Computational protein design enables a novel one-carbon assimilation pathway

We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.Novel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1–3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a one-carbon fixation pathway (5–8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based on existing enzymes.Open in a separate windowFig. 1.Overview of formolase pathway reactions. (A) Benzaldehyde lyase couples two benzaldehydes into benzoin through an acyloin addition reaction. (B) Acetyl-CoA synthase (ACS) catalyzes the ATP-dependent conversion of acetate into acyl-CoA. (C) Acetaldehyde dehydrogenase (ACDH) catalyzes the NADH-dependent reduction of acetyl-CoA to acetaldehyde. (D) Conversion of formate to dihydroxyacetone phosphate (DHAP) by the formolase pathway. To generate reducing equivalents in the cell, formate is oxidized by formate dehydrogenase (FDH) to produce CO₂ and NADH (stage 1). To use formate as a carbon source, activation (stage 2) and carbon-carbon coupling (stage 3) to form dihydroxyacetone (DHA) are carried out by the enzymes ACS, ACDH, and formolase (FLS). DHA is phosphorylated to DHAP, a glycotic intermediate by a dihydroxyacetone kinase (DHAK) (stage 4). The novel enzyme functions identified here are underlined.