Browsing by Subject "Whole-cell biotransformation"

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    Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon
    (Springer Verlag, 2012) Gudiminchi, Rama Krishna; Randall, Charlene; Opperman, Diederik J; Olaofe, Oluwafemi A; Harrison, Susan T L; Albertyn, Jacobus; Smit, Martha S
    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 gDCW−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 LBRM−1 was obtained within 24 h (0.34 g LBRM−1 h−1 ) with IPTG-induced cells containing only 0.20 μmol P450 gDCW−1 , when glucose (22 g LBRM−1 ) was added for cofactor regeneration.