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1.
Metab Eng ; 79: 49-65, 2023 09.
Article in English | MEDLINE | ID: mdl-37414134

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 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 ß-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.


Subject(s)
Cupriavidus necator , Cupriavidus necator/genetics , Crotonates/metabolism , Metabolic Engineering/methods , Formates/metabolism , Carbon/metabolism
2.
Biotechnol Bioeng ; 117(11): 3422-3434, 2020 11.
Article in English | MEDLINE | ID: mdl-32658302

ABSTRACT

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.


Subject(s)
Biosensing Techniques/methods , Escherichia coli , NAD/metabolism , Adenosine Triphosphate/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Energy Metabolism/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Ethanol/metabolism , Formate Dehydrogenases/genetics , Formate Dehydrogenases/metabolism , Models, Biological
3.
Nat Chem Biol ; 16(5): 538-545, 2020 05.
Article in English | MEDLINE | ID: mdl-32042198

ABSTRACT

Engineering a biotechnological microorganism for growth on one-carbon intermediates, produced from the abiotic activation of CO2, is a key synthetic biology step towards the valorization of this greenhouse gas to commodity chemicals. Here we redesign the central carbon metabolism of the model bacterium Escherichia coli for growth on one-carbon compounds using the reductive glycine pathway. Sequential genomic introduction of the four metabolic modules of the synthetic pathway resulted in a strain capable of growth on formate and CO2 with a doubling time of ~70 h and growth yield of ~1.5 g cell dry weight (gCDW) per mol-formate. Short-term evolution decreased doubling time to less than 8 h and improved biomass yield to 2.3 gCDW per mol-formate. Growth on methanol and CO2 was achieved by further expression of a methanol dehydrogenase. Establishing synthetic formatotrophy and methylotrophy, as demonstrated here, paves the way for sustainable bioproduction rooted in CO2 and renewable energy.


Subject(s)
Escherichia coli/growth & development , Escherichia coli/metabolism , Formates/metabolism , Glycine/metabolism , Methanol/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Carbon Dioxide/metabolism , Escherichia coli/genetics , Genome, Bacterial , Metabolic Engineering/methods , Metabolic Networks and Pathways , Microorganisms, Genetically-Modified , Mutation , Synthetic Biology/methods
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