大肠杆菌rhtA缺失对血红素合成的影响

The effect of rhtA deletion on heme synthesis in Escherichia coli

摘要:

【背景】Escherichia coli BL21(DE3)是基因工程的常用宿主，以C5途径合成5-氨基乙酰丙酸(5-aminolevulinic acid，ALA)，ALA是合成血红素的重要前体物质，但ALA分泌对血红素合成的影响尚不清楚。【目的】阐明参与ALA外运的RhtA在血红素合成途径中的作用。【方法】利用Red同源重组，敲除Escherichia coli BL21(DE3)的rhtA，同时构建重组质粒pEA过表达血红素合成途径中的关键酶基因hemA，检测分析血红素及其前体物质含量，以及血红素合成途径中10个关键基因的表达水平。【结果】敲除rhtA对菌体生长没有显著影响，敲除菌株BL21(DE3)ΔrhtA与原始菌株BL21(DE3)比较，ALA的胞外含量下降23%，血红素含量提高12%，尿卟啉III (Uroporphyrin III，UIII)、粪卟啉III (Coproporphyrin III，CIII)和原卟啉IX (Protoporphyrin IX，PPIX)的含量分别提高25%、15%和18%；敲除rhtA同时过表达hemA的菌株BL21(DE3)ΔrhtA/pEA与仅过表达hemA的菌株BL21(DE3)/pEA比较，胞外ALA减少了16%，血红素含量提高了24%，UIII和CIII含量分别提高55%和64%，PPIX含量显著增加，约为4.7倍。实时定量PCR结果表明，rhtA缺失后，hemC基因转录水平下调，其余9个基因转录水平均有不同程度的上调。【结论】rhtA敲除减少了ALA的外运，使得胞内血红素产量得到提高。

Abstract:

[Background] Escherichia coli BL21(DE3) is a commonly used host for genetic engineering. It synthesizes 5-aminolevulinic (ALA) through C5 pathway. ALA is an important precursor, while its secretion on the heme synthesis is unclear. [Objective] To elucidate the role of RhtA in the export of ALA in the heme synthesis pathway. [Methods] rhtA was knocked out by Red homologous recombination, and plasmid pEA was constructed to overexpress hemA, a key enzyme gene in the heme biosynthesis pathway. The contents of heme and its precursors as well as the transcription levels of 10 key genes in the heme synthesis pathway were analyzed. [Results] The deletion of rhtA had no significant effect on cell growth. Compared with parental strain BL21(DE3), the extracellular content of ALA decreased by 23% while the content of heme increased by 12% in the knockout strain BL21(DE3)ΔrhtA. The contents of uroporphyrin III (UIII), coproporphyrin III (CIII) and protoporphyrin IX (PPIX) increased by 25%, 15% and 18%, respectively in BL21(DE3)ΔrhtA. Compared with the strain BL21(DE3)/pEA in which hemA was overexpressed, the content of extracellular ALA reduced by 16% and the content of heme increased by 24% in the strain BL21(DE3)ΔrhtA/pEA. And the contents of UIII and CIII increased by 55% and 64%, respectively. The content of PPIX increased significantly, about 4.7 times in BL21(DE3)ΔrhtA/pEA compared with that in BL21(DE3)/pEA. The results of real-time quantitative PCR showed that after the deletion of rhtA, the transcription level of hemC was down-regulated, while those of the other 9 genes were up-regulated to various degrees. [Conclusion] The rhtA knockout reduces the export of ALA and leads to an increase in intracellular heme production.