Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease
Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease
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DOI：
10.1128/aem.02310-14
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发表时间：
2014-12-01
影响因子：
4.4
通讯作者：
Jin, Yong-Su
中科院分区：
文献类型：
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作者：
Zhang, Guo-Chang;Kong, In Iok;Jin, Yong-Su
工业多倍体酵母菌株具有许多有益的性状，但缺乏可用于遗传操作的营养缺陷型标记。在这里，我们证明了一种快速有效的策略，用RNA指导的Cas9核酸酶在工业多倍体酵母菌株中产生营养缺陷型标记。我们通过RNA指导的Cas9核酸酶成功构建了一种流行的工业多倍体酵母菌株酿酒酵母ATCC 4124的四重营养缺陷型突变体，其具有ura3，trp1，leu2和his3营养缺陷型。尽管营养缺陷型标记基因的多个等位基因必须同时被破坏，但我们观察到靶向基因破坏后高达60%的阳性菌落被敲除。此外，基于生长的斑点试验和发酵实验表明，营养缺陷型突变体继承了亲本菌株的有益性状，例如对主要发酵抑制剂和高温的耐受性。此外，营养缺陷型突变体可以用含有选择标记基因的质粒转化。这些结果表明，基于RNA引导的Cas9核酸酶的精确基因破坏现在能够实现多倍体S的代谢工程。酿酒酵母菌株已广泛用于葡萄酒、啤酒和发酵工业。
Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.