Abstract:
: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.
journal_name
Biotechnol Bioengjournal_title
Biotechnology and bioengineeringauthors
Schneider S,Wubbolts MG,Oesterhelt G,Sanglard D,Witholt Bdoi
10.1002/(sici)1097-0290(19990805)64:3<333::aid-bitsubject
Has Abstractpub_date
1999-08-05 00:00:00pages
333-41issue
3eissn
0006-3592issn
1097-0290pii
10.1002/(SICI)1097-0290(19990805)64:3<333::AID-BITjournal_volume
64pub_type
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