
306.3

PIR3_CBDAs_L651M
418
L
M
303

PIR3_CBDAs_H281Q
48
H
Q
301

PIR3_CBDAs_R554K
321
R
K
299

PIR3_CBDAs_K706E
473
K
E
297.7

PIR3_CBDAs_Q555E
322
Q
E
294.3

PIR3_CBDAs_R755H
522
R
H
294.2

PIR3_CBDAs_R753P
520
R
P
292.5

PIR3_CBDAs_V736A
503
V
A
290.7

PIR3_CBDAs_A393V
160
A
V
285.6

PIR3_CBDAs_S286T
53
S
T
285.5

PIR3_CBDAs_D572N
339
D
N
285.4

wild type control
wild type
N/A
N/A
283.7

construct

PIR3_CBDAs_Q610K
377
Q
K
277.8

PIR3_CBDAs_G398S
165
G
S
276.9

PIR3_CBDAs_L499V
266
L
V
274.1

PIR3_CBDAs_Q513H
280
Q
H
274

PIR3_CBDAs_D574A
341
D
A
270.7

PIR3_CBDAs_L735K
502
L
K
269.3

PIR3_CBDAs_N577K
344
N
K
265.7

PIR3_CBDAs_Q588K
355
Q
K
264.1

PIR3_CBDAs_L383F
150
L
F
261.3

PIR3_CBDAs_T259A
26
T
A
260

PIR3_CBDAs_D635E
402
D
E
256.4

PIR3_CBDAs_V527I
294
V
I
253.2

PIR3_CBDAs_V374I
141
V
I
252.8

PIR3_CBDAs_R348H
115
R
H
250.4

PIR3_CBDAs_S380N
147
S
N
248.9

PIR3_CBDAs_S606T
373
S
T
248.6

PIR3_CBDAs_A258P
25
A
P
243.2

PIR3_CBDAs_R507K
274
R
K
243

PIR3_CBDAs_A384P
151
A
P
237.4

PIR3_CBDAs_L670I
437
L
I
227

PIR3_CBDAs_S408N
175
S
N
224.3

PIR3_CBDAs_T522G
289
T
G
220.4

PIR3_CBDAs_N268H
35
N
H
215.2

PIR3_CBDAs_V607A
374
V
A
213.7

PIR3_CBDAs_N707S
474
N
S
210.9

PIR3_CBDAs_I702K
469
I
K
203.1

PIR3_CBDAs_I520V
287
I
V
199.5

PIR3_CBDAs_H301N
68
H
N
195.1

PIR3_CBDAs_F608M
375
F
M
186.4

PIR3_CBDAs_A519T
286
A
T
181.3

PIR3_CBDAs_D727N
494
D
N
174.8

PIR3_CBDAs_N589K
356
N
K
172.2

PIR3_CBDAs_D704N
471
D
N
162

PIR3_CBDAs_T308S
75
T
S
161.7

PIR3_CBDAs_I657T
424
I
T
160.2

PIR3_CBDAs_V484F
251
V
F
160

PIR3_CBDAs_I673V
440
I
V
157.8

PIR3_CBDAs_A385G
152
A
G
145.6

PIR3_CBDAs_I474N
241
I
N
141.2

PIR3_CBDAs_A447G
214
A
G
127.5

PIR3_CBDAs_L529H
296
L
H
112.1

PIR3_CBDAs_M680T
447
M
T
100.3

PIR3_CBDAs_P270Q
37
P
Q
96.2

PIR3_CBDAs_N703T
470
N
T
96.1

PIR3_CBDAs_L556F
323
L
F
92.9

PIR3_CBDAs_L381F
148
L
F
76.8

PIR3_CBDAs_E479G
246
E
G
75.3

PIR3_CBDAs_I562T
329
I
T
10.5

TABLE 24

yCBGA_0513 mutant strains

SEQ

ID

Constructs
CBDA amino acid sequence
NO:

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
153

DAs_S449
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAENFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
154

DAs_G30
TIHNLRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGG

7A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
155

DAs_H42
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5D
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVDGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
156

DAs_P464
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

PS
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPSKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDK

DLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMN

KSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSA

GQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPY

GGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRN

IYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNS
157

DAs_N26
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9D
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
158

DAs_V45
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIA

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
159

DAs_M46
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

81
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTIFSVKKIMEIHELVKLVNKWQNIAYKYDKDL

LLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKS

FPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
160

DAs_I700
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

L
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDLGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
161

DAs_Y57
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

1F
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNFDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
162

DAs_L44
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2I
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWAIRGGGAESFGIIVA

WKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDL

LLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKS

FPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
163

DAs_S328
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

A
HDAEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
164

DAs_I620
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDVGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
165

