基因重组与外源铁离子对大肠杆菌合成血红素的影响

Effects of genetic recombination and exogenous iron on heme synthesis in Escherichia coli

摘要:

【背景】大肠杆菌通过C5途径合成卟啉及血红素，5-氨基乙酰丙酸(5-aminolevulinic acid, 5-ALA)是C5途径中关键的前体物质，血红素由原卟啉IX (protoporphyrin IX, PPIX)螯合一个铁离子所形成，目前5-ALA与PPIX的外泌对卟啉的积累和血红素合成的影响尚不清楚。【目的】构建5-ALA外泌蛋白基因rhtA和卟啉外泌蛋白基因tolC双缺失的大肠杆菌以积累卟啉，同时外源添加铁离子，并过表达亚铁螯合酶基因hemH及参与铁摄取的基因efeB，促进卟啉向血红素的转化。【方法】通过Red同源重组敲除大肠杆菌BL21(DE3)的rhtA和tolC，并外源添加不同浓度的FeSO₄及Fe₂(SO₄)₃，同时构建重组质粒pEHE过表达hemH和efeB，检测卟啉和血红素含量，分析卟啉向血红素的转化。【结果】敲除rhtA和tolC对菌体生长无显著影响，与野生菌WT相比，敲除菌株WT-RT的卟啉含量增加，血红素合成略有提升。外源添加100μmol/L Fe²⁺时，菌株WT-RT的血红素含量最高为29.44μmol/g-DCW。外源添加25 μmol/L的Fe³⁺时，菌株WT-RT的血红素含量达到了38.22μmol/g-DCW，是野生菌WT的1.78倍。过表达efeB的菌株RT-pEE血红素含量显著下降，而共表达hemH和efeB的菌株RT-pEHE的血红素含量相较于菌株RT-pEE显著提高。【结论】tolC和rhtA的缺失导致卟啉的积累，适量添加Fe²⁺和Fe³⁺、共表达hemH和efeB可促进PPIX向血红素的转化。该结果为利用重组大肠杆菌生产血红素提供了新策略。

Abstract:

[Background] Escherichia coli synthesizes porphyrins through the C5 pathway. 5-aminolevulinic acid (5-ALA) is an important precursor for the synthesis of porphyrins through the C5 pathway. Heme is formed by the chelation of an iron to protoporphyrin IX (PPIX). However, it is still not clear how the secretion of 5-ALA and porphyrin affects the accumulation and conversion of porphyrin to heme. [Objective] To construct E. coli without rhtA and tolC, which encode 5-ALA and porphyrin secretion proteins, respectively, to accumulate porphyrins. Iron was added exogenously, and the ferrochelatase gene hemH and efeB involved in iron uptake were over-expressed to promote the conversion of porphyrins to heme. [Methods] The rhtA and tolC of E. coli BL21(DE3) were knocked out by Red homologous recombination, and different concentrations of FeSO₄ and Fe₂(SO₄)₃ were supplemented. Meanwhile, the recombinant plasmid pEHE for overexpressing hemH and efeB was constructed. The content of porphyrin and heme was analyzed to evaluate the conversion of porphyrins to heme. [Results] The removal of rhtA and tolC did not affect the strain growth significantly. As compared with wild-type strain WT, the porphyrin content of knockout strain WT-RT increased, and the synthesis of heme increased slightly. When 100 μmol/L Fe²⁺ was added exogenously, the heme content in WT-RT strain was 29.44 μmol/g-DCW. When 25 μmol/L Fe³⁺ was added exogenously, the heme content in WT-RT reached 38.22 μmol/g-DCW, which was 1.78 times compared with that in WT. The heme content in efeB-overexpressed strain RT-pEE decreased significantly, while that increased significantly in RT-pEHE strain with over-expressed efeB and hemH. [Conclusion] The deletion of tolC and rhtA leads to the accumulation of porphyrins. The addition of Fe²⁺ and Fe³⁺ at appropriate amount and the co-expression of hemH and efeB can promote the conversion of PPIX to heme. The results provide a new strategy for producing heme by recombinant E. coli.