Cannabinoid production and engineered cells therefor

Abstract

Disclosed herein are novel cells that are engineered to catabolize a reduced number of acyl-CoA and branched-chain amino acids, and methods for improved cannabinoid production using these cells.In addition, disclosed are cells that are engineered to produce rare cannabinoids, methods for producing these novel rare cannabinoids, and the novel rare cannabinoid compounds thus produced.Some aspects of the disclosure are directed to cells that are engineered to provide increased production of cannabinoids and / or their derivatives compared to control cells.

Description

[Technical Field]

[0001] Inventors: Diep Minh Ngoc Nguyen, Caleb Marshall Walker, Nicky Christopher Caiazza, Jun Urano Related Applications This application claims priority to and the benefit of co-pending U.S. Provisional Application No. 63 / 434,892, filed December 22, 2022. The disclosure of said provisional application is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Plants in the Cannabaceae family produce numerous different cannabinoids (>=120) in variable relative amounts over a 7-10 week flowering period. Many of these cannabinoids have been and are currently being studied as therapeutic agents in chordates (e.g., mammals), and as a result, most are approved (in over 35 states) for either medical and / or recreational use in the United States (Abrams DI Eur J Int Med 2018, 49, 7-11). Specifically, the most sought-after (phyto)cannabinoids are tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA), as well as related minor cannabinoids. These phytocannabinoids and their related chemical analogs are all biosynthesized in varying amounts from the same precursor: cannabigerolic acid (CBGA). As a result, achieving high titers in the biosynthesis of THC(A), CBD(A), and CBC(A) in either plants or recombinant host organisms is of paramount importance. However, efficient biosynthesis of these cannabinoids in recombinant hosts such as yeast has been hindered by endogenous pathways that catabolize precursor molecules and prevent their efficient use in the cannabinoid biosynthetic pathway. Therefore, there remains a need to provide engineered recombinant hosts that provide increased availability of cannabinoid biosynthetic precursor molecules, including acyl-CoA molecules, by deleting, inactivating or otherwise mutating genes that affect the cellular metabolism of these precursor molecules. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Abrams DI Eur J Int Med 2018,49,7-11 Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention Some embodiments of the present disclosure are directed to cells engineered to provide increased production of cannabinoids and / or derivatives thereof compared to control cells, wherein the engineered cells express one or more enzymes of a cannabinoid biosynthetic pathway and at least one gene encoding one or more enzymes or other proteins that affect the metabolism of at least one acyl-CoA molecule in the cell has been deleted, inactivated or otherwise mutated, thereby allowing the redirection of acyl-CoA molecules from the cellular metabolic pathway to the cannabinoid biosynthetic pathway to achieve increased production of cannabinoids and / or derivatives thereof in the engineered cells compared to control cells lacking the same deletion, inactivation or other mutation of at least one gene.

[0005] In some embodiments, the cellular metabolic pathway is a beta-oxidation pathway. In some embodiments, the beta-oxidation pathway is one or both of the mitochondrial beta-oxidation pathway and / or the peroxisomal beta-oxidation pathway. In other embodiments, the cellular metabolic pathway is an amino acid catabolic pathway. In some embodiments, the amino acid catabolic pathway is one or more branched-chain amino acid catabolic pathways selected from the group consisting of leucine, isoleucine, and / or valine catabolic pathways that produce branched acyl-CoA intermediates.

[0006] In some embodiments, the acyl-CoA comprises one or more of butyryl-CoA, valeryl-CoA, isovaleryl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-CoA, and / or hexanoyl-CoA.

[0007] In some embodiments, the at least one gene encodes peroxisomal acyl-CoA oxidase, peroxisomal acyl-CoA dehydrogenase, mitochondrial acyl-CoA dehydrogenase, mitochondrial isovaleryl-CoA dehydrogenase, mitochondrial branched-chain α-keto acid dehydrogenase subunit, isovaleryl-CoA dehydrogenase, branched-chain aminotransferase, glutaryl-CoA dehydrogenase, keto acid decarboxylase, multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase, peroxisomal oxoacylthiolase, fatty acid CoA ligase, peroxisomal adenine nucleotide transporter, cytosolic carnitine acetyltransferase, or peroxisome biogenesis factor.

[0008] In some embodiments, the at least one gene is selected from one or more of the group consisting of POX1, POX2, POX3, POX4, POX5, POX6, BAT1, BAT2, YAT1, FAA1, ANT1, POT1, ACD1, IVD1, MFE1, PEX10, ARO10, GCDH1, GCDH2, ACD2, BCKD-E1α, BCKD-E1β, BCKD-E2 and homologs or orthologs thereof. In some embodiments, at least one gene is selected from the group consisting of YALI0_D15708g, YALI0_B10406g, YALI0_D01265g, YALI0_F19910g, YALI0_D08690g, YALI0_F05038g, YALI0_D23815g, YALI0_D20768g, YALI0_D06930g, YALI0_E32835g, YALI0_F10857g, YALI0_D24750g, YALI0_E27654g, YALI0_C23859g, YALI0_E06567g, YALI0_C16797g, YALI0_F2 3749g, YALI0_B04906g, YALI0_D02387g, YALI0_E15378g, YALI0_E18568g, YALI0_D17864g, YALI0_E03058g, YALI0_F21197g, YALI0_E12573g, YALI0_F28567g, YALI0_F22121g, YALI0_D00671g, YALI0_D06215g, YALI0_D09383g, YALI0_D09493g, YALI0_A07733g and homologs or orthologs thereof.

[0009] In some embodiments, the engineered cells are capable of expressing cannabigerolic acid (CBGA) synthase, unsaturated cannabigerolic acid (CBGA-uns) synthase, ketolized cannabigerolic acid (CBGA-keto) synthase, cannabigerol (iso)butyrate (CBG(i)BA) synthase, cannabigerolic acid (CBGVA) synthase, tetrahydrocannabic acid (THCA) synthase, unsaturated tetrahydrocannabic acid (THCA-uns) synthase, ketolized tetrahydrocannabic acid (THCA ... tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic Tetrahydrocannabinic acid (THCA-keto) synthase, tetrahydrocannabi(iso)butolic acid (THC(i)BA) synthase, tetrahydrocannabivaric acid (THCVA) synthase, cannabichromenic acid (CBCA) synthase, unsaturated cannabichromenic acid (CBCA-uns) synthase, ketolized cannabichromenic acid (CBCA-keto) synthase, cannabichromene(iso)butyric acid (CBC(i)BA) synthase, cannabichromevaric acid (CBCVA) synthase, cannabidiolic acid (CBDA) synthase, unsaturated cannabidiolic acid (CBDA-uns) synthase, ketolized cannabidiolic acid (CBDA-keto) synthase, cannabib(iso)butolic acid (CBD(i)BA) synthase, cannabidivalic acid (CBDVA) synthase, hexanoyl-CoA synthetase, (iso)valeryl-CoA synthetase, butyryl-CoA synthetase, polyketide synthase, polyketide cyclase, geranyl-CoA The cannabinoid biosynthetic pathway expresses two or more enzymes of the cannabinoid biosynthesis pathway selected from the group consisting of mevalonate-5-phosphate decarboxylase, isopentenyl phosphokinase, and / or hydroxymethylglutaryl-CoA reductase.

[0010] In some embodiments, the cells, when cultured under appropriate conditions, are capable of producing one or more cannabinoid derivatives selected from the group consisting of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA, and CBDVA, and their decarboxylated derivatives. In some embodiments, the appropriate conditions include the addition of one or more of butyric acid, valeric acid, isovaleric acid, hexanoic acid, glucose, glycerol, hexanol, butanol, and / or oleic acid.

[0011] In some embodiments, at least one gene comprises a combination of POX3, POX5 and one or both of ACD1 and IVD1, and the cells exhibit synergistic production of cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations or mutations of only ACD1, IVD1 or the POX3 / POX5 combination, hi some embodiments, at least one gene comprises a combination of YALI0_D15708g and YALI0_E12573g.

[0012] In some embodiments, at least one gene comprises POX3, POX5, ACD1 in combination with one or more of POX2, POX6, GCDH2, GCDH1, MFE1, YAT1, FAA1, ANT1, POT1, ACD2 and PEX10, and the cells exhibit enhanced production of cannabinoids compared to the production of cannabinoids achieved in a cell comprising deletion, inactivation or mutation of only the combination of POX3, POX5 and ACD1. In some embodiments, at least one gene comprises IVD1 in combination with at least one of BAT1 or BAT2, and the cells exhibit enhanced production of one or more C5 branched-chain cannabinoids compared to the production of the same one or more C5 branched-chain cannabinoids achieved in a cell comprising deletion, inactivation or mutation of only IVD1. In some preferred embodiments, the one or more C5 branched chain cannabinoids include one or more of CBGiBA, CBDiBA, CBCiBA and / or THCiBA.

[0013] In some embodiments, the at least one gene comprises a combination of at least two of POX3, POX5, and ACD1. In some embodiments, the at least one gene comprises a combination of POX3, POX5, ACD1, and FAA1, and the cells exhibit significantly improved production of C4 cannabinoids compared to the production of C4 cannabinoids achieved in cells comprising deletions, inactivations, or mutations of only POX3, POX5, and ACD1. In other embodiments, the at least one gene comprises a combination of POX3, POX5, ACD1, and YAT1, and the cells exhibit significantly improved production of cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations, or mutations of only POX3, POX5, and ACD1.

[0014] In some embodiments, the at least one gene comprises a combination of IVD1 and at least one of BCKD-E1α and BCKD-E2, and the cells exhibit reduced production of C5 branched cannabinoid products compared to the production of C5 branched cannabinoids achieved in cells comprising deletion, inactivation, or mutation of IVD1 alone. In some embodiments, the cells further exhibit significantly increased production of CBGVA compared to the production of CBGVA achieved in cells comprising deletion, inactivation, or mutation of IVD1 alone. In some embodiments, the at least one gene further comprises at least one of POX3, POX5, and ACD1. In some embodiments, the at least one gene further comprises FAA1.

[0015] In some embodiments, the at least one gene includes FAA1, ACD1, IVD1, POX3, POX5, and one or both of BCKD-BCKD-E1α and BCKD-E2. In some embodiments, the cells exhibit significantly improved production of C4 cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations, or mutations of only POX3, POX5, ACD1, IVD1, and BCKD-E1α.

[0016] In some embodiments, the at least one gene comprises FAA1. In some embodiments, the at least one gene further comprises ACD1 or IVD1.

[0017] In some embodiments, the cell is a yeast cell or a bacterial cell. In some embodiments, the yeast cell is a Yarrowia strain or a Saccharomyces strain.

[0018] Another aspect of the present disclosure is directed to a method for producing a cannabinoid or a derivative thereof, comprising culturing the cells disclosed herein under suitable conditions to produce the cannabinoid or a derivative thereof. In some embodiments, the cannabinoid or derivative thereof comprises one or more of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA, and CBDVA, or their decarboxylated derivatives. In some embodiments, the suitable conditions include adding at least one of butyric acid, butyl butyrate, valeric acid, isovaleric acid, hexanoic acid, hexanol, hexyl hexanoate, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured. In some embodiments, the method includes isolating the cannabinoid or derivative thereof from the culture.

[0019] Another aspect of the present invention is directed to cells engineered to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, wherein the cells comprise an inactivated or otherwise mutated IVD1, POT1 and / or MFE1 gene, and the cells, when cultured under appropriate conditions, further express sufficient enzymes in the cannabinoid synthesis pathway to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof. In some embodiments, suitable conditions include supplementing the culture medium in which the cells are cultured with butyric acid, butyl butyrate, valeric acid, isovaleric acid, butanol, hexanol, hexanoic acid, hexyl hexanoate, oleic acid, glycerol, or glucose.

[0020] Another aspect of the present disclosure is directed to a method for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns, and / or CBD-keto, or decarboxylated derivatives thereof, by culturing a cell disclosed herein under conditions suitable for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns, and / or CBD-keto, or decarboxylated derivatives thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, butyl butyrate, valeric acid, isovaleric acid, butanol, hexanol, hexanoic acid, hexyl hexanoate, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured.

[0021] In some embodiments, the method includes isolating CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, from the culture.

[0022] Some other aspects of the present disclosure include the following structure: [ka] [ka] [ka] [ka] [ka] The present invention is directed to a compound according to any one of the preceding claims.

[0023] These and additional embodiments, along with their features, advantages and uses, will be further appreciated from the following description, claims and drawings. [Brief explanation of the drawings]

[0024] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1-1] FIG. 1 illustrates the biosynthetic pathways for CBGA and the major cannabinoids derived therefrom, including THC(V)A, CBD(V)A, and CBC(V)A. [Figure 1-2] Same as above.

[0025] [Figure 2] FIG. 2 illustrates the fatty acid β-oxidation pathway.

[0026] [Figure 3] FIG. 3 illustrates the leucine catabolic pathway.

[0027] [Figure 4A] 4A-4B illustrate the expression of ACD1 and IVD1 in butyrate-fed fermentations. [Figure 4B] Same as above.

[0028] [Figure 5A] 5A-5B illustrate the expression of ACD1 and IVD1 in hexanoic acid-fed fermentations. [Figure 5B] Same as above.

[0029] [Figure 6] FIG. 6 illustrates the metabolic pathways of monounsaturated cannabinoid species.

[0030] [Figure 7] FIG. 7 illustrates the metabolic pathways of ketolized cannabinoid species. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention Some definitions "Identity" or "homology" refers to the degree to which two or more nucleic acid or polypeptide sequences are identical. In some embodiments, the percent identity or homology between a sequence of interest and a second sequence over a window of evaluation, for example, the length of the sequence of interest, can be calculated by aligning these sequences, determining the number of residues (nucleotides or amino acids) within the window of evaluation that are identical, allowing for the introduction of gaps to maximize identity, dividing by the total number of residues in the sequence of interest or the second sequence that fall within that window (whichever is greater), and multiplying by 100. When calculating the number of identical residues required to achieve a specific percent identity or homology, fractions should be rounded to the nearest integer. Percent identity or homology can be calculated using various computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, and Gapped BLAST generate alignments and provide percent identity between sequences of interest. The algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:22264-2268, 1990), as modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993, has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (Altschul et al., J. Mol. Biol. 215:403-410, 1990). To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (Altschul, et al. Nucleic Acids Res. 25:3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program can be used. PAM250 or BLOSUM62 matrices can be used.Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). For these programs, see the website with the URL ncbi.nlm.nih.gov. In certain embodiments, percent identity or homology is calculated using BLAST2 with the default parameters provided by NCBI.

