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WO 2015/017866 Al 5 February 2015 (05.02.2015) 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 2015/017866 Al 5 February 2015 (05.02.2015) P O P C T (51) International Patent Classification: Gregory, B. [US/US]; C/o 83 Cambridge Parkway, Cam C12N 15/63 (2006.01) C40B 40/06 (2006.01) bridge, MA 02142 (US). C12N 15/87 (2006.01) (74) Agents: ALTIERI, Stephen, L. et al; Bingham Mc- (21) International Application Number: cutchen LLP, 2020 K Street, NW, Washington, DC 20006- PCT/US20 14/049649 1806 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 4 August 2014 (04.08.2014) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (25) Filing Language: English BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (26) Publication Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (30) Priority Data: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 61/861,805 2 August 2013 (02.08.2013) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/883,13 1 26 September 2013 (26.09.2013) US OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 61/935,265 3 February 2014 (03.02.2014) US SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 61/938,933 12 February 2014 (12.02.2014) US TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (71) Applicant: ENEVOLV, INC. [US/US]; 83 Cambridge ZW. Parkway, Cambridge, MA 02142 (US). (84) Designated States (unless otherwise indicated, for every (72) Inventors; and kind of regional protection available): ARIPO (BW, GH, (71) Applicants : KONIECZKA, Jay, H. [US/US]; C/o 83 GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Cambridge Parkway, Cambridge, MA 02142 (US). SPOO- UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, NAMORE, James, E. [US/US]; C/o 83 Cambridge Park TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, way, Cambridge, MA 02142 (US). WAPINSKI, Ilan, N. EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, [US/US]; C/o 83 Cambridge Parkway, Cambridge, MA 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, 02142 (US). ISAACS, Farren, J. [US/US]; C/o 83 Cam KM, ML, MR, NE, SN, TD, TG). bridge Parkway, Cambridge, MA 02142 (US). FOLEY, [Continued on nextpage] (54) Title: PROCESSES AND HOST CELLS FOR GENOME, PATHWAY, AND BIOMOLECULAR ENGINEERING Shuttle MAGE Plasmid Oesic 2u NA (57) Abstract: The present disclosure provides compositions and methods for genomic engineering. WO 2015/017866 Al llll II II 11III II Published: before the expiration of the time limit for amending the — with international search report (Art. 21(3)) claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) PROCESSES AND HOST CELLS FOR GENOME, PATHWAY, AND BSOMOLECULAR ENGINEERING FIELD OF THE INVENTION [0001] The invention relates to, inter alia, methods and compositions for genome-scale editing of genetic information. PRIORITY [0002] This application claims benefit of and priority to US Provisional Application Nos. 61/861 ,805 filed August 2, 201 3, 61/883,1 3 1 filed September 26, 2013, 61/985,265 filed February 3, 2014, and 6 1/938,933 filed February 12, 2014, each of which is hereby incorporated by reference In its entirety. BACKGROUND [0003] Successful genomic and pathway engineering requires that metabolic flux be increased through select pathways, while not substantially interfering with viability and/or growth of the organism, or a desired pnenotype. This can be especially pertinent for substrates or intermediates of the desired pathway that are Involved in core or primary metabolism, or for branch intermediates involved in more than one pathway. Stephanopouios, Metabolic Fluxes and Metabolic Engineering, Metabolic Engineering 1, 1- 11 ( 1999). In fact, genetic alterations, or combinations of genetic alterations, that increase metabolic flux through a desired pathway are difficult to predict, limiting the usefulness of rational engineering approaches. Kern A, et a!., Engineering primary metabolic pathways of industrial microorganisms, J. Biotechnology 129: 6-29 (2007). Further, it is often impractical to generate random and discrete mutational events in vivo and screen or select for improved metabolic flux or biomoiecular function. [0004] Methods are needed for screening the genetic space of a host organism to identify changes in endogenous genes and/or heterologous recombinant genes that provide improved phenotypes, such as in metabolic flux and balance, so as to improve or optimize microbial processes, including production of desired chemicals and biomoiecuies at industrial levels, or bioremediation applications. SUMMARY OF THE INVENTION [0005] n various aspects, the invention provides methods for genomic