Difference between revisions of "Part:BBa K4759271"

Revision as of 17:38, 11 October 2023

T7-RBS6-pntAB-RBS2-nadK

Design Notes

We cloned nadK and pntAB genes to construct the plasmid pACYCDuet-pntAB-nadK.

Usage and Biology

P450 enzyme catalysis required redox partners to transfer NADPH electrons to heme to catalyze the substrate. The content of NADPH in E. coli cells was low, which can only meet its own growth needs. By constructing a cofactor circulatory system, the effect of NADPH on whole-cell activity was investigated. However, the cost of exogenous NADPH addition is higher and the catalytic efficiency is low. NAD+ kinase (NadK) and membrane-bound hydrogenase (PntAB) can be used to enhance the circulation of NADPH. Thus, 3 recombinant strains are constructed. The recombinant plasmid pACYCDuet-nadK is expressed in E. coli C1 (C2 strain). The recombinant plasmid pACYCDuet-pntAB (BBa_K4759269) was expressed in E. coli C1 (C3 strain). The recombinant plasmid pACYCDuet-pntAB-nadK(BBa_K4759271) was expressed in E. coli C1 (C4 strain). Without the addition of NADPH, the conversion rate of whole-cell catalysts prepared by the C4 strain was as high as 99.1%. Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 714
Illegal EcoRI site found at 1353
Illegal EcoRI site found at 1626
Illegal PstI site found at 2233
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 714
Illegal EcoRI site found at 1353
Illegal EcoRI site found at 1626
Illegal PstI site found at 2233
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 714
Illegal EcoRI site found at 1353
Illegal EcoRI site found at 1626
Illegal BglII site found at 2158
Illegal BamHI site found at 1620
Illegal XhoI site found at 720
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 714
Illegal EcoRI site found at 1353
Illegal EcoRI site found at 1626
Illegal PstI site found at 2233
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 714
Illegal EcoRI site found at 1353
Illegal EcoRI site found at 1626
Illegal PstI site found at 2233
Illegal NgoMIV site found at 1190
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 ===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 <i>hemA</i> and the other was <i>hemL</i>. So we constructed plasmids containing hemA and hemL, and overexpressed them by transforming recombinant plasmids to <i>E. coli</i>.
-The downstream pathway, that was, the synthesis pathway of heme, involved 7 genes, of which 4 genes (<i>hemB</i>, <i>hemD</i>, <i>hemC</i>, <i>hemH</i>) were more critical, according to the literature. Therefore, we also constructed and overexpressed plasmids containing <i>hemB</i> , <i> hemD</i>, <i>hemC</i> , and <i>hemH</i> .
-https://static.igem.wiki/teams/4759/wiki/hem.png
-Fig2: Heme biosynthetic pathways in <i>E. coli</i> . 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, <i>E. coli</i> AL strain(The recombinant plasmid pCDFDuet-hemA-hemL was expressed in <i>E. coli</i> O2), <i>E. coli</i> BCDH strain (The recombinant plasmid pETDuet-hemB-hemC-hemD-hemH was expressed in <i>E. coli</i>O2), <i>E. coli</i> AL-BCDH strain(The recombinant plasmids pCDFDuet-hemA-hemL and pETDuet-hemB-hemC-hemD-hemH were expressed in <i>E. coli</i> O2).
-https://static.igem.wiki/teams/4759/wiki/hem2.png
-Fig3: The color of engineered strains and pure enzyme. 1: <i>E. coli</i> O1 strain; 2: <i>E. coli</i> O2 strain; 3: <i>E. coli</i> O2 strain cultivated with ALA and FeCl<sub>3<sub>; 4: <i>E. coli</i> AL strain; 5: Olep enzyme purified from <i>E. coli</i>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 FeCl<sub>3<sub>, the heme binding rate of Olep increased to 53.9%, and the conversion rate of deoxycholic acid increased to 41.4%.
-https://static.igem.wiki/teams/4759/wiki/hem3.png
-Fig4: The effect of enhancing heme biosynthesis on the Olep catalysis
+P450 enzyme catalysis required redox partners to transfer NADPH electrons to heme to catalyze the substrate. The content of NADPH in E. coli cells was low, which can only meet its own growth needs. By constructing a cofactor circulatory system, the effect of NADPH on whole-cell activity was investigated. However, the cost of exogenous NADPH addition is higher and the catalytic efficiency is low. NAD+ kinase (NadK) and membrane-bound hydrogenase (PntAB) can be used to enhance the circulation of NADPH. Thus, 3 recombinant strains are constructed. The recombinant plasmid pACYCDuet-nadK is expressed in E. coli C1 (C2 strain).  The recombinant plasmid pACYCDuet-pntAB (BBa_K4759269) was expressed in E. coli C1 (C3 strain). The recombinant plasmid pACYCDuet-pntAB-nadK(BBa_K4759271) was expressed in E. coli C1 (C4 strain). Without the addition of NADPH, the conversion rate of whole-cell catalysts prepared by the C4 strain was as high as 99.1%.
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>