谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成

Enhancing heme synthesis in Escherichia coli by regulating glutamic acid metabolism and over-expressing hemA

摘要:

【背景】大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hemA)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid，ALA)和血红素合成的影响。【方法】通过Red同源重组敲除与谷氨酸代谢有关的mscS与aroG，构建hemA表达载体并导入基因缺失菌株中。【结果】mscS单敲除或mscS与aroG双敲除对菌体生长无显著影响。与出发菌株相比，单敲除与双敲除菌株的谷氨酸含量均有所增加，ALA含量略微下降，血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后，胞内血红素含量增至47.603 μmol/L。【结论】通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成，该结果为增强C5途径的血红素合成提供了新的思路。

Abstract:

[Background] Escherichia coli uses glutamic acid as a precursor to synthesize limited heme through the C5 pathway. [Objective] The purpose of this study was to investigate the effects of the regulation of intracellular glutamic acid metabolism and over-expression of glutamyl-tRNA reductase gene (hemA) on 5-aminolevulinic acid (ALA) and heme synthesis. [Methods] Through Red homologous recombination, mscS and aroG which are related to glutamate metabolism were knocked out, and the hemA expression vector was constructed and introduced into the gene-knockout strains. [Results] The single and double gene-knockout had no significant effect on cell growth, but the content of glutamic acid increased, whereas that of ALA decreased slightly. The heme content in single and double gene-knockout strains increased by 11.6% and 35.7% compared with that in the parental strain respectively. [Conclusion] The over-expression of hemA in the double gene-knockout strain caused the increase of heme to 47.603 μmol/L. Heme synthesis can be promoted by regulation of intracellular glutamate metabolic flow and over-expression of hemA which provides a new approach for heme enhancement through C5 pathway.