Patent Application
20220002764
The application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 13, 2019, is named MAN-021PC_Sequence_Listing.txt and is 393,114 bytes in size.
Cannabis sativa (cannabis) is a flowering plant that has been cultivated for over 10,000 years. It is best known as a source for cannabinoids with psychoactive effects, such as tetrahydrocannabinol (THC). Cannabis is an annual, usually dioecious wind-pollinated herb, with male and female flowers growing on separate plants. Cannabinoids are found throughout the plant, with the exception of its seeds, but are mainly concentrated in the glandular trichomes of female flowers.
The beneficial properties of less-abundant natural cannabinoids have been discovered more recently. Cannabidiol (CBD), for example, has been investigated for the treatment of a variety of ailments, and has been approved by the Federal Drug Administration (FDA) for the treatment of seizures associated with two rare and severe forms of epilepsy: Lennox-Gastaut syndrome and Dravet syndrome. Additional potentially useful cannabinoids include cannabinol (CBN), a non-psychoactive cannabinoid with promise as a sedative and sleep aid; Δ8-THC, an isomer being investigated for treatment of the nausea associated with chemotherapy; and Tetrahydrocannabivarin (THCV), which has energizing and appetite suppressing activities.
Given the recognized and potential value of these and other rare cannabinoids, cost effective, scalable, and/or sustainable processes are needed for their production.
The present invention is concerned with the production of cannabinoids. In various aspects, the invention provides enzymes for cannabinoid biosynthesis, polynucleotides encoding said enzymes, recombinant host cells expressing said enzymes, and recombinant host cells that produce cannabinoids. In other aspects, the invention provides methods of producing cannabinoids using the enzymes or host cells. For example, cannabinoids may be produced by fermentation of recombinant host cells, or by biotransformation of cannabinoid precursors by whole cells, disrupted cells, or isolated or partially purified enzymes. Isolated cannabinoids produced according to the present invention may have higher purity and/or yield than natural cannabinoids because recombinant cells can be engineered to produce specific cannabinoid compounds by expressing particular biosynthetic enzymes. The cannabinoids thus produced may be incorporated into products such as pharmaceuticals, dietary supplements, baked goods, and others.
In some embodiments, the present invention provides methods, enzymes, and recombinant host cells for producing cannabinoids such as Δ9-tetrahydrocannbinol (THC or Δ9-THC), cannabigerol (CBG), cannabicyclol (CBL), cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), Δ8-tetrahydrocannbinol (Δ8-THC), cannabinerol (CBNR), Δ9-tetrahydrocannabivarol (THCV), cannabidivarin (CBDV) and/or cannabichrovarin (CBCV), as well as derivatives thereof. In some embodiments, recombinant host cells are fed with a cannabinoid biosynthetic intermediate, such as olivetol, olivetolic acid (OA), divarin, divarinic acid (DA), hexanoic acid, butyric acid, hexanoyl-CoA, butyryl-CoA, GPP precursor, or derivative thereof. Alternatively, host cells produce the cannabinoid from C1-C6 carbon substrates, such as glucose. In some embodiments, cannabinoids are recovered from recombinant host cells or their culture medium.
In some embodiments, the host cell recombinantly expresses a prenylating enzyme having cannabigerolic acid synthase (CBGAS) and/or cannabigerovarinic acid synthase (CBGVAS) activity, central enzymes for the biosynthesis of all cannabinoids, and one or more additional enzymes, such as geranyl diphosphate synthase (GPPS), acyl-activating enzyme (AAE), olivetol synthase (OLS), olivetolic acid cyclase (OAC), divarin synthase (DS), divaric acid cyclase (DAS), that increase the availability of CBGAS reactants. The host cell may also express enzymes such as tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and cannabichromenic acid synthase (CBCAS), that act on CBGAS and/or CBGVAS products. In some embodiments, one or more of the enzymes expressed in the host cell is derived from a cannabinoid-producing plant such as Cannabis sativa.
In some embodiments, the host cell further expresses or overexpresses one or more enzymes in the methylerythritol phosphate (MEP) and/or the mevalonic acid (MVA) pathway to catalyze the conversion of glucose to isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP). In some embodiments, the host cell further expresses an enzyme catalyzing the conversion of IPP and/or DMAPP to geranyl diphosphate (GPP), allowing for one or more cannabinoids to be produced from sugar or other carbon sources (carbon substrates such as C1, C2, C3, C4, C5, and/or C6 carbon substrates). In some embodiments, the host cell may express one or more enzymes capable of converting isoprenol to IPP and/or prenol to DMAPP.
