Patent Application
20230193329
The present disclosure relates generally to recombinant host cells comprising nucleic acids derived from Cannabis trichome mRNA that enhance the ability of the host cells to produce cannabinoids, associated nucleic acid compositions, and methods for using the recombinant host cells for cannabinoid production.
The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center as an WIPO Standard ST.26 formatted XML file with file name “13421-001WO1_SeqList_ST26.xml”, a creation date of Sep. 26, 2022, and a size of 747,339 bytes. This Sequence Listing filed via USPTO Patent Center is part of the specification and is incorporated in its entirety by reference herein. This sequence listing corresponds to the ST.26 formatted version of the ST.25 formatted sequence listing file, “13421-001WO1_SeqList_ST25.txt” that was filed with the parent application International Appl. No. PCT/US2021/024390 on Mar. 26, 2021.
Cannabinoids are a class of compounds that act on endocannabinoid receptors and include the phytocannabinoids naturally produced by Cannabis sativa. Cannabinoids include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and more than 80 related metabolites and synthetically produced compounds. Cannabinoids are increasingly used to treat a range of diseases and conditions such as multiple sclerosis and chronic pain. Current large-scale production of cannabinoids for pharmaceutical or other use is through extraction from plants. These plant-based production processes, however, have several challenges including susceptibility of the plants to inconsistent production caused by variance in biotic and abiotic factors, difficulty reproducing identical cannabinoid accumulation profiles, and difficulty in producing a single cannabinoid compound with purity high enough for pharmaceutical applications. While some cannabinoids can be produced as a single pure product via chemical synthesis, these processes have proven very costly and too costly for large-scale production.
More economical biosynthetic approaches to cannabinoid production are being developed using microbial hosts. These processes have the potential to be robust, scalable, and capable of producing single cannabinoid compound with higher purity compared to other current processes. Several biosynthetic systems for cannabinoid compound have been reported (see e.g., WO2019071000, WO2018200888, WO2018148849, WO2019014490, US20180073043, US20180334692, and WO2019046941). However, these biosynthetic systems still are not efficient in the biosynthesis of cannabinoids. The possible reasons for the low-yield conversion of these systems include poor protein expression, stressed cell growth, suboptimal metabolite transport, and lack of accessory genes required for optimal cannabinoid biosynthesis.
There exists a need for improved methods for the production of cannabinoid compounds. In particular, there is a need to improve the performance of microbial hosts in carrying out the recombinant biosynthesis of cannabinoid compounds.
The present disclosure relates generally to recombinant host cells comprising nucleic acids derived from Cannabis trichome mRNA that enhance the ability of the host cells to produce cannabinoids, associated nucleic acid compositions, and methods for using the recombinant host cells for cannabinoid production.
This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.
In at least one embodiment, the present disclosure provides recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway.
In at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide having a protein function selected from: amino acid biosynthesis, amino acid degradation, carbohydrate metabolism, carrier-mediated transport, cell cycle organization, cellular respiration, chromatin organization, circadian clock system regulation, coenzyme metabolism, oxidoreductase, transferase, hydrolase, lyase, effector-triggered immunity network co-regulatory protein, fatty acid biosynthesis, lipid degradation, lipid transfer type protein, messenger ribonucleoparticle (mRNP) export, mulatexin-like, organelle machinery ribonuclease activity, phytohormone action, protein folding, protein phosphorylation, protein S-glutathionylation, protein translocation, redox homeostasis, RNA biosynthesis, RNA processing, solute transport, terpenoid metabolism, and/or vesicle trafficking.
In at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide selected from: 1-acyl-sn-glycerol-3-phosphate acyltransferase 2; 1-aminocyclopropane-1-carboxylate oxidase homolog 1-like; acyl-acyl carrier protein thioesterase ATL3, chloroplastic-like; barwin-like; beta-adaptin-like protein B; BURP domain protein RD22-like; cationic peroxidase 2-like; cell division control protein 2 homolog 2; chloroplast stem-loop binding protein of 41 kDa b, chloroplastic; cysteine-rich receptor-like protein kinase 19; cytochrome B5-like protein; delta(12)-fatty-acid desaturase FAD2-like; desiccation-related protein PCC13-62-like; dormancy-associated protein 2-like; E3 ubiquitin-protein ligase SDIR1; gamma-carbonic anhydrase-like 2, mitochondrial; germacrene-A synthase-like; glucan endo-1,3-beta-glucosidase 12; glucose-6-phosphate 1-dehydrogenase 6, cytoplasmic-like; glycine-rich RNA-binding protein-like; major allergen Pru av 1-like; malonate--CoA ligase; mannose-1-phosphate guanylyltransferase 1; mediator of RNA polymerase II transcription subunit 11; MLP-like protein 423; NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial; NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; NDR1/HIN1-like protein 1; non-specific lipid-transfer protein 1-like; non-specific lipid-transfer protein 2-like; ornithine aminotransferase, mitochondrial; peptidyl-prolyl cis-trans isomerase CYP19-3; peroxidase 12-like; phosphoinositide phosphatase SAC1; probable gamma-secretase subunit PEN-2; probable protein phosphatase 2C 60; programmed cell death protein 2-like; protein CASP; protein ELF4-LIKE 4-like; protein REVEILLE 8; protein SRC1; protein TIC 56, chloroplastic; pyruvate decarboxylase 1-like; small acidic protein 1; sphingoid long-chain bases kinase 1; structural maintenance of chromosomes protein 1; translationally-controlled tumor protein homolog; tubulin beta-2 chain; ubiquitin-conjugating enzyme E2-17 kDa-like; U-box domain-containing protein 34; upstream activation factor subunit UAF30; uridine kinase-like protein 1, chloroplastic; V-type proton ATPase subunit B 1; and YTH domain-containing protein ECT4-like.
In at least one embodiment, the nucleic acid derived from Cannabis trichome mRNA encodes a polypeptide comprising (a) an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs: 16-540; (b) an amino acid sequence sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 36, 74, 88, 94, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 14, 150, 168, 174, 184, 188, 206, 210, 232, 270, 274, and 396; or (c) an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs:
In at least one embodiment, the nucleic acid derived from Cannabis trichome mRNA comprises: (a) a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 15-539; (b) a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 35, 73, 87, 93, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 167, 173, 183, 187, 205, 209, 231, 269, 273, and 395; or (c) a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 121-149.
In at least one embodiment, the pathway capable of producing a cannabinoid comprises enzymes capable of converting hexanoic acid to CBGA. In at least one embodiment, the pathway capable of producing a cannabinoid comprises at least the following enzymes: AAE, OLS, OAC, and PT4; optionally, wherein AAE has an amino acid sequence of at least 90% identity to SEQ ID NO: 2, OLS has an amino acid sequence of at least 90% identity to SEQ ID NO: 4, OAC has an amino acid sequence of at least 90% identity to SEQ ID NO: 6, and PT4 has an amino acid sequence of at least 90% identity to SEQ ID NO: 8 or 10.
In at least one embodiment, the pathway further comprises an enzyme capable of catalyzing the conversion of CBGA to Δ9-THCA, CBDA, and/or CBCA. In at least one embodiment, the pathway comprising AAE, OLS, OAC, and PT4, further comprises: THCA synthase, CBDA synthase, and/or CBCA synthase; optionally, wherein the pathway further comprises (a) a CBDA synthase having an amino acid sequence of at least 90% identity to SEQ ID NO: 12 or 14, and/or (b) a THCA synthase having an amino acid sequence of at least 90% identity to SEQ ID NO: 542 or 544.
