Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide-cofactor specificity for increasing l-lysine production.

l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L-1 ), carbon yield (from 0.35 to 0.44 g [g glucose]-1 ) and productivity (from 2.07 to 2.93 g L-1 hr-1 ) of l-lysine in JL-6 ΔdapB::Ec-dapBC115G,G116C in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapBC115G,G116C , followed by JL-6 Cg-dapBC37G,G38C (21.4%) and JL-6 ΔdapB::Ec-dapBC46G,G47C (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity.

NADPH metabolism: a survey of its theoretical characteristics and manipulation strategies in amino acid biosynthesis.

Reduced nicotinamide adenine nucleotide phosphate (NADPH), which is one of the key cofactors in the metabolic network, plays an important role in the biochemical reactions, and physiological function of amino acid-producing strains. The manipulation of NADPH availability and form is an efficient and easy method of redirecting the carbon flux to the amino acid biosynthesis in industrial strains. In this review, we survey the metabolic mode of NADPH. Furthermore, we summarize the research developments in the understanding of the relationship between NADPH metabolism and amino acid biosynthesis. Detailed strategies to manipulate NADPH availability are addressed based on this knowledge. Finally, the uses of NADPH manipulation strategies to enhance the metabolic function of amino acid-producing strains are discussed.