(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.

L -Threonine aldolases (LTAs) are attractive biocatalysts for synthesizing β-hydroxy-α-amino acids (HAAs) via C–C bond formation in pharmaceuticals, although their industrial applications suffer from low activity and diastereoselectivity. Herein, we describe the discovery of a new LTA from Neptunomonas marine ( Nm LTA) that displays both ideal enzymatic activity (64.8 U/mg) and diastereoselectivity (89.5% diastereomeric excess; de) for the desired product l - threo -4-methylsulfonylphenylserine ( l - threo -MPTS). Using X-ray crystallography, site-directed mutagenesis, and computational modeling, we propose a “dual-conformation” mechanism for the diastereoselectivity control of Nm LTA, whereby the incoming 4-methylsulfonylbenzaldehyde (4-MTB) could potentially bind at the Nm LTA active site in two distinct orientations, potentially forming two diastereoisomers ( threo - or erythro -form products). Importantly, two key Nm LTA residues H140 and Y319 play critical roles in fine-tuning the binding mode of 4-MTB, supported by our site-mutagenesis assays. Uncovering of the catalytic mechanism in Nm LTA guides us to further improve the diastereoselectivity of this enzyme. A triple variant of Nm LTA (N18S/Q39R/Y319L; SRL) exhibited both improved diastereoselectivity (de value > 99%) and enzymatic activity (95.7 U/mg) for the synthesis of l - threo -MPTS compared with that of wild type. The preparative gram-scale synthesis for l - threo -MPTS with the SRL variant produced a space-time yield of up to 9.0 g L ⁻¹ h ⁻¹ , suggesting a potential role as a robust C–C bond synthetic tool for industrial synthesis of HAAs at a preparative scale. Finally, the SRL variant accepted a wider range of aromatic aldehyde derivatives as substrates and exhibited improved diastereoselectivity toward para -site substituents. This work provides deep structural insights into the molecular mechanism underlying the catalysis in Nm LTA and pinpoints the key structural motifs responsible for regulating the diastereoselectivity control, thereby guiding future attempts for protein engineering of various LTAs from different sources.