In some embodiments, the host cell is engineered for increased synthesis of cannabinoid precursors. In some embodiments, the host cell is engineered for decreased utilization of cannabinoid precursors by competing biosynthetic pathways. The host cell may be engineered to increase carbon flux through the MEP pathway or for increased production of acetyl-CoA, malonyl-CoA, fatty acids, and/or other biomolecules.
In some embodiments, the host cell is a microbial cell, which may be prokaryotic or a eukaryotic (e.g. a bacterium or a yeast). For example, the host cell may be an Escherichia coli, Saccharomyces cerevisiae or Yarrowia lipolytica cell.
Other aspects and embodiments of the invention will be apparent from the following detailed disclosure.
The structures of various cannabinoids produced in the female flowers of Cannabis sativa are shown in
In accordance with various embodiments, the invention provides a microbial cell for producing one or more cannabinoids, where the microbial cell expresses a cannabinoid biosynthetic pathway that comprises a heterologous prenyltransferase having cannabigerolic acid synthase (CBGAS) activity or cannabigerovarinic acid synthase (CBGVAS) enzyme. The microbial cell further comprises one or more modifications that increase carbon flux to geranyl diphosphate (GPP) and/or carbon flux to hexanoic acid, hexanoyl-CoA, butyric acid, butyryl-CoA, and/or acetyl-CoA. Alternatively, or in addition to comprising one or more modifications that increase carbon flux to GPP, the microbial cell produces the cannabinoid from a fed precursor selected from olivetol, olivetolic acid, divarin, divarinic acid, hexanoic acid, butyric acid, hexanoyl-CoA, butyryl-CoA, GPP precursor, or derivative thereof.
CBGAS, also known as geranylpyrophosphate:olivetolate geranyltransferase, is a prenyl transferase that catalyzes the C-prenylation of OA or DA (CBGVAS activity) using GPP. In some embodiments, the CBGAS or CBGVAS enzyme may be Cannabis sativa CBGAS having SEQ ID NO: 60, or a derivative thereof. Alternatively, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence selected from SEQ ID NOs: 61 to 94, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 60 to 94. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 60 to 94. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 63, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 63. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 74, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 74. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 77, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 77. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 84, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 84. Amino acid modifications can be independently selected from substitutions, deletions, and insertions. In some embodiments, the derivative comprises a mutation at position corresponding to G286 of SEQ ID NO: 84. In some embodiments, the mutation at the position corresponding to G286 with respect to SEQ ID NO: 84 is a substitution with a polar amino acid. In embodiments, the substitution at position corresponding to G286 with respect to SEQ ID NO: 84 is selected from Arginine, Asparagine, Aspartic acid, Glutamine, Glutamic acid, Histidine, Lysine, Serine, Threonine, and Tyrosine. In one embodiment, the substitution at position corresponding to G286, with respect to SEQ ID NO: 84, is Serine.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 85, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 85. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 86, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 86. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 87, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 87. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 88, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 88. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 89, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 89. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 90, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 90. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 91, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 91. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the prenyl transferase activity may be provided by an enzyme comprising an amino acid sequence of SEQ ID NO: 93, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NO: 93. Amino acid modifications can be independently selected from substitutions, deletions, and insertions. In various embodiments, the enzymatic pathway further comprises one or more enzymes involved in the production of GPP, such as a GPP synthase (GPPS) and/or enzymes of the methylerythritol phosphate (MEP) and/or mevalonic acid (MVA) pathways. In various embodiments, the enzymatic pathway further comprises one or more enzymes involved in the production of OA, such as an acyl-activating enzyme (AAE), an olivetol synthase (OLS), and/or an olivetolic acid cyclase (OAC). In various embodiments, the enzymatic pathway further comprises one or more enzymes involved in the production of DA, such as an acyl-activating enzyme (AAE), a Divarin synthase (DS) and/or a Divarinic Acid Cyclase (DAC).
In some embodiments, the CBGAS or CBGVAS efficiently directs the flow of precursors into cannabinoids rather than other compounds. For example, in some embodiments, at least 50%, 60%, 70%, 80% or 90% of OA is converted to CBGA. Likewise, at least 50%, 60%, 70%, 80% or 90% of DA may be converted to CBGVA.