DAs_I676
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNV

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSEYIPNNATNLKLVYTQNNPLYMSVLNS
166

DAs_Q25
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2E
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
167

DAs_C39
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2G
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVGAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
168

DAs_R45
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIKLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
169

DAs_I341
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

V
HDSEGMSYISQVPFVVVDLRNMRSIKIDVHSQTAWVEAGATLG

EVYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYG

LAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
170

DAs_A62
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

6V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYVLYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNPKLVYTQNNPLYMSVLNS
171

DAs_L26
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

1P
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
172

DAs_G59
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

0T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNTAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
173

DAs_C65
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYIASWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLIYTQNNPLYMSVLNS
174

DAs_V26
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4I
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
175

DAs_A62
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGVGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
176

DAs_N67
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5S
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRSIY

NFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPQENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
177

DAs_R24
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3Q
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
178

DAs_L65
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

1M
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGIMYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
179

DAs_H28
TIQNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

1Q
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
180

DAs_R55
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCKQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
181

DAs_K70
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

6E
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPENPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
182

DAs_Q55
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5E
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRELSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
183

DAs_R75
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5H
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHHH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
184

DAs_R75
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3P
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPPHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
185

DAs_V73
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

6A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLADPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
186

DAs_A39
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCVGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
187

DAs_S286
TIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
188

DAs_D57
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYNTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

wild type
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
189

control
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

construct
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
190

DAs_Q61
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

0K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVKILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
191

DAs_G39
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8S
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFSGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
192

DAs_L49
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LVLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
193

DAs_Q51
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3H
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNHGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
194

DAs_D57
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTANFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
195

DAs_L73
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTKVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
196

DAs_N57
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFKKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
197

DAs_Q58
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

KNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
198

DAs_L38
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3F
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSFAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNAANLKLVYTQNNPLYMSVLNS
199

DAs_T25
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
200

DAs_D63
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5E
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMEEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIY

NFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
201

DAs_V52
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7I
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSIFLGGVDSLVDLMNKS

FPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
202

DAs_V37
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4I
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWINEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
203

DAs_R34
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8H
HDSEGMSYISQVPFVIVDLRNMHSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
204

DAs_S380
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNENLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
205

DAs_S606
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPETVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNPTNLKLVYTQNNPLYMSVLNS
206

DAs_A25
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8P
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
207

DAs_R50
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITKNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
208

DAs_A38
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4P
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLPAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
209

DAs_L67
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

0I
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHINWIRNIY

NFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
210

DAs_S408
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRNYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
211

DAs_T52
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

2G
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHGYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQHNPLYMSVLNS
212

DAs_N26
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

8H
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
213

DAs_V60
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7A
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESAFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
214

DAs_N70
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7S
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKSPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
215

DAs_I702
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGKNDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
216

DAs_I520
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAVHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
217

DAs_H30
TIHNLRFSSDTTPKPLVIVTPSNVSHIQGTILCSKKVGLQIRTRSGG

1N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
218

DAs_F608
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

M
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVMVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
219

DAs_A51
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTTIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
220

DAs_D72
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFNRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
221

DAs_N58
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9K
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QKGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
222

DAs_D70
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINNPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
223

DAs_T30
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGSILCSKKVGLQIRTRSGG

8S
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
224

DAs_I657
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYTCSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
225

DAs_V48
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

4F
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLFNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
226

DAs_I673
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

V
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWVRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
227

DAs_A38
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

5G
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAGGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
228

DAs_I474
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

N
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKNMEIHELVKLVNKWQNIAYKYDK

DLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMN

KSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSA

GQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPY

GGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRN

IYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
229

DAs_A44
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

7G
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGGESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
230

DAs_L52
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9H
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFHGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
231

DAs_M68
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

0T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFTTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNQLYMSVLNS
232

DAs_P270
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

Q
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
233

DAs_N70
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

3T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGITDPKNPNNYTQARIWGEK

YFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
234

DAs_L55
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

6F
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQFSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
235

DAs_L38
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

1F
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESFSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
236

DAs_E47
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

9G
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHGLVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAG

QNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNI

YNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNS
237

DAs_I562
TIHNLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGG

T
HDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGE

VYYWVNEKNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGL

AADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIV

AWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKD

LLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNK

SFPELGIKKTDCRQLSWIDTTIFYSGVVNYDTDNFNKEILLDRSA

GQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPY

GGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRN

IYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGE

KYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

TABLE 25

yCBGA_0523 mutant strains

First

Second

mutation

mutation

site on

site on

CBDAs

CBDAs

coding

coding

sequence

sequence

Average

without
native
new
without
native
new
titer of

the signal
amino
amino
the signal
amino
amino
CBDA

Constructs
sequence
acid
acid
sequence
acid
acid
mg/L

wild type control
wild type
N/A
N/A
N/A
N/A
N/A
104.9

construct
control

construct

PIR3_CBDAS_G307A
74
G
A
N/A
N/A
N/A
244.8

PIR3_CBDAS_H425D
192
H
D
N/A
N/A
N/A
199.5

PIR3_CBDAS_I620V
387
I
V
N/A
N/A
N/A
181.3

PIR3_CBDAS_I676V
443
I
V
N/A
N/A
N/A
155.6

PIR3_CBDAS_I700L
467
I
L
N/A
N/A
N/A
141.8

PIR3_CBDAS_L442I
209
L
I
N/A
N/A
N/A
169.6

PIR3_CBDAS_M468I
235
M
I
N/A
N/A
N/A
191.4

PIR3_CBDAS_N269D
36
N
D
N/A
N/A
N/A
210.6

PIR3_CBDAS_P464P5
231
P
PS
N/A
N/A
N/A
201.3

PIR3_CBDAS_R243Q
10
R
Q
N/A
N/A
N/A
162.8

PIR3_CBDAS_S328A
95
S
A
N/A
N/A
N/A
125.2

PIR3_CBDAS_S449N
216
S
N
N/A
N/A
N/A
156.5

PIR3_CBDAS_V454A
221
V
A
N/A
N/A
N/A
145.6

PIR3_CBDAS_Y571F
338
Y
F
N/A
N/A
N/A
137.2

PIR3_CBDAS_G307A_
74
G
A
192
H
D
371.8

H425D

PIR3_CBDAS_G307A_
74
G
A
387
I
V
373.2

1620V

PIR3_CBDAS_G307A_
74
G
A
443
I
V
173.2

I676V

PIR3_CBDAS_G307A_
74
G
A
467
I
L
327.4

1700L

PIR3_CBDAS_G307A_
74
G
A
209
L
I
353.6

L442I

PIR3_CBDAS_G307A_
74
G
A
235
M
I
391.7

M468I

PIR3_CBDAS_G307A_
74
G
A
36
N
D
281.2

N269D

PIR3_CBDAS_G307A_
74
G
A
231
P
PS
395.3

P464PS

PIR3_CBDAS_G307A_
74
G
A
10
R
Q
270.2

R243Q

PIR3_CBDAS_G307A_
74
G
A
95
S
A
306.8

S328A

PIR3_CBDAS_G307A_
74
G
A
216
S
N
346.8

S449N

PIR3_CBDAS_G307A_
74
G
A
221
V
A
314.2

V454A

PIR3_CBDAS_G307A_
74
G
A
338
Y
F
307.5

Y571F

PIR3_CBDAS_H425D_
192
H
D
235
M
I
324.1

M468I

PIR3_CBDAS_H425D_
192
H
D
36
N
D
351.5

N269D

PIR3_CBDAS_H425D_
192
H
D
221
V
A
277.8

V454A

PIR3_CBDAS_N269D_
36
N
D
235
M
I
332.8

M468I

PIR3_CBDAS_P464P5
231
P
PS
192
H
D
317.6

H425D

PIR3_CBDAS_P464P5
231
P
PS
36
N
D
348.0

N269D

PIR3_CBDAS_P464P5
231
P
PS
235
M
I
21.4

T468I

PIR3 CBDAS_P464PS
231
P
PS
221
V
A
268.0

V454A

PIR3_CBDAS_S449N_
216
S
N
192
H
D
292.2

H425D

PIR3_CBDAS_S449N_I
216
S
N
387
I
V
280.7

620V

PIR3_CBDAS_S449N_I
216
S
N
443
I
V
202.2

676V

PIR3_CBDAS_S449N_I
216
S
N
467
I
L
147.3

700L

PIR3_CBDAS_S449N_
216
S
N
209
L
I
248.3

L442I

PIR3_CBDAS_S449N_
216
S
N
235
M
I
201.7

M468I

PIR3_CBDAS_S449N_
216
S
N
36
N
D
312.3

N269D

PIR3_CBDAS_S449N_
216
S
N
231
P
PS
238.2

P464PS

PIR3_CBDAS_S449N_
216
S
N
10
R
Q
229.1

R243Q

PIR3_CBDAS_S449N_
216
S
N
95
S
A
88.1

S328A

PIR3_CBDAS_S449N_
216
S
N
221
V
A
202.7

V454A

PIR3_CBDAS_S449N_
216
S
N
338
Y
F
148.7

Y571F

PIR3_CBDAS_V454A_
221
V
A
235
M
I
256.8

M468I

PIR3_CBDAS_V454A_
221
V
A
36
N
D
275.4

N269D

TABLE 26

yCBGA_0523 mutant strains

SEQ ID

Constructs
CBDA amino acid sequence
NO:

wild type
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
238

control
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

construct
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
239

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNIFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
240

