WO 2017/087846 Al 26 May 20 17 (26.05.2017) W P O P C T
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(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 2017/087846 Al 26 May 20 17 (26.05.2017) W P O P C T (51) International Patent Classification: (74) Agents: VEITENHEIMER, Erich et al; Cooley LLP, A01M 29/12 (201 1.01) C12N 15/53 (2006.01) 1299 Pennsylvania Avenue, N.W., Suite 700, Washington, A 7/46 (2006.01) C12N 15/63 (2006.01) District of Columbia 20004-2400 (US). A61K 9/42 (2006.01) C12P 7/64 (2006.01) (81) Designated States (unless otherwise indicated, for every C07C 57/66 (2006.01) kind of national protection available): AE, AG, AL, AM, (21) International Application Number: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, PCT/US20 16/062852 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, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 18 November 2016 (18.1 1.2016) KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (25) Filing Language: English MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (30) Priority Data: TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, 62/257,054 18 November 2015 (18. 11.2015) US ZW. 62/35 1,605 17 June 2016 (17.06.2016) US (84) Designated States (unless otherwise indicated, for every (71) Applicant: PROVIVI, INC. [US/US]; 1701 Colorado Av kind of regional protection available): ARIPO (BW, GH, enue, Santa Monica, California 90404 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (72) Inventors: EFFENDI, Leonard; c/o PROVIVI, INC., TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 1701 Colorado Avenue, Santa Monica, California 90404 DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (US). MEINHOLD, Peter; c/o PROVIVI, INC., 1701 LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, Colorado Avenue, Santa Monica, California 90404 (US). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, WAMPLER, Keith; c/o PROVIVI, INC., 1701 Colorado GW, KM, ML, MR, NE, SN, TD, TG). Avenue, Santa Monica, California 90404 (US). COELHO, Declarations under Rule 4.17 : Pedro; c/o PROVIVI, INC., 1701 Colorado Avenue, Santa Monica, California 90404 (US). SHEPPARD, Micah; c/o — as to applicant's entitlement to applyfor and be granted a PROVIVI, INC., 1701 Colorado Avenue, Santa Monica, patent (Rule 4.1 7(H)) California 90404 (US). [Continued on nextpage] (54) Title: MICROORGANISMS FOR THE PRODUCTION OF INSECT PHEROMONES AND RELATED COMPOUNDS i FIGURE 3 1 (57) Abstract: The present application relates to recombinant microorganisms useful in the biosynthesis of unsaturated -C 2 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The - C24 fatty alcohols, aldehydes, and acetates described herein may be used as substrates for metathesis reactions to expand the reper toire of target compounds and pheromones. The application further relates to recombinant microorganisms co-expressing a pher- omone pathway and a pathway for the production of a toxic protein, peptide, oligonucleotide, or small molecule suitable for use in an attract-and-kill pest control approach. Also provided are methods of producing unsaturated C -C fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising the recombinant microorgamsms and/or option ally one or more of the product alcohols, aldehydes, or acetates. w o 2017/087846 Al II 11 II I 1 I 1 II III I III I III! Ill II I II Published. with sequence listing part of description (Rule 5.2(a)) [0001] This application claims a priority benefit to U.S. Provisional Application Serial No. 62/257,054, filed November 18 , 201 5 , and claims a priority benefit to U.S. Provisional Application Serial No. 62/351 ,605, filed June 17 , 2016; each of the aforementioned applications is herein expressly incorporated by reference ra l ! ί n i [0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is PRVI_007_02WO_SeqList_ST25.txt. The text file is about 54 KB, was created on November 15 , 2016, and is being submitted electronically via EFS-Web. TECHNICAL FIELD [0003] This application relates to recombinant microorganisms useful in the biosynthesis of unsaturated C -C 4 fatty alcohols, aldehydes, and acetates which may be useful as insect pheromones, fragrances, flavors, and polymer intermediates. The application further relates to methods of producing unsaturated C6-C24 fatty alcohols, aldehydes, and acetates using the recombinant microorganisms, as well as compositions comprising one or more of these compounds and/or the recombinant microorganisms. BACKGROUND [0004] As the global demand for food grows, there is an increasing need for effective pest control. Conventional insecticides