Premium
Rational modification of Corynebacterium glutamicum dihydrodipicolinate reductase to switch the nucleotide‐cofactor specificity for increasing l ‐lysine production
Author(s) -
Xu JianZhong,
Yang HanKun,
Liu LiMing,
Wang YingYu,
Zhang WeiGuo
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26591
Subject(s) - corynebacterium glutamicum , cofactor , lysine , biochemistry , reductase , nucleotide , escherichia coli , nad+ kinase , biology , fermentation , enzyme , amino acid , chemistry , stereochemistry , gene
Abstract 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 ( Cg DHDPR) 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 Δ dapB::Ec ‐ dapB C115G,G116C in fed‐batch fermentation. To do this, we comparatively analyzed the characteristics of Cg DHDPR and Escherichia coli DHDPR ( Ec DHDPR), indicating that hetero‐expression of NADH‐dependent Ec DHDPR 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 Cg DHDPR and introducing the mutation R16A or R39A in Ec DHDPR 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 Δ dapB::Ec ‐ dapB C115G,G116C , followed by JL‐6 Cg ‐ dapB C37G,G38C (21.4%) and JL‐6 Δ dapB::Ec ‐ dapB C46G,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.