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

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 15ß-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.

Directed Reaction Engineering Boosts Succinate Formation of Synechocystis sp. PCC 6803_Δsll1625.

It is known that Synechocystis sp. PCC 6803 carrying a partial deletion of the succinate dehydrogenase (Synechocystis_∆sll1625) secretes succinate during aerobic cultivation with continuous illumination and in the presence of CO2 . Maximal succinate titers of 2 mM (236 mg L-1 ) are reported. CO2 is identified as a crucial parameter for product formation, however, a detailed characterization of different cultivation conditions is still missing. Here the focus is on further reaction engineering to improve the photoautotrophic production of succinate using Synechocystis_∆sll1625. Therefore the impact of light availability, illumination regimes, nutrient availability, and external pH on product formation are investigated. Results obtained in this study reveal the importance of these parameters on the formation of succinate and cultivation with light/dark cycles increases the succinate concentration to 3 mM (354 mg L-1 ) after 28 days of cultivation. Furthermore, cultivation in unbuffered medium under ambient CO2 conditions even doubled the final succinate titer to 4 mM (472 mg L-1 ) after 28 days. Taking biomass concentrations into account, a maximal yield of succinate on biomass of 215 mgSucc  gCDW -1 is achieved, which is the highest so far reported for the production of succinate utilizing Synechocystis as host organism.