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

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.