Optimizing E. coli as a formatotrophic platform for bioproduction via the reductive glycine pathway

Kim, Seohyoung; Giraldo, Néstor David; Rainaldi, Vittorio; Machens, Fabian; Collas, Florent; Kubis, Armin; Kensy, Frank; Bar‐Even, Arren; Lindner, Steffen N.

doi:10.3389/fbioe.2023.1091899

Cited by 22 publications

(15 citation statements)

References 41 publications

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“…When further combined with methanol oxidation to formic acid, the pathway may also be altered to co-assimilate methanol and CO 2 , providing a exible platform for bioconversion of various C1-compounds. Based on this pathway, engineered E. coli strains growing formic acid, methanol, and CO 2 alone have been developed 21,24 . The core module of the rGly pathway has also been engineered in S. cerevisiae to convert formate to glycine 31 .…”

Section: Resultsmentioning

confidence: 99%

“…The reductive glycine pathway, a linear route for directly assimilating formate and CO 2 into the central metabolism, has been commonly engineered for C1 compound co-utilization 23 . As a result, several mixotrophic E. coli strains able to grow on CO 2 and formic acid alone have been produced 20,21,24 . One mixotrophic E. coli strain capable of growth on CO 2 and methanol alone was also reported, however, the growth rate was extremely low due to limited methanol oxidation rate 21 .…”

Section: Introductionmentioning

confidence: 99%

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Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

Guo

Zhang

Wang

et al. 2023

Preprint

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Synthetic microorganisms capable of using one-carbon compounds, such as methanol, formic acid or carbon dioxide, are of increasing interest. In this study, we converted the yeasts of Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotroph and formatotroph, allowing them to grow on methanol and formic acid alone coupled with CO2 fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consists of a methanol-formic acid oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using only native enzymes, followed by overexpression of genes in the reductive glycine pathway, blocking the native methanol assimilation pathway, and compartmentalizing the methanol oxidation module. These modifications successfully redesigned the native methylotrophic yeast P. pastoris to grow on both methanol and formic acid, where higher growth rate and yield on methanol was obtained compared to the wild-type strain. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing full synthetic methylotrophy and formatotrophy in this organism. The resulting strain was able to successfully grow on methanol or formic acid alone with consumption rates of 24 mg/L*h and 15.2 mg/L*h, respectively. The CO2 fixation ability of synthetic P. pastoris and S. cerevisiae through the MFORG pathway was confirmed by 13C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid with methanol as the sole carbon source was demonstrated using synthetic P. pastoris and S. cerevisiae, indicating the potential of yeasts as promising hosts for biochemical production from various one-carbon compounds.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

Guo

Zhang

Wang

et al. 2023

Preprint

View full text Add to dashboard Cite

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“…In addition, a strain of E. coli that grows on CO 2 and formate was obtained by equipping with rGlyP 16 and optimised to produce lactate. 17 Recently, Sánchez-Andrea et al 18 showed that Desulfovibrio desulfuricans assimilates CO 2 by rGlyP, which is a previously proposed yet unconfirmed natural CO 2 fixation pathway.…”

Section: Introductionmentioning

confidence: 96%

“…In addition, a strain of E . coli that grows on CO 2 and formate was obtained by equipping with rGlyP 16 and optimised to produce lactate 17 . Recently, Sánchez‐Andrea et al 18 .…”

Section: Introductionmentioning

confidence: 99%

When green carbon plants meet synthetic biology

Wang,

Zhang,

Dai

et al. 2023

Modern Agriculture

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Recycling carbon dioxide (CO2) into chemicals or fuels presents a promising avenue for mitigating carbon emissions and addressing the energy crisis. Plants serve as the ideal platform for the production of materials and chemicals, thanks to their innate capacity to directly use CO2 in the synthesis of various organic compounds. While conventional methods for enhancing plant CO2 fixation may reach their limits, novel technological solutions are imperative. Synthetic biology has illuminated the potential for biosynthesising multiple carbon sources through artificial CO2 fixation pathways in vitro. Recent breakthroughs in photorespiratory bypasses and artificial carboxylation modules offer significant promise for engineering plants to improve carbon fixation, guiding the design and development of plants with more efficient CO2 utilisation. In this context, we begin by summarising recent progress in designing or engineering in vitro CO2 fixation pathways, as well as those solely established in microbes. Subsequently, we delineate strategies employed to enhance CO2 fixation in plants. Finally, we explore potential methods for introducing artificial CO2 fixation pathways into plants. These advancements are critical in advancing synthetic biology's efforts to tackle future challenges related to food and energy scarcity.

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“…Yet, biological conversion of formate into value-added molecules has seldom been explored ( Table 1 ). Apart from the endogenous polymer (PHB), the only platform chemicals derived from formate that are reported in literature are the alcohols isobutanol and 3-methyl-1-butanol (Li et al, 2012), the organic acids mesaconate, 2S-methylsuccinate (Hegner et al, 2020), and lactate (Kim et al, 2023). Most likely, the limited exploration of formate-based bioprocesses has a two-fold explanation: i) the lack of metabolic engineering endeavors targeted for formatotrophic bioproduction combined with ii) several cultivation challenges related to the use of formate as substrate.…”

Section: Introductionmentioning

confidence: 99%

Engineering the biological conversion of formate into crotonate inCupriavidus necator

Collas¹,

Dronsella

Kubis³

et al. 2023

Preprint

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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.

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Optimizing E. coli as a formatotrophic platform for bioproduction via the reductive glycine pathway

Cited by 22 publications

References 41 publications

Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

When green carbon plants meet synthetic biology

Engineering the biological conversion of formate into crotonate inCupriavidus necator

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