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A2) Patent Application Publication Co) Pub. No.: US 2018/0127784 Al ALEXANDER Et Al US 20180127784A1 as) United States a2) Patent Application Publication co) Pub. No.: US 2018/0127784 Al ALEXANDER et al. (43) Pub. Date: May 10, 2018 (54) SYNTHETIC YEAST CELLS AND METHODS Publication Classification OF MAKING AND USING THE SAME (51) Int. Cl. CI2N 15/0 (2006.01) Applicant: Wisconsin Alumni Research (71) CI2N 15/1 (2006.01) Foundation, Madison, WI (US) CI2N 15/65 (2006.01) CI2N 1/18 (2006.01) (72) Inventors: William Gerald ALEXANDER, Madison, WI (US); David Peris (52) U.S. Cl. NAVARRO, Madison, WI (US); CPC oe. C1I2N 15/905 (2013.01); C12N 15/81 Christopher Todd HITTINGER, (2013.01); C12N 2840/002 (2013.01); CI2N Madison, WI (US) I/18 (2013.01); C12N 2800/80 (2013.01); CI2N 15/65 (2013.01) (73) Assignee: Wisconsin Alumni Research Foundation, Madison, WI (US) (21) Appl. No.: 15/805,950 (57) ABSTRACT (22) Filed: Nov. 7, 2017 Methods of making synthetic yeast cells by mating together Related U.S. Application Data two diploid (or higher ploidy) yeast species or hybrids to (60) Provisional application No. 62/418,444, filed on Nov. generate multi-ploid yeast hybrids are provided herein. The 7, 2016, provisional application No. 62/524,700, filed synthetic yeast cells made by this process and kits for on Jun. 26, 2017. performing the process are also provided. 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Figure yHRVM356 Patent Application Publication May 10,2018 Sheet 14 0f 15 US 2018/0127784 Al 11B Figure L1A Figure Patent Application Publication May 10, 2018 Sheet 15 of 15 US 2018/0127784 Al et panenle kb TREES 348 RHRVV4AD V4 sige ae «et 12 yet kor wher Figure kb Whe pHRAWS? YO9144 eID: ae. «eh US 2018/0127784 Al May 10, 2018 SYNTHETIC YEAST CELLS AND METHODS Libkind et al., 2011). Two major lineages are used in lager OF MAKING AND USING THE SAME production (Dunn and Sherlock, 2008; Nakao et al., 2009; Walther et al., 2014), and recent evidence indicates that CROSS-REFERENCE TO RELATED these two lineages arose from independent hybridization APPLICATIONS events, suggesting that genetic diversity from the parental populations may be one contributor to the phenotypic dif- [0001] This patent application claims the benefit of prior- ferences seen in modern industrial strains (Baker et al., ity of US. Provisional Patent Application No. 62/418,444, filed Nov. 7, 2016 and United States Provisional Patent 2015). Many other fermented beverages also make use of Saccharomyces hybrids: S. cerevisiaexSaccharomyces Application No. 62/524,700, filed Jun. 26, 2017, both of which are incorporated herein by reference in their entire- uvarum hybrids are used in production of some ciders and cold-fermented wines (Le Jeune et al., 2007; Masneuf et al., ties. 1998; Perez-Través et al., 2014b), while many Belgian ale STATEMENT REGARDING FEDERALLY and some European wine yeasts are S. cerevisiaexSaccha- SPONSORED RESEARCH romyces kudriavzevii hybrids (Peris et al., 2012a, 2012b). [0007] Given the importance of interspecies yeast hybrids [0002] This invention was made with United States gov- in many commercial fermentations, there is an interest in the ernment support under grant number DE-FC02-07ER64494 art of developing new synthetic interspecies hybrids that awarded by the US Department of Energy and 1253634 may possess novel properties and allow for strain improve- awarded by the National Science Foundation. The govern- ment. However, current methods for making synthetic inter- ment has certain rights in this invention. species hybrids are cumbersome and/or require genomic modifications. New hybrid brewing strains have been gen- SEQUENCE LISTING erated by a laborious process of isolating auxotrophic [0003] This application is being filed electronically via mutants, which arise spontaneously at low frequency, fol- EFS-Web and includes an electronically submitted Sequence lowed by sporulation, dissection, and crossing MAT-com- Listing in .txt format. The .txt file contains a sequence listing patible spores to obtain hybrids (Krogerus et al., 2015; entitled “2017-11-07 5671-00073_ST25.txt” created on Perez-Través et al., 2012). Although these methods lack Nov. 7, 2017 and is 39,919 bytes in size. The Sequence markers, which would likely streamline approval for food Listing contained in this -txt file is part of the specification and beverage applications, the strains contain mutations in and is hereby incorporated by reference herein in its entirety. important biosynthetic pathways. Furthermore, using genetic markers can generate a reduction in genetic diversity INTRODUCTION by co-selecting for physically linked chromosomal sequences that could inadvertently remove interesting indus- [0004] Yeasts are critical biocatalysts commonly used in trial traits. An easier method is to first generate stable many types of commercial fermentations. One potential way heterothallic haploids for one or both parents, such as by of developing new yeast biocatalysts takes advantage of the replacing HO with drug markers, followed by interspecies natural mechanism of reproduction exhibited by many yeast crosses (Bullard et al., 2010; Swain Lenz et al., 2014; Tirosh species. For example, numerous yeast species are able to et al., 2009). Variations of this strategy have used comple- reproduce sexually, wherein meiosis serves to both increase mentary drug markers and auxotrophic mutants in one genetic diversity and to repair genetic material. As with species and spore dissection of wild-type diploids from other eukaryotes, yeast meiosis generally initiates in diploid another (Hebly et al., 2015; Piatkowska et al., 2013). How- cells (John, 1990). Regulation of this process involves ever, the persistence of drug markers in the latter hybrids specialized genetic loci called the mating-type (MAT) loci. raises legitimate concerns about their safety that would need Among yeast species, Saccharomyces yeast species, for to be addressed prior to introducing them into the food and example, possess two different mating type loci, MATa and beverage industry. MATa, which each may contain two different genes. These [0008] Accordingly, there is a need in the art for new, mating-type genes encode transcription factors that regulate easier, and “scarless” methods of creating genetic diversity the expression of meiosis-specific genes, as well as other within a yeast cell as well as new synthetic yeast strains that genes that function in self-recognition, pheromone produc- may be used for commercial fermentations such as in the tion, and non-homologous end joining. beverage and biofuel industries. [0005] In addition to performing sexual reproduction among members of the same species (“intraspecies sexual SUMMARY reproduction”), Saccharomyces yeasts may also perform sexual reproduction among members of different species [0009] In one aspect, methods of making synthetic yeast (“interspecies sexual reproduction”). All seven species of cells are provided. The methods may be directed to mating Saccharomyces yeasts possess the same mating-type locus together two diploid (or higher ploidy) yeast species or organization and a predominantly diploid or diplontic life- hybrids. The methods may include i) introducing a first style. Only limited pre-zygotic speciation barriers exist polynucleotide comprising a first promoter operably con- between Saccharomyces species, making hybridization pos- nected to an HO polynucleotide encoding a yeast Ho protein, sible between different Saccharomyces yeast species (“inter- a first selectable marker cassette, and a first yeast origin of species hybrids”). replication into a first yeast cell from a first yeast species or [0006] Interestingly, interspecies yeast hybrids produce hybrid to produce a first transformed yeast cell, 11) intro- many commercially important fermentation products.
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