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Llllllllllllllllllllllllllllllll^ (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization llllllllllllllllllllllllllllllll^ International Bureau (10) International Publication Number (43) International Publication Date WO 2018/144996 Al 09 August 2018 (09.08.2018) WIPO I PCT (51) International Patent Classification: GHOSH, Souvik; c/o Manus Bio, Inc., 1030 Massachusetts C12N1/00 (2006.01) C12N15/00(2006.01) Avenue, Cambridge, MA 02138 (US). PIRIE, Christo­ C12N1/20 (2006.01) C12N15/52 (2006.01) pher; c/o Manus Bio, Inc., 1030 Massachusetts Avenue, C12N1/21 (2006.01) C12P 5/00 (2006.01) Cambridge, MA 02138 (US). DONAUD, Jason; c/o Manus C12N 9/00 (2006.01) Bio, Inc., 1030 Massachusetts Avenue, Cambridge, MA 02138 (US). UOVE, Aaron; c/o Manus Rio, Inc., 1030 (21) International Application Number: Massachusetts Avenue, Cambridge, MA 02138 (US). NAN, PCT/US2018/016848 Hong; c/o Manus Rio, Inc., 1030 Massachusetts Avenue, (22) International Filing Date: Cambridge, MA 02138 (US). TSENG, Hsien-chung; c/ 05 February 2018 (05.02.2018) o Manus Rio, Inc., 1030 Massachusetts Avenue, Cam­ bridge, MA 02138 (US). SANTOS, Christine Nicole, S.; (25) Filing Language: English c/o Manus Rio, Inc., 1030 Massachusetts Avenue, Cam­ (26) Publication Language: English bridge, MA 02138 (US). PHIUIPPE, Ryan; c/o Manus Rio, Inc., 1030 Massachusetts Avenue, Cambridge, MA 02138 (30) Priority Data: (US). 62/454,121 03 February 2017 (03.02.2017) US (74) Agent: HAYMAN, Mark, U. et al.; Morgan, Lewis & (71) Applicant: MANUS BIO, INC. [US/US]; 1030 Massachu­ Bockius LLP, 1111 Pennsylvania Avenue, NW, Washing­ setts Avenue, Cambridge, MA 02138 (US). ton, DC 20004 (US). (72) Inventors: KUMARAN, Ajikumar, Parayil; c/o Manus (81) Designated States (unless otherwise indicated, for every Bio, Inc., 1030 Massachusetts Avenue, Cambridge, MA kind of national protection available): AE, AG, AL, AM, 02138 (US). UIM, Chin-giaw; c/o Manus Bio, Inc., AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, 1030 Massachusetts Avenue, Cambridge, MA 02138 (US). CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (54) Title: METABOLIC ENGINEERING FOR MICROBIAL PRODUCTION OF TERPENOID PRODUCTS FIGURE I Ghicose Extracellular ..... " * Ubiquinones Giixmi? ............. g·.... Nggggg, gyygfff .... Per xs f xl ^giggia^g agyafgS tsigis» SSPPS ... ................... β ..... .. ................... et (■iK .... ♦ «♦τκρ<>ί>« φ TwpenokSs ggiggg^g asagsa dxr /spD.......................................................................................................................ispG .... .................... J. :--:::11::..... :4^ " ΙΗΗΗιιιιβ* ... .....VT US^gg z (TCACvdi .................................... ggggll Al DOX ME MEcPP Farnesol Terpenes Terpenoids (57) Abstract: In various aspects and embodiments, the invention relates to bacterial strains and methods for making terpene and terpenoid products. The invention provides bacterial strains with improved carbon flux through the MEP pathway, to thereby increase terpene and/or terpenoid product yield by fermentation with carbon sources such as glucose. 2018/144996 WO [Continued on next page] WO 2018/144996 Al IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIN DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 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, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 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, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) WO 2018/144996 PCT/US2018/016848 METABOLIC ENGINEERING FOR MICROBIAL PRODUCTION OF TERPENOID PRODUCTS CROSS-REFERENCE TO RELATED APPLICATIONS 5 This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/454,121, filed February 3, 2017, the content of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING 10 The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 2, 2018, is named MAN-008PC_ST25.txt and is 20,480 bytes in size. 15 BACKGROUND The food and beverage industries as well as other industries such as the perfume, cosmetic and health care industries routinely use terpenes and/or terpenoid products, including for use as flavors and fragrances. However, factors such as: (i) the availability and high price of the plant raw material; (ii) the relatively low terpene 20 content in plant; and (iii) the tedious and inefficient extraction processes to produce sufficient quantities of terpene products on an industrial scale all have stimulated research on the biosynthesis of terpenes using plant-independent systems. Consequently, effort has been expended in developing technologies to engineer microorganisms for converting renewable resources such as glucose into terpenoid 25 products. By comparison with traditional methods, microorganisms have the advantage of fast growth without the need for land to sustain development. There are two major biosynthetic routes for the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the mevalonate (MVA) pathway and the methyl erythritol phosphate (MEP) pathway. The 30 MVA pathway is found in most eukaryotes, archaea and a few eubacteria. The MEP pathway is found in eubacteria, the chloroplasts of plants, cyanobacteria, algae and apicomplexan parasites. E. coli and other Gram-negative bacteria utilize the MEP to WO 2018/144996 PCT/US2018/016848 synthesize JPP and DMAPP metabolic precursors. While the MEP pathway provides a theoretically better stoichiometric yield over the MV A pathway, the MEP pathway in E. coli and in other bacteria has a variety of intrinsic regulation mechanisms that control and/or limit carbon flux through the pathway. See, Zhao et al., Methyl erythritol 5 Phosphate Pathway of Isoprenoid Biosynthesis, Annu Rev. Biochem. 2013; 82:497-530; Ajikumar PK, et al., Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 2010; 330-70-74. Microbial strains and methods for improving carbon flux through the MEP pathway are needed for industrial-scale production of terpenes and terpenoids in 10 bacterial systems. SUMMARY OF THE INVENTION In various aspects, the invention relates to methods and bacterial strains (such as E. coli) for making terpene and terpenoid products. In certain aspects, the invention provides bacterial strains with improved carbon flux through the MEP pathway, to 15 thereby increase terpene and/or terpenoid product yield by fermentation with carbon sources such as glucose. For example, in some embodiments the method comprises providing a bacterial strain that produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through the MEP pathway, and converts the IPP and DMAPP to a terpene or terpenoid product through a downstream synthesis 20 pathway. The bacterial strain, when cultured with a carbon source such as glucose, metabolizes greater than 1% of the carbon entering glycolysis through the MEP pathway, and in various embodiments metabolizes greater than 15% of the carbon entering glycolysis through the MEP pathway (greater than 15% “MEP carbon”). In various embodiments, the invention involves “tuning” down expression or 25 activity of one or more competing enzymes or pathways in the bacterial production strain, such as the ubiquinone synthesis pathway, without substantial or measurable impact on strain growth or viability. In some embodiments, the expression or activity of the IspB enzyme is decreased, optionally by modification to the ribosomal binding sequence (RBS), promoter, or amino acid sequence, or replacement with an IspB 30 ortholog. 2 WO 2018/144996 PCT/US2018/016848 Alternatively, or in addition, the invention involves increasing the availability or activity of Fe-S cluster proteins, so as to support higher activity of the Fe-S enzymes IspG and/or IspH, optionally by altering expression of the isc operon, and/or by deletion of the ryhB small RNA. 5 Alternatively, or in addition, in some embodiments the invention involves tuning the activity of IspG and/or IspH by overexpression and/or by selection of beneficial mutants or ortholog(s). Such mutants or orthologs can increase MEP carbon by pulling carbon further down the MEP pathway. Alternatively, or in addition, MEP enzyme complementation as evaluated by metabolomics can identify MEP enzyme 10 complementation that results in high MEP carbon, with carbon pulled further down the pathway to the MEcPP intermediate. In certain embodiments, the bacterial cell produces one or more terpenoid compounds, such as monoterpenoids, sesquiterpenoids, and diterpenoids, among others. Such terpenoid compounds find use in perfumery (e.g., patchoulol), in the flavor 15 industry (e.g., nootkatone), as sweeteners
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