Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations.

Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450BM-3 monooxygenase [E.C. 1. 14.14.1], multiple oxidations of saturated and unsaturated fatty acids were performed by the enzyme under conditions of excess oxygen. The amount of oxygen dissolved in the culture medium strongly influenced the regioselectivity of the reaction, 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 P450BM-3 are identical to those formed in cell-free extracts containing the enzyme. The formation of keto- and dihydroxy acids, side products which are characteristic for in vitro conversions with purified cytochrome P450BM-3 in the presence of excess oxygen, has been observed as well. Thus, by varying the oxygen concentration, we could control the regioselectivity of oxidation and the number of products made. Under oxygen limiting conditions, only monooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained. Consequently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion. Furthermore, whole cell oxidation of two unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexadec-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids. Although we obtained higher activities of C15:0 conversion in vitro, the whole cell biocatalyst proved to be useful for specific oxidations of long-chain fatty acids since there is no need to add the costly cofactor NADPH. This biooxidation by E. coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonstrated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range.

[1]  M. Wubbolts,et al.  Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase , 1998, Applied and Environmental Microbiology.

[2]  M. Wubbolts,et al.  Biosynthesis of synthons in two-liquid-phase media. , 2000, Biotechnology and bioengineering.

[3]  J. Falck,et al.  The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450BM-3* , 1996, The Journal of Biological Chemistry.

[4]  S. D. Black,et al.  Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium. , 1994, Archives of biochemistry and biophysics.

[5]  B. Witholt,et al.  Continuous production of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans in a high-cell-density, two-liquid-phase chemostat , 1993 .

[6]  C. Slaughter,et al.  Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions. , 1992, Archives of biochemistry and biophysics.

[7]  W. Deckwer,et al.  High cell density cultivation of Escherichia coli at controlled specific growth rate. , 1991, Journal of biotechnology.

[8]  S. Boddupalli,et al.  Fatty acid monooxygenation by cytochrome P-450BM-3. , 1990, The Journal of biological chemistry.

[9]  A. Fulco,et al.  Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. , 1989, The Journal of biological chemistry.

[10]  G. Huisman,et al.  Formation of Polyesters by Pseudomonas oleovorans: Effect of Substrates on Formation and Composition of Poly-(R)-3-Hydroxyalkanoates and Poly-(R)-3-Hydroxyalkenoates , 1988, Applied and environmental microbiology.

[11]  B. Witholt,et al.  Controlled and functional expression of the Pseudomonas oleovorans alkane utilizing system in Pseudomonas putida and Escherichia coli. , 1987, The Journal of biological chemistry.

[12]  A. Fulco,et al.  Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts. , 1987, The Journal of biological chemistry.

[13]  L. Narhi,et al.  Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. , 1986, The Journal of biological chemistry.

[14]  H. Kleber,et al.  Effect of oxygen Iimitation on the content of n‐hexadecane‐inducible cytochrome P‐450 in Acinetobacter calcoaceticus strain EB 104 , 1986, Journal of basic microbiology.

[15]  K. Wimalasena,et al.  Mechanistic studies on non-heme iron monooxygenase catalysis: epoxidation, aldehyde formation and demethylation by the .omega.-hydroxylation system of Pseudomonas oleovorans , 1984 .

[16]  B. Witholt,et al.  Synthesis of 1,2-Epoxyoctane by Pseudomonas oleovorans During Growth in a Two-Phase System Containing High Concentrations of 1-Octene , 1981, Applied and environmental microbiology.

[17]  A. Fulco,et al.  Epoxidation of unsaturated fatty acids by a soluble cytochrome P-450-dependent system from Bacillus megaterium. , 1981, The Journal of biological chemistry.

[18]  B. Witholt.  Method for Isolating Mutants Overproducing Nicotinamide Adenine Dinucleotide and Its Precursors , 1972, Journal of bacteriology.

[19]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. II. SOLUBILIZATION, PURIFICATION, AND PROPERTIES. , 1964, The Journal of biological chemistry.

[20]  R. E. Laramy,et al.  Flame Response in the Quantitative Determination of High Molecular Weight Paraffins and Alcohols by Gas Chromatography. , 1963 .