In at least one embodiment, the cannabinoid produced by the host cell is selected from cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinolic acid (Δ8-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabinolic acid (CBNA), cannabinol (CBN), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), Δ9-tetrahydrocannabivann (Δ9-THCV), cannabidibutolic acid (CBDBA), cannabidibutol (CBDB), Δ9-tetrahydrocannabutolic acid (Δ9-THCBA), Δ9-tetrahydrocannabutol (Δ9-THCB), cannabidiphorolic acid (CBDPA), cannabidiphorol (CBDP), Δ9-tetrahydrocannabiphorolic acid (Δ9-THCPA), Δ9-tetrahydrocannabiphorol (Δ9-THCP), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabielsoinic acid (CBEA), cannabielsoin (CBE), cannabicitranic acid (CBTA), cannabicitran (CBT), and any combination thereof.
In at least one embodiment, the recombinant host cell produces the cannabinoid with a titer that is increased at least 1.2-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more relative to a control recombinant host cell comprising the pathway and not the nucleic acid derived from a Cannabis trichome mRNA.
In at least one embodiment, the present disclosure also provides a method for producing a cannabinoid comprising: (a) culturing in a suitable medium a recombinant host cell of the present disclosure (e.g., a host comprising a pathway of enzymes capable of converting hexanoic acid to CBGA and a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway); and (b) recovering the produced cannabinoid. In at least one embodiment, the method further comprises contacting a cell-free extract of the culture with a biocatalytic reagent or chemical reagent.
In at least one embodiment, the present disclosure also provides method for making a recombinant host cell for producing a cannabinoid comprising introducing into a host cell: (a) a first set of nucleic acids that encode a pathway of enzymes capable of producing a cannabinoid; and (b) a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway. In at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide having a protein function selected from: amino acid biosynthesis, amino acid degradation, carbohydrate metabolism, carrier-mediated transport, cell cycle organization, cellular respiration, chromatin organization, circadian clock system regulation, coenzyme metabolism, oxidoreductase, transferase, hydrolase, lyase, effector-triggered immunity network co-regulatory protein, fatty acid biosynthesis, lipid degradation, lipid transfer type protein, messenger ribonucleoparticle (mRNP) export, mulatexin-like, organelle machinery ribonuclease activity, phytohormone action, protein folding, protein phosphorylation, protein S-glutathionylation, protein translocation, redox homeostasis, RNA biosynthesis, RNA processing, solute transport, terpenoid metabolism, and/or vesicle trafficking. In at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs: 16-540. In at least one embodiment, the nucleic acid derived from Cannabis trichome mRNA comprises a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 15-539.
A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:
For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.
Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011, and now available in journal format online as Current Protocols in Molecular Biology, Vols. 00 -130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).
All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.
Definitions
“Cannabis trichome mRNA” refers to an mRNA molecule produced in the glandular trichome tissue of a cannabis plant, e.g., Cannabis sativa.
“Cannabinoid” refers to a compound that acts on cannabinoid receptor, and is intended to include the endocannabinoid compounds that are produced naturally in animals, the phytocannabinoid compounds produced naturally in cannabis plants, and the synthetic cannabinoids compounds. Exemplary cannabinoids of the present disclosure include those compounds listed in Table 3 (below).
“Pathway” refers an ordered sequence of enzymes that act in a linked series to convert an initial substrate molecule into final product molecule. As used herein, “pathway” is intended to encompass naturally-occurring pathways and non-naturally occurring, recombinant pathways. Accordingly, a pathway of the present disclosure can include a series of enzymes that are naturally-occurring and/or non-naturally occurring, and can include a series of enzymes that act in vivo or in vitro.
“Pathway capable of producing a cannabinoid” refers to a pathway that can convert an initial substrate compound into a cannabinoid. For example, the four enzymes AAE, OLS, OAC, and PT4 which convert hexanoic acid (HA) to cannabigerolic acid (CBGA) form a pathway capable of producing a cannabinoid.
“Conversion” as used herein refers to the enzymatic conversion of the substrate(s) to the corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an enzymatic conversion can be expressed as “percent conversion” of the substrate to the product.
“Substrate” as used herein in the context of an enzyme mediated process refers to the compound or molecule acted on by the enzyme.
“Product” as used herein in the context of an enzyme mediated process refers to the compound or molecule resulting from the activity of the enzyme.
“Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides.
“Nucleic acid,” or “polynucleotide” as used herein refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2′-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2′-deoxyribonucleosides. The nucleoside units of the nucleic acid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include single-stranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), or molecules comprising that include one or more modified and/or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.
“Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.
“Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
“Recombinant,” “engineered,” or “non-naturally occurring” when used herein with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but is produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
“Nucleic acid derived from Cannabis trichome mRNA” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of an mRNA found in Cannabis trichome cells or tissue. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from Cannabis trichome cells, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to, a Cannabis trichome mRNA sequence.
“Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
“Heterologous” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.
“Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression.
“Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin, “Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).
“Control sequence” as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
“Operably linked” as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.
“Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
“Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.
“Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence typically is at least 20 nucleotide or amino acid residue units in length, but can also be the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
“Substantial identity” or “substantially identical” refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
“Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
“Isolated” as used herein in reference to a molecule means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).
“Substantially pure” refers to a composition in which a desired molecule is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.
“Recovered” as used herein in relation to an enzyme, protein, or cannabinoid compound, refers to a more or less pure form of the enzyme, protein, or cannabinoid.
Recombinant Host Cells with Enhanced Cannabinoid Production
The present disclosure provides recombinant host cells (e.g., S. cerevisiae) already transformed with a cannabinoid biosynthesis pathway, which further comprise an introduced heterologous nucleic acid derived from Cannabis trichome mRNA. In the context of the recombinant host cells already transformed with a cannabinoid biosynthesis pathway, an exemplary cannabinoid biosynthesis pathway is one capable of converting hexanoic acid (HA) cannabigerolic acid (CBGA) as depicted in
The presence of the additional nucleic acid derived from Cannabis trichome mRNA integrated in the recombinant host cells results in substantially enhanced yields of the cannabinoid (e.g., 2-fold or more increased) relative to a recombinant host cell comprising the same cannabinoid synthesis pathway but without a nucleic acid derived from Cannabis trichome mRNA. Furthermore, the additional nucleic acid derived from Cannabis trichome mRNA does not encode an enzyme in the cannabinoid biosynthesis pathway already present in the recombinant host cell. Indeed, the nucleic acids derived from Cannabis trichome mRNA that exhibit this effect on the cannabinoid biosynthesis do not encode enzymes or proteins that would be predicted a priori to enhance cannabinoid production in a recombinant host cell. For example, as disclosed elsewhere herein, nucleic acid derived from a Cannabis trichome mRNA capable of enhancing cannabinoid biosynthesis can include a nucleic acid encoding a polypeptide having a protein function selected from: amino acid biosynthesis, amino acid degradation, carbohydrate metabolism, carrier-mediated transport, cell cycle organization, cellular respiration, chromatin organization, circadian clock system regulation, coenzyme metabolism, oxidoreductase, transferase, hydrolase, lyase, effector-triggered immunity network co-regulatory protein, fatty acid biosynthesis, lipid degradation, lipid transfer type protein, messenger ribonucleoparticle (mRNP) export, mulatexin-like, organelle machinery ribonuclease activity, phytohormone action, protein folding, protein phosphorylation, protein S-glutathionylation, protein translocation, redox homeostasis, RNA biosynthesis, RNA processing, solute transport, terpenoid metabolism, and/or vesicle trafficking.