[0032] The term "homolog" is intended to mean a nucleic acid sequence that has close sequence identity to the nucleic acid sequence of a described gene, where both nucleic acid sequences are determined to be derived from the same ancestral gene, such as through speciation, either through phylogenetic analysis or statistical analysis of alignments between sequences. When a determination that two nucleic acid sequences are homologous is made through statistical analysis of alignments between sequences, widely known and online available tools, such as BLAST, can be used to make this determination. For purposes of this definition, an alignment in BLAST given an expectation value (E-value) of less than 1 x 10 is considered sufficient to determine that both nucleic acids are derived from the same ancestral gene. The term "homolog" can also be used to identify two amino acid sequences that have close sequence homology and / or function, and are similarly determined to be encoded by and derived from the same ancestral gene. "Ortholog" is defined similarly to "homolog," except that any nucleic acid sequence that shares close sequence identity with the nucleic acid sequence of the described gene has been determined to be derived from the same ancestral gene through speciation.

[0033] The term "exogenous" is intended to mean that the referenced molecule or referenced activity is introduced into a cell. The molecule can be introduced by introduction of an encoding nucleic acid into the host genetic material, for example, by integration into a host chromosome, or as non-chromosomal genetic material such as a plasmid. Thus, when used with respect to expression of an encoding nucleic acid, the term refers to the introduction of an expressible form of the encoding nucleic acid into a cell. When used with respect to a biosynthetic activity, the term refers to an activity introduced into a host. The source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity after introduction into the cell. Thus, the term "endogenous" refers to the referenced molecule or activity present in a cell. Similarly, when used with respect to expression of an encoding nucleic acid, the term refers to expression of an encoding nucleic acid contained within a microorganism. The term "heterologous" refers to a molecule or activity derived from a source other than the referenced species, whereas "homologous" refers to a molecule or activity derived from the host microorganism. Thus, exogenous expression of an encoding nucleic acid can utilize either or both heterologous or homologous encoding nucleic acid.

[0034] The terms "reduce," "decreased," "reduction," "reduction," and "inhibit" are all generally used herein to mean a statistically significant reduction. However, for the avoidance of doubt, "decreased," "reduction," or "reduction" or "inhibit" means a reduction of at least 5% compared to a reference level, for example, a reduction of at least 10% compared to a reference level, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction up to and including 100% (i.e., a level that is absent compared to a reference sample), or any reduction between 10 and 100%.

[0035] The terms "increased," "increase," "enhance," "improve," or "activate" are all used herein to generally mean an increase by a statically significant amount, and for the avoidance of doubt, the terms "increased," "increase," "enhance," or "activate" mean an increase of at least 5% compared to a reference level, an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase up to and including 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase between 2-fold and 10-fold or more. The terms "increased production," "enhanced production," and "improved production" refer to an increase, enhancement, or improvement in the amount, yield, titer, concentration, quantity, volume, output, or flux of a particular measured product, including cannabinoids and derivatives thereof, compared to a reference level achieved by another method or in a control cell.

[0036] The term "statistically significant" or "significantly" refers to statistical significance, generally meaning two standard deviations (2SD) below the normal or lower concentration of the marker. The term refers to statistical evidence that a difference exists. This is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using a p-value.

[0037] The term "acyl-CoA" generally refers to a coenzyme A bound to an RC(O) group, where R refers to an aliphatic chain that may be saturated or unsaturated and optionally carbonyl-substituted (e.g., ketolized). The term is defined to include at least butyryl-CoA, valeryl-CoA, isovaleryl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-CoA, and hexanoyl-CoA.

[0038] The term "C4 cannabinoid" is defined as a cannabinoid derived from a four-carbon starting acyl compound, such as butyric acid. The resulting C4 cannabinoids include CBGVA, THCVA, CBDVA, and CBCVA, as well as their decarboxylated derivatives.

[0039] The term "C5 cannabinoid" is defined as a cannabinoid derived from a five-carbon starting acyl compound, such as valeric acid or isovaleric acid. The resulting cannabinoids include CBGBA, THCBA, CBDBA, CBCBA, CBGiBA, THCiBA, CBDiBA, CBCiBA, and their decarboxylated derivatives.

[0040] The term "iC5 cannabinoids" is defined as a subset of C5 cannabinoids derived from branched-chain five-carbon starting acyl compounds such as isovaleric acid. The resulting cannabinoids include CBGiBA, THCiBA, CBDiBA, CBCiBA, and their decarboxylated derivatives.

[0041] The term "C6 cannabinoids" is defined as cannabinoids derived from a six-carbon starting acyl compound, such as hexanoic acid. The resulting cannabinoids include CBGA, THCA, CBDA, CBCA, CBGA-uns, THCA-uns, CBDA-uns, CBCA-uns, CBGA-keto, THCA-keto, CBDA-keto, CBCA-keto, and their decarboxylated derivatives.

[0042] The term "at least one gene" is defined as an open-ended range requiring a minimum of one gene, but allowing for combinations of two or more genes, three or more genes, four or more genes, five or more genes, six or more genes, seven or more genes, eight or more genes, etc. To further identify and define the specific genes described in this application, the following index is provided below, citing the abbreviated gene name, corresponding Yarrowia lipolytica gene identification number, accession number, and description. [Table 35]

[0043] Engineered cells that provide increased production of cannabinoids and their derivatives Some aspects of the disclosure are directed to cells engineered to provide increased production of cannabinoids and / or derivatives thereof compared to control cells, wherein the engineered cells express one or more enzymes of a cannabinoid biosynthetic pathway and at least one gene encoding one or more enzymes or other proteins that affect the metabolism of at least one acyl-CoA molecule in the cell has been deleted, inactivated or otherwise mutated, thereby allowing the redirection of acyl-CoA molecules from the cellular metabolic pathway to the cannabinoid biosynthetic pathway to achieve increased production of cannabinoids and / or derivatives thereof compared to the control cells, wherein the control cells lack the same deletion, inactivation or other mutation in at least one gene as the cells engineered to produce increased production of cannabinoids and / or derivatives thereof.

[0044] In some embodiments, at least one gene encoding one or more enzymes or other proteins that affect the metabolism of at least one acyl-CoA molecule through a cellular metabolic pathway is deleted. The deletion can include the deletion of the entire at least one gene or the deletion of a portion of at least one gene. In some embodiments, the deletion includes the deletion of a single allele, two alleles, or alleles of the same gene in the cell. In addition, the deletion can also include the deletion of one or more alleles of a gene homologous to the at least one deleted gene in the cell. In some embodiments, more than one gene is deleted. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve or more genes are deleted. In some embodiments, the deletion of at least one gene does not result in cell death.

[0045] The technique for gene deletion is not limited, and includes all techniques known to those skilled in the art for achieving partial or complete deletion of gene.In some embodiments, deletion can be carried out by a one-step process that produces a selectable phenotype, such as the method described in U.S. Patent No. 4,963,487 and U.S. Patent No. 4,713,337, both of which are incorporated herein by reference in their entirety.In some embodiments, deletion can be carried out by other protocols, including those described in U.S. Patent No. 6,989,265, U.S. Patent No. 8,026,098 and U.S. Patent No. 9,249,428, which are also incorporated herein by reference in their entirety.

[0046] In some embodiments, at least one gene encoding one or more enzymes or other proteins that affect the metabolism of at least one acyl-CoA molecule in a cellular metabolic pathway is inactivated or otherwise mutated. In some embodiments, the inactivation or mutation provides a gene knockout, where the inactivated or mutated gene encodes an inactive protein or can no longer be transcribed or translated. In some embodiments, the inactivation or mutation provides a gene knockdown, where the protein encoded by the inactivated or mutated gene has reduced activity compared to the protein encoded by the non-inactivated or non-mutated gene. Such a reduction in activity may constitute a reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96% or more. In some embodiments, inactivation and / or mutation of at least one gene will not result in cell death. In other embodiments, a reduction in activity of the protein encoded by the inactivated or mutated gene will not result in cell death.

[0047] In some embodiments, inactivation or mutation includes inactivation or mutation of a single allele, two alleles, or all alleles of the same gene in a cell.In addition, inactivation or mutation can also include inactivation or mutation of one or more alleles of at least one gene that is homologous to at least one gene that is inactivated or mutated in a cell.In some embodiments, more than one gene is inactivated or mutated.In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve or more genes are inactivated or mutated.The technique used to inactivate or mutate at least one gene is not limited and includes techniques known to those skilled in the art. In some embodiments, the inactivation and / or mutation is achieved by selective nucleic acid DNA insertion or deletion, such as through the use of one or more gene disruption cassettes as described in U.S. Pat. Nos. 7,449,179 and 6,534,315, through suppressing gene transcription as described in U.S. Pat. No. 6,576,469, or through the use of nucleases, including zinc finger nucleases (ZFNs), I-CreI meganucleases, transcription activator-like effector nucleases (TALENs), or the like. This can be achieved through methods including, but not limited to, recombination, through providing deletions, and through the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems as described in U.S. Patent No. 7,951,925, U.S. Patent No. 8,106,255, U.S. Patent No. 7,897,372, U.S. Patent Application Publication No. 2011 / 0225664 and U.S. Patent Application Publication No. 2014 / 0170753, all of which are incorporated by reference in their entirety.

[0048] In some embodiments, the enzyme or protein encoded by the at least one gene affects acyl-CoA catabolism and / or affects one or both of the mitochondrial beta-oxidation pathway and the peroxisomal beta-oxidation pathway. For purposes of the present invention, affecting acyl-CoA and / or mitochondrial or peroxisomal beta-oxidation pathway catabolism is defined as the ability of an enzyme or other protein to directly or indirectly regulate, control, facilitate, modulate, bias, or otherwise affect the catabolism of cellular acyl-CoA molecules, 2-trans-enoyl-CoA molecules, L-3-hydroxyacyl-CoA molecules, or 3-ketoacyl-CoA molecules.

[0049] In some embodiments, the at least one gene may encode an enzyme that directly affects the mitochondrial or peroxisomal beta-oxidation pathway of the cell or that directly affects the catabolism of acyl-CoA molecules. In such embodiments, the at least one gene may encode peroxisomal acyl-CoA oxidase, mitochondrial acyl-CoA oxidase, peroxisomal acyl-CoA dehydrogenase, mitochondrial acyl-CoA dehydrogenase, mitochondrial enoyl-CoA hydratase, peroxisomal enoyl-CoA hydratase, mitochondrial 3-hydroxyacyl-CoA dehydrogenase, peroxisomal 3-hydroxyacyl-CoA dehydrogenase, mitochondrial 3-ketoacyl-CoA thiolase, peroxisomal 3-ketoacyl-CoA thiolase, or the multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase.

[0050] In some embodiments, the at least one gene may encode an enzyme or protein that indirectly affects the mitochondrial or peroxisomal beta-oxidation pathway and / or catabolism of acyl-CoA molecules in the cell. In such embodiments, the at least one gene may encode a peroxisomal oxoacylthiolase, a fatty acid CoA ligase, a peroxisomal adenine nucleotide transporter, a cytosolic carnitine acetyltransferase, or a peroxisome biogenesis factor.

[0051] In some embodiments, the enzyme or protein encoded by at least one gene affects a branched-chain amino acid catabolic pathway. In some embodiments, the branched-chain amino acid catabolic pathway is a leucine, isoleucine, or valine catabolic pathway. In such embodiments, the at least one gene may encode a mitochondrial isovaleryl-CoA dehydrogenase, a mitochondrial branched-chain α-keto acid dehydrogenase subunit, an isovaleryl-CoA dehydrogenase, a branched-chain aminotransferase, or a keto acid decarboxylase.

[0052] In some embodiments, the cells are able to redirect acyl-CoA molecules from cellular metabolic pathways into the cannabinoid biosynthetic pathway to achieve increased production of cannabinoids and / or derivatives thereof compared to control cells. This redirection of acyl-CoA molecules into the cannabinoid biosynthetic pathway results in at least a 5% increased level of one or more cannabinoids or derivatives thereof compared to control cells. In other embodiments, the cells have at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more increase in one or more cannabinoids and / or derivatives thereof compared to control cells. In some embodiments the cells have a 1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold or more increase in cannabinoids and / or derivatives thereof compared to control cells.

[0053] In some embodiments, the at least one gene encodes peroxisomal acyl-CoA oxidase, peroxisomal acyl-CoA dehydrogenase, mitochondrial acyl-CoA dehydrogenase, mitochondrial isovaleryl-CoA dehydrogenase, mitochondrial branched-chain α-keto acid dehydrogenase subunit, isovaleryl-CoA dehydrogenase, branched-chain aminotransferase, glutaryl-CoA dehydrogenase, keto acid decarboxylase, multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase, peroxisomal oxoacylthiolase, fatty acid CoA ligase, peroxisomal adenine nucleotide transporter, cytosolic carnitine acetyltransferase, or peroxisome biogenesis factor.

[0054] In some embodiments, the at least one gene is selected from one or more of the group consisting of POX1, POX2, POX3, POX4, POX5, POX6, BAT1, BAT2, YAT1, FAA1, ANT1, POT1, ACD1, IVD1, MFE1, PEX10, ARO10, GCDH1, GCDH2, ACD2, BCKD-E1α, BCKD-E1β, BCKD-E2 and homologs or orthologs thereof. In some embodiments, at least one gene is selected from the group consisting of YALI0_D15708g, YALI0_B10406g, YALI0_D01265g, YALI0_F19910g, YALI0_D08690g, YALI0_F05038g, YALI0_D23815g, YALI0_D20768g, YALI0_D06930g, YALI0_E32835g, YALI0_F10857g, YALI0_D24750g, YALI0_E27654g, YALI0_C23859g, YALI0_E06567g, YALI0_C16797g, YALI0_F2 3749g, YALI0_B04906g, YALI0_D02387g, YALI0_E15378g, YALI0_E18568g, YALI0_D17864g, YALI0_E03058g, YALI0_F21197g, YALI0_E12573g, YALI0_F28567g, YALI0_F22121g, YALI0_D00671g, YALI0_D06215g, YALI0_D09383g, YALI0_D09493g, YALI0_A07733g and homologs or orthologs thereof.

