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WO 2017/139496 Al 17 August 2017 (17.08.2017) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/139496 Al 17 August 2017 (17.08.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 1/15 (2006.01) C12N 15/52 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/29 (2006.01) C12P 7/40 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 15/31 (2006.01) C12P 7/22 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, PCT/US20 17/0 17246 KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, (22) International Filing Date: MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, ' February 2017 (09.02.2017) NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, (25) Filing Language: English TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, (26) Publication Language: English ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 62/293,050 February 2016 (09.02.2016) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: CEVOLVA BIOTECH, INC. [US/US]; 111 TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Queensbury Street, Suite 2, Boston, Massachusetts 02205 TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (US). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventor: ABIDI, Syed Hussain Iman; 111 Queensbury SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Street, Suite 2, Boston, Massachusetts 02215 (US). GW, KM, ML, MR, NE, SN, TD, TG). (74) Agents: VAN GOOR, David et al; Wilson Sonsini Published: Goodrich & Rosati, 650 Page Mill Road, Palo Alto, Cali fornia 94304 (US). — with international search report (Art. 21(3)) (54) Title: MICROBIAL ENGINEERING FOR THE PRODUCTION OF CANNABINOIDS AND CANNABINOID PRECURS ORS (57) Abstract: Disclosed herein are compositions and methods for producing cannabinoids and cannabinoid precursors in microor ganisms from a carbohydrate source. The methods described herein involve genetic engineering of microorganisms for large-scale production of cannabinoids. 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
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