基因atpA、purHD和ndh的过量表达对大肠杆菌DH5a生长的影响

Effects of overexpression of atpA, purHD and ndh on the growth of Escherichia coli DH5a

摘要:

【目的】理解大肠杆菌DH5a及其耐乙酸突变株DA19在基本培养基中生长和乙酸积累差异的机理。【方法】根据前期针对两个菌株蛋白质组和培养中物料及能量平衡的分析，通过PCR分别扩增大肠杆菌DH5a及DA19的atpIBEFHAGDC基因、purHD基因和与NADH氧化代谢相关ndh基因及其各自的调节区，以及NADH脱氢酶Ⅰ基因（nuoA-N）的调节区。将atpA、purHD和ndh基因分别克隆到载体pTrc99a中，转化DH5a，研究基因过量表达对生长的影响。【结果】测序结果表明两个菌株的这些DNA序列与公布的大肠杆菌K-12的相应序列完全一致，在DH5a菌株中分别过量表达atpA、purHD或ndh基因可以在一定程度上改善菌体的生长。【结论】过量表达purHD或ndh基因比过量表达atpA基因的效果更好，但与DA19的生长相比仍然存在较大差距，反映了代谢全局调节的重要性。

Abstract:

[Objective] To study the mechanism of the differences in cell growth and acetate production between Escherichia coli DH5a and its acetate-tolerant mutant DA19. [Methods] Based on our previous analysis of the proteome and mass and energy balances for both strains, we first amplified the genes of atpIBEFHAGDC, purHD, and ndh, their regulation regions, as well as the regulation region of nuoA-N in both strains by PCR, and compared the sequences. Then, we constructed the recombinant strains DH5a (pTrc99a-atpA), DH5a (pTrc99a-purHD)?and DH5α (pTrc99a-ndh) to study the effect of overexpression of atpA, purHD and ndh in DH5a on growth. [Results] The sequences of the structural genes and the regulation regions in the two strains were completely identical with those of E. coli K-12. Overexpression of atpA, purHD and ndh separately in DH5a improved cell growth to some extent. [Conclusion] Overexpression of ndh, purHD and atpA resulted in improved growth of DH5a, but the growth of these recombinant strains was still lower than that of DA19, indicating the importance of global regulation to the cellular metabolism.