[0055] In some embodiments, the cells comprise an exogenous polynucleotide that expresses multiple enzymes of a cannabinoid synthesis pathway, including enzymes of the mevalonate, olivetolic acid (OA) / hexanoate, 2,4-dihydroxy-(iso)butylbenzoic acid (OA-(i)B) / (iso)valerate, and divaleric acid (DVA) / butyrate pathways. These cannabinoid synthesis pathway enzymes expressed in the cells include acyl-CoA synthetases, such as hexanoyl-CoA, (iso)valeryl-CoA, and butyryl-CoA synthetases, polyketide synthases, polyketide cyclases, geranyl pyrophosphate (GPP) synthases, prenyltransferases, such as cannabigerolic acid (CBGA) synthase, unsaturated cannabigerolic acid (CBGA-uns) synthase, ketolized cannabigerolic acid (CBGA-uns) synthase, and ketolized cannabigerolic acid (CBGA-uns) synthase. GA-keto) synthase, cannabigerol (iso)butyrate (CBG(i)BA) synthase, cannabigerovaric acid (CBGVA) synthase, tetrahydrocannabic acid (THCA) synthase, unsaturated tetrahydrocannabic acid (THCA-uns) synthase, ketolized tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabi(iso)butolic acid (THC(i)BA) synthase, tetrahydrocannabivaric acid (THCVA) synthase, Cannabichromenic acid (CBCA) synthase, unsaturated cannabichromenic acid (CBCA-uns) synthase, ketolized cannabichromenic acid (CBCA-keto) synthase, cannabichromene(iso)butyrate (CBC(i)BA) synthase, cannabichromen-valeric acid (CBCVA) synthase, cannabidiolic acid (CBDA) synthase, unsaturated cannabidiolic acid (CBDA-uns) synthase, ketolized cannabidiolic acid (CBDA-keto) synthase, cannabi (iso)butanoic acid (CBD(i)BA) synthase, and / or cannabidivarinic acid (CBDVA) synthase, as well as HMG-CoA synthase, mevalonate kinase, acetyl-CoA synthase, acetyl-CoA carboxylase, acetyl-CoA acetyltransferase / HMG-CoA reductase, mevalonate-5-phosphate decarboxylase, isopentenyl phosphokinase, and hydroxymethylglutaryl-CoA reductase.

[0056] An expression vector or vectors can be constructed containing exogenous nucleotide sequences encoding these enzymes and other polypeptides described herein, operably linked to expression control sequences functional in cells. Applicable expression vectors include, for example, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which contain vectors operable for stable integration into the host chromosome and selection sequences or markers. In addition, the expression vector can contain one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes that provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not present in the culture medium can also be included. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, all of which are well known in the art. When two or more exogenous encoding nucleic acids are co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single vector expression, the encoding nucleic acids can be operably linked to one common expression control sequence, or can be linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. Transformation of the exogenous nucleic acid sequence can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the exogenous nucleic acid will be expressed in an amount sufficient to produce the desired enzyme or other product, and it will further be understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and disclosed herein.

[0057] In some embodiments, the cells express an acyl-CoA synthetase capable of producing acyl-CoA from carboxylic acids having 2 to 22 carbons. In some embodiments, the acyl-CoA synthetase can produce butyryl-CoA, (iso)valeryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoleyl-CoA, linoleyl-CoA, palmityl-CoA, and oleyl-CoA from (iso)valeric acid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, myristic acid, palmitic acid, linoleic acid, palmitic acid, and oleic acid. The acyl-CoA synthase can be a wild-type enzyme or can be engineered to produce acyl-CoA at a rate higher than wild-type acyl-CoA from Cannabis sativa. In some embodiments, the cells express a fusion protein comprising a protein with acyl-CoA synthetase activity fused to a protein with polyketide cyclase and / or a protein with polyketide synthase activity.

[0058] In some embodiments, the cells express a polyketide synthase capable of producing tetraketides from one or more acyl-CoA substrates selected from carboxylic acids having 2 to 22 carbons, such as, for example, acetyl-CoA, butyryl-CoA, (iso)valeryl-CoA, hexanoyl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoleyl-CoA, linoleyl-CoA, palmityl-CoA, and oleyl-CoA. The polyketide synthase can be a wild-type enzyme or can be engineered to produce tetraketides from acyl-CoA substrates at a rate greater than that of wild-type polyketide synthases from Cannabis sativa. In some embodiments, the cells express a fusion protein comprising a protein with polyketide synthase activity fused to a protein with polyketide cyclase and / or a protein with hexanoyl-CoA synthetase activity.

[0059] In some embodiments, the cells express a polyketide cyclase that can cyclize a tetraketide produced by the polyketide synthase to the corresponding 6-alkyl-2,4-dihydroxybenzoic acid. In some embodiments, cyclization of the tetraketide produces olivetolic acid (OA), an OA analog, 2,3-dihydroxy-6-(iso)butylbenzoic acid (OA-(i)B), an OA-(i)B analog, divalanic acid (DVA) or a DVA analog from the tetraketide, 2,4-dihydroxy-6-[(1E)-penten-1-yl]benzoic acid (OA-uns) or an OA-uns analog from the tetraketide, or 2,4-dihydroxy-6-(2-oxopentyl)benzoic acid (OA-keto) or an OA-uns analog from the tetraketide. In some embodiments, the polyketide cyclase is capable of producing OA, an OA analog, OA-(i)B, an OA-(i)B analog, DVA or a DVA analog, OA-uns or an OA-uns analog, OA-keto or an OA-keto analog at a rate greater than that of a wild-type polyketide cyclase from Cannabis sativa. In some embodiments, the cells express a fusion protein comprising a protein with polyketide synthase activity, a protein with acyl-CoA synthetase activity, a protein with prenyltransferase activity, and / or a protein with CBGA, CBG(i)BA, CBGVA, CBGA-uns, or CBGA-keto synthase activity.

[0060] In some embodiments, the cells express an aromatic soluble or membrane-bound prenyltransferase capable of transferring a prenyl group, such as a geranyl group, to pyrophosphate to produce geranyl pyrophosphate (GPP). In some embodiments, the prenyltransferase exhibits a preference for transferring a geranyl group to a pyrophosphate-containing group over a farnesyl group. In some embodiments, the prenyltransferase can produce GPP at a higher rate than wild-type prenyltransferases from Cannabis sativa. In some embodiments, the cells express a fusion protein comprising a protein with prenyltransferase activity fused to a protein with polyketide synthase activity, a protein with acyl-CoA synthetase activity, a protein with polyketide cyclase activity, and / or a protein with CBGA, CBG(i)BA, CBGVA, CBGA-uns, or CBGA-ketosynthase activity.

[0061] In some embodiments, the cells express wild-type or engineered cannabigerolic acid (CBGA), unsaturated cannabigerolic acid (CBGA-uns), ketolized cannabigerolic acid (CBGA), cannabigerol (iso)butyrate (CBG(i)BA), and / or cannabigerovalic acid (CBGVA) synthases that can form CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, or CBGVA from GPP and at least one of OA, OA-(i)B, or DVA. In some embodiments, the CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, and / or CBGVA synthases can produce CBGA and / or CBGVA at a higher rate than wild-type CBGA or CBGBA synthases from Cannabis sativa.

[0062] In some embodiments, the cells express wild-type or engineered cannabichromenic acid (CBCA), unsaturated cannabichromenic acid (CBCA-uns), ketolized cannabichromenic acid (CBCA-keto), cannabichromene(iso)butyric acid (CBC(i)BA), and cannabichromevaleric acid (CBCVA) synthases that can form CBCA, CBCA-uns, CBCA-keto, CBC(i)BA, or CBCVA from CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, or CBCVA. In some embodiments, the CBCA or CBCVA synthase can produce CBCA and / or CBCVA at a higher rate than wild-type CBCA or CBCBA synthase from Cannabis sativa.

[0063] In some embodiments, the cells express wild-type or engineered tetrahydrocannabic acid (THCA), unsaturated tetrahydrocannabic acid (THCA-uns), ketolized tetrahydrocannabic acid (THCA-keto), tetrahydrocannabi(iso)butolic acid (THC(i)BA), and tetrahydrocannabivaric acid (THCVA) synthases that can form THCA, THCA-uns, THCA-keto, THC(i)BA, or THCVA from CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, or CBGVA. In some embodiments, the THCA or THCVA synthase can produce THCA or THCVA at a higher rate than wild-type THCA or THCVA synthase from Cannabis sativa.

[0064] In some embodiments, the cells express wild-type or engineered cannabidiolic acid (CBDA), unsaturated cannabidiolic acid (CBDA-uns), ketolized cannabidiolic acid (CBDA-keto), cannabi(iso)butolic acid (CBD(i)BA), and / or cannabidivaric acid (CBDVA) synthases that can form CBDA, CBDA-uns, CBGA-keto, CBG(i)BA, or CBDVA from CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, or CBGVA. In some embodiments, the CBDA or CBDVA synthase can produce CBDA or CBDVA at a higher rate than wild-type CBDA or CBDVA synthase from Cannabis sativa.

[0065] In some embodiments, the engineered cells are capable of expressing cannabigerolic acid (CBGA) synthase, unsaturated cannabigerolic acid (CBGA-uns) synthase, ketolized cannabigerolic acid (CBGA-keto) synthase, cannabigerol (iso)butyrate (CBG(i)BA) synthase, cannabigerolic acid (CBGVA) synthase, tetrahydrocannabic acid (THCA) synthase, unsaturated tetrahydrocannabic acid (THCA-uns) synthase, ketolized tetrahydrocannabic acid (THCA ... tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic acid (THCA-keto) synthase, tetrahydrocannabic Tetrahydrocannabinic acid (THCA-keto) synthase, tetrahydrocannabi(iso)butolic acid (THC(i)BA) synthase, tetrahydrocannabivaric acid (THCVA) synthase, cannabichromenic acid (CBCA) synthase, unsaturated cannabichromenic acid (CBCA-uns) synthase, ketolized cannabichromenic acid (CBCA-keto) synthase, cannabichromene(iso)butyric acid (CBC(i)BA) synthase, cannabichromevaric acid (CBCVA) synthase, cannabidiolic acid (CBDA) synthase, unsaturated cannabidiolic acid (CBDA-uns) synthase, ketolized cannabidiolic acid (CBDA-keto) synthase, cannabib(iso)butolic acid (CBD(i)BA) synthase, cannabidivalic acid (CBDVA) synthase, hexanoyl-CoA synthetase, (iso)valeryl-CoA synthetase, butyryl-CoA synthetase, polyketide synthase, polyketide cyclase, geranyl-CoA The cannabinoid biosynthetic pathway expresses two or more enzymes of the cannabinoid biosynthesis pathway selected from the group consisting of mevalonate-5-phosphate decarboxylase, isopentenyl phosphokinase, and / or hydroxymethylglutaryl-CoA reductase.

[0066] In some embodiments, when cultured under appropriate conditions, the cells are capable of producing one or more cannabinoid derivatives selected from the group consisting of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA, and CBDVA, and their decarboxylated derivatives. In some embodiments, the appropriate conditions include the addition of a carbon source and / or nutrients in the presence of the cannabinoid derivatives. The carbon source or nutrients are not particularly limited and may include any carbon source or nutrient described herein or known in the art. In some embodiments, the carbon source or nutrient includes one or more of butyric acid, butanol, valeric acid, isovaleric acid, hexanoic acid, hexanol, glucose, glycerol, and / or oleic acid.

[0067] In some embodiments, the cells produce increased production of cannabinoid derivatives compared to control cells that have not been engineered to metabolize a reduced number of acyl-CoA molecules and / or a reduced number of amino acids by deletion, inactivation, or other mutation of at least one gene that encodes an enzyme or other protein that affects the beta-oxidation pathway and affects the acyl-CoA catabolic and / or branched-chain amino acid catabolic pathways. In some embodiments, the cells have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more increased production of cannabinoid derivatives compared to control cells. In some embodiments, the cells have a 1.1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold or more increased flux of a cannabinoid derivative compared to control cells.

[0068] In some embodiments, the at least one gene comprises a combination of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more genes that directly or indirectly affect the beta-oxidation pathway, directly or indirectly affect acyl-CoA metabolism, and / or directly or indirectly affect branched-chain amino acid catabolism.

[0069] In some embodiments, at least one gene comprises a combination of POX3, POX5 and one or both of ACD1 and IVD1, and the cells exhibit synergistic production of cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations or mutations of only ACD1, IVD1 or the POX3 / POX5 combination, hi some embodiments, at least one gene comprises a combination of YALI0_D15708g and YALI0_E12573g.

[0070] In some embodiments, at least one gene comprises POX3, POX5, ACD1 in combination with one or more of POX2, POX6, GCDH1, GCDH2, MFE1, YAT1, FAA1, ANT1, POT1, ACD2 and PEX10, and the cells exhibit enhanced production of cannabinoids compared to the production of cannabinoids achieved in a cell comprising a deletion, inactivation or mutation of only the combination of POX3, POX5 and ACD1. In some embodiments, at least one gene comprises IVD1 in combination with at least one of BAT1 or BAT2, and the cells exhibit enhanced production of one or more C5 branched-chain cannabinoids compared to the production of the same one or more C5 branched-chain cannabinoids achieved in a cell comprising a deletion, inactivation or mutation of only IVD1. In some preferred embodiments, the one or more C5 branched chain cannabinoids include one or more of CBGiBA, CBDiBA, CBCiBA and / or THCiBA.

[0071] In some embodiments, the at least one gene comprises a combination of at least two of POX3, POX5, and ACD1. In some embodiments, the at least one gene comprises a combination of POX3, POX5, ACD1, and FAA1, and the cells exhibit significantly improved production of C4 cannabinoids compared to the production of C4 cannabinoids achieved in cells comprising deletions, inactivations, or mutations of only POX3, POX5, and ACD1. In other embodiments, the at least one gene comprises a combination of POX3, POX5, ACD1, and YAT1, and the cells exhibit significantly improved production of cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations, or mutations of only POX3, POX5, and ACD1.

