Engineering the biological conversion of formate into crotonate in<i>Cupriavidus necator</i>
Abstract 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 CO2and 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 bacteriumC. necatoras 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 growingC. necatorin 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 bacteriumviaa 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..
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Preprint| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
bioRxiv.org - (2023) vom: 18. März Zur Gesamtaufnahme - year:2023 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Collas, Florent [VerfasserIn] |
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Volltext [kostenfrei] |
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| doi: |
10.1101/2023.03.14.532570 |
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| PPN (Katalog-ID): |
XBI038965410 |
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| 520 | |a Abstract 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 CO2and 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 bacteriumC. necatoras 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 growingC. necatorin 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 bacteriumviaa 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. | ||
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| 700 | 1 | |a Dronsella, Beau B. |0 (orcid)0000-0002-5541-3289 |4 aut | |
| 700 | 1 | |a Kubis, Armin |4 aut | |
| 700 | 1 | |a Schann, Karin |0 (orcid)0000-0003-1548-7643 |4 aut | |
| 700 | 1 | |a Binder, Sebastian |4 aut | |
| 700 | 1 | |a Arto, Nils |4 aut | |
| 700 | 1 | |a Claassens, Nico J. |0 (orcid)0000-0003-1593-0377 |4 aut | |
| 700 | 1 | |a Kensy, Frank |4 aut | |
| 700 | 1 | |a Orsi, Enrico |0 (orcid)0000-0002-4741-0344 |4 aut | |
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