WO2021042057A1 - Systems and methods for preparing cannabinoids and derivatives - Google Patents

Abstract

Provided herein are systems and methods for the production of cannabinoid derivatives in host cells, such as e.g., those containing or utilizing OLS and other enzymes provided or utilized herein. As used herein, cannabinoids include olivetol; olivetolic acid; olivetolic acid derived compounds obtainable according to the enzymatic transformations of the cannabinoid pathway such as that shown in Figure 1; compounds of formula (I); compounds of formula (I) chemically transformed according to the enzymatic transformations of the cannabinoid pathway such as that shown in Figure 1; and the likes.

Description

SYSTEMS AND METHODS FOR PREPARING CANNABINOIDS AND DERIVATIVES PRIORITY CLAIM [0001] This application claims priority to US provisional application No. 62/894,552, filed August 30, 2019, the content of which is incorporated herein in its entirety by reference. REFERENCE TO SEQUENCE LISTING [0002] This application contains a Sequence Listing submitted via EFS-web which is hereby incorporated by reference in its entirety for all purposes. The ASCII copy, created on August 27, 2020, is named Lygos_0035_01_WO_ST25.txt and is 52 KB in size. BACKGROUND OF THE INVENTION [0003] Cannabis sativa L. (cannabis, hemp, marijuana) is now used as a source of medicinal, food, cosmetic and industrial products. However, it is also known for its use as an illicit drug owing to containing certain psychoactive cannabinoids (e.g. D9-tetrahydrocannabinol, D9-THC). Cannabinoids and other drugs that act through mammalian cannabinoid receptors can be beneficial for the treatment of diverse conditions such as chronic pain, multiple sclerosis, and epilepsy. SUMMARY [0004] Provided herein are systems and methods for producing cannabinoids and derivatives. As used herein, cannabinoids include olivetol; olivetolic acid; olivetolic acid derived compounds obtainable according to the enzymatic transformations of the cannabinoid pathway such as that shown in Figure 1; compounds of formula (I); compounds of formula (I) chemically transformed according to the enzymatic transformations of the cannabinoid pathway such as that shown in Figure 1; and the likes. As used herein, derivatives include limitation, salts; esters such as carboxy and phosphate esters; 1-3, preferably 1-2 carbon homologs; and isosteres. Isosteric replacement of functional groups, and moieties such as phenyl rings are reported for other molecules, and can be adapted by a skilled artisan based on the disclosure. [0005] In some aspects and embodiments, provided herein are polyketide synthases, nucleic acids encoding said polyketide synthases, expression vectors comprising said nucleic acids, host cells comprising said expression vectors, methods for preparing said polyketide synthases, methods of preparing olivetol, olivetolic acid, and cannabinoids and derivatives, and methods for isolating the olivetol, olivetolic acid, and cannabinoids and derivatives from fermentation broth. [0006] Further provided herein are polyketide synthases having k_cat values that are greater than k_cat values of wild type polyketide synthases. In some aspects and embodiments, provided herein are recombinant host cells, materials, and methods for the biological production of cannabinoids and derivatives. In some embodiments, the cannabinoids include without limitation, olivetol, olivetolic acid, and an olivetolic acid-derived compound, including, but not limited to, cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), D⁹-tetrahydrocannabinolic acid, cannabichromenic acid (CBCA), cannabichromene (CBC), D⁹-tetrahydrocannabinol, and D⁶-tetrahydrocannabinol.

[0007] A technical and commercial challenge to producing olivetol, olivetolic acid, and/or OLA-derived compounds is the low activity level of the native C. sativa OLS enzyme. The k_cat of the native C. sativa OLS enzyme is approximately 1-4 turnovers/minute. Without being bound by theory, the impact of a low OLS k_cat value is multi-fold. First, low OLS k_cat values reduce cel1- specific productivities since carbon flux through the biosynthesis pathway is limited to the maximal activity of the OLS and its expression level within the cell. Unless an abundance of biomass is generated in the fermentation, a low k_cat value will also result in low volumetric productivity and thus higher production costs. Second, low OLS enzyme activity leads to a buildup of pathway intermediates, ultimately resulting in high concentrations of undesired byproducts and potentially decreased cell growth and/or viability.

[0008] An enzyme in the biosynthesis of olivetol is a polyketide synthase (“PKS”) capable of converting three molecules of malony1-CoA and one molecule of hexanoy1-CoA to olivetol. In 2009, Taura et al. reported a clone of a type III PKS named olivetol synthase (“OLS”, Uni Prot ID B1Q2B6, SEQ ID NO: 1) from Cannabis sativa (Futoshi et al., 2009, “Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway”. FEBS Letters. 583 (12): 2061-6). OLS is a homodimeric protein that consists of a 385 amino acid polypeptide that has (60-70%) sequence similarity to plant PKSs (id.). When expressed with an accessory enzyme, namely olivetolic acid cyclase (OLAC), OLS is useful to biosynthesize olivetolic acid (“OLA”) and a variety of OLA-derived compounds and cannabinoids. In one aspect, provided herein is a polyketide synthase having at least 97% sequence identity to SEQ ID NO: 1 or a salt thereof. [0009] In another aspect, provided herein is a polyketide synthase is provided comprising an amino acid sequence of SEQ ID NO: 2, or salt thereof, wherein X is defined as follows. In one embodiment, X at position 198 is Asp, His, Ser, Thr, or Arg. In another embodiment, X at position 199 is any amino acid. In another embodiment, X at position 200 is any amino acid. In another embodiment, X at position 201 any amino acid. In another embodiment, X at position 202 is any amino acid. In another embodiment, X at position 203 is lie, Leu, Val, Ala, Met, or Phe. In another embodiment, X at position 204 is Ala, Cys, Gly, Pro, Ser, Thr, or Val. In another embodiment, X at position 205 is Ala, Asn, Asp, Gin, His, Met, or Ser. In another embodiment, X at position 206 is Ala, Cys, His, Leu, Pro, Ser, Thr, or Val. In another embodiment, X at position 207 is Ala, lie, Leu, or Val. In another embodiment, X at position 208 is Leu, lie, or Phe. In another embodiment, X at position 209 is Ala, Gly, or Ser. Preferably, as used herein, any amino acid refers to an amino acid selected from twenty naturally occurring amino acids.

[0010] In other embodiments, the OLS employed is of SEQ ID NO: 3. In other embodiments, the OLS employed is SEQ ID NO: 3, which is mutated according to the mutations of SEQ ID NO:

2.

[0011] In another aspect, provided herein is a polyketide synthase having a k_cat value greater than wild type Cannabis sativa olivetol synthase. Lor example, a polyketide synthase provided or utilized herein comprises a k_cat value greater than 5 turnovers per minute, greater than 10 turnovers per minute, greater than 50 turnovers per minute, greater than 100 turnovers per minute, or greater than 500 turnovers per minute.

[0012] In another aspect, a heterologous nucleic acid is provided which encodes a polyketide synthase comprising a mutated form of olivetol synthase derived from C. sativa. In certain embodiments, provided herein are mutated olivetol synthases, nucleic acids encoding them, and recombinant expression vectors comprising those nucleic acids. In some embodiments, the heterologous nucleic acid codes for the overexpression of an endogenous enzyme. Lurther, in some embodiments, the heterologous nucleic acid encodes a wild type or mutant enzyme heterologous to (not natively expressed in) the host cell.

[0013] In another aspect, a host cell is provided comprising an expression vector of a polyketide synthase provided or utilized herein. In some embodiments, the expression vector is a yeast expression vector; in other embodiments, the expression vector is a bacterial expression vector. In some embodiment, the host cells produce olivetol, olivetolic acid, an olivetolic acid- derived compound, and such other cannabinoids and derivatives at a faster rate than counterpart cells that do not comprise a polyketide synthase disclosed herein.

[0014] In some embodiments, the host cell further expresses or overexpresses a functional hexanoy1-CoA synthetase protein. In some embodiments, the host cell further expresses or overexpresses an enzyme to provide increased malony1-CoA production. In some embodiments, the overexpressed enzyme is an acety1-CoA carboxylase, an aldehyde dehydrogenase, an acety1- CoA synthetase, and/or a pymvate decarboxylase.

[0015] In another aspect, provided herein are recombinant host cells suitable for the biosynthetic production of cannabinoids and derivatives, such as without limitation, olivetol, olivetolic acid, and/or an olivetolic acid-derived compound at levels enabling isolation. In some embodiments, a host cell is a prokaryotic organism, a eukaryotic organism, a fungal organism, a yeast organism, a bacterial organism, or a unicellular organism. In other embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a eukaryote. In some embodiments, the host cell is a yeast cell. In various embodiments, the yeast is selected from the non-limiting list of example genera: Candida, Cryptococcus, Hansenula, lssatchenkia, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces or Yarrowia. In some embodiments, the eukaryotic host cell is a fungus. In some embodiments, the host cell is an algae. In some instance, a host cell is selected from the group consisting of P. kudriavzevii, P. pastoris, S. cerevisiae, and Y. lipolytica organisms.

[0016] In other embodiments, the host cell is a bacterial cell. In various embodiments, the host cell is a bacterial cell selected from the group consisting of Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas, and Streptomyces. In some embodiments, the host cell is selected from the group consisting E. coli and C. glutamicum.

[0017] In some embodiments, the recombinant host cells of the disclosure have been genetically modified for improved olivetol, olivetolic acid, an olivetolic acid-derived compound, or another cannabinoid or cannabinoid derivative yield, titer, and/or productivity. In some embodiments, the host cells provided or utilized herein are modified to include a polyketide synthase having a k_cat value that is greater than k_cat values of wild type polyketide synthases. In various embodiments, the host cells are further genetically modified for increased or improved production through one or more host cell modifications selected from the group consisting of modifications that result in increased acety1-CoA biosynthesis, increased malony1-CoA biosynthesis, increased hexanoy1-CoA synthetase biosynthesis, increased acety1-CoA carboxylase biosynthesis, increased aldehyde dehydrogenase biosynthesis, increased acety1-CoA synthetase biosynthesis, and increased pyruvate decarboxylase biosynthesis.

[0018] In another aspect, a method is provided for producing a cannabinoid or a derivative, in a recombinant host cell in fermentation broth. In some embodiment, the compound produced is selected from olivetol, olivetolic acid, and an olivetolic acid-derived compound. In some embodiments, the host cell is engineered to express more or less of an endogenous enzyme that results in the production of more cannabinoid and derivatives, such as without limitation, olivetol, olivetolic acid, and/or an olivetolic acid-derived compound than a corresponding cell that has not been engineered. In some embodiments, the method comprises culturing a recombinant host cell expressing a heterologous (foreign or non-native) enzyme that results in the increased production of cannabinoid and derivatives, such as without limitation, olivetol, olivetolic acid, and/or an olivetolic acid-derived compound. In some embodiments, the host cells comprise one or more expression vectors comprising one or more heterologous polyketide synthases provided or utilized herein.

[0019] In one embodiment, the host cell, preferably a heterologous host cell, provided herein further comprises an enzyme selected from an acety1-CoA carboxylase (ACC such as a P. kudriavzevii (Pk) ACC, such as PkACCl), an aldehyde dehydrogenase (ALD such as an ALD selected from S. cerevisiae (Sc) ALD2-6, such as ScALD6), an acety1-CoA synthetase (ACS such as a Y. lipolytica ACS such as Y1ACS), and a pyruvate decarboxylase (PDC such as a S. cerevisiae PDC such as ScPDC6). Certain ACC, ALD, ACS, PDC, and other enzymes useful in increasing malony1-CoA production, and useful in accordance with this disclosure, is disclosed in US patent application no. 20160177345, which is incorporated herein by reference.

[0020] In one embodiment, provided herein is a host cell further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate: olivetolic acid geranyltransferase polypeptide, wherein said geranyl pyrophosphate: olivetolic acid geranyltransferase polypeptide catalyzes production of cannabigerolic acid from geranyl pyrophosphate and olivetolic acid in an amount at least about ten times higher than a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0021] In one embodiment, provided herein is a host cell further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate: olivetolic acid geranyltransferase polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 5.

[0022] In one embodiment, provided herein is a host cell, further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate:lolivetolic acid geranyltransferase polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 6.

[0023] In one embodiment, provided herein is a host cell further comprising one or more heterologous nucleic acids encoding an olivetolic acid (OAC) polypeptide, or one or more heterologous nucleic acids encoding a fusion OLS and OAC polypeptide.

[0024] In one embodiment, the TKS polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 3.

[0025] In one embodiment, the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8. [0026] In one embodiment, provided herein is a host cell further comprising one or more of the following: a) one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative; b) one or more heterologous nucleic acids encoding a polypeptide that generates geranyl pyrophosphate; or c) one or more heterologous nucleic acids encoding a polypeptide that generates malony1-CoA. [0027] In one embodiment, provided herein is a host cell further comprising one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative, wherein the polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative is an acy1-activating enzyme (AAE) polypeptide. In one embodiment, the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 9. In one embodiment, the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11.

[0028] In one embodiment, provided herein is a host cell further comprising one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative. In one embodiment, the polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative is a fatty acy1-CoA ligase polypeptide. In one embodiment, the fatty acy1-CoA ligase polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 13. [0029] In one embodiment, the polypeptide that generates an acy1-CoA compound or an acy1- CoA compound derivative is a fatty acy1-CoA synthetase (FAA) polypeptide.

[0030] In another aspect, provided herein is a process of either (i) producing olivetol or a compound of formula (I), or (ii) chemically transforming olivetol or a compound of formula (I):

wherein R¹ is: an optionally substituted hydrocarbyl group, preferably an optionally substituted C1-C12 hydrocarbyl group, such as without limitation, optionally substituted alkyl, preferably, optionally substituted C₁-C₈ alkyl; optionally substituted alkenyl, preferably, optionally substituted C₂-C₈ alkenyl; optionally substituted alkynyl, preferably, optionally substituted C₂-C₈ alkynyl;

R² is H or CO₂H or a salt thereof;

R³ is hydrogen, geranyl, or

R³ and the OH* together with the intervening carbon atoms form

said method comprising: for (i), culturing a host cell provided or utilized herein under conditions that result in production of olivetol or a compound of formula (I), wherein for producing olivetol, the host cell utilizes hexanol, hexanoic acid, or hexanoy1-CoA, and for producing a compound of formula (I), the host cell utilizes R¹-CoA or a precursor thereof such as without limitation R¹CH₂OH R¹CO₂H, and the likes; or for (ii), culturing a host cell provided or utilized herein with olivetol or a compound of formula (I), thereby chemically transforming olivetol or the compound of formula (I).

[0031] As used herein, chemically transforming refers to changing the chemical structure of a compound, e.g., and without limitation, enzymatically according to the cannabinoid pathway(s) illustrated in Figure 1. E.g., and without limitation, optional substituents include alkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, halo, fluoro, fluoroalkyl, fluorocycloalkyl, cyano, and ester group(s), provided that the substitution(s) do not result in polymers. In one embodiment, R¹ is pentyl. In another embodiment, R¹ is propyl. In another embodiment, R² is CO₂H or a salt thereof. In another embodiment, R² is hydrogen. In another embodiment, R³ is hydrogen. In another embodiment, R³ is geranyl. In another embodiment, R³ is

In another embodiment, R³ and the OH* together with the intervening carbon atoms form

[0032] In one embodiment, the process further comprises isolating olivetol or the compound of formula (I) from the fermentation broth, or further comprising isolating chemically transformed olivetol or a chemically transformed compound of formula (I) from the fermentation broth.