and pathway engineering. The methods are useful in E. co!i, as well as bacterial cells that are harder to genetically manipulate but are otherwise valuable for production of chemicals, including Baaiius sp. (e.g. , Baciiius subti!is), Strepiomycetes (e.g., Streptomyces avermitilis, Streptomyces coeiicolor, Streptomyces iividins, Streptomyces cinnamonensis, Streptomyces coiiinus, etc.), Cyanobacteria (e.g., Synechocystis spp., Proch!orococcus spp , Nostoc punctiforme, Caiothrk spp , Aphanizomenon flos-aquae, Arthrospira piatensis, etc.), and Corynebacteria (e.g., Corynebacteria giutamicum, Corynebacteria ammoniagenes, etc.), among others. The methods are further applicable in some embodiments to Eukaryotic cells, such as yeasts (e.g., P c ia sp., Saccharomyc.es sp., Schizosaccharomyces sp., ! yvero yces sp, etc.), filamentous fungi (e.g., Neurospora sp. and Aspergiiius sp., Penici!!ium, etc.), algae (e.g., Botryococcus, C ore a, Dunaiieiia, Graciiaria, Pieurochr/sis, and Sargassum, etc.), and plants. [0006] In various aspects, the invention is applicable to phenotype engineering, including but not limited to: change in carbon substrate utilization, Increased cell growth rate, increased production of a desired chemical, redox cofactor balance, reduced production of one or more undesired byproducts, increased resistance to industrial fermentation, and increased recombinant protein production, among others. In some embodiments, the phenotype relates to pathway engineering, such as where metabolic flux through a core or primary metabolic pathway is significantly altered (e.g., significantly altered as compared to wild type strain, an unengineered strain, or a starting strain), including alteration of metabolic flux through one or more intermediates that represent metabolic branch points. In various embodiments, the invention is applicable to introducing and balancing of heterologous recombinant enzyme activity with endogenous metabolism, to limit effects on viability and growth, for example in some embodiments, the phenotype relates to engineering alterations among one or more proteins, such as, by way oif illustration, enzymes, for improved biochemical or biophysical properties. [0007] In various aspects, the invention provides methods for pathway engineering, in which metabolic flux is altered through one or more intermediates of glycolysis, pentose phosphate pathway, TCA cycle, one or more secondary biosynthesis pathways (e.g., amino acid or nucleotide biosynthesis pathway), the mevalonate or non- mevaionate pathway, pathways involved in sulfur or nitrogen metabolism, and others. The invention relates to, in various embodiments, engineering cells to result in one or more of increased or diversified carbon substrate utilization, increased or maintained growth rate, modified enzyme activity at metabolic branch points, decreased metabolic flux through one or more competing secondary pathways and/or increase n flux through a desired secondary biosynthetlc pathway, balanced cellular redox chemistry (e.g., redox cofactor balance), balanced heterologous enzyme activity with the endogenous metabolism, including reduction in toxic intermediates, increased resistance to environmental (e.g., industrial) conditions, increased recombinant protein production, and increased yield of desired product. [0008] in various aspects, the invention engineers cells using one or a combination of recombineerlng systems. The recombineerlng systems each offer distinct advantages in engineering cells. In some embodiments, the method produces a library of mutants ceils using a ssDNA recombinase system, which may include a single- stranded annealing protein (SSAP), such as the A Red recombineering system (e.g., Seta protein) or RecET system (e.g., reel), or homologous system, which in some embodiments offers advantages in Identifying pathways, genes, or regions of genes for alteration, as well as targeting specific regions for genetic diversification. The A Red operon encodes ssDNA annealing protein Beta, which promotes annealing of single stranded oligonucleotides at the lagging strand of the replication fork. The A Red recombineering system, as with other SSAP systems, may further involve a deletion, inactivation, or reduction in mismatch repair (e.g., deletion, inactivation, or reduction in activity or level of mutS). Such a system s useful for engineering E. coii and other prokaryotic systems, and may be engineered into other species described herein. Specifically, an oligonucleotide library designed to screen for beneficial mutations (or combinations of mutations) in a target
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