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Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation
Author(s) -
Wu Dianhui,
Li Xiaomin,
Shen Chao,
Lu Jian,
Chen Jian,
Xie Guangfa
Publication year - 2013
Publication title -
journal of the institute of brewing
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.523
H-Index - 51
eISSN - 2050-0416
pISSN - 0046-9750
DOI - 10.1002/jib.97
Subject(s) - isolation (microbiology) , yeast , wine , metabolic engineering , yeast in winemaking , food science , microbiology and biotechnology , ploidy , industrial biotechnology , biology , chemistry , biochemical engineering , biochemistry , saccharomyces cerevisiae , engineering , enzyme , gene
The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre– loxP system and a removable G418 r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop‐out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling