EP4031657A1 - Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides - Google Patents

Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides

Info

Publication number
EP4031657A1
EP4031657A1 EP20786122.0A EP20786122A EP4031657A1 EP 4031657 A1 EP4031657 A1 EP 4031657A1 EP 20786122 A EP20786122 A EP 20786122A EP 4031657 A1 EP4031657 A1 EP 4031657A1
Authority
EP
European Patent Office
Prior art keywords
seq
polypeptide
host cell
modified host
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20786122.0A
Other languages
German (de)
English (en)
French (fr)
Inventor
Andrew HORWITZ
Jeff Wong
Darren Platt
Jeff UBERSAX
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Demetrix Inc
Original Assignee
Demetrix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Demetrix Inc filed Critical Demetrix Inc
Publication of EP4031657A1 publication Critical patent/EP4031657A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12P7/40—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
    • C12Y121/00—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21)
    • C12Y121/03—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21) with oxygen as acceptor (1.21.3)
    • C12Y121/03007—Tetrahydrocannabinolic acid synthase (1.21.3.7)

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.
  • THC tetrahydrocannabinol
  • CBD cannabidiol
  • 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.
  • 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.
  • secreted enzymes such as the cannabinoid synthases
  • Engineered variants of cannabinoid synthases, modified host cells, and new methods are needed to address these challenges.
  • the present disclosure provides engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing cannabinoids or cannabinoid derivatives, methods of producing cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide.
  • THCAS tetrahydrocannabinolic acid synthase
  • the engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids).
  • the modified host cells of the disclosure may be useful for producing 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.
  • An aspect of the disclosure relates to an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions.
  • the engineered 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%, 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:44.
  • the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing. In some embodiments, the engineered variant comprises substitution of at least one surface exposed amino acid.
  • FAD flavin adenine dinucleotide
  • BBE berberine bridge enzyme
  • the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, N168, E167, S170, F171, P172, Y175, G180, N196, H208,
  • the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F, T109V, Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, W161R, W161Y, W161K, K165A, N168S, E167P, Y175F, G180A,
  • the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
  • the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, to least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.
  • the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the FAD binding domain.
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the BBE domain.
  • the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, E117, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178,
  • the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, El 17, S120, F127, D131, G156, E157, Y159, G174, C176, G182, G183, F185, G187, G188, G189, Y190, G191, P192, R195,
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids.
  • the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase (THCAS) polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDGA cannabigerolic acid
  • THCAS tetrahydrocannabinolic acid synthase
  • the engineered variant produces THCA from CBGA 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 THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDA cannabichromenic acid
  • the engineered variant produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • the engineered variant comprises a truncation at an N- terminus, at a C-terminus, or at both the N- and C-termini.
  • the truncated engineered variant comprises a signal polypeptide or a membrane anchor.
  • the engineered variant lacks a native signal polypeptide.
  • the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus.
  • the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus.
  • nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID N0:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID N0:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:
  • the nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:
  • nucleotide sequence is codon-optimized.
  • An aspect of the disclosure relates to a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure into a host cell.
  • Another aspect of the disclosure relates to a vector comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • An aspect of the disclosure relates to a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more vectors comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure into a host cell.
  • Another aspect of the disclosure relates to a modified host cell for producing a cannabinoid or a cannabinoid derivative, wherein the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide.
  • GOT polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 17.
  • the modified host cell comprises two or more heterologous nucleic acids comprising the nucleotide sequence encoding the GOT polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide.
  • the NphB polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 188.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tetraketide synthase (TKS) polypeptide and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase (OAC) polypeptide.
  • TKS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 19.
  • the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide.
  • the OAC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:21 or SEQ ID NO:48.
  • the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acyl activating enzyme (AAE) polypeptide.
  • the AAE polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23.
  • the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide.
  • the modified host cell comprises one or more of the following: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMG-CoA synthase (HMGS) polypeptide; b) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a truncated 3- hydroxy-3-methyl-glutaryl-CoA reductase (tHMGR) polypeptide; c) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate kinase (MK) polypeptide; d) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a phosphomevalonate kinase (PMK) polypeptide; e) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate pyr
  • the IDI1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:25.
  • the tHMGR polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:27.
  • the HMGS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:29.
  • the MK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:39.
  • the PMK polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:37.
  • the MVD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:33.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide.
  • the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:31.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide.
  • PDC pyruvate decarboxylase
  • the PDC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS) polypeptide.
  • GPPS geranyl pyrophosphate synthetase
  • the GPPS polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:41.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the KAR2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:5.
  • the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide.
  • the PDI1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:9.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IRE1 polypeptide.
  • the IRE1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 190.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide.
  • the EROl polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:7.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a FAD1 polypeptide.
  • the FAD1 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 192.
  • the modified host cell comprises a deletion or downregulation of one or more genes encoding a PEP4 polypeptide.
  • the PEP4 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 15.
  • the modified host cell comprises a deletion or downregulation of one or more genes encoding a ROT2 polypeptide.
  • the ROT2 polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13.
  • the modified host cell is a eukaryotic cell.
  • the eukaryotic cell is a yeast cell.
  • the yeast cell is Saccharomyces cerevisiae.
  • the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae.
  • At least one of the one or more nucleic acids are integrated into the chromosome of the modified host cell. In some embodiments of the disclosure, at least one of the one or more nucleic acids are maintained extrachromosomally. In some embodiments of the disclosure, at least one of the one or more nucleic acids are operably-linked to an inducible promoter. In some embodiments of the disclosure, at least one of the one or more nucleic acids are operably-linked to a constitutive promoter.
  • 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered 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 150% at least 200%, at least 500%, or at least 1000% 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid syntha
  • the modified host cell has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered 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 higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a
  • the modified host cell produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under similar culture conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • the modified host cell produces
  • THCA from CBGA in a ratio of THCA over another cannabinoid e.g., cannabichromenic acid (CBCA)
  • cannabinoid e.g., cannabichromenic acid (CBCA)
  • CBCA cannabichromenic acid
  • CBCA cannabichromenic acid
  • Another aspect of the disclosure relates to 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.
  • the method comprises: b) recovering the produced cannabinoid or cannabinoid derivative.
  • the culture medium comprises a carboxylic acid.
  • the carboxylic acid is an unsubstituted or substituted C3-C18 carboxylic acid.
  • the unsubstituted or substituted C3-C18 carboxylic acid is an unsubstituted or substituted hexanoic acid.
  • the culture medium comprises olivetolic acid or an olivetolic acid derivative.
  • the cannabinoid is cannabidiolic acid, cannabidiol, cannabidivarinic acid, or cannabidivarin.
  • the culture medium comprises a fermentable sugar.
  • the culture medium comprises a pretreated cellulosic feedstock.
  • the culture medium comprises a non-fermentable carbon source.
  • the non-fermentable carbon source comprises ethanol.
  • the cannabinoid or the cannabinoid derivative is produced in an amount of more than 100 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 culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered 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
  • 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 culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence en
  • the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA)) compared to that produced in a method comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the modified host cell of the disclosure, wherein the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 lacks a nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure, grown under
  • An aspect of the disclosure relates to a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of the disclosure.
  • the method comprises recovering the produced cannabinoid or cannabinoid derivative.
  • the cannabinoid is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin.
  • 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.
  • the cannabinoid is tetrahydrocannabinolic acid (THCA), and wherein the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., cannabichromenic acid (CBCA) compared to that produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instread of the engineered variant of the disclosure, wherein the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBCA cannabichromenic acid
  • the method produces
  • CBCA cannabinoid
  • Another aspect of the disclosure relates to a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, 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 a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a comparison cannabinoid synthase polypeptide, wherein the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 can convert cannabigerolic acid (CBGA) to a first cannabinoid, tetrahydrocannabinolic acid (THCA), and the comparison cannabinoid synthase polypeptide can convert the same CBGA to a different second cannabinoid; c) co-expressing in the test population the engineered variant and the comparison
  • the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase (THCAS) polypeptide having an amino acid sequence of SEQ ID NO:44, wherein improved in vivo performance is demonstrated by an increase in the ratio of the first cannabinoid over the second cannabinoid produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time.
  • THCAS tetrahydrocannabinolic acid synthase
  • the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO: 3 by producing the first cannabinoid 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 cannabinoid synthase polypeptide is a cannabidiolic acid synthase polypeptide.
  • the cannabidiolic acid synthase (CBDAS) polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:3.
  • the second cannabinoid is cannabidiolic acid (CBDA).
  • the engineered variant is an engineered variant of the disclosure.
  • FIGS. 1 A, IB, and 1C depict expression constructs used in the production of the S29 strain.
  • the expression constructs depicted in FIGS. 1 A, IB, and 1C were also used in the production of the following strains: S61, S122, S171, S181, S220, S241, S270, S487, S951, S1000-S1059, S1072-1079, and S1081.
  • 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 Table 1 with 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.
  • FIG. 2 depicts an expression construct used in the production of the S181 strain.
  • the expression construct depicted in FIG. 2 was also used in the production of following strains: S220, S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and SI 081.
  • FIG. 3 depicts an expression construct used in the production of the S220 strain. The expression construct depicted in FIG. 3 was also used in the production of following strains: S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and SI 081.
  • FIG. 4 depicts expression constructs used in the production of the S241 strain.
  • FIG. 5 depicts a landing pad construct used in the production of the S61 strain.
  • the construct depicted in FIG. 5 was also used in the production of the following strains: S122, S171, S181, S220, S241, S270, S487, S951, S1000-S1059, S1072-1079, and S 1081.
  • FIG. 6 depicts expression constructs used in the production of the S122 strain.
  • FIG. 7 depicts an expression construct used in the production of the S 171 strain.
  • the expression construct depicted in FIG. 7 was also used in the production of the following strains: S181, S220, S241, S270, S487, S951, S1000-S1059, SI 072- 1079, and S 1081.
  • FIG. 8 depicts expression constructs used in the production of the S270 strain.
  • FIGS. 9A and 9B depict an expression construct used in the production of the
  • S487 strain The expression constructs depicted in FIGS. 9A and 9B were also used in the production of the following strains: S951 S1000-S1059, S1072-1079, and S1081.
  • FIG. 10 depicts an expression construct used in the production of the SI 042 strain.
  • FIG. 11 depicts an expression construct used in the production of the following strains: S951, S1000-S1041, S1043-S1059, S1072-1079, and S1081. Detailed Description
  • 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 a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells for producing cannabinoids or cannabinoid derivatives, methods of producing cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the THCAS polypeptide.
  • the engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids).
  • the modified host cells of the disclosure may be useful for producing 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.
  • Cannabinoid synthase polypeptides such as tetrahydrocannabinolic acid synthase, cannabichromenic acid synthase, or cannabidiolic acid synthase polypeptides, play an important role in the biosynthesis of cannabinoids.
  • reconstituting their activity in a modified host cell has proven challenging, hampering progress in the production of cannabinoids or cannabinoid derivatives.
  • Cannabinoid synthases must successfully traverse the secretory pathway to fold and function properly.
  • CBDAS cannabigerolic acid
  • CBDA cannabidiolic acid
  • THCA tetrahydrocannabinolic acid
  • the natural CBDAS or THCAS enzymes are 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.
  • 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.
  • the present disclosure provides engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide.
  • THCAS tetrahydrocannabinolic acid synthase
  • various engineered variants were screened.
  • THCA titers were improved (outside standard deviation of wild type) in 58 distinct variants.
  • These engineered variants of the disclosure may be useful for producing cannabinoids or cannabinoid derivatives (e.g., non-naturally occurring cannabinoids).
  • the engineered variants of the disclosure may produce tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDA cannabigerolic acid
  • the engineered variants of the disclosure may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 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
  • Naturally- occurring 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 cannabinoids or cannabinoid derivatives from simple precursors such as sugars and carboxylic acids.
  • nucleic acids e.g., heterologous nucleic acids
  • 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: cannabinoids or cannabinoid derivatives.
  • 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 cannabinoids or cannabinoid derivatives with defined stereochemistries, which is challenging to do using chemical synthesis.
  • modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of a THCAS polypeptide of the disclosure 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.
  • GPP geranylpyrophosphate
  • prenyl phosphates e.g., olivetolic acid, or hexanoyl-CoA
  • nucleotide sequences encoding the polypeptides involved in cannabinoid or cannabinoid precursor are codon- optimized.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, 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 a THCAS polypeptide of the disclosure.
  • nucleic acids e.g., heterologous nucleic acids
  • the nucleotide sequence encoding the engineered variant of a THCAS polypeptide is codon-optimized. Modification of the secretory pathway in the host cell may improve expression and solubilization of the engineered variants of the disclosure, as these variants are processed through the secretory pathway.
  • a modified host cell such as a modified yeast cell
  • the expressed engineered variants may be misfolded or mislocalized, resulting in low expression, expressed engineered variants lacking activity, engineered variant aggregation, reduced host cell viability, and/or cell death. Additionally, a backlog of misfolded or mislocalized expressed engineered variants can induce metabolic stress within the modified host cell, harming the modified host cell.
  • the expressed engineered variants 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.
  • 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 (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding an engineered variant of a THCAS polypeptide of the disclosure may improve expression, folding, and enzymatic activity of the engineered variant as well as viability of the modified yeast host cell, such as modified Saccharomyces cerevisiae.
  • 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.
  • the present disclosure also provides a platform for the economical production of high-value cannabinoids, including THC, as well as derivatives thereof. It also provides for the production of different 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, over 100 g per liter of culture medium.
  • the disclosure provides engineered variants of a THCAS polypeptide, methods, modified host cells, and nucleic acids to produce cannabinoids or cannabinoid derivatives in vivo or in vitro from simple precursors.
  • Nucleic acids e.g., heterologous nucleic acids
  • the in vitro methods are cell-free.
  • nucleic acids e.g., heterologous nucleic acids
