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

The recombinant whole cell biocatalyst Escherichia coli TOPIO [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, (—)-(IS,5-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(LR,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. By employing a bicyclo[3.2.0]hept-2-en6-one feed rate below that of the theoretical volumetric biocatalyst activity (275 moiʹmin L ), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2—0.4 g*L In this way approximately 3.5 g.L 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.