Engineering the biological conversion of formate into crotonate in Cupriavidus necator
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved..
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 CO2 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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:79 |
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| Enthalten in: |
Metabolic engineering - 79(2023) vom: 01. Sept., Seite 49-65 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Collas, Florent [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 12.09.2023 Date Revised 18.09.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ymben.2023.06.015 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 CO2 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a C1 bioeconomy | |
| 650 | 4 | |a Crotonate | |
| 650 | 4 | |a Cupriavidus necator | |
| 650 | 4 | |a Fed-batch | |
| 650 | 4 | |a Formate | |
| 650 | 4 | |a Formate toxicity | |
| 650 | 4 | |a Modular pathway engineering | |
| 650 | 4 | |a Reverse beta-oxidation | |
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| 650 | 7 | |a Crotonates |2 NLM | |
| 650 | 7 | |a Formates |2 NLM | |
| 650 | 7 | |a Carbon |2 NLM | |
| 650 | 7 | |a 7440-44-0 |2 NLM | |
| 700 | 1 | |a Dronsella, Beau B |e verfasserin |4 aut | |
| 700 | 1 | |a Kubis, Armin |e verfasserin |4 aut | |
| 700 | 1 | |a Schann, Karin |e verfasserin |4 aut | |
| 700 | 1 | |a Binder, Sebastian |e verfasserin |4 aut | |
| 700 | 1 | |a Arto, Nils |e verfasserin |4 aut | |
| 700 | 1 | |a Claassens, Nico J |e verfasserin |4 aut | |
| 700 | 1 | |a Kensy, Frank |e verfasserin |4 aut | |
| 700 | 1 | |a Orsi, Enrico |e verfasserin |4 aut | |
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