WO2017139496A1 - Microbial engineering for the production of cannabinoids and cannabinoid precursors - Google Patents

Abstract

Disclosed herein are compositions and methods for producing cannabinoids and cannabinoid precursors in microorganisms from a carbohydrate source. The methods described herein involve genetic engineering of microorganisms for large-scale production of cannabinoids.

Description

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CANNABINOIDS AND

CANNABINOID PRECURSORS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/293,050, filed February 9, 2016, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Cannabis sativa (cannabis, hemp, marijuana) is one of the oldest and most versatile domesticated plants that produces cannabinoids used in medicinal, food, cosmetic, and industrial products. Cannabinoids and cannabinoid precursors can be effective for the treatment of a wide range of medical conditions, including neuropathic pain, AIDS wasting, anxiety, epilepsy, glaucoma, and cancer. Current methods of producing cannabinoids include the growth of the cannabis plant and industrial production of synthetic cannabinoids. However, these methods are severely limited due to high operational and economic costs.

INCORPORATION BY REFERENCE

[0003] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually.

SUMMARY OF THE INVENTION

[0004] Disclosed herein are genetically engineered microorganisms comprising one or more genetic modifications that increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP) relative to a microorganism of the same species without the one or more genetic modifications, wherein the genetically modified microorganism has increased production of hexanoic acid relative to an unmodified organism of the same species.

[0005] Also disclosed herein are genetically engineered microorganisms comprising one or more genetic modification that enable production of olivetolic acid in the absence of an external source of hexanoic acid.

[0006] Also disclosed herein are genetically engineered microorganisms comprising one or more genetic modifications that enable production of olivetolic acid from a carbohydrate source with an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

[0007] The genetically engineered microorganisms disclosed herein can have one or more further genetic modifications that enable production of cannabinoid precursors, cannabinoids, and/or cannabinoid derivatives. [0008] Also disclosed herein are methods of producing the genetically engineered microorganisms.

[0009] Also disclosed herein are methods of producing one or more fermentation end- productions (e.g., cannabinoid precursors, cannabinoids, and/or cannabinoid derivatives) using the genetically engineered microorganisms disclosed herein.

[0010] The present disclosure also provides products (e.g., cannabinoid precursors, cannabinoids, and/or cannabinoid derivatives) produced by the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 illustrates an exemplary metabolic pathway and genetic engineering strategy for the production of cannabinoid precursors and cannabinoids in a microorganism.

[0012] FIG. 2 illustrates another view of an exemplary metabolic pathway and genetic engineering strategy for the production of cannabinoid precursors in a microorganism.

[0013] FIG. 3 illustrates an exemplary metabolic pathway and genetic engineering strategy for the production of cannabinoid precursors and cannabinoids in a microorganism.

[0014] FIG. 4 illustrates an exemplary process for the production and purification of

cannabinoid precursors, cannabinoids, and/or cannabinoid derivatives.

[0015] FIG. 5 illustrates an exemplary semi-synthetic process for the conversion of methyl olivetolate to cannabidiol.

[0016] FIG. 6 illustrates the production of C6 and C8 fatty acids in a genetically-engineered yeast strain with increased expression of FASa and FASp.

[0017] FIG. 7 illustrates cell growth tolerance to cannabidiol (CBD) of genetically-engineered yeast and wild-type yeast grown on glucose.

[0018] FIG. 8 illustrates biomass production of genetically-engineered yeast and wild-type yeast grown on glucose.

[0019] FIG. 9 illustrates lipid production of genetically-engineered yeast and wild-type yeast grown on glucose.

[0020] FIG. 10 illustrates biomass production profile (left) and substrate production profile (right) of genetically-engineered yeast grown on glucose.

[0021] FIG. 11 illustrates the HPLC profile for olivetolic acid production in the genetically- engineered yeast strain after 24 hours (A), 48 hours (B), and 96 hours (C) of growth.

[0022] FIG. 12 illustrates fluorescent images of wild-type yeast (A) and genetically-engineered yeast (B) grown on glucose for 96 hours under nitrogen-limiting conditions. DETAILED DESCRIPTION OF THE INVENTION

[0023] In view of the rapidly growing demand for cannabinoids for medical and recreational use, numerous research efforts have been directed to develop a cost-effective supply chain for cannabinoids. These efforts include developing new strains of the Cannabis sativa plant that produce a higher content of cannabinoids and using organic chemistry methods for the production of synthetic cannabinoids.

[0024] Another approach is genetic engineering of microorganisms for the production of cannabinoids or cannabinoid precursors from renewable carbon sources. Non-limiting examples of renewable carbon sources include biomass-derived fermentable sugars, such as glucose or sugars from corn or sugarcane; non-fermentable carbohydrate polymers, such as cellulose or hemicellulose; and cannabinoid precursors produced from dark fermentation processes.

Engineering methods for economically-viable production of cannabinoids can involve the identification of a suitable microorganism and engineering of desirable phenotypes in the microorganism. Non-limiting examples of desirable phenotypes include rapid and efficient biomass production, increased fatty acid flux, growth advantage over unsuitable microbes, efficient carbohydrate-to-oil and carbohydrate-to-cannabinoid conversion, high substrate tolerance, and end-product tolerance as compared to the unmodified microbe. The engineered microorganism can display a combination of beneficial traits that allow for efficient conversion of an abundant carbon source to cannabinoid products in a scalable, cost-efficient manner.

[0025] Nitrogen can be essential for growth of microorganisms, and the ability to metabolize a wide variety of nitrogen sources can enable microorganisms (e.g., fungi, e.g., yeast) to colonize different environmental niches and survive under nutrient limitations. Primary nitrogen sources that can be used for growth include, for example, ammonium and glutamine. Secondary nitrogen metabolites subject to diverse regulatory controls through a regulatory expression mechanism called nitrogen metabolite repression can be utilized by microorganisms under specific conditions. Modified promoters and genetic elements responsive to nitrogen for the endogenous and heterologous gene expression can be used to increase cannabinoid and cannabinoid precursor synthesis. In some microorganisms, nitrogen depletion during the stationary phase of growth can trigger increased fatty acid flux.

[0026] The term "cannabinoid" refers to a compound that is derived from a biological source, such as a living cell, microbe, fungus, or plant. A cannabinoid includes, for example, a compound directly obtained from a biological source including, for example, by conventional extraction, distillation, or refining methods, and compound produced by processing a

cannabinoid precursor obtained from a biological source by chemical synthesis procedures. Non- limiting examples of cannabinoids and cannabinoid products include cannabigerol (CBG), tetrahydrocannabidiol or A⁹-tetrahydrocannabidiol (THC), zso-tetrahydrocannabinol (iso-THC), 1 l-hydroxy-A⁹-tetrahydrocannabinol (1 l-OH-A⁹-THC or 1 1-OH-THC), cannabichromene (CBC), cannabidiol (CBD), cannabielsoin (CBE), cannabicyclol (CBL), cannabinol (CBN), cannabicitran (CBT), tetrahydrocannabivarin (THCV), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), nabilone, and other cannabinoid analogs. Non-limiting examples of cannabinoid precursors include mevalonic acid, hydroxylmethylglutaryl-CoA (HMG-CoA), isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl diphosphate (geranyl pyrophosphate), citric acid, acetyl-CoA, malonyl-CoA, hexanoic acid, hexanoyl-CoA, olivetol, pentyl diacetic acid lactone (PDAL), hexanoyl triacetic acid lactone (HTAL), olivetolic acid, cannabigerolic acid (CBGA), cannabichromic acid (CBCA), cannabidiolic acid (CBDA), and tetrahydrocannolic acid (THCA).

[0027] The term "biomass" can refer to material produced by and/or propagation of a living cell or organism, for example, a microorganism. Biomass can contain cells, microbes and/or intracellular contents including, for example, cellular cannabinoids or cannabinoid precursors, fatty acids (FA), and triglycerides (TAG). Biomass can also contain extracellular material including, for example, compounds that are secreted by a cell, such as secreted cannabinoid or cannabinoid precursors. Biomass used for the production of cannabinoids and cannabinoid precursors can be derived from bacteria, fungus, yeast, and algae.

[0028] Biomass yield can refer to total lipid free yeast cells predominately seen in growth phase measured as mass-to-mass percentage or gram of biomass produced per gram of substrate (g/g).

[0029] In some embodiments, the biomass yield can range from about 0.5% w/w to about 30%> w/w, from about 1%> w/w to about 30%> w/w, from about 1%> w/w to about 25% w/w, om about 1%) w/w to about 20%) w/w, om about 1%> w/w to about 10%> w/w, from about 2% w/w to about 30%) w/w, from about 2% w/w to about 25% w/w, from about 2% w/w to about 2% w/w, from about 2%) w/w to about 15% w/w, from about 2% w/w to about 10% w/w, from about 2% w/w to about 5%) w/w, from about 5% w/w to about 30% w/w, about 5% w/w to about 25% w/w, about 5%) w/w to about 20% w/w, about 5% w/w to about 10% w/w, about 10% w/w to about 25% w/w, about 10%) w/w to about 20% w/w, or about 10% w/w to about 15% w/w. In some embodiments, the biomass yield can be at least about 1% w/w, at least about 2% w/w, at least about 3%) w/w, at least about 4% w/w, at least about 5% w/w, at least about 6% w/w, at least about 7%) w/w, at least about 8% w/w, at least about 9% w/w, at least about 10% w/w, at least about 1 1%) w/w, at least about 12% w/w, at least about 13% w/w, at least about 14% w/w, at least about 15% w/w, at least about 16%> w/w, at least about 17% w/w, at least about 18%> w/w, at least about 19% w/w, at least about 20% w/w, at least about 21% w/w, at least about 22% w/w, at least about 23% w/w, at least about 24% w/w, at least about 25% w/w, at least about 26% w/w, at least about 27% w/w, at least about 28% w/w, at least about 29% w/w, or at least about 30% w/w. In some embodiments, the biomass yield can be about 1% w/w, about 2% w/w, about 3% w/w, about 4%) w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8% w/w, about 9% w/w, about 10%) w/w, about 11% w/w, about 12% w/w, about 13% w/w, about 14% w/w, about 15%) w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20% w/w, about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25% w/w, about 26% w/w, about 27%) w/w, about 28% w/w, about 29% w/w, or about 30% w/w.

[0030] In some embodiments, the biomass yield can range from about 5 g/g to about 35 g/g, from about 10 g/g to about 35 g/g, from about 10 g/g to about 30 g/g, from about 15 g/g to about 35 g/g, or from about 15 g/g to about 30 g/g. In some embodiments, the biomass yield can be at least about 5 g/g, at least about 10 g/g, at least about 15 g/g, at least about 20 g/g, at least about 25 g/g, at least about 30 g/g, at least about 35 g/g, at least about 40 g/g, at least about 45 g/g, or about 50 g/g. In some embodiments, the biomass yield can be about 5 g/g, about 10 g/g, about 15 g/g, about 20 g/g, about 25 g/g, about 30 g/g, about 35 g/g, about 40 g/g, about 45 g/g, or about 50 g/g.

[0031] Biomass productivity can refer to total yeast biomass with or without lipid (as per context) in a defined fermenting vessel during the fermentation period. Quantified as gram of biomass per unit volume and time (g/l/h). In some embodiments, the biomass productivity can range from about 0.1 g/l/h to about 1 g/l/h. In some embodiments, the biomass productivity can be at least about 0.1 g/l/h, at least about 0.2 g/l/h, at least about 0.3 g/l/h, at least about 0.4 g/l/h, at least about 0.5 g/l/h, at least about 0.6 g/l/h, at least about 0.7 g/l/h, at least about 0.8 g/l/h, at least about 0.9 g/l/h, or at least about 1 g/l/h. In some embodiments, the biomass productivity can be about 0.1 g/l/h, about 0.2 g/l/h, about 0.3 g/l/h, about 0.4 g/l/h, about 0.5 g/l/h, about 0.6 g/l/h, about 0.7 g/l/h, about 0.8 g/l/h, about 0.9 g/l/h, or about 1 g/l/h.

[0032] Substrate consumption rate can refer to substrate depletion over time to produce yeast biomass in growth phase or oil during stationary phase measured as gram substrate consumed per unit volume and time (g/l/h). In some embodiments, the substrate consumption rate can range from about 1 g/l/h to about 10 g/l/h. In some embodiments, the substrate consumption rate can be at least about 1 g/l/h, at least about 2 g/l/h, at least about 3 g/l/h, at least about 4 g/l/h, at least about 5 g/l/h, at least about 6 g/l/h, at least about 7 g/l/h, at least about 8 g/l/h, at least about 9 g/l/h, or at least about 10 g/l/h. In some embodiments, the substrate consumption rate can be about 1 g/l/h, about 2 g/l/h, about 3 g/l/h, about 4 g/l/h, about 5 g/l/h, about 6 g/l/h, about 7 g/l/h, about 8 g/l/h, about 9 g/l/h, or about 10 g/l/h.

[0033] Lipid yield and productivity in growth phase or in stationary phase where lipid synthesis rate is at maximum can be measured as gram of lipid produced per gram substrate consumed and expressed as g/g unit. Lipid produced per unit time and volume and unit measurement are in g/L/h. In some embodiments, the lipid yield can range from about 0.01 to about 2 g/g, from about 0.01 to about 2 g/g, from about 0.01 to about 2 g/g, In some embodiments, the lipid yield can be about 0.05 g/g, about 0.1 g/g, about 0.15 g/g, about 0.2 g/g, about 0.25 g/g, about 0.3 g/g, about 0.35 g/g, about 0.4 g/g, about 0.45 g/g, about 0.5 g/g, about 0.55 g/g, about 0.6 g/g, about 0.65 g/g, about 0.7 g/g, about 0.75 g/g, about 0.8 g/g, about 0.85 g/g, about 0.9 g/g, about 0.95 g/g, about 1 g/g, about 1.05 g/g, about 1.1 g/g, about 1.15 g/g, about 1.2 g/g, about 1.25 g/g, about 1.3 g/g, about 1.35 g/g, about 1.4 g/g, about 1.45 g/g, about 1.5 g/g, about 1.55 g/g, about 1.6 g/g, about 1.65 g/g, about 1.7 g/g, about 1.75 g/g, about 1.8 g/g, about 1.85 g/g, about 1.9 g/g, about 1.95 g/g, or about 2 g/g. In some embodiments, the lipid productivity can range from about 0.1 to about 2 g/l/h, from about 0.1 to about 1.5 g/l/h, or from about 0.1 to about 1 g/l/h. In some embodiments, the lipid productivity can be at least about 0.1 g/L/h, at least about 0.15 g/L/h, at least about 0.2 g/L/h, at least about 0.25 g/L/h, at least about 0.3 g/L/h, at least about 0.35 g/L/h, at least about 0.4 g/L/h, at least about 0.45 g/L/h, at least about 0.5 g/L/h, at least about 0.55 g/L/h, at least about 0.6 g/L/h, at least about 0.65 g/L/h, at least about 0.7 g/L/h, at least about 0.75 g/L/h, at least about 0.8 g/L/h, at least about 0.85 g/L/h, at least about 0.9 g/L/h, at least about 0.95 g/L/h, at least about 1 g/L/h, at least about 1.1 g/L/h, at least about 1.2 g/L/h, at least about 1.3 g/L/h, at least about 1.4 g/L/h, or at least about 1.5 g/L/h. In some embodiments, the lipid productivity can be about 0.1 g/L/h, about 0.15 g/L/h, about 0.2 g/L/h, about 0.25 g/L/h, about 0.3 g/L/h, about 0.35 g/L/h, about 0.4 g/L/h, about 0.45 g/L/h, about 0.5 g/L/h, about 0.55 g/L/h, about 0.6 g/L/h, about 0.65 g/L/h, about 0.7 g/L/h, about 0.75 g/L/h, about 0.8 g/L/h, about 0.85 g/L/h, about 0.9 g/L/h, about 0.95 g/L/h, about 1 g/L/h, about 1.1 g/L/h, about 1.2 g/L/h, about 1.3 g/L/h, about 1.4 g/L/h, or about 1.5 g/L/h.

[0034] Lipid content can be expressed as the mass percentage of oil in dry cell mass. In some embodiments, the lipid content of a microbe can be 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w, 35% w/w, 40% w/w, 45% w/w, 50% w/w, 55% w/w, 60% w/w, 65% w/w, 70% w/w, 75% w/w, 80% w/w, 85% w/w, 90% w/w, 95% w/w, or 100% w/w dry cell mass.

[0035] Fermentation efficiency or just efficiency can be used to describe the yield of a

fermentation end-product based on the input feed source for the microorganism. Efficiency can be on a weight basis by g product per g feed (e.g., g product per g carbohydrate). [0036] The efficiency of production of olivetolic acid from carbohydates using the genetically engineered microorganisms disclosed herein can be from about 0.5% to about 30%> on a weight basis. For example, the efficiency of olivetolic acid production from carbohydrates can be: from about 0.5%) w/w to about 30%> w/w, from about 1%> w/w to about 30%> w/w, from about 1%> w/w to about 25%o w/w, om about 1%> w/w to about 20% w/w, om about 1%> w/w to about 10%> w/w, from about 2% w/w to about 30%> w/w, from about 2% w/w to about 25% w/w, from about 2% w/w to about 2%o w/w, from about 2% w/w to about 15% w/w, from about 2% w/w to about 10% w/w, from about 2% w/w to about 5% w/w, from about 5% w/w to about 30% w/w, about 5% w/w to about 25%o w/w, about 5% w/w to about 20% w/w, about 5% w/w to about 10% w/w, about 10%o w/w to about 25% w/w, about 10% w/w to about 20% w/w, or about 10% w/w to about 15%o w/w. In some embodiments, the biomass yield can be at least about 1% w/w, at least about 2%o w/w, at least about 3% w/w, at least about 4% w/w, at least about 5% w/w, at least about 6%o w/w, at least about 7% w/w, at least about 8% w/w, at least about 9% w/w, at least about 10%o w/w, at least about 1 1% w/w, at least about 12% w/w, at least about 13% w/w, at least about 14% w/w, at least about 15% w/w, at least about 16% w/w, at least about 17% w/w, at least about 18% w/w, at least about 19% w/w, at least about 20% w/w, at least about 21% w/w, at least about 22% w/w, at least about 23% w/w, at least about 24% w/w, at least about 25% w/w, at least about 26% w/w, at least about 27% w/w, at least about 28% w/w, at least about 29% w/w, or at least about 30% w/w. In some embodiments, the biomass yield can be about 1% w/w, about 2%o w/w, about 3%) w/w, about 4% w/w, about 5% w/w, about 6% w/w, about 7% w/w, about 8%o w/w, about 9%) w/w, about 10% w/w, about 1 1% w/w, about 12% w/w, about 13% w/w, about 14%o w/w, about 15% w/w, about 16% w/w, about 17% w/w, about 18% w/w, about 19% w/w, about 20%o w/w, about 21% w/w, about 22% w/w, about 23% w/w, about 24% w/w, about 25%o w/w, about 26% w/w, about 27% w/w, about 28% w/w, about 29% w/w, or about 30% w/w.

[0037] In some embodiments, the present disclosure relates to the identification of a microbe for cannabinoid or cannabinoid precursor production based on a suitable lipid metabolism of the microbe. Microbes can use lipids to store energy including, for example, in the form of triacylglycerols in lipid vacuoles and/or lipid droplets. The term "lipid metabolism" can refer to the molecular processes that involve the creation or degradation of lipids. The term "lipid" can refer to fatty acids and fatty acids derivatives. Non-limiting examples of lipids include saturated and unsaturated fatty acids, diglycerides, diacylglycerols, triglycerides, triacylglycerols, neutral fats, prenol lipids, terpenoids, fatty alcohols, polyketides, and complex lipid derivatives.

[0038] Non-limiting examples of processes of lipid metabolism of a cell include fatty acid synthesis, fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAG storage, and TAG degradation. The term "fatty acid metabolism" can refer to all cellular or organismic processes that involve the synthesis, anabolism, creation, transformation, or degradation of fatty acids. Examples of processes of fatty acid metabolism of a cell can include, for example, fatty acid synthesis, fatty acid oxidation, TAG synthesis, and TAG degradation.

[0039] The term "triacylglycerol", "triglyceride", and "TAG" can refer to a molecule comprising a single molecule of glycerol covalently bound to three fatty acid molecules that are aliphatic monocarboxylic acids via ester bonds with one fatty acid molecule on each of the three hydroxyl groups of glycerol. Due to the reduced, anhydrous environment of T AGs, highly concentrated levels of inert lipids can be stored in TAGs. In some embodiments, TAGs can be used to store lipophilic cannabinoids and cannabinoid precursors.

[0040] In some embodiments, the present disclosure relates to the engineering of microbial fatty acid and polyketide metabolism to induce desirable phenotypes for cannabinoid production.

Desirable phenotypes can include, for example, increased malonyl-CoA pool for fatty acid and cannabinoid biosynthesis. In some embodiments, the present disclosure utilizes the increased malonyl-CoA in a condensation reaction with hexanoic acid to yield a first-committed

cannabinoid precursor, olivetolic acid. The term "cannabinoid precursor" can refer to olivetolic acid and cannabigerolic acid that are produced via polyketide and terpene prenylation pathways.

[0041] In some embodiments, the present disclosure relates methods for the manipulation of a gene in a microorganism for increased cannabinoid and cannabinoid precursor production in the microbe. In some embodiments, manipulation of a gene product is increased expression of the gene that results increased the activity of the gene product. In some embodiments,

overexpression of a gene can increase the activity of the gene product in a microbe by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 95%, or about 200%.

Microbial Production of Cannabinoid Precursors, Cannabinoids, and Cannabinoid

Derivatives

[0042] Described herein are compositions, methods, and systems for the production of cannabinoid precursors, cannabinoids, or cannabinoid derivatives from a carbohydrate source in a microorganism. Also described herein are the products produced using the compositions, methods, and systems disclosed herein. The methods described herein can involve genetic engineering of microorganisms to produce cannabinoid precursors, cannabinoids, or cannabinoid derivatives with high yield and low cost.

[0043] In some aspects, the disclosure herein relates to microbe-mediated production of a cannabinoid precursor, a cannabinoid, or a cannabinoid derivative. The disclosure herein also relates to the identification, engineering, and development of a microbial source of cannabinoid precursors, cannabinoids, and cannabinoid derivatives for economically-viable, industrial-scale production.

[0044] In some embodiments, increased cannabinoids and cannabinoid precursors produced by a genetically-modified microbe can be the result of modification of the activity of one or more enzymes associated with cannabinoid biosynthesis. Modification of an enzyme activity in a microbe can include, for example, the overexpression of a gene that encodes for the enzyme and introduction of a gene encoding for the enzyme from another organism. In some embodiments, introduction of a gene encoding for the enzyme from another organism to the microbe can be accomplished by homologous recombination. Non-limiting examples of target enzymes associated with cannabinoid biosynthesis include ATP citrate lyase (ACL), acetyl-CoA carboxylase (ACC), type-I fatty acid synthase (FAS), type-I fatty acid synthase alpha (FASa), type-I fatty acid synthase beta (FASP), hexanoyl-CoA synthetase (HS; also known as hexonate synthase or acyl -activating enzymes (AAE)), polyketide synthase (PKS), tetraketide synthase (TKS), olivetolic acid cyclase (OAC), HMG-CoA reductase 1 (HMGR1), truncated HMG-CoA reductase 1 (tUMGRl), mevalonate kinase, isopentenyl-diphosphate del ta-isom erase 1 (IDI1), geranyl pyrophosphate synthase (GPPS), aromatic prenyltransferase, geranyl

pyrophosphate:olivetolic acid geranyltransferase (GOGT), tetrahydrocannabidiol synthase (THCS), cannabichromene synthase (CBCS), and cannabidiol synthase (CBDS).

Overview of metabolic pathways and genetic engineering strategy

[0045] FIGS. 1, 2, and 3 illustrate exemplary metabolic pathways and genetic engineering strategies for the production of cannabinoid precursors and cannabinoids in a microorganism. As illustrated in FIG. 1, an exemplary metabolic pathway can be sub-divided into three distinct but connected pathways. Pathway 1 illustrates the production of acetyl-CoA from the citric acid cycle, which is also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle. Pathway 2 illustrates the production of malonyl-CoA, short-chain fatty acids, and cannabinoid precursors (e.g., olivetolic acid) from the acetyl-CoA of Pathway 1. Pathway 3 illustrates the production of cannabinoids (e.g., cannabergolic acid (CBG), tetrahydrocannabidiol or Δ⁹- tetrahydrocannabidiol (THC), cannabidiol (CBD), cannabichromene (CBC)) from the olivetolic acid of Pathway 2. A genetically-engineered microorganism disclosed herein can comprise one or more genetic modifications that increase the expression or activity of one or more of the enzymes involved in the metabolic pathways illustrated in FIGS. 1 and/or 2. The genetically- engineered microorganisms disclosed herein can further comprise one or more genetic

modifications to increase the activity and/or expression of one or more enzymes that can produce cannabinoid derivatives.

[0046] Depending upon the microorganism, some of the metabolic pathways or components of the pathways, illustrated in FIG. 1 and 2, will be native to the wild-type microorganism.

However, some of the pathways, or components of the pathways, illustrated in FIG. 1 and 2 will be non-native, and must be engineered into the microorganism. Accordingly, when this disclosure refers to genetic modifications that increase the activity or expression of an enzyme or protein, it can mean, for example, providing one or more extra copies of an endogenous gene, introducing one or more copies of an exogenous gene or protein coding polynucleotide, putting an endogenous or exogenous gene under the control of a strong promoter, mutating an

endogenous or exogenous gene to encode a higher activity enzyme, or any combination thereof. Whenever an exogenous gene or protein coding nucleotide is introduced to a microorganism, that exogenous gene or protein coding polynucleotide can be codon-optimized for expression in the microorganism.

[0047] Acetyl-CoA can serve as an intermediate in several important biosynthetic pathways, including lipogenesis, polyketide biosynthesis, isoprenoid biosynthesis, and cannabinoid synthesis. Acetyl-CoA can also serve as a precursor for the biosynthesis of many industrial chemicals including lipids (dietary supplements and biodiesels), polyketides (antibiotics and anticancer drugs), and isoprenoids (flavors and fragrances, biodiesels, anti-microbial and anticancer drugs, rubber, cosmetic additives and vitamins). Accordingly, disclosed herein are genetically-engineered microorganisms that comprise one or more genetic modifications that increase the production of acetyl-CoA relative to an unmodified microorganism of the same species. The one or more genetic modifications can include modifications that increase expression or activity of an ACL, a citryl-CoA lyase, a citryl-CoA synthase, or a combination thereof. Methods of making such genetically-engineered microorganisms are also disclosed.

[0048] As illustrated in FIG. 1, acetyl-CoA can be produced by in the citric acid cycle from citrate. Acetyl-CoA can be produced directly from citrate by an ATP citrate lyase (ACL) enzyme. As illustrated in FIG. 2, ACL can catalyze the conversion of citrate (citric acid) and coenzyme A (CoA) to acetyl-CoA and oxaloacetate, with concomitant hydrolysis of ATP to ADP and phosphate. As illustrated in FIG. 1, acetyl-CoA can also be produced from citrate through a citryl-CoA intermediate by the actions of a citryl-CoA lyase enzyme and a citryl-CoA synthetase enzyme. Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of ACL, citryl-CoA lyase, citryl-CoA synthetase, or a combination thereof. The genetically-modified

microorganisms can produce increased levels of acetyl-CoA in comparison to microorganisms of the same species without the genetic modifications.

[0049] In addition, increased expression of aconitase can shift the product ratio to enhance citric acid production in a microorganism (e.g., a microorganism suitable for cannabinoid or cannabinoid precursor production). In some embodiments, excessive citrate production is inhibited in a microbe for cannabinoid or cannabinoid precursor production.

[0050] Some microorganisms (e.g., oleaginous microorganisms, e.g., Yarrowia lipolytica) can accumulate large amounts of storage lipids during secondary metabolism stage of fermentation. For example, in the oleaginous yeast Yarrowia lipolytica, large amounts of storage lipids in the form of triglycerides can be produced during secondary metabolism stage of fermentation. Lipid content in some microorganisms (e.g., oleaginous microorganisms, e.g., Yarrowia lipolytica) can range from about 20% to about 70% of cell mass, depending upon culture conditions. Lipid accumulation can be triggered by a nutrient limitation combined with an excess of carbon. For example, when the nitrogen source is exhausted in the medium, production of acetyl-CoA can be induced for lipid accumulation. Some microorganisms (e.g., Saccharomyces cerevisiae) may not produce significant intracellular lipid droplets. Such microorganisms can lack ACL activity. Accordingly, introducing or increasing the activity or expression of acetyl-CoA producing enzymes (e.g., ACL, citryl-CoA lyase, citryl-CoA synthetase, or a combination thereof) can increase the metabolic flux towards lipid biosynthesis and cannabinoid production. Introducing or increasing the activity of acetyl-CoA producing enzymes (e.g., ACL, citryl-CoA lyase, citryl- CoA synthetase, or a combination thereof) can increase intracellular accumulation of lipid droplets. An increase in intracellular lipid droplets can increase a microorganism' s tolerance to concentrations of cannabinoids, cannabinoid precursors, or a toxic substance.

[0051] In some microorganisms (e.g., in the oleaginous yeast Yarrowia lipolytica) citrate can be secreted into the culture medium. Increasing the activity or expression of acetyl-CoA producing enzymes (e.g., ACL, citryl-CoA lyase, citryl-CoA synthetase, or a combination thereof) can increase the amount of acetyl-CoA produced (and decrease the loss of citrate to the medium).

[0052] The present disclosure includes methods and compositions for increasing the expression of an ATP citrate lyase (ACL) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous ACL gene, putting an endogenous ACL gene under the control of a stronger promoter, mutating an endogenous ACL gene to encode a higher activity enzyme, introducing an exogenous ACL gene, or any combination thereof. Exemplary ACL polynucleotide and polypeptide sequences are shown in TABLE 1. A genetic modification that increases the expression of an ACL can comprise a polynucleotide comprising an open reading frame that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. A genetic modification that increases the expression of an ACL can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to SEQ ID NO: 2. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon- optimized for expression of an encoded protein in a particular microorganism.

[0053] Increased ACL expression can confer on a genetically-modified microorganism one or more beneficial phenotypes for large-scale conversion of a carbon source (e.g., carbohydrate(s)) to cannabinoid precursor(s), cannabinoid(s), cannabinoid derivative(s), or a combination thereof. Non-limiting examples of beneficial phenotypes can include one or more of: increased fatty acid synthesis rate (e.g., increased hexanoic acid synthesis rate); increased carbohydrate to olivetolic acid conversion efficiency; increased lipid storage; increased growth rate; increased tolerance to concentrations of a cannabinoid, a cannabinoid precursor, or a toxic substance; and increased cannabinoid and cannabinoid precursor production relative to an unmodified organism of the same species.

