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WO 2018/187754 Al 11 October 2018 (11.10.2018) W !P O PCT

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WO 2018/187754 Al 11 October 2018 (11.10.2018) W !P O PCT (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 2018/187754 Al 11 October 2018 (11.10.2018) W !P O PCT (51) International Patent Classification: (74) Agent: HAN, Jong-Jin et al; Cooley LLP, 1299 Penn syl AOlH l/00 (2006.01) C12N 15/12 (2006.01) vania Avenue, NW, Suite 700, Washington, District of C o AOlH S/00 (2018.01) C12N 15/63 (2006.01) lumbia 20004 (US). A23J3/04 (2006.01) C12N 15/82 (2006.01) (81) Designated States (unless otherwise indicated, for every C12N 15/11 (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, BZ, PCT/US2018/026572 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, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 06 April 2018 (06.04.2018) KR, 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, TN, (30) Priority Data: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 62/483,157 07 April 2017 (07.04.2017) US (84) Designated States (unless otherwise indicated, for every 62/539,786 0 1 August 2017 (01.08.2017) US kind of regional protection available): ARIPO (BW, GH, (71) Applicant: ALPINE ROADS, INC. [US/US]; 953 Indiana GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, Street, San Francisco, California 94107 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (72) Inventors: EL-RICHANI, Magi; 388 Beale Street, Apart EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, ment 1615, San Francisco, California 94105 (US). LI, Shu; MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 230 Dolores Street, Apartment 304, San Francisco, Califor TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, nia 94103 (US). KM, ML, MR, NE, SN, TD, TG). (54) Title: MILK PROTEIN PRODUCTION IN TRANSGENIC PLANTS FIG. 2A pCambia 1305.1 O C : optimized kappa casein version 1 binary vector 1 -T: optimized kappa casein truncated version 1 (without signal peptide) O : optimized beta casein version 1 * U8C1-T: optimized beta casein truncated version 1 {without signal peptide) (57) Abstract: The disclosure describes a transgenic dicot or monocot plant having bovine milk protein(s) and methods of producing the transgenic dicot or monocot plant containing bovine milk protein(s). These transgenic dicot or monocot plants can express and produce bovine milk protein(s). The methods involve introducing a recombinant DNA construct expressing a bovine milk protein into a dicot or monocot plant, obtaining the dicot or monocot plant containing the bovine milk protein(s) from a recombinant DNA construct, cultivating and harvesting the transgenic dicot or monocot plant, and extracting and purifying the bovine milk protein(s) from transgenic dicot or monocotyledonous plants. [Continued on nextpage] WO 2018/187754 Al llll II II 11III II I I II I II III I Hill II I II Declarations under Rule 4.17: — as to applicant's entitlement to applyfor and be granted a patent (Rule 4.1 7(H)) — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.17(in)) Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) MILK PROTEIN PRODUCTION IN TRANSGENIC PLANTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. provisional application No. 62/539,786 filed on August 1, 2017, and U.S. provisional application No. 62/483,157 filed on April 7, 2017, both of which are hereby incorporated by reference in their entirety for all purposes. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY [0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing filename: ALRO_002_01WO_SeqList_ST25.txt, date recorded, March 26, 2018, file size ~ 89.1 kilobytes. FIELD [0003] The present disclosure generally relates to production, extraction, and purification of milk proteins from transgenic plants. BACKGROUND [0004] Globally, more than 6 billion people around the world consume milk and milk products. Demand for cow milk and dairy products is expected to keep increasing due to increased reliance on these products in developing countries as well as growth in human population which is expected to exceed 9 billion people by 2050. [0005] Relying on animal agriculture to meet growing demand for food is not a sustainable solution. According to the Food & Agriculture Organization of the United Nations, animal agriculture is responsible for 18% of all greenhouse gases, more than the entire transportation sector combined. Dairy cows alone account for 3% of this total. [0006] In addition to impacting the environment, animal agriculture poses a serious risk to human health. A startling 80% of antibiotics used in the United States go towards treating animals, resulting in the development of antibiotic resistant microorganisms also known as