[0033] In one embodiment, the olivetolic acid-derived compound is cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), D⁹- tetrahydrocannabinolic acid, cannabichromenic acid (CBCA), cannabichromene (CBC), D⁹- tetrahydrocannabinol, and D⁶-tetrahydrocannabinol.

[0034] In another aspect, provided herein is a purified cannabinoid or a derivative, such as without limitation, olivetol, olivetolic acid, and/or an olivetolic acid-derived compound from the fermentation broth comprising a host cell producing these materials. Also provided herein are methods for purifying cannabinoid or a derivative, such as without limitation, olivetol, olivetolic acid, and/or olivetolic acid-derived compounds from the fermentation broth of a host cell producing these materials, the methods generally comprising culturing a host cell in fermentation broth under conditions that enable the host cell to produce these materials, and purifying these materials from the fermentation broth. In some embodiments, the purification is achieved by adding an organic solvent to the host cell fermentation broth, and extracting the olivetol, olivetolic acid, and/or olivetolic acid-derived compounds from the fermentation broth. Non-limiting examples of an olivetolic acid-derived compound include cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), D⁹-tetrahydrocannabinolic acid, cannabichromenic acid (CBCA), cannabichromene (CBC), D⁹-tetrahydrocannabinol, and D⁶- tetrahydrocannabinol. In some embodiments, the organic solvent is hydrophobic. Any organic solvent that easily phase separates from the fermentation broth and preferentially separates the cannabinoid from the fermentation broth may be used. Non-limiting examples of an organic solvent include 2-ethylhexanol, 1-decanol, 1-dodecanol, isodecanol, n-octyl acetate, ethyl dodecanoate, 2-octy1- 1-dodecanol, 2-buty1- 1-octanol, oleyl alcohol, dimethyl adipate, diethyl adipate, dioctyl adipate, butyl oleate, 2-hexy1- 1-decanol, diisobutyl adipate, dibutyl phthalate, tributyl citrate, dibutyl sebacate, dodecane, hexadecane, silicone oil, toluene, paraffin oil, soybean oil, and mixtures thereof.

[0035] These and other aspects and embodiments of the disclosure are described in more detail below.

BRIEF DESCRIPTON OF THE FIGURES

[0036] Figure 1 schematically illustrates certain enzymatic pathways, the cannabinoid pathways, useful in preparing and chemically transforming cannabinoids and derivatives.

[0037] Figure 2 schematically illustrates certain enzymatic pathways, the 1-hexanol catabolic pathways, useful in preparing and chemically transforming cannabinoids and derivatives. (1) NADH-dependent 1-hexanol dehydrogenase; (2) NADPH-dependent 1-hexanol dehydrogenase; (3) NADH-dependent hexanal dehydrogenase; (4) NADPH-dependent hexanal dehydrogenase; (5) hexanoy1-CoA synthetase; (6) hexanoate kinase; (7) phosphate hexyltransferase; (8) hexanal dehydrogenase (acylating).

[0038] Figure 3 schematically illustrates certain enzymatic pathways, the geranio1-dependent GPP pathways, useful in preparing and chemically transforming cannabinoids and derivatives. (1) geraniol kinase; (2) geranyl monophosphate kinase; (3) geraniol isomerase; (4) linalool dehydratase; (5) geranyl acetate esterase.

DETAILED DESCRIPTION

[0039] While the present disclosure is described herein with reference to aspects and specific embodiments thereof, those skilled in the art will recognize that various changes may be made and equivalents may be substituted without departing from the disclosure. The present disclosure is not limited to particular nucleic acids, expression vectors, enzymes, host microorganisms, or processes, as such may vary. The terminology used herein is for purposes of describing particular aspects and embodiments only, and is not to be construed as limiting. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, in accordance with the disclosure. All such modifications are within the scope of the claims appended hereto.

Definitions

[0040] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0041] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “expression vector” includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to “cell” includes a single cell as well as a plurality of cells; and the like.

[0042] Amino acids in a protein coding sequence are identified herein by the following abbreviations and symbols. Specific amino acids are identified by a three-letter abbreviation, as follows: Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine, Gin is glutamine, Glu is glutamic acid, Gly is glycine, His is histidine, Leu is leucine, lle is isoleucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, Trp is tryptophan, Tyr is tyrosine, and Val is valine, or by a one-letter abbreviation, as follows: A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G is glycine, H is histidine, L is leucine, I is isoleucine, K is lysine, O is pyrrolysine, M is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is tryptophan, Y is tyrosine, and V is valine. A dash (-) in a consensus sequence indicates that there is no amino acid at the specified position. A plus (+) in a consensus sequence indicates any amino acid may be present at the specified position. Thus, a plus in a consensus sequence herein indicates a position at which the amino acid is generally non-conserved; a homologous enzyme sequence, when aligned with the consensus sequence, can have any amino acid at the indicated “+” position. Specific amino acids in a protein coding sequence are identified by their respective one-letter abbreviation followed by the amino acid position in the protein coding sequence where 1 corresponds to the amino acid (typically methionine) at the N-terminus of the protein. For example, G204 in C. sativa wild type OFS refers to the glycine at position 204 from the OLS N-terminal methionine (i.e., Ml). Amino acid substitutions (i.e., point mutations) are indicated by identifying the mutated (i.e., progeny) amino acid after the one-letter code and number in the parental protein coding sequence; for example, G204A in C. sativa OLS refers to substitution of alanine for glycine at position 204 in the OLS protein coding sequence. The mutation may also be identified in parentheticals, for example OLS (G204A). Multiple point mutations in the protein coding sequence are separated by a backslash (/); for example, OLS G204A/Q205N indicates that mutations G204A and Q205N are both present in the OLS protein coding sequence. The number of mutations introduced into some examples has been annotated by a dash followed by the number of mutations, preceding the parenthetical identification of the mutation (e.g., B1Q2B6-1 (G204A)). The Uniprot IDs with and without the dash and number are used interchangeably herein (i.e., B1Q2B6-1 (G204A) = B1Q2B6 (G204A)).

[0043] As used herein, the term “express”, when used in connection with a nucleic acid encoding an enzyme or an enzyme itself in a cell, means that the enzyme, which may be an endogenous or exogenous (heterologous) enzyme, is produced in the cell. The term “overexpress”, in these contexts, means that the enzyme is produced at a higher level, i.e., enzyme levels are increased, as compared to the wild type, in the case of an endogenous enzyme. Those skilled in the art appreciate that overexpression of an enzyme can be achieved by increasing the strength or changing the type of the promoter used to drive expression of a coding sequence, increasing the strength of the ribosome binding site or Kozak sequence, increasing the stability of the mRNA transcript, altering the codon usage, increasing the stability of the enzyme, and the like.

[0044] The term “expression vector” or “vector” refer to a nucleic acid and/or a composition comprising a nucleic acid that can be introduced into a host cell, e.g., by transduction, transformation, or infection, such that the cell then produces (“expresses”) nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell, that are contained in or encoded by the nucleic acid so introduced. Thus, an “expression vector” contains nucleic acids (ordinarily DNA) to be expressed by the host cell. Optionally, the expression vector can be contained in materials to aid in achieving entry of the nucleic acid into the host cell, such as the materials associated with a virus, liposome, protein coating, or the like. Expression vectors suitable for use in various aspects and embodiments include those into which a nucleic acid sequence can be, or has been, inserted, along with any preferred or required operational elements. Thus, an expression vector can be transferred into a host cell and, typically, replicated therein (although, one can also employ, in some embodiments, non-replicable vectors that provide for “transient” expression). In some embodiments, an expression vector that integrates into chromosomal, mitochondrial, or plastid DNA is employed. In other embodiments, an expression vector that replicates extrachromasomally is employed. Typical expression vectors include plasmids, and expression vectors typically contain the operational elements required for transcription of a nucleic acid in the vector. Such plasmids, as well as other expression vectors, are described herein or are well known to those of ordinary skill in the art.

[0045] The terms “ferment”, “fermentative”, and “fermentation” are used herein to describe culturing host cells and microbes under conditions to produce useful chemicals, including but not limited to conditions under which microbial growth, be it aerobic or anaerobic, occurs.

[0046] The term “heterologous” as used herein refers to a material that is non-native to a cell. For example, a nucleic acid is heterologous to a cell, and so is a “heterologous nucleic acid” with respect to that cell, if at least one of the following is true: (a) the nucleic acid is not naturally found in that cell (that is, it is an “exogenous” nucleic acid); (b) the nucleic acid is naturally found in a given host cell (that is, “endogenous to”), but the nucleic acid or the RNA or protein resulting from transcription and translation of this nucleic acid is produced or present in the host cell in an unnatural (e.g., greater or lesser than naturally present) amount; (c) the nucleic acid comprises a nucleotide sequence that encodes a protein endogenous to a host cell but differs in sequence from the endogenous nucleotide sequence that encodes that same protein (having the same or substantially the same amino acid sequence), typically resulting in the protein being produced in a greater amount in the cell, or in the case of an enzyme, producing a mutant version possessing altered (e.g., higher or lower or different) activity; and/or (d) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to each other in the cell. As another example, a protein is heterologous to a host cell if it is produced by translation of RNA or the corresponding RNA is produced by transcription of a heterologous nucleic acid; a protein is also heterologous to a host cell if it is a mutated version of an endogenous protein, and the mutation was introduced by genetic engineering.

[0047] The terms “host cell” and “host microorganism” are used interchangeably herein to refer to a living cell that can perform one or more steps of the cannabinoid pathway, e.g., as illustrated in Figure 1. A host cell can be (or is) transformed via insertion of an expression vector. A host microorganism or cell as described herein may be a prokaryotic cell (e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0048] The terms “isolated” or “pure” refer to material that is substantially, e.g. greater than 50% or greater than 75%, or essentially, e.g., greater than 90%, 95%, 98% or 99%, free of components that normally accompany it in its native state, e.g., the state in which it is naturally found or the state in which it exists when it is first produced.

[0049] Polyketide synthases (PKSs) are a family of multi-domain enzymes or enzyme complexes that produce polyketides, a large class of secondary metabolites, in bacteria, fungi, plants, and a few animal lineages. The terms “polyketide synthase”, “PKS”, “olivetol synthase” (“OLS”), “tetraketide synthase”, TKS, and olivetolic synthase as described herein or elsewhere typically refers to any enzyme capable of converting three molecules of malony1-CoA and one molecule of hexanoy1-CoA to olivetol. A wild type example of an OLS is the native C. sativa OLS enzyme (UniProt ID: B1Q2B6; SEQ ID NO: 1).

[0050] Olivetol (also referred to as 5-(1-pentyl)resorcinol, 5-pentylbenzene-l,3-diol, and 3,5- dihydroxyamylbenzene, CAS# 500-66-3) is a precursor in various syntheses of tetrahydrocannabinol (“THC”).

[0051] Olivetolic acid (“OLA”, also referred to as olivetolate, 2,4-dihydroxy-6-pentylbenzoic acid, and olivetolcarboxylic acid, CAS# 491-72-5) is a polyketide, derived from olivetol, that is used as a precursor in various syntheses of THC.

[0052] Olivetolic acid cyclase (“OAC”, EC: 4.4.1.26) is a polyketide cyclase derived from C. sativa which functions in concert with an OLS enzyme or a tetraketide synthase (“TKS”) to form OLA.

[0053] The term “hexanoy1-CoA synthetase” (“HCS”) as used herein refers to any enzyme capable of catalyzing the conversion of hexanoate (a short-chain fatty acid anion that is the conjugate base of hexanoic acid, also known as caproic acid) or hexanoic acid, and a free CoA to hexanoy1-CoA. A non-limiting example of a hexanoy1-CoA synthetase is the FadK protein derived from E. coli (SEQ ID NO: 15).

[0054] The terms “cannabinoid pathway”, “cannabinoid production”, “cannabinoid compound production”, “cannabinoid synthesis”, “THC synthesis”, and the like, refer generally to a biosynthetic pathway that facilitates the synthesis and production of olivetol, olivetolic acid, and olivetolic acid-derived compounds. This biosynthetic pathway utilizes a variety of enzymes, catalysts, and intermediate compounds. For example, cannabigerolic acid synthase (EC: 2.5.1.102) is used to convert OLA to cannabigerolic acid, which is a key intermediate acted upon by a variety of enzymes during THC synthesis. Cannabidiolic acid synthase (EC: 1.21.3.7) is used to convert cannabigerolic acid into cannabidiolic acid. Tetrahydrocannabinolic acid synthase (EC: 1.21.3.8) is used to convert cannabigerolic acid into D⁹-tetrahydrocannabinolic acid. A cannabichromenic acid synthase is used to convert cannabigerolic acid into cannabichromenic acid (CAS# 20408-52- 0). These three olivetolic acid-derived compounds (i.e., cannabidiolic acid, D⁹- tetrahydrocannabinolic acid, and cannabichromenic acid) are themselves converted to even more diverse cannabinoids via a combination of oxidation, decarboxylation, and isomerization reactions, which can be catalyzed using either biological or synthetic catalysts, or can also occur spontaneously following heating and/or application of uv light. For example, cannabidiol results from cannabidiolic acid decarboxylation, D⁹-tetrahydrocannabinol results from D⁹- tetrahydrocannabinolic acid decarboxylation, and subsequent isomerization of D⁹- tetrahydrocannabinol results in D⁶-tetrahydrocannabinol. [0055] The term “olivetolic acid-derived compounds” as used herein refers to any small molecules derived through, or by, enzymatic or synthetic conversion of olivetolic acid. Non- limiting examples of olivetolic acid-derived compounds include cannabigerolic acid (also referred to as CBGA), cannabigerol (also referred to as CBG), cannabidiol (also referred to as CBD), cannabidiolic acid (also referred to as CBDA, cannabidiol acid, and 2,4-dihydroxy-3-[(1r,6r)-3- methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-6-pentylbenzoic acid, CAS# 1244-58-2), D⁹- tetrahydrocannabinolic acid (also referred to as 9-carboxy-delta(9)-THC, 9-carboxy-THC, delta(9)-tetrahydrocannabinolic acid, THC-9-COOH, and THCA-A compound, CAS# 23978-85- 0), cannabichromene (also referred to as CBC), cannabichromenic acid (also referred to as CBCA, (+)-cannabichromenic acid, 5-hydroxy-2-methyl-(4-methylpent-3-enyl)-7-pentylchromene-6- carboxylic acid, and 2H-1-benzopyran-6-carboxylic acid, 5-hydroxy-2-methyl-2-(4-methyl-3- pentenyl)-7-pentyl-, (+)-, CAS# 20408-52-0), D⁹-tetrahydrocannabinol (also referred to as D⁹- THC, 8 alpha-hdroxy-delta (9)-THC, and 8-OHTHC, CAS# 34984-78-6), and D⁶- tetrahydrocannabinol (also referred to as D⁶-THC, and 6-teterahydrocannabinol, PubChem CID: 91751589). The olivetolic acid-derived compounds can be produced synthetically from olivetolic acid and are themselves valuable compounds, but are also useful substrates in the chemical synthesis of a number of other valuable compounds. 15 [0056] As used herein, the term “nucleic acid” and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and to polyribonucleotides (containing D-ribose). “Nucleic acid” can also refer to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970). A “nucleic acid” may also be referred to herein with respect to its sequence, the order in which different nucleotides occur in the nucleic acid, as the sequence of nucleotides in a nucleic acid typically defines its biological activity, e.g., as in the sequence of a coding region, the nucleic acid in a gene composed of a promoter and coding region, which encodes the product of a gene, which may be an RNA, e.g. a rRNA, tRNA, or mRNA, or a protein (where a gene encodes a protein, both the mRNA and the protein are “gene products” of that gene).

