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WO 2019/086583 Al 09 May 2019 (09.05.2019) W 1P O P C T

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WO 2019/086583 Al 09 May 2019 (09.05.2019) W 1P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/086583 Al 09 May 2019 (09.05.2019) W 1P O P C T (51) International Patent Classification: (74) Agent: WP THOMPSON; REES, Kerry, 138 Fetter Lane, C12N 15/63 (2006.0 1) C12N 5/10 (2006.0 1) London EC4A 1BT (GB). C07C 49/385 (2006.01) (81) Designated States (unless otherwise indicated, for every (21) International Application Number: kind of national protection available): AE, AG, AL, AM, PCT/EP20 18/079960 AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (22) International Filing Date: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 0 1 November 2018 (01. 11.2018) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (25) Filing Language: English KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (26) Publication Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (30) Priority Data: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 62/580,037 0 1 November 2017 (01. 11.2017) US TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicants: EVOLVA SA [CH/CH] ;Duggingerstrasse 23, (84) Designated States (unless otherwise indicated, for every CH-4153 Reinach (CH). TAKASAGO INTERNATION¬ kind of regional protection available): ARIPO (BW, GH, AL CORPORATION [JP/JP]; Nissay Aroma Square 17F, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, 5-37-1, Kamata, Ota-ku, Tokyo 144-8721 (JP). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (72) Inventors: NIELSEN, Curt Aime Friis; Bruderholzs- EE, ES, FI, FR, GB, GR, HR, HU, ΓΕ , IS, IT, LT, LU, LV, trasse 60, CH-4153 Reinach (BL) (CH). HEAL, Jon; 9 MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, the Strand, Attenborough, Nottingham NG9 6AU (GB). TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, HAERLE, Johannes; Steinrebenstrasse 92, 4153 Reinach KM, ML, MR, NE, SN, TD, TG). (CH). (54) Title: PRODUCTION OF MACROCYCLIC KETONES IN RECOMBINANT HOSTS (57) Abstract: The invention relates to recombinant microorganisms and methods for producing macrocyclic ketones and macrocyclic ketone precursors. 2-Methylbutyric acid HICCL4 o r StCCL 14-Methylhexadecanedioic acid 3-Methylhexadecanedioic acid CoA MCC028 3-Methylhexadecanedioic acid-CoA 0 ©0 o o [Continued on nextpage] W O 2019/086583 A l Illlll II lllll lllll lllll llll III III lllll lllll lllll lllll lllll llll llll llll llll Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) PRODUCTION OF MACROCYCLIC KETONES IN RECOMBINANT HOSTS BACKGROUND OF THE INVENTION Field of the Invention [0001] This disclosure relates to recombinant production of macrocylic ketones and macrocylic ketone precursors, such as muscone, civetone and precursors thereof, in recombinant hosts. In particular, this disclosure relates to production of muscone molecules such as I- and/or nor-muscone, and muscone precursors comprising hexadecanedioic acid, (S)-2-methylbutyryl acid-CoA, 14-methylhexadecanoic acid, and 3-methylhexadecanedioic acid in recombinant hosts and/or excretion of such muscone, civetone and/or precursors thereof into the culture medium of the recombinant host cell. The disclosure further relates to producing muscone, civetone and precursors thereof by bioconversion or in vitro reactions. Description of Related Art [0002] Macrocyclic ketones have applications in the fragrance industry, specifically perfume. Macrocyclic ketones include, but are not limited to, muscone and civetone. Both muscone and civetone are characterized as having a musky odor. Civetone is a commercially useful pheromone obtained from the African civet. Muscone is naturally obtained from a glandular secretion of the musk deer, extraction of which often results in the death of the animal. Because musk deer are endangered species, muscone must be made synthetically. Because muscone and civetone are large molecules, they are involatile and act as fixatives which reduce the evaporation rate of lighter molecules found in fragrances. [0003] As recovery and purification of muscone has proven to be labor-intensive and inefficient, there remains a need for a recombinant production system that can produce high yields of desired muscone and muscone precursors, such as l-muscone, nor-muscone, (R)-2- methylbutyryl-CoA and/or (S)-2-methylbutyryl-CoA (Figure 1A-1C). There also remains a need for improved production of muscone in recombinant hosts for commercial uses. SUMMARY OF THE INVENTION [0004] It is against the above background that the present