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Induction of production and secretion β(1→4) glucanase with Saccharomyces cerevesiae by replacing the MET10 gene with egl1 gene from Trichoderma reesei
Author(s) -
Lu Y.,
Wang T.H.,
Ding X.L.
Publication year - 2009
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2009.02730.x
Subject(s) - glucanase , laminarin , trichoderma reesei , plasmid , brewing , yeast , biology , fermentation , microbiology and biotechnology , saccharomyces cerevisiae , gene , cellulase , biochemistry , food science , enzyme
Aims: To construct novel brewer’s yeast strains with the ability to degrade β‐glucan and increase sulfite levels in beer brewing by genetic manipulation. Methods and Results: The recombinant plasmid pA15ME containing P met10 ‐egl1‐T met10 expression cassette was constructed. Bam HI‐linearized target plasmid pA15ME was transformed into the industrial brewer’s yeast strain Z0103 to replace the MET10 locus through one‐step gene replacement. The recombinants Z8, Z7 and Z3 with the ability to secrete active endo‐β‐1,4‐glucanase I into the culture medium were isolated by Congo red dyeing. The enzymatic activities of EG I of Z8, Z7 and Z3 were 3·3, 1·5, 1·3 U l −1 , and the hydrolysing degrees of β‐glucans in wort were increased 11·9%, 8·6% and 6·9%, respectively, than that of original strain Z0103. The MET10 gene deletions were confirmed by real‐time PCR, and the sulfite levels of the culture mediums inoculated with Z8, Z7 and Z3 were increased 26%, 16% and 17%, respectively, compared to that of Z0103. Conclusions: The novel endoglucanase‐producing brewer’s yeast strains with inserted endoglucanase gene and deficient MET10 gene led to reduced content of barley β‐glucans, enhanced filterability and increased sulfur dioxide in fermenting wort. Thus, the cost for addition of microbial β‐glucanase enzyme and sulfite preparations in normal beer brewing processes could be reduced. Significance and Impact of the Study: These results suggested that genetic engineering approach is a powerful tool to construct the novel recombinant brewer’s yeast strains with different properties to reduce the cost of beer brewing and improve the flavour of a beer, and the strains obtained have potential application value in beer brewing.