一株耐高乙醇和低pH己酸产生菌Lysinibacillus fusiformis-pBE_3-ep-SigB的构建  

Construction of a caproic acid-producing strain Lysinibacillus fusiformis-pBE_3-ep-SigB with high resistance to ethanol and low pH

作　　者：薛正楷[1,2] 杨根庆 张宿义[4] 倪斌 XUE Zhengkai;YANG Gengqing;ZHANG Suyi;NI Bin(l.School of Liquor-Making Engineering,Luzhou Vocat ional ＆ Technical College,Luzhou 646001,China;Institute of Biomedical Engineering , Luzhou 64600 , C hina;Department of Inspect ion,the Third Affiliated Hospi tal ofXingxiang Medical University,Xinxiang 453003, China;Luzhou Laoj iao Group Co., Ltd. ,Luzhou 646002, China, 5.Luzhou Jiangtanjiao Co., Ltd. ,Luzhou 646005, China)

机构地区：[1]泸州职业技术学院白酒学院,四川泸州646001 [2]泸州市生物医学工程研究所,四川泸州646000 [3]新乡医学院第三附属医院检验科,河南新乡453003 [4]泸州老窖股份有限责任公司,四川泸州646002 [5]泸州江潭窖酒业有限公司,四川泸州646005

出　　处：《中国酿造》2018年第5期162-168,共7页China Brewing

基　　金：四川省科技支撑计划项目(NO.2014FZ0018);四川省教育厅理工重点项目(18ZA0250);泸州市科学技术和知识产权局社会发展重点项目(2017-S-45(3/3))

摘　　要：以Lysinibacillus fusiformis为出发菌,分别转染Sig A和Sig B突变基因,获得耐高乙醇和低p H胁迫的优良己酸产生菌。以p ET32a(+)-Sig A/Sig B为模板,采用均匀试验优化Sig A/Sig B易错聚合酶链式反应(ep PCR)扩增体系中的Mn^(2+)和Mg^(2+)浓度,通过ep PCR获得ep-Sig A/Sig B突变基因,与载体p BE_3-MCS连接后,构建L.fusiformis-p BE_3-ep-Sig A/Sig B突变菌体库,将两种菌体库在p H值2~5和乙醇含量2%~12%条件下进行筛选。结果表明,L.fusiformis-p BE_3-ep-Sig B重组菌株耐受最高乙醇含量为8%左右,最低耐受p H值为2.5左右;当乙醇含量为8%、p H值为3.5时,L.fusiformis-p BE_3-ep-Sig B重组菌株的己酸产量达到(254.35±39.74)mg/100 m L,显著高于工程菌株L.fusiformis-p BE_3-ep-Sig A的己酸产量(P<0.01),且为出发菌L.fusiformis己酸产量(93.62±10.33)mg/100 m L的271.68%,表明L.fusiformis-p BE_3-ep-Sig B重组菌株具有较强的抗高乙醇浓度和低p H值胁迫产己酸的能力。The Lysinibacillus fusiformis was used as the original strain to be transfected by mutant genes SigA and SigB to obtain the excellent caproic acid-producing strain which showed the characteristics of resistance to high ethanol and low pH stress. With pET32a（＋）-SigA/SigB as the template, the reaction conditions of error-prone PCR （ep PCR） for S ig A /B amplification system in Mn2＋ and Mg2＋ concentrations were optimized by uniform ex-periments. The mutant gene ep-SigA/SigB was obtained by the amplification of ep PCR, after connecting with the carrier pBE3 -MCS, the mutant pool L. fusiformis-pBE3 -ep-SigA/SigB was constructed, the two mutant pools were screened under the conditions of pH 2-5 and ethanol contents 2%-12%. The results showed that the maximum tolerable ethanol content for L . fusiform is-pBE3-ep -SigB recombination strain was about 8%, the minimum tolerable pH value was about 2.5. When the ethanol content was 8% and the pH value was 3.5, the caproic acid yield of recombination strain L. fusiformis-pBE3 -ep-SigB was up to （254.35±39.74） mg/100 ml, which was significantly higher than caproic acid yield of recombination strain L. fusifbrmis-pBE3 -ep-SigA （P〈0.01）, and was 271.68% of the caproic acid yield of original strain L. fusifbmis （93.62±10.33） mg/100 ml. It indicated that L. fusi formis-pBE3-ep-SigB recombination strain had strong resistance capacity to high ethanol concentration and low pH value for caproic acid production.

关 键 词：SigA基因 SigB基因 易错PCR 高抗性 产己酸菌 

分 类 号：TS275.4[轻工技术与工程—农产品加工及贮藏工程]