In various embodiments, the enzymatic pathway further comprises one or more enzymes that use CBGA as a substrate and catalyze the oxidative cyclization of the monoterpene moiety of CBGA, and such enzyme may be stereoselective. Such enzymes include tetrahydrocannabinolic acid synthase (THCAS), which produces tetrahydrocannabinolic acid (THCA); cannabidiolic acid synthase (CBDAS), which produces cannabidiolic acid (CBDA); and cannabichromenic acid synthase (CBCAS), which produces cannabichromenic acid (CBCA).
In various embodiments, the enzymatic pathway further comprises one or more enzymes that use CBGVA as a substrate and catalyze the oxidative cyclization of the monoterpene moiety of GBGVA, which in some embodiments is stereoselective. Such enzymes include THCAS, which produces tetrahydrocannabivarinic acid (THCVA), CBDAS, which produces cannabidivarinic acid (CBDVA), and CBCAS, which produces cannabichrovarinic acid (CBCVA).
In various embodiments, the enzymatic pathway further comprises enzymes involved in the production of geranyl diphosphate (GPP), such as a GPPS and enzymes in the methylerythritol phosphate (MEP) and/or mevalonic acid (MVA) pathways. GPPS catalyzes a reaction between isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP) to form GPP. The GPPS activity may be provided by an enzyme comprising an amino acid sequence selected from SEQ ID NOS: 1 to 25, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 1 to 25. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 1 to 25. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes in the MEP and/or MVA pathways to catalyze IPP and DMAPP biosynthesis from glucose or other carbon source. In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes of the MEP pathway. In some embodiments, the MEP pathway is increased and balanced with downstream pathways by providing duplicate copies of certain rate-limiting enzymes. The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/l-deoxy-D-xylulose 5-phosphate) pathway or the non-mevalonate pathway or the mevalonic acid-independent pathway refers to the pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The pathway typically involves action of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), and isopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genes and enzymes that make up the MEP pathway, are described in U.S. Pat. No. 8,512,988, which is hereby incorporated by reference in its entirety. For example, genes that make up the MEP pathway include dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, the microbial host cell expresses or overexpresses of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, GPP is produced at least in part by metabolic flux through an MEP pathway, and wherein the microbial host cell has at least one additional gene copy of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof.
In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes of the MVA pathway. The MVA pathway refers to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway typically comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA (e.g., by action of acetoacetyl-CoA thiolase); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoA synthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by action of HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by action of phosphomevalonate kinase (PMK)); and (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by action of mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, and the genes and enzymes that make up the MVA pathway, are described in U.S. Pat. No. 7,667,017, which is hereby incorporated by reference in its entirety. In some embodiments, the microbial host cell expresses or overexpresses one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, and MPD or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, GPP is produced at least in part by metabolic flux through an MVA pathway, and wherein the microbial host cell has at least one additional gene copy of one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, or modified variants thereof.
In some embodiments, the MEP pathway of the microbial host cell is engineered to increase production of IPP and DMAPP from glucose as described in US 2018/0245103 or US 2018/0216137, the contents of which are hereby incorporated by reference in their entireties. For example, in some embodiments the microbial host cell overexpresses MEP pathway enzymes, with balanced expression to push/pull carbon flux to IPP and DMAPP. In some embodiments, the microbial host cell is engineered to increase the availability or activity of Fe—S cluster proteins, so as to support higher activity of IspG and IspH, which are Fe—S enzymes. In some embodiments, the host cell is engineered to overexpress IspG and IspH, so as to provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, but with balanced expression to prevent accumulation of HMBPP at an amount that reduces cell growth or viability, or at an amount that inhibits MEP pathway flux.
In alternative embodiments, the microbial host cell is not engineered to increase production of GPP from MEP or MVA pathway precursors, but GPP or precursor compound (e.g., a terpene or terpene precursor) is fed to the cells to provide GPP substrate for CBD production.
In various embodiments, the enzymatic pathway further comprises enzymes involved in the production of OA, such as OAC, OLS, or an AAE.
