WO2022204007A2

WO2022204007A2 - Recombinant polypeptides for enhanced biosynthesis of cannabinoids

Info

Publication number: WO2022204007A2
Application number: PCT/US2022/021113
Authority: WO
Inventors: Amy LUM; Xueyang FENG; Matthew WORKENTINE
Original assignee: Willow Biosciences, Inc.; Epimeron Usa, Inc.
Priority date: 2021-03-22
Filing date: 2022-03-21
Publication date: 2022-09-29
Also published as: WO2022204007A3

Abstract

The present disclosure provides recombinant polypeptides having cannabinoid synthase activity, such as CBDA synthase, THCA synthase, and/or CBCA synthase activity, and N-terminal secretion peptides that can be used with cannabinoid synthase polypeptides in recombinant host cells for enhanced biosynthesis of cannabinoids, such as CBDA, THCA, and CBCA.

Description

RECOMBINANT POLYPEPTIDES FOR ENHANCED BIOSYNTHESIS OF CANNABINOIDS FIELD [0001] The present disclosure relates generally to recombinant polypeptides having cannabinoid synthase activity, and associated N-terminal secretion peptides. The recombinant polypeptides and secretion peptides are useful incorporated into a cannabinoid biosynthesis pathway of a recombinant host cell, and can enhance the ability of the cell to produce cannabinoids, such as CBDA, THCA, and CBCA. REFERENCE TO SEQUENCE LISTING [0002] The official copy of the Sequence Listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “13421- 011WO1_SeqList_ST25.txt”, a creation date of March 18, 2022, and a size of 408,156 bytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein. BACKGROUND [0003] Cannabinoids are a class of compounds that act on endocannabinoid receptors and include the phytocannabinoids naturally produced by Cannabis sativa. Cannabinoids include the more prevalent and well-known compounds, Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), as well as 80 or more less prevalent cannabinoids, cannabinoid precursors, related metabolites, and synthetically produced derivative 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. [0004] 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). These biosynthetic systems are production of the downstream cannabinoid compounds, CBDA and THCA. [0005] There exists a need for improved recombinant polypeptides, recombinant host cells, and methods for their use in the biosynthetic production of cannabinoid compounds, such as CBDA, THCA, and CBCA. SUMMARY [0006] The present disclosure provides recombinant cannabinoid synthase polypeptides and recombinant secretion peptides, that can be used with the cannabinoid synthase polypeptides, and that are useful for enhanced in vitro cannabinoid biosynthesis systems and/or that can be incorporated into a cannabinoid biosynthesis pathway in a recombinant host cell to enhance the in vivo biosynthesis of cannabinoids. [0007] 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. [0008] In at least one embodiment, the present disclosure provides a recombinant host cell which produces Δ⁹-tetrahydrocannabinolic acid (THCA) when cultured in the presence of hexanoic acid (HA) or olivetolic acid (OA), wherein the cell comprises a pathway of enzymes capable of producing cannabigerolic acid (CBGA) and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having THCAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 90, 92, 72, and 84. [0009] In at least one embodiment, the recombinant host cell which produces Δ⁹- tetrahydrocannabinolic acid (THCA): (a) the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Escherichia coli; (b) the polypeptide having THCAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18; (c) the N-terminal secretion peptide linked to the polypeptide having THCAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 124, 126, 128, 130, 132, 134, and 136; (d) the amount of THCA produced by the cell when cultured in the presence of HA or OA is increased relative to a control host cell comprising a pathway of enzymes AAE, TKS, OAC, and PT4, and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having THCAS activity comprising an amino acid sequence of SEQ ID NO: 122; and/or (e) the cell also produces cannabidiolic acid (CBDA), and/or cannabichromenic acid (CBCA). [0010] In at least one embodiment, the present disclosure provides a recombinant host cell which produces cannabidiolic acid (CBDA) when cultured in the presence of hexanoic acid (HA) or olivetolic acid (OA), wherein the cell comprises a pathway of enzymes capable of producing to a polypeptide having CBDAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 70, and 72. [0011] In at least one embodiment, the recombinant host cell which produces cannabidiolic acid (CBDA): (a) the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Escherichia coli; (b) the polypeptide having CBDAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 16 or SEQ ID NO: 20; (c) the N-terminal secretion peptide linked to the polypeptide having CBDAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 94, 96, 98, 108, 110, 112, 114, and 46; (d) the amount of CBDA produced by the cell when cultured in the presence of HA or OA is increased relative to a control host cell comprising a pathway of enzymes AAE, TKS, OAC, and PT4, and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having CBDAS activity comprising an amino acid sequence of SEQ ID NO: 44; and/or (e) the cell also produces Δ⁹- tetrahydrocannabinolic acid (THCA), and/or cannabichromenic acid (CBCA). [0012] In at least one embodiment of the recombinant host cells of the present disclosure: (a) the pathway of enzymes is capable of catalyzing reactions (i) – (iv): (i) O O H₃ , H₃ ,

and (iv) COOH 3 ;