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

H425D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_CB
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
241

DAS_I620
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDV

GAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEK

HLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
242

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

I676V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNVYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
243

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

I700L
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDLGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
244

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

L442I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWAIRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
245

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
246

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

N269D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
247

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

P464PS
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPQENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
248

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

R243Q
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
249

CBDAS-
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDAEGMS

S328A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
250

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
251

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

V454A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
252

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

Y571F
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNF

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
253

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

H425D
LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
254

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_I620V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDV

GAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEK

HLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
255

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_I676V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNVYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
256

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_I700L
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDLGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
257

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_L442I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWAIRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
258

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
259

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

N269D
LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
260

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_P464PS
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPQENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
261

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_R243Q
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
262

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDAEGMS

G307A_S328A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
263

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_S449N
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFIRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
264

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_V454A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
265

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQATILCSKKVGLQIRTRSGGHDSEGMS

G307A_Y571F
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNF

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
266

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

H425D_M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
267

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

H425D_N269D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
268

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

H425D_V454A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
269

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

N269D_M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
270

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

P464PS_H425D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
271

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

P464PS_N269D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
272

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

P464PS_T468
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

I
LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPSKSIMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
273

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

P464PS_V454A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
274

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_H425D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVDGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
275

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_I620V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDV

GAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEK

HLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
276

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_I676V
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNVYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARI

WGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
277

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_I700L
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDLGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
278

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_L442I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWAIRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
279

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
280

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_N269D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
281

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

P464PS
LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPSKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPQENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
282

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_R243Q
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
283

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDAEGMS

S449N_S328A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
284

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_V454A
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIAAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
285

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

S449N_Y571F
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAENFGIIVAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNF

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHN
286

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

V454A_M468I
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPKSTIFSVKKIM

EIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTY

FSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYD

TDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGA

GMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHL

NWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIWG

EKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

PIR3_
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNDPLYMSVLNSTIHN
287

CBDAS_
LRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMS

V454A_N269D
YISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNES

LSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLVNVHGK

VLDRKSMGEDLFWALRGGGAESFGIIAAWKIRLVAVPKSTMFSVKKI

MEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHT

YFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNY

DTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIG

AGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKH

LNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

GEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH

Example 25—CBCA Synthase Mutagenesis

Fifty-three (53) positions around the predicted active site of the Cannabis sativa native CBDA synthase enzyme were mutagenized in a random manner to increase the synthesis of CBCA. The parental plasmid for mutagenesis was the 0285/asn080-2 plasmid. Plasmids carrying mutant CBDA synthase genes were isolated and screened for CBCA synthase activity. The screen identified amino acid positions where substitutions for certain amino acids result in the formation of a highly specific CBCA synthase or CBDA synthase with elevated CBCA synthase activity. The parental plasmid and plasmids with mutant CBDA synthase gene were transformed into the yCBGA0513 strain.

Mutant CBDA synthases with CBCA activity were screened using the following high throughput screening process: Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan, histidine and Hygromycin B). The inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter. After a 48 hour growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH was added to the samples. Finally, samples were grown for an additional 42 hours and were analyzed for cannabinoids.

The CBCA titer in the above described screen ranged from 18.0 to 69.6 mg/L. Sample titers for the transformed strains, along with each strain's respective amino acid mutations and amino acid sequences, are included in Tables 27-28 below. (All mutation sites refer to the mutation site on CBDA's coding sequence without the wild-type signal sequence.)

TABLE 27

yCBGA_0513 mutant strains

mutation

site on

CBDAs

coding

sequence

Average
Average

without the
native
new
titer of
titer of

signal
amino
amino
CBDA
CBCA

Constructs
sequence
acid
acid
mg/L
mg/L

SP_CBGA1137_10_B10
237
S
Q
0.0
20.9

SP_CBGA1137_03_H01
268
M
T
0.0
60.0

SP_CBGA1137_11_H10
292
S
N
23.3
32.9

SP_CBGA1136_04_F03
330
I
R
23.2
26.8

SP_CBGA1138_12_G06
332
Y
L
26.8
20.9

SP_CBGA1135_11_F02
334
G
Q
35.9
35.9

SP_CBGA1139_01_D07
338
Y
K
81.9
69.6

SP_CBGA1135_10_B02
359
F
H
26.5
21.5

SP_CBGA1136_05_A05
361
I
Y
0.0
22.1

0285/asn080-2
wild type
N/A
N/A
134.0
18.0

control

construct

TABLE 28

yCBGA_0513 mutants

SEQ ID

Constructs
CBCAs amino acid sequence without the signal sequence
NO:

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
288

1137_10_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

B10
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FQVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQG

KNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTI

IFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQ

ILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICS

WEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDP

KNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLP

RHRH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
289

1137_03_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

H01
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLTTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQIL

EKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW

EKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKN

PNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRH

RH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
290

1137_11_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

H10
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFNSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTII

FYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQI

LEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICS

WEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDP

KNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLP

RHRH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
291

1136_04_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

F03
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIR

FYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQI

LEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICS

WEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDP

KNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLP

RHRH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
292

1138_12_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

G06
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

LSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQIL

EKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW

EKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKN

PNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRH

RH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
293

1135_11_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

F02
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSQVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQIL

EKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW

EKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKN

PNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRH

RH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
294

1139_01_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

D07
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQ
GK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNKDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQIL

EKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW

EKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKN

PNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRH

RH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
295

1135_10_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

B02
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNYDTDNFNKEILLDRSAGQNGAHKIKLDYVKKPIPESVFVQI

LEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICS

WEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDP

KNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLP

RHRH

SP_CBGA
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
296

1136_05_
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

A05
MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNYDTDNFNKEILLDRSAGQNGAFKYKLDYVKKPIPESVFVQI

LEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICS

WEKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDP

KNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLP

RHRH

0285/asn0
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIH
297

80-2
NLRFSSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEG

MSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNE

KNESLSLAAGYCPTVCAGGHFGGGGYGPLMRSYGLAADNIIDAHLV

NVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTM

FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGK

NKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQIL

EKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW

EKQEDNEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDLDIGINDPKN

PNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRH

RH

Example 26—CBCA Synthase Mutagenesis

To improve the performance of CBCA synthase, an enzyme mutagenesis experiment was conducted. The mutant CBCA C-terminal core enzyme contains 3 differences as the compared to CBDA Cannabis sativa C-terminal core enzyme, G74A, S insertion at 232, and M269T (when numbering the core sequence (without the signal sequence).

The starting CBCA N-terminal signal sequence and C-terminal coding sequence are included Table 29 below:

TABLE 29

length of

amino acid

SEQ ID

sequence
CBCA amino acid sequence
NO:

N-terminal
251
MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYA
SEQ ID

signal

RGAYAPKDPWSTLTPSATYKGGITDYSSSFGIAI
No: 434

sequence

EAVATSASSVASSKAKRAASQIGDGQVQAATTT

AAVSKKSTAAAVSQITDGQVQAAKSTAAAVSQI

TDGQVQAAKSTAAAVSQITDGQVQAAKSTAAA

VSQITDGQVQAAKSTAAAASQISDGQVQATTST

KAAASQITDGQIQASKTTSGASQVSDGQVQATA

EVKDANDPVDVVSCNNNST

C-terminal
524
NIQTSIANPRENFLKCFSQYIPNNATNLKLVYTQ
SEQ ID

core

NNPLYMSVLNSTIHNLRFSSDTTPKPLVIVTPSH
No: 304

enzyme:

VSHIQATILCSKKVGLQIRTRSGGHDSEGMSYIS

(signal

QVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLG

sequence

EVYYWVNEKNESLSLAAGYCPTVCAGGHFGGG

removed)

GYGPLMRSYGLAADNIIDAHLVNVHGKVLDRK

SMGEDLFWALRGGGAESFGIIVAWKIRLVAVPS

KSTMFSVKKIMEIHELVKLVNKWQNIAYKYDK

DLLLTTHFITRNITDNQGKNKTAIHTYFSSVFLG

GVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIF

YSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLD

YVKKPIPESVFVQILEKLYEEDIGAGMYALYPYG

GIMDEISESAIPFPHRAGILYELWYICSWEKQED

NEKHLNWIRNIYNFMTPYVSQNPRLAYLNYRDL

DIGINDPKNPNNYTQARIWGEKYFGKNFDRLVK

VKTLVDPNNFFRNEQSIPPLPRHRH

Positions around the predicted active site of the Cannabis sativa CBDA enzyme were mutagenized in a random manner to increase the synthesis of CBCA. The parental plasmid for mutagenesis was the 0285/asn080-2 plasmid. Plasmids carrying mutant CBCA synthase genes were isolated and screened for CBCA synthase activity. The screen identified amino acid positions where substitutions for certain amino acids result in the formation of a highly specific CBCA synthase with elevated CBCA synthase activity. The parental plasmid and plasmids with mutant CBDA synthase gene were transformed into the yCBGA0513 or the yCBGA0523 strain.

