Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450(BM-3) monooxygenase under varied dissolved oxygen concentrations

Utilising whole cells of recombinant Escherichia coil K27 (pCYP102, pGEc47) containing active cytochrome P450(BM-3) monooxygenase [E.C. 1.14.14.1], mu ltiple oxidations of saturated and unsaturated fatty acids were performed b y the enzyme under conditions of excess oxygen. The amount of oxygen dissol ved in the culture medium strongly influenced the regioselectivity of the r eaction, as reflected in the distribution and amount of oxidised products. We have verified by gas chromatography/mass spectrometry that the products of in vivo biotransformation of pentadecanoic acid by cytochrome P450(BM-3) are identical to those formed in cell-free extracts containing the enzyme. The formation of keto- and dihydroxy acids, side products which are charac teristic for in vitro conversions with purified cytochrome P450(BM-3) in th e presence of excess oxygen, has been observed as well. Thus, by varying th e oxygen concentration, we could control the regioselectivity of oxidation and the number of products made. Under oxygen limiting conditions, only mon ooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained. Conse quently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion. Furthermore, whole cell oxidation of t wo unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexade c-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids. Although we obt ained higher activities of C-15:0 conversion in vitro, the whole cell bioca talyst proved to be useful for specific oxidations of long-chain fatty acid s since there is no need to add the costly cofactor NADPH. This biooxidatio n by E. coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonst rated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range. (C) 1999 John Wiley & Sons, Inc.