, , , T4; and/or (c) the pathway of enzymes comprises (i) AAE having an amino acid sequence of at least 90% identity to SEQ ID NO: 2; (ii) TKS having an amino acid sequence of at least 90% identity to SEQ ID NO: 4; (iii) OAC having an amino acid sequence of at least 90% identity to SEQ ID NO: 6; and (iv) PT4 having an amino acid sequence of at least 90% identity to SEQ ID NO: 8 or 10. [0013] In at least one embodiment the present disclosure also provides a method for producing a cannabinoid selected from cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), and/or cannabichromenic acid (CBCA), wherein the method comprises: (a) culturing in a suitable medium comprising hexanoic acid (HA) or olivetolic acid (OA) and a recombinant host cell of the present disclosure that produces THCA and/or CBDA; and (b) recovering the produced cannabinoid. [0014] In at least one embodiment the present disclosure also provides a recombinant polypeptide having cannabinoid synthase activity, wherein the polypeptide comprises an amino acid sequence of at least 85% identity to SEQ ID NO: 14 and comprises at least one amino acid residue difference relative to SEQ ID NO: 14 selected from: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, P164A, H186D, L203I, A208G, S210N, V215A, R220K, M229I, I235N, E240G, V245F, K447Q, and R516H. In at least one embodiment, the amino acid sequence has at least 90% identity to SEQ ID NO: 14 and the following amino acid residue differences relative to SEQ ID NO: 14: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, P164A, H186D, L203I, A208G, S210N, V215A, R220K, M229I, I235N, E240G, V245F, K447Q, and R516H. In at least one embodiment, the amino acid sequence has at least 90% identity to SEQ ID NO: 20. In at least one embodiment, the polypeptide has cannabinoid synthase activity capable of converting cannabigerolic acid (CBGA) to cannabidiolic acid (CBDA), and/or Δ⁹-tetrahydrocannabinolic acid (THCA). In at least one embodiment, the polypeptide further comprises N-terminal secretion peptide comprising an amino acid sequence selected from SEQ ID NO: 86, 88, 90, 92, 70, 72, 78, and 84. polypeptide comprising an N-terminal secretion peptide linked to a polypeptide having CBDAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 86, 88, 90, 92, 72, 78, and 84. In at least one embodiment, the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 94, 96, 98, 108, 110, 112, and 114. In at least one embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 20. In at least one embodiment, the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 108, 110, 112, and 114. [0016] In at least one embodiment the present disclosure also provides a recombinant polypeptide comprising an N-terminal secretion peptide linked to a polypeptide having THCAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 90, 92, 72, and 84. In at least one embodiment, the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 124, 126, 128, 130, 132, 134, and 136. In at least one embodiment, the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18. [0017] In at least one embodiment, the present disclosure also provides recombinant polynucleotides that encode the recombinant polypeptides of the present disclosure. In at least one embodiment, the disclosure also provides expression vectors and host cells comprising any of the polynucleotides of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS [0018] 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: [0019] FIG.1 depicts an exemplary four enzyme pathway capable of converting hexanoic acid (HA) to the cannabinoid precursor, olivetolic acid (OA), and then further converting OA to the cannabinoid, cannabigerolic acid (CBGA). The four enzymes catalyzing the steps in the biosynthetic pathway, AAE, TKS, OAC, PT4, are indicated. [0020] FIG.2 depicts three exemplary two step pathways for converting the cannabinoid, CBGA, to one or more of the cannabinoids, THCA, CBDA, and/or CBCA, and then, optionally, further converting them to the decarboxylated cannabinoids, Δ⁹-THC, CBD, and/or CBC. The first conversion from CBGA to THCA, CBDA, and/or CBCA can be catalyzed by a single herein, in some embodiments the single cannabinoid synthase (e.g., CBDAs) is capable of catalyzing not only the conversion of CBGA to its preferred product (e.g., CBDAs preferentially converts CBGA to CBDA), but also converts CBGA to one or both of the other cannabinoid acid products, typically in lesser amounts. [0021] FIG.3 depicts an exemplary pathway capable of converting butyric acid (BA) to the cannabinoid precursory, divarinic acid (DA), and further converting DA to the rare cannabinoid, cannabigerovarinic acid (CBGVA). [0022] FIG.4 depicts three exemplary two step pathways for converting the rare cannabinoid, CBGVA, to one or more of the rare cannabinoids, Δ⁹-THCVA, CBDVA, and/or CBCVA, and then, optionally, further converting them to the decarboxylated cannabinoids, Δ⁹-THCV, CBDV, and/or CBCV. The first conversion from CBGVA to Δ⁹-THCVA, CBDVA, and/or CBCVA can be catalyzed by a single cannabinoid synthase, CBDAs, THCAs and/or CBCAs, respectively. As described elsewhere herein, in some embodiments the single cannabinoid synthase (e.g., CBDAs) is capable of catalyzing not only the conversion of CBGVA to its preferred product (e.g., CBDAs preferentially converts CBGVA to CBDVA), but also converts CBGVA to one or both of the other cannabinoid acid products, typically in lesser amounts. [0023] FIG.5 depicts a BLAST alignment of the polypeptide sequences of the d28_CBDAS of SEQ ID NO: 14 from C. sativa with the variant homolog, CBDAS_Var95 of SEQ ID NO: 20. [0024] FIG.6 depicts a BLAST alignment of the polypeptide sequences of the d28_THCAS of SEQ ID NO: 18 from C. sativa with the variant homolog, CBDAS_Var95 of SEQ ID NO: 20. [0025] FIG.7 depicts an exemplary plasmid wbPlasmid089 used as template to generate linear DNA by PCR to transform yeast strains with yeast-optimized CBDAS or THCAS genes via homologous recombination. The yeast strains were already engineered with a pathway of the enzymes capable of converting either olivetolic acid (OA) or hexanoic acid (HA) to the cannabinoid CBGA. The newly recombined yeast strains were tested for the presence of the CBDAS or THCAS gene using PCR and sequencing and then screened for the ability to convert OA or HA to CBDA, THCA, and/or CBCA as described in Examples 1 and 2. DETAILED DESCRIPTION [0026] 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 “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.” [0027] 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. [0028] 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”). [0029] 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. [0030] 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. [0031] Definitions 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). [0033] “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. [0034] “Pathway capable of producing a cannabinoid” refers to a pathway that can convert an initial substrate molecule, such as hexanoic acid, into a final product molecule that is a cannabinoid, such as cannabigerolic acid (CBGA). For example, the four enzymes AAE, TKS, OAC, and PT4 which convert hexanoic acid to CBGA, form a pathway capable of producing a cannabinoid. [0035] “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. [0036] “Substrate” as used herein in the context of an enzyme mediated process refers to the compound or molecule acted on by the enzyme. [0037] “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. [0038] “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. [0039] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to 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 nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. [0040] “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. [0041] “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. [0042] “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. [0043] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, 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 nucleic sequence found in an organism. [0044] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein. [0045] “Heterologous nucleic acid” 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. [0046] “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 embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression. [0047] “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). [0048] “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. [0049] “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 regulates the expression of the sequence of interest. [0050] “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. [0051] “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 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. [0052] “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. 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. [0054] “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. [0055] “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). [0056] “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. [0057] “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. [0058] Recombinant Cannabinoid Biosynthesis [0059] The present disclosure provides recombinant nucleic acid constructs and recombinant polypeptides, including enzymes and secretion peptides, that can be used for enhanced in vivo cannabinoid biosynthesis in recombinant host cells, as well as enhance in vitro cannabinoid biosynthesis systems. In the context of recombinant host cells, the recombinant nucleic acids and recombinant polypeptides can be incorporated into a cannabinoid pathway to enhance the capable of converting hexanoic acid (HA) to the cannabinoid, cannabigerolic acid (CBGA) is depicted in FIG.1. The biosynthetic conversion of HA to CBGA as shown in FIG.1 is carried out by the sequence of the enzymes, Acyl Activating Enzyme (AAE), Tetraketide Synthase (TKS), Olivetolic Acid Cyclase (OAC), and Aromatic Prenyltransferase (PT4). Although FIG.1 depicts a four enzyme pathway from HA to CBGA, it is contemplated that a shorter pathway comprising only the three enzyme, AAE, TKS, and OAC, could be incorporated into a host cell for the biosynthetic production of the cannabinoid precursor olivetolic acid (OA) from HA. [0060] As shown in FIG.2, an extension of the four enzyme exemplary pathway of FIG.1 with a cannabinoid synthase (e.g., CBDAS, THCAS, and/or CBCAS) allows for the biosynthetic production of one or more of the cannabinoids, THCA, CBDA, and/or CBCA. These cannabinoids are capable of further conversion by decarboxylation to provide the cannabinoids, Δ⁹-THC, CBD, and/or CBC. It is contemplated, that in some embodiments this further decarboxylation reaction can be carried out under in vitro reaction conditions using the cannabinoid acids separated and/or isolated from the recombinant host cells. [0061] As is described throughout the present disclosure, the recombinant host cells comprise a recombinant biosynthetic pathway for producing a cannabinoid. Generally, the pathway comprises a sequence of linked enzymes that convert a cannabinoid precursor substrate to a final cannabinoid compound product. As described elsewhere herein (e.g., Table 3), there are a wide range of cannabinoid products that are contemplated for such biosynthetic production in a recombinant host cell. In at least one embodiment, the recombinant pathway comprises at least an aromatic prenyl transferase (e.g., PT4), which catalyzes the final enzymatic conversion to produce the cannabinoid acid intermediate (e.g.,cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabinolic acid (CBNA), etc), that is a common intermediate in the biosynthesis of a wide range of cannabinoid products. [0062] In at least one embodiment, the recombinant pathway comprises at least enzymes capable of converting hexanoic acid to cannabigerolic acid (CBGA). One such a pathway capable of converting hexanoic acid to CBGA is illustrated in FIG.1. Accordingly, in at least one embodiment of the recombinant host cell, the pathway capable of producing a cannabinoid comprises enzymes capable of catalyzing reactions (i) – (iv): (i) O O H₃ ,

O 3 , 3 ,

and (iv) OH COOH H₃ .

[0063] As shown in FIG.1, exemplary enzymes capable of catalyzing reactions (i) – (iv) are: (i) acyl activating enzyme (AAE); (ii) tetraketide synthase (TKS); (iii) olivetolic acid cyclase (OAC); and (iv) aromatic prenyl transferase (PT4). Exemplary AAE, TKS, OAC, and PT4 enzymes include, but are not limited to, the naturally occurring enzymes from C. sativa, such as AAE of SEQ ID NO: 2, TKS of SEQ ID NO: 4, OAC of SEQ ID NO: 6, and PT4 of SEQ ID NO: 8. [0064] As shown in FIG.2, the cannabinoid compound, CBGA, that is produced by the pathway of FIG.1, can be further converted to at least three other different cannabinoid compounds, Δ⁹-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and/or cannabichromenic acid (CBCA). Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of converting hexanoic acid to CBGA and further comprising an enzyme capable of catalyzing the conversion of (v) CBGA to THCA; (vi) CBGA to CBDA; and/or (vii) CBGA to CBCA. Thus, in at least one embodiment, the recombinant host cell comprises pathway capable of converting hexanoic acid and/or (vii): (v) CH₃ CH₃ OH A₎ , . [0

) THCA synthase (THCAS); (vi) CBDA synthase (CBDAS); and (vii) CBCA synthase (CBCAS). [0066] Exemplary cannabinoid pathway enzymes that can be introduced into a recombinant host cell to provide the pathways illustrated in FIGS.1 and 2 include, but are not limited to, the cannabinoid pathway enzymes listed in Table 1 (below) with sequences provided in the accompanying Sequence Listing. [0067] TABLE 1: Exemplary cannabinoid pathway enzymes SEQ SEQ ID ID