[0057] The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, ribosome-binding site, and transcription terminator) and a second nucleic acid sequence, the coding sequence or coding region, wherein the expression control sequence directs or otherwise regulates transcription and/or translation of the coding sequence.

[0058] The term “optional” or “optionally” as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes embodiments where a particular feature or structure is present and embodiments where the feature or structure is absent, or embodiments where the event or circumstance occurs and embodiments where it does not. [0059] As used herein, “recombinant” refers to the alteration of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or RNA in a cell or virus or expression vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination. Recombinant can also refer to manipulation of DNA or RNA in a cell or virus by random or directed mutagenesis. A “recombinant” cell or nucleic acid can typically be described with reference to how it differs from a naturally occurring counterpart (the “wild type”). In addition, any reference to a cell or nucleic acid that has been “engineered” or “modified” and variations of those terms, is intended to refer to a recombinant cell or nucleic acid.

[0060] The terms “transduce”, “transform”, “transfect”, and variations thereof as used herein refers to the introduction of one or more nucleic acids into a cell. For practical purposes, the nucleic acid must be stably maintained or replicated by the cell for a sufficient period of time to enable the function(s) or product(s) it encodes to be expressed for the cell to be referred to as “transduced”, “transformed”, or “transfected”. As will be appreciated by those of skill in the art, stable maintenance or replication of a nucleic acid may take place either by incorporation of the sequence of nucleic acids into the cellular chromosomal DNA, e.g., the genome, as occurs by chromosomal integration, or by replication extrachromosomally, as occurs with a freely-replicating plasmid. A virus can be stably maintained or replicated when it is “infective”: when it transduces a host microorganism, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.

Systems and Methods OLS Enzymes

[0061] The present disclosure results in part from the discovery that various amino acid substitutions in an active site of wild type OLS can increase OLS enzyme activity. According to one aspect of the disclosure, an OLS polypeptide is provided having at least 97% identity to the C. sativa OLS enzyme, the amino acid sequence of which is shown below.

Amino acid sequence of C. sativa OLS enzyme (SEQ ID NO: 1)

[0062] In some embodiments, the OLS polypeptide further comprises conserved, wild type amino acids at positions 157, 297 and 330, namely C157, H297 and N330.

[0063] In some embodiments, the OLS polypeptide has at least one amino acid substitution in an active site (i.e., XI 98 to X209) of the wild type C. sativa OLS enzyme, as represented by the amino acid sequence shown below. Amino acid sequence of C. sativa OLS enzyme (SEQ ID NO: 2)

1 MNHLRAEGPA SVLAIGTANP ENILLQDEFP DYYFRVTKSE HMTQLKEKFR KICDKSMIRK

61 RNCFLNEEHL KQNPRLVEHE MQTLDARQDM LW EVPKLGK DACAKAIKEW GQPKSKITHL

121 IFTSASTTDM PGADYHCAKL LGLSPSVKRV MMYQLGCYGG GTVLRIAKDI AENNKGARVL

181 AVCCDIMACL FRGPSESXXX XXXXXXXXXD GAAAVIVGAE PDESVGERPI FELVSTGQTI

241 LPNSEGTIGG HIREAGLIFD LHKDVPMLIS NNIEKCLIEA FTPIGISDWN SIFWITHPGG

301 KAILDKVEEK LHLKSDKFVD SRHVLSEHGN MSSSTVLFVM DELRKRSLEE GKSTTGDGFE

361 WGVLFGFGPG LTVERW VRS VPIKY

[0064] In some embodiments, one or more amino acid substitutions in the wild type C. sativa OLS enzyme (i.e., XI 98 to X209) provides an OLS polypeptide having an increased k_cat value as compared to wild type k_cat values. In one embodiment, an OLS polypeptide is provided having at least one amino acid substitution in an active site of the wild type C. sativa OLS enzyme, wherein the OLS polypeptide has a k_cat value greater than 5 turnovers per minute, a k_cat value greater than 10 turnovers per minute, greater than 50 turnovers per minute, greater than 100 turnovers per minute, or greater than 500 turnovers per minute.

[0065] In some embodiments, provided herein is an OLS polypeptide of SEQ ID NO: 2 including one or more of the following mutations. In one embodiment, X at position 198 is Asp, His, Ser, Thr, or Arg. In another embodiment, X at position 199 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala. In another embodiment, X at position 200 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala. In another embodiment, X at position 201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala. In another embodiment, X at position 202 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala. In another embodiment, X at position 203 is lle, Leu, Val, Ala, Met, or Phe. In another embodiment, X at position 204 is Ala, Cys, Gly, Pro, Ser, Thr, or Val. In another embodiment, X at position 205 is Ala, Asn, Asp, Gin, His, Met, or Ser. In another embodiment, X at position 206 is Ala, Cys, His, Leu, Pro, Ser, Thr, or Val. In another embodiment, X at position 207 is Ala, lle, Leu, or Val. In another embodiment, X at position 208 is Leu, lle, or Phe. In another embodiment, X at position 209 is Ala, Gly, or Ser. In one embodiment, X at position 204 is Ala, Cys, Ser, Val, or Thr. In some embodiments, X at position 204 is Cys. In some embodiments, X at position 204 is Ser.

[0066] In some embodiments, an OLS polypeptide according to SEQ ID NO: 2 is provided having an increased k_cat value as compared to wild type k_cat values. Lor example, in some embodiments an OLS polypeptide according to SEQ ID NO: 2 is provided having a k_cat value greater than 5 turnovers per minute, a k_cat value greater than 10 turnovers per minute, greater than 50 turnovers per minute, greater than 100 turnovers per minute, or greater than 500 turnovers per minute.

[0067] In some embodiments, provided herein is a polypeptide comprising a 12-mer corresponding to an active region of the wild type C. sativa OLS (i.e., positions D198 to G209), as represented by the amino acid sequence below (SEQ ID NO: 14).

1 DLELLVGQAI FG

[0068] In some embodiments, provided herein is a polypeptide according to SEQ ID NO: 14, including one or more of the following mutations. In one embodiment, D1 is D, H, S, T or R. In another embodiment, L2 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In another embodiment, E3 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In another embodiment, L4 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In another embodiment, L5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In another embodiment, V6 is I, L, V, A, M, or F. In another embodiment, G7 is A, C, P, S, T, G, or V. In another embodiment, Q8 is A, N, D, Q, H, M, or S; In another embodiment, A9 is A, C, H, L, P, S, T, or V. In another embodiment, 110 A, I, L, or V. In another embodiment, FI 1 is L, I, or F. In another embodiment, G12 is A, G, or S. In some embodiments, G7 is selected from the group consisting of A, C, S, V, and T. In one embodiment, G7 is C. In one embodiment, G7 is S.

[0069] Also provide herein are a number of genes that are counterparts to wild type genes that have been mutated to facilitate biosynthesis of olivetol, OLA, OLA-derived compound, and other cannabinoids or cannabinoid derivatives. The host cell making these materials is a recombinant host cell; in many embodiments, the host cell has been genetically modified to comprise heterologous nucleic acid(s) encoding a polyketide synthase provided or utilized herein.

[0070] In certain embodiments, and OLS provided herein include those that are homologous to consensus sequences provided by the disclosure. As noted above, any enzyme substantially homologous to an enzyme specifically described herein can be used in a host cell of the disclosure. One enzyme is homologous to another (the “reference enzyme”) when it exhibits the same activity of interest and can be used for substantially similar purposes. Generally, homologous enzymes share substantial sequence identity. Sets of homologous enzymes generally possess one or more specific amino acids that are conserved across all members of the consensus sequence protein class.

[0071] The percent sequence identity of an enzyme relative to a consensus sequence is determined by aligning the enzyme sequence against the consensus sequence. Those skilled in the art will recognize that various sequence alignment algorithms are suitable for aligning an enzyme with a consensus sequence. See, for example, Needleman, SB, et al, “A general method applicable to the search for similarities in the amino acid sequence of two proteins.” Journal of Molecular Biology 48 (3): 443-53 (1970). Following alignment of the enzyme sequence relative to the consensus sequence, the percentage of positions where the enzyme possesses an amino acid (or dash) described by the same position in the consensus sequence determines the percent sequence identity.

[0072] Provided herein are consensus sequences useful in identifying and constructing OLSs of the disclosure. In various embodiments, these OLS consensus sequences contain active site amino acid residues believed to be necessary (although the disclosure is not to be limited by any theory of mechanism of action) for increased enzymatic activity (i.e., increased k_cat values), namely the amino acid residues at D198 to G209 of wild type OLS from C. sativa. An OLS enzyme encompassed by a consensus sequence provided herein has an enzymatic activity that is identical, or essentially identical, or at least substantially similar with respect to ability to produce olivetol to that of one of the OLS enzymes exemplified herein. An OLS enzyme may be found in nature or, more typically, is an engineered mutant of a wild type enzyme modified in accordance with the disclosure to have increased enzymatic activity. A OLS enzyme may be identified or constructed from another enzyme by mutating the sequence of the other enzyme to create a sequence encompassed by a consensus sequence herein; if an enzyme shares substantial homology to a consensus sequence herein but has suboptimal, including no, polyketide synthase activity, then, in accordance with the disclosure, it is mutated to conform to a consensus sequence provided herein to provide a OLS of the disclosure.

[0073] In other aspects, increased olivetol, OLA, an OLA-derived compound, or another cannabinoid or cannabinoid derivative yield, titer, and/or productivity is achieved by employing host cells provided by the disclosure that have been genetically modified in ways other than, or in addition to, introduction of a heterologous OLS and/or by employing fermentation conditions provided by certain methods of the disclosure. In brief, the recombinant host cell of the disclosure comprises genetic modifications that increase olivetol biosynthesis, increase malony1-CoA biosynthesis, increase hexanoy1-CoA synthetase biosynthesis, increased available hexanoic acid, increase available carbohydrates (i.e., for example glucose, sucrose, etc.), and the likes.

[0074] In accordance with the disclosure, increased olivetol titer, yield, and/or productivity can be achieved by genetic modifications that increase biosynthesis of OLSs having increased k_cat values, wherein the disclosure provides OLS enzymes, vectors for expressing OLS enzymes, host cells expressing OLS enzymes to increase olivetol titer, yield, and/or productivity, and methods relating thereto. As described above, olivetol is comprised of three molecules of malony1-CoA and one molecule of hexanoy1-CoA, wherein the process for converting these molecules to olivetol requires a polyketide synthase enzyme. Thus, increases in OLS enzymatic activity (i.e., turnover/k_cat value), increases in malony1-CoA biosynthesis, and increases in hexanoy1-CoA synthetase biosynthesis can improve olivetol production, olivetolic acid production, and/or olivetolic acid-derived compound production.

Hexanoy1-CoA biosynthesis

[0075] In many embodiments, the host cell is genetically modified to comprise heterologous nucleic acid(s) encoding a hexanoy1-CoA synthetase. One non-limiting example of a functional hexanoly-CoA synthetase (HCS) is the E. coli FadK protein (UniProt ID: P38135), as represented by the amino acid sequence below (SEQ ID NO: 15).

1 MHPTGPHLGP DVLFRESNMK VTLTFNEQRR AAYRQQGLWG DASLADYWQQ TARAMPDKIA

61 W DNHGASYT YSALDHAASC LANWMLAKGI ESGDRIAFQL PGWCEFTVIY LACLKIGAVS

121 VPLLPSWREA ELVWVLNKCQ AKMFFAPTLF KQTRPVDLIL PLQNQLPQLQ QIVGVDKLAP

181 ATSSLSLSQI IADNTSLTTA ITTHGDELAA VLFTSGTEGL PKGVMLTHNN ILASERAYCA

241 RLNLTWQDVF MMPAPLGHAT GFLHGVTAPF LIGARSVLLD IFTPDACLAL LEQQRCTCML

301 GATPFVYDLL NVLEKQPADL SALRFFLCGG TTIPKKVARE CQQRGIKLLS VYGSTESSPH

361 AVVNLDDPLS RFMHTDGYAA AGVEIKW DD ARKTLPPGCE GEEASRGPNV FMGYFDEPEL

421 TARALDEEGW YYSGDLCRMD EAGYIKITGR KKDIIVRGGE NISSREVEDI LLQHPKIHDA

481 CVVAMSDERL GERSCAYW L KAPHHSLSLE EW AFFSRKR VAKYKYPEHI W IEKLPRTT

541 SGKIQKFLLR KDIMRRLTQD VCEEIE

[0076] In accordance with the disclosure, additional hexanoy1-CoA can be heterologously expressed to increase olivetol biosynthesis. In some embodiments, the feedstock for the fermentation broth is a carbohydrate (i.e., for example glucose, sucrose, etc) and, optionally, hexanoic acid (or hexanoate salt, depending upon the fermentation pH). If the hexanoic acid is produced microbially from the carbohydrate in the fermentation broth, the fermentation broth does not need to be supplemented with hexanoic acid. When supplemented, the hexanoic acid (or a salt of hexanoic acid) serves as the substrate for hexanoy1-CoA biosynthesis. The pKa for hexanoic acid is 4.88 and the solubility in water is relatively low (ca. 10 g/1). These characteristics are advantageous for supplementation to a fermentation broth when using a microbe capable of growth and/or product formation at relatively low pH values. For example, P. kudriavzevii (as well as many other yeast strains) grows and consumes sugar at pH values near the hexanoic acid pKa. These types of microbes will generally only uptake the acid form and not the salt hexanoate. Thus, for these embodiments, at least a substantial fraction of the hexanoate supplemented to the fermentation broth is in the acid form. A fermentation in which the pH is controlled at the pKa of hexanoic acid, for example, would ensure that 50% of the hexanoate is in the acid form. Operating the fermentation at a lower pH would further increase hexanoic acid availability in vivo. Second, the relatively low solubility of hexanoic acid in the fermentation broth is helpful for preventing substrate-induced toxicity. Importantly, in some embodiments, toxicity can further be mitigated by operating the fermentation at a higher pH to decrease the concentration of free acid present. Thus, in various embodiments of the disclosure the fermentation pH is maintained between 4.0 and 6.0. In some embodiments, the fermentation pH is approximately 5.0.

[0077] In addition to fermentation pH, another process parameter altered or controlled according to this disclosure is the oxygen transfer rate, which is typically expressed as mmol/l/hr of oxygen transferred into the fermentation broth from gas being sparged into the fermenter. Production of malony1-CoA from glucose results in the net formation of NAD(P)H, which must be re-oxidized to NAD(P)⁺ to maintain redox balance and continued pathway activity over time. The requirement for NAD(P)H re-oxidation necessitates the fermentation process being aerobic. As such, some embodiments provide an aerobic fermentation process having an oxygen transfer rate greater that 0 mmol/l/hr. and preferably greater than 20 mmol/l/hr, greater than 50 mmol/l/hr, greater than 75 mmol/l/hr, or greater than 100 mmol/l/hr.

[0078] In the methods of the disclosure, carbon feedstocks are utilized for production of olivetol or another compound produced herein. Suitable carbon sources include, without limitation, those selected from the group consisting of purified sugars (e.g., dextrose, sucrose, xylose, arabinose, lactose, etc.); plant-derived, mixed sugars (e.g., sugarcane, sweet sorghum, molasses, cornstarch, potato starch, beet sugar, wheat, etc.), plant oils, fatty acids, glycerol, cellulosic biomass, alginate, ethanol, carbon dioxide, methanol, and synthetic gas (“syn gas”).

