Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica

Abstract

Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

EngineeringYarrowia lipolyticaforde novoproduction of tetraacetyl phytosphingosine

“…To develop a diploid strain from the haploid TAPS‐producing Y. lipolytica strain, the mating‐type region of CE3 strain was switched with the opposite mating type via a homologous recombination‐mediated method described in our previous study (Han et al . 2020), thereby yielding CE5 strain. CE6 strain (MatA, leu − and ura + ), the leu2 auxotroph obtained from CE3 strain, was mated with the mating‐type‐switched CE5 (MatB, leu + , ura − ).…”

“…To integrate the Sli1 and Atf2 expression cassettes into the ALK9 and ALK10 loci of Y. lipolytica , respectively, the bipartite gene‐targeting method was performed as we previously described (Han et al . 2020). The 5′ and 3′ regions of the ALK9 and ALK10 genes were amplified from Y. lipolytica genome using gene‐specific primer pairs (ALK X _1F/ALK X _1R and ALK X _2F/ALK X _2R).…”

“…Mating‐type switching of the TAPS‐producing strain and sexual hybridization were performed as we previously described (Han et al . 2020).…”

“…Afterward, yeast cells were transformed with the obtained bipartite fragments as previously described (Han et al . 2020). The transformants were selected on UD‐plated medium and confirmed via PCR using their genomic DNA as templates.…”

“…[Colour figure can be viewed at wileyonlinelibrary.com] named pTAPS-TEF-SLI1 and pTAPS-TEF-ATF2. To integrate the Sli1 and Atf2 expression cassettes into the ALK9 and ALK10 loci of Y. lipolytica, respectively, the bipartite gene-targeting method was performed as we previously described (Han et al 2020). The 5 0 and 3 0 regions of the ALK9 and ALK10 genes were amplified from Y. lipolytica…”