Mutant CBCA synthases were screened using the following high throughput screening process: Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan, histidine and Hygromycin B). The inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter. After a 48 hour growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter and 8 μl of 1200 mg/l OLA dissolved in EtOH was added to the samples. Finally, samples were grown for an additional 42 hours and were analyzed for cannabinoids.

Using the CBCA mutants of Table 30 in the above described screen resulted in improved titers as compared to yCBGA0513 or yCBGA0523 strains encoding the native Cannabis sativa CBCA. Each mutant's respective amino acid mutations and amino acid sequences, are included in Tables 30 below. (All mutation sites refer to the mutation site on CBCA's coding sequence without the signal sequence.)

TABLE 30

yCBGA_0523 mutant strains

mutation site

on CBCAs

coding

sequence

without the
native
new

signal
amino
amino

sequence
acid
acid

1
Asn
Asn
SEQ ID NO: 305

94
Asp
Ser
SEQ ID NO: 306

94
Asp
Asn
SEQ ID NO: 307

267
Leu
Thr
SEQ ID NO: 308

461
Leu
Arg
SEQ ID NO: 309

461
Leu
Lys
SEQ ID NO: 310

1
Asn
Leu
SEQ ID NO: 311

1
Asn
Gly
SEQ ID NO: 312

54
Ser
Arg
SEQ ID NO: 313

54
Ser
Thr
SEQ ID NO: 314

95
Ser
Gly
SEQ ID NO: 315

95
Ser
Ala
SEQ ID NO: 316

82
Val
Ala
SEQ ID NO: 317

82
Val
Thr
SEQ ID NO: 318

Example 27—Prenyl Transferase Site Directed Mutagenesis

To improve the performance of prenyl transferase (PT), a site directed mutagenesis experiment was conducted. The base gene for mutagenesis was a fusion construct: N-terminal part: yEVenus (a modified GFP protein); C-terminal part: GFP-dPT of strain 314 (SEQ ID NO: 27), a truncated Saccharomyces cerevisiae prenyl-transferase (the N-terminal 98 amino acid of the original prenyl-transferase was truncated), and the sequences are included Table 31 below:

TABLE 31

length of

amino acid

SEQ ID

sequence
prenyltransferase amino acid sequence
NO:

Coding
539
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGE
SEQ ID

sequence

GDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGL
NO: 319

QCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD

DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL

GHKLEYNYNSHNVYITADKQKNGIKANFKIRHNI

EDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQS

ALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

KILNFGHTCWKLQRPYVVKGMISIACGLFGRELF

NNRHLFSWGLMWKAFFALVPILSFNFFAAIMNQI

YDVDIDRINKPDLPLVSGEMSIETAWILSIIVALTG

LIVTIKLKSAPLFVFIYIFGIFAGFAYSVPPIRWKQY

PFTNFLITISSHVGLAFTSYSATTSALGLPFVWRPA

FSFIIAFMTVMGMTIAFAKDISDIEGDAKYGVSTV

ATKLGARNMTFVVSGVLLLNYLVSISIGIIWPQVF

KSNIMILSHAILAFCLIFQTRELALANYASAPSRQF

FEFIWLLYYAEYFVYVFI

Positions around the predicted active site of the native Saccharomyces cerevisiae prenyltransferase synthase enzyme were mutagenized in a random manner to increase the synthesis of prenyltransferase. The parental plasmid for mutagenesis was the 0285/asn080-2 plasmid. Plasmids carrying mutant prenyltransferase synthase genes were isolated and screened for prenyltransferase synthase activity. The screen identified amino acid positions where substitutions for certain amino acids result in the formation of a highly specific prenyltransferase synthase with elevated prenyltransferase synthase activity. The parental plasmid and plasmids with mutant CBDA synthase gene were transformed into the yCBGA0537 strain (yCBGA0537=yCBGA0523 where all PT were deleted).

Mutant prenyltransferase synthases were screened using the following high throughput screening process: Colonies were inoculated into wells of a 96-well deep well plate. Each well contains 400 μl SC liquid medium (6.7 g/L Yeast Nitrogen Base, 1.6 g/L Amino Acid Drop Out mix without leucine, uracil, tryptophan and histidine, 22 g/L glucose, buffered to pH 6.0, supplemented with leucine, tryptophan, histidine and Hygromycin B). The inoculums were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter. After a 48 hour growth period, 40 μl samples of these cultures were inoculated into 360 μl YPD-2400LA (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose and 240 mg/L olivetolic acid) medium. Then samples were grown for 48 hours at 30° C. and shaken at 300 rpm with 50 mm shaking diameter and 8 μl of 12000 mg/l OLA dissolved in EtOH was added to the samples. Finally, samples were grown for an additional 42 hours and were analyzed for cannabinoids.

