SCRaMbLE of heterozygous diploid yeast
Inventors
Shen, Michael • Boeke, Jef D. • Wu, Yi
Assignees
Publication Number
US-12221617-B2
Publication Date
2025-02-11
Expiration Date
2037-07-07
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Abstract
Provided are compositions and methods for making a diploid yeast having heterozygosity for at least one chromosome. The method includes mating haploid yeast having at least a first modified chromosome comprising a synthetic chromosome suitable for recombination-site-mediated evolution (as a SCRaMbLE-ready modification). The SCRaMbLE-ready modification includes introduced site-specific recombinase recognition sites that can be recognized by a recombinase. Yeast that have at least one SCRaMbLE-ready modification of a chromosome are mated with a haploid yeast devoid of the SCRaMbLE-ready modification to obtain diploid SCRaMbLE-ready yeast. Subsequent to mating the haploid SCRaMbLE-ready yeast to yeast devoid of the SCRaMbLE-ready modification the method includes using the recombinase to recombine the first modified chromosome to obtain heterozygous diploid yeast comprising at least one recombined (SCRaMbLEd) chromosome and a homologous non-SCRaMbLEd chromosome. The method further includes identifying heterozygous diploid yeast that comprise the at least one SCRaMbLEd chromosome that have a changed phenotype that is different from the phenotype of the diploid SCRaMbLE-ready yeast. Yeast made by the methods, compositions of matter made by the yeast, spores made by the yeast, and kits containing the yeast and/or their spores are included.
Core Innovation
The invention provides compositions and methods for making diploid yeast that are heterozygous for at least one chromosome by mating a haploid yeast with at least one synthetic, recombination-site-modified chromosome (SCRaMbLE-ready modification) with a haploid yeast lacking this modification. The SCRaMbLE-ready chromosome features site-specific recombinase recognition sites, such as loxPsym, that can be acted upon by a recombinase. Induction of the recombinase activity after mating enables targeted and controlled recombination events on the synthetic chromosome within the diploid, generating genetic rearrangements only in the synthetic homolog while keeping the wild-type homolog intact.
This approach allows the creation of heterozygous diploid yeast consisting of one modified (SCRaMbLEd) and one non-modified (wild type) chromosome, which can be screened or selected based on phenotypic changes compared to the unmodified parental diploid. The process includes identifying heterozygous diploids that exhibit altered phenotypes, such as improved growth rate, resistance to chemicals, or production of a particular product, following recombination. The disclosure encompasses the engineered yeast strains, their spores, and kits containing these yeasts or spores.
The problem addressed by the invention arises from limitations of the SCRaMbLE system when used in haploid yeast, where random deletions of essential genes frequently result in high lethality, masking potential phenotype improvements and restricting SCRaMbLE to laboratory strains rather than industrially relevant backgrounds. By implementing controlled recombination in heterozygous diploids, the invention overcomes lethality associated with essential gene loss, enables broader genotype and phenotype exploration, and extends applicability to additional Saccharomyces species and industrial strains.
Claims Coverage
The patent has one independent claim that defines a method for the generation and identification of heterozygous diploid yeast with SCRaMbLEd chromosomes. The following inventive features are extracted from the independent claim.
Generation of heterozygous diploid yeast by mating a SCRaMbLE-ready haploid with a non-modified haploid
This inventive feature is the process of obtaining a diploid yeast heterozygous for at least one chromosome, where one haploid yeast has a synthetic chromosome capable of recombination-site-mediated evolution (SCRaMbLE-ready modification with introduced site-specific recombinase recognition sites), and the other haploid yeast is devoid of the SCRaMbLE-ready modification.
Induction of recombination exclusively on the modified chromosome in the diploid
After formation of the diploid, the method includes the step of using a recombinase to recombine only the first modified (synthetic) chromosome, resulting in a heterozygous diploid with at least one recombined (SCRaMbLEd) chromosome and a homologous chromosome that remains non-SCRaMbLEd.
Identification of heterozygous diploid yeast with changed phenotype due to SCRaMbLE
The method further encompasses identifying the heterozygous diploid yeast that have undergone SCRaMbLE and exhibit at least one SCRaMbLEd chromosome, with a phenotype that is distinct from the diploid SCRaMbLE-ready yeast prior to recombination.
In summary, the independent claim covers the steps of creating heterozygous diploid yeast via specific mating schemes, inducing targeted recombination on synthetic chromosomes, and identifying diploids with novel phenotypes resulting from these recombination events.
Stated Advantages
Heterozygous diploid strains exhibit reduced lethality from essential gene deletions during SCRaMbLE, allowing for recovery of viable cells with diverse genomic rearrangements.
The approach enables the generation of larger and more diverse populations for phenotype screening, overcoming limitations of previous haploid-only SCRaMbLE implementations.
SCRaMbLE of heterozygous diploid yeasts can rapidly yield desired or improved phenotypes after short induction periods without extensive evolutionary cycles.
The method permits application of SCRaMbLE to a variety of industrially relevant and interspecies yeast backgrounds, extending its utility beyond laboratory strains.
The process facilitates identification of genotype–phenotype relationships, including discovery of previously unlinked genes contributing to phenotypic traits.
Documented Applications
Generation of yeast strains with improved tolerance to extreme environmental conditions, such as elevated temperatures (thermotolerance) or chemical stresses including caffeine.
Construction of industrial yeast strains with enhanced properties for applications in fermentation, biofuel production, or processes that value specific phenotypes such as chemical resistance or altered product output.
Discovery and characterization of the genetic basis for trait improvements, enabling biological discovery and functional genomics in yeast.
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