OAC is a polyketide cyclase that can convert olivetol to OA by catalyzing a C2→C7 intramolecular aldol condensation upon which the carboxylate moiety is preserved. The OAC may comprise the amino acid sequence of SEQ ID NO: 52, or a derivative thereof. Alternatively, the OAC activity may be provided by an enzyme comprising an amino acid sequence selected from SEQ ID NOs: 53 to 59, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 52 to 59. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 52 to 59. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
OLS catalyzes the formation of olivetol by the aldol condensation of hexanoyl-CoA with three molecules of malonyl-CoA. The OLS may comprise the amino acid sequence of SEQ ID NO: 49, or a derivative thereof. Alternatively, the OLS activity may be provided by an enzyme comprising an amino acid sequence selected from SEQ ID NOs: 49-51, or a derivative thereof. The OLS enzyme may additionally have, or alternatively have, or be engineered to have, DS activity, and therefore useful for production of C3 cannabinoids. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 49 to 51. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 49 to 51. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
The acyl-activating enzyme (AAE), also called hexanoyl-CoA synthetase, synthesizes hexanoyl-CoA from hexanoate and CoA. Alternatively, the AAE may have or be engineered to have activity for producing Butyric acid instead of Hexanoic acid, and therefore useful for the production of C3 cannabinoids. The AAE may comprise the amino acid sequence of SEQ ID NO: 26, or may be a derivative thereof. Alternatively, the AAE may comprise the amino acid sequence of SEQ ID NO: 27, or a derivative thereof. Alternatively, the AAE activity may be provided by an enzyme comprising an amino acid sequence selected from SEQ ID NOS: 26 to 48, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 26 to 48. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 26 to 48. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In various embodiments, the enzymatic pathway further comprises enzymes involved in the production of DA, such as a DAC, DS, or an AAE. An enzyme having OAC activity may also have, or be engineered to have, DAC activity, and therefore be useful for production of C3 cannabinoids. Likewise, an enzyme having OLS activity may also have or be engineered to have DS activity; and an enzyme having AAE activity on Hexanoic Acid may also have or be engineered to have AAE activity on Butyric Acid.
In some embodiments, the enzymatic pathway for production of a C5 or C3 cannabinoid comprises an OAC or DAC enzyme comprising an amino acid sequence selected from SEQ ID NOS: 52-59, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 52 to 59. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 52 to 59. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the enzymatic pathway for production of a C5 or C3 cannabinoid comprises an OLS or DS enzyme, which may comprise an amino acid sequence selected from SEQ ID NOS: 49 to 51, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 49 to 51. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 49 to 51. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In various embodiments, the enzymatic pathway further comprises one or more enzymes that convert CBGA or CBGVA into cannabinoid derivatives that are optionally converted by a non-enzymatic process into additional cannabinoid compounds. In various embodiments, one or more nonenzymatic reactions convert THCA to THC, CBDA to CBD, CBCA to CBC, THCVA to THCV, CBDVA to CBDV, and/or CBCVA to CBCV.
In some embodiments, a combination of enzymes are expressed in the pathway to produce a plurality of cannabinoid compounds. Each of the diverse cannabinoid compounds created by these processes has unique and potentially beneficial biological activities.
Enzymes with substrate specificity for CBGA or CBGVA include THCAS, CBDAS, and CBCAS, including derivatives described herein. These enzymes may be derived or engineered from a plant that produces cannabinoids, such as Cannabis sativa.
In some embodiments, the enzymatic pathway comprises a THCAS enzyme comprising the amino acid sequence of SEQ ID NO: 99, or a derivative thereof. Alternatively, the enzymatic pathway comprises a THCAS enzyme comprising an amino acid sequence selected from SEQ ID NOS: 99 to 101, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 99 to 101. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 99 to 101. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the enzymatic pathway comprises a CBDAS enzyme comprising the amino acid sequence of SEQ ID NO: 95, or a derivative thereof. Alternatively, the CBDAS enzyme comprises an amino acid sequence selected from SEQ ID NOS: 96 or 97, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to an amino acid sequence selected from SEQ ID NOS: 95 to 97. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to a sequence selected from SEQ ID NOS: 95 to 97. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
In some embodiments, the enzymatic pathway comprises a CBCAS enzyme, which may comprise the amino acid sequence of SEQ ID NO: 98, or a derivative thereof. In some embodiments, the derivative comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of SEQ ID NO:98. In some embodiments, the derivative comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to the sequence of SEQ ID NOS: 98. Amino acid modifications can be independently selected from substitutions, deletions, and insertions.