TABLE 1

Exemplary ACL sequences

SEQ ID NO: 1 Homo sapiens (human) ACL polynucleotide

GCGAGCCGATGGGGGCGGGGAAAAGTCCGGCTGGGCCGGGACAAAAGCCGGATCCCGGGAAGCT ACCGGCTGCTGGGGTGCTCCGGAT T T TGCGGGGT TCGTCGGGCCTGTGGAAGAAGCTGCCGCGC ACGGACT TCGGCAGAGGTAGAGCAGGTCTCTCTGCAGCCATGTCGGCCAAGGCAAT T TCAGAGC AGACGGGCAAAGAACTCCT T TACAAGT TCATCTGTACCACCTCAGCCATCCAGAATCGGT TCAA GTATGCTCGGGTCACTCCTGACACAGACTGGGCCCGCT TGCTGCAGGACCACCCCTGGCTGCTC AGCCAGAACT TGGTAGTCAAGCCAGACCAGCTGATCAAACGTCGTGGAAAACT TGGTCTCGT TG GGGTCAACCTCACTCTGGATGGGGTCAAGTCCTGGCTGAAGCCACGGCTGGGACAGGAAGCCAC AGT TGGCAAGGCCACAGGCT TCCTCAAGAACT T TCTGATCGAGCCCT TCGTCCCCCACAGTCAG GCTGAGGAGT TCTATGTCTGCATCTATGCCACCCGAGAAGGGGACTACGTCCTGT TCCACCACG AGGGGGGTGTGGACGTGGGTGATGTGGACGCCAAGGCCCAGAAGCTGCT TGT TGGCGTGGATGA GAAAC T GAAT C C T GAG GAC AT C AAAAAAC AC CTGT TGGTC C AC G C C C C T GAAGAC AAGAAAGAA AT TCTGGCCAGT T T TATCTCCGGCCTCT TCAAT T TCTACGAGGACT TGTACT TCACCTACCTCG AGATCAATCCCCTTGTAGTGACCAAAGATGGAGTCTATGTCCTTGACTTGGCGGCCAAGGTGGA CGCCACTGCCGACTACATCTGCAAAGTGAAGTGGGGTGACATCGAGTTCCCTCCCCCCTTCGGG CGGGAGGCATATCCAGAGGAAGCCTACATTGCAGACCTCGATGCCAAAAGTGGGGCAAGCCTGA AGCTGACCTTGCTGAACCCCAAAGGGAGGATCTGGACCATGGTGGCCGGGGGTGGCGCCTCTGT CGTGTACAGCGATACCATCTGTGATCTAGGGGGTGTCAACGAGCTGGCAAACTATGGGGAGTAC TCAGGCGCCCCCAGCGAGCAGCAGACCTATGACTATGCCAAGACTATCCTCTCCCTCATGACCC GAGAGAAGCACCCAGATGGCAAGATCCTCATCATTGGAGGCAGCATCGCAAACTTCACCAACGT GGCTGCCACGTTCAAGGGCATCGTGAGAGCAATTCGAGATTACCAGGGCCCCCTGAAGGAGCAC GAAGTCACAATCTTTGTCCGAAGAGGTGGCCCCAACTATCAGGAGGGCTTACGGGTGATGGGAG AAGTCGGGAAGACCACTGGGATCCCCATCCATGTCTTTGGCACAGAGACTCACATGACGGCCAT TGTGGGCATGGCCCTGGGCCACCGGCCCATCCCCAACCAGCCACCCACAGCGGCCCACACTGCA AACTTCCTCCTCAACGCCAGCGGGAGCACATCGACGCCAGCCCCCAGCAGGACAGCATCTTTTT CTGAGTCCAGGGCCGATGAGGTGGCGCCTGCAAAGAAGGCCAAGCCTGCCATGCCACAAGATTC AGTCCCAAGTCCAAGATCCCTGCAAGGAAAGAGCACCACCCTCTTCAGCCGCCACACCAAGGCC ATTGTGTGGGGCATGCAGACCCGGGCCGTGCAAGGCATGCTGGACTTTGACTATGTCTGCTCCC GAGACGAGCCCTCAGTGGCTGCCATGGTCTACCCTTTCACTGGGGACCACAAGCAGAAGTTTTA CTGGGGGCACAAAGAGATCCTGATCCCTGTCTTCAAGAACATGGCTGATGCCATGAGGAAGCAC CCGGAGGTAGATGTGCTCATCAACTTTGCCTCTCTCCGCTCTGCCTATGACAGCACCATGGAGA CCATGAACTATGCCCAGATCCGGACCATCGCCATCATAGCTGAAGGCATCCCTGAGGCCCTCAC GAGAAAGCTGATCAAGAAGGCGGACCAGAAGGGAGTGACCATCATCGGACCTGCCACTGTTGGA GGCATCAAGCCTGGGTGCTTTAAGATTGGCAACACAGGTGGGATGCTGGACAACATCCTGGCCT CCAAACTGTACCGCCCAGGCAGCGTGGCCTATGTCTCACGTTCCGGAGGCATGTCCAACGAGCT CAACAATATCATCTCTCGGACCACGGATGGCGTCTATGAGGGCGTGGCCATTGGTGGGGACAGG TACCCGGGCTCCACATTCATGGATCATGTGTTACGCTATCAGGACACTCCAGGAGTCAAAATGA TTGTGGTTCTTGGAGAGATTGGGGGCACTGAGGAATATAAGATTTGCCGGGGCATCAAGGAGGG CCGCCTCACTAAGCCCATCGTCTGCTGGTGCATCGGGACGTGTGCCACCATGTTCTCCTCTGAG GTCCAGTTTGGCCATGCTGGAGCTTGTGCCAACCAGGCTTCTGAAACTGCAGTAGCCAAGAACC AGGCTTTGAAGGAAGCAGGAGTGTTTGTGCCCCGGAGCTTTGATGAGCTTGGAGAGATCATCCA GTCTGTATACGAAGATCTCGTGGCCAATGGAGTCATTGTACCTGCCCAGGAGGTGCCGCCCCCA ACCGTGCCCATGGACTACTCCTGGGCCAGGGAGCTTGGTTTGATCCGCAAACCTGCCTCGTTCA TGACCAGCATCTGCGATGAGCGAGGACAGGAGCTCATCTACGCGGGCATGCCCATCACTGAGGT CTTCAAGGAAGAGATGGGCATTGGCGGGGTCCTCGGCCTCCTCTGGTTCCAGAAAAGGTTGCCT AAGTACTCTTGCCAGTTCATTGAGATGTGTCTGATGGTGACAGCTGATCACGGGCCAGCCGTCT CTGGAGCCCACAACACCATCATTTGTGCGCGAGCTGGGAAAGACCTGGTCTCCAGCCTCACCTC GGGGCTGCTCACCATCGGGGATCGGTTTGGGGGTGCCTTGGATGCAGCAGCCAAGATGTTCAGT AAAGCCTTTGACAGTGGCATTATCCCCATGGAGTTTGTGAACAAGATGAAGAAGGAAGGGAAGC TGATCATGGCATTGGTCACCGAGTGAAGTCGATAAACAACCCAGACATGCGAGTGCAGATCCTC AAAGATTACGTCAGGCAGCACTTCCCTGCCACTCCTCTGCTCGATTATGCACTGGAAGTAGAGA AGATTACCACCTCGAAGAAGCCAAATCTTATCCTGAATGTAGATGGTCTCATCGGAGTCGCATT TGTAGACATGCTTAGAAACTGTGGGTCCTTTACTCGGGAGGAAGCTGATGAATATATTGACATT GGAGCCCTCAATGGCATCTTTGTGCTGGGAAGGAGTATGGGGTTCATTGGACACTATCTTGATC AGAAGAGGCTGAAGCAGGGGCTGTATCGTCATCCGTGGGATGATATTTCATATGTTCTTCCGGA ACACATGAGCATGTAA

SEQ ID NO: 2 | Homo Sapiens ACL polypeptide

MGAGKSPAGPGQKPDPGKLPAAGVLRILRGSSGLWKKLPRTDFGRGRAGLSAAMSAKAISEQTG KELLYKFICTTSAIQNRFKYARVTPDTDWARLLQDHPWLLSQNLWKPDQLIKRRGKLGLVGVN LTLDGVKSWLKPRLGQEATVGKATGFLKNFLIEPFVPHSQAEEFYVCIYATREGDYVLFHHEGG VDVGDVDAKAQKLLVGVDEKLNPEDIKKHLLVHAPEDKKEILASFISGLFNFYEDLYFTYLEIN PLWTKDGVYVLDLAAKVDATADYICKVKWGDIEFPPPFGREAYPEEAYIADLDAKSGASLKLT LLNPKGRIWTMVAGGGASWYSDTICDLGGVNELANYGEYSGAPSEQQTYDYAKTILSLMTREK HPDGKIL11GGS IANFTNVAATFKGIVRAIRDYQGPLKEHEVTI FVRRGGPNYQEGLRVMGEVG KTTGIPIHVFGTETHMTAIVGMALGHRPIPNQPPTAAHTANFLLNASGSTSTPAPSRTASFSES RADEVAPAKKAKPAMPQDSVPSPRSLQGKSTTLFSRHTKAIVWGMQTRAVQGMLDFDYVCSRDE PSVAAMVYPFTGDHKQKFYWGHKEILIPVFKNMADAMRKHPEVDVLINFASLRSAYDSTMETMN YAQIRTIAI IAEGIPEALTRKLIKKADQKGVTI IGPATVGGIKPGCFKIGNTGGMLDNILASKL YRPGSVAYVSRSGGMSNELNNIISRTTDGVYEGVAIGGDRYPGSTFMDHVLRYQDTPGVKMIW LGEIGGTEEYKICRGIKEGRLTKPIVCWCIGTCATMFSSEVQFGHAGACANQASETAVAKNQAL KEAGVFVPRSFDELGEI IQSVYEDLVANGVIVPAQEVPPPTVPMDYSWARELGLIRKPASFMTS ICDERGQELIYAGMPITEVFKEEMGIGGVLGLLWFQKRLPKYSCQFIEMCLMVTADHGPAVSGA HNTI ICARAGKDLVSSLTSGLLTIGDRFGGALDAAAKMFSKAFDSGI IPMEFVNKMKKEGKLIM ALVTE

[0054] Increased ACL expression can also provide an additional source of acetyl-CoA that can be converted malonyl-CoA, for example, by an acetyl-CoA carboxylase (ACC).

[0055] As shown in FIGS. 1 and 2, malonyl-CoA can be a building block for lipogenesis. If the right enzymes are present, lipogenesis can produce short-chain fatty acids like the C6 fatty acid hexanoic acid. As shown in FIGS. 1 and 2, malonyl-CoA can also be co-substrate, with hexanoyl-CoA, for olivetolic acid production. Olivetolic acid can be a committed precursor for cannabinoid biosynthesis.

[0056] Increased production of malonyl-CoA in a genetically-modified microorganism can have one or more beneficial phenotypes. Non-limiting examples of beneficial phenotypes can include: an increase in lipid production; an increase in the accumulation of intracellular lipid droplets; an increase in tolerance to concentrations of cannabinoids, cannabinoid precursors, or a toxic substance; an increase in production of cannabinoid precursors, cannabinoids, cannabinoid derivatives; or any combination of the foregoing. Accordingly, disclosed herein are genetically- engineered microorganisms that comprise one or more genetic modifications that increase production of malonyl-CoA relative to an unmodified microorganism of the same species. The one or more genetic modifications can comprise modifications that increase expression of an ACC, an ACL, or a combination thereof. Also disclosed are methods of making such genetically- engineered microorganisms.

[0057] As illustrated in FIGS. 1 and 2, an ACC (e.g., an ACC encoded by ACC1 and/or HFA1 in yeast) can catalyze the carboxylation of acetyl-CoA to malonyl-CoA. ACC can be a trifunctional enzyme, harboring a biotin carboxyl carrier protein (BCCP) domain, a biotin- carboxylase (BC) domain, and a carboxyltransferase (CT) domain. In some bacteria and plants, these domains can be expressed as individual polypeptides and assembled into a heteromeric complex. In most eukaryotic ACC, including mitochondrial ACC variants (e.g., Hfal in yeast), these three functions can be contained on a single polypeptide. ACC catalysis can be a rate- limiting step in fatty acid biosynthesis, and thus, can act as a metabolic regulator of lipogenesis. [0058] The present disclosure includes methods for increasing the expression and/or activity of an ACC gene product in a genetically-modified microorganism (e.g., a genetically-modified microorganism that produces cannabinoid(s), cannabinoid precursor(s), cannabinoid derivatives, or a combination thereof) relative to an unmodified organism of the same species. The present disclosure also includes genetically-engineered microorganisms produced by such methods. Such methods can include providing one or more extra copies of an endogenous ACC gene, putting an endogenous ACC gene under the control of a stronger promoter, mutating an endogenous ACC gene to encode a higher activity enzyme, introducing an exogenous ACC gene, or any combination thereof. Exemplary ACC sequences are disclosed in TABLE 2 and can also be found under the entry for GenelD: 855750 in the NCBI database. A genetic modification that increases the expression of an ACC can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3. A genetic modification that increases the expression of an ACC comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism.

[0059] Increased expression of ACC in a genetically-modified microorganism can increase the rate of fatty acid synthesis and/or can confer a beneficial phenotype for large-scale carbohydrate to cannabinoid precursor and cannabinoid conversion in the microbe. Non-limiting examples of beneficial phenotypes include increased rate of olivetolic acid synthesis, increased conversion efficiency of carbohydrate to CBGA, increased cannabinoid precursor and cannabinoid storage capacity in lipids, increased growth rate, and increased tolerance to a cannabinoid precursor or a toxic substance, as compared to an unmodified organism of the same species.

TABLE 2

Exemplary ACC sequences

SEQ ID NO: 3 Mus musculus (mouse) ACC polynucleotide

CTCTGAGAGCT TAT T T TGAAAGAATAATGGATGAACCATCTCCGT TGGCCAAAACTCTGGAGCT AAAC C AG C AC TCCCGAT TCATAAT TGGGTCTGTGTCT GAAGAC AC T C AGAAGAT GAGAT C AG T AACCTGGTGAAGCTGGACCTAGAAGAGAAGGAGGGCTCCCTGTCACCAGCCTCCGTCAGCTCAG ATACACT T TCTGAT T TGGGGATCTCTGGCT TACAGGATGGT T TGGCCT T TCACATGAGATCCAG CATGTCTGGCT TG C AC C T AG T AAAC AG G T C GAGAC AGAAAGAAAAT AGAC T C AC AC GAGAT TTCACTGTGGCT TCTCCAGCAGAAT T TGT TACTCGT T T TGGGGGAAATAAAGTAAT TGAGAAGG TTCT TATCGCCAACAATGGTAT TGCAGCAGTGAAATGCATGCGATCTATCCGTCGGTGGTCT TA TGAAATGT T TCGAAATGAACGTGCAATCCGAT T TGT TGTCATGGT TACACCTGAAGACCT TAAA GC C AAT G C AGAAT AC AT T AAGAT G G C AGAC C AC T AT G T T C C AG T G C C T G GAG GAC C C AAC AAC A AC AAT T AC G C AAAT G T G GAG T T GAT T C T T GAT AT T G C T AAAAG GAT AC C T G T AC AAG C AG T G T G GGCTGGCTGGGGTCATGCCTCTGAGAACCCGAAACTCCCAGAACTGCTCTTAAAAAATGGCATT GCTTTCATGGGCCCTCCAAGCCAGGCCATGTGGGCTTTGGGGGATAAGATTGCATCTTCTATTG TGGCTCAAACTGCAGGTATCCCAACTCTTCCCTGGAGTGGCAGTGGTCTTCGAGTGGATTGGCA AGAAAAT GAT T T T T C GAAAC G T AT C T T AAAT G T T C C AC AG GAT C T G T AT GAGAAAG G C T AT G T G AAGGATGTGGATGATGGTCTGAAGGCAGCTGAGGAAGTTGGCTATCCAGTGATGATCAAGGCCT CAGAGGGAGGAGGAGGGAAAGGGAT CAGAAAAGT TAACAAT GCAGAT GAC T T CCC TAACC T C T T CAGAC AG G T T C AAG C T GAAG TTCCTGGAT C AC CTATATTTG T AAT GAGAC TAG C AAAAC AAT C T CGACATCTGGAGGTCCAGATTCTGGCAGATCAGTATGGCAATGCTATTTCTTTGTTTGGTCGTG ACTGCTCTGTGCAGCGCAGGCATCAGAAGATCATTGAAGAAGCTCCTGCTGCGATTGCTACCCC AGCAGTATTTGAACACATGGAACAGTGTGCAGT GAAAC TTGCCAAAATGGTTGGT TAT GTGAGT GCTGGGACTGTGGAATACTTGTACAGCCAGGATGGAAGCTTCTACTTTTTGGAACTGAACCCTC GGCTACAGGTTGAACATCCTTGTACAGAGATGGTGGCTGATGTCAATCTTCCTGCAGCACAGCT CCAGATTGCCATGGGGATCCCTCTATTTAGGATCAAGGATATTCGTATGATGTATGGGGTATCT CCTTGGGGAGATGCTCCCATTGATTTTGAAAATTCTGCTCATGTTCCTTGCCCAAGGGGCCACG TGATTGCTGCTCGGATCACCAGTGAAAACCCAGATGAGGGGTTTAAGCCCAGCTCTGGAACAGT TCAGGAACTTAATTTTCGTAGCAATAAGAACGTTTGGGGTTATTTCAGTGTTGCTGCTGCTGGA GGACTTCATGAATTTGCTGATTCTCAGTTCGGGCACTGCTTTTCCTGGGGAGAAAACAGGGAGG AAGCAATCTCAAATATGGTGGTGGCACTGAAGGAGCTGTCTATTCGGGGTGACTTTCGAACTAC AG T G GAAT AC C T CAT C AAAC T G C T G GAGAC AGAAAG C T T T C AG C T T AAC AGAAT C GAC AC T G G C T G G C T G GAC AGAC T GAT C G C AGAGAAAG T G C AG G C AGAG C GAC C T GAC AC CAT G T T G G GAG T T G TGTGTGGGGCTCTCCATGTAGCAGATGTGAGCCTGAGGAACAGCATCTCTAACTTCCTTCACTC C T T AGAGAG G G G T C AAG TCCTTCCTGCT C AC AC AC T T C T GAAC AC AG T AGAT G T T GAAC T T AT C T AT GAAG GAAT C AAAT AT G T AC T T AAG G T GAC T C G G C AG T C T C C C AAC T C C T AC G T AG T GAT AA TGAATGGCTCGTGTGTGGAAGTGGATGTGCATCGGCTGAGTGATGGTGGCCTGCTCTTGTCTTA T GAC G G C AG C AG T T AC AC C AC AT AC AT GAAG GAAGAG G T G GAC AGAT AT C GAAT C AC AAT T G G C AATAAAACCTGTGTGTTTGAGAAGGAAAATGACCCATCTGTAATGCGCTCACCGTCTGCTGGGA AGTTAATCCAGTATATTGTGGAAGATGGCGGACATGTGTTTGCTGGCCAGTGCTATGCTGAGAT T GAG G T AAT GAAGAT G G T GAT GAC T T T AAC AG C T G T AGAAT C T G G C T G CAT C CAT T AT G T C AAA CGACCTGGAGCAGCACTTGACCCTGGCTGTGTGATAGCCAAAATGCAACTGGACAACCCCAGTA AAG T T C AAC AG G C T GAG C T T C AC AC G G G C AG T C T AC C AC AGAT C C AGAG C AC AG C T C T C AGAG G CGAGAAGCTCCATCGAGTTTTCCACTATGTCCTGGATAACCTGGTCAATGTGATGAATGGATAC TGCCTTCCAGACCCTTTCTTCAGCAGCAGGGTAAAAGACTGGGTAGAAAGATTGATGAAGACTC TGAGAGACCCCTCCTTGCCTCTGCTAGAGCTGCAGGATATCATGACCAGTGTCTCTGGCCGGAT C C C C C T C AAT G T G GAGAAG T C T AT T AAGAAG GAAAT G G C T C AG T AT G C TAG C AAC AT C AC AT C A GTCCTGTGT C AG T T T C C C AG C C AG C AGAT T G C C AAC AT C C T AGAT AG T CAT G C AG C T AC AC T GA ACCGGAAATCTGAGCGGGAAGTCTTCTTCATGAACACCCAGAGCATTGTCCAGCTGGTGCAGAG GTACCGAAGTGGCATCCGTGGCCACATGAAGGCTGTGGTGATGGATCTGCTGCGGCAGTACCTG C GAG T AGAGAC AC AG T T T C AGAAC G G C C AC T AC GAC AAAT GTGTATTCGCCCTTCGG GAAGAGA AC AAAAG C GAC AT GAAC AC C G T AC T GAAC T AC AT C T T C T C C C AC G C T C AG G T C AC C AAAAAGAA TCTCCTGGTGACAATGCTTATTGATCAGTTATGTGGCCGGGACCCTACACTTACTGATGAGCTG CTAAATATCCTCACAGAGCTAACCCAACTCAGCAAGACCACCAACGCTAAAGTGGCGCTGCGCG CTCGTCAGGTTCTTATTGCTTCCCATTTGCCATCATATGAGCTTCGCCATAACCAAGTAGAGTC TATCTTCCTAT C AG C C AT T GAC AT G T AT G GAC AC C AG TTTTGCATT GAGAAC C T G C AGAAAC T C ATCCTCTCGGAAACATCTATTTTCGATGTCCTCCCAAACTTTTTTTACCACAGCAACCAGGTGG TGAGGATGGCAGCTCTGGAGGTGTATGTTCGAAGGGCTTACATTGCCTATGAACTCAACAGCGT ACAACACCGCCAGCTTAAGGACAACACCTGTGTGGTGGAATTTCAGTTCATGCTGCCCACATCC CAT C C AAAC AGAG G GAAC AT C C C C AC G C T AAAC AGAAT G T C C T T T G C C T C C AAC C T C AAC C AC T ATGGCATGACTCATGTAGCTAGTGTCAGCGATGTTCTGTTGGACAACGCCTTCACGCCACCTTG TCAGCGGATGGGCGGAATGGTCTCTTTCCGGACCTTTGAAGATTTTGTCAGGATCTTTGATGAA ATAATGGGCTGCTTCTGTGACTCCCCACCCCAAAGCCCCACATTCCCAGAGTCTGGTCATACTT CGCTCTAT GAT GAAGAC AAG G T C C C C AG G GAT GAAC C AAT AC AT AT T C T GAAT GTGGCTAT C AA GAC T GAT G G C GAT AT T GAG GAT GAC AG G C T T G C AG C T AT G T T C AGAGAG T T C AC C C AG C AGAAT AAAGCTACTTTGGTTGAGCATGGCATCCGGCGACTTACGTTCCTAGTTGCACAAAAGGATTTCA GAAAAC AAG T C AAC T G T GAG G T G GAT C AGAGAT T T C AT AGAGAAT T C C C CAAAT T T T T C AC AT T CCGAGCAAGGGATAAGTTTGAGGAGGACCGCATTTATCGACACCTGGAGCCTGCTCTGGCTTTC CAG T T AGAG T T GAAC C G GAT GAGAAAT T T T GAC CTTACTGCCATCCCATGTGCTAAT C AC AAGA TGCACCTGTACCTTGGGGCTGCTAAGGTGGAAGTAGGCACAGAAGTGACTGACTACAGGTTCTT TGTTCGTGCGATCATCAGGCACTCTGATCTGGTCACAAAGGAAGCTTCTTTCGAATATCTACAA AAT GAAGGGGAACGAC T GC T CC T GGAAGC TAT GGAT GAAT T GGAAGT T GC T T T TAATAATACAA ATGTCCGCACTGACTGTAACCACATCTTCCTCAACTTTGTGCCCACGGTCATCATGGACCCATC AAAGATTGAAGAATCTGTGCGGAGCATGGTAATGCGCTATGGAAGTCGGCTATGGAAATTGCGG GTCCTCCAGGCAGAACTGAAAATCAACATTCGCCTGACAACAACTGGAAAAGCAATTCCCATCC GCCTCTTCCTGACAAACGAGTCTGGCTACTACTTGGACATCAGCCTGTATAAGGAAGTGACTGA C T C CAG GAC AG C AC AGAT CAT G T T T CAG G CAT AT G GAGAC AAG CAG G GAC C AC T G CAT G GAAT G T T AAT T AAT AC T C CAT AT G T GAC CAAAGAC C T T C T T C AAT CAAAGAG G T T C CAG G C AC AG T C C T TAG GAAC AAC AT AT AT AT AT GAT AT C C C AGAGAT G T T T C G G CAG T C AC T CAT C AAAC T C T G G GA GTCCATGTCCACCCAAGCATTTCTTCCTTCGCCTCCTTTGCCTTCCGACATCCTGACGTATACT GAAC TGGTGTTG GAT GAT C AAG G C CAG C T G G T C CAT AT GAAC AGAC T T C CAG GAG GAAAT GAGA TTGGCATGGTAGCCTGGAAAATGAGCCTTAAAAGCCCTGAATATCCAGATGGCCGAGATATCAT TGTCATCGGCAATGACATTACATATCGGATCGGTTCCTTTGGGCCTCAGGAGGATTTGCTGTTT CTCAGAGCTTCTGAACTTGCCAGAGCAGAAGGCATCCCACGCATCTACGTGGCAGCGAACAGTG GAGCTAGAATTGGACTTGCAGAAGAAATACGCCATATGTTCCATGTGGCCTGGGTAGATCCTGA AGAT C C C T AC AAG G GAT AC AAG TAT T TAT AT C T GAC AC C C CAG GAT T AT AAGAGAG T CAG T G C C C T C AAT T C T G T C C AC T G T GAAC AT G T G GAG GAT GAAG G G GAAT C CAG G T AC AAGAT AAC AGAT A TTATCGGGAAAGAAGAAGGACTTGGAGCAGAGAACCTTCGGGGTTCTGGAATGATTGCTGGGGA ATCCTCATTGGCTTACGATGAGGTCATCACCATCAGCCTGGTTACATGCCGGGCCATTGGTATT GGGGCTTACCTTGTCCGGCTGGGACAAAGAACCATCCAGGTTGAGAATTCTCACTTAATTCTGA CAGGAGCAGGTGCCCTCAACAAAGTCCTTGGTCGGGAAGTATACACCTCCAACAACCAGCTTGG GGGCATCCAGATTATGCACAACAATGGGGTTACCCACTCCACTGTTTGTGATGACTTTGAGGGA GTCTTCACAGTCTTACACTGGCTGTCATACATGCCGAAGAGCGTACACAGTTCAGTTCCTCTCC T GAAT T C C AAG GAT C C TAT AGAT AGAAT C AT C GAG TTTGTTCC C AC AAAG GCCCCATATGATCC TCGGTGGATGCTAGCAGGCCGTCCTCACCCAACCCAGAAAGGCCAATGGTTGAGTGGGTTTTTT GACTATGGATCTTTCTCAGAAATCATGCAGCCCTGGGCACAGACCGTGGTAGTTGGCAGAGCCA GGTTAGGGGGAATACCCGTGGGAGTAGTTGCTGTAGAAACCCGAACAGTGGAACTCAGTATCCC AGCTGATCCTGCGAACCTGGATTCTGAAGCCAAGATAATCCAGCAGGCCGGCCAGGTTTGGTTC C C AGAC T C T G CAT T T AAGAC C T AT C AAG C T AT C AAG GAC T T T AAC C G T GAAG G G C T AC C T C T AA TGGTCTTTGCCAACTGGAGAGGTTTCTCTGGTGGGATGAAAGATATGTATGACCAAGTGCTCAA GTTTGGCGCTTACATTGTGGATGGCTTGCGGGAATGTTCCCAGCCTGTAATGGTTTACATCCCG CCCCAGGCTGAGCTTCGGGGTGGTTCTTGGGTTGTGATCGACCCCACCATCAACCCTCGGCACA T G GAGAT G T AC G C T GAC C GAGAAAG CAG G G GAT CTGTTCTG GAG C C AGAAG G GAC AG T AGAAAT CAAAT TCCGTAAAAAGGATCTGGTGAAAACCATGCGTCGGGTAGATCCAGTTTACATCCGCTTG GCTGAGCGATTGGGCACCCCAGAGCTAAGCCCCACTGAGCGGAAGGAGCTGGAGAGCAAGTTGA AGGAGCGGGAGGAGTTCCTAATTCCCATTTACCATCAGGTAGCTGTGCAGTTTGCTGACTTGCA C GAC AC AC CAG G C C G GAT G CAG GAGAAG G G T G T CAT T AAC GAT AT C T T G GAT T G GAAAAC AT C C CGCACCTTCTTCTACTGGAGGCTGAGGCGCCTCCTGCTGGAGGACCTGGTCAAGAAGAAAATCC ACAATGCCAACCCTGAGCTGACTGACGGCCAGATCCAGGCCATGTTGAGACGCTGGTTTGTAGA AGTTGAAGGCACAGTGAAGGCTTACGTCTGGGACAATAATAAGGACCTGGTGGAGTGGCTGGAG AAG C AAC T GAC AGAG GAAGAT GGCGTCCGCTCTGT GAT AGAG GAGAAC AT CAAAT AC AT CAG C A GGGACTATGTCCTGAAGCAGATCCGCAGCTTGGTCCAGGCCAACCCAGAAGTCGCCATGGACTC CAT C G T C C AC AT GAC C CAG C AC AT C T C AC C C AC C CAG C GAG C AGAAG T T G T AAG GATCCTCTCC ACTATGGATTCCCCTTCCACGTAGGAGGAGCTTCCCGCCCACCCCTGCCCTGTCTCTGGAGAAG AGAGTCGGGCTGCCTCTCCCATCTGAGACCACTGTAATGAGAAGGCACCGGAGGCCTGAGACTG GATCAGTGGCATTTGCTTCCCTTGAGTGTTTCAGGCTCTGCATGACATCCTGGGCTATAGGATC ACACAGCCCAGTCACACATACCCGATTCAGTATTTATTAGCCCAGCTATGATGACAGTCCTCTT CCCATGCACAGGACTGAGAAGGCAATGAAAGGTACTTGCCTGTACCATGAGGTCTTACTAGACT AAAGCAGGACTGCCCTCCGTCCTGCCTGCCCCAGCATAGGGTCGTGTGAACAGTGTCCAAGTGT CTGCAGCCCCTGCCCCATGAGCCACAGCCAACAGGGGAGGGCTGGGGCTGCCAGGAGACGCAAG TCCATCGATCACTACCCCACATTGCAACCAAGCACATGCCAGGCACTGGAGAGAGACAGTCCCA TCTAACCACAGCAAGATAACTGAGAAATGTCAGAGCAGAAAATCTGTCGCAAGCCCGTGAGAAC ACAGAGATAAAGGCAAAGCCATGATCTAACAGGCAGGCTCAAAACCAGATCATGTCATTGTTCC TGGTCAAACACACTCAACCACCTCAGTGGCGTCTCAGTATCCCTGGGGAGACGGTCACCCTGCC ACCACCTTATCACTATGTAATCACATAGTGACTATACCTAGGAGCTAAAGGCCTGTGCATAAGG GCCCTAGCCTCTGCGGGTGGATGGAGACAGGCAGGCAGTGCAGGAGCCTGAGGTGGCTAAGAGG AGGCTCTTTTCTGTTGGTGATAGGGGAAGAGTACTTCAAAACAGGAGAAGTCTCTTCTGGGGAC AGGTTGTCACTATGGGGCCATGCCTATTCCTGGCAGAAGGTATGGCGGGTCACCCACCTGATGA CACCTGTGCTTTCAGCACCAGAAACTGGCCTTCTTGATGTTAGGAGGCTTCCAGGGAACAAGAA TTCACCTGCCCTTAAGATACCTTACAGTTCAGTCTCGGTGCTGGTGTTTCCAGACTTCTGGGCC CTATGTGGCCAGCAACACGGCTTCCTCAGGGGTGGCCTGAAAAGTTTAACTACCCATTTGTCCA AGAAAGCAGCTGATGGTTCAGCATTTGTGTTCATAAAGCAAGAATGAAGATGATCATAACTCGT ATAACATCCAGTTTCAACTTTATGACTAATACATCTGTCGCTGAAGATCGAGAAGGAAAAAGAT ACCTGCCACAGAAACCATTCAAGTGAGTACTTCAGTCTGAGCCTTAACCTGGATTCCACGAAAA GAGCTGACCTCCCCTTCAGGAGCACCTCAAGCAGATATTCCAGAGGACCTTCTCAATGGTGCCA CATTGCAAGTGGACATCCAAGGGACTATAGATGCAAGTTGCCCCACCCCCACAAGCCCTGGTGG AAGGAACAGGAGGGCCAGAGCTGGATCAGGTGGGGAGCATCCTTAGTCCAGAAGGAAGCTCCCT GGCCCCTGGTTCCCTGCTTACCTGGTAGGTAAGGATGGGATTTATCTCTGGCCTCCACTTTTGC TACAGCACGTCCAATCCCACACCCCCGGTGGTATGAAAGCTGCTTTCCTGGAGAGGTGGAGTGG GCTGGGCTTGTATAAGTCCCTTTTCCCTGCTGCCCCATCCCCGGGACCGGGCCGTCTCAATACG GAGAAGTCAGAGCCACGGCACATGGGCAGTGTGATGGTACCACAGCCCATTACACATGCAGGGT TACTGAGGAGGAGGGTGAATGATGTATTGACCCAGACTGGCTTGAACTGAGATTGCCATATTAT CTGTCCACTTGTTCACAAAGCAGCCTTCACACGTGTCAGTGAGAGTTCGATGGACACACACGCC AGCGAGCAGGGGCTTCAGTCCAATGCACTCTTCTCACGTTTTGTTGAAATAAACCTCCACATTT GTAGAAGAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 4 | Mus musculus (mouse) ACC Protein

MDEPSPLAKTLELNQHSRFI IGSVSEDNSEDEISNLVKLDLEEKEGSLSPASVSSDTLSDLGIS GLQDGLAFHMRSSMSGLHLVKQGRDRKKIDSQRDFTVASPAEFVTRFGGNKVIEKVLIANNGIA AVKCMRS IRRWSYEMFRNERAIRFWMVTPEDLKANAEYIKMADHYVPVPGGPNNNNYANVELI LDIAKRIPVQAVWAGWGHASENPKLPELLLKNGIAFMGPPSQAMWALGDKIASS IVAQTAGIPT LPWSGSGLRVDWQENDFSKRILNVPQDLYEKGYVKDVDDGLKAAEEVGYP\/MIKASEGGGGKGI RKVNNADDFPNLFRQVQAEVPGSPI F\/MRLAKQSRHLEVQILADQYGNAISLFGRDCSVQRRHQ KIIEEAPAAIATPAVFEHMEQCAVKLAKMVGYVSAGTVEYLYSQDGSFYFLELNPRLQVEHPCT EMVADVNLPAAQLQIAMGIPLFRIKDIRMMYGVSPWGDAPIDFENSAHVPCPRGHVIAARITSE NPDEGFKPSSGTVQELNFRSNKNVWGYFSVAAAGGLHEFADSQFGHCFSWGENREEAISNMWA LKELSIRGDFRTTVEYLIKLLETESFQLNRIDTGWLDRLIAEKVQAERPDTMLGWCGALHVAD VSLRNSISNFLHSLERGQVLPAHTLLNTVDVELIYEGIKYVLKVTRQSPNSYWIMNGSCVEVD VHRLSDGGLLLSYDGSSYTTYMKEEVDRYRITIGNKTCVFEKENDPSVMRSPSAGKLIQYIVED GGHVFAGQCYAEIE\/MKM\/MTLTAVESGCIHYVKRPGAALDPGCVIAKMQLDNPSKVQQAELHT GSLPQIQSTALRGEKLHRVFHYVLDNLVN\/MNGYCLPDPFFSSRVKDWVERLMKTLRDPSLPLL ELQDIMTSVSGRIPLNVEKS IKKEMAQYASNITSVLCQFPSQQIANILDSHAATLNRKSEREVF FMNTQS IVQLVQRYRSGIRGHMKAVVMDLLRQYLRVETQFQNGHYDKCVFALREENKSDMNTVL NYIFSHAQVTKKNLLVTMLIDQLCGRDPTLTDELLNILTELTQLSKTTNAKVALRARQVLIASH LPSYELRHNQVES I FLSAIDMYGHQFCIENLQKLILSETS I FDVLPNFFYHSNQVVRMAALEVY VRRAYIAYELNSVQHRQLKDNTCWEFQFMLPTSHPNRGNIPTLNRMSFASNLNHYGMTHVASV SDVLLDNAFTPPCQRMGGMVSFRTFEDFVRIFDEIMGCFCDSPPQSPTFPESGHTSLYDEDKVP RDEPIHILNVAIKTDGDIEDDRLAAMFREFTQQNKATLVEHGIRRLTFLVAQKDFRKQVNCEVD QRFHREFPKFFTFRARDKFEEDRIYRHLEPALAFQLELNRMRNFDLTAIPCANHKMHLYLGAAK VEVGTEVTDYRFFVRAI IRHSDLVTKEASFEYLQNEGERLLLEAMDELEVAFNNTNVRTDCNHI FLNFVPTVIMDPSKIEESVRSM\/MRYGSRLWKLRVLQAELKINIRLTTTGKAIPIRLFLTNESG YYLDISLYKEVTDSRTAQIMFQAYGDKQGPLHGMLINTPYVTKDLLQSKRFQAQSLGTTYIYDI PEMFRQSLIKLWESMSTQAFLPSPPLPSDILTYTELVLDDQGQLVHMNRLPGGNEIGMVAWKMS LKSPEYPDGRDI IVIGNDITYRIGSFGPQEDLLFLRASELARAEGIPRIYVAANSGARIGLAEE IRHMFHVAWVDPEDPYKGYKYLYLTPQDYKRVSALNSVHCEHVEDEGESRYKITDI IGKEEGLG AENLRGSGMIAGESSLAYDEVITISLVTCRAIGIGAYLVRLGQRTIQVENSHLILTGAGALNKV LGREVYTSNNQLGGIQIMHNNGVTHSTVCDDFEGVFTVLHWLSYMPKSVHSSVPLLNSKDPIDR IIEFVPTKAPYDPRWMLAGRPHPTQKGQWLSGFFDYGSFSEIMQPWAQTWVGRARLGGIPVGV VAVETRTVELSIPADPANLDSEAKI IQQAGQVWFPDSAFKTYQAIKDFNREGLPLMVFANWRGF SGGMKDMYDQVLKFGAYIVDGLRECSQPVMVYIPPQAELRGGSWWIDPTINPRHMEMYADRES RGSVLEPEGTVEIKFRKKDLVKTMRRVDPVYIRLAERLGTPELSPTERKELESKLKEREEFLIP IYHQVAVQFADLHDTPGRMQEKGVINDILDWKTSRTFFYWRLRRLLLEDLVKKKIHNANPELTD GQIQAMLRRWFVEVEGTVKAYVWDNNKDLVEWLEKQLTEEDGVRSVIEENIKYISRDYVLKQIR SLVQANPEVAMDSIVHMTQHISPTQRAEWRILSTMDSPST

[0060] The present disclosure also includes methods for increasing the expression and/or activity of both an ACL and an ACC in a genetically-modified microorganism relative to an unmodified organism of the same species. The present disclosure also includes genetically-engineered microorganisms produced by such methods. Such methods can include providing one or more extra copies of an endogenous ACL and/or ACC gene, putting an endogenous ACL and/or ACC gene under the control of a stronger promoter, mutating an endogenous ACL and/or ACC gene to encode a higher activity enzyme, introducing an exogenous ACL and/or ACC gene, or any combination thereof. Increased expression of ACL and ACC in a genetically-modified microorganism can increase synthesis of C6 fatty acids (e.g., hexanoic acid). Increased expression of ACL and ACC in a genetically-modified microorganism can confer one or more beneficial phenotypes for cannabinoid production. Non-limiting examples of beneficial phenotypes can include hyperactivation of the TAG storage pathway, growth advantage, continuous fatty acid production, elevated tolerance to cannabinoid precursors in the culture medium, modification of fatty acid profile as compared to the unmodified microbe, or a combination thereof. Fatty acid profile modification can include, for example, an equilibrium shift in the ratio of saturated fatty acids to unsaturated fatty acids that is favorable for cannabinoid or cannabinoid precursor production.

[0061] Malonyl-CoA can be an intermediate that serves as the building block for lipogenesis. During lipogenesis, malonyl-CoA can serve as a two-carbon donor unit in a cyclic series of reactions catalyzed by a fatty acid synthase (FAS) and elongases.

[0062] The present disclosure relates to the metabolic engineering of nitrogen metabolism in a genetically-engineered microorganism to increase synthesis rate and accumulation of cannabinoid precursor and cannabinoid derivatives in the genetically-engineered microorganism relative to an unmodified organism of the same species. In some embodiments, nitrogen depletion during stationary phase can trigger increased fatty acid flux through type-I FAS.

[0063] Type-I FAS can be a multifunctional enzyme that elongates the fatty acid chain in the fatty acid biosynthesis pathway. In yeast, the individual functions involved in cytosolic fatty acid synthesis can be performed by discrete domains on a single polynucleotide, or by two different polypeptide chains. Yeast cytosolic FAS can be a multienzyme complex composed of two subunits, Fasl (β subunit) and Fas2 (a subunit) which can be organized as a hexameric α₆β₆ complex. Fasl can harbor acetyl transferase, enoyl reductase, dehydratase, and malonyl- palmitoyl transferase activities. Fas2 can contain acyl carrier protein, 3-ketoreductase, 3- ketosynthase, and the phosphopantheteine transferase activities.

[0064] Mitochondrial fatty acid synthesis in yeast can be carried out by a type-II FAS system, which can include Acpl, acyl-carrier protein that carries the phosphopantetheine prosthetic group; Ceml, β-ketoacyl-ACP synthase; Oarl, 3-oxoacyl-[acyl-carrier protein] reductase; Htd2, 3-hydroxyacyl-thioester dehydratase; and Etrl, enoyl-ACP reductase. Ppt2 encodes for phosphopantetheine: protein transferase, which catalyzes the attachment of the

phosphopantetheine prosthetic group to the ACP apoprotein. The immediate products of de novo fatty acid synthesis are typically saturated fatty acids.

[0065] Many microorganisms (including oleaginous microorganisms such as Y. lipolytica) typically produce long-chain fatty acids (e.g., C14 or longer) instead of short-chain fatty acids, such as hexanoic acid (C6). Accordingly, disclosed herein are methods of genetically- engineering a microorganism to produce short-chain fatty acids. Non-limiting examples of short- chain fatty acids include ethanoic acid (acetic acid), propanoic acid (propionic acid), butanoic acid (butyric acid), and pentenoic acid (valeric acid). Non-limiting examples of medium chain fatty acids include hexanoic acid (caproic acid), hepantoic acid (enanthic acid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), decanoic acid (capric acid), undecanoic acid (undecyclic acid), and dodecanoic acid (lauric acid). Fatty acid derivatives can include short- chain fatty acids and medium chain fatty acids thioesters, including, for example, fatty acids that carry coenzyme A (CoA). Also disclosed herein are genetically-engineered microorganisms that comprise one or more genetic modifications that increase the production of a short-chain fatty acid relative to an unmodified organism of the same species. The short-chain fatty acid can be hexanoic acid.

[0066] As illustrated in FIGS. 1 and 2, malonyl-CoA be converted to short-chain fatty acids (e.g., C6 fatty acids, e.g., hexanoic acid) by the actions of a type-I fatty acid synthase alpha (FASa) and a fatty acid synthase beta (FASP) capable of producing short-chain fatty acids (e.g., hexanoic acid specific FASa and FASP). For example, FASa and FASP from Aspergillus species (e.g., Aspergillus parasiticus) can produce short-chain fatty acids such as hexanoic acid.

[0067] In some embodiments, the present disclosure relates to the engineering of a

microorganism for cannabinoid or cannabinoid precursor production based on the ability of the microbe to synthesize short-chain fatty acids and fatty acid derivatives using a dedicated type-I fatty acid synthase (FAS). Increased expression of a dedicated type-I fungal FAS can produce high concentrations of hexanoic acid in a microbe.

[0068] Enzymes involved in the synthesis of short-chain fatty acids can be engineered into a microorganism to increase the production or flux of hexanoic acid, for example, for cannabinoid biosynthesis in the microorganism. Accordingly, the present disclosure includes genetically- engineered microorganisms comprising one or more genetic modifications that increase the expression of FASa and FASp. The FASa and FASP can be hexanoic acid specific Type-I fatty acid synthases. The FASa and FASP can be from an Aspergillus species. In some embodiments, the FASa and FASP can be from an Aspergillus parasiticus species.

[0069] The present disclosure also includes methods and compositions for increasing the expression of FASa and FASP in a genetically-engineered microorganism relative to an unmodified organism of the same species. Such methods can include providing one or more extra copies of endogenous FASa and FASP genes, putting endogenous FASa and FASP genes under the control of a stronger promoter, mutating endogenous FASa and FASp genes to encode a higher activity enzyme, introducing exogenous FASa and FASP genes or coding sequences, or any combination thereof. The FASa and FASP can be hexanoic acid specific Type-I fatty acid synthases. The FASa and FASP can be from an Aspergillus species. The FASa and FASp can be from an Aspergillus parasiticus species. Exemplary FASa and FASP polynucleotides and polypeptide sequences are disclosed in TABLE 3 and can include GenelD: 853653 and GenelD: 855845, which can be found in the NCBI database. A genetic modification that increases the expression of FASa and/or FASP can comprise a polynucleotide that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an open reading frame of SEQ ID NO: 5, a polynucleotide that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to an open reading frame of SEQ ID NO: 7, or both. A genetic modification that increases the expression of FASa and/or FASP can comprise a polynucleotide that encodes a polypeptide that is at least 80%, at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, a polynucleotide that encodes a polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8, or both. The polynucleotide can be integrated into the genome of a genetically-engineered microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism.

[0070] Modifying the fatty acid metabolism in a microorganism in accordance with methods provided herein (for example, by increasing expression of FAS alpha and beta alone or in combination with other genetic or non-genetic modifications provided herein) can allow for the generation of a genetically-engineered microorganism that has one or more advantages for large- scale cannabinoid or cannabinoid precursor production. In some embodiments, the increased expression of FAS alpha and beta genes of the type-I FAS pathway in a suitable microorganism can drive heterologous production of C6 fatty acids, which can enable increased synthesis of olivetolic acid in the genetically-engineered microorganism, as compared to an unmodified organism of the same species. Increased expression of a type-I FAS can confer one or more beneficial phenotypes for cannabinoid and cannabinoid precursor production. For example, increased expression of type-I FAS enzymes described herein can give the genetically- engineered microorganism the ability to synthesize short-chain fatty acids. Increased expression of type-I FAS enzymes described herein can increase fatty acid synthesis rate and/or confer a beneficial phenotypes for large-scale carbohydrate to cannabinoid or cannabinoid precursor conversion. Non-limiting examples of beneficial phenotypes can include, for example, increased fatty acid precursor synthesis rate, increased carbohydrate to olivetolic acid and CBG conversion efficiency, increased cannabinoid storage and/or secretion, increased growth rate, increased tolerance to concentrations of a cannabinoid precursor or a toxic substance, or a combination thereof as compared to an unmodified organism of the same species.

TABLE 3

Exemplary FAS sequences

SEQ ID NO: 5 Aspergillus parasiticus FAS-a polynucleotide

ATGGTCATCCAAGGGAAGAGAT TGGCCGCCTCCTCTAT TCAGCT TCTCGCAAGCTCGT TAGACG C GAAGAAG CT T TGT TAT GAG TAT GAC GAGAG G C AAG C C C C AG G T G T AAC C C AAAT C AC C GAG GA GGCGCCTACAGAGCAACCGCCTCTCTCTACCCCTCCCTCGCTACCCCAAACGCCCAATAT T TCG CCTATAAGTGCT TCAAAGATCGTGATCGACGATGTGGCGCTATCTCGAGTGCAAAT TGT TCAGG CTCTTGTTGCCAGAAAGCTGAAGACGGCAATTGCTCAGCTTCCTACATCAAAGTCAATCAAAGA GTTGTCGGGTGGTCGGTCTTCTCTGCAGAACGAGCTCGTGGGGGATATACACAACGAGTTCAGC TCCATCCCGGATGCACCAGAGCAGATCCTGCTGCGGGACTTTGGCGACGCCAACCCAACAGTGC AACTGGGGAAAACGTCCTCCGCGGCAGTTGCCAAACTAATCTCGTCCAAGATGCCTAGTGACTT CAACGCCAACGCTATTCGAGCCCACCTAGCAAACAAGTGGGGTCTAGGACCCTTGCGACAAACA GCGGTGCTGCTCTACGCCATTGCGTCAGAACCCCCATCGCGTTTAGCTTCATCGAGCGCAGCGG AAGAGTACTGGGACAACGTGTCATCCATGTACGCCGAATCGTGTGGCATCACCCTCCGCCCGAG ACAAGACACTATGAATGAAGATGCTATGGCATCGTCGGCGATTGATCCGGCTGTGGTAGCCGAG TTTTCCAAGGGGCACCGTAGGCTCGGAGTTCAACAGTTCCAAGCGCTAGCAGAATACTTACAAA TTGATTTGTCGGGGTCTCAAGCCTCTCAGTCGGATGCTTTGGTGGCGGAACTTCAGCAGAAAGT CGATCTCTGGACGGCCGAAATGACCCCCGAGTTTCTCGCCGGGATATCACCAATGCTGGATGTA AAGAAGTCGCGACGCTATGGCTCGTGGTGGAACATGGCACGGCAGGATGTCTTGGCCTTCTATC GCCGTCCTTCCTACAGTGAATTCGTGGACGACGCCCTGGCCTTCAAAGTTTTTCTCAATCGTCT CTGTAACCGAGCTGATGAGGCCCTCCTCAACATGGTACGCAGTCTTTCCTGTGACGCCTACTTC AAGCAAGGTTCTTTGCCCGGATATCATGCCGCCTCGCGACTCCTTGAGCAGGCCATCACATCCA CAGTGGCGGATTGCCCGAAGGCACGCCTCATTCTCCCGGCGGTGGGCCCCCACACCACCATTAC AAAGGACGGCACGATTGAATACGCGGAGGCACCGCGCCAGGGAGTGAGTGGTCCCACTGCGTAC ATCCAGTCTCTCCGCCAAGGCGCATCTTTCATTGGTCTCAAGTCAGCCGACGTCGATACTCAGA GCAACTTGACCGACGCTCTGCTTGACGCCATGTGCTTAGCACTCCATAATGGAATCTCGTTTGT TGGTAAAACCTTTCTGGTGACGGGAGCGGGTCAGGGGTCAATAGGAGCGGGAGTGGTGCGTCTA T T G T T AGAG G GAG GAG C C C GAG T AC T G G T GAC GAC GAG C AG G GAG C C G G C GAC GAC AT C C AGAT ACTTCCAGCAGATGTACGATAATCACGGTGCGAAGTTCTCCGAGCTGCGGGTAGTTCCTTGCAA TCTAGCCAGCGCCCAAGATTGCGAAGGGCTGATCCGGCACGTCTACGATCCCCGTGGGCTAAAT TGGGATCTGGATGCCATCCTTCCCTTCGCTGCCGCGTCCGACTACAGCACCGAGATGCATGACA TTCGGGGACAGAGCGAGCTGGGCCACCGGCTAATGCTGGTCAATGTCTTCCGCGTGCTGGGGCA TATCGTCCACTGTAAACGAGATGCCGGGGTTGACTGCCATCCGACGCAGGTGCTGCTGCCACTG TCGCCAAATCACGGCATCTTCGGTGGCGATGGGATGTATCCGGAGTCAAAGCTAGCCCTTGAGA GCCTGTTCCATCGCATCCGATCAGAGTCTTGGTCAGACCAGTTATCTATATGCGGCGTTCGTAT CGGTTGGACCCGGTCGACCGGTCTAATGACGGCGCATGATATCATAGCCGAAACGGTCGAGGAA CACGGAATACGCACATTTTCCGTGGCCGAGATGGCACTCAACATAGCCATGCTGTTAACCCCCG ACTTTGTGGCCCATTGTGAAGATGGACCTTTGGATGCCGATTTCACCGGCAGCTTGGGAACATT GGGTAGCATCCCCGGTTTCCTAGCCCAATTGCACCAGAAAGTCCAGCTGGCAGCCGAGGTGATC CGTGCCGTGCAGGCCGAGGATGAGCATGAGAGATTCTTGTCTCCGGGAACAAAACCTACCCTGC AAGCACCCGTGGCCCCAATGCACCCCCGCAGTAGCCTTCGTGTAGGCTATCCCCGTCTCCCCGA TTATGAGCAAGAGATTCGCCCGCTGTCCCCACGGCTGGAAAGGTTGCAAGATCCGGCCAATGCT GTGGTGGTGGTCGGGTACTCGGAGCTGGGGCCATGGGGTAGCGCGCGATTACGGTGGGAAATAG AGAGCCAGGGCCAGTGGACTTCAGCCGGTTATGTCGAACTTGCCTGGCTGATGAACCTCATCCG CCACGTCAACGATGAATCCTACGTCGGCTGGGTGGATACTCAGACCGGAAAGCCAGTGCGGGAT GG C GAGAT C C AG G C AC T G T AC G G G GAC C AC AT T GAC AAC C AC AC CGGTATCCGTCCTATC C AG T CCACCTCGTACAACCCAGAGCGCATGGAGGTCCTGCAGGAGGTCGCTGTCGAGGAGGATCTGCC CGAGTTTGAAGTATCTCAACTTACCGCCGACGCCATGCGTCTCCGCCATGGAGCTAACGTTTCC ATCCGCCCCAGTGGAAATCCCGACGCATGCCACGTGAAGCTTAAACGAGGCGCTGTTATCCTTG TTCCCAAGACAGTTCCCTTTGTTTGGGGATCGTGTGCCGGTGAGTTGCCGAAGGGATGGACTCC AGCCAAGTACGGCATCCCTGAGAACCTAATTCATCAGGTCGACCCCGTCACGCTCTATACAATT TGCTGCGTGGCGGAGGCATTTTACAGTGCCGGTATAACTCACCCTCTTGAGGTCTTTCGACACA TTCACCTCTCGGAACTAGGCAACTTTATCGGATCCTCCATGGGTGGGCCGACGAAGACTCGTCA GCTCTACCGAGATGTCTACTTCGACCATGAGATTCCGTCGGATGTTCTGCAAGACACTTATCTC AACACACCTGCTGCCTGGGTTAATATGCTACTCCTTGGCTGCACGGGGCCGATCAAAACTCCCG TCGGCGCATGTGCCACCGGGGTCGAGTCGATCGATTCCGGCTACGAGTCAATCATGGCGGGCAA GACAAAGATGTGTCTTGTGGGTGGCTACGACGATCTGCAGGAGGAGGCATCGTATGGATTCGCA CAACTTAAGGCCACGGTCAACGTTGAAGAGGAGATCGCCTGCGGTCGACAGCCCTCGGAGATGT CGCGCCCCATGGCTGAGAGTCGTGCTGGCTTTGTCGAGGCGCATGGCTGCGGTGTACAGCTGCT GTGTCGAGGTGACATCGCCCTGCAAATGGGTCTTCCTATCTATGCGGTCATTGCCAGCTCAGCC ATGGCCGCCGACAAGATCGGTTCCTCGGTGCCAGCACCGGGCCAGGGCATTCTAAGCTTCTCCC GTGAGCGCGCTCGATCCAGTATGATATCCGTCACGTCGCGCCCGAGTAGCCGTAGCAGCACATC ATCTGAAGTCTCGGACAAATCATCCCTGACCTCAATCACCTCAATCAGCAATCCCGCTCCTCGT GCACAACGCGCCCGATCCACCACTGATATGGCTCCGCTGCGAGCAGCGCTTGCGACTTGGGGGC TGACTATCGACGACCTGGATGTGGCCTCATTGCACGGCACCTCGACGCGCGGTAACGATCTCAA TGAGCCCGAGGTGATCGAGACGCAGATGCGCCATTTAGGTCGCACTCCTGGCCGCCCCCTGTGG GCCATCTGCCAAAAGTCAGTGACGGGACACCCTAAAGCCCCAGCGGCCGCATGGATGCTCAATG GATGCCTGCAAGTACTGGACTCGGGGTTGGTGCCGGGCAACCGCAATCTTGACACGCTGGACGA GGCCCTGCGCAGCGCGTCTCATCTCTGCTTCCCTACGCGCACCGTGCAGCTACGTGAGGTCAAG GCATTCCTGCTGACCTCATTTGGCTTCGGACAGAAGGGGGGCCAAGTCGTCGGCGTTGCCCCCA AGTACTTCTTTGCTACGCTCCCCCGCCCCGAGGTTGAGGGCTACTATCGCAAGGTGAGGGTTCG AACCGAGGCGGGTGATCGCGCCTACGCCGCGGCGGTCATGTCGCAGGCGGTGGTGAAGATCCAG ACGCAAAACCCGTACGACGAGCCGGATGCCCCCCGCATTTTTCTCGATCCCTTGGCACGTATCT CCCAGGATCCGTCGACGGGCCAGTATCGGTTTCGTTCCGATGCCACTCCCGCCCTCGATGATGA TGCTCTGCCACCTCCCGGCGAACCCACCGAGCTAGTGAAGGGCATCTCCTCCGCCTGGATCGAG GAGAAGGTGCGACCGCATATGTCTCCCGGCGGCACGGTGGGCGTGGACCTGGTTCCTCTCGCCT CCTTCGACGCATACAAGAATGCCATCTTTGTTGAGCGCAATTATACGGTAAGGGAGCGCGATTG GGCTGAAAAGAGTGCGGATGTGCGCGCGGCCTATGCCAGTCGGTGGTGTGCAAAAGAGGCGGTG T T C AAAT G T C T C C AGAC AC AT T C AC AG GGCGCGGGGG C AG C CAT GAAAGAGAT T GAGAT C GAG C ATGGAGGTAACGGCGCACCGAAAGTCAAGCTCCGGGGTGCTGCGCAAACAGCGGCGCGGCAACG AGGATTGGAAGGAGTGCAACTGAGCATCAGCTATGGCGACGATGCGGTGATAGCGGTGGCGCTG GGGCTGATGTCTGGTGCTTCATAA

SEQ ID NO: 6 | Aspergillus parasiticus FAS-a polypeptide

MVIQGKRLAASS IQLLASSLDAKKLCYEYDERQAPGVTQITEEAPTEQPPLSTPPSLPQTPNIS PISASKIVIDDVALSRVQIVQALVARKLKTAIAQLPTSKS IKELSGGRSSLQNELVGDIHNEFS SIPDAPEQILLRDFGDANPTVQLGKTSSAAVAKLISSKMPSDFNANAIRAHLANKWGLGPLRQT AVLLYAIASEPPSRLASSSAAEEYWDNVSSMYAESCGITLRPRQDTMNEDAMASSAIDPAWAE FSKGHRRLGVQQFQALAEYLQIDLSGSQASQSDALVAELQQKVDLWTAEMTPEFLAGISPMLDV KKSRRYGSWWNMARQDVLAFYRRPSYSEFVDDALAFKVFLNRLCNRADEALLNMVRSLSCDAYF KQGSLPGYHAASRLLEQAITSTVADCPKARLILPAVGPHTTITKDGTIEYAEAPRQGVSGPTAY IQSLRQGASFIGLKSADVDTQSNLTDALLDAMCLALHNGISFVGKTFLVTGAGQGSIGAGWRL LLEGGARVLVTTSREPATTSRYFQQMYDNHGAKFSELRWPCNLASAQDCEGLIRHVYDPRGLN WDLDAILPFAAASDYSTEMHDIRGQSELGHRLMLVNVFRVLGHIVHCKRDAGVDCHPTQVLLPL SPNHGI FGGDGMYPESKLALESLFHRIRSESWSDQLS ICGVRIGWTRSTGLMTAHDI IAETVEE HGIRTFSVAEMALNIAMLLTPDFVAHCEDGPLDADFTGSLGTLGS IPGFLAQLHQKVQLAAEVI RAVQAEDEHERFLSPGTKPTLQAPVAPMHPRSSLRVGYPRLPDYEQEIRPLSPRLERLQDPANA

λΑΑΑ GYSELGPWGSARLRWEIESQGQWTSAGYVELAWLMNLIRHVNDESYVGWVDTQTGKPVRD GEIQALYGDHIDNHTGIRPIQSTSYNPERMEVLQEVAVEEDLPEFEVSQLTADAMRLRHGANVS IRPSGNPDACHVKLKRGAVILVPKTVPFVWGSCAGELPKGWTPAKYGIPENLIHQVDPVTLYTI CCVAEAFYSAGITHPLEVFRHIHLSELGNFIGSSMGGPTKTRQLYRDVYFDHEIPSDVLQDTYL NTPAAWVNMLLLGCTGPIKTPVGACATGVES IDSGYES IMAGKTKMCLVGGYDDLQEEASYGFA QLKATVNVEEEIACGRQPSEMSRPMAESRAGFVEAHGCGVQLLCRGDIALQMGLPI YAVIASSA MAADKIGSSVPAPGQGILSFSRERARSSMISVTSRPSSRSSTSSEVSDKSSLTSITSISNPAPR AQRARSTTDMAPLRAALATWGLTIDDLDVASLHGTSTRGNDLNEPEVIETQMRHLGRTPGRPLW AICQKSVTGHPKAPAAAWMLNGCLQVLDSGLVPGNRNLDTLDEALRSASHLCFPTRTVQLREVK AFLLTSFGFGQKGGQWGVAPKYFFATLPRPEVEGYYRKVRVRTEAGDRAYAAAVMSQAWKIQ TQNPYDEPDAPRI FLDPLARISQDPSTGQYRFRSDATPALDDDALPPPGEPTELVKGISSAWIE EKVRPHMSPGGTVGVDLVPLASFDAYKNAI FVERNYTVRERDWAEKSADVRAAYASRWCAKEAV FKCLQTHSQGAGAAMKEIEIEHGGNGAPKVKLRGAAQTAARQRGLEGVQLS ISYGDDAVIAVAL GLMSGAS

SEQ ID NO: 7 | Aspergillus parasiticus FAS-β polynucleotide

ATGGGTTCCGTTAGTAGGGAACATGAGTCAATCCCCATCCAGGCCGCCCAGAGAGGCGCTGCCC GGATCTGCGCTGCTTTTGGAGGTCAAGGGTCTAACAATCTGGACGTGTTAAAAGGTCTACTGGA GTTATACAAGCGGTATGGCCCAGATCTGGATGAGCTACTAGACGTGGCATCCAACACGCTTTCG CAGCTGGCATCTTCCCCTGCTGCAATAGACGTCCACGAACCCTGGGGTTTCGACCTCCGACAAT GGCTGACCACACCGGAGGTTGCTCCTAGCAAAGAAATTCTTGCCCTGCCACCACGAAGCTTTCC CTTAAATACGTTACTTAGCCTGGCGCTCTATTGTGCAACTTGTCGAGAGCTTGAACTTGATCCT GGGCAATTTCGATCCCTCCTTCATAGTTCCACGGGGCATTCCCAAGGCATATTGGCGGCGGTGG CCATCACCCAAGCCGAGAGCTGGCCAACCTTTTATGACGCCTGCAGGACGGTGCTCCAGATCTC TTTCTGGATTGGACTCGAGGCTTACCTCTTCACTCCATCCTCCGCCGCCTCGGATGCCATGATC CAAGATTGCATCGAACATGGCGAGGGCCTTCTTTCCTCAATGCTAAGTGTCTCCGGGCTCTCCC GCTCCCAAGTTGAGCGAGTAATTGAGCACGTCAATAAAGGGCTCGGAGAATGCAACCGATGGGT TCACTTGGCCCTGGTTAACTCCCACGAAAAGTTCGTCTTAGCGGGACCACCTCAATCCTTATGG GCCGTTTGTCTTCATGTCCGACGGATCAGAGCAGACAATGACCTCGACCAGTCGCGTATCCTGT T C C G C AAC C GAAAG C C TAT AG T G GAT AT AT TATTTCTTCC CAT AT C C G C AC C AT T T C AC AC AC C GTACTTGGACGGTGTTCAAGATCGCGTTATCGAGGCTTTGAGCTCTGCTTCGTTGGCTCTCCAT T C CAT C AAAAT CCCCCTCTAT C AC AC G G G C AC T G G GAG C AAC C T AC AAGAAC T AC AAC C AC AT C AGCTAATCCCGACTCTTATCCGCGCCATTACCGTGGACCAATTGGACTGGCCGCTGGTTTGCCG GGGCTTGAACGCAACGCACGTGTTGGACTTTGGACCTGGACAAACATGCAGTCTTATTCAGGAG C T C AC AC AAG GAAC AG G T G T AT C AG T GAT C C AG T T GAC T AC T CAAT C G G GAC CAAAAC C C G T T G GAGGCCATCTGGCGGCAGTGAACTGGGAGGCCGAGTTTGGCTTACGACTTCATGCCAATGTCCA CGGTGCAGCTAAATTGCACAACCGTATGACAACACTGCTTGGGAAGCCTCCTGTGATGGTAGCC GGAATGACACCTACTACGGTGCGCTGGGACTTTGTCGCTGCCGTTGCTCAAGCTGGATACCACG TCGAACTGGCTGGTGGTGGCTACCACGCAGAGCGCCAGTTCGAGGCCGAGATTCGGCGCCTGGC AACTGCCATCCCAGCAGATCATGGCATCACCTGCAATCTCCTCTACGCCAAGCCTACGACTTTT TCCTGGCAGATCTCTGTCATCAAGGATCTGGTGCGCCAGGGAGTTCCCGTGGAAGGAATCACCA TCGGCGCCGGCATCCCTTCTCCGGAGGTCGTCCAAGAATGTGTACAGTCCATCGGACTCAAGCA CATCTCATTCAAGCCTGGGTCTTTCGAAGCCATTCACCAAGTCATACAGATCGCGCGTACCCAT CCTAACTTTTTGATCGGGTTGCAATGGACCGCAGGACGAGGGGGAGGACATCATTCCTGGGAAG ACTTCCATGGACCTATTCTGGCAACCTACGCTCAAATCCGATCATGTCCGAATATTCTCCTCGT TGTAGGTAGTGGATTCGGTGGAGGCCCGGACACGTTTCCCTACCTCACGGGCCAATGGGCCCAG GCCTTTGGCTATCCATGCATGCCCTTCGACGGAGTGTTGCTCGGCAGTCGCATGATGGTGGCTC GGGAAGCCCATACGTCAGCCCAGGCAAAACGCCTGATTATAGATGCGCAAGGCGTGGGAGATGC AGATTGGCACAAGTCTTTCGATGAGCCTACCGGCGGCGTAGTGACGGTCAACTCGGAATTCGGT CAACCTATCCACGTTCTAGCTACTCGCGGAGTGATGCTGTGGAAAGAACTCGACAACCGGGTCT T T T CAAT C AAAGAC AC T T C T AAG C G C T T AGAAT AT C T G C G C AAC C AC C G G C AAGAAAT T G T GAG CCGTCTTAACGCAGACTTTGCCCGTCCCTGGTTTGCCGTTGACGGACACGGACAGAATGTGGAG CTGGAGGACATGACCTACCTCGAGGTTCTCCGCCGTCTGTGCGATCTCACGTATGTTTCCCACC AGAAGCGATGGGTAGATCCATCATATCGAATATTACTGTTGGACTTCGTTCATCTGCTTCGAGA ACGATTCCAATGCGCTATTGACAACCCCGGCGAATATCCACTCGACATCATCGTCCGGGTGGAA GAGAG C C T GAAG GAT AAAG CAT AC C G C AC GCTTTATC C AGAAGAT GTCTCTCTTC T AAT G CAT T TGTTCAGCCGACGTGACATCAAGCCCGTACCATTCATCCCCAGGTTGGATGAGCGTTTTGAGAC CTGGTTTAAAAAAGACTCATTGTGGCAATCCGAAGATGTGGAGGCGGTAATTGGACAGGACGTC CAGCGAATCTTCATCATTCAAGGGCCTATGGCCGTTCAGTACTCAATATCCGACGATGAGTCTG T TAAAGACAT T T TACACAATAT T TGTAAT CAT TACGT GGAGGC T C TACAGGC T GAT T CAAGAGA AACTTCTATCGGCGATGTACACTCGATCACGCAAAAACCTCTCAGCGCGTTTCCTGGGCTCAAA GTGACGACAAATAGGGTCCAAGGGCTCTATAAGTTCGAGAAAGTAGGAGCAGTCCCCGAAATGG ACGTTCTTTTTGAGCATATTGTCGGACTGTCGAAGTCATGGGCTCGGACATGTTTGATGAGTAA ATCGGTCTTTAGGGACGGTTCTCGTCTGCATAACCCCATTCGCGCCGCACTCCAGCTCCAGCGC GG S GAC AC CAT C GAG G T G C T T T T AAC AG C AGAC T C G GAAAT T C G C AAGAT T C GAC T T AT T T C AC CCACGGGGGATGGTGGATCCACTTCTAAGGTCGTATTAGAGATAGTCTCTAACGACGGACAAAG AGTTTTCGCCACCTTGGCCCCTAACATCCCACTCAGCCCCGAGCCCAGCGTCGTCTTTTGCTTC AAGGTCGACCAGAAGCCGAATGAGTGGACCCTTGAGGAGGATGCGTCTGGCCGGGCAGAGAGGA TCAAGGCATTATACATGAGTCTGTGGAACTTGGGCTTTCCGAACAAGGCCTCTGTTTTGGGTCT TAATTCGCAATTCACGGGAGAAGAACTGATGATCACAACGGACAAGATTCGTGATTTCGAAAGG GTACTGCGGCAAACCAGTCCTCTTCAGCTGCAGTCATGGAACCCCCAAGGATGTGTACCTATCG ACTACTGCGTGGTCATCGCCTGGTCTGCTCTTACCAAGCCTCTGATGGTCTCCTCTCTGAAATG CGACCTCCTGGATCTGCTCCACAGCGCTATAAGCTTCCACTATGCTCCATCTGTCAAACCATTG CGGGTGGGCGATATTGTCAAAACCTCATCCCGTATCCTAGCGGTCTCGGTGAGACCTAGGGGAA CTATGCTGACGGTGTCGGCGGACATTCAGCGCCAGGGACAACATGTAGTCACTGTCAAATCAGA TTTCTTTCTCGGAGGCCCCGTTCTGGCATGTGAAACCCCTTTCGAACTCACTGAGGAGCCTGAA ATGGTTGTCCATGTCGACTCTGAAGTGCGCCGTGCTATTTTACACAGCCGCAAGTGGCTCATGC GAGAAGATCGCGCGCTAGATCTGCTAGGGAGGCAGCTCCTCTTCAGATTAAAGAGCGAAAAATT GTTCAGGCCAGACGGCCAGCTAGCACTGTTACAGGTAACAGGTTCCGTGTTCAGCTACAGCCCC GATGGGTCAACGACAGCATTCGGTCGCGTATACTTCGAAAGCGAGTCTTGTACAGGGAACGTGG TGATGGACTTCCTGCACCGCTACGGTGCACCTCGGGCGCAGCTGCTGGAGCTGCAACATCCCGG GTGGACGGGCACCTCTACTGTGGCAGTAAGAGGTCCTCGACGCAGCCAATCCTACGCACGCGTC TCCCTCGATCATAATCCCATCCATGTTTGTCCGGCCTTTGCGCGATACGCTGGTCTCTCGGGTC CCATTGTCCATGGGATGGAAACCTCTGCCATGATGCGCAGAATTGCCGAATGGGCCATCGGAGA TGCAGACCGGTCTCGGTTCCGGAGCTGGCATATCACCTTGCAAGCACCCGTCCACCCCAACGAC CCTCTGCGGGTGGAGCTGCAGCATAAGGCCATGGAGGACGGGGAAATGGTTTTGAAAGTACAAG CATTTAACGAAAGGACGGAAGAACGCGTAGCGGAGGCAGATGCCCATGTTGAGCAGGAAACTAC GGCTTACGTCTTCTGTGGCCAGGGCAGTCAACGACAGGGGATGGGAATGGACTTGTACGTCAAC TGTCCGGAGGCTAAAGCGTTGTGGGCTCGCGCCGACAAGCATTTGTGGGAGAAATATGGGTTCT CCATCTTGCACATTGTGCAAAACAACCCTCCAGCCCTCACTGTTCACTTTGGCAGCCAGCGAGG GCGCCGTATTCGTGCCAACTATCTGCGCATGATGGGACAGCCACCGATAGATGGTAGACATCCG CCCATACTGAAGGGATTGACGCGGAATTCGACCTCGTACACCTTCTCCTATTCCCAGGGGCTGT TGATGTCCACCCAGTTCGCCCAGCCCGCACTGGCGCTGATGGAAATGGCTCAGTTCGAATGGCT CAAAGCCCAGGGAGTCGTTCAGAAGGGTGCGCGGTTCGCGGGACATTCGTTGGGAGAATATGCC GCCCTTGGAGCTTGTGCTTCCTTCCTCTCATTTGAAGATCTCATATCTCTCATCTTTTATCGGG GCTTGAAGATGCAGAATGCGCTGCCGCGCGATGCCAACGGCCACACCGACTATGGAATGTTGGC TGCCGATCCATCGCGGATAGGAAAAGGTTTCGAGGAAGCGAGTCTGAAATGTCTTGTCCATATC ATTCAACAGGAGACCGGCTGGTTCGTGGAAGTCGTCAACTACAACATCAACTCGCAGCAATACG TCTGTGCAGGCCATTTCCGAGCCCTTTGGATGCTGGGTAAGATATGCGATGACCTTTCATGCCA CCCTCAACCGGAGACTGTTGAAGGCCAAGAGCTACGGGCCATGGTCTGGAAGCATGTCCCGACG GTGGAGCAGGTGCCCCGCGAGGATCGCATGGAACGAGGTCGAGCGACCATTCCGCTGCCGGGGA TCGATATCCCATACCATTCGACCATGTTACGAGGGGAGATTGAGCCTTATCGTGAATATCTGTC TGAACGTATCAAGGTGGGGGATGTGAAGCCGTGCGAATTGGTGGGACGCTGGATCCCTAATGTT GTTGGCCAGCCTTTCTCCGTCGATAAGTCTTACGTTCAGCTGGTGCACGGCATCACAGGTAGTC CTCGGCTTCATTCCCTGCTTCAACAAATGGCGTGA