  • 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 cannabinoids or cannabinoid derivatives.
  • Cannabinoid precursors may include, for example, geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or 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. This tetraketide intermediate is then cyclized by an olivetolic acid cyclase (OAC) polypeptide to produce olivetolic acid.
  • GPP geranylpyrophosphate
  • hexanoyl-CoA and malonyl-CoA are combined to afford a 12-carbon tetraketide intermediate by a tetraketide synthase (TKS) polypeptide. 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.”
  • GOT geranyl pyrophosphate:olivetolic acid geranyltransferase
  • the engineered variants of a THCAS polypeptide of the disclosure then convert CBGA into other cannabinoids, e.g., THCA, etc. In the presence of heat or light, the acidic cannabinoids can undergo decarboxylation, e.g., THCA producing THC.
  • 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.
  • 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 polypeptides e.g., one or more MEV pathway polypeptides.
  • MEV pathway mevalonate pathway
  • MAPP dimethylallyl pyrophosphate
  • the mevalonate pathway comprises polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoAto generate acetoacetyl-CoA(e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoAto 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.
  • 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);
  • Cannabinoid or “cannabinoid compound” as used herein may refer to a member of a class of unique meroterpenoids found until now only in Cannabis sativa.
  • Cannabinoids may include, but are not limited to, cannabichromene (CBC) type (e.g. cannabichromenic acid), cannabigerol (CBG) type (e.g. cannabigerolic acid), cannabidiol (CBD) type (e.g. cannabidiolic acid), A 9 -trans-tetrahydrocannabinol (D 9 -THC) type (e.g.
  • CBC cannabichromene
  • CBG cannabigerol
  • CBD cannabidiol
  • D 9 -THC A 9 -trans-tetrahydrocannabinol
  • D 9 - tetrahydrocannabinolic acid A 8 -trans-tetrahydrocannabinol (D 8 -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, cannabigerolic acid (CBGA), cannabigerolic acid monomethylether (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidiolic acid (CBD), cannabidio
  • 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).
  • 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.
  • Functional and reactive groups may be unsubstituted or substituted with one or more functional or reactive groups.
  • 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.
  • Such chemical moieties may include, but are not limited to, methyl, alkyl, alkenyl, methoxy, alkoxy, acetyl, carboxyl, carbonyl, oxo, ester, hydroxyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkenylalkyl, cycloalkenylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, arylalkenyl, heterocyclyl, aralkyl, cycloalkylalkyl, heterocyclylalkyl, heteroarylalkyl, and the like.
  • a cannabinoid derivative or olivetolic acid derivative may also comprise one
  • nucleic acid 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.
  • polypeptide 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.
  • An “engineered variant of a tetrahydrocannabinolic acid synthase polypeptide” or “engineered variant of the disclosure” may indicate a non-wild type polypeptide having tetrahydrocannabinolic acid synthase activity.
  • One skilled in the art can measure the tetrahydrocannabinolic acid synthase 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 tetrahydrocannabinolic acid synthase sequence, such as the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44.
  • engineered variants may comprise truncations, additions, and/or deletions, and/or other mutations compared to a wild type tetrahydrocannabinolic acid synthase sequence, such as the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44.
  • Engineered variants may have substitutions compared a non-wild type tetrahydrocannabinolic acid synthase sequence.
  • engineered variants may comprise truncations, additions, and/or deletions and/or other mutations compared to a non-wild type tetrahydrocannabinolic acid synthase sequence.
  • the engineered variants described herein contain at least one amino acid residue substitution from a parent tetrahydrocannabinolic acid synthase polypeptide.
  • the parent tetrahydrocannabinolic acid synthase polypeptide is a wild type sequence.
  • the parent tetrahydrocannabinolic acid synthase polypeptide is a non-wild type sequence.
  • heterologous may refer to what is not normally found 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.
  • 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 codon-optimized nucleotide sequence may be an example of a heterologous nucleotide sequence.
  • 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.
  • 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.
  • 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.
  • these one or more heterologous nucleic acids may comprise a nucleotide sequence encoding one polypeptide.
  • these one or more heterologous nucleic acids may comprise nucleotide sequences encoding more than one polypeptide.
  • these one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of the same polypeptide.
  • these one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of different polypeptides.
  • “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 THCA over CBCA produced by an engineered variant disclosed herein would be an increased ratio of THCA over CBCA compared to an 11:1 ratio of THCA over CBCA 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 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.
  • a modified host cell disclosed herein produced 4 mM THCA and 1 mM CBCA, the ratio of THCA over CBCA would be 4:1.
  • 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 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 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.
  • 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 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.
  • Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and aspara
  • 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.
  • THCAS tetrahydrocannabinolic acid synthase
  • the inventors have identified amino acid locations of the THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44 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 THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa.
  • the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa comprises the following domains:
  • FAD binding domain amino acids 77-251.
  • BBE domain amino acids 480-538.
  • the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa also comprises the following domains surface exposed amino acids: 28-33, 35, 36, 39-45, 47-50,
  • THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa (shown herein in Table 1; UniProtKB/Swiss-Prot: Q8GTB6). Accordingly, a reference to “F317” identifies an amino acid that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 th amino acid from the N-terminus, wherein the methionine is the first amino acid.
  • the 317 th amino acid is a phenylalanine (F) in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa.
  • F317 amino acid may have a different position in the THCAS polypeptides from different species or in different isoforms.
  • Xn amino acid sequence position at which a particular amino acid or amino acid change (“residue difference”) is present
  • Xn amino acid sequence position at which a particular amino acid or amino acid change (“residue difference”) is present
  • position n refers to the amino acid position with respect to the reference sequence.
  • X317 identifies an amino acid that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 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.
  • a reference to “F317Y” identifies a substitution that, in the THCAS polypeptide of SEQ ID NO:44 from Cannabis sativa , is the 317 th amino acid from the N- terminus, phenylalanine, being replaced by tyrosine.
  • Cannabinoid synthase polypeptides secreted polypeptides, have structural features that may hinder expression in modified host cells, such as modified yeast cells.
  • Cannabinoid synthase polypeptides comprise disulfide bonds, numerous glycosylation sites, including A -glycosylation sites, and a bicovalently attached flavin adenine dinucleotide (FAD) cofactor moiety. Accordingly, reconstituting the activity of or expressing cannabinoid synthase polypeptides in a modified host cell, such as a modified yeast cell, can be challenging and unreliable.
  • FAD flavin adenine dinucleotide
  • engineered variants may have improved expression, folding, and enzymatic activity compared to the THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44. Additionally, expression of the engineered variants of the disclosure may enhance viability of the modified host cells disclosed herein compared to modified host cells expressing a THCAS polypeptide comprising an amino acid sequence of SEQ ID NO:44.
  • the disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions.
  • the engineered 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%, 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:44 .
  • 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%, or at least 84% sequence identity to SEQ ID NO:44.
  • the disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing. In some embodiments, at least one amino acid substitution is present in the signal polypeptide.
  • THCAS tetrahydrocannabinolic acid synthase
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 the signal polypeptide. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6,
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 the FAD binding domain. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the FAD binding domain.
  • the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X100, X103, X109, X124, X125, X132, X137, X143, X149, X161,
  • the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, E167, N168, S170, F171, P172, Y175, G180, N196, H208,
  • the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132M, S170T, F 1711, N196T, N196Q, and N196V. In some embodiments, at least one amino acid substitution is present in the BBE domain. In certain such embodiments, the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 the BBE domain. In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X500 and X528.
  • the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of Y500 and N528.
  • the engineered variant comprises at least one amino acid substitution selected from the group consisting of Y500M, Y500V, and N528E.
  • the disclosure provides for an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the engineered variant comprises substitution of at least one surface exposed amino acid.
  • THCAS tetrahydrocannabinolic acid synthase
  • the engineered variant comprises substitution of at least one surface exposed amino acid.
  • at least one hydrophobic surface exposed amino acid is substituted with a hydrophilic amino acid.
  • at least one hydrophilic surface exposed amino acid is substituted with a hydrophobic amino acid.
  • the engineered variant comprises substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 surface exposed amino acids. In some embodiments, the engineered variant comprises substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 surface exposed amino acids. In some embodiments, wherein the engineered variant comprises substitution of at least one surface exposed amino acid, the engineered variant comprises at least one amino acid substitution selected from the group consisting of X132, X170, X171, X196, X261, X269, X317, and X539. In some embodiments, wherein the engineered variant comprises substitution of at least one surface exposed amino acid, the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132, S170, F171, N196, K261,
  • the engineered variant comprises substitution of at least one surface exposed amino acid
  • the engineered variant comprises at least one amino acid substitution selected from the group consisting of L132M, S170T,
  • Substitution of hydrophobic surface exposed amino acids with hydrophilic amino acids may increase the hydrophilicity of solvent-exposed amino acids, which may improve solubility of the engineered variants of the disclosure in an aqueous (non-trichome) environment.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of X31, X43, X49, X50, X 51, X55, X56, X59, X61, X62, X71, X100, X103, X109, X124, X125, X132, X137, X143, X149, X161, X165, X168, X167, X170,
  • Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • CBCA cannabinoid
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, W161, K165, N168, E167, Y175, G180, N196, H208, A250, 1257, K261, G311, F317, L327, K390, T379, D429, Y500, N528, P542, H543, H544, and H545.
  • an amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, W161, K165, N168, E167,
  • Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • CBCA cannabinoid
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F,T109V,Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, W161R, W161Y, W161K, K165A, N168S, E167P, Y175F, G180A, N196Q, N196V, H208T, A250T, I257V, K261C, G311A, F317Y, L327I, K390E, T3797S, Y500M, Y500V, N528E, P542E, P542V, H
  • Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • CBCA cannabinoid
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO:
  • SEQ ID NO: 134 SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, and SEQ ID NO:186.
  • Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time and may produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g. CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO: 44 under similar conditions for the same length of time.
  • CBCA cannabinoid
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the amino acid substitutions described herein.
  • the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, to 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the amino acid substitutions described herein.
  • the engineered variant comprises 1, 2, 3, 4, 5, 6, 7,
  • the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the amino acid substitutions described herein (e.g., 1-15 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acid substitutions described herein (e.g., 1-10 of the amino acid substitutions described herein).
  • the engineered variant comprises 1, 2, 3, 4, or 5 of the amino acid substitutions described herein (e.g., 1-5 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, 3, or 4 of the amino acid substitutions described herein (e.g., 1-4 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1, 2, or 3 of the amino acid substitutions described herein (e.g., 1-3 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1 or 2 of the amino acid substitutions described herein (e.g., 1-2 of the amino acid substitutions described herein). In some embodiments, the engineered variant comprises 1 of the amino acid substitutions described herein.
  • the engineered variant comprises 2 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 3 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 4 of the amino acid substitutions described herein. In some embodiments, the engineered variant comprises 5 of the amino acid substitutions described herein. [0124]
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.
  • FAD flavin adenine dinucleotide
  • BBE berberine bridge enzyme
  • the engineered variant comprises at least one immutable amino acid in the FAD binding domain.
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the FAD binding domain.
  • the engineered variant comprises at least one immutable amino acid in the FAD binding domain, wherein the at least one immutable amino acid is selected from the group consisting of X87, X93, X99, X108, X110, X112, X117, X118, X120, X126, X127, X131, X141, X148, X152, X153, X155, X156, X157, X159, X160, X163, X170, X171, X172, X173, X174, X176,
  • mutation of one or more of these immutable amino acids reduces titer of one or more cannabinoids.
  • mutation of one or more of amino acids XI 70, X171, and/or XI 72 reduces titer of one or more cannabinoids.
  • the at least one immutable amino acid is selected from the group consisting of P87, 193, C99, R108, R110, G112, E117, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, S170, F171, P172, G173, G174, C176, P177, T178, V179, G182, G183, H184, F185, G187, G188, G189, Y190, G191, A192, L193, R195, A201,
  • mutation of one or more of amino acids SI 70, F171, and/or PI 72 reduces titer of one or more cannabinoids.
  • the engineered variant comprises at least one immutable amino acid in the BBE domain.
  • the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the BBE domain.
  • the at least one immutable amino acid is selected from the group consisting of X485, X499, X503, X514, X515, X522, X529, X530, X534, X535, and X536.
  • the engineered variant comprises at least one immutable amino acid in the BBE domain
  • the at least one immutable amino acid is selected from the group consisting of R485, N499, A503, N514, F515, K522, N529, F530, E534, Q535, and S536.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of X28, X34, X35, X37, X64, X70, X87, X93, X99, X108, XI 10, XI 12, XI 17, XI 18, X120, X126, X127, X131, X141, X148, X152, X153, X155, X156, X157, X159, X160, X163,
  • the engineered variant comprises at least one immutable amino acid selected from the group consisting ofX37, X70, X93, X99, XI 17, X120, X127, X131, X156, X157, X159, X174, X176, X182, X183, X185, X187, X188, X189, X190, X191, X192, X195, X202, X206,
  • the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, El 17, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178, V179, G182, G183, H184, F185,
  • the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, E117, S120, F127, D131, G156, E157, Y159, G174, C176, G182,
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids, provided that the engineered variant has at least one amino acid substitution compared to SEQ ID NO:44.
  • Engineered variants with combinations of the immutable amino acids and substitiutions described herein can be made and the resulting engineered variants screened for improved tetrahydrocannabinolic acid synthase (THCAS) properties.
  • Engineered variants comprising combinations of all of the substitutions and immutable amino acids described herein are intended to be encompassed by this disclosure.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises at least one amino acid substitution at the C-terminus.
  • a hydrophilic amino acid is replaced with a hydrophobic amino acid.
  • a hydrophobic amino acid is replaced with a hydrophilic amino acid.
  • Such engineered variants may produce THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • the disclosure provides for an engineered variant, wherein the engineered variant comprises a truncation at the N-terminus, at the C-terminus, or at both the N- and C- termini.
  • the engineered variant comprises a truncation at the N- terminus.
  • the engineered variant comprises a truncation at the C- terminus.
  • the engineered variant comprises a truncation at both the Isl and C-termini.
  • the engineered variant lacks a native signal polypeptide (i.e., amino acids 1-28 of SEQ ID NO:44).
  • the engineered variant comprises 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 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:44.
  • the engineered variant comprises 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%, or at least 84% sequence identity to SEQ ID NO:44.
  • the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the C-terminus. In some embodiments, the engineered variant comprises a truncation of at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids at the C-terminus.
  • the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus (e.g., 1-10 amino acids at the C-terminus). In some embodiments, the engineered variant comprises 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 a truncation of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the C-terminus (e.g., 21-30 amino acids at the C-terminus).
  • the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the N-terminus. In some embodiments, the engineered variant comprises a truncation of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus. In some embodiments, the engineered variant comprises a truncation of at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids at the N-terminus.
  • the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus (e.g., 1-10 amino acids at the N-terminus). In some embodiments, the engineered variant comprises a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the N-terminus (e.g., 11- 20 amino acids at the N-terminus). In some embodiments, the engineered variant comprises a truncation of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the N-terminus (e.g., 21-30 amino acids at the N-terminus).
  • a truncated engineered variant of the disclosure may comprise a signal polypeptide.
