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WO 2017/100377 Al 15 June 2017 (15.06.2017) W P O P C T

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WO 2017/100377 Al 15 June 2017 (15.06.2017) W 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 2017/100377 Al 15 June 2017 (15.06.2017) W P O P C T (51) International Patent Classification: land, California 94609 (US). FLASHMAN, Michael; 926 G06F 19/22 (201 1.01) C12N 15/10 (2006.01) 15th Street, Eureka, California 95501 (US). SHELLMAN, G06F 19/28 (201 1.01) Erin; 812 5th Avenue North, #207, Seattle, Washington 98109 (US). KIMBALL, Aaron; 33 Rosemont Place, San (21) International Application Number: Francisco, California 94103 (US). SZYJKA, Shawn; 2330 PCT/US20 16/065465 New Lake Place, Martinez, California 94553 (US). (22) International Filing Date: FREWEN, Barbara; 3017 Thompson Avenue, Alameda, 7 December 2016 (07.12.2016) California 94501 (US). TREYNOR, Thomas; 1370 Ada Street, Berkeley, California 94702 (US). (25) Filing Language: English (74) Agents: HOLLY, David C. et al; Cooley LLP, 1299 (26) Publication Language: English Pennsylvania Avenue, NW, Suite 700, Washington, Dis (30) Priority Data: trict of Columbia 20004 (US). 62/264,232 7 December 2015 (07. 12.2015) US (81) Designated States (unless otherwise indicated, for every 15/140,296 27 April 2016 (27.04.2016) US kind of national protection available): AE, AG, AL, AM, 62/368,786 29 July 2016 (29.07.2016) US AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (71) Applicant: ZYMERGEN, INC. [US/US]; 6121 Hollis BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, Street, Suite 700, Emeryville, California 94608 (US). 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, (72) Inventors: SERBER, Zach; 100 Ebbtide Avenue, #230, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, Sausalito, California 94965 (US). DEAN, Erik Jed; 3392 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, Orchard Valley Lane, Lafayette, California 94549 (US). OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, MANCHESTER, Shawn; 278 38th Street, Oakland, Cali SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, fornia 946 11 (US). GORA, Kasia; 649 60th Street, Oak [Continued on nextpage] (54) Title: MICROBIAL STRAIN IMPROVEMENT BY A HTP GENOMIC ENGINEERING PLATFORM (57) Abstract: The present disclosure provides a Fig. 5 HTP microbial genomic engineering platform that is computationally driven and integrates molecular bio logy, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. The HTP genomic engineering platform described herein is microbial strain host agnostic and therefore can be implemented across taxa. Furthermore, the disclosed platform can be implemented to modulate or improve any microbial host parameter of interest. W O 2017/100377 A l Illlll II lllll Hill Hill llll III III Hill Hill Hill Hill lllll llll llll i l llll TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, Declarations under Rule 4.17: zw. — as to applicant's entitlement to apply for and be granted (84) Designated States (unless otherwise indicated, for every a patent (Rule 4.1 7(H)) kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, Published: TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, — with international search report (Art. 21(3)) TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, — before the expiration of the t ne limit for amending the DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, claims and to be republished in the event of receipt of LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, amendments (Rule 48.2(h)) SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). — with sequence listing part of description (Rule 5.2(a)) IN THE UNITED STATES PATENT & TRADEMARK OFFICE PCT PATENT APPLICATION MICROBIAL STRAIN IMPROVEMENT BY A HTP GENOMIC ENGINEERING PLATFORM CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. provisional application No. 62/264,232, filed on December 07, 2015, U.S. nonprovisional application No. 15/140,296, filed on April 27, 2016, and U.S. provisional application No. 62/368,786, filed on July 29, 2016, each of which are hereby incorporated by reference in their entirety, including all descriptions, references, figures, and claims for all purposes. FIELD [0002] The present disclosure is directed to high-throughput (HTP) microbial genomic engineering. The disclosed HTP genomic engineering platform is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. STATEMENT REGARDING SEQUENCE LISTING [0003] 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 ZYMR_001_01WO_SeqList_ST25.txt. The text file is ~ 5 KB, was created on December 7, 2016, and is being submitted electronically via EFS-Web. BACKGROUND [0004] Humans have been harnessing the power of microbial cellular biosynthetic pathways for millennia to produce products of interest, the oldest examples of which include alcohol, vinegar, cheese, and yogurt. These products are still in large demand today and have also been accompanied by an ever increasing repertoire of products producible by microbes. The advent of genetic engineering technology has enabled scientists to design and program novel biosynthetic pathways into a variety of organisms to produce a broad range of industrial, medical, and consumer products. Indeed, microbial cellular cultures are now used to produce products ranging from small molecules, antibiotics, vaccines, insecticides, enzymes, fuels, and industrial chemicals. [0005] Given the large number of products produced by modern industrial microbes, it comes as no surprise that engineers are under tremendous pressure to improve the speed and efficiency by which a given microorganism is able to produce a target product. [0006] A variety of approaches have been used to improve the economy of biologically -based industrial processes by "improving" the microorganism involved. For example, many pharmaceutical and chemical industries rely on microbial strain improvement programs in which the parent strains of a microbial culture are continuously mutated through exposure to chemicals or UV radiation and are subsequently screened for performance increases, such as in productivity, yield and titer. This mutagenesis process is extensively repeated until a strain demonstrates a suitable increase in product performance. The subsequent "improved" strain is then utilized in commercial production. [0007] As alluded to above, identification of improved industrial microbial strains through mutagenesis is time consuming and inefficient. The process, by its very nature, is haphazard and relies upon one stumbling upon a mutation that has a desirable outcome on product output. [0008] Not only are traditional microbial strain improvement programs inefficient, but the process can also lead to industrial strains with a high degree of detrimental mutagenic load. The accumulation of mutations in industrial strains subjected to these types of programs can become significant and may lead to an eventual stagnation in the rate of performance improvement. [0009] Thus, there is a great need in the art for new methods of engineering industrial microbes, which do not suffer from the aforementioned drawbacks inherent with traditional strain improvement programs and greatly accelerate the process of discovering and consolidating beneficial mutations. [0010] Further, there is an urgent need for a method by which to "rehabilitate" industrial strains that have been developed by the antiquated and deleterious processes currently employed in the field of microbial strain improvement. SUMMARY OF THE DISCLOSURE [0011] The present disclosure provides a high-throughput (HTP) microbial genomic engineering platform that does not suffer from the myriad of problems associated with traditional microbial strain improvement programs. [0012] Further, the HTP platform taught herein is able to rehabilitate industrial microbes that have accumulated non-beneficial mutations through decades of random mutagenesis-based strain improvement programs. [0013] The disclosed HTP genomic engineering platform is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. This integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition. [0014] The taught HTP genetic design libraries function as drivers of the genomic engineering process, by providing libraries of particular genomic alterations for testing in a microbe. The microbes engineered utilizing a particular library, or combination of libraries, are efficiently screened in a HTP manner for a resultant outcome, e.g. production of a product of interest. This process of utilizing the HTP genetic design libraries to define
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