IIRGKKRIPLYSRVVEAKSPMAIVIPCSGSNIGAELRDGDI

QSIPPLPRHRH

q p y p y y , , , 4, CBDAS, and THCAS listed in Table 1 are naturally occurring sequences from the plant source, Cannabis sativa. It also is contemplated, however, that heterologous cannabinoid pathway enzymes incorporated in a recombinant host cell of the present disclosure can include naturally occurring homologs and/or non-naturally occurring variants (e.g., engineered enzymes) having the relevant enzymatic activity of the AAE, TKS, OAC, PT4, CBDAS, THCAS, CBCAS polypeptides. For example, recombinant polypeptides with amino acid sequences engineered to function optimally in a particular enzyme pathway, and/or function optimally for production of a particular intermediate or product compound, and/or function optimally in a particular host. the art and include methods for enzyme engineering such as directed evolution. [0069] Other modifications of cannabinoid pathway enzymes contemplated by the present disclosure include modification of the enzyme’s amino acid sequence of 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: 4 (TKS), SEQ ID NO: 6 (OAC), and SEQ ID NOs: 10 (PT4). Similarly, the CBDAS enzyme of SEQ ID NO: 12 can be truncated at the N-terminus by 28 amino acids to delete the native signal peptide and thereby provide the d28_CBDAS enzyme of SEQ ID NO: 14. 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: 4 (TKS), SEQ ID NO: 6 (OAC), SEQ ID NOs: 10 (PT4), and SEQ ID NO: 14 (d28_CBDAS). [0070] It also is contemplated that functional homologs and/or engineered versions of the AAE, TKS, OAC, PT4, THCAS, 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, functional homologs of AAE that can be used in a cannabinoid pathway of a recombinant host cell of the present disclosure are disclosed in PCT application PCT/US2021/062910, entitled “Recombinant Acyl Activating Enzyme (AAE) Genes For Enhanced Biosynthesis Of Cannabinoids And Cannabinoid Precursors,” filed December 10, 2021, are hereby incorporated by reference herein. Additionally, engineered variants of TKS, OAC, PT4, CBDAS, and THCAS that can be used in a cannabinoid pathway of a recombinant host cell of the present disclosure are disclosed in: WO2020/176547A1, entitled “Biosynthesis of Cannabinoids and Cannabinoid Precursors,” published September 3, 2020; WO2020/236789A1, entitled “Optimized Cannabinoid Synthase Polypeptides,” published November 26, 2020; US Prov. Appl. No. 63/227,747, entitled “Recombinant Prenyltransferase Polypeptides Engineered For Enhanced Biosynthesis Of Cannabinoids,” filed July 30, 2021; WO2021183448A1, entitled “Optimized Olivetolic Acid Cyclase Polypeptides,” published September 19, 2021; WO2021195520A1, entitled “Biosynthesis of Cannabinoids and Cannabinoid Precursors,” published September 30, 2021; US Prov. Appl. No.63/257,523, entitled “Recombinant THCA Synthase Polypeptides Engineered For Enhanced Biosynthesis Of Cannabinoids,” filed October 19, 2021; and US Prov. Appl. No.63/320,421, entitled “Recombinant Olivetolic Acid Cyclase Polypeptides is hereby incorporated by reference herein. [0071] 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, 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. [0072] Although the cannabinoid pathways of FIGS.1-2 depict the production of the more common naturally occurring cannabinoids, CBGA, THCA, CBDA, and CBCA, it is also contemplated that the recombinant polypeptides, cannabinoid pathways, recombinant host cells, and associated methods of the present disclosure can also be used to biosynthesize a range of additional rarely occurring, and/or synthetic cannabinoid compounds. Table 2 (below) depicts the names and structures of a wide range of exemplary cannabinoid compounds that are contemplated for production using the recombinant polypeptides, host cells, compositions and methods of the present disclosure. [0073] TABLE 2: Exemplary cannabinoid compounds Abbrev. Compound Name Name Chemical Structure

acid

^{3 C} _CH3

tetrahydrocannabivarinic THCVA

[0074] In at least one embodiment, the compositions and methods of the present disclosure can be used for the production of the varin series of cannabinoids, CBGVA, Δ⁹-THCVA, CBDVA, and CBCVA. As shown in Table 2, the varin cannabinoids feature a 3 carbon propyl side-chain rather than the 5 carbon pentyl side chain found in the common cannabinoids, CBGA, THCA, CBDA, and CBCA. An exemplary cannabinoid pathway capable of producing the rare naturally occurring cannabinoid, cannabigerovarinic acid (CBGVA), is depicted in FIG. 3. Instead of starting with hexanoic acid, the pathway of FIG.3 is fed butyric acid (BA) which is converted to divarinic acid (DA) via the same three enzyme pathway of AAE, TKS, and OAC. The precursor DA is then converted by PT4 to the rare cannabinoid, CBGVA. Accordingly, in at least one embodiment of the recombinant host cell, the pathway capable of producing a cannabinoid comprises enzymes capable of catalyzing reactions (i) – (iv): (i) O O H₃ ,

OH O O O O COOH H₃ .

and (iv) OH COOH CH₃ OH COOH H₃ P^P

.

[0075] As shown in FIG.3, exemplary enzymes capable of catalyzing reactions (i) – (iv) are: (i) acyl activating enzyme (AAE); (ii) tetraketide synthase (TKS); (iii) olivetolic acid cyclase (OAC); and (iv) aromatic prenyltransferase (PT4). Exemplary enzymes, AAE, TKS, OAC, and PT4 derived from C. sativa are known in the art and also provided in Table 1 and the accompanying Sequence Listing. [0076] As further illustrated in FIG.4, the heterologous pathway depicted in FIG.3 which is capable of producing a rare cannabinoid, such as CBGVA, can be further modified to include one or more cannabinoid synthase enzymes (e.g., CBDAS, THCAS, CBCAS). As shown by the exemplary pathway of FIG.4, with the incorporation of one or more synthase enzymes, the rare varin cannabinoid, CBGVA, can be converted to the rare varin cannabinoids, cannabidivarinic acid (CBDVA), Δ⁹-tetrahydrocannabivarinic acid (Δ⁹-THCVA), and cannabichromevarinic acid (CBCVA). Enzymes capable of carrying out these conversions include the C. sativa CBDA synthase, THCA synthase, and CBCA synthase, respectively. Accordingly, in at least one embodiment, the present disclosure provides a recombinant host cell comprising a pathway capable of converting BA to CBGVA and further comprising an enzyme capable of catalyzing the conversion of (v) CBGVA to Δ⁹-THCVA; (vi) CBGVA to CBDVA; and/or (vii) CBGVA to CBCVA. Thus, in at least one embodiment, the recombinant host cell comprises pathway capable of converting BA to CBGVA further comprises further comprises enzymes capable of catalyzing a reaction (v), (vi), and/or (vii): (v) ₃ COOH H₃ THCVA)

, (vi) CH₃ H₃

, (vii) CH₃ OH COOH ^{H C} OH H₃

. [0077] Exemplary enzymes capable of catalyzing reaction (v)-(vii) as shown above are: (v) THCA synthase (THCAS); (vi) CBDA synthase (CBDAS); and (vii) CBCA synthase (CBCAS). Exemplary THCAS, CBDAS, and CBCAS enzymes are provided in Table 1. [0078] Furthermore, as shown in FIG.4, the rare cannabinoid acids, CBDVA, Δ⁹-THCVA, and CBCVA, can undergo a further decarboxylation reaction to provide the varin cannabinoid products, cannabidivarin (CBDV), Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), and cannabichromevarin (CBCV), respectively. In some embodiments, this further decarboxylation can be carried out under in vitro reaction conditions using the cannabinoid acids isolated from the recombinant host cells. [0079] As shown by the pathways depicted in FIGS.1 and 3, a heterologous cannabinoid pathway comprising the sequence of at least the three enzymes AAE, TKS, and OAC, is capable of converting a precursor substrate compound, such as hexanoic acid (HA) to the cannabinoid precursor compound, such as olivetolic acid (OA). Accordingly, it is contemplated that a heterologous cannabinoid pathway of the present disclosure can be used to produce a cannabinoid precursor compound. Such precursor compounds can be useful products, and/or can be used to prepare other derivative compounds, either synthetically or biosynthetically. Other cannabinoid precursor compounds, or derivatives that can be produced by such a three- 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 Rimoldi, J. M., “The Structure- Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation,” Perspectives in Medicinal Chemistry 2016:817-39 doi: 10.4137/PMC.S32171.) 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. [0080] Similarly, as shown in FIG.1 and 3, a heterologous cannabinoid pathway comprising the sequence of at least the four enzymes AAE, TKS, OAC, and PT4, is capable of converting a precursor substrate compound, such as hexanoic acid (HA) to an initial cannabinoid compound, such as cannabigerolic acid (CBGA) or CBGVA. These initial cannabinoid product compounds can themselves be used as a substrate for the in vitro biosynthesis of a range of further cannabinoid product compounds, such as THCA and THCVA, as shown in FIGS.2 and 4. A wide range of cannabinoid compounds, such as those shown in Table 2, are contemplated for in vivo biosynthetic production in a recombinant host cell of the present disclosure or via a partial or full in vitro biosynthesis process using recombinant polypeptides of the present disclosure. [0081] As described herein, the heterologous cannabinoid pathways of the present disclosure can be incorporated (e.g., by recombinant transformation) into a range of host cells to provide a system for biosynthetic production of cannabinoids (e.g., CBGA, CBDA, THCA, CBCA) or cannabinoid precursor compounds. Generally, the host cell used in the recombinant host cells of the present disclosure can be any cell that can be recombinantly modified with nucleic acids and 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 transformed with a 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. acids encoding a pathway of enzymes capable of producing the cannabinoid, CBGA, and a heterologous nucleic acid comprising a sequence encoding a polypeptide having CBDAS and/or THCAS activity. As described elsewhere herein, nucleic acid sequences encoding the cannabinoid pathway enzymes, 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 which form a part of a cannabinoid pathway, further include one or more additional nucleic acid sequences, for example, a nucleic acid sequence controlling expression of the proteins which form a part of a cannabinoid biosynthetic enzyme pathway, and these one or more additional nucleic acid sequences together with the nucleic acid sequence encoding a protein which form a part of an cannabinoid biosynthetic enzyme 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 heterologous nucleic acid sequences, such as nucleic acid sequences encoding the cannabinoid pathway enzymes (e.g., CBDA synthase), into a host cell so as to attain expression the host cell. Such techniques are well known to the skilled artisan and can, for example, be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. [0083] One of ordinary skill will recognize that the heterologous nucleic acids encoding the recombinant CBDA synthase enzymes and associated N-terminal secretion peptides (or other pathway enzymes) will further comprise transcriptional promoters capable of controlling expression of the enzymes in the recombinant host cell. [0084] 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 Gal1 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 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. [0085] 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. Such techniques are described in e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. [0086] 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 nucleic acid sequence encoding an AAE enzyme, and, as necessary, other enzymes constituting a cannabinoid pathway (e.g., TKS, OAC, PT4, CBDAS). This heterologous nucleic acid sequence 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. [0087] It is also contemplated that in some embodiments an expression vector comprising a heterologous nucleic acid 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). [0088] Recombinant Polypeptides with Enhanced Cannabinoid Synthase Activity [0089] The present disclosure provides recombinant host cells (e.g., S. cerevisiae) that comprise a functional pathway capable of production of a cannabinoid CBGA and further comprising a recombinant polypeptide having CBDA synthase (CBDAS) activity capable of converting CBGA to CBDA, and in some cases, also THCA synthase (THCAS) activity, capable CBGA to CBCA. [0090] In at least one embodiment, the recombinant host cell comprises a cannabinoid pathway that includes at least the enzymes AAE, OAC, TKS, and PT4, and a recombinant polypeptide having a cannabinoid synthase (CBDAS, THCAS, and/or CBCAS) activity, wherein the polypeptide is a homolog of the CBDAS of SEQ ID NO: 12 from C. sativa comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12. [0091] A library of 12 homologs of the d28_CBDAS polypeptide of SEQ ID NO: 14 was generated by bioinformatics analysis of C. sativa and related genome databases. The library of CBDAS homologs along with their nucleotide and amino acid sequences are disclosed below in Table 3, as well as in the accompanying Sequence Listing. Also disclosed in Table 3 are the sequences of the d28_CBDAS and the 12 CBDAS homologs fused with the N-terminal yeast secretion peptide, SP-Alpha (SEQ ID NO: 70), and the sequences of a library of secretion peptides and their N-terminal fusions with d28_CBDAS, the homolog, CBDAS_Var95, and the THCA synthase polypeptide d28_THCAs of SEQ ID NO: 18. [0092] TABLE 3 SEQ SEQ ID ID