  • the truncated engineered variant lacks a native signal polypeptide.
  • the signal polypeptide is a secretory signal polypeptide.
  • the secretory signal polypeptide is a native secretory signal polypeptide.
  • the secretory signal polypeptide is a synthetic secretory signal polypeptide.
  • the secretory signal polypeptide is an endoplasmic reticulum retention signal polypeptide.
  • the endoplasmic reticulum retention signal polypeptide is a HDEL polypeptide or a KDEL polypeptide.
  • the secretory signal polypeptide is a mitochondrial targeting signal polypeptide.
  • the secretory signal polypeptide is a Golgi targeting signal polypeptide.
  • the secretory signal polypeptide is a vacuolar localization signal polypeptide.
  • the vacuolar localization signal polypeptide is a PEP4t polypeptide or a PRC It polypeptide.
  • the vacuolar localization signal polypeptide is a PEP4t polypeptide.
  • the secretory signal polypeptide is a plasma membrane localization signal polypeptide. In some embodiments, the secretory signal polypeptide is a peroxisome targeting signal polypeptide. In some embodiments, the peroxisome targeting signal polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal polypeptide is a mating factor secretory signal polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal polypeptide is linked to the N-terminus of the engineered variant.
  • a truncated engineered variant of the disclosure may comprise a membrane anchor.
  • a membrane anchor may be a sequence that inserts into a membrane in the cell and anchor an attached polypeptide there.
  • a membrane anchor may be present in a membrane external to the cell (e.g., GPI polypeptides) or internal to the cell (e.g., tail anchors, ER anchoring).
  • Examples of membrane anchors include, but are not limited to, glycosylphosphatidylinositol membrane anchors (GPI polypeptides, e.g. AGA1), CAAX box polypeptides (get prenylated, e.g.
  • tail anchored polypeptides with a hydrophobic C-terminus e.g. phosphatidylinositol 4,5-bisphosphate 5-phosphatase (INP54) has a hydrophobic tail anchor in ER membrane or synaptobrevin 2 (VAMP2) has a hydrophobic poly-I tail anchor in vesicle membranes.
  • VAMP2 synaptobrevin 2
  • Engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of THCA from CBGA in a greater amount, as measured in mg/L or mM, than an amount of THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • Similar conditions may refer to reaction conditions at the same temperature, pH, buffer, and/or fermentation conditions and in the same culture medium and/or reaction solvent.
  • the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) 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 THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDG cannabigerolic acid
  • the engineered variant produces
  • CBCA cannabinoid
  • THCA THCA, 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.
  • the modified host cell expresses polypeptides involved in the MEV pathway and/or polypeptides involved in cannabinoid biosynthesis and/or comprises modifications to the secretory pathway. It is contemplated that engineered variants of the disclosure having various degrees of stability, solubility, activity, and/or expression level in one or more of the test conditions will find use in the present disclosure for the production of cannabinoids or cannabinoid derivatives in a diversity of host cells.
  • engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, production of cannabinoids or cannabinoid derivatives 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant have a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant produce THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time. Similar culture conditions may refer to host cells grown in the same culture medium at the same temperature, pH, and/or fermentation conditions.
  • engineered variants of a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant do not have significantly decreased growth or viability compared to modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 a THCAS polypeptide can be made and screened for improved properties, such as, modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant do not have significantly decreased growth or viability compared to an unmodified host cell.
  • Nucleic Acids Comprising Nucleotide Sequences Encoding Engineered Variants of the Tetrahydrocannabinolic Acid Synthase (THCAS) Polypeptide and Expression Vectors and Constructs
  • nucleic acids comprising nucleotide sequences encoding engineered variants of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein and expression vectors and constructs comprising said nucleic acids.
  • THCAS tetrahydrocannabinolic acid synthase
  • 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 amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:
  • nucleic acid comprising a nucleotide sequence encoding an engineered variant of the disclosure comprising an amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, S
  • the disclosure also provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding an engineered variant, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO
  • 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), 5xDenhardt’s solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in O.lxSSC at about 65 °C.
  • Other known hybridization conditions are well known and are described in Sambrook et ah, 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.
  • the disclosure provides a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions.
  • the method involves a competition assay wherein the engineered variant of the disclosure is expressed in a modified host cells alongside a related enzyme.
  • THCAS tetrahydrocannabinolic acid synthase
  • Some embodiments of the disclosure relate to a method of screening an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, 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 a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a comparison cannabinoid synthase polypeptide, wherein the THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 can convert CBGA to a first cannabinoid, THCA, and the comparison cannabinoid synthase polypeptide can convert the same CBGA to a different second cannabinoid; c) co-expressing in the test population the engineered variant and the comparison cannabinoid synthase polypeptide, wherein the engineered variant may convert C
  • the test population is identified as comprising an engineered variant having improved in vivo performance compared to the tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 by producing the first cannabinoid 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 tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, wherein improved in vivo performance is demonstrated by an increase in the ratio of the first cannabinoid over the second cannabinoid produced by the test population compared to that produced by the control population under similar culture conditions for the same length of time.
  • the cannabinoid synthase polypeptide is a cannabidiolic acid synthase (CBDAS) polypeptide.
  • CBDAS polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:3.
  • a nucleotide sequence encoding the CBDAS polypeptide is the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, a nucleotide sequence encoding the CBDAS polypeptide is the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:2, or a codon degenerate nucleotide sequence thereof.
  • a nucleotide sequence encoding the CBDAS polypeptide has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:l or SEQ ID NO:2.
  • the second cannabinoid is CBDA.
  • THCAS Tetrahydrocannabinolic Acid Synthase
  • 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 a cannabinoid or a cannabinoid derivative.
  • the nucleotide sequence encoding the engineered variant is codon-optimized.
  • the disclosure also provides nucleic acids (e.g., heterologous nucleic acids), which can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of the engineered variants of the disclosure, which can then be utilized in vitro (e.g., cell-free) or in vivo for the production of cannabinoids or cannabinoid derivatives.
  • these nucleic acids comprise a codon-optimized nucleotide sequence encoding the engineered variant.
  • Cannabinoid synthase polypeptides secreted polypeptides, such as the engineered variants of the disclosure, have structural features that may hinder expression in modified host cells, such as modified yeast cells.
  • Cannabinoid synthase polypeptides, including the engineered variants of the disclosure comprise disulfide bonds, numerous glycosylation sites, including A-glycosylation sites, and a bicovalently attached flavin adenine dinucleotide (FAD) cofactor moiety.
  • FAD flavin adenine dinucleotide
  • manipulation of secretory pathway in host cells modified with one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may improve expression, folding, and enzymatic activity of the engineered variant of the disclosure as well as viability of the modified host cell.
  • the nucleotide sequence encoding the engineered variant is codon-optimized.
  • modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant 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, olivetolic acid, or hexanoyl-CoA) biosynthesis.
  • GPP geranylpyrophosphate
  • prenyl phosphates e.g., olivetolic acid, or hexanoyl-CoA
  • the nucleotide sequences encoding the polypeptides involved in cannabinoid or cannabinoid precursor are codon- optimized.
  • the modified host cells of the disclosure for producing cannabinoid or cannabinoid derivatives comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant 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 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 nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.
  • the modified host cell for producing 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. In some embodiments, the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides.
  • culturing of a modified host cell for producing cannabinoids or cannabinoid derivatives in a culture medium provides for synthesis of the cannabinoid or the cannabinoid derivative.
  • the modified host cells may express or overexpress one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant.
  • the nucleotide sequences encoding the engineered variants are codon-optimized.
  • the modified host cells of the disclosure for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant 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 expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant 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 nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.
  • the modified host cell for expressing an engineered variant of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant 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, aPDIl polypeptide, an EROl polypeptide, FAD1 polypeptide, aPEP4 polypeptide, and an IREl 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 nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.
  • the modified host cells of the disclosure comprise a deletion or downregulation of one or more of the following genes: a ROT2 gene or a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of one or more of the following genes: a ROT2 gene or a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of one or more of the following genes: a ROT2 gene or a PEP4 gene.
  • the secretory pathway polypeptides and heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant, or mouse.
  • the secretory pathway polypeptides and heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides may be derived from Pichia pastoris (now known as Komagataella phaffii ), Pichia fmlandica , Pichia trehalophila , Pichia koclamae , Pichia memhranaefaciens , 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 pomhe , Scheffer somyces stipites, Dekkera hruxellensis , 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.
  • the disclosure also encompasses orthologous genes encoding the secretory pathway polypeptides disclosed herein.
  • Exemplary secretory pathway polypeptides disclosed herein may also include a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide.
  • Exemplary KAR2 polypeptides disclosed herein may include a full-length
  • KAR2 polypeptide a fragment of a KAR2 polypeptide, a variant of a KAR2 polypeptide, a truncated KAR2 polypeptide, or a fusion polypeptide that has at least one activity of a KAR2 polypeptide.
  • Exemplary ROT2 polypeptides disclosed herein may include a full-length
  • ROT2 polypeptide a fragment of a ROT2 polypeptide, a variant of a ROT2 polypeptide, a truncated ROT2 polypeptide, or a fusion polypeptide that has at least one activity of a ROT2 polypeptide.
  • Exemplary PDI1 polypeptides disclosed herein may include a full-length
  • PDI1 polypeptide a fragment of a PDI1 polypeptide, a variant of a PDI1 polypeptide, a truncated PDI1 polypeptide, or a fusion polypeptide that has at least one activity of a PDI1 polypeptide.
  • Exemplary EROl polypeptides disclosed herein may include a full-length
  • EROl polypeptide a fragment of an EROl polypeptide, a variant of an EROl polypeptide, a truncated EROl polypeptide, or a fusion polypeptide that has at least one activity of an EROl polypeptide.
  • Exemplary FAD1 polypeptides disclosed herein may include a full-length
  • FAD1 polypeptide a fragment of a FAD1 polypeptide, a variant of a FAD1 polypeptide, a truncated FAD1 polypeptide, or a fusion polypeptide that has at least one activity of a FAD1 polypeptide.
  • Exemplary PEP4 polypeptides disclosed herein may include a full-length
  • PEP4 polypeptide a fragment of a PEP4 polypeptide, a variant of a PEP4 polypeptide, a truncated PEP1 polypeptide, or a fusion polypeptide that has at least one activity of a PEP4 polypeptide.
  • Exemplary IREl polypeptides disclosed herein may include a full-length
  • Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more of a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, a PEP4 polypeptide, or an IREl polypeptide.
  • the one or more modifications to modulate the expression of one or more of a KAR2 polypeptide, a ROT2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, a PEP4 polypeptide, or an IREl polypeptide may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of the KAR2 polypeptide, the PDI1 polypeptide, the EROl polypeptide, the FAD1 polypeptide, or the IREl polypeptide and/or deletion or downregulation of one or more genes encoding one or more of the ROT2 polypeptide or the PEP4 polypeptide in a host cell.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide resulting in expression or overexpression of the KAR2 polypeptide, the PDI1 polypeptide, the EROl polypeptide, the FAD1 polypeptide, or the IREl polypeptide.
  • the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, reducing or eliminating the expression of the ROT2 polypeptide or PEP4 polypeptide.
  • the one or more modifications to modulate the expression of one or more secretory pathway polypeptides may improve modified host cell viability. Improving modified host cell viability may improve the industrial fermentation process.
  • the EROl polypeptide may serve as a partner to the PDI1 polypeptide, a protein disulfide isomerase polypeptide. Modulating the expression of an IREl polypeptide may prevent degradation of expressed engineered variants of the disclosure.
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO:5 (a KAR2 polypeptide), SEQ ID NO:9 (a PDI1 polypeptide), SEQ ID NO:7 (an EROl polypeptide), SEQ ID NO: 192 (a FAD1 polypeptide), SEQ ID NO: 11 (an IRE1 polypeptide), or SEQ ID NO: 190 (a fragment IRE1 polypeptide).
  • SEQ ID NO:5 a KAR2 polypeptide
  • SEQ ID NO:9 a PDI1 polypeptide
  • SEQ ID NO:7 an EROl polypeptide
  • SEQ ID NO: 192 a FAD1 polypeptide
  • SEQ ID NO: 11 an IRE1 polypeptide
  • SEQ ID NO: 190 a fragment IRE1 polypeptide
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO:5 (a KAR2 polypeptide), SEQ ID NO:9 (a PDI1 polypeptide), SEQ ID NO:7 (an EROl polypeptide), SEQ ID NO: 192 (a FAD1 polypeptide), SEQ ID NO: 11 (an IRE1 polypeptide), or SEQ ID NO: 190 (a fragment IRE1 polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.
  • SEQ ID NO:5 a KAR2 polypeptide
  • SEQ ID NO:9 a PDI1 polypeptide
  • SEQ ID NO:7 an EROl polypeptide
  • SEQ ID NO: 192 a FAD1 polypeptide
  • SEQ ID NO: 11 an IRE1 polypeptide
  • SEQ ID NO: 190
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 5 (a KAR2 polypeptide), SEQ ID NO: 9 (a PDI1 polypeptide), SEQ ID NO: 5 (a KAR2 poly
  • the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides comprising the amino acid sequences set forth in SEQ ID NO: 13 (a ROT2 polypeptide) or SEQ ID NO: 15 (a PEP4 polypeptide).
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IREl polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding a FAD1 polypeptide.
  • the nucleotide sequences encoding the one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide are codon-optimized.
  • the modified host cells of the disclosure 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 modified host cells of the disclosure comprise a deletion or downregulation of genes encoding a ROT2 polypeptide and a PEP4 polypeptide.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a secretory pathway polypeptide, such as, a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide.
  • the nucleotide sequence is codon-optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a KAR2 polypeptide, such as, a full- length KAR2 polypeptide, a fragment of a KAR2 polypeptide, a variant of a KAR2 polypeptide, a truncated KAR2 polypeptide, or a fusion polypeptide that has at least one activity of a KAR2 polypeptide.
  • the nucleotide sequence is codon- optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a ROT2 polypeptide, such as, a full- length ROT2 polypeptide, a fragment of a ROT2 polypeptide, a variant of a ROT2 polypeptide, a truncated ROT2 polypeptide, or a fusion polypeptide that has at least one activity of a ROT2 polypeptide.
  • the nucleotide sequence is codon- optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PDI1 polypeptide, such as, a full- length PDI1 polypeptide, a fragment of a PDI1 polypeptide, a variant of a PDI1 polypeptide, a truncated PDI1 polypeptide, or a fusion polypeptide that has at least one activity of a PDI1 polypeptide.
  • the nucleotide sequence is codon-optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an EROl polypeptide, such as, a full- length EROl polypeptide, a fragment of an EROl polypeptide, a variant of an EROl polypeptide, a truncated EROl polypeptide, or a fusion polypeptide that has at least one activity of an EROl polypeptide.
  • the nucleotide sequence is codon- optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a FAD1 polypeptide, such as, a full- length FAD1 polypeptide, a fragment of a FAD1 polypeptide, a variant of a FAD1 polypeptide, a truncated FAD1 polypeptide, or a fusion polypeptide that has at least one activity of a FAD1 polypeptide.
  • the nucleotide sequence is codon- optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PEP4 polypeptide, such as, a full- length PEP4 polypeptide, a fragment of a PEP4 polypeptide, a variant of a PEP4 polypeptide, a truncated PEP1 polypeptide, or a fusion polypeptide that has at least one activity of a PEP4 polypeptide.
  • the nucleotide sequence is codon- optimized.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an IRE1 polypeptide, such as, a full- length IRE1 polypeptide, a fragment of an IRE1 polypeptide (e.g., missing the first 7 amino acids), a variant of an IRE1 polypeptide, a truncated IRE1 polypeptide, or a fusion polypeptide that has at least one activity of an IRE1 polypeptide.
  • the nucleotide sequence is codon-optimized.
  • one or more secretory pathway polypeptides such as a
  • KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide 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 nucleotide sequences encoding one or more secretory pathway polypeptides, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, 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 sequences encoding one or more secretory pathway polypeptides, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptid
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, a FAD1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide.
  • the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a secretory pathway polypeptide, such as a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide.
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences set forth in SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO:8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide), SEQ ID NO: 191 (encodes a FADl polypeptide), SEQ ID NO: 10 (encodes an IREl polypeptide), and SEQ ID NO: 189 (encodes a fragment IREl polypeptide).
  • SEQ ID NO:4 encodes a KAR2 polypeptide
  • SEQ ID NO:8 encodes a PDI1 polypeptide
  • SEQ ID NO:6 encodes an EROl polypeptide
  • SEQ ID NO: 191 encodes a FADl polypeptide