invention provides certain advantages and advancements over the prior art. [0005] Although this invention disclosed herein is not limited to specific advantages or functionalities the invention provides a recombinant host cell producing one or more macrocyclic ketone precursors and/or one or more macrocyclic ketones, comprising: (a) a gene encoding a polypeptide capable of synthesizing 3-methyl-2-oxopentanoate from L-isoleucine; (b) a gene encoding a polypeptide capable of synthesizing (S)-2-methylbutanal from 3-methyl-2-oxopentanoate; (c) a gene encoding a polypeptide capable of synthesizing (S)-2-methylbutyric acid from (S)-2-methylbutanal; (d) a gene encoding a polypeptide capable of synthesizing (S)-2-methylbutyryl-CoA from (S)-2-methylbutyric acid; (e) a gene encoding a polypeptide capable of synthesizing an anteiso fatty acid from (S)-2-methylbutyryl-CoA; (f) a gene encoding a polypeptide capable of synthesizing a dicarboxylic acid (DCA) from the anteiso fatty acid or from an iso fatty acid; (g) a gene encoding a polypeptide capable of synthesizing a CoA activated DCA (DCA-CoA) from the DCA; (h) a gene encoding a polypeptide having cyclisation activity capable of synthesizing a muscone from the DCA; and/or (i) a gene encoding a polypeptide having cyclisation activity capable of synthesizing the muscone from the DCA-CoA; wherein at least one of the genes is a recombinant gene. [0006] In one aspect, the recombinant host cell disclosed herein comprises: (a) the gene encoding the polypeptide capable of synthesizing 3-methyl-2- oxopentanoate from L-isoleucine; (b) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutanal from 3-methyl-2-oxopentanoate; (c) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyric acid from (S)-2-methylbutanal; (d) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyryl- CoA from (S)-2-methylbutyric acid; (e) the gene encoding the polypeptide capable of synthesizing the anteiso fatty acid from (S)-2-methylbutyryl-CoA; (f) the gene encoding the polypeptide capable of synthesizing the DCA from the anteiso fatty acid or from an iso fatty acid; and (g) the gene encoding the polypeptide capable of synthesizing the DCA-CoAfrom the DCA; wherein the recombinant host cell produces the one or more macrocyclic ketone precursors. [0007] In one aspect, the recombinant host cell disclosed herein comprises: (a) the gene encoding the polypeptide capable of synthesizing 3-methyl-2- oxopentanoate from L-isoleucine; (b) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutanal from 3-methyl-2-oxopentanoate; (c) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyric acid from (S)-2-methylbutanal; (d) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyryl- CoA from (S)-2-methylbutyric acid; (e) the gene encoding the polypeptide capable of synthesizing the anteiso fatty acid from (S)-2-methylbutyryl-CoA; (f) the gene encoding the polypeptide capable of synthesizing the DCA from the anteiso fatty acid or from an iso fatty acid; (g) the gene encoding the polypeptide capable of synthesizing the DCA-CoAfrom the DCA; and (h) the gene encoding the polypeptide capable of synthesizing the muscone from the DCA; wherein the recombinant host cell produces the one or more macrocyclic ketones. [0008] In one aspect, the recombinant host cell disclosed herein comprises: (a) the gene encoding the polypeptide capable of synthesizing 3-methyl-2- oxopentanoate from L-isoleucine; (b) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutanal from 3-methyl-2-oxopentanoate; (c) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyric acid from (S)-2-methylbutanal; (d) the gene encoding the polypeptide capable of synthesizing (S)-2-methylbutyryl- CoA from (S)-2-methylbutyric acid; (e) the gene encoding the polypeptide capable of synthesizing the anteiso fatty acid from (S)-2-methylbutyryl-CoA; (f) the gene encoding the polypeptide capable of synthesizing the DCA from the anteiso fatty acid or from an iso fatty acid; (g) the gene encoding the polypeptide capable of synthesizing the DCA-CoAfrom the DCA; and (i) the gene encoding the polypeptide capable of synthesizing the muscone from the DCA-CoA; wherein the recombinant host cell produces the one or more macrocyclic ketones. [0009] In one aspect, the recombinant host cell disclosed herein has a deletion in a genetic locus encoding a polypeptide capable of oxidizing one or more muscone precursors. [0010] In one aspect of the recombinant host cell disclosed herein, the genetic locus encoding a polypeptide capable of oxidizing one or more muscone precursors comprises
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