[0072] In some embodiments, the at least one gene comprises a combination of IVD1 and at least one of BCKD-E1α and BCKD-E2, and the cells exhibit reduced production of C5 branched cannabinoid products compared to the production of C5 branched cannabinoids achieved in cells comprising a deletion, inactivation, or mutation of IVD1 alone. In some embodiments, the cells further exhibit significantly increased production of CBGVA compared to the production of CBGVA achieved in cells comprising a deletion, inactivation, or mutation of IVD1 alone. In some embodiments, the at least one gene further comprises a combination of at least two of POX3, POX5, ACD1, and FAA1.

[0073] In some embodiments, the cells express a mutant farnesyl pyrophosphate synthase protein (FPPS). In some embodiments, the mutant FPPS is ERG20.A28 and is capable of producing an increased ratio of GPP to FPP. In some embodiments, the cells have elevated levels of GPP compared to control cells.

[0074] In some embodiments, the cell may be a microorganism or a plant cell. Suitable cells are Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia species, Saccharomyces cerevisiae, Saccharomyces species, Hansenula polymorpha (now known as Pichia angusta), Kluyveromyces species, Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pompe, Dekkera bruxellensis, Arxula adeninivorans, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, Yarrowia lipolytica, and the like. In some embodiments, the cell is a protease-deficient strain of Saccharomyces cerevisiae. In some embodiments, the cell is Yarrowia lipolytica. In some embodiments, the cell is a eukaryotic cell other than a plant cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a plant cell that does not normally produce cannabinoids, cannabinoid derivatives or analogs, cannabinoid precursors, or cannabinoid precursor derivatives or analogs.In some embodiments, the cells disclosed herein are cultured in vitro.

[0075] In some embodiments, the cell is a prokaryotic cell. Suitable prokaryotic cells may include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus species, Salmonella species, Shigella species, and the like. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains may include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, laboratory strains are non-pathogenic strains. Non-limiting examples of other suitable bacteria may include, but are not limited to, Bacillus subtilis, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus species, and the like.

[0076] How to Produce Cannabinoids Some aspects of the present disclosure are directed to methods of producing a cannabinoid or derivative thereof, comprising culturing the cells disclosed herein under suitable conditions to produce the cannabinoid or derivative thereof.

[0077] Depending on the cell, an appropriate culture medium can be used. For example, descriptions of various culture media can be found in the "Manual of Methods for General Bacteriology" (Washington DC, USA, 1981) of the American Society for Bacteriology. As used herein, "culture medium" in relation to a growth source refers to the starting medium, whether in solid or liquid form. On the other hand, as used herein, "cultured medium" refers to a medium (e.g., a liquid medium) containing fermentatively grown microorganisms and may contain other cellular biomass. A culture medium generally includes one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and / or trace elements.

[0078] Exemplary carbon sources include sugar carbons such as sucrose, glucose, galactose, fructose, mannose, isomaltose, xylose, panose, maltose, arabinose, cellobiose, and their 3-, 4-, or 5-oligomers. Other carbon sources include alcohol carbon sources such as methanol, ethanol, glycerol, butanol, and hexanol. Other carbon sources include acids and esters such as acetate esters, formate esters, and fatty acids having 4 to 22 carbon atoms or their fatty acid esters. Other carbon sources can include renewable feedstocks and biomass. Exemplary renewable feedstocks include cellulosic biomass, hemicellulosic biomass, and lignin feedstocks. Mixed carbon sources, such as fatty acids and sugars, as described herein, can also be used. In some embodiments, the appropriate conditions, including adding to the culture medium in which the cells are cultured, include adding at least one of butyric acid, butanol, butyl butyrate, valeric acid, isovaleric acid, hexanoic acid, hexanol, hexyl hexanoate, oleic acid, glycerol, or glucose.

[0079] Culture conditions can include, for example, liquid culture procedures and fermentation and other large-scale culture procedures.Under aerobic culture conditions, useful yields of products can be obtained.Exemplary growth conditions for achieving one or more cannabinoid products include aerobic culture or fermentation conditions.In certain embodiments, microorganisms can be maintained, cultured or fermented under aerobic conditions.

[0080] Substantially aerobic conditions include, for example, cultures, batch fermentations, or continuous fermentations in which the dissolved oxygen concentration in the medium remains between 5% and 100% of saturation. The percentage of dissolved oxygen can be maintained, for example, by sparging with air, pure oxygen, or a mixture of air and oxygen.

[0081] Culture conditions can be scaled up and continuously grown to produce cannabinoid products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation; or continuous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of cannabinoid products. Generally, and similar to non-continuous culture procedures, continuous and / or near-continuous production of cannabinoid products involves culturing a cannabinoid-producing organism on nutrients and medium sufficient to maintain and / or nearly maintain exponential growth. Continuous culture under such conditions can include, for example, 1, 2, 3, 4, 5, 6, or 7 days or more. In addition, continuous culture can include 1, 2, 3, 4, or 5 weeks or more, and up to several months. Alternatively, if suitable for a particular application, the desired microorganism can be cultured for several hours. It should be understood that continuous and / or near-continuous culture conditions can also include all time intervals between these exemplary periods. It is further understood that the time for culturing the microorganism is for a period of time sufficient to produce a sufficient amount of product for the desired purpose.

[0082] Fermentation procedures are well known in the art. Briefly, for example, fermentation for the biosynthetic production of cannabinoid products can be used in fed-batch fermentation and batch separation; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[0083] In some embodiments, cells are grown in stirred tank fermenters with feed addition (sugars with or without organic acids), where dissolved oxygen, temperature, and pH are adjusted according to optimal growth and production processes. In some embodiments, aqueous-immiscible organic solvents are added to dissolve the added organic acids or to extract the cannabinoid product as it is synthesized. In some embodiments, these solvents may include, but are not limited to, isopropyl myristate (IPM), diisobutyl adipate, decane, dodecane, hexadecane, or other organic solvents with a logP > 5. The latter number (logP) is defined as the logarithm of the compound's partition between water and octanol and is a standard parameter of a compound's hydrophobicity (the higher the logP, the less soluble it is in water). Depending on the fermentation process, the product may be isolated and purified using different methods.

[0084] If no organic co-solvents are used and the target cannabinoids are secreted into the culture supernatant, different methods can be applied. In one embodiment, an aqueous-miscible organic solvent (ethanol, acetonitrile, etc.) is added to dissolve the product. In some embodiments, simple filtration, ultrafiltration, or centrifugation can remove the cells, and the aqueous medium is evaporated to dryness or to a small volume where the cannabinoid product precipitates or crystallizes. Alternatively, to extract the cannabinoids, the cell supernatant can be extracted with an aqueous-immiscible organic solvent (ethyl acetate, heptane, decane, etc.). Evaporation of the organic solvent and possible recrystallization produces pure cannabinoids. If the cannabinoid products are not secreted into the culture medium but are trapped inside the cells, different methods for their extraction and purification can be utilized. In some embodiments, the cells are disrupted using mechanical methods or by suspension in an appropriate lysis buffer, which allows cannabinoids to be extracted with an organic-aqueous-immiscible solvent (ethyl acetate, hexane, decane, methylene chloride, etc.). In other embodiments, the cells can be suspended in an organic solvent (ethanol, methanol, methylene chloride, etc.) to extract the cannabinoids from the cells.

[0085] In some embodiments, an organic solvent is required during growth, which is separated at the end of fermentation. Back-extraction with a basic aqueous solvent or a different organic solvent with a low boiling point and high polarity (ethanol, acetonitrile, etc.) removes the cannabinoids. Isolation can then involve a simple pH shift if water is used, or evaporation if an organic solvent is used. In either case, a final recrystallization step may be required to improve the purity of the product.

[0086] In some embodiments, the method further comprises purifying or isolating cannabinoids, derivatives or analogs thereof from the culture.Isolation method is not limited and can be any suitable method known in the art.Purification method includes, for example, extraction procedures and methods including continuous liquid-liquid extraction, pervaporation, evaporation, filtration, membrane filtration (including reverse osmosis, nanofiltration, ultrafiltration and microfiltration), membrane filtration with diafiltration, membrane separation, reverse osmosis, electrodialysis, distillation, extractive distillation, reactive distillation, azeotropic distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, carbon adsorption, hydrogenation and ultrafiltration.

[0087] In some embodiments, the cannabinoids, cannabinoid derivatives, and cannabinoid analogs described herein produced by the methods are not limited. In some embodiments, cannabinoids may include, but are not limited to, cannabichromene (CBC) types (e.g., cannabichromene acid), cannabigerol (CBG) types (e.g., cannabigerolic acid), cannabidiol (CBD) types (e.g., cannabidiolic acid), Δ9-trans-tetrahydrocannabinol (Δ9-THC) types (e.g., Δ9-tetrahydrocannabinolic acid), Δ8-trans-tetrahydrocannabinol (Δ8-THC) types, cannabicyclol (CBL) types, cannabielsoin (CBE) types, cannabinol (CBN) types, cannabinodiol (CBND) types, and cannabiditriol (CBT) types. In some embodiments, cannabinoids, cannabinoid derivatives and cannabinoid analogs include cannabigerolic acid (CBGA), unsaturated cannabigerolic acid (CBGA-uns), ketolized cannabigerolic acid (CBGA-keto), cannabigerolic acid monomethyl ether (CBGAM), cannabigerol (CBG), unsaturated cannabigerol (CBG-uns), ketolized cannabigerol (CBG-keto), cannabigerol monomethyl ether (CBGM), cannabigerolic acid (CBGVA), unsaturated cannabigerolic acid (CBGVA-uns), ketolized cannabigerolic acid (CBGVA-keto), cannabigerolic acid (CBGV), unsaturated cannabigerolic acid (CBGVA-uns), ketolized cannabigerolic acid (CBGVA-keto), cannabigerolic acid (CBGV), unsaturated cannabigerolic acid (CBGVA-uns), cannabigerolic acid (CBGVA-keto), cannabigerolic acid (CBGV), cannabigerolic acid (CBGVA ... Phosphorus (CBGV-uns), ketolized cannabichromenic acid (CBGV-keto), cannabichromenic acid (CBCA), unsaturated cannabichromenic acid (CBCA-uns), ketolized cannabichromenic acid (CBCA-keto), cannabichromene (CBC), unsaturated cannabichromene (CBC-uns), ketolized cannabichromene (CBC-keto), cannabichromevaric acid (CBCVA), unsaturated cannabichromevaric acid (CBCVA-uns), ketolized cannabichromevaric acid (CBCVA-keto), cannabichromevarin (CBCV), unsaturated cannabichromevarin (CBCV-uns), ketolized cannabichromevarin (CBCV-keto), cannabidiolic acid (CBDA),Unsaturated cannabidiolic acid (CBDA-uns), ketolized cannabidiolic acid (CBDA-keto), cannabidiol (CBD), unsaturated cannabidiol (CBD-uns), ketolized cannabidiol (CBD-keto), cannabidiol monomethyl ether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivaric acid (CBDVA), unsaturated cannabidivaric acid (CBDVA-uns), ketolized cannabidivaric acid (CBDVA-keto), cannabidivarin (CBDV), cannabidiolchol (CBD-C1), Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinolic acid B (THCA-B), unsaturated Δ9-tetrahydrocannabinolic acid A (THCA-A-uns), ketolized Δ9-tetrahydrocannabinolic acid A (THCA-A-keto), unsaturated Δ9-tetrahydrocannabinolic acid B (THCA-B-uns), ketolized Δ9-tetrahydrocannabinolic acid B (THCA-B-keto), Δ9-tetrahydrocannabinol (THC), unsaturated Δ9-tetrahydrocannabinol (THC-uns), ketolized Δ 9-Tetrahydrocannabinol (THC-keto), Δ9-tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9-tetrahydrocannabinol-C4 (THC-C4), Δ9-tetrahydrocannabivarinic acid (THCVA), unsaturated Δ9-tetrahydrocannabivarinic acid (THCVA-uns), ketolized Δ9-tetrahydrocannabivarinic acid (THCVA-keto), Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabiorcholic acid (THCA-C1), Δ9-tetrahydrocannabiorcholic acid (THC-C1) ), Δ7-cis-iso-tetrahydrocannabivarin, Δ8-tetrahydrocannabinolic acid (Δ8-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovalin (CBLV), cannabielsonic acid A (CBEA-A), cannabielsonic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methyl ether (CBNM),Cannabinol-C4 (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiocol (CBN-C1), cannabinodiol (CBND), cannabinodivarine (CBVD), cannabidiol (CBT), 10-ethyoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a-tetrahydrocannabinol, cannabidiol valine (CBTVE), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabi Contains one or more of chromanone (CBCN), cannabiditran (CBT), 10-oxo-delta-6a-tetrahydrocannabinol (OTHC), delta-9-cis-tetrahydrocannabinol (cis-THC), 3,4,5,6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n-propyl-2,6-methano-2H-1-benzoxocine-5-methanol (OH-iso-HHCV), cannabilipsol (CBR), and trihydroxy-delta-9-tetrahydrocannabinol (tri-OH-THC). In a preferred embodiment, the cannabinoids or derivatives thereof produced by the method include one or more of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA and CBDVA, and their decarboxylated derivatives.

[0088] In some embodiments, production of one or more of these cannabinoids is increased by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more compared to use of the method with control cells that do not have a deletion, inactivation, or other mutation in at least one gene that affects the beta-oxidation pathway and affects the catabolism of acyl-CoA molecules and / or the catabolism of branched-chain amino acids. In some embodiments, the production of at least one of these cannabinoids is increased by 1.1 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold or more compared to use of the method with a control cell that does not have a deletion, inactivation or other mutation in at least one gene that affects the beta-oxidation pathway and affects the catabolism of acyl-CoA molecules and / or the catabolism of branched chain amino acids.