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences set forth in SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO:8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide), SEQ ID NO: 191 (encodes a FADl polypeptide), SEQ ID NO: 10 (encodes an IREl polypeptide), and SEQ ID NO: 189 (encodes a fragment IREl polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.
  • SEQ ID NO:4 encodes a KAR2 polypeptide
  • SEQ ID NO:8 encodes a PDI1 polypeptide
  • SEQ ID NO:6 encodes an EROl polypeptide
  • the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides selected from the group consisting of nucleotide sequences having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:4 (encodes a KAR2 polypeptide), SEQ ID NO: 8 (encodes a PDI1 polypeptide), SEQ ID NO:6 (encodes an EROl polypeptide
  • the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 12 (encodes a ROT2 polypeptide) and SEQ ID NO: 14 (encodes a PEP4 polypeptide).
  • the modified host cells of the disclosure comprise a deletion or downregulation of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a ROT2 gene.
  • the modified host cells of the disclosure comprise a deletion or downregulation of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PEP4 gene.
  • the modified host cells of the disclosure comprise a deletion or downregulation of a PEP4 gene and a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PEP4 gene and a ROT2 gene.
  • the modified host cells of the disclosure comprise a downregulation of a PEP4 gene and a ROT2 gene.
  • a modified host cell of the present disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may also comprise 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
  • such polypeptides may include, but are not limited to: a geranyl pyrophosphate: olivetolic acid geranyltransferase (GOT) polypeptide, a tetraketide synthase (TKS) polypeptide, an olivetolic acid cyclase (OAC) 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 poly
  • GPP mevalonate
  • nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor are codon-optimized.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA
  • polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis and heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant (e.g., Cannabis), or mouse.
  • the disclosure also encompasses orthologous genes encoding the polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis disclosed herein.
  • 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 disclosed herein.
  • THCAS tetrahydrocannabinolic acid synthase
  • the tetrahydrocannabinolic acid synthase polypeptide has an amino acid sequence of SEQ ID NO:44.
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, wherein the engineered variant comprises the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 96,
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, wherein the engineered variant comprises the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 50, SEQ ID NO
  • 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.
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,
  • a modified host cell of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) polypeptide disclosed herein, wherein the nucleotide sequence is that set forth in SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO NO:
  • At least one of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure is operably linked to an inducible promoter. In some embodiments, at least one of the one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure is operably linked to a constitutive promoter.
  • a 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.
  • the GOT polypeptide has aromatic prenyltransferase (PT) activity.
  • PT aromatic prenyltransferase
  • the GOT polypeptide modifies a cannabinoid precursor or a cannabinoid precursor derivative.
  • the GOT polypeptide modifies olivetolic acid or an olivetolic acid derivative.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 17.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 17.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 17.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the GOT polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 17.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a GOT polypeptide, such as, 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 nucleotide sequence is codon-optimized.
  • the GOT polypeptide is 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 GOT polypeptide, 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 GOT polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide.
  • the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GOT polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 16.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 16, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 16.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 16.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 16.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GOT polypeptide, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 16.
  • 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.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 188, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 188.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 188.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the NphB polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 188.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a NphB polypeptide, such as, 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 nucleotide sequence is codon- optimized.
  • the NphB polypeptide is 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 NphB polypeptide, 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 NphB polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide.
  • the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the NphB polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 187.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 187, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 187.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 187.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 187.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 187. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide, wherein the nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 187.
  • 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.
  • 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 CsAAEl 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).
  • cannabinoid derivatives e.g., cannabigerolic acid derivatives
  • 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 cannabinoids or cannabinoid derivatives.
  • the cell culture medium comprising the modified host cells comprises unsubstituted or substituted hexanoate.
  • the cell culture medium comprising the modified host cells comprises a carboxylic acid other than unsubstituted or substituted hexanoate.
  • 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 disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:23.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:23.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the AAE polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:23.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an AAE, FAA, or fatty acyl-CoA ligase polypeptide, such as, 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.
  • the nucleotide sequence is codon- optimized.
  • one or more AAE, FAA, or fatty acyl-CoA ligase polypeptide 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 AAE, FAA, or fatty acyl-CoA ligase polypeptide, 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 a nucleotide sequence encoding the AAE, FAA, or fatty acyl-CoA ligase polypeptide 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 heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide.
  • the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide.
  • the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding an AAE, FAA, or fatty acyl-CoA ligase polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:22.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:22, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:22.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AAE polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:22.
  • 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 and OAC polypeptides.
  • TKS and OAC polypeptides have been found to have broad substrate specificity, enabling production of cannabinoid derivatives or cannabinoids.
  • 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide.
  • Exemplary TKS or OAC polypeptides disclosed herein may include a full- length TKS or OAC polypeptide, a fragment of a TKS or OAC polypeptide, a variant of a TKS or OAC polypeptide, a truncated TKS or OAC polypeptide, or a fusion polypeptide that has at least one activity of a TKS or OAC polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 19.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 19.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the TKS polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 19.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:48, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:21 or SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:21.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:21.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:21.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:48, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:48.
  • OAC Y27F variant
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:48.
  • OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:48.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide comprising an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:48.
  • OAC Y27F variant
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a TKS or OAC polypeptide, such as, a full-length TKS or OAC polypeptide, a fragment of a TKS or OAC polypeptide, a variant of a TKS or OAC polypeptide, a truncated TKS or OAC polypeptide, or a fusion polypeptide that has at least one activity of a TKS or OAC polypeptide.
  • the nucleotide sequence is codon-optimized.
  • the TKS polypeptide is 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 TKS polypeptide, 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 TKS polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide.
  • the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has nine copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide.
  • the modified host cell has ten copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has eleven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has twelve copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide. In some embodiments, the modified host cell has twelve or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the TKS polypeptide.
  • the OAC polypeptide is 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 OAC polypeptide, 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 OAC polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide.
  • the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has nine copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide.
  • the modified host cell has ten copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has eleven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has twelve copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide. In some embodiments, the modified host cell has twelve or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the OAC polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 18.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 18, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 18.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 18.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20 or SEQ ID NO:47.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20 or SEQ ID NO:47, or a codon degenerate nucleotide sequence of any of the foregoing.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:20 or SEQ ID NO:47.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:20 or SEQ ID NO:47.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:20, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:20.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:20.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:47.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:47, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:47.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an OAC polypeptide, wherein the OAC polypeptide is a variant OAC (Y27F variant) polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:47.
  • 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.
  • 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.
  • 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.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:41, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:41.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:41.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide comprising an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:41.
  • the mutation in this amino acid sequence shifts the ratio of GPP to farnesyl diphosphate (FPP), increasing the production of the GPP required to produce CBDA or THCA.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a GPPS polypeptide, such as, 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.
  • the nucleotide sequence is codon- optimized.
  • the GPPS polypeptide is 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 GPPS polypeptide, 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 GPPS polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide.
  • the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the GPPS polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:40.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:40, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:40.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a GPPS polypeptide, wherein the GPPS polypeptide is a variant GPPS (ERG20mut, F96W, N127W) polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:40.
  • 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:35.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:35.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the PDC polypeptide comprises an amino acid sequence having at least
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a PDC polypeptide, such as, 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.
  • the nucleotide sequence is codon-optimized.
  • the PDC polypeptide is 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 PDC polypeptide, 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 PDC polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the PDC polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:34.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:34, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:34.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDC polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:34.
  • 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 disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl- CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:31.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the acetoacetyl-CoA thiolase polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:31.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes an acetoacetyl-CoA thiolase polypeptide, such as, 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.
  • the nucleotide sequence is codon-optimized.
  • the acetoacetyl-CoA thiolase polypeptide is 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 acetoacetyl-CoA thiolase polypeptide, 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 acetoacetyl- CoA thiolase polypeptide 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 heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide.
  • the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the acetoacetyl-CoA thiolase polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl- CoA thiolase polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:30.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:30, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:30.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:30.
  • 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-CoAto generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoAthiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoAto 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 (
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding a MEV pathway polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than one MEV pathway polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than two MEV pathway polypeptides.
  • a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than three MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than four MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than five MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding more than six MEV pathway polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding all MEV pathway polypeptides.
  • Exemplary MEV pathway polypeptides disclosed herein may include a full- length MEV pathway polypeptide, a fragment of a MEV pathway polypeptide, a variant of a MEV pathway polypeptide, a truncated MEV pathway polypeptide, or a fusion polypeptide that has at least one activity of a MEV pathway polypeptide.
  • the one or more MEV pathway polypeptides are selected from the group consisting of an acetoacetyl-CoA thiolase polypeptide, a HMGS polypeptide, a HMGR polypeptide, an MK polypeptide, a PMK polypeptide, an MVD1 polypeptide, and an IDI1 polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:29.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:29.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the HMGS polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:29.
  • the HMGR polypeptide is a truncated HMGR
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:27.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:27.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the tHMGR polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:27.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:39, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:39.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:39.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the MK polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:39.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises the amino acid sequence set forth in SEQ ID NO:37, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:37.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:37.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the PMK polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:37.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:33.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:33.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the MVD1 polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:33.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the IDI1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25, or a conservatively substituted amino acid sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:25.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the IDIl polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:25.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the IDI1 polypeptide comprises an amino acid sequence having 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:25.
  • Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a MEV pathway polypeptide, such as, a full-length MEV pathway polypeptide, a fragment of a MEV pathway polypeptide, a variant of a MEV pathway polypeptide, a truncated MEV pathway polypeptide, or a fusion polypeptide that has at least one activity of a polypeptide that is part of the MEV pathway.
  • a MEV pathway polypeptide such as, a full-length MEV pathway polypeptide, a fragment of a MEV pathway polypeptide, a variant of a MEV pathway polypeptide, a truncated MEV pathway polypeptide, or a fusion polypeptide that has at least one activity of a polypeptide that is part of the MEV pathway.
  • the nucleotide sequence is codon-optimized.
  • one or more MEV pathway 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 nucleotide sequences encoding a MEV pathway polypeptide, 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 sequences encoding a MEV pathway polypeptide to a strong promoter.
  • the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide.
  • the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a MEV pathway polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:28.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:28, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:28.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMGS polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:28.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:26.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:26, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:26.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:26.
