Publication Details

Category Text Publication
Reference Category Journals
DOI 10.3389/fctls.2022.887458
Licence creative commons licence
Title (Primary) Hydrophobic outer membrane pores boost testosterone hydroxylation by cytochrome P450 BM3 containing cells
Author Bertelmann, C.; Mock, M.; Koch, R.; Schmid, A.; Bühler, B.
Source Titel Frontiers in Catalysis
Year 2022
Department SOMA
Volume 2
Page From art. 887458
Language englisch
Topic T7 Bioeconomy
Keywords Whole-cell biocatalysis; steroid hydroxylation; Substrate uptake; Cytochrome P450 monooxygenase; hydrophobic membrane pores; AlkL
Abstract The implementation of biocatalytic steroid hydroxylation processes at an industrial scale still suffers from low conversion rates. In this study, we selected variants of the self-sufficient cytochrome P450 monooxygenase BM3 from Bacillus megaterium (BM3) for the hydroxylation of testosterone either at the 2β- or 15β-position. Recombinant Escherichia coli cells were used as biocatalysts to provide a protective environment for recombinant enzymes and to ensure continuous cofactor recycling via glucose catabolism. However, only low initial whole-cell testosterone conversion rates were observed for resting cells. Results obtained with different biocatalyst formats (permeabilized cells, cell-free extracts, whole cells) indicated a limitation in substrate uptake, most likely due to the hydrophilic character of the outer membrane of Escherichia coli. Thus, we co-expressed nine genes encoding hydrophobic outer membrane proteins potentially facilitating steroid uptake. Indeed, the application of four candidates led to increased initial testosterone hydroxylation rates. Respective whole-cell biocatalysts even exceeded activities obtained with permeabilized cells or cell-free extracts. The highest activity of 34 U gCDW-1 was obtained for a strain containing the hydrophobic outer membrane protein AlkL from Pseudomonas putida GPo1 and the BM3 variant KSA14m. Overall, we show that the straightforward application of hydrophobic outer membrane pores can boost whole-cell steroid conversion rates and thus be game-changing with regard to industrial steroid production efficiency.
Persistent UFZ Identifier
Bertelmann, C., Mock, M., Koch, R., Schmid, A., Bühler, B. (2022):
Hydrophobic outer membrane pores boost testosterone hydroxylation by cytochrome P450 BM3 containing cells
Front. Catal. 2 , art. 887458 10.3389/fctls.2022.887458