TY - JOUR
T1 - Comprehensive screening strategy coupled with structure-guided engineering of l-threonine aldolase from Pseudomonas putida for enhanced catalytic efficiency towards l-threo-4-methylsulfonylphenylserine
AU - Li, Lihong
AU - Zhang, Rongzhen
AU - Xu, Yan
AU - Zhang, Wenchi
N1 - Funding Information:
This work was supported by the National Key research and Development Program of China (2018YFA0900302), the National Science Foundation of China (32271487, 31970045), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE 2018-12), the Program of Introducing Talents of Discipline to Universities (111-2-06).
Publisher Copyright:
Copyright © 2023 Li, Zhang, Xu and Zhang.
PY - 2023/1/30
Y1 - 2023/1/30
N2 - l-Threonine aldolases (TAs) can catalyze aldol condensation reactions to form β-hydroxy-α-amino acids, but afford unsatisfactory conversion and poor stereoselectivity at the Cβ position. In this study, a directed evolution coupling high-throughput screening method was developed to screen more efficient l-TA mutants based on their aldol condensation activity. A mutant library with over 4000 l-TA mutants from Pseudomonas putida were obtained by random mutagenesis. About 10% of mutants retained activity toward 4-methylsulfonylbenzaldehyde, with five site mutations (A9L, Y13K, H133N, E147D, and Y312E) showing higher activity. Iterative combinatorial mutant A9V/Y13K/Y312R catalyzed l-threo-4-methylsulfonylphenylserine with a 72% conversion and 86% diastereoselectivity, representing 2.3-fold and 5.1-fold improvements relative to the wild-type. Molecular dynamics simulations illustrated that additional hydrogen bonds, water bridge force, hydrophobic interactions, and π-cation interactions were present in the A9V/Y13K/Y312R mutant compared with the wild-type to reshape the substrate-binding pocket, resulting in a higher conversion and Cβ stereoselectivity. This study provides a useful strategy for engineering TAs to resolve the low Cβ stereoselectivity problem and contributes to the industrial application of TAs.
AB - l-Threonine aldolases (TAs) can catalyze aldol condensation reactions to form β-hydroxy-α-amino acids, but afford unsatisfactory conversion and poor stereoselectivity at the Cβ position. In this study, a directed evolution coupling high-throughput screening method was developed to screen more efficient l-TA mutants based on their aldol condensation activity. A mutant library with over 4000 l-TA mutants from Pseudomonas putida were obtained by random mutagenesis. About 10% of mutants retained activity toward 4-methylsulfonylbenzaldehyde, with five site mutations (A9L, Y13K, H133N, E147D, and Y312E) showing higher activity. Iterative combinatorial mutant A9V/Y13K/Y312R catalyzed l-threo-4-methylsulfonylphenylserine with a 72% conversion and 86% diastereoselectivity, representing 2.3-fold and 5.1-fold improvements relative to the wild-type. Molecular dynamics simulations illustrated that additional hydrogen bonds, water bridge force, hydrophobic interactions, and π-cation interactions were present in the A9V/Y13K/Y312R mutant compared with the wild-type to reshape the substrate-binding pocket, resulting in a higher conversion and Cβ stereoselectivity. This study provides a useful strategy for engineering TAs to resolve the low Cβ stereoselectivity problem and contributes to the industrial application of TAs.
KW - Pseudomonas putida
KW - high-throughput screening
KW - product enantioselectivity
KW - structure-guided engineering
KW - threonine aldolases
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U2 - 10.3389/fbioe.2023.1117890
DO - 10.3389/fbioe.2023.1117890
M3 - Article
C2 - 36793440
AN - SCOPUS:85147949196
SN - 2296-4185
VL - 11
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
M1 - 1117890
ER -