[0089] Engineered cells and methods for producing C5 branched cannabinoids, unsaturated cannabinoids and ketolized cannabinoids Some embodiments of the present disclosure are also directed to cells engineered to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, wherein the cells comprise an inactivated or otherwise mutated IVD1, POT1 and / or MFE1 gene, and the cells, when cultured under appropriate conditions, such as those previously described herein, further express sufficient enzymes in the cannabinoid synthesis pathway to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof. In some embodiments, suitable conditions include supplementing the culture medium in which the cells are cultured with butyric acid, valeric acid, isovaleric acid, hexanoic acid, oleic acid, butanol, hexanol, glycerol, or glucose.

[0090] Some embodiments of the present disclosure are directed to methods of producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, by culturing a cell disclosed herein under conditions suitable for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof.

[0013] The present invention is directed to a method for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns, and / or CBD-keto, or decarboxylated derivatives thereof, by culturing the cells disclosed herein under conditions suitable for producing CBCA-uns, CBCA-keto, CBDiBA, CBD-uns, and / or CBD-keto, or decarboxylated derivatives thereof. In some embodiments, the suitable conditions include adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured. In some embodiments, the method includes isolating CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, from the culture using methods previously described herein.

[0091] Some other aspects of the present disclosure include the following structure: [ka] [ka] [ka] [ka] [ka] The present invention is directed to a compound according to any one of the preceding claims.

[0092] Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The detailed description and examples herein are representative of certain embodiments and are exemplary and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0093] As used in this specification and claims, the articles "a" and "an" should be understood to include plural referents unless a contrary meaning is clearly indicated. Unless otherwise stated or apparent from context, a claim or description including "or" between one or more elements of a group is considered satisfied if one, more than one, or all of the group elements are present in, utilized in, or otherwise associated with a given object or process. The invention includes embodiments in which exactly one element of the group is present in, utilized in, or otherwise associated with a given object or process. The invention also includes embodiments in which more than one or all of the group elements are present in, utilized in, or otherwise associated with a given object or process. Furthermore, it should be understood that the present invention provides all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims introduced into another claim (or any other related claim) dependent on the same base claim, unless otherwise indicated or unless a contradiction or inconsistency would arise, apparent to one skilled in the art. All embodiments described herein are contemplated as being applicable to all different aspects of the invention, where appropriate. It is also contemplated that any of the embodiments or aspects may be freely combined, where appropriate, with one or more other such embodiments or aspects. Where elements are presented as lists, e.g., in a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed, and that any one or more elements can be removed from the group. In general, when the invention or aspects of the invention are referred to as including particular elements, features, etc., it should be understood that a particular embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc.For simplicity, the embodiments herein are not specifically and specifically described in so many words in every instance. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is described herein. For example, any one or more nucleic acids, polypeptides, cells, species or types of organisms, disorders, subjects, or combinations thereof may be excluded.

[0094] Where a claim or description is directed to a composition, e.g., a nucleic acid, polypeptide, or cell, it should be understood that methods of making or using the composition in accordance with any of the methods disclosed herein, and methods of using the composition for any of the purposes disclosed herein, are aspects of the invention, unless otherwise indicated or unless a contradiction or inconsistency would arise, apparent to one of ordinary skill in the art. Where a claim or description is directed to a method, it should be understood that, for example, methods of making compositions useful for practicing the method, and products produced in accordance with the method, are aspects of the invention, unless otherwise indicated or unless a contradiction or inconsistency would arise, apparent to one of ordinary skill in the art.

[0095] When ranges are given herein, the invention includes embodiments in which multiple endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Unless otherwise indicated, both endpoints should be assumed to be inclusive. Furthermore, unless otherwise stated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to encompass any specific value or subrange within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. When a series of numerical values ​​is described herein, it is also understood that the invention includes embodiments similar to any intervening value or range defined by any two values ​​in the series, and that the smallest value may be taken as a minimum value and the largest value may be taken as a maximum value. Numerical values ​​used herein include values ​​expressed as percentages. For any embodiment of the invention in which a numerical value is preceded by "about" or "approximately," the invention includes embodiments in which the exact value is recited. For any embodiment of the invention where a numerical value is not preceded by "about" or "approximately," the invention includes embodiments in which the value is preceded by "about." "Approximately" or "about" generally includes numbers that fall within 1% of the number in either direction (greater or less than the number), or in some embodiments, within 5% of the number, or in some embodiments, within 10% of the number, unless otherwise stated or clear from context (except where such number would unacceptably exceed 100% of possible values). Unless expressly indicated otherwise, in any method claimed herein that includes more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are described, but it is understood that the invention includes embodiments in which the order is so limited. It is also understood that any product or composition described herein can be considered "isolated" unless otherwise indicated or clear from context.

[0096] The present invention and its advantages are further illustrated by the foregoing non-limiting examples. [Example]

[0097] Example 1. Rationale for deleting ACD1 and ECH1 in Yarrowia lipolytica cannabinoid producing strains. One aspect of the present invention is metabolic engineering to conserve cellular pools of acyl-CoA molecules for improved cannabinoid production. An overview of the cannabinoid metabolic pathway, including some of the key acyl-CoA molecules (hexanoyl-CoA, butyryl-CoA, (iso)valeryl-CoA, trans-2-hexenoyl-CoA, and 3-oxohexanoyl-CoA), is shown in Figure 1 and Table 1. Hexanoyl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-CoA, (iso)valeryl-CoA, and butyryl-CoA are key precursors for the formation of the intermediates olivetolic acid (OA), OA-uns, OA-keto, OA-(i)B, and divalanic acid (DVA), respectively, which condense with GPP to form the initial cannabinoids CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, and CBGVA, respectively. These initial cannabinoids (CBGA, CBGA-uns, CBGA-keto, CBG(i)BA, and CBGVA) can be converted to various final cannabinoids, as shown in Figure 1. To improve microbial cannabinoid production, we hypothesized that cells would need to direct hexanoyl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-coA, (iso)valeryl-CoA, or butyryl-CoA toward OA, OA-(i)B, or DVA, and away from competing pathways. [Table 1]

[0098] Fatty acid β-oxidation is the process of degrading long-chain acyl-CoA molecules into acetyl-CoA molecules and is a major pathway that competes with the cannabinoid formation pathway for hexanoyl-CoA and butyryl-CoA. This process involves various enzymes, and the four key enzymes are, in order, acyl-CoA dehydrogenase, enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, and ketoacyl-CoA thiolase (Figure 2).

[0099] In mammals, β-oxidation occurs in both mitochondria and peroxisomes, whereas in plants and most fungi, the β-oxidation cycle occurs exclusively in peroxisomes. Indeed, the literature states that "yeast and fungi, including the ascomycete fungi Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida tropicalis, appear to possess peroxisomal β-oxidation capacity, specifically the D-isomer pathway" (Kunau et al., 1988; Hiltunen et al., 1992; Kurihara et al., 1992; Smith et al., 2000).

[0100] Y. lipolytica contains six acyl-CoA oxidases, encoded by the POX1–POX6 genes, that catalyze the rate-limiting step (acyl-CoA dehydrogenase) of peroxisomal β-oxidation. Because the POX genes have been characterized and the acyl chain specificity determined for each encoded enzyme (Wang et al., 1999), a subset of these genes can be targeted for disruption to generate engineered cells with increased acyl-CoA availability for cannabinoid production. In contrast, to our knowledge, there are no reports confirming mitochondrial β-oxidation gene targets in Y. lipolytica. However, mitochondrial β-oxidation has been reported in the filamentous fungus Aspergillus nidulans and shown to be important for the catabolism of short-chain (C4–C6) fatty acids, such as butyryl-CoA and hexanoyl-CoA (Maggio-Hall and Keller, 2004). For this purpose, we used human mitochondrial short-chain / branched-chain-specific acyl-CoA dehydrogenase (GenBank: AAH13756.1) in a BLAST search against the Y. lipolytica genome. The top hit in the BLAST search was YALI0_D15708g, which has 58.7% sequence identity (amino acid) with human mitochondrial short-chain / branched-chain-specific acyl-CoA dehydrogenase. This gene is called ACD1 (Acyl-CoA dehydrogenase). Furthermore, we used human mitochondrial short-chain / branched-chain-specific enoyl-CoA hydratase (GenBank: EAA57779) in a BLAST search against the Y. lipolytica genome. YALI0_B10406g was the top hit in the BLAST search and is 52.5% identical (amino acid) to a human mitochondrial short-chain / branched-chain specific enoyl-CoA hydratase, which we refer to as ECH1 (Enoyl-CoA hydratase).Despite the low % amino acid identity with both the ACD and ECH homologs, we decided to disrupt these genes in Y. lipolytica strains engineered to produce cannabinoids to assess whether inactivating these enzyme activities would increase the ability of the engineered cells to make cannabinoids, possibly through increased availability of hexanoyl-CoA and butyryl-CoA.

[0101] Example 2. Assays used to assess cannabinoid formation from glucose with or without added fatty acids. Individual clones from the agar plates were used to inoculate, in triplicate, 0.5 mL of YDCM medium (yeast nitrogen base plus nitrogen, 6.71 g / L; casamino acids, 10 g / L; dextrose monohydrate, 66 g / L; MES hydrate, 19.5 g / L; pH adjusted to 6.5 with KOH) in 96-deep-well blocks grown at 30°C with shaking at 990 rpm. After 24 h, 2 μL from each preculture was used to inoculate 0.5 mL of YDCM medium in 96-deep-well blocks. At the time of inoculation, the cultures were supplemented with 2.5 mM of either butyric acid, butyl butyrate, butanol, valeric acid, hexanoic acid, hexyl hexanoate, hexanol, or no fatty acids and incubated at 30°C with shaking at 990 rpm. After 24 h of growth, the cultures were supplemented with an additional 3 mM of the same fatty acid and incubated at 30° C. with shaking at 990 rpm. After another 24 h (48 h total), the cultures were quenched with quench solution (0.5 mL of ethanol containing 0.1 mg / mL pentylbenzoic acid) and subjected to LC analysis.

[0102] Example 3. Analytical chemistry methods for cannabinoids. All samples after quenching with an equal volume of quench solution were centrifuged and analyzed by HPLC-MS.

[0103] Column: 2.1 x 50 mm COSMOCORE PBr (Nacalai USA, Inc.)

[0104] Buffer:

[0105] Buffer A: water, 0.1% formic acid

[0106] Buffer B: acetonitrile, 0.1% formic acid

[0107] Flow rate: 0.45mL / min

[0108] Column temperature: 50℃

[0109] Injection volume: 1 μL [Table 2] [Table 3-1] [Table 3-2]

[0110] The retention times for all cannabinoid compounds listed in Table 1 are recorded in Table 3. All compounds were confirmed by LC-MS analysis using mass spectrometry of the theoretical molecular weights.

[0111] Example 4. ACD1 and ECH1 deletion strains SB-01213, SB-01209, SB-01215 and SB-01211. ACD1 and ECH1 were disrupted in two cannabinoid-producing strains, SB-00889 and SB-01052. The genotypes of these strains are listed in Table 34. Both strains were engineered to convert added butyrate or hexanoate to CBGVA or CBGA, respectively. One key difference between the two strains is that SB-0889 has an intact peroxisomal β-oxidation pathway, while SB-01052 has certain acyl-CoA oxidases (POX3 and POX5) inactivated. Standard molecular biology cloning techniques were used to disrupt ACD1 and ECH1 in strains SB-00889 and SB-01052. Deletion of ACD1 in strains SB-00889 and SB-01052 resulted in strains SB-01213 and SB-01209, respectively, and deletion of ECH1 in strains SB-00889 and SB-01052 resulted in strains SB-01215 and SB-01211, respectively.

[0112] Example 5. Evaluating CBGA formation from a glucose + hexanoic acid feed in an ACD1 deletion strain. Clones derived from SB-01209 and SB-01213 were evaluated, along with the parental control strain, under the conditions described in Example 2. As shown in Figures 1 and 2, activation of hexanoic acid to hexanoyl-CoA occurs, which is the direct precursor to OA, the direct precursor to the cannabinoid CBGA, and a substrate for β-oxidation. In the assays performed, all hexanoic acid was consumed by the strains in question; therefore, the amount of OA and CBGA produced is a measure of how efficiently these strains convert hexanoyl-CoA to cannabinoids, compared to degrading hexanoyl-CoA via β-oxidation. The amounts of OA and CBGA produced by these strains are shown in Table 4. The data in Table 4 show that ACD1 inactivation in cannabinoid-producing strains increased both OA and CBGA titers from hexanoic acid (compare SB-01213 with SB-00889). This indicates that inactivation of mitochondrial β-oxidation can improve cannabinoid production. Comparison of SB-01052 (Δpox3Δpox5) with SB-00889 indicates that disruption of pox3 and pox5 results in improved cannabinoid production. Additionally, the SB-01209 clone inactivated both mitochondrial (ACD1) and peroxisomal (POX3 and POX5) β-oxidation genes, indicating that this combination has a synergistic effect on increasing cannabinoid production. ECH1-inactivated strains were evaluated in the same manner as the ACD1-inactivated strains, but none of the clones produced more OA and / or CBGA (data not shown). [Table 4]

[0113] Example 6. Inactivation of IVD1 in Yarrowia lipolytica cannabinoid-producing strains. Building on the success of the ACD1 deletion strain, additional mitochondrial acyl-CoA dehydrogenases were targeted for inactivation to further preserve short- and medium-chain acyl-CoA molecules and increase cannabinoid production. Specifically, 12 ACD and ECH homologs (Table 5) were identified and then disrupted in cannabinoid-producing strains using standard molecular biology techniques. One targeted gene, when disrupted, resulted in a strain capable of producing rare cannabinoids. This gene is a likely ACD homolog encoding isovaleryl-CoA dehydrogenase (IVD1; YALI0_E12573g), which is likely part of the leucine catabolic pathway (Figure 3). Leucine is deaminated by aminotransferase to α-ketoisocaproic acid, which is subsequently decarboxylated by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKD complex) to isovaleryl-CoA, which is subsequently dehydrated by isovaleryl-CoA dehydrogenase and finally decarboxylated to acetyl-CoA and acetoacetate. Inactivation of IVD1 should result in the accumulation of isovaleryl-CoA, a potential substrate for producing rare cannabinoids. [Table 5]

[0114] Using standard molecular biology cloning techniques, IVD1 was disrupted in SB-01052 (POX3 and POX5 inactivated) and SB-01268 (POX3, POX5, and ACD1 inactivated), which were engineered to produce CBGVA and CBGA from added butyrate and hexanoate, respectively, resulting in strains SB-01343 and SB-01345. The genotypes of these strains are listed in Table 34.

