产气肠杆菌fhlA基因克隆、表达及重组菌株产氢量

Cloning of Enterobacter aerogenes fhlA gene and overexpression of hydrogen production

摘要:

摘要：【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象，克隆甲酸-氢裂解酶 (formate hydrogen lyase, FHL) 系统的转录激活蛋白FHL activator (fhlA)基因，构建过表达重组菌株，以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术，克隆fhlA的全长基因，将该基因连接到改造质粒pGEX-4T-2-Cat中，电击转化得到重组菌株，用厌氧发酵方法测定重组细菌的产氢量。【结果】 E. aerogenes ATCC13408 fhlA ORF全长2073 bp，编码一个含690个氨基酸残基的蛋白（GenBank accession GU188474）。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定，结果表明：底物产氢潜力由原来的1.23±0.08 mol H2/mol 葡萄糖提高到了1.48±0.04 mol H2/mol 葡萄糖，提高了20.36%。【结论】本研究首次克隆了E. aerogenes ATCC13408的fhlA基因，并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高，为进一步研究和开发利用E. aerogenes ATCC13408的fhlA基因提供了基础。 关键词：产气肠杆菌；fhlA；克隆；过量表达；发酵产氢 摘要：【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象，克隆甲酸-氢裂解酶 (formate hydrogen lyase, FHL) 系统的转录激活蛋白FHL activator (fhlA)基因，构建过表达重组菌株，以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术，克隆fhlA的全长基因，将该基因连接到改造质粒pGEX-4T-2-Cat中，电击转化得到重组菌株，用厌氧发酵方法测定重组细菌的产氢量。【结果】 E. aerogenes ATCC13408 fhlA ORF全长2073 bp，编码一个含690个氨基酸残基的蛋白（GenBank accession GU188474）。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定，结果表明：底物产氢潜力由原来的1.23±0.08 mol H2/mol 葡萄糖提高到了1.48±0.04 mol H2/mol 葡萄糖，提高了20.36%。【结论】本研究首次克隆了E. aerogenes ATCC13408的fhlA基因，并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高，为进一步研究和开发利用E. aerogenes ATCC13408的fhlA基因提供了基础。

Abstract:

Abstract: [Objective] We amplified and overexpressed the FHL activator (fhlA) in E. aerogenes ATCC13408 to enhance hydrogen production. [Methods] By using universal primers and genome walking, we cloned the full open reading frame (ORF) of fhlA gene. We inserted it into the glutathion S-transferase (GST) fusion expression vector pGEX-4T-2-Cat, and transformed the recombinant plasmid into E. aerogenes ATCC13408 via electroporation for expression. Then we measured the hydrogen production of the recombinant strain in a batch culture. [Results] We found that the ORF of fhlA was 2073 base pair in length, potential to encode a 690 amino acid peptide (GenBank accession GU188474). The FhlA protein from E. aerogenes ATCC13408 shared high amino acid identities with those from other bacterial species. By using SDS-PAGE and Western blot analysis, we confirmed that the fhlA gene had successfully expressed in the strain. The hydrogen yield of the recombinant strain was increased from 1.23 to 1.48 mol H2/mol glucose. [Conclusion] Enhancement of hydrogen productivity was attained under anaerobic conditions with the recombinant strain.