The term “or a derivative thereof” indicates some degree of similarity between the derivative and a “parent” enzyme having the recited sequence. A derivative may have at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a parent enzyme. A derivative may also share structural similarity with a parent enzyme, such as similarity in secondary, tertiary, or quaternary structure. In various embodiments, a derivative and parent enzyme have similar substrate and/or cofactor binding sites, active sites, or reaction mechanisms.
The identity of amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, such as with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. BLAST protein searches may be performed with the BLASTP program, score=50, word length=3. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.
In various embodiments, two or more heterologous enzymes are expressed together in an operon, or are expressed individually. The enzymes may be expressed from extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
The amounts of various cannabinoids and cannabinoid precursors can be measured in a recombinant host cell to identify rate limiting steps in the biosynthetic pathway. Once a rate-limiting step has been identified, expression or activity of the limiting enzyme can be increased by various methods known in the art, such as codon optimization, use of a stronger promotor, expressing multiple copies of the corresponding gene, and constructing variants with increase stability and/or activity.
In some embodiments, one or more cannabinoids produced by a recombinant host cell are partially or completely exported to the culture medium. In other embodiments, one or more cannabinoids produced by a recombinant host cell are retained within the recombinant cell. Cannabinoids can be recovered from the culture medium or from the recombinant host cell.
In various embodiments, the microbe cell is a bacterium, and may be of a genus selected from Escherichia, Bacillus, Corynebacterium, Rhodobacter, Zymomonas, Vibrio, Pseudomonas, Agrobacterium, Brevibacterium, and Paracoccus. In some embodiments, the bacterium is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterium is E. coli. In various embodiments, the microbial cell is a yeast cell, which is a species of Saccharomyces, Pichia, or Yarrowia. For example, the microbial cell may be a species selected from Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.
In various embodiments, a recombinant host cell incorporates modifications that increase the pool of acyl-CoA precursors to enable high-titer production of OA and DA pathway intermediates. In these or other embodiments, the host cell is modified for enhanced GPP production. In some embodiments, a recombinant E. coli cell overexpresses one or more enzymes of the MEP pathway. The E. coli may have engineered expression of MEP pathway enzymes and other modifications as described in US 2018/0245103 or US 2018/0216137, the contents of which are hereby incorporated by reference in their entireties.
In some embodiments, the microbial host cell is a species of Saccharomyces, Pichia, or Yarrowia, including, but not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.
In some embodiments, the host cell is the oleaginous yeast Yarrowia lipolytica, which can utilize a wide variety of carbon sources and has the potential for high flux through key cannabinoid precursors, acetyl-CoA and malonyl-CoA. PCT/US2017/022252, which is hereby incorporated by reference in its entirety, presents various methods for increasing the biosynthesis of polyketides such as OA and DA in yeast by metabolic engineering. Polyketide synthesis is enhanced by reducing or eliminating the expression of certain genes, and by overexpressing other genes.
In yeast species such as Y. lipolytica, coordinated overexpression of pyruvate dehydrogenase complex components PDA1, PDE2, PDE3, and PDB1 with ACC1, the enzyme that converts acetyl-CoA to malonyl-CoA, is useful to increase polyketide synthesis. Enhanced expression of pyruvate bypass pathway enzymes further increase polyketide synthesis. These enzymes convert pyruvate to acetaldehyde through pyruvate decarboxylase (PDC1, PDC2), and then to acetate through acetylaldehde dehydrogenase (ALD2, ALD3, ALD5), and finally to acetyl-CoA via acetyl-CoA synthase (ACS1). For example, polyketide synthesis can be increased in some embodiments upon overexpression of various combinations of ACS1, ALD2, ALD3, ALD5, PDC1, PDC2 and ACC1. Additionally, genetic modifications such as overproduction of peroxisomal matrix protein 10 (PEX10), multifunctional β oxidation protein (MFE1), primary oleate regulator (POR1) or phosphatidate phosphatase (PAH) can increase β-oxidation of fatty acids and thereby increase the availability of acetyl-CoA and malonyl-CoA.
