In-Depth Computational Analysis of Natural and Artificial Carbon Fixation Pathways

Abstract

In the recent years, engineering new-to-nature CO₂- and C1-fixing metabolic pathways made a leap forward. New, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham (CBB) cycle. The question remains how to best predict their in vivo performance and what actually makes one pathway "better" than another. In this context, we explore aerobic carbon fixation pathways