Preparative scale Baeyer–Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process Iris Hilker 1 , Maria C Gutie ´rrez 1 , Roland Furstoss 1 , John Ward 2 , Roland Wohlgemuth 3 & Ve ´ronique Alphand 1 1 Biosciences CNRS 3005, Aix-Marseille Universite ´, Avenue Escadre Normandie-Niemen, 13397 Marseille, France. 2 Department of Biochemistry and Molecular Biology, University College of London, Gower Street, London WC1E 6BT, UK. 3 Research Specialties, Sigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland. Correspondence should be addressed to V.A. (v.alphand@univ-cezanne.fr). Published online 6 March 2008; doi:10.1038/nprot.2007.532 An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer–Villiger biooxidation of ()-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75–80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column. INTRODUCTION Biotransformations have moved from laboratory to industrial large-scale operations to produce chiral building blocks 1,2 . Besides green and sustainable aspects like renewable biocatalysts and generally lower toxicity of processes, biocatalysis is attractive because of the outstanding power of enzymes for chemo-, regio- and enantioselective reactions. Thus, biocatalysts are increasingly being used in catalytic asymmetric syntheses on industrial scale 3 . Biotransformations can be carried out with either isolated enzymes or whole-cell systems. The choice depends on several parameters like commercial availability of the enzyme, price and stability of the enzyme, reaction scale, complexity of the enzymatic system itself (requirement for cofactors, enzyme isoform displaying different selectivities, multicomponent system) as well as on the question how to fit the biotransformation step into a synthetic route in organic synthesis 4 . However, a weakness frequently met in any type of biocatalytic process is the limitation to rather small substrate concentrations. This can be due to one or several of the following reasons: low substrate solubility, inhibitory effect of substrate (or product) on enzyme and toxic effect of substrate (or product) on whole cells. This drawback can considerably hamper the synthetic interest of biocatalysis and several strategies can be implemented to overcome this, as very clearly described by Woodley and co-workers 5 . Besides organic solvents or ionic liquids, controlled substrate supply or downstream product removal, a method based on the use of an adsorbent polymeric resin enables a simultaneous substrate supply and product removal, provided the physicochemical properties of compounds are appropriate. The principle of this method is quite simple (see Fig. 1). The substrate is adsorbed onto the resin. After introduction of the solid phase into the cell broth or the enzyme suspension, the substrate is released from the resin into the broth according to the adsorption/ desorption equilibrium. Then it is enzymatically transformed to the product, which is readsorbed onto the solid resin, thus avoiding both substrate and product accumulation in the aqueous phase. Interestingly, by proper choice of substrate/resin ratio (based on adsorption/desorption equilibria), it is possible to tune up substrate and product concentrations below their inhibitory level. This method, called in situ substrate feeding and product removal (SFPR) or formerly extractive biocatalysis, was successfully applied to sulfoxidation 6 , ketone reduction 7–10 and Baeyer–Villiger (BV) oxidation 11,12 , the object of this protocol. Chemical BV oxidation has been known for more than a century but in spite of many efforts only a small number of asymmetric metal-based catalysts have been described, leading mostly to moderate success 13 . In comparison, biocatalytic BV oxidation is an efficient method to access highly optically active lactones starting from racemic or prochiral ketones 13,14 . Also, it often presents the advantage of high regioselectivity. Safety and health aspects are also improved because the combination of high-energy oxidants and flammable solvents in chemical BV oxidation should be preferably avoided as per risk management strategies for large- scale production. In the biocatalytic BV oxidation, the enzymes replace classical oxidants by air, and flammable solvents by water, thereby substantially lowering safety risks 15 . The enzymes involved are BV monooxygenases (BVMOs), many of which have been identified recently 16 . They are able to oxidize ketones, sulfides 17,18 and even nitrogen. They are cofactor-dependent p u o r G g n i h s i l b u P e r u t a N 8 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h Desorption O 2 Substrate Product Adsorbent resin Air bubbles Biotransformation Cell Enzyme NADPH recycling Cell metabolism Glycerol S S P P P O 2 Bioreactor Adsorption Figure 1 | Principle of resin-based in situ SFPR. 546 | VOL.3 NO.3 | 2008 | NATURE PROTOCOLS PROTOCOL