[0079] In some embodiments, one or multiple intermediates and precursors of the cannabinoid pathways, including sugar, malonic acid, hexanoic acid, mevalonate, olivetol, and olivetolic acid are employed as feedstock. In one embodiment the one or multiple intermediates and precursors of the cannabinoid pathway are utilized in a cell free system, e.g., and without limitation, with a prenyl transferase that condenses olivetol/olivetolic acid and GPP. In some embodiments, another enzyme of the cannabinoid pathway is incorporated in a host cell (or added to a cell free system) to further process CBGA or CBG into THCA, CBDA, THC, CBD or other CBG(A) derivative. Without being bound by theory, in some embodiments, such a feedstock would result in a commercially relevant process without the limitations and timelines associated with careful balancing of full pathway enzymes in a cell or cel1-free system.

[0080] A given host cell may catabolize a particular feedstock efficiently or inefficiently. If a host cell inefficiently catabolizes a feedstock, then one can modify the host cell to enhance or create a catabolic pathway for that feedstock. Additional embodiments of the disclosure include the use of methanol catabolizing host strains. In some embodiments, the host is a yeast strain. In some embodiments, the host is selected from Pichia kudriavzevii, Komagataella pastoris, Pichia methanolica, or Pichia pastoris.

[0081] The disclosure provides host cells comprising genetic modifications that increase titer, yield, and/or productivity of olivetol or another compound produced herein through the increased ability to catabolize non-native carbon sources. Wild type S. cerevisiae cells are unable to catabolize pentose sugars, lignocellulosic biomass, or alginate feedstocks. In some embodiments, the disclosure provides a S. cerevisiae cell comprising a heterologous nucleic acid encoding enzymes enabling catabolism of pentose sugars useful in production of olivetol, as described herein. In other embodiments, the heterologous nucleic acid encodes enzymes enabling catabolism of lignocellulosic feedstocks. In yet other embodiments of the disclosure, the heterologous nucleic acid encodes enzymes increasing catabolism of alginate feedstocks.

[0082] Those skilled in the art will recognize that the individual manipulations to increase production of olivetol or another compound produced herein from a given host cell can be used in combinations and often confer simultaneous benefits resulting in a much higher output than a single manipulation and in cases, the sum of the individual manipulations. Thus, the disclosure encompasses not only the single manipulations described herein, it also is embodied by any combination of these perturbations resulting in the increase of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative produced in the host strain. 1-Hexanol catabolic pathway enzymes [0083] In one embodiment, provided herein is a host cell, which host cell utilizes hexanol to prepare hexanoy1-CoA. In some embodiments, the hexanol is added externally. In some embodiments, the host cell comprises a 1 -hexanol catabolic pathway enzyme, defined herein as comprising one or more enzymes catalyzing the conversion of 1 -hexanol to hexanoy1-CoA with a net production of ATP. As illustrated in Figure 1, hexanoy1-CoA is one of the metabolites required to produce cannabinoids.

[0084] As disclosed herein, olivetol synthase (OLS; EC 2.3.1.206), catalyzes the conversion of 1 hexanoy1-CoA and 2 malony1-CoA to olivetol or olivetolic acid in the case where an olivetolic acid cyclase (OAC; EC 4.4.1.26) is expressed with the olivetol synthase. One olivetol synthase substrate, hexanoy1-CoA, is not present at high concentrations in the cytosol of most microbes, and in particular yeast cells. Therefore, without being bound by theory, native hexanoy1-CoA biosynthetic pathways need to be over expressed, or non-native biosynthetic pathways introduced into the host cell to increase hexanoy1-CoA biosynthesis enabling increased production of cannabinoid products.

[0085] As provided herein, several biosynthetic pathways to hexanoy1-CoA can be used. One embodiment relates to a direct synthesis of hexanoy1-CoA from glucose, utilizing fatty acid biosynthetic enzymes. Without being bound by theory, this route can be inefficient, low yielding, and difficult to optimize as fatty acid biosynthesis generally does not result in a single alky1-CoA product but rather a number of alky1-CoA products of different chain lengths. These undesired byproducts (e.g., butyry1-CoA and octanoy1-CoA) may be inadvertently used as substrates by the OLS, resulting in undesired cannabinoid impurities.

[0086] A second embodiment relates to supplementing exogenous hexanoic acid to a fermentation of a host cell expressing a hexanoy1-CoA synthetase. This route addresses many of the short-comings described above for the in vivo biosynthesis of hexanoic acid from glucose. E.g., a low pH fermentation (defined herein as below pH 6.0) can allow for passive diffusion of hexanoic acid across the cell membrane. However, it is not always possible or desirable to run a low pH fermentation; for example, some microbes, including E. coli and S. cerevisiae, perform better at a neutral pH. In these cases, the fermentation broth pH will be above the pKa of hexanoic acid (4.88) and a hexanoate importer will be required to transport the monoanionic species (i.e., hexanoate) from the broth into the cytosol. [0087] Thus, in some embodiments, provided herein are host cells comprising a 1-hexanol catabolic pathway comprising enzymes capable of converting cytosolic 1-hexanol to hexanoy1- CoA. Without being bound by theory, the advantages to using a 1-hexanol catabolic pathway are many-fold. First, 1-hexanol is soluble in water to about 6 g/1 at 25°C irrespective of pH and thus can be found in the aqueous phase at sufficient concentrations to enable passive diffusion across the cell membrane. Since the energy required for passive diffusion comes from the extracellular: intracellular concentration gradient, import of hexanol occurs without cellular expenditure of energy, as is required for import of hexanoate under neutral pH conditions. Second, as described in more detail below, the activity of a 1-hexanol catabolic pathway for conversion of 1-hexanol to hexanoy1-CoA results in a net production of NAD(P)H, which can be used by the cell for ATP production under aerobic fermentation conditions. This net production of ATP both provides selective pressure for the cell to maintain the 1-hexanol catabolic pathway and the resulting ATP is useful for improved cellular health and vitality, and increased cannabinoid product biosynthesis.

[0088] There are two steps in the hexano1-based hexanoy1-CoA biosynthetic pathway. In the first step, 1-hexanol is oxidized to hexanal by a 1-hexanol dehydrogenase with concomitant reduction of a redox cofactor. In the second step, hexanal is converted to hexanoy1-CoA using a variety of enzyme catalyzed reactions, as outlined in Figure 2.

[0089] In the first step, the 1-hexanol dehydrogenase utilizes a redox cofactor, including but not limited to NAD⁺, NADP⁺, FAD, and FMN. Due to their relative abundance in the cytosol of most microbes, NAD⁺ and NADP⁺ utilizing 1-hexanol dehydrogenases are preferred. NAD⁺ and NADP⁺ utilizing 1-hexanol dehydrogenases include those in the following enzyme classes 1.1.1.1 and 1.1.1.2:

(i) An NADH-dependent 1 -hexanol dehydrogenase (EC 1.1.1.1 ) capable of catalyzing the reaction:

1-hexanol + NADP⁺ ® NADPH + Hexanal

(ii) A NADPH-dependent alcohol dehydrogenase (EC 1.1.1.2) capable of catalyzing the reaction:

1-hexanol + NADP⁺ ® NADPH + Hexanal

[0090] In the second step of the hexano1-based hexanoy1-CoA biosynthetic pathway, hexanal (from the first step described above) can be converted to hexanoy1-CoA using a variety of enzyme catalyzed reactions, as outlined in Figure 2. These enzymes include the following:

A NADH-dependent hexanal dehydrogenase capable of catalyzing the reaction: Hexanal + NAD⁺ + H₂O ® NADH + Hexanoate (Figure 2; enzyme 3)

This reaction is irreversible under physiological conditions.

A NADPH-dependent hexanal dehydrogenase capable of catalyzing the reaction: Hexanal + NADP⁺ + H₂O ® NADPH + Hexanoate (Figure 2; enzyme 4)

This reaction is irreversible under physiological conditions.

A hexanoy1-CoA synthetase capable of catalyzing the reaction:

Hexanoate + ATP + CoA ® AMP + Hexanoy1-CoA (Figure 2; enzyme 5)

This reaction is irreversible under physiological conditions.

A hexanoate kinase capable of catalyzing the reaction:

Hexanoate + ATP ® ADP + Hexyl phosphate (Figure 2; enzyme 6)

A phosphate hexyltransferase capable of catalyzing the reaction:

Hexyl phosphate + Co ® Hexanoy1-CoA (Figure 2; enzyme 7)

A hexanal dehydrogenase (acylating) capable of catalyzing the reaction:

Hexanal + NAD(P) ⁺ + Co ® NADPH + Hexanoy1-CoA (Figure 2; enzyme 8) [0091] Thus, various 1-hexanol catabolic pathways are possible and several different combinations of 1-hexanol catabolic pathway enzymes are useful to convert 1-hexanol to hexanoy1-CoA. A particularly efficient 1-hexanol catabolic pathway is one comprising the following enzymes: one or more NAD(P)H-dependent 1-hexanol dehydrogenases; one or more NAD(P)H-dependent hexanal dehydrogenases; and one or more Hexanoy1-CoA synthetases. This route is contemplated to be efficient because two of the three enzyme-catalyzed reactions are irreversible under physiological conditions and hexanoy1-CoA formation is strongly favored thermodynamically. The combination of these three enzymes results in 2 mol NAD(P)H per mol hexanoy1-CoA, which can be used by the electron transport for ATP production. Under aerobic conditions in yeast where the 1-hexanol catabolic pathway enzymes are expressed in the cytosol, 1.5 ATP are generated per NAD(P)H oxidized. Thus, there is a net gain of 1 ATP per hexanoy1- CoA produced using this pathway. Because the hexanoy1-CoA synthetase converts 1 ATP to 1 AMP, it is equivalent to a net loss of 2 ATP.

[0092] Another efficient 1-hexanol catabolic pathway comprises the following enzymes: one or more NAD(P)H-dependent 1-hexanol dehydrogenases; one or more hexanoate kinases; and one or more phosphate hexyltransferases. Without being bound by theory, this route is useful because the conversion of 1-hexanol to hexanoy1-CoA can be thermodynamically favored under physiological conditions by increasing the concentration 1-hexanol and decreasing the concentration of hexanoy1-CoA. The combination of these three enzymes results in 2 mol NAD(P)H per mol hexanoy1-CoA. Accounting for NAD(P)H oxidation and ATP formation, under aerobic conditions in yeast where the 1-hexanol catabolic pathway enzymes are expressed in the cytosol, this pathway results in a net gain of 2 ATP per hexanoy1-CoA produced.

[0093] A third 1-hexanol catabolic pathway comprises the following enzymes: one or more NAD(P)H-dependent 1-hexanol dehydrogenases; and one hexanal dehydrogenase (acylating).

The pathway can be pushed in the forward direction under conditions of high concentration 1- hexanol and low concentration NAD(P)H. The combination of these two enzymes results in 2 mol NAD(P)H per mol hexanoy1-CoA produced. Accounting for NAD(P)H oxidation and ATP formation, under aerobic conditions in yeast where the 1-hexanol catabolic pathway enzymes are expressed in the cytosol, this pathway results in a net gain of 3 ATP per hexanoy1-CoA produced. [0094] The kinetic parameters for all the 1-hexanol catabolic pathway enzymes used are sufficient to support the rapid conversion of substrate to product at reasonable enzyme concentrations. Therefore, Km of each 1-hexanol catabolic pathway enzyme for its target substrate is contemplated to be less than 10 mM, and ideally below 1 mM. A low Km is necessary to ensure that pathway intermediate concentrations do not reach toxic levels.

[0095] The k_cat value of each 1-hexanol catabolic pathway enzyme with its target substrate is contemplated to be greater than 1 turnover/second, more preferably greater than 10 turnovers/second, and ideally greater than 100 tumovers/second. At lower than 1 turnover/second, an exorbitant amount of enzyme must be expressed to support efficient pathway flux. Generally speaking, a higher k_cat value means less enzyme need be expressed to sustain the same pathway flux, translating into increased microbial health and efficiency.

[0096] 1-hexanol catabolic pathway enzymes can be selected for improved kinetic constants ( i.e ., lower Km values and higher k_ra, values) in a growth-based selection. In short, first a microbe is identified (e.g., a wild type microbe) or engineered that displays no growth, or low levels of growth, on 1-hexanol as a sole carbon source. The microbe is then transformed with nucleic acids encoding one or more 1-hexanol catabolic pathway enzymes and grown for a period of time on 1- hexanol as the sole carbon source. Over time, the faster growing cells are those which express mutant 1-hexanol catabolic pathways with decreased Km and/or increased k_cat kinetic constants. By choosing different promoters, a desired concentration of each pathway enzyme can be achieved, and thus selective pressure can be increased by choosing weaker promoters resulting in decreased expression of one or more target 1-hexanol catabolic pathway enzymes.

A geranio1-dependent GPP pathway enzymes

[0097] In one embodiment, provided herein is a host cell comprising a geranio1-dependent GPP pathway. A geranio1-dependent GPP pathway provides a route to produce geranyl diphosphate (GPP) in the cell. As illustrated in Figure 1, GPP is one of the metabolites required to produce cannabinoids.

[0098] Various cannabinoid products are biosynthetically produced from cannabigerolic acid (CBGA), which in turn is generated from the activity of cannabigerolic acid synthase (CGAS), an aromatic prenyltransferase which converts its substrates olivetolic acid and geranyl diphosphate (GPP) to cannabigerolic acid as follows:

Olivetolic acid + GP ® C₂nnabigerolic acid.

Microbial production of GPP from glucose requires the optimization of a multi-step pathway from glucose to GPP. For example, the mevalonate pathway, which comprises enzymes catalyzing the conversion of acety1-CoA to GPP, has eight enzyme-catalyzed steps and many of the intermediates are reported to be toxic to microbes. The pathway may be low yielding, and six molecules of acety1-CoA are required to produce one molecule of GPP. Lastly, the mevalonate pathway competes for acety1-CoA with acety1-CoA carboxylase and olivetolic acid synthase, and balancing of the relative activities of these proteins is challenging.

[0099] Therefore, there is need for an alternative pathway to GPP from non-glucose feedstocks. Accordingly, in some embodiments, provided herein are host cells comprising a pathway comprising enzymes capable of converting geraniol or various geraniol derivatives to GPP. The expression of said geranio1-dependent GPP pathway enzymes results in increased production of cannabigerolic acid or downstream derivatives of cannabigerolic acid as compared to a parental, control cell that does not express said geranio1-dependent GPP pathway enzymes. The geranio1-dependent GPP pathway requires no exotic cofactors or cosubstrates and thus can be used in a variety of microbes engineered for production of cannabigerolic acid or downstream cannabinoids. Non-limiting examples of host cells the geranio1-dependent GPP pathway can be expressed in include E. coli, S. cerevisiae, and P. kudriavzevii. [0100] The geranio1-dependent GPP pathway (Figure 3) comprises at least two enzymes: a geraniol kinase and a geranyl monophosphate kinase.

Geraniol kinase (GK) catalyzes the conversion of geraniol to geranyl monophosphate with concomitant hydrolysis of ATP: geraniol + ATP ® ADP + geranyl monophosphate (Figure 3; enzyme 1) Geranyl monophosphate kinase (GPK) catalyzes the conversion of geranyl monophosphate to geranyl diphosphate with concomitant hydrolysis of ATP: geranyl monophosphate + ATP ® ADP + geranyl diphosphate (Figure 3; enzyme 2)

[0101] The geranio1-dependent GPP pathway may optionally include one or more enzymes catalyzing the conversion of various geraniol derivatives to geraniol. These geraniol derivatives include, but are not limited to, (3S)-linalool, (3R)-linalool, myrcene, geranyl acetate, and geranial. As depicted in Figure 3, linalool is converted to geraniol through the activity of a geraniol isomerase catalyzing the reaction: linaloo ® geraniol (Figure 3; enzyme 3)

[0102] Myrcene can in turn be converted into linalool through the activity of a linalool dehydratase catalyzing the reaction: myrcene + H₂O ® linalool (Figure 3; enzyme 4)

Thus, myrcene can be converted to geraniol by expressing a linalool dehydratase and a geraniol isomerase. Lastly, geranyl acetate can be converted to geraniol and acetate by expressing a geranyl acetate esterase catalyzing the reaction: geranyl acetate + H₂O ® geraniol + acetate (Figure 3; enzyme 5)

Thus, as provided herein, various combinations of geranio1-dependent GPP pathway enzymes are useful to convert geraniol or geraniol derivatives to GPP.

