A synthetic pathway for the fixation of carbon dioxide in vitro

Abstract

Carbon dioxide (CO₂) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO₂ in vitro. The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO₂ into organic molecules at a rate of 5 nanomoles of CO₂ per minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO₂ fixation pathways, thereby opening the way for in vitro and in vivo applications.

Fig. 1. Structure guided engineering of Mcd into a Mco.

(A) Active site comparison of the human short-chain acyl-CoA dehydrogenase (PDB: 2VIG, green backbone) with a model of Mcd from Rhodobacter sphaeroides (blue backbone, modeled by the SWISS-MODEL server with 2VIG as template) and the short-chain acyl-CoA oxidase 4 from Arabidopsis thaliana (PDB: 2IX5, orange backbone). The three residues that were targeted to introduce oxidase activity into Mcd are highlighted. (B) HPLC-MS based analysis of wild-type and different single active-site mutants for oxidation of methylsuccinyl-CoA into mesaconyl-CoA with molecular oxygen as electron acceptor. The screen identified three substitutions (W315F, T317G and E377N) that were combined into double and triple mutants. (C) Michaelis-Menten graphs of single, double and triple mutants characterized in more detail. The triple mutant showed best kinetic parameters.

Fig. 2. The CETCH cycle.

(A) Topology of the CETCH cycle (version 5.4), including proofreading and cofactor regenerating enzymes. Numbering of reaction steps and enzyme abbreviations according to Table S3. (B) Dynamics of key intermediates of CETCH 5.4 over 90 minutes. Shown are the levels of six different intermediates, as well as their fractional labeling patterns from the incorporation of ¹³CO₂ for each turn of the cycle (see Fig. S6 for the expected labeling pattern). Intermediate coloring according to panel A. (C) Left x-axis: CO₂-fixation efficiency of the CETCH cycle over the course of its optimization (see Supplementary text). CO₂-fixation efficiency is defined as the CO₂-equivalents fixed per acceptor molecule in the cycle (i.e., starting amount of propionyl-CoA). Right x-axis: Absolute malate concentration formed over the course of 90 minutes in CETCH 5.4. The final assay (0.52 ml) contained 2.3 mg ml^-1 protein of cycle core enzymes plus 0.8 mg ml^-1 auxillary enzymes and produced 540 μM malate over 90 min, which corresponds to 1080 μM fixed CO₂.