Using the prenyltransferase mutants of Table 32 in the above described screen resulted in similar or improved CBGA titers as compared to yCBGA0537 strains encoding the prenyltransferase. Each mutant's respective amino acid mutations and amino acid sequences, are included in Tables 32 below. (All mutation sites refer to the mutation site to the prenyltransferase in table 31 (SEQ ID 319).)

TABLE 32

yCBGA_0537 mutant strains

First
Last

position
position

of the
of the

Mutants

mutation
mutation

titer of

site on
site on

Wild

CBGA/

PT's
PT's
Length
type
mutant
Wild Type
SEQ

Plasmid
coding
coding
of
amino
amino
titer of
ID

Constructs
sequence
sequence
mutation
acid(s)
acid(s)
CBGA
NO:

bCBGA_0537
NA
NA
NA
NA
NA
1.000
320

0383/asn001-1
239
241
3
KIL
RVA
0.889
321

0382/asn002-3
242
242
1
N
D
0.983
322

0382/asn001-2
242
242
1
N
Q
1.158
323

0383/asn002-3
244
245
2
GH
LK
0.957
324

0382/asn003-4
249
249
1
K
R
0.978
325

0382/asn009-3
264
264
1
C
S
1.316
326

0382/asn013-2
272
272
1
F
I
0.858
327

0382/asn016-4
275
275
1
R
P
0.877
328

0382/asn015-2
275
275
1
R
K
1.002
329

0382/asn019-2
283
283
1
M
I
0.894
330

0383/asn008-3
283
284
2
MW
CF
1.145
331

0382/asn020-1
287
287
1
F
L
1.071
332

0382/asn023-4
295
295
1
s
c
0.713
333

0382/asn026-1
298
298
1
F
G
1.003
334

0382/asn028-3
309
309
1
V
I
1.338
335

0382/asn029-2
314
314
1
I
V
0.729
336

0382/asn034-2
323
323
1
S
A
0.965
337

0382/asn033-3
323
323
1
S
T
1.057
338

0382/asn035-3
326
326
1
M
I
0.744
339

0382/asn036-1
329
329
1
E
Q
0.975
340

0382/asn037-4
333
333
1
I
L
0.789
341

0382/asn039-2
343
343
1
L
F
0.788
342

0382/asn041-2
348
348
1
K
G
0.796
343

0382/asn042-2
350
350
1
K
N
1.196
344

0382/asn044-2
354
354
1
L
F
0.870
345

0383/asn018-4
354
356
3
LFV
VYI
1.252
346

0382/asn045-3
357
357
1
F
Y
0.829
347

0382/asn047-2
360
360
1
I
c
1.043
348

0382/asn048-2
361
361
1
F
L
1.715
349

0382/asn049-3
363
363
1
I
L
1.098
350

0382/asn052-1
374
374
1
I
L
1.417
351

0382/asn055-3
378
378
1
Q
R
0.947
352

0382/asn057-3
382
382
1
T
A
0.821
353

0382/asn061-3
398
398
1
S
V
1.138
354

0382/asn062-5
402
402
1
T
S
0.787
355

0382/asn065-4
417
417
1
S
T
1.029
356

0382/asn066-1
421
421
1
A
L
0.791
357

0382/asn068-4
426
426
1
M
F
1.050
358

0382/asn069-1
428
428
1
M
L
0.819
359

0362/asn046-4
447
450
4
VSTV
ISTI
1.058
360

(SEQ ID
(SEQ

NO: 430)
ID NO:

431)

0382/asn075-1
448
448
1
S
T
0.972
361

0382/asn076-1
450
450
1
V
L
0.996
362

0383/asn032-3
460
462
3
TFV
SWL
1.057
363

0382/asn079-2
473
473
1
V
A
1.069
364

0382/asn080-1
476
476
1
S
L
1.088
365

0382/asn081-3
481
481
1
W
M
0.926
366

0382/asn082-4
484
484
1
V
A
0.974
367

0362/asn053-2
484
491
8
VFKSNI
LFKSN
1.142
368

MI (SEQ
VMV

ID NO:
(SEQ

432)
433)

0382/asn084-4
488
488
1
N
S
1.093
369

0382/asn085-2
489
489
1
I
V
0.994
370

0382/asn088-2
493
493
1
S
A
1.117
371

0382/asn089-3
495
495
1
A
I
0.821
372

0382/asn090-2
499
499
1
F
S
0.745
373

0382/asn091-3
500
500
1
C
S
1.092
374

0382/asn092-3
503
503
1
F
Y
1.421
375

0382/asn094-1
510
510
1
L
K
0.799
376

0382/asn098-2
520
520
1
Q
S
0.769
377

0382/asn101-3
525
525
1
I
L
1.378
378

0382/asn102-2
527
527
1
L
I
0.741
379

Using the prenyltransferase mutant combinations of Tables 33-41 in the above described screen resulted in similar or improved CBGA titers as compared to yCBGA0537 strains encoding the native Saccharomyces cerevisiae prenyltransferase. Each mutant's respective amino acid mutations and amino acid sequences, are included in Tables 33-41 below. (All mutation sites refer to the mutation site on prenyltransferase's coding sequence without the wild-type signal sequence.)