superbugs. For years, food companies and farmers have used antibiotics not only to treat sick animals, but also to feed them a steady diet of the drugs to prevent illnesses. In September 2016, the United Nations announced the use of antibiotics in the food system as a crisis on par with Ebola and HIV. [0007] According to the World Dairy Situation Report 201 1 published by the International Dairy Federation, cow milk accounted for 83 % of global milk production. At present, there is a need of providing bovine milk and/or producing essential high-quality proteins from bovine milk in a more sustainable and humane manner, instead of solely relying on animal farming, in order to produce milk extracts and essential milk protein concentrates or isolates. Also, there is a need for selectively producing the high-quality proteins that are more beneficial than others nutritionally and clinically. Recombinant proteins have been produced and marketed in numerous agricultural, industrial, and pharmaceutical uses. The sustainable production of important recombinant proteins is necessary to provide abundant amounts of the high-quality proteins for commercial applications. These valuable proteins can be efficiently produced in living cells such as bacteria, mammalian, and even plant cells. The subject disclosure described herein provides for a solution to produce essential milk proteins in transgenic plants in a safe, humane and sustainable way. SUMMARY OF THE DISCLOSURE [0008] The present disclosure is based, in part, on the observation that transgenic plants having nucleic acid sequences coding for mammalian milk proteins can produce the milk proteins. In some embodiments, the mammalian milk proteins used in the present invention can be from any mammal that produces milk, including but not limited to a mammal selected from the group consisting of bovine, human, goat, sheep, camel, buffalo, water buffalo, dromedary, llama and any combination thereof. [0009] The present disclosure is based, in part, on the observation that transgenic plants having bovine milk proteins can be generated by processes of producing transgenic plants containing bovine milk proteins. Bovine casein and whey proteins that are efficiently expressed from chimeric genes in plants are valuable in terms of producing milk proteins in the plants. Appropriate construction of recombinant constructs/vectors/plasmids having milk protein- coding nucleic acid sequences is critical in order to produce high-quality milk proteins. Codon- optimized nucleic acids can be synthetized based on the genetic and genomic information of a host plant, thus decreasing the risks associated with expressing milk proteins of mammal origin in non-mammal species. Also, the present disclosure involves methods of obtaining, cultivating, and harvesting the transgenic plants by introducing recombinant constructs/vectors/plasmids containing milk protein-coding sequences into the host plants, as well as extracting and purifying the milk proteins expressed in the transgenic plants. [0010] In some embodiments, the present disclosure teaches production, extraction, and purification of bovine milk proteins from transgenic plants. In other embodiments, the present disclosure teaches production, extraction, and purification of milk proteins from the transgenic plants that are genetically engineered. In some embodiments the bovine milk proteins produced and obtained as provided herein can be consumed directly or can be incorporated into any food composition, any feed composition or any beverage in place of or in addition to bovine milk products obtained directly from bovines. [0011] In other embodiments, the present disclosure teaches a transgenic plant comprising a recombinant DNA construct, said construct comprising (i) a promoter, (ii) a nucleic acid sequence encoding a bovine milk protein and/or a functional fragment thereof, which is operably linked to said promoter, and (iii) a termination sequence; wherein the bovine milk protein and/or the functional fragment thereof is expressed in the transgenic plant and/or a part thereof. [0012] In some embodiments, the present disclosure teaches a transgenic plant comprising a recombinant DNA construct, said construct comprising (i) a promoter, (ii) a nucleic acid sequence encoding a bovine milk protein and/or a functional fragment thereof, which is operably linked to said promoter, and (iii) a termination sequence; wherein the bovine milk protein and/or the functional fragment thereof is expressed in the transgenic plant and/or a part thereof, wherein the promoter is selected from the group consisting of a Cauliflower Mosaic Virus (CaMV) 35S promoter, plant constitutive promoters, and plant tissue-specific promoters.
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