Part:BBa_K4759025:Experience

Applications of BBa_K4759025

First, three different combinations of exogenous additions (direct addition of Heme, addition of precursor ALA+FeSO4, addition of precursor ALA+FeCl3) are tested. The results showed that the addition of 0.64 mM ALA and 0.3 mM FeCl3 during the induction of Olep increased the heme binding rate to 67.7% and the conversion rate of deoxycholic acid to 40.7%. Through the above experiments, we verified that the increase in heme content has a positive effect on P450 enzyme activity, but the exogenous addition of ALA increases the cost of whole-cell catalysis by 60%, so we tried to increase intracellular heme content by strengthening the heme synthesis pathway of E. coli itself.

Fig1: The effect of supplements on the Olep catalysis

Endogenous synthesis of heme

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.

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 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).

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 FeCl3; 4: E. coli AL strain; 5: Olep enzyme purified from E. coli AL 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 FeCl3, 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