Efficiency aspects of regioselective testosterone hydroxylation with highly active CYP450‐based whole‐cell biocatalysts

• Published in: Microbial biotechnology Vol. 17; no. 1; pp. e14378 - n/a
• Main Authors: Bertelmann, Carolin, Mock, Magdalena, Schmid, Andreas, Bühler, Bruno
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.01.2024; John Wiley and Sons Inc; Wiley
• Subjects: Biocatalysts; Catalysis; Cytochrome P450; Cytochromes P450; E coli; Efficiency; Enzymes; Gene expression; Glucose; Hydrophobicity; Hydroxylation; Instability; Membrane proteins; Physiology; Plasmids; Process engineering; Stability; Stability analysis; Steroids; Testosterone; Toxicity; Turnover rate
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.14378

Steroid hydroxylations belong to the industrially most relevant reactions catalysed by cytochrome P450 monooxygenases (CYP450s) due to the pharmacological relevance of hydroxylated derivatives. The implementation of respective bioprocesses at an industrial scale still suffers from several limitations commonly found in CYP450 catalysis, that is low turnover rates, enzyme instability, inhibition and toxicity related to the substrate(s) and/or product(s). Recently, we achieved a new level of steroid hydroxylation rates by introducing highly active testosterone‐hydroxylating CYP450 BM3 variants together with the hydrophobic outer membrane protein AlkL into Escherichia coli‐based whole‐cell biocatalysts. However, the activity tended to decrease, which possibly impedes overall productivities and final product titres. In this study, a considerable instability was confirmed and subject to a systematic investigation regarding possible causes. In‐depth evaluation of whole‐cell biocatalyst kinetics and stability revealed a limitation in substrate availability due to poor testosterone solubility as well as inhibition by the main product 15β‐hydroxytestosterone. Instability of CYP450 BM3 variants was disclosed as another critical factor, which is of general significance for CYP450‐based biocatalysis. Presented results reveal biocatalyst, reaction and process engineering strategies auguring well for industrial implementation of the developed steroid hydroxylation platform. Poor stability was found to critically affect testosterone (T) hydroxylation efficiency of E. coli‐based whole‐cell biocatalysts equipped with highly active BM3 variants and AlkL. Beside hydrophobic substrate solubility and product inhibition, inherent instability of BM3 variants was identified as most critical for T conversion into 2β‐ and 15β‐hydroxytestosterone (2/15β‐OH‐T) involving NADPH recycling via glucose catabolism.