WO2021183448A1 - Optimized olivetolic acid cyclase polypeptides - Google Patents

Optimized olivetolic acid cyclase polypeptides Download PDF

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Publication number
WO2021183448A1
WO2021183448A1 PCT/US2021/021388 US2021021388W WO2021183448A1 WO 2021183448 A1 WO2021183448 A1 WO 2021183448A1 US 2021021388 W US2021021388 W US 2021021388W WO 2021183448 A1 WO2021183448 A1 WO 2021183448A1
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acid
amino acid
host cell
seq
cannabinoid
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PCT/US2021/021388
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French (fr)
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Andrew HORWITZ
Rathin BECTOR
Stacey SHIIGI
Darren Platt
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Demetrix, Inc.
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Publication of WO2021183448A1 publication Critical patent/WO2021183448A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01026—Olivetolic acid cyclase (4.4.1.26)

Definitions

  • Cannabisbinoids Plants from the genus Cannabis have been used by humans for their medicinal properties for thousands of years. In modern times, the bioactive effects of Cannabis are attributed to a class of compounds termed “cannabinoids,” of which there are hundreds of structural analogs including tetrahydrocannabinol (THC) and cannabidiol (CBD). These molecules and preparations of Cannabis material have recently found application as therapeutics for chronic pain, multiple sclerosis, cancer-associated nausea and vomiting, weight loss, appetite loss, spasticity, seizures, and other conditions. [0004] The physiological effects of certain cannabinoids are thought to be mediated by their interaction with two cellular receptors found in humans and other animals.
  • Cannabinoid receptor type 1 (CB1) is common in the brain, the reproductive system, and the eye.
  • Cannabinoid receptor type 2 (CB2) is common in the immune system and mediates therapeutic effects related to inflammation in animal models.
  • THC and CBD hundreds of other cannabinoids have been identified in Cannabis. However, many of these compounds exist at low levels and alongside more abundant cannabinoids, making it difficult to obtain pure samples from plants to study their therapeutic potential. Similarly, methods of chemically synthesizing these types of products have been cumbersome and costly, and tend to produce insufficient yield.
  • One possible method is production via fermentation of engineered microbes, such as yeast.
  • engineered microbes such as yeast.
  • By engineering production of the relevant plant enzymes in microbes it may be possible to achieve conversion of various feedstocks into a range of cannabinoids, potentially at much lower cost and with much higher purity than what is available from the plant.
  • a key challenge to this effort is the difficulty of expressing plant enzymes in the microbe, particularly secreted enzymes such as the cannabinoid synthases, which must successfully traverse the microbe’s secretory pathway to fold and function properly.
  • Variants of cannabinoid synthases, modified host cells, and new methods are needed to address these challenges.
  • the present disclosure provides engineered variants of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, methods of producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the olivetolic acid cyclase (OAC) polypeptide.
  • OAC olivetolic acid cyclase
  • the engineered variants of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives or precursors (e.g., non-naturally occurring cannabinoids).
  • the modified host cells of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure.
  • the disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure.
  • the present disclosure provides a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions.
  • the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, E67, Q70, D71, V84, S87, I94, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, N29, A36, Q48, K49, T56, I58, E64, I94, and R100
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, E64, and I94.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, V8, L9, E64, I94, R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K10, N29, T56, I58, and I94.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of L9, A36, Q28, I58, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of K49, E64, I94, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, K12, A36, Q48, and I58. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of K49, E64, I94, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, K12, A36, Q48, and I58. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is at amino acid V8.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8 and L9. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are at amino acids V8 and L9. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are at amino acids V8, L9, and T56.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six or seven of the amino acid substitutions are at amino acids selected from the group consisting of: [0013] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids A2, V8, L9, E64, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K10, N29, T56, I58, and I94.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9, A36, Q48, I58, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K49, T56, I58, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K12, K49, T56, and D71.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids K12, A36, and D71. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, A36, K49, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, N49, I58, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K49, I58, Q70, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, A36, T62, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, E22, A36, Q48, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K10, T56, I58, and Q70.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids L9, K10, K25, Q48, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, T56, I58, and E64. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, A36, Q48, K49, and R100.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, E22, A36, Q48, and R100. [0014] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions occurs in the beta sheet 1 chain, the beta sheet 2 chain, and/or the alpha helix 2 chain.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E52H, E53V, T56S, I58C, I58V, T62
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, F23I, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, E67S, Q70A, Q70K, D71T, V84D, S87P, I94K, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, K49R, T56S, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, and T56S. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I and L9I.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is V8I. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are V8I and L9I. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are V8I, L9I, and T56S. [0018] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions, wherein six or seven of the amino acid substitutions are selected from the group consisting of:
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions A2P, V8I, L9I, E64D, I94K, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K10A, N29D, T56S, I58V, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions L9I, A36Q, Q48C, I58V, E64D, and I94K.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K49R, T56S, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K12N, K49R, T56S, and D71T. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids V8I, L9I, K12N, A36E, K49R, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids V8I, L9I, K12N, N49R, I58V, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising six amino acid substitutions.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising seven amino acid substitutions. [0021] In some embodiments, the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 3-146. In some embodiments, the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 36-45 and 94-98.
  • the variant produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the variant produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100%, greater than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the variant produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a variant of the disclosure.
  • the nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 147-290.
  • the variant nucleotide sequence is selected from a nucleotide of any one of SEQ ID NOs: 180-189 and 238-242.
  • the nucleotide sequence is codon-optimized.
  • the present disclosure provides a method of making a modified host cell, the method comprising introducing one or more nucleic acids of the disclosure into a host cell.
  • the present disclosure provides a vector comprising one or more nucleic acids of the disclosure.
  • the present disclosure provides a method of making a modified host cell, the method comprising introducing one or more vectors of the disclosure into a host cell.
  • the present disclosure provides a modified host cell comprising one or more nucleic acids of the disclosure.
  • the modified host cell produces olivetolic acid or an olivetolic acid derivative.
  • the modified host cell produces a cannabinoid or a cannabinoid derivative.
  • the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 160%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least
  • the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time.
  • the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • the present disclosure provides a method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative.
  • the cannabinoid is cannabigerolic acid (CBGA), cannbigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • CBDA cannabigerolic acid
  • cannbigerol cannabidiolic acid
  • cannabidiol cannabidivarinic acid
  • cannabidivarin cannabidivarin.
  • the present disclosure provides a method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: a) culturing a modified host cell of the disclosure in a culture medium.
  • the method comprises: b) recovering the produced olivetolic acid or an olivetolic acid derivative.
  • the culture medium comprises a carboxylic acid.
  • the carboxylic acid is an unsubstituted or substituted C 3 -C 18 carboxylic acid. In some embodiments, the unsubstituted or substituted C 3 -C 18 carboxylic acid is an unsubstituted or substituted hexanoic acid.
  • the present disclosure provides a method of producing an olivetolic acid or olivetolic acid derivative, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding
  • the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of S
  • the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1,
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence en
  • the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of
  • the method produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • the present disclosure provides a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an OAC variant of the disclosure.
  • the method comprises recovering the produced cannabinoid or cannabinoid derivative.
  • the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • the present disclosure provides a method of producing olivetolic acid or an olivetolic acid derivative, the method comprising use of an OAC variant of the disclosure. In some embodiments, the method comprises recovering the produced olivetolic acid or olivetolic acid derivative.
  • the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3-butyl- resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3-methylpentyl)-resorcylic acid, 3-(3- pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3- (trans-1-hexyl)-resorcylic acid, 3-octyl-resorcylic acid, 3-(trans-1-octenyl)-resorcylic acid, 3- nonyl-resorcylic acid
  • the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OACvariant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the method produces olivetolic acid or a derivative thereof (e.g. CBGA or CBDA) in an increased ratio of olivetolic acid or a derivative thereof (e.g. CBGA or CBDA) over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OACvariant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OACvariant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the method produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • FIGS.1A and 1B depict expression constructs used in the production of the S21 strain.
  • Construct maps also depict genes denoted with a preceding “m” (e.g., mERG13), which specify open reading frames from SEQ ID NOs: 147-290with 200-250 base pairs (bp) of downstream regulatory (terminator) sequence.
  • FIG.2 depicts an expression construct used in the production of the S434 strain.
  • the expression construct depicted in FIG.2 was also used in the production of following strains: S929, S955, S1540, S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840.
  • FIGS.3A and 3B depict an expression construct and a landing pad used in the production of the S929 strain.
  • FIG.3 depicts expression constructs used in the production of the S955 strain.
  • the expression constructs depicted in FIG.4 were also used in the production of following strains: S1540, S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840.
  • FIG.5 depicts an expression construct used in the production of the S1540 strain.
  • FIG.5 depicts expression constructs used in the production of the following strains: S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840.
  • FIG.6 depicts expression constructs used in the production of the S1696 strain. The expression constructs depicted in FIG.6 were also used in the production of the following strains: S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840.
  • FIG.7 depicts a mutant OAC expression construct (2 copies) used in the production of the S1789 strain. The expression construct depicted in FIG.7 was also used in the production of the following strains: S1837 and S1839.
  • FIG.8 depicts expression constructs used in the production of the S1835 strain.
  • FIG.9 depicts a mutant OAC construct expression construct (1 copy) used in the production of the S1791 strain. The expression constructs depicted in FIG.9 were also used in the production of the following strains: S1838, and S1840.
  • FIG.10 depicts expression constructs used in the production of the S1836 strain.
  • FIG.11 depicts expression constructs used in the production of the S927 strain.
  • FIG.12A and 12B depict expression constructs used in the production of the S952 strain.
  • the expression constructs depicted in FIG.12 were also used in the production of the following strains: S983, S1273, S1277, S1278, S1322, S1323, S1324, S1325, S1327, S1328, S1329, S1331, S1333, S1334, S1335, S1391, S1397, S1403, S1405, S1406, S1407, S1409, S1410, S1438, S1479, S1480, S1481, S1482, S1483, S1485, S1486, S1487, S1488, S1489, S1490, S1491, S1492, S1493, S1494, S1495, S1496, S1497, S1498, S1499, S1500, S1501, S1502, S1503, S1504, S1505, S1506, S1507, S1508, S1509, S1510, S1511, S1512, S1513, S1514, S150
  • FIG.13 depicts an expression construct used in the production of the S983 strain.
  • FIG.14 depicts an expression construct used in the production of S1322.
  • FIG.15 depicts expression constructs used in the production of S1323.
  • FIG.16 depicts an expression construct used in the production of the following strains: S1273, S1277, S1278, S1322, S1323, S1324, S1325, S1327, S1328, S1329, S1331, S1333, S1334, S1335, S1391, S1397, S1403, S1405, S1406, S1407, S1409, S1410, S1438, S1479, S1480, S1481, S1482, S1483, S1485, S1486, S1487, S1488, S1489, S1490, S1491, S1492, S1493, S1494, S1495, S1496, S1497, S1498, S1499, S1500, S1501, S1502, S1503, S1504, S1505, S
  • Synthetic biology allows for the engineering of industrial host organisms — e.g., microbes — to convert simple sugar feedstocks into medicines.
  • This approach includes identifying genes that produce the target molecules and optimizing their activities in the industrial host.
  • Microbial production can be significantly cost-advantaged over agriculture and chemical synthesis, less variable, and allow tailoring of the target molecule.
  • reconstituting or creating a pathway to produce a target molecule in an industrial host organism can require significant engineering of both the pathway genes and the host.
  • the present disclosure provides engineered variants of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing olivetolic acid or derivatives thereof, and/or cannabinoids or cannabinoid derivatives, methods of producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the OAC polypeptide.
  • OAC olivetolic acid cyclase
  • the engineered variants of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids).
  • the modified host cells of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure.
  • the disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure.
  • the Olivetolic Acid Cyclase (OAC) enzyme of Cannabis sativa is a polyketide cyclase enzyme that, catalyzes the formation of olivetolic acid or derivative thereof from a malonyl-CoA and hexanoyl-CoA-derived pre-cursor molecule, 3,5,7- trioxododecanoyl-CoA or derivative thereof.
  • Olivetolic acid forms the polynucleotide core of the cannabinoids and thus is an important enzyme for biosynthetic production of cannabinoids.
  • the 3,5,7-trioxododecanoyl-CoA produced by the TKS enzyme converts intoOlivetol, a “dead-end” byproduct that cannot be converted into the desired cannabinoid products.
  • Olivetol in particular, is a skin sensitizer and can be difficult to separate from the desired cannabinoid products in downstream processing.
  • OAC enzymes with improved catalytic activity via improvement in actual catalysis, or expression in yeast) can be used not only to increase titers of the desired cannabinoids in an engineered microbe, but also to reduce the amount of off-product formation, resulting in a better product profile.
  • the natural OAC enzyme e.g., wild type
  • improved enzymes are required. Parameters of interest include catalytic activity, product profile, enzyme stability, and pH and temperature optima. Enzyme improvement is typically accomplished by coupling the generation of diversity (a library of engineered variants) to a screen or selection for the properties of interest.
  • DNA libraries encoding engineered variants can be generated in a variety of ways. For example, libraries can be generated using error prone PCR using the wild type gene sequence as a template. The resulting library can be quite large, consisting of genes with variable numbers of mutations at random positions. Error prone PCR is inexpensive and convenient but has several drawbacks.
  • the engineered variants of the disclosure may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the engineered variants of the disclosure may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA in an increased ratio of OA over olivetol or derivatives thereof compared to that produced by an olivetolic acid cyclase (OAC) polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Similar conditions may include the same temperature, pH, buffer, and/or fermentation conditions and in the same culture medium and/or reaction solvent.
  • the methods of the disclosure may include using engineered microorganisms (e.g., modified host cells) or engineered variants of an OAC polypeptide of the disclosure to produce naturally-occurring and non-naturally occurring olivetolic acid, olivetolic acid derivatives, cannabinoids, cannabinoid derivatives, and/or cannabinoid precursors.
  • Naturally-occurring occurring olivetolic acid, olivetolic acid derivatives, cannabinoids and non-naturally occurring cannabinoids are challenging to produce using chemical synthesis due to their complex structures.
  • the methods of the disclosure enable the construction of metabolic pathways inside living cells to produce bespoke olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives from simple precursors such as sugars and carboxylic acids.
  • One or more nucleic acids e.g., heterologous nucleic acids
  • nucleic acids disclosed herein comprising nucleotide sequences encoding one or more polypeptides or engineered variants disclosed herein can be introduced into host microorganisms allowing for the stepwise conversion of inexpensive feedstocks, e.g., sugar, into final products: olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives and/or precursors.
  • These products can be specified by the choice and construction of expression constructs or vectors comprising one or more nucleic acids (e.g., heterologous nucleic acids) disclosed herein, allowing for the efficient bioproduction of chosen cannabinoids, such as THC and THCA and less common cannabinoid species found at low levels in Cannabis; or cannabinoid derivatives.
  • Bioproduction also enables synthesis of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis.
  • the modified host cell may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • GPP geranylpyrophosphate
  • prenyl phosphates e.g., olivetolic acid, or hexanoyl-CoA
  • the disclosure also provides for modification of the secretory pathway of a host cell modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding an engineered variant of an OAC polypeptide of the disclosure.
  • nucleic acids e.g., heterologous nucleic acids
  • the nucleotide sequence encoding the engineered variant of an OAC polypeptide is codon-optimized.
  • Modification of the secretory pathway in the host cell may improve expression and solubilization of cannabinoids or cannabinoid derivatives synthesized using pathways incorporating the engineered variants of the disclosure, as cannabinoids or cannabinoid derivatives are processed through the secretory pathway.
  • a modified host cell such as a modified yeast cell
  • cannabinoids or cannabinoid derivatives may be misfolded or mislocalized, resulting in low expression, polypeptides lacking activity, polypeptide aggregation, reduced host cell viability, and/or cell death.
  • a backlog of misfolded or mislocalized polypeptides can induce metabolic stress within the modified host cell, harming the modified host cell.
  • the polypeptides may lack necessary posttranslational modifications for folding and activity, such as disulfide bonds, glycosylation and trimming, and cofactors, affording inactive polypeptides or polypeptides with reduced enzymatic activity. Accordingly, modification of the secretory pathway in the modified host cell may improve expression and activity of cannabinoids or cannabinoid derivatives and viability of the modified host cell.
  • the modified host cell of the disclosure may be a modified yeast cell. Yeast cells may be cultured using known conditions, grow rapidly, and are generally regarded as safe. Yeast cells contain the secretory pathway common to all eukaryotes.
  • manipulation of that secretory pathway in yeast host cells modified with one or more nucleic acids may improve expression, folding, and enzymatic activity of cannabinoids or cannabinoid derivatives as well as viability of the modified yeast host cell, such as modified Saccharomyces cerevisiae.
  • nucleic acids e.g., heterologous nucleic acids
  • use of codon- optimized nucleotide sequences encoding engineered variants of the disclosure may improve expression and activity of the engineered variant and viability of modified yeast host cells, such as modified Saccharomyces cerevisiae.
  • the present disclosure provides a more reliable and economical process than agriculture-based production.
  • Microbial fermentations can be completed in days versus the months necessary for an agricultural crop, are not affected by climate variation or soil contamination (e.g., by heavy metals), and can produce pure products at high titer.
  • Moroever, olivetolic acid is very expensive to produce by synthesis.
  • novel sources of olivetolic acid allows for economical production of cannabinoids, cannabinoid derivatives, and/or cannabinoid precursors as disclosed herein.
  • the present disclosure also provides a platform for the economical production of high-value cannabinoids, including THC and/or CBD, as well as derivatives thereof. It also provides for the production of different olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives for which no viable method of production exists.
  • cannabinoids and cannabinoid derivatives may be produced in an amount of over 100 mg per liter of culture medium, over 1 g per liter of culture medium, over 10 g per liter of culture medium, or over 100 g per liter of culture medium.
  • the disclosure provides engineered variants of an OAC polypeptide, methods, modified host cells, and nucleic acids to produce olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives in vivo or in vitro from simple precursors.
  • Nucleic acids e.g., heterologous nucleic acids
  • Nucleic acids disclosed herein can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of one or more polypeptides, such as the engineered variants of the disclosure, which can then be utilized in vitro or in vivo for the production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives.
  • the in vitro methods are cell-free.
  • Cannabinoid Biosynthesis [0084]
  • one or more nucleic acids e.g., heterologous nucleic acids
  • one or more nucleic acids e.g., heterologous nucleic acids
  • one or more nucleic acids encoding one or more polypeptides having at least one activity of a polypeptide present in the cannabinoid or cannabinoid precursor biosynthetic pathway may be useful in the methods and modified host cells for the synthesis of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives.
  • Cannabinoid precursors may include, for example, geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA.
  • GPP geranylpyrophosphate
  • prenyl phosphates olivetolic acid
  • hexanoyl-CoA hexanoyl-CoA
  • cannabinoids are produced from the common metabolite precursors geranylpyrophosphate (GPP) and hexanoyl-CoA by the action of three polypeptides. Hexanoyl-CoA and malonyl-CoA are combined to afford a 12-carbon tetraketide intermediate by a tetraketide synthase (TKS) polypeptide (3,5,7- trioxododecanoyl-CoA).
  • TTS tetraketide synthase
  • This tetraketide intermediate is then cyclized by an olivetolic acid cyclase (OAC) polypeptide to produce olivetolic acid.
  • Olivetolic acid is then prenylated with the common isoprenoid precursor GPP by a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide (e.g., a CsPT4 polypeptide) to produce CBGA, the cannabinoid also known as the “mother cannabinoid.”
  • GTT geranyl pyrophosphate:olivetolic acid geranyltransferase
  • the engineered variants of an OAC polypeptide of the disclosure then converts 3,5,7-trioxododecanoyl-CoA to olivetolic acid or a derivative thereof.
  • GPP and hexanoyl-CoA can be generated through several pathways.
  • One or more nucleic acids e.g., heterologous nucleic acids
  • Polypeptides that generate GPP or are part of a biosynthetic pathway that generates GPP may be one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides).
  • MEV mevalonate
  • MEV pathway may refer to the biosynthetic pathway that converts acetyl-CoA to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • the mevalonate pathway comprises polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMG-CoA synthase (HMGS) polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of a HMG-CoA reductase (HMGR) polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a mevalonate kinase (MK) polypeptide); (e) converting mevalonate 5-phosphate
  • Polypeptides that generate hexanoyl-CoA may include polypeptides that generate acyl-CoA compounds or acyl-CoA compound derivatives (e.g., an acyl-activating enzyme polypeptide, a fatty acyl-CoA synthetase polypeptide, or a fatty acyl-CoA ligase polypeptide). Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such polypeptides.
  • GPP and hexanoyl-CoA may also be generated through pathways comprising polypeptides that condense two molecules of acetyl-CoA to generate acetoacetyl-CoA and pyruvate decarboxylase polypeptides that generate acetyl-CoA from pyruvate via acetaldehyde.
  • Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such pathways.
  • Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabidiol (CBD) type (e.g., cannabidiolic acid), ⁇ 9 -trans-tetrahydrocannabinol ( ⁇ 9 -THC) type (e.g., ⁇ 9 -tetrahydrocannabinolic acid), ⁇ 8 -trans-tetrahydrocannabinol ( ⁇ 8 -THC) type, cannabicyclol
  • CBC cannabichromene
  • CBD cannabidiol
  • CBD cannabidiolic acid
  • ⁇ 9 -trans-tetrahydrocannabinol ⁇ 9 -THC
  • ⁇ 8 -trans-tetrahydrocannabinol ⁇ 8 -TH
  • Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, cannabimovone (CBM) , cannabimovonic acid (CBMA), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBDA, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C 4 (CBD-C 4 ), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C 1 ), ⁇ 9 –tetrahydrocannabinolic acid A (THCA-A), ⁇ 9 –tetrahydrocannabinolic acid A (THCA-A), ⁇ 9 –tetrahydrocannabin
  • Olivetolic acid derivatives include, but are not limited to, : divarinolic acid, orsellinic acid, 3-butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3- methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-resorcylic acid, 3-octyl-resorcylic acid, 3-(trans- 1-octenyl)-resorcylic acid, 3-nonyl-resorcy
  • An acyl-CoA compound as detailed herein may include compounds with the following structure: , wherein R may be an unsubstituted fatty acid side chain or a fatty acid side chain substituted with or comprising one or more functional and/or reactive groups as disclosed herein (i.e., an acyl-CoA compound derivative).
  • R may be an unsubstituted fatty acid side chain or a fatty acid side chain substituted with or comprising one or more functional and/or reactive groups as disclosed herein (i.e., an acyl-CoA compound derivative).
  • a hexanoyl CoA derivative, an acyl-CoA compound derivative, a cannabinoid derivative, or an olivetolic acid derivative may refer to hexanoyl CoA, an acyl-CoA compound, a cannabinoid, or olivetolic acid substituted with or comprising one or more functional and/or reactive groups.
  • Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl (including branched and straight chain alkyl groups), alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkenyl, heteroarylalkenyl, arylal
  • Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like.
  • amine e.g., alkyl amine (e.g., lower alkyl amine), aryl amine
  • ester e.g., alkyl ester (e.g., lower alkyl ester, benzy
  • a reactive group may facilitate covalent attachment of a molecule of interest.
  • Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type; molecules that target to a particular tissue; molecules that provide for crossing the blood- brain barrier; molecules to facilitate selective attachment to a surface; and the like.
  • a cannabinoid derivative or olivetolic acid derivative may also refer to a compound lacking one or more chemical moieties found in naturally-occurring cannabinoids or olivetolic acid, yet retains the core structural features (e.g., cyclic core) of a naturally- occurring cannabinoid or olivetolic acid.
  • Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, and the like.
  • a cannabinoid derivative or olivetolic acid derivative may also comprise one or more of any of the functional and/or reactive groups described herein.
  • Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.
  • nucleic acid or “nucleic acids” used herein, may refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides.
  • this term may include, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, genes, synthetic DNA or RNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other naturally-occurring, chemically or biochemically modified, non- naturally-occurring, or derivatized nucleotide bases.
  • the terms “peptide,” “polypeptide,” and “protein” may be used interchangeably herein, and may refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids.
  • polypeptides disclosed herein may include full- length polypeptides, fragments of polypeptides, truncated polypeptides, fusion polypeptides, or polypeptides having modified peptide backbones.
  • the polypeptides disclosed herein may also be variants differing from a specifically recited “reference” polypeptide (e.g., a wild- type polypeptide) by amino acid insertions, deletions, mutations, and/or substitutions.
  • An “engineered variant of an olivetolic acid cyclase polypeptide” or “engineered variant of the disclosure” may indicate a non-wild type polypeptide having olivetolic acid cyclase activity.
  • Engineered variants may have amino acid substitutions compared to a wild type olivetolic acid cyclase sequence, such as the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO: 1.
  • engineered variants may comprise truncations, additions, and/or deletions, and/or other mutations compared to a wild type olivetolic acid cyclase sequence, such as the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1.
  • Engineered variants may have substitutions compared to a non-wild type sequence (e.g., reference sequence) of olivetolic acid cyclase.
  • engineered variants may comprise truncations, additions, and/or deletions and/or other mutations compared to a non-wild type olivetolic acid cyclase sequence.
  • the engineered variants described herein contain at least one amino acid residue substitution from a reference olivetolic acid cyclase polypeptide.
  • the reference olivetolic acid cyclase polypeptide is a wild type sequence.
  • the reference olivetolic acid cyclase polypeptide is a non-wild type sequence.
  • heterologous may refer to what is not normally found in nature.
  • heterologous nucleotide sequence or the term “heterologous nucleic acid” may refer to a nucleic acid or nucleotide sequence not normally found in a given cell in nature.
  • a heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus; or (d) be naturally found in the host cell, but with introns removed or added.
  • a heterologous nucleic acid may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.
  • a heterologous nucleic acid may comprise a codon-optimized nucleotide sequence.
  • a codon-optimized nucleotide sequence may be an example of a heterologous nucleotide sequence.
  • the heterologous nucleic acids disclosed herein may comprise nucleotide sequences that encode a polypeptide disclosed herein, such as an engineered variant of the disclosure, but do not comprise nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements).
  • the heterologous nucleic acids disclosed herein may comprise nucleotide sequences encoding a polypeptide disclosed herein, such as an engineered variant of the disclosure, along with nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements).
  • heterologous enzyme or “heterologous polypeptide” may refer to an enzyme or polypeptide that is not normally found in a given cell in nature.
  • the term encompasses an enzyme or polypeptide that is: (a) exogenous to a given cell (i.e., encoded by a nucleic acid that is not naturally present in the host cell or not naturally present in a given context in the host cell); or (b) naturally found in the host cell (e.g., the enzyme or polypeptide is encoded by a nucleic acid that is endogenous to the cell) but that is produced in an unnatural amount (e.g., greater or lesser than that naturally found) in the host cell.
  • a heterologous polypeptide may include a mutated version of a polypeptide naturally occurring in a host cell.
  • the term “one or more heterologous nucleic acids” or “one or more heterologous nucleotide sequences” may refer to heterologous nucleic acids comprising one or more nucleotide sequences encoding one or more polypeptides.
  • the one or more heterologous nucleic acids may comprise a nucleotide sequence encoding one polypeptide.
  • the one or more heterologous nucleic acids may comprise nucleotide sequences encoding more than one polypeptide.
  • the nucleotide sequences encoding the more than one polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof.
  • the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of the same polypeptide.
  • the nucleotide sequences encoding the multiple copies of the same polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof.
  • the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of different polypeptides.
  • the nucleotide sequences encoding the multiple copies of the different polypeptides may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof.
  • “increased ratio” may refer to an increase in the molar ratio, an increase in the mass (or weight) ratio, an increase in the molarity ratio, or an increase in the mass concentration (e.g., mg/L or mg/mL) ratio between two products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein compared to the molar ratio, mass (or weight) ratio, molarity ratio, or mass concentration ratio between the same two products produced by another polypeptide, engineered variant, method, and/or modified host cell disclosed herein (e.g., a comparative polypeptide, engineered variant, method, and/or modified host cell disclosed herein).
  • a 100:1 ratio of olivetolic acid or derivatives thereof over olivetol produced by an engineered variant disclosed herein would be an increased ratio of olivetolic acid or derivatives thereof over olivetol compared to an 11:1 ratio of olivetolic acid or derivatives thereof over olivetol produced by a different engineered variant disclosed herein.
  • a ratio of products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein, such as the ratio of olivetolic acid over olivetol may refer to a molar ratio, a mass (or weight) ratio, molarity ratio, or a mass concentration (e.g., mg/L or mg/mL) ratio.
  • “Operably linked” may refer to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • isolated may refer to polypeptides or nucleic acids that are substantially or essentially free from components that normally accompany them in their natural state.
  • An isolated polypeptide or nucleic acid may be other than in the form or setting in which it is found in nature. Isolated polypeptides and nucleic acids therefore may be distinguished from the polypeptides and nucleic acids as they exist in natural cells.
  • An isolated nucleic acid or polypeptide may be purified from one or more other components in a mixture with the isolated nucleic acid or polypeptide, if such components are present.
  • a “modified host cell” may refer to a host cell into which has been introduced a nucleic acid (e.g., a heterologous nucleic acid), e.g., an expression vector or construct.
  • a modified eukaryotic host cell may be produced through introduction into a suitable eukaryotic host cell of a nucleic acid (e.g., a heterologous nucleic acid).
  • a “cell-free system” may refer to a cell lysate, cell extract or other preparation in which substantially all of the cells in the preparation have been disrupted or otherwise processed so that all or selected cellular components, e.g., organelles, proteins, nucleic acids, the cell membrane itself (or fragments or components thereof), or the like, are released from the cell or resuspended into an appropriate medium and/or purified from the cellular milieu.
  • Cell-free systems can include reaction mixtures prepared from purified and/or isolated polypeptides and suitable reagents and buffers.
  • conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide.
  • Conservative substitutions may be accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. The term “conservative amino acid substitution” may refer to the interchangeability in proteins of amino acid residues having similar side chains.
  • a group of amino acids having aliphatic side chains may consist of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains may consist of serine, valine, isoleucine, methionine, and threonine; a group of amino acids having amide containing side chains may consist of asparagine and glutamine; a group of amino acids having aromatic side chains may consist of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains may consist of lysine, arginine, and histidine; a group of amino acids having acidic side chains may consist of glutamate and aspartate; and a group of amino acids having sulfur containing side chains may consist of cysteine and methionine; a group of hydrophililic amino acids may consist of alanine, proline, glycine, glutamic acid, aspartic acid, glutamine
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different manners.
  • sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST,ebi.ac.uk/Tools/msa/tcoffee/ebi.ac.uk/ Tools/msa/muscle/mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol.215:403-10.
  • references to “a cannabinoid compound” or “cannabinoid” may include a plurality of such compounds and reference to “the modified host cell” may include reference to one or more modified host cells and equivalents thereof known to those skilled in the art, and so forth.
  • the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0108] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.
  • OAC olivetolic Acid Cyclase
  • the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution.
  • the inventors have identified amino acid locations of the OAC polypeptide comprising an amino acid sequence of SEQ ID NO:1 that when substituted, may result in one or more improved properties of the engineered variant.
  • the substitution is at a location corresponding to the position in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa.
  • Residue positions in the engineered variants discussed herein may be identified with respect to a reference amino acid sequence, for example, the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa (UniProtKB/Swiss-Prot: Q8GTB6). Accordingly, a reference to “I94” identifies an amino acid that, in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa, is the 94 th amino acid from the N-terminus, wherein the methionine is the first amino acid. The 94 th amino acid is a isoleucine (I) in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa.
  • I isoleucine
  • the 94 th amino acid may have a different position in the OAC polypeptides from different species or in different isoforms. These engineered variants are intended to be encompassed by this disclosure.
  • the polypeptide sequence position at which a particular amino acid or amino acid change (“residue difference”) is present is sometimes described herein as “Xn”, or “position n”, where n refers to the amino acid position with respect to the reference sequence. Accordingly, a reference to “X94” identifies an amino acid that, in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa, is the 94 th amino acid from the N- terminus.
  • a specific substitution mutation which is a replacement of the specific amino acid in a reference sequence with a different specified residue may be denoted by the conventional notation “X (number)Y”, where X is the single letter identifier of the amino in the reference sequence, “number” is the amino acid position in the reference sequence, and Y is the single letter identifier of the amino acid substitution in the engineered sequence.
  • X is the single letter identifier of the amino in the reference sequence
  • number is the amino acid position in the reference sequence
  • Y is the single letter identifier of the amino acid substitution in the engineered sequence.
  • the disclosure provides for an engineered variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions.
  • the engineered variant comprises an amino acid sequence between about 75% to about 99.9% sequence identity with SEQ ID NO:1.
