- Kim, S., Lindner, S.N., Aslan, S. et al. Growth of E. coli on formate and methanol via the reductive glycine pathway. Nat Chem Biol (2020). doi:10.1038/s41589-020-0473-5
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.
The integration of electrochemical and microbial processes offers a unique opportunity to displace fossil carbon with CO2 and renewable energy as the primary feedstocks for carbon-based chemicals. Yet, it is unclear which strategy for CO2 activation and electron transfer to microbes has the capacity to transform the chemical industry. Here, we systematically survey experimental data for microbial growth on compounds that can be produced electrochemically, either directly or indirectly. We show that only a few strategies can support efficient electromicrobial production, where formate and methanol seem the best electron mediators in terms of energetic efficiency of feedstock bioconversion under both anaerobic and aerobic conditions. We further show that direct attachment of microbes to the cathode is highly constrained due to an inherent discrepancy between the rates of the electrochemical and biological processes. Our quantitative perspective provides a data-driven roadmap towards an economically and environmentally viable realization of electromicrobial production.
Archived at the Zenodo repository at: https://zenodo.org/record/3516562#.Xd_JD1dKiUk
- Krüsemann J.L., Lindner S.N., Dempfle M., Widmer J., Arrivault S., Debacker M., He H., Kubis A., Chayot R., Anissimova M., Marlière P., Cotton C.A.R., Bar-Even A. 2018. Artificial pathway emergence in central metabolism from three recursive phosphoketolase reactions. doi: 10.1111/febs.14682
The promiscuous activities of a recursive, generalist enzyme provide raw material for the emergence of metabolic pathways. Here, we use a synthetic biology approach to recreate such an evolutionary setup in central metabolism and explore how cellular physiology adjusts to enable recursive catalysis. We generate an Escherichia coli strain deleted in transketolase and glucose 6‐phosphate dehydrogenase, effectively eliminating the native pentose phosphate pathway. We demonstrate that the overexpression of phosphoketolase restores prototrophic growth by catalyzing three consecutive reactions, cleaving xylulose 5‐phosphate, fructose 6‐phosphate, and, notably, sedoheptulose 7‐phosphate. We find that the activity of the resulting synthetic pathway becomes possible due to the recalibration of steady‐state concentrations of key metabolites, such that the in vivo cleavage rates of all three phosphoketolase substrates are similar. This study demonstrates our ability to rewrite one of nature's most conserved pathways and provides insight into the flexibility of cellular metabolism during pathway emergence.
- During V., Darii E., Yishai O., Bar-Even A., Bouton M. 2018. Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution. doi: 10.1021/acssynbio.8b00167
Endowing biotechnological platform organisms with new carbon assimilation pathways is a key challenge for industrial biotechnology. Here we report progress toward the construction of formatotrophic Escherichia coli strains. Glycine and serine, universal precursors of one-carbon compounds oxidized during heterotrophic growth, are produced from formate and CO2 through a reductive route. An adaptive evolution strategy was applied to optimize the enzymatic steps of this route in appropriate selection strains. Metabolic labeling experiments with 13C-formate confirm the redirected carbon-flow. These results demonstrate the high plasticity of the central carbon metabolism of E. coli and the applicative potential of directed evolution for implementing synthetic pathways in microorganisms.
- Yishai O., Blouson M., Dring V., Bar-Even A. 2018. In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli. doi: 10.1021/acssynbio.8b00131
Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2. Importantly, the biosynthesis of serine from formate and CO2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.