  • a modified host cell of the present disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence that encodes a tHMGR polypeptide. In some embodiments, a modified host cell of the present disclosure comprises two heterologous nucleic acids comprising a nucleotide sequence that encodes a tHMGR polypeptide.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:38.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:38, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:38.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MK polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:38.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:36.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:36, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:36.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a PMK polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:36.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:32.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:32, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:32.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a MVD1 polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:32.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:24.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:24, or a codon degenerate nucleotide sequence thereof.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:24.
  • a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding an IDIl polypeptide, wherein the nucleotide sequence has 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%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:24.
  • 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure may be for producing cannabinoids or cannabinoid derivatives and/or for expressing an engineered variant of the disclosure.
  • modified host cells for producing cannabinoids or cannabinoid derivatives.
  • modified host cells disclosed herein 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 EROl 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.
  • GPP geranylpyrophosphate
  • prenyl phosphates olivetolic acid
  • a modified host cell for producing cannabinoids or cannabinoid derivatives may comprise 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 for producing cannabinoids or cannabinoid derivatives may comprise a deletion of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell for producing cannabinoids or cannabinoid derivatives may comprise a downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a codon- optimized nucleotide sequence encoding an engineered variant of the disclosure.
  • GPP geranylpyrophosphate
  • prenyl phosphates olivetolic acid, or hexanoyl-CoA
  • the disclosure also provides modified host cells modified to express or overexpress one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and one or more heterologous nucleic acids disclosed herein comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, aFADl polypeptide, or an IRE1 polypeptide.
  • the modified host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding the 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 may comprise a deletion of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell may comprise a downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the nucleotide sequence encoding the engineered variant of the disclosure is a codon-optimized nucleotide sequence.
  • the nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide are codon-optimized.
  • expression or overexpression of one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure in a modified host cell may be done in combination with expression or overexpression by the modified host cell of one or more heterologous nucleic acids disclosed herein (e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide) and/or with deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • heterologous nucleic acids disclosed herein e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide,
  • the nucleotide sequence encoding the engineered variant of the disclosure is a codon-optimized nucleotide sequence.
  • expression or overexpression of one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant in a modified host cell may be done in combination with expression or overexpression by the modified host cell of one or more heterologous nucleic acids disclosed herein (e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide) and/or with deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the nucleotide sequence encoding the engineered variant is
  • a modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for producing cannabinoids or cannabinoid derivatives 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 150% at least 200%, at least 500%, or at least 1000% 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure 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 150% at least 200%, at least 500%, or at least 1000% 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence en
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure
  • the modified host cell 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FADl polypeptide, or an IRE1 polypeptide 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide 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 150% at least 200%, at least 500%, or at least 1000% greater than an amount of the cannabinoid or the cannabinoid derivative produced by a modified host cell comprising one or more
  • the modified host cell of the disclosure comprising 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, a FAD1 polypeptide, an EROl polypeptide, or an IREl polypeptide
  • the modified host cell 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 hex
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engine
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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 150% at least 200%, at least 500%, or at least 1000% 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 a tetrahydrocannabinolic acid
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide 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 150% at least 200%, at least 500%, or at least 100
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, 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 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., ger
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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), pren
  • GPP
  • the modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for producing cannabinoids or cannabinoid derivatives has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for producing cannabinoids or cannabinoid derivatives has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for producing cannabinoids or cannabinoid derivatives has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for expressing an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 expressing an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for expressing an engineered variant of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 for expressing an engineered variant of the disclosure has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or biomass yield similar to, or lower than, a growth rate and/or biomass yield and an increased titer of THCA compared to a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, 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 comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure
  • the modified host cell 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptid
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FADl polypeptide, or an IREl polypeptide has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydro
  • the modified host cell of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide
  • the modified host cells 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 hex
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising 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 has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, 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 comprising one or more nucleic acids comprising 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 has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a faster growth rate and/or higher biomass yield compared to a growth rate and/or higher biomass yield of a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a growth rate and/or higher 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 than a growth rate and/or higher biomass yield of a modified host cell comprising
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, 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 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., ger
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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), pren
  • GPP
  • a modified host cell of the disclosure for producing cannabinoids or cannabinoid derivatives produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • CBCA cannabinoid
  • the modified host cell for producing cannabinoids or cannabinoid derivatives produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • a modified host cell of the disclosure for expressing an engineered variant of the disclosure produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • CBCA cannabinoid
  • the modified host cell for expressing an engineered variant of the disclosure produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • CBCA cannabinoid
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure
  • the modified host cell 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a
  • a modified host cell of the disclosure comprising 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 EROl polypeptide, a FADl polypeptide, or an IREl polypeptide produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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:
  • the modified host cell of the disclosure comprising 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 EROl polypeptide, a FADl polypeptide, or an IRE1 polypeptide
  • the modified host cell 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 hex
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising 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 produces THCA from CBGA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, 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 comprising one or more nucleic acids comprising 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 produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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, or hexanoyl-CoA
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in an increased ratio of THCA over another a cannabinoid (e.g., CBCA) compared to that produced by a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucle
  • a modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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:
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, 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 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., ger
  • a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, 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), pren
  • GPP
  • the growth and/or viability of modified host cells of the disclosure for producing cannabinoids or cannabinoid derivatives is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure for producing cannabinoids or cannabinoid derivatives has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same
  • the growth and/or viability of modified host cells of the disclosure for expressing an engineered variant of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure for expressing an engineered variant of the disclosure has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under
  • the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to 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 modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure
  • the modified host cell 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 growth and/or viability of modified host cells of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IRE1 polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least
  • the modified host cell of the disclosure comprising 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 EROl polypeptide, a FAD1 polypeptide, or an IREl polypeptide
  • the modified host cell 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 hex
  • the growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising 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 is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising 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 has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 110% or more, at least 120% or more, at least 130% or more, at least 140% or more, or at least 150% or more compared to the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising 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 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 growth and/or viability of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell.
  • a culture of modified host cells of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide, and a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide has a cell density of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more at least 90% or more, at least 95% or more, at least 100% or more, at least 11
  • the modified host cell of the disclosure comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, 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 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., ger
  • Parent host cells that are suitable for use in generating a modified host cell of the present disclosure may include eukaryotic cells.
  • the eukaryotic cells are yeast cells.
  • Host cells are in some embodiments unicellular organisms, or are grown in culture as single cells.
  • 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 fmlandica , 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
  • 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: PEP 4 , PRBf 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. For instance, modified Komagataella phaffli 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 modified host cells comprising one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant 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 nucleic acids comprise codon-optimized nucleotide sequences.
  • the present disclosure provides for modified host cells and methods of making modified host cells for producing a cannabinoid or a cannabinoid derivative, comprising introducing into a host cell one or more nucleic acids (e.g., heterologous) disclosed herein.
  • the nucleic acids comprise codon-optimized nucleotide sequences.
  • the disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising a) introducing into a host cell one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure.
  • the method comprises b) introducing into the host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide.
  • the method comprises b) introducing into the host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a codon-optimized nucleotide sequence encoding an engineered variant of the disclosure.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more heterologous nucleic acids comprising nucleotide sequences 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 EROl polypeptide, or an IREl polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more heterologous nucleic acids comprising nucleotide sequences 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 EROl polypeptide or a FAD1 polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising 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 ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell 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 for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing into a host cell one or more nucleic acids comprising 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 for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide and a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, 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 EROl polypeptide, or an IRE1 polypeptide, and c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the disclosure provides a method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, 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 and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and 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.
  • GPP geranylpyrophosphate
  • prenyl phosphates e.g., olivetolic acid, or hexanoyl-CoA
  • the methods of making a modified host cell for producing a cannabinoid or a cannabinoid derivative comprise introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, and 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
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IRE1 polypeptide and a deletion or downregulation of the genes encoding the ROT2 polypeptide and the PEP4 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide, and 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide.
  • the modified host cell for producing a cannabinoid or a cannabinoid derivative comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the present disclosure provides for a method of making a modified host cell for expressing an engineered variant of the disclosure, the method comprising introducing into a host cell one or more nucleic acids disclosed herein.
  • the disclosure provides a method of making a modified host cell for expressing an engineered variant 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 and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IRE1 polypeptide.
  • the disclosure provides a method of making a modified host cell for expressing an engineered variant 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 and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a codon-optimized nucleotide sequence encoding the engineered variant of the disclosure.
  • the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered variant of the disclosure and comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure 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 of a ROT2 polypeptide or a PEP4 polypeptide.
  • the modified host cell 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 for expressing an engineered variant 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 and b) 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 for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, and one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IREl polypeptide and a deletion or downregulation of one or more genes encoding the ROT2 polypeptide and the PEP4 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the disclosure provides a method of making a modified host cell for expressing an engineered variant 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 EROl polypeptide, or an IRE1 polypeptide, and c) a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide.
  • the disclosure provides a method of making a modified host cell for expressing an engineered variant 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 and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure may comprise one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure and 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.
  • cannabinoid or cannabinoid precursor e.g., geranylpyrophosphate (GPP), prenyl phosphates, olivetolic acid, or hexanoyl-CoA
  • the methods of making a modified host cell for expressing an engineered variant of the disclosure comprise introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in cannabinoid or cannabinoid precursor biosynthesis.
  • the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or an IREl polypeptide, a deletion or downregulation of one or more genes encoding one or more of a ROT2 polypeptide or a PEP4 polypeptide, and 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
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the IRE1 polypeptide and a deletion or downregulation of the genes encoding the ROT2 polypeptide and the PEP4 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered variant of the disclosure, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more of a KAR2 polypeptide, a PDI1 polypeptide, an EROl polypeptide, or a FAD1 polypeptide and 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 comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding the KAR2 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the PDI1 polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the EROl polypeptide, one or more heterologous nucleic acids comprising a nucleotide sequence encoding the FAD1 polypeptide.