  • the engineered variant comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.
  • the engineered variant comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1.
  • the disclosure provides for an engineered variant comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein the at least one of the amino acid substitutions occurs at an amino acid in a beta sheet and/or an alpha helix of the polypeptide.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions occurs at an amino acid in the beta sheet 1 (e.g., amino acids 4-12), alpha helix 1 (e.g., amino acids 17-33), beta sheet 2 (e.g., amino acids 39-44), beta sheet 3 (e.g., amino acids 57-62), alpha helix 2 (e.g., amino acids 66-73), alpha helix 3 (e.g., amino acids 76-88), or beta sheet 4 (e.g., amino acids 91-97) or combinations of the foregoing.
  • beta sheet 1 e.g., amino acids 4-12
  • alpha helix 1 e.g., amino acids 17-33
  • beta sheet 2 e.g., amino acids 39-44
  • beta sheet 3 e.g., amino acids 57-62
  • alpha helix 2 e.g., amino acids 66-73
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X22, X23, X25, X29, X31, X36, X41, X47, X48, X49, X50, X52, X53, X56, X58, X62, X64, X66, X67, X70, X71, X74, X84, X87, X90, X94, and X100.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X22, X23, X25, X29, X31, X36, X41, X47, X48, X49, X50, X53, X56, X58, X62, X64, X66, X67, X70, X71, X74, X84, X87, X90, X94, and X100.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X22, X
  • Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase (OAC) polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • OAC olivetolic acid cyclase
  • such engineered variants may produce OAC from 3,5,7- trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of OAC produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce OAC from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivative thereof over olivetol compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • Such engineered variants may produce olivetolic acid or a derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid or derivative thereof produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid or a derivative thereof produced from 3,5,7-trioxododecanoyl-CoA or a derivative thereof by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in an increased ratio of olivetolic acid or derivatives thereof over olivetol or derivatives thereof compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions selected from the group consisting of A2P, A2S, V8I, L9I, L9V, K10A, K12A, K12C, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47R, T47S, Q48C, Q48H, Q48M, K49R, N50Y, E52H, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K,
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions selected from the group consisting of A2P, A2S, V8I, L9I, L9V, K10A, K12A, K12C, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47R, T47S, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M
  • Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivative thereof over olivetol compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids A2P, V8I,L9I, E64D, I94K, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K10A, N29D, T56S, I58V, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9I, A36Q, Q48C, I58V, E64D, and I94K.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K49R, T56S, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, A36E, K49R, and R100G. [0119] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, K49R, T56S, and D71T.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, K49R, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K49R, I58V, Q70A, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K10A, T56S, I58V, and Q70K.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids . L9I, K10A, K25N, Q48H, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12V, T56S, I58V, and E64D. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, A36E, Q48H, K49R, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9V, E22L, A36E, Q48M, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, A36S, T62C, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, E22L, A36E, Q48M, and R100G.
  • the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions V8I, L9I, K12N, A36E, D71, and R100G.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO: 1 comprising between about 6 to about 16 amino acid substitutions.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid substitutions.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO: 1 comprising 6 amino acid substitutions.
  • the engineered variant comprises an amino acid sequence having between about 75% to about 99.9%, or about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant comprises an amino acid sequence having at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 2 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 of the amino acid substitutions described herein.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the amino acid substitutions described herein (e.g., 2-16 of the amino acid substitutions described herein).
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6 of the amino acid substitutions described herein.
  • the engineered variant comprises an amino acid sequence comprising amino acid substituions as disclosed herein and having between about 75% to about 99.9%, or between about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant comprises an amino acid sequence amino acid substituions as disclosed herein and having about at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions, wherein at least one of the amino acid substitutions is at the C-terminus.
  • a hydrophilic amino acid is replaced with a hydrophobic amino acid.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 substitutions, wherein at least one of the amino acid substitutions is at the C-terminus, a hydrophobic amino acid is replaced with a hydrophilic amino acid.
  • Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the engineered variant comprises an amino acid sequence having between about 75% to about 99.9%, or between about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1, wherein at least one of the amino acid substitutions is at the C-terminus.
  • the engineered variant comprises an amino acid sequence having at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein at least one of the amino acid substituteions is at the C-terminus.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 and a truncation at the C-terminus.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 ions and a truncation at both the N- and C-termini.
  • the engineered variant lacks a native signal polypeptide.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini, and comprises an amino acid sequence with between about 75% to about 99.9%, or about 85% to about 95% sequence identity to SEQ ID NO:1.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini, and comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus (e.g., 1-10 amino acids at the C-terminus).
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the C-terminus (e.g., 11-20 amino acids at the C-terminus).
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus and comprises an amino acid sequence having about between about 75% to about 99.9%, or about 85% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus and comprises an amino acid sequence having about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, aobut 83%, about
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution and an addition and/or deletion of at least one amino acid.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the N-terminus and comprises an amino acid sequence having about between about 75% to about 99.9%, or about 85% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the N-terminus and comprises an amino acid sequence having about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, aobut 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • the engineered variant produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • the engineered variant produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetol or a derivative thereof of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.
  • the engineered variants of the disclosure can be made and screened for improved properties, such as, production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives or precursors by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • engineered variants of the disclosure can be made and screened for improved properties, such as, a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • the engineered variants of the disclosure exhibit similar and/or not significantly decreased growth and/or viability compared to modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • Nucleic Acids Comprising Nucleotide Sequences Encoding Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide and Expression Vectors and Constructs
  • the disclosure provides for nucleic acids comprising nucleotide sequences encoding engineered variants of the olivetolic acid cyclase (OAC) polypeptide disclosed herein and expression vectors and constructs comprising said nucleic acids.
  • the disclosure provides nucleic acids comprising nucleotide sequences encoding engineered variants of the disclosure.
  • nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an olivetolic acid cyclase variant amino acid sequence of any of SEQ ID NOs: 3-146.
  • the disclosure relates to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an olivetolic acid cyclase variant amino acid sequence of any of SEQ ID NOs: 36-45 and 94-98.
  • the disclosure also provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs: 147-290.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs:180-189 and 138-242.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs: 147-290 or a codon degenerate sequence of anyof the foregoing.
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs:180-189 and 138-242, or a codon degenerate sequence of any of the foregoing.
  • nucleic acids that hybridize to the nucleic acids disclosed herein.
  • Hybridization conditions may be stringent in that hybridization will occur if there is at least a 90%, at least a 95%, or at least a 97% sequence identity with the nucleotide sequence present in the nucleic acid encoding the polypeptides disclosed herein.
  • the stringent conditions may include those used for known Southern hybridizations such as, for example, incubation overnight at 42 °C in a solution having 50% formamide, 5 ⁇ SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt’s solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in 0.1 ⁇ SSC at about 65 °C.
  • Other known hybridization conditions are well known and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001).
  • the length of the nucleic acids disclosed herein may depend on the intended use. For example, if the intended use is as a primer or probe, for example for PCR amplification or for screening a library, the length of the nucleic acid will be less than the full length sequence, for example, 15-50 nucleotides.
  • the primers or probes may be substantially identical to a highly conserved region of the nucleotide sequence or may be substantially identical to either the 5’ or 3’ end of the nucleotide sequence. In some cases, these primers or probes may use universal bases in some positions so as to be “substantially identical” but still provide flexibility in sequence recognition. It is of note that suitable primer and probe hybridization conditions are well known in the art.
  • Some embodiments of the disclosure relate to a vector comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to an expression construct comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to nucleic acids comprising codon-optimized nucleotide sequences encoding the engineered variants of the disclosure. In some embodiments, the nucleic acids disclosed herein are heterologous.
  • Methods of Screening Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide OFC
  • the disclosure provides a method of screening an engineered variant of an olivetolic acid cyclase polypeptide comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution.
  • the method involves a competition assay wherein the engineered variant of the disclosure is expressed in a modified host cells alongside a related enzyme.
  • Some embodiments of the disclosure relate to a method of screening an engineered variant of an olivetolic acid cyclase polypeptide comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, the method comprising: a) dividing a population of host cells into a control population and a test population; b) co-expressing in the control population an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 and a comparison olivetolic acid cyclase polypeptide or TKS enzyme, wherein the OAC polypeptide having an amino acid sequence of SEQ ID NO:1 can convert 3,5,7-trioxododecanoyl-CoA or a derivative thereof to olivetolic acid or a derivative thereof and the comparison olivetolic acid cyclase polypeptide or TKS enzyme can convert the same 3,5,7-triox
  • the engineered variant is an engineered variant of the disclosure.
  • the test population is identified as comprising an engineered variant having improved in vivo performance compared to the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 by producing the first molecule in a greater amount, as measured in mg/L or mM, by the test population compared to the amount produced by the control population under similar culture conditions for the same length of time.
  • the test population is identified as comprising an engineered variant having improved in vivo performance compared to the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, wherein improved in vivo performance is demonstrated by an increase in the ratio of olivetolic acid or derivatives thereof over olivetol or a byproduct thereof produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time.
  • Modified Host Cells for Expressing Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide and for Producing Cannabinoids and Cannabinoid Derivatives [0145]
  • the present disclosure provides modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cells of the disclosure are for expressing an engineered variant and/or for producing an olivetolic acid, an olivetolic acid derivative, a cannabinoid or a cannabinoid derivative.
  • modified host cells of the disclosure may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, or hexanoyl-CoA) biosynthesis.
  • the modified host cells of the disclosure comprise one or more modifications to modulate the expression of one or more secretory pathway polypeptides.
  • the one or more modifications to modulate the expression of one or more secretory pathway polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell.
  • a modified host cell of the present disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, resulting in expression or overexpression of the one or more secretory pathway polypeptides.
  • the modified host cell for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, reducing or eliminating the expression of the one or more secretory pathway polypeptides.
  • the modified host cells comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides.
  • the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides.
  • Secretory pathway polypeptides with modulated expression in the modified host cells of the disclosure may include, but are not limited to: a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, FAD1 polypeptide, a PEP4 polypeptide, and an IRE1 polypeptide.
  • Expression of secretory pathway polypeptides may be modulated by introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell.
  • the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway modifications as disclosed in PCT/US2019/053292, the contents of which are herein incorporated by reference in their entirety for all purposes.
  • the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, or hexanoyl-CoA) biosynthesis.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, or hexanoyl-CoA
  • such polypeptides may include, but are not limited to: a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide, a tetraketide synthase (TKS) polypeptide, one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides), an acyl-activating enzyme (AAE) polypeptide, a polypeptide that generates GPP (e.g., a geranyl pyrophosphate synthetase (GPPS) polypeptide), a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., an acetoacetyl-CoA thiolase polypeptide), and a pyruvate decarboxylase poly
  • GPP mevalonate
  • the modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide.
  • GOT polypeptides disclosed herein may include a full-length GOT polypeptide, a fragment of a GOT polypeptide, a variant of a GOT polypeptide, a truncated GOT polypeptide, or a fusion polypeptide that has at least one activity of a GOT polypeptide.
  • the GOT polypeptide has aromatic prenyltransferase (PT) activity.
  • the GOT polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative. In certain such embodiments, the GOT polypeptide modifies olivetolic acid or an olivetolic acid derivative. In some embodiments, the GOT polypeptide is disclosed in US 2019-0300888, the contents of which are incorporated herein in their entireties for all purposes. [0151] In some embodiments, a NphB polypeptide is used instead of a GOT polypeptide to generate cannabigerolic acid from GPP and olivetolic acid.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide.
  • Exemplary NphB polypeptides disclosed herein may include a full-length NphB polypeptide, a fragment of a NphB polypeptide, a variant of a NphB polypeptide, a truncated NphB polypeptide, or a fusion polypeptide that has at least one activity of a NphB polypeptide.
  • the NphB polypeptide has aromatic prenyltransferase (PT) activity.
  • PT aromatic prenyltransferase
  • the NphB polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative.
  • the NphB polypeptide modifies olivetolic acid or an olivetolic acid derivative.
  • the NphB polypeptide is disclosed in US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300, the contents of which are incorporated herein in their entireties for all purposes.
  • the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more comprises involved in cannabinoid or cannabinoid precursor biosynthesis as disclosed in US 2019/0300888; PCT/US2019/053173; US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300 filed September 18, 2019, the contents of which are incorporated by reference in their entirety for all purposes.
  • a modified host cell of the present disclosure may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide and/or cannabinolic acid synthase (CBDAS).
  • THCAS tetrahydrocannabinolic acid synthase
  • CBDAS cannabinolic acid synthase
  • the THCAS and/or CBDAS polypeptides and variants are disclosed in US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300 filed September 18, 2019 the contents of which are incorporated by reference in their entirety for all purposes.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acyl-CoA compounds or acyl-CoA compound derivatives.
  • polypeptides may include, but are not limited to, acyl-activating enzyme (AAE) polypeptides, fatty acyl-CoA synthetases (FAA) polypeptides, or fatty acyl-CoA ligase polypeptides.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide.
  • AAE polypeptides, FAA polypeptides, and fatty acyl-CoA ligase polypeptides can convert carboxylic acids to their CoA forms and generate acyl-CoA compounds or acyl-CoA compound derivatives.
  • Promiscuous acyl-activating enzyme polypeptides such as CsAAE1 and CsAAE3 polypeptides, FAA polypeptides, or fatty acyl- CoA ligase polypeptides, may permit generation of cannabinoid derivatives (e.g., cannabigerolic acid derivatives), as well as cannabinoids (e.g., cannabigerolic acid).
  • unsubstituted or substituted hexanoic acid or carboxylic acids other than unsubstituted or substituted hexanoic acid are fed to modified host cells expressing an AAE polypeptide, FAA polypeptide, or fatty acyl-CoA ligase polypeptide (e.g., are present in the culture medium in which the cells are grown) to generate hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl-CoA compounds.
  • the hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl- CoA compounds can then be further utilized by a modified host cell to generate olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives.
  • the cell culture medium comprising the modified host cells comprises unsubstituted or substituted hexanoic acid.
  • the cell culture medium comprising the modified host cells comprises a carboxylic acid other than unsubstituted or substituted hexanoic acid.
  • Exemplary AAE, FAA, or fatty acyl-CoA ligase polypeptides disclosed herein may include a full-length AAE, FAA, or fatty acyl-CoA ligase polypeptide; a fragment of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a variant of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a truncated AAE, FAA, or fatty acyl-CoA ligase polypeptide; or a fusion polypeptide that has at least one activity of an AAE, FAA, or fatty acyl-CoA ligase polypeptide.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides that condense an acyl-CoA compound, such as hexanoyl-CoA, or an acyl-CoA compound derivative, such as a hexanoyl-CoA derivative, with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid.
  • Polypeptides that react an acyl-CoA compound or an acyl-CoA compound derivative with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid may include TKS polypeptides.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates GPP.
  • the polypeptide that generates GPP is a geranyl pyrophosphate synthetase (GPPS) polypeptide.
  • GPPS geranyl pyrophosphate synthetase
  • the GPPS polypeptide also has farnesyl diphosphate synthase (FPPS) polypeptide activity.
  • FPPS farnesyl diphosphate synthase
  • the GPPS polypeptide is modified such that it has reduced FPPS polypeptide activity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than at least 90%, less FPPS polypeptide activity) than the corresponding wild-type or parental GPPS polypeptide from which the modified GPPS polypeptide is derived.
  • the GPPS polypeptide is modified such that it has substantially no FPPS polypeptide activity.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide.
  • GPPS polypeptides disclosed herein may include a full-length GPPS polypeptide, a fragment of a GPPS polypeptide, a variant of a GPPS polypeptide, a truncated GPPS polypeptide, or a fusion polypeptide that has at least one activity of a GPPS polypeptide.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acetyl-CoA from pyruvate.
  • Polypeptides that generate acetyl-CoA from pyruvate may include a pyruvate decarboxylase (PDC) polypeptide.
  • PDC pyruvate decarboxylase
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide.
  • Exemplary PDC polypeptides disclosed herein may include a full-length PDC polypeptide, a fragment of a PDC polypeptide, a variant of a PDC polypeptide, a truncated PDC polypeptide, or a fusion polypeptide that has at least one activity of a PDC polypeptide.
  • a modified host cell of the disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA.
  • the polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA is an acetoacetyl-CoA thiolase polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide.
  • Exemplary acetoacetyl-CoA thiolase polypeptides disclosed herein may include a full-length acetoacetyl-CoA thiolase polypeptide, a fragment of an acetoacetyl- CoA thiolase polypeptide, a variant of an acetoacetyl-CoA thiolase polypeptide, a truncated acetoacetyl-CoA thiolase polypeptide, or a fusion polypeptide that has at least one activity of an acetoacetyl-CoA thiolase polypeptide.
  • a modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway.
  • the one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway comprise one or more MEV pathway polypeptides.
  • the one or more polypeptides that are part of a biosynthetic pathway that generates GPP are one or more polypeptides having at least one activity of a polypeptide present in the mevalonate pathway.
  • the mevalonate pathway may comprise polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMGS polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of an HMGR polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a MK polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by action of a PMK polypeptide); (f) converting
  • the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell. In some embodiments, the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell, yet displays increased activity compared to a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time. In some embodiments, the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell, yet produces more olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA compared to a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time.
  • the engineered variant of the disclosure expressed in the modified host cell produces more olivetolic acid or derivative thereof from 3,5,7- trioxododecanoyl-CoA compared to multiple copies of a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time.
  • the engineered variant of the disclosure is expressed from a single copy in the modified host cell, yet produces more olivetolic acid or derivative thereof from 3,5,7- trioxododecanoyl-CoA or derivative thereof compared to two, three, four, five, six, seven, eight, nine, or ten wild type polypeptides expressed in a corresponding host cell under similar culture conditions for the same length of time.
  • the engineered variant of the disclosure is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the engineered variant of the disclosure to a strong promoter.
  • the modified host cell has one copy of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure.
  • the modified host cell has two copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has three copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has four copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has five copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure.
  • the modified host cell has six copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has seven copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight or more copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure.
  • modified host cells of the disclosure may be modified to express or overexpress one or more nucleic acids disclosed herein comprising nucleotide sequences encoding an engineered variant of the disclosure, one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and/or one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA
  • a modified host cell of the disclosure may comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA
  • a modified host cell of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • a modified host cell of the disclosure has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • the growth and/or viability of modified host cells of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, the growth and/or viability of modified host cells of the disclosure is about the same as the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments, the growth and/or viability of modified host cells of the disclosure is less than the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions.
  • the growth and/or viability of modified host cells of the disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives is greater than the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions.
  • a culture of modified host cells of the disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions.
  • the one or more heterologous polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the one or more heterologous nucleic acids to a strong promoter.
  • a high copy number expression vector e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell
  • the modified host cell has one copy of a one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptide. In some embodiments, the modified host cell has two copies, three copies, four copies, five copies, six copies, seven copies, or eight copies or more of one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptides. Increased copy number of the one or more heterologous nucleic acids and/or codon optimization of the one or more heterologous nucleotide acids may result in an increase in the desired enzyme catalytic activity in the modified host cell.
  • a modified host cell of the disclosure produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivatives thereof over olivetol thereof compared to that produced by a modified host cell comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • the modified host cell of the disclosure produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA in an increased ratio of olivetolic acid or derivatives thereof over olivetol of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1.
  • the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • the modified host cell of the disclosure comprises nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, and/or an IRE1 polypeptide.
  • the modified host cell of the disclosure comprises a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • GPP geranylpyrophosphate
  • prenyl phosphates e.g., olivetolic acid, or hexanoyl-CoA
  • the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA
  • the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide.
  • the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and further comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • GPP geranylpyrophosphate
  • prenyl phosphates olivetolic acid
  • the modified host cell of the disclosure further comprises a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • Suitable Host Cells may include eukaryotic cells. In some embodiments, the eukaryotic cells are yeast cells. [0176] Host cells (including parent host cells and modified host cells) are in some embodiments unicellular organisms, or are grown in culture as single cells. In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells may include, but are not limited to, yeast cells and fungal cells.
  • Suitable eukaryotic host cells may include, but are not limited to, Pichia pastoris (now known as Komagataella phaffii), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta), Yarrowia lipolytica, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pombe, Scheffersomyces stipites, Dekkera brux
  • the modified host cell disclosed herein is cultured in vitro.
  • the host cell of the disclosure is a yeast cell.
  • the host cell is a protease-deficient strain of Saccharomyces cerevisiae.
  • Protease-deficient yeast strains may be effective in reducing the degradation of expressed heterologous proteins. Examples of proteases deleted in such strains may include one or more of the following: PEP4, PRB1, and KEX1.
  • the host cell is Saccharomyces cerevisiae.
  • the host cell for use in generating a modified host cell of the present disclosure may be selected because of ease of culture; rapid growth; availability of tools for modification, such as promoters and vectors; and the host cell’s safety profile.
  • the host cell for use in generating a modified host cell of the present disclosure may be selected because of its ability or inability to introduce certain posttranslational modifications onto expressed polypeptides, such as engineered variants of the disclosure.
  • modified Komagataella phaffii host cells may hyperglycosylate engineered variants of the disclosure and hyperglycosylation may alter the activity of the resultant expressed polypeptide.
  • the present disclosure provides for modified host cells and methods of making the modified host cells of the disclosure.
  • the method of making a modified host cell of the disclosure comprises introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cell comprises two or more heterologous nucleic acids each comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell further comprises a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide.
  • the disclosure provides a method of making a modified host cell of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, and/or an IRE1 polypeptide, and/or c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • nucleic acids e.g., heterologous
  • Such techniques may include, but are not limited to, electroporation, calcium phosphate precipitation, DEAE- dextran mediated transfection, liposome-mediated transfection, the lithium acetate method, and the like. See Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD.
  • nucleic acids comprising one or more nucleic acids (e.g., heterologous) disclosed herein will generally include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.
  • the selectable marker gene to provide a phenotypic trait for selection of transformed host cells is dihydrofolate reductase.
  • a parent host cell is modified to produce a modified host cell of the present disclosure using a CRISPR/Cas9 system to modify a parent host cell with one or more nucleic acids (e.g., heterologous) disclosed herein.
  • varying polypeptide expression level such as engineered variant expression level, and/or the production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives in a modified host cell may be done by changing the gene copy number, promoter strength, and/or promoter regulation and/or by codon-optimization.
  • One or more nucleic acids (e.g., heterologous) disclosed herein, such as one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, can be present in an expression vector or construct.
  • Suitable expression vectors may include, but are not limited to, plasmids, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as yeast).
  • one or more nucleic acids e.g., heterologous
  • Such vectors may include chromosomal, non-chromosomal, and synthetic DNA sequences.
  • the present disclosure provides for a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell one or more vectors disclosed herein.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides.
  • the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.
  • the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon- optimized.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.
  • the nucleotide sequences encoding the one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis are codon- optimized.
  • the present disclosure provides for a method of making a modified host cell for expressing an olivetolic acid cyclase, the method comprising introducing into a host cell one or more vectors disclosed herein.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides.
  • the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.
  • the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.
  • the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.
  • the nucleotide sequences encoding the one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis are codon-optimized.
  • Numerous additional suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for yeast, the low copy CEN ARS and high copy 2 micron plasmids. However, any other plasmid or other vector may be used so long as it is compatible with the host cell.
  • one or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector.
  • two, three, four, five, six, seven, or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, two, three, four, five, six, seven, eight, nine, ten or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, the expression vector is an expression construct. [0189] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a high copy number plasmid, e.g., a plasmid that exists in about 10-50 copies per cell, or more than 50 copies per cell.
  • one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a low copy number plasmid. In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a medium copy number plasmid.
  • the copy number of the plasmid may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression by limiting the copy number of the plasmid may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.
  • the modified host cell has one copy of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has two, three, four, five, six, seven, eight, nine, ten, eleven, or tweleve or more copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. [0191] Depending on the host/vector or host/construct system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc.
  • the nucleic acids (e.g., heterologous) disclosed herein are operably linked to a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is functional in a eukaryotic cell.
  • the promoter can be a strong driver of expression.
  • the promoter can be a weak driver of expression.
  • the promoter can be a medium driver of expression.
  • the promoter may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression through promoter selection may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.
  • one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into the genome of the modified host cell disclosed herein.
  • one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into a chromosome of the modified host cell disclosed herein.
  • one or more nucleic acids (e.g., heterologous) disclosed herein remains episomal (i.e., is not integrated into the genome or a chromosome of the modified host cell). In some embodiments, at least one of the one or more nucleic acids (e.g., heterologous) disclosed herein is maintained extrachromosomally (e.g., on a plasmid or artificial chromosome).
  • the gene copy number of one or more genes encoding one or more polypeptides disclosed herein, such as an engineered variant of the disclosure may be selected to reduce expression of the one or more polypeptides disclosed herein, such as an engineered variant of the disclosure.
  • Reducing expression by limiting the gene copy number may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.
  • slight changes in nucleotide sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. It will be appreciated by persons skilled in the art that changes in the identities of nucleotides in a specific gene sequence that change the amino acid sequence of the encoded polypeptide may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g., anti-sense, co-suppression, or RNAi), partial sequences often work as effectively as full length versions.
  • nucleotide sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. In certain embodiments, effectiveness may easily be tested by, for example, conventional gas chromatography. All such variations of the genes are therefore included as part of the present disclosure.
  • Genomic deletion of the open reading frame encoding the protein may abolish all expression of a gene. Downregulation of a gene can be accomplished in several ways at the DNA, RNA, or protein level, with the result being a reduction in the amount of active protein in the cell.
  • Engineering of the regulatory regions of the gene can also be used to change gene expression. Alteration of the promoter sequence or replacement with a different promoter is one method.
  • Truncation of the terminator known as decreased abundance of mRNA perturbation (DAmP), is also known to reduce gene expression.
  • Other methods that reduce the stability of the mRNA include the use of cis- or trans-acting ribozymes, e.g., self-cleaving ribozymes, or RNA elements that recruit an exonuclease, or antisense DNA.
  • RNAi may be used to silence genes in budding yeast strains via import of the required protein factors from other species, e.g., Drosha or Dice (Drinnenberg et al 2009). Gene expression may also be silenced in S. cerevisiae via recruitment of native or heterologous silencing factors or repressors, which may be accomplished at arbitrary loci using the D-Cas9 CRISPR system (Qi et al 2013). Protein level can also be reduced by engineering the amino acid sequence of the target protein. A variety of degron sequences may be used to target the protein for rapid degradation, including, but not limited to, ubiquitin fusions and N-end rule residues at the amino terminus. These methods may be implemented in a constitutive or conditional fashion.
  • the disclosure provides methods for expressing an engineered variant of the olivetolic acid cyclase (OAC) polypeptide of the disclosure.
  • the methods comprise culturing a modified host cell of the disclosure in a culture medium.
  • the disclosure also provides methods for preparing an engineered variant of the olivetolic acid cyclase (OAC) polypeptide of the disclosure.
  • the disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of the disclosure.
  • the methods may involve culturing a modified host cell of the present disclosure in a culture medium and recovering the produced cannabinoid or cannabinoid derivative.
  • the methods may also involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives using one or more polypeptides disclosed herein, such as an engineered variant of the disclosure, expressed or overexpressed by a modified host cell of the disclosure.
  • the methods may also involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives using an engineered variant disclosed herein.
  • Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabidiol (CBD) type (e.g., cannabidiolic acid), ⁇ 9 -trans-tetrahydrocannabinol ( ⁇ 9 -THC) type (e.g., ⁇ 9 -tetrahydrocannabinolic acid), ⁇ 8 -trans-tetrahydrocannabinol ( ⁇ 8 -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, derivatives of any of the foregoing, and others as listed in Elsohly
  • Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, cannabimovone (CBM) , cannabimovonic acid (CBMA), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBDA, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C 4 (CBD-C 4 ), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C 1 ), ⁇ 9 –tetrahydrocannabinolic acid A (THCA-A), ⁇ 9 –tetrahydrocannabinolic acid A (THCA-A), ⁇ 9 –tetrahydrocannabin
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.
  • the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is ⁇ 9 - tetrahydrocannabinolic acid, ⁇ 9 -tetrahydrocannabinol, ⁇ 8 -tetrahydrocannabinolic acid, ⁇ 8 - tetrahydrocannabinol, cannabidiolic acid, cannabidiol, cannabichromenic acid, cannabichromene, cannabinolic acid, cannabinol, cannabidivarinic acid, cannabidivarin, tetrahydrocannabivarinic acid, tetrahydrocannabivarin, cannabichromevarinic acid, cannabichromevarin, cannabigerovarinic acid, cannabigerovarin, cannabicyclolic acid, cannabicyclol, cannabielsoinic acid, cannabielsoin
  • the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium. [0199] In some embodiments, the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin. In some embodiments, the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium.
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the engineered variant of the disclosure.
  • the engineered variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the engineered variant of the disclosure.
  • the engineered variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, CBDA, CBD, CBGA, CBG, THC, THCA, THCVA, THCV, CBDVA, CBDV, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-butyl-6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-3-butyl-6,6,9-trimethyl-6a,7,8,10a- tetrahydro-6H-benzo[c]chromen-1-ol, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-(
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.
  • Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, (1'R,2'R)-4-(hexan-2-yl)-5'-methyl-2'-(prop-1-en-2-yl)- 1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-hexyl-5'-methyl-2'-(prop-1-en-2- yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(3-methylpentyl)-2'- (prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-bi
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.
  • a cannabinoid derivative may also refer to a compound lacking one or more chemical moieties found in naturally-occurring cannabinoids, yet retains the core structural features (e.g., cyclic core) of a naturally-occurring cannabinoid.
  • a cannabinoid derivative may also comprise one or more of any of the functional and/or reactive groups described herein.
  • Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups such as those described in PCT/US2019/053292, the contents of which are herein incorporated by reference in their entirety for all purposes.
  • a cannabinoid derivative may be a cannabinoid substituted with or comprising one or more functional and/or reactive groups.
  • Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocycly
  • Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, acetyl, and the like.
  • amine e.g., alkyl amine (e.g., lower alkyl amine), aryl amine
  • ester e.g., alkyl ester (e.g., lower alkyl
  • the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, a thioester, a thioether, a sulfonyl halide, an alcohol, a thiol, an alkyne, alkene, an azide, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, and a hydrazine.
  • Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.
  • Alkyl may refer to a straight or branched chain saturated hydrocarbon.
  • C 1 -C 6 alkyl groups contain 1 to 6 carbon atoms.
  • Examples of a C 1 -C 6 alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec- butyl and tert-butyl, isopentyl, and neopentyl.
  • Alkenyl may include an unbranched (i.e., straight) or branched hydrocarbon chain containing 2-12 carbon atoms.
  • the “alkenyl” group contains at least one double bond. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group.
  • alkenyl groups may include, but are not limited to, ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2- ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl and the like.
  • Compounds disclosed herein, such as cannabinoids and cannabinoid derivatives may be substituted with one or more substituents, such as those illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the present disclosure.
  • substituted refers to the replacement of a hydrogen atom in a given structure with a specified substituent. Combinations of substituents envisioned by the present disclosure are typically those that result in the formation of stable or chemically feasible compounds.
  • unsubstituted may mean that the specified group bears no substituents beyond the moiety recited (e.g., where valency satisfied by hydrogen).
  • a reactive group may facilitate covalent attachment of a molecule of interest.
  • Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; and the like.
  • a linker may be a peptide linker or a non-peptide linker.
  • a cannabinoid derivative substituted with an azide may be reacted with a compound comprising an alkyne group via “click chemistry” to generate a product comprising a heterocycle, also known as an azide-alkyne cycloaddition.
  • a cannabinoid derivative substituted with an alkyne may be reacted with a compound comprising an azide group via click chemistry to generate a product comprising a heterocycle.
  • Additional molecules of interest that may be desirable for attachment to a cannabinoid derivative may include, but are not necessarily limited to, detectable labels (e.g., spin labels, fluorescence resonance energy transfer (FRET)-type dyes, e.g., for studying structure of biomolecules in vivo); small molecule drugs; cytotoxic molecules (e.g., drugs); imaging agents; ligands for binding by a target receptor; tags to aid in purification by, for example, affinity chromatography (e.g., attachment of a FLAG epitope); molecules that increase solubility (e.g., poly(ethylene glycol)); molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type (e.g., an antibody specific for an epitope on a target cell); molecules that target to a particular tissue; molecules that provide for crossing the blood-brain barrier; and molecules to facilitate selective attachment to a surface, and the like.
  • detectable labels e.g., spin labels,
  • a molecule of interest comprises an imaging agent.
  • Suitable imaging agents may include positive contrast agents and negative contrast agents.