The unexpected and surprising technical effect of enhanced cannabinoid biosynthesis associated with the introduction of one or more such nucleic acids into a recombinant host cell already capable of biosynthesizing a cannabinoid provides an improved recombinant host cell, and associated improved methods of cannabinoid production. Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway. As described further below, an exemplary pathway capable of biosynthesizing a cannabinoid can include four enzymes that convert hexanoic acid to cannabigerolic acid (CBGA).
In at least one embodiment, the recombinant host cell comprising a nucleic acid derived from a Cannabis trichome mRNA is capable of producing the cannabinoid with a titer that is increased relative to a control recombinant host cell comprising the pathway and not the nucleic acid derived from a Cannabis trichome mRNA. In at least one embodiment, the titer of cannabinoid produced is increased by at least 1.1-fold. 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 10-fold, or more relative to a control recombinant host cell comprising the pathway and not the nucleic acid derived from a Cannabis trichome mRNA.
Without intending to be bound by theory, it believed that the nucleic acids derived from a Cannabis trichome mRNA disclosed herein facilitate a range of metabolic mechanisms that can enhance cannabinoid biosynthesis across a range of recombinant host cells that are engineered with cannabinoid pathways. For example, it is believed that these nucleic acids contribute to higher cannabinoid production through the following off-pathway functions: increase heterologous protein expression, improve host stress response, stabilize heterologous proteins, mediate cannabinoid or precursor molecule transport, modify host metabolic regulation, facilitating protein-protein interaction (metabolon formation). In at least one embodiment, the recombinant host cell comprises a cannabinoid producing pathway comprising the enzymes capable of converting hexanoic acid to cannabigerolic acid (CBGA). One such a pathway capable of converting hexanoic acid to CBGA is illustrated in
As shown in
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As shown in
Cannabinoid pathway enzymes that can be introduced into a recombinant host cell to provide the pathways illustrated in
In at least one embodiment, the recombinant host cell the pathway capable of producing a cannabinoid comprises at least the exemplary enzymes, wherein the enzymes have the amino acid sequences of SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (OLS), SEQ ID NO: 6 (OAC), and SEQ ID NO: 8 (PT4). In at least one embodiment, the recombinant host cell the pathway capable of producing a cannabinoid further comprises the enzyme of SEQ ID NO: 12 (CBDAS).
The exemplary cannabinoid pathway enzymes listed in Table 1 are the naturally occurring sequences from C. sativa, it also is contemplated, however, that cannabinoid pathway enzymes used in the recombinant host can include naturally occurring sequence homologs of these enzymes and/or enzymes having non-naturally occurring sequences. For example, enzymes with amino acid sequences engineered to function optimally in a particular enzyme pathway, and/or optimally for production of particular cannabinoid, and/or optimally in a particular host. Methods for preparing such non-naturally occurring enzyme sequences are known in the art and include methods for enzyme engineering such as directed evolution. In at least one embodiment, the amino acid sequence of non-naturally occurring enzyme can be modified at either its N- or C-terminus by truncation, or fusion. For example, in at least one embodiment of the pathway of producing a cannabinoid, the naturally occurring amino acid sequence of the PT4 enzyme of SEQ ID NO: 8 can be truncated at the N-terminus by up to 82 amino acids to provide the PT4 of SEQ ID NO: 10 (also referred to herein as “d82_PT4”), which is capable of functioning to produce the cannabinoid CBGA in a recombinant host cell. Accordingly, in at least one embodiment of the recombinant host cell, the pathway capable of producing a cannabinoid comprises at least enzymes having an amino acid sequence at least 90% identity to SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (OLS), SEQ ID NO: 6 (OAC), and SEQ ID NOs: 10 (PT4). Similarly, it is contemplated that engineered versions of the AAE, OLS, OAC, and CBDAS enzymes can be prepared using methods known in the art, and used in the compositions and methods of the present disclosure. For example, the CBDAS enzyme of SEQ ID NO: 12 or the THCAS enzyme of SEQ ID NO: 542 can be truncated at the N-terminus by up to 28 amino acids to provide the d28_CBDAS enzyme of SEQ ID NO: 14 and the d28_THCAS enzyme of SEQ ID NO: 544. Accordingly, in at least one embodiment of the recombinant host cell, the pathway capable of producing a cannabinoid comprises at least enzymes having an amino acid sequence at least 90% identity to SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (OLS), SEQ ID NO: 6 (OAC), SEQ ID NOs: 10 (d82_PT4), and SEQ ID NO: 14 (d28_CBDAS). Or in at least one embodiment of the recombinant host cell, the pathway capable of producing a cannabinoid comprises at least enzymes having an amino acid sequence at least 90% identity to SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (OLS), SEQ ID NO: 6 (OAC), SEQ ID NOs: 10 (d82_PT4), and SEQ ID NO: 544 (d28_THCAS).
Other cannabinoid pathway enzymes useful in the recombinant host cells and associated methods of the present disclosure are known in the art, and can include naturally occurring enzymes obtained or derived from cannabis plants, (e.g., Cannabis sativa, and its sub-species, sativa, indica, and ruderalis), or non-naturally occurring enzymes that have been engineered based on the naturally occurring cannabis plant sequences. It is also contemplated that enzymes obtained or derived from other organisms (e.g., microorganisms) having a catalytic activity related to a desired conversion activity useful in a cannabinoid pathway can be engineered for use in a recombinant host cell of the present disclosure.
The heterologous nucleic acids derived from C. sativa trichome mRNA of the present disclosure can be incorporated (e.g., by recombinant transformation, or Cas9 integration) into a range of host cells already comprising a cannabinoid biosynthesis pathway to provide a system for enhanced production of cannabinoids (e.g., CBGA, CBDA, THCA, CBCA) or cannabinoid precursor compounds. Generally, the host cell used for the recombinant host cells of the present disclosure can be any cell that can be recombinantly modified with nucleic acids and then cultured to express the recombinant products of those nucleic acids, including polypeptides and metabolites produced by the activity of the recombinant polypeptides. A wide range of suitable sources of host cells are known in the art, and exemplary host cell sources useful as recombinant host cells of the present disclosure include, but are not limited to, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Escherichia coli. It is also contemplated that the host cell source for a recombinant host cell of the present disclosure can include a non-naturally occurring cell source, e.g., an engineered host cell. For example, a non-naturally occurring source host cell, such as a yeast cell previously engineered for improved production of recombinant genes, may be used to prepare the recombinant host cell of the present disclosure. Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell previously engineered with an integrated functional cannabinoid biosynthesis pathway and a heterologous nucleic acid encoding a protein that is not part of the pathway, wherein the host cell source is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli, or an engineered cell derived from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli.