SEQ ID NO: 8 | Aspergillus parasiticus FAS-β polypeptide

MGSVSREHES IPIQAAQRGAARICAAFGGQGSNNLDVLKGLLELYKRYGPDLDELLDVASNTLS QLASSPAAIDVHEPWGFDLRQWLTTPEVAPSKEILALPPRSFPLNTLLSLALYCATCRELELDP GQFRSLLHSSTGHSQGILAAVAITQAESWPTFYDACRTVLQISFWIGLEAYLFTPSSAASDAMI QDCIEHGEGLLSSMLSVSGLSRSQVERVIEHVNKGLGECNRWVHLALVNSHEKFVLAGPPQSLW AVCLHVRRIRADNDLDQSRILFRNRKPIVDILFLPISAPFHTPYLDGVQDRVIEALSSASLALH SIKIPLYHTGTGSNLQELQPHQLIPTLIRAITVDQLDWPLVCRGLNATHVLDFGPGQTCSLIQE LTQGTGVSVIQLTTQSGPKPVGGHLAAVNWEAEFGLRLHANVHGAAKLHNRMTTLLGKPP\/MVA GMTPTTVRWDFVAAVAQAGYHVELAGGGYHAERQFEAEIRRLATAIPADHGITCNLLYAKPTTF SWQISVIKDLVRQGVPVEGITIGAGIPSPEWQECVQSIGLKHISFKPGSFEAIHQVIQIARTH PNFLIGLQWTAGRGGGHHSWEDFHGPILATYAQIRSCPNILLWGSGFGGGPDTFPYLTGQWAQ AFGYPCMPFDGVLLGSRMMVAREAHTSAQAKRLIIDAQGVGDADWHKSFDEPTGGWTVNSEFG QPIHVLATRG\ LWKELDNRVFSIKDTSKRLEYLRNHRQEIVSRLNADFARPWFAVDGHGQNVE LEDMTYLEVLRRLCDLTYVSHQKRWVDPSYRILLLDFVHLLRERFQCAIDNPGEYPLDI IVRVE ESLKDKAYRTLYPEDVSLLMHLFSRRDIKPVPFIPRLDERFETWFKKDSLWQSEDVEAVIGQDV QRIFI IQGPMAVQYSISDDESVKDILHNICNHYVEALQADSRETSIGDVHSITQKPLSAFPGLK VTTNRVQGLYKFEKVGAVPEMDVLFEHIVGLSKSWARTCLMSKSVFRDGSRLHNPIRAALQLQR XDTIEVLLTADSEIRKIRLISPTGDGGSTSKWLEIVSNDGQRVFATLAPNIPLSPEPSWFCF KVDQKPNEWTLEEDASGRAERIKALYMSLWNLGFPNKASVLGLNSQFTGEELMITTDKIRDFER VLRQTSPLQLQSWNPQGCVPIDYCWIAWSALTKPLMVSSLKCDLLDLLHSAISFHYAPSVKPL RVGDIVKTSSRILAVSVRPRGTMLTVSADIQRQGQHWTVKSDFFLGGPVLACETPFELTEEPE MWHVDSEVRRAILHSRKWLMREDRALDLLGRQLLFRLKSEKLFRPDGQLALLQVTGSVFSYSP DGSTTAFGRVYFESESCTGNVVMDFLHRYGAPRAQLLELQHPGWTGTSTVAVRGPRRSQSYARV SLDHNPIHVCPAFARYAGLSGPIVHGMETSAMMRRIAEWAIGDADRSRFRSWHITLQAPVHPND PLRVELQHKAMEDGEMVLKVQAFNERTEERVAEADAHVEQETTAYVFCGQGSQRQGMGMDLYVN CPEAKALWARADKHLWEKYGFSILHIVQNNPPALTVHFGSQRGRRIRANYLRMMGQPPIDGRHP PILKGLTRNSTSYTFSYSQGLLMSTQFAQPALALMEMAQFEWLKAQGWQKGARFAGHSLGEYA ALGACASFLSFEDLISLIFYRGLKMQNALPRDANGHTDYGMLAADPSRIGKGFEEASLKCLVHI IQQETGWFVEWNYNINSQQYVCAGHFRALWMLGKICDDLSCHPQPETVEGQELRAMVWKHVPT VEQVPREDRMERGRATIPLPGIDIPYHSTMLRGEIEPYREYLSERIKVGDVKPCELVGRWIPNV VGQPFSVDKSYVQLVHGITGSPRLHSLLQQMA

[0071] Hexanoyl-CoA can be a co-substrate with malonyl-CoA for the production of olivetolic acid, which can be a precursor for cannabinoid synthesis. As illustrated in FIGS. 1 and 2, hexanoyl-CoA can be produced by a hexanoate synthase enzyme from hexanoic acid.

Accordingly, disclosed herein are genetically-engineered microorganisms that comprise one or more genetic modifications that increase the production of hexanoyl-CoA relative to an unmodified microorganism of the same species. The one or more genetic modifications can include modifications that increase expression or activity of an HS. Methods of making such genetically-engineered microorganisms are also disclosed.

[0072] The present disclosure includes methods and compositions for increasing the expression of a hexanoate synthase in a genetically-modified microorganism relative to an unmodified organism of the same species. Such methods can include providing one or more extra copies of an endogenous HS gene, putting an endogenous HS gene under the control of a stronger promoter, mutating an endogenous HS gene to encode a higher activity enzyme, introducing an exogenous HS gene, or any combination thereof. An exogenous HS can be from a Cannabis species (e.g., a Cannabis sativa species). Exemplary HS polynucleotide and polypeptide sequences are shown in TABLE 4. A genetic modification that increases the expression of an

HS can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9. A genetic modification that increases the expression of an HS can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism.

TABLE 4

Exemplary HS sequences

SEQ ID NO: 9 | Cannabis sativa acyl-activating enzyme polynucleotide

ATGGGAAGGAAGAGCATAAGTGAGGTAGGAGTGGAGGACCTGGTTCAGGCTGGTCTAACCACTG AGGAAGCCACTGGCTTCCAAAGGGTCCTTAAAGATTCACTCAGCTGCACCAAAGGGTCCGACCC AAGTGAGGTCTGGAGGCACCTGGTGGCTCGGAGAGTGCTCAAACCTTGGCACCCACATGGGCTA CATCAGCTGGTTTACTACTCTGTTTATGCTCATTGGGATGTCTCCTCCAAAGGCCCTCCACCTT ATTGGTTTCCTTCTTTATATGAGTCTAAACATACAAACATGGGAGGCATCATGGAGAAGCATGG TTCAAGCCTTCTTGGCCCTTTATATAAGGATCCTATAACAAGTTATAGCCTCTTCCAGAAGTTC TCTGCTCAGCACCCTGAGGCTTATTGGTCTATTGTTCTGAAAGAGCTTTCAGTTTCATTCCAAG AAGAACCAAAATGCATTCTAGACAGATCTGATCTTAAATCGAAGCATGGCGGATCATGGCTTCC TGGCTCAGTTTTGAACATTGCTGAATGTTGTTTGCTGCCTACTGCATATCCAAGAAAAGATGAT GATAGTTTGGCTATTGTATGGAGAGATGAAGGTTGTGATGATTCTGGTATAAACATAATTACAC TAAAGCAACTCCGGGAGCAAGTAATCTCGGTTGCCAAGGCGCTTGATGCCATGTTTTCCAAGGG CGATGCAATTGCAATAGACATGCCAATGACAGCTAATGCAGTTATAATATACTTAGCAATTATA TTATCAGGTCTTGTTGTTGTATCAATAGCTGACAGCTTTGCTCCAAAGGAAATTTCAATTCGAT TGCGTGTCTCACAAGCCAAGGCTATCTTTACCCAGGATTTCATACTAAGAGGCAGTCGAAAGTT TCCTCTATACAGTCGAGTTGTGGAAGCTGCACCAGATAAAGTTATTGTCCTCCCTGCAATTGGG AGCAATGTAGGCATTCAGCTAAGAGAACAGGATATGTCATGGGGAGACTTCCTCTCCAGTGTTG GCACTCGTTCAAGAAATTACTCGCCATGCTATCAACCAGTTGACACTTTGATCAATATACTATT TTCATCTGGAACAACTGGAGAACCAAAAGCTATTCCATGGACGCAACTTTCTCCCATTAGGTGT GCAGCGGAGTCATGGGCTCATATGGATATGCAAGTTGGAGATGTTTTCTGTTGGCCTACAAATT TAGGATGGGTGATGGGTCCAATTCTAATTTTCTCAAGCTTTTTGTCTGGTGCAACACTTGCGCT CTATCATGGATCTCCTCTAGGCTATGGCTTTGGCAAATTTGTTCAGGATGCAGGTGTGACTAAA TTAGGTACAGTGCCAAGCCTGGTGAAAGCTTGGAAGAATACGCAGTGTATGAATGGCCTAGATT GGACAAAAATAAAGTGCTTTGCTTCCACAGGAGAAACATCTAATGTCGATGATGACCTTTGGCT ATCTTCGCGAGCATATTACAAGCCCGTTATTGAATGTTGTGGAGGGACAGAACTTTCATCATCT TACATACAAGGAAGTCTACTGCAACCTCAAGCTTTTGGTGCATTCAGCACAACATCAATGACAA CAAGCCTTGTCATACTTGATGAACATGGAAATCCTTTTCCAGATGATCAAGCTTGTATAGGTGA GGTGGGGTTATTCCCTCTATATCTAGGTGCGACTGATAGGTTGCTTAACGCTGATCATGAAGAA GTTTACTTTAAGGGAATGCCATTATACAAAGGAATGCGCCTCAGGAGACATGGAGATATTATCA AGAGAACTGTTGGAGGCTATTTCATTGTTCAGGGCAGAGCTGATGACACCATGAACCTTGGGGG CATTAAGACAAGTTCTGTTGAAATTGAGCGTGTATGTGACCGAGCTGATGAAAGCATTGTAGAG ACAGCGGCAGTTAGCGTGTCTCCAGTTGATGGTGGTCCAGAACAGCTGGTTATGTTTGTGGTAT TAAAGAATGGATATAACTCTGAAGCTGAAAATCTTAGGACTAAATTCTCAAAAGCCATTCAAAG TAATCTTAATCCATTATTCAAGGTTAGATTTGTGAAGATTGTTCCAGAGTTTCCTCGAACAGCA TCGAACAAGTTACTAAGGAGAGTATTGAGGGATCAAATAAAGCATGAATTGTCGGCTCATAGTA GAATTTAA

SEQ ID NO: 10 | Cannabis sativa acyl-activating enzyme polypeptide

MGRKS ISEVGVEDLVQAGLTTEEATGFQRVLKDSLSCTKGSDPSEVWRHLVARRVLKPWHPHGL HQLVYYSVYAHWDVSSKGPPPYWFPSLYESKHTNMGGIMEKHGSSLLGPLYKDPITSYSLFQKF SAQHPEAYWS IVLKELSVSFQEEPKCILDRSDLKSKHGGSWLPGSVLNIAECCLLPTAYPRKDD DSLAIVWRDEGCDDSGINI ITLKQLREQVISVAKALDAMFSKGDAIAIDMPMTANAVI IYLAI I LSGLWVSIADSFAPKEISIRLRVSQAKAIFTQDFILRGSRKFPLYSRWEAAPDKVIVLPAIG SNVGIQLREQDMSWGDFLSSVGTRSRNYSPCYQPVDTLINILFSSGTTGEPKAIPWTQLSPIRC AAESWAHMDMQVGDVFCWPTNLGWVMGPILIFSSFLSGATLALYHGSPLGYGFGKFVQDAGVTK LGTVPSLVKAWKNTQCMNGLDWTKIKCFASTGETSNVDDDLWLSSRAYYKPVIECCGGTELSSS YIQGSLLQPQAFGAFSTTSMTTSLVILDEHGNPFPDDQACIGEVGLFPLYLGATDRLLNADHEE VYFKGMPLYKGMRLRRHGDI IKRTVGGYFIVQGRADDTMNLGGIKTSSVEIERVCDRADESIVE TAAVSVSPVDGGPEQL\/MFWLKNGYNSEAENLRTKFSKAIQSNLNPLFKVRFVKIVPEFPRTA SNKLLRRVLRDQIKHELSAHSRI

[0073] Olivetolic acid can form the polyketide nucleus of cannabinoids and cannabinoid precursors. Fatty acids and polyketides are structurally dissimilar molecules that are synthesized by the evolutionarily-related enzymes, FAS and polyketide synthase (PKS), respectively. Both types of enzymes can facilitate the reiterative condensation of simple carboxylic acids using acetyl-CoA as the starter unit and malonyl-CoA as the extender unit.

[0074] As illustrated in FIGS. 1 and 2, olivetolic acid can be synthesized from hexanoyl-CoA and malonyl-CoA, for example by an aldol condensation reaction catalyzed by a polyketide synthase (PKS) and an olivetolic acid cyclase (OAC). Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of PKS, OAC, or both. The genetically-modified

microorganisms can produce increased levels of olivetolic acid in comparison to microorganisms of the same species without the genetic modifications.

[0075] The present disclosure includes methods and compositions for increasing the expression of a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or both in a genetically- engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous PKS and/or OAC gene, putting an endogenous PKS and/or OAC gene under the control of a stronger promoter, mutating an endogenous PKS and/or OAC gene to encode a higher activity enzyme, introducing an exogenous PKS and/or OAC gene, or any combination thereof. Exogenous PKC and/or OAC genes can be from a Cannabis species (e.g., from a Cannabis sativa species). Exemplary PKS and OAC polynucleotide and polypeptide sequences are shown in TABLE 5. A genetic modification that increases the expression of a PKS can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. A genetic modification that increases the expression of a PKS can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to SEQ ID NO: 12. A genetic modification that increases the expression of an OAC can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) identical to SEQ ID NO: 13. A genetic modification that increases the expression of an OAC can comprise a polynucleotide encoding a polypeptide at least 80%>, at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. The polynucleotide(s) can be integrated into the genome of a genetically- modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide(s) can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-modified microorganism can have increased production of olivetolic acid relative to a microorganism of the same species without the genetic modifications that increase the expression of the PKS, the OAC, or both.

TABLE 5

Exemplary PKS and OAC sequences

SEQ ID NO: 11 | Cannabis sativa PKS polynucleotide

ATGAATCATCTTCGTGCTGAGGGTCCGGCCTCCGTTCTCGCCATTGGCACCGCCAATCCGGAGA AC AT T T T AAT AC AAGAT GAG T T T C C C GAC TACTACTTTCGGGT C AC CAAAAG T GAAC AC AT GAC T C AAC T C AAAGAAAAG T T T C GAAAAAT AT G T GAC AAAAG TAT GAT AAG GAAAC G T AAC T G T T T C T T AAAT GAAGAAC AC C T AAAG C AAAAC C C AAGAT T G G T G GAG C AC GAGAT G C AAAC T C T G GAT G CACGTCAAGACATGTTGGTAGTTGAGGTTCCAAAACTTGGGAAGGATGCTTGTGCAAAGGCCAT CAAAGAAT G G G G T C AAC C C AAG T C T AAAAT C AC T CAT T T AAT C T T C AC TAG C G CAT C AAC C AC T GACATGCCCGGTGCAGACTACCATTGCGCTAAGCTTCTCGGACTCAGTCCCTCAGTGAAGCGTG TGATGATGTATCAACTAGGCTGTTATGGTGGTGGAACAGTTCTACGCATTGCCAAGGACATAGC AGAGAATAACAAAGGCGCACGAGTTCTCGCCGTGTGTTGTGACATAATGGCTTGCTTGTTTCGT GGGCCTTCAGATTCTGACCTCGAATTACTAGTGGGACAAGCTATCTTTGGTGATGGGGCTGCTG CTGTCATTGTTGGAGCCGAACCCGATGAGTCAGTTGGCGAAAGGCCGATATTTGAGTTAGTGTC AAC T G G G C AGAC AAT C T T AC C AAAC T C G GAAG GAAC TATTGGGG GAC AT AT AAG G GAAG C AG GA C T GAT AT T T GAT T T AC AT AAAGAT GTGCCTATGTT GAT C T C T AAT AAT AT T GAGAAAT G T T T GA T T GAG G CAT T T AC TCCTATTGG GAT TAG T GAT T G GAAC T C CAT AT T T T G GAT T AC AC AC C C AG G T G G GAAAG CTATTTTG GAC AAAG T G GAG GAGAAG T T G GAT C T GAAGAAG GAGAAG T T T G T G GAT TCACGTCATGTGCTGAGTGAGCATGGGAATATGTCTAGCTCAACTGTCTTGTTTGTTATGGATG AGTTGAGGAAGAGGTCGTTGGAGGAAGGGAAGTCTACCACTGGAGATGGATTTGAGTGGGGTGT TCTTTTTGGGTTTGGACCAGGTTTGACTGTCGAAAGAGTGGTCGTGCGTAGTGTTCCCATCAAA TATTAA

SEQ ID NO: 12 | Cannabis sativa PKS polypeptide

MNHLRAEGPASVLAIGTANPENILIQDEFPDYYFRVTKSEHMTQLKEKFRKICDKSMIRKRNCF LNEEHLKQNPRLVEHEMQTLDARQDMLWEVPKLGKDACAKAIKEWGQPKSKITHLIFTSASTT DMPGADYHCAKLLGLSPSVKR\/MMYQLGCYGGGTVLRIAKDIAENNKGARVLAVCCDIMACLFR GPSDSDLELLVGQAI FGDGAAAVIVGAEPDESVGERPI FELVSTGQTILPNSEGTIGGHIREAG LI FDLHKDVPMLISNNIEKCLIEAFTPIGISDWNS I FWITHPGGKAILDKVEEKLDLKKEKFVD SRHVLSEHGmSSSTVLFVMDELRKRSLEEGKSTTGDGFEWGVLFGFGPGLTVERWVRSVPIK Y

SEQ ID NO: 13 | Cannabis sativa OAC polynucleotide

AT G G C AG T GAAG CAT T T GAT T G T AT T GAAG T T C AAAGAT GAAAT C AC AGAAG C C CAAAAG GAAG AATTTTTCAAGACGTATGTGAATCTTGTGAATATCATCCCAGCCATGAAAGATGTATACTGGGG TAAAGATGTGACTCAAAAGAATAAGGAAGAAGGGTACACTCACATAGTTGAGGTAACATTTGAG AGTGTGGAGACTATTCAGGACTACATTATTCATCCTGCCCATGTTGGATTTGGAGATGTCTATC GTTCTTTCTGGGAAAAACTTCTCATTTTTGACTACACACCACGAAAGTAG

SEQ ID NO: 14 | Cannabis sativa OAC polypeptide

MAVKHLIVLKFKDEITEAQKEEFFKTYVNLVNI IPAMKDVYWGKDVTQKNKEEGYTHIVEVTFE SVETIQDYI IHPAHVGFGDVYRSFWEKLLI FDYTPRK

[0076] As illustrated in FIGS. 1 and 2, olivetolic acid and geranyldiphosphate are co-substrates in the synthesis of cannabigerolic acid (CBGA). This reaction can be mediated by a

geranylpyrophosphate olivetolate geranyltransferase (GOGT) enzyme. Although a single enzyme can facilitate olivetolic acid and geranyldiphophate conversion to cannabigerolic acid, increased expression of one or more enzymes involved in the synthesis of geranyldiphosphate can increase the forward flux towards the biosynthesis of cannabinoids and cannabinoid precursors. As illustrated in FIG. 2, a HMG-CoA Reductase 1 (HMGR1) enzyme can catalyze the conversion of HMG-CoA to mevalonate, which can be a substrate for the production of isopentenyl pyrophosphate (IPP). IPP can be interconverted into dimethylallyl pyrophosphate (DMAPP) by an isopentenyl-diphosphate delta isomerase 1 (TDI1) enzyme. IPP and DMAPP are co-substrates in the synthesis of geranyldiphosphate, which can be produced through the actions of a geranyl pyrophosphate synthase (GPPS). Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of a HMG-CoA Reductase 1 (HMGRl), an isopentenyl-diphosphate delta isomerase 1 (IDI1), a geranyl pyrophosphate synthase (GPPS), a geranylpyrophosphate olivetolate geranyltransferase (GOGT), or a combination thereof. The genetically-modified microorganisms can produce increased levels of cannabigerolic acid in comparison to microorganisms of the same species without the genetic modifications.

[0077] HMG-CoA reductase is an endoplasmic reticulum membrane protein that can catalyze the synethsis of mevalonic acid (mevalonate), which can be a key intermediate in sterol and isoprenoid biosynthesis. The reduction of HMG-CoA to mevalonic acid by HMG-CoA reductase (HMGR) can be the rate-limiting step of the mevalonate pathway, and thus, can act as a metabolic regulator of isoprenoid biosynthesis. Accordingly, disclosed herein are genetically- modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of HMGR. The HMGR can be a truncated version of HMGR. The genetically-modified microorganisms can produce increased levels of mevalonate in comparison to microorganisms of the same species without the genetic modifications.

[0078] In some yeast, there are two isozymes that encode for HMG-CoA reductase: HMGl and HMG2. The HMG-CoA reductase protein can consist of two domains: a sterol-sensing polytopic membrane domain and a cytosolic catalytic domain. An N-terminal transmembrane domain can serve as the regulatory domain, for example, by inhibiting activity of the catalytic domain when sterol concentrations in the cell are high. Accordingly, a truncated version of HMGRl (tHMGRl) can have increased activity because such tHMGRl enzymes may not be susceptible to feedback inhibition. Polynucleotide and polypeptide sequences for a truncated HMG-CoA reductase gene (tHGMl) can be found in TABLE 6 (truncated from full-length HGM1 from GenBank:

M22002.1).

[0079] The present disclosure includes methods and compositions for increasing the expression of a HMG-CoA Reductase 1 (HMGRl) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous HMGRl gene, putting an endogenous HMGRl gene under the control of a stronger promoter, mutating an endogenous HMGRl gene to encode a higher activity enzyme, introducing an exogenous HMGRl gene, or any combination thereof. The HMGRl can be a truncated version of HMGRl lacking a regulatory transmembrane domain. Exemplary truncated HMGRl polynucleotide and polypeptide sequences are shown in TABLE 6. A genetic modification that increases the expression of an tHMGRl can comprise a

polynucleotide comprising an open reading frame at 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15. A genetic modification that increases the expression of an tHMGRl can comprise a

polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of mevalonate relative to a microorganism of the same species without the genetic modifications that increase the expression of the HMGRl .

TABLE 6

Exemplary truncated HGM1 sequences

SEQ ID NO: 15 | Truncated Saccharomyces cerevisiae HGM1 polynucleotide

AT GAC C AAT AAAAC AG TCAT T TCTGGATC GAAAG T C AAAG T T TATCATCTGCG CAAT C GAG C T CAT C AG GAC C T T CAT CAT C TAG T GAG GAAGAT GAT T C C C G C GAT AT T GAAAG C T T G GAT AGAA AATACGTCCT T T AGAAGAAT TAGAAG CAT TAT TAAG T AG T G GAAAT AC AAAC AT T GAAGAAC AAAGAGGTCGCTGCCT TGGT TAT TCACGGTAAGT TACCT T TGTACGCT T TGGAGAAAAAAT TAG GTGATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAATTTTGGCAGAAGCTCCTGT ATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACCGCGTATTTGGCGCTTGTTGT GAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGTGTTATAGGCCCCTTGGTTATCGATGGTA CATCTTATCATATACCAATGGCAACTACAGAGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTG TAAGGCAATCAATGCTGGCGGTGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGC CCAGTAGTCCGTTTCCCAACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGACTCAGAAG AG G GAC AAAAC G C AAT TAAAAAAGC T T T TAAC T C TACAT CAAGAT T TGCACGTC T GCAACATAT T CAAAC T T G T C TAG C AG GAGAT T T AC T C T T CAT GAGAT T T AGAACAAC T AC T GG T GAC GCAAT G GG T AT GAAT AT GAT T T C T AAAG G T G T C GAAT AC T CAT T AAAG C AAAT G G T AGAAGAG T AT G G C T GGGAAGATATGGAGGTTGTCTCCGTTTCTGGTAACTACTGTACCGACAAAAAACCAGCTGCCAT CAACTGGATCGAAGGTCGTGGTAAGAGTGTCGTCGCAGAAGCTACTATTCCTGGTGATGTTGTC AGAAAAGTGTTAAAAAGTGATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTG GATCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAATTTAGTGACAGCTGT TTTCTTGGCATTAG GAC AAGAT C C T G C AC AAAAT G T T GAAAG T T C C AAC T G TAT AAC AT T GAT G AAAGAAG T G GAC G G T GAT T T GAGAAT TTCCGTATCCATGCCATCCATC GAAG T AG G T AC C AT C G GTGGTGGTACTGTTCTAGAACCACAAGGTGCCATGTTGGACTTATTAGGTGTAAGAGGCCCGCA TGCTACCGCTCCTGGTACCAACGCACGTCAATTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCA GGTGAATTATCCTTATGTGCTGCCCTAGCAGCCGGCCATTTGGTTCAAAGTCATATGACCCACA AC AG GAAAC C T G C T GAAC C AAC AAAAC C TAAC AAT T T G GAC G C C AC T GAT AT AAAT C G T T T GAA AGATGGGTCCGTCACCTGCATTAAATCCTAA

SEQ ID NO: 16 | Truncated Saccharomyces cerevisiae HGM1 polypeptide

MTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKN KEVAALVIHGKLPLYALEKKLGDTTRAVAVRRKALS ILAEAPVLASDRLPYKNYDYDRVFGACC ENVIGYMPLPVGVIGPLVIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTVLTKDGMTRG PWRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTCLAGDLLFMRFRTTTGDAM GMNMISKGVEYSLKQMVEEYGWEDMEWSVSGNYCTDKKPAAINWIEGRGKSWAEATIPGDW RKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVESSNCITLM KEVDGDLRISVSMPS IEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLA GELS LCAALAAGHLVQSHMTHNRKPAEPTKPNNLDATDINRLKDGSVTC IKS

[0080] As illustrated in FIG.2, isopentenyl-diphosphate delta isomerase 1 (IDI1) can catalyze the interconversion of isopentenyl pyrophosphate (IPP) to its isomer dimethylallyl

pyrophosphate (DMAPP). IPP and DMAPP can be five-carbon building blocks used in the successive reactions for isoprenoid biosynthesis, for example in the synthesis of

geranyldiphosphate. Increased expression of IDI1 can significantly enhance monoterpene titers by increasing the IPP and DMAPP pool. Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of IDI1. The genetically-modified microorganisms can produce increased levels of DMAPP, IPP, or both in comparison to microorganisms of the same species without the genetic modifications.

[0081] The present disclosure includes methods and compositions for increasing the expression of an isopentenyl-diphosphate delta isomerase 1 (IDI1) in a genetically-engineered

microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous IDI1 gene, putting an endogenous IDI1 gene under the control of a stronger promoter, mutating an endogenous IDI1 gene to encode a higher activity enzyme, introducing an exogenous IDI1 gene, or any combination thereof. Exemplary IDI1 polynucleotide and polypeptide sequences are shown in TABLE 7. A genetic modification that increases the expression of an IDI1 can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 17. A genetic modification that increases the expression of an IDI1 can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to SEQ ID NO: 18. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon- optimized for expression of an encoded protein in a particular microorganism. The genetically- engineered microorganism can have increased production of IPP, DMAPP, or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the IDIl.

TABLE 7

Exemplary IDIl sequences

SEQ ID NO: 17 | Variant 1 Homo sapiens (human) IDIl polynucleotide

ATGTGGCGTGGACTGGCGCTGGCGCGAGCGATTGGCTGCGCGGCCCGGGGGCGGGGCCAGTGGG CGGTGCGCGCCGCAGACTGTGCTCAAAGCGGGCGCCATCCGGGACCGGCGGTTGTCTGTGGCCG GAG G C T GAT C AG T G T T C T AGAAC AGAT C AGAC AT TTTGTAATGATGCCT GAAAT AAAC AC T AAC CAC C T C GACAAG C AAC AG G T T C AAC T C C T G G C AGAGAT GTGTATCCTTATTGAT GAAAAT GAC A AT AAAAT T G GAG C T GAGAC C AAGAAGAAT T G T CAC C T GAAC GAGAAC AT T GAGAAAG GAT TAT T GC AT C GAG CTTTTAGTGTCTTCTTATT C AAC AC C GAAAAT AAG C T T C T G C T AC AG CAAAGAT C A GATGCTAAGATTACCTTTCCAGGTTGTTTTACGAATACGTGTTGTAGTCATCCATTAAGCAATC C AG C C GAG C T T GAG G AAAG T GAC G C C C T T G GAG T GAG G C GAG C AG CAC AG AG AC G G C T G AAAG C T GAG C T AG GAAT T C C C T T G GAAGAG G T T C C T C C AGAAGAAAT T AAT TAT T T AAC AC GAAT T CAC TACAAAGCTCAGTCTGATGGTATCTGGGGTGAACATGAAATTGATTACATTTTGTTGGTGAGGA AGAAT G T AAC T T T GAAT C C AGAT C C C AAT GAGAT T AAAAG CTATTGTTATGTGT C AAAG GAAGA AC T AAAAGAAC T T C T GAAAAAAG C AG C C AG T G G T GAAAT T AAGAT AAC G C CAT G G T T T AAAAT T ATTGCAGCGACTTTTCTCTTTAAATGGTGGGATAACTTAAATCATTTGAATCAGTTTGTTGACC AT GAGAAAAT AT AC AGAAT G T GA

SEQ ID NO: 18 | Variant 1 Homo sapiens (human) IDIl polypeptide

MWRGLALARAIGCAARGRGQWAVRAADCAQSGRHPGPAWCGRRLISVLEQIRHF\/MMPEINTN HLDKQQVQLLAEMCILIDENDNKIGAETKKNCHLNENIEKGLLHRAFSVFLFNTENKLLLQQRS DAKITFPGCFTNTCCSHPLSNPAELEESDALGVRRAAQRRLKAELGIPLEEVPPEEINYLTRIH YKAQSDGIWGEHEIDYILLVRKNVTLNPDPNEIKSYCYVSKEELKELLKKAASGEIKITPWFKI IAATFLFKWWDNLNHLNQFVDHEKI YRM [0082] As illustrated in FIG. 2, geranyl pyrophosphate synthase (GPPS) can catalyze the synthesis of geranyldiphosphate (GPP) from isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). Farnesyl pyrophosphate synthase (FPPS) can also catalyze the synthesis of geranyldiphosphate. The FPPS can be mutated to increase the selectivity of the enzyme for GPP synthesis. For example, mutations in the binding pocket of the enzyme can increase the selectivity of an FPPS for GPP production over farnesyl pyrophosphate. Geranyldiphosphate can be a co-substrate, with olivetolic acid, in the synthesis of cannabigerolic acid. Increased expression of GPPS, FPPS, and/or mutated FPPS can significantly enhance cannabigerolic acid production by increasing the geranyldiphosphate pool. Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of GPPS, FPPS, and/or mutated FPPS. The genetically-modified microorganisms can produce increased levels of geranyldiphosphate in comparison to microorganisms of the same species without the genetic modifications.

[0083] The present disclosure includes methods and compositions for increasing the expression of a geranyl pyrophosphate synthase (GPPS), Farnesyl pyrophosphate synthase (FPPS), and/or mutated Farnesyl pyrophosphate synthase (mFPPS) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous GPPS, FPPS, and/or mFPPS, putting an endogenous GPPS, FPPS, and/or mFPPS under the control of a stronger promoter, mutating an endogenous GPPS, FPPS, and/or mFPPS to encode a higher activity enzyme, introducing an exogenous GPPS, FPPS, and/or mFPPS, or any combination thereof. Exemplary FPPS, mFPPS, and GPPS polynucleotide and polypeptide sequences are shown in TABLE 8. A genetic modification that increases the expression of an GPPS, FPPS, and/or mFPPS can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19, 21, 25, or 27. A genetic modification that increases the expression of an GPPS can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20, 22, 23, 24, 26, or 28. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of geranyldiphosphate relative to a microorganism of the same species without the genetic modifications that increase the expression of the GPPS, FPPS, and/or mFPPS.

TABLE 8

Exemplary FPDS and GPPS sequences

SEQ ID NO: 19 Gallus gallus FPPS polynucleotide isoform X2

ATGGCCTGGGTTGAAAAGGACCACAATGGTCACCAGTTCCAACCCGCTGCTGTGTGTAGGGTCG CCAAGCACCAGCCCAGGCTGCCCAGAGAACCCATAGGGCGCCACAGGGACCCAAAGGGAGCCAT AGGGACCCATCACCATAGAGCTATAGGATGGCCTGGGTTGAAAAGGACCACAGTGGTCATCCCC TTCCAACCCCCTGCTGTGTGTAGGGTCACCAACCAGCAGCCCAGGCTGCCCAGAGAACCCATAG GGCACCATGGGGACCCGCAGGGTGCTGTTAGGACCTGTAGGGCACCATTAGGACCAATAGAATC ACTGACTCATAGGATGGTCTGGGTTGAAAAGGACCACAATGACCATCAGTTCCAACCCCCTGCT GTGTGTAGGGTCACCAACCAGCAGCCCAGGCTGCCCAGAGCCACATCCAGCCTGGCCTGGAATG CCTGCAGGGATGGGGCATCCAGCCTTCTCTTCTCCAAGCTAAACGAGCCCGGTTCCCTCCACCC TTCCTCACAGGAGAGCTGCTCCAGCCCTGTGACCACCTTAACGGACCTCCACGTCCTTCCTGTG CTGGGACCCCCAGCTCTGAACGCAGCACTGCAGAGGGGGCCTCCCAACAGCCGAGCAGAGGGGC CCAATCCCCTCCCTCTCCCCGCTGCCCCCTCCCCATCTCACGCAGCCCAGCACACCGTTGGCCC TCGGGGTTGCAGGCGCACACTGCTGCCTCACGTGCAGCTCCTCACCCCCCAGGACCCCCAGGTC CTTCCCCGCAGGGCTGCTCTCCAGGAGATCTTCCCCAGCCCCTCTCAACACCTGGGGCCGCCCC GACCCCACTGCAGCACTTTGCACTCGGCCTTATTGAACCCACAGCATCCGGAGCTGCAGCGGCC CCACGCAGCCCCCCCACCTCCCCACGCTGTGTTTGCAGGACAACTACGGCCG

SEQ ID NO: 20 Gallus gallus FPPS polypeptide isoform X2

MAWVEKDHNGHQFQPAAVCRVAKHQPRLPREPIGRHRDPKGAIGTHHHRAIGWPGLKRTTWIP FQPPAVCRVTNQQPRLPREPIGHHGDPQGAVRTCRAPLGPIESLTHRMVWVEKDHNDHQFQPPA VCRVTNQQPRLPRATSSLAWNACRDGASSLLFSKLNEPGSLHPSSQESCSSPVTTLTDLHVLPV LGPPALNAALQRGPPNSRAEGPNPLPLPAAPSPSHAAQHTVGPRGCRRTLLPHVQLLTPQDPQV LPRRAALQEI FPSPSQHLGPPRPHCSTLHSALLNPQHPELQRPHAAPPPPHAVFAGQLRP

SEQ ID NO: 21 Gallus gallus mutated FPPS polynucleotide isoform X2

ATGGCCTGGGTTGAAAAGGACCACAATGGTCACCAGTTCCAACCCGCTGCTGTGTGTAGGGTCG CCAAGCACCAGCCCAGGCTGCCCAGAGAACCCATAGGGCGCCACAGGGACCCAAAGGGAGCCAT AGGGACCCATCACCATAGAGCTATAGGATGGCCTGGGTTGAAAAGGACCACAGTGGTCATCCCC TTCCAACCCCCTGCTGTGTGTAGGGTCACCAACCAGCAGCCCAGGCTGCCCAGAGAACCCATAG GGCACCATGGGGACCCGCAGGGTGCTGTTAGGACCTGTAGGGCACCATTAGGACCAATAGAATC ACTGACTCATAGGATGGTCTGGGTTGAAAAGGACCACAATGACCATCAGTTCCAACCCCCTGCT GTGTGTAGGGTCACCTGGCAGCAGCCCAGGCTGCCCAGAGCCACATCCAGCCTGGCCTGGAATG CCTGCAGGGATGGGGCATCCAGCCTTCTCTTCTCCAAGCTAAACGAGCCCGGTTCCCTCCACCC TTCCTCACAGGAGAGCTGCTCCAGCCCTGTGACCACCTTAACGGACCTCCACGTCCTTCCTGTG CTGGGACCCCCAGCTCTGAACGCAGCACTGCAGAGGGGGCCTCCCAACAGCCGAGCAGAGGGGC CCAATCCCCTCCCTCTCCCCGCTGCCCCCTCCCCATCTCACGCAGCCCAGCACACCGTTGGCCC TCGGGGTTGCAGGCGCACACTGCTGCCTCACGTGCAGCTCCTCACCCCCCAGGACCCCCAGGTC CTTCCCCGCAGGGCTGCTCTCCAGGAGATCTTCCCCAGCCCCTCTCAACACCTGGGGCCGCCCC GACCCCACTGCAGCACTTTGCACTCGGCCTTATTGAACCCACAGCATCCGGAGCTGCAGCGGCC CCACGCAGCCCCCCCACCTCCCCACGCTGTGTTTGCAGGACAACTACGGCCG

SEQ ID NO: 22 Gallus gallus mutated FPPS polypeptide isoform X2

MAWVEKDHNGHQFQPAAVCRVAKHQPRLPREPIGRHRDPKGAIGTHHHRAIGWPGLKRTTWIP FQPPAVCRVTNQQPRLPREPIGHHGDPQGAVRTCRAPLGPIESLTHRMVWVEKDHNDHQFQPPA VCRVTWQQPRLPRATSSLAWNACRDGASSLLFSKLNEPGSLHPSSQESCSSPVTTLTDLHVLPV LGPPALNAALQRGPPNSRAEGPNPLPLPAAPSPSHAAQHTVGPRGCRRTLLPHVQLLTPQDPQV LPRRAALQEI FPSPSQHLGPPRPHCSTLHSALLNPQHPELQRPHAAPPPPHAVFAGQLRP

SEQ ID NO: 23 Gallus gallus FPPS polypeptide P08836 MHKFTGVNAKFQQPALRNLSPWVEREREEFVGFFPQIVRDLTEDGIGHPEVGDAVARLKEVLQ YNAPGGKCNRGLTWAAYRELSGPGQKDAESLRCALAVGWCIELFQAFFLVADDIMDQSLTRRG QLCWYKKEGVGLDAINDSFLLESSVYRVLKKYCRQRPYYVHLLELFLQTAYQTELGQMLDLITA PVSKVDLSHFSEERYKAIVKYKTAFYSFYLPVAAAMYMVGIDSKEEHENAKAILLEMGEYFQIQ DDYLDCFGDPALTGKVGTDIQDNKCSWLWQCLQRVTPEQRQLLEDNYGRKEPEKVAKVKELYE AVGMRAAFQQYEESSYRRLQELIEKHSNRLPKEI FLGLAQKI YKRQK

SEQ ID NO: 24 Gallus gallus mutated FPPS polypeptide P08836

MHKFTGVNAKFQQPALRNLSPWVEREREEFVGFFPQIVRDLTEDGIGHPEVGDAVARLKEVLQ YNAPGGKCNRGLTWAAYRELSGPGQKDAESLRCALAVGWCIELFQAFFLVADDIMDQSLTRRG QLCWYKKEGVGLDAIWDSFLLESSVYRVLKKYCRQRPYYVHLLELFLQTAYQTELGQMLDLITA PVSKVDLSHFSEERYKAIVKYKTAFYSFYLPVAAAMYMVGIDSKEEHENAKAILLEMGEYFQIQ DDYLDCFGDPALTGKVGTDIQDNKCSWLWQCLQRVTPEQRQLLEDNYGRKEPEKVAKVKELYE AVGMRAAFQQYEESSYRRLQELIEKHSNRLPKE I FLGLAQKI YKRQK

SEQ ID NO: 25 Solarium lycopersicum GPPS polynucleotide

ATGATATTTTCAAAGGGTTTAGCTCAGATTTCCAGAAACCGCTTCAGCAGATGCCGATGGTTAT TTTCATTACGTCCCATCC C AC AAT T AC AT C AAT C C AAT C AC AT C C AC GAT C C T C CAAAG G T T C T GGGTTGCAGAGTAATTCATTCATGGGTTTCTAATGCTCTTAGTGGTATTGGGCAACAAATTCAT CAG CAAAG C AC T G C T G TAG C AGAG GAG C AAG T G GAC C CAT TTTCCCTTGTTG C AGAT GAAT TAT CCCTTCTGACAAACAGGCTGAGATCAATGGTAGTCGCGGAGGTCCCAAAGCTGGCTTCAGCTGC T GAAT AT T T C T T C AAAC T G G GAG T AGAAG GAAAGAG G T T T C GAC C AAC AG TTTTGCTATT GAT G GCAACTGCATTGAACGTACAGATTCCTAGATCTGCTCCGCAGGTGGATGTTGATTCTTTTTCCG GG GAT T T G C G T AC AAG G CAG CAG T G TAT AG C T GAGAT C AC T GAGAT GAT C CAT G T T G C TAG C C T ACTTCATGATGATGTACTGGATGATGCTGACACAAGACGTGGGATAGGTTCTTTAAACTTTGTG ATGGGAAATAAGCTAGCTGTACTAGCCGGAGACTTTTTGCTTTCCCGAGCATGTGTGGCACTTG CCTCCTTGAAGAACACAGAGGTTGTATGTCTTCTGGCAACTGTTGTGGAACATCTTGTTACTGG AGAGAC AAT G C AAAT GAC GAC TTCTTCTGAT GAAC GTTGTAGCATG GAG TATTATATG C AGAAA AC AT AT T AC AAGAC T G CAT CAT T GAT T T C AAAT AG T T G CAAAG C AAT T G C AC T AC TTGCTGGGC ATAGTGCTGAAGTCTCCGTGCTGGCTTTTGACTACGGAAAAAATCTGGGATTGGCATTTCAATT AATAGATGATGTTCTTGATTTCACGGGCACATCTGCAACTCTTGGCAAGGGTTCATTGTCTGAT ATTCGTCATGGGATTGTAACTGCCCCGATATTGTATGCCATGGAGGAATTTCCTCAACTGCGTA CGCTGGTGGACCGAGGTTTTGATGATCCTGTCAATGTGGAGATCGCTCTGGACTACCTTGGGAA GAG C AGAG G GAT AC AGAGAAC AAGAGAAC T T G C GAGAAAG CAT G C TAG CCTTGCGT CAG C G G C A AT T GAC T C T C T T C CAGAAAG C GAT GAC GAG GAAG T T C AGAGAT C AAGAC GAG C AC T T G T AGAAC T T AC T C AC AGAG T C AT C AC AAGAAC AAAA

SEQ ID NO: 26 Solarium lycopersicum GPPS polypeptide

MI FSKGLAQISRNRFSRCRWLFSLRPIPQLHQSNHIHDPPKVLGCRVIHSWVSNALSGIGQQIH QQSTAVAEEQVDPFSLVADELSLLTNRLRSMWAEVPKLASAAEYFFKLGVEGKRFRPTVLLLM ATALNVQIPRSAPQVDVDSFSGDLRTRQQCIAEITEMIHVASLLHDDVLDDADTRRGIGSLNFV MGNKLAVLAGDFLLSRACVALASLKNTEWCLLATWEHLVTGETMQMTTSSDERCSMEYYMQK TYYKTASLISNSCKAIALLAGHSAEVSVLAFDYGKNLGLAFQLIDDVLDFTGTSATLGKGSLSD IRHGIVTAPILYAMEEFPQLRTLVDRGFDDPVNVEIALDYLGKSRGIQRTRELARKHASLASAA IDSLPESDDEEVQRSRRALVELTHRVITRTK

SEQ ID NO: 27 Catharanthus roseus GPPS polynucleotide

ATGTTGTTTTCCAGAGGATTGTATAGGATCGCAAGGACGAGTTTGAACAGAAGTCGATTGCTTT ACCCGTTACAAAGTCAGTCGCCGGAGCTGCTGCAGTCTTTTCAGTTTCGCTCTCCTATTGGTTC TTCTCAAAAGGTTTCAGGTTTCAGAGTAATCTATTCATGGGTCTCAAGTGCCCTGGCCAATGTT GGAC AG CAG G T AC AG C G C C AGAG C AAC TCTGTTGCC GAG GAG C C AC T AGAT C CAT T T T C AC T T G TTGCTGATGAATTGTCCATTCTTGCTAATAGACTGAGGTCAATGGTAGTTGCAGAGGTCCCGAA GCTTGCTTCAGCTGCCGAATATTTTTTTAAGTTAGGGGTGGAAGGAAAGAGGTTTCGACCAACA GT T T T GC TAT T GAT G G C GAC AG C TAT AGAT G C AC C AAT AT C T AGAAC AC C T C C T GATACAT CAC T T GAT AC T T TAT CCACAGAAC TACGCC TAAGGCAGCAGACGAT T GC T GAGAT CAC TAAGAT GAT CCATGTTGCTAGTCTTCTTCATGACGATGTATTAGATGATGCTGAAACAAGGCGAGGGATTGGT TCTCTAAATTTTGTGATGGGAAATAAGTTAGCAGTGTTGGCTGGTGATTTCCTGCTATCAAGAG CCTGTGTTGCACTTGCCTCTTTGAAAAACACAGAGGTCGTGTCCCTCTTGGCAACAGTTGTGGA GC AT C T T G T T AC G G G T GAAAC GAT G C AAAT GAC CAC CAC AT C T GAT C AAC G T T G TAG CAT G GAG TAC TAT AT GCAAAAGACATAC TAT AT GACGGCAT CC T T GAT C T CAAACAGT T GCAAAGCAAT T G CCCTTCTTGCTGGGCAAACATCAGAAGTTGCAATGTTGGCTTATGAGTATGGAAAAAATCTGGG ATTGGCGTTTCAGTTAATAGATGATGTTCTTGATTTCACCGGCACATCAGCTTCCCTTGGCAAG GGCTCTCTGTCTGACATTCGCCACGGAATTGTTACTGCTCCAATATTATTTGCCATAGAAGAGT TCCCTGAACTACGTGCTGTTGTTGACGAGGGATTTGAAAATCCATATAATGTAGATCTTGCTCT AC AT TAC C T T G GAAAGAG T AGAG GAAT AC AAC GAAC GAG G GAAC T G G C AAT AAAG CAT G C T AAC CTTGCCTCTGATGCAATCGACTCTCTTCCGGTGACTGATGATGAACATGTTTTAAGGTCAAGAA GAG CTCTTGTG GAAC T T AC T C AAC G C G T T AT T AC AAGAAGAAAG

SEQ ID NO: 28 Catharanthus roseus GPPS polypeptide

MLFSRGLYRIARTSLNRSRLLYPLQSQSPELLQSFQFRSPIGSSQKVSGFRVIYSWVSSALANV GQQVQRQSNSVAEEPLDPFSLVADELSILANRLRSMWAEVPKLASAAEYFFKLGVEGKRFRPT VLLLMATAIDAPISRTPPDTSLDTLSTELRLRQQTIAEITKMIHVASLLHDDVLDDAETRRGIG SLNF\/MGNKLAVLAGDFLLSRACVALASLKNTEWSLLATWEHLVTGETMQMTTTSDQRCSME YYMQKTYYMTASLISNSCKAIALLAGQTSEVAMLAYEYGKNLGLAFQLIDDVLDFTGTSASLGK GSLSDIRHGIVTAPILFAIEEFPELRAWDEGFENPYNVDLALHYLGKSRGIQRTRELAIKHAN LASDAIDSLPVTDDEHVLRSRRALVELTQRVITRRK

[0084] As illustrated in FIG.2, geranylpyrophosphate olivetolate geranyltransferase (GOGT) can catalyze the synthesis of cannabigerolic acid from olivetolic acid and geranyldiphosphate. Increased expression of GOGT can significantly enhance production of cannabigerolic acid (CBGA) and other cannabinoids that can be produced from CBGA (e.g., THCA, THC, CBDA, CBD, CBCA, CBC). Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of GOGT. The genetically-modified microorganisms can produce increased levels of CBGA (and/or other downstream cannabinoids) in comparison to microorganisms of the same species without the genetic modifications.

[0085] The present disclosure includes methods and compositions for increasing the expression of a geranylpyrophosphate olivetolate geranyltransferase (GOGT) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous GOGT, putting an endogenous GOGT under the control of a stronger promoter, mutating an endogenous GOGT to encode a higher activity enzyme, introducing an exogenous GOGT, or any combination thereof. The GOGT can be from a Cannabis species (e.g., from a Cannabis sativa species). Exemplary GOGT polynucleotide and polypeptide sequences are shown in TABLE 9. A genetic modification that increases the expression of an GOGT can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 29. A genetic modification that increases the expression of an GOGT can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 30. The polynucleotide can be integrated into the genome of a genetically-modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of CBGA (and/or other downstream cannabinoids) relative to a microorganism of the same species without the genetic modifications that increase the expression of the GOGT.

TABLE 9

Exemplary GOGT sequences

SEQ ID NO: 29 Cannabis sativa GOGT polynucleotide

ATGGGACTCTCATCAGTTTGTACCTTTTCATTTCAAACTAATTACCATACTTTATTAAATCCTC ACAATAATAATCCCAAAACCTCATTATTATGTTATCGACACCCCAAAACACCAATTAAATACTC TTACAATAATTTTCCCTCTAAACATTGCTCCACCAAGAGTTTTCATCTACAAAACAAATGCTCA GAATCATTATCAATCGCAAAAAATTCCATTAGGGCAGCTACTACAAATCAAACTGAGCCTCCAG AATCTGATAATCATTCAGTAGCAACTAAAATTTTAAACTTTGGGAAGGCATGTTGGAAACTTCA AAGACCATATACAATCATAGCATTTACTTCATGCGCTTGTGGATTGTTTGGGAAAGAGTTGTTG CATAACACAAATTTAATAAGTTGGTCTCTGATGTTCAAGGCATTCTTTTTTTTGGTGGCTATAT TATGCATTGCTTCTTTTACAACTACCATCAATCAGATTTACGATCTTCACATTGACAGAATAAA CAAGCCTGATCTACCACTAGCTTCAGGGGAAATATCAGTAAACACAGCTTGGATTATGAGCATA ATTGTGGCACTGTTTGGATTGATAATAACTATAAAAATGAAGGGTGGACCACTCTATATATTTG GCTACTGTTTTGGTATTTTTGGTGGGATTGTCTATTCTGTTCCACCATTTAGATGGAAGCAAAA TCCTTCCACTGCATTTCTTCTCAATTTCCTGGCCCATATTATTACAAATTTCACATTTTATTAT GCCAGCAGAGCAGCTCTTGGCCTACCATTTGAGTTGAGGCCTTCTTTTACTTTCCTGCTAGCAT TTATGAAATCAATGGGTTCAGCTTTGGCTTTAATCAAAGATGCTTCAGACGTTGAAGGCGACAC TAAATTTGGCATATCAACCTTGGCAAGTAAATATGGTTCCAGAAACTTGACATTATTTTGTTCT GGAATTGTTCTCCTATCCTATGTGGCTGCTATACTTGCTGGGATTATCTGGCCCCAGGCTTTCA ACAGTAACGTAATGTTACTTTCTCATGCAATCTTAGCATTTTGGTTAATCCTCCAGACTCGAGA TTTTGCGTTAACAAATTACGACCCGGAAGCAGGCAGAAGATTTTACGAGTTCATGTGGAAGCTT TATTATGCTGAATATTTAGTATATGTTTTCATATAA

SEQ ID NO: 30 Cannabis sativa GOGT polypeptide

MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPSKHCSTKSFHLQNKCS ESLS IAKNS IRAATTNQTEPPESDNHSVATKILNFGKACWKLQRPYTI IAFTSCACGLFGKELL HNTNLISWSLMFKAFFFLVAILCIASFTTTINQIYDLHIDRINKPDLPLASGEISVNTAWIMSI IVALFGLI ITIKMKGGPLYI FGYCFGI FGGIVYSVPPFRWKQNPSTAFLLNFLAHI ITNFTFYY ASRAALGLPFELRPSFTFLLAFMKSMGSALALIKDASDVEGDTKFGISTLASKYGSRNLTLFCS GIVLLSYVAAILAGI IWPQAFNSN\ LLSHAILAFWLILQTRDFALTNYDPEAGRRFYEFMWKL YYAEYLVYVFI

[0086] As illustrated in FIGS. 1 and 2, cannabigerolic acid (CBGA) can be a precursor in the production of other cannabinoids. THC synthase (THCS) can produce A⁹-tetrahydrocannabinolic acid (THCA) from CBGA, which can subsequently be used in the synthesis of Δ⁹- tetrahydrocannabinol (THC). CBD synthase (CBDS) can produce cannabidiolic acid (CBDA from CBGA, which can subsequently be used in the synthesis of cannabidiol (CBD). CBC synthase can produce cannabichromenic acid (CBCA) from CBGA, which can subsequently be used in the synthesis of cannabichromene (CBC). Accordingly, disclosed herein are genetically- modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of THCS, CBDS, CBCS, or a combination thereof. The genetically- modified microorganisms can produce increased levels of THCA, THC, CBDA, CBD, CBCA, CBC, or a combination thereof in comparison to microorganisms of the same species without the genetic modifications.

[0087] The present disclosure includes methods and compositions for increasing the expression of a THC synthase (THCS) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous THCS, putting an endogenous THCS under the control of a stronger promoter, mutating an endogenous THCS to encode a higher activity enzyme, introducing an exogenous THCS, or any combination thereof. The THCS can be from a Cannabis species (e.g., from a Cannabis sativa species). Exemplary THCS polynucleotide and polypeptide sequences are shown in TABLE 10 and GenBank: AB057805.1. A genetic modification that increases the expression of an THCS can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) identical to SEQ ID NO: 31. A genetic modification that increases the expression of an THCS can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 32. The polynucleotide can be integrated into the genome of a genetically- modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of THCA, THC, or a combination thereof relative to a microorganism of the same species without the genetic modifications that increase the expression of the THCS.

[0088] The present disclosure includes methods and compositions for increasing the expression of a CBD synthase (CBDS) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous CBDS, putting an endogenous CBDS under the control of a stronger promoter, mutating an endogenous CBDS to encode a higher activity enzyme, introducing an exogenous CBDS, or any combination thereof. The CBDS can be from a Cannabis species (e.g., from a Cannabis sativa species). Exemplary CBDS polynucleotide and polypeptide sequences are shown in TABLE 10 and GenBank: AB292682.1. A genetic modification that increases the expression of an CBDS can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) identical to SEQ ID NO: 33 or 35. A genetic modification that increases the expression of an CBDS can comprise a polynucleotide encoding a polypeptide at least 80%>, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 34 or 36. The polynucleotide can be integrated into the genome of a genetically -modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of CBDA, CBD, or a combination thereof relative to a

microorganism of the same species without the genetic modifications that increase the expression of the CBDS.

[0089] The present disclosure includes methods and compositions for increasing the expression of a CBC synthase (CBCS) in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of an endogenous CBCS, putting an endogenous CBCS under the control of a stronger promoter, mutating an endogenous CBCS to encode a higher activity enzyme, introducing an exogenous CBCS, or any combination thereof. The CBCS can be from a Cannabis species (e.g., from a Cannabis sativa species).

TABLE 10

Exemplary THCS and CBDS sequences

SEQ ID NO: 31 Cannabis sativa THCA synthase polynucleotide

ATGAAT TGCTCAGCAT T T TCCT T T TGGT T TGT T TGCAAAATAATAT T T T TCT T TCTCTCAT TCC AT AT C C AAAT T T C AAT AG C T AAT C C T C GAGAAAAC T T C C T T AAAT G C T T C T C AAAAC AT AT T C C CAAC AAT G T AG C AAAT C C AAAAC T C G TAT AC AC T C AAC AC GAC C AAT TGTATATGTCTATCCTG AAT T C GAC AAT AC AAAAT C T T AGAT T C AT C T C T GAT AC AAC C C C AAAAC C AC TCGT TAT TGTCA CTCCT TCAAATAACTCCCATATCCAAGCAACTAT T T TATGCTCTAAGAAAGT TGGCT TGCAGAT TCGAACTCGAAGCGGTGGCCATGATGCTGAGGGTATGTCCTACATATCTCAAGTCCCAT T TGT T G TAG T AGAC T T GAGAAAC AT G CAT T C GAT C AAAAT AGAT G T T CAT AG C C AAAC TGCGTGGGT TG AAG C C G GAG C T AC C C T T G GAGAAG T T TAT TAT TG GAT C AAT GAGAAGAAT GAGAAT C T TAG T T T TCCTGGTGGGTAT TGCCCTACTGT TGGCGTAGGTGGACACT T TAGTGGAGGAGGCTATGGAGCA TTGATGCGAAAT TATGGCCT TGCGGCTGATAATAT TAT TGATGCACACT TAGTCAATGT TGATG GAAAAGT TCTAGATCGAAAATCCATGGGAGAAGATCTGT T T TGGGCTATACGTGGTGGTGGAGG AGAAAACT T TGGAATCAT TGCAGCATGGAAAATCAAACTGGT TGCTGTCCCATCAAAGTCTACT AT AT T CAGT GT TAAAAAGAACAT GGAGATACAT GGGC T T GT CAAGT TAT T TAACAAAT GGCAAA ATATTGCTTACAAGTATGACAAAGATTTAGTACTCATGACTCACTTCATAACAAAGAATATTAC AGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTCTTCAATTTTTCATGGTGGA GTGGATAGTCTAGTCGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTATTAAAAAAACTGATT GCAAAGAATTTAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAATTTTAACACTGC TAATTTTAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACGGCTTTCTCAATTAAG TTAGACTATGTTAAGAAACCAATTCCAGAAACTGCAATGGTCAAAATTTTGGAAAAATTATATG AAGAAGATGTAGGAGCTGGGATGTATGTGTTGTACCCTTACGGTGGTATAATGGAGGAGATTTC AGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATAATGTATGAACTTTGGTACACTGCTTCC TGGGAGAAGCAAGAAGATAATGAAAAGCATATAAACTGGGTTCGAAGTGTTTATAATTTTACGA CTCCTTATGTGTCCCAAAATCCAAGATTGGCGTATCTCAATTATAGGGACCTTGATTTAGGAAA AACTAATCATGCGAGTCCTAATAATTACACACAAGCACGTATTTGGGGTGAAAAGTATTTTGGT AAAAATTTTAACAGGTTAGTTAAGGTGAAAACTAAAGTTGATCCCAATAATTTTTTTAGAAACG AACAAAGTATCCCACCTCTTCCACCGCATCATCATTAA

SEQ ID NO: 32 Cannabis sativa THCA synthase polypeptide

MNCSAFSFWFVCKI I FFFLSFHIQIS IANPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMS IL NSTIQNLRFISDTTPKPLVIVTPSNNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFV WDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGA LMRNYGLAADNI IDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGI IAAWKIKLVAVPSKST IFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLVLMTHFITKNITDNHGKNKTTVHGYFSS I FHGG VDSLVDLMNKSFPELGIKKTDCKEFSWIDTTIFYSGWNFNTANFKKEILLDRSAGKKTAFSIK LDYVKKPIPETAMVKILEKLYEEDVGAGMYVLYPYGGIMEEISESAIPFPHRAGIMYELWYTAS WEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLGKTNHASPNNYTQARIWGEKYFG KNFNRLVKVKTKVDPNNFFRNEQS IPPLPPHHH

SEQ ID NO: 33 Cannabis sativa CBDA synthase polynucleotide

ATGAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAACAAATC TAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAA TCTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCT CATATCCAAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTG GTCATGATTCTGAGGGCATGTCCTACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAA CATGCGTTCAATCAAAATAGATGTTCATAGCCAAACTGCATGGGTTGAAGCCGGAGCTACCCTT GGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTTGGCGGCTGGGTATTGCC CTACTGTTTGCGCAGGTGGACACTTTGGTGGAGGAGGCTATGGACCATTGATGAGAAACTATGG CCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATGGAAAAGTGCTAGATCGA AAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGCAGAAAGCTTCGGAATCA TTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTTTAGTGTTAAAAAGAT CATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTACAAGTATGAC AAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGAATA AGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTT GATGAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATT GATACTATCATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTT TGCTTGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACC AATTCCAGAATCTGTATTTGTCCAAATTTTGGAAAAATTATATGAAGAAGATATAGGAGCTGGG ATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGAGATTTCAGAATCAGCAATTCCATTCC CTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGTTGGGAGAAGCAAGAAGATAA CGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTCCTTATGTGTCCAAAAAT CCAAGATTGGCATATCTCAATTATAGAGACCTTGATATAGGAATAAATGATCCCAAGAATCCAA ATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATTTTGACAGGCTAGT AAAAGTGAAAACCCTGGTTGATCCCAATAACTTTTTTAGAAACGAACAAAGCATCCCACCTCTT CCACGGCATCGTCATTAA SEQ ID NO: 34 Cannabis sativa CBDA synthase polypeptide

MNPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVS HIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRS IKIDVHSQTAWVEAGATL GEVYYWVNEKNENLSLAAGYCPTVCAGGHFGGGGYGPLMRNYGLAADNI IDAHLVNVHGKVLDR KSMGEDLFWALRGGGAESFGI IVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYD KDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWI DTIIFYSGWNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAG MYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSKN PRLAYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQS IPPL PRHRH

SEQ ID NO: 35 Cannabis sativa CBDAS polynucleutide

ATGAAGTGCTCAACATTCTCCTTTTGGTTTGTTTGCAAGATAATATTTTTCTTTTTCTCATTCA ATATCCAAACTTCCATTGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCC CAATAATGCAACAAATCTAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTA AATTCGACAATACACAATCTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCA CTCCTTCACATGTCTCTCATATCCAAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGAT TCGAACTCGAAGTGGTGGTCATGATTCTGAGGGCATGTCCTACATATCTCAAGTCCCATTTGTT ATAGTAGACTTGAGAAACATGCGTTCAATCAAAATAGATGTTCATAGCCAAACTGCATGGGTTG AAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTT GGCGGCTGGGTATTGCCCTACTGTTTGCGCAGGTGGACACTTTGGTGGAGGAGGCTATGGACCA TTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATG GAAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGC AGAAAGCTTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATG TTTAGTGTTAAAAAGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATA TTGCTTACAAGTATGACAAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGA TAATCAAGGGAAGAATAAGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTG GATAGTCTAGTCGACTTGATGAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCA GACAATTGAGCTGGATTGATACTATCATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAA TTTTAACAAGGAAATTTTGCTTGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGATTAAGTTA GACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGTCCAAATTTTGGAAAAATTATATGAAG AAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGAGATTTCAGA ATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGTTGG GAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTC CTTATGTGTCCAAAAATCCAAGATTGGCATATCTCAATTATAGAGACCTTGATATAGGAATAAA TGATCCCAAGAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAA AATTTTGACAGGCTAGTAAAAGTGAAAACCCTGGTTGATCCCAATAACTTTTTTAGAAACGAAC AAAGCATCCCACCTCTTCCACGGCATCGTCATTAA

SEQ ID NO: 36 Cannabis sativa CBDAS Protein

MKCSTFSFWFVCKI I FFFFSFNIQTS IANPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVL NSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFV IVDLRNMRS IKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYCPTVCAGGHFGGGGYGP LMRNYGLAADNI IDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGI IVAWKIRLVAVPKSTM FSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITDNQGKNKTAIHTYFSSVFLGGV DSLVDLMNKSFPELGIKKTDCRQLSWIDTI I FYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKL DYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSW EKQEDNEKHLNWIRNIYNFMTPYVSKNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGK NFDRLVKVKTLVDPNNFFRNEQS IPPLPRHRH

[0090] Cannabinoids can be subject to hepatic metabolism. The cannabinoid derivatives produced during hepatic metabolism can have increased potency, increased solubility in aqueous environments (e.g., in blood), increased ability to pass the blood brain barrier, or a combination thereof. Heptatic enzymes, such as cytochrome P450 enzymes (CYP), can be used to produce such cannabinoid derivatives. For example, CYP2C9 can catalyze the 11 -hydroxylation of Δ⁸- THC, A⁹-THC, CBN, or a combination thereof. CYP3 A4 can be involved in the hydroxylation of cannabinoids at the 8- or 7-position, and can produce 7a- and 7P-hydroxylations of A⁸-THC, 9a,10a-epoxidation of A⁹-THC, or a combination thereof.

