Coding

Part:BBa_K4759042

Designed by: Shiyue Zhao   Group: iGEM23_Jiangnan-China   (2023-10-05)


hemB-RBS2-hemD-RBS2-hemC

The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities.


Usage and Biology

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 .

hem.png

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).

hem2.png

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. 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 FeCl3, the heme binding rate of Olep increased to 53.9%, and the conversion rate of deoxycholic acid increased to 41.4%.

hem3.png

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 982
    Illegal EcoRI site found at 1759
    Illegal EcoRI site found at 2050
    Illegal PstI site found at 467
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 982
    Illegal EcoRI site found at 1759
    Illegal EcoRI site found at 2050
    Illegal PstI site found at 467
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 982
    Illegal EcoRI site found at 1759
    Illegal EcoRI site found at 2050
    Illegal BamHI site found at 976
    Illegal BamHI site found at 1753
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 982
    Illegal EcoRI site found at 1759
    Illegal EcoRI site found at 2050
    Illegal PstI site found at 467
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 982
    Illegal EcoRI site found at 1759
    Illegal EcoRI site found at 2050
    Illegal PstI site found at 467
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2662


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