The recombinant host cells of the present disclosure comprise heterologous nucleic acids encoding a pathway of enzymes capable of producing a cannabinoid, such as CBGA, and a heterologous nucleic acid derived from C. sativa trichome mRNA that is “off-pathway” (does not encode a pathway enzyme). As described elsewhere herein, cannabinoid pathway enzymes and the nucleic acid sequences encoding them are known in the art and provided herein, and can readily be used in accordance with the present disclosure. Typically, the nucleic acid sequence encoding enzymes in the cannabinoid pathway further include one or more nucleic acid sequences controlling expression of these pathway enzymes. These one or more additional nucleic acid sequences together with the nucleic acid sequences encoding the enzymes which form a part of an cannabinoid biosynthetic pathway can be considered a heterologous nucleic acid sequence. A variety of techniques and methodologies are available and well known in the art for introducing such heterologous nucleic acid sequences encoding the cannabinoid pathway enzymes into a host cell so as to attain expression in the host cell. Techniques well known to the skilled artisan include, for example, those techniques found in the well known Sambrook and Ausubel references cited elsewhere herein.
One of ordinary skill will recognize that the heterologous nucleic acids encoding the cannabinoid pathway enzymes and/or the nucleic acids derived from C. sativa mRNA can further comprise transcriptional promoters capable of controlling expression of the enzymes in the recombinant host cell. Generally, the transcriptional promoters are selected to be compatible with the host cell, so that promoters obtained from bacterial cells are used when a bacterial host cell is selected in accordance herewith, while a fungal promoter is used when a fungal host cell is selected, a plant promoter is used when a plant cell is selected, and so on.
Promoters useful in the recombinant host cells of the present disclosure may be constitutive or inducible, provided such promoters are operable in the host cells. Promoters that may be used to control expression in fungal host cells, such as Saccharomyces cerevisiae, are well known in the art and include, but are not limited to: inducible promoters, such as a Gall promoter or Gal10 promoter, a constitutive promoter, such as an alcohol dehydrogenase (ADH) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, or an S. pombe Nmt, or ADH promoter. Exemplary promoters that may be used to control expression in bacterial cells can include the Escherichia coli promoters lac, tac, trc, trp or the T7 promoter. Exemplary promoters that may be used to control expression in plant cells include, for example, a Cauliflower Mosaic Virus 35S promoter (Odell et al. (1985) Nature 313:810-812), a ubiquitin promoter (U.S. Pat. No. 5,510,474; Christensen et al. (1989)), or a rice actin promoter (McElroy et al. (1990) Plant Cell 2:163-171). Exemplary promoters that can be used in mammalian cells include, a viral promoter such as an SV40 promoter or a metallothionine promoter. All of these host cell promoters are well known by and readily available to one of ordinary skill in the art. Further nucleic acid control elements useful for controlling expression in a recombinant host cell can include transcriptional terminators, enhancers and the like, all of which may be used with the heterologous nucleic acids incorporate in the recombinant host cells of the present disclosure.
A wide variety of techniques are well known in the art for linking transcriptional promoters and other control elements to heterologous nucleic acid sequences encoding pathway genes or other heterologous off-pathway nucleic acids. Such techniques are described in e.g., the Sambrook and Ausubel references cited elsewhere herein. Accordingly, in at least one embodiment, the heterologous nucleic acid sequences of the present disclosure comprise a promoter capable of controlling expression in a host cell, wherein the promoter is linked to a heterologous nucleic acid sequence derived from C. sativa trichome mRNA, and/or an enzyme of a cannabinoid pathway (e.g., AAE, OLS, OAC, PT4, or CBDAS). Such heterologous nucleic acid sequences can be integrated into a recombinant expression vector which ensures good expression in the desired host cell, wherein the expression vector is suitable for expression in a host cell, meaning that the recombinant expression vector comprises the heterologous nucleic acid sequence linked to any genetic elements required to achieve expression in the host cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication, and the like. In some embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell's genome.
It is also contemplated that in some embodiments an expression vector comprising a heterologous nucleic acid derived from C. sativa trichome mRNA of the present disclosure may further contain a marker gene. Marker genes useful in accordance with the present disclosure include any genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable markers that may be employed to identify transformants through visual inspection include β-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14: 403).
Nucleic Acids Derived from Cannabis trichome mRNA that Enhance Recombinant Cannabinoid Biosynthesis
The glandular trichome tissue of C. sativa plants are known to produce and secrete high quantities of metabolites including cannabinoid compounds. Although heterologous enzymes capable of acting as a functional cannabinoid biosynthesis pathway have been transformed recombinantly into host cells to produce cannabinoid compounds, the effect, if any, of other “off-pathway” genes that are expressed in the C. sativa trichome tissue on cannabinoid production has remained unknown. As described elsewhere herein (including the Examples), high-throughput screening of cDNA derived C. sativa trichome mRNA sequences in a recombinant host system containing a biosynthetic pathway that converts hexanoic acid (HA) to the cannabinoid, cannabigerolic acid (CBGA), has identified numerous off-pathway genes that substantially enhance production of CBGA in transformed host cells.
Table 2 below provides a summary description of exemplary off-pathway Cannabis sativa genes derived from C. sativa trichome mRNA screening that result in at least 2-fold increased CBGA yield in a recombinant host cell system, and their associated sequence identifiers. The nucleotide and amino acid sequences are included in the accompanying Sequence Listing.
C. sativa trichome genes that enhance cannabinoid biosynthesis
1“Protein Function” correspond to plant protein BIN Descriptions described in Schwacke et al., (2019). “MapMan4: A Refined Protein Classification and Annotation Framework Applicable to Multi-Omics Data Analysis.” Mol. Plant. 12, 879-892.
2“Annotation” correspond to known annotations of the C. sativa genome based on the cs10 cannabis assembly (see at: www.ncbi.nlm.nih.gov/assembly/GCF_900626175.1/) with those specific locations of the genome not yet annotated indicated as “uncharacterized.”
3“FIOPC” is “fold-improvement relative to positive control” which was determined by screening as described in Example 1. FIOPC ranges are indicated as follows: + > 2.0; ++ > 3.0; +++ > 4.0; ++++ > 5.0
All of the C. sativa genes derived from trichome mRNAs shown in Table 2 were capable of providing at least 2-fold improvement of CBGA production in a recombinant host cell system that converts hexanoic acid to CBGA via a pathway comprising the enzymes AAE, OLS, OAC and PT4 (see Table 1). The trichome mRNA derived genes of Table 2, however, encode a wide range of proteins that do not appear to be directly involved with that with the hexanoic acid to CBGA pathway. Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid comprises a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 15-539. In at least one embodiment, the nucleic acid comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of odd-numbered SEQ ID NOs: 15-539. In at least one embodiment, the nucleic acid comprises a nucleotide sequence of any one of odd-numbered SEQ ID NOs: 15-539.
In at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid comprises a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 35, 73, 87, 93, 103, 105, 107, 109, 111, 113, 115, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 167, 173, 183, 187, 205, 209, 231, 269, 273, and 395.
In at least one embodiment, the present disclosure provides an isolated nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs: 16-540. In at least one embodiment, the nucleotide sequence of the isolated nucleic acid is codon-optimized for expression in a recombinant host cell, wherein the host cell source is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli, or an engineered cell derived from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli. In at least one embodiment, the isolated nucleic acid derived from Cannabis trichome mRNA comprises a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 15-539.
In at least one embodiment, the present disclosure provides a vector comprising a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs: 16-540. In at least one embodiment, the vector comprises nucleic acid that is codon-optimized for expression in a recombinant host cell, wherein the host cell source is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli, or an engineered cell derived from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli. In at least one embodiment, the vector comprising the nucleic acid derived from Cannabis trichome mRNA comprises a nucleotide sequence having at least 90% identity to any one of odd-numbered SEQ ID NOs: 15-539.
