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

• Published in: Biotechnology and bioengineering Vol. 115; no. 7; pp. 1764 - 1777
• Main Authors: Xu, Jian‐Zhong, Yang, Han‐Kun, Liu, Li‐Ming, Wang, Ying‐Yu, Zhang, Wei‐Guo
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2018
• Subjects: Affinity; Amino acids; Corynebacterium glutamicum; Dihydrodipicolinate reductase; E coli; Fermentation; Lysine; l‐lysine production; Mutation; NAD; NADH; NADP; Nicotinamide adenine dinucleotide; nucleotide‐cofactor specificity; nucleotide-cofactor specificity; dihydrodipicolinate reductase; Corynebacterium glutamicum; l-lysine production
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.26591

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. DHDPR is vital for maximize the efficiency of L‐lysine fermentation. However, CgDHDPR is inhibited by NADH at high concentrations of DHDP and NADPH. Therefore, replacement of CgDHDPR by EcDHDPR R39A mutant benefits to L‐lysine production.