Exploring alternative pathways for the in vitro establishment of the HOPAC cycle for synthetic CO2 fixation

Abstract

Nature has evolved eight different pathways for the capture and conversion of CO₂, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO₂-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO₂ fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning-guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO₂ into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.

Fig. 1.. The HOPAC cycle and its alternative designs explored in this study.

(A) Overall scheme of the HOPAC cycle. The cycle converts two molecules of inorganic carbon (HCO₃⁻ and/or CO₂) into one molecule of glyoxylate. The HOPAC cycle can be divided into a reductive and an oxidative part, for each of which two different variants were developed in this study. Color coding indicates key metabolites that were analyzed in this study. Enzyme abbreviations are expanded in table S6. Option 1: Scs, Smt, or Sch; option 2: Smt or Mcs; option 3: GabT or βapt; option 4: βcl or βct. (B) Variants of the reductive part of the HOPAC cycle tested in this study with the respective cofactors (C) Variants of the oxidative part of the HOPAC cycle variants tested in this study with the respective cofactors. ATP and NADPH balances for all possible combinations of the reductive and oxidative parts of the HOPAC cycle are given in Table 1.

Fig. 2.. Construction of the β-alanine route.

(A) Reaction sequence includes reduction of malonyl-CoA into malonic semialdehyde by the C-terminal domain of Mcr (McrC), transamination of malonic semialdehyde to β-alanine by GabT (or βApt), CoA ligation of β-alanine by βCl (or βCt), and lastly deamination of β-alanyl-CoA by βCal. (B and C) Michaelis-Menten kinetics of Chloroflexus aurantiacus McrC for Malonyl-CoA (B) and NADPH (C), respectively (D) Michaelis-Menten kinetics of S. aurantiaca βCal on β-alanyl-CoA. (E and F) Michaelis-Menten kinetics of Escherichia coli GabT for malonic semialdehyde (E) and l-glutamate (F), respectively. (G) Validation of the β-alanine route using GabT and Gdh (assay with βApt and Adh can be found in fig. S18). Values refer to relative concentrations of intermediates as determined by LCMS. Colors correspond to compounds in (A).

Fig. 3.. Construction of the 3HP route.

(A) Reaction sequence of the 3HP route includes reduction of malonyl-CoA to malonic semialdehyde and 3-hydroxypropionate by Mcr, CoA ligation of 3-hydroxypropionate by Pcl (or Pct), and dehydration of 3HP by Ech. (B and C) Michaelis-Menten kinetics of Chloroflexus aurantiacus Mcr for Malonyl-CoA (B) and NADPH (C), respectively. (D) Michaelis–Menten kinetics of P. aeruginosa Ech on 3HP. Michaelis-Menten kinetics of Cupriavidus necator Pcl (E) 3-hydroxypropionate, (F) CoA, and (G) ATP. (H) Validation of the 3HP route using Ccr (assay with Pcs/Pcc can be found in fig. S19). Values refer to relative concentrations of intermediates as determined by LCMS. Colors correspond to compounds in (A).

Fig. 4.. Construction of the free acid route.

(A) Reaction sequence, cleavage of succinyl-CoA to succinate by #1 (Scs, Smt, or Sch), oxidation of succinate to fumarate by Sdh, hydration of fumarate to malate by Fuh, and ligation of malate to malyl-CoA by #2 (Smt or Mcs). Michaelis-Menten kinetics of Chloroflexus aurantiacus Smt (B) malate and (C) succinyl-CoA. (D) Michaelis-Menten kinetics of Escherichia coli Sdh, circles are SdhA, squares are SdhAB on succinate. (E) Michaelis-Menten kinetics of Mus musculus Sch on succinyl-CoA. (F) Promiscuity assay of Sch. (G) Michaelis-Menten kinetics of E. coli Fuh on fumarate.

Fig. 5.. Construction of the fumaryl-CoA route.

(A) Reaction sequence, oxidation of succinyl-CoA to fumaryl-CoA by Mcd and hydration of fumaryl-CoA to malyl-CoA by Mch. (B) Michaelis-Menten kinetics of P. migulae Mcd on succinyl-CoA (triangles) and methylsuccinyl-CoA (circles). (C) Michaelis-Menten kinetics of C. sphaeroides Mch on fumaryl-CoA. (D) Validation of the fumaryl-CoA route using Etf and EtfQO. Values refer to relative concentrations of intermediates as determined by LCMS. Colors correspond to compounds in (A).

Fig. 6.. Active site of methylsuccinyl-CoA dehydrogenase (Mcd).

(A) P. migulae Mcd in complex with FAD and methylsuccinyl-CoA, as determined in this study (PDB: 8CIW). (B) Paracoccus denitrificans Mcd in complex with FAD (PDB: 6ES9) (methylsuccinyl-CoA modeled). Residues in cyan are responsible for substrate binding, orange is the catalytic glutamate, and green was targeted for mutagenesis to improve succinyl-CoA dehydrogenase activity. (C) Reaction scheme for electron transport. FADH₂ can be oxidized by ferrocenium, or as part of a chain through Etf terminating in molecular O₂. (D) Fumaryl-CoA production by Mcd alone, with Etf, with Etf and EtfQO, or with ferrocenium.

Fig. 7.. Uniting and optimizing the HOPAC cycle.

(A) Scheme of the different final HOPAC cycle variants and versions. The best-performing reductive parts (HOPAC_Ccr and HOPAC_Pcs/Pcc) were combined with the CoA route of the oxidative part to create HOPAC_Ccr version 1.0 and HOPAC_Pcs/Pcc version 1.0. Both variants follow the same topology except that HOPAC_Pcs/Pcc uses two ATP-dependent carboxylation reactions (green reactions), while HOPAC_Ccr uses one ATP-dependent carboxylation and one reductive carboxylation reaction (blue reactions). In HOPAC_Ccr version 2.0, ETFQ was added to improve ETF oxidation (dark purple reactions). In HOPAC_Ccr version 3.0, AdoCBl recycling (light purple reactions) was introduced to maintain high Mcm activities. HOPAC_Ccr version 3.1, Mcm was added in regular intervals (red reactions) to maintain high Mcm activities. (B) Carbon fixation efficiency of the HOPAC cycle. Right axis: Number of CO₂/HCO₃⁻ fixed per molecule of acetyl-CoA in the assay. Left axis: Absolute concentration of glycolate produced. Concentrations were determined with triplicate assays measured by LCMS. Colors correspond to variants and versions of the HOPAC cycles as described in (A). (C) Further optimization of HOPAC_Ccr (starting from version 3.0) through the active learning–based workflow “METIS” over eight rounds featuring 240 different combinations of individual enzyme and cofactor concentrations. Right axis: Absolute glycolate concentrations of triplicate assays after 2 hours measured by LCMS. Each dot represents a HOPAC_Ccr assay with different setup. Per round, 30 different combinations of individual enzyme and cofactor concentrations were tested. The best combination (HOPAC_Ccr version 4.0), producing almost 1500 μM glycolate in 2 hours is highlighted in hot pink. (D) Compositions and changes between HOPAC_Ccr version 3.0 and 4.0. All enzymes, factors, and their respective concentrations are provided for version 3.0 and 4.0, and the respective fold changes are indicated in color. Concentrations are given in mM (gray shading) and μM (no shading).