In at least one embodiment, the present disclosure provides a vector comprising a nucleic acid encoding a pathway of enzymes capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of even-numbered SEQ ID NOs: 16-540. In at least one embodiment, the pathway capable of producing a cannabinoid comprises at least the exemplary enzymes AAE, OLS, OAC, and PT4, wherein the enzymes have the amino acid sequences of SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (OLS), SEQ ID NO: 6 (OAC), and SEQ ID NO: 8 (PT4). In at least one embodiment, the vector comprises nucleic acid sequences encoding the pathway of enzymes that are codon-optimized for expression in a recombinant host cell, wherein the host cell source is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coli, or an engineered cell derived from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Escherichia coll.
In at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 80% identity to any one of even-numbered SEQ ID NOs: 16-540. In at least one embodiment, the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of even-numbered SEQ ID NOs: 16-540.
In at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID NOs: 36, 74, 88, 94, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 168, 174, 184, 188, 206, 210, 232, 270, 274, and 396.
The amino acid sequences of even-numbered SEQ ID NOs: 16-540 provided in the present disclosure begin with initiating methionine (M) residue at position 1, although it will be understood by the skilled artisan that this initiating methionine residue may be removed by biological processing machinery, such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue. Accordingly, it is contemplated that any embodiment of the present disclosure comprising an enzyme of Table 2 can comprising an amino acid sequence of even-numbered SEQ ID NOs: 16-540 with the methionine residue at position 1 deleted.
As further described in the Examples, the off-pathway trichome mRNA derived genes of Table 2 are capable of providing at least 2-fold, and up to 5-fold or greater increased cannabinoid production when introduced into a recombinant host cell comprising a heterologous cannabinoid biosynthesis pathway. Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the recombinant host cell produces the cannabinoid with a titer that is increased at least 1.2-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or more relative to a control recombinant host cell comprising the pathway and not the nucleic acid derived from a Cannabis trichome mRNA. For example, a control recombinant yeast cell that can convert hexanoic acid to CBGA but that has not been transformed with a heterologous nucleic acid derived from a C. sativa trichome mRNA of Table 2.
The nucleic acids derived from C. sativa trichome mRNA that are identified in Table 2 encode a wide range of protein functions that generally would not be identified a priori as capable of enhancing cannabinoid production in a recombinant host cell. The exemplary protein functions include: amino acid biosynthesis, amino acid degradation, carbohydrate metabolism, carrier-mediated transport, cell cycle organization, cellular respiration, chromatin organization, circadian clock system regulation, coenzyme metabolism, EC_1 oxidoreductase, EC_2 transferase, EC_3 hydrolase, EC_4 lyase, effector-triggered immunity (ETI) network co-regulatory protein (RAR1), fatty acid biosynthesis, lipid degradation, lipid transfer type protein, messenger ribonucleoparticle (mRNP) export, mulatexin-like, organelle machinery ribonuclease activity, phytohormone action, protein folding, protein phosphorylation, protein S-glutathionylation, protein translocation, redox homeostasis, RNA biosynthesis, RNA processing, solute transport, terpenoid metabolism, and vesicle trafficking.
Without intending to be bound by theory, it is believed that these off-pathway proteins that are encoded by nucleic acids derived from Cannabis trichome mRNA can enhance cannabinoid production in a recombinant host via a range of mechanisms including but not limited to: (i) lipid transfer proteins can mediate cannabinoid transfer across the cytoplasm to cell membranes in the host cell; (ii) peroxidases can scavenge the peroxides that are formed as a result of THCA and CBDA synthesis, alleviating damage to the host cell; (iii) acyl-acyl carrier protein thioesterases can determine the chain length of fatty acids during their formation, thereby producing fatty acids with chain lengths favourable for cannabinoid synthesis; (iv) desaturases can introduce double-bonds in acyl lipids, eventually leading to the formation of hexanoyl-CoA, a key precursor in the cannabinoid pathway; (v) malonate-CoA ligase utilizes malonate and CoA to form malonyl-CoA, another key precursor in the cannabinoid pathway; (vi) major allergen Pru av 1-like proteins, which include major latex protein-like proteins, bind hydrophobic compounds, that can assist in intracellular trafficking of cannabinoid precursors and/or prevent degradation of these precursors; and (vii) patatin-like proteins, acting as lipases, break down phospholipids from into fatty acids which can serve as precursors for cannabinoid synthesis.
Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway, wherein the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide having a protein function corresponding to a MapMan4 Bin Description selected from: amino acid biosynthesis, amino acid degradation, carbohydrate metabolism, carrier-mediated transport, cell cycle organization, cellular respiration, chromatin organization, circadian clock system regulation, coenzyme metabolism, EC_1 oxidoreductase, EC_2 transferase, EC_3 hydrolase, EC_4 lyase, effector-triggered immunity (ETI) network co-regulatory protein (RAR1), fatty acid biosynthesis, lipid degradation, lipid transfer type protein, messenger ribonucleoparticle (mRNP) export, mulatexin-like protein, organelle machinery ribonuclease activity, phytohormone action, protein folding, protein phosphorylation, protein S-glutathionylation, protein translocation, redox homeostasis, RNA biosynthesis, RNA processing, solute transport, terpenoid metabolism, and/or vesicle trafficking.
In at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide having a protein function selected from: carbohydrate metabolism, EC_1 oxidoreductase, EC_2 transferase, fatty acid biosynthesis, lipid degradation, lipid transfer type protein, and mulatexin-like protein.
For example, polypeptides having a protein function of carbohydrate metabolism can include pyruvate decarboxylase 1-like, and/or can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 36; polypeptides having a protein function of EC_1 oxidoreductase can include 2-oxoglutarate-dependent dioxygenase, and/or can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 74; polypeptides having a protein function of EC_2 transferase can include protein ECERIFERUM 26-like, or stemmadenine 0-acetyltransferase-like, and/or can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 88, or 94; polypeptides having a protein function of fatty acid biosynthesis can include 3-ketoacyl-CoA synthase 6, acyl carrier protein 1 (chloroplastic-like), or malonate—CoA ligase, and/or can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 104, 106, 108, 110, 112, or 114; polypeptides having a protein function of lipid degradation can include acyl-acyl carrier protein thioesterase ATL3, chloroplastic-like, or patatin-like protein 1, and/or can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 116, or 120; polypeptides having a protein function of lipid transfer type protein can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 122, 124, 126, 128, 130, 132, 134, 136, 138, or 140; and polypeptides having a protein function of mulatexin-like protein can include a polypeptide comprising an amino acid sequence having at 90% identity to an amino acid sequence of SEQ ID NO: 142, 144, 146, 148, or 150. Accordingly, in at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide comprising an amino acid sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID NOs: 36, 74, 88, 94, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, and 150.
In at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide having a gene annotation (e.g., based on cs10 assembly) selected from: ECERIFERUM 26-like, stemmadenine O-acetyltransferase-like, 3-ketoacyl-CoA synthase 6, acyl carrier protein 1 (chloroplastic-like), malonate-CoA ligase, acyl-acyl carrier protein thioesterase ATL3, chloroplastic-like, patatin-like protein 1, mulatexin-like, non-specific lipid-transfer protein 1-like, non-specific lipid-transfer protein 2-like, BURP domain protein RD22-like, cationic peroxidase 2-like, disease resistance protein RGA2-like, MLP-like protein 423, peroxidase 12-like, pyruvate decarboxylase 1-like, desiccation-related protein PCC13-62-like, or dormancy-associated protein 2-like. Accordingly, in at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide comprising an amino acid sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID NOs: 36, 74, 88, 94, 104, 106, 108, 110, 112, 114, 116, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 168, 174, 184, 188, 206, 210, 232, 270, or 274.
