Difference between revisions of "Part:BBa K4759269"
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===Usage and Biology===
 
===Usage and Biology===
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The use of purified P450 enzyme for biotransformation reaction is not convenient, on the one hand, the purification process of pure enzyme-catalyzed reaction requires complex requirements, on the other hand, it also requires expensive NAD (P)H cofactors
 
  
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P450 enzyme catalysis required redox partners to transfer NADPH electrons to heme to catalyze the substrate. The content of NADPH in  <i>E. coli </i> 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  <i>E. coli </i> C1 (C2 strain).  The recombinant plasmid pACYCDuet-pntAB (BBa_K4759269) was expressed in  <i>E. coli </i> C1 (C3 strain). The recombinant plasmid pACYCDuet-pntAB-nadK(BBa_K4759271) was expressed in  <i>E. coli</i> 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%.
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https://static.igem.wiki/teams/4759/wiki/5-5.png
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Fig. 1: Effect of NADPH addition and intracellular NADPH regeneration system
  
 
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Revision as of 17:43, 11 October 2023


T7-RBS6-pntAB

pntAB is a gene coding for NAD(P) transhydrogenase subunit alpha


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%.

5-5.png

Fig. 1: Effect of NADPH addition and intracellular NADPH regeneration system

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal XhoI site found at 720
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal NgoMIV site found at 1190
  • 1000
    COMPATIBLE WITH RFC[1000]