Search

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]

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans -4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli . The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23-55 fold increase in space-time yield when using Synechocystis . Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria., (© 2020 Published by Elsevier B.V. on behalf of International Metabolic Engineering Society.)