  • Suitable positive contrast agents may include, but are not limited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA); gadolinium- diethylenetriaminepentaacetic acid (Gd-DTPA); gadolinium-1,4,7-tris(carbonylmethyl)-10- (2'-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (Gd-HP-DO3A); Manganese(II)- dipyridoxal diphosphate (Mn-DPDP); Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); and the like.
  • Gd-DOTA gadolinium tetraazacyclododecanetetraacetic acid
  • Gd-DTPA gadolinium- diethylenetriaminepentaacetic acid
  • Suitable negative contrast agents may include, but are not limited to, a superparamagnetic iron oxide (SPIO) imaging agent; and a perfluorocarbon, where suitable perfluorocarbons may include, but are not limited to, fluoroheptanes, fluorocycloheptanes, fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes, fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes, fluorodimethylcyclopentanes, fluoromethylcyclobutanes, fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes, fluorocyclobutanes, fluoropropanes, fluoroethers, fluoropolyethers, fluorotriethylamines, perfluorohexanes, perfluoropentanes, perfluorobutanes, perfluoropropanes, sulfur
  • Additional cannabinoid derivatives that can be produced with an engineered variant, method, or modified host cell of the present disclosure may include derivatives that have been modified via organic synthesis or an enzymatic route to modify drug metabolism and pharmacokinetics (e.g., solubility, bioavailability, absorption, distribution, plasma half- life and metabolic clearance). Modification examples may include, but are not limited to, halogenation, acetylation, and methylation.
  • the cannabinoids or cannabinoid derivatives described herein further include all pharmaceutically acceptable isotopically labeled cannabinoids or cannabinoid derivatives.
  • an “isotopically-” or “radio-labeled” compound is a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • hydrogen atoms are replaced or substituted by one or more deuterium or tritium.
  • Certain isotopically labeled cannabinoids or cannabinoid derivatives of this disclosure for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies.
  • radioactive isotopes tritium, i.e., 3 H, and carbon 14, i.e., 14 C are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • Suitable isotopes that may be incorporated in cannabinoids or cannabinoid derivatives described herein include but are not limited to 2 H (also written as D for deuterium), 3 H (also written as T for tritium), 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl , 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, and 131 I. Substitution with positron emitting isotopes, such as 11 C, 18 F, 15 O, and 13 N, can be useful in Positron Emission Topography (PET) studies.
  • PET Positron Emission Topography
  • Cannabinoids or cannabinoid derivatives disclosed herein may be enantiomers or disastereomers.
  • the term “enantiomers” may refer to a pair of stereoisomers which are non-superimposable mirror images of one another.
  • the cannabinoids or cannabinoid derivatives may be the (S)-enantiomer.
  • the cannabinoids or cannabinoid derivatives may be the (R)-enantiomer.
  • the cannabinoids or cannabinoid derivatives may be the (+) or (-) enantiomers.
  • the term “diastereomers” may refer to the set of stereoisomers which cannot be made superimposable by rotation around single bonds. For example, cis- and trans- double bonds, endo- and exo- substitution on bicyclic ring systems, and compounds containing multiple stereogenic centers with different relative configurations may be considered to be diastereomers.
  • the term “diastereomer” may refer to any member of this set of compounds.
  • Cannabinoids or cannabinoid derivatives disclosed herein may include a double bond or a fused ring.
  • the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the cannabinoid or cannabinoid derivative contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined.
  • the cannabinoid or cannabinoid derivative when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium; or from a cell- free reaction mixture comprising one or more polypeptides and/or engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt.
  • the salt is a pharmaceutically acceptable salt.
  • the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • composition includes pharmaceutically acceptable salts of the cannabinoids or cannabinoid derivatives described herein.
  • “Pharmaceutically acceptable salts” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable.
  • Representative pharmaceutically acceptable salts include, but are not limited to, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate, lactate, lactobionate, laurate,
  • “Pharmaceutically acceptable salt” also includes both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulf
  • “Pharmaceutically acceptable base addition salt” may refer to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. For example, inorganic salts include, but are not limited to, ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • basic ion exchange resins such as am
  • the disclosure provides methods of producing an olivetolic acid, olivetolic acid derivatives,cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced olivetolic acid, olivetolic acid derivative, cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • culturing of the modified host cells of the disclosure in a culture medium provides for synthesis of an olivetolic acid, olivetolic acid derivative, cannabinoid or a cannabinoid derivative, such as those described herein, in an increased amount compared to an unmodified host cell cultured under similar conditions.
  • the disclosure provides methods of producing a cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium comprising a carboxylic acid. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative.
  • the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; or from the cell lysate, the modified host cell, and the culture medium.
  • the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • the recovered cannabinoid or cannabinoid derivative is in the form of a salt.
  • the salt is a pharmaceutically acceptable salt.
  • the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid.
  • the carboxylic acid may be substituted with or comprise one or more functional and/or reactive groups.
  • Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, heteroarylalkyn
  • Reactive groups may include, but are not necessarily limited to, azide, halogen, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like.
  • amine e.g., alkyl amine (e.g., lower alkyl amine), aryl amine
  • ester e.g., alkyl ester (e.g., lower alkyl ester, benzyl
  • the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, thioester, thioether, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, an azide, an alkyne, an alkene, and a hydrazine.
  • Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.
  • the carboxylic acid is isotopically- or radio-labeled.
  • the carboxylic acid may be an enantiomer or disastereomer. In some embodiments the carboxylic acid may be the (S)-enantiomer. In some embodiments, the carboxylic acid may be the (R)-enantiomer. In some embodiments, the carboxylic acid may be the (+) or (-) enantiomer. In some embodiments, the carboxylic acid may include a double bond or a fused ring. In certain such embodiments, the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the carboxylic acid contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined.
  • the carboxylic acid comprises an alkyl group. In some embodiments, the carboxylic acid comprises an aryl group.
  • Carboxylic acids may include, but are not limited to, unsubstituted or substituted C 3 -C 18 fatty acids, C 3 -C 18 carboxylic acids, C 1 -C 18 carboxylic acids, butyric acid, isobutyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, myristic acid, C 15 -C 18 fatty acids, C 15 -C 18 carboxylic acids, fumaric acid, itaconic acid, malic acid, succinic acid, maleic acid, malonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebac
  • Carboxylic acids may include unsubstituted or substitutedC 1 -C 18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C 3 -C 18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C 3 -C 12 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C 4 -C 10 carboxylic acids. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 4 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 5 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 6 carboxylic acid.
  • the carboxylic acid is an unsubstituted or substituted C 7 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 8 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 9 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C 10 carboxylic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted butyric acid. In some embodiments, carboxylic acid is unsubstituted or substituted valeric acid. In some embodiments, the carboxylic acid is unsubstituted or substituted hexanoic acid.
  • the carboxylic acid is unsubstituted or substituted heptanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted octanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted nonanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted decanoic acid.
  • Carboxylic acids may include, but are not limited to, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3- hexenoic acid, 4-hexenoic acid, 5-hexenoic acid, 5-chlorovaleric acid, 5-aminovaleric acid, 5-cyanovaleric acid, 5-(methylsulfanyl)valeric acid, 5-hydroxyvaleric acid, 5-phenylvaleric acid, 2,3-dimethylhexanoic acid, d 3 -hexanoic acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, trans-2- nonenoic acid, 4-phenylbutyric acid, 6-phenylhexanoic acid, 7-phenylheptanoic
  • the carboxylic acid is 2-methylhexanoic acid. In some embodiments, the carboxylic acid is 3-methylhexanoic acid. In some embodiments, the carboxylic acid is 4-methylhexanoic acid. In some embodiments, the carboxylic acid is 5- methylhexanoic acid. In some embodiments, the carboxylic acid is 2-hexenoic acid. In some embodiments, the carboxylic acid is 3-hexenoic acid. In some embodiments, the carboxylic acid is 4-hexenoic acid. In some embodiments, the carboxylic acid is 5-hexenoic acid. In some embodiments, the carboxylic acid is 5-chlorovaleric acid.
  • the carboxylic acid is 5-aminovaleric acid. In some embodiments, the carboxylic acid is 5-cyanovaleric acid. In some embodiments, the carboxylic acid is 5- (methylsulfanyl)valeric acid. In some embodiments, the carboxylic acid is 5-hydroxyvaleric acid. In some embodiments, the carboxylic acid is 5-phenylvaleric acid. In some embodiments, the carboxylic acid is 2,3-dimethylhexanoic acid. In some embodiments, the carboxylic acid is d3-hexanoic acid. In some embodiments, the carboxylic acid is 4- pentynoic acid. In some embodiments, the carboxylic acid is trans-2-pentenoic acid.
  • the carboxylic acid is 5-hexynoic acid. In some embodiments, the carboxylic acid is trans-2-hexenoic acid. In some embodiments, the carboxylic acid is 6-heptynoic acid. In some embodiments, the carboxylic acid is trans-2-octenoic acid. In some embodiments, the carboxylic acid is trans-2-nonenoic acid. In some embodiments, the carboxylic acid is 4- phenylbutyric acid. In some embodiments, the carboxylic acid is 6-phenylhexanoic acid. In some embodiments, the carboxylic acid is 7-phenylheptanoic acid.
  • the carboxylic acid is an unsubstituted or substituted C 3 -C 18 carboxylic acid.
  • the unsubstituted or substituted C 3 -C 18 carboxylic acid is an unsubstituted or substituted hexanoic acid.
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.
  • the carboxylic acid is butyric acid, valeric acid, hexanoic acid, octanoic acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4- hexenoic acid, 5-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5- (methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myr
  • Suitable media for culturing modified host cells of the disclosure may include standard culture media (e.g., Luria-Bertani broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where nucleic acids disclosed herein are under the control of an inducible promoter, etc.); standard yeast culture media; and the like).
  • standard culture media e.g., Luria-Bertani broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where nucleic acids disclosed herein are under the control of an inducible promoter, etc.); standard yeast culture media; and the like).
  • the culture medium can be supplemented with a fermentable sugar (e.g., a hexose sugar or a pentose sugar, e.g., glucose, xylose, galactose, and the like).
  • Sugars fermentable by yeast may include, but are not limited to, sucrose, dextrose, glucose, fructose, mannose, galactose, and maltose.
  • the culture medium can be supplemented with unsubstituted or substituted hexanoic acid, carboxylic acids other than unsubstituted or substituted hexanoic acid, olivetolic acid, or olivetolic acid derivatives.
  • the culture medium can be supplemented with pretreated cellulosic feedstock (e.g., wheat grass, wheat straw, barley straw, sorghum, rice grass, sugarcane straw, bagasse, switchgrass, corn stover, corn fiber, grains, or any combination thereof).
  • the culture medium can be supplemented with oleic acid.
  • the culture medium comprises a non-fermentable carbon source.
  • the non-fermentable carbon source comprises ethanol.
  • the suitable media comprises an inducer.
  • the inducer comprises galactose.
  • the inducer comprises KH 2 PO 4 , galactose, glucose, sucrose, maltose, an amino acid (e.g., methionine, lysine), CuSO4, a change in temperature (e.g., 30 °C to 37 °C), a change in pH (e.g., pH 6 to pH 4), a change in oxygen level (e.g., 20% to 1% dissolved oxygen levels), addition of hydrogen peroxide or superoxide-generating drug menadione, tunicamycin, expression of proteins prone to misfolding (e.g., cannabinoid synthases), estradiol, or doxycycline. Additional induction systems are detailed herein.
  • the carbon source in the suitable media can vary significantly, from simple sugars like glucose to more complex hydrolysates of other biomass, such as yeast extract.
  • the addition of salts generally provides essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize polypeptides and nucleic acids.
  • the suitable media can also be supplemented with selective agents, such as antibiotics, to select for the maintenance of certain plasmids and the like. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent cells lacking the resistance from growing.
  • modified host cells disclosed herein are grown in minimal medium or minimal media.
  • minimal medium or minimal media may refer to media comprising a defined composition of nutrients, generally chosen for minimal cost, while still allowing for robust growth and production.
  • minimal medium or “minimal media” may refer to media containing: (1) one or more carbon sources for cellular (e.g., bacterial or yeast) growth; (2) various salts, which can vary among cellular (e.g., bacterial or yeast) species and growing conditions; (3) vitamins and trace elements; and (4) water.
  • minimal media lacks one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids).
  • Minimal media may also comprise growth factors, inducers, and repressors.
  • minimal media or minimal medium affords higher biomass formation in a fermentation tank compared to rich medium or rich media.
  • the minimal medium or minimal media comprises a carboxylic acid (e.g., 1 mM olivetolic acid, 1 mM olivetolic acid derivative, 2 mM unsubstituted or substituted hexanoic acid, or 2 mM of a carboxylic acid other than unsubstituted or substituted hexanoic acid).
  • modified host cells disclosed herein are grown in rich medium or rich media.
  • the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, yeast extract, Bacto peptone, and dextrose (glucose).
  • the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, 10 g/L yeast extract, 20 g/L Bacto peptone, and 20 g/L dextrose (glucose).
  • the rich medium or rich media comprises YP + galactose and glucose.
  • the rich medium or rich media comprises YP + 20 g/L galactose or YP + 40 g/L galactose and 1 g/L glucose.
  • the rich medium or rich media comprises a carboxylic acid (e.g., 1 mM olivetolic acid, 1 mM olivetolic acid derivative, 2 mM unsubstituted or substituted hexanoic acid, or 2 mM of a carboxylic acid other than unsubstituted or substituted hexanoic acid).
  • rich medium or rich media affords greater cell density in fermentation compared to minimal media or minimal medium.
  • Standard cell culture conditions can be used to culture the modified host cells disclosed herein (see, for example, WO 2004/033646 and references cited therein).
  • cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 °C to about 37 °C, at about 0.04% to about 84% CO 2 , at about 0% to about 100% dissolved oxygen, and at a pH between about 2.0 to about 9.0).
  • modified host cells disclosed herein are grown at about 34 °C in a suitable cell culture medium. In some embodiments, modified host cells disclosed herein are grown at about 20 °C to about 37 °C in a suitable cell culture medium. While the growth optimum for S. cerevisiae is about 30 °C, culturing cells at a higher temperature, e.g., 34 °C may be advantageous by reducing the costs to cool industrial fermentation tanks.
  • modified host cells disclosed herein are grown at about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, or about 37 °C in a suitable cell culture medium.
  • the pH ranges for fermentation are between about pH 3.0 to about pH 9.0 (such as about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 6.0 to about pH 8.0 or about pH 6.5 to about pH 7.0).
  • the pH ranges for fermentation are between about pH 4.5 to about pH 5.5.
  • the pH ranges for fermentation are between about pH 4.0 to about pH 6.0.
  • the pH ranges for fermentation are between about pH 3.0 to about pH 6.0.
  • the pH ranges for fermentation are between about pH 3.0 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.0. In some embodiments, the dissolved oxygen is between about 0% to about 10%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% to about 80%, about 0% to about 90%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40% or about 10% to about 50%.
  • the CO 2 level is between about 0.04% to about 0.1% CO 2 , about 0.04% to about 1% CO 2 , about 0.04% to about 5% CO 2 , about 0.04% to about 10% CO 2 , about 0.04% to about 20% CO 2 , about 0.04% to about 30% CO 2 , about 0.04% to about 40% CO 2 , about 0.04% to about 50% CO 2 , about 0.04% to about 60% CO 2 , about 0.04% to about 70% CO 2 , about 0.1% to about 5% CO 2 , about 0.1% to about 10% CO 2 , about 0.1% to about 20% CO 2 , about 0.1% to about 30% CO 2 , about 0.1% to about 40% CO 2 , about 0.1% to about 50% CO 2 , about 1% to about 5% CO 2 , about 1% to about 10% CO 2 , about 1% to about 20% CO 2 , about 1% to about 30% CO 2 , about 1% to about 40% CO 2 , about 1% to about 50% CO 2 , about 1% to about 5% CO 2 , about
  • Modified host cells disclosed herein can be grown under aerobic, anoxic, microaerobic, or anaerobic conditions based on the requirements of the cells.
  • Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in International Publication No. WO 2009/076676, U.S. Patent Application No.12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No.2010/0048964, WO 2009/132220, US Publ. No.2010/0003716, the contents of each of which are incorporated by reference herein in their entireties.
  • the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor, such as those disclosed herein, by modified host cells of the disclosure in an amount of from about 1 mg/L culture medium to about 1 g/L culture medium.
  • the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor, such as those disclosed herein, by modified host cells of the disclosure in an amount of from about 1 ng/L to about 50 ng/L, from about 50 ng/L to about 100 ng/L, from about 100 ng/L to about 500 ng/L, about from 500 ng/L to about 1 ⁇ g/L, from about 1 ⁇ g/L to about 50 ⁇ g/L, from about 50 ⁇ g/L to about 100 ⁇ g/L, from about 100 ⁇ g/L to about 500 ⁇ g/L, from about 500 ⁇ g/L to about 1 mg/L, from about 1 mg/L to about 50 mg/L, from about 50 mg/L to about 100 mg/L, from about 100 mg/L to about 500 mg/L, or from about 500 mg/L to about 1 g/L, of from about 1 mg/L culture medium to about 1 g/L culture medium, from about 1 mg/L
  • the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor in an amount of from about 1 mg/L culture medium to about 5 mg/L culture medium, from about 5 mg/L culture medium to about 10 mg/L culture medium, from about 10 mg/L culture medium to about 25 mg/L culture medium, from about 25 mg/L culture medium to about 50 mg/L culture medium, from about 50 mg/L culture medium to about 75 mg/L culture medium, or from about 75 mg/L culture medium to about 100 mg/L culture medium, from about 100 mg/L culture medium to about 150 mg/L culture medium, from about 150 mg/L culture medium to about 200 mg/L culture medium, from about 200 mg/L culture medium to about 250 mg/L culture medium, from about 250 mg/L culture medium to about 500 mg/L culture medium, from about 500 mg/L culture medium to about 750 mg/L culture medium, or from about 750 mg/L culture medium to about 1 g/L culture medium.
  • the modified host cell disclosed herein is cultured in a liquid medium comprising a carboxylic acid, olivetolic acid, or an olivetolic acid derivative.
  • a method of producing a cannabinoid or a cannabinoid derivative may involve culturing a modified yeast cell of the present disclosure under conditions that favor fermentation of a sugar, and under conditions that favor production of a cannabinoid or a cannabinoid derivative; wherein the cannabinoid or the cannabinoid derivative is produced by the modified yeast cell and is present in alcohol produced by the modified yeast cell.
  • an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 ⁇ g/L, from 1 ⁇ g/L to 50 ⁇ g/L, from 50 ⁇ g/L to 100 ⁇ g/L, from 100 ⁇ g/L to 500 ⁇ g/L, from 500 ⁇ g/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from
  • an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L.
  • the present disclosure provides a beverage produced by the modified yeast cell, where the beverage comprises the cannabinoid or cannabinoid derivative, such as those disclosed herein, produced by the modified yeast cell.
  • a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 ⁇ g/L, from 1 ⁇ g/L to 50 ⁇ g/L, from 50 ⁇ g/L to 100 ⁇ g/L, from 100 ⁇ g/L to 500 ⁇ g/L, from 500 ⁇ g/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L).
  • 1 ng/L to 1 g/L e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L
  • a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L. In some embodiments, a beverage of the present disclosure is non-alcoholic. [0243] In some embodiments, a method of the present disclosure provides for increased production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein. In certain such embodiments, culturing of the modified host cell disclosed herein in a culture medium provides for synthesis of a cannabinoid or a cannabinoid derivative in an increased amount compared to an unmodified host cell cultured under similar conditions.
  • the production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 5% to about 1,000,000 folds compared to an unmodified host cell cultured under similar conditions.
  • the production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 10% to about 1,000,000 folds (e.g., about 50% to about 1,000,000 folds, about 1 to about 500,000 folds, about 1 to about 50,000 folds, about 1 to about 5,000 folds, about 1 to about 1,000 folds, about 1 to about 500 folds, about 1 to about 100 folds, about 1 to about 50 folds, about 5 to about 100,000 folds, about 5 to about 10,000 folds, about 5 to about 1,000 folds, about 5 to about 500 folds, about 5 to about 100 folds, about 10 to about 50,000 folds, about 50 to about 10,000 folds, about 100 to about 5,000 folds, about 200 to about 1,000 folds, about 50 to about 500 folds, or about
  • the production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10,000 folds, 20,000 folds, 50,000 folds, 100,000 folds, 200,000 folds, 500,000 folds, or 1,000,000 folds or more compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions.
  • the production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, by modified host cells of the disclosure may also be increased by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions.
  • the production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 1-20%, 2-20%, 5-20%, 10-20%, 15- 20%, 1-15%, 1-10%, 2-15%, 2-10%, 5-15%, 10-15%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 50-100%, 50-60%, 50-70%, 50-80%, 50-90%, or 50-100% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions.
  • production of a cannabinoid or a cannabinoid derivative by modified host cells of the disclosure is determined by LC-MS analysis.
  • each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition.
  • MRM multiple reaction monitoring
  • the modified host cell of the disclosure is a yeast cell.
  • the modified host cell disclosed herein is cultured in a bioreactor.
  • the modified host cell is cultured in a culture medium supplemented with unsubstituted or substituted hexanoic acid, a carboxylic acid other than unsubstituted or substituted hexanoic acid, olivetolic acid, or an olivetolic acid derivative.
  • the modified yeast cell is a modified S. cerevisiae.
  • the cannabinoid or cannabinoid derivative such as those disclosed herein, is recovered from a cell lysate, e.g., by lysing the modified host cell disclosed herein and recovering the cannabinoid or cannabinoid derivative derivative from the lysate.
  • the cannabinoid or cannabinoid derivative is recovered from the culture medium in which the modified host cell disclosed herein is cultured. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the cell lysate and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from a modified host cell. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the modified host cell and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from the cell lysate, the modified host cell, and the culture medium.
  • the recovered cannabinoid or cannabinoid derivative when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; or from the cell lysate, the modified host cell, and the culture medium, the recovered cannabinoid or cannabinoid derivative is in the form of a salt.
  • the salt is a pharmaceutically acceptable salt.
  • the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0248] In some embodiments, the recovered cannabinoid or cannabinoid derivative, such as those disclosed herein, is then purified.
  • whole-cell broth from cultures comprising modified host cells of the disclosure may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives.
  • suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate.
  • the suitable organic solvent comprises hexane.
  • the suitable organic solvent may be added to the whole-cell broth from fermentations comprising modified host cells of the disclosure at a 10:1 ratio (10 parts whole-cell broth – 1 part organic solvent) and stirred for 30 minutes.
  • the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5).
  • the organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals.
  • the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative.
  • the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative.
  • the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material.
  • a suitable solvent e.g., n-pentane
  • the solvent may then be removed e.g., by rotary evaporation, to produce pure crystalline product.
  • the cannabinoid or cannabinoid derivative is pure, e.g., at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98%, or more than 98% pure, where “pure” in the context of a cannabinoid or a cannabinoid derivative may refer to a cannabinoid or a cannabinoid derivative that is free from other cannabinoids or cannabinoid derivatives, macromolecules, contaminants, etc.
  • the methods may comprise culturing a modified host cell of the disclosure in a culture medium.
  • the modified host cell of the disclosure is a Pichia sp.
  • the method can comprise isolating and/or purifying the expressed engineered variants, as described herein.
  • the method for preparing engineered variants comprises the step of isolating or purifying the engineered variants.
  • the engineered variants of the disclosure can be expressed in modified host cells, as described herein, and isolated from the modified host cells and/or culture medium using any one or more of the well known techniques used for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.
  • Chromatographic techniques for isolation of the engineered variants of the disclosure may include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. In some embodiments, affinity chromatography is used.
  • the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in various forms including but not limited to crude extracts (e.g., cell-free lysates), powders (e.g., shake-flask powders), lyophilizates, frozen stocks made with glycerol or another cryoprotectant, and substantially pure preparations (e.g., DSP powders).
  • the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in purified form.
  • conditions for purifying a particular engineered variant will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.
  • Cell-Free Methods of Producing Cannabinoids or Cannabinoid Derivatives may involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, such as those disclosed herein, using engineered variants disclosed herein expressed or overexpressed by a modified host cell of the disclosure.
  • an engineered variant disclosed herein is used in a cell-free system for the production of cannabinoids or cannabinoid derivatives.
  • the engineered variant of the disclosure is isolated and/or purified.
  • appropriate starting materials for use in producing cannabinoids or cannabinoid derivatives may be mixed together with engineered variants disclosed herein in a suitable reaction vessel to effect the reaction.
  • the engineered variants disclosed herein may be used in combination to effect a complete synthesis of a cannabinoid or cannabinoid derivative from the appropriate starting materials.
  • the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising engineered disclosed herein.
  • the recovered cannabinoids or cannabinoid derivatives are then purified.
  • a cell-free reaction mixture comprising an engineered variant disclosed herein may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives.
  • suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate.
  • the suitable organic solvent comprises hexane.
  • the suitable organic solvent may be added to the cell-free reaction mixture comprising one or more of the polypeptides disclosed herein at a 10:1 ratio (10 parts reaction mixture – 1 part organic solvent) and stirred for 30 minutes.
  • the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5).
  • the organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals.
  • the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative.
  • the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative.
  • the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material.
  • the solvent may then be removed e.g., by rotary evaporation, to produce pure crystalline product.
  • the recovered cannabinoid or cannabinoid derivative is in the form of a salt.
  • the salt is a pharmaceutically acceptable salt.
  • the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • cell-free production of a cannabinoid or a cannabinoid derivative by engineered variants disclosed herein is determined by LC-MS analysis.
  • each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition.
  • MRM multiple reaction monitoring
  • the recovered cannabinoid or cannabinoid derivative is in the form of a salt.
  • the salt is a pharmaceutically acceptable salt.
  • the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • Embodiments of the present subject matter disclosed herein may be beneficial alone or in combination with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure, numbered I-1 to III-38 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below. [0260] Embodiments of the Disclosure: [0261] Embodiment I-1.
  • a variant of an olivetolic acid cyclase (OAC) polypeptide wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitutions.
  • Embodiments I-2 The variant of Embodiment I-1, wherein the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1.
  • Embodiment I-3 Embodiment I-3.
  • Embodiment I-1 or I-2 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitutions, wherein the at least one of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • Embodiment II-1 Embodiment II-1.
  • a variant of an olivetolic acid cyclase (OAC) polypeptide wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions.
  • Embodiment II-2 The variant of any one of Embodiments I-1 to Embodiment II- 1, wherein the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1.
  • Embodiment II-3 Embodiment II-3.
  • any one of Embodiments I-1 to Embodiment II- 4 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, E64, and I94.
  • Embodiment II-6 The variant of any one of Embodiments I-1 to Embodiment II- 5, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56.
  • Embodiment II-7 Embodiment II-7.
  • Embodiment II-8 The variant of any one of Embodiments I-1 to Embodiment II- 6, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8 and L9.
  • Embodiment II-8 The variant of any one of Embodiments I-1 to Embodiment II- 7, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is at amino acid V8.
  • Embodiment II-9 Embodiment II-9.
  • any one of Embodiments I-1 to Embodiment II- 10 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six or seven of the amino acid substitutions are at amino acids selected from the group consisting of: A L V V L V A V A V V V V V V V V V V L A V V V A L L V A V V V L V [0275] Embodiment II-12.
  • Embodiment II-14 The variant of any one of Embodiments I-1 to Embodiment II- 13, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9, A36, Q48, I58, E64, and I94.
  • Embodiment II-15 The variant of any one of Embodiments I-1 to Embodiment II- 12, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K10, N29, T56, I58, and I94.
  • any one of Embodiments I-1 to Embodiment II-16 wherein the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions occurs in the beta 1 chain, the beta 2 chain, and/or the alpha 2 chain.
  • Embodiment II-18 The variant of any one of Embodiments I-1 to Embodiment II- 17, wherein the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids K12, A36, and D71.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E53V, T56S, I
  • Embodiment II-20 The variant of any one of Embodiments I-1 to Embodiment II- 19, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, F23I, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G.
  • Embodiment II-21 The variant of any one of Embodiments I-1 to Embodiments II-20, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, E67S, Q70A, Q70K, D71T, V84D, S87P, I94K, and R100G.
  • Embodiment II-22 The variant of any one of Embodiments I-1 to Embodiments II-21, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, K49R, T56S, E64D, and I94K.
  • Embodiment II-23 The variant of any one of Embodiments I-1 to Embodiments II-22, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, and T56S.
  • Embodiment II-24 The variant of any one of Embodiments I-1 to Embodiments II-23, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I and L9I.
  • Embodiment II-25 The variant of any one of Embodiments I-1 to Embodiments II-24, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is V8I.
  • Embodiment II-26 The variant of any one of Embodiments I-1 to Embodiments II-23, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is V8I.
  • variants any one of Embodiments I-1 to Embodiments II-7, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions, wherein six or seven of the amino acid substitutions are selected from the group consisting of:
  • Embodiment II-29 The variant of any one of Embodiments I-1 to Embodiments II-289, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions A2P, V8I, L9I, E64D, I94K, and R100G.
  • Embodiment II-30 The variant of any one of Embodiments I-1 to Embodiments II-31, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K10A, N29D, T56S, I58V, and I94K.
  • Embodiment II-32 The variant of any one of Embodiments I-1 to Embodiments II-31, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K49R, T56S, I58V, and R100G. [0296] Embodiment II-33.
  • Embodiment II-34 The variant of any one of Embodiments I-1 to Embodiments II-33, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions.
  • Embodiment II-35 The variant of any one of Embodiments I-1 to Embodiments II-32, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions.
  • Embodiment II-34 The variant of any one of Embodiments I-1 to Embodiments II-34, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions.
  • Embodiment II-36 The variant of any one of Embodiments I-1 to Embodiments II-35, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising six amino acid substitutions.
  • Embodiment II-37 The variant of any one of Embodiments I-1 to Embodiments II-36, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising seven amino acid substitutions.
  • Embodiment II-38 The variant of any one of Embodiments I-1 to Embodiments II-36, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising seven amino acid substitutions.
  • any one of Embodiments I-1 to Embodiments II-37 wherein the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 3 to 93.
  • Embodiment II-39 The variant of any one of Embodiments I-1 to Embodiments II-38, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment II-40 The variant of any one of Embodiments I-1 to Embodiments II-39, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment II-41 Embodiment II-41.
  • Embodiment II-42 The variant of any one of Embodiments I-1 to Embodiments II-41, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment II-43 Embodiment II-43.
  • Embodiment II-44 A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to Embodiments II-43.
  • Embodiment II-45 A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to Embodiments II-43.
  • a nucleic acid comprising a nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 94-184.
  • OAC olivetolic acid cyclase
  • a method of making a modified host cell comprising introducing one or more nucleic acids of any one of Embodiments II-44 to II- 46 into a host cell.
  • Embodiment II-48 A vector comprising one or more nucleic acids of any one of Embodiments II-44 to II-46.
  • Embodiment II-49 A method of making a modified host cell, the method comprising introducing one or more vectors of Embodiment II-48 into a host cell.
  • Embodiment II-50 A modified host cell comprising one or more nucleic acids of any one of Embodiments II-44 to II-46.
  • Embodiment II-51 Embodiment II-51.
  • Embodiment II-50 wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative.
  • Embodiment II-52 The modified host cell of Embodiment II-50 or II-51, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative.
  • Embodiment II-53 The modified host cell of Embodiment II-50, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative.
  • the modified host cell of any of Embodiments II-50 to II-52 wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-54 The modified host cell of any one of Embodiments II-50 to II- 53, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-55 The modified host cell of any of Embodiments II-50 to II-54, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence
  • Embodiment II-56 The modified host cell of any of Embodiments II-50 to II-55, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-57 The modified host cell of any one of Embodiments II-50 to II- 56, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-58 The modified host cell of any one of Embodiments II-50 to II-57, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising
  • Embodiment II-59 The modified host cell of any one of Embodiments II-50 to II- 58, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-60 The modified host cell of any one of Embodiments II-50 to II- 59, wherein the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43,
  • Embodiment II-61 The modified host cell of any one of Embodiments II-50 to II- 60, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time.
  • Embodiment II-62 The modified host cell of any one of Embodiments II-50 to II- 61, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • Embodiment II-63 A method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of any one of Embodiments II-50 to II-62 in a culture medium.
  • Embodiment II-64 The method of Embodiment II-63, wherein the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative.
  • Embodiment II-65 The method of Embodiment II-63 or II-64, wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • Embodiment II-66 Embodiment II-66.
  • a method of producing olivetolic acid or an olivetolic acid derivative comprising: a) culturing a modified host cell of any one of Embodiments II-50 to II-62 in a culture medium.