Among the off-pathway nucleic acids derived from Cannabis trichome mRNAs that resulted in over 4-fold improvement of CBGA yield were nucleic acids encoding protein types including: cell cycle organization, cellular respiration, EC_1 oxidoreductase, EC_2 transferase, lipid degradation, lipid transfer type protein, circadian clock system regulation, protein folding, protein translocation, RNA processing, and vesicle trafficking.
More specifically, the off-pathway nucleic acids derived from Cannabis trichome mRNAs that resulted in over 3-fold improvement of CBGA yield encoded proteins having the following annotations: 1-acyl-sn-glycerol-3-phosphate acyltransferase 2; 1-aminocyclopropane-1-carboxylate oxidase homolog 1-like; acyl-acyl carrier protein thioesterase ATL3, chloroplastic-like; barwin-like; beta-adaptin-like protein B; BURP domain protein RD22-like; cationic peroxidase 2-like; cell division control protein 2 homolog 2; chloroplast stem-loop binding protein of 41 kDa b, chloroplastic; cysteine-rich receptor-like protein kinase 19; cytochrome B5-like protein; delta(12)-fatty-acid desaturase FAD2-like; desiccation-related protein PCC13-62-like; dormancy-associated protein 2-like; E3 ubiquitin-protein ligase SDIR1; gamma carbonic anhydrase-like 2, mitochondrial; germacrene-A synthase-like; glucan endo-1,3-beta-glucosidase 12; glucose-6-phosphate 1-dehydrogenase 6, cytoplasmic-like; glycine-rich RNA-binding protein-like; major allergen Pru av 1-like; malonate—CoA ligase; mannose-1-phosphate guanylyltransferase 1; mediator of RNA polymerase II transcription subunit 11; MLP-like protein 423; NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial; NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; NDR1/HIN1-like protein 1; non-specific lipid-transfer protein 1-like; non-specific lipid-transfer protein 2-like; ornithine aminotransferase, mitochondrial; peptidyl-prolyl cis-trans isomerase CYP19-3; peroxidase 12-like; phosphoinositide phosphatase SAC1; probable gamma-secretase subunit PEN-2; probable protein phosphatase 2C 60; programmed cell death protein 2-like; protein CASP; protein ELF4-LIKE 4-like; protein REVEILLE 8; protein SRC1; protein TIC 56, chloroplastic; pyruvate decarboxylase 1-like; small acidic protein 1; sphingoid long-chain bases kinase 1; structural maintenance of chromosomes protein 1; translationally-controlled tumor protein homolog; tubulin beta-2 chain; ubiquitin-conjugating enzyme E2-17 kDa-like; U-box domain-containing protein 34; upstream activation factor subunit UAF30; uridine kinase-like protein 1, chloroplastic; V-type proton ATPase subunit B 1; and YTH domain-containing protein ECT4-like.
Accordingly, it is contemplated that recombinant host engineered with a pathway of heterologous enzymes capable of producing a cannabinoid can have the biosynthetic yield of the cannabinoid increased or otherwise enhance by introduction a heterologous off-pathway nucleic acid that encodes a recombinant polypeptide having a protein function or enzymatic activity selected from the above list. Thus, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA, wherein the nucleic acid encodes a recombinant polypeptide having a protein function or enzymatic activity selected from: 1-acyl-sn-glycerol-3-phosphate acyltransferase 2; 1-aminocyclopropane-1-carboxylate oxidase homolog 1-like; acyl-acyl carrier protein thioesterase ATL3, chloroplastic-like; barwin-like; beta-adaptin-like protein B; BURP domain protein RD22-like; cationic peroxidase 2-like; cell division control protein 2 homolog 2; chloroplast stem-loop binding protein of 41 kDa b, chloroplastic; cysteine-rich receptor-like protein kinase 19; cytochrome B5-like protein; delta(12)-fatty-acid desaturase FAD2-like; desiccation-related protein PCC13-62-like; dormancy-associated protein 2-like; E3 ubiquitin-protein ligase SDIR1; gamma-carbonic anhydrase-like 2, mitochondrial; germacrene-A synthase-like; glucan endo-1,3-beta-glucosidase 12; glucose-6-phosphate 1-dehydrogenase 6, cytoplasmic-like; glycine-rich RNA-binding protein-like; major allergen Pru av 1-like; malonate—CoA ligase; mannose-1-phosphate guanylyltransferase 1; mediator of RNA polymerase II transcription subunit 11; MLP-like protein 423; NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial; NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; NDR1/HIN1-like protein 1; non-specific lipid-transfer protein 1-like; non-specific lipid-transfer protein 2-like; ornithine aminotransferase, mitochondrial; peptidyl-prolyl cis-trans isomerase CYP19-3; peroxidase 12-like; phosphoinositide phosphatase SAC1; probable gamma-secretase subunit PEN-2; probable protein phosphatase 2C 60; programmed cell death protein 2-like; protein CASP; protein ELF4-LIKE 4-like; protein REVEILLE 8; protein SRC1; protein TIC 56, chloroplastic; pyruvate decarboxylase 1-like; small acidic protein 1; sphingoid long-chain bases kinase 1; structural maintenance of chromosomes protein 1; translationally-controlled tumor protein homolog; tubulin beta-2 chain; ubiquitin-conjugating enzyme E2-17 kDa-like; U-box domain-containing protein 34; upstream activation factor subunit UAF30; uridine kinase-like protein 1, chloroplastic; V-type proton ATPase subunit B 1; and YTH domain-containing protein ECT4-like.
Additionally, the nucleic acids derived from C. sativa trichome mRNA of Table 2 include the following non-annotated (or “uncharacterized”) proteins that are identified by their location in the Cannabis sativa genome. The nucleic acids also resulted in 2-fold or greater improvement of CBGA yield. Accordingly, it is contemplated that a recombinant host engineered with a pathway of heterologous enzymes capable of producing a cannabinoid can have the biosynthetic yield of the cannabinoid increased or otherwise enhanced by introduction a nucleic acid that encodes any of the polypeptides listed as “uncharacterized” in Table 2. Accordingly, in at least one embodiment, the nucleic acid derived from a Cannabis trichome mRNA encodes a polypeptide comprising an amino acid sequence having at least 80% identity, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to any one of SEQ ID NOs: 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, or 418.
Use of Recombinant Host Cells in Preparation of Cannabinoids
As described elsewhere herein, the recombinant host cells provided by the present disclosure comprise a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA that does not encode an enzyme in the pathway. Furthermore, the recombinant host cells are capable of producing the cannabinoid with a titer that is increased (e.g., 2-fold or more) relative to a control recombinant host cell comprising the same pathway but without the off-pathway nucleic acid derived from a Cannabis trichome mRNA. As described elsewhere herein, the off-pathway nucleic acid can encode a protein described in Table 2 and the associate Sequence Listing.