DCKQLSWIDTIIFYSGVVNYNTTYFKKEILLDRSGGRKAAFSIKLDYVKKPI

PNNYTQARIWGEKYFGKNFNRLVKVKTKVDP

NEKHLNWIRNIYNFMTPYVSKNPRLAYLNYRDLDIGINDPKNPNNYTQARIW

MRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWK

WGEKYFGKNFNRLVKVKTKVDPDNFFRNEQSIPPLPLRHH

Alpha-factor full DFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA

FRNEQSIPPLPRHRH

RNEQSIPPLPRHRH

PRLAYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPN

ISESAIPFPHRAGIMYELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNP

SAIPFPHRAGIMYELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLA

y q g . . , synthase, CBDAS_Var95 (SEQ ID NO: 20) has an amino acid sequence that has 95% sequence identity to CBDAS synthase d28_CBDAS (SEQ ID NO: 14) of C. sativa, and 89% sequence identity to the THCAS synthase d28_THCAS (SEQ ID NO: 18) of C. sativa. As described elsewhere herein, the CBDAS_Var95 (SEQ ID NO: 20) enzyme when engineered in a cannabinoid pathway is capable of producing CBDA, THCA, and CBCA (see Example 1). CBDAS_Var95 represents a surprising polypeptide having cannabinoid synthase activity and a chimeric amino acid sequence structure not previously known in the art. [0094] Accordingly, in at least one embodiment, the present disclosure also provides a recombinant polypeptide having CBDA synthase activity, wherein the polypeptide comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the naturally occurring d28_CBDAS of SEQ ID NO: 14 and also comprises at least one of the amino acid residue differences relative to SEQ ID NO: 14 found in the sequence of SEQ ID NO: 20 (CBDAS_Var95). This includes any one or more the following amino acid differences: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, P164A, H186D, L203I, A208G, S210N, V215A, R220K, M229I, I235N, E240G, V245F, K447Q, and R516H. In at least one embodiment, the polypeptide can comprise an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 14 and all of the amino acid residue differences relative to SEQ ID NO: 14 found in the sequence of SEQ ID NO: 20. [0095] In at least one embodiment, the present provides a recombinant polypeptide having CBDA synthase activity wherein the polypeptide comprises an amino acid sequence of at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20, wherein the amino acid sequence includes one or more amino acid differences relative to SEQ ID NO: 14 selected from: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, and R516H. [0096] As noted above, it is surprising feature of CBDAS_Var95 of SEQ ID NO: 20 that it has THCAS activity and converts CBGA to THCA despite having only 89% amino acid sequence identity to the C. sativa THCAS of SEQ ID NO: 18 and the following 59 amino acid differences relative to SEQ ID NO: 18: K13Q, H14Y, V19A, A20T, P22L, H29N, D30N, Q31P, I36V, Q42H, I47S, N62H, N63V, A68G, V261L, K269R, H275Q, T281A, V282I, G284T, I289V, H291L, K316R, E317Q, F318L, T324I, F333Y, N334D, A336D, K339N, K350Q, K351N, K352G, S355K, T368S, A369V, M370F, K372Q, V382I, V388A, E397D, M413L, T419I, A420C, I432L, V435I, S437N, V438I, T442M, L462I, K464I, T465N, N466D, H467P, A468K, S469N, N489D, K497L, P515R. Accordingly, in at least one embodiment, the present disclosure also provides a recombinant polypeptide with THCA synthase activity, wherein the polypeptide comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the naturally occurring d28_THCAS of SEQ ID NO: 18 and also comprises at least one of the amino acid residue differences relative to SEQ ID NO: 18 found in the sequence of SEQ ID NO: 20 (CBDAS_Var95). This includes any one or more the following amino acid differences: K13Q, H14Y, V19A, A20T, P22L, H29N, D30N, Q31P, I36V, Q42H, I47S, N62H, N63V, A68G, V261L, K269R, H275Q, T281A, V282I, G284T, I289V, H291L, K316R, E317Q, F318L, T324I, F333Y, N334D, A336D, K339N, K350Q, K351N, K352G, S355K, T368S, A369V, M370F, K372Q, V382I, V388A, E397D, M413L, T419I, A420C, I432L, V435I, S437N, V438I, T442M, L462I, K464I, T465N, N466D, H467P, A468K, S469N, N489D, K497L, P515R. In at least one embodiment, the polypeptide can comprise an amino acid sequence of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 18 and all of the amino acid residue differences relative to SEQ ID NO: 18 found in the sequence of SEQ ID NO: 20. [0097] In at least one embodiment, the present disclosure provides a recombinant polypeptide having THCA synthase activity wherein the polypeptide comprises an amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20, wherein the amino acid sequence includes one or more amino acid differences relative to SEQ ID NO: 18 selected from: K13Q, H14Y, V19A, A20T, P22L, H29N, D30N, Q31P, I36V, Q42H, I47S, N62H, N63V, A68G, V261L, K269R, H275Q, T281A, V282I, G284T, I289V, H291L, K316R, E317Q, F318L, T324I, F333Y, N334D, A336D, K339N, K350Q, K351N, K352G, S355K, T368S, A369V, M370F, K372Q, V382I, V388A, E397D, M413L, T419I, A420C, I432L, V435I, S437N, V438I, T442M, L462I, K464I, T465N, N466D, H467P, A468K, S469N, N489D, K497L, P515R. [0098] As described elsewhere herein, the recombinant CBDAS synthase polypeptides of even-numbered SEQ ID NOs: 20-42 were modified with an N-terminal linked yeast secretion engineered yeast host cells that produce CBGA, to further convert the CBGA to one or more of the cannabinoid compounds, CBDA, CBCA, or THCA. Recombinant polynucleotide constructs encoding the SP-Alpha-CBDAS homolog polypeptides (even-numbered SEQ ID NOs: 46-68) were synthesized and cloned under control of the Gal1 promoter. The 12 recombinant CBDA synthase homolog constructs (odd-numbered SEQ ID NOs: 45-67) were integrated into the genome of a Saccharomyces cerevisiae strain already comprising a cannabinoid pathway capable of biosynthesizing CBGA. As described in elsewhere herein (e.g., Example 1) the library variants were cultured in rich media and screened using LC/MS for cannabinoid synthase activity capable of producing, CBDA, THCA, and/or CBCA. [0099] Pooled screening of the transformed yeast strains followed by sequencing identified at least 3 of the 12 putative CBDAS homologs modified with the yeast secretion peptide SP- Alpha were capable of producing CBDA, THCA, and/or CBCA from either HA or OA feedstock. Moreover, the yeast strains capable of producing CBGA from HA or OA when transformed with the variant CBDAS homolog “CBDAS_Var95” (polypeptide of SEQ ID NO: 20 or SEQ ID NO: 46 with SP-Alpha) were able to produce all three cannabinoids, CBDA, THCA, and CBCA. This indicates that CBDAS_Var95 exhibits CBDAS activity in converting CBGA to CBDA, as well as THCAS activity, and CBCAS activity. Additionally, two other of the 12 putative CBDAS homologs, “Cs10_4” and “CBDAS2” (SEQ ID NO: 22 and 24) when recombinantly transformed in the yeast strains capable of producing CBGA from OL were found to exhibit production of the cannabinoid, CBCA, but not CBDA or THCA. [00100] Based on what is known regarding the relatively high level of production of the cannabinoids, CBDA or THCA, in the trichome cells of the C. sativa plant, it is believed that production of these cannabinoids in a recombinant host cell (e.g., yeast) may depend on proper secretion of the CBDA synthase or THCA synthase enzyme that converts CBGA to CBDA and CBGA to THCA, respectively. In order to further explore enhanced in vivo CBDA and THCA production from recombinantly transformed yeast, a range of secretion peptide fusions of the CBDA synthase, “d28_CBDAS” (SEQ ID NO: 14), the CBDAS homolog “CBDAS_Var95” (SEQ ID NO: 20), and the THCA synthase, “d28_THCAS” (SEQ ID NO: 18), were constructed and screened as described in the Examples. It is a surprising technical effect of the present disclosure that the secretion peptides SP_AA (SEQ ID NO: 72), SP_AT (SEQ ID NO: 78), SP_IV (SEQ ID NO: 86), and SP_KP (SEQ ID NO: 88) when combined as N-terminal fusions with these CBDAS and THCAS polypeptides and integrated into yeast strains capable of producing CBGA resulted in enhanced production of CBDA or THCA. Additionally, the secretion peptides SP_IN (SEQ ID NO: 84), SP_LZ (SEQ ID NO: 90), or SP_SA (SEQ ID NO: 92) when combined as N-terminal fusions with d28_THCAS polypeptide and integrated into a yeast strain capable of producing CBGA resulted in enhanced production of THCA. THCA by a recombinant host cell with the integration of secretion peptide constructs of the cannabinoid synthases of CBDAS of SEQ ID NO: 14 or 20, or a THCAS of SEQ ID NO: 18 into a heterologous cannabinoid pathway, provides a distinct and unexpected advantage of these recombinant host cells for use in the production of the cannabinoids, CBDA and/or THCA. [0101] In at least one embodiment, a recombinant host cell capable of producing CBGA and further comprising a recombinant SP-CBDAS construct, wherein the SP is selected from SP_AA (SEQ ID NO: 72), SP_AT (SEQ ID NO: 78), SP_IV (SEQ ID NO: 86), and SP_KP (SEQ ID NO: 88), and is capable of producing CBDA with a titer that is increased relative to a control recombinant host cell comprising the same cannabinoid biosynthesis pathway but with an SP- CBDAS construct wherein the SP is SP-Alpha of SEQ ID NO: 70. In at least one embodiment, the titer of CBDA produced by the host cell 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 the SP-Alpha- CBDAS control recombinant host cell. [0102] In at least one embodiment, a recombinant host cell capable of producing CBGA and further comprising a recombinant SP-THCAS construct, wherein the SP is selected from SP_AA (SEQ ID NO: 72), SP_AT (SEQ ID NO: 78), SP_IV (SEQ ID NO: 86), and SP_KP (SEQ ID NO: 88), SP_IN (SEQ ID NO: 84), SP_LZ (SEQ ID NO: 90), or SP_SA (SEQ ID NO: 92), is capable of producing THCA with a titer that is increased relative to a control recombinant host cell comprising the same cannabinoid biosynthesis pathway but with an SP-THCAS construct wherein the SP is SP-Alpha of SEQ ID NO: 70. In at least one embodiment, the titer of THCA produced by the host cell 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 the SP-Alpha-THCAS control recombinant host cell. [0103] In at least one embodiment, the present disclosure also provides a recombinant host cell comprising a pathway capable of producing CBGA, wherein the pathway comprises the enzymes AAE, TKS, OAC, and PT4, and a SP-CBDAS construct integrated into the host cell that encodes an amino acid 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 NO: 44, 46, 94, 96, 98, 108, 110, 112, and 114. In at least one embodiment, the SP-CBDAS polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 44, 46, 94, 96, 98, 108, 110, 112, and 114. In at least one embodiment, the SP-CBDAS construct comprises a nucleic acid 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 NO: 43, 45, 93, 95, 97, 107, 109, 111, and 113. In at least one embodiment, the SP-CBDAS construct comprises a polynucleotide sequence of any one of SEQ ID NO: 43, 45, 93, 95, 97, 107, 109, 111, and 113. [0104] In at least one embodiment, the present disclosure also provides a recombinant host cell comprising a pathway capable of producing CBGA, wherein the pathway comprises the that encodes an amino acid 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 NO: 122, 124, 126, 128, 130, 132, 134, and 136. In at least one embodiment, the SP-THCAS polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 122, 124, 126, 128, 130, 132, 134, and 136. In at least one embodiment, the SP-THCAS construct integrated in the host cell comprises a nucleic acid 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 NO: 121, 123, 125, 127, 129, 131, 133, and 135, and 139. In at least one embodiment, the SP-THCAS construct comprises a nucleotide sequence of any one of SEQ ID NO: 121, 123, 125, 127, 129, 131, 133, and 135. [0105] In at least one embodiment, the polynucleotide sequence of the nucleic acid encoding the heterologous cannabinoid pathway in the recombinant host cell 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. [0106] In at least one embodiment, the present disclosure provides an isolated nucleic acids, wherein the nucleic acid encodes a pathway comprising the enzymes AAE, TKS, OAC, and PT4, and a recombinant CBDAS homolog or SP-CBDAS polypeptide of the present disclosure, wherein the portion of the nucleic acid encoding the CBDAS enzyme 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: 19, 21, 23, 25, 43, 45, 47, 49, 93, 95, 97, 107, 109, 111, and 113. In at least one embodiment, the nucleic acid encoding the pathway comprising the enzymes AAE, TKS, OAC, PT4, and a recombinant CBDAS homolog or SP- CBDAS polypeptide, the portion of the nucleic acid encoding the recombinant CBDAS homolog or SP-CBDAS polypeptide comprises a nucleotide sequence of any one of SEQ ID NOs: 19, 21, 23, 25, 43, 45, 47, 49, 93, 95, 97, 107, 109, 111, and 113. [0107] In at least one embodiment, the present disclosure provides a vector comprising a heterologous nucleic acid encoding a pathway comprising the enzymes AAE, TKS, OAC, PT4, and a recombinant SP-CBDAS polypeptide, wherein the portion of the nucleic acid encoding the recombinant SP-CBDAS polypeptide 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: 43, 45, 47, 49, 93, 95, 97, 107, 109, 111, and 113. [0108] In at least one embodiment, the present disclosure provides a vector comprising a heterologous nucleic acid encoding a pathway comprising the enzymes AAE, TKS, OAC, and PT4, and a recombinant CBDAS homolog or SP-CBDAS polypeptide of the present disclosure, wherein the portion of the nucleic acid encoding the recombinant CBDAS homolog or SP- CBDAS polypeptide encodes an amino acid sequence having at least 90%, at least 95%, at 22, 24, 26, 44, 46, 48, 50, 94, 96, 98, 108, 110, 112, and 114. In at least one embodiment, the vector comprises a 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. [0109] In at least one embodiment, the present disclosure provides an isolated nucleic acid, wherein the nucleic acid encodes a pathway comprising the enzymes AAE, TKS, OAC, and PT4, and a recombinant SP-THCAS polypeptide of the present disclosure, wherein the portion of the nucleic acid encoding the SP-THCAS polypeptide 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: 121, 123, 125, 127, 129, 131, 133, and 135. In at least one