  • Embodiment II-67 The method of Embodiment II-66, wherein the method comprises: b) recovering the produced olivetolic acid or an olivetolic acid derivative.
  • Embodiment II-68 The method of any one of Embodiments II-63 to II-67, wherein the culture medium comprises a carboxylic acid.
  • Embodiment II-69 Embodiment II-69.
  • Embodiment II-68 wherein the carboxylic acid is an unsubstituted or substituted C 3 -C 18 carboxylic acid.
  • Embodiment II-70 The method of Embodiment II-68 or II-69, wherein the unsubstituted or substituted C 3 -C 18 carboxylic acid is an unsubstituted or substituted hexanoic acid.
  • Embodiment II-71 Embodiment II-71.
  • any one of Embodiments II-63 to II-70 wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising a
  • Embodiment II-72 The method of any one of Embodiments II-63 to II-71, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host
  • Embodiment II-73 The method of any of Embodiments II-63 to II-72, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid
  • Embodiment II-745 The method of any one of Embodiments II-63 to II-73, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of
  • Embodiment II-75 The method of any one of Embodiments II-63 to II-74, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and where
  • Embodiment II-76 The method of any one of Embodiments II-63 to II-75, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC poly
  • Embodiment II-77 The method of any one of Embodiments II-63 to II-76, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising
  • Embodiment II-78 The method of any one of Embodiments II-63 to II-77, wherein the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • Embodiment II-79 A method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43.
  • Embodiment II-80 A method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43.
  • Embodiment II-79 wherein the method comprises recovering the produced cannabinoid or cannabinoid derivative.
  • Embodiment II-81 The method of Embodiment II- 79 or II-80, wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • Embodiment II-82 A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43.
  • Embodiment II-83 A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43.
  • Embodiment II-84 The method of Embodiment II-82 or II-83 wherein the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3- butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3-methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)- resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-res
  • Embodiment II-85 The method of any one of Embodiments II-63 to II-84 wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment II-86 Embodiment II-86.
  • any of Embodiments II-63 to II-86 wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment II-88 The method of any one of Embodiments II-63 to II-87, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment II-89 Embodiment II-89.
  • any of Embodiments II-63 to II-89 wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment II-91 The method of any one of Embodiments II-63 to II-90, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment II-93 Embodiment II-93.
  • Embodiment III-1 A variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant has between 85% and 95% sequence identity to SEQ ID NO: 1, and comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions [0357] Embodiment III-2.
  • OAC olivetolic acid cyclase
  • Embodiment III-1 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56.
  • Embodiment III-3 [0358] Embodiment III-3.
  • Embodiment III-1 or III-2 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
  • Embodiment III-4 Embodiment III-4.
  • any one of Embodiments III-1 to III-3 wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, I58, E64, I94, and R100.
  • Embodiment III-5 The variant of any one of Embodiments III-1 to III-4, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six of the amino acid substitutions are at amino acids selected from the group consisting of: [0361] Embodiment III-6.
  • the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E52A, E53V, T56S, I58
  • Embodiment III-7 The variant of any one of Embodiments III-1 to III-6, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 6 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of:
  • Embodiment III-8 The variant of claim any one of Embodiments III-1 to III-7, wherein the variant comprises an amino acid sequence selected from an amino acid sequence of any of SEQ ID NOs: 3-146.
  • Embodiment III-9 The variant of any one of Embodiments III-1 to III-8, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment III-10 The variant of any one of Embodiments III-1 to III-9, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment III-11 The variant of any one of Embodiments III-1 to III-9, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
  • Embodiment III-10 wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • Embodiment III-12 A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11.
  • Embodiment III-13 A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11.
  • a nucleic acid comprising a nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 147-290.
  • OAC olivetolic acid cyclase
  • Embodiment III-14 A method of making a modified host cell, the method comprising introducing one or more nucleic acids of Embodiment III-12 or Embodiment III-13 into a host cell.
  • Embodiment III-15 A vector comprising one or more nucleic acids of Embodiment III-12 or Embodiment III-13.
  • Embodiment III-16 A method of making a modified host cell, the method comprising introducing one or more vectors of Embodiment III-15 into a host cell.
  • Embodiment III-17 A modified host cell comprising one or more nucleic acids of Embodiment III-12 or Embodiment III-13 or the vector of Embodiment III-15.
  • Embodiment III-18 The modified host cell of Embodiment III-17, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative.
  • Embodiment III-19 The modified host cell of Embodiment III-17 or Embodiment III-18, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative.
  • Embodiment III-20 The modified host cell of any one of Embodiments III-17 to III-19, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time.
  • Embodiment III-21 The modified host cell of any one of Embodiments III-17 to III-20, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time.
  • Embodiment III-22 The modified host cell of any one of Embodiments III-17 to III-21, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time.
  • Embodiment III-23 The modified host cell of any one of Embodiments III-17 to III-22, wherein the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 or III-11, grown under
  • Embodiment III-24 The modified host cell of any one of Embodiments III-17 to III-22, wherein the modified host cell produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time.
  • Embodiment III-25 The modified host cell of Embodiment III-24, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • Embodiment III-26 A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: [0382] a) culturing a modified host cell of any one of Embodiments III-15 to III-25 in a culture medium.
  • Embodiment III-27 Embodiment III-27.
  • Embodiment III-25 wherein the method comprises: [0384] b) recovering the produced olivetolic acid or an olivetolic acid derivative.
  • Embodiment III-28 The method of Embodiment III-26 or Embodiment III-27, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III-11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment III-29 The method of any one of Embodiments III-26 to III-28, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III- 11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
  • Embodiment III-30 Embodiment III-30.
  • Embodiment III-29 The method of Embodiment III-29, wherein the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • Embodiment III-31 the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
  • the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3-butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3- methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)- resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)- resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-resorcylic acid, 3-octyl- resorcylic acid, 3-(trans-1-octenyl)-resorcylic acid, 3-resorcylic acid, 3-oct
  • Embodiment III-32 A method of producing a cannabinoid or a cannabinoid derivative, the method comprising: [0390] a) culturing a modified host cell of any one of Embodiments III-17 to III-25 in a culture medium. [0391] Embodiment III-33. The method of Embodiment III-32, wherein the method comprises: [0392] b) recovering the produced cannabinoid or cannabinoid derivative. [0393] Embodiment III-34.
  • Embodiment III-32 or Embodiment III-33 wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III-11, and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0394] Embodiment III-35.
  • Embodiment III-36 The method of any one of Embodiments III-26 to III-35, wherein the culture medium comprises a carboxylic acid.
  • Embodiment III-37 The method of Embodiment III-36, wherein the carboxylic acid is an unsubstituted or substituted C 3 -C 18 carboxylic acid.
  • Embodiment III-38 The method of any one of Embodiments III-32 to III-34 wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • the sequences provided in SEQ ID NOs: 1-290 are olivetolic acid cyclase (OAC) amino acid and nucleotide sequences disclosed herein. Where a genus and/or species is noted, the sequence should not be construed to be limited only to the specified genus and/or species, but also includes other genera and/or species expressing said sequence. Orthologs of the sequences disclosed in SEQ ID NOs: 1-290 may also be encompassed by this disclosure.
  • Example 1 OAC mutations, Construction, and Transformations [0402] A library of DNA constructs encoding OAC mutants was synthesized and each codon encoding an amino acid was mutated to encode all other amino acids (saturation mutagenesis).
  • This library was combined and transformed into a screening strain containing all other necessary engineering to make olivetolic acid (S983), and the resulting strains bearing mutant OAC constructs were benchmarked against a wild type OAC control (S1322) in a 96-well plate assay.
  • OAC and other heterologous genes were added into S983 under the control of a galactose-inducible promoter (e.g. the promoter for GAL1) thereby enabling coordinated expression of the genes required for olivetolic acid production in galactose-containing media.
  • Strains containing OAC mutants were cultured for 2 days in 360 ⁇ L YP media containing 2% dextrose in 96-well plates at 30°C.
  • Combinatorial Library [0407] To further increase OAC performance, a combinatorial library containing 6 or more mutations was assessed in the same assay. To increase diversity in the library, additional potential mutations were identified in silico. A BLAST search using the OAC protein sequence was used to identify a family of similar enzymes that were aligned to identify positions with low conservation, with the rationale that these positions could introduce additional diversity without loss of function. In total, 500 OAC variants were ordered for synthesis, combining successful mutations from the initial saturation library with mutations of the variable residues identified in the alignment for a total of 6 or more mutations per construct.
  • strains bearing these mutant OAC constructs performed significantly better than the WT control and, in many cases, better than any strains bearing single mutations (Tables 2-3).
  • the strain bearing the highest performing construct, S1502 produced 1.5-fold more OA than the WT control strain, and improved the OA/Olivetol ratio from 1.23 to 3.01. More modest improvements in OD600 were also observed, e.g. from 3.2 to 3.34 for S1502.
  • S1535 strains that outperformed the WT control bore OAC constructs that exclusively drew mutations from the saturation mutagenesis library.
  • one strain, S1500, identified in this screen contained an OAC variant with an additional mutation, N29D, that likely arose from errors during DNA synthesis of the variant prior to integration.
  • a strain has a parent strain, it is a child strain. All of the constructs present in the parent strain are also all present in the child strain. ** S4 is CEN.PK113-1A with genotype MATalpha; URA3; TRP1; LEU2; HIS3; MAL2-8C; SUC2 *** S487 is the base strain used to test THCA synthase constructs. In this strain, the nucleotide sequence encoding a pGAL1_tTDH1 empty expression cassette is added and the i33 locus is returned to its native sequence by deleting the CBDAS construct there.

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Abstract

The present disclosure provides engineered variants of an olivetolic acid cyclase polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells expressing said engineered variants, methods of producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the olivetolic acid cyclase polypeptide.

Description

OPTIMIZED OLIVETOLIC ACID CYCLASE POLYPEPTIDES CROSS-REFERENCE TO RELATED APPLICTIONS [0001] This application claims priority to Provisional U.S. Application No. 62/987,274 filed March 9, 2020, which is incorporated by reference in its entirety. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY [0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: a computer readable format copy of the sequence listing (filename: DEMT-005_01WO_SeqList_ST25.txt, date recorded: March 8, 2021, file size 259 kb). INTRODUCTION [0003] Plants from the genus Cannabis have been used by humans for their medicinal properties for thousands of years. In modern times, the bioactive effects of Cannabis are attributed to a class of compounds termed “cannabinoids,” of which there are hundreds of structural analogs including tetrahydrocannabinol (THC) and cannabidiol (CBD). These molecules and preparations of Cannabis material have recently found application as therapeutics for chronic pain, multiple sclerosis, cancer-associated nausea and vomiting, weight loss, appetite loss, spasticity, seizures, and other conditions.
Figure imgf000003_0001
[0004] The physiological effects of certain cannabinoids are thought to be mediated by their interaction with two cellular receptors found in humans and other animals. Cannabinoid receptor type 1 (CB1) is common in the brain, the reproductive system, and the eye. Cannabinoid receptor type 2 (CB2) is common in the immune system and mediates therapeutic effects related to inflammation in animal models. The discovery of cannabinoid receptors and their interactions with plant-derived cannabinoids predated the identification of endogenous ligands. [0005] Besides THC and CBD, hundreds of other cannabinoids have been identified in Cannabis. However, many of these compounds exist at low levels and alongside more abundant cannabinoids, making it difficult to obtain pure samples from plants to study their therapeutic potential. Similarly, methods of chemically synthesizing these types of products have been cumbersome and costly, and tend to produce insufficient yield. Accordingly, additional methods of making pure cannabinoids or cannabinoid derivatives are needed. [0006] One possible method is production via fermentation of engineered microbes, such as yeast. By engineering production of the relevant plant enzymes in microbes, it may be possible to achieve conversion of various feedstocks into a range of cannabinoids, potentially at much lower cost and with much higher purity than what is available from the plant. A key challenge to this effort is the difficulty of expressing plant enzymes in the microbe, particularly secreted enzymes such as the cannabinoid synthases, which must successfully traverse the microbe’s secretory pathway to fold and function properly. Variants of cannabinoid synthases, modified host cells, and new methods are needed to address these challenges. SUMMARY [0007] The present disclosure provides engineered variants of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, methods of producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the olivetolic acid cyclase (OAC) polypeptide. The engineered variants of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives or precursors (e.g., non-naturally occurring cannabinoids). The modified host cells of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure. The disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure. [0008] In some aspects, the present disclosure provides a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions. In some embodiment, the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1. [0009] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. [0010] In some embodiments, variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, E67, Q70, D71, V84, S87, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, N29, A36, Q48, K49, T56, I58, E64, I94, and R100In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, V8, L9, E64, I94, R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K10, N29, T56, I58, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of L9, A36, Q28, I58, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of K49, E64, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, K12, A36, Q48, and I58. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of K49, E64, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of A2, K12, A36, Q48, and I58. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is at amino acid V8. [0011] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8 and L9. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are at amino acids V8 and L9. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are at amino acids V8, L9, and T56. [0012] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six or seven of the amino acid substitutions are at amino acids selected from the group consisting of:
Figure imgf000006_0001
Figure imgf000007_0001
[0013] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids A2, V8, L9, E64, I94, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K10, N29, T56, I58, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9, A36, Q48, I58, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K49, T56, I58, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K12, K49, T56, and D71. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids K12, A36, and D71. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, A36, K49, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, N49, I58, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K49, I58, Q70, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, A36, T62, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, E22, A36, Q48, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K10, T56, I58, and Q70. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids L9, K10, K25, Q48, E64, and I94. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, K12, T56, I58, and E64. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, A36, Q48, K49, and R100. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8, L9, E22, A36, Q48, and R100. [0014] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions occurs in the beta sheet 1 chain, the beta sheet 2 chain, and/or the alpha helix 2 chain. [0015] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E52H, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71A, D71E, D71K, D71N, D71Q, D71S, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71A, D71E, D71K, D71N, D71Q, D71S, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. [0016] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, F23I, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. [0017] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, E67S, Q70A, Q70K, D71T, V84D, S87P, I94K, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, K49R, T56S, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, and T56S. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I and L9I. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is V8I. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are V8I and L9I. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are V8I, L9I, and T56S. [0018] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions, wherein six or seven of the amino acid substitutions are selected from the group consisting of:
Figure imgf000010_0001
Figure imgf000011_0001
[0019] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions A2P, V8I, L9I, E64D, I94K, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K10A, N29D, T56S, I58V, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions L9I, A36Q, Q48C, I58V, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K49R, T56S, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K12N, K49R, T56S, and D71T. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids V8I, L9I, K12N, A36E, K49R, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids V8I, L9I, K12N, N49R, I58V, and R100G. [0020] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising six amino acid substitutions. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising seven amino acid substitutions. [0021] In some embodiments, the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 3-146. In some embodiments, the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 36-45 and 94-98. [0022] In some embodiments, the variant produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, the variant produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100%, greater than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, the variant produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0023] In some aspects, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a variant of the disclosure. In some embodiments, the nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 147-290. In some embodiments, the variant nucleotide sequence is selected from a nucleotide of any one of SEQ ID NOs: 180-189 and 238-242. In some embodiments, the nucleotide sequence is codon-optimized. [0024] In some aspects, the present disclosure provides a method of making a modified host cell, the method comprising introducing one or more nucleic acids of the disclosure into a host cell. [0025] In some aspects, the present disclosure provides a vector comprising one or more nucleic acids of the disclosure. [0026] In some aspects, the present disclosure provides a method of making a modified host cell, the method comprising introducing one or more vectors of the disclosure into a host cell. [0027] In some aspects, the present disclosure provides a modified host cell comprising one or more nucleic acids of the disclosure. In some embodiments, the modified host cell produces olivetolic acid or an olivetolic acid derivative. In some embodiments, the modified host cell produces a cannabinoid or a cannabinoid derivative. In some embodiments, the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0028] In some embodiments, the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0029] In some embodiments, the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0030] In some embodiments, the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0031] In some embodiments, the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 160%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0032] In some embodiments, the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0033] In some embodiments, the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0034] In some embodiments, the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, grown under similar culture conditions for the same length of time. [0035] In some embodiments, the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0036] In some aspects, the present disclosure provides a method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative. In some embodiments, the cannabinoid is cannabigerolic acid (CBGA), cannbigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. [0037] In some aspects, the present disclosure provides a method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: a) culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the method comprises: b) recovering the produced olivetolic acid or an olivetolic acid derivative. [0038] In some embodiments, the culture medium comprises a carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid. In some embodiments, the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. [0039] In some aspects, the present disclosure provides a method of producing an olivetolic acid or olivetolic acid derivative, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0040] In some embodiments, the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0041] In some embodiments, the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0042] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0043] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0044] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0045] In some embodiments, the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, and wherein the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an OAC variant of the disclosure, are cultured under similar culture conditions for the same length of time. [0046] In some embodiments, the method produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0047] In some aspects, the present disclosure provides a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an OAC variant of the disclosure. In some embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In some embodiments, the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. [0048] In some aspects, the present disclosure provides a method of producing olivetolic acid or an olivetolic acid derivative, the method comprising use of an OAC variant of the disclosure. In some embodiments, the method comprises recovering the produced olivetolic acid or olivetolic acid derivative. In some embodiments, the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3-butyl- resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3-methylpentyl)-resorcylic acid, 3-(3- pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3- (trans-1-hexyl)-resorcylic acid, 3-octyl-resorcylic acid, 3-(trans-1-octenyl)-resorcylic acid, 3- nonyl-resorcylic acid, 3-(3-phenylpropyl)-resorcylic acid, 3-(4-phenylbutyl)-resorcylic acid, 3-(5-phenylpentyl)-resorcylic acid, and 3-(6-phenylhexyl)-resorcylic acid. [0049] In some embodiments, the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0050] In some embodiments, the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0051] In some embodiments, the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OACvariant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0052] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0053] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0054] In some embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OAC variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0055] In some embodiments, the method produces olivetolic acid or a derivative thereof (e.g. CBGA or CBDA) in an increased ratio of olivetolic acid or a derivative thereof (e.g. CBGA or CBDA) over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the OAC variant of the disclosure, wherein the OACvariant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0056] In some embodiments, the method produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. BRIEF DESCRIPTION OF THE DRAWINGS [0057] FIGS.1A and 1B depict expression constructs used in the production of the S21 strain. The expression constructs depicted in FIGS.1A and 1B, were also used in the production of the following strains: S434, S929, S955, S1540, S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. Throughout the figures, in addition to the specified coding sequences from SEQ ID NOs: 147-290, construct maps depict regulatory, non-coding and genomic cassette sequences described in Table 5. Construct maps also depict genes denoted with a preceding “m” (e.g., mERG13), which specify open reading frames from SEQ ID NOs: 147-290with 200-250 base pairs (bp) of downstream regulatory (terminator) sequence. Arrows in construct maps indicate the directionality of certain DNA parts. The “!” preceding a part name is an output of the DNA design software used, is redundant with the arrow directionality, and can be ignored. [0058] FIG.2 depicts an expression construct used in the production of the S434 strain. The expression construct depicted in FIG.2 was also used in the production of following strains: S929, S955, S1540, S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. [0059] FIGS.3A and 3B depict an expression construct and a landing pad used in the production of the S929 strain. The landing pad and expression construct depicted in FIG.3 were also used in the production of following strains: S955, S1540, and S1696. The expression construct, but not the landing pad, was used in the construction of S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. [0060] FIG.4 depicts expression constructs used in the production of the S955 strain. The expression constructs depicted in FIG.4 were also used in the production of following strains: S1540, S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. [0061] FIG.5 depicts an expression construct used in the production of the S1540 strain. The construct depicted in FIG.5 was also used in the production of the following strains: S1696, S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. [0062] FIG.6 depicts expression constructs used in the production of the S1696 strain. The expression constructs depicted in FIG.6 were also used in the production of the following strains: S1789, S1791, S1835, S1836, S1837, S1838, S1839 and S1840. [0063] FIG.7 depicts a mutant OAC expression construct (2 copies) used in the production of the S1789 strain. The expression construct depicted in FIG.7 was also used in the production of the following strains: S1837 and S1839. [0064] FIG.8 depicts expression constructs used in the production of the S1835 strain. [0065] FIG.9 depicts a mutant OAC construct expression construct (1 copy) used in the production of the S1791 strain. The expression constructs depicted in FIG.9 were also used in the production of the following strains: S1838, and S1840. [0066] FIG.10 depicts expression constructs used in the production of the S1836 strain. [0067] FIG.11 depicts expression constructs used in the production of the S927 strain. The expression constructs depicted in FIG.11 were also used in the production of the following strains: S952, S983, S1273, S1277, S1278, S1322, S1323, S1324, S1325, S1327, S1328, S1329, S1331, S1333, S1334, S1335, S1391, S1397, S1403, S1405, S1406, S1407, S1409, S1410, S1438, S1479, S1480, S1481, S1482, S1483, S1485, S1486, S1487, S1488, S1489, S1490, S1491, S1492, S1493, S1494, S1495, S1496, S1497, S1498, S1499, S1500, S1501, S1502, S1503, S1504, S1505, S1506, S1507, S1508, S1509, S1510, S1511, S1512, S1513, S1514, S1515, S1516, S1517, S1518, S1519, S1520, S1521, S1522, S1523, S1524, S1525, S1526, S1527, S1528, S1529, S1530, S1531, S1532, S1534, S1535, S1536, S1537, S1538, S1602, S1609, S1610, S1617, S1623, S1638, S1639, S1640, S1644, S1651, S1652 and S1671. [0068] FIG.12A and 12B depict expression constructs used in the production of the S952 strain. The expression constructs depicted in FIG.12 were also used in the production of the following strains: S983, S1273, S1277, S1278, S1322, S1323, S1324, S1325, S1327, S1328, S1329, S1331, S1333, S1334, S1335, S1391, S1397, S1403, S1405, S1406, S1407, S1409, S1410, S1438, S1479, S1480, S1481, S1482, S1483, S1485, S1486, S1487, S1488, S1489, S1490, S1491, S1492, S1493, S1494, S1495, S1496, S1497, S1498, S1499, S1500, S1501, S1502, S1503, S1504, S1505, S1506, S1507, S1508, S1509, S1510, S1511, S1512, S1513, S1514, S1515, S1516, S1517, S1518, S1519, S1520, S1521, S1522, S1523, S1524, S1525, S1526, S1527, S1528, S1529, S1530, S1531, S1532, S1534, S1535, S1536, S1537, S1538 , S1602, S1609, S1610, S1617, S1623, S1638, S1639, S1640, S1644, S1651, S1652 and S1671. [0069] FIG.13 depicts an expression construct used in the production of the S983 strain. [0070] FIG.14 depicts an expression construct used in the production of S1322. [0071] FIG.15 depicts expression constructs used in the production of S1323. [0072] FIG.16 depicts an expression construct used in the production of the following strains: S1273, S1277, S1278, S1322, S1323, S1324, S1325, S1327, S1328, S1329, S1331, S1333, S1334, S1335, S1391, S1397, S1403, S1405, S1406, S1407, S1409, S1410, S1438, S1479, S1480, S1481, S1482, S1483, S1485, S1486, S1487, S1488, S1489, S1490, S1491, S1492, S1493, S1494, S1495, S1496, S1497, S1498, S1499, S1500, S1501, S1502, S1503, S1504, S1505, S1506, S1507, S1508, S1509, S1510, S1511, S1512, S1513, S1514, S1515, S1516, S1517, S1518, S1519, S1520, S1521, S1522, S1523, S1524, S1525, S1526, S1527, S1528, S1529, S1530, S1531, S1532, S1534, S1535, S1536, S1537, S1538, , S1602, S1609, S1610, S1617, S1623, S1638, S1639, S1640, S1644, S1651, S1652 and S1671. DETAILED DESCRIPTION [0073] Synthetic biology allows for the engineering of industrial host organisms — e.g., microbes — to convert simple sugar feedstocks into medicines. This approach includes identifying genes that produce the target molecules and optimizing their activities in the industrial host. Microbial production can be significantly cost-advantaged over agriculture and chemical synthesis, less variable, and allow tailoring of the target molecule. However, reconstituting or creating a pathway to produce a target molecule in an industrial host organism can require significant engineering of both the pathway genes and the host. The present disclosure provides engineered variants of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing olivetolic acid or derivatives thereof, and/or cannabinoids or cannabinoid derivatives, methods of producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the OAC polypeptide. The engineered variants of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids). The modified host cells of the disclosure may be useful for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids) and/or for expressing engineered variants of the disclosure. The disclosure also provides for modified host cells for expressing the engineered variants of the disclosure. Additionally, the disclosure provides for preparation of engineered variants of the disclosure. [0074] The Olivetolic Acid Cyclase (OAC) enzyme of Cannabis sativa is a polyketide cyclase enzyme that, catalyzes the formation of olivetolic acid or derivative thereof from a malonyl-CoA and hexanoyl-CoA-derived pre-cursor molecule, 3,5,7- trioxododecanoyl-CoA or derivative thereof. Olivetolic acid forms the polynucleotide core of the cannabinoids and thus is an important enzyme for biosynthetic production of cannabinoids. In the absence of OAC, the 3,5,7-trioxododecanoyl-CoA produced by the TKS enzyme converts intoOlivetol, a “dead-end” byproduct that cannot be converted into the desired cannabinoid products. Olivetol, in particular, is a skin sensitizer and can be difficult to separate from the desired cannabinoid products in downstream processing. Thus, OAC enzymes with improved catalytic activity (via improvement in actual catalysis, or expression in yeast) can be used not only to increase titers of the desired cannabinoids in an engineered microbe, but also to reduce the amount of off-product formation, resulting in a better product profile. [0075] For these reasons, the natural OAC enzyme (e.g., wild type) is not optimal for industrial purposes, and improved enzymes are required. Parameters of interest include catalytic activity, product profile, enzyme stability, and pH and temperature optima. Enzyme improvement is typically accomplished by coupling the generation of diversity (a library of engineered variants) to a screen or selection for the properties of interest. DNA libraries encoding engineered variants can be generated in a variety of ways. For example, libraries can be generated using error prone PCR using the wild type gene sequence as a template. The resulting library can be quite large, consisting of genes with variable numbers of mutations at random positions. Error prone PCR is inexpensive and convenient but has several drawbacks. First, instead of a precise number of mutations per construct, a distribution is obtained. This presents an unfortunate trade-off. A distribution centered around a low number of mutations will include a significant amount of zero-mutation wild- type constructs that waste screening capacity. A distribution centered around a higher number of mutations is likely to generate constructs that have accumulated loss of function mutations that would prevent identification of the desired gain of function mutations. Second, error prone PCR introduces mutational bias (an intrinsic property of the low fidelity polymerases used) which means that the library underrepresents certain types of mutation. A powerful alternative to error prone PCR is saturation mutagenesis, which involves synthesis of a library containing every possible amino acid at every position in the protein. Recent advances in DNA synthesis technologies have improved the quality of these libraries significantly. [0076] Once a library encoding engineered variants is generated, it is necessary to select or screen for engineered variants with the properties of interest. This can be accomplished by using a protein production host to express and purify the engineered variants, followed by testing in vitro. Such an approach allows careful measurement of the engineered variants’ kinetic parameters and assessment of performance under carefully controlled conditions. However, for application in an engineered microbial strain, in vitro data can be highly misleading as no in vitro system can represent the cellular milieu accurately. In this case, the best option is to test the engineered variants in the exact context they must eventually perform—inside an engineered production strain. In the case of the olivetolic acid cyclase, such a production strain would be engineered to produce the substrate 3,5,7-trioxododecanoyl-CoA in excess. [0077] The engineered variants of the disclosure may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. Additionally, the engineered variants of the disclosure may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA in an increased ratio of OA over olivetol or derivatives thereof compared to that produced by an olivetolic acid cyclase (OAC) polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. Similar conditions may include the same temperature, pH, buffer, and/or fermentation conditions and in the same culture medium and/or reaction solvent. [0078] The methods of the disclosure may include using engineered microorganisms (e.g., modified host cells) or engineered variants of an OAC polypeptide of the disclosure to produce naturally-occurring and non-naturally occurring olivetolic acid, olivetolic acid derivatives, cannabinoids, cannabinoid derivatives, and/or cannabinoid precursors. Naturally-occurring occurring olivetolic acid, olivetolic acid derivatives, cannabinoids and non-naturally occurring cannabinoids (e.g., cannabinoid derivatives) are challenging to produce using chemical synthesis due to their complex structures. The methods of the disclosure enable the construction of metabolic pathways inside living cells to produce bespoke olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives from simple precursors such as sugars and carboxylic acids. One or more nucleic acids (e.g., heterologous nucleic acids) disclosed herein comprising nucleotide sequences encoding one or more polypeptides or engineered variants disclosed herein can be introduced into host microorganisms allowing for the stepwise conversion of inexpensive feedstocks, e.g., sugar, into final products: olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives and/or precursors. These products can be specified by the choice and construction of expression constructs or vectors comprising one or more nucleic acids (e.g., heterologous nucleic acids) disclosed herein, allowing for the efficient bioproduction of chosen cannabinoids, such as THC and THCA and less common cannabinoid species found at low levels in Cannabis; or cannabinoid derivatives. Bioproduction also enables synthesis of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis. To produce cannabinoids or cannabinoid derivatives and create biosynthetic pathways within the modified host cells, the modified host cell may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. [0079] The disclosure also provides for modification of the secretory pathway of a host cell modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding an engineered variant of an OAC polypeptide of the disclosure. In some embodiments, the nucleotide sequence encoding the engineered variant of an OAC polypeptide is codon-optimized. Modification of the secretory pathway in the host cell may improve expression and solubilization of cannabinoids or cannabinoid derivatives synthesized using pathways incorporating the engineered variants of the disclosure, as cannabinoids or cannabinoid derivatives are processed through the secretory pathway. Reconstituting the activity of polypeptides processed through the secretory pathwayin a modified host cell, such as a modified yeast cell, can be challenging and unreliable. Often the cannabinoids or cannabinoid derivatives may be misfolded or mislocalized, resulting in low expression, polypeptides lacking activity, polypeptide aggregation, reduced host cell viability, and/or cell death. Additionally, a backlog of misfolded or mislocalized polypeptides can induce metabolic stress within the modified host cell, harming the modified host cell. The polypeptides may lack necessary posttranslational modifications for folding and activity, such as disulfide bonds, glycosylation and trimming, and cofactors, affording inactive polypeptides or polypeptides with reduced enzymatic activity. Accordingly, modification of the secretory pathway in the modified host cell may improve expression and activity of cannabinoids or cannabinoid derivatives and viability of the modified host cell. [0080] The modified host cell of the disclosure may be a modified yeast cell. Yeast cells may be cultured using known conditions, grow rapidly, and are generally regarded as safe. Yeast cells contain the secretory pathway common to all eukaryotes. As disclosed herein, manipulation of that secretory pathway in yeast host cells modified with one or more nucleic acids (e.g., heterologous nucleic acids) may improve expression, folding, and enzymatic activity of cannabinoids or cannabinoid derivatives as well as viability of the modified yeast host cell, such as modified Saccharomyces cerevisiae. Further, use of codon- optimized nucleotide sequences encoding engineered variants of the disclosure, may improve expression and activity of the engineered variant and viability of modified yeast host cells, such as modified Saccharomyces cerevisiae. [0081] Besides allowing for the production of desired olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives and/or precursors, the present disclosure provides a more reliable and economical process than agriculture-based production. Microbial fermentations can be completed in days versus the months necessary for an agricultural crop, are not affected by climate variation or soil contamination (e.g., by heavy metals), and can produce pure products at high titer. Moroever, olivetolic acid is very expensive to produce by synthesis. The development of novel sources of olivetolic acid allows for economical production of cannabinoids, cannabinoid derivatives, and/or cannabinoid precursors as disclosed herein. [0082] The present disclosure also provides a platform for the economical production of high-value cannabinoids, including THC and/or CBD, as well as derivatives thereof. It also provides for the production of different olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives for which no viable method of production exists. Using the engineered variants, methods, and modified host cells disclosed herein, cannabinoids and cannabinoid derivatives may be produced in an amount of over 100 mg per liter of culture medium, over 1 g per liter of culture medium, over 10 g per liter of culture medium, or over 100 g per liter of culture medium. [0083] Additionally, the disclosure provides engineered variants of an OAC polypeptide, methods, modified host cells, and nucleic acids to produce olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives in vivo or in vitro from simple precursors. Nucleic acids (e.g., heterologous nucleic acids) disclosed herein can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of one or more polypeptides, such as the engineered variants of the disclosure, which can then be utilized in vitro or in vivo for the production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives. In some embodiments, the in vitro methods are cell-free. Cannabinoid Biosynthesis [0084] In addition to one or more nucleic acids (e.g., heterologous nucleic acids) encoding an engineered variant of an OAC polypeptide, one or more nucleic acids (e.g., heterologous nucleic acids) encoding one or more polypeptides having at least one activity of a polypeptide present in the cannabinoid or cannabinoid precursor biosynthetic pathway may be useful in the methods and modified host cells for the synthesis of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives. Cannabinoid precursors may include, for example, geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA. [0085] In Cannabis, cannabinoids are produced from the common metabolite precursors geranylpyrophosphate (GPP) and hexanoyl-CoA by the action of three polypeptides. Hexanoyl-CoA and malonyl-CoA are combined to afford a 12-carbon tetraketide intermediate by a tetraketide synthase (TKS) polypeptide (3,5,7- trioxododecanoyl-CoA). This tetraketide intermediate is then cyclized by an olivetolic acid cyclase (OAC) polypeptide to produce olivetolic acid. Olivetolic acid is then prenylated with the common isoprenoid precursor GPP by a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide (e.g., a CsPT4 polypeptide) to produce CBGA, the cannabinoid also known as the “mother cannabinoid.” The engineered variants of an OAC polypeptide of the disclosure then converts 3,5,7-trioxododecanoyl-CoA to olivetolic acid or a derivative thereof. [0086] GPP and hexanoyl-CoA can be generated through several pathways. One or more nucleic acids (e.g., heterologous nucleic acids) encoding one or more polypeptides having at least one activity of a polypeptide present in these pathways can be useful in the methods and modified host cells for the synthesis of cannabinoids or cannabinoid derivatives. [0087] Polypeptides that generate GPP or are part of a biosynthetic pathway that generates GPP may be one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides). The term “mevalonate pathway” or “MEV pathway,” as used herein, may refer to the biosynthetic pathway that converts acetyl-CoA to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mevalonate pathway comprises polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMG-CoA synthase (HMGS) polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of a HMG-CoA reductase (HMGR) polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a mevalonate kinase (MK) polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5- pyrophosphate (e.g., by action of a phosphomevalonate kinase (PMK) polypeptide); (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by action of a mevalonate pyrophosphate decarboxylase (MVD1) polypeptide); and (g) converting isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP) (e.g., by action of an isopentenyl pyrophosphate isomerase (IDI1) polypeptide). A geranyl pyrophosphate synthetase (GPPS) polypeptide then acts on IPP and/or DMAPP to generate GPP. [0088] Polypeptides that generate hexanoyl-CoA may include polypeptides that generate acyl-CoA compounds or acyl-CoA compound derivatives (e.g., an acyl-activating enzyme polypeptide, a fatty acyl-CoA synthetase polypeptide, or a fatty acyl-CoA ligase polypeptide). Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such polypeptides.
