3-Phosphoglycerate dehydrogenase (PGDH) is the rate-limiting enzyme in the biosynthetic pathway of L-serine, playing a crucial role in efficient L-serine synthesis.In this study, the enzymatic characteristics of PGDH from Corynebacterium.glutamicum was investigated.The results indicated that the enzyme activity of Cg-PGDH was only 1.98 U/mg with poor thermal stability, which significantly hindered the high-efficiency biosynthesis of L-serine.To address this issue, directed evolution and high-throughput screening were employed to enhance the catalytic activity and thermal stability of Cg-PGDH.Through multiple rounds of screening, five mutants with significantly improved catalytic activity were obtained, including P85Q, D365Y, A389T, A183V, and I231V.Notably, the P85Q mutant exhibited a 1.55-fold increase in enzyme activity and significantly improved temperature stability, demonstrating an optimum catalytic temperature of 60 ℃.Subsequently, the combined mutations on the mutation sites of five mutants including P85Q, D365Y, A389T, A183V, and I231V were conducted, leading to the mutant P85Q-D365Y with enhanced temperature stability.Structural analysis and molecular dynamics simulations of the mutated sites within Cg-PGDH revealed that, despite P85Q and D365Y not being located in the catalytic center, they enhanced the stability of the loop in the 130-140 region, thereby increasing substrate affinity and catalytic activity.These research findings provided a significant foundation and direction for further elucidating the catalytic mechanism of Cg-PGDH and enhancing its catalytic activity.
HUANG Xinyan
,
SUN Mujiao
,
DU Muhua
,
PAN Yue
,
ZHANG Xinyu
,
ZHANG Yaohan
,
XU Ning
,
LIU Jun
,
JU Jiansong
,
WEI Liang
. Directed evolution of 3-phosphoglycerate dehydrogenase from Corynebacterium glutamicum[J]. Food and Fermentation Industries, 2025
, 51(3)
: 89
-96
.
DOI: 10.13995/j.cnki.11-1802/ts.038795
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