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Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450 BM‐3 monooxygenase under varied dissolved oxygen concentrations
Author(s) -
Schneider Silke,
Wubbolts Marcel G.,
Oesterhelt Gottfried,
Sanglard Dominique,
Witholt Bernard
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990805)64:3<333::aid-bit9>3.0.co;2-2
Subject(s) - monooxygenase , regioselectivity , chemistry , cytochrome , biochemistry , cytochrome p450 , fatty acid , oxygen , stereochemistry , organic chemistry , enzyme , catalysis
Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450 BM‐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 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 characteristic for in vitro conversions with purified cytochrome P450 BM‐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 C 15:0 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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 333–341, 1999.