embodiment, the nucleic acid encoding the pathway comprising the enzymes AAE, TKS, OAC, PT4, and a recombinant SP-THCAS polypeptide, the portion of the nucleic acid encoding the recombinant SP-THCAS polypeptide comprises a nucleotide sequence of any one of SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, and 135. [0110] In at least one embodiment, the present disclosure provides a vector comprising a heterologous nucleic acid encoding a pathway comprising the enzymes AAE, TKS, OAC, PT4, and a recombinant SP-THCAS polypeptide, wherein the portion of the nucleic acid encoding the recombinant SP-THCAS polypeptide 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: 121, 123, 125, 127, 129, 131, 133, and 135. [0111] In at least one embodiment, the present disclosure provides a vector comprising a heterologous nucleic acid encoding a pathway comprising the enzymes AAE, TKS, OAC, and PT4, and a recombinant SP-THCAS polypeptide of the present disclosure, wherein the portion of the nucleic acid encoding the recombinant SP-THCAS polypeptide encodes an amino acid 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: 122, 124, 126, 128, 130, 132, 134, and 136. In at least one embodiment, the vector comprises a 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. [0112] In at least one embodiment, the nucleic acids and vectors encoding pathway capable of producing a CBDA, THCA, and/or CBCA of the present disclosure comprise the enzymes AAE, TKS, OAC, and PT4, wherein the enzymes AAE, TKS, OAC, PT4, have the amino acid sequences of SEQ ID NO: 2 (AAE), SEQ ID NO: 4 (TKS), SEQ ID NO: 6 (OAC), and SEQ ID NO: 10 (PT4). In at least one embodiment, the nucleotide sequences encoding the pathway of 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. [0113] In some embodiments, the amino acid sequences of the AAE, TKS, OAC, PT4, CBDAS, and THCAS polypeptides of even-numbered SEQ ID NOs: 2-42 and the SP-CBDAS and SP- THCAS recombinant secretion peptide fusion polypeptides of even-numbered SEQ ID NOs: 44- 68 and 94-136 provided in the present disclosure include an initiating methionine (M) residue at position 1. It will be understood by the skilled artisan, however, 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 in any embodiment of the present disclosure comprising an amino acid sequence of an AAE, TKS, OAC, PT4, and/or CBDAS enzyme can comprise an amino acid sequence of SEQ ID NO: 2-42, 44-68, or 94-136, wherein the methionine residue at position 1 is deleted. It is also understood that nucleotides encoding the initiating methionine (M) residue at position 1 of any of the CBDAS or THCAS enzyme sequences should not be included in preparing a nucleic acid construct of SP-CBDAS or SP-THCAS for integration into a cannabinoid pathway of a recombinant host cell. [0114] Generally, the host cell source used in the recombinant host cell of the present disclosure can be any cell that can be recombinantly modified with nucleic acids and 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 transformed with a 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. [0115] The recombinant hosts of the present disclosure comprise heterologous nucleic acids encoding a cannabinoid pathway capable of producing CBDA, THCA, and/or CBCA, wherein the heterologous nucleic acids comprise sequences encoding the recombinant SP-CBDAS or acid sequences encoding SP-CBDAS, or SP-THCAS, and the other cannabinoid pathway enzymes, 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 which form a part of a cannabinoid pathway, further include one or more additional nucleic acid sequences, for example, a nucleic acid sequence controlling expression of the proteins which form a part of a cannabinoid biosynthetic enzyme pathway, and these one or more additional nucleic acid sequences together with the nucleic acid sequence encoding a protein which form a part of an cannabinoid biosynthetic enzyme 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 heterologous nucleic acid sequences, such as nucleic acid sequences encoding the SP-CBDAS or SP-THCAS, into a host cell so as to attain expression of the corresponding enzymes in a cannabinoid pathway. Such techniques are well known to the skilled artisan and can, for example, be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. [0116] One of ordinary skill will recognize that the heterologous nucleic acids encoding the the SP-CBDAS or SP-THCAS polypeptides(and other pathway enzymes) will 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 GAL1 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 disclosure. [0117] 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. Such techniques are described in e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. [0118] 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 nucleic acid sequence encoding an SP-CBDAS or SP- THCAS polypeptide, and, as necessary, other enzymes constituting a cannabinoid pathway (e.g., AAE, TKS, OAC, PT4). This heterologous nucleic acid sequence 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. [0119] It is also contemplated that in some embodiments an expression vector comprising a heterologous nucleic acid 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). [0120] Use of Recombinant Host Cells in Preparation of Cannabinoids [0121] As described elsewhere herein, the present disclosure provides recombinant host cells capable of producing CBDA, THCA, and/or CBCA, wherein the host cell comprises a pathway of at least the enzymes AAE, TKS, OAC, and PT4, which produce the cannabinoid, CBGA, and a recombinant SP-CBDAS or SP-THCAS capable of converting the CBGA to CBDA, THCA, and/or CBCA. In at least one embodiment of such a recombinant host cell, the recombinant SP-CBDAS or SP-THCAS polypeptide comprises an amino acid 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: 44, 46, 94, 96, 98, 108, 110, 112, 114, 116, 118, 126, 128, 130, 132, 134, and with a titer that is increased relative to a control recombinant host cell comprising the same pathway but with the SP-CBDAS of SEQ ID NO: 44, or the SP-THCAS of SEQ ID NO: 122. Accordingly, the recombinant host cell of the present disclosure can be used for improved biosynthetic production of CBDA or THCA. It is also contemplated that such a recombinant host cell can be used for the enhanced production of other rare cannabinoid compounds that are structural analogs to CBDA or THCA, including, but not limited to, the exemplary rare CBDA and THCA analog compounds provided in Table 2. [0122] In at least one embodiment, the present disclosure provides a method for producing a rare CBDA analog or a rare THCA analog comprising: (a) culturing in a suitable medium a recombinant host cell of the present disclosure; and (b) recovering the produced rare CBDA analog or THCA analog. [0123] In at least one embodiment, a recombinant host cell of the present disclosure can be used to produce a rare CBDA analog or THCA analog that is a varin series cannabinoid selected from cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), Δ⁹- tetrahydrocannabivarinic acid (THCVA), Δ⁹-tetrahydrocannabivarin (THCV), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), and any combination thereof. However, it is also contemplated that the recombinant host cells of the present disclosure can be used to produce other CBDA analog or THCA analog compounds of Table 2 that do not include a varin group, including any of the cannabinoids selected from Δ⁸-tetrahydrocannabinolic acid (Δ⁸-THCA), Δ⁸- tetrahydrocannabinol (Δ⁸-THC), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabinolic acid (CBNA), cannabinol (CBN), 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), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabielsoinic acid (CBEA), cannabielsoin (CBE), cannabicitranic acid (CBTA), cannabicitran (CBT), and any combination thereof. [0124] It is also contemplated that the method for producing CBDA of the present disclosure can further comprise contacting a cell-free extract of the culture containing the produced cannabinoid, CBGA or CBGVA, with CBDAS homolog polypeptide of the present disclosure, such as CBDAS_Var95 (SEQ ID NO: 20), as a biocatalytic reagent. In such an embodiment of the method, the biocatalytic reagent used is an isolated CBDAS enzyme capable of converting the produced cannabinoid, CBGA or CBGVA, to a different cannabinoid compound, CBDA or CBDVA. [0125] In another embodiment of such a method using a cell-free extract, a chemical reagent is contacted with the culture, wherein the reagent is capable of chemically modifying the produced cannabinoid, e.g., CBDA, can be used to produce a derivative compound of the cannabinoid, comprising a nucleic acid encoding a recombinant SP-CBDAS polypeptide of the present disclosure, which provide improved CBDA production in terms of titer, yield, and production rate can be used in the production of a downstream CBDA analog or derivative (e.g., CBD or other compounds of Table 2) by contacting the cell-free extract of the culture medium containing CBDA with a chemical reagent. Such derivative compounds of CBDA can include a wide range of naturally-occurring and non-naturally occurring compounds. [0126] For example, cannabinoid derivative compounds produced using the recombinant host cells and methods of the present disclosure can include any compound structurally related to a cannabinoid compound (e.g., compounds of Table 2) 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. [0127] Alternatively, cannabinoid derivative compounds using the recombinant host cells and methods of the present disclosure can include one or more additional chemical moieties that are 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. [0128] Accordingly, in at least one embodiment, the present disclosure provides a method of producing a derivative compound of a cannabinoid precursor or cannabinoid, wherein the method comprises: (a) culturing in a suitable medium a recombinant host cell of the present disclosure; and (b) recovering the produced derivative compound. In at least one embodiment, the method of producing a derivative compound of a cannabinoid precursor or cannabinoid can further contacting a cell-free extract of the culture of the recombinant host cell containing the produced cannabinoid precursor or cannabinoid with a biocatalytic reagent or chemical reagent capable of converting the cannabinoid precursor or cannabinoid to a derivative compound. [0129] Derivative compounds of cannabinoid precursor and cannabinoid compounds that can be produced with improved yield using a recombinant host cell of the present disclosure can 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 derivative compounds of cannabinoids produced by the methods disclosed herein can include pharmaceutically acceptable isotopically labeled compounds. For example, a cannabinoid compound wherein the hydrogen atoms are replaced or substituted by one or more deuterium or tritium atoms. Such isotopically labeled derivatives of cannabinoids can be useful in studies of in vivo pharmacokinetics and tissue distribution. [0130] Upon production by the host cells or in the cell-free mixture of the rare cannabinoid precursors or rare cannabinoid compounds in accordance with the compositions, 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 rare cannabinoid compounds may be obtained in a more or less pure form, for example, the desired rare 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). [0131] It also 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. [0132] As described elsewhere herein, the rare cannabinoid compounds provided by the recombinant host cells and methods of the present disclosure are contemplated to have 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 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 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. EXAMPLES [0133] 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. Example 1: Biosynthesis of the cannabinoids, CBDA, THCA, and/or CBCA, from HA or OA by engineered Saccharomyces cerevisiae [0134] This example illustrates a study showing that Saccharomyces cerevisiae strains derived from CEN.PK2-1D with a pathway capable of converting hexanoic acid (HA) or the olivetolic acid (OA) to the cannabinoid, CBGA, can be further engineered to incorporate CBDA synthase (CBDAS) homologs and variants. Some of the resulting engineered strains when cultured in the presence of HA (or the cannabinoid precursor, OA) are capable of converting the CBGA to the further downstream cannabinoids, CBDA, THCA, and/or CBCA. [0135] Materials and Methods [0136] A. Identification of candidate CBDAS homologs [0137] Public and internal cannabis genomes were searched for proteins >50% identical to THCAS-like synthases. Unique hits were aligned and clustered. 12 potential CBDAS homologs were selected from the cluster tree for further screening. [0138] B. CBDAS library construction [0139] Sequences encoding the “wild-type” d28_CBDAS and the 12 potential CBDAS homolog polypeptides with a N-terminal SP-Alpha secretion peptide of SEQ ID NOs: 44, 46, 48, 50, 52, sequences of SEQ ID NOs: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, and 67. The yeast codon optimized polynucleotide sequences encoding the fused SP-Alpha_CBDAS polypeptides were synthesized (Twist Bioscience Corporation, South San Francisco, CA, USA). The optimized polynucleotide of SEQ ID NO: 43 encoding SP-Alpha_d28_CBDAS (SEQ ID NO: 44) was subcloned using NEBuilder® HiFi DNA Assembly Master Mix (NEB) with the Gal1 promoter into an E. coli construct containing the ALD4 terminator along with homology regions to the X-3 integration site (EasyClone 2.0) needed for integration into the yeast genome to generate wbPlasmid089 shown in FIG.7. Linear DNA for integration for the Alpha_d28_CBDAS control was PCR amplified from wbPlasmid089. Linear DNA for integration of all other synthesized homologs were assembled by SOE-PCR to contain the Gal1 promoter, ALD4 terminator, and X-3 homology regions for integration. Primers used to assemble the linear DNA for integration are provided in Table 4 below [0140] TABLE 4 SEQ ID Name Sequence NO:

