Rational Design of l‑Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production

l-threo-p-methylsulfonylphenylserine (compound 1b) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa l-threonine transaldolase (BuLTTA) was rationally designed based on the sequence–structure–function relations...

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Published inJournal of agricultural and food chemistry Vol. 72; no. 1; pp. 461 - 474
Main Authors Xi, Zhiwen, Li, Lihong, Liu, Zhiyong, Wu, Xiaolong, Xu, Yan, Zhang, Rongzhen
Format Journal Article
LanguageEnglish
Published WASHINGTON American Chemical Society 10.01.2024
Amer Chemical Soc
Subjects
Agriculture
Agriculture, Multidisciplinary
Biotechnology and Biological Transformations
Chemistry
Chemistry, Applied
Food Science & Technology
Life Sciences & Biomedicine
Physical Sciences
Science & Technology
rational design
cofactor-recycling system
l-threo-p-methylsulfonylphenylserine
l-threonine transaldolase
molecular dynamics simulation
byproduct elimination system
ALDOLASE
ACID
TOOLBOX
BIOSYNTHESIS
SUBSTRATE
ENZYMES
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Summary:l-threo-p-methylsulfonylphenylserine (compound 1b) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa l-threonine transaldolase (BuLTTA) was rationally designed based on the sequence–structure–function relationship. A mutant M4 (Asn35Ser/Thr352Asn) could produce 35.5 mM 1b with 88.8% conversion and 93.8% diastereoselectivity, 314 and 129% of the values observed for wild-type BuLTTA. Molecular dynamics simulations indicated that the shortened distance between key active site residues and the transition state (PLP-1b) and the improved hydrogen bond force enhanced the catalytic performance of the M4 variant. Then, the mutant M4 was combined with K. kurtzmanii alcohol dehydrogenase (KkADH) to eliminate the BuLTTA-inhibiting byproduct acetaldehyde, and a cosubstrate was added to regenerate the ADH cofactor NADH. Under optimized conditions, the yield of 1b reached 115.2 mM with a conversion of 96% and a diastereoselectivity of 95.5%. This work provides a new strategy for the efficient and sustainable production of 1b.
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ISSN:0021-8561
1520-5118
1520-5118
DOI:10.1021/acs.jafc.3c05267