  • the modified host cell for expressing an engineered variant of the disclosure comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 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.
  • a nucleic acid e.g., heterologous
  • 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.
  • 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 engineered variant expression level and/or the production of cannabinoids or cannabinoid derivatives in a modified host cell may be done by changing the gene copy number, promoter strength, and/or promoter regulation.
  • nucleic acids e.g., heterologous
  • 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).
  • nucleic acids e.g., heterologous
  • 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.
  • 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 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 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 a cannabinoid synthase polypeptide, 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 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.
  • nucleic acids e.g., heterologous
  • yeast the low copy CEN ARS and high copy 2 micron plasmids.
  • 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 or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector.
  • nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, four or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, five or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, six or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector. In some embodiments, seven or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression vector.
  • two or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.
  • three or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.
  • four or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.
  • five or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.
  • six or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression vectors.
  • nucleic acids e.g., heterologous
  • eight or more nucleic acids e.g., heterologous
  • nine or more nucleic acids e.g., heterologous
  • ten or more nucleic acids e.g., heterologous
  • one or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • two or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • three or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • four or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • five or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • nucleic acids e.g., heterologous
  • six or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • seven or more of the nucleic acids (e.g., heterologous) disclosed herein are present in a single expression construct.
  • two or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.
  • three or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.
  • four or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.
  • five or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.
  • six or more nucleic acids (e.g., heterologous) disclosed herein are in separate expression constructs.
  • nucleic acids e.g., heterologous
  • eight or more nucleic acids e.g., heterologous
  • nine or more nucleic acids e.g., heterologous
  • ten or more nucleic acids e.g., heterologous
  • 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.
  • 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.
  • the modified host cell has one copy of a nucleic acid
  • the modified host cell has two copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has three copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has four copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein.
  • the modified host cell has five copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has six copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has seven copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein.
  • the modified host cell has eight copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has nine copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has ten copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein.
  • the modified host cell has eleven copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve or more copies of a nucleic acid (e.g., heterologous) comprising a nucleotide sequence encoding a polypeptide disclosed herein.
  • 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 Emymology , 153:516-544).
  • 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.
  • strong constitutive promoters include, but are not limited to: pTDH3 and pFBAl.
  • medium constitutive promoters include, but are not limited to: pACTl and pCYCl.
  • An example of a weak constitutive promoter includes, but is not limited to: pSLNl.
  • strong inducible promoters include, but are not limited to: pGALl and pGALlO.
  • An example of a medium inducible promoter includes, but is not limited to: pGAL7.
  • a weak inducible promoter includes, but is not limited to: pGAL3.
  • suitable eukaryotic promoters may include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector, construct, and promoter is well within the level of ordinary skill in the art.
  • the expression vector or construct may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector or construct may also include appropriate sequences for amplifying expression.
  • yeast a number of vectors or constructs containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et al., 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
  • a constitutive yeast promoter such as pADH, pTDH3, pFBAl, pACTl, pCYCl, and pSLNl or an inducible promoter such as pGALl, pGALlO, pGAL7, and pGAL3 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the S. cerevisiae TRPl gene or a gene cassette encoding resistance to an antibiotic, etc.; and a promoter derived from a highly-expressed gene to direct transcription of the coding sequence.
  • promoters can be derived from genetic sequences encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.
  • inducible promoters are well known in the art. Suitable inducible promoters may include, but are not limited to, a tetracycline-inducible promoter; an estradiol inducible promoter, a sugar inducible promoter, e.g, pGall or pSUC2, an amino acid inducible promoter, e.g. pMet25; a metal inducible promoter, e.g. pCupl, a methanol-inducible promoter, e.g. pAOXl, and the like.
  • the expression vectors or constructs will in many embodiments contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture.
  • 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.
  • 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.
  • 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.
  • 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.
  • DmP mRNA perturbation
  • 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.
  • microbes such as yeast have evolved a wide range of natural inducible promoter systems. Any promoter that is regulated by a small molecule or change in environment (temperature, pH, oxygen level, osmolarity, oxidative damage) can in principle be converted into an inducible system for the expression of heterologous genes.
  • the best known system in S. cerevisiae is the galactose regulon, which is strongly repressed by glucose and activated by galactose.
  • galactose-inducible promoters are regulated in the same way, and thus an engineered strain can be grown in glucose media to build biomass, and then switched to galactose to induce pathway expression.
  • a range of expression levels can be achieved, from very strong pGALl to relatively weak pGAL3.
  • galactose may be expensive and a poor carbon source for S. cerevisiae. Therefore, for industrial applications, it may be advantageous to re-engineer the regulon such that the cells can be induced in a non galactose media.
  • the galactose regulon can be modified for this purpose in many ways, including:
  • GAL80 Overexpressing the negative regulator of GAL80, GAIA, from an inducible promoter, e.g. pSUC2-GAL3, such that switching from glucose to sucrose relieves GAL80 expression and activates the pathway.
  • an inducible promoter e.g. pSUC2-GAL3
  • the pSUC2 promoter is used to induce the galactose regulon in sucrose media.
  • any inducible promoter can be used for this purpose, or for control of individual genes outside of the context of the galactose regulon. The list below provides some examples:
  • Phosphate regulated promoters e.g. pPH05
  • Carbon source regulated promoters e.g. pADH2
  • Temperature regulated promoters e.g. pHSP12, pHSP26
  • pH regulated promoters e.g. pHSP12, pHSP26
  • Oxygen level regulated promoters e.g. pDANl
  • Oxidative stress regulated promoters e.g. promoters from AHPl, TRR1, TRX2, TSAI, GPX2, GSH1, GSH2, GLR1, SOD1, or SOD2 genes.
  • ER stress regulated promoters e.g. unfolded protein response element promoters.
  • Estradiol-inducible systems involving fusion of the estradiol receptor to DNA-binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites.
  • tTA tet Trans Activator
  • rtTA reverse tet Trans Activator
  • one of the above inducible promoter systems is used in a modified host cell of the disclosure.
  • the inducible promoter system is a natural inducible promoter system.
  • the inducible promoter system is a synthetic inducible promoter system.
  • a suitable media for culturing modified host cells of the disclosure comprises one or more of the inducers disclosed herein. Possible inducers include:
  • Phosphate regulated promoters e.g. pPH05 o KH2PO4
  • Carbon source regulated promoters e.g. pADH2 o
  • Galactose e.g. pGALl
  • Glucose e.g. pADH2
  • Sucrose e.g. pSUC2, pGPHl, pMAL12
  • Maltose e.g. pMAL12, pMAL32
  • Amino acid regulated promoters e.g. pMET25 o Methionine (e.g. pMET25) o Lysine (e.g. pLYS9) o Other amino acids
  • Metal ion induced promoters e.g. pCUPl o CuS0 4
  • Temperature regulated promoters e.g. pHSP12, pHSP26 o Change in temperature, e.g. 30 °C to 37 °C
  • pH regulated promoters e.g. pHSP12, pHSP26 o Change in pH, e.g. pH 6 to pH 4
  • Oxygen level regulated promoters e.g. pDANl o Change in oxygen level, e.g. 20% to 1% dissolved oxygen levels
  • Oxidative stress regulated promoters e.g. pSODl o Addition of hydrogen peroxide or superoxide-generating drug menadione
  • ER stress regulated promoters e.g. unfolded protein response element promoters.
  • Tunicamycin or expression of proteins prone to misfolding (e.g., cannabinoid synthases)
  • estradiol-inducible systems involving fusion of the estradiol receptor to DNA- binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites.
  • a nucleic acid disclosed herein is modified or optimized such that the nucleotide sequence reflects the codon preference for the particular host cell.
  • the nucleotide sequence will in some embodiments be modified or optimized for yeast codon preference.
  • a nucleotide sequence disclosed herein is codon- optimized. See, e.g., Bennetzen and Hall (1982) J Biol. Chem. 257(6): 3026-3031.
  • the codon usage of a nucleotide sequence is modified or optimized such that the level of translation of the encoded mRNA is decreased.
  • a codon-optimized nucleotide sequence may be optimized such that the level of translation of the encoded mRNA is decreased. Reducing the level of translation of an mRNA by modifying codon usage may be achieved by modifying the nucleotide sequence to include codons that are rare or not commonly used by the host cell. Codon usage tables for many organisms are available that summarize the percentage of time a specific organism uses a specific codon to encode for an amino acid. Certain codons are used more often than other, “rare” codons.
  • a nucleotide sequence is modified by introducing one or more rare codons, which affect the rate of translation, but not the amino acid sequence of the polypeptide translated.
  • rare codons there are six codons that encode for arginine: CGT, CGC, CGA, CGG, AGA, and AGG.
  • CGT and CGC are used far more often (encoding approximately 40% of the arginines in E. coli each) than the codon AGG (encoding approximately 2% of the arginines in E. coli). Modifying a CGT codon within the sequence of a gene to an AGG codon would not change the sequence of the polypeptide, but would likely decrease the gene’s rate of translation.
  • a codon-optimized nucleotide sequence may be optimized for expression in a yeast cell.
  • the yeast cell is Saccharomyces cerevisiae.
  • the disclosure provides methods for expressing an engineered variant of the tetrahydrocannabinolic acid synthase (THCAS) 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 tetrahydrocannabinolic acid synthase (THCAS) 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 present disclosure also provides methods of producing a cannabinoid or a cannabinoid derivative.
  • the methods of the present disclosure may involve production of cannabinoids or cannabinoid derivatives using an engineered variant disclosed herein.
  • 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 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 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), A 9 -trans-tetrahydrocannabinol (D 9 -THC) type (e.g.
  • CBC cannabichromene
  • CBD cannabidiol
  • D 9 -THC A 9 -trans-tetrahydrocannabinol
  • a 9 -tetrahydrocannabinolic acid A 8 -trans-tetrahydrocannabinol (D 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 M.A. and Slade D., Life Sci. 2005 Dec 22;78(5):539-48. Epub 2005 Sep 30.
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 100 mg/L culture medium.
  • the cannabinoid or cannabinoid derivative is produced in an amount of more than 50 mg/L culture medium.
  • 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, cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), CBD A, cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-Ci), D 9 - tetrahydrocannabinolic acid A (THCA-A), D 9 -tetrahydrocannabinolic acid B (THCA-B), D 9 - tetra
  • 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 D 9 - tetrahydrocannabinolic acid, A 9 -tetrahydrocannabinol, A 8 -tetrahydrocannabinolic acid, D 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, canna
  • the cannabinoid produced with the engineered variants, methods, or modified host cells of the present disclosure is tetrahydrocannabinolic acid, tetrahydrocannabivarinic acid, or tetrahydrocannabivarin.
  • 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.
  • 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, THC, THCA, THCVA, CBDVA, (6aR,10aR)-l-hydroxy-6,6,9-trimethyl-3-butyl-6a,7,8,10a-tetrahydro-6H-dibenzo[b,d]pyran-
  • 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, (l'R,2'R)-4-(hexan-2-yl)-5'-methyl-2'-(prop-l-en-2-yl)- 1 ',2',3 ',4'-tetrahydro-[ 1 , 1 '-biphenyl]-2,6-diol, (l'R,2'R)-4-hexyl-5'-methyl-2'-(prop- 1 -en-2- yl)-l',2',3',4'-tetrahydro-[l,r-biphenyl]-2,6-diol, (rR,2'R)-5'-methyl-4-(3-methylpentyl)-2'- (prop- 1 -en-2-yl)- 1 ',2',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. [0380] A cannabinoid derivative may lack one or more chemical moieties found in 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, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylalkenyl, cycloalkenylalkyl, cycloalkenylalkenyl, heterocyclylalkenyl, heteroarylalkenyl, arylalkenyl, heterocyclyl, aralkyl, cycloalkylalkyl, heterocyclylalkyl, heteroarylalkyl, and the like.
  • a cannabinoid derivative lacking one or more chemical moieties found in a naturally-occurring cannabinoid 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.
  • 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, heteroarylalken
  • 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.
  • Ci-C6alkyl groups contain 1 to 6 carbon atoms.
  • Examples of a Ci-C6alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec- butyl and /e/V-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.
  • 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.
  • the term “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., polyethylene 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, flu
  • 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-D03 A); 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
  • Gd-HP-D03 A Manganes
  • 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, fluorocyclobutanse, 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.
  • isotopically labeled cannabinoids or cannabinoid derivatives of this disclosure are useful in drug and/or substrate tissue distribution studies.
  • the 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), U C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F, 35 S, 36 C1 , 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, and 131 I. Substitution with positron emitting isotopes, such as U C, 18 F, 15 0, 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.
  • 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 fV)-enantiomer In some embodiments the cannabinoids or cannabinoid derivatives may be the (//(-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.
  • 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.
  • 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-di sulfonate), 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, laur
  • “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 a method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of an engineered variant of of the disclosure.
  • 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 a THCAS polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure.
  • the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 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 a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence
  • the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.
  • the cannabinoid is THCA and the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced in a method comprising use of a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 instead of the engineered variant of the disclosure.
  • the engineered variant of the disclosure and the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44 are used under similar conditions for the same length of time.
  • the cannabinoid is THCA and the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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,
  • 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. 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.
  • culturing of the modified host cells of the disclosure in a culture medium provides for synthesis of a 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.
  • 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; 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 cannabinoid or cannabinoid derivative 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; from the cell lysate, the modified host cell, and the culture medium; or from the cell-free reaction mixture comprising one or more polypeptides 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.
  • 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.
  • the carboxylic acid may be the fV)-enantiomer
  • the carboxylic acid may be the (//(-enantiomer
  • the carboxylic acid may be the (+) or (-) enantiomer.
  • 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 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, dode
  • Carboxylic acids may include unsubstituted or substituted C1-C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C 3 -C18 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C 3 -C12 carboxylic acids. Carboxylic acids may include unsubstituted or substituted C4-C1 0 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 Cs carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted G carboxylic acid.
  • the carboxylic acid is an unsubstituted or substituted C7 carboxylic acid. In some embodiments, the carboxylic acid is an unsubstituted or substituted G 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.
  • 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-phenylyheptanoic acid, and the like.
  • 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 -phenyl valeric 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 C3-C18 carboxylic acid.
  • the unsubstituted or substituted C3-C18 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
  • 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
  • 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 2- methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-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 -phenyl valeric acid, 6-phenylhexanoic 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 2- methylhexanoic acid, 3-methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-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 -phenyl valeric acid, 6-phenylhexanoic 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 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, 4-phenylbutyric acid, 5-phenylvaleric acid, 6-phenylhexanoic acid, or 7-phenylheptanoic 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 2- methylhexanoic acid, 4-methylhexanoic acid, 5-methylhexanoic acid, 2-hexenoic acid, 3- hexenoic acid, 4-hexenoic acid, heptanoic acid, 5 -chi or oval eric acid, or 5- (methylsulfanyl)valeric 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 disclosure also 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 olivetolic acid or an olivetolic acid derivative.