[0103] One geranio1-dependent GPP pathway comprises GK and GPK and is capable of converting geraniol to GPP. Geraniol (MW = 154 g/mol) is slightly soluble in water (0.7 g/1; 4.5 mM). Thus, the fermentation broth can be exogenously supplemented with geraniol, which can diffuse into the host cell and be used for GPP production. In order to maintain a sufficiently high intracellular concentration of geraniol, which may need to be between 1-4.5 mM, an extracellular concentration of between 1 - 4.5 mM - or higher if elevated temperatures are used or solubilizing reagents such as DMSO are used - must be maintained throughout the course of the fermentation. [0104] A second geranio1-dependent GPP pathway comprises three enzymes - Linalool isomerase, GK and GPK - and is capable of converting linalool to GPP. Either (3S)-linalool, (3R)- linalool, or a racemic mixture of the two stereoisomers are exogenously supplemented to the fermentation broth and linalool is converted to GPP. Advantages of this route include the low cost of the racemic linalool mixture and linalool’ s higher solubility (10 mM) as compared to geraniol. The higher solubility can be used to increase the rate of diffusion across the cell membrane and promote increased geranio1-dependent GPP pathway flux and therefore increased GPP, cannabigerolic acid, and downstream cannabinoid production.

[0105] The measured kinetic constants for all the geranio1-dependent GPP pathway enzymes used are contemplated to be sufficient to support efficient conversion of its respective substrate to product without requiring high (see below) enzyme copy numbers per cell. Enzymes with low activity necessitate either a high enzyme load, which detracts from cell growth and vitality, or the enzyme becomes the rate limiting step in GPP, cannabigerolic acid, or downstream cannabinoid biosynthesis.

[0106] A low measured Km value is necessary to ensure that pathway intermediate concentrations do not reach toxic levels. The Km of each pathway enzyme for its respective pathway substrate is contemplated to be less than 10 mM, and ideally below 1 mM.

[0107] Generally speaking, a higher k_cat value means improved enzyme activity, which translates both to higher pathway flux and a lower enzyme load. The k_cat value of each geranio1- dependent GPP pathway enzyme with its target substrate is contemplated to be greater than 1 tumover/second, more preferably greater than 10 turnovers/second, and ideally greater than 100 tumovers/second.

[0108] Lastly, the intracellular enzyme concentration, measured as protein copy number per cell or protein copy number per gram dry cell weight, is contemplated to be low. Either measurement is valid and those skilled in the art will recognize that one can readily determine the number of cells per gram dry cell weight using a flow cytometer or other standard measurement techniques. The enzyme copy number per cell is contemplated to be no more than 500,000 copies per cell, more preferably less than 250,000 copies per cell, and ideally less than 100,000 copies per cell.

[0109] A specific geraniol kinase has not been reported to date and occurs at only low levels in related enzymes, for example, farnesol kinases (EC 2.7.1.216). Famesol kinases are defined as those enzymes with a higher catalytic efficiency (kcat/Km) for farnesol as compared to geraniol. Farnesol kinases can be converted to geraniol kinases by mutagenesis of the active site amino acids resulting in a protein with a higher catalytic efficiency for geraniol as compared to farnesol. [0110] A specific geraniol monophosphate kinase has not been reported to date and occurs at only low levels in related enzymes, for example, isopentenyl phosphate kinases. Farnesol kinases are defined as those enzymes with a higher catalytic efficiency (kcat/Km) for farnesol as compared to geraniol. Isopentenyl phosphate kinases (EC 2.7.4.26), are defined as having a higher catalytic efficiency (kcat/Km) for isopentenyl phosphate as compared to geraniol. An isopentenyl phosphate kinase is converted to a geraniol kinase by mutagenesis of the active site amino acids resulting in a protein with a higher catalytic efficiency for gemanyl phosphate as compared to isopentenyl phosphate. For example, Mabanglo et al, (ACS Chem. Biol. 2012, Jul 20; 7(7): DOI 10.1021/cb3000106e; (incorporated herein by reference)) reports the introduction of point mutations into an isopentenyl kinase derived from Thermoplasma acidophilum (UniProt ID: 026153) that result in increased activity using geranyl monophosphate as the substrate. Namely, mutant proteins comprising the mutations Y70A/V73A/V130A/I140A, Y70A/V73A/I140A, or V73A/V130A/I140A have increased activity toward geranyl monophosphate as compared to the wild type, control enzyme. Similar mutations are incorporated into the corresponding positions of enzymes homologous to this enzyme, including those enzymes derived from Methanocaldococcus jannaschii (UniProt ID: Q60352) and Methanothermobacter thermautotrophicus (UniProt ID: Q9HLX1).

[0111] Similarly, as disclosed here, other acyl monophosphate kinases can be converted to geraniol monophosphate kinases. For example, a farnesol monophosphate kinases can be converted to geraniol monophosophate kinase.

[0112] In one embodiment, provided herein is a method of selecting for increased geranio1- dependent GPP pathway enzyme activity and/or specificity is through a GPP selection. Since GPP is required for cellular growth, a strain made auxotrophic for GPP by knocking out one or more enzymes in the native biosynthetic pathway is used to select for improved geranio1-dependent GPP pathway activity. In yeast, ERG20 is the enzyme responsible for production of GPP and thus a conditional or permanent deletion of the ERG20 gene can be used to select for increased geranio1- dependent GPP pathway activity. Expression Vectors

[0113] In various aspects, provided herein is a recombinant host cell modified by “genetic engineering” as disclosed herein in one embodiment, a recombinant polyketide synthase enzyme is introduced. In another embodiment, an aromatic prenyitransferase is introduced. In another embodiment, the modification increases the production of malony1-CoA, hexanoly-CoA or a R¹- CoA. In some embodiments, the host cell is engineered via recombinant DNA technology to express heterologous nucleic acids that encode a cannahinoid pathway enzyme such as an OLS enzyme, which is either a mutated version of a naturally occurring enzyme, or a non-naturally occurring enzyme as provided herein.

[0114] In one preferred embodiment, the disclosure includes methods of generating a polynucleotide that expresses one or more of the SEQ IDs related to a modified OLS provided or utilized herein. In certain preferred embodiments, the proteins of the disclosure are expressed using any of a number of systems, such as in whole plants, as well as plant cell and/or yeast suspension cultures. E.g., the polynucleotide that encodes the OLS is placed under the control of a promoter that is functional in the desired host cell. An extremely wide variety of promoters may be available and can be used in the expression vectors of the disclosure, depending on the particular application. Ordinarily, the promoter selected depends on the cell in which the promoter is to be active. Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included.

[0115] Nucleic acid constructs provided and utilized herein include expression vectors that comprise nucleic acids encoding one or more polyketide synthase enzymes. The nucleic acids encoding the enzymes are operably linked to promoters and optionally other control sequences such that the subject enzymes are expressed in a host cell containing the expression vector when cultured under suitable conditions. The promoters and control sequences employed depend on the host cell selected for the production of olivetol, OLA, OLA-derived compound, or another cannahinoid or cannahinoid derivative. Thus, the disclosure provides not only expression vectors but also nucleic acid constructs useful in the construction of expression vectors. Methods for designing and making nucleic acid constructs and expression vectors generally are well known to those skilled in the art and so are only briefly reviewed herein.

[0116] Nucleic acids encoding the polyketide synthase enzymes can be prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis and cloning. Further, nucleic acid sequences for use in the disclosure can be obtained from commercial vendors that provide de novo synthesis of the nucleic acids.

[0117] A nucleic acid encoding the desired enzyme can be incorporated into an expression vector by known methods that include, for example, the use of restriction enzymes to cleave specific sites in an expression vector, e.g., plasmid, thereby producing an expression vector of the disclosure. Some restriction enzymes produce single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. The ends are then covalently linked using an appropriate enzyme, e.g., DNA ligase. DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.

[0118] A set of individual nucleic acid sequences can also be combined by utilizing polymerase chain reaction (PCR)-based methods known to those of skill in the art. For example, each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and can act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are “spliced” together. In this way, a series of individual nucleic acid sequences may be joined and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is affected.

[0119] A typical expression vector contains the desired nucleic acid sequence preceded and optionally followed by one or more control sequences or regulatory regions, including a promoter and, when the gene product is a protein, ribosome binding site, e.g., a nucleotide sequence that is generally 3-9 nucleotides in length and generally located 3-11 nucleotides upstream of the initiation codon that precede the coding sequence, which is followed by a transcription terminator in the case of E. coli or other prokaryotic hosts. See Shine el al, Nature. 254:34 (1975) and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349 (1979) Plenum Publishing, N.Y. In the case of eukaryotic hosts like yeast, a typical expression vector contains the desired nucleic acid coding sequence preceded by one or more regulatory regions, along with a Kozak sequence to initiate translation and followed by a terminator. See Kozak, Nature 308:241-246 (1984). [0120] Regulatory regions or control sequences include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid coding sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a transcription factor can bind. Transcription factors activate or repress transcription initiation from a promoter. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding transcription factor. Non-limiting examples for prokaryotic expression include lactose promoters ( Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator). Non- limiting examples of promoters to use for eukaryotic expression include pTDH3, pTEF1, pTEF2, pRNR2, pRPL18B, pREV1, pGAL1, pGAL10, pGAPDH, pCUP1, pMET3, pPGK1, pPYK1, pHXT7, pPDC , pFBA1, pTDH2, pPGI1, pPDC1, pTPI1, pEN02, pADH1, and pADH2. As will be appreciated by those of ordinary skill in the art, a variety of expression vectors and components thereof are useful.

[0121] Although any suitable expression vector are useful to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pESC, pTEF, p414CYC1, p414GALS, pSC101, pBR322, pBBRlMCS-3, pUR, pEX, pMRIOO, pCR4, pBAD24, pUC19, pRS series; and bacteriophages, such as M13 phage and l phage. Of course, such expression vectors may only be suitable for particular host cells or for expression of particular polyketide synthases. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell or protein. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, relevant texts and literature describe expression vectors and their suitability to any particular host cell. In addition to the use of expression vectors, strains are built where expression cassettes are directly integrated into the host genome.

[0122] The expression vectors are introduced or transferred, e.g., by transduction, transfection, or transformation, into the host cell. Such methods for introducing expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming P. kudriavzevii with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate.

[0123] For identifying whether a nucleic acid has been successfully introduced or into a host cell, a variety of methods are available. For example, potentially transformed host cells in a culture are separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of a desired gene product of a gene contained in the introduced nucleic acid. For example, an often-used practice involves the selection of cells based upon antibiotic resistance that has been conferred by antibiotic resistance-conferring genes in the expression vector, such as the beta lactamase (amp), aminoglycoside phosphotransferase (neo), and hygromycin phosphotransferase (hyg, hph, hpt) genes.

[0124] In one embodiment, a host cell of the disclosure is transformed with at least one expression vector. When only a single expression vector is used, the vector will typically contain a polyketide synthase gene. Once the host cell has been transformed with the expression vector, the host cell is cultured in a suitable medium containing a carbon source, such as a sugar (e.g., glucose). As the host cell is cultured, expression of the polyketide synthase enzyme(s) occurs. Once expressed, these OLS(s) and other enzymes provided and utilized herein convert three molecules of malony1-CoA and one molecule of hexanoy1-CoA or R¹Co-A, wherein R¹ is defined as herein, to olivetol or a compound of formula (I).

[0125] If a host cell of the disclosure is to include more than one heterologous gene, the multiple genes can be expressed from one or more vectors. For example, a single expression vector can comprise one, two, or more genes encoding one, two, or more mutant OLS enzyme(s), other enzymes of the cannabinoid pathway, e.g., improved malony1-CoA production, hexanoly-CoA, or R¹-CoA production, etc. The heterologous genes can be contained in a vector replicated episomally or in a vector integrated into the host cell genome, and where more than one vector is employed, then all vectors may replicate episomally (extrachromasomally), or all vectors may integrate, or some may integrate and some may replicate episomally. While a “gene” is generally composed of a single promoter and a single coding sequence, in certain host cells, two or more coding sequences are controlled by one promoter in an operon. In some embodiments, a two or three operon system is used.

[0126] In some embodiments, the coding sequences employed have been modified, relative to some reference sequence, to reflect the codon preference of a selected host cell. Codon usage tables for numerous organisms are readily available and can be used to guide sequence design. The use of prevalent codons of a given host organism generally improves translation of the target sequence in the host cell. As one non-limiting example, in some embodiments the subject nucleic acid sequences will be modified for yeast codon preference (see, for example, Bennetzen et al, J. Biol. Chem. 257: 3026-3031 (1982)). In some embodiments, the nucleotide sequences will be modified for P. kudriavzevii codon preference (see, for example, Nakamura el al.. Nucleic Acids Res. 28:292 (2000)). In other embodiments, the nucleotide sequences are modified to include codons optimized for S. cerevisiae codon preference.

[0127] Nucleic acids can be prepared by a variety of routine recombinant techniques. Briefly, the subject nucleic acids can be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which can be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and rt-PCR. Subject nucleic acids can also be prepared by a direct chemical synthesis.

[0128] The nucleic acid transcription levels in a host microorganism can be increased (or decreased) using numerous techniques. For example, the copy number of the nucleic acid can be increased through use of higher copy number expression vectors comprising the nucleic acid sequence, or through integration of multiple copies of the desired nucleic acid into the host microorganism’s genome. Non-limiting examples of integrating a desired nucleic acid sequence onto the host chromosome include recA-mediated recombination, lambda phage recombinase- mediated recombination and transposon insertion. Nucleic acid transcript levels can be increased by changing the order of the coding regions on a polycistronic mRNA or breaking up a polycistronic operon into multiple poly- or mono-cistronic operons each with its own promoter. RNA levels can be increased (or decreased) by increasing (or decreasing) the strength of the promoter to which the protein-coding region is operably linked.

[0129] The translation level of a desired polypeptide sequence in a host microorganism can also be increased in a number of ways. Non-limiting examples include increasing the mRNA stability, modifying the ribosome binding site (or Kozak) sequence, modifying the distance or sequence between the ribosome binding site (or Kozak sequence) and the start codon of the nucleic acid sequence coding for the desired polypeptide, modifying the intercistronic region located 5' to the start codon of the nucleic acid sequence coding for the desired polypeptide, stabilizing the 3 '-end of the mRNA transcript, modifying the codon usage of the polypeptide, altering expression of low- use/rare codon tRNAs used in the biosynthesis of the polypeptide. Determination of preferred codons and low-use/rare codon tRNAs can be based on a sequence analysis of genes derived from the host microorganism.

[0130] The polypeptide half-life, or stability, can be increased through mutation of the nucleic acid sequence coding for the desired polypeptide, resulting in modification of the desired polypeptide sequence relative to the control polypeptide sequence. When the modified polypeptide is an enzyme, the activity of the enzyme in a host is altered due to increased solubility in the host cell, improved function at the desired pH, removal of a domain inhibiting enzyme activity, improved kinetic parameters (lower Km or higher k_cat values) for the desired substrate, removal of allosteric regulation by an intracellular metabolite, and the like. Altered/modified enzymes can also be isolated through random mutagenesis of an enzyme, such that the altered/modified enzyme can be expressed from an episomal vector or from a recombinant gene integrated into the genome of a host microorganism.

