Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease

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 nu...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2014-12, Vol.80 (24), p.7694-7701
Main Authors: Zhang, Guo-Chang, Kong, In Iok, Kim, Heejin, Liu, Jing-Jing, Cate, Jamie H D, Jin, Yong-Su
Format: Article
Language:English
Subjects:
3-Isopropylmalate Dehydrogenase - genetics
3-Isopropylmalate Dehydrogenase - metabolism
Aldose-Ketose Isomerases - genetics
Aldose-Ketose Isomerases - metabolism
Autotrophic Processes
Biotechnology
Endonucleases - metabolism
Fermentation
Genes
Hydro-Lyases - genetics
Hydro-Lyases - metabolism
Industrial Microbiology
Metabolic Engineering
Microbiology
Plasmids
Plasmids - genetics
Polyploidy
RNA, Guide, CRISPR-Cas Systems
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - growth & development
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Vitaceae
Yeast
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Summary: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.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/aem.02310-14