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

Abstract

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.

Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential

“…They exhibit high catalytic activity and are frequently used for aldehyde reduction . In our prior investigation, ADH from Komagataella kurtzmanii ( Kk ADH) was introduced into the Bd LTTA reaction to eliminate acetaldehyde while facilitating coenzyme recycling via the introduction of the cosubstrate isopropyl alcohol . However, isopropyl alcohol competitively inhibits acetaldehyde consumption, thereby limiting the enhancement of the desired product.…”