Abstract

BackgroundNAD(H/+) and NADP(H/+) are the most important redox cofactors in bacteria. However, the intracellular redox balance is in advantage of the cell growth and production of NAD(P)H-dependent products.ResultsIn this paper, we rationally engineered glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and isocitrate dehydrogenase (IDH) to switch the nucleotide-cofactor specificity resulting in an increase in final titer [from 85.6 to 121.4 g L−1] and carbon yield [from 0.33 to 0.46 g (g glucose)−1] of l-lysine in strain RGI in fed-batch fermentation. To do this, we firstly analyzed the production performance of original strain JL-6, indicating that the imbalance of intracellular redox was the limiting factor for l-lysine production. Subsequently, we modified the native GAPDH and indicated that recombinant strain RG with nonnative NADP-GAPDH dramatically changed the intracellular levels of NADH and NADPH. However, l-lysine production did not significantly increase because cell growth was harmed at low NADH level. Lastly, the nonnative NAD-IDH was introduced in strain RG to increase the NADH availability and to equilibrate the intracellular redox. The resulted strain RGI showed the stable ratio of NADPH/NADH at about 1.00, which in turn improved cell growth (μmax. = 0.31 h−1) and l-lysine productivity (qLys, max. = 0.53 g g−1 h−1) as compared with strain RG (μmax. = 0.14 h−1 and qLys, max. = 0.42 g g−1 h−1).ConclusionsThis is the first report of balancing the intracellular redox state by switching the nucleotide-cofactor specificity of GAPDH and IDH, thereby improving cell growth and l-lysine production.

Highlights

  • NAD(H/+) and NADP(H/+) are the most important redox cofactors in bacteria

  • 4 mol of NADPH must be supplied for 1 mol of l-lysine biosynthesis from oxaloacetate, and numerous studies have focused on engineering the metabolism of NADPH for improving l-lysine producing strains [9, 16,17,18,19]

  • This is the first report of balancing the intracellular redox state by switching the nucleotide-cofactor specificity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) combined with isocitrate dehydrogenase (IDH), thereby improving cell growth and l-lysine production

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Summary

Introduction

NAD(H/+) and NADP(H/+) are the most important redox cofactors in bacteria. the intracellu‐ lar redox balance is in advantage of the cell growth and production of NAD(P)H-dependent products. Corynebacterium glutamicum has three major redox systems, i.e., NAD(H/+), NADP(H/+) and reduced/oxidized glutathione (GSH/GSSG), which are used to adjust the intracellular redox state [1]. They are involved in other physiological functions, including regulating the energy transfer, controlling the cell life cycle, monitoring the cellular signaling and modulating the microbial. Genetic modification of the native glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been proven to improve the l-lysine production because of the increase of NADPH availability [17, 19, 20].

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