[0141] C. Expression of SP-Alpha-CBDAS homolog library in yeast [0142] The linear donor DNA for each SP-Alpha-CBDAS homolog was pooled and transformed for integration into the X-3 site of two Saccharomyces cerevisiae strains using CRISPR-Cas9: (1) a strain incorporating only the 82 aa truncated PT4 prenyltransferase, d82_PT4 (SEQ ID NO: 10); or (2) a strain incorporating the cannabinoid biosynthesis pathway including genes encoding the enzymes C. sativa AAE1 (SEQ ID NO: 2), TKS (SEQ ID NO: 4), OAC (SEQ ID NO: 6), and d82_PT4 (SEQ ID NO: 10). Linear donor DNA for the SP-Alpha_d28_CBDAS reference construct was transformed separately. Colonies from the SP-Alpha_CBDAS homolog library and SP-Alpha_d28_CBDAS control build were picked for screening. [0143] D. Screening of CBDAS homolog library for cannabinoid biosynthesis [0144] Yeast strains were grown in 0.3 mL YPD media for 24 hr at 30°C. The parent strain to each integration was used a baseline control. Cultures were then sub-cultured into 0.3 mL YPD media with feeding of OA for the strains containing only PT and feeding of HA for the strains containing the four enzyme cannabinoid biosynthesis pathway of enzymes, AAE1 (SEQ ID NO: 2), TKS (SEQ ID NO: 4), OAC (SEQ ID NO: 6), and PT4 (SEQ ID NO: 10). OA or HA were fed to a final concentration of 1 mM and the cultures grown for 72 hours at 30°C. [0145] Samples for LC/MS analysis were prepared by extracting whole broth of the culture and diluting with acetonitrile. The prepared samples were loaded onto Agilent LC/MS (1290 Infinity II LC system with 6470B Triple quadrupole mass spectrometry) for analysis. The UPLC solid phase was a C18 column, and the mobile phase was the Acetonitrile/Water system. [0146] THCA was identified with primary mass transition 357.2/313.2 and secondary transition 357.2/245.1; CBCA was identified with primary mass transition 357.2/313.2 and secondary transition 357.2/339.1. CBDA was identified with primary mass transition 357.2/339.1 and secondary transition 357.2/245.1, The peak identities are also confirmed by their LC/MS retention time. The concentrations of THCA, CBCA, and CBDA were quantified by referring to a standard curve. The top CBDAS homologs showing activity were confirmed by LC/MS. [0147] The library clones from the pooled screening showing detectable activity were sequenced to identify the integrated SP-Alpha-CBDAS homolog showing activity. [0148] Results: As shown by the results summarized in Table 5 (below), the pooled screening followed by hit sequencing identified three SP-Alpha CBDAS homolog polypeptides producing detectable levels of the downstream cannabinoids, CBDA, THCA, and/or CBCA: SP- Alpha_CBDAS_Var95 (SEQ ID NO: 46), SP-Alpha_Cs10_4 (SEQ ID NO: 46) and SP- Alpha_CBDAS2 (SEQ ID NO: 48). Only the engineered yeast strains expressing the homolog, CBDAS_Var95 produced all three cannabinoids, CBDA, THCA, and CBCA when fed either OA, in the case of the strain only incorporating a heterologous PT4 enzyme, or HA, in the case of the strain incorporating the complement of four heterologous cannabinoid pathway enzymes, AAE1, TKS, OAC, and PT4. The other nine SP-Alpha-CBDAS homologs tested not showing up expression, or simply not enough screening of the pool in this study. [0149] TABLE 5 AA Sequence SP-CBDAS (SEQ ID NO: ) CBDA THCA CBCA

