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We divided the synthesis pathway of heme into two parts, one is the synthesis pathway of heme premise ALA (upstream), and the other part is the synthesis pathway of ALA to heme (downstream). For the upstream pathway, there were two pathways from glutamate to ALA, one was the C4 pathway and the other was the C5 pathway. The C4 pathway had been enhanced by the existing team, and the experimental results showed that the effect was not as good as the C5 pathway, so we chose the C5 pathway for modification. The C5 pathway had two key genes, one was hemA and the other was hemL. So we constructed plasmids containing hemA and hemL, and overexpressed them by transforming recombinant plasmids to E. coli. The downstream pathway, that was, the synthesis pathway of heme, involved 7 genes, of which 4 genes (hemB, hemD, hemC, hemH) were more critical, according to the literature. Therefore, we also constructed and overexpressed plasmids containing hemB , hemD, hemC , and hemH .

Fig2: Heme biosynthetic pathways in E. coli . The purple arrow represents the C5 pathway and the pink arrow represents the downstream biosynthetic pathway of heme. The pCDFDuet-hemA-hemL plasmid was constructed to enhance C5 pathway; the pETDuet-hemBDC-hemH plasmid was constructed to enhance downstream biosynthetic pathway

Therefore, 3 recombinant strains were constructed, E. coli AL strain(The recombinant plasmid pCDFDuet-hemA-hemL was expressed in E. coli O2), E. coli BCDH strain (The recombinant plasmid pETDuet-hemB-hemC-hemD-hemH was expressed in E. coliO2), E. coli AL-BCDH strain(The recombinant plasmids pCDFDuet-hemA-hemL and pETDuet-hemB-hemC-hemD-hemH were expressed in E. coli O2).

Fig3: The color of engineered strains and pure enzyme. 1: E. coli O1 strain; 2: E. coli O2 strain; 3: E. coli O2 strain cultivated with ALA and FeCl_{3; 4: E. coli AL strain; 5: Olep enzyme purified from E. coliAL strain. The lower values represent the content of intracellular heme in different engineered strains}

The experimental results showed that, without the addition of ALA and FeCl_{3, the heme binding rate of Olep increased to 53.9%, and the conversion rate of deoxycholic acid increased to 41.4%.}

Fig4: The effect of enhancing heme biosynthesis on the Olep catalysis

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