Figure imgf000031_0001
Cannabidiol / CBD Tetrahydrocannabinol / Dronabinol / Marinol / THC Biosynthetic pathways to cannabinoids [0089] GPP and hexanoyl-CoA may also be generated through pathways comprising polypeptides that condense two molecules of acetyl-CoA to generate acetoacetyl-CoA and pyruvate decarboxylase polypeptides that generate acetyl-CoA from pyruvate via acetaldehyde. Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such pathways. General Information [0090] In certain aspects, the practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature: “Molecular Cloning: A Laboratory Manual,” second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994). Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y.1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y.1992), provide one skilled in the art with a general guide to many of the terms used in the present application [0091] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabidiol (CBD) type (e.g., cannabidiolic acid), Δ9-trans-tetrahydrocannabinol (Δ9 -THC) type (e.g., Δ9-tetrahydrocannabinolic acid), Δ8-trans-tetrahydrocannabinol (Δ8 -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, derivatives of any of the foregoing, and others as listed in Elsohly M.A. and Slade D., Life Sci.2005 Dec 22;78(5):539-48. Epub 2005 Sep 30. [0092] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, cannabimovone (CBM) , cannabimovonic acid (CBMA), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBDA, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C1), Δ9 –tetrahydrocannabinolic acid A (THCA-A), Δ9 –tetrahydrocannabinolic acid B (THCA-B), Δ9 –tetrahydrocannabinol (Δ9- THC), Δ9 –tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9 –tetrahydrocannabinol-C4 (THC- C4), Δ9 –tetrahydrocannabivarinic acid (THCVA), Δ9 –tetrahydrocannabivarin (THCV), Δ9 – tetrahydrocannabiorcolic acid (THCA-C1), Δ9 –tetrahydrocannabiorcol (THC-C1), Δ7 –cis- iso-tetrahydrocannabivarin, Δ8 –tetrahydrocannabinolic acid (Δ8 –THCA), Δ8 – tetrahydrocannabinol (Δ8 –THC), tetrahydrocannabiphorol (THCP), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA- A), cannabielsoic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methylether (CBNM), cannabinol-C4, (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiorcol (CBN-C1), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a- tetrahydrocannabinol, cannabitriolvarin (CBTV), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanon (CBCN), cannabicitran (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-benzoxocin-5- methanol (OH-iso-HHCV), cannabiripsol (CBR), trihydroxy-delta-9-tetrahydrocannabinol (triOH-THC), CBGA-hydrocinnamic acid (3‐[(2E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl]‐2,4‐ dihydroxy‐6‐(2‐phenylethyl)benzoic acid), CBG-hydrocinnamic acid (2‐[(2E)‐3,7‐ dimethylocta‐2,6‐dien‐1‐yl]‐5‐(2‐phenylethyl)benzene‐1,3‐diol), CBDA-hydrocinnamic acid (2,4‐dihydroxy‐3‐[3‐methyl‐6‐(prop‐1‐en‐2‐yl)cyclohex‐2‐en‐1‐yl]‐6‐(2‐ phenylethyl)benzoic acid), CBD-hydrocinnamic acid (2‐[3‐methyl‐6‐(prop‐1‐en‐2‐ yl)cyclohex‐2‐en‐1‐yl]‐5‐(2‐phenylethyl)benzene‐1,3‐diol), THCA-hydrocinnamic acid (1‐ hydroxy‐6,6,9‐trimethyl‐3‐(2‐phenylethyl)‐6H,6aH,7H,8H,10aH‐benzo[c]isochromene‐2‐ carboxylic acid), THC-hydrocinnamic acid (6,6,9‐trimethyl‐3‐(2‐phenylethyl)- 6H,6aH,7H,8H,10aH‐benzo[c]isochromen‐1‐ol, perrottetinene), and derivatives of any of the foregoing. [0093] Olivetolic acid derivatives include, but are not limited to, : divarinolic acid, orsellinic acid, 3-butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3- methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-resorcylic acid, 3-octyl-resorcylic acid, 3-(trans- 1-octenyl)-resorcylic acid, 3-nonyl-resorcylic acid, 3-(3-phenylpropyl)-resorcylic acid, 3-(4- phenylbutyl)-resorcylic acid, 3-(5-phenylpentyl)-resorcylic acid, and 3-(6-phenylhexyl)- resorcylic acid. [0094] An acyl-CoA compound as detailed herein may include compounds with the following structure:
Figure imgf000034_0001
, wherein R may be an unsubstituted fatty acid side chain or a fatty acid side chain substituted with or comprising one or more functional and/or reactive groups as disclosed herein (i.e., an acyl-CoA compound derivative). [0095] As used herein, a hexanoyl CoA derivative, an acyl-CoA compound derivative, a cannabinoid derivative, or an olivetolic acid derivative may refer to hexanoyl CoA, an acyl-CoA compound, a cannabinoid, or olivetolic acid substituted with or comprising one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl (including branched and straight chain alkyl groups), alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, heterocyclyl, spirocyclyl, heterospirocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like. A reactive group may facilitate covalent attachment of a molecule of interest. Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type; molecules that target to a particular tissue; molecules that provide for crossing the blood- brain barrier; molecules to facilitate selective attachment to a surface; and the like. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups. [0096] A cannabinoid derivative or olivetolic acid derivative may also refer to a compound lacking one or more chemical moieties found in naturally-occurring cannabinoids or olivetolic acid, yet retains the core structural features (e.g., cyclic core) of a naturally- occurring cannabinoid or olivetolic acid. Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, and the like. In some embodiments, a cannabinoid derivative or olivetolic acid derivative may also comprise one or more of any of the functional and/or reactive groups described herein. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups. [0097] The term “nucleic acid” or “nucleic acids” used herein, may refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term may include, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, genes, synthetic DNA or RNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other naturally-occurring, chemically or biochemically modified, non- naturally-occurring, or derivatized nucleotide bases. [0098] The terms “peptide,” “polypeptide,” and “protein” may be used interchangeably herein, and may refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The polypeptides disclosed herein may include full- length polypeptides, fragments of polypeptides, truncated polypeptides, fusion polypeptides, or polypeptides having modified peptide backbones. The polypeptides disclosed herein may also be variants differing from a specifically recited “reference” polypeptide (e.g., a wild- type polypeptide) by amino acid insertions, deletions, mutations, and/or substitutions. [0099] An “engineered variant of an olivetolic acid cyclase polypeptide” or “engineered variant of the disclosure” may indicate a non-wild type polypeptide having olivetolic acid cyclase activity. One skilled in the art can measure the olivetolic acid cyclase activity of the engineered variants using known methods. For example, by GC-MS or LC- MS or as described in the examples provided herein. Engineered variants may have amino acid substitutions compared to a wild type olivetolic acid cyclase sequence, such as the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO: 1. In addition to substitutions, engineered variants may comprise truncations, additions, and/or deletions, and/or other mutations compared to a wild type olivetolic acid cyclase sequence, such as the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1. Engineered variants may have substitutions compared to a non-wild type sequence (e.g., reference sequence) of olivetolic acid cyclase. In addition to substitutions, engineered variants may comprise truncations, additions, and/or deletions and/or other mutations compared to a non-wild type olivetolic acid cyclase sequence. The engineered variants described herein contain at least one amino acid residue substitution from a reference olivetolic acid cyclase polypeptide. In some embodiments, the reference olivetolic acid cyclase polypeptide is a wild type sequence. In some embodiments, the reference olivetolic acid cyclase polypeptide is a non-wild type sequence. [00100] As used herein, the term “heterologous” may refer to what is not normally found in nature. The term “heterologous nucleotide sequence” or the term “heterologous nucleic acid” may refer to a nucleic acid or nucleotide sequence not normally found in a given cell in nature. A heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus; or (d) be naturally found in the host cell, but with introns removed or added. A heterologous nucleic acid may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus. In some embodiments, a heterologous nucleic acid may comprise a codon-optimized nucleotide sequence. A codon-optimized nucleotide sequence may be an example of a heterologous nucleotide sequence. In some embodiments, the heterologous nucleic acids disclosed herein may comprise nucleotide sequences that encode a polypeptide disclosed herein, such as an engineered variant of the disclosure, but do not comprise nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements). In some embodiments, the heterologous nucleic acids disclosed herein may comprise nucleotide sequences encoding a polypeptide disclosed herein, such as an engineered variant of the disclosure, along with nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements). [00101] The term “heterologous enzyme” or “heterologous polypeptide” may refer to an enzyme or polypeptide that is not normally found in a given cell in nature. The term encompasses an enzyme or polypeptide that is: (a) exogenous to a given cell (i.e., encoded by a nucleic acid that is not naturally present in the host cell or not naturally present in a given context in the host cell); or (b) naturally found in the host cell (e.g., the enzyme or polypeptide is encoded by a nucleic acid that is endogenous to the cell) but that is produced in an unnatural amount (e.g., greater or lesser than that naturally found) in the host cell. For example, a heterologous polypeptide may include a mutated version of a polypeptide naturally occurring in a host cell. [00102] As used herein, the term “one or more heterologous nucleic acids” or “one or more heterologous nucleotide sequences” may refer to heterologous nucleic acids comprising one or more nucleotide sequences encoding one or more polypeptides. In some embodiments, the one or more heterologous nucleic acids may comprise a nucleotide sequence encoding one polypeptide. In other embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding more than one polypeptide. In certain such embodiments, the nucleotide sequences encoding the more than one polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof. In some embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of the same polypeptide. In certain such embodiments, the nucleotide sequences encoding the multiple copies of the same polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof. In some embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of different polypeptides. In certain such embodiments, the nucleotide sequences encoding the multiple copies of the different polypeptides may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof. [00103] As used herein, “increased ratio” may refer to an increase in the molar ratio, an increase in the mass (or weight) ratio, an increase in the molarity ratio, or an increase in the mass concentration (e.g., mg/L or mg/mL) ratio between two products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein compared to the molar ratio, mass (or weight) ratio, molarity ratio, or mass concentration ratio between the same two products produced by another polypeptide, engineered variant, method, and/or modified host cell disclosed herein (e.g., a comparative polypeptide, engineered variant, method, and/or modified host cell disclosed herein). For example, a 100:1 ratio of olivetolic acid or derivatives thereof over olivetol produced by an engineered variant disclosed herein would be an increased ratio of olivetolic acid or derivatives thereof over olivetol compared to an 11:1 ratio of olivetolic acid or derivatives thereof over olivetol produced by a different engineered variant disclosed herein. [00104] As used herein, a ratio of products produced by a polypeptide, engineered variant, method, and/or modified host cell disclosed herein, such as the ratio of olivetolic acid over olivetol, may refer to a molar ratio, a mass (or weight) ratio, molarity ratio, or a mass concentration (e.g., mg/L or mg/mL) ratio. For example, if a modified host cell disclosed herein produced 4 mM olivetolic acid and 1 mM olivetol, the ratio of olivetolic acid or derivatives thereof over olivetol would be 4:1. [00105] “Operably linked” may refer to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. [00106] “Isolated” may refer to polypeptides or nucleic acids that are substantially or essentially free from components that normally accompany them in their natural state. An isolated polypeptide or nucleic acid may be other than in the form or setting in which it is found in nature. Isolated polypeptides and nucleic acids therefore may be distinguished from the polypeptides and nucleic acids as they exist in natural cells. An isolated nucleic acid or polypeptide may be purified from one or more other components in a mixture with the isolated nucleic acid or polypeptide, if such components are present. [0100] A “modified host cell” (also may be referred to as a “recombinant host cell”) may refer to a host cell into which has been introduced a nucleic acid (e.g., a heterologous nucleic acid), e.g., an expression vector or construct. For example, a modified eukaryotic host cell may be produced through introduction into a suitable eukaryotic host cell of a nucleic acid (e.g., a heterologous nucleic acid). [0101] As used herein, a “cell-free system” may refer to a cell lysate, cell extract or other preparation in which substantially all of the cells in the preparation have been disrupted or otherwise processed so that all or selected cellular components, e.g., organelles, proteins, nucleic acids, the cell membrane itself (or fragments or components thereof), or the like, are released from the cell or resuspended into an appropriate medium and/or purified from the cellular milieu. Cell-free systems can include reaction mixtures prepared from purified and/or isolated polypeptides and suitable reagents and buffers. [0102] In some embodiments, conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions may be accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. The term “conservative amino acid substitution” may refer to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains may consist of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains may consist of serine, valine, isoleucine, methionine, and threonine; a group of amino acids having amide containing side chains may consist of asparagine and glutamine; a group of amino acids having aromatic side chains may consist of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains may consist of lysine, arginine, and histidine; a group of amino acids having acidic side chains may consist of glutamate and aspartate; and a group of amino acids having sulfur containing side chains may consist of cysteine and methionine; a group of hydrophililic amino acids may consist of alanine, proline, glycine, glutamic acid, aspartic acid, glutamine, asparagine, serine, and threonine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. [0103] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST,ebi.ac.uk/Tools/msa/tcoffee/ebi.ac.uk/ Tools/msa/muscle/mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol.215:403-10. [0104] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0105] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [0106] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0107] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cannabinoid compound” or “cannabinoid” may include a plurality of such compounds and reference to “the modified host cell” may include reference to one or more modified host cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. [0108] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide [0109] Disclosed herein are engineered variants of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variants comprise an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution. The inventors have identified amino acid locations of the OAC polypeptide comprising an amino acid sequence of SEQ ID NO:1 that when substituted, may result in one or more improved properties of the engineered variant. In one aspect of the disclosure, the substitution is at a location corresponding to the position in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa. [0110] Residue positions in the engineered variants discussed herein may be identified with respect to a reference amino acid sequence, for example, the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa (UniProtKB/Swiss-Prot: Q8GTB6). Accordingly, a reference to “I94” identifies an amino acid that, in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa, is the 94th amino acid from the N-terminus, wherein the methionine is the first amino acid. The 94th amino acid is a isoleucine (I) in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa. Those of skill in the art appreciate that the 94th amino acid may have a different position in the OAC polypeptides from different species or in different isoforms. These engineered variants are intended to be encompassed by this disclosure. [0111] The polypeptide sequence position at which a particular amino acid or amino acid change (“residue difference”) is present is sometimes described herein as “Xn”, or “position n”, where n refers to the amino acid position with respect to the reference sequence. Accordingly, a reference to “X94” identifies an amino acid that, in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa, is the 94th amino acid from the N- terminus. [0112] A specific substitution mutation, which is a replacement of the specific amino acid in a reference sequence with a different specified residue may be denoted by the conventional notation “X (number)Y”, where X is the single letter identifier of the amino in the reference sequence, “number” is the amino acid position in the reference sequence, and Y is the single letter identifier of the amino acid substitution in the engineered sequence. Accordingly, a reference to “I94K” identifies a substitution that, in the OAC polypeptide of SEQ ID NO:1 from Cannabis sativa, is the 94th amino acid from the N-terminus, isoleucine, being replaced by lysine. [0113] The disclosure provides for an engineered variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions. In certain such embodiments, the engineered variant comprises an amino acid sequence between about 75% to about 99.9% sequence identity with SEQ ID NO:1. In some embodiments, the engineered variant comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the engineered variant comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1. [0114] The disclosure provides for an engineered variant comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein the at least one of the amino acid substitutions occurs at an amino acid in a beta sheet and/or an alpha helix of the polypeptide. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions occurs at an amino acid in the beta sheet 1 (e.g., amino acids 4-12), alpha helix 1 (e.g., amino acids 17-33), beta sheet 2 (e.g., amino acids 39-44), beta sheet 3 (e.g., amino acids 57-62), alpha helix 2 (e.g., amino acids 66-73), alpha helix 3 (e.g., amino acids 76-88), or beta sheet 4 (e.g., amino acids 91-97) or combinations of the foregoing. [0115] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X22, X23, X25, X29, X31, X36, X41, X47, X48, X49, X50, X52, X53, X56, X58, X62, X64, X66, X67, X70, X71, X74, X84, X87, X90, X94, and X100. The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of X2, X8, X9, X10, X12, X22, X23, X25, X29, X31, X36, X41, X47, X48, X49, X50, X53, X56, X58, X62, X64, X66, X67, X70, X71, X74, X84, X87, X90, X94, and X100. Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase (OAC) polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce OAC from 3,5,7- trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of OAC produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce OAC from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivative thereof over olivetol compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0116] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions at an amino acid selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. Such engineered variants may produce olivetolic acid or a derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid or derivative thereof produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid or a derivative thereof produced from 3,5,7-trioxododecanoyl-CoA or a derivative thereof by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA or a derivative thereof in an increased ratio of olivetolic acid or derivatives thereof over olivetol or derivatives thereof compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0117] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions selected from the group consisting of A2P, A2S, V8I, L9I, L9V, K10A, K12A, K12C, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47R, T47S, Q48C, Q48H, Q48M, K49R, N50Y, E52H, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, wherein at least one of the amino acid substitutions selected from the group consisting of A2P, A2S, V8I, L9I, L9V, K10A, K12A, K12C, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47R, T47S, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time and may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivative thereof over olivetol compared to that produced by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0118] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids A2P, V8I,L9I, E64D, I94K, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K10A, N29D, T56S, I58V, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9I, A36Q, Q48C, I58V, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K49R, T56S, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, A36E, K49R, and R100G. [0119] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, K49R, T56S, and D71T. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12N, K49R, I58V, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K49R, I58V, Q70A, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K10A, T56S, I58V, and Q70K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids . L9I, K10A, K25N, Q48H, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, K12V, T56S, I58V, and E64D. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, A36E, Q48H, K49R, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9V, E22L, A36E, Q48M, and R100G. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, A36S, T62C, E64D, and I94K. In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising amino acid substitutions at least at amino acids V8I, L9I, E22L, A36E, Q48M, and R100G. [0120] In some embodiments, the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions V8I, L9I, K12N, A36E, D71, and R100G. [0121] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO: 1 comprising between about 6 to about 16 amino acid substitutions. The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO: 1 comprising 6 amino acid substitutions. In some embodiments, the engineered variant comprises an amino acid sequence having between about 75% to about 99.9%, or about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered variant comprises an amino acid sequence having at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. [0122] Combinations of the amino acid substitutions described herein can be made and the resulting engineered variants screened for improved olivetolic acid cyclase (OAC) properties. Engineered variants comprising combinations of all of the substitutions described herein are intended to be encompassed by this disclosure. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 2 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the amino acid substitutions described herein (e.g., 2-16 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises an amino acid sequence comprising amino acid substituions as disclosed herein and having between about 75% to about 99.9%, or between about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered variant comprises an amino acid sequence amino acid substituions as disclosed herein and having about at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. [0123] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions, wherein at least one of the amino acid substitutions is at the C-terminus. In certain such embodiments, a hydrophilic amino acid is replaced with a hydrophobic amino acid. In some embodiments, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 substitutions, wherein at least one of the amino acid substitutions is at the C-terminus, a hydrophobic amino acid is replaced with a hydrophilic amino acid. Such engineered variants may produce olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. In some embodiments, the engineered variant comprises an amino acid sequence having between about 75% to about 99.9%, or between about 85% to about 95% identity to the amino acid sequence of SEQ ID NO: 1, wherein at least one of the amino acid substitutions is at the C-terminus. In some embodiments, the engineered variant comprises an amino acid sequence having at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein at least one of the amino acid substituteions is at the C-terminus. [0124] The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 and a truncation at the C-terminus. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 ions and a truncation at both the N- and C-termini. In some embodiments, the engineered variant lacks a native signal polypeptide. [0125] In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini, and comprises an amino acid sequence with between about 75% to about 99.9%, or about 85% to about 95% sequence identity to SEQ ID NO:1. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation at the N-terminus, at the C-terminus, or at both the N- and C-termini, and comprises an amino acid sequence with at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% , at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1. [0126] In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus (e.g., 1-10 amino acids at the C-terminus). In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the C-terminus (e.g., 11-20 amino acids at the C-terminus). In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus and comprises an amino acid sequence having about between about 75% to about 99.9%, or about 85% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the C-terminus and comprises an amino acid sequence having about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, aobut 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% identity to the amino acid sequence of SEQ ID NO: 1 [0127] In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 one amino acid substitutions and a truncation of between 1 and 20 amino acids at the N- terminus (e.g., 1-10 amino acids at the N-terminus). The disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution and an addition and/or deletion of at least one amino acid. In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the N-terminus and comprises an amino acid sequence having about between about 75% to about 99.9%, or about 85% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. [0128] In some embodiments, the engineered variant comprises an amino acid sequence of SEQ ID NO:1 comprising between about 1 to about 16, or about 2 to about 14, or about 3 to about 12, or about 4 to about 10, or about 5 to about 9, or about 6 to about 8 amino acid substitutions and a truncation of between 1 and 20 amino acids at the N-terminus and comprises an amino acid sequence having about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, aobut 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% identity to the amino acid sequence of SEQ ID NO: 1. [0129] In some embodiments of the disclosure, the engineered variant produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0130] In some embodiments of the disclosure, the engineered variant produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetol or a derivative thereof of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1. [0131] These improved properties may be assessed by the conversion of 3,5,7- trioxododecanoyl-CoA to olivetolic acid, or alternatively the conversion of another starting material to a desired cannabinoid or cannabinoid derivative, in vitro with isolated and/or purified engineered variants of the disclosure or in vivo in the context of a modified host cell expressing the engineered variant. [0132] In some embodiments, the engineered variants of the disclosure can be made and screened for improved properties, such as, production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives or precursors by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. [0133] Additionally, engineered variants of the disclosure can be made and screened for improved properties, such as, a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. [0134] In some embodiments, the engineered variants of the disclosure exhibit similar and/or not significantly decreased growth and/or viability compared to modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 , but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. Nucleic Acids Comprising Nucleotide Sequences Encoding Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide and Expression Vectors and Constructs [0135] The disclosure provides for nucleic acids comprising nucleotide sequences encoding engineered variants of the olivetolic acid cyclase (OAC) polypeptide disclosed herein and expression vectors and constructs comprising said nucleic acids. [0136] The disclosure provides nucleic acids comprising nucleotide sequences encoding engineered variants of the disclosure. Some embodiments of the disclosure relate to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an olivetolic acid cyclase variant amino acid sequence of any of SEQ ID NOs: 3-146. In some embodiments, the disclosure relates to a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an olivetolic acid cyclase variant amino acid sequence of any of SEQ ID NOs: 36-45 and 94-98. [0137] The disclosure also provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs: 147-290. In some embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs:180-189 and 138-242. [0138] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs: 147-290 or a codon degenerate sequence of anyof the foregoing. In some embodiments, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an olivetolic acid cyclase engineered variant, wherein the nucleotide sequence is any one of SEQ ID NOs:180-189 and 138-242, or a codon degenerate sequence of any of the foregoing. [0139] Further included are nucleic acids that hybridize to the nucleic acids disclosed herein. Hybridization conditions may be stringent in that hybridization will occur if there is at least a 90%, at least a 95%, or at least a 97% sequence identity with the nucleotide sequence present in the nucleic acid encoding the polypeptides disclosed herein. The stringent conditions may include those used for known Southern hybridizations such as, for example, incubation overnight at 42 °C in a solution having 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt’s solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in 0.1×SSC at about 65 °C. Other known hybridization conditions are well known and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001). [0140] The length of the nucleic acids disclosed herein may depend on the intended use. For example, if the intended use is as a primer or probe, for example for PCR amplification or for screening a library, the length of the nucleic acid will be less than the full length sequence, for example, 15-50 nucleotides. In certain such embodiments, the primers or probes may be substantially identical to a highly conserved region of the nucleotide sequence or may be substantially identical to either the 5’ or 3’ end of the nucleotide sequence. In some cases, these primers or probes may use universal bases in some positions so as to be “substantially identical” but still provide flexibility in sequence recognition. It is of note that suitable primer and probe hybridization conditions are well known in the art. [0141] Some embodiments of the disclosure relate to a vector comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to an expression construct comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to nucleic acids comprising codon-optimized nucleotide sequences encoding the engineered variants of the disclosure. In some embodiments, the nucleic acids disclosed herein are heterologous. Methods of Screening Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide [0142] The disclosure provides a method of screening an engineered variant of an olivetolic acid cyclase polypeptide comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution. In certain such embodiments, the method involves a competition assay wherein the engineered variant of the disclosure is expressed in a modified host cells alongside a related enzyme. [0143] Some embodiments of the disclosure relate to a method of screening an engineered variant of an olivetolic acid cyclase polypeptide comprising an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitution, the method comprising: a) dividing a population of host cells into a control population and a test population; b) co-expressing in the control population an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 and a comparison olivetolic acid cyclase polypeptide or TKS enzyme, wherein the OAC polypeptide having an amino acid sequence of SEQ ID NO:1 can convert 3,5,7-trioxododecanoyl-CoA or a derivative thereof to olivetolic acid or a derivative thereof and the comparison olivetolic acid cyclase polypeptide or TKS enzyme can convert the same 3,5,7-trioxododecanoyl-CoA or a derivative thereof to a olivetol or derivative; c) co-expressing in the test population the engineered variant and the comparison olivetolic acid cyclase polypeptide, wherein the engineered variant may convert 3,5,7- trioxododecanoyl-CoA or a derivative thereof to the same molecule, olivetolic acid or a derivative thereof, as the OAC polypeptide having an amino acid sequence of SEQ ID NO:1 , and wherein the comparison olivetolic acid cyclase polypeptide or TKS enzyme can convert the same 3,5,7-trioxododecanoyl-CoA derivative thereof to the different byproduct or derivative and is expressed at similar levels in the test population and in the control population; d) measuring a ratio of the first molecule, olivetolic acid, over the different byproduct or derivative produced by both the test population and the control population (e.g. olivetolic if using TKS); and e) measuring an amount, in mg/L or mM, of the olivetolic acid and byproduct or derivative produced by both the test population and the control population. In cetain such embodiments, the engineered variant is an engineered variant of the disclosure. [0144] In some embodiments, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 by producing the first molecule in a greater amount, as measured in mg/L or mM, by the test population compared to the amount produced by the control population under similar culture conditions for the same length of time. In some embodiments, the test population is identified as comprising an engineered variant having improved in vivo performance compared to the olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, wherein improved in vivo performance is demonstrated by an increase in the ratio of olivetolic acid or derivatives thereof over olivetol or a byproduct thereof produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time. Modified Host Cells for Expressing Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide and for Producing Cannabinoids and Cannabinoid Derivatives [0145] The present disclosure provides modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the modified host cells of the disclosure are for expressing an engineered variant and/or for producing an olivetolic acid, an olivetolic acid derivative, a cannabinoid or a cannabinoid derivative. [0146] To produce olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives and create biosynthetic pathways within modified host cells, modified host cells of the disclosure may express or overexpress combinations of heterologous nucleic acids comprising nucleotide sequences encoding polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, or hexanoyl-CoA) biosynthesis. In some embodiments, the modified host cells of the disclosure comprise one or more modifications to modulate the expression of one or more secretory pathway polypeptides. The one or more modifications to modulate the expression of one or more secretory pathway polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, resulting in expression or overexpression of the one or more secretory pathway polypeptides. In some embodiments, the modified host cell for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, reducing or eliminating the expression of the one or more secretory pathway polypeptides. In certain such embodiments, the modified host cells comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides. [0147] Secretory pathway polypeptides with modulated expression in the modified host cells of the disclosure may include, but are not limited to: a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, FAD1 polypeptide, a PEP4 polypeptide, and an IRE1 polypeptide. Expression of secretory pathway polypeptides may be modulated by introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. [0148] In some embodiments, the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway modifications as disclosed in PCT/US2019/053292, the contents of which are herein incorporated by reference in their entirety for all purposes. [0149] In some embodiments, the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, or hexanoyl-CoA) biosynthesis. In addition to engineered variants of the disclosure, such polypeptides may include, but are not limited to: a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide, a tetraketide synthase (TKS) polypeptide, one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway (e.g., one or more MEV pathway polypeptides), an acyl-activating enzyme (AAE) polypeptide, a polypeptide that generates GPP (e.g., a geranyl pyrophosphate synthetase (GPPS) polypeptide), a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., an acetoacetyl-CoA thiolase polypeptide), and a pyruvate decarboxylase polypeptide. [0150] In some embodiments, the modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide. Exemplary GOT polypeptides disclosed herein may include a full-length GOT polypeptide, a fragment of a GOT polypeptide, a variant of a GOT polypeptide, a truncated GOT polypeptide, or a fusion polypeptide that has at least one activity of a GOT polypeptide. In some embodiments, the GOT polypeptide has aromatic prenyltransferase (PT) activity. In some embodiments, the GOT polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative. In certain such embodiments, the GOT polypeptide modifies olivetolic acid or an olivetolic acid derivative. In some embodiments, the GOT polypeptide is disclosed in US 2019-0300888, the contents of which are incorporated herein in their entireties for all purposes. [0151] In some embodiments, a NphB polypeptide is used instead of a GOT polypeptide to generate cannabigerolic acid from GPP and olivetolic acid. A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide. Exemplary NphB polypeptides disclosed herein may include a full-length NphB polypeptide, a fragment of a NphB polypeptide, a variant of a NphB polypeptide, a truncated NphB polypeptide, or a fusion polypeptide that has at least one activity of a NphB polypeptide. In some embodiments, the NphB polypeptide has aromatic prenyltransferase (PT) activity. In some embodiments, the NphB polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative. In certain such embodiments, the NphB polypeptide modifies olivetolic acid or an olivetolic acid derivative. In some embodiments, the NphB polypeptide is disclosed in US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300, the contents of which are incorporated herein in their entireties for all purposes. [0152] In some embodiments, the modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more comprises involved in cannabinoid or cannabinoid precursor biosynthesis as disclosed in US 2019/0300888; PCT/US2019/053173; US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300 filed September 18, 2019, the contents of which are incorporated by reference in their entirety for all purposes. [0153] A modified host cell of the present disclosure may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide and/or cannabinolic acid synthase (CBDAS). In some embodiments, the THCAS and/or CBDAS polypeptides and variants are disclosed in US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300 filed September 18, 2019 the contents of which are incorporated by reference in their entirety for all purposes. [0154] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acyl-CoA compounds or acyl-CoA compound derivatives. Such polypeptides may include, but are not limited to, acyl-activating enzyme (AAE) polypeptides, fatty acyl-CoA synthetases (FAA) polypeptides, or fatty acyl-CoA ligase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide. [0155] AAE polypeptides, FAA polypeptides, and fatty acyl-CoA ligase polypeptides can convert carboxylic acids to their CoA forms and generate acyl-CoA compounds or acyl-CoA compound derivatives. Promiscuous acyl-activating enzyme polypeptides, such as CsAAE1 and CsAAE3 polypeptides, FAA polypeptides, or fatty acyl- CoA ligase polypeptides, may permit generation of cannabinoid derivatives (e.g., cannabigerolic acid derivatives), as well as cannabinoids (e.g., cannabigerolic acid). In some embodiments, unsubstituted or substituted hexanoic acid or carboxylic acids other than unsubstituted or substituted hexanoic acid are fed to modified host cells expressing an AAE polypeptide, FAA polypeptide, or fatty acyl-CoA ligase polypeptide (e.g., are present in the culture medium in which the cells are grown) to generate hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl-CoA compounds. The hexanoyl-CoA, acyl-CoA compounds, derivatives of hexanoyl-CoA, or derivatives of acyl- CoA compounds can then be further utilized by a modified host cell to generate olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives. In certain such embodiments, the cell culture medium comprising the modified host cells comprises unsubstituted or substituted hexanoic acid. In some embodiments, the cell culture medium comprising the modified host cells comprises a carboxylic acid other than unsubstituted or substituted hexanoic acid. [0156] Exemplary AAE, FAA, or fatty acyl-CoA ligase polypeptides disclosed herein may include a full-length AAE, FAA, or fatty acyl-CoA ligase polypeptide; a fragment of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a variant of an AAE, FAA, or fatty acyl-CoA ligase polypeptide; a truncated AAE, FAA, or fatty acyl-CoA ligase polypeptide; or a fusion polypeptide that has at least one activity of an AAE, FAA, or fatty acyl-CoA ligase polypeptide. [0157] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides that condense an acyl-CoA compound, such as hexanoyl-CoA, or an acyl-CoA compound derivative, such as a hexanoyl-CoA derivative, with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid. Polypeptides that react an acyl-CoA compound or an acyl-CoA compound derivative with malonyl-CoA to generate olivetolic acid, or a derivative of olivetolic acid, may include TKS polypeptides. TKS polypeptides have been found to have broad substrate specificity, enabling production of cannabinoid derivatives or cannabinoids. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide. [0158] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates GPP. In some embodiments, the polypeptide that generates GPP is a geranyl pyrophosphate synthetase (GPPS) polypeptide. In some embodiments, the GPPS polypeptide also has farnesyl diphosphate synthase (FPPS) polypeptide activity. In some embodiments, the GPPS polypeptide is modified such that it has reduced FPPS polypeptide activity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than at least 90%, less FPPS polypeptide activity) than the corresponding wild-type or parental GPPS polypeptide from which the modified GPPS polypeptide is derived. In some embodiments, the GPPS polypeptide is modified such that it has substantially no FPPS polypeptide activity. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide. [0159] Exemplary GPPS polypeptides disclosed herein may include a full-length GPPS polypeptide, a fragment of a GPPS polypeptide, a variant of a GPPS polypeptide, a truncated GPPS polypeptide, or a fusion polypeptide that has at least one activity of a GPPS polypeptide. [0160] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that generates acetyl-CoA from pyruvate. Polypeptides that generate acetyl-CoA from pyruvate may include a pyruvate decarboxylase (PDC) polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide. [0161] Exemplary PDC polypeptides disclosed herein may include a full-length PDC polypeptide, a fragment of a PDC polypeptide, a variant of a PDC polypeptide, a truncated PDC polypeptide, or a fusion polypeptide that has at least one activity of a PDC polypeptide. [0162] A modified host cell of the disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA. In some embodiments, the polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA is an acetoacetyl-CoA thiolase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide. [0163] Exemplary acetoacetyl-CoA thiolase polypeptides disclosed herein may include a full-length acetoacetyl-CoA thiolase polypeptide, a fragment of an acetoacetyl- CoA thiolase polypeptide, a variant of an acetoacetyl-CoA thiolase polypeptide, a truncated acetoacetyl-CoA thiolase polypeptide, or a fusion polypeptide that has at least one activity of an acetoacetyl-CoA thiolase polypeptide. [0164] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway. In certain such embodiments, the one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway comprise one or more MEV pathway polypeptides. [0165] In some embodiments, the one or more polypeptides that are part of a biosynthetic pathway that generates GPP are one or more polypeptides having at least one activity of a polypeptide present in the mevalonate pathway. The mevalonate pathway may comprise polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMGS polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of an HMGR polypeptide); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of a MK polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by action of a PMK polypeptide); (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by action of a mevalonate pyrophosphate decarboxylase (MPD or MVD1) polypeptide); and (g) converting isopentenyl pyrophosphate to dimethylallyl pyrophosphate (e.g., by action of an isopentenyl pyrophosphate isomerase (IDI1) polypeptide). [0166] In some embodiments, the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell. In some embodiments, the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell, yet displays increased activity compared to a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time. In some embodiments, the engineered variant of the disclosure is expressed at endogenous levels in the modified host cell, yet produces more olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA compared to a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time. [0167] In some embodiments, the engineered variant of the disclosure expressed in the modified host cell produces more olivetolic acid or derivative thereof from 3,5,7- trioxododecanoyl-CoA compared to multiple copies of a wild type polypeptide expressed in a corresponding host cell under similar culture conditions for the same length of time. In some embodiments, the engineered variant of the disclosure is expressed from a single copy in the modified host cell, yet produces more olivetolic acid or derivative thereof from 3,5,7- trioxododecanoyl-CoA or derivative thereof compared to two, three, four, five, six, seven, eight, nine, or ten wild type polypeptides expressed in a corresponding host cell under similar culture conditions for the same length of time. [0168] In some embodiments, the engineered variant of the disclosure is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the engineered variant of the disclosure to a strong promoter. In some embodiments, the modified host cell has one copy of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has two copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has three copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has four copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has five copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has six copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has seven copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. In some embodiments, the modified host cell has eight or more copies of a nucleic acid comprising a nucleotide sequence encoding the engineered variant of the disclosure. Increased copy number of the nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell. [0169] The disclosure provides for modified host cells of the disclosure may be modified to express or overexpress one or more nucleic acids disclosed herein comprising nucleotide sequences encoding an engineered variant of the disclosure, one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and/or one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. A modified host cell of the disclosure may comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the nucleotide sequences encoding the one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and/or one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis are codon-optimized. [0170] In some embodiments, a modified host cell of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1 but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, a modified host cell of the disclosure has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. [0171] In some embodiments, the growth and/or viability of modified host cells of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, the growth and/or viability of modified host cells of the disclosure is about the same as the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments, the growth and/or viability of modified host cells of the disclosure is less than the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments, the growth and/or viability of modified host cells of the disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives is greater than the growth and/or viability of an unmodified host cell grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments, a culture of modified host cells of the disclosure for producing olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. [0172] In some embodiments, the one or more heterologous polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the one or more heterologous nucleic acids to a strong promoter. In some embodiments, the modified host cell has one copy of a one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptide. In some embodiments, the modified host cell has two copies, three copies, four copies, five copies, six copies, seven copies, or eight copies or more of one or more heterologous nucleic acids comprising a nucleotide sequence encoding the one or more heterologous polypeptides. Increased copy number of the one or more heterologous nucleic acids and/or codon optimization of the one or more heterologous nucleotide acids may result in an increase in the desired enzyme catalytic activity in the modified host cell. [0173] In some embodiments, a modified host cell of the disclosure produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid or derivatives thereof over olivetol thereof compared to that produced by a modified host cell comprising a nucleotide sequence encoding an olivetolic acid cyclase polypeptide having an amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. In some embodiments, the modified host cell of the disclosure produces olivetolic acid or derivative thereof from 3,5,7-trioxododecanoyl- CoA in an increased ratio of olivetolic acid or derivatives thereof over olivetol of about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, about 15:1, about 15.5:1, about 16:1, about 16.5:1, about 17:1, about 17.5:1, about 18:1, about 18.5:1, about 19:1, about 19.5:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 150:1, about 200:1, about 500:1, or greater than about 500:1. In some embodiments, the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the modified host cell of the disclosure comprises nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, and/or an IRE1 polypeptide. In some embodiments, the modified host cell of the disclosure comprises a nucleotide sequence encoding an engineered variant of the disclosure and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. In some embodiments, the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. [0174] In some embodiments, the modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide. In some embodiments the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, and further comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor (e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA) biosynthesis. In some embodiments, the modified host cell of the disclosure further comprises a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. Suitable Host Cells [0175] Parent host cells that are suitable for use in generating a modified host cell of the present disclosure may include eukaryotic cells. In some embodiments, the eukaryotic cells are yeast cells. [0176] Host cells (including parent host cells and modified host cells) are in some embodiments unicellular organisms, or are grown in culture as single cells. In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells may include, but are not limited to, yeast cells and fungal cells. Suitable eukaryotic host cells may include, but are not limited to, Pichia pastoris (now known as Komagataella phaffii), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta), Yarrowia lipolytica, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pombe, Scheffersomyces stipites, Dekkera bruxellensis, Blastobotrys adeninivorans (formerly Arxula adeninivorans), Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, and the like. In some embodiments, the modified host cell disclosed herein is cultured in vitro. [0177] In some embodiments, the host cell of the disclosure is a yeast cell. In some embodiments, the host cell is a protease-deficient strain of Saccharomyces cerevisiae. Protease-deficient yeast strains may be effective in reducing the degradation of expressed heterologous proteins. Examples of proteases deleted in such strains may include one or more of the following: PEP4, PRB1, and KEX1. [0178] In some embodiments, the host cell is Saccharomyces cerevisiae. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of ease of culture; rapid growth; availability of tools for modification, such as promoters and vectors; and the host cell’s safety profile. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of its ability or inability to introduce certain posttranslational modifications onto expressed polypeptides, such as engineered variants of the disclosure. For instance, modified Komagataella phaffii host cells may hyperglycosylate engineered variants of the disclosure and hyperglycosylation may alter the activity of the resultant expressed polypeptide. Genetic Modification of Host Cells and Exemplary Modified Host Cells of the Disclosure [0179] The present disclosure provides for modified host cells and methods of making the modified host cells of the disclosure. In some embodiments, the method of making a modified host cell of the disclosure comprises introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure. In some embodiments, the modified host cell comprises two or more heterologous nucleic acids each comprising a nucleotide sequence encoding a KAR2 polypeptide. In some embodiments, the modified host cell further comprises a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide. [0180] The disclosure provides a method of making a modified host cell of the disclosure, the method comprising introducing into a host cell: a) one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an ERO1 polypeptide, and/or an IRE1 polypeptide, and/or c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide. [0181] To modify a parent host cell to produce a modified host cell of the present disclosure, one or more nucleic acids (e.g., heterologous) disclosed herein may be introduced stably or transiently into a host cell, using established techniques. Such techniques may include, but are not limited to, electroporation, calcium phosphate precipitation, DEAE- dextran mediated transfection, liposome-mediated transfection, the lithium acetate method, and the like. See Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. For stable transformation, a plasmid, vector, expression construct, etc. comprising one or more nucleic acids (e.g., heterologous) disclosed herein will generally include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like. In some embodiments, the selectable marker gene to provide a phenotypic trait for selection of transformed host cells is dihydrofolate reductase. In some embodiments, a parent host cell is modified to produce a modified host cell of the present disclosure using a CRISPR/Cas9 system to modify a parent host cell with one or more nucleic acids (e.g., heterologous) disclosed herein. [0182] In some embodiments, varying polypeptide expression level, such as engineered variant expression level, and/or the production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives in a modified host cell may be done by changing the gene copy number, promoter strength, and/or promoter regulation and/or by codon-optimization. [0183] One or more nucleic acids (e.g., heterologous) disclosed herein, such as one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, can be present in an expression vector or construct. Suitable expression vectors may include, but are not limited to, plasmids, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as yeast). Thus, for example, one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding a mevalonate pathway gene product(s) is included in any one of a variety of expression vectors for expressing the mevalonate pathway gene product(s). Such vectors may include chromosomal, non-chromosomal, and synthetic DNA sequences. [0184] The present disclosure provides for a method of making a modified host cell of the disclosure, the method comprising introducing into a host cell one or more vectors disclosed herein. [0185] The present disclosure provides for a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell one or more vectors disclosed herein. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In some embodiments, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon- optimized. In some embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis. In some embodiments, the nucleotide sequences encoding the one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis are codon- optimized. [0186] The present disclosure provides for a method of making a modified host cell for expressing an olivetolic acid cyclase, the method comprising introducing into a host cell one or more vectors disclosed herein. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising a nucleotide sequence encoding an engineered variant of the disclosure. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the one or more vectors comprise one or more vectors comprising one or more nucleic acids (e.g., heterologous) comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis. In some embodiments, the nucleotide sequences encoding the one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis are codon-optimized. [0187] Numerous additional suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for yeast, the low copy CEN ARS and high copy 2 micron plasmids. However, any other plasmid or other vector may be used so long as it is compatible with the host cell. [0188] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, two, three, four, five, six, seven, or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, two, three, four, five, six, seven, eight, nine, ten or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors. In some embodiments, the expression vector is an expression construct. [0189] In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a high copy number plasmid, e.g., a plasmid that exists in about 10-50 copies per cell, or more than 50 copies per cell. In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a low copy number plasmid. In some embodiments, one or more of the nucleic acids (e.g., heterologous) disclosed herein is present in a medium copy number plasmid. The copy number of the plasmid may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression by limiting the copy number of the plasmid may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. [0190] In some embodiments, the modified host cell has one copy of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has two, three, four, five, six, seven, eight, nine, ten, eleven, or tweleve or more copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. [0191] Depending on the host/vector or host/construct system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector or construct (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). [0192] In some embodiments, the nucleic acids (e.g., heterologous) disclosed herein are operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is functional in a eukaryotic cell. In some embodiments, the promoter can be a strong driver of expression. In some embodiments, the promoter can be a weak driver of expression. In some embodiments, the promoter can be a medium driver of expression. The promoter may be selected to reduce expression of one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression through promoter selection may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. [0193] In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into the genome of the modified host cell disclosed herein. In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into a chromosome of the modified host cell disclosed herein. In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein remains episomal (i.e., is not integrated into the genome or a chromosome of the modified host cell). In some embodiments, at least one of the one or more nucleic acids (e.g., heterologous) disclosed herein is maintained extrachromosomally (e.g., on a plasmid or artificial chromosome). The gene copy number of one or more genes encoding one or more polypeptides disclosed herein, such as an engineered variant of the disclosure, may be selected to reduce expression of the one or more polypeptides disclosed herein, such as an engineered variant of the disclosure. Reducing expression by limiting the gene copy number may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. [0194] As will be appreciated by the skilled artisan, slight changes in nucleotide sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. It will be appreciated by persons skilled in the art that changes in the identities of nucleotides in a specific gene sequence that change the amino acid sequence of the encoded polypeptide may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g., anti-sense, co-suppression, or RNAi), partial sequences often work as effectively as full length versions. The ways in which the nucleotide sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. In certain embodiments, effectiveness may easily be tested by, for example, conventional gas chromatography. All such variations of the genes are therefore included as part of the present disclosure. [0195] Genomic deletion of the open reading frame encoding the protein may abolish all expression of a gene. Downregulation of a gene can be accomplished in several ways at the DNA, RNA, or protein level, with the result being a reduction in the amount of active protein in the cell. Truncations of the open reading frame or the introduction of mutations that destabilize the protein or reduce catalytic activity achieve a similar goal, as does fusing a “degron” polypeptide that destabilizes the protein. Engineering of the regulatory regions of the gene can also be used to change gene expression. Alteration of the promoter sequence or replacement with a different promoter is one method. Truncation of the terminator, known as decreased abundance of mRNA perturbation (DAmP), is also known to reduce gene expression. Other methods that reduce the stability of the mRNA include the use of cis- or trans-acting ribozymes, e.g., self-cleaving ribozymes, or RNA elements that recruit an exonuclease, or antisense DNA. RNAi may be used to silence genes in budding yeast strains via import of the required protein factors from other species, e.g., Drosha or Dice (Drinnenberg et al 2009). Gene expression may also be silenced in S. cerevisiae via recruitment of native or heterologous silencing factors or repressors, which may be accomplished at arbitrary loci using the D-Cas9 CRISPR system (Qi et al 2013). Protein level can also be reduced by engineering the amino acid sequence of the target protein. A variety of degron sequences may be used to target the protein for rapid degradation, including, but not limited to, ubiquitin fusions and N-end rule residues at the amino terminus. These methods may be implemented in a constitutive or conditional fashion. Methods of Producing a Cannabinoid or a Cannabinoid Derivative or of Expressing and/or Preparing Engineered Variants of the Olivetolic Acid Cyclase (OAC) Polypeptide [0196] The disclosure provides methods for expressing an engineered variant of the olivetolic acid cyclase (OAC) polypeptide of the disclosure. In certain such embodiments, the methods comprise culturing a modified host cell of the disclosure in a culture medium. The disclosure also provides methods for preparing an engineered variant of the olivetolic acid cyclase (OAC) polypeptide of the disclosure. The disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of the disclosure. [0197] The methods may involve culturing a modified host cell of the present disclosure in a culture medium and recovering the produced cannabinoid or cannabinoid derivative. The methods may also involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives using one or more polypeptides disclosed herein, such as an engineered variant of the disclosure, expressed or overexpressed by a modified host cell of the disclosure. The methods may also involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives using an engineered variant disclosed herein. [00107] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabidiol (CBD) type (e.g., cannabidiolic acid), Δ9-trans-tetrahydrocannabinol (Δ9 -THC) type (e.g., Δ9-tetrahydrocannabinolic acid), Δ8-trans-tetrahydrocannabinol (Δ8 -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, derivatives of any of the foregoing, and others as listed in Elsohly M.A. and Slade D., Life Sci.2005 Dec 22;78(5):539-48. Epub 2005 Sep 30. [00108] Cannabinoids or cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, cannabimovone (CBM) , cannabimovonic acid (CBMA), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBDA, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C1), Δ9 –tetrahydrocannabinolic acid A (THCA-A), Δ9 –tetrahydrocannabinolic acid B (THCA-B), Δ9 –tetrahydrocannabinol (Δ9- THC), Δ9 –tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9 –tetrahydrocannabinol-C4 (THC- C4), Δ9 –tetrahydrocannabivarinic acid (THCVA), Δ9 –tetrahydrocannabivarin (THCV), Δ9 – tetrahydrocannabiorcolic acid (THCA-C1), Δ9 –tetrahydrocannabiorcol (THC-C1), Δ7 –cis- iso-tetrahydrocannabivarin, Δ8 –tetrahydrocannabinolic acid (Δ8 –THCA), Δ8 – tetrahydrocannabinol (Δ8 –THC), tetrahydrocannabiphorol (THCP) and the acidic form thereof, cannabidiphorol” (CBDP) and the acidic form thereof, cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA- A), cannabielsoic acid B (CBEA-B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methylether (CBNM), cannabinol-C4, (CBN-C4), cannabivarin (CBV), cannabinol-C2 (CNB-C2), cannabiorcol (CBN-C1), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a-tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a- tetrahydrocannabinol, cannabitriolvarin (CBTV), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanon (CBCN), cannabicitran (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-benzoxocin-5- methanol (OH-iso-HHCV), cannabiripsol (CBR), trihydroxy-delta-9-tetrahydrocannabinol (triOH-THC), CBGA-hydrocinnamic acid (3‐[(2E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl]‐2,4‐ dihydroxy‐6‐(2‐phenylethyl)benzoic acid), CBG-hydrocinnamic acid (2‐[(2E)‐3,7‐ dimethylocta‐2,6‐dien‐1‐yl]‐5‐(2‐phenylethyl)benzene‐1,3‐diol), CBDA-hydrocinnamic acid (2,4‐dihydroxy‐3‐[3‐methyl‐6‐(prop‐1‐en‐2‐yl)cyclohex‐2‐en‐1‐yl]‐6‐(2‐ phenylethyl)benzoic acid), CBD-hydrocinnamic acid (2‐[3‐methyl‐6‐(prop‐1‐en‐2‐ yl)cyclohex‐2‐en‐1‐yl]‐5‐(2‐phenylethyl)benzene‐1,3‐diol), THCA-hydrocinnamic acid (1‐ hydroxy‐6,6,9‐trimethyl‐3‐(2‐phenylethyl)‐6H,6aH,7H,8H,10aH‐benzo[c]isochromene‐2‐ carboxylic acid), THC-hydrocinnamic acid (6,6,9‐trimethyl‐3‐(2‐phenylethyl)- 6H,6aH,7H,8H,10aH‐benzo[c]isochromen‐1‐ol, perrottetinene), and derivatives of any of the foregoing. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0198] In some embodiments, the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is Δ9- tetrahydrocannabinolic acid, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinolic acid, Δ8- tetrahydrocannabinol, cannabidiolic acid, cannabidiol, cannabichromenic acid, cannabichromene, cannabinolic acid, cannabinol, cannabidivarinic acid, cannabidivarin, tetrahydrocannabivarinic acid, tetrahydrocannabivarin, cannabichromevarinic acid, cannabichromevarin, cannabigerovarinic acid, cannabigerovarin, cannabicyclolic acid, cannabicyclol, cannabielsoinic acid, cannabielsoin, cannabicitranic acid, or cannabicitran. In some embodiments, the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium. [0199] In some embodiments, the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin. In some embodiments, the cannabinoid is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid is produced in an amount of more than 50 mg/L culture medium. [0200] In certain such embodiments, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the engineered variant of the disclosure. In certain such embodiments, the engineered variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. In some embodiments of the methods of producing a cannabinoid or a cannabinoid derivative of the disclosure, the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the engineered variant of the disclosure. In certain such embodiments, the engineered variant of the disclosure and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0201] Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, CBDA, CBD, CBGA, CBG, THC, THCA, THCVA, THCV, CBDVA, CBDV, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-butyl-6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-3-butyl-6,6,9-trimethyl-6a,7,8,10a- tetrahydro-6H-benzo[c]chromen-1-ol, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-(3- methylpentyl)-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)- 6,6,9-trimethyl-3-(3-methylpentyl)-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3-(4-pentenyl)-6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-6,6,9-trimethyl-3-(pent-4-en-1-yl)- 6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol, (6aR,10aR)-1-hydroxy-6,6,9-trimethyl-3- hexyl-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-3-hexyl- 6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol, (6aR,10aR)-1-hydroxy- 6,6,9-trimethyl-3-(5-hexynyl)-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-3-(hex-5-yn-1-yl)-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H- benzo[c]chromen-1-ol, and others as listed in Bow, E. W. and Rimoldi, J. M., “The Structure–Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation,” Perspectives in Medicinal Chemistry 2016:817–39 doi: 10.4137/PMC.S32171, incorporated by reference herein. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0202] Additional cannabinoids and cannabinoid derivatives that can be produced with the engineered variants, methods, or modified host cells of the present disclosure may also include, but are not limited to, (1'R,2'R)-4-(hexan-2-yl)-5'-methyl-2'-(prop-1-en-2-yl)- 1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-hexyl-5'-methyl-2'-(prop-1-en-2- yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(3-methylpentyl)-2'- (prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-(4-chlorobutyl)-5'- methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl- 4-(4-methylpentyl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(4-(methylthio)butyl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-((E)-pent-1-en-1-yl)-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-((E)-pent-3-en-1-yl)-2'-(prop-1-en- 2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-((E)-pent-2-en-1- yl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-(but-3-yn-1- yl)-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4- ((E)-but-1-en-1-yl)-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6- diol, (1'R,2'R)-5'-methyl-4-(pent-4-yn-1-yl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-2'-(prop-1-en-2-yl)-4-undecyl-1',2',3',4'-tetrahydro- [1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-(hex-5-yn-1-yl)-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-((E)-hept-1-en-1-yl)-5'-methyl-2'-(prop-1-en- 2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-octyl-2'-(prop-1-en- 2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-((E)-oct-1-en-1-yl)- 2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4- nonyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl- 4-(3-phenylpropyl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(4-phenylbutyl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(5-phenylpentyl)-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(6-phenylhexyl)-2'-(prop-1-en-2- yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(2-methylpentyl)-2'- (prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-isopropyl-5'- methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-4-decyl- 5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'- methyl-2'-(prop-1-en-2-yl)-4-tridecyl-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (E)-3- ((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]- 4-yl)acrylic acid, (Z)-3-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-4-yl)acrylic acid, 7-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)heptanoic acid, 8-((1'R,2'R)-2,6- dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)octanoic acid, 9-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-4-yl)nonanoic acid, 11-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)- 1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)undecanoic acid, (1''R,2''R)-3',5'-dihydroxy-5''- methyl-2''-(prop-1-en-2-yl)-1'',2'',3'',4''-tetrahydro-[1,1':4',1''-terphenyl]-2-carboxylic acid, (1''R,2''R)-3',5'-dihydroxy-5''-methyl-2''-(prop-1-en-2-yl)-1'',2'',3'',4''-tetrahydro-[1,1':4',1''- terphenyl]-3-carboxylic acid, (1''R,2''R)-3',5'-dihydroxy-5''-methyl-2''-(prop-1-en-2-yl)- 1'',2'',3'',4''-tetrahydro-[1,1':4',1''-terphenyl]-4-carboxylic acid, (1''R,2''R)-3',5'-dihydroxy-5''- methyl-2''-(prop-1-en-2-yl)-1'',2'',3'',4''-tetrahydro-[1,1':4',1''-terphenyl]-3,5-dicarboxylic acid, (1'R,2'R)-4-(4-hydroxybutyl)-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-2,6-diol, (1'R,2'R)-4-(4-aminobutyl)-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-2,6-diol, 5-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2- yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)pentanenitrile, (1'R,2'R)-5'-methyl-4-(3- methylhexan-2-yl)-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-2'-(prop-1-en-2-yl)-4-propyl-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6- diol, (1'R,2'R)-4-butyl-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]- 2,6-diol, (1'R,2'R)-5'-methyl-4-pentyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'- biphenyl]-2,6-diol, (1'R,2'R)-4-heptyl-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro- [1,1'-biphenyl]-2,6-diol, (1'R,2'R)-5'-methyl-4-(pent-4-en-1-yl)-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-2,6-diol, 3-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2- yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)propanoic acid, (1'R,2'R)-4,5'-dimethyl-2'- (prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-2,6-diol, 2-((1'R,2'R)-2,6-dihydroxy-5'- methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)acetic acid, 4- ((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]- 4-yl)butanoic acid, (1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'- tetrahydro-[1,1'-biphenyl]-4-carboxylic acid, 5-((1'R,2'R)-2,6-dihydroxy-5'-methyl-2'-(prop- 1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)pentanoic acid, and 6-((1'R,2'R)-2,6- dihydroxy-5'-methyl-2'-(prop-1-en-2-yl)-1',2',3',4'-tetrahydro-[1,1'-biphenyl]-4-yl)hexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0203] A cannabinoid derivative may also refer to a compound lacking one or more chemical moieties found in naturally-occurring cannabinoids, yet retains the core structural features (e.g., cyclic core) of a naturally-occurring cannabinoid. Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, and the like. In some embodiments, a cannabinoid derivative may also comprise one or more of any of the functional and/or reactive groups described herein. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups such as those described in PCT/US2019/053292, the contents of which are herein incorporated by reference in their entirety for all purposes. [0204] A cannabinoid derivative may be a cannabinoid substituted with or comprising one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocyclyl, heterospirocyclyl, heterocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Suitable reactive groups may include, but are not necessarily limited to, azide, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), halide, ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, acetyl, and the like. In some embodiments, the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, a thioester, a thioether, a sulfonyl halide, an alcohol, a thiol, an alkyne, alkene, an azide, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, and a hydrazine. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups. [0205] “Alkyl” may refer to a straight or branched chain saturated hydrocarbon. For example, C1-C6alkyl groups contain 1 to 6 carbon atoms. Examples of a C1-C6alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec- butyl and tert-butyl, isopentyl, and neopentyl. [0206] “Alkenyl” may include an unbranched (i.e., straight) or branched hydrocarbon chain containing 2-12 carbon atoms. The “alkenyl” group contains at least one double bond. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Examples of alkenyl groups may include, but are not limited to, ethylenyl, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2- ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl and the like. [0207] Compounds disclosed herein, such as cannabinoids and cannabinoid derivatives, may be substituted with one or more substituents, such as those illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the present disclosure. In general, the term “substituted” refers to the replacement of a hydrogen atom in a given structure with a specified substituent. Combinations of substituents envisioned by the present disclosure are typically those that result in the formation of stable or chemically feasible compounds. [0208] As used herein, the term “unsubstituted” may mean that the specified group bears no substituents beyond the moiety recited (e.g., where valency satisfied by hydrogen). [0209] A reactive group may facilitate covalent attachment of a molecule of interest. Suitable molecules of interest may include, but are not limited to, a detectable label; imaging agents; a toxin (including cytotoxins); a linker; a peptide; a drug (e.g., small molecule drugs); a member of a specific binding pair; an epitope tag; ligands for binding by a target receptor; tags to aid in purification; molecules that increase solubility; and the like. A linker may be a peptide linker or a non-peptide linker. [0210] In some embodiments, a cannabinoid derivative substituted with an azide may be reacted with a compound comprising an alkyne group via “click chemistry” to generate a product comprising a heterocycle, also known as an azide-alkyne cycloaddition. In some embodiments, a cannabinoid derivative substituted with an alkyne may be reacted with a compound comprising an azide group via click chemistry to generate a product comprising a heterocycle. [0211] Additional molecules of interest that may be desirable for attachment to a cannabinoid derivative may include, but are not necessarily limited to, detectable labels (e.g., spin labels, fluorescence resonance energy transfer (FRET)-type dyes, e.g., for studying structure of biomolecules in vivo); small molecule drugs; cytotoxic molecules (e.g., drugs); imaging agents; ligands for binding by a target receptor; tags to aid in purification by, for example, affinity chromatography (e.g., attachment of a FLAG epitope); molecules that increase solubility (e.g., poly(ethylene glycol)); molecules that enhance bioavailability; molecules that increase in vivo half-life; molecules that target to a particular cell type (e.g., an antibody specific for an epitope on a target cell); molecules that target to a particular tissue; molecules that provide for crossing the blood-brain barrier; and molecules to facilitate selective attachment to a surface, and the like. [0212] In some embodiments, a molecule of interest comprises an imaging agent. Suitable imaging agents may include positive contrast agents and negative contrast agents. Suitable positive contrast agents may include, but are not limited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA); gadolinium- diethylenetriaminepentaacetic acid (Gd-DTPA); gadolinium-1,4,7-tris(carbonylmethyl)-10- (2'-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (Gd-HP-DO3A); Manganese(II)- dipyridoxal diphosphate (Mn-DPDP); Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); and the like. Suitable negative contrast agents may include, but are not limited to, a superparamagnetic iron oxide (SPIO) imaging agent; and a perfluorocarbon, where suitable perfluorocarbons may include, but are not limited to, fluoroheptanes, fluorocycloheptanes, fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes, fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes, fluorodimethylcyclopentanes, fluoromethylcyclobutanes, fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes, fluorocyclobutanes, fluoropropanes, fluoroethers, fluoropolyethers, fluorotriethylamines, perfluorohexanes, perfluoropentanes, perfluorobutanes, perfluoropropanes, sulfur hexafluoride, and the like. [0213] Additional cannabinoid derivatives that can be produced with an engineered variant, method, or modified host cell of the present disclosure may include derivatives that have been modified via organic synthesis or an enzymatic route to modify drug metabolism and pharmacokinetics (e.g., solubility, bioavailability, absorption, distribution, plasma half- life and metabolic clearance). Modification examples may include, but are not limited to, halogenation, acetylation, and methylation. [0214] The cannabinoids or cannabinoid derivatives described herein further include all pharmaceutically acceptable isotopically labeled cannabinoids or cannabinoid derivatives. An “isotopically-” or “radio-labeled” compound is a compound where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). For example, in some embodiments, in the cannabinoids or cannabinoid derivatives described herein, hydrogen atoms are replaced or substituted by one or more deuterium or tritium. Certain isotopically labeled cannabinoids or cannabinoid derivatives of this disclosure, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., 3H, and carbon 14, i.e., 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Suitable isotopes that may be incorporated in cannabinoids or cannabinoid derivatives described herein include but are not limited to 2H (also written as D for deuterium), 3H (also written as T for tritium), 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 18F, 35S, 36Cl , 82Br, 75Br, 76Br, 77Br, 123I, 124I, 125I, and 131I. Substitution with positron emitting isotopes, such as 11C, 18F, 15O, and 13N, can be useful in Positron Emission Topography (PET) studies. [0215] The methods of bioproduction, modified host cells, and engineered variants disclosed herein enable synthesis of cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis. Cannabinoids or cannabinoid derivatives disclosed herein may be enantiomers or disastereomers. The term “enantiomers” may refer to a pair of stereoisomers which are non-superimposable mirror images of one another. In some embodiments the cannabinoids or cannabinoid derivatives may be the (S)-enantiomer. In some embodiments the cannabinoids or cannabinoid derivatives may be the (R)-enantiomer. In some embodiments, the cannabinoids or cannabinoid derivatives may be the (+) or (-) enantiomers. The term “diastereomers” may refer to the set of stereoisomers which cannot be made superimposable by rotation around single bonds. For example, cis- and trans- double bonds, endo- and exo- substitution on bicyclic ring systems, and compounds containing multiple stereogenic centers with different relative configurations may be considered to be diastereomers. The term “diastereomer” may refer to any member of this set of compounds. Cannabinoids or cannabinoid derivatives disclosed herein may include a double bond or a fused ring. In certain such embodiments, the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the cannabinoid or cannabinoid derivative contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined. [0216] In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; from the cell lysate, the modified host cell, and the culture medium; or from a cell- free reaction mixture comprising one or more polypeptides and/or engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0217] The disclosure includes pharmaceutically acceptable salts of the cannabinoids or cannabinoid derivatives described herein. “Pharmaceutically acceptable salts” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable. Representative pharmaceutically acceptable salts include, but are not limited to, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, sethionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p- toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. [0218] “Pharmaceutically acceptable salt” also includes both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” may refer to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo- glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4- aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p- toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. [0219] “Pharmaceutically acceptable base addition salt” may refer to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. For example, inorganic salts include, but are not limited to, ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Methods of Culturing Host Cells to Generate Cannabinoids or Cannabinoid Derivatives [0220] The disclosure provides methods of producing an olivetolic acid, olivetolic acid derivatives,cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced olivetolic acid, olivetolic acid derivative, cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0221] In some embodiments, culturing of the modified host cells of the disclosure in a culture medium provides for synthesis of an olivetolic acid, olivetolic acid derivative, cannabinoid or a cannabinoid derivative, such as those described herein, in an increased amount compared to an unmodified host cell cultured under similar conditions. [0222] The disclosure provides methods of producing a cannabinoid or a cannabinoid derivative, such as those described herein, the method comprising: culturing a modified host cell of the disclosure in a culture medium comprising a carboxylic acid. In certain such embodiments, the method comprises recovering the produced cannabinoid or cannabinoid derivative. In certain such embodiments, the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0223] In some embodiments, the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; or from the cell lysate, the modified host cell, and the culture medium. In certain such embodiments, the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. In some embodiments when the cannabinoid or cannabinoid derivative is recovered from the cell lysate; from the culture medium; from the modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; or from the cell lysate, the modified host cell, and the culture medium, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0224] In some embodiments, the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid. In some embodiments, the carboxylic acid may be substituted with or comprise one or more functional and/or reactive groups. Functional groups may include, but are not limited to, azido, halo (e.g., chloride, bromide, iodide, fluorine), methyl, alkyl, alkynyl, alkenyl, methoxy, alkoxy, acetyl, amino, carboxyl, carbonyl, oxo, ester, hydroxyl, thio (e.g., thiol), cyano, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkylalkynyl, cycloalkenylalkyl, cycloalkenylalkenyl, cycloalkenylalkynyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroarylalkenyl, heteroarylalkynyl, arylalkenyl, arylalkynyl, spirocyclyl, heterospirocyclyl, heterocyclyl, thioalkyl (or alkylthio), arylthio, heteroarylthio, sulfone, sulfonyl, sulfoxide, amido, alkylamino, dialkylamino, arylamino, alkylarylamino, diarylamino, N-oxide, imide, enamine, imine, oxime, hydrazone, nitrile, aralkyl, cycloalkylalkyl, haloalkyl, heterocyclylalkyl, heteroarylalkyl, nitro, thioxo, and the like. Reactive groups may include, but are not necessarily limited to, azide, halogen, carboxyl, carbonyl, amine (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine), ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted aryl ester), cyano, thioester, thioether, sulfonyl halide, alcohol, thiol, succinimidyl ester, isothiocyanate, iodoacetamide, maleimide, hydrazine, alkynyl, alkenyl, and the like. In some embodiments, the reactive group is selected from a carboxyl, a carbonyl, an amine, an ester, thioester, thioether, a sulfonyl halide, an alcohol, a thiol, a succinimidyl ester, an isothiocyanate, an iodoacetamide, a maleimide, an azide, an alkyne, an alkene, and a hydrazine. Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups. [0225] In some embodiments, the carboxylic acid is isotopically- or radio-labeled. In some embodiments, the carboxylic acid may be an enantiomer or disastereomer. In some embodiments the carboxylic acid may be the (S)-enantiomer. In some embodiments, the carboxylic acid may be the (R)-enantiomer. In some embodiments, the carboxylic acid may be the (+) or (-) enantiomer. In some embodiments, the carboxylic acid may include a double bond or a fused ring. In certain such embodiments, the double bond or fused ring may be cis or trans, unless the configuration is specifically defined. If the carboxylic acid contains a double bond, the substituent may be in the E or Z configuration, unless the configuration is specifically defined. [0226] In some embodiments, the carboxylic acid comprises a C=C group. In some embodiments, the carboxylic acid comprises an alkyne group. In some embodiments, the carboxylic acid comprises an N3 group. In some embodiments, the carboxylic acid comprises a halogen. In some embodiments, the carboxylic acid comprises a CN group. In some embodiments, the carboxylic acid comprises iodo. In some embodiments, the carboxylic acid comprises bromo. In some embodiments, the carboxylic acid comprises chloro. In some embodiments, the carboxylic acid comprises fluoro. In some embodiments, the carboxylic acid comprises a carbonyl. In some embodiments, the carboxylic acid comprises an acetyl. In some embodiments, the carboxylic acid comprises an alkyl group. In some embodiments, the carboxylic acid comprises an aryl group. [0227] Carboxylic acids may include, but are not limited to, unsubstituted or substituted C3-C18 fatty acids, C3-C18 carboxylic acids, C1-C18 carboxylic acids, butyric acid, isobutyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, myristic acid, C15-C18 fatty acids, C15-C18 carboxylic acids, fumaric acid, itaconic acid, malic acid, succinic acid, maleic acid, malonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, glutaconic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, citric acid, isocitric acid, aconitic acid, tricarballylic acid, and trimesic acid. Carboxylic acids may include unsubstituted or substitutedC1-C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C3-C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C3-C12 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C4-C10 carboxylic acids. In some embodiments, the carboxylic acid is an unsubstituted or substituted C4 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C5 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C6 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C7 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C8 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C9 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted C10 carboxylic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted butyric acid. In some embodiments, carboxylic acid is unsubstituted or substituted valeric acid. In some embodiments, the carboxylic acid is unsubstituted or substituted hexanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted heptanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted octanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted nonanoic acid. In some embodiments, the carboxylic acid is unsubstituted or substituted decanoic acid. [0228] Carboxylic acids may include, but are not limited to, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3- hexenoic acid, 4-hexenoic acid, 5-hexenoic acid, 5-chlorovaleric acid, 5-aminovaleric acid, 5-cyanovaleric acid, 5-(methylsulfanyl)valeric acid, 5-hydroxyvaleric acid, 5-phenylvaleric acid, 2,3-dimethylhexanoic acid, d3-hexanoic acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, trans-2- nonenoic acid, 4-phenylbutyric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, and the like. In some embodiments, the carboxylic acid is 2-methylhexanoic acid. In some embodiments, the carboxylic acid is 3-methylhexanoic acid. In some embodiments, the carboxylic acid is 4-methylhexanoic acid. In some embodiments, the carboxylic acid is 5- methylhexanoic acid. In some embodiments, the carboxylic acid is 2-hexenoic acid. In some embodiments, the carboxylic acid is 3-hexenoic acid. In some embodiments, the carboxylic acid is 4-hexenoic acid. In some embodiments, the carboxylic acid is 5-hexenoic acid. In some embodiments, the carboxylic acid is 5-chlorovaleric acid. In some embodiments, the carboxylic acid is 5-aminovaleric acid. In some embodiments, the carboxylic acid is 5-cyanovaleric acid. In some embodiments, the carboxylic acid is 5- (methylsulfanyl)valeric acid. In some embodiments, the carboxylic acid is 5-hydroxyvaleric acid. In some embodiments, the carboxylic acid is 5-phenylvaleric acid. In some embodiments, the carboxylic acid is 2,3-dimethylhexanoic acid. In some embodiments, the carboxylic acid is d3-hexanoic acid. In some embodiments, the carboxylic acid is 4- pentynoic acid. In some embodiments, the carboxylic acid is trans-2-pentenoic acid. In some embodiments, the carboxylic acid is 5-hexynoic acid. In some embodiments, the carboxylic acid is trans-2-hexenoic acid. In some embodiments, the carboxylic acid is 6-heptynoic acid. In some embodiments, the carboxylic acid is trans-2-octenoic acid. In some embodiments, the carboxylic acid is trans-2-nonenoic acid. In some embodiments, the carboxylic acid is 4- phenylbutyric acid. In some embodiments, the carboxylic acid is 6-phenylhexanoic acid. In some embodiments, the carboxylic acid is 7-phenylheptanoic acid. [0229] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid. In certain such embodiments, the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. [0230] In some embodiments wherein the modified host cell of the present disclosure is cultured in a culture medium comprising a carboxylic acid, the carboxylic acid is butyric acid, valeric acid, hexanoic acid, octanoic acid, 2-methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4- hexenoic acid, 5-hexenoic acid, heptanoic acid, 5-chlorovaleric acid, 5- (methylsulfanyl)valeric acid, 4-pentynoic acid, trans-2-pentenoic acid, 5-hexynoic acid, trans-2-hexenoic acid, 6-heptynoic acid, trans-2-octenoic acid, nonanoic acid, trans-2- nonenoic acid, decanoic acid, undecanoic acid, dodecanoic acid, myristic acid, 4- phenylbutyric acid, 5-phenylvaleric acid, 6-phenylhexanoic acid, 7-phenylheptanoic acid, isobutyric acid, fumaric acid, itaconic acid, malic acid, succinic acid, maleic acid, malonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecandioic acid, glutaconic acid, ortho-phthalic acid, isophthalic acid, terephthalic acid, citric acid, isocitric acid, aconitic acid, tricarballylic acid, trimesic acid, 5- aminovaleric acid, 5-cyanovaleric acid, 5-hydroxyvaleric acid, or 2,3-dimethylhexanoic acid. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium. In some embodiments, the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium. Exemplary Cell Culture Conditions [0231] Suitable media for culturing modified host cells of the disclosure may include standard culture media (e.g., Luria-Bertani broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where nucleic acids disclosed herein are under the control of an inducible promoter, etc.); standard yeast culture media; and the like). In some embodiments, the culture medium can be supplemented with a fermentable sugar (e.g., a hexose sugar or a pentose sugar, e.g., glucose, xylose, galactose, and the like). Sugars fermentable by yeast may include, but are not limited to, sucrose, dextrose, glucose, fructose, mannose, galactose, and maltose. [0232] In some embodiments, the culture medium can be supplemented with unsubstituted or substituted hexanoic acid, carboxylic acids other than unsubstituted or substituted hexanoic acid, olivetolic acid, or olivetolic acid derivatives. In some embodiments, the culture medium can be supplemented with pretreated cellulosic feedstock (e.g., wheat grass, wheat straw, barley straw, sorghum, rice grass, sugarcane straw, bagasse, switchgrass, corn stover, corn fiber, grains, or any combination thereof). In some embodiments, the culture medium can be supplemented with oleic acid. In some embodiments, the culture medium comprises a non-fermentable carbon source. In certain such embodiments, the non-fermentable carbon source comprises ethanol. In some embodiments, the suitable media comprises an inducer. In certain such embodiments, the inducer comprises galactose. In some embodiments, the inducer comprises KH2PO4, galactose, glucose, sucrose, maltose, an amino acid (e.g., methionine, lysine), CuSO4, a change in temperature (e.g., 30 °C to 37 °C), a change in pH (e.g., pH 6 to pH 4), a change in oxygen level (e.g., 20% to 1% dissolved oxygen levels), addition of hydrogen peroxide or superoxide-generating drug menadione, tunicamycin, expression of proteins prone to misfolding (e.g., cannabinoid synthases), estradiol, or doxycycline. Additional induction systems are detailed herein. [0233] The carbon source in the suitable media can vary significantly, from simple sugars like glucose to more complex hydrolysates of other biomass, such as yeast extract. The addition of salts generally provides essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize polypeptides and nucleic acids. The suitable media can also be supplemented with selective agents, such as antibiotics, to select for the maintenance of certain plasmids and the like. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent cells lacking the resistance from growing. The suitable media can be supplemented with other compounds as necessary to select for desired physiological or biochemical characteristics, such as particular amino acids and the like. [0234] In some embodiments, modified host cells disclosed herein are grown in minimal medium or minimal media. As used herein, the terms “minimal medium” or “minimal media” may refer to media comprising a defined composition of nutrients, generally chosen for minimal cost, while still allowing for robust growth and production. As used herein, the terms “minimal medium” or “minimal media” may refer to media containing: (1) one or more carbon sources for cellular (e.g., bacterial or yeast) growth; (2) various salts, which can vary among cellular (e.g., bacterial or yeast) species and growing conditions; (3) vitamins and trace elements; and (4) water. Generally, but not always, minimal media lacks one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids). Minimal media may also comprise growth factors, inducers, and repressors. In some embodiments, minimal media or minimal medium affords higher biomass formation in a fermentation tank compared to rich medium or rich media. In some embodiments, the minimal medium or minimal media comprises a carboxylic acid (e.g., 1 mM olivetolic acid, 1 mM olivetolic acid derivative, 2 mM unsubstituted or substituted hexanoic acid, or 2 mM of a carboxylic acid other than unsubstituted or substituted hexanoic acid). [0235] In some embodiments, modified host cells disclosed herein are grown in rich medium or rich media. In certain such embodiments, the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, yeast extract, Bacto peptone, and dextrose (glucose). In certain such embodiments, the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, 10 g/L yeast extract, 20 g/L Bacto peptone, and 20 g/L dextrose (glucose). In some embodiments, the rich medium or rich media comprises YP + galactose and glucose. In some embodiments, the rich medium or rich media comprises YP + 20 g/L galactose or YP + 40 g/L galactose and 1 g/L glucose. In some embodiments, the rich medium or rich media comprises a carboxylic acid (e.g., 1 mM olivetolic acid, 1 mM olivetolic acid derivative, 2 mM unsubstituted or substituted hexanoic acid, or 2 mM of a carboxylic acid other than unsubstituted or substituted hexanoic acid). In some embodiments, rich medium or rich media affords greater cell density in fermentation compared to minimal media or minimal medium. [0236] Materials and methods suitable for the maintenance and growth of the recombinant cells of the disclosure are described herein, e.g., in the Examples section. Other materials and methods suitable for the maintenance and growth of cell (e.g., bacterial or yeast) cultures are well known in the art. Exemplary techniques can be found in International Publication No. WO2009/076676, U.S. Patent Application No.12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No.2010/0048964, WO 2009/132220, US Publ. No.2010/0003716, Manual of Methods for General Bacteriology Gerhardt et al, eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA. [0237] Standard cell culture conditions can be used to culture the modified host cells disclosed herein (see, for example, WO 2004/033646 and references cited therein). In some embodiments, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 °C to about 37 °C, at about 0.04% to about 84% CO2, at about 0% to about 100% dissolved oxygen, and at a pH between about 2.0 to about 9.0). In some embodiments, modified host cells disclosed herein are grown at about 34 °C in a suitable cell culture medium. In some embodiments, modified host cells disclosed herein are grown at about 20 °C to about 37 °C in a suitable cell culture medium. While the growth optimum for S. cerevisiae is about 30 °C, culturing cells at a higher temperature, e.g., 34 °C may be advantageous by reducing the costs to cool industrial fermentation tanks. In some embodiments, modified host cells disclosed herein are grown at about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, or about 37 °C in a suitable cell culture medium. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 9.0 (such as about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 6.0 to about pH 8.0 or about pH 6.5 to about pH 7.0). In some embodiments, the pH ranges for fermentation are between about pH 4.5 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 4.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.0. In some embodiments, the dissolved oxygen is between about 0% to about 10%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% to about 80%, about 0% to about 90%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40% or about 10% to about 50%. In some embodiments, the CO2 level is between about 0.04% to about 0.1% CO2, about 0.04% to about 1% CO2, about 0.04% to about 5% CO2, about 0.04% to about 10% CO2, about 0.04% to about 20% CO2, about 0.04% to about 30% CO2, about 0.04% to about 40% CO2, about 0.04% to about 50% CO2, about 0.04% to about 60% CO2, about 0.04% to about 70% CO2, about 0.1% to about 5% CO2, about 0.1% to about 10% CO2, about 0.1% to about 20% CO2, about 0.1% to about 30% CO2, about 0.1% to about 40% CO2, about 0.1% to about 50% CO2, about 1% to about 5% CO2, about 1% to about 10% CO2, about 1% to about 20% CO2, about 1% to about 30% CO2, about 1% to about 40% CO2, about 1% to about 50% CO2, about 5% to about 10% CO2, about 10% to about 20% CO2, about 10% to about 30% CO2, about 10% to about 40% CO2, about 10% to about 50% CO2, about 10% to about 60% CO2, about 10% to about 70% CO2, about 10% to about 80% CO2, about 50% to about 60% CO2, about 50% to about 70% CO2, or about 50% to about 80% CO2. Modified host cells disclosed herein can be grown under aerobic, anoxic, microaerobic, or anaerobic conditions based on the requirements of the cells. [0238] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in International Publication No. WO 2009/076676, U.S. Patent Application No.12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No.2010/0048964, WO 2009/132220, US Publ. No.2010/0003716, the contents of each of which are incorporated by reference herein in their entireties. Batch and Fed-Batch fermentations are common and well known in the art and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc. Production and Recovery of Produced Cannabinoids or Cannabinoid Derivatives [0239] In some embodiments, the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor, such as those disclosed herein, by modified host cells of the disclosure in an amount of from about 1 mg/L culture medium to about 1 g/L culture medium. In some embodiments, the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor, such as those disclosed herein, by modified host cells of the disclosure in an amount of from about 1 ng/L to about 50 ng/L, from about 50 ng/L to about 100 ng/L, from about 100 ng/L to about 500 ng/L, about from 500 ng/L to about 1 μg/L, from about 1 μg/L to about 50 μg/L, from about 50 μg/L to about 100 μg/L, from about 100 μg/L to about 500 μg/L, from about 500 μg/L to about 1 mg/L, from about 1 mg/L to about 50 mg/L, from about 50 mg/L to about 100 mg/L, from about 100 mg/L to about 500 mg/L, or from about 500 mg/L to about 1 g/L, of from about 1 mg/L culture medium to about 1 g/L culture medium, from about 1 mg/L culture medium to about 500 mg/L culture medium, from about 1 mg/L culture medium to about 100 mg/L culture medium. For example, in some embodiments, the present disclosure provides for production of a cannabinoid or a cannabinoid derivative or precursor in an amount of from about 1 mg/L culture medium to about 5 mg/L culture medium, from about 5 mg/L culture medium to about 10 mg/L culture medium, from about 10 mg/L culture medium to about 25 mg/L culture medium, from about 25 mg/L culture medium to about 50 mg/L culture medium, from about 50 mg/L culture medium to about 75 mg/L culture medium, or from about 75 mg/L culture medium to about 100 mg/L culture medium, from about 100 mg/L culture medium to about 150 mg/L culture medium, from about 150 mg/L culture medium to about 200 mg/L culture medium, from about 200 mg/L culture medium to about 250 mg/L culture medium, from about 250 mg/L culture medium to about 500 mg/L culture medium, from about 500 mg/L culture medium to about 750 mg/L culture medium, or from about 750 mg/L culture medium to about 1 g/L culture medium. [0240] In some embodiments, the modified host cell disclosed herein is cultured in a liquid medium comprising a carboxylic acid, olivetolic acid, or an olivetolic acid derivative. [0241] In some embodiments, a method of producing a cannabinoid or a cannabinoid derivative, such as those disclosed herein, may involve culturing a modified yeast cell of the present disclosure under conditions that favor fermentation of a sugar, and under conditions that favor production of a cannabinoid or a cannabinoid derivative; wherein the cannabinoid or the cannabinoid derivative is produced by the modified yeast cell and is present in alcohol produced by the modified yeast cell. The present disclosure provides an alcoholic beverage produced by the modified yeast cell, where the alcoholic beverage comprises the cannabinoid or cannabinoid derivative produced by the modified yeast cell. Alcoholic beverages may include beer, wine, and distilled alcoholic beverages. In some embodiments, an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 μg/L, from 1 μg/L to 50 μg/L, from 50 μg/L to 100 μg/L, from 100 μg/L to 500 μg/L, from 500 μg/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L). In some embodiments, an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L. [0242] The present disclosure provides a beverage produced by the modified yeast cell, where the beverage comprises the cannabinoid or cannabinoid derivative, such as those disclosed herein, produced by the modified yeast cell. In some embodiments, a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount of from 1 ng/L to 1 g/L (e.g., from 1 ng/L to 50 ng/L, from 50 ng/L to 100 ng/L, from 100 ng/L to 500 ng/L, from 500 ng/L to 1 μg/L, from 1 μg/L to 50 μg/L, from 50 μg/L to 100 μg/L, from 100 μg/L to 500 μg/L, from 500 μg/L to 1 mg/L, from 1 mg/L to 50 mg/L, from 50 mg/L to 100 mg/L, from 100 mg/L to 500 mg/L, or from 500 mg/L to 1 g/L). In some embodiments, a beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L. In some embodiments, a beverage of the present disclosure is non-alcoholic. [0243] In some embodiments, a method of the present disclosure provides for increased production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein. In certain such embodiments, culturing of the modified host cell disclosed herein in a culture medium provides for synthesis of a cannabinoid or a cannabinoid derivative in an increased amount compared to an unmodified host cell cultured under similar conditions. The production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 5% to about 1,000,000 folds compared to an unmodified host cell cultured under similar conditions. The production of a cannabinoid or a cannabinoid derivative by the modified host cells disclosed herein may be increased by about 10% to about 1,000,000 folds (e.g., about 50% to about 1,000,000 folds, about 1 to about 500,000 folds, about 1 to about 50,000 folds, about 1 to about 5,000 folds, about 1 to about 1,000 folds, about 1 to about 500 folds, about 1 to about 100 folds, about 1 to about 50 folds, about 5 to about 100,000 folds, about 5 to about 10,000 folds, about 5 to about 1,000 folds, about 5 to about 500 folds, about 5 to about 100 folds, about 10 to about 50,000 folds, about 50 to about 10,000 folds, about 100 to about 5,000 folds, about 200 to about 1,000 folds, about 50 to about 500 folds, or about 50 to about 200 folds) compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. The production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10,000 folds, 20,000 folds, 50,000 folds, 100,000 folds, 200,000 folds, 500,000 folds, or 1,000,000 folds or more compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. [0244] In some embodiments, the production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, by modified host cells of the disclosure may also be increased by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. In some embodiments, the production of a cannabinoid or a cannabinoid derivative by modified host cells disclosed herein may also be increased by at least about any of 1-20%, 2-20%, 5-20%, 10-20%, 15- 20%, 1-15%, 1-10%, 2-15%, 2-10%, 5-15%, 10-15%, 1-50%, 10-50%, 20-50%, 30-50%, 40-50%, 50-100%, 50-60%, 50-70%, 50-80%, 50-90%, or 50-100% compared to the production of a cannabinoid or a cannabinoid derivative by unmodified host cells cultured under similar conditions. [0245] In some embodiments, production of a cannabinoid or a cannabinoid derivative by modified host cells of the disclosure is determined by LC-MS analysis. In certain such embodiments, each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition. [0246] In some embodiments, the modified host cell of the disclosure is a yeast cell. In certain such embodiments, the modified host cell disclosed herein is cultured in a bioreactor. In some embodiments, the modified host cell is cultured in a culture medium supplemented with unsubstituted or substituted hexanoic acid, a carboxylic acid other than unsubstituted or substituted hexanoic acid, olivetolic acid, or an olivetolic acid derivative. In some embodiments, the modified yeast cell is a modified S. cerevisiae. [0247] In some embodiments, the cannabinoid or cannabinoid derivative, such as those disclosed herein, is recovered from a cell lysate, e.g., by lysing the modified host cell disclosed herein and recovering the cannabinoid or cannabinoid derivative derivative from the lysate. In other cases, the cannabinoid or cannabinoid derivative is recovered from the culture medium in which the modified host cell disclosed herein is cultured. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the cell lysate and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from a modified host cell. In other cases, the cannabinoid or cannabinoid derivative is recovered from both the modified host cell and the culture medium. In other cases, the cannabinoid or cannabinoid derivative is recovered from the cell lysate, the modified host cell, and the culture medium. In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell lysate; from a culture medium; from a modified host cell; from both the cell lysate and the culture medium; from both the modified host cell and the culture medium; or from the cell lysate, the modified host cell, and the culture medium, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0248] In some embodiments, the recovered cannabinoid or cannabinoid derivative, such as those disclosed herein, is then purified. In some embodiments, whole-cell broth from cultures comprising modified host cells of the disclosure may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate. In some embodiments, the suitable organic solvent comprises hexane. In some embodiments, the suitable organic solvent may be added to the whole-cell broth from fermentations comprising modified host cells of the disclosure at a 10:1 ratio (10 parts whole-cell broth – 1 part organic solvent) and stirred for 30 minutes. In certain such embodiments, the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals. In certain such embodiments, the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material. In certain such embodiments, the solvent may then be removed e.g., by rotary evaporation, to produce pure crystalline product. [0249] In some embodiments, the cannabinoid or cannabinoid derivative is pure, e.g., at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98%, or more than 98% pure, where “pure” in the context of a cannabinoid or a cannabinoid derivative may refer to a cannabinoid or a cannabinoid derivative that is free from other cannabinoids or cannabinoid derivatives, macromolecules, contaminants, etc. Methods of Preparing Engineered Variants of an Olivetolic Acid Cyclase (OAC) Polypeptide [0250] In certain such embodiments, the methods may comprise culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the modified host cell of the disclosure is a Pichia sp. The method can comprise isolating and/or purifying the expressed engineered variants, as described herein. [0251] In some embodiments, the method for preparing engineered variants comprises the step of isolating or purifying the engineered variants. The engineered variants of the disclosure can be expressed in modified host cells, as described herein, and isolated from the modified host cells and/or culture medium using any one or more of the well known techniques used for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Chromatographic techniques for isolation of the engineered variants of the disclosure may include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. In some embodiments, affinity chromatography is used. [0252] In some embodiments, the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in various forms including but not limited to crude extracts (e.g., cell-free lysates), powders (e.g., shake-flask powders), lyophilizates, frozen stocks made with glycerol or another cryoprotectant, and substantially pure preparations (e.g., DSP powders). [0253] In some embodiments, the engineered variants of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in purified form. Generally, conditions for purifying a particular engineered variant will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. Cell-Free Methods of Producing Cannabinoids or Cannabinoid Derivatives [0254] The methods of the disclosure may involve cell-free production of olivetolic acid, olivetolic acid derivatives, cannabinoids or cannabinoid derivatives, such as those disclosed herein, using engineered variants disclosed herein expressed or overexpressed by a modified host cell of the disclosure. In some embodiments, an engineered variant disclosed herein is used in a cell-free system for the production of cannabinoids or cannabinoid derivatives. In certain such embodiments, the engineered variant of the disclosure is isolated and/or purified. In some embodiments, appropriate starting materials for use in producing cannabinoids or cannabinoid derivatives may be mixed together with engineered variants disclosed herein in a suitable reaction vessel to effect the reaction. The engineered variants disclosed herein may be used in combination to effect a complete synthesis of a cannabinoid or cannabinoid derivative from the appropriate starting materials. In some embodiments, the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising engineered disclosed herein. [0255] In some embodiments, the recovered cannabinoids or cannabinoid derivatives, such as those disclosed herein, are then purified. In certain such embodiments, a cell-free reaction mixture comprising an engineered variant disclosed herein may be extracted with a suitable organic solvent to afford cannabinoids or cannabinoid derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate. In some embodiments, the suitable organic solvent comprises hexane. In some embodiments, the suitable organic solvent may be added to the cell-free reaction mixture comprising one or more of the polypeptides disclosed herein at a 10:1 ratio (10 parts reaction mixture – 1 part organic solvent) and stirred for 30 minutes. In certain such embodiments, the organic fraction may be separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer may then be separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid or cannabinoid derivative crystals. In certain such embodiments, the crude crystals may be heated or exposed to light to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystals may be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate the crude cannabinoid or cannabinoid derivative. In certain such embodiments, the crude crystalline product may be re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered to remove any insoluble material. In certain such embodiments, the solvent may then be removed e.g., by rotary evaporation, to produce pure crystalline product. [0256] In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising one or more engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. [0257] In some embodiments, cell-free production of a cannabinoid or a cannabinoid derivative by engineered variants disclosed herein is determined by LC-MS analysis. In certain such embodiments, each cannabinoid or cannabinoid derivative is identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition. [0258] In some embodiments when the cannabinoid or cannabinoid derivative is recovered from a cell-free reaction mixture comprising one or more polypeptides and/or engineered variants disclosed herein, the recovered cannabinoid or cannabinoid derivative is in the form of a salt. In certain such embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt of the recovered cannabinoid or cannabinoid derivative is then purified as disclosed herein. EXAMPLES OF NON-LIMITING EMBODIMENTS OF THE DISCLOSURE [0259] Embodiments of the present subject matter disclosed herein may be beneficial alone or in combination with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure, numbered I-1 to III-38 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below. [0260] Embodiments of the Disclosure: [0261] Embodiment I-1. A variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitutions. [0262] Embodiments I-2. The variant of Embodiment I-1, wherein the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1. [0263] Embodiment I-3. The variant of Embodiment I-1 or I-2, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least one amino acid substitutions, wherein the at least one of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. [0264] Embodiment II-1. A variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions. [0265] Embodiment II-2. The variant of any one of Embodiments I-1 to Embodiment II- 1, wherein the variant comprises an amino acid sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% sequence identity, but less than 95% sequence identity, to SEQ ID NO:1. [0266] Embodiment II-3. The variant of any one of Embodiments I-1 to Embodiment II- 2, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. [0267] Embodiment II-4. The variant of any one of Embodiments I-1 to Embodiment II- 3, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E53, T56, I58, T62, E64, E67, Q70, D71, V84, S87, I94, and R100. [0268] Embodiment II-5. The variant of any one of Embodiments I-1 to Embodiment II- 4, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, E64, and I94. [0269] Embodiment II-6. The variant of any one of Embodiments I-1 to Embodiment II- 5, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56. [0270] Embodiment II-7. The variant of any one of Embodiments I-1 to Embodiment II- 6, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8 and L9. [0271] Embodiment II-8. The variant of any one of Embodiments I-1 to Embodiment II- 7, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is at amino acid V8. [0272] Embodiment II-9. The variant of any one of Embodiments I-1 to Embodiment II- 8, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are at amino acids V8 and L9. [0273] Embodiment II-10. The variant of any one of Embodiments I-1 to Embodiment II- 9, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are at amino acids V8, L9, and T56. [0274] Embodiment II-11. The variant of any one of Embodiments I-1 to Embodiment II- 10, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six or seven of the amino acid substitutions are at amino acids selected from the group consisting of: A L V V L V A V A V V V V V V V V L A V V A L L