[0115] Example 7. Assessing cannabinoid formation in IVD1 deletion strains without fatty acid supplementation As described in Example 2, the IVD1-inactivated strains were assayed for their ability to produce cannabinoids without added fatty acids. The results are shown in Table 6. These data indicate that SB-01343 and SB-01355, in which IVD1 was inactivated, produced the C5 branched-chain cannabinoids OA-iB and CBGiBA, whereas the parent strains did not. In addition to the parent strains in which POX3 and POX5 were inactivated, SB-01268 also had ACD1 inactivation. Based on the strain genotypes and the data generated, it appears that inactivation of IVD1 is required to produce branched-chain cannabinoids, whereas inactivation of ACD1, POX3, and POX5 is not sufficient. [Table 6]

[0116] Example 8: Evaluating cannabinoid formation in strain IVD1 with fatty acid supplementation To assess whether IVD1 and ACD1 disruption has synergistic consequences on final cannabinoid titer, SB-01343 and SB-01355, along with the parental control strains SB-01052 and SB-01268, were assayed under the fermentation conditions described in Example 2. The results are recorded in Tables 7, 8, and 9. In the assays performed, all of the added butyric, valeric, and hexanoic acids were consumed by the strains in question; therefore, the amount of DVA+CBGVA, OA-B+CBGBA, and OA+CBGA produced is a measure of how efficiently these strains convert these molecules into cannabinoids compared to breaking down butyryl-CoA, valeryl-CoA, and hexanoyl-CoA via various catabolic processes.

[0117] Because inactivation of POX3 and POX5 is common to all strains, SB-01052 can be used as a baseline. When ACD1 (SB-01268) or IVD1 (SB-01343) are inactivated in SB-01052, these strains produce fewer total cannabinoids when butyrate or valerate is added. However, when both ACD1 and IVD1 (SB-01355) are inactivated in SB-01052, these strains produce more total cannabinoids when butyrate or valerate is added. These data suggest that ACD1 and IVD1 have overlapping activities toward butyryl-CoA and valeryl-CoA, and that inactivation of both is required to improve cannabinoid production derived from butyrate or valerate.

[0118] When ACD1 (SB-01268) or IVD1 (SB-01343) are inactivated in SB-01052, these strains produce more total cannabinoids when hexanoic acid is added. These data suggest that deletion of either ACD1 or IVD1 can improve production of cannabinoids derived from hexanoic acid. However, when ACD1 and IVD1 (SB-01355) are inactivated in SB-01052, these strains do not produce more total cannabinoids when hexanoic acid is added than the individual deletions. [Table 7] [Table 8] [Table 9]

[0119] Example 9: Experimental procedures used to assess the expression of the IVD1 and ACD1 genes. Individual clones from the agar plates were used to inoculate, in triplicate, 2 mL of YDCM medium in 24-deep-well blocks grown at 30°C with shaking at 250 rpm. After 24 hours, 10 μL from each preculture was used to inoculate 2 mL of YDCM medium in 24-deep-well blocks incubated at 30°C with shaking at 250 rpm. After 24 hours, either 3 mM butyrate or hexanoic acid was added to the cultures. After an additional 6 hours (30 hours total), cells from 1 mL of culture were harvested by centrifugation at 6000 g for 3 minutes. The supernatant was removed, and the cell pellets were frozen and stored in a -80°C freezer. RNA was extracted from individual cell pellets using a commercially available Quick-RNA Fungal / Bacterial Miniprep Kit (Zymo Research). RNA samples were reverse transcribed into cDNA using the LunaScript® RT SuperMix Kit (NEB), which was then used for qPCR analysis. The qPCR primer sequences used for ACD1, IVD1 and the reference gene TAF10 are SEQ ID NOs: 1 to 6.

[0120] Example 10: Evaluating the expression of ACD1 and IVD1 genes in cannabinoid producing strains. A subset of strains from Table 34 was used to perform transcriptional analysis of ACD1 and IVD1 under fermentation conditions supplemented with either butyrate or hexanoic acid. The results are shown in Figures 4 and 5. In response to butyrate supplementation, ACD1 showed higher expression when IVD1 was inactivated (SB-01343, in which POX3, POX5, and IVD1 were inactivated, whereas SB-01052, in which POX3 and POX5 were inactivated). Only baseline signals were observed in the ACD1-disrupted strains SB-01268 and SB-01355. Similarly, IVD1 showed higher expression when ACD1 was inactivated (SB-01268, in which POX3, POX5, and ACD1 were inactivated, whereas SB-01052, in which POX3 and POX5 were inactivated). Only baseline signals were observed in the IVD1-disrupted strains SB-01343 and SB-01355. These data indicate that both ACD1 and IVD1 are expressed when cells are grown in the presence of butyrate, and that expression of one gene increases upon inactivation of the other. This is consistent with these genes having overlapping activities and the need to inactivate both to improve cannabinoid production from butyrate or valerate. The same trend was observed for hexanoate (Figure 5B).

[0121] Example 11: Inactivation of both ACD1 and IVD1 in THCA-producing strains. Using standard molecular biology cloning techniques, IVD1 was disrupted in SB-01542, which had ACD1 inactivated and was engineered to produce THCVA and THCA from added butyryl-CoA and hexanoyl-CoA, respectively, resulting in strain SB-01703. The genotypes of these strains are listed in Table 34.

[0122] Example 12: Evaluating SB-01703 in fermentations without added fatty acids Twenty-seven individual clones of SB-01703, along with the parent strain SB-01542, were assayed as described in Example 2 without added fatty acids. The data are shown in Tables 10 and 11. Surprisingly, we found that our strain, which contains functional genes for producing the terminal molecule THCA, was able to produce butyryl-CoA-derived cannabinoids without added butyrate. Furthermore, SB-01703 appeared to have increased availability of butyryl-CoA, resulting in a two-fold increase in DVA yield over SB-01542. Both SB-01542 and SB-01703 produced similar amounts of the terminal molecule THCVA (13 vs. 17 μM). In the same fermentation, SB-01703 also produced OA-iB (697 ± 34 μM), and this strain was able to convert OA-iB to CBGiBA and ultimately to THCiBA (50 and 30 μM, respectively). On the other hand, the parent strain SB-01542 did not produce any of these branched-chain cannabinoids. Here, we provide an example of utilizing ACD1 and IVD1 disruptions to produce minor cannabinoids from sugars without an external fatty acid feedstock. [Table 10] [Table 11-1] [Table 11-2]

[0123] Example 13: Evaluating SB-01703 in fermentation with butyric and valeric acid supplementation The 27 individual clones of SB-01703 were assessed for their ability to produce THCVA and THCBA using the assay described in Example 2. The results for butyrate and valerate addition are recorded in Tables 12 and 13. Under both feeding conditions, total C4 products (DVA, CBGVA, and THCVA) and / or total C5 products (OAB, CBGBA, and THCBA) increased approximately 4-fold and 2-fold, respectively, in SB-01703 (ACD1 and IVD1 inactivated) compared to SB-01542 (ACD1 only inactivated). These results indicate that when both ACD1 and IVD1 were disrupted, more butyryl-CoA and valeryl-CoA were diverted into our engineered pathway, increasing the production of CBG(V)(B)A and THC(V)(B)A. [Table 12] [Table 13]

[0124] Example 14: Combining the "A28" genotype with ACD1 inactivation improves cannabinoid production. As previously reported in U.S. Patent Application No. 63 / 256,398, the entire contents of which are incorporated herein by reference, introduction of the "A28" genotype (expression of the ERG20.A28 allele and subsequent disruption of the native ERG20 gene) results in a significant increase in GPP production. The combination of the "A28" genotype with ACD1 inactivation was examined by introducing the "A28" genotype into SB-01268 (in which ACD1 was inactivated) using standard molecular biology techniques (as described in CELB-5). The resulting strain, SB-01554, was examined in a 96-well deep-well assay as detailed in Example 2, except that plates were quenched at 40 hours instead of 48 hours (Tables 14 and 15). The data show that the combination of the "A28" genotype with ACD1 disruption resulted in a significant improvement in the production of both CBGA and CBGVA compared to the ACD1 disruption alone. [Table 14] [Table 15]

[0125] Example 15: Combination of ACD1 inactivation, IVD1 inactivation and expression of the A28 allele improves cannabinoid production To test whether double inactivation of ACD1 and IVD1 in the A28 background strain (CELB-5) could further improve C4 cannabinoid production, the IVD1 gene was disrupted in SB-01787 (in which the A28 alleles, POX3, POX5, and ACD1 were inactivated) using standard molecular biology techniques, resulting in SB-01890. Nineteen individual clones of SB-01890 were screened in fermentations fed either butyric acid, valeric acid, or hexanoic acid. The average results for these 19 clones are reported in Tables 16, 17, and 18.

[0126] Under all conditions, disruption of IVD1 in the SB-01787 strain background (POX3, POX5, ACD1, inactivation + A28) showed improved cannabinoid production. DVA and CBGVA produced by SB-01890 (POX3, POX5, ACD1, and IVD1 inactivation + A28) increased threefold. OA-B and CBGBA improved by 51%. OA and CBGA titers improved by 34% compared to the parent strain with only ACD1 inactivation. Based on these results, inactivation of both ACD1 and IVD1 is also beneficial for improving titers in strains with an engineered A28 allele background by preserving pools of butyryl-CoA, valeryl-CoA, and hexanoyl-CoA and redirecting these starting CoA molecules into our engineered pathway. [Table 16] [Table 17] [Table 18]

[0127] Example 16: Assessing the function of POX3 and POX5 in maintaining improved cannabinoid production in DVA producers To determine whether inactivation of POX3 and POX5 was required to maintain improved DVA and CBGVA titers, POX3 and / or POX5 were disrupted in SB-02101 (ACD1 and IVD1 inactivation only) to generate SB-02403 (ACD1, IVD1, and POX5 inactivated) and SB-02404 (ACD1, IVD1, POX3, and POX5 inactivated). The resulting strains were assayed for DVA and CBGVA production using butyrate-fed fermentation as described in Example 2, and their fermentation data, along with those of the parent strain, are recorded in Table 19. Disruption of POX5 alone (SB-02403) showed an 8% improvement in DVA and CBGVA production over the parent strain, SB-02101, while double inactivation of POX3 and POX5 resulted in a strain (SB-02404) with a 58% improvement. Taken together, dual POX3 and POX5 inactivation significantly increased the intracellular butyryl-CoA pool, thereby increasing our C4 cannabinoid yield. Additionally, POX3 and POX5 inactivation was synergistic with ACD1 and IVD1 disruption, suggesting that alterations of both mitochondrial and peroxisomal β-oxidation genes are necessary to achieve high C4 cannabinoid titers. [Table 19]

[0128] Example 17: Disruption of BAT1 and BAT2 to reduce C5 branched-chain cannabinoids To obtain an engineered strain with increased availability of butyryl-CoA and valeryl-CoA to our cannabinoid pathway without the accumulation of isovaleryl-CoA, which has been shown to be an equivalent substrate for PKS / PKC to produce branched-chain versions of cannabinoid intermediates, we attempted to eliminate the accumulation of isovaleryl-CoA by disrupting enzymes upstream of IVD1. S. cerevisiae contains two functionally divergent branched-chain aminotransferases (termed BAT1 and BAT2) responsible for the final steps of leucine, isoleucine, and valine biosynthesis and the first step of catabolism of these branched-chain amino acids (Gonzalez et al., 2011). Using the S. cerevisiae BAT1 and BAT2 sequences, we identified their homologs in Y. lipolytica, encoded by YALI0_D01265g and YALI0_F19910g, respectively. We hypothesize that disruption of either or both BAT1 and BAT2 will prevent branched-chain amino acid catabolism (especially leucine) and halt the accumulation of isovaleryl-CoA caused by IVD1 inactivation, while maintaining the benefits of increasing butyryl-CoA and valeryl-CoA flux toward our engineered pathways to produce THCVA and THCBA (when combined with ACD1 inactivation).

[0129] BAT1 and BAT2 were individually disrupted in SB-01268 (in which POX3, POX5, and ACD1 were inactivated) to generate SB-01892 and SB-01895, respectively. Similarly, disruption of BAT1 and BAT2 in SB-01861 (in which POX3, POX5, ACD1, and IVD1 were inactivated) resulted in SB-01894 and SB-01897. Up to 11 clones of the BAT1 and BAT2 disruptants, along with their parent strains, were assayed for C5 branched-chain cannabinoid production as described in Example 2, and the results are recorded in Tables 20 and 21. [Table 20] [Table 21]

[0130] Our results showed that disruption of BAT1 or BAT2 in SB-01268 (in which POX3, POX5 and ACD1 are inactivated) did not alter the C4 or C6 cannabinoid profile (data not shown) or induce the production of iC5 cannabinoids (as shown in Table 20), strengthening our conclusion that IVD1 inactivation is required for the production of these rare branched-chain cannabinoids.

[0131] When BAT1 or BAT2 was inactivated in SB-01861 (in which POX3, POX5, ACD1, and IVD1 were inactivated), iC5 cannabinoid production increased by 29% and 12%, respectively. This result suggested that when BAT1 or BAT2 was disrupted, there was increased accumulation of isovaleryl-CoA in the cells, causing increased iC5 cannabinoid production. Therefore, contrary to our initial hypothesis, deleting BAT1 and / or BAT2 is a way to increase branched-chain cannabinoids.

[0132] Example 18: Inactivation of BCKD-E1α, E1β, or E2 subunits to reduce C5 branched-chain cannabinoids Another approach to reducing isovaleryl-CoA accumulation is to inactivate the branched-chain α-ketoacid dehydrogenase complex (BCKD), which is responsible for generating isovaleryl-CoA from α-ketoisocaproate (Figure 3). This complex is composed of four subunits: E1α (YALI0_D08690g), E1β (YALI0_F05038g), E2 (YALI0_D23815g), and E3 (YALI0D20768g). The E3 subunit is shared with the pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex. Therefore, to specifically inactivate BCKD, subunits E1α, E1β, or E2 can be disrupted. These genes were inactivated in SB-02218 (in which POX3, POX5, ACD1, and IVD1 were inactivated) to generate SB-02225, SB-02226, and SB-02227, respectively. Eleven clones for each disruptant were assayed for C4 and iC5 cannabinoid production as described in Example 2. The data are recorded in Tables 22 and 23.

