Growth of E. coli on formate and methanol via the reductive glycine pathway.

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.

NADPH-Auxotrophic E. coli: A Sensor Strain for Testing in Vivo Regeneration of NADPH.

Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.

An Engineering Approach for Rewiring Microbial Metabolism.

The introduction of synthetic pathways into microbes often requires substantial modifications of the host metabolism. Here, we present and discuss key experimental aspects required for modifying microbial central metabolism. We introduce the concept of dividing pathways into metabolic modules, the activity of which can be selected for and optimized in dedicated gene-deletion strains. We provide a comprehensive methodology for systematic pathway implementation in vivo, ranging from gene-deletion methods for the creation of selection strains to cloning strategies that allow fine-tuned expression of individual pathway enzymes in synthetic operons. We further describe pathway testing and validation via high-throughput growth experiments and 13C-labeling measurements. While we focus on Escherichia coli as bacterial host, the holistic approach we present could be easily adapted for the metabolic engineering of other microbes.

Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution.

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.

In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli.

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.

Engineered Assimilation of Exogenous and Endogenous Formate in Escherichia coli.

Decoupling biorefineries from land use and agriculture is a major challenge. As formate can be produced from various sources, e.g., electrochemical reduction of CO2, microbial formate-assimilation has the potential to become a sustainable feedstock for the bioindustry. However, organisms that naturally grow on formate are limited by either a low biomass yield or by a narrow product spectrum. The engineering of a model biotechnological microbe for growth on formate via synthetic pathways represents a promising approach to tackle this challenge. Here, we achieve a critical milestone for two such synthetic formate-assimilation pathways in Escherichia coli. Our engineering strategy involves the division of the pathways into metabolic modules; the activity of each module-providing at least one essential building block-is selected for in an appropriate auxotrophic strain. We demonstrate that formate can serve as a sole source of all cellular C1-compounds, including the beta-carbon of serine. We further show that by overexpressing the native threonine cleavage enzymes, the entire cellular glycine requirement can be provided by threonine biosynthesis and degradation. Together, we confirm the simultaneous activity of all pathway segments of the synthetic serine-threonine cycle. We go beyond the formate bioeconomy concept by showing that, under anaerobic conditions, formate produced endogenously by pyruvate formate-lyase can replace exogenous formate. The resulting prototrophic strain constitutes a substantial rewiring of central metabolism in which C1, glycine, and serine metabolism proceed via a unique set of pathways. This strain can serve as a platform for future metabolic-engineering efforts and could further pave the way for investigating the plasticity of metabolic networks.

The formate bio-economy.

In this review we discuss the concept of the formate bio-economy: formate can be produced efficiently from various available resources and can be consumed by microbes as the sole carbon source for the production of value-added chemicals, directly addressing major challenges in energy storage and chemical production. We show that the formate assimilation pathways utilized by natural formatotrophs are either inefficient or are constrained to organisms that are difficult to cultivate and engineer. Instead, adapting model industrial organisms to formatotrophic growth using synthetic, specially tailored formate-assimilation routes could prove an advantageous strategy. Several studies have started to tackle this challenge, but a fully active synthetic pathway has yet to be established, leaving room for future undertakings.