Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer, using a kar1 mutant as vector

Abstract

A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an auxotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; (alpha ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and then, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.

Improving industrial yeast strains: exploiting natural and artificial diversity

“…Alternatively, it can be used to transfer flocculation characteristics (Barre et al ., 1993), factors influencing carbon source utilization (Spencer et al ., 1992) or yeast artificial chromosomes (YACs; Spencer et al ., 1994). Cytoduction is also applied in fundamental research when studying amyloids (e.g.…”

“…Cytoduction is frequently used to obtain industrial strains with a positive killer phenotype, a trait encoded by a dsRNA virus-like particle (Ouchi et al, 1979;Young, 1983;Hammond & Eckersley, 1984;Seki et al, 1985;Yoshiuchi et al, 2000). Alternatively, it can be used to transfer flocculation characteristics (Barre et al, 1993), factors influencing carbon source utilization (Spencer et al, 1992) or yeast artificial chromosomes (YACs; Spencer et al, 1994). Cytoduction is also applied in fundamental research when studying amyloids (e.g.…”

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

“…Kar1 mutant-mediated abortive mating allows the transfer of a single chromosome from one parent to another while losing the remaining chromosomes. ,,, To test the ability of the chromosome transfer method for delivering synthetic yeast chromosomes to nonsynthetic hosts, synthetic chromosomes III, V, X, and XII were respectively transferred into BY4741 or BY4742 based on their mating types (Figure a). To construct a kar1Δ15 mutant (deletion of the region between residues 106 and 192) for the essential gene KAR1 , a two-step method was applied in the wild-type strain BY4741, the synV strain (yML007), and the synX strain (yML004).…”

“…The essential gene KAR1 is involved in karyogamy during yeast mating, , and mutations of the KAR1 genes in parent strains can delay the process of nuclear fusion . In the progeny of zygote, occasionally one or more chromosomes are transferred from one parental nucleus to another. , This kar1 mutant-mediated method has been used to transfer YAC plasmids or to generate disomic yeast strains. − Here, using kar1 mutant-mediated abortive mating strategy, synthetic yeast chromosomes were directly transferred to wild-type strains BY4741 and an industrial strain Y12. Four synthetic yeast chromosomes (synIII, synV, synX, synXII) were separately transferred to BY4741, resulting in various tolerant traits.…”

Cross-species microbial genome transfer: a Review

“…In this case, it is possible that the target chromosome, such as YAC, could be transferred from one nucleus to the other ( Torres et al, 2007 ). 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 ).…”