Host Cells

[0131] Provided herein are host cells, preferably recombinant host cells, more preferably heterologous recombinant host cells for performing one or more steps of the cannabinoid pathway. In some embodiments, the recombinant host cell is a eukaryote. In various embodiments, the eukaryote is a yeast strain selected from the non-limiting list of example genera: Candida, Cryptococcus , Hansenula, Issatchenkia , Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, or Yarrowia. Those skilled in the art will recognize that these genera broadly encompass yeast, including those distinguished as oleaginous yeast. In some embodiments, the host cell is Saccharomyces cerevisiae. In other embodiments, the host cell is Pichia kudriavzevii. In other embodiments of the disclosure, the eukaryotic host cell is a fungus or algae. In yet other embodiments, the recombinant host cell is a prokaryote selected from the non-limited example genera: Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas, Rhodobacter, and Streptomyces. In various embodiments, the host cell is P. kudriavzevii.

[0132] As utilized herein, a number of genetic modifications are further useful for increasing microbial biosynthesis of malony1-CoA. For example, in some embodiments a host cell provided or utilized herein is further engineered to include a genetic modification useful for converting pyruvate to malony1-CoA, wherein the genetic modification produces and/or provides a pyruvate decarboxylase, an acetaldehyde dehydrogenase, an acety1-CoA synthetase, an acety1-CoA carboxylase, and a carbonic anhydrase.

[0133] In some embodiments, an engineered host cell provided or utilized herein is a P. kudriavzevii host cell. In some embodiments, an engineered host cell comprises heterologous enzymes that are overexpressed to increase malony1-CoA production, thereby facilitating production of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative. In some embodiments, the engineered host cell comprises heterologous enzymes selected from the group consisting of P. kudriavzevii acety1-CoA carboxylase, S. cerevisiae aldehyde dehydrogenase, Yarrowia lipolytica acety1-CoA synthetase, and S. cerevisiae pyruvate decarboxylase.

[0134] In some embodiments, the host cell is a P. kudriavzevii host cell. In some embodiments, a yeast host cell expressing an OLS is used to produce olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative. In some embodiments, an oleaginous yeast host cell expressing an OLS is used to produce olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative.

[0135] In other embodiments, the recombinant host cell expresses an OLS nucleophilic amino acid point mutation ( i.e an amino acid substitution in the active OLS region comprising positions D 198 to G209 of the wild type C. sativa OLS enzyme; SEQ ID NO: 1) which provides an OLS, wherein D198 is Asp, His, Ser, Thr, or Arg; L199 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala; E200 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; L201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; L201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala; L202 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala; V203 is lle, Leu, Val, Ala, Met, or Phe; G204 is Ala, Cys, Gly, Pro, Ser, Thr, or Val; Q205 is Ala, Asn, Asp, Gin, His, Met, or Ser; A206 is Ala, Cys, His, Leu, Pro, Ser, Thr, or Val; 1207 is Ala, lle, Leu, or Val; F208 is Leu, lle, or Phe; and/or G209 is Ala, Gly, or Ser.

[0136] Also provided herein is a mutated OLS comprising a mutated active site, vectors for expressing the mutant, and host cells that express the mutant. In another embodiment, the host cell further produces olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative. Introduction of mutations in the region comprising D198 to G209 of OLA increases the turnover rate (i.e., k_cat values) of the mutated OLS. One or more point mutations at amino acid positions D198 to G209 can be introduced alone or in any desired combination. In these embodiments, the recombinant host cell can be, without limitation, a P. kudriavzevii or yeast, including but not limited to S. cerevisiae or other yeast, host cell.

[0137] In some aspects, provided herein are recombinant host cells, preferably host cells suitable for producing olivetol (including OLA and/or OLA-derived compounds) and other cannabinoids and cannabinoid derivatives in accordance with the methods provided herein, the host cells comprising one or more heterologous OLS enzymes, preferably OLS enzymes having an increased kcat value as compared to wild type or homologous OLS enzymes, wherein the recombinant host cells provide increase olivetol titer, yield, and/or productivity relative to a host cell not comprising a heterologous OLS enzyme. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased malony1-CoA biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased hexanoy1-CoA synthetase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased pyruvate dehydrogenase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased acetaldehyde dehydrogenase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased acety1-CoA synthetase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased acety1-CoA carboxylase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the disclosure comprising increased carbonic anhydrase biosynthesis.

[0138] In accordance with the disclosure, increased olivetol titer, yield, and/or productivity can be achieved through increased OLS enzymatic activity, which may require increased malony1- CoA biosynthesis, and the disclosure provides host cells, vectors, enzymes, and methods relating thereto. Malony1-CoA is produced in host cells through the activity of an acety1-CoA carboxylase (EC 6.4.1.2) catalyzing the formation of malony1-CoA from acety1-CoA and carbon dioxide. The disclosure provides recombinant host cells for producing olivetol that express a heterologous acety1-CoA carboxylase (ACC). In some embodiments, the host cell is a S. cerevisiae cell comprising a heterologous S. cerevisiae acety1-CoA carboxylase ACC1 or an enzyme homologous thereto. In some embodiments, the host cell modified for heterologous expression of an ACC such as S. cerevisiae ACC1 is further modified to eliminate ACC1 post-translational regulation by genetic modification of S. cerevisiae SNF1 protein kinase or an enzyme homologous thereto. The disclosure also provides a recombinant host cell suitable for producing olivetol in accordance with the disclosure that is an E. coli cell that comprises a heterologous nucleic acid coding for expression of E. coli acety1-CoA carboxylase complex proteins AccA, AccB, AccC and AccD or one or more enzymes homologous thereto.

[0139] Thus, in one aspect of the disclosure, the recombinant host cell comprises a heterologous nucleic acid encoding a mutant OLS enzyme or another mutant cannabinoid pathway enzyme, that results in increased production of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative relative to host cells not comprising the mutant OLS enzyme and/or an OLS enzyme.

[0140] Thus, in accordance with the disclosure an OLS enzyme other than, or in addition to, OLS derived from C. sativa can be used for biological synthesis of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative in a recombinant host. In some embodiments, the recombinant host is P. kudriavzevii. In some embodiments, the recombinant host is S. cerevisiae. In other embodiments, the recombinant host is E. coli. In other embodiments, the recombinant host is a yeast other than P. kudriavzevii. In various embodiments, the host is modified to express a mutated OLS enzyme and/or an OLS enzyme provided or utilized herein. In various embodiments, the host is further modified to express one or more heterologous enzymes that are overexpressed to increase malony1-CoA production. In various embodiments, the host is further modified to express or overexpress a functional hexanoy1-CoA synthetase.

[0141] Moreover, additional enzymes and catalysts other than those specifically disclosed herein can be utilized in mutated or heterologously expressed form. It will be well understood to those skilled in the art in view of this disclosure how other appropriate enzymes can be identified, modified, and expressed to achieve the desired olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative production, as disclosed herein.

[0142] In one aspect, provided herein are recombinant host cells suitable for biological production of cannabinoids and derivatives, such as without limitation olivetol, OLA, OLA- derived compound, or another cannabinoid or cannabinoid derivative. Any suitable host cell is useful in practice of the methods provided herein. In some embodiments, the host cell is a recombinant host microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), either to produce olivetol, or to increase yield, titer, and/or productivity of olivetol relative to a “wild type”, “control cell”, “parental cell”, or “reference cell”. A “control cell” can be used for comparative purposes, and is typically a wild type or recombinant parental cell that does not contain one or more of the modification(s) made to the host cell of interest.

[0143] In some embodiments, the disclosure provides a recombinant host cell that has been modified to produce one or more enzymes that facilitate malony1-CoA production. In some embodiments, the disclosure provides a recombinant host cell that has been modified to produce one or more enzymes of the cannabinoid pathway. In some embodiments, the disclosure provides a recombinant host cell that has been modified to produce an OLS, such as, without limitation, an engineered or modified OLS, for example, olivetolic acid synthase, having improved k_cat values. Thus, various embodiments of the disclosure provide recombinant host cells capable of producing increased amounts of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative (i.e., product) per unit time. Accordingly, various embodiments of the disclosure provide recombinant host cells capable of achieving higher titers of product over shorter fermentation run times.

[0144] With respect to production titer levels, the recombinant host cells provided or utilized herein produce titer levels that exceed production titer levels of control cells. In some embodiments, the recombinant host cells provided or utilized herein produce titer levels that are suitable for commercial production, for example approximately 10 g/L, or greater. The recombinant host cells described herein promote high titer levels of product(s) in at least two ways. First, the recombinant host cells produce mutated OLS enzymes having improved synthetase kinetics (i.e., an increase in kcat), which allows for faster product production, thereby increasing the rate and ease at which a desired titer level can be achieved. Secondly, the materials and methods provided or utilized herein provide and facilitate in situ extraction of the product into an organic phase, as described below. By adding the organic phase directly to the broth during the fermentation, the product can be quickly and continuously separated from the fermentation process, thereby decreasing undesirable effects of the product on the fermentation process, such as toxicity and product inhibition feedback on the pathway enzymes, thereby further increasing the titer levels of the product(s). Additionally, various genetic modifications provided or utilized herein are useful for increasing the provision of malony1-CoA, which is a substrate for OLS. [0145] In one embodiment, provided herein are recombinant yeast cells suitable for the production of cannabinoids and derivatives such as, without limitation, olivetol, at levels sufficient for subsequent purification and use as described herein. Yeast host cells are excellent host cells for construction of recombinant metabolic pathways comprising heterologous enzymes catalyzing production of small molecule products. There are established molecular biology techniques and nucleic acids encoding genetic elements necessary for construction of yeast expression vectors, including, but not limited to, promoters, origins of replication, antibiotic resistance markers, auxotrophic markers, terminators, and the like. Second, techniques for integration of nucleic acids into the yeast chromosome are well established. Yeast also offers a number of advantages as an industrial fermentation host. Yeast can tolerate high concentrations of organic acids and maintain cell viability at low pH and can grow under both aerobic and anaerobic culture conditions, and there are established fermentation broths and fermentation protocols. The ability of a strain to propagate and/or produce desired product under low pH provides a number of advantages. First, this characteristic provides tolerance to the environment created by the production of malonic acid. Second, from a process standpoint, the ability to maintain a low pH environment limits the number of organisms that are able to contaminate and spoil a batch.

[0146] In some embodiments of the disclosure, the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is a eukaryote. In various embodiments, the eukaryote is a yeast selected from the non-limiting list of genera; Candida, Cryptococcus , Hansenula, Issatchenki, Kluyveromyces, Komagataella , Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces or Yarrowia species. In various embodiments, the yeast is of a species selected from the group consisting of Candida albicans, Candida ethanolica, Candida krusei , Candida methanosorbosa, Candida sonorensis , Candida tropicalis. Cryptococcus curvatus , Hansenula polymorpha , Issatchenkia orientalis , Kluyveromyces lactis, Kluyveromyces marxianus , Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi, Pichia angusta , Pichia deserticola, Pichia galeiformis, Pichia kodamae, Pichia kudriavzevii, Pichia membranaefaciens , Pichia methanolica, Pichia pastoris, Pichia salictaria , Pichia stipitis , Pichia thermotolerans , Pichia trehalophila, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula graminis, Saccharomyces bay an us.. Saccharomyces boulardi, Saccharomyces cerevisiae, Saccharomyces kluyveri, and Yarrowia lipolytica. One skilled in the art will recognize that this list encompasses yeast in the broadest sense, including both oleaginous and non- oleaginous strains.

[0147] Other recombinant host cells provided or utilized herein include without limitation, eukaryotic, prokaryotic, and archaea cells. Illustrative examples of eukaryotic cells include, but are not limited to: Aspergillus niger, Aspergillus oryzae, Crypthecodinium cohnii, Cunninghamella japonica, Entomophthora coronata, Mortierella alpina, Min or circinelloides, Neurospora crassa, Pythium ultimum, Schizochytrium limacinum, Thraustochytrium aureum, Trichoderma reesei and Xanthophyllomyces dendrorhous. In general, if a eukaryotic cell is used, a non-pathogenic strain is employed. Illustrative examples of non-pathogenic strains include, but are not limited to: Pichia pastoris and Saccharomyces cerevisiae. In addition, certain strains. Including Saccharomyces cerevisiae , have been designated by the Food and Drug Administration as Generally Regarded As Safe (or GRAS) and so can be conveniently employed in various embodiments of the methods of the disclosure,

[0148] Illustrative and non-limiting examples of recombinant prokaryotic host cell s provided or utilized herein include, Bacillus subtilis, Brevibacterium ammoniagen.es, Clostridium beigerinckii, Corynebacterium glutamicum, Escherichia coli, Enterobacter sakazakii , Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Streptomyces fungicidicus, Streptomyces griseochromogenes, Streptomyces griseus, Streptomyces lividans, Streptomyces olivogriseus, Streptomyces rarneus, Streptomyces tanashiensis, and Streptomyces vinaceus. Certain of these cells, including Bacillus subtilis, Corynebacterium glutamicum, and Lactobacillus acidophilus , have been designated by the Food and Drug Administration as Generally Regarded As Safe (or GRAS) and so are employed in various embodiments of the methods of the disclosure. While desirable from public safety and regulatory standpoints, GRAS status does not impact the ability of a host strain to be used in the practice of this disclosure; hence, non- GRAS and even pathogenic organisms are included in the list of illustrative host strains suitable for use in the practice of this disclosure.

[0149] Escherichia coli and Corynebacterium glutamicum are suitable prokaryotic host cells for metabolic pathway construction. Wild type E. coli can catabolize both pentose and hexose sugars as carbon sources. Provided herein are variety of E. coli host cells suitable for the production of malonate as described herein. In various embodiments, the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is an E. coli cell. In various embodiments of the methods of the disclosure, the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is a C. glutamicum cell.

Synthesis. Utilization, and Purification of C₂nnabinoids and Derivatives [0150] In some aspects, provided herein are methods of producing a cannabinoid, a cannabinoid derivative a cannabinoid precursor or a cannabinoid precursor derivative. In some embodiments, the methods may involve culturing a genetically modified host cell of the present disclosure in a suitable medium and recovering the produced cannabinoid, the cannabinoid precursor, the cannabinoid precursor derivative, or the cannabinoid derivative. The methods may also involve cel1-free production of cannabinoids, cannabinoid precursors, cannabinoid precursor derivatives, or cannabinoid derivatives using one or more polypeptides disclosed herein expressed or overexpressed by a genetically modified host cell of the disclosure.

[0151] In some embodiments, provided herein are methods of producing a cannabinoid or a cannabinoid derivative. The methods may involve culturing a genetically modified host cell of the present disclosure in a suitable medium and recovering the produced cannabinoid or cannabinoid derivative. The methods may also involve cel1-free production of cannabinoids or cannabinoid derivatives using one or more polypeptides disclosed herein expressed or overexpressed by a genetically modified host cell of the disclosure.

[0152] C₂nnabinoids, cannabinoid derivatives, cannabinoid precursors, or cannabinoid precursor derivatives that can be produced according to the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabigerol. (CBG) type (e.g., camiabigerolic acid), cannabidiol (CBD) type {e.g., cannabidiolic acid), D⁹-irans- tetrahydrocannabinol ( D⁹- THC) type (e.g., D⁹-tetrahydrocannabinolic acid), D⁸-trans- tetrahydrocannabinol ( D⁸- THC) type, eannabicyclol (CBL) type, cannabielsoin (CBE) type, cannahinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, olivetolic acid, GPP. derivatives of any of the foregoing, and others as listed in Elsohly M.A. and Slade I)., Life Sci.2005 Dec 22;78(5):539-48. Epub 2005 Sep 30.

