Enantiodivergent biosynthesis of β-hydroxy esters by self-sufficient heterogeneous biocatalysts in a continuous flow

β-Hydroxy esters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. The asymmetric reduction of β-keto esters using cell-free enzymes presents a viable approach to manufacture enantiomerically pure β-hydroxy esters. However, the unbearable economic costs underlying enzymes and cofactors call for innovative approaches to maximise their reusability. Herein, we develop two self-sufficient Heterogeneous Biocatalysts (ssHBs) for the enantiodivergent reduction of β-keto esters to yield enantiomerically pure β-hydroxy esters. A thermophilic (S)-3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (TtHBDH) and an (R)-specific ketoreductase from Lactobacillus kefir (LkKRED) are selected, kinetically characterised, and immobilised onto macroporous agarose beads. Finally, the immobilised enzymes are coated with cationic polymers to co-immobilise the required redox cofactors. The resulting ssHBs catalyse the asymmetric reduction of β-keto esters without the exogenous supply of NAD(P)H and using 2-propanol as an ancillary electron donor. Then, we construct two enantiodivergent packed bed reactors (PBRs) integrating these two ssHBs and determine their optimal operational parameters through condition screening and kinetic simulations. The ssHBs in continuous flow operation exhibit good operational stability, illustrated by a maximum Space-Time Yield (STY) of 49.5 g L−1 h−1 for the continuous production of enantiopure ethyl 3-(R)-hydroxybutyrate over 21 days. Under these conditions, LkKRED and NADPH achieve total turnover numbers of 9.3 × 105 and 2.7 × 104, respectively. Upon mass metric analysis, we conclude that these ssHBs meet the efficiency and sustainability standards to be implemented in some industrial processes, advancing the concept of self-sufficient biocatalysis for process intensification.