  • the method comprises recovering the produced cannabinoid or cannabinoid derivative.
  • the produced cannabinoid or cannabinoid derivative is then purified as disclosed herein.
  • Olivetolic acid derivatives used herein may be substituted with or comprise one or more reactive and/or functional groups as disclosed herein.
  • an olivetolic acid derivative may lack one or more chemical moieties found in olivetolic acid.
  • the culture medium comprises an olivetolic acid derivative, the olivetolic acid derivative is orsellinic acid.
  • the culture medium comprises an olivetolic acid derivative, the olivetolic acid derivative is divarinic 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 disclosure provides methods of using a modified host cell of the disclosure for producing a cannabinoid or cannabinoid derivative.
  • 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 instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant.
  • the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant are cultured under similar culture 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 instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising
  • the modified host cell of the disclosure and the modified host cell comprising one or more nucleic acids comprising the nucleotide sequence encoding the tetrahydrocannabinolic acid synthase polypeptide having the amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant are cultured under similar culture conditions for the same length of time.
  • the cannabinoid is THCA and the method produces THCA in an increased ratio of THCA over another cannabinoid (e.g., CBCA) compared to that produced in a method instead comprising culturing a modified host cell comprising one or more nucleic acids comprising a nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44, but lacking a nucleic acid comprising a nucleotide sequence encoding an engineered variant, grown under similar culture conditions for the same length of time.
  • CBCA cannabinoid
  • the cannabinoid is THCA and the method produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • 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).
  • the culture medium can be supplemented with a fermentable sugar (e.g., a hexose sugar, e.g., glucose, xylose, 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 hexanoate, carboxylic acids other than unsubstituted or substituted hexanoate, 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, com 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 KH2PO4, galactose, glucose, sucrose, maltose, an amino acid (e.g., methionine, lysine), CuSCri, 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.
  • 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 provide 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.
  • minimal medium may refer to growth medium containing the minimum nutrients possible for cell growth, generally, but not always, without the presence of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids).
  • Minimal medium typically contains: (1) a carbon source 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; and (3) water.
  • modified host cells disclosed herein are grown in rich medium.
  • 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).
  • YPD yeast extract peptone dextrose
  • the rich medium or rich media comprises YP + 20 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).
  • 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 more rapid cell growth 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% CO2, at about 0% to about 100% dissolved oxygen, and at a pH between about 2 to about 9).
  • modified host cells disclosed herein are grown at about 34 °C in a suitable cell culture medium.
  • 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.
  • 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 6.5 to about 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 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 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,
  • 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%
  • Modified host cells disclosed herein 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.
  • 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.
  • the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, 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.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 mg/L culture medium to about 500 mg/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 mg/L culture medium to about 100 mg/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative 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.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of 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.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about from about 50 mg/L culture medium to about 100 mg/L culture medium, 50 mg/L culture medium to about 150 mg/L culture medium, from about 50 mg/L culture medium to about 200 mg/L culture medium, from about 50 mg/L culture medium to about 250 mg/L culture medium, from about 50 mg/L culture medium to about 500 mg/L culture medium, or from about 50 mg/L culture medium to about 750 mg/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of from about 50 mg/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of from about 50 mg/L culture medium to about 100 mg/L culture medium, or more than 100 mg/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of more than 50 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein, in an amount of more than 100 mg/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 100 mg/L culture medium to about 500 mg/L culture medium, or more than 500 mg/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 500 mg/L culture medium to about 1 g/L culture medium, or more than 1 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 10 g/L culture medium, or more than 10 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 20 g/L culture medium, or more than 20 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 1 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 20 g/L culture medium, or more than 20 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 10 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 50 g/L culture medium to about 90 g/L culture medium, or more than 90 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 100 g/L culture medium, or more than 100 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 30 g/L culture medium, or more than 30 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 40 g/L culture medium, or more than 40 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 50 g/L culture medium, or more than 50 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 60 g/L culture medium, or more than 60 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 70 g/L culture medium, or more than 70 g/L culture medium. In some embodiments, a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 80 g/L culture medium, or more than 80 g/L culture medium.
  • a method of the present disclosure provides for production of a cannabinoid or a cannabinoid derivative in an amount of from about 20 g/L culture medium to about 90 g/L culture medium, or more than 90 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 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 the culture medium (e.g., a liquid culture medium) in which the modified yeast cell is cultured.
  • the culture medium in which the modified yeast cell is cultured 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 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/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
  • the modified yeast cell is a modified S. cerevisiae.
  • the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 50 mg/L to 100 mg/L.
  • the modified yeast cell is a modified S. cerevisiae.
  • the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 100 mg/L to 500 mg/L.
  • the modified yeast cell is a modified S. cerevisiae.
  • the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount from 500 mg/L to 1 g/L.
  • the modified yeast cell is a modified S. cerevisiae.
  • the culture medium in which the modified yeast cell is cultured comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L.
  • the modified yeast cell is a modified S. cerevisiae.
  • 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.
  • 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.
  • 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 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/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).
  • an alcoholic beverage of the present disclosure comprises a cannabinoid or a cannabinoid derivative in an amount more than 1 g/L.
  • 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 pg/L, from 1 pg/L to 50 pg/L, from 50 pg/L to 100 pg/L, from 100 pg/L to 500 pg/L, from 500 pg/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 (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 pg/L
  • a method of the present disclosure provides for increased production of a cannabinoid or a cannabinoid derivative, such as those disclosed herein.
  • 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, 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%, or 90% 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%, or 50-90% 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 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.
  • the cannabinoid or cannabinoid derivative is recovered from both the cell lysate and the culture medium.
  • the cannabinoid or cannabinoid derivative is recovered from a modified host cell.
  • 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 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 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 recovered cannabinoid or cannabinoid derivative 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.
  • 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 present disclosure provides methods for preparing engineered variants of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide.
  • 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).
  • crude extracts e.g., cell-free lysates
  • powders e.g., shake-flask powders
  • lyophilizates e.g., frozen stocks made with glycerol or another cryoprotectant
  • frozen stocks made with glycerol or another cryoprotectant e.g., frozen stocks made with glycerol or another cryoprotectant
  • 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 [0445] The methods of the disclosure may involve cell-free production of 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
  • 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 1-1 to 1-121 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.
  • Embodiment I Some embodiments of the disclosure are of Embodiment I:
  • Embodiment 1-1 An engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions.
  • THCAS tetrahydrocannabinolic acid synthase
  • Embodiment 1-2 The engineered variant of Embodiment 1-1, wherein the engineered 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%, 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: 44.
  • Embodiment 1-3 Embodiment 1-3.
  • the engineered variant of Embodiment I- 1 or 1-2 wherein the engineered variant comprises at least one amino acid substitution in a signal polypeptide, a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.
  • FAD flavin adenine dinucleotide
  • BBE berberine bridge enzyme
  • Embodiment 1-4 The engineered variant of Embodiment 1-3, wherein the engineered variant comprises at least one amino acid substitution in the signal polypeptide.
  • Embodiment 1-5 The engineered variant of Embodiment 1-3 or 1-4, wherein the engineered variant comprises at least one amino acid substitution in the FAD binding domain.
  • Embodiment 1-6 The engineered variant of any one of Embodiments 1-3 to 1-5, wherein the engineered variant comprises at least one amino acid substitution in the BBE domain.
  • Embodiment 1-7 The engineered variant of any one of Embodiments 1-3 to 1-6, wherein the engineered variant comprises substitution of at least one surface exposed amino acid.
  • Embodiment 1-8 The engineered variant of Embodiment I- 1 or 1-2, wherein the engineered variant comprises at least one amino acid substitution at an amino acid selected from the group consisting of R31, P43, P49, K50, L51, Q55, H56, L59, M61, S62, L71, S100, V103, T109, Q124, V125, L132, S137, H143, V149, W161, K165, N168, E167, S170, F171, P172, Y175, G180, N196, H208, G235, A250, 1257, K261, L269, G311, F317, L327, K390, T379, S429, N467, Y500, N528, P539, P542, H543, H544, and H545 [0459] Embodiment 1-9.
  • the engineered variant of Embodiment 1-8 wherein the engineered variant comprises at least one amino acid substitution selected from the group consisting of R31Q, P43E, P49E, P49K, P49Q, K50T, L51I, Q55E, Q55P, H56E, L59E, M61W, M61H, M61S, S62Q, L71A, S100A, V103F,T109V,Q124D, Q124E, Q124N, V125E, V125Q, L132M, S137G, H143D, V149I, W161R, W161Y, W161K, K165A, N168S, E167P, S170T, F171I, P172V, Y175F, G180A, N196Q, N196V, H208T, G235P, A250T, 1257V, K261W, K261C, L269I, G311A, G311C, F317Y, L327I, K
  • Embodiment I- 10 The engineered variant of Embodiment I- 1 or 1-2, wherein the engineered variant comprises an amino acid sequence selected from the group consisting of SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80 SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO
  • Embodiment 1-11 The engineered variant of any one of Embodiments 1-1 to 1-9, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.
  • SEQ ID NO:44 amino acid sequence of SEQ ID NO:44 with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acid substitutions.
  • Embodiment 1-12 The engineered variant of any one of Embodiments 1-1 to 1-9, wherein the engineered variant comprises an amino acid sequence of SEQ ID NO:44 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • Embodiment 1-13 The engineered variant of any one of Embodiments 1-1 to 1-12, wherein the engineered variant comprises at least one immutable amino acid in a flavin adenine dinucleotide (FAD) binding domain, a berberine bridge enzyme (BBE) domain, or a combination of the foregoing.
  • FAD flavin adenine dinucleotide
  • BBE berberine bridge enzyme
  • Embodiment 1-14 The engineered variant of Embodiment 1-13, wherein the engineered variant comprises at least one immutable amino acid in the FAD binding domain.
  • Embodiment 1-15 The engineered variant of Embodiment 1-14, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the FAD binding domain.
  • Embodiment 1-16 The engineered variant of any one of Embodiments I-
  • the engineered variant comprises at least one immutable amino acid in the BBE domain.
  • Embodiment 1-17 The engineered variant of Embodiment 1-16, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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 immutable amino acids in the BBE domain.
  • Embodiment 1-18 The engineered variant of any one of Embodiments 1-1 to 1-17, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, El 17, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160, N163, A173, G174, C176, P177, T178, V179, G182, G183, H184, F185,
  • immutable amino acid selected from the group consisting of A28, F34, L35, C37, L64, N70, P87, 193, C99, R108, R110, G112, El 17, G118, S120, P126, F127, D131, D141, W148, G152, A153, L155, G156, E157, Y159, Y160
  • Embodiment 1-19 The engineered variant of Embodiment 1-18, wherein the engineered variant comprises at least one immutable amino acid selected from the group consisting of C37, N70, 193, C99, El 17, S120, F127, D131, G156, E157, Y159, G174,
  • Embodiment 1-20 The engineered variant of any one of Embodiments 1-1 to 1-19, wherein the engineered variant comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 immutable amino acids.
  • Embodiment 1-21 The engineered variant of any one of Embodiments 1-1 to 1-20, wherein the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in a greater amount, as measured in mg/L or mM, than an amount of THC A produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDA cannabigerolic acid
  • Embodiment 1-22 The engineered variant of any one of Embodiments 1-1 to 1-21, wherein the engineered variant produces tetrahydrocannabinolic acid (THC A) from cannabigerolic acid (CBGA) 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 THCA produced from CBGA by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THC A tetrahydrocannabinolic acid
  • CBDA cannabigerolic acid
  • CBGA cannabigero
  • Embodiment 1-23 The engineered variant of any one of Embodiments 1-1 to 1-22, wherein the engineered variant produces tetrahydrocannabinolic acid (THCA) from cannabigerolic acid (CBGA) in an increased ratio of THCA over another cannabinoid (e.g. cannabichromenic acid (CBCA)) compared to that produced by a tetrahydrocannabinolic acid synthase polypeptide having an amino acid sequence of SEQ ID NO:44 under similar conditions for the same length of time.
  • THCA tetrahydrocannabinolic acid
  • CBDA cannabichromenic acid
  • Embodiment 1-24 The engineered variant of any one of Embodiments 1-1 to 1-23, wherein the engineered variant produces THCA from CBGA in a ratio of THCA over another cannabinoid (e.g., CBCA) 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.
  • CBCA cannabinoid
  • Embodiment 1-25 The engineered variant of any one of Embodiments 1-1 to 1-24, wherein the engineered variant comprises a truncation at an N-terminus, at a C- terminus, or at both the N- and C-termini.
  • Embodiment 1-26 The engineered variant of Embodiment 1-25, wherein the truncated engineered variant comprises a signal polypeptide or a membrane anchor.
  • Embodiment 1-27 The engineered variant of Embodiment 1-25 or 1-26, wherein the engineered variant lacks a native signal polypeptide.
  • Embodiment 1-28 The engineered variant of any one of Embodiments I-
  • the engineered variant comprises a truncation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the C-terminus.
  • Embodiment 1-2 The engineered variant of any one of Embodiments I-
  • the engineered variant comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus.
  • Embodiment 1-30 A nucleic acid comprising a nucleotide sequence encoding an engineered variant of any one of Embodiments 1-1 to 1-29.
  • a nucleic acid comprising a nucleotide sequence encoding an engineered variant of a tetrahydrocannabinolic acid synthase (THCAS) polypeptide comprising an amino acid sequence of SEQ ID NO:44 with one or more amino acid substitutions, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:
  • Embodiment 1-32 The nucleic acid of Embodiment 1-30 or 1-31, wherein the nucleotide sequence is codon-optimized.
  • Embodiment 1-33. A method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more nucleic acids of any one of Embodiments 1-30 to 1-32 into a host cell.
  • Embodiment 1-34 A vector comprising one or more nucleic acids of any one of Embodiments 1-30 to 1-32.
  • Embodiment 1-35 A method of making a modified host cell for producing a cannabinoid or a cannabinoid derivative, the method comprising introducing one or more vectors of Embodiment 1-34 into a host cell.
  • Embodiment 1-36 A modified host cell for producing a cannabinoid or a cannabinoid derivative, wherein the modified host cell comprises one or more nucleic acids of any one of Embodiments 1-30 to 1-32.
  • Embodiment 1-37 The modified host cell of Embodiment 1-36, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT) polypeptide.
  • GAT geranyltransferase
  • Embodiment 1-38 The modified host cell of Embodiment 1-37, wherein the GOT polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:17.
  • Embodiment 1-39 The modified host cell of Embodiment 1-37 or 1-38, wherein the modified host cell comprises two or more heterologous nucleic acids comprising the nucleotide sequence encoding the GOT polypeptide.
  • Embodiment 1-40 The modified host cell of Embodiment 1-36, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a NphB polypeptide.
  • Embodiment 1-4 The modified host cell of Embodiment 1-40, wherein the NphB polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:188.
  • Embodiment 1-4 The modified host cell of any one of Embodiments I-
  • the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a tetraketide synthase (TKS) polypeptide and one or more heterologous nucleic acids comprising a nucleotide sequence encoding an olivetolic acid cyclase (OAC) polypeptide.
  • TKS tetraketide synthase
  • OAC olivetolic acid cyclase
  • Embodiment 1-44 The modified host cell of Embodiment 1-42 or 1-43, wherein the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a TKS polypeptide.
  • Embodiment 1-45 The modified host cell of any one of Embodiments I-
  • OAC polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:21 or SEQ ID NO:48.
  • Embodiment 1-46 The modified host cell of any one of Embodiments I-