[0133] Disruption of BCKD-E1α (SB-02225) or the E2 subunit (SB-02227) significantly reduced iC5 production by nearly 8-fold compared to the parent strain SB-02218, whereas disruption of BCKD-E1β did not reduce C5 branched-chain cannabinoids. Furthermore, surprisingly, strains in which BCKD-E1α or BCKD-E2 was inactivated also had significantly increased CBGVA production, converting over 95% of the DVA produced from the butyrate feed to CBGVA and THCVA. This result suggests that, in addition to reducing iC5 production, disruption of BCKD-E1α or BCKD-E2 also results in (1) more GPP being available for the conversion of DVA to CBGVA and / or (2) reducing substrate competition for CBGA synthase by C5 branched-chain cannabinoids. [Table 22-1] [Table 22-2] [Table 23] Example 19: Inactivation of ARO10 to reduce C5 branched-chain cannabinoids [1] As shown in Figure 3, the first step in the degradation of leucine produces α-ketoisocaproate. This compound can be decarboxylated in the cytoplasm by ketoacid decarboxylase (part of the Ehrlich pathway). The resulting isoamylaldehyde can be oxidized to isovalerate, which can then be utilized by the cannabinoid synthesis pathway in our organism. To eliminate this possible pathway to isovalerate, ketoacid decarboxylase (ARO10, YALI0_D06930g) can be disrupted. To test this hypothesis, we inactivated this gene in SB-02218 (in which POX3, POX5, ACD1, and IVD1 are inactivated) and assayed for cannabinoid production as described in Example 2.

[0134] Example 20: Inactivation of peroxisomal acyl-CoA oxidase genes (POX1, POX2, POX4, POX6) in combination with ACD1, POX3, POX5 inactivation and the A28 allele improves cannabinoid production. Acyl-CoA oxidase is the first committed step in peroxisomal β-oxidation, a competing pathway for hexanoyl-CoA and butyryl-CoA molecules. Therefore, we attempted to increase cannabinoid production titers by inactivating peroxisomal β-oxidation through individual disruption of acyl-CoA genes, including POX1 (YALI0_E32835g), POX2 (YALI0_F10857g), POX4 (YALI0_E27654g), or POX6 (YALI0_E06567g). These individual gene disruptions were performed in the strain background SB-01983, which contains the A28 allele and is inactivated for the ACD1, POX3, and POX5 genes. The resulting engineered strains (SB-02257, ΔPOX1; SB-02258, ΔPOX2; SB-02259, ΔPOX4; SB-02265, ΔPOX6) were assayed in quadruplicate for cannabinoid production as described in Example 2. The data in Table 24 confirm that disruption of POX2 or POX6 in combination with inactivation of the A28 allele and ACD1, POX3 and POX5 significantly improved cannabinoid product titer over the background strain SB-01983, whereas disruption of POX1 or POX4 did not have a significant improvement in product titer. [Table 24]

[0135] Example 21: Inactivation of ACD homologs (GCDH1, GCDH2, ACD2) in combination with ACD1, POX3, POX5 inactivation and the A28 allele improves cannabinoid production.

[0136] Mitochondrial β-oxidation is another competing pathway for hexanoyl-CoA and butyryl-CoA molecules. Because inactivation of mitochondrial β-oxidation via ACD1 disruption increased product titers, we attempted to further increase product titers by also disrupting ACD1 homologs identified by BLAST searches (see Example 5, Table 6). To test this, we individually disrupted GCDH1 (YALI0_C16797g), GCDH2 (YALI0_F23749g), or ACD2 (YALI0_B04906g) in strain SB-01983 (inactivated for the A28 allele, ACD1, POX3, and POX5). The resulting engineered strains (SB-02260, ΔGCDH1; SB-02261, ΔGCDH2; SB-02262, ΔACD2) were assayed in quadruplicate for cannabinoid production as described in Example 2. The data in Table 25 confirm that disruption of GCDH2 in combination with inactivation of the A28 allele and ACD1, POX3 and POX5 improves cannabinoid product titer by 16% over the background strain SB-01983, while disruption of GCDH1 or ACD2 does not show a significant improvement in product titer. [Table 25]

[0137] Example 22: Inactivation of peroxisome biogenesis through PEX10 in combination with ACD1, POX3, POX5 inactivation and the A28 allele improves cannabinoid production. Because peroxisomal β-oxidation is a competing pathway for hexanoyl-CoA and butyryl-CoA molecules, we attempted to eliminate peroxisome biogenesis to inhibit the peroxisomal β-oxidation pathway. To achieve this, we inactivated peroxisome biogenesis factor 10 (PEX10, YALI0_D02387g) in strain background SB-01983, which contains the A28 allele and is inactivated for the ACD1, POX3, and POX5 genes. The resulting engineered strain (SB-02264) was assayed in quadruplicate for cannabinoid production as described in Example 2. The data in Table 26 confirmed that disruption of PEX10 in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 significantly increased cannabinoid product titers by 41% over the background strain SB-01983. [Table 26]

[0138] Example 23: Inactivation of multifunctional beta-oxidation enzyme (MFE1) in combination with ACD1, POX3, POX5 inactivation and the A28 allele improves cannabinoid production. The second and third steps of peroxisomal beta-oxidation are accomplished by the multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (MFE1, YALI0_E15378G) (Beopoulos et al., 2008). Therefore, this gene is responsible for converting 2-trans-enoyl-CoA molecules to L-3-hydroxyacyl-CoA and subsequently to 3-ketoacyl-CoA, as seen in Figure 2. Inactivation of MFE1 was performed in strain background SB-01983, which contains the A28 allele and is inactivated for ACD1, POX3, and POX5. The resulting engineered strain, SB-02263, also produced a rare cannabinoid in which the hexanoyl-CoA molecule was replaced with trans-2-hexenoyl-CoA, as confirmed by LC-MS (Figure 6). The data in Table 27 confirmed that disruption of MFE1 in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 significantly increased cannabinoid product titers over the background strain SB-01983. The data in Table 28 confirmed that disruption of MFE1 in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 produced rare cannabinoids in which hexanoyl-CoA molecules were replaced with trans-2-hexenoyl-CoA. Taken together, these results indicate that inactivation of the peroxisomal β-oxidation pathway by disrupting MFE1 not only increased cannabinoid product titers, but also produced rare cannabinoid species. [Table 27] [Table 28-1] [Table 28-2]

[0139] Example 24: Assessing disruption of POT1 to reduce beta-oxidation and improve cannabinoid production The fourth and final step in peroxisomal β-oxidation is accomplished by peroxisomal oxoacylthiolase (POT1, YALI0_E18568g) (Beopoulos et al., 2008). Therefore, inactivation of POT1 should inhibit this pathway from degrading hexanoyl-CoA or butyryl-CoA to acetyl-CoA. Because POT1 is required to convert ketolized β-oxidation products to acyl-CoA molecules, inactivation of POT1 may also enable the production of rare ketolized cannabinoid species in which the hexanoyl-CoA molecule is replaced by 3-oxohexanoyl-CoA (Figure 7). POT1 is disrupted in SB-01983 (POX3, POX5, and ACD1 are inactivated and the A28 allele is present). The resulting strains are assayed for C4 and C6 cannabinoid production as well as production of rare ketolized cannabinoids as described in Example 2.

[0140] Example 25: Assessing disruption of ANT1 and FAA1 to inhibit fatty acid activation and improve cannabinoid production Fatty acids (e.g., hexanoic acid, butyric acid) are activated by reacting with CoA molecules to form fatty acyl-CoA. Fatty acids are activated in the cytoplasm by long-chain fatty acid CoA ligase (FAA1) (YALI0_D17864g) or in peroxisomes by an enzyme (YALI0_E03058g) that requires ATP generated by peroxisomal adenine nucleotide transporter 1 (ANT1) (Dulermo et al., 2015). Activated fatty acids are either stored in the lipid body or degraded by beta-oxidation. Therefore, disruption of FAA1 and / or ANT1 inhibits fatty acid activation, preventing their degradation by beta-oxidation or storage in the lipid body, improving cannabinoid production. To test this, we individually disrupted FAA1 or ANT1 in strain SB-01983 (POX3, POX5, and ACD1 are inactivated, and the A28 allele is present). The resulting engineered strains (SB-02456, ΔFAA1; SB-02500, ΔANT1) were assayed for cannabinoid production as described in Example 2. Disruption of FAA1 (SB-02456) in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 significantly improved C4 cannabinoid product titers over the background strain SB-01983 with the addition of both hexanoic acid (Table 29) or butyric acid (Table 30), while no improvement in C6 cannabinoid product titers was observed. Disruption of ANT1 in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 (SB-02500) improved C6 cannabinoid production but had no effect on C4 cannabinoid titers (Tables 29 and 30). [Table 29] [Table 30]

[0141] Example 26: Assessing disruption of YAT1 to reduce beta-oxidation and improve cannabinoid production. Activated hexanoyl-CoA and butyryl-CoA in the cytoplasm can be transported into peroxisomes and mitochondria and metabolized by beta-oxidation. Cytosolic carnitine acetyltransferase YAT1 (YALI0_F21197g) may attach carnitine to hexanoyl-CoA or butyryl-CoA. To test this, we inactivated YAT1 in strain background SB-01983, which contains the A28 allele and is inactivated for the ACD1, POX3, and POX5 genes. The resulting engineered strain (SB-02457) was assayed in triplicate for cannabinoid production, as described in Example 2. The data in Table 31 confirmed that disruption of YAT1 in combination with inactivation of the A28 allele and ACD1, POX3, and POX5 increased cannabinoid product titers over the background strain SB-01983. [Table 31]

[0142] Example 27: Inactivation of FAA1, ACD1, IVD1 and BCKD to further improve cannabinoid production. In the preceding examples, disruption of either IVD1 (with or without BCKD inactivation) or FAA1 increases C4 cannabinoid product titers. To test whether the positive improvements of IVD and FAA1 disruption are synergistic and additive, we attempted to disrupt FAA1 in strain SB-02762, which has deletions of POX3, POX5, ACD1, IVD1, and BCKD-E1α in combination with the A28 allele. The resulting engineered strain (SB-03603) was assayed in triplicate for cannabinoid production as described in Example 2. SB-03603 significantly improved C4 cannabinoid product titers over the background strain (SB-02762) without any supplementation (Table 32) and with butyrate supplementation (Table 33). [Table 32] [Table 33]

[0143] Example 28: Inactivation of FAA1 alone to further improve cannabinoid production. In the above example, disruption of FAA1 increases C4 cannabinoid production with or without butyrate supplementation. To test whether FAA1 inactivation for improved C4 cannabinoid production is dependent on other gene disruptions (ACD, POX3, POX5, IVD, BCKD-E1α), we attempted to inactivate FAA1 in strain SB-03715, which does not have other gene disruptions or the A28 allele. The resulting strain is assayed for C4 cannabinoid production as previously described in Example 2. [Table 34-1] [Table 34-2] [Table 34-3] [Table 34-4]

[0144] Nucleic acid sequence enumeration Primer sequences Acyl-CoA dehydrogenase FW primer (SEQ ID NO: 1)

[0145] [ka]

[0146] Acyl-CoA dehydrogenase BW primer (SEQ ID NO: 2)

[0147] [ka]

[0148] Isovaleryl-CoA dehydrogenase FW primer (SEQ ID NO: 3)

[0149] [ka]

[0150] Isovaleryl-CoA dehydrogenase BW primer (SEQ ID NO: 4)

[0151] [ka]

[0152] TAF10 FW primer (SEQ ID NO: 5)

[0153] [ka]

[0154] TAF10 BW primer (SEQ ID NO: 6)

[0155] [ka]

[0156] Amino acid sequence enumeration YALI0_D15708g, mitochondrial acyl-CoA dehydrogenase (SEQ ID NO: 7)

[0157] [ka]

[0158] YALI0_B10406g, enoyl-CoA hydratase (SEQ ID NO: 8)

[0159] [ka]

[0160] YALI0_D01265g, branched-chain aminotransferase 1 (SEQ ID NO: 9)

[0161] [ka]

[0162] YALI0_F19910g, branched-chain aminotransferase 2 (SEQ ID NO: 10)

[0163] [ka]

[0164] YALI0_D08690g, branched-chain α-ketoacid dehydrogenase, subunit 1 alpha (SEQ ID NO: 11)

[0165] [ka]

[0166] YALI0_F05038g, branched-chain α-ketoacid dehydrogenase, subunit 1 beta (SEQ ID NO: 12)

[0167] [ka] [ka]

[0168] YALI0_D23815g, branched-chain α-ketoacid dehydrogenase, subunit 2 (SEQ ID NO: 13)

[0169] [ka]

[0170] YALI0_D20768g, branched-chain α-ketoacid dehydrogenase, subunit 3 (SEQ ID NO: 14)

[0171] [ka]

[0172] YALI0_D06930g, keto acid decarboxylase (SEQ ID NO: 15)

[0173] [ka] [ka]

[0174] YALI0_E32835g, peroxisomal acyl-CoA oxidase 1 (SEQ ID NO: 16)

[0175] [ka]

[0176] YALI0_F10857g, peroxisomal acyl-CoA oxidase 2 (SEQ ID NO: 17)

[0177] [ka] [ka]

[0178] YALI0_D24750g, peroxisomal acyl-CoA oxidase 3 (SEQ ID NO: 18)

[0179] [ka]

[0180] YALI0_E27654g, peroxisomal acyl-CoA oxidase 4 (SEQ ID NO: 19)

[0181] [ka]

[0182] YALI0_C23859g, peroxisomal acyl-CoA oxidase 5 (SEQ ID NO: 20)

[0183] [ka]

[0184] YALI0_E06567g, peroxisomal acyl-CoA oxidase 6 (SEQ ID NO: 21)