[0091] Accordingly, disclosed herein are genetically-modified microorganisms that comprise one or more genetic modifications that increase the expression or activity of one or more cytochrome P450 enzymes. The cytochrome P450 enzymes can be from a mammalian species. The cytochrome P450 enzymes can be from Homo sapiens. The cytochrome P450 enzymes can comprise cytochrome P450 2C9 (CYP2C9), cytochrome P450 3 A4 (CYP3A4), or a combination thereof. The genetically-modified microorganisms can produce increased levels of cannabinoid derivatives (e.g., 1 l-OH-A⁹-THC) in comparison to microorganisms of the same species without the genetic modifications.

[0092] The present disclosure includes methods and compositions for increasing the expression of one or more cytochrome P450 enzymes in a genetically-engineered microorganism relative to an unmodified microorganism of the same species. Such methods can include providing one or more extra copies of endogenous cytochrome P450 enzyme(s), putting endogenous cytochrome P450 enzyme(s) under the control of a stronger promoter, mutating endogenous cytochrome P450 enzyme(s) to encode a higher activity enzyme, introducing an exogenous cytochrome P450 enzyme(s), or any combination thereof. The cytochrome P450 enzyme(s) can be from a mammalian species (e.g., from Homo sapiens). The cytochrome P450 enzymes can comprise cytochrome P450 2C9 (CYP2C9), cytochrome P450 3 A4 (CYP3A4), or a combination thereof. Exemplary cytochrome P450 enzyme polynucleotide and polypeptide sequences are shown in TABLE 11. A genetic modification that increases the expression of an CYP can comprise a polynucleotide comprising an open reading frame at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 37, a polynucleotide comprising an open reading frame at least 880%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 39, or a combination thereof. A genetic modification that increases the expression of an CYP can comprise a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 38, a polynucleotide encoding a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 40, or a combination thereof. The polynucleotide can be integrated into the genome of a genetically -modified microorganism, maintained in the genetically-modified microorganism on plasmid, or a combination thereof. The polynucleotide can be codon-optimized for expression of an encoded protein in a particular microorganism. The genetically-engineered microorganism can have increased production of one or more cannabinoid derivatives (e.g., 1 l-OH-A⁹-THC) relative to a microorganism of the same species without the genetic modifications that increase the expression of the CYP(s).

TABLE 11

Exemplary Cytochrome P450 sequences

SEQ ID NO: 37 Homo sapiens cytochrome P4502C9 polynucleotide

GAAGGCTTCAATGGATTCTCTTGTGGTCCTTGTGCTCTGTCTCTCATGTTTGCTTCTCCTTTCA CTCTGGAGACAGAGCTCTGGGAGAGGAAAACTCCCTCCTGGCCCCACTCCTCTCCCAGTGATTG GAAAT AT C C T AC AGAT AG G T AT TAAG GAC AT C AG CAAAT C C T T AAC CAAT C T C T CAAAG G T C T A TGGCCCTGTGTTCACTCTGTATTTTGGCCTGAAACCCATAGTGGTGCTGCATGGATATGAAGCA GTGAAGGAAGCCCTGATTGATCTTGGAGAGGAGTTTTCTGGAAGAGGCATTTTCCCACTGGCTG AAAGAGC TAACAGAGGAT T T GGAAT T GT T T T CAGCAAT GGAAAGAAAT GGAAGGAGAT CCGGCG TTTCTCCCTCATGACGCTGCGGAATTTTGGGATGGGGAAGAGGAGCATTGAGGACCGTGTTCAA GAGGAAGCCCGCTGCCTTGTGGAGGAGTTGAGAAAAACCAAGGCCTCACCCTGTGATCCCACTT TCATCCTGGGCTGTGCTCCCTGCAATGTGATCTGCTCCATTATTTTCCATAAACGTTTTGATTA TAAAGAT CAGCAAT T T C T TAAC T T AAT G GAAAAG T T GAAT GAAAACAT CAAGAT T T T GAGCAGC CCCTGGATCCAGATCTGCAATAATTTTTCTCCTATCATTGATTACTTCCCGGGAACTCACAACA AAT T AC T T AAAAAC GTTGCTTTTAT GAAAAG TTATATTTTG GAAAAAG T AAAAGAAC AC C AAGA AT CAAT GGACAT GAACAACCC T CAGGAC T T TAT T GAT TGCTTCCT GAT GAAAAT GGAGAAGGAA AAGCACAAC CAAC CAT C T GAAT T T AC TAT T GAAAGCT T GGAAAACAC TGCAG T T GAC T T G T T T G GAGCTGGGACAGAGACGACAAGCACAACCCTGAGATATGCTCTCCTTCTCCTGCTGAAGCACCC AG AG GTCACAGC T AAAG T C C AG G AAG AG AT T G AAC G T G T GAT T G G C AG AAAC C G GAG C C C C T G C AT G C AAGAC AG GAG C C AC AT G C C C T AC AC AGAT GCTGTGGTG C AC GAG G T C C AGAGAT AC AT T G ACCTTCTCCCCACCAGCCTGCCCCATGCAGTGACCTGTGACATTAAATTCAGAAACTATCTCAT T C C C AAG G G C AC AAC CATATTAATTTCCCT GAC TTCTGTGC T AC AT GAC AAC AAAGAAT T T C C C AAC C C AGAGAT G T T T GAC C C T C AT C AC TTTCTGGAT GAAG G T G G CAAT T T T AAGAAAAG TAAAT ACTTCATGCCTTTCTCAGCAGGAAAACGGATTTGTGTGGGAGAAGCCCTGGCCGGCATGGAGCT GTTTTTATTCCTGACCTCCATTTTACAGAACTTTAACCTGAAATCTCTGGTTGACCCAAAGAAC CTTGACACCACTCCAGTTGTCAATGGATTTGCCTCTGTGCCGCCCTTCTACCAGCTGTGCTTCA TTCCTGTCTGAAGAAGAGCAGATGGCCTGGCTGCTGCTGTGCAGTCCCTGCAGCTCTCTTTCCT CTGGGGCATTATCCATCTTTCACTATCTGTAATGCCTTTTCTCACCTGTCATCTCACATTTTCC CTTCCCTGAAGATCTAGTGAACATTCGACCTCCATTACGGAGAGTTTCCTATGTTTCACTGTGC AAAT AT AT CTGCTATTCTCCATACTCTG TAAC AG T T G C AT T GAC T G T C AC AT AAT G C T C AT AC T TATCTAATGTT GAG T T AT T AAT AT GTTATTAT TAAAT AGAGAAAT AT GAT TTGTGTAT TAT AAT TCAAAGGCAT T T C T T T T C T GCAT GT T C TAAAT AAAAAGCAT TAT TAT T T GC T G

SEQ ID NO: 38 Homo sapiens Cytochrome P4502C9 polypeptide

MDSLWLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLPVIGNILQIGIKDISKSLTNLSKVYGPV FTLYFGLKPIWLHGYEAVKEALIDLGEEFSGRGIFPLAERANRGFGIVFSNGKKWKEIRRFSL MTLRNFGMGKRS IEDRVQEEARCLVEELRKTKASPCDPTFILGCAPCNVICS I I FHKRFDYKDQ QFLNLMEKLNENIKILSSPWIQICNNFSPI IDYFPGTHNKLLKNVAFMKSYILEKVKEHQESMD MNNPQDFIDCFLMKMEKEKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRYALLLLLKHPEVT AKVQEEIERVIGRNRSPCMQDRSHMPYTDAWHEVQRYIDLLPTSLPHAVTCDIKFRNYLIPKG TTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKKSKYFMPFSAGKRICVGEALAGMELFLF LTSILQNFNLKSLVDPKNLDTTPWNGFASVPPFYQLCFIPV

SEQ ID NO: 39 Homo sapiens CYP3 A4 polynucleotide

ATGGCTCTCATCCCAGACTTGGCCATGGAAACCTGGCTTCTCCTGGCTGTCAGCCTGGTGCTCC TCTATCTATATG GAAC C CAT T C AC AT G GAC T T T T T AAGAAG C T T G GAAT T C C AG G G C C C AC AC C TCTGCCTTTTTTGGGAAATATTTTGTCCTACCATAAGGGCTTTTGTATGTTTGACATGGAATGT CATAAAAAGTATGGAAAAGTGTGGGGCTTTTATGATGGTCAACAGCCTGTGCTGGCTATCACAG AT C C T GAC AT GAT C AAAAC AG T G C T AG T GAAAGAAT G T T AT T C T G T C T T C AC AAAC C G GAG G C C TTTTGGTC C AG T G G GAT T T AT GAAAAG T G C CAT C T C TAT AG C T GAG GAT GAAGAG T G GAAGAGA TTACGATCATTGCTGTCTCCAACCTTCACCAGTGGAAAACTCAAGGAGATGGTCCCTATCATTG CCCAGTATGGAGATGTGTTGGTGAGAAATCTGAGGCGGGAAGCAGAGACAGGCAAGCCTGTCAC C T T GAAAGAC GTCTTTGGGGCC T AC AG CAT G GAT G T GAT C AC TAG C AC AT CAT T T G GAG T GAAC AT C GAC T C T C T C AAC AAT C C AC AAGAC CCCTTTGTG GAGAAC AC C AAGAAG C T T T T AAGAT T T G ATTTTTTGGATCCATTCTTTCTCTCAATAACAGTCTTTCCATTCCTCATCCCAATTCTTGAAGT AT T AAAT AT C T G T G T G T T T C C AAGAGAAG T T AC AAAT T T T T T AAGAAAAT C T G T AAAAAG GAT G AAAGAAAG T C G C C T C GAAGAT AC AC AAAAG C AC C GAG TGGATTTCCTT C AG C T GAT GAT T GAC T CTCAGAATTCAAAAGAAACTGAGTCCCACAAAGCTCTGTCCGATCTGGAGCTCGTGGCCCAATC AATTATCTTTATTTTTGCTGGCTATGAAACCACGAGCAGTGTTCTCTCCTTCATTATGTATGAA C T G G C C AC T C AC C C T GAT G T C C AG C AGAAAC T G C AG GAG GAAAT T GAT G C AG T T T T AC C C AAT A AG G C AC C AC C C AC C T AT GAT AC T G T G C T AC AGAT G GAG T AT C T T GAC AT G G T G G T GAAT GAAAC GC T CAGAT TAT T C C CAAT T GC T AT GAGAC T T GAGAGGG T C T GCAAAAAAGAT G T T GAGAT CAAT GGGATGTTCATTCCCAAAGGGGTGGTGGTGATGATTCCAAGCTATGCTCTTCACCGTGACCCAA AG T AC T G GAC AGAG C C T GAGAAG TTCCTCCCT GAAAGAT T C AG C AAGAAGAAC AAG GAC AAC AT AGAT C C T T AC AT AT AC AC AC C C T T T G GAAG T G GAC C C AGAAAC T G CAT T G G CAT GAG G T T T G C T CTCATGAACATGAAACTTGCTCTAATCAGAGTCCTTCAGAACTTCTCCTTCAAACCTTGTAAGG AAAC AC AGAT C C C C C T GAAAT T AAG C T TAG GAG GAC T T C T T C AAC CAGAAAAAC C C G T T G T T C T AAAGGTTGAGTCAAGGGATGGCACCGTAAGTGGAGCCTGA

SEQ ID NO: 40 Homo sapiens CYP3 A4 polypeptide

MALIPDLAMETWLLLAVSLVLLYLYGTHSHGLFKKLGIPGPTPLPFLGNILSYHKGFCMFDMEC HKKYGKVWGFYDGQQPVLAITDPDMIKTVLVKECYSVFTNRRPFGPVGFMKSAIS IAEDEEWKR LRSLLSPTFTS GKLKEMVP 11 AQ YGDVLVRNLRREAE T GKPVT LKDVFGAYSMDVI T S T S FGVN IDSLNNPQDPFVENTKKLLRFDFLDPFFLS ITVFPFLIPILEVLNICVFPREVTNFLRKSVKRM KESRLEDTQKHRVDFLQLMIDSQNSKETESHKALSDLELVAQS I I FI FAGYETTSSVLSFIMYE LATHPDVQQKLQEEIDAVLPNKAPPTYDTVLQMEYLDMWNETLRLFPIAMRLERVCKKDVEIN GMFIPKG\AA/MIPSYALHRDPKYWTEPEKFLPERFSKKNKDNIDPYI YTPFGSGPRNCIGMRFA LMNMKLALIRVLQNFSFKPCKETQIPLKLSLGGLLQPEKPWLKVESRDGTVSGA

Microbes Suitable for Cannabinoid Production

[0093] In some embodiments, the present disclosure relates to the identification of a suitable microorganism for industrial-scale carbohydrate-to-terpenophenolic compound conversion for the production of cannabinoid and cannabinoid precursors in the microorganism. Suitable microorganisms can include fungus (e.g., yeast), bacteria, or algae. Non-limiting examples of suitable fungus species include Aspergillus shirousamii, Aspergillus niger, or Trichoderma reesei. Suitable yeast include, for example, Yarrowia lipolytica, Cryptococcus curvatus, Lipomyces starkeyi, Rhodosporidium toruloides, Trichosporon fermentans, Trichosporon pullulan , Lipomyces lipofer, Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, S. bayanus, S. K. lactis, Waltomyces lipofer, Mortierella alpine, Mortierella isabellina, Hansenula polymorpha, Mucor rouxii, Trichosporon cutaneu, Rhodotorula glutinis, Saccharomyces diastasicus, Schwanniomyces occidentalis, S. cerevisiae, Pichia stipitis,

Schwanniomyces occidentalis, or Schizosaccharomyces pombe . Suitable bacteria include, for example, Bacillus subtilis, Salmonella sp., an Escherichia coli, Vibrio cholerae, Streptomyces sp., Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas sp., Rhodococcus sp.,

Streptomyces sp., or Alcaligenes sp. Suitable algae include, for example, Neochloris

oleoabundans, Scenedesmus obliquus, Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, or Spirulina maxima.

[0094] The present disclosure also relates to the identification of an oleaginous yeast species as a suitable microorganism for genetic engineering for cannabinoid, cannabinoid derivative, or cannabinoid precursor production based on the base metabolism of the oleaginous yeast species. The term "oleaginous" can refer to a microbe that can accumulate at least 20% lipid by dry cell mass. Y. lipolytica is an obligate aerobe that can assimilate carbohydrates as the sole carbon source. Y. lipolytica can be a suitable microorganism for cannabinoid, cannabinoid derivative, or cannabinoid precursor production. Y. lipolytica can be a non-pathogenic oleaginous yeast that can metabolize a variety of carbon sources, including, for example, organic acids, hydrocarbons, fats, and oils.

[0095] Compared to other yeast strains, Y. lipolytica can have a higher glucose to fatty acid and triacylglycerol flux and can have a higher lipid storage capacity. In wild-type Y. lipolytica, fatty acid and TAG synthesis from a carbon source can be triggered during the stationary growth phase, suggesting a tight regulatory mechanism of lipid metabolism. The regulatory mechanism of lipid metabolism in Y. lipolytica can limit the availability of fatty acid flux to cannabinoid synthesis and the storage of cannabinoid or cannabinoid precursors in lipid vacuoles. In some embodiments, genetic engineering of the heterologous hexanoic fatty acid pathway for polyketide coupling reactions with fatty acid precursors from malonyl-CoA, and the prenylation reaction with geranyl pyrophosphate can be used to promote the synthesis of cannabinoids and cannabinoid precursors.

Genetic Modification of Microbes

[0096] The production of genetically-engineered microorganism can be a multistage process that can involve identification of a gene of interest; isolation of the gene of interest; amplification of the gene of interest; association of the gene with an appropriate promoter, poly A sequence (terminator region), and a selectable marker; insertion into plasmids; and transformation into the microorganism.

[0097] An expression vector suitable for use in Y. lipolytica can include a selection marker or a defective URA3 marker, which is derived from the URA3 gene of Y. lipolytica. A defective URA3 marker, such as the URA3d, allows complementation of auxotrophy for uracil. The sequences for controlling the gene expression can include, for example, promoter and terminator sequences that are active in Yarrowia species. In some embodiments, the vector comprises an inducible or constitutive promoter. In some embodiments, genes can be overexpressed in microbes from pYLEXl . For example, genetic overexpression can be accomplished by cloning a construct of interest into pYLEXl under the control of a promoter.

[0098] Non-limiting examples of a construct of interest include ACL cDNA, ACC cDNA, type-I FAS cDNA, HS cDNA, OAC cDNA, PKS cDNA, tUMGCR cDNA, IDI1 cDNA, GPP synthase cDNA, GOGT cDNA, THCA synthase cDNA, CBDA synthase cDNA, and CBCA synthase cDNA.

[0099] Methods used to deliver expression vectors or expression constructs into microbes are well known to those of skill in the art. Nucleic acids, including expression vectors, can be delivered to prokaryotic and eukaryotic microbes by various methods well known to those of skill in the relevant biological arts. Methods for the delivery of nucleic acids to a microbe in accordance to some embodiments described herein can include chemical, electrochemical, and biological approaches. Vector delivery methods can include, for example, heat shock

transformation, electroporation, in vivo transfection, co-transfection, transient transfection, stable transfection, DEAE-Dextran transfection, liposome-mediated transfection, cationic lipid transfection, calcium phosphate transfection, CRISPR transfection, RNAi transfection, and siRNA transfection.

[0100] In some embodiments, a nucleic acid construct can be introduced into the host microorganism using a vehicle or vector to transfer the genetic material. Non-limiting examples of vectors for transferring genetic material to microbes include plasmids, artificial chromosomes, and viral vectors. Non-limiting examples of nucleic acid constructs include expression constructs comprising constitutive or inducible heterologous promoters, knockout constructs, and knockdown constructs. Methods and vectors for the delivery of a nucleic acid or nucleic acid construct to a microbe are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 15 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), AcademicPress; 1st edition (July 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002); Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998); and Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), all of which are incorporated by reference herein.

[0101] In some embodiments, a native promoter of a gene encoding a gene product conferring a desirable phenotype to a microbe is modified in the microbe to alter the regulation of the transcriptional activity of the promoter. The native promoter can be, for example, the native TEF promoter. In some embodiments, the modified promoter can exhibit an increased transcriptional activity as compared to the unmodified promoter. The term "modified promoter" can refer to a promoter the nucleotide sequence of which has been artificially altered. Non-limiting examples of artificial alterations include nucleotide deletions, nucleotide insertions, and nucleotide mutations, alone or in combination. Artificial promoter alterations can be affected in a targeted fashion, which can include, for example, homologous recombination approaches, gene targeting, gene knockout, gene knock-down, gene knock-in, site-directed mutagenesis, and artificial zinc finger nuclease-mediated strategies.

[0102] In some embodiments, promoters can be induced by an exogenous carbon source and/or regulatory element. Non-limiting examples of galactose promoters include GALl, GAL7, GALIO, PISl, and LAC4. Non-limiting examples of maltose promoters include MALI, MAL62, and AGTl . Non-limiting examples of ethanol promoters include ICLl, FBPl, PCKl, GUTl, and ADH4. Non-limiting examples of methanol promoters include AOX1, AOX2, AUG1, AUG2, DAS1, FDH, and FDL1.

Microbe Fermentation

[0103] Fermentation processes for large-scale microorganism-mediated carbohydrate to cannabinoid conversion can be carried out in bioreactors. The terms "bioreactor" and

"fermenter", which are interchangeably used, can refer to an enclosure or a partial enclosure in which a biological and/or chemical reaction takes place and at least part of which involves a living organism or part of a living organism. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product, for example, a cannabinoid or cannabinoid precursor on a commercial or quasi-commercial scale. Large scale bioreactors can have a volume in the range of liters, hundreds of liters, thousands of liters, or more.

[0104] A bioreactor described herein can comprise a microorganism (e.g., a genetically-modified microorganism disclosed herein) or a microorganism culture. In some embodiments, a bioreactor can comprise a spore and/or any kind of dormant cell type of any isolated microorganism described herein, for example, in a dry state. In some embodiments, the addition of a

carbohydrate source to such bioreactors can lead to activation of the dormant cell, for example, to the germination of a yeast spore, and subsequent conversion of the carbohydrate source to a cannabinoid or cannabinoid precursor. In some embodiments, bioreactors described herein can include cell culture systems where the microbes are in contact with moving liquids and/or gas bubbles.

[0105] Microorganisms or microorganism cultures described herein can be grown in suspension or attached to solid phase carriers. Non-limiting examples of carrier systems include

microcarriers, polymer spheres, microbeads, porous or non-porous microdisks, cross-linked beads, dextran beads charged with specific chemical groups, 2D microcarriers, microcarriers trapped in nonporous polymer fibers, 3D carriers, carrier fibers, hollow fibers, multi cartridge reactors, semipermeable membranes with porous fibers, microcarriers with reduced ion exchange capacity, encapsulation cells, capillaries, and aggregates. Carriers can be fabricated from various materials including, for example, dextran, gelatin, glass, and cellulose.

[0106] Industrial-scale carbohydrate-to-cannabinoid conversion processes described herein can be operated in continuous, semi-continuous, or non-continuous modes. Non-limiting examples of operation modes described herein include batch, fed-batch, extended-batch, repetitive-batch, draw/fill, rotating-wall, spinning flask, and perfusion modes of operation. In some embodiments, bioreactors can be used that allow continuous or semi-continuous replenishment of the substrate stock (a carbohydrate source), separation of the product (a secreted cannabinoid or precursor) from an organic phase comprising a cannabinoid and/or cells exhibiting a desired cannabinoid content from the reactor.

[0107] Non-limiting examples of bioreactors described herein include stirred tank fermenters, bioreactors agitated by rotating mixing devices, chemostats, bioreactors agitated by shaking devices, airlift fermenters, packed-bed reactors, fixed-bed reactors, fluidized bed bioreactors, bioreactors employing wave induced agitation, centrifugal bioreactors, roller bottles, hollow fiber bioreactors, benchtop roller apparatuses, cart-mounted roller apparatuses, automated roller apparatuses, vertically-stacked plates, spinner flasks, stirring flasks, rocking flasks, shaken multi- well plates, MD bottles, T-flasks, Roux bottles, multiple-surface tissue culture propagators, modified fermenters, and coated beads. Bioreactors and fermenters described herein can, optionally, comprise a sensor and/or a control system to measure and/or adjust reaction parameters.

[0108] Non-limiting examples of reaction parameters include biological parameters, chemical parameters, nutrient concentrations, metabolite concentration, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, concentration of an oligopeptide, amino acid concentration of an, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of buffering agents, concentration of adjuvants, concentration of reaction by-products, physical/mechanical parameters, and

thermodynamic parameters.

[0109] Non-limiting examples of biological parameter include growth rate, cell size, cell number, cell density, cell type, and cell state. Non-limiting examples of chemical parameters include pH, redox potential, concentration of reaction substrate and/or product, and concentration of dissolved gases, including, for example, oxygen and C0₂. Non-limiting examples of

physical/mechanical parameters include density, conductivity, degree of agitation, pressure, flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, and conversion rate. Non-limiting examples of thermodynamic parameters include temperature, light intensity, and light quality.

[0110] Sensors that are used to measure parameters as described herein are well known to those of skill in the relevant mechanical and electronic arts. Control systems that can be used to adjust the parameters in a bioreactor based on the inputs from a sensor as described herein are well known to those of skill in the art of bioreactor engineering.

[0111] A variety of different microorganisms as described herein can be cultured in a suitable bioreactor to perform large-scale carbohydrate to cannabinoid or cannabinoid precursor conversion as described herein. Non-limiting examples of suitable microbes include yeast, oleaginous microbes, bacteria, algae, and fungi. Non-limiting examples of yeast include

Yarrow ia lipolytica, Hansenula polymorpha, Pichia deserticolab, Pichia pastor is, Pichia stipitis, Saccharomyces cerevisiae, Saccharomyces bay anus, Saccharomyces diastasicus, Kluyveromyces lactis, Mortierella alpine, Mortierella isabellina, Hansenula polymorpha, Mucor rouxii,

Trichosporon cutaneu, Rhodotorula glutinis, Schwanniomyces occidentalis,

Schizosaccharomyces pombe, and Waltomyces lipofer. Non-limiting examples of oleaginous microbes include from Yarrowia lipolytica, Yarrowia lipolynca, Cryptococcus curvatus, Lipomyces starkeyi, Lipomyces lipofer, Rhodosporidium babjevae, Rhodosporidium diobovatum, Rhodosporidium ci.fluviale, Rhodotorula glutinis, Rhodosporidium toruloides, Rhodosporidium kratochvilovae, Rhodosporidium paludigenum, Rhodosporidium sphaerocarpum,

Rhodosporidium sphaerocarpum, Rhodotorula araucariae, Rhodotorula colostri, Rhodotorula aff. lusitaniae, Rhodotorula dairenensis, Rhodotorula mucilaginosa, Rhodotorula graminis, Rhodotorula aff. hylophila, Rhodotorula bogoriensis, Rhodotorula minuta, Sporidiobolus salmonicolor, Sporidiobolus johnsonii, Sporidiobolus pararoseus, Sporobolomyces carnicolor, Sporobolomyces bannaensis, Sporidiobolus ruineniae, Sporidiobolus salmonicolor,

Sporobolomyces aff. beijingensis, Sporobolomyces odoratus, Sporobolomyces poonsookiae, Sporobolomyces aff. inositophilus, Sporobolomyces singularis, Starmerella bombicola,

Trichosporon pullulan, and Trichosporon fermentans.

[0112] In some embodiments, a typical fermentation can utilize glucose and trace amounts of nutrients to support the cell growth and efficient cannabinoid product generation. TABLE 12 shows an example of the general makeup of a fermentation culture of Y. lipolytica.

TABLE 12

[0113] Non-limiting examples of bacteria include Bacillus subtilis, Salmonella, Escherichia coli, Vibrio cholerae, Streptomyces, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas sp, Rhodococcus sp, Streptomyces sp, and Alcaligenes sp. Non-limiting examples of fungi include Aspergillus shirousamii, Aspergillus niger, and Trichoderma reesei. Non-limiting examples of algae include Neochloris oleoabundans, Scenedesmus obliquus, Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, and Spirulina maxima.

[0114] The carbohydrate source used for conversion to a cannabinoid or cannabinoid precursor according to aspects described herein can depend on the specific microbe. In some embodiment, a microbe described herein can efficiently convert a specific carbohydrate source. In some embodiments, the same microbe cannot metabolize a different carbohydrate source at high efficiency or at all.

[0115] Non-limiting examples of sugars that can be efficiently metabolized by a microorganism described herein include glucose, galactose, maltose, sucrose, lactose, xylose, glycerol, acetate, molasses, and plant fibers. In some embodiments, a cannabinoid or cannabinoid precursor can be generated from a carbon source feedstock and is secreted, at least partially, by a microbe described herein.

[0116] In some embodiments, a microbe described herein can be contacted with a carbohydrate source in an aqueous solution in a bioreactor, and the secreted cannabinoid or cannabinoid precursor forms an organic phase that can be separated from the aqueous phase. The term

"organic phase" can refer to a liquid phase comprising a non-polar, organic compound, including, for example, a cannabinoid, a cannabinoid precursor, and/or a non-polar lipid. An organic phase described herein can further contain a microbe, a carbohydrate, or a compound found in other phases found in a respective bioreactor.

[0117] Methods useful for industrial-scale phase separation are well known to those of ordinary skill in the art. In some embodiments, the organic phase is continuously or semi-continuously siphoned off. In some embodiments, a bioreactor can comprise a separator that can be used, to continuously or semi-continuously extract the organic phase from the inorganic phase.

[0118] In some embodiments, a cannabinoid or cannabinoid precursor can accumulate in a cell according to aspects described herein. In some embodiments, a cell that accumulates a desirable amount of a cannabinoid or cannabinoid precursor can be separated continuously or semi- continuously from a bioreactor. Non-limiting chemical separation methods include

centrifugation, sedimentation, and filtration. Cell separation can further be affected based on a change in physical cell characteristic, such as cell size and cell density, by methods well known to those skilled in the art.

Downstream Processing and Recovery of Cannabinoids

[0119] The accumulated cannabinoid or cannabinoid precursor can subsequently be extracted from the respective cells using standard methods of extraction well known to those skilled in the art. Non-limiting extraction methods include liquid-liquid solvent extraction. In some

embodiments, microbial cells are collected and extracted with 3x the collected cell volume of solvent. In some embodiments, the extracted cannabinoid or cannabinoid precursor can be further refined using additional purification methods. In some embodiments, a cannabinoid or cannabinoid precursor can be converted to a cannabinoid by a semi-synthetic conversion procedure.

[0120] A cannabinoid or cannabinoid precursor molecule can be recovered from a suitable genetically-modified microorganism producer described herein, using processing steps that minimize product contamination and simplify recovery of the final product from the fermentation broth. Different end products can require different chemical and biological methodologies to capture, collect, and purify the final product. Cannabinoids and cannabinoid precursors are largely water insoluble. Crude cannabinoid product can be recovered using separation methods including, for example, centrifugation and solvent extraction.

[0121] Extraction of cannabinoids and cannabinoid precursors from a suitable microbe source can involves cell wall disruption of the microbial cell. Non-limiting examples of method include the application of enzymes, detergents, heat, pressure, and mechanical action. Example methodologies for cell wall disruption are summarized in TABLE 13.

TABLE 13

[0122] Mechanical cell disruption involves forcing open the cell wall to cause the contents of the cell to leak or flush into the surrounding media. Compared to chemical approaches, mechanical disruption can be advantageous because chemicals that might interfere with the extracted product are not introduced to the mixture. Mechanical disruption can be disadvantageous because high amounts of mechanical energy can be too abrasive and destroy that molecule or product of interest.

[0123] Mechanical disruption can involve grinding cells using a mortar and pestle. The cells can be in suspension or frozen in liquid nitrogen. Once the material has been disrupted, intracellular products can be extracted by solvent extraction methods. Mechanical disruption can also involve bead milling in which glass or ceramic beads are used to crack open cells. This mechanical shearing technique is gentle enough to keep organelles intact.

[0124] Cell lysis can also be accomplished by sonication and ultrasonic homogenization. These methods involve the introducing ultrasonic vibrations to a cell suspension. The ultrasonic process induces cavitation in the solution creating localized Shockwave to disrupt the integrity of the cell wall. Homogenizers use shearing forces on the cell similar to the bead milling method.

Homogenization can be performed by pressurizing cells through a tube that is slightly smaller than the size of the cells, thereby shearing away the outer layer (French Press) or by using a rotating blade similar to a blender (Rotor-Stator Processors).

[0125] Freezing is another mechanical method of cell disruption. Freeze-thaw cycles that involve the formation of ice crystals upon freezing and expansion the cells upon thawing lead to cell wall rupture. The freeze-thaw approach is typically used for algae and soft plant material. The freeze- thaw method can be time-consuming, can require large freezer space, and can be difficult to scale cost effectively.

[0126] High temperatures and pressure can also be used to disrupt cell wall structures and release the contents of the cells. Non-limiting examples of high temperature-high pressure methods include microwaving and autoclaving. The application of heat and pressure can be fast, but can damage to heat-sensitive products.

[0127] Non-mechanical methods of cell disruption can involve the addition of enzymes or chemicals that specifically break down cell wall components. Non-limiting examples of naturally-occurring enzymes that degrade cell wall include cellulases, chitinase, bacteriolytic enzymes, lysozyme, mannase, and glycanase. Non-mechanical methods can be used in combination with mechanical methods to ensure complete disruption of the cell. For example, solvent extraction also be combined with any of the mechanical approaches to improve cell wall disruption and product extraction.

[0128] Organic solvents including, for example, alcohols, ether, esters, and chlorinated solvents can also disrupt the cell wall by permeating and degrading cell walls and membranes. The use of organic solvents can be especially effective if the desirable product is hydrophobic because the products can accumulate in the solvent and be isolated by solvent extraction. For example, EDTA (ethylenediaminetetraacetic acid) can be used in Gram-negative bacteria that contain cell walls made of lipopolysaccharides. EDTA can disrupt the integrity of the cell walls by chelating divalent and trivalent cations that stabilize lipopolysaccharide cell walls.

[0129] In some embodiments, the cannabinoid or cannabinoid precursor product is mostly or fully-contained within the cells of the microbe. Following cell lysis, the cell mass can be separated from the fermentation broth by methods including, for example, centrifugation, filtration, and membrane separation.

[0130] In some embodiments, the cannabinoid or cannabinoid precursor product is at least partially excreted by the microbe. The cannabinoid product can also be extracted from the fermentation media by separation methods, including, for example, centrifugation and solvent extraction.

[0131] Centrifugation involves separation of the light phase (hydrophobic cannabinoid product) from the heavy phase (hydrophilic water and nutrients) using centrifugal force. Solvent extraction, such as liquid-liquid extraction, involves the use of a suitable solvent that can phase- separate the hydrophobic phase from the aqueous phase. Non-limiting examples of suitable solvents that can be used for cannabinoid extraction include ethyl acetate, hexane, chloroform, methanol, and long-chain fatty alcohols. In some embodiments, the solvent has a low boiling point and the solvent can subsequently be separated from the cannabinoid product by distillation methods. Fractional distillation allows for the removal of the product at a set boiling point and results in recovery of product with 99.9% purity or higher. Fractional distillation can also provide an effective way to separate and purify multiple products. The cannabinoid product can be further purified by UPLC.

Semi-synthetic Production of Cannabinoids

[0132] In some embodiments, the present disclosure utilizes semi -synthetic approaches for conversion of microbial-derived olivetolic acid, olivetol, and cannabigerolic acid precursors to produce cannabinoids and cannabinoid precursors. Non-limiting examples of semi-synthetic approaches include enzymatic methods and cell-free, organic chemistry methods.

[0133] In some embodiments, the cannabinoid precursors produced by a genetically-engineered microbe can be isolated to produce other cannabinoid precursors and/or cannabinoid products.

[0134] For example, cannabidiol (CBD) can be prepared using methyl olivetolate as the starting material, for example as illustrated in FIG. 5. The identity and purity of the CBD product can be confirmed by NMR, HPLC/MS, and TLC.

Specific Embodiments

[0135] Without limiting the foregoing disclosure, specific embodiments of the disclosure are presented herein.

[0136] Embodiment 1. A genetically engineered microorganism comprising one or more genetic modifications that increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP) relative to a microorganism of the same species without the one or more genetic modifications, wherein the genetically modified microorganism has increased production of hexanoic acid relative to an unmodified organism of the same species.

[0137] Embodiment 2. The microorganism of embodiment 1, wherein the FASa and

FASP are hexanoic acid specific Type I fatty acid synthases.

[0138] Embodiment 3. The microorganism of any one of embodiments 1-2, wherein the

FASa and FASP are from an Aspergillus species.

[0139] Embodiment 4. The microorganism of any one of embodiments 1-2, wherein the

FASa and FASP are from an Aspergillus parasiticus species.

[0140] Embodiment 5. The microorganism of any one of embodiments 1-2, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 5, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 7, or both.

[0141] Embodiment 6. The microorganism of any one of embodiments 1-2, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 6, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 8, or both.

[0142] Embodiment 7. The microorganism of any one of embodiments 5-6, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0143] Embodiment 8. The microorganism of any one of embodiments 1-7, further comprising one or more genetic modifications that increase the expression of an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), or both relative to a microorganism of the same species with the one or more genetic modifications.

[0144] Embodiment 9. The microorganism of any one of embodiments 1-7, further comprising one or more genetic modifications that increase the expression of an ATP Citrate Lyase (ACL) and an Acetyl-coA Carboxylase (ACC) relative to a microorganism of the same species with the one or more genetic modifications.

[0145] Embodiment 10. The microorganism of embodiment 8 or 9, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 1, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 3, or both.

[0146] Embodiment 11. The microorganism of embodiment 8 or 9, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 2, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 4, or both.

[0147] Embodiment 12. The microorganism of embodiment 10 or 11, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0148] Embodiment 13. The microorganism of any one of embodiments 8-12, wherein the genetically modified microorganism has increased production of acetyl-CoA, malonyl-CoA, or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the ATP Citrate Lyase (ACL), the Acetyl-coA Carboxylase (ACC), or both.

[0149] Embodiment 14. The microorganism of any one of embodiments 1-13, wherein the microorganism does not comprise a genetic modification that increases expression of a stearoyl- CoA desaturase (SCD).

[0150] Embodiment 15. The microorganism of any one of embodiments 1-14, wherein the microorganism does not comprise a genetic modification that increases expression of a diacylglycerol acyltransferase (DGA1).

[0151] Embodiment 16. The microorganism of any one of embodiments 1-15, further comprising one or more genetic modifications that increase the expression of a hexanoate synthase (HS) relative to a microorganism of the same species with the one or more genetic modifications.

[0152] Embodiment 17. The microorganism of embodiment 16, wherein the HS is from a Cannabis species.

[0153] Embodiment 18. The microorganism of embodiment 16, wherein the HS is from a Cannabis sativa species.

[0154] Embodiment 19. The microorganism of embodiment 16, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 9.

[0155] Embodiment 20. The microorganism of embodiment 16, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide at least 80%> identical so SEQ ID NO: 10.

[0156] Embodiment 21. The microorganism of embodiment 19 or 20, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0157] Embodiment 22. The microorganism of any one of embodiments 16-21, wherein the genetically modified microorganism has increased production of hexanoyl-CoA relative to a microorganism of the same species without the genetic modifications that increase the expression of the HS.

[0158] Embodiment 23. The microorganism of any one of embodiments 1-22, further comprising one or more genetic modifications that increase the expression of a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or both relative to a microorganism of the same species with the one or more genetic modifications.

[0159] Embodiment 24. The microorganism of any one of embodiments 1-22, further comprising one or more genetic modifications that increase the expression of a polyketide synthase (PKS) and an olivetolic acid cyclase (OAC) relative to a microorganism of the same species with the one or more genetic modifications.

[0160] Embodiment 25. The microorganism of embodiment 23 or 24, wherein the PKS, the OAC, or both are from a Cannabis species.

[0161] Embodiment 26. The microorganism of embodiment 23 or 24, wherein the PKS, the OAC, or both are from a Cannabis sativa species.

[0162] Embodiment 27. The microorganism of embodiment 23 or 24, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 11, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 13, or both.

[0163] Embodiment 28. The microorganism of embodiment 23 or 24, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%) identical to SEQ ID NO: 12, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 14, or both.

[0164] Embodiment 29. The microorganism of embodiment 27 or 28, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0165] Embodiment 30. The microorganism of any one of embodiments 23-29, wherein the genetically modified microorganism has increased production of olivetolic acid relative to a microorganism of the same species without the genetic modifications that increase the expression of the PKS, the OAC, or both.

[0166] Embodiment 31. The microorganism of any one of embodiments 1-30, further comprising one or more genetic modifications that increase the expression of a HMG-CoA Reductase 1 (HMGR1), an isopentenyl-diphosphate delta isomerase 1 (IDIl), a geranyl pyrophosphate synthase (GPPS), a farnesyl pyrophosphate synthase (FPPS), a mutated farnesyl pyrophosphate synthase (mFPPS), a geranylpyrophosphate olivetolate geranyltransferase (GOGT), or a combination thereof relative to a microorganism of the same species with the one or more genetic modifications.

[0167] Embodiment 32. The microorganism of embodiment 31, comprising the genetic modification that increases expression of HMGR1.

[0168] Embodiment 33. The microorganism of embodiment 32, wherein the HMGR1 is a truncated HMGR1 (tHMGRl) lacking a regulatory transmembrane domain.

[0169] Embodiment 34. The microorganism of embodiment 33, wherein the genetic modification comprises a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 15.

[0170] Embodiment 35. The microorganism of embodiment 33, wherein the genetic modification comprises a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 16.

[0171] Embodiment 36. The microorganism of embodiment 34 or 35, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0172] Embodiment 37. The microorganism of any one of embodiments 32-36, wherein the genetically modified microorganism has increased production of mevalonate relative to a microorganism of the same species without the genetic modifications that increase the expression of the HMGR1.

[0173] Embodiment 38. The microorganism of any one of embodiments 31-37, comprising the genetic modification that increases expression of IDI1.

[0174] Embodiment 39. The microorganism of embodiment 38, wherein the genetic modification comprises a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 17.

[0175] Embodiment 40. The microorganism of embodiment 38, wherein the genetic modification comprises a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 18.

[0176] Embodiment 41. The microorganism of embodiment 39 or 40, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0177] Embodiment 42. The microorganism of any one of embodiments 38-41, wherein the genetically modified microorganism has increased production of isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the IDI1. [0178] Embodiment 43. The microorganism of any one of embodiments 31-42, comprising the genetic modification that increases expression of the GPPS, the FPPS, the mFPPS, or a combination thereof.

[0179] Embodiment 44. The microorganism of embodiment 43, wherein the genetic modification comprises a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 19, a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 21, a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 25, a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 27, or a combination thereof.

[0180] Embodiment 45. The microorganism of embodiment 43, wherein the genetic modification comprises a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 20, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 22, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 23, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 24, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 26, a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 28, or a combination thereof.

[0181] Embodiment 46. The microorganism of embodiment 44 or 45, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0182] Embodiment 47. The microorganism of any one of embodiments 43-46, wherein the genetically modified microorganism has increased production of geranyl diphosphate relative to a microorganism of the same species without the genetic modifications that increases the expression of the GPPS, the FPPS, the mFPPS, or the combination thereof.

[0183] Embodiment 48. The microorganism of any one of embodiments 31-47, comprising the genetic modification that increases expression of the geranylpyrophosphate olivetolate geranyltransferase (GOGT).

[0184] Embodiment 49. The microorganism of embodiment 48, wherein the GOGT is from a Cannabis species.

[0185] Embodiment 50. The microorganism of embodiment 48, wherein the GOGT is from a Cannabis sativa species.

[0186] Embodiment 51. The microorganism of embodiment 48, wherein the genetic modification comprises a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 29. [0187] Embodiment 52. The microorganism of embodiment 48, wherein the genetic modification comprises a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 30.

[0188] Embodiment 53. The microorganism of embodiment 51 or 52, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0189] Embodiment 54. The microorganism of any one of embodiments 48-53, wherein the genetically modified microorganism has increased production of cannabigerolic acid (CBGA) relative to a microorganism of the same species without the genetic modifications that increases the expression of the GOGT.

[0190] Embodiment 55. The microorganism of any one of embodiments 1-54, further comprising one or more genetic modifications that increase the expression of a

tetrahydrocannabidiol synthase (THCS), a cannabidiol synthase (CBDS), cannabichromene synthase (CBCS), or a combination thereof relative to a microorganism of the same species with the one or more genetic modifications.

[0191] Embodiment 56. The microorganism of embodiment 55, comprising the genetic modification that increases expression of the THCS.

[0192] Embodiment 57. The microorganism of embodiment 56, wherein the THCS is from a Cannabis species.

[0193] Embodiment 58. The microorganism of embodiment 56, wherein the THCS is from a Cannabis sativa species.

[0194] Embodiment 59. The microorganism of embodiment 56, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 31.

[0195] Embodiment 60. The microorganism of embodiment 56, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide at least 80% identical to SEQ ID NO: 32.

[0196] Embodiment 61. The microorganism of embodiment 59 or 60, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0197] Embodiment 62. The microorganism of any one of embodiments 55-61, wherein the genetically modified microorganism has increased production of A⁹-tetrahydrocannabinolic acid

(THCA), A⁹-tetrahydrocannabinol (THC), or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the THCS.

[0198] Embodiment 63. The microorganism of any one of embodiments 55-62, comprising the genetic modification that increases expression of the CBDS. [0199] Embodiment 64. The microorganism of embodiment 63, wherein the CBDS is from a Cannabis species.

[0200] Embodiment 65. The microorganism of embodiment 63, wherein the CBDS is from a Cannabis sativa species.

[0201] Embodiment 66. The microorganism of embodiment 63, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 33, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 35, or both.

[0202] Embodiment 67. The microorganism of embodiment 63, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide at least 80% identical to SEQ ID NO: 34, a polynucleotide that encodes a polypeptide at least 80% identical to SEQ ID NO: 36, or both.

[0203] Embodiment 68. The microorganism of embodiment 66 or 67, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0204] Embodiment 69. The microorganism of any one of embodiments 63-68, wherein the genetically modified microorganism has increased production of cannabidiolic acid (CBD A), cannabidiol (CBD), or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the CBDS.

[0205] Embodiment 70. The microorganism of any one of embodiments 55-69, comprising the genetic modification that increases expression of the CBCS.

[0206] Embodiment 71. The microorganism of embodiment 70, wherein the CBCS is from a Cannabis species.

[0207] Embodiment 72. The microorganism of embodiment 70, wherein the CBCS is from a Cannabis sativa species.

[0208] Embodiment 73. The microorganism of any one of embodiments 70-72, wherein the genetically modified microorganism has increased production of cannabichromenic acid (CBCA), cannabichromene (CBC), or both relative to a microorganism of the same species without the genetic modifications that increase the expression of the CBCS.

[0209] Embodiment 74. The microorganism of any one of embodiments 1-73, wherein the genetically modified microorganism further comprises one or more genetic modifications that increase the expression of one or more cytochrome P450 enzymes relative to a microorganism of the same species with the one or more genetic modifications. [0210] Embodiment 75. The microorganism of embodiment 74, wherein the one or more cytochrome P450 enzymes comprise cytochrome P450 2C9 (CYP2C9), cytochrome P450 3 A4 (CYP3 A4), or a combination thereof.

[0211] Embodiment 76. The microorganism of embodiment 74 or 75, wherein the one or more cytochrome P450 enzymes are from a mammalian species.

[0212] Embodiment 77. The microorganism of embodiment 74 or 75, wherein the one or more cytochrome P450 enzymes are from Homo sapiens.

[0213] Embodiment 78. The microorganism of any one of embodiment 74-77, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 37, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 39, or a combination thereof.

[0214] Embodiment 79. The microorganism of any one of embodiment 74-77, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide at least 80%) identical to SEQ ID NO: 38, a polynucleotide t that encodes a polypeptide at least 80%> identical to SEQ ID NO: 40, or a combination thereof.

[0215] Embodiment 80. The microorganism of embodiment 78or 79, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

[0216] Embodiment 81. The microorganism of any one of embodiments 74- 80, wherein the genetically modified microorganism has increased production of one or more cannabinoid derivatives relative to a microorganism of the same species without the genetic modifications that increase the expression of the one or more cytochrome P450 enzymes.

[0217] Embodiment 82. The microorganism of embodiment 81, wherein the one or more cannabinoid derivatives comprise l l-OH-A⁹-THC.

[0218] Embodiment 83. The microorganism of any one of embodiments 1-82, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae.

[0219] Embodiment 84. The microorganism of any one of embodiments 1-82, wherein the genetically engineered microorganism is a yeast.

[0220] Embodiment 85. The microorganism of 84, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a Hansenula polymorpha, a Pichia pastoris, a Saccharomyces cerevisiae, a S. bayanus, a S. K. lactis, a Waltomyces lipofer, a Mortierella alpine, a Mortierella isabellina, a Mucor rouxii, a

Trichosporon cutaneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a Schizosaccharomyces pombe. [0221] Embodiment 86. The microorganism of 84, wherein the yeast is Yarrowia lipolytica.

[0222] Embodiment 87. The microorganism of any one of embodiments 1-82, wherein the genetically engineered microorganism is a bacterium.

[0223] Embodiment 88. The microorganism of embodiment 87, wherein the bacterium is a Bacillus subtilis, a Salmonella sp., an Escherichia coli, a Vibrio cholerae, a Streptomyces sp., a Pseudomonas fluorescens, a Pseudomonas putida, a Pseudomonas sp., a Rhodococcus sp., or a Alcaligenes sp.

[0224] Embodiment 89. The microorganism of any one of embodiments 1-82, wherein the genetically engineered microorganism is a fungus.

[0225] Embodiment 90. The microoganism of embodiment 89, wherein the fungus is a Aspergillus shirousamii, a Aspergillus niger, or a Trichoderma reesei.

[0226] Embodiment 91. The microorganism of any one of embodiments 1-82, wherein the genetically engineered microorganism is an algae.

[0227] Embodiment 92. The microorganism of embodiment 91, wherein the algae is a Neochloris oleoabundans, a Scenedesmus obliquus, a Nannochloropsis sp., a Dunaliella tertiolecta, a Chlorella vulgaris, a Chlorella emersonii, or a Spirulina maxima.

[0228] Embodiment 93. A method of producing one or more fermentation end-products comprising contacting the genetically engineered microorganism of any one of embodiments 1- 92 with a carbohydrate source under culture conditions and for a time sufficient to produce the one or more fermentation end products.

[0229] Embodiment 94. The method of embodiment 93, wherein the one or more fermentation end-products comprise one or more cannabinoid precursors, one or more cannabinoids, one or more cannabinoid derivatives, or a combination thereof.

[0230] Embodiment 95. The method of embodiment 94, wherein the one or more fermentation end products comprise the one or more cannabinoid precursors that are hexanoic acid, hexanoyl-CoA, olivetolic acid, geranyldiphosphase, cannabigerolic acid (CBGA), or a combination thereof.

[0231] Embodiment 96. The method of embodiment 95, wherein the one or more fermentation end products comprise the cannabinoid precursor olivetolic acid.

[0232] Embodiment 97. The method of embodiment 95-96, further comprising synthesizing one or more cannabinoids from the cannabinoid precursor.

[0233] Embodiment 98. The method of embodiment 97, wherein the one or more cannabinoids comprise cannabigerolic acid (CBGA), A⁹-tetrahydrocannabinolic acid (THCA), A⁹-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabichromenic acid (CBCA), cannabichromene (CBC), or a combination thereof.

[0234] Embodiment 99. The method of embodiment 97, wherein the one or more cannabinoids comprise cannabidiol (CBD), A9-tetrahydrocannabinol (THC), cannabichromene (CBC), or a combination thereof.

[0235] Embodiment 100. The method of embodiment 97, wherein the one or more cannabinoids comprise cannabidiol (CBD).

[0236] Embodiment 101. The method of embodiment 94, wherein the one or more fermentation end-products comprise the one or more cannabinoids that are cannabigerolic acid (CBGA), A⁹-tetrahydrocannabinolic acid (THCA), A⁹-tetrahydrocannabinol (THC),

cannabidiolic acid (CBDA), cannabidiol (CBD), cannabichromenic acid (CBCA),

cannabichromene (CBC), or a combination thereof.

[0237] Embodiment 102. The method of embodiment 94, wherein the one or more fermentation end-products comprise the one or more cannabinoid derivatives that are 11-ΟΗ-Δ⁹- THC.

[0238] Embodiment 103. The method of any one of embodiments 93-102, wherein the carbohydrate source comprises one or more fermentable sugars.

[0239] Embodiment 104. The method of embodiment 103, wherein the one or more fermentable sugars comprise glucose.

[0240] Embodiment 105. The method of any one of embodiments 93-104, wherein the culture conditions comprise nitrogen depletion conditions.

[0241] Embodiment 106. The fermentation end-product produced by the method of any one of embodiments 93-105.

[0242] Embodiment 107. A genetically engineered microorganism comprising one or more genetic modification that enable production of olivetolic acid in the absence of an external source of hexanoic acid.

[0243] Embodiment 108. The microorganism of embodiment 107, wherein the one or more genetic modifications enable production of the olivetolic acid from a carbohydrate source at an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

[0244] Embodiment 109. A genetically engineered microorganism comprising one or more genetic modifications that enable production of olivetolic acid from a carbohydrate source with an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

[0245] Embodiment 110. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 2%. [0246] Embodiment 111. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 3%.

[0247] Embodiment 112. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 4%.

[0248] Embodiment 113. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 5%.

[0249] Embodiment 114. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 6%.

[0250] Embodiment 115. The microorganism of embodiment 108 or 109, wherein the efficiency is at least 7%.

[0251] Embodiment 116. The microorganism of embodiment 108 or 109, wherein the efficiency is about 1% to about 30%.

[0252] Embodiment 117. The microorganism of embodiment 108 or 109, wherein the efficiency is about 2% to about 15%.

[0253] Embodiment 118. The microorganism of embodiment 108 or 109, wherein the efficiency is about 5% to about 10%.

[0254] Embodiment 119. The microorganism of any one of embodiments 107-118, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or a combination thereof relative to an unmodified microorganism of the same species.

[0255] Embodiment 120. The microorganism of any one of embodiments 107-118, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), and an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

[0256] Embodiment 121. The microorganism of any one of embodiments 107-120, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP) relative to an unmodified microorganism of the same species.

[0257] Embodiment 122. The microorganism of embodiment 121, wherein the FASa and FASP are hexanoic acid specific Type I fatty acid synthases. [0258] Embodiment 123. The microorganism of any one of embodiments 121-122, wherein the FASa and FASP are from an Aspergillus species.

[0259] Embodiment 124. The microorganism of any one of embodiments 121-122, wherein the FASa and FASP are from an Aspergillus parasiticus species.

[0260] Embodiment 125. The microorganism of any one of embodiments 121-122, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 5, a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 7, or both.

[0261] Embodiment 126. The microorganism of any one of embodiments 121-122, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 6, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 8, or both.

[0262] Embodiment 127. The microorganism of any one of embodiments 125-126, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0263] Embodiment 128. The microorganism of any one of embodiments 107-127, wherein the one or more genetic modifications increase the expression of an ATP Citrate Lyase (ACL) relative to an unmodified microorganism of the same species.

[0264] Embodiment 129. The microorganism of embodiment 128, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 1.

[0265] Embodiment 130. The microorganism of embodiment 128, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 2.

[0266] Embodiment 131. The microorganism of any one of embodiments 129-130, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0267] Embodiment 132. The microorganism of any one of embodiments 107-131, wherein the one or more genetic modifications increase the expression of an Acetyl-coA Carboxylase (ACC) relative to an unmodified microorganism of the same species.

[0268] Embodiment 133. The microorganism of embodiment 128, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 3.

[0269] Embodiment 134. The microorganism of embodiment 128, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 4. [0270] Embodiment 135. The microorganism of any one of embodiments 133-134, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0271] Embodiment 136. The microorganism of any one of embodiments 107-135, wherein the one or more genetic modifications increase the expression of a polyketide synthase (PKS) relative to an unmodified microorganism of the same species.

[0272] Embodiment 137. The microorganism of embodiment 136, wherein the PKS is from a Cannabis species.

[0273] Embodiment 138. The microorganism of embodiment 136, wherein the PKS is from a Cannabis sativa species.

[0274] Embodiment 139. The microorganism of embodiment 136, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 11.

[0275] Embodiment 140. The microorganism of embodiment 136, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 12.

[0276] Embodiment 141. The microorganism of any one of embodiments 139-140, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0277] Embodiment 142. The microorganism of any one of embodiments 107-141, wherein the one or more genetic modifications increase the expression of an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

[0278] Embodiment 143. The microorganism of embodiment 142, wherein the OAC is from a Cannabis species.

[0279] Embodiment 144. The microorganism of embodiment 142, wherein the OAC is from a Cannabis sativa species.

[0280] Embodiment 145. The microorganism of embodiment 142, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 13.

[0281] Embodiment 146. The microorganism of embodiment 142, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 14.

[0282] Embodiment 147. The microorganism of any one of embodiments 145-146, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0283] Embodiment 148. The microorganism of any one of embodiments 107-147, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae. [0284] Embodiment 149. The microorganism of any one of embodiments 107-147, wherein the genetically engineered microorganism is a yeast.

[0285] Embodiment 150. The microorganism of embodiment 149, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a

Hansenula polymorpha, a Pichia pastor is, a Saccharomyces cerevisiae, a S. bay anus, a S. K. lactis, a Waltomyces lipofer, aMortierella alpine, aMortierella isabellina, aMucor rouxii, a Trichosporon cutoneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a Schizosaccharomyces pombe.

[0286] Embodiment 151. The microorganism of embodiment 149, wherein the yeast is a Yarrowia lipolytica.

[0287] Embodiment 152. The microorganism of any one of embodiments 107-147, wherein the genetically engineered microorganism is a bacterium.

[0288] Embodiment 153. The microorganism of embodiment 152, wherein the bacterium is a Bacillus subtilis, a Salmonella sp., an Escherichia coli, a Vibrio cholerae, a Streptomyces sp., a Pseudomonas fluorescens, a Pseudomonas putida, a Pseudomonas sp., a Rhodococcus sp., or a Alcaligenes sp.

[0289] Embodiment 154. The microorganism of any one of embodiments 107-147, wherein the genetically engineered microorganism is a fungus.

[0290] Embodiment 155. The microoganism of embodiment 154, wherein the fungus is a Aspergillus shirousamii, a Aspergillus niger, or a Trichoderma reesei.

[0291] Embodiment 156. The microorganism of any one of embodiments 107-147, wherein the genetically engineered microorganism is an algae.

[0292] Embodiment 157. The microorganism of embodiment 156, wherein the algae is Neochloris oleoabundans, Scenedesmus obliquus, Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, or Spirulina maxima.

[0293] Embodiment 158. A method of producing olivetolic acid comprising: contacting the genetically modified microorganism of any one of embodiments 107-157 with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate).

[0294] Embodiment 159. The method of embodiment 158, wherein the yield of olivetolic acid is at least about 2%.

[0295] Embodiment 160. The method of embodiment 158, wherein the yield of olivetolic acid is at least 3%. [0296] Embodiment 161. The method of embodiment 158, wherein the yield of olivetolic acid is at least 4%.

[0297] Embodiment 162. The method of embodiment 158, wherein the yield of olivetolic acid is at least 5%.

[0298] Embodiment 163. The method of embodiment 158, wherein the yield of olivetolic acid is at least 6%.

[0299] Embodiment 164. The method of embodiment 158, wherein the yield of olivetolic acid is at least 7%.

[0300] Embodiment 165. The method of embodiment 158, wherein the yield of olivetolic acid is about 1% to about 30%.

[0301] Embodiment 166. The method of embodiment 158, wherein the yield of olivetolic acid is about 2% to about 15%.

[0302] Embodiment 167. The method of embodiment 158, wherein the yield of olivetolic acid is about 5% to about 10%.

[0303] Embodiment 168. The method of any one of embodiments 158-167, wherein the carbohydrate source comprises one or more fermentable sugars.

[0304] Embodiment 169. The method of any one of embodiments 158-167, wherein the carbohydrate source comprises glucose.

[0305] Embodiment 170. The method of any one of embodiments 158-169, wherein the culture conditions comprise nitrogen depletion conditions.

[0306] Embodiment 171. The method of any one of embodiments 158-170, wherein the culture conditions do not comprise an external source of hexanoic acid.

[0307] Embodiment 172. The olivetolic acid produced by the method of any one of embodiments 158-171.

[0308] Embodiment 173. The method of any one of embodiments 158-171, further comprising purifying the olivetolic acid.

[0309] Embodiment 174. The method of embodiment 173, further comprising producing cannabidiol from the purified olivetolic acid using a semisynthetic approach.

[0310] Embodiment 175. The cannabidiol produced by the method of embodiment 174.

[0311] Embodiment 176. A method of producing olivetolic acid comprising: contacting a genetically engineered microorganism comprising one or more genetic modification that enable production of olivetolic acid in the absence of an external source of hexanoic acid with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate). [0312] Embodiment 177. The method of embodiment 176, wherein the one or more genetic modifications enable production of the olivetolic acid from a carbohydrate source at an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

[0313] Embodiment 178. A method of producing olivetolic acid comprising: contacting a genetically engineered microorganism comprising one or more genetic modification that enable production of olivetolic acid from a carbohydrate source with an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate) with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate).

[0314] Embodiment 179. The method of embodiment 177 or 178, wherein the efficiency is at least 2%.

[0315] Embodiment 180. The method of embodiment 177 or 178, wherein the efficiency is at least 3%.

[0316] Embodiment 181. The method of embodiment 177 or 178, wherein the efficiency is at least 4%.

[0317] Embodiment 182. The method of embodiment 177 or 178, wherein the efficiency is at least 5%.

[0318] Embodiment 183. The method of embodiment 177 or 178, wherein the efficiency is at least 6%.

[0319] Embodiment 184. The method of embodiment 177 or 178, wherein the efficiency is at least 7%.

[0320] Embodiment 185. The method of embodiment 177 or 178, wherein the efficiency is about 1% to about 30%.

[0321] Embodiment 186. The method of embodiment 177 or 178, wherein the efficiency is about 2% to about 15%.

[0322] Embodiment 187. The method of embodiment 177 or 178, wherein the efficiency is about 5% to about 10%.

[0323] Embodiment 188. The method of any one of embodiments 176-187, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA

Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or a combination thereof relative to an unmodified microorganism of the same species. [0324] Embodiment 189. The method of any one of embodiments 176-187, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA

Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), and an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

[0325] Embodiment 190. The method of any one of embodiments 176-189, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP) relative to an unmodified microorganism of the same species.

[0326] Embodiment 191. The method of embodiment 190, wherein the FASa and FASP are hexanoic acid specific Type I fatty acid synthases.

[0327] Embodiment 192. The method of any one of embodiments 190-191, wherein the FASa and FASP are from an Aspergillus species.

[0328] Embodiment 193. The method of any one of embodiments 190-191, wherein the FASa and FASP are from an Aspergillus parasiticus species.

[0329] Embodiment 194. The method of any one of embodiments 190-191, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 5, a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 7, or both.

[0330] Embodiment 195. The method of any one of embodiments 190-191, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%) identical to SEQ ID NO: 6, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 8, or both.

[0331] Embodiment 196. The method of any one of embodiments 194-195, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0332] Embodiment 197. The method of any one of embodiments 176-196, wherein the one or more genetic modifications increase the expression of an ATP Citrate Lyase (ACL) relative to an unmodified microorganism of the same species.

[0333] Embodiment 198. The method of embodiment 197, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 1.

[0334] Embodiment 199. The method of embodiment 197, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 2. [0335] Embodiment 200. The method of any one of embodiments 198-199, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0336] Embodiment 201. The method of any one of embodiments 176-200, wherein the one or more genetic modifications increase the expression of an Acetyl-coA Carboxylase (ACC) relative to an unmodified microorganism of the same species.

[0337] Embodiment 202. The method of embodiment 201, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 3.

[0338] Embodiment 203. The method of embodiment 201, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 4.

[0339] Embodiment 204. The method of any one of embodiments 202-203, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0340] Embodiment 205. The method of any one of embodiments 176-204, wherein the one or more genetic modifications increase the expression of a polyketide synthase (PKS) relative to an unmodified microorganism of the same species.

[0341] Embodiment 206. The method of embodiment 205, wherein the PKS is from a Cannabis species.

[0342] Embodiment 207. The method of embodiment 205, wherein the PKS is from a Cannabis sativa species.

[0343] Embodiment 208. The method of embodiment 205, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80%> identical to an open reading frame of SEQ ID NO: 11.

[0344] Embodiment 209. The method of embodiment 205, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80%> identical to SEQ ID NO: 12.

[0345] Embodiment 210. The method of any one of embodiments 208-209, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0346] Embodiment 211. The method of any one of embodiments 176-210, wherein the one or more genetic modifications increase the expression of an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

[0347] Embodiment 212. The method of embodiment 211, wherein the OAC is from a Cannabis species. [0348] Embodiment 213. The method of embodiment 211, wherein the OAC is from a Cannabis sativa species.

[0349] Embodiment 214. The method of embodiment 211, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 13.

[0350] Embodiment 215. The method of embodiment 211, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 14.

[0351] Embodiment 216. The method of any one of embodiments 214-215, wherein the polynucleotide is integrated into the genetically engineered microorganism's genome.

[0352] Embodiment 217. The method of any one of embodiments 176-216, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae.

[0353] Embodiment 218. The method of any one of embodiments 176-216, wherein the genetically engineered microorganism is a yeast.

[0354] Embodiment 219. The method of embodiment 218, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a Hansenula polymorpha, a Pichia pastoris, a Saccharomyces cerevisiae, a S. bayanus, a S. K. lactis, a Waltomyces lipofer, a Mortierella alpine, a Mortierella isabellina, a Mucor rouxii, a

Trichosporon cutaneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a Schizosaccharomyces pombe.

[0355] Embodiment 220. The method of embodiment 218, wherein the yeast is a Yarrowia lipolytica.

[0356] Embodiment 221. The method of any one of embodiments 176-216, wherein the genetically engineered microorganism is a bacterium.

[0357] Embodiment 222. The method of embodiment 222, wherein the bacterium is a Bacillus subtilis, a Salmonella sp., an Escherichia coli, a Vibrio cholerae, a Streptomyces sp., a Pseudomonas fluorescens, a Pseudomonas putida, a Pseudomonas sp., a Rhodococcus sp., or a Alcaligenes sp.

[0358] Embodiment 223. The method of any one of embodiments 176-216, wherein the genetically engineered microorganism is a fungus.

[0359] Embodiment 224. The microoganism of embodiment 223, wherein the fungus is a Aspergillus shirousamii, a Aspergillus niger, or a Trichoderma reesei. [0360] Embodiment 225. The method of any one of embodiments 176-216, wherein the genetically engineered microorganism is an algae.

[0361] Embodiment 226. The method of embodiment 225, wherein the algae is Neochloris oleoabundans, Scenedesmus obliquus, Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, or Spirulina maxima.

[0362] Embodiment 227. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least about 2%.

[0363] Embodiment 228. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least 3%.

[0364] Embodiment 229. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least 4%.

[0365] Embodiment 230. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least 5%.

[0366] Embodiment 231. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least 6%.

[0367] Embodiment 232. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is at least 7%.

[0368] Embodiment 233. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is about 1% to about 30%.

[0369] Embodiment 234. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is about 2% to about 15%.

[0370] Embodiment 235. The method of any one of embodiments 176-226, wherein the yield of olivetolic acid is about 5% to about 10%.

[0371] Embodiment 236. The method of any one of embodiments 176-235, wherein the carbohydrate source comprises one or more fermentable sugars.

