Reactor Operation and Scale-Up of Whole Cell Baeyer-Villiger Catalyzed Lactone Synthesis

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5‐ and 55‐L fed‐batch processes to oxidize bicyclo[3.2.0]hept‐2‐en‐6‐one to its corresponding regioisomeric lactones, (–)‐(1 S,5 R)‐2‐oxabicyclo[3.3.0]oct‐6‐en‐3‐one and (–)‐(1 R,5 S)‐3‐oxabicyclo[3.3.0]oct‐6‐en‐2‐one. By employing a bicyclo[3.2.0]hept‐2‐en‐6‐one feed rate below that of the theoretical volumetric biocatalyst activity (275 μmol·min−1·L−1), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2–0.4 g·L−1. In this way approximately 3.5 g·L−1 of the combined regioisomeric lactones was produced with a yield of product on reactant of 85–90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5‐fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.