are among the most popular chemical control agents because they are readily available, rapid acting, and highly reliable. However, the overuse, misuse, and abuse of these chemicals have led to resistant pests, alteration of the natural ecology, and in some cases, environmental damage. [0005] The use of insect pheromones to control pest populations has gained increasing popularity as a viable, safe, and environmentally-friendly alternative to conventional insecticides. Since their discovery in the late 1950s, these molecules have shown efficacy in reducing insect populations through a variety of methods, including mass trappings, attract and kill, and mating disruption. The latter method in particular represents a non-toxic means of pest control and utilizes the ability of synthetic pheromones to mask naturally occurring pheromones, thereby causing confusion and mating disruption. [0006] Although pheromones have significant potential in agricultural insect control, the cost of synthesizing pheromones using currently available techniques is very high, which prohibits widespread use of this sustainable technology beyond high-value crops. Thus, there is an existing need to develop novel technologies for the cost-efficient production of insect pheromones and related fragrances, flavors, and polymer intermediates. The present inventors address this need with the development of recombinant microorganisms capable of producing a wide-range of unsaturated C -C24 fatty alcohols, aldehydes, and acetates including synthetic insect pheromones from low-cost feedstocks. MARY OF THE DISCLOSURE [0007] The present application relates to recombinant microorganisms having a biosynthesis pathway for the production of one or more compounds 4 selected from unsaturated C6-C2 fatty alcohols, aldehydes, and acetates. The recombinant microorganisms described herein may be used for the production of at least one compound, such as an insect pheromone, a fragrance, or a flavoring agent, selected from unsaturated C6-C24 fatty alcohols, aldehydes, and acetates. [0008] In one embodiment, the recombinant microorganism comprises a biosynthesis pathway for the production of an unsaturated C6-C24 fatty alcohol. Accordingly, in a first aspect, the application relates to a recombinant microorganism capable of producing an unsaturated C6--C24 fatty alcohol from an endogenous or exogenous source of saturated C -C2 4 fatty acyi-CoA, wherein the recombinant microorganism expresses (a): at least one exogenous nucleic acid molecule encoding a fatty-acyl desaturase that catalyzes the conversion of a saturated C6-C24 fatty acyl-CoA to a corresponding mono- or poly-unsaturated C6-C24 fatty acyl-CoA; and (b): at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase that catalyzes the conversion of the mono- or poly unsaturated C6-C2 4 fatty acyl-CoA from (a) into the corresponding mono- or poly-unsaturated -C24 fatty alcohol. In some embodiments, the mono- or poly-unsaturated C-6-C24 fatty alcohol is an insect pherornone. In some embodiments, the mono- or poly-unsaturated C6-C24 fatty alcohol is a fragrance or flavoring agent. In some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an alcohol oxidase or an alcohol dehydrogenase, wherein the alcohol oxidase or alcohol dehydrogenase is capable of catalyzing the conversion of the mono- or poly-unsaturated C6-C24 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C2 fatty aldehyde n some embodiments, the recombinant microorganism further comprises at least one endogenous or exogenous nucleic acid molecule encoding an acetyl transferase capable of catalyzing the conversion of the mono- or poly-unsaturated C 6-C2 fatty alcohol from (b) into a corresponding mono- or poly-unsaturated C6-C2 4 fatty acetate. [0009] n some embodiments, the fatty-acyl desaturase is a desaturase capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6 , 7 , 8 , 9 , 10 , 11, 12, 13 , 14, 15 , 16, 17 , 18, 19 , 20, 2 1, 22, 23, or 24 carbon atoms. [0010] In some embodiments, the fatty-acyl desaturase is capable of generating a double bond at position C5, C6, C7, C8, C9, C 10 , C , C12, or C 13 in the fatty acid or its derivatives, such as, for example, fatty acid CoA esters. [001 1] In one exemplary embodiment, the fatty-acyl desaturase is a Z 11 desaturase. In various embodiments described herein, the Z 11 desaturase, or the nucleic acid sequence that encodes it, can be isolated from organisms of the species Agrotis segetum, Amye!ois transitel!a, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capiteila, Trichopiusia ni, Helicoverpa zea, or Thalassiosira pseudonana.