大肠杆菌MetD运输系统缺失对蛋氨酸积累的影响

Effect of inactivating MetD transporter system of Escherichia coli W3110 on L-methionine production

摘要:

【目的】构建MetD运输系统缺失突变株，研究该运输系统功能缺失对Escherichia coli W3110蛋氨酸吸收和积累的影响。【方法】通过RT-qPCR比较MetJ阻遏调控解除菌株和野生株E. coli metNIQ表达量的变化，并分析蛋氨酸吸收速度的变化；利用Red同源重组系统分别敲除metNIQ基因簇、metN、metI和metQ，构建MetD运输功能缺失的突变株，研究蛋氨酸吸收速度的变化及对蛋氨酸积累的影响。【结果】MetJ阻遏调控解除后，metNIQ的表达量和蛋氨酸吸收速度显著增加。通过敲除E. coli W3110和Me05的metNIQ，MetD运输系统缺失导致蛋氨酸吸收速度下降。另外，分别敲除用于产蛋氨酸基座菌株Me06的metNIQ基因簇、metN、metI和metQ。生长曲线和摇瓶发酵结果表明，metI的敲除促进菌体的生长和蛋氨酸的合成，蛋氨酸的产量从0.39 g/L提高到0.45 g/L，提高了15.4%，蛋氨酸产率从0.14 g/g DCW提高到0.15 g/g DCW。【结论】E. coli MetD功能的缺失能够降低蛋氨酸的吸收速度，敲除metNIQ基因簇上的metI能够提高蛋氨酸产量。

Abstract:

[Objective] To study the effect of inactivating MetD transporter system of Escherichia coli W3110 on the production of L-methionine. [Methods] We compared the metNIQ genes’ expression ratio of wild type E. coli W3110 and the deregulation of MetJ repression strains by Quantitative real-time PCR (RT-qPCR). The methionine uptake capacity of E. coli W3110 and Me05 were analyzed. To construct the inactivation of MetD transporter system mutants, metNIQ genes cluster, metN, metI, and metQ were deleted by Red recombinase system respectively. We analyzed their effects on the uptake capacity and methionine production of these mutants. [Results] The metNIQ genes expression ratio and the uptake capacity of methionine were significantly increased by the deregulation of MetJ repression. By deleting the metNIQ genes cluster of E. coli W3110 and Me05, the MetD transport capacity were inactivated which resulted in the reduced uptake capacity of methionine. Besides, we deleted the metNIQ genes cluster, metN, metI, and metQ of methionine-producing chassis strain Me06. Growth curves and flask batch fermentation showed that, the growth and production of methionine were improved by the deletion of metI. The methionine production improved from 0.39 g/L to 0.45 g/L, increased by 15.4%. The methionine yield on biomass improved from 0.14 to 0.15 g/g DCW. [Conclusion] Inactivation of the function of MetD transport system in E. coli could lead to the decreased uptake capacity of methionine. Deletion of the metI can improve the methionine-producing capacity of recombination E. coli strain.