[0153]] Cannabinoids or cannabinoid derivatives that can be produced with the methods or genetically modified host cells of the present disclosure may also include, but are not limited to, catinabigerolic acid (CBGA), caimabigerolic acid monomethylether, (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBG), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidioi (CBD), eannabidiol monomethylether (CBDM), eannabidio1-C₄ (CBD-C₄ cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidioreol (CBD-C₁), D⁹- tetrahydrocaimabinolic acid A (THCA-A), A⁹ tetrahydrocannabinolic acid B (THCA-B), D⁹-- tetrahydrocannabinol (THC), D⁹-tetrahydrocannabinolic acid-Ch (THCA-CU), D⁹- tetrahydfocannabino1-C₄, (THCO), D⁹- tetrahydrocannabivarinic acid (THCVA), D⁹- tetrahydrocannabivarin (THCV), D⁹- tetrahydrocannabiorcolic acid (THCA-C₁), D⁹- tetraliydrocannabiorcol (THC-C₁), D⁷-cis- iso-tetrahydrocannabivarin, D⁸- tetraliydrocannabinolic acid ( D⁸-THCA), D⁸- tetrahydrocannabinol D⁸- THC). carmabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA-A), cannabielsoic acid B (CBEA- B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methyiether (CBNM), eannabino1-C₄, (CBN-C₄), cannabivarin (CBV), cannabino1-C₂ (CNB-C₂), cannabiorcol (CBN-C₁), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitxiol (CBT), 10-ethyosy-9-hydroxy-delta-6a- tetrahydrocannabinol, 8,9-dihydroxy1-delta-6a-tetrahydrocannabinol, cannabitriolvarin (CBTVE), dehydrocannabifuran (DCBF), carmabifuran (CBF), cannabichromanon (CBGN), cannabiciiran (CBT), 10-oxo-delta-6a-tetraliydrocannabinol (OTHC), delta-9-cis- tetrahydrocannabinol (cis- THC), 3,4,5,6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethy1-9-n- propy1-2, 6-methano-2H- 1 - benzoxocin-5 -methanol (OH-iso-HHCV), cannabiripsol (CBR), trihydroxy-delta-9- tetrahydrocannabinol (triOH-THC), and derivatives of any of the foregoing.

[0154] Additional cannabinoid derivatives that can be produced with the methods or genetically modified host cells of the present disclosure may also include, but are not limited to, 2-gerany1-5- penty1-resorcylic acid, 2-gerany1-5-(4-pentynyl)-resorcylic acid, 2-gerany1-5- (trans-2-pentenyl)- resorcylic acid, 2-gerany1-5-(4-methylhexyl)-resorcylic acid, 2-gerany1-5- (5-hexynyl) resorcylic acid, 2-gerany 1-5-(tran s-2-bexenyl)-resorcylic acid, 2-geranyI-5-(5- hexenyl)-resorcylic acid. 2- gerany1-5-hepty1-resorcylie acid, 2-gerany1-5-(6-heptynoic)- resorcylic acid. 2-gerany1-5-oetyI- resorcylic acid, 2-geranyi-5-(trans-2-octenyl)-resorcylic acid, 2-gerany1-5 -nonyl -resorcylic acid, 2-gerany1-5 -(trans-2-nonenyl) resorcylic acid, 2- gerany1-5-decy1-resorcylic acid, 2-gerany1-5-(4- phenylbutyl)-resorcylic acid, 2-gerany 1-5-(5- phenylpentyl)-resorcylic acid, 2-gerany1-5-(6- phenylhexyl)-resorcyiic acid, 2-geranyl-5-(7- phenylheptyl)-resorcylic acid, (6aR,10aR)-1- hydroxy-6,6, 9-trirnethyl-3-propy1-6a,7, 8, 10a- ietrahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid (6aR, 10aR)- 1 -hydroxy-6, 6, 9-trimethy1- 3-(4-methylhexyl)-6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-1-hydroxy-6,6,9-trimethy1-3-(5-hexenyl)- 6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, (6aR,l 0aR)-1-hydroxy-6,6,9- trimethy1-3-(5-hexenyl)- 6a,7,8,10a-tetahydro-6H-dibenzo[b,d]pyran-2-carboxylic acid, (6aR,10aR)-l -hydroxy- 6,6,9-trimethy]-3-(6-hepiynyl)-6a,7,8,10a-tetrahydro-6H- dibenzo[b,d]pyran-2-carboxylic acid, 3-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-6-(hexan-2-yl)- 2,4-dihydroxybenzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- ylj-2,4-dihydroxy-6-(2- methylpentyl)benzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-(3- methylpentyl)benzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-(4- methylpentyl)benzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-[(1E)-pent- 1-en-1- yl] benzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-[(2E)-pent-2- en-1 -yl]benzoic acid, 3- [(2E)-3,7-dimetfaylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-[(2E)-pent-3-en- 1 -yljbenzoic acid, 3- [(2E)-3,7 -dimethylocta-2,6-dien- -1-yl]-24dihydroxy-6-(pent-4-en- 1 - yl)benzoic acid, 3- [(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6-propylbenzoic acid, 3- [(2E)-3,7- dimethyiocta-2,6-dien- -1-yl]-24dihydroxy-6-butylbenzoic acid, 3-[(2E)-3,7- dimethylocta- 2,6-dien- 1 -yl]-2,4-dihydroxy-6-hexylbenzoic acid, 3-[(2E)-3,7-dimethylocta-2,6- dien- 1 -yl] - 2,4-dihydroxy-6 -hepiylbenzoic acid, 3- [(2E)-3 ,7 -dimethylocta-2,6-dien--1-yl]-24 dihydroxy- 6-octylbenzoic acid, 3-[(2E)-3,7-dimethylocta-2,6-dien-1- yl]-2,4-dihydroxy-6- nonanylbenzoic acid, 3-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-24-dihydroxy-6- decanylbenzoic acid, 3-[(2E)-3,7-dimethylocta-2,6-dien-1- yi]-2,4-dihydroxy-6- undecanyibenzoie acid, 6-(4- chlorobutyl)-3- ((2E)-3 ,7 -dimethylocta-2,6-dien- -1-yl]-24 dihydroxybenzoic acid, 3-[(2E)-3,7- dimethy locta-2,6-dien- -1-yl]-24dihydroxy-6- [4- (methylsulfanyl)butyl]benzoic acid, and others as listed in Bow, E. W. and Rimoldi, J. ML, “The Structure-Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation,^" Perspectives in Medicinal Chemistry 2016:8, 17-39 doi: 10.4137/PMC. S32171, incorporated herein by reference. Methods of determining the activity and properties of cannabinoids and cannabinoid derivatives are well known (see, e.g., Bow and Rimoldi, supra), and can be adapted in view of the present disclosure by the skilled artisan.

[0155] In certain embodiments, provided herein are methods to isolate olivetol or another cannabinoid or cannabinoid derivate produced biologically. As used herein, “isolate”, “purify”, and “recover” are used to refer to separation of the olivetol from other substances present. “Isolation”, “purification”, or “recovery” as used in this context is intended to convey a preparation of olivetol that is enriched in olivetol relative to the cell or fermentation broth that produced it but that may or may not be substantially (i.e., more than 50%) pure on a weight/weight (w/w) basis. [0156] The disclosure further provides methods for purifying olivetol from fermentation broth. On advantage of fermentative production of olivetol over olivetolic acid (as well as many of the downstream intermediates and OLA-derived compounds) is its very low solubility in water. With a solubility of about 1 g/1 at room temperature, nearly all olivetol produced in the fermentation will phase- separate from the broth. The calculated/predicted olivetol LogP is 3.6; thus, olivetol will readily phase separate into most hydrophobic, organic solvents (i.e., liquid-liquid extraction), thereby providing a facile method to separate olivetol from the aqueous fermentation broth. [0157] Organic solvents, utilized herein, have a number of characteristics that facilitate downstream processing and olivetol purification. First, they are low cost. Second, they are non toxic. Third, they have a low solubility in water. Fourth, the density differential between the organic solvent and water is substantial, a feature that can facilitate efficient separation of the organic solvent from water in some downstream purification processes. Fifth, the boiling point differential between the organic solvent and olivetol is substantial, wherein the boiling point of the organic solvent is higher than that for olivetol, thereby facilitating efficient separation of olivetol from the organic solvent by distillation.

[0158] The boiling point of olivetol is approximately 163°C, thus preferred organic solvents utilized herein have a boiling point greater than 163°C, and more preferably greater than 180°C. Non-limiting examples of hydrophobic solvents meeting the abovementioned criteria, and which are suitable for extracting olivetol from fermentation broth include, but are not limited to, those provided in Table 1. [0159] Table 1: Organic solvents suitable for use as olivetol extractants

[0160] In addition to the organic solvents provided in Table 1, additional organic solvents suitable for extracting olivetol include, but are not limited to, butyl oleate, 2-hexy1- 1-decanol, diisobutyl adipate, dibutyl phthalate, tributyl citrate, dibutyl sebacate, dodecane, hexadecane, silicon oil, toluene, paraffin oil, and soybean oil.

[0161] In the absence of an organic overlay during the fermentation process, the olivetol may precipitate out of solution or phase separate into an immiscible organic layer. During or after conclusion of the fermentation process, the fermentation broth containing olivetol is brought into contact with one or more hydrophobic organic solvents, resulting in mass transport of the olivetol from the aqueous fermentation broth into the organic solvent. In some embodiments, a particularly efficient approach for liquid-liquid extraction is counter-current extraction in which a semi- permeable membrane is used to avoid mixing of the aqueous and organic layers.

[0162] In some embodiments of the purification methods of disclosure, the fermentation broth is concentrated to increase the working concentration of olivetol and decrease the volume of liquid that requires processing. In various embodiments of the purification methods of the disclosure, this concentration is achieved by evaporation, including vacuum and heat, reverse osmosis, “high pass” membrane dewatering, and/or thin film evaporation. [0163] The methods provided herein for downstream process or purification f of olivetol are also useful for downstream process or purification of compounds of formula (I), wherein R² and R³ are hydrogen. While the purification methods provided and utilized herein can be used as single-step purification methods for purification of olivetol from the fermentation broth, two or more purification methods can be used in series in accordance with the disclosure.

[0164] Organisms, nucleotides, and proteins reported elsewhere can be adapted by a skilled artisan to practice various aspects and embodiments of the disclosure provided herein, in view of the present disclosure. See PCT publication Nos. WO 2012/158466; WO 2018/148848; WO 2018/1488849; WO 2018/176055; WO 2018/200888; and WO 2019/014395; U.S. Pat. Nos. 8,884,100; 9,359,625; 9,611,460; 9,822,384; and 10,093,949; U.S. Published Patent application Nos. 2008/0233628; 2014/40141476; 2018/0334692; 2018/0371507; and 2019/0002848; C₂rvalho, et al, 2017, FEMS Yeast Res., 17(4): fox037; Russo, et al, 2019, Front Plant Sci., 9:1969; Richins etal, 2018, PLoS One, 13(7): e0201119; Walker, et al, 2018, Genes, 9(7), 340; and Goold, et al, 2018, Genes, 9(7), 341, each of which is incorporated herein by reference. [0165] The disclosure, having been described in detail, is illustrated by the following examples, which should not be constmed as limiting the disclosure, given its diverse aspects, embodiments, and applications.

EXAMPLES

Example 1: Production of olivetol and other cannabinoids and derivatives in an E, coli host [0166] Provided herein are methods for producing olivetol and other cannabinoids and derivatives in an E. coli host, as well as E. coli host cells that produce olivetol and express a polyketide synthase enzyme according to SEQ ID NO: 2. This example describes the construction of protein coding sequences for OLS enzymes useful in the disclosure, expression vectors containing those coding sequences, and host cells comprising those expression vectors. The nucleic acid encoding wild type C. sativa OLS is provided by a DNA synthesis service provider. Point mutations are introduced at the active site corresponding to D 198 to G209 appropriate primer pairs. The resulting nucleic acids are cloned into an E. coli expression vector containing the pSC101 origin of replication, a chloramphenicol resistance cassette, and a P_LacO1 promoter using standard techniques. The resulting vectors are transformed into an E. coli DHlOb host and plated on Luria-Bertani (LB) agar plates containing 50 mg/ml chloramphenicol (Cm⁵⁰) and 2% w/v glucose. Individual colonies are then inoculated into 3 ml LB media in 48-well plates; following 6 hours growth, plasmids are isolated and the OLS protein-coding region sequenced. The OLS enzyme strains are used for olivetol production.

Example 2: In vivo production of olivetol in E. coli using OLS enzymes [0167] This example describes the host cells and culture conditions resulting in the in vivo production of olivetol using a heterologous OLS enzymes in an E. coli host cell. E. coli strain K12 is transformed with vectors containing OLS enzyme strains according to SEQ ID NO: 2. Transformants are streaked on LB agar plates (Cm⁵⁰, 2% glucose). Following overnight growth at 37°C, individual colonies are inoculated into 3 ml LB (Cm⁵⁰, 2% glucose) in a 48-well plate. Cultures are incubated on a plate shaker at 37°C for 6 hours, at which point each culture is inoculated 1% v/v into M9 minimal medium supplemented with Cm⁵⁰ and a mixed carbon source (0.5% glycerol, 0.05% glucose, 0.2% lactose) in a 48-well plate. Cultures are incubated on a plate shaker at 30°C, and a 500 mL sample of the fermentation broth is removed for analysis after 48 hours incubation.

[0168] Samples are centrifuged (6000xg, 1 min) and the supernatant analyzed for olivetol quantification. Chemical standards are prepared in 20 mM of water. The separation of olivetol is conducted by liquid-liquid phase separation and distillation using a hydrophobic organic solvent listed in Table 1.

Example 3: Integration of a hexanoy1-CoA synthetase encoding gene to the S. cerevisiae genome for the production of olivetol

[0169] In this example, S. cerevisiae strain BY4741 is used as a host strain, for the genomic integration of the hexanoy1-CoA synthetase afforded by the disclosure. Three sites in the S. cerevisiae genome are chosen for integration of the synthetic nucleic acid constructs in this strain.

Example 4: Integration of a pyruvate decarboxylase for increased production of olivetol from an engineered host cell

[0170] In this example, a compatible strain of a suitable host cell such as E. coli, P. pastoris, or P. kudriavzevii is used for the genomic integration of the pyruvate decarboxylase, such as S. cerevisiae PDC6, utilized by the disclosure. Three sites in the genome of the suitable host cell are chosen for integration of the synthetic nucleic acid constructs in this strain.

[0171] This example illustrates the benefits of genomic integration of pyruvate decarboxylase encoding gene utilized by the disclosure in that a single integrated copy of the gene results in higher olivetol titers than the same gene expressed from a plasmid.

Example 5: Integration of an aldehyde dehydrogenase for increased production of olivetol from an engineered host cell

[0172] In this example, a compatible strain of a suitable host cell such as E. coli, P. pastoris, or P. kudriavzevii is used for the genomic integration of the aldehyde dehydrogenase, such as S. cerevisiae ALD6 utilized by the disclosure. Three sites in the genome of the suitable host cell are chosen for integration of the synthetic nucleic acid constructs in this strain.

[0173] This example illustrates the benefits of genomic integration of acetaldehyde dehydrogenase encoding gene utilized by the disclosure in that a single integrated copy of the gene results in higher olivetol titers than the same gene expressed from a plasmid.

