Biosynthesis\nof Methyl (2<i>S</i>,3<i>R</i>)‑2-[(Benzoylamino)methyl]-3-hydroxybutanoate\nin\nHigh Space-Time Yield with Immobilized Engineered Carbonyl Reductase

4-Acetoxy-azacyclobutanone\n(4AA) is a highly demanded chemical\ncompound used in the production of Penem and Carbapenem antibiotics.\nHowever, its synthesis is constrained by the preparation of methyl\n(2<i>S</i>,3<i>R</i>)-2-[(benzoylamino)methyl]-3-hydroxybutanoate\n[(2<i>S</i>,3<i>R</i>)-BHME]. In light of stringent\nenvironmental regulations, there is an urgent need to develop an effective\nenzymatic method using 2-benzoylaminomethyl-3-oxy-butyrate methyl\nester (BOME) as the substrate. This study mined a carbonyl reductase\nAxSDR from Algoriella xinjiangensis, which asymmetrically reduces BOME to (2<i>S</i>,3<i>R</i>)-BHME using isopropanol (IPA) as a cosubstrate. The mechanisms\nunderlying the high stereoselectivity, substrate selectivity, and\nlimited activity of AxSDR toward BOME were analyzed using computer-aided\ntechnology. Based on these analyses, AxSDR was rationally designed,\nleading to the identification of a triple-point variant, G94T/H145Y/Y188L\n(Mu3), which exhibited a 2-fold increase in catalytic efficiency.\nAfter condition optimization, Mu3 cells were able to convert 300 mM\nBOME, achieving a space-time yield of 15.1 g/L/h. The sustainability\nof the (2<i>S</i>,3<i>R</i>)-BHME biosynthesis\nmethod was further enhanced by immobilizing Mu3 on IPA-tolerant amino\nresin. The space-time yield of the immobilized enzyme Mu3-imm increased\nto 75.3 g/(L·h) and was maintained at 50.2 g/(L·h) after\n100 uses. These results demonstrate the significant industrial application\npotential of Mu3-imm in reducing the costs and environmental risks\nassociated with the preparation of (2<i>S</i>,3<i>R</i>)-BHME and its downstream products such as 4-AA, Penem, and Carbapenem\nantibiotics.