Although the cannabinoid pathways of
In at least one embodiment, it is also contemplated that the nucleic acids derived from a Cannabis sativa trichome mRNA of the present disclosure (e.g., as in Table 2) can be introduced into a recombinant host cell to provide a method for the improved biosynthesis of cannabinoid precursor compounds or cannabinoid precursor derivatives in terms of titer, yield, and production rate. Cannabinoid precursors, or cannabinoid precursor derivatives can include, but are not limited to olivetolic acid, olivetol, divarin, PDAL, HTAL, GPP, polyketides, polyketide derivatives, and others known in the art (see e.g,, Elsohly and Slade, Life Sci. 2005 Dec. 22; 78(5):539-48, Epub 2005 Sep. 30; Bow, E. W. and Rimoidi, J. M., “The Structure-Function Relationships of Classical Cannabinoids: CB1/C62 Modulation,” Perspectives in Medicinal Chemistry 2016; 8 17-39 doi:10.4137/PMC.S32171.) Such precursor compounds can be useful products, and/or can be used to prepare other derivative compounds, either synthetically or biosynthetically. In at least one embodiment, a cannabinoid precursor compound, such as OA or divarinic acid (DA), can be produced, and then further modified or derivatized using an in vitro enzymatic biosynthesis, using e.g., a cannabinoid synthase.
In at least one embodiment, the present disclosure provides a method for producing a cannabinoid or cannabinoid precursor comprising: (a) culturing in a suitable medium a recombinant host cell of the present disclosure; and (b) recovering the produced cannabinoid or cannabinoid precursor.
In at least one embodiment of the method for producing a cannabinoid, a nucleic acid derived from a Cannabis sativa trichome mRNA of Table 2, can be introduced into a recombinant host cell comprising a pathway capable of producing a cannabinoid (e.g., CBGA) to provide an recombinant host cell that has improved biosynthesis of the cannabinoid in terms of titer, yield, and production rate.
In at least one embodiment, a recombinant host cell of the present disclosure can produce a cannabinoid compound, or a composition comprising a cannabinoid compound, wherein the cannabinoid is selected from cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ9-tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (Δ8-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabinolic acid (CBNA), cannabinol (CBN), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), Δ9-tetrahydrocannabivarin (Δ9-THCV), cannabidibutolic acid (CBDBA), cannabidibutol (CBDB), Δ9-tetrahydrocannabutolic acid (Δ9-THCBA), Δ9-tetrahydrocannabutol (Δ9-THCB), cannabidiphorolic acid (CBDPA), cannabidiphorol (CBDP), Δ9-tetrahydrocannabiphorolic acid (Δ9-THCPA), Δ9-tetrahydrocannabiphorol (Δ9-THCP), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabielsoinic acid (CBEA), cannabielsoin (CBE), cannabicitranic acid (CBTA), cannabicitran (CBT), and any combination thereof. In at least one embodiment, a recombinant host cell of the present disclosure can be used to produce a cannabinoid selected from cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabichromenic acid (CBCA), and any combination thereof.
In at least one embodiment of the method for producing a cannabinoid, the method can further comprise contacting a cell-free extract of the culture containing the produced cannabinoid with a biocatalytic reagent or chemical reagent.
In at least one embodiment, the biocatalytic reagent is an enzyme capable of converting the produced cannabinoid to a different cannabinoid or a cannabinoid derivative compound. In at least one embodiment, the chemical reagent is capable of chemically modifying the produced cannabinoid to produce a different cannabinoid or a cannabinoid derivative compound.
Accordingly, in at least one embodiment of the method, the recombinant host cell with improved cannabinoid production in terms of titer, yield, and production rate can be used in the production of a cannabinoid (see e.g., compounds of Table 3), or a cannabinoid derivative compound. Cannabinoid derivative compounds can include a wide range of naturally-occurring and non-naturally occurring compounds.
Cannabinoid derivative compounds produced using the recombinant host cells of the present disclosure can include any compound structurally related to a cannabinoid compound (e.g., compounds of Table 3) but which lacks one or more of the chemical moieties present in the cannabinoid compound from which it derives. Exemplary chemical moieties that may be lacking in a cannabinoid derivative include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkenylalkyl, cycloalkenylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, arylalkenyl, heterocyclyl, aralkyl, cycloalkylalkyl, heterocyclylalkyl, heteroarylalkyl, and the like.
Alternatively, cannabinoid derivative compounds using the recombinant host cells of the present disclosure can include one or more additional chemical moieties not present in the cannabinoid compound from which it derives. Exemplary chemical moieties that may be added in a cannabinoid derivative include, but are not limited to azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio, cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocyclyl, heterospirocyclyl, heterocyclyl, thioalkyl, sulfone, sulfonyl, sulfoxide, amino, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like.
Accordingly, in at least one embodiment, the present disclosure provides a method of producing a cannabinoid derivative, wherein the method comprises: (a) culturing in a suitable medium a recombinant host cell of the present disclosure; and (b) recovering the produced carinabirioid derivative. In at least one embodiment, the method of producing a cannabinoid derivative further contacting a cell-free extract of the culture containing the produced cannabinoid with a biocatalytic reagent or chemical reagent capable of converting the cannabinoid to a cannabinoid derivative. In at least one embodiment, the biocatalytic reagent is an enzyme capable of converting the produced cannabinoid to a different cannabinoid or a cannabinoid derivative compound. In at least one embodiment, the chemical reagent is capable of chemically modifying the produced cannabinoid to produce a different cannabinoid or a cannabinoid derivative compound.
Cannabinoid derivatives that can be produced with improved yield using a recombinant host cell of the present disclosure can include cannabinoid derivatives modified (e.g., biocatalytically or synthetically) to provide improved properties of pharmaceutical metabolism and/or pharmacokinetics (e.g. solubility, bioavailability, absorption, distribution, plasma half-life and metabolic clearance). Modifications typically providing such improved pharmaceutical properties can include, but are not limited to, halogenation, acetylation and methylation, It is also contemplated that the cannabinoids and cannabinoid derivatives produced by the methods disclosed herein can include pharmaceutically acceptable isotopically labeled cannabinoid and cannabinoid derivative compounds. For example, cannabinoid and cannabinoid derivative compounds wherein the hydrogen atoms are replaced or substituted by one or more deuterium or tritium atoms. Such isotopically labeled cannabinoids and derivatives can be useful in studies of in vivo pharmacokinetics and tissue distribution,
Upon production by the host cells or in the cell-free mixture of the cannabinoid precursors or cannabinoids in accordance with the recombinant host cells, and methods of the present disclosure, the desired compounds may be recovered from the host cell suspension or cell-free mixture and separated from other constituents, such as media constituents, cellular debris, etc. Techniques for separation and recovery of the desired compounds are known to those of skill in the art and can include, for example, solvent extraction (e.g. butane, chloroform, ethanol), column chromatography-based techniques, high-performance liquid chromatography (HPLC), for example, and/or countercurrent separation (CCS) based systems. The recovered cannabinoid compounds may be obtained in a more or less pure form, for example, the desired cannabinoid compound of purity of at least about 60% (w/v), about 70% (w/v), about 80% (w/v), about 90% (w/v), about 95% (w/v) or about 99% (w/v).
It is contemplated that the cannabinoid, cannabinoid precursor, cannabinoid precursor derivative, or cannabinoid derivative recovered using the methods of the present disclosure can be in the form of a salt. In at least one embodiment, the recovered salt of the cannabinoid, cannabinoid precursor, cannabinoid precursor derivative, or cannabinoid derivative is a pharmaceutically acceptable salt. Such pharmaceutically acceptable salts retain the biological effectiveness and properties of the free base compound,
As described elsewhere herein, the rare or synthetic derivatives of cannabinoid compounds that can be produced by the recombinant host cells and methods of the present disclosure are contemplated to exhibit biological and pharmacological properties like those of the more well-studied cannabinoids such as THC and CBD. Accordingly, in at least one embodiment, the present disclosure also provides a composition comprising a rare or synthetic cannabinoid, such as a varin cannabinoid, prepared using the recombinant host cells and methods disclosed herein. It is contemplated that the rare cannabinoid compositions provided by the recombinant host cells and methods of the present disclosure can include pharmaceutical compositions, food compositions, and beverage compositions, containing a rare cannabinoid. Generally, compositions comprising rare cannabinoid compounds can further comprise any of the well-known vehicles, excipients and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, used in the art of formulating pharmaceutical, food, or beverage compositions. For example, pharmaceutical compositions can contain any of the typical pharmaceutically acceptable excipients including, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, benzoates, and the like. In at least one embodiment, a pharmaceutical composition can comprise a pharmaceutically acceptable excipient that serves as a stabilizer of the rare cannabinoid composition. Examples of suitable excipients that also act as stabilizers include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, sorbitol, inositol, dextran, and the like. Other suitable pharmaceutical excipients can include, without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, glycine, polyethylene glycols (PEGs), and combinations thereof.
Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.
This example illustrates the generation of a cDNA library from mRNA extracted from isolated trichomes of Cannabis sativa flowers, transformation of the cDNA library into a strain of Saccharomyces cerevisiae already comprising an integrated cannabinoid biosynthesis pathway of genes encoding the enzymes AAE (SEQ ID NO: 2), OLS (SEQ ID NO: 4), OAC (SEQ ID NO: 6), and PT4 (SEQ ID NO: 10) capable of synthesizing cannabigerolic acid (CBGA), and screening the transformants for CBGA production. This cDNA library provides a useful tool for identifying genes and variations (alleles) of the cannabinoid biosynthesis genes that can be used to recreate the biosynthetic pathway in recombinant microorganisms such as yeast.
Materials and Methods
A. cDNA library creation: Total mRNA was extracted from isolated Cannabis sativa trichomes derived from flower tissues, and subsequently reverse-transcribed to cDNA using standard molecular biology techniques. Upon second strand cDNA synthesis and adapter ligation, the library was cloned into a plasmid cloning vector (pDONR-222) and subsequently sub-cloned into a yeast expression vector with auxotrophic marker (pAG425; LEU2 gene), as shown
B. cDNA library verification: In order to ascertain the quality (gene diversity and integrity) of the cDNA library that was cloned, we performed PacBio Single Molecule, Real-Time (SMRT) Sequencing of the pAG425 cDNA library. After trimming away vector sequences and sequence artifacts, a total of 142,367 cDNA sequences were identified in this library using the PacBio sequencing approach. A total of 129,263 cDNAs (90.8% of total) of these sequences were successfully mapped back to a cannabis genome assembly from which the trichomes and resulting cDNAs were isolated. This demonstrates that there is a large number of cannabis genes in the library. RNAseq analysis has identified hundreds of genes that are specifically expressed in the glandular trichomes where cannabinoid biosynthesis takes place. Moreover, the known cannabinoid pathway biosynthesis genes, AAE, OLS, OAC, PT4, were also found in the trichome specific RNAseq dataset, providing additional positive evidence that the unknown genes in the cDNA library contribute to promoting cannabinoid biosynthesis in cannabis.
In order confirm that the cDNA library was enriched in genes specifically expressed in cannabis glandular trichomes we performed conditional reciprocal best blast hit analysis between PacBio cDNA library and the trichome specific transcriptome assemblies that we had previously generated. A total of 105,871 cDNA sequences representing 74.4% of the total cDNA reads were successfully matched to a transcript in the Cannabis trichome specific RNAseq assembly. This data demonstrates that the cDNA library that was screened to identify nucleic acid sequences that aid a yeast host biosynthesize cannabinoids as described in the present disclosure, is derived from cDNAs that are expressed in the glandular trichomes of Cannabis sativa.
Finally, an EviGene gene prediction and analysis approach was used to evaluate the ‘completeness’ of the sequenced cDNA transcripts (see e.g., EvidentialGene described at web-site: arthropods.eugenes.org/EvidentialGene/plants/). A total of 99918, 37624, 4336, and 599 sequences were binned into the “complete” (complete reading frame, from amino acid conversion); “partial5” (partial reading frames including 5prime utr); “partial3” (partial reading frames including 3prime utr); and “partial” (partial reading frames), respectively. This analysis shows that the vast majority (70.2%) of sequences that were cloned are predicted to be full length protein coding sequences.
C. Express cDNA library in yeast: The pAG425 based cDNA library was transformed into a Saccharomyces cerevisiae strain that included a cannabinoid pathway for biosynthesis of CBGA from hexanoic acid (HA). The pathway included genes derived from C. sativa encoding the following enzymes expressed under constitutive promoters: AAE, OLS, OAC, and PT4. A constitutive promoter (GAP promoter) in the yeast expression vector (pAG425) was used to express the cDNA library.
D. Screen cDNA library for cannabinoid biosynthesis: Transformed yeast strains were plated on selective SC-LEU plates and 11,789 single colonies were using Molecular Devices Q-Pix 420 into 96-well mid well plates with 300 μL of SC-LEU+glucose media and incubated for 48-hr. Each plate is also inoculated with media blank, empty vector (negative) and parent strain (positive) controls. These pre-culture plates are then sub-cultured (1-10× dilution) into fresh SC-Leu +glucose media with 0.2 mM hexanoic acid and grown for another 48 hr at 30° C. for CBGA production. The whole cell broth production samples are extracted by adding equal volumes of acetonitrile and shaking for 30 min at 250 rpm. The plates are centrifuged and the supernatant is appropriately diluted for analysis by RapidFire 365 HTP-Mass Spectrometry instrument by Agilent. The CBGA titers in 96-well plates are quantified based on the calibration curve generated with pure standards prior to running the samples. The selection criteria for the top hits is 2x or higher fold improvement over the positive control.
Results
From a total of 11,789 independent yeast transformants a total of 619 yeast strains were identified as having a >2× fold improvement in CBGA titer. Furthermore, a total of 263 yeast strains were selected from these 619 strain hits and were sequenced to identify the causative cDNA that resulted in the increased CBGA titer. As summarized in Table 2, a total of 263 cDNAs capable of at least 2-fold improved CBGA titer production were identified and categorized based on plant protein function BIN Description and annotated.
Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following clauses, which may be beneficial alone or in combination, with one or more other causes or embodiments. Without limiting the foregoing description, certain non-limiting clauses of the disclosure numbered as below are provided, wherein each of the individually numbered clauses may be used or combined with any of the preceding or following clauses. Thus, this is intended to provide support for all such combinations and is not necessarily limited to specific combinations explicitly provided below:
While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.
Additional embodiments of the invention are set forth in the following claims.
The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.
This application is a continuation of International Appl. No. PCT/US2021/024390, filed Mar. 26, 2021, which claims priority benefit to U.S. Provisional Appl. No. 63/001,202, filed Mar. 27, 2020, each of which is hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63001202 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US21/24390 | Mar 2021 | US |
| Child | 17935491 | US |
