Shuya Sun, Yiquan Zhou, Feiyang Deng, Yifang Meng, Xinglu Zhu, Hualei Wang, Di Wei
l -Threonine aldolase (LTA) is an attractive biocatalyst for the synthesis of l - syn - p -nitrophenylserine ( l - syn -1b), a key intermediate in chloramphenicol synthesis. However, low diastereoselectivity has limited its broader application in stereospecific C–C bond formation. To overcome this limitation, a metagenomic library constructed from non-natural amino acid-enriched environments was screened, leading to the identification of an LTA from Staphylococcus epidermidis ( Se LTA) that exhibits the highest diastereoselectivity toward l - syn -1b among naturally occurring LTAs reported to date. To further enhance its diastereoselectivity, structural comparison, alanine scanning, and tunnel analysis were employed to identify hotspots that modulate the diastereoselectivity of Se LTA. Subsequent saturation mutagenesis and iterative saturation mutagenesis at these positions yielded the quadruple variant A176G/Y202S/N7C/F129E (Mut4), which increased the diastereoselectivity from 32.5% syn to 92.7% syn . Furthermore, Mut4 exhibits markedly improved diastereoselectivity toward para- and meta-substituted benzaldehyde derivatives. Molecular dynamics (MD) simulations further elucidated the molecular basis underlying the enhanced diastereoselectivity of Mut4. This study provides a potential biocatalyst for the sustainable and efficient synthesis of a chloramphenicol intermediate.