Example 2: Secretion peptide screening for enhanced cannabinoid biosynthesis by recombinant CBDAS and THCAS in Saccharomyces cerevisiae [0150] Efficient secretion of the cannabinoid synthases, CBDAS and THCAS in the trichome cells of the plant C. sativa is believed to be necessary to facilitate efficient production of the cannabinoids, CBDA, THCA, and/or CBCA. This example illustrates a study that screens a library of various secretion peptides in combination with CBDAS and THCAS for the ability to enhance cannabinoid production of CBDA and THCA when the cannabinoid synthases are expressed in yeast strains engineered with a cannabinoid pathway that produces CBGA. [0151] Materials and Methods [0152] A. Secretion peptide library construction [0153] The secretion peptides from various source organisms selected for library construction and screening are summarized below in Table 6. [0154] TABLE 6 SP Name SEQ ID NO: SEQ ID NO: Abbrev. (source organism) (nt) (aa)

(S. cerevisiae)

ucleotide pools (Opools, Integrated DNA Technologies, San Diego, CA, USA) with a 5’ flanking region to the Gal1 promoter and a 3’ flanking region to the yeast optimized polynucleotide encoding d28_CBDAS (SEQ ID NO: 14) and d28_THCAS (SEQ ID NO: 18). Each synthase was PCR amplified with their respective oligo pools then subcloned using NEBuilder® HiFi DNA Assembly Master Mix (NEB) into an E. coli construct containing the Gal1 promoter and ALD4 terminator cassette along with homology regions needed for integration into the X-3 site (EasyClone 2.0) of the yeast genome. E. coli colonies from the cloning transformation for each synthase were pooled and plasmid DNA was isolated from the pools. Linear donor DNA was amplified from the plasmid DNA pools and used to transform yeast. The N-terminal SP-Alpha constructs were built separately from the pool and used as a reference control for both CBDAS and THCAS. Oligo pools and primers used to assemble the linear donor DNA for integration are provided in Table 7 below. [0156] TABLE 7 SEQ ID Name Sequence NO:

