Engineering the biological conversion of formate into crotonate in Cupriavidus necator.

Autor: Collas F; b.fab GmbH, Cologne, Germany., Dronsella BB; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany., Kubis A; b.fab GmbH, Cologne, Germany., Schann K; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany., Binder S; b.fab GmbH, Cologne, Germany., Arto N; b.fab GmbH, Cologne, Germany., Claassens NJ; Laboratory of Microbiology, Wageningen University, Wageningen, the Netherlands., Kensy F; b.fab GmbH, Cologne, Germany., Orsi E; Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany. Electronic address: enricoo@biosustain.dtu.dk.
Jazyk: angličtina
Zdroj: Metabolic engineering [Metab Eng] 2023 Sep; Vol. 79, pp. 49-65. Date of Electronic Publication: 2023 Jul 04.
DOI: 10.1016/j.ymben.2023.06.015
Abstrakt: To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO 2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse β-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 ± 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.
Competing Interests: Declaration of competing interest F.C., A.K. and F.K. are employed by b.fab GmbH, a German biotech company aiming for the biomanufacturing of proteins and chemicals from C1 feedstocks. The other authors declare no competing interest.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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