转运系统集成改造提升大肠杆菌胞外蛋氨酸积累

Effect of Amino Acid Transporter System Modification on Extracellular L-methionine Accumulation in Escherichia coli

通过构建大肠杆菌(Escherichia coli)W3110 MetD吸收系统编码基因metI、metN、metQ单个缺损菌株,获得胞外蛋氨酸吸收速率最小菌株;游离及染色体整合表达YjeH和YeaS分泌系统编码基因yjeH和yeaS,研究增强蛋氨酸分泌与弱化蛋氨酸吸收集成改造策略对菌体胞外蛋氨酸积累和生物量的影响,为理性构建蛋氨酸高产菌株提供可行策略。实验结果表明,基因metI的缺损对胞外蛋氨酸的吸收影响最大,吸收速率降低了16.7%;游离表达基因yjeH和yeaS,胞外蛋氨酸开始积累,胞外蛋氨酸最大积累量为0.21 g/L和0.18 g/L;染色体整合表达基因yjeH和yeaS,胞外蛋氨酸的最大积累量为0.48 g/L和0.25 g/L,与游离表达相比提高了128%和38.9%。增强蛋氨酸分泌与弱化蛋氨酸吸收集成改造策略对于促进胞外蛋氨酸的积累效果显著,可以作为理性构建蛋氨酸高产菌株的策略。

For the research of amino acid transporter system,it is important to construct a rational amino acid production strain.In this study,Escherichia coli 3110 mutants with MetD methionine absorption system pertinent genes metI,metN or metQ deleted were constructed to confirm the main factor in extracellular methionine absorption.Then genes yjeH and yeaS of methionine secretion system were overexpressed by plasmids and genomic integration to investigate the comprehensive effects on extracellular methionine accumulation and biomass.Compared with wild type strain W3110,the methionine uptake capacity of metI gene damaged strain decreased 16.7%.Combining yjeH and yeaS overexpression through episomal plasmid,the methionine accumulation could reach 0.21 g/L and 0.18 g/L,and the concentration increased 128%and 38.9%after yjeH and yeaS overexpression through genomic integration,reached 0.48 g/L and 0.25 g/L,respectively.In conclusion,to improve amino acid secretion and absorption simultaneously is effective for extracellular methionine accumulation and this mode can regarded as a strategy for constructing rational amino acid production strains.