Figure imgf000099_0001
V A V V L V
Figure imgf000100_0001
[0275] Embodiment II-12. The variant of any one of Embodiments I-1 to Embodiment II- 11, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids A2, V8, L9, E64, I94, and R100. [0276] Embodiment II-13. The variant of any one of Embodiments I-1 to Embodiment II- 12, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K10, N29, T56, I58, and I94. [0277] Embodiment II-14. The variant of any one of Embodiments I-1 to Embodiment II- 13, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids L9, A36, Q48, I58, E64, and I94. [0278] Embodiment II-15. The variant of any one of Embodiments I-1 to Embodiment II- 14, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K49, T56, I58, and R100. [0279] Embodiment II-16. The variant of any one of Embodiments I-1 to Embodiment II- 15, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising amino acid substitutions at least at amino acids V8, L9, K12, K49, T56, and D71. [0280] Embodiment II-17. The variant of of any one of Embodiments I-1 to Embodiment II-16, wherein the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions occurs in the beta 1 chain, the beta 2 chain, and/or the alpha 2 chain. [0281] Embodiment II-18. The variant of any one of Embodiments I-1 to Embodiment II- 17, wherein the variant comprises an amino acid sequence of SEQ ID NO: 1 comprising at least six amino acid substitutions, wherein at least one of the six amino acid substitutions is at amino acids K12, A36, and D71. [0282] Embodiment II-19. The variant of any one of Embodiments I-1 to Embodiment II-18, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71A, D71E, D71K, D71N, D71Q, D71S, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. [0283] Embodiment II-20. The variant of any one of Embodiments I-1 to Embodiment II- 19, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, F23I, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. [0284] Embodiment II-21. The variant of any one of Embodiments I-1 to Embodiments II-20, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9I, K10A, K12N, K12Q, K12V, E22L, K25N, N29D, V31M, A36E, A36F, A36P, A36Q, A36S, Y41E, T47R, Q48C, Q48H, Q48M, K49R, N50Y, E53V, T56S, I58C, I58V, T62C, E64D, E67S, Q70A, Q70K, D71T, V84D, S87P, I94K, and R100G. [0285] Embodiment II-22. The variant of any one of Embodiments I-1 to Embodiments II-21, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, K49R, T56S, E64D, and I94K. [0286] Embodiment II-23. The variant of any one of Embodiments I-1 to Embodiments II-22, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I, L9I, and T56S. [0287] Embodiment II-24. The variant of any one of Embodiments I-1 to Embodiments II-23, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is selected from the group consisting of V8I and L9I. [0288] Embodiment II-25. The variant of any one of Embodiments I-1 to Embodiments II-24, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein one of the amino acid substitutions is V8I. [0289] Embodiment II-26. The variant of any one of Embodiments I-1 to Embodiments II-22, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein two of the amino acid substitutions are V8I and L9I. [0290] Embodiment II-27. The variant of any one of Embodiments I-1 to Embodiments II-26, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein three of the amino acid substitutions are V8I, L9I, and T56S. [0291] Embodiment II-28. The variant of any one of Embodiments I-1 to Embodiments II-7, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions, wherein six or seven of the amino acid substitutions are selected from the group consisting of:
Figure imgf000102_0001
Figure imgf000103_0001
[0292] Embodiment II-29. The variant of any one of Embodiments I-1 to Embodiments II-289, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions A2P, V8I, L9I, E64D, I94K, and R100G. [0293] Embodiment II-30. The variant of any one of Embodiments I-1 to Embodiments II-31, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K10A, N29D, T56S, I58V, and I94K. [0294] Embodiment II-31. The variant of any one of Embodiments I-1 to Embodiments II-30, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions L9I, A36Q, Q48C, I58V, E64D, and I94K. [0295] Embodiment II-32. The variant of any one of Embodiments I-1 to Embodiments II-31, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K49R, T56S, I58V, and R100G. [0296] Embodiment II-33. The variant of any one of Embodiments I-1 to Embodiments II-32, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least amino acid substitutions V8I, L9I, K12N, K49R, T56S, and D71T. [0297] Embodiment II-34. The variant of any one of Embodiments I-1 to Embodiments II-33, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions. [0298] Embodiment II-35. The variant of any one of Embodiments I-1 to Embodiments II-34, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. [0299] Embodiment II-36. The variant of any one of Embodiments I-1 to Embodiments II-35, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising six amino acid substitutions. [0300] Embodiment II-37. The variant of any one of Embodiments I-1 to Embodiments II-36, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising seven amino acid substitutions. [0301] Embodiment II-38. The variant of any one of Embodiments I-1 to Embodiments II-37, wherein the variant comprises an amino acid sequence selected from an amino acid sequence of any one of SEQ ID NOs: 3 to 93. [0302] Embodiment II-39. The variant of any one of Embodiments I-1 to Embodiments II-38, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a greater amount, as measured in mg/L or mM, than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0303] Embodiment II-40. The variant of any one of Embodiments I-1 to Embodiments II-39, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0304] Embodiment II-41. The variant of any one of Embodiments I-1 to Embodiments II- 40, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100%, greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0305] Embodiment II-42. The variant of any one of Embodiments I-1 to Embodiments II-41, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0306] Embodiment II-43. The variant of any one of Embodiments I-1 to Embodiments II-42, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0307] Embodiment II-44. A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to Embodiments II-43. [0308] Embodiment II-45. A nucleic acid comprising a nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 94-184. [0309] Embodiment II-46. The nucleic acid of Embodiment II-44 or Embodiment II-45, wherein the nucleotide sequence is codon-optimized. [0310] Embodiment II-47. A method of making a modified host cell, the method comprising introducing one or more nucleic acids of any one of Embodiments II-44 to II- 46 into a host cell. [0311] Embodiment II-48. A vector comprising one or more nucleic acids of any one of Embodiments II-44 to II-46. [0312] Embodiment II-49. A method of making a modified host cell, the method comprising introducing one or more vectors of Embodiment II-48 into a host cell. [0313] Embodiment II-50. A modified host cell comprising one or more nucleic acids of any one of Embodiments II-44 to II-46. [0314] Embodiment II-51. The modified host cell of Embodiment II-50, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative. [0315] Embodiment II-52. The modified host cell of Embodiment II-50 or II-51, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative. [0316] Embodiment II-53. The modified host cell of any of Embodiments II-50 to II-52, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0317] Embodiment II-54. The modified host cell of any one of Embodiments II-50 to II- 53, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0318] Embodiment II-55. The modified host cell of any of Embodiments II-50 to II-54, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0319] Embodiment II-56. The modified host cell of any of Embodiments II-50 to II-55, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0320] Embodiment II-57. The modified host cell of any one of Embodiments II-50 to II- 56, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0321] Embodiment II-58. The modified host cell of any one of Embodiments II-50 to II-57, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0322] Embodiment II-59. The modified host cell of any one of Embodiments II-50 to II- 58, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0323] Embodiment II-60. The modified host cell of any one of Embodiments II-50 to II- 59, wherein the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0324] Embodiment II-61. The modified host cell of any one of Embodiments II-50 to II- 60, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, grown under similar culture conditions for the same length of time. [0325] Embodiment II-62. The modified host cell of any one of Embodiments II-50 to II- 61, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl- CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0326] Embodiment II-63. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of any one of Embodiments II-50 to II-62 in a culture medium. [0327] Embodiment II-64. The method of Embodiment II-63, wherein the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative. [0328] Embodiment II-65. The method of Embodiment II-63 or II-64, wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. [0329] Embodiment II-66. A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: a) culturing a modified host cell of any one of Embodiments II-50 to II-62 in a culture medium. [0330] Embodiment II-67. The method of Embodiment II-66, wherein the method comprises: b) recovering the produced olivetolic acid or an olivetolic acid derivative. [0331] Embodiment II-68. The method of any one of Embodiments II-63 to II-67, wherein the culture medium comprises a carboxylic acid. [0332] Embodiment II-69. The method of Embodiment II-68, wherein the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid. [0333] Embodiment II-70. The method of Embodiment II-68 or II-69, wherein the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. [0334] Embodiment II-71. The method of any one of Embodiments II-63 to II-70, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0335] Embodiment II-72. The method of any one of Embodiments II-63 to II-71, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0336] Embodiment II-73 The method of any of Embodiments II-63 to II-72, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0337] Embodiment II-745. The method of any one of Embodiments II-63 to II-73, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0338] Embodiment II-75. The method of any one of Embodiments II-63 to II-74, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0339] Embodiment II-76. The method of any one of Embodiments II-63 to II-75, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0340] Embodiment II-77. The method of any one of Embodiments II-63 to II-76, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the modified host cell of any one of Embodiments II-50 to II-62, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, and wherein the modified host cell of any one of Embodiments II-50 to II-62 and the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1, but lacking a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments I-1 to II-43, are cultured under similar culture conditions for the same length of time. [0341] Embodiment II-78. The method of any one of Embodiments II-63 to II-77, wherein the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0342] Embodiment II-79. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43. [0343] Embodiment II-80. The method of Embodiment II-79, wherein the method comprises recovering the produced cannabinoid or cannabinoid derivative. [0344] Embodiment II-81. The method of Embodiment II- 79 or II-80, wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. [0345] Embodiment II-82. A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising use of a variant of any one of Embodiments I-1 to II- 43. [0346] Embodiment II-83. The method of Embodiment II-82, wherein the method comprises recovering the produced olivetolic acid or olivetolic acid derivative. [0347] Embodiment II-84. The method of Embodiment II-82 or II-83 wherein the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3- butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3-methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)- resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-resorcylic acid, 3-octyl-resorcylic acid, 3-(trans-1-octenyl)- resorcylic acid, 3-nonyl-resorcylic acid, 3-(3-phenylpropyl)-resorcylic acid, 3-(4- phenylbutyl)-resorcylic acid, 3-(5-phenylpentyl)-resorcylic acid, and 3-(6-phenylhexyl)- resorcylic acid. [0348] Embodiment II-85. The method of any one of Embodiments II-63 to II-84 wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0349] Embodiment II-86. The method of any one of Embodiments II-63 to II-85, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0350] Embodiment II-87. The method of any of Embodiments II-63 to II-86, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0351] Embodiment II-88. The method of any one of Embodiments II-63 to II-87, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0352] Embodiment II-89. The method of any one of Embodiments II-63 to II-88, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0353] Embodiment II-90. The method of any of Embodiments II-63 to II-89, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0354] Embodiment II-91. The method of any one of Embodiments II-63 to II-90, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments I-1 to II-43, wherein the variant of any one of Embodiments I-1 to II-43 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0355] Embodiment II-93. The method of any one of Embodiments II-63 to II-91, wherein the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0356] Embodiment III-1. A variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant has between 85% and 95% sequence identity to SEQ ID NO: 1, and comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions [0357] Embodiment III-2. The variant of Embodiment III-1, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56. [0358] Embodiment III-3. The variant of Embodiment III-1 or III-2, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100. [0359] Embodiment III-4. The variant of any one of Embodiments III-1 to III-3, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, I58, E64, I94, and R100. [0360] Embodiment III-5. The variant of any one of Embodiments III-1 to III-4, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six of the amino acid substitutions are at amino acids selected from the group consisting of:
Figure imgf000116_0001
Figure imgf000117_0002
[0361] Embodiment III-6. The variant of any of Embodiments III-1 to III-5, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E52A, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71A, D71E, D71K, D71N, D71Q, D71S, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G. [0362] Embodiment III-7. The variant of any one of Embodiments III-1 to III-6, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 6 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of:
Figure imgf000117_0001
Figure imgf000118_0001
[0363] Embodiment III-8. The variant of claim any one of Embodiments III-1 to III-7, wherein the variant comprises an amino acid sequence selected from an amino acid sequence of any of SEQ ID NOs: 3-146. [0364] Embodiment III-9. The variant of any one of Embodiments III-1 to III-8, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7-trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0365] Embodiment III-10. The variant of any one of Embodiments III-1 to III-9, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time. [0366] Embodiment III-11. The variant of Embodiment III-10, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0367] Embodiment III-12. A nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11. [0368] Embodiment III-13. A nucleic acid comprising a nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 147-290. [0369] Embodiment III-14. A method of making a modified host cell, the method comprising introducing one or more nucleic acids of Embodiment III-12 or Embodiment III-13 into a host cell. [0370] Embodiment III-15. A vector comprising one or more nucleic acids of Embodiment III-12 or Embodiment III-13. [0371] Embodiment III-16. A method of making a modified host cell, the method comprising introducing one or more vectors of Embodiment III-15 into a host cell. [0372] Embodiment III-17. A modified host cell comprising one or more nucleic acids of Embodiment III-12 or Embodiment III-13 or the vector of Embodiment III-15. [0373] Embodiment III-18. The modified host cell of Embodiment III-17, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative. [0374] Embodiment III-19. The modified host cell of Embodiment III-17 or Embodiment III-18, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative. [0375] Embodiment III-20. The modified host cell of any one of Embodiments III-17 to III-19, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time. [0376] Embodiment III-21. The modified host cell of any one of Embodiments III-17 to III-20, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time. [0377] Embodiment III-22. The modified host cell of any one of Embodiments III-17 to III-21, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time. [0378] Embodiment III-23. The modified host cell of any one of Embodiments III-17 to III-22, wherein the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 or III-11, grown under similar culture conditions for the same length of time. [0379] Embodiment III-24. The modified host cell of any one of Embodiments III-17 to III-22, wherein the modified host cell produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of Embodiments III-1 to III-11, grown under similar culture conditions for the same length of time. [0380] Embodiment III-25. The modified host cell of Embodiment III-24, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0381] Embodiment III-26. A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: [0382] a) culturing a modified host cell of any one of Embodiments III-15 to III-25 in a culture medium. [0383] Embodiment III-27. The method of Embodiment III-25 wherein the method comprises: [0384] b) recovering the produced olivetolic acid or an olivetolic acid derivative. [0385] Embodiment III-28. The method of Embodiment III-26 or Embodiment III-27, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III-11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0386] Embodiment III-29. The method of any one of Embodiments III-26 to III-28, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III- 11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0387] Embodiment III-30. The method of Embodiment III-29, wherein the method produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1. [0388] Embodiment III-31. The method of any one of Embodiments III- 26 to III-30, wherein the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3-butyl-resorcylic acid, 3-(trans-1-butenyl)-resorcylic acid, 3-(3- methylpentyl)-resorcylic acid, 3-(3-pentynyl)-resorcylic acid, 3-(trans-1-pentenyl)- resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl-resorcylic acid, 3-(5-hexynyl)- resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)-resorcylic acid, 3-octyl- resorcylic acid, 3-(trans-1-octenyl)-resorcylic acid, 3-nonyl-resorcylic acid, 3-(3- phenylpropyl)-resorcylic acid, 3-(4-phenylbutyl)-resorcylic acid, 3-(5-phenylpentyl)- resorcylic acid, and 3-(6-phenylhexyl)-resorcylic acid. [0389] Embodiment III-32. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising: [0390] a) culturing a modified host cell of any one of Embodiments III-17 to III-25 in a culture medium. [0391] Embodiment III-33. The method of Embodiment III-32, wherein the method comprises: [0392] b) recovering the produced cannabinoid or cannabinoid derivative. [0393] Embodiment III-34. The method of Embodiment III-32 or Embodiment III-33, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of Embodiments III-1 to III-11, wherein the variant of any one of Embodiments III-1 to III-11, and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time. [0394] Embodiment III-35. The method of any one of Embodiments III-32 to III-34 wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin. [0395] Embodiment III-36. The method of any one of Embodiments III-26 to III-35, wherein the culture medium comprises a carboxylic acid. [0396] Embodiment III-37. The method of Embodiment III-36, wherein the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid. [0397] Embodiment III-38. The method of Embodiment III-37, wherein the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid. [0398] The sequences provided in SEQ ID NOs: 1-290 are olivetolic acid cyclase (OAC) amino acid and nucleotide sequences disclosed herein. Where a genus and/or species is noted, the sequence should not be construed to be limited only to the specified genus and/or species, but also includes other genera and/or species expressing said sequence. Orthologs of the sequences disclosed in SEQ ID NOs: 1-290 may also be encompassed by this disclosure. Nucleotide sequences indicated as codon optimized in SEQ ID NOs: 2 and 147-290 are codon optimized for expression in S. cerevisiae INCORPORATION BY REFERENCE [0399] All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes. [0400] This application incorporates by reference the following publications in their entirety for all purposes: WO 2018/200888, PCT/US2019/053292, PCT/US2019/053173, US 62/851,560 filed May 22, 2019; US 62/906,017 filed September 25, 2019; US 62/906,551 filed September 26, 2019, and US 62/902,300 filed September 18, 2019. EXAMPLES [0401] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); bp, base pair(s); nt, nucleotide(s); and the like. Example 1: OAC mutations, Construction, and Transformations [0402] A library of DNA constructs encoding OAC mutants was synthesized and each codon encoding an amino acid was mutated to encode all other amino acids (saturation mutagenesis). This library was combined and transformed into a screening strain containing all other necessary engineering to make olivetolic acid (S983), and the resulting strains bearing mutant OAC constructs were benchmarked against a wild type OAC control (S1322) in a 96-well plate assay. [0403] OAC and other heterologous genes were added into S983 under the control of a galactose-inducible promoter (e.g. the promoter for GAL1) thereby enabling coordinated expression of the genes required for olivetolic acid production in galactose-containing media. Strains containing OAC mutants were cultured for 2 days in 360µL YP media containing 2% dextrose in 96-well plates at 30°C. Cultures were diluted 25-fold into 360µL of minimal media containing 50mM succinate, 1.33mM hexanoic acid, 0.1% glucose, and 4% galactose, and cultured for 2 days at 30°C. Each experiment was benchmarked to its own WT construct-bearing strain to account for small week to week changes in absolute plate assay titers. The constructs from strains that were significantly improved over WT were PCR amplified and submitted for Sanger sequencing to identify the exact mutation. [0404] In many instances, identical mutations, or multiple different mutations at the same residue were recovered, which indicates the screen is working well: A2S/P, L9C/I/V, K12V/N/Q, A36Q/E/P/S, T47R x 3, Q48H/C/M, I58C/V, Q70K/A, D71T x 2, R100G/A. One hit recovered in the initial screen contained three mutations (F23I, V66I, I74M). These additional mutations likely arose during DNA synthesis or PCR amplification of the original library before integration. [0405] Mutant constructs in the initial screen (n=1) with increased titers of Olivetolic acid were re-tested at higher replication (Table 1) and also re-transformed into the same library strain to confirm that the OAC construct was responsible, and not a background mutation in the genome. [0406] These enzymes demonstrate several types of improvements. Improvements in OA titer likely result from increased catalytic activity and/or better expression or stability in S. cerevisiae. Reduced production of Olivetol likely results from improved competition by the OAC enzyme for the substrate. The biomass yield of production cells bearing the most improved OACs increased, which may indicate decreased production of byproducts or pre- cursors of the reactions that make OA and can cause toxicity. Thus, in addition to improved titers of final products and reduced byproduct formation, improvement in cell health and growth is another beneficial feature of these mutant OAC enzymes. Combinatorial Library: [0407] To further increase OAC performance, a combinatorial library containing 6 or more mutations was assessed in the same assay. To increase diversity in the library, additional potential mutations were identified in silico. A BLAST search using the OAC protein sequence was used to identify a family of similar enzymes that were aligned to identify positions with low conservation, with the rationale that these positions could introduce additional diversity without loss of function. In total, 500 OAC variants were ordered for synthesis, combining successful mutations from the initial saturation library with mutations of the variable residues identified in the alignment for a total of 6 or more mutations per construct. A number of strains bearing these mutant OAC constructs performed significantly better than the WT control and, in many cases, better than any strains bearing single mutations (Tables 2-3). The strain bearing the highest performing construct, S1502, produced 1.5-fold more OA than the WT control strain, and improved the OA/Olivetol ratio from 1.23 to 3.01. More modest improvements in OD600 were also observed, e.g. from 3.2 to 3.34 for S1502. With the exception of S1535 strains that outperformed the WT control bore OAC constructs that exclusively drew mutations from the saturation mutagenesis library. Additionally, one strain, S1500, identified in this screen contained an OAC variant with an additional mutation, N29D, that likely arose from errors during DNA synthesis of the variant prior to integration. Effects of increased number of copies of OAC: [0408] Mutant constructs were benchmarked against a WT construct to give fold improvement. To determine whether a similar improvement could be obtained by simply increasing the copy number of the WT OAC, we compared strains bearing 1 or 2 copies of the WT OAC to strains bearing a single copy of the improved OAC construct (Table 4). Doubling the copy number of WT OAC only increased OA titer by 1.34 fold. Surprisingly, a single copy of the most improved OAC mutant, S1502, increased OA titer 1.52 fold over WT levels. This shows that one copy of an improved enzyme is more effective than two copies of the WT enzyme and confirms that increasing copy number of the WT OAC enzyme is not a viable strategy for achieving large improvements in OA titers. CBGA Production Strain Development [0409] Next, selected improved OAC constructs were tested in a strain with all necessary engineering for production of CBGA to determine how improved OA production might affect CBGA titers. One or two copies of the top performing mutant constructs from the combinatorial library were integrated into S1696, a strain bearing all necessary engineering for production of CBGA from sugar and hexanoic acid except for the the OAC construct. These strains were compared to control strains bearing zero, one or two copies of the WT OAC construct (Table 4). We observed that strains bearing a single copy of the improved OAC enzymes produced significantly more CBGA than the WT control, e.g. S1791 which had 2.23-fold higher titer. S1791 titers also exceeded that of the strain bearing two copies of the WT synthase. [0410] Engineered strains were cultured for 2 days in 96-well plates containing 360µL YP media and 2% dextrose at 30°C. Cultures were diluted 25-fold into 360µL of minimal media containing 50mM succinate, 1.33mM hexanoic acid, 0.05% galactose, and 4% sucrose, and cultured for 5 days at 30°C.
a b l e d et s et-e r d n a n o i t a t u m yfit n e d i ot d e c n e u q e s e r e w n 7 e e 4 r 0 c s 2-4 1 3 = 9 n 6 l a 2 i 3 t i n i O 5W m 1 o 2 1 0 r / f e r 5 st 0 i e h 0- H n T . t w e o M s h s E a t D a e d r a 4 l = o r n t . n s t o c n a T i r a W v t t n e na m t i r u e p m x el e- g n i ni s r e v C o A st O n d e e m v e o v r o p r p m I m i :1 d l 1 v e o 1 l F 0 3 b . 1 a 4 7 3 T = n 6 4 2
Figure imgf000127_0001
7 4 0 2-4 3 9 6 2 3 O 6W 2 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3 6 4 2
Figure imgf000128_0001
. 4 = n ) t a T d W e ( ts e et p - y t er d d l i na wt n a oi h t t a e t t u o N m . y r fi e t t i n t ed A i O ot n i d t ec n e ne m u e q v e o s r p e m 7 r i 4 e d 0 w l 2 o - n f 4 e y 3 e r b 9 c 6 s d 2 1 e t 3 = r o O nl s , 7W a i 1 ti e r 2 e 1 0 / n i h 5 0 0 m n w - o r T f o h . s y M t s i e l t h E H r D . a s l g il t o n r t s a y i n r r o a a c v v T d t n n W t a , a t n e t u e a l m m i p r l n o e l i t p e x w a n e i - 6 n 9 b i r h m e c v a o e c o C st n o A n e n m u O r d e e v e r v o r e o r p w s p m i l o m I d l r t : 2 o n F o c 1 v e 1 l 0 3 b 1 a 7 3 T 6
Figure imgf000129_0001
4 2
7 4 0 2-4 3 9 6 2 3 O 8W 2 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3
Figure imgf000130_0001
6 4 2 7 4 0 2-4 3 9 6 2 3 O 9W 2 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3 6 4
Figure imgf000131_0001
2 7 4 0 2-4 3 9 6 2 3 O 0W 3 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3 6
Figure imgf000132_0001
4 2 . 4 = nt sl a o r d t e n ts o e c t- ) e T r d W n ( a e p n y o t it a dl t i u w m t a y f h i t t n e t e o d i N. o t r e d t i e t cn A e O uq n e i s t e n r e 7 e m e 4 w v 0 o 2 n r - e p 4 e r m 3 9 c s i d6 2 1 l o 3 = n f O l a y i b 1W t 1 i d e t 3 1 0 n i r / o 5 m s 0 o 0 r , e - f r T s t e h M i E H. n w D s t o n h a i s r e a r v a l t o n r a t . y t n lt u o c h m T g il n i W s o t t a n y r a n e v i b m i r d n m e a o p c x , e e t a C -n l p A i r l l O e d v e e o w v s 6 o t n 9 r e h p m c m I e a v e - o r n 3 p o 1 v e l m n i u r 1 0 3 b a d l e o r 1 e 7 3 6
Figure imgf000133_0002
4
Figure imgf000133_0001
T F w 2
7 4 0 2-4 3 9 6 2 3 O 2W 3 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3
Figure imgf000134_0001
6 4 2 7 4 0 2-4 3 9 6 2 3 O 3W 3 1 1 0 / 5 0 0-T M E D C A Of o se i po C e l pi t l u Mf o t c eff E- 4 1 v e 1 l 0 3 b 1 a 7 3 T 6 4
Figure imgf000135_0001
2
Figure imgf000135_0002
7 4 0 2-4 3 9 6 2 3 O 4W 3 1 1 0 / 5 0 0-T M E D 1v 1 0 3 1 7 3 6 4 2
Figure imgf000136_0001
Table 5: Constructs and strains used in the Examples S4 is wild type Saccharomyces cerevisiae, CEN.PK2
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
* If a strain has a parent strain, it is a child strain. All of the constructs present in the parent strain are also all present in the child strain. ** S4 is CEN.PK113-1A with genotype MATalpha; URA3; TRP1; LEU2; HIS3; MAL2-8C; SUC2 *** S487 is the base strain used to test THCA synthase constructs. In this strain, the nucleotide sequence encoding a pGAL1_tTDH1 empty expression cassette is added and the i33 locus is returned to its native sequence by deleting the CBDAS construct there. [0411] Although the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims
CLAIMS What is claimed is: 1. A variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant has between 85% and 95% sequence identity to SEQ ID NO: 1, and comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions 2. The variant of claim 1, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, and T56.
3. The variant of claim 1 or claim 2, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are at amino acids selected from the group consisting of A2, V8, L9, K10, K12, E22, F23, K25, N29, V31, A36, Y41, T47, Q48, K49, N50, E52, E53, T56, I58, T62, E64, V66, E67, Q70, D71, I74, V84, S87, E90, I94, and R100.
4. The variant of any one of claims 1-3, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least one of the amino acid substitutions is at an amino acid selected from the group consisting of V8, L9, K49, T56, I58, E64, I94, and R100.
5. The variant of any one of claims 1-4, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein six of the amino acid substitutions are at amino acids selected from the group consisting of:
Figure imgf000143_0001
Figure imgf000144_0001
6. The variant of any of claims 1-5, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein at least six of the amino acid substitutions are selected from the group consisting of A2P, A2S, V8I, L9C, L9I, L9V, K10A, K12A, K12C, K12H, K12L, K12M, K12N, K12Q, K12T, K12V, E22L, F23I, F23L, K25N, N29D, V31M, A36D, A36E, A36F, A36H, A36M, A36N, A36P, A36Q, A36S, A36W, A36Y, Y41E, T47C, T47K, T47N, T47R, T47S, Q48C, Q48F, Q48H, Q48M, K49R, N50Y, E52A, E53V, T56S, I58C, I58V, T62C, E64D, V66I, E67S, Q70A, Q70K, D71A, D71E, D71K, D71N, D71Q, D71S, D71T, I74M, V84D, S87P, E90D, I94K, R100A, and R100G.
7. The variant of any one of claims 1-6, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least 6 amino acid substitutions, wherein the amino acid substitutions are selected from the group consisting of:
Figure imgf000145_0001
Figure imgf000146_0001
8. The variant of claim any one of claims 1-7, wherein the variant comprises an amino acid sequence selected from an amino acid sequence of any of SEQ ID NOs: 3-146.
9. The variant of any one of claims 1-8, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of olivetolic acid produced from 3,5,7- trioxododecanoyl-CoA by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
10. The variant of any one of claims 1-9, wherein the variant produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 under similar conditions for the same length of time.
11. The variant of claim 10, wherein the variant produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
12. A nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11.
13. A nucleic acid comprising a nucleotide sequence encoding a variant of an olivetolic acid cyclase (OAC) polypeptide, wherein the variant comprises an amino acid sequence of SEQ ID NO:1 comprising at least six amino acid substitutions, wherein the nucleotide sequence is selected from a nucleotide sequence of any one of SEQ ID NOs: 147-290.
14. A method of making a modified host cell, the method comprising introducing one or more nucleic acids of claim 12 or claim 13 into a host cell.
15. A vector comprising one or more nucleic acids of claim 12 or claim 13.
16. A method of making a modified host cell, the method comprising introducing one or more vectors of claim 15 into a host cell.
17. A modified host cell comprising one or more nucleic acids of claim 12 or claim 13 or the vector of claim 15.
18. The modified host cell of claim 17, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative.
19. The modified host cell of claim 17 or 18, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative.
20. The modified host cell of any one of claims 17-19, wherein the modified host cell produces olivetolic acid or an olivetolic acid derivative in an amount, as measured in mg/L or mM at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11, grown under similar culture conditions for the same length of time.
21. The modified host cell of any one of claims 17-20, wherein the modified host cell produces a cannabinoid or a cannabinoid derivative in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11, grown under similar culture conditions for the same length of time.
22. The modified host cell of any one of claims 17-21, wherein the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11, grown under similar culture conditions for the same length of time.
23. The modified host cell of any one of claims 17-22, wherein the modified host cell has a growth rate and/or biomass yield 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 60%, at least 70%, at least 80%, at least 90%, or at least 100% faster or higher than a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11, grown under similar culture conditions for the same length of time.
24. The modified host cell of any one of claims 17-22, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in an increased ratio of olivetolic acid over olivetol compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 lacks a nucleic acid comprising a nucleotide sequence encoding a variant of any one of claims 1-11, grown under similar culture conditions for the same length of time.
25. The modified host cell of claim 24, wherein the modified host cell produces olivetolic acid from 3,5,7-trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
26. A method of producing olivetolic acid or an olivetolic acid derivative, the method comprising: a) culturing a modified host cell of any one of claims 15-25 in a culture medium.
27. The method of claim 25 wherein the method comprises: b) recovering the produced olivetolic acid or an olivetolic acid derivative.
28. The method of claim 26 or 27, wherein the olivetolic acid or the olivetolic acid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the olivetolic acid or the olivetolic acid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of claims 1-11, wherein the variant of any one of claims 1-11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
29. The method of any one of claims 26-28, wherein the method produces olivetolic acid in an increased ratio of olivetolic acid over olivetol compared to that produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of claims 1-11, wherein the variant of any one of claims 1- 11 and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
30. The method of claim 29, wherein the method produces olivetolic acid from 3,5,7- trioxododecanoyl-CoA in a ratio of olivetolic acid over olivetol of about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, or greater than about 5:1.
31. The method of any one of claims 26-30, wherein the olivetolic acid derivative is selected from the group consisting of divarinolic acid, orsellinic acid, 3-butyl-resorcylic acid, 3- (trans-1-butenyl)-resorcylic acid, 3-(3-methylpentyl)-resorcylic acid, 3-(3-pentynyl)- resorcylic acid, 3-(trans-1-pentenyl)-resorcylic acid, 3-(4-pentenyl)-resorcylic acid, 3-hexyl- resorcylic acid, 3-(5-hexynyl)-resorcylic acid, 3-heptyl-resorcylic acid, 3-(trans-1-hexyl)- resorcylic acid, 3-octyl-resorcylic acid, 3-(trans-1-octenyl)-resorcylic acid, 3-nonyl- resorcylic acid, 3-(3-phenylpropyl)-resorcylic acid, 3-(4-phenylbutyl)-resorcylic acid, 3-(5- phenylpentyl)-resorcylic acid, and 3-(6-phenylhexyl)-resorcylic acid.
32. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising: a) culturing a modified host cell of any one of claims 17-25 in a culture medium.
33. The method of claim 32, wherein the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative.
34. The method of claim 32 or 33, wherein the cannabinoid or the cannabinoid derivative is produced in an amount, as measured in mg/L or mM, at least 5% to at least 500% greater than an amount of the cannabinoid or the cannabinoid derivative produced in a method comprising use of an OAC polypeptide having an amino acid sequence of SEQ ID NO:1 instead of the variant of any one of claims 1-11, wherein the variant of any one of claims 1- 11, and the OAC polypeptide having the amino acid sequence of SEQ ID NO:1 are used under similar conditions for the same length of time.
35. The method of any one of claims 32-34 wherein the cannabinoid is cannabigerolic acid, cannabigerol, cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
36. The method of any one of claims 26-35, wherein the culture medium comprises a carboxylic acid.
37. The method of claim 36, wherein the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid.
38. The method of claim 37, wherein the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid.
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