Overexpression Of NAD Kinase And Glucose-6-phosphate Dehydrogenase Gene Improve NADPH Supply And L-isoleucine Biosynthesis In Coiynebacterium Glutamicum Ssp. Lactofermentum

L-isoleucine (L-Ile) is an essential amino acid of commercial interest that can be used asa food and feed additive and as an intravenous infusion agent. Corynebacterium glutamicum ssp.lactofermentum JHI3-156is an L-Ile producing strain. NADPH is a key cofactor of L-Ilesynthesis. In this study, POS5gene of Saccharomyces cerevisiae BY4742and zwf, ppnK,zwf-ppnK genes of JHI3-156was overexpressed in JHI3-156. Their impact on NADPHsupply and L-Ile biosynthesis was researched. The main contents and results are as follows.The enzyme activity, catalytic efficiency, and stability of its ATP-NADH kinase weregreater than those of its ATP-NAD+kinase, indicating it as an NADH kinase, different fromPpnK, an NAD kinase of JHI3-156, which showed a stronger NAD+kinase activity. TheATP-NADH kinase activity of Pos5was only inhibited weakly by NADPH (Ki=1.15mmol/L),while the ATP-NAD+kinase activity of Pos5was strongly inhibited by NADH (Ki=0.038mmol/L) and NADPH (Ki=0.043mmol/L).Intracellular NADPH concentration of JHI/p8-POS5, JHI/p9-POS5and JHI/p10-POS5increased by71μmol/L (226%),17μmol/L (41%) and36μmol/L (96%), respectively ascompared to the control strains. L-Ile production in the fermentation medium of JHI/p8-POS5,JHI/p9-POS5and JHI/p10-POS5increased to3.87±0.68g/L,3.30±0.21g/L, and3.02±0.52g/L, about75.6%,57.2%, and54.1%higher than that produced by the control strains,respectively. Overexpression of POS5improves the supply of NADPH and L-Ile biosynthesisin JHI3-156.Intracellular NADPH concentration of JHI/p8-ppnK, JHI/p8-zwf, and JHI/p8-zwf-ppnKincreased by57μmol/L (184%),201μmol/L (648%), and251μmol/L (810%), respectivelyas compared to the control strain. L-Ile production of JHI/p8-ppnK, JHI/p8-zwf, andJHI/p8-zwf-ppnK increased to3.17±0.52g/L,4.01±0.01g/L, and4.10±0.44g/L, about43.6%,81.7%,85.9%higher than that produced by the control strain (2.21±0.28g/L), respectively.Intracellular NADPH concentration of JHI/p9-zwf and JHI/p9-zwf-ppnK increased by94μmol/L (235%) and122μmol/L (305%), respectively as compared to the control strain. L-Ileproduction of JHI/p9-ppnK, JHI/p9-zwf, and JHI/p9-zwf-ppnK increased to3.08±0.41g/L,2.68±0.14g/L, and3.72±0.35g/L, about46.7%,27.8%, and77.0%higher than that producedby the control strain (2.10±0.42g/L), respectively. These results indicated that overexpressionof ppnK and zwf gene can increase the NADPH supply and L-Ile biosynthesis effectively, withcoexpression of zwf and ppnK the most effectively. Meanwhile, the inducible expressionmanner was better than the constitutive expression manner.Finally, three constitutive expression strains were fermented in a fermentor. IntracellularNADPH concentration of JHI/p9-POS5and JHI/p9-zwf-ppnK increased by11%and45%ascompared to the control strain, respectively. L-Ile production of JHI/p9-POS5andJHI/p9-zwf-ppnK increased to16.17g/L and16.74g/L, about26.1%and30.6%higher thanthat produced by the control strain (12.82g/L), respectively. Such result indicated thatco-expression of zwf and ppnK is more effectively than overexpression of POS5for NADPHsupply and L-Ile biosynthesis. The results indicate that overexpression NADH kinase, NAD+kinase, G6PDH, andco-expression of NAD+kinase and G6PDH is a useful metabolic engineering strategy toimprove NADPH supply and L-Ile biosynthesis.