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We divide the synthesis pathway of heme into two parts, one is the synthesis pathway of heme premise ALA, and the other part is

the synthesis pathway of ALA to heme, one upstream and one downstream.

  For the upstream pathway, there are two pathways from glutamate to ALA, one is the C4 pathway and the other is the C5 pathway.

The C4 pathway has been enhanced by the existing team, and the experimental results show that the effect is not as good as the C5

pathway, so we chose the C5 pathway for modification. The C5 pathway has two key genes, one is hemA and the other is hemL. By 

constructing plasmid containing hemA and hemL and transforming them, we overexpress hemA and hemL genes.

The downstream pathway, that is, the synthesis pathway of heme, involves 7 genes, of which 4 genes (hemB, hemD, hemC, hemH) are

more critical, according to the literature. Therefore, we also constructed plasmid to overexpress hemB, D, C, and H.

Fig1: 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 are constructed, E. coli AL strain(The recombinant plasmid pCDFDuet-hemA-hemL is expressed in

E. coli O2), E. coli BCDH strain (The recombinant plasmid pETDuet-hemB-hemC-hemD-hemH is expressed in E. coli O2), E. coli 

AL-BCDH strain(The recombinant plasmids pCDFDuet-hemA-hemL and pETDuet-hemB-hemC-hemD-hemH are expressed in E. coli O2).

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