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Journal of Zhejiang University SCIENCE B 2008 Vol.9 No.7 P.527~535


Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

Author(s):  Qiang ZHANG, Qi-he CHEN, Ming-liang FU, Jin-ling WANG, Hong-bo ZHANG, Guo-qing HE

Affiliation(s):  Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China

Corresponding email(s):   chenqh@zju.edu.cn, gqhe@zju.edu.cn

Key Words:  Endo-l, 3-1, 4-&beta, -glucanase (bglS), Gene replacement, Homologous recombination, Bacillus subtilis, PEP4 gene, Saccharomyces cerevisiae

Qiang ZHANG, Qi-he CHEN, Ming-liang FU, Jin-ling WANG, Hong-bo ZHANG, Guo-qing HE. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination[J]. Journal of Zhejiang University Science B, 2008, 9(7): 527~535.

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author="Qiang ZHANG, Qi-he CHEN, Ming-liang FU, Jin-ling WANG, Hong-bo ZHANG, Guo-qing HE",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination
%A Qiang ZHANG
%A Qi-he CHEN
%A Ming-liang FU
%A Jin-ling WANG
%A Hong-bo ZHANG
%A Guo-qing HE
%J Journal of Zhejiang University SCIENCE B
%V 9
%N 7
%P 527~535
%@ 1673-1581
%D 2008
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B0820019

T1 - Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination
A1 - Qiang ZHANG
A1 - Qi-he CHEN
A1 - Ming-liang FU
A1 - Jin-ling WANG
A1 - Hong-bo ZHANG
A1 - Guo-qing HE
J0 - Journal of Zhejiang University Science B
VL - 9
IS - 7
SP - 527
EP - 535
%@ 1673-1581
Y1 - 2008
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B0820019

The bglS gene encoding Endo-l,3-1,4-&beta;-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1S), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different Endo-l,3-1,4-&beta;-glucanase assay methods showed that the recombinant strain SC-βG had high Endo-l,3-1,4-&beta;-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of Endo-l,3-1,4-&beta;-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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