Difference between revisions of "Part:BBa K4759269"

Revision as of 17:45, 11 October 2023

T7-RBS6-pntAB

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

Design Notes

We cloned pntAB gene to construct the plasmid pACYCDuet-pntAB

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%.

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

Sequence and Features