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
EP20786122.0A 2019-09-18 2020-09-17 Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides Withdrawn EP4031657A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962902300P 2019-09-18 2019-09-18
PCT/US2020/051261 WO2021055597A1 (en) 2019-09-18 2020-09-17 Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides

Publications (1)

Publication Number Publication Date
EP4031657A1 true EP4031657A1 (en) 2022-07-27

Family

ID=72744852

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20786122.0A Withdrawn EP4031657A1 (en) 2019-09-18 2020-09-17 Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides

Country Status (9)

Country Link
EP (1) EP4031657A1 (ja)
JP (1) JP2022548904A (ja)
CN (1) CN115038786A (ja)
AU (1) AU2020349513A1 (ja)
BR (1) BR112022004797A2 (ja)
CA (1) CA3152803A1 (ja)
IL (1) IL291328A (ja)
MX (1) MX2022003203A (ja)
WO (1) WO2021055597A1 (ja)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020176547A1 (en) 2019-02-25 2020-09-03 Ginkgo Bioworks, Inc. Biosynthesis of cannabinoids and cannabinoid precursors
WO2023168266A2 (en) * 2022-03-02 2023-09-07 Genomatica, Inc. Flavin-dependent oxidases having cannabinoid synthase activity

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60335574D1 (de) 2002-10-04 2011-02-10 Du Pont Verfahren zur biologischen herstellung von 1,3-propandiol mit hoher ausbeute
CN102027124B (zh) 2007-12-13 2015-11-25 丹尼斯科美国公司 用于生产异戊二烯的组合物和方法
EP2279251A2 (en) 2008-04-23 2011-02-02 Danisco US Inc. Isoprene synthase variants for improved microbial production of isoprene
ES2716231T3 (es) 2008-07-02 2019-06-11 Danisco Us Inc Procedimientos para producir isopreno
ES2898272T3 (es) * 2017-04-27 2022-03-04 Univ California Microorganismos y métodos para producir cannabinoides y derivados de cannabinoides
US11149291B2 (en) * 2017-07-12 2021-10-19 Biomedican, Inc. Production of cannabinoids in yeast
EP3724322A1 (en) * 2017-12-14 2020-10-21 Medicinal Genomics Corporation Methods and kits for classifying cannabinoid production in cannabis plants
WO2020069214A2 (en) * 2018-09-26 2020-04-02 Demetrix, Inc. Optimized expression systems for producing cannabinoid synthase polypeptides, cannabinoids, and cannabinoid derivatives

Also Published As

Publication number Publication date
CA3152803A1 (en) 2021-03-25
IL291328A (en) 2022-05-01
CN115038786A (zh) 2022-09-09
BR112022004797A2 (pt) 2022-06-21
WO2021055597A1 (en) 2021-03-25
MX2022003203A (es) 2022-06-08
AU2020349513A1 (en) 2022-05-05
JP2022548904A (ja) 2022-11-22

Similar Documents

Publication Publication Date Title
EP3615667B1 (en) Microorganisms and methods for producing cannabinoids and cannabinoid derivatives
CA3140079A1 (en) Optimized cannabinoid synthase polypeptides
IL270214B1 (en) Anti-sortilin antibodies and methods of using them
Scalcinati et al. Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae
WO2020069214A2 (en) Optimized expression systems for producing cannabinoid synthase polypeptides, cannabinoids, and cannabinoid derivatives
BRPI0513837B1 (pt) Célula hospedeira saccharomyces cerevisiae geneticamente modificada; composição compreendendo a mesma e método para aumentar a produção de isoprenoides
EP4031657A1 (en) Optimized tetrahydrocannabinolic acid (thca) synthase polypeptides
JP2022533449A (ja) 植物性カンナビノイド及び植物性カンナビノイド前駆体の産生のための方法及び細胞
WO2020069142A1 (en) Optimized expression systems for expressing berberine bridge enzyme and berberine bridge enzyme-like polypeptides
US20230137139A1 (en) Biosynthesis of cannabinoids and cannabinoid precursors
WO2021183448A1 (en) Optimized olivetolic acid cyclase polypeptides
Jiang et al. Engineering Saccharomyces cerevisiae for enhanced (–)-α-bisabolol production
KR20240005708A (ko) 이소프레노이드 및 그의 전구체의 생합성
US20230257785A1 (en) Production of bioactive bibenzylic acid or derivatives thereof by genetically modified microbial hosts
AU2018256863B2 (en) Microorganisms and methods for producing cannabinoids and cannabinoid derivatives
US20230340446A1 (en) Biosynthesis of cannabinoids and cannabinoid precursors

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220413

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20221117