Computational protein design enables a novel one-carbon assimilation pathway

Abstract

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.

Keywords: carbon fixation; computational protein design; pathway engineering.

Fig. 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.

Fig. 2.

Comparison of design model and crystal structure. (A) Native BAL (PDB ID code 2AG0) with TPP and a docked model of benzoin in the active site. The residues that were mutated in the designed formose enzyme (FLS) are highlighted in brown. (B) Model of FLS active site with the four active site mutations (brown) around the DHA bound intermediate (purple). (C) Overlay of the Des1 crystal structure (blue) and the FLS model (green, with mutated residues brown) with the docked DHA product (purple). The four active site mutations (BAL vs. Des1) are shown in sticks, conserved amino acids in lines. Figure made with PyMol (38).

Fig. 3.

Formolase pathway conversion of formate into dihydroxyacetone-phosphate by purified enzymes. (A) Production of ¹³C-DHAP from ¹³C-Formate by the combination of purified ecACS, lmACDH, FLS, FDH, and DHAK. Production of DHAP absolutely requires FLS. (B) (Upper) Dependence of ¹³C-DHAP production on reaction components. (Lower) The SDS/PAGE gel illustrates the protein levels for each component; the concentration of FDH is too low to be evident on the gel. Each rate is based on a linear fit to five independent measurements. Error bars represent SE.

Fig. 4.

Conversion of formate into the central metabolites DHAP and 2/3-PG by cell lysates. Protein-normalized concentrations of ¹³C-DHAP (DHAP M+3), and ¹³C-2/3-PG (2/3-PG M+3) in clarified cell lysates with the pathway genes for ecACS, lmACDH, FLS, and yDHAK (pTrcCO₂-3, pSB3K3 yDHAK), or in the absence of the key formate assimilation enzymes with only yDHAK (pTrcHis2, pSB3K3 yDHAK), after incubation with ¹³C-formate for 24 h. Commercial FDH was added to balance the net NADH oxidation rate of the test extracts in a 1:1 ratio as described in Materials and Methods. Error bars represent the SE of measurements for three biological replicates.

Fig. 5.

Thermodynamics and carbon utilization efficiencies. Biomass yield, in gram cellular dry weight (gCDW) per mole of formate consumed, was calculated using flux balance analysis and the core metabolic model of E. coli supplemented with pathway enzymes and without considering ATP maintenance (6). The MDF is the lowest value of −Δ_rG′ in the pathway [for the reaction(s) with the smallest chemical driving force force] (37). Squares correspond to pathways that can directly assimilate formate, whereas circles mark carbon fixation pathways that can indirectly assimilate formate following oxidation. Numbers in parentheses indicate the number of foreign enzymes that need to be expressed in E. coli to establish an active pathway. Pathway abbreviations are as follows: DCHB, dicarboxylate-4-hydroxypropionate cycle; FLS, formolase pathway; HPBC, 3-hydroxypropionate bicycle; HPHB, 3-hydroxypropionate-4-hydroxybutyrate cycle; rAC, reductive acetyl-CoA pathway; rPP, reductive pentose phosphate cycle/Calvin-Benson-Bassham cycle; rTCA, reductive TCA cycle; RuMP, ribulose 4-phosphate cycle; SER, serine cycle; Xy5P, xylulose 5-phosphate cycle/dihydroxyacetone cycle).