In some embodiments, a recombinant yeast (e.g., Y. lipolytica) host cell is engineered to incorporate modifications that increase the pool of acyl-CoA precursors to enable high-titer production of OA or DA pathway intermediates. In various embodiments, the recombinant yeast cell is modified for enhanced GPP production, which can be through overexpression of one or more enzymes of the MVA pathway. In alternative embodiments, the yeast cell does not overexpress enzymes of the MVA pathway, or is not engineered for increased production of MVA pathway products, and instead the cell may be fed GPP or terpene or terpene precursor compounds to support cannabinoid biosynthesis. In some embodiments, the cell produces GPP from IPP and/or DMAPP. In embodiments, the microbial cell expresses one or more enzymes for converting fed isoprenol and/or prenol to isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP), and, in some embodiments, the one or more enzymes are optionally kinases.
In some embodiments, recombinant host cells can produce cannabinoids from sugar (e.g., glucose) and other components present in growth media. In other embodiments, cannabinoids are produced by bioconversion from precursors, such as, olivetol, OA, divarin, DA, hexanoic acid, butyric acid, hexanoyl-CoA, butyryl-CoA and GPP precursor, which are fed to recombinant cells. In various embodiments, cannabinoids are produced from one or more alternative carbon sources including, for example, C1, C2, C3, C4, C5, and/or C6 carbon substrates, glycerol, xylose, fructose, mannose, ribose, sucrose, lignocellulosic biomass, ethanol, acetate, beet pulp, black liquor, corn starch, or switchgrass.
In some embodiments, the recombinant host cell expresses enzymes having CBGAS and CBDAS activity, and thus produces CBDA, which can be converted to CBD.
In some embodiments, the recombinant host cell expresses enzymes having CBGAS and CBDAS activity, and produces CBDA and/or CBD when fed with media comprising sugar such as glucose, or other carbon C1 to C6 carbon substrates. Such recombinant host cells may further express enzymes having GPPS, OAC, OLS, and/or AAE activity. In some embodiments, the recombinant host cell expressing CBGAS and CBDAS enzymes produces CBDA and/or CBD when fed with olivetol or OA. In some embodiments, CBDA recovered from a recombinant host cell is converted to CBD by exposure to heat and/or UV light.
In some embodiments, a recombinant host cell expresses enzymes having CBGAS and THCAS activity, the host cell producing THCA, which can be converted to THC. In some embodiments, the recombinant host cell expressing enzymes having CBGAS and THCAS activity produces THCA, which can convert to THC, when fed with media comprising sugar such as glucose or other C1 to C6 carbon substrates. In such embodiments, the recombinant host cell further expresses GPPS, OLS and/or OAC enzymes. In some embodiments the recombinant host cell expresses enzymes having CBGAS and THCAS activity, the host cell producing THCA, which can convert to THC, when fed with olivetol or OA. In some embodiments, THCA recovered from a recombinant host cell is converted to THC by exposure to heat and/or UV light.
In some embodiments, a recombinant host cell expresses enzymes having CBGAS and CBCAS activity, the host cell producing CBCA, which can be converted to CBC. In some embodiments, the recombinant host cell expressing enzymes having CBGAS and CBCAS activity produces CBCA, which can convert to CBC, when fed with media comprising sugar such as glucose or other C1 to C6 carbon substrates. In such embodiments, the recombinant host cell further expresses GPPS, OLS and/or OAC enzymes. In some embodiments the recombinant host cell expresses enzymes having CBGAS and CBCAS activity, the host cell producing CBCA, which can convert to CBC, when fed with olivetol or OA. In some embodiments, CBCA recovered from a recombinant host cell is converted to CBC by exposure to heat and/or UV light.
In some embodiments, a recombinant host cell expresses enzymes having CBGVAS and THCAS activity, the host cell producing THCVA, which can be converted to THCV. In some embodiments, the recombinant host cell expressing enzymes having CBGVAS and THCAS activity produces THCVA, which can convert to THCV, when fed with media comprising sugar such as glucose or other C1 to C6 carbon substrates. In such embodiments, the recombinant host cell further expresses GPPS, DS and/or DAC enzymes. In some embodiments the recombinant host cell expresses enzymes having CBGVAS and THCAS activity, the host cell producing THCVA, which can convert to THCV, when fed with divarin or DA. In some embodiments, THCVA recovered from a recombinant host cell is converted to THCV by exposure to heat and/or UV light.
