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Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into pep4 locus by homologous recombination

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2008年第7期9卷 527-535页

作者： Qiang ZHANG Qi-he CHEN Ming-liang FU Jin-ling WANG Hong-bo ZHANG Guo-qing HE Department of Food Science and Nutrition Zhejiang University Hangzhou 310029 China

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ 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 a-factor （MFals）, 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-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-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-1,3-1,4-β-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.

关键词： Endo-1 3-1 4-β-glucanase (bglS) gene replacement Homologous recombination Bacillus subtilis pep4 gene Saccharomyces cerevisiae

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Effects of proteinase A on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2009年第10期10卷 769-776页

作者： Hong-bo ZHANG Hai-feng ZHANG Qi-he CHEN Hui RUAN Ming-liang FU Guo-qing HE Department of Food Science and Nutrition Zhejiang University Hangzhou 310029 China

Proteinase A （PrA）, encoded by pep4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial pep4 gene deletion mutant （36 h） and PrA-negative mutant （48 h） was significantly extended, compared with the wild type strain （24 h）（P〈0.05）, but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking cultivation. The effects of glucose starvation on cell growth of partial pep4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate （RCVR） was 10% for the wild type S. cerevisiae and 90% for both mutant strains （P〈0.01）, suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate （RGCR）（61.0% and 80.0%） than the partial mutant （78.0% and 98.5%） and the complete mutant （80.0% and 98.0%）（P〈0.05）, suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.

关键词： Proteinase A (PrA) pep4 gene Saccharomyces cerevisiae WZ65 Cell metabolism Viability

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