Physiologic roles of soluble pyridine nucleotide transhydrogenase inEscherichia coli as determined by homologous recombination

摘要

The soluble transhydrogenase is an energy-independent flavoprotein and important in cofactor regenerating system. In order to understand its physiologic roles, the recombinant strain with the deletion of soluble transhydrogenase gene (ΔudhA) inEscherichia coli was constructed using homologous recombination. Then the different genetic back-grounds containing eithericdNADP oricdNAD, which encodes NADP-dependent isocitrate dehydrogenase (IDH) or engineered NAD-dependent IDH, were transduced into ΔudhA, creating two strains (icdNADP/ΔudhA, icdNAD/ΔudhA). During growth on acetate,icdNADP/ΔudhA grew poorly and its growth rate was remarkably reduced by 75% as compared with the wild type. However,icdNAD/ΔudhA showed significantly better growth thanicdNADP/ΔudhA. Its growth rate was about 3.7 fold oficdNADP/ΔudhA, which was equivalent to the wild type. These results indicated that UdhA is an essential NADH resource for acetate-grownE. coli and is a dominant factor for bacteria to adapt to the stress environment. Furthermore, when UdhA was absence,icdNAD/ΔudhA displayed about 1.5 fold increase in the IDH activity after switching the carbon source from glucose to acetate. And RT-PCR showed that the expression of NADH dehydrogenase II (NDH-2) inicdNAD/ΔudhA was remarkably up-regulated by about 2.8 fold as compared withicdNADP/ΔudhA. The increase of IDH activity and NDH-2 expression can be explained by the reducing excess NADPH production and restoring higher levels of NADH generation in cells.