Identification of CDC25-P1306L, a novel mutant allele of CDC25, conferring tolerance to multiple stresses associated with food production on Saccharomyces cerevisiae
Identification of CDC25-P1306L, a novel mutant allele of CDC25, conferring tolerance to multiple stresses associated with food production on Saccharomyces cerevisiae
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DOI：
10.1080/13102818.2018.1559094
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发表时间：
2019-01
影响因子：
1.4
通讯作者：
Y. Nakagawa;R. Fujita;Hideki Yamamura;M. Hayakawa
中科院分区：
文献类型：
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作者：
Y. Nakagawa;R. Fujita;Hideki Yamamura;M. Hayakawa
摘要酿酒酵母芽生菌株对不同胁迫的耐受性(即多重胁迫耐受性)突变对面包酵母和葡萄酒酵母等工业酵母菌株是有用的。然而，关于赋予多重压力耐受性的突变知之甚少。在此之前，我们发展了一种简单的方法来分离酿酒酵母耐多重胁迫的半显性突变体，我们称之为LCH法。在这项研究中，我们应用遗传学和下一代测序(NGS)技术相结合的方法来鉴定使用LCH方法分离的突变体的多重耐受原因突变。将单倍体突变菌株与野生型菌株杂交，得到的二倍体进行孢子萌发。将20个表现耐多抗的单倍体后代菌株混合后进行DNA提取，以获得NGS。CDC25-P1306L是编码Ras鸟氨酸核苷酸交换因子的CDC25的新的突变等位基因，共检测到86次，但在耐多胁迫后代菌株的NGS数据中没有检测到野生型CDC25等位基因，这表明所有表现出多胁迫耐性的后代菌株都具有CDC25-P1306L等位基因，而不是野生型CDC25等位基因。用CDC25-P1306L替换野生型菌株中的CDC25，使该菌株对乙醇、热休克、冻融、时间老化和高浓度葡萄糖具有耐受性。这些结果表明，CDC25-P1306L是一个耐多重胁迫的突变株，有望用于选育耐多重胁迫的酿酒酵母生产菌株。
Abstract Mutations conferring tolerance to diverse stresses (i.e. multistress tolerance) on budding yeast Saccharomyces cerevisiae are useful for industrial yeast strains such as baker’s and wine yeast. However, little is known about the mutations conferring multistress tolerance. Previously, we developed a simple method for isolating multistress-tolerant semidominant mutants of S. cerevisiae by one-step selection under lethal hydrogen peroxide stress, which we named the LCH method. In this study, we applied a combination of genetics and next-generation sequencing (NGS) technology to identify the causal mutation for multistress tolerance of the mutant isolated using the LCH method. The haploid mutant strain was crossed with the wild-type strain and the resulting diploids were sporulated. The 20 haploid progeny strains showing multistress tolerance were mixed and subjected to DNA extraction for NGS. CDC25-P1306L, a novel mutant allele of CDC25 encoding a Ras guanine nucleotide exchange factor, was detected 86 times; however, a wild-type CDC25 allele was not detected in the NGS data from the mixture of the multistress-tolerant progeny strains, suggesting that all of the progeny strains showing multistress tolerance have a CDC25-P1306L allele instead of a wild-type CDC25 allele. Substitution of CDC25 in the wild-type strain with CDC25-P1306L rendered the strain tolerant to ethanol, heat shock, freeze-thaw, chronological aging and high concentrations of glucose. These results indicate that CDC25-P1306L is a multistress-tolerant mutation and is promising for breeding multistress-tolerant S. cerevisiae strains for food production.