BIOCATALYST ENGINEERING BY ASSEMBLY OF FATTY-ACID TRANSPORT AND OXIDATION ACTIVITIES FOR IN-VIVO APPLICATION OF CYTOCHROME P-450(BM-3) MONOOXYGENASE

The application of whole cells containing cytochrome P-450(BM-3) monoo xygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to omega-1, omega-2, and omega-3 hydroxy fatty acids was i nvestigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, a nd 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants contain ing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450(BM-3) were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid upta ke system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system off. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenz yme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47? which has been shown to effect facilitated uptake of o leic acid in E. coli W3110 (M. Nieboer, Ph.D, thesis, University of Gr oningen, Groningen, The Netherlands, 1996), By using a double recombin ant off. coli K27, which is a fadD mutant and therefore unable to cons ume substrates or products via the beta-oxidation cycle, a twofold inc rease in productivity was achieved. Applying cytochrome P-350(BM-3) mo nooxygenase as a biocatalyst in whole cells does not require the exoge nous addition of the costly cofactor NADPH. In combination with the co enzyme A-independent fatty acid uptake system from P. oleovorans, cyto chrome P-450(BM-3) recombinants appear to be useful alternatives to th e enzymatic approach for the bioconversion of long-chain fatty acids t o subterminal hydroxylated fatty acids.