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

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.