In some embodiments, a recombinant host cell expresses enzymes having CBGVAS and CBDAS activity, the host cell producing CBDVA, which can be converted to CBDV. In some embodiments, the recombinant host cell expressing enzymes having CBGVAS and CBDAS activity produces CBDVA, which can convert to CBDV, when fed with media comprising sugar such as glucose or other C1 to C6 carbon substrates. In such embodiments, the recombinant host cell further expresses GPPS, DS and/or DAC enzymes. In some embodiments the recombinant host cell expresses enzymes having CBGVAS and CBDAS activity, the host cell producing CBDVA, which can convert to CBDV, when fed with divarin or DA. In some embodiments, CBDVA recovered from a recombinant host cell is converted to CBDV by exposure to heat and/or UV light.
In some embodiments, a recombinant host cell expresses enzymes having CBGVAS and CBCAS activity, the host cell producing CBCVA, which can be converted to CBCV. In some embodiments, the recombinant host cell expressing enzymes having CBGVAS and CBCAS activity produces CBCVA, which can convert to CBCV, when fed with media comprising sugar such as glucose or other C1 to C6 carbon substrates. In such embodiments, the recombinant host cell further expresses GPPS, DS and/or DAC enzymes. In some embodiments the recombinant host cell expresses enzymes having CBGVAS and CBCAS activity, the host cell producing CBCVA, which can convert to CBCV when fed with divarin or DA. In some embodiments, CBCVA recovered from a recombinant host cell is converted to CBCV by exposure to heat and/or UV light.
In various embodiments, the host cell is cultured at a temperature between 22° C. and 37° C. While commercial biosynthesis in host cells such as E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes (e.g., enzymes derived from plants) are stable, recombinant enzymes (including the terpenoid synthase) may be engineered to allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity. In some embodiments, the host cell (bacterial or yeast host cell) is cultured at about 22° C. or greater, about 23° C. or greater, about 24° C. or greater, about 25° C. or greater, about 26° C. or greater, about 27° C. or greater, about 28° C. or greater, about 29° C. or greater, about 30° C. or greater, about 31° C. or greater, about 32° C. or greater, about 33° C. or greater, about 34° C. or greater, about 35° C. or greater, about 36° C. or greater, or about 37° C.
Cannabinoids can be extracted from media and/or whole cells, and recovered. In some embodiments, the cannabinoids are recovered and optionally enriched by fractionation (e.g. fractional distillation). The product can be recovered by any suitable process, including partitioning the desired product into an organic phase. Various methods of cannabinoid preparation are known in the art, such as centrifugal partition chromatography. The production of the desired product can be determined and/or quantified, for example, by gas chromatography (e.g., GC-MS) or high pressure liquid chromatography (HPLC-MS).
The desired product can be produced in batch or continuous bioreactor systems. Production of product, recovery, and/or analysis of the product can be done as described in US 2012/0246767, which is hereby incorporated by reference in its entirety. For example, in some embodiments, oxidized oil is extracted from aqueous reaction medium, which may be done by partitioning into an organic phase, followed by fractional distillation. Cannabinoid components of fractions may be measured quantitatively by GC/MS or HPLC/MS, followed by blending of the fractions.
In some embodiments, the microbial host cells and methods disclosed herein are suitable for commercial production of one or more cannabinoids, that is, the microbial host cells and methods are productive at commercial scale. In some embodiments, the size of the culture is at least about 100 L, at least about 200 L, at least about 500 L, at least about 1,000 L, at least about 10,000 L, at least about 100,000 L, or at least about 1,000,000 L. In some embodiment, the culturing may be conducted in batch culture, continuous culture, or semi-continuous culture.
In some aspects, the present disclosure provides methods for making a product comprising one or more cannabinoids. In various aspects, the product is a pharmaceutical composition, a dietary supplement or a baked good. A cannabinoid of the present invention can be mixed with one or more excipients to form a pharmaceutical product, which may be a pill, a capsule, a mouth spray, or an oral solution.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.
Several candidate prenyltransferases (Table 1) were screened using liquid chromatography (LC) mass spectrometry (MS/MS) for their ability to generate cannabigerolic acid (CBGA).
Olivetolic acid (OA) and geranyl pyrophosphate (GPP) (both substrates) were mixed with each candidate prenyltransferase and reactions were performed under conditions suitable for production of CBGA. Products generated from the reaction of each candidate prenyl transferase were identified by multiple reaction monitoring and their retention times were compared to the authentic CBGA standard. The results obtained for each candidate prenyltransferase is shown in Table 1 below.
Each panel in
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/767,056, filed Nov. 14, 2018, the entire contents of all of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/061487 | 11/14/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62767056 | Nov 2018 | US |
