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The implementation of biocatalytic steroid hydroxylation processes plays a crucial role in the pharmaceutical industry due to a plethora of medicative effects of hydroxylated steroid derivatives and their crucial role in drug approval processes. Cytochrome P450 monooxygenases (CYP450s) typically constitute the key enzymes catalyzing these reactions, but commonly entail drawbacks such as poor catalytic rates and the dependency on additional redox proteins for electron transfer from NAD(P)H to the active site. Recently, these bottlenecks were overcome by equipping Escherichia coli cells with highly active variants of the self-sufficient single-component CYP450 BM3 together with hydrophobic outer membrane proteins facilitating cellular steroid uptake. The combination of the BM3 variant KSA14m and the outer membrane pore AlkL enabled exceptionally high testosterone hydroxylation rates of up to 45 U gCDW-1 for resting (i.e., living but non-growing) cells. However, a rapid loss of specific activity heavily compromised final product titers and overall space-time yields. In this study, several stabilization strategies were evaluated on enzyme-, cell-, and reaction level. However, neither changes in biocatalyst configuration nor variation of cultivation media, expression systems, or inducer concentrations led to considerable improvement. This qualified the so-far used genetic construct pETM11-ksa14m-alkL, M9 medium, and the resting-cell state as the best options enabling comparatively efficient activity along with fast growth prior to biotransformation. In summary, we report several approaches not enabling a stabilization of the high testosterone hydroxylation rates, providing vital guidance for researchers tackling similar CYP450 stability issues. A comparison with more stable natively steroid-hydroxylating CYP106A2 and CYP154C5 in equivalent setups further highlighted the high potential of the investigated CYP450 BM3-based whole-cell biocatalysts. The immense and continuously developing repertoire of enzyme engineering strategies provides promising options to stabilize the highly active biocatalysts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]