Expression of NAD(H) kinase and glucose-6-phosphate dehydrogenase improve NADPH supply and L-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum.

Corynebacterium glutamicum is the workhorse for the production of amino acids, including L-isoleucine (Ile). During Ile biosynthesis, NADPH is required as a crucial cofactor. In this study, four NADPH-supplying strategies based on NAD kinase, NADH kinase, glucose-6-phosphate dehydrogenase, and NAD kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on Ile biosynthesis were examined. PpnK is a NAD kinase of C. glutamicum ssp. lactofermentum JHI3-156 that predominantly phosphorylates NAD(+) to produce NADP(+). Pos5 is a NADH kinase of Saccharomyces cerevisiae that predominantly phosphorylates NADH to produce NADPH. Zwf is a glucose-6-phosphate dehydrogenase of JHI3-156. The ppnK, POS5, zwf, and zwf-ppnK genes were overexpressed in the Ile-producing strain JHI3-156. The expression of all four genes increased intracellular NADPH concentration and Ile production. The increase of NADPH concentration and Ile production in a POS5-expressing strain (229 and 75.6 %, respectively) was higher than that in a ppnK-expression strain. The expression of zwf also increased NADPH supply and Ile biosynthesis, but the constitutive expression of zwf was not as effective as the inducible expression of zwf. Coexpression of zwf and ppnK genes greatly enhanced NADPH supply and thus improved Ile production by up to 85.9 %, indicating that this strategy was the most effective one. These results are helpful for improving Ile biosynthesis and other biosynthetic processes.

Overexpression of NAD kinases improves the L-isoleucine biosynthesis in Corynebacterium glutamicum ssp. lactofermentum.

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.

[Overexpression of Corynebacterium glutamicum NAD kinase improves L-isoleucine biosynthesis].

NAD kinase catalyzes the phosphorylation of coenzyme I [NAD(H)] to form coenzyme II [NADP(H)], and NADPH is an important cofactor in L-isoleucine biosynthesis. In order to improve NADPH supply, ppnK, the gene encoding NAD kinase in Corynebacterium glutamicum was cloned and separately expressed in an L-isoleucine synthetic strain, Brevibacterium lactofermentum JHI3-156, by an inducible expression vector pDXW-8 and a constitutive expression vector pDXW-9. Compared with the control strain JHI3-156/pDXW-8, NAD kinase activity of the inducible ppnK-expressing strain JHI3-156/pDXW-8-ppnK was increased by 83.5%. NADP(H)/NAD(H) ratio was also increased by 63.8%. L-isoleucine biosynthesis was improved by 82.9%. Compared with the control strain JHI3-156/pDXW-9, NAD kinase activity of the constitutive ppnK-expressing strain JHI3-156/pDXW-9-ppnK was increased by 220%. NADP(H)/ NAD(H) ratio and NADPH concentration were increased by 134% and 21.7%, respectively. L-isoleucine biosynthesis was increased by 41.7%. These results demonstrate that NAD kinase can improve the coenzyme II supply and L-isoleucine biosynthesis, which would also be useful for biosynthesis of other amino acids.

Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer.

Brevibacterium flavum has been developed to produce amino acids l-valine, l-lysine and l-threonine. However, there are not enough vectors available for the research on metabolic engineering in Brevibacterium flavum. Here we have constructed a shuttle expression vector pDXW-8 between Escherichia coli and B. flavum. The vector harbors an origin oriE for replicating in E. coli, a second origin oriC for replicating in B. flavum, a large multiple cloning site including 11 single restriction enzyme sites and suitable for cloning multiple genes or large DNA fragments, a tac promoter and a lacI(PF104) fragment which tightly controls the tac promoter. The applicability of pDXW-8 was confirmed by the expression of the vhb gene in B. flavum. The vector pDXW-8 will be very useful for research on metabolic engineering in corynebacteria.