Biosynthesis of Methyl (2<i>S</i>,3<i>R</i>)-2-[(Benzoylamino)methyl]-3-hydroxybutanoate in High Space-Time Yield with Immobilized Engineered Carbonyl Reductase

4-Acetoxy-azacyclobutanone (4AA) is a highly demanded chemical compound used in the production of Penem and Carbapenem antibiotics. However, its synthesis is constrained by the preparation of methyl (2S,3R)-2-[(benzoylamino)methyl]-3-hydroxybutanoate [(2S,3R)-BHME]. In light of stringent environmental regulations, there is an urgent need to develop an effective enzymatic method using 2-benzoylaminomethyl-3-oxy-butyrate methyl ester (BOME) as the substrate. This study mined a carbonyl reductase AxSDR from Algoriella xinjiangensis, which asymmetrically reduces BOME to (2S,3R)-BHME using isopropanol (IPA) as a cosubstrate. The mechanisms underlying the high stereoselectivity, substrate selectivity, and limited activity of AxSDR toward BOME were analyzed using computer-aided technology. Based on these analyses, AxSDR was rationally designed, leading to the identification of a triple-point variant, G94T/H145Y/Y188L (Mu3), which exhibited a 2-fold increase in catalytic efficiency. After condition optimization, Mu3 cells were able to convert 300 mM BOME, achieving a space-time yield of 15.1 g/L/h. The sustainability of the (2S,3R)-BHME biosynthesis method was further enhanced by immobilizing Mu3 on IPA-tolerant amino resin. The space-time yield of the immobilized enzyme Mu3-imm increased to 75.3 g/(L·h) and was maintained at 50.2 g/(L·h) after 100 uses. These results demonstrate the significant industrial application potential of Mu3-imm in reducing the costs and environmental risks associated with the preparation of (2S,3R)-BHME and its downstream products such as 4-AA, Penem, and Carbapenem antibiotics.