Example 6: Integration of an acety1-CoA synthetase for increased production of olivetol from an engineered host cell

[0174] In this example, a compatible strain of a suitable host cell such as E. coli, P. pastoris, or P. kudriavzevii is used for the genomic integration of the acety1-CoA synthetase, such as Y. lipolytica ACS, utilized by the disclosure. Three sites in the genome of the suitable host cell are chosen for integration of the synthetic nucleic acid constructs in this strain.

[0175] This example illustrates the benefits of genomic integration of acety1-CoA synthetase encoding gene provided by the disclosure in that a single integrated copy of the gene results in higher olivetol titers than the same gene expressed from a plasmid.

Example 7: Integration of an acety1-CoA carboxylase for increased production of olivetol from an engineered host cell

[0176] In this example, a compatible strain of a suitable host cell such as E. coli or P. pastoris, is used for the genomic integration of the acety1-CoA carboxylase, such as P. kudriavzevii ACC1, afforded by the disclosure. Three sites in the genome of the suitable host cell are chosen for integration of the synthetic nucleic acid constructs in this strain.

[0177] This example illustrates the benefits of genomic integration of acety1-CoA carboxylase encoding gene utilized by the disclosure in that a single integrated copy of the gene results in higher olivetol titers than the same gene expressed from a plasmid.

Example 8: Integration of a carbonic anhydrase for increased production of olivetol from an engineered host cell

[0178] In this example, a compatible strain of a suitable host cell such as E. coli, P. pastoris, or P. kudriavzevii is used for the genomic integration of the carbonic anhydrase utilized by the disclosure. Three sites in the genome of the suitable host cell are chosen for integration of the synthetic nucleic acid constructs in this strain.

[0179] This example illustrates the benefits of genomic integration of carbonic anhydrase encoding gene utilized by the disclosure in that a single integrated copy of the gene results in higher olivetol titers than the same gene expressed from a plasmid.

Example 9: Bio-reactor based production of olivetol

[0180] In this example, a yeast strain, preferably a recombinant heterologous yeast strain, provided or utilized herein, such as P. kudriavzevii or P. pastoris is grown in fed-batch control in a 0.5 L bioreactor. A single colony of a host cell having one or more point mutations introduced at the active site corresponding to D198 to G209 of OLS (i.e., modified OLS) is isolated from a Synthetic Complete (SC) plate and cultured in 5 mL of a compatible media. A single colony of a host cell having wild type OLS is also isolated from a SC plate and cultured in 5 mL of the compatible media. Both cultures are maintained at 30°C overnight, shaking at 200 rpm. 4 mL of each culture is used to inoculate 50 mL of fresh media in separate 250 mL non-baffled flasks, and each grown overnight at 30°C, 200 rpm. The time zero OD 600 nm absorbance is recorded for each culture. After overnight growth (16 hours), each culture is used to inoculate 1 L of the compatible media. Both cultures are split into 2 separate 500 mL aliquots and added to four separate bioreactors. All four fermentations are maintained at 30°C, with a single impeller mn at 400 rpm, and a sparge rate of 1 vessel volumes per minute (VVM) using compressed air. The cultures are grown overnight (21 hours) to allow for glucose consumption prior to starting the fed- batch phase. The feed (recipe below) is delivered for 2 secones, every 980 seconds. 0.5 mL samples are taken daily and analyzed for production of olivetol.

[0181] The batch feed media consists of 17g/L Difco YNB ; 50 g/L ammonium sulfate; 49.8 g/L SC supplement lacking histidine, methionine, and leucine; 2.57 g/L methionine; 8.85 g/L succinic acid, and 20 g/L glucose; the pH of the fermentation broth is adjusted to 4.0.

[0182] The fermentation process of the modified OLS cultures provides increased oxygen transfer rate (OTR) as compared the wild type OLS cultures.

Example 10: Bio-reactor based production of olivetol

[0183] In a further example, the batch feed of example 9 further comprises hexanoic acid (or an acceptable salt thereof), wherein one modified OLS culture is pH adjusted to approximately 5.0, and the other modified OLS culture is pH adjusted to approximately 7.0, and wherein one wild type OLS cultures is pH adjusted to approximately 5.0, and the other wild type OLS culture is pH adjusted to approximately 7.0.

[0184] The fermentation process of the modified OLS culture pH adjusted to approximately 5.0 performs better than the remaining cultures in this example.

Example 11: Purification of biologically derived olivetol from fermentation broth by reactive extraction with an organic solvent

[0185] In this example, endogenously produced olivetol from cultures comprising a host cell harboring an olivetol biosynthesis pathway is purified from fermentation broth by liquid-liquid phase separation followed by distillation. Following the fermentation process, an organic solvent is added to the fermentation broth. The organic solvent is selected based on an aggregate of preferred characteristics, namely, (i) cost; (ii) toxicity; (iii) low solubility in water; (iv) substantial density differential between organic solvent and water, and v) substantial difference in boiling point between organic solvent and olivetol. Non-limiting examples of suitable organic solvents are provided herein. Upon addition of the organic solvent to the fermentation broth, the olivetol is extracted from the broth and into the organic solvent. The low solubility of the organic solvent in water results in a phase separation or partitioning of the fermentation broth and the organic solvent. The organic solvent phase of the fermentation broth is removed and subjected to distillation, wherein the endogenously produced olivetol is purified from the organic solvent based on the substantial difference in boiling point of the organic solvent and olivetol.

Example 12: Biosynthetic Production of Cannabidiolic Acid in S. cerevisiae [0186] In this example, the CBDA metabolic pathway found in C. sativa is reconstituted into S. cerevisiae through the method outlined in U.S. Pat. No. 10,093,949 (‘“949 method”). However, in this example the wild type olivetolic acid synthase of the ‘949 method is substituted with an OLS provided or utilized herein, wherein the substitution increased production of CBDA.

Example 13: Biosynthetic Production of Olivetol in Yeast

[0187] In this example, the methods and cell lines outlined in PCT Publication Nos. WO 2018/148848 and WO 2018/148849 are modified to include a coding sequence which produces an OLS provided or utilized herein, wherein the modification increases production of olivetol.

Example 14: Biosynthetic Cannabinoid Production

[0188] In this example, a modified recombinant host cell is provided according to the methods outlined is U.S. Published Patent application No. 2018/0334692 (‘“692 methods”). However, in this example the olivetolic acid synthase of the ‘692 method is substituted with an OLS provided or utilized herein, wherein the substitution increase cannabinoid production.

Example 14: Generation of Water-Soluble Cannabinoid Compounds in Cell Suspension Cultures

[0189] In this example, cannabinoid compounds are generated according to the methods outlined in PCT Publication Nos. WO 2019/014395 and WO 2018/176055. However, in this example the host cells are transformed with a nucleotide sequence expressing an OLS provided or utilized herein, wherein the substitution provides increased cannabinoid generation.

Example 15: Production of Cannabinoids and Cannabinoid Derivatives

[0190] In this example, a genetically modified host cell according to the systems and methods outlined in PCT Publication No. WO 2018/200888 is transformed with a nucleotide sequence expressing an OLS provided or utilized herein, wherein expression of the OLS provides increased cannabinoid and cannabinoid derivative production.

APPENDIX A

Claims

1. An olivetol synthase polypeptide (OLS) comprising an amino acid sequence of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

2. A polypeptide of claim 1, wherein:

X at position 198 is Asp, His, Ser, Thr, or Arg;

X at position 199 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala;

X at position 200 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala;

X at position 201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala;

X at position 202 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala;

X at position 203 is lle, Leu, Val, Ala, Met, or Phe;

X at position 204 is Ala, Cys, Gly, Pro, Ser, Thr, or Val;

X at position 205 is Ala, Asn, Asp, Gin, His, Met, or Ser;

X at position 206 is Ala, Cys, His, Leu, Pro, Ser, Thr, or Val;

X at position 207 is Ala, lle, Leu, or Val;

X at position 208 is Leu, lle, or Phe; and X at position 209 is Ala, Gly, or Ser.

3. The polypeptide of claim 1, wherein X at position 204 is Ala.

4. The polypeptide of claim 1, wherein X at position 204 is Cys.

5. The polypeptide of claim 1, wherein X at position 204 is Ser.

6. The polypeptide of claim 1, wherein X at position 204 is Val.

7. The polypeptide of claim 1, wherein X at position 204 is Thr.

8. The polypeptide of any of claims 1 to 7, wherein the protein has a k_cat value greater than a wild type olivetol synthase.

9. The polypeptide of claim 8, wherein the protein has a k_cat value greater than 10 turnovers per minute.

10. The polypeptide of claim 8, wherein the protein has a k_cat value greater than 50 turnovers per minute.

11. The polypeptide of claim 8, wherein the protein has a k_cat value greater than 100 turnovers per minute.

12. The polypeptide of claim 8, wherein the protein has a k_cat value greater than 500 turnovers per minute.

13. A host cell, which is an organism per se or part of a multi-cellular organism, said host cell comprising an expression vector for expressing a polypeptide of any of claims 1 to 12, or comprising another wild type or mutant OLS.

14. The host cell according to claim 13, wherein the host cell is selected from the group consisting of P. kudriavzevii, P. pastoris, S. cerevisiae, E. coli, C. glutamicum, and Y. lipolytica.

15. The host cell according to claim 13 that further expresses a function hexanoy1-CoA synthetase protein.

16. The host cell according to claim 15, wherein the functional hexanoy1-CoA synthetase is SEQ ID NO: 15.

17. The host cell according to claim 13, further comprising an expressed or overexpressed enzyme to increase malony1-CoA production.

18. The host cell according to claim 17, wherein the enzyme is selected from the group consisting of an acety1-CoA carboxylase (ACC such as a P. kudriavzevii (Pk) ACC, such as PkACCl), an aldehyde dehydrogenase (ALD such as an ALD selected from S. cerevisiae (Sc) ALD2-6, such as ScALD6), an acety1-CoA synthetase (ACS such as a Y. lipolytica ACS such as Y1ACS), a pyruvate dehydrogenase complex (PDC such as a S. cerevisiae PDC such as ScPDC6) and a pyruvate decarboxylase.

19. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide, wherein said geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide catalyzes production of cannabigerolic acid from geranyl pyrophosphate and olivetolic acid in an amount at least ten times higher than a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4.

20. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 5.

21. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 6.

22. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding an olivetolic acid (OAC) polypeptide, or one or more heterologous nucleic acids encoding a fusion OLS and OAC polypeptide.

23. The genetically modified host cell of claim 13, wherein the TKS polypeptide comprises an amino acid sequence having at least 50%, at least 755, or at least 95% sequence identity to SEQ ID NO: 3.

24. The host cell according to claim 22, wherein the OAC polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 8.

25. The host cell according to claim 13, further comprising one or more of the following: a) one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative; b) one or more heterologous nucleic acids encoding a polypeptide that generates geranyl pyrophosphate; or c) one or more heterologous nucleic acids encoding a polypeptide that generates malony1-CoA.

26. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative, wherein the polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative is an acy1-activating enzyme (AAE) polypeptide.

27. The host cell according to claim 26, wherein the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 9.

28. The host cell for producing a cannabinoid or a cannabinoid derivative of claim 26, wherein the AAE polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11.

29. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative, wherein the polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative is a fatty acy1-CoA ligase polypeptide.

30. The host cell according to claim 29, wherein the fatty acy1-CoA ligase polypeptide comprises an amino acid sequence having at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 13.

31. The host cell according to claim 13, further comprising one or more heterologous nucleic acids encoding a polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative, wherein the polypeptide that generates an acy1-CoA compound or an acy1-CoA compound derivative is a fatty acy1-CoA synthetase (FAA) polypeptide.

32. A method of either (i) producing olivetol or a compound of formula (I), or (ii) chemically transforming olivetol or a compound of formula (I):

wherein R¹ is: an optionally substituted hydrocarbyl group, preferably an optionally substituted C₁-C₁₂ hydrocarbyl group, such as without limitation, optionally substituted alkyl, preferably, optionally substituted C₁-C₈ alkyl; optionally substituted alkenyl, preferably, optionally substituted C₂-C₈ alkenyl; optionally substituted alkynyl, preferably, optionally substituted C₂-C₈ alkynyl; R² is H or CO₂H or a salt thereof;

R³ is hydrogen, geranyl, or

R³ and the OH* together with the intervening carbon atoms form

said method comprising: for (i), culturing a host cell of any one of claims 13 to 31 under conditions that result in production of olivetol or a compound of formula (I), wherein for producing olivetol, the host cell utilizes hexanol, hexanoic acid, or hexanoy1-CoA, and for producing a compound of formula (I), the host cell utilizes R-CoA or a precursor thereof such as without limitation RCH₂OH, RCO₂H, and the likes; or for (ii), culturing a host cell of any one of claims 13 to 31 with olivetol or a compound of formula (I), thereby chemically transforming olivetol or the compound of formula (I).

33. The method of claim 32, further comprising isolating olivetol or the compound of formula (I) from the fermentation broth, or further comprising isolating chemically transformed olivetol or a chemically transformed form of the compound of formula (I) from the fermentation broth.

34. The method of claim 32, wherein the compound produced or chemically transformed is cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), D⁹-tetrahydrocannabinolic acid, cannabichromenic acid (CBCA), cannabichromene (CBC), D⁹-tetrahydrocannabinol, and D⁶-tetrahydrocannabinol.

35. A polyketide synthase polypeptide having improved k_cat activity compared to native C. sativa olivetol synthase (SEQ ID NO: 1).

36. The polyketide synthase polypeptide of claim 35, wherein the polyketide polypeptide comprises one or more amino acid substitutions compared to the native amino acid at position 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209 of SEQ ID NO: 1.

37. The polyketide synthase polypeptide of claim 35, wherein the amino acid at position 198 is Asp, His, Ser, Thr or Arg; the amino acid at position 199 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; the amino acid at position 200 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala; the amino acid at position 201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala; the amino acid at position 202 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala; the amino acid at position 203 is lle, Leu, Val, Ala, Met or Phe; the amino acid at position 204 is Ala, Cys, Gly, Pro, Ser, Thr or Val; the amino acid at position 205 is Ala, Asn, Asp, Gin, His, Met or Ser; the amino acid at position 206 is Ala, Cys, His, Leu, Pro, Ser, Thr or Val; the amino acid at position 207 is Ala, lle, Leu or Val; the amino acid at position 208 is Leu, lle or Phe; and the amino acid at position 209 is Ala, Gly or Ser.

38. The polyketide polypeptide of claim 35, wherein the polyketide synthase polypeptide comprises an amino acid sequence of SEQ ID NO: 2, wherein

X at position 198 is Asp, His, Ser, Thr, or Arg; X at position 199 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala;

X at position 200 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lie, His, Asn, Cys, Gly or Ala;

X at position 201 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala;

X at position 202 is Lys, Leu, Val, Pro, Glu, Gin, Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, lle, His, Asn, Cys, Gly or Ala;

X at position 203 is lle, Leu, Val, Ala, Met, or Phe;

X at position 204 is Ala, Cys, Gly, Pro, Ser, Thr, or Val;

X at position 205 is Ala, Asn, Asp, Gin, His, Met, or Ser;

X at position 206 is Ala, Cys, His, Leu, Pro, Ser, Thr, or Val;

X at position 207 is Ala, lle, Leu, or Val;

X at position 208 is Leu, lle, or Phe; and

X at position 209 is selected from the group consisting of Ala, Gly, and Ser.

39. The polyketide synthase polypeptide of claim 38, wherein X at position 204 is Ala, Cys, Ser, Val, or Thr.

40. The polyketide synthase polypeptide of claim 38, wherein the amino acids at positions 157, 297 and 330 are wild type.