[0372] Embodiment 237. The method of any one of embodiments 176-235, wherein the carbohydrate source comprises glucose.

[0373] Embodiment 238. The method of any one of embodiments 176-237, wherein the culture conditions comprise nitrogen depletion conditions.

[0374] Embodiment 239. The method of any one of embodiments 176-238, wherein the culture conditions do not comprise an external source of hexanoic acid.

[0375] Embodiment 240. The olivetolic acid produced by the method of any one of embodiments 176-239. [0376] Embodiment 241. The method of any one of embodiments 176-239, further comprising purifying the olivetolic acid.

[0377] Embodiment 242. The method of embodiment 241, further comprising producing cannabidiol from the purified olivetolic acid using a semisynthetic approach.

[0378] Embodiment 243. The cannabidiol produced by the method of embodiment 242.

[0379]

EXAMPLES

[0380] Example 1: Production of cannabinoid precursors from Yarrowia lipolytica.

[0381] The production of cannabinoids and cannabinoid precursors from genetically-modified microbe is summarized in FIG. 4. First, a suitable microorganism that has been genetically- modified is fermented in a culture to generate cell mass. The cell mass containing the

cannabinoid product is then isolated from the fermentation broth through centrifugation. The cell mass was further processed by cell lysis. The cannabinoid product is then separated from components of the cell mass by various separation and distillation methods.

Generating genetically-modified Y. lipolytica

[0382] The expression vector, pYLEXl, is used for transgene expression in Y. lipolytica. The respective genes are cloned into the pYLEX plasmid between Pmll and Kpn restriction sites. All cDNA can be sequenced and mapped to genomic databases. Exemplary, representative sequence database entries to include Mus musculus (mouse) ACC (GenelD: 107476) and Homo sapiens (human) ACL (GenelD: 47) in Y. lipolytica. FAS alpha and beta, PKS, HS, and OAC genes are synthesized in vitro and cloned into the pYLEX plasmid for direct genomic integration using homologous recombination.

fPropagation

[0383] Propagation utilizes a seed culture grown on a rich media to create initial cell mass to eventually pitch into the fermentation broth. Propagation requires a rich media, whereas the fermentation media only requires minimal media to support growth of yeast in a way to allow efficient catabolism of sugar to a cannabinoid product. The formation of cell mass limits product generation by diverting cellular energy to making new cells rather than synthesizing the desired cannabinoid product.

[0384] The engineered yeast is grown in either YPD full media containing yeast extract, peptone, and dextrose, or YNB minimal media containing all nutrients except amino acids, nitrogen, and carbon. When grown in YNB media, nitrogen is provided as ammonium sulfate and carbon was provided as glucose at a carbon to nitrogen ratio of 75-150. This carbon-to-nitrogen ratio is necessary for triggering oil accumulation. Upon depletion of the nitrogen source, excess sugar is channeled to lipogenesis and lipid accumulation.

[0385] Yeast strains are maintained on YPD slants containing 1% yeast extract, 2% peptone, and 2% glucose and stored at 4 °C prior to use. A small amount of cells from the slants is re- suspended in water, followed by centrifugation to remove any media components. This cell suspension is used as the inoculum for seed culture (2 mL) and other succeeding growth experiments.

[0386] Next, 2 mL of the seed is inoculated in 50 mL of fermentation medium at 30 °C in 125 mL flasks and grown for 24 hours. The C/N 75 seed media (75 mol C/mol N) contains 33 g/L aqueous glucose, 0.139 g/L NH₄C1, 1.5 g/1 yeast extract, 3.2 g/L KH₂P0₄, and 1.0 g/L

MgS0₄-7H₂0. Media ingredients are sterile-filtered separately from glucose, which is sterilized by autoclaving at 121 °C for 20 min. Biotin is sterile-filtered and added to the media at a concentration of 0.02 mg/L. Adjustment of the C/N content of the medium is accomplished by decreasing the NH₄C1 content. A C/N ratio of 75-90 mol C/mol N can be obtained as needed without varying the yeast extract content.

[0387] After 24 hours, 10 mL of growth phase culture obtained from a single colony is used to inoculate 100 mL of C/N 75 medium. The fermentation media contains 5.0 g/L (NH₄)₂S0₄, 3.0 g/L KH₂P0₄, 0.5 g/L MgS0₄-7H₂0, 0.05 mL/L Antifoam 298 (Sigma-Aldrich), 1 mL/L trace metal solution containing 3 g/L FeS0₄-7H₂0, 4.5 g/L ZnS0₄-7H₂0, 4.5 g/L CaCl₂-6H₂0, 0.84 g/L MnCl₂-2H₂0, 0.3 g/L CoCl₂-6H₂0, 0.3 g/L CuS0₄-5H₂0, 0.4 g/L Na₂Mo0₄-2H₂0, 1 g/L H₃BO₃, 0.1 g/L KI, 15 g/L Na₂EDTA-2H₂0, and 1 mL/L vitamin solution containing 25 mg/L D-biotin, 0.5 g/L calcium pantothenate, 0.5 g/L thiamine HC1, 0.5 g/L pyridoxine HC1, 0.5 g/L nicotinic acid, 0.1 g/L p-aminobenzoic acid, and 12.5 g/L m-inositol. All chemicals are of analytical grade.

Fermentation

[0388] After the propagation of the inoculum, the engineered Y. lipolytica is pitched into the 1 L fermenter. The inoculum culture is fermented at 30 °C with 250 rpm agitation. After 24 hours, the inoculum is transferred into a 10-L fermenter and fermented until the OD reached 135 within 120 hours. Fed-batch fermentation is performed in 1-10 L BioFlo 115 fermenter using the C/N 75 medium. The fermentation is maintained at 30 °C for 72-120 hours. During fermentation, the pH is maintained at 6.5 and adjusted by adding 2 M NaOH or 1 M H₃P0₄. After depletion of the initial glucose concentration to 10 g/L, a glucose bolus of 50 g/L is established. The aeration is maintained at 0.9 vvm and dissolved oxygen concentration is at about 40-50% saturation.

Throughout fermentation, 4 mL samples are removed from the culture for analysis and extraction.

[0389] After the 72-120 hour period, the fermentation is terminated. While the product itself can inhibit growth and product formation after the product reaches a certain concentration in the wild-type organism, product inhibition is not observed during fermentation of the engineered strain.

Product extraction

[0390] A 50-mL sample of the culture broth is centrifuged for 20 min and the collected wet cells are washed twice with distilled water. The cell dry mass is determined by drying the washed cells and drying the biomass in an oven at 60 °C until a constant mass is achieved. The cells are lysed by adding 2 M HC1 with the dried cells in capped glass tubes and mixing. The mixture is incubated overnight at 50 °C. The product is isolated by solvent extracting by adding 5 mL of ethyl acetate and vortexing, followed by centrifugation at 2000 g for 5 min. The top layer is then transferred to another tube and the excess ethyl acetate is evaporated by an air stream at 50 °C. After drying is completed, 100 μL of ethyl acetate is added to dissolve the isolated residue for further analysis. Results from HPLC profiling of the isolated residue are shown in FIG. 11.

[0391] Separately, the top layer supernatant is acidified and extracted with ethyl acetate in a liquid-liquid extraction process. A 1 :3 ratio of watenethyl acetate mixture is used for extraction and the mixture was stirred for 4 hours at room temperature. The resulting top layer containing ethyl acetate is evaporated and the isolated residue is re-suspended in the dried cell ethyl acetate extract for final analytical profiling.

[0392] Example 2: Strain engineering for the production of short-chain fatty acid.

[0393] Wild-type yeast (e.g., Y. lipolytica) do not normally produce shorter chain fatty acids (e.g., C6 or C8 fatty acids). This experiment was conducted to show that expression of heterologous and codon-optimized Fatty Acid Synthase (FAS) alpha and beta genes can cause such yeast to produce a fatty acid profile that is conducive to production of cannabinoid precursors, cannabinoids, cannabinoid derivatives, or a combination thereof. Genetically- engineered Y. lipolytica yeast was produced essentially as described. This strain contains a genomic integration of heterologous and codon-optimized Fatty Acid Synthase (FAS) alpha and beta genes.

[0394] This experiment was conducted under nitrogen depleting growth conditions to promote cellular fatty acid production. The setup includes shake flasks in duplicate and the genetically- engineered strain was grown in Y B media (pH 7.0) without amino acids (yeast extract, ammonium sulfate and dextrose) at about 30 °C. This time-course experiment was designed such that the yeast cells were expected to enter stationary phase metabolism in about 72 hours. This is the stage where maximum cellular fatty acid production is usually seen in oleaginous yeast such a Y. lipolytica.

[0395] As shown in FIG. 6, a genetically-engineered yeast strain that expresses heterologous FASa and FASP was capable of producing both C6 and C8 fatty acids. The data shown in FIG. 6 was from a sample taken after about 114 hours of growth. The over-expressed FAS genes alters the total fatty acid profile of the yeast and produces significant quantities of cellular C6 fatty acid which can be used for the de novo synthesis of, for example, the cannabinoid precursor olivetolic acid.

[0396] Example 3: Biological characterization of the genetically-engineered Y. lipolytica with overexpressed ACL, ACC, and FAS alpha and beta genes.

[0397] A genetically-modified strain of Y. lipolytica with overexpressed ACL, ACC, and FAS alpha and beta genes was prepared using methods described above.

Cannabinoid Toxicity

[0398] The engineered strain was assessed for growth toxicity to high concentrations of cannabidiol (CBD) by measuring the optical density of yeast cells from the engineered strain and the wild-type strain grown on glucose and treated with 5 g/L CBD. FIG. 7 shows the optical density (OD) at 600 nm of the engineered strain (Engineered) compared to the unmodified, wild- type strain (WT). The optical density pattern of the engineered strain and the wild-type strain was essentially identical with an exponential phase between 0 and about 48 hours followed by a stationary phase/death phase in OD between about 48 and 96 hours. However, the engineered strain exhibited a higher maximum OD of about 3.5-4.5, compared to the wild-type strain, which exhibited a maximum OD of about 1.8-2.3. Despite the difference in growth capacities, both yeast strains were able to grow and sustain in high concentrations of CBD. The growth profiles suggest that both oleaginous yeast strains were resistant to CBD toxicity. The higher growth rate of the engineered strain suggests that the engineered strain exhibited greater growth efficiency than the wild-type strain.

Biomass Production

[0399] Biomass production was assessed by measuring the dry cell mass (DCW) of yeast cells grown from the engineered strain and the wild-type strain on glucose over time. FIG. 8 shows the biomass production of genetically-engineered yeast (Engineered) compared to the unmodified, wild-type strain (WT). DCW was measured as grams per liter of media (g/L) at 24 hour-time points after an initial time point of 48 hours.

[0400] The engineered strain exhibited an exponential biomass increase from about 20 g/L to about 28 g/L between about 48 hours and 55 hours, followed by a short 24-hour stationary phase and another exponential increase from about 28 g/L to about 40 g/L between about 96 hours and 144 hours. The engineered strain exhibited a maximum biomass content of about 40 g/L during the stationary phase after about 144 hours.

[0401] The wild-type strain exhibited an exponential biomass increase from about 6 g/L to about 17 g/L between about 48 hours and 120 hours, followed by a short 24-hour stationary phase and a slow biomass decline after 144 hours.

[0402] The engineered strain exhibited two growth phases that collectively resulted in a maximum biomass content of about 40 g/L after 150 hours. The wild-type strain exhibited only one growth phase that resulted in a maximum biomass content of about 17 g/L after 120 hours. The engineered strain had greater biomass productivity and a longer growth phase than the wild- type strain. The biomass production profiles suggest that the overexpression of ACL, ACC, and FAS alpha and beta genes resulted in a growth advantage.

Lipid Production

[0403] Lipid production was assessed by measuring the total lipid content (triglycerides) of yeast cells grown from the engineered strain and the wild-type strain on glucose over time. FIG. 9 shows the lipid production of genetically-engineered yeast (Engineered) compared to the unmodified, wild-type strain (WT) when grown on glucose. Lipid content was measured as percentage mass by mass (% w/w) at 24 hour-time points after an initial time point of 48 hours.

[0404] The engineered strain exhibited an initial lag phase between 48 hours and 72 hours, followed by an exponential lipid content increase from about 25% w/w to about 60% w/w between about 72 hours and 168 hours. The engineered strain produced a maximum lipid content of about 60% w/w during the stationary phase after about 150 hours.

[0405] The wild-type strain exhibited linear lipid accumulation phase from 48 hours to about 96 hours, followed by a stationary phase/decline phase in lipid production from about 96 hours to about 192 hours.

[0406] The engineered strain exhibited a substantial increase in lipid content between about 72 hours and 168 hours with a maximum lipid content of about 60% w/w, whereas the wild-type strain exhibited only a modest increase in lipid content between about 48 hours and 96 hours. The engineered strain accumulated a substantially greater concentration of lipid than the wild- type strain. These results suggest that that the overexpression of ACL, ACC, and FAS alpha and beta genes leads to efficient lipid accumulation. [0407] Example 4: Biological characterization of the genetically-engineered Y. lipolytica with overexpressed ACL, ACC, FAS alpha and beta genes, PKS, OAC, and HS.

[0408] A genetically-modified strain of Y. lipolytica with overexpressed H. sapiens ACL, M musculus ACC, A. parasiticus FAS alpha and beta genes, C. sativa PKS, C. sativa OAC, and C. sativa HS was prepared using methods described above.

[0409] The fermentation profile of the genetically-engineered yeast was characterized by measuring the optical density (OD) at 600 nm and glucose substrate concentration over time. FIG. 10 illustrates biomass profile (left) and glucose substrate profile (right) of the genetically- engineered yeast grown in 10 L culture on glucose under aerobic conditions. The biomass profile (left panel) showed an initial lag phase during the first 16 hours, followed by an exponential growth phase between about 16 hours and about 48 hours. This exponential growth was predominantly attributed to initial cell growth prior to lipid synthesis. At 48 hours, lipid synthesis was initiated by nitrogen depletion (vertical gray line). After about 48 hours after lipid synthesis was triggered, the biomass profile exhibited two humped growth phases. The first hump exhibited a maximum OD₆oo of about 200 and the second hump exhibited a maximum OD₆oo of about 235. This growth pattern suggests that the engineered strain has sustained biomass production, particularly for lipid biosynthesis. The fatty acid flux is maximum during later growth stage (second hump), which suggested that the cells are able produce the target metabolite (C6 fatty acids) in large quantities when cells are not in growth phase. This growth pattern can be advantageous because the cells can accumulate high cellular biomass necessary to produce C6 fatty acids and other end products, and the potential toxicity from short-chain fatty acids and other end products can be circumvented. The fermentation profile demonstrated that an engineered strain can be grown to high densities and fatty acid synthesis can be triggered by nitrogen depletion to produce an essential early component of the cannabinoid pathway, i.e. hexanoic acid. The C6 fatty acid can then be utilized to produce the olivetolic acid, the first committed precursor of cannabinoid pathway.

[0410] The glucose substrate formation profile (right) exhibited fluctuating consumption and formation patterns between 0 hours and about 60 hours. After about 60 hours, the glucose substrate profile exhibited a decline phase from about 78 g/L to about 0 g/1 at about 96 hours. The glucose substrate profile suggested that the engineered strain has robust growth properties with rapid consumption of glucose at about 80 hours consisting of multiple phases of glucose consumption and formation. The fluctuations in substrate profile were consistent with the biomass profile. For example, the initial 20 hours of growth is characterized by increasing OD₆oo and decreasing glucose concentration. This observation suggested that glucose was being consumed for cell production without cellular imbalances in engineered strain. The humped growth phases that followed can be attributed to formation of lipids, adaption to an inhibitory lipid synthesis intermediate, and subsequent resolution of the inhibition. Subsequent resolution of the growth inhibition, characterized by the valley between the two humps, can be attributed to an adaptation process that enhanced the ability of the yeast to produce intracellular lipids and store hydrophobic target metabolites in lipids and overcome short-chain fatty acid and/or cannabinoid or derivatives toxicity.

Fluorescent Imaging

[0411] Fluorescent microscopic imaging was used to visualize the wild-type and engineering yeast cells. Results are shown in FIG. 12. The cells were grown on glucose for 96 hours under nitrogen-limiting conditions. The oleaginous cells were stained with a lipophilic stain, Nile Red. The wild-type yeast cells (A) exhibited about 2-4 cytoplasmic lipid droplets that are

characterized by the bright white dots. The engineered yeast cells (B) exhibited one large lipid droplet that encompassed almost the entirety of cytoplasmic space. These images suggest that the engineered yeast cells had significantly greater ability for lipid accumulation and lipid storage capacity than the wild-type cells.

[0412] Example 5: Generating genetically-modified Y. lipolytica that produces CBGA.

[0413] The expression vector, pYLEXl, will be used for transgene expression in Y. lipolytica. The respective genes will be cloned into the pYLEX plasmid between Pmll and Kpn restriction sites. All cDNA will be sequenced and mapped to genomic databases. Exemplary, representative sequence database entries to include Mus musculus (mouse) ACC (GenelD: 107476) and Homo sapiens (human) ACL (GenelD: 47) in Y. lipolytica. FAS alpha and beta, PKS, HS, OAC, FDVIGRl, IDIl, GPPS, and GOGT genes will be synthesized in vitro and cloned into the pYLEX plasmid for direct genomic integration using homologous recombination.

[0414] Example 6: Generating genetically-modified Y. lipolytica that produces THCA, CBDA, CBCA.

[0415] The expression vector, pYLEXl, will be used for transgene expression in Y. lipolytica. The respective genes will be cloned into the pYLEX plasmid between Pmll and Kpn restriction sites. All cDNA will be sequenced and mapped to genomic databases. Exemplary, representative sequence database entries to include Mus musculus (mouse) ACC (GenelD: 107476) and Homo sapiens (human) ACL (GenelD: 47) in Y. lipolytica. FAS alpha and beta, PKS, HS, OAC, HMGRl, IDIl, GPPS, GOGT, THCS, CBDS, and CBCS genes will be synthesized in vitro and cloned into the pYLEX plasmid for direct genomic integration using homologous recombination.

[0416] Example 7: Generating genetically-modified Y. lipolytica that produces 11-OH-THC.

[0417] The expression vector, pYLEXl, will be used for transgene expression in Y. lipolytica. The respective genes will be cloned into the pYLEX plasmid between Pmll and Kpn restriction sites. All cDNA will be sequenced and mapped to genomic databases. Exemplary, representative sequence database entries to include Mus musculus (mouse) ACC (GenelD: 107476) and Homo sapiens (human) ACL (GenelD: 47) in Y. lipolytica. FAS alpha and beta, PKS, HS, OAC, HMGRl, IDIl, GPPS, GOGT, THCS, CBDS, CBCS, and p450 genes will be synthesized in vitro and cloned into the pYLEX plasmid for direct genomic integration using homologous recombination.

[0418] Example 8: Solubility of olivetolic acid.

[0419] The solubility of olivetolic acid was studied using various solvents, including water, chloroform, methanol, ethyl acetate, and vegetable cooking oil (canola oil). The identity and purity of olivetolic acid was confirmed by MR and MS. Physical properties of some common organic solvents are shown in TABLE 14.

TABLE 14

M, completely miscible.

[0420] 2.5 mL of canola oil (Wesson, commercial-grade) was added to 3.9 mg of olivetolic acid (Santa Cruz Biotech, SC-484998). The olivetolic acid solid was gradually dissolved with stirring, and after 5 minutes, a clear solution was obtained.

[0421] 1 mL of water (distilled) was added to 5.5 mg of olivetolic acid. After 5 min of stirring, a suspension was obtained. After an additional 10 min of stirring, no change was observed. A few drops of a saturated aqueous sodium bicarbonate solution were then added, resulting in a clear solution. The pH of the clear solution was pH 8.

[0422] 1.0 mL of ethyl acetate (reagent grade) was added to 5.6 mg of olivetolic acid. A clear solution was obtained immediately. [0423] 1.5 mL of chloroform (reagent grade) was slowly added to 4.0 mg of olivetolic acid. After about 0.5-0.75 mL of solvent was added, a suspension was obtained. After the additional of 1.5 mL, a clear solution was obtained.

[0424] 0.25 mL of methanol (reagent grade) was added to 3.8 mg of olivetolic acid. A clear solution was obtained immediately.

[0425] Conclusions: Olivetolic acid was most soluble in methanol, followed by ethyl acetate, chloroform, and canola oil. Olivetolic acid was insoluble in water, but was freely soluble in dilute aqueous base at pH 8.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A genetically engineered microorganism comprising one or more genetic

modifications that increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP) relative to a microorganism of the same species without the one or more genetic modifications, wherein the genetically modified microorganism has increased production of hexanoic acid relative to an unmodified organism of the same species.

2. The microorganism of claim 1, wherein the FASa and FASP are hexanoic acid specific Type I fatty acid synthases.

3. The microorganism of any one of claims 1-2, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 5, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 7, or both.

4. The microorganism of any one of claims 1-2, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 6, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 8, or both.

5. The microorganism of any one of claims 3-4, wherein the polynucleotide is integrated into the genetically modified microorganism's genome.

6. The microorganism of any one of claims 1-5, further comprising one or more genetic modifications that increase the expression of an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), or both relative to a microorganism of the same species with the one or more genetic modifications.

7. The microorganism of claim 6, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 1, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 3, or both.

8. The microorganism of claim 6, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 2, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 4, or both.

9. The microorganism of any one of claims 1-8, wherein the microorganism does not comprise a genetic modification that increases expression of a stearoyl-CoA desaturase (SCD).

10. The microorganism of any one of claims 1-9, wherein the microorganism does not comprise a genetic modification that increases expression of a diacylglycerol acyltransferase (DGA1).

11. The microorganism of any one of claims 1-10, further comprising one or more genetic modifications that increase the expression of a hexanoate synthase (HS) relative to a microorganism of the same species with the one or more genetic modifications.

12. The microorganism of claim 11, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 9.

13. The microorganism of claim 11, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide at least 80% identical so SEQ ID NO: 10.

14. The microorganism of any one of claims 11-13, wherein the genetically modified microorganism has increased production of hexanoyl-CoA relative to a microorganism of the same species without the genetic modifications that increase the expression of the HS.

15. The microorganism of any one of claims 1-14, further comprising one or more genetic modifications that increase the expression of a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or both relative to a microorganism of the same species with the one or more genetic modifications.

16. The microorganism of claim 15, wherein the one or more genetic modifications comprise a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 11, a polynucleotide that is at least 80% identical to an open reading frame of SEQ ID NO: 13, or both.

17. The microorganism of claim 15, wherein the one or more genetic modifications comprise a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 12, a polynucleotide that encodes a polypeptide that is at least 80% identical to SEQ ID NO: 14, or both.

18. The microorganism of any one of claims 15-17, wherein the genetically modified microorganism has increased production of olivetolic acid relative to a microorganism of the same species without the genetic modifications that increase the expression of the PKS, the OAC, or both.

19. The microorganism of any one of claims 1-18, further comprising one or more genetic modifications that increase the expression of a HMG-CoA Reductase 1 (HMGR1), an isopentenyl-diphosphate delta isomerase 1 (IDI1), a geranyl pyrophosphate synthase (GPPS), a farnesyl pyrophosphate synthase (FPPS), a mutated farnesyl pyrophosphate synthase (mFPPS), a geranylpyrophosphate olivetolate geranyltransferase (GOGT), or a combination thereof relative to a microorganism of the same species with the one or more genetic modifications.

20. The microorganism claim 19, wherein the genetically modified microorganism has increased production of cannabigerolic acid (CBGA) relative to a microorganism of the same species without the genetic modifications that increases the expression of the HMGR1, IDI1, GPPS, FPPS, mFPPS, GOGT, or a combination thereof.

21. The microorganism of any one of claims 1-20, further comprising one or more genetic modifications that increase the expression of a tetrahydrocannabidiol synthase (THCS), a cannabidiol synthase (CBDS), cannabichromene synthase (CBCS), or a combination thereof relative to a microorganism of the same species with the one or more genetic modifications.

22. The microorganism of claim 21, wherein the genetically modified microorganism has increased production of A⁹-tetrahydrocannabinolic acid (THCA), A⁹-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabichromenic acid (CBCA), cannabichromene (CBC), or a combination thereof relative to a microorganism of the same species without the genetic modifications that increase the expression of the THCS, CBDS, CBCS, or a combination thereof.

23. The microorganism of any one of claims 1-22, wherein the genetically modified microorganism further comprises one or more genetic modifications that increase the expression of one or more cytochrome P450 enzymes relative to a microorganism of the same species with the one or more genetic modifications.

24. The microorganism of claim 23, wherein the one or more cytochrome P450 enzymes comprise cytochrome P450 2C9 (CYP2C9), cytochrome P450 3 A4 (CYP3A4), or a combination thereof.

25. The microorganism of claim 23 or 24, wherein the genetically modified

microorganism has increased production of one or more cannabinoid derivatives relative to a microorganism of the same species without the genetic modifications that increase the expression of the one or more cytochrome P450 enzymes.

26. The microorganism of claim 25, wherein the one or more cannabinoid derivatives comprise 1 l-OH-A⁹-THC.

27. The microorganism of any one of claims 1-26, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae.

28. The microorganism of any one of claims 1-26, wherein the genetically engineered microorganism is a yeast.

29. The microorganism of 28, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a Hansenula polymorpha, a Pichia pastoris, a Saccharomyces cerevisiae, a S. bay anus, a S. K. lactis, a Waltomyces lipofer, a Mortierella alpine, a Mortierella isabellina, aMucor rouxii, a Trichosporon cutaneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a

Schizosaccharomyces pombe.

30. The microorganism of 28, wherein the yeast is Yarrowia lipolytica.

31. A method of producing one or more fermentation end-products comprising contacting the genetically engineered microorganism of any one of claims 1-30 with a carbohydrate source under culture conditions and for a time sufficient to produce the one or more fermentation end products.

32. The method of claim 31, wherein the one or more fermentation end-products comprise one or more cannabinoid precursors, one or more cannabinoids, one or more

cannabinoid derivatives, or a combination thereof.

33. The method of claim 32, wherein the one or more fermentation end products comprise the one or more cannabinoid precursors that are hexanoic acid, hexanoyl-CoA, olivetolic acid, geranyldiphosphase, cannabigerolic acid (CBGA), or a combination thereof.

34. The method of claim 33, wherein the one or more fermentation end products comprise the cannabinoid precursor olivetolic acid.

35. The method of claim 33-34, further comprising synthesizing one or more

cannabinoids from the cannabinoid precursor.

36. The method of claim 35, wherein the one or more cannabinoids comprise

cannabigerolic acid (CBGA), A⁹-tetrahydrocannabinolic acid (THCA), A⁹-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabichromenic acid (CBCA), cannabichromene (CBC), or a combination thereof.

37. The method of claim 35, wherein the one or more cannabinoids comprise cannabidiol (CBD), A⁹-tetrahydrocannabinol (THC), cannabichromene (CBC), or a combination thereof.

38. The method of claim 35, wherein the one or more cannabinoids comprise cannabidiol

(CBD).

39. The method of claim 32, wherein the one or more fermentation end-products comprise the one or more cannabinoids that are cannabigerolic acid (CBGA), Δ⁹- tetrahydrocannabinolic acid (THCA), A⁹-tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabichromenic acid (CBCA), cannabichromene (CBC), or a combination thereof.

40. The method of claim 32, wherein the one or more fermentation end-products comprise the one or more cannabinoid derivatives that are l l-OH-A⁹-THC.

41. The method of any one of claims 31-40, wherein the carbohydrate source comprises one or more fermentable sugars.

42. The method of claim 41, wherein the one or more fermentable sugars comprise glucose.

43. The method of any one of claims 31-42, wherein the culture conditions comprise nitrogen depletion conditions.

44. The fermentation end-product produced by the method of any one of claims 31-43.

45. A genetically engineered microorganism comprising one or more genetic

modification that enable production of olivetolic acid in the absence of an external source of hexanoic acid.

46. The microorganism of claim 45, wherein the one or more genetic modifications enable production of the olivetolic acid from a carbohydrate source at an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

47. A genetically engineered microorganism comprising one or more genetic

modifications that enable production of olivetolic acid from a carbohydrate source with an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

48. The microorganism of claim 46 or 47, wherein the efficiency is at least 5%.

49. The microorganism of claim 46 or 47, wherein the efficiency is about 1% to about

30%.

50. The microorganism of claim 46 or 47, wherein the efficiency is about 2% to about

15%.

51. The microorganism of any one of claims 45-50, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or a combination thereof relative to an unmodified microorganism of the same species.

52. The microorganism of any one of claims 45-50, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), and an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

53. The microorganism of any one of claims 45-52, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae.

54. The microorganism of any one of claims 45-52, wherein the genetically engineered microorganism is a yeast.

55. The microorganism of claim 54, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a Hansenula polymorpha, a Pichia pastoris, a Saccharomyces cerevisiae, a S. bayanus, a S. K. lactis, a Waltomyces lipofer, a Mortierella alpine, aMortierella isabellina, aMucor rouxii, a Trichosporon cutoneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a Schizosaccharomyces pombe.

56. The microorganism of claim 54, wherein the yeast is a Yarrowia lipolytica.

57. A method of producing olivetolic acid comprising: contacting the genetically modified microorganism of any one of claims 45-56, with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate).

58. The method of claim 57, wherein the yield of olivetolic acid is at least 5%.

59. The method of claim 57, wherein the yield of olivetolic acid is about 1% to about

30%.

60. The method of claim 57, wherein the yield of olivetolic acid is about 2% to about

15%.

61. The method of any one of claims 57-60, wherein the carbohydrate source comprises one or more fermentable sugars.

62. The method of any one of claims 57-60, wherein the carbohydrate source comprises glucose.

63. The method of any one of claims 57-62, wherein the culture conditions comprise nitrogen depletion conditions.

64. The method of any one of claims 57-63, wherein the culture conditions do not comprise an external source of hexanoic acid.

65. The olivetolic acid produced by the method of any one of claims 57-64.

66. The method of any one of claims 57-64, further comprising purifying the olivetolic acid.

67. The method of claim 66, further comprising producing cannabidiol from the purified olivetolic acid using a semisynthetic approach.

68. The cannabidiol produced by the method of claim 67.

69. A method of producing olivetolic acid comprising: contacting a genetically engineered microorganism comprising one or more genetic modification that enable production of olivetolic acid in the absence of an external source of hexanoic acid with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate).

70. The method of claim 69, wherein the one or more genetic modifications enable production of the olivetolic acid from a carbohydrate source at an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate).

71. A method of producing olivetolic acid comprising: contacting a genetically engineered microorganism comprising one or more genetic modification that enable production of olivetolic acid from a carbohydrate source with an efficiency of at least 1% on a weight basis (g olivetolic acid/g carbohydrate) with a carbohydrate source under culture conditions and for a time sufficient to produce olivetolic acid in a yield that is at least about 1% on a weight basis (g olivetolic acid/ g carbohydrate).

72. The method of claim 70 or 71, wherein the efficiency is at least 5%.

73. The method of claim 70 or 71, wherein the efficiency is about 1% to about 30%.

74. The method of claim 70 or 71, wherein the efficiency is about 2% to about 15%.

75. The method of any one of claims 69-74, wherein the one or more genetic modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), an olivetolic acid cyclase (OAC), or a combination thereof relative to an unmodified microorganism of the same species.

76. The method of any one of claims 69-74, wherein the one or more genetic

modifications increase expression of a Type I Fatty Acid Synthase alpha (FASa) and a Fatty Acid Synthase beta (FASP), an ATP Citrate Lyase (ACL), an Acetyl-coA Carboxylase (ACC), a hexanoate synthase (HS), a polyketide synthase (PKS), and an olivetolic acid cyclase (OAC) relative to an unmodified microorganism of the same species.

77. The method of any one of claims 69-76, wherein the genetically engineered microorganism is a fungus, a bacterium, or an algae.

78. The method of any one of claims 69-76, wherein the genetically engineered microorganism is a yeast.

79. The method of claim 78, wherein the yeast is a Yarrowia lipolytica, a Cryptococcus curvatus, a Lipomyces starkeyi, a Rhodosporidium toruloides, a Trichosporon fermentans, a Trichosporon pullulan, a Lipomyces lipofer, a Hansenula polymorpha, a Pichia pastoris, a Saccharomyces cerevisiae, a S. bay anus, a S. . lactis, a Waltomyces lipofer, a Mortierella alpine, a Mortierella isabellina, a Mucor rouxii, a Trichosporon cutaneu, a Rhodotorula glutinis, a Saccharomyces diastasicus, a Schwanniomyces occidentalis, Pichia stipitis, or a

Schizosaccharomyces pombe.

80. The method of claim 78, wherein the yeast is a Yarrowia lipolytica.

81. The method of any one of claims 69-80, wherein the yield of olivetolic acid is at least about 2%.

82. The method of any one of claims 69-80, wherein the yield of olivetolic acid is at least

5%.

83. The method of any one of claims 69-80, wherein the yield of olivetolic acid is about 1% to about 30%.

84. The method of any one of claims 69-80, wherein the yield of olivetolic acid is about 2% to about 15%.

85. The method of any one of claims 69-84, wherein the carbohydrate source comprises one or more fermentable sugars.

86. The method of any one of claims 69-84, wherein the carbohydrate source comprises glucose.

87. The method of any one of claims 69-86, wherein the culture conditions comprise nitrogen depletion conditions.

88. The method of any one of claims 69-87, wherein the culture conditions do not comprise an external source of hexanoic acid.

89. The olivetolic acid produced by the method of any one of claims 69-88.

90. The method of any one of claims 69-88, further comprising purifying the olivetolic acid.

91. The method of claim 90, further comprising producing cannabidiol from the purified olivetolic acid using a semisynthetic approach.

92. The cannabidiol produced by the method of claim 91.