title: Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon.
abstract: CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 μM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6-0.8 μM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5-1.0 μmol P450 g (DCW)⁻¹, for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol L (BRM)⁻¹ was obtained within 24 h (0.34 g L (BRM)⁻¹  h⁻¹) with IPTG-induced cells containing only 0.20 μmol P450 g (DCW)⁻¹, when glucose (22 g L (BRM)⁻¹) was added for cofactor regeneration.
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa.
authors.forename: Rama Krishna
authors.initials: RK
authors.lastname: Gudiminchi
authors.forename: Charlene
authors.initials: C
authors.lastname: Randall
authors.forename: Diederik J
authors.initials: DJ
authors.lastname: Opperman
authors.forename: Oluwafemi A
authors.initials: OA
authors.lastname: Olaofe
authors.forename: Susan T L
authors.initials: ST
authors.lastname: Harrison
authors.forename: Jacobus
authors.initials: J
authors.lastname: Albertyn
authors.forename: Martha S
authors.initials: MS
authors.lastname: Smit
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D042926:Cytochrome P-450 CYP4A / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006900:Hydroxylation; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics; D009795:Octanes / Q000378:metabolism*; D009876:Operon
chemical_list: D001426:Bacterial Proteins; D009795:Octanes; D042926:Cytochrome P-450 CYP4A; C026728:octane