[0185] [ka]

[0186] YALI0_C16797g, glutaryl-CoA dehydrogenase 1 (SEQ ID NO: 22)

[0187] [ka]

[0188] YALI0_F23749g, glutaryl-CoA dehydrogenase 2 (SEQ ID NO: 23)

[0189] [ka]

[0190] YALI0_B04906g, acyl-CoA dehydrogenase 2 (SEQ ID NO: 24)

[0191] [ka]

[0192] YALI0_D02387g, peroxisome biogenesis factor 10 (SEQ ID NO: 25)

[0193] [ka] [ka]

[0194] YALI0_E15378g, multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (SEQ ID NO: 26)

[0195] [ka]

[0196] YALI0_E18568g, peroxisomal oxoacylthiolase (SEQ ID NO: 27)

[0197] [ka] [ka]

[0198] YALI0_D17864g, long-chain fatty acid CoA ligase (SEQ ID NO: 28)

[0199] [ka]

[0200] YALI0_E03058g, peroxisomal adenine nucleotide transporter 1 (SEQ ID NO: 29)

[0201] [ka]

[0202] YALI0_F21197g, cytoplasmic carnitine acetyltransferase (SEQ ID NO: 30)

[0203] [ka]

[0204] YALI0_E12573g, isovaleryl-CoA dehydrogenase (SEQ ID NO: 31)

[0205] [ka]

[0206] YALI0_F28567g, enoyl-CoA hydratase homolog (SEQ ID NO: 32)

[0207] [ka] [ka]

[0208] YALI0_F22121g, enoyl-CoA hydratase homolog (SEQ ID NO: 33)

[0209] [ka]

[0210] YALI0_D00671g, enoyl-CoA hydratase homolog (SEQ ID NO: 34)

[0211] [ka]

[0212] YALI0_D06215g, enoyl-CoA hydratase homolog (SEQ ID NO: 35)

[0213] [ka]

[0214] YALI0_D09383g, enoyl-CoA hydratase homolog (SEQ ID NO: 36)

[0215] [ka]

[0216] YALI0_D09493g, enoyl-CoA hydratase homolog (SEQ ID NO: 37)

[0217] [ka]

[0218] YALI0_A07733g, enoyl-CoA hydratase homolog (SEQ ID NO: 38)

[0219] [ka]

Claims

1. 1. A cell engineered to provide increased production of cannabinoids and / or derivatives thereof compared to a control cell, wherein the engineered cell expresses one or more enzymes of a cannabinoid biosynthetic pathway; at least one gene encoding one or more enzymes or other proteins that affect the metabolism of at least one acyl-CoA molecule in the cell has been deleted, inactivated or otherwise mutated, thereby allowing the redirection of acyl-CoA molecules from cellular metabolic pathways into the cannabinoid biosynthetic pathway to achieve increased production of cannabinoids and / or derivatives thereof compared to control cells; The control cell lacks the same deletion, inactivation or other mutation of the at least one gene as the engineered cell.

2. The cell of claim 1 , wherein the cellular metabolic pathway is the beta-oxidation pathway.

3. The cell of claim 2 , wherein the beta-oxidation pathway is one or both of the mitochondrial beta-oxidation pathway and / or the peroxisomal beta-oxidation pathway.

4. The cell of claim 1 , wherein the cellular metabolic pathway is an amino acid catabolic pathway.

5. 5. The cell of claim 4, wherein the amino acid catabolic pathway is one or more branched-chain amino acid catabolic pathways selected from the group consisting of leucine, isoleucine, and / or valine catabolic pathways that produce branched acyl-CoA intermediates.

6. 6. The cell of any one of claims 1 to 5, wherein the acyl-CoA comprises one or more of butyryl-CoA, valeryl-CoA, isovaleryl-CoA, trans-2-hexenoyl-CoA, 3-oxohexanoyl-CoA and / or hexanoyl-CoA.

7. 7. The cell of any one of claims 1 to 6, wherein the at least one gene encodes peroxisomal acyl-CoA oxidase, peroxisomal acyl-CoA dehydrogenase, mitochondrial acyl-CoA dehydrogenase, mitochondrial isovaleryl-CoA dehydrogenase, mitochondrial branched-chain α-keto acid dehydrogenase subunit, isovaleryl-CoA dehydrogenase, branched-chain aminotransferase, glutaryl-CoA dehydrogenase, keto acid decarboxylase, multifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase, peroxisomal oxoacylthiolase, fatty acid CoA ligase, peroxisomal adenine nucleotide transporter, cytosolic carnitine acetyltransferase, or peroxisome biogenesis factor.

8. 8. The cell of any one of claims 1 to 7, wherein the at least one gene is selected from one or more of the group consisting of POX1, POX2, POX3, POX4, POX5, POX6, BAT1, BAT2, YAT1, FAA1, ANT1, POT1, ACD1, IVD1, MFE1, PEX10, ARO10, GCDH1, GCDH2, ACD2, BCKD-E1α, BCKD-E1β, BCKD-E2, and homologs or orthologs thereof.

9. The at least one gene is selected from the group consisting of YALI0_D15708g, YALI0_B10406g, YALI0_D01265g, YALI0_F19910g, YALI0_D08690g, YALI0_F05038g, YALI0_D23815g, YALI0_D20768g, YALI 0_D06930g, YALI0_E32835g, YALI0_F10857g, YALI0_D24750g, YALI0_E27654g , YALI0_C23859g, YALI0_E06567g, YALI0_C16797g, YALI0_F23749g, YALI0_B04 906g, YALI0_D02387g, YALI0_E15378g, YALI0_E18568g, YALI0_D17864g, YALI0_E03058g, YALI0_F21197g, YALI0_E12573g, YALI0_F28567g, YALI0_F22121g, YALI0_D00671g, YALI0_D06215g, YALI0_D09383g, YALI0_D09493g, YALI0_A07733g and homologs or orthologs thereof.

10. The engineered cells may express cannabigerolic acid (CBGA) synthase, unsaturated cannabigerolic acid (CBGA-uns) synthase, ketolized cannabigerolic acid (CBGA-keto) synthase, cannabigerol (iso)butyrate (CBG(i)BA) synthase, cannabigerolic acid (CBGVA) synthase, tetrahydrocannabic acid (THCA) synthase, unsaturated tetrahydrocannabic acid (THCA-uns) synthase, ketolized tetrahydrocannabic acid (T HCA-keto) synthase, tetrahydrocannabi(iso)butolic acid (THC(i)BA) synthase, tetrahydrocannabivaric acid (THCVA) synthase, cannabichromenic acid (CBCA) synthase, unsaturated cannabichromenic acid (CBCA-uns) synthase, ketolized cannabichromenic acid (CBCA-keto) synthase, cannabichromene(iso)butyric acid (CBC(i)BA) synthase, cannabichromevaric acid (CBCVA) synthase, cannabidiolic acid ( CBDA) synthase, unsaturated cannabidiolic acid (CBDA-uns) synthase, ketolized cannabidiolic acid (CBDA-keto) synthase, cannabi(iso)butolic acid (CBD(i)BA) synthase, cannabidivalic acid (CBDVA) synthase, hexanoyl-CoA synthetase, (iso)valeryl-CoA synthetase, butyryl-CoA synthetase, polyketide synthase, polyketide cyclase, geranyl pyrophosphate (GPP) synthase, HMG- 10. The cell of any one of claims 1 to 9, wherein the cell expresses two or more enzymes of the cannabinoid biosynthetic pathway selected from the group consisting of: acetyl-CoA synthase, mevalonate kinase, acetyl-CoA synthase, acetyl-CoA carboxylase, acetyl-CoA acetyltransferase / HMG-CoA reductase, mevalonate-5-phosphate decarboxylase, isopentenyl phosphokinase, and / or hydroxymethylglutaryl-CoA reductase.

11. 11. The cell of claims 1-10, wherein the cell, when cultured under suitable conditions, is capable of producing one or more cannabinoid derivatives selected from the group consisting of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA and CBDVA, and decarboxylated derivatives thereof.

12. 12. The cell of claim 11, wherein the suitable conditions include the addition of one or more of butyric acid, butyl butyrate, valeric acid, isovaleric acid, hexanoic acid, hexyl hexanoate, glucose, glycerol, hexanol, butanol, and / or oleic acid.

13. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises POX3, POX5 in combination with one or both of ACD1 and IVD1, and wherein the cell exhibits synergistic production of cannabinoids compared to the production of cannabinoids achieved in a cell comprising deletions, inactivations or mutations of only ACD1, IVD1 or a combination of POX3 / POX5.

14. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises a combination of YALI0_D15708g and YALI0_E12573g.

15. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises a combination of POX3, POX5, ACD1 and one or more of POX2, POX6, GCDH2, GCDH1, MFE1, YAT1, FAA1, ANT1, POT1, ACD2 and PEX10, and wherein the cell exhibits enhanced production of cannabinoids compared to the production of cannabinoids achieved in a cell comprising deletions, inactivations or mutations of only a combination of POX3, POX5 and ACD1.

16. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises a combination of IVD1 and at least one of BAT1 or BAT2, and wherein the cell exhibits enhanced production of one or more C5 branched chain cannabinoids compared to the production of the same one or more C5 branched chain cannabinoids achieved in a cell comprising a deletion, inactivation or mutation of IVD1 alone.

17. 17. The cell of claim 16, wherein the one or more C5 branched chain cannabinoids comprise one or more of CBGiBAs, CBDiBAs, CBCiBAs and / or THCiBAs and their decarboxylated derivatives.

18. The cell of any one of claims 1 to 12, wherein the at least one gene comprises a combination of at least two of POX3, POX5 and ACD1.

19. 19. The cell of claim 18, wherein the at least one gene comprises a combination of POX3, POX5, ACD1 and FAA1, and the cell exhibits significantly improved production of C4 cannabinoids compared to the production of C4 cannabinoids achieved in cells comprising deletions, inactivations or mutations of only POX3, POX5 and ACD1.

20. 19. The cell of claim 18, wherein the at least one gene comprises a combination of POX3, POX5, ACD1 and YAT1, and the cell exhibits significantly improved production of cannabinoids compared to the production of cannabinoids achieved in cells comprising deletions, inactivations or mutations of only POX3, POX5 and ACD1.

21. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises IVD1 in combination with one or both of BCKD-E1α and BCKD-E2, and the cell exhibits reduced production of C5 branched cannabinoid products compared to the production of C5 branched cannabinoids achieved in a cell comprising a deletion, inactivation or mutation of IVD1 alone.

22. 22. The cell of claim 21, wherein the cell additionally exhibits significantly increased production of CBGVA compared to the production of CBGVA achieved in cells containing a deletion, inactivation or mutation of IVD1 alone.

23. 23. The cell of claim 21 or 22, wherein the at least one gene further comprises at least one of POX3, POX5, and ACD1.

24. 24. The cell of claim 23, wherein the at least one gene further comprises FAA1.

25. 13. The cell of any one of claims 1 to 12, wherein the at least one gene comprises FAA1, ACD1, IVD1, POX3, POX5, and one or both of BCKD-E1α and BCKD-E2, and wherein the cell exhibits significantly improved production of C4 cannabinoids compared to the production of cannabinoids achieved in a cell comprising deletions, inactivations or mutations of only POX3, POX5, ACD1, IVD1 and BCKD-E1α.

26. The cell of any one of claims 1 to 12, wherein the at least one gene comprises FAA1.

27. 27. The cell of claim 26, wherein the at least one gene further comprises ACD1 or IVD1.

28. The cell of any one of claims 1 to 27, wherein the cell is a yeast cell or a bacterial cell.

29. 29. The cell of claim 28, wherein the yeast cell is a Yarrowia or Saccharomyces strain.

30. 30. A method for producing a cannabinoid or a derivative thereof, comprising culturing a cell according to any one of claims 1 to 29 under suitable conditions to produce the cannabinoid or derivative thereof.

31. 31. The method of claim 30, wherein the cannabinoid or derivative thereof comprises one or more of CBGA, CBGA-uns, CBGA-keto, CBGBA, CBGiBA, CBGVA, THCA, THCA-uns, THCA-keto, THCBA, THCiBA, THCVA, CBCA, CBCA-uns, CBCA-keto, CBCBA, CBCiBA, CBCVA, CBDA, CBDA-uns, CBDA-keto, CBDBA, CBDiBA and CBDVA, or decarboxylated derivatives thereof.

32. 32. The method of claim 30 or 31, wherein the suitable conditions comprise adding at least one of butyric acid, valeric acid, isovaleric acid, hexanoic acid, hexanol, butanol, oleic acid, glycerol, or glucose to a culture medium in which the cells are cultured.

33. 33. The method of any one of claims 30 to 32, further comprising isolating the cannabinoid or derivative thereof from the culture.

34. 1. A cell engineered to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, said cell comprising an inactivated or otherwise mutated IVD1, POT1 and / or MFE1 gene. and wherein the cells further express sufficient enzymes in the cannabinoid synthesis pathway to produce CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, when cultured under appropriate conditions.

35. 35. The cell of claim 34, wherein the suitable conditions comprise the addition of butyric acid, butyl butyrate, valeric acid, isovaleric acid, butanol, hexanol, hexanoic acid, hexyl hexanoate, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured.

36. 35. A method for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof, by culturing the cell of claim 34 under conditions suitable for producing CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof.

37. 37. The method of claim 36, wherein the suitable conditions comprise adding at least one of butyric acid, butyl butyrate, valeric acid, isovaleric acid, hexanoic acid, hexyl hexanoate, hexanol, butanol, oleic acid, glycerol, or glucose to the culture medium in which the cells are cultured.

38. 38. The method of claim 36 or 37, further comprising isolating CBGiBA, CBGA-uns, CBGA-keto, THCiBA, THCA-uns, THCA-keto, CBCiBA, CBCA-uns, CBCA-keto, CBDiBA, CBD-uns and / or CBD-keto, or decarboxylated derivatives thereof from the culture.

39. The following structure: 【Chemistry 41-1】 【Chemistry 41-2】 【Chemistry 41-3】 【Chemistry 41-4】 【Chemistry 41-5】 The compound according to any one of the preceding claims.

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