Ogata Tomoo, Koide Kotori, Kudou Shiori, Suto Miu, Uehara Kotaro, Kaneko Teruya
Department of Life Engineering, Maebashi Institute of Technology.
J Gen Appl Microbiol. 2025 Jul 15;70(5). doi: 10.2323/jgam.2025.04.002. Epub 2025 Apr 30.
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.
基因组修饰对于开发用于米曲霉和酱油生产中使用的单倍体鲁氏接合酵母和天然杂种异源二倍体接合酵母属酵母菌株的育种技术将是有用的。在本研究中，尝试在接合酵母属菌株中使用CRISPR-Cas9进行基因组编辑。基于酿酒酵母中的技术，Cas9基因和引导RNA（gRNA）从同一质粒表达。对单倍体鲁氏接合酵母菌株DA2的ZygoLEU2基因进行靶向导致开放阅读框（ORF）中出现单核苷酸插入，导致翻译在10个氨基酸处终止。该单碱基插入位于原间隔序列邻近基序（PAM）序列上游3个碱基对处，表明它发生在Cas9诱导的双链断裂后的修复过程中。该转化体对亮氨酸营养缺陷，证实已发生使用CRISPR-Cas9的基因组编辑。将CRISPR-Cas9系统应用于具有T和P亚基因组的异源二倍体接合酵母属菌株，产生了在PAM序列上游有碱基插入或缺失，或不同亚基因组序列插入的转化体。获得了亮氨酸营养缺陷型转化体，其中两个亚基因组中的ZygoLEU2基因的ORF均发生了突变。在一些基因组编辑菌株中，一个亚基因组染色体的一个重要区域缺失。最后，我们将CRISPR-Cas9应用于编码Hog1的基因，Hog1是一种参与适应高盐和高渗透压条件的蛋白激酶。通过CRISPR-Cas9对T和P亚基因组的HOG1基因进行突变，显著降低了在高盐和高渗透压条件下的生长。
