| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 17248 | Enzyme and Microbial Technology | 2012 | 8 Pages |
NADPH is the key cofactor in l-isoleucine (Ile) biosynthetic pathway. To increase the Ile biosynthesis in Corynebacterium glutamicum ssp. lactofermentum JHI3-156, NADPH supply needs to be enhanced. Here NAD kinase, the key enzyme for the de novo biosynthesis of NADP+ and NADPH, were cloned and expressed in JHI3-156, and their influences on Ile production were analysed. Meanwhile, enzyme properties of NAD kinase from JHI3-156 (CljPpnK) were compared with that from C. glutamicum ssp. lactofermentum ATCC 13869 (ClPpnK). Four variations existed between CljPpnK and ClPpnK. Both PpnKs were poly(P)/ATP-dependent NAD kinases that used ATP as the preferred phosphoryl donor and NAD+ as the preferred acceptor. CljPpnK exhibited a higher activity and stability than ClPpnK and less sensitivity towards the effectors NADPH, NADP+, and NADH, partly due to the variations between them. The S57P variation decreased their activity. Expression of CljppnK and ClppnK in JHI3-156 increased the ATP-NAD+ kinase activity by 69- and 47-fold, respectively, the intracellular NADP+ concentration by 36% and 101%, respectively, the NADPH concentration by 95% and 42%, respectively, and Ile production by 37% and 24%, respectively. These results suggest that overexpressing NAD kinase is a useful metabolic engineering strategy to improve NADPH supply and isoleucine biosynthesis.
► We compared the enzyme properties of two NAD kinases derived from Corynebacterium. ► We examined the protein variation of the two enzymes. ► We researched the influence of two NAD kinases on NADP(H) supply and Ile production. ► We demonstrated NAD kinase as a useful strategy to improve Ile biosynthesis.
