Direct Transfer and Consolidation of Synthetic Yeast Chromosomes by Abortive Mating and Chromosome Elimination

Abstract

Large DNA transfer technology has been challenged with the rapid development of large DNA assembly technology. The research and application of synthetic yeast chromosomes have been mostly limited in the assembled host itself. The mutant of KAR1 prevents nuclear fusion during yeast mating, and occasionally single chromosome can be transferred from one parental nucleus to another. Using the kar1 mutant method, four synthetic yeast chromosomes of Sc2.0 (synIII, synV, synX, synXII) were transferred to wild-type yeasts separately. SynIII was also transferred into an industrial strain Y12, resulting in an improvement of thermotolerance. Moreover, by combining abortive mating and chromosome elimination by CRISPR-Cas9, which has been reported in our previous study, we developed a strategy for consolidation of multiple synthetic yeast chromosomes. Compared to the previous pyramidal strategy using endoreduplication backcross, our method is a linear process independent of meiosis, providing a convenient path for accelerating consolidation of Sc2.0 chromosomes. Overall, the method of transfer and consolidation of synthetic yeast chromosomes by abortive mating and chromosome elimination enables a novel route that large DNA was assembled in donor yeast and then in vivo directly transferred to receptor yeasts, enriching the manipulation tools for synthetic genomics.

YLC-assembly: large DNA assembly via yeast life cycle

“…This strengthens the feasibility of using YLC-assembly for even larger DNA assembly, which presents an attractive avenue for the design and construction of synthetic mammalian chromosomes ( 57 , 58 ). Moreover, with the development of large DNA delivery technologies, large DNA assembled in S. cerevisiae can also be delivered to other cells by various delivery technologies such as mating-based chromosome delivery ( 59 , 60 ), and spheroplasts fusion ( 61–63 ), which has a promising prospect for large DNA manipulation for more species.…”

Application and Technical Challenges in Design, Cloning, and Transfer of Large DNA

“…Xu et al [68] have developed a CRISPR-Cas9 chromosome driver system capable of deleting entire chromosomes by targeting the centromere region. Additionally, Guo et al [69] utilized an abortive mating method that hinders nuclear fusion, facilitating chromosome transfer between synthetic and wild-type yeasts. This approach, combined with CRISPR-Cas9 chromosome elimination, streamlines the process by avoiding traditional mating complexities like sporulation and spore disassembly.…”

Cross-species microbial genome transfer: a Review

“…For example, Spencer et al used a kar1 mutant as a vector to transfer starch ( sta2 ) and melibiose ( mel )-utilizing genes into industrial strains of S. cerevisiae by single-chromosomal transfer ( Spencer et al, 1992 ). In another study, Guo et al used the kar1 mutant approach; the four synthetic yeast chromosomes (synII, synV, synX, synXII) from the Synthetic Yeast Genome Project (Sc2.0) were transferred separately into wild-type yeast ( Guo et al, 2022 ). In addition, Xu et al used chromosome elimination via CRISPR-Cas9 to enable the chromosome transfer and demonstrated that chromosome XIV (chrXIV) is critical for the thermotolerance trait of the industrial strain Y12.…”