GGGTGTATCTTTAGAGAAGAGAGAAGCTGAAGCCAATCCTAGAGAA

GTTTGGAGAAGAGGGAAGCAGAGGCTAACCCAAGGGAGAACTTCCT

p p g _ _ secretion peptide libraries (SP_AA, SP_AT, SP_IV, and SP_KP) were fused to CBDAS_Var95. Linear DNA for these four SP-CBDAS_Var95 fusions were constructed using SOE-PCR to contain the Gal1 promoter, appropriate secretion peptide, ALD4 terminator, and X-3 homology regions for integration. For a list of primers see Table 7. [0158] B. Transformation and expression of secretion peptide library in yeast [0159] The pooled SP library linear donor DNA for d28_CBDAS and d28_THCAS were transformed for integration into the X-3 site of various Saccharomyces cerevisiae strains using CRISPR-Cas9. All Saccharomyces cerevisiae strains transformed had the pathway of cannabinoid biosynthesis, which included the following genes from Cannabis sativa: AAE1 (SEQ ID NO: 2), TKS (SEQ ID NO: 4), OAC (SEQ ID NO: 6), and PT4 (SEQ ID NO: 10). builds were picked for screening. Linear DNA for each of the four SP-CBDAS_Var95 fusions were transformed separately for integration into the X-3 site of a HA-fed Saccharomyces cerevisiae strain using CRISPR-Cas9. The SP-Alpha_CBDAS_Var95 fusion was used as a reference control for the four tested SP-CBDAS_Var95 fusions. Colonies from each SP- CBDAS_Var95 fusion were picked for screening. [0160] C. Screening secretion peptide library for cannabinoid biosynthesis [0161] Yeast strains were grown in 0.3 mL YPD media for 24 hr at 30°C. The parent strain to each integration was used a baseline control. Cultures were then sub-cultured into 0.3 mL YPD media with feeding of HA to 1 mM final concentration and grown for 72 hours at 30°C. The whole broth of the culture was extracted and diluted with acetonitrile for sample preparation. The prepared samples were loaded onto Agilent LC/MS (1290 Infinity II LC system with 6470B Triple quadrupole mass spectrometry) for analysis. The UPLC solid phase was using C18 column, and the mobile phase was using the Acetonitrile/Water system. THCA was identified with primary mass transition 357.2/313.2 and secondary transition 357.2/245.1; CBCA was identified with primary mass transition 357.2/313.2 and secondary transition 357.2/339.1; CBDA was identified with primary mass transition 357.2/339.1 and secondary transition 357.2/245.1; Their identities are also confirmed by their LC/MS retention time. The concentrations of THCA, CBCA, and CBDA were quantified by referring to a standard curve. Top variants showing activity were confirmed by LC-MS. [0162] D. Sequence identification [0163] Secretion peptide sequences from selected top library clones showing activity from both the SP-d28_CBDAS and SP-d28_THCAS libraries were identified by PCR and sequencing. [0164] Results: [0165] The combinations of secretion peptides fused with either THCAS, CBDAS, or CBDAS_Var95 that resulted in production of THCA, CBDA, and CBDA, respectively, are summarized in Table 8 below. [0166] TABLE 8 SEQ ID SEQ ID NO: NO:

SP KP CBDAS Var95 ++ 111 112

ited 3-fold to 4- fold increased levels of THCA or CBDA production, respectively, relative to the control SP- Alpha secretion peptide, fused with the same enzymes. The secretion peptide SP-KP when fused with CBDAS resulted in engineered yeast exhibiting 3-fold to 4-fold increased levels of CBDA production relative to the control secretion peptide, SP-Alpha fused with CBDAS. The combination of the SP_IV and SP_KP secretion peptides when fused with the CBDAS homolog, CBDAS_Var95, exhibited 2-fold to 3-fold increased levels of CBDA production relative to the control secretion peptide, SP-Alpha fused with CBDAS_Var95. [0168] 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. [0169] Additional embodiments of the invention are set forth in the following claims. [0170] 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.

Claims

What is claimed is: 1. A recombinant host cell which produces Δ⁹-tetrahydrocannabinolic acid (THCA) when cultured in the presence of hexanoic acid (HA) or olivetolic acid (OA), wherein the cell comprises a pathway of enzymes capable of producing cannabigerolic acid (CBGA) and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having THCAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 90, 92, 72, and 84. 2. The cell of claim 1, wherein: (a) the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Escherichia coli; (b) the polypeptide having THCAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18; (c) the N-terminal secretion peptide linked to the polypeptide having THCAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 124, 126, 128, 130, 132, 134, and 136; (d) the amount of THCA produced by the cell when cultured in the presence of HA or OA is increased relative to a control host cell comprising a pathway of enzymes AAE, TKS, OAC, and PT4, and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having THCAS activity comprising an amino acid sequence of SEQ ID NO: 122; and/or (e) the cell also produces cannabidiolic acid (CBDA), and/or cannabichromenic acid (CBCA). 3. A recombinant host cell which produces cannabidiolic acid (CBDA) when cultured in the presence of hexanoic acid (HA) or olivetolic acid (OA), wherein the cell comprises a pathway of enzymes capable of producing cannabigerolic acid (CBGA) and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having CBDAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 70, and 72. 4. The cell of claim 3, wherein: (a) the source organism of the recombinant host cell is selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Escherichia coli; (b) the polypeptide having CBDAS activity comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 16 or SEQ ID NO: 20; (c) the N-terminal secretion peptide linked to the polypeptide having CBDAS activity 108, 110, 112, 114, and 46; (d) the amount of CBDA produced by the cell when cultured in the presence of HA or OA is increased relative to a control host cell comprising a pathway of enzymes AAE, TKS, OAC, and PT4, and a nucleic acid encoding an N-terminal secretion peptide linked to a polypeptide having CBDAS activity comprising an amino acid sequence of SEQ ID NO: 44; and/or (e) the cell also produces Δ⁹-tetrahydrocannabinolic acid (THCA), and/or cannabichromenic acid (CBCA). 5. The cell of any one of claims 1-4, wherein: (a) the pathway of enzymes is capable of catalyzing reactions (i) – (iv): (i) O O H₃ ,

and (iv)

COOH 3 ; nd PT4;

and/or (c) the pathway of enzymes comprises (i) AAE having an amino acid sequence of at least 90% identity to SEQ ID NO: 2; (ii) TKS having an amino acid sequence of at least 90% identity to SEQ ID NO: 4; (iii) OAC having an amino acid sequence of at least 90% identity to SEQ ID NO: 6; and (iv) PT4 having an amino acid sequence of at least 90% identity to SEQ ID NO: 8 or 10. 6. A method for producing a cannabinoid selected from cannabidiolic acid (CBDA), Δ⁹- tetrahydrocannabinolic acid (THCA), and/or cannabichromenic acid (CBCA), the method comprising: (a) culturing in a suitable medium comprising hexanoic acid (HA) or olivetolic acid (OA) and a recombinant host cell of any one of claims 1-5; and (b) recovering the produced cannabinoid. 7. A recombinant polypeptide having cannabinoid synthase activity, wherein the polypeptide comprises an amino acid sequence of at least 85% identity to SEQ ID NO: 14 and comprises at least one amino acid residue difference relative to SEQ ID NO: 14 selected from: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, P164A, H186D, L203I, A208G, S210N, V215A, R220K, M229I, I235N, E240G, V245F, K447Q, and R516H. 8. The recombinant polypeptide of claim 7, wherein the amino acid sequence has at least 90% identity to SEQ ID NO: 14 and the following amino acid residue differences relative to SEQ ID NO: 14: T47S, S89A, I102V, R109H, V135I, L144F, A145P, A146G, C152G, A153V, G158S, P164A, H186D, L203I, A208G, S210N, V215A, R220K, M229I, I235N, E240G, V245F, K447Q, and R516H. identity to SEQ ID NO: 20. 10. The polypeptide of any one of claims 7-9, wherein the polypeptide has cannabinoid synthase activity capable of converting cannabigerolic acid (CBGA) to cannabidiolic acid (CBDA), and/or Δ⁹-tetrahydrocannabinolic acid (THCA). 11. The polypeptide of any one of claims 7-10, wherein the polypeptide further comprises N- terminal secretion peptide comprising an amino acid sequence selected from SEQ ID NO: 86, 88, 90, 92, 70, 72, 78, and 84. 12. A recombinant polypeptide comprising an N-terminal secretion peptide linked to a polypeptide having CBDAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 86, 88, 90, 92, 72, 78, and 84. 13. The polypeptide of claim 12, wherein the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 94, 96, 98, 108, 110, 112, and 114. 14. The polypeptide of claim 12, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 20. 15. The polypeptide of claim 14, wherein the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 108, 110, 112, and 114. 16. A recombinant polypeptide comprising an N-terminal secretion peptide linked to a polypeptide having THCAS activity, wherein the N-terminal secretion peptide comprises an amino acid sequence selected from SEQ ID NO: 78, 86, 88, 90, 92, 72, and 84. 17. The polypeptide of claim 16, wherein the N-terminal secretion peptide linked to the polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 124, 126, 128, 130, 132, 134, and 136 18. The polypeptide of claim 16, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18. 19. A polynucleotide encoding a recombinant polypeptide of any one of claims 7-18. 20. An expression vector comprising a polynucleotide of claim 19.