TABLE 33

Combination “Combination Mutant Strain 1”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn001-2
242
242
1
N
Q

0382/asn003-4
249
249
1
K
R

0383/asn008-3
283
284
2
MW
CF

0382/asn020-1
287
287
1
F
L

TABLE 34

Combination “Combination Mutant Strain 2”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0383/asn001-1
239
241
3
KIL
RVA

0382/asn001-2
242
242
1
N
Q

0383/asn002-3
244
245
2
GH
LK

0382/asn003-4
249
249
1
K
R

0382/asn009-3
264
264
1
C
S

0382/asn013-2
272
272
1
F
I

0382/asn015-2
275
275
1
R
K

0383/asn008-3
283
284
2
MW
CF

0382/asn020-1
287
287
1
F
L

TABLE 35

Combination “Combination Mutant Strain 3”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn026-1
298
298
1
F
G

0382/asn028-3
309
309
1
V
I

0382/asn033-3
323
323
1
S
T

0382/asn037-4
333
333
1
I
L

TABLE 36

Combination “Combination Mutant Strain 4”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn023-4
295
295
1
S
C

0382/asn026-1
298
298
1
F
G

0382/asn028-3
309
309
1
V
I

0382/asn029-2
314
314
1
I
V

0382/asn033-3
323
323
1
S
T

0382/asn035-3
326
326
1
M
I

0382/asn036-1
329
329
1
E
Q

0382/asn037-4
333
333
1
I
L

0382/asn039-2
343
343
1
L
F

TABLE 37

Combination “Combination Mutant Strain 5”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0383/asn018-4
354
356
3
LFV
VYI

0382/asn048-2
361
361
1
F
L

0382/asn049-3
363
363
1
I
L

0382/asn052-1
374
374
1
I
L

TABLE 38

Combination “Combination Mutant Strain 6”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn062-5
402
402
1
T
S

0382/asn065-4
417
417
1
S
T

0382/asn066-1
421
421
1
A
L

0382/asn068-4
426
426
1
M
F

TABLE 39

Combination “Combination Mutant Strain 7”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without

without
the
Length
Wild type
mutant

Plasmid
the signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn075-1
448
448
1
S
T

0382/asn076-1
450
450
1
V
L

0383/asn032-3
460
462
3
TFV
SWL

0382/asn079-2
473
473
1
V
A

0382/asn085-2
489
489
1
I
V

TABLE 40

Combination “Combination Mutant Strain 8”

yCBGA_0537 combination mutant strains with multiple plasmid

constructs

Last

First
position

position
of the

of the
mutation

mutation
site on

site on
PT's

PT's
coding

coding
sequence

sequence
without
Length

without
the
of
Wild type
mutant

Plasmid
the signal
signal
muta-
amino
amino

Constructs
sequence
sequence
tion
acid
acid

0362/asn046-4
447
450
4
VSTV
ISTI

0383/asn032-3
460
462
3
TFV
SWL

0382/asn079-2
473
473
1
V
A

0382/asn080-1
476
476
1
S
L

0382/asn081-3
481
481
1
W
M

0362/asn053-2
484
491
8
VFKSNI
LFKSN

MI
VMV

TABLE 41

Combination “Combination Mutant Strain 9”

yCBGA_0527 comination mutant strains with multiple plasmid

constructs

First
Last

position
position

of the
of the

mutantion
mutation

site on
site on

PT's
PT's

coding
coding

sequence
sequence

without
without

the
the
Length
Wild type
mutant

Plasmid
signal
signal
of
amino
amino

Constructs
sequence
sequence
mutation
acid
acid

0382/asn089-3
495
495
1
A
I

0382/asn091-3
500
500
1
C
S

0382/asn092-3
503
503
1
F
Y

0382/asn101-3
525
525
1
I
L

Number	Date	Country
62832852	Apr 2019	US
62861667	Jun 2019	US
62861992	Jun 2019	US
62899378	Sep 2019	US

MICROORGANISMS AND METHODS FOR THE FERMENTATION OF CANNABINOIDS

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