The mating-type locus of the miso and soy sauce yeast Zygosaccharomyces sp. contains a gene encoding the transcription factor Mat a2 (hereafter ZygoMat a2), which has a DNA-binding domain containing a high mobility group (HMG)-box not found in the baker’s yeast Saccharomyces cerevisiae. To investigate the role of ZygoMat a2 in mating-type a expression, we constructed mutant strains of ZygoMAT a2 using CRISPR-Cas9. The resulting mutants showed reduced expression of ZygoSTE6, which is specific to mating-type a, and did not mate, suggesting that ZygoMat a2 regulates the expression of mating-type a in Zygosaccharomyces sp. Expression of ZygoSTE6 was observed when the HMG-box of ZygoMat a2 was exchanged with that of Mat1-Mc, which regulates mating-type expression in Schizosaccharomyces pombe, indicating that ZygoMat a2 recognizes its target DNA sequence via the HMG-box. Substitution of the predicted upstream activation sequence (UAS) in ZygoSTE6 with the UAS of a mating-type a-specific gene from another yeast species led to mating-type a-specific gene expression in transformed Zygosaccharomyces sp. yeast, indicating that ZygoMat a2 can recognize the UAS of mating-type a-specific genes of other species. We speculated that the flexibility in the binding sequences of ZygoMat a2 is due to synergistic or cooperative binding with the DNA-binding protein ZygoMcm1. This hypothesis was supported by replacement of the UAS of ZygoSTE6 with the complete palindrome sequence, P(PAL), and by the results of protein and DNA structure prediction using AlphaFold. Collectively, our study indicates that ZygoMat a2 regulates mating-type a-specific gene expression via its HMG-box, which shows flexible recognition of the binding sequence of other HMG-box transcription factors.

Genome modification would be useful for developing breeding techniques for haploid Zygosaccharomyces rouxii and natural hybrid allodiploid Zygosaccharomyces sp. yeast strains used in miso and soy sauce production. In this study, genome editing using CRISPR-Cas9 was attempted in Zygosaccharomyces sp. strains. Based on techniques in Saccharomyces cerevisiae, the Cas9 gene and guide RNA (gRNA) were expressed from the same plasmid. Targeting of the ZygoLEU2 gene of haploid Z. rouxii strain DA2 led to of a single-nucleotide insertion in the ORF, resulting in termination of translation at 10 amino acids. This single-base insertion was 3-bp upstream of the protospacer-associated motif (PAM) sequence, suggesting that it occurred during the repair process following the Cas9-induced double-strand break. The transformant was auxotrophic for leucine, verifying that genome editing using CRISPR-Cas9 had occurred. Application of the CRISPR-Cas9 system to allodiploid Zygosaccharomyces sp. strains, which have T- and P-subgenomes, resulted in transformants with base insertions or deletions upstream of the PAM sequence, or insertions of different subgenome sequences. Leucine-auxotrophic transformants were obtained in which the ORF of the ZygoLEU2 gene in both subgenomes were mutated. In some genome-edited strains, a significant region of one subgenome chromosome was missing. Lastly, we applied CRISPR-Cas9 to the gene encoding Hog1, a protein kinase involved in adaptation to high-salt and high-osmolarity conditions. Mutation of the HOG1 genes of both the T- and P-subgenomes by CRISPR-Cas9 significantly reduced growth in high salt and high osmolarity conditions.