Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination
Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination
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DOI：
10.1631/jzus.b0820019
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发表时间：
2008-07
期刊：
影响因子：
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通讯作者：
Qiang Zhang;Qihe Chen;Ming-liang Fu;Jin-ling Wang;Hong-bo Zhang;G. He
中科院分区：
文献类型：
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作者：
Qiang Zhang;Qihe Chen;Ming-liang Fu;Jin-ling Wang;Hong-bo Zhang;G. He
克隆了枯草芽孢杆菌（Bacillus subtilis）内切1，3 - 1，4-β-葡聚糖酶基因bglS，并对其进行了序列测定。构建了含有PGK 1启动子、与酵母交配信息素α-因子（MFα 1 s）信号序列融合的bglS基因和以G418抗性为选择标记的ADH 1终止子的bglS表达盒。然后利用PCR介导的同源重组技术，将酿酒酵母WZ 65菌株的PEP 4等位基因替换为bglS表达盒，并同时表达bglS基因。重组菌株S.利用大麦β-葡聚糖在平板中水解后形成的透明水解带，以及发酵液中未测到蛋白酶A（PrA）活性，初步筛选出一株产酶良好的酿酒酵母（SC-βG）。基因组DNA PCR分析结果表明，重组菌株的PEP 4基因位点发生了置换，bglS基因已插入到PEP 4基因位点上。不同的内切1，3 - 1，4-β-葡聚糖酶活性测定方法表明，重组菌SC-βG具有较高的内切1，3 - 1，4-β-葡聚糖酶表达量，培养60 h后，表达量达到最大值69.3 U/（h·ml）。同时，刚果红法适用于实际酿造过程中内切-1，3-1，4-β-葡聚糖酶活性的测定。本研究表明，所构建的酵母菌可用于提高啤酒的工业酿造性能。
The bglS gene encoding endo-1,3-1,4-β-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-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.