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, (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-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 micromol x min(-1) x L(-1)), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2-0.4 g x L(-1). In this way approximately 3.5 g x 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.
D016954:Acinetobacter calcoaceticus / Q000145:classification / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007659:Ketones / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D010865:Pilot Projects; D011786:Quality Control; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013237:Stereoisomerism
D001643:Bridged Bicyclo Compounds; D005420:Flavoproteins; D007659:Ketones; D007783:Lactones; D011994:Recombinant Proteins; C472066:bicyclo(3,2,0)hept-2-en-6-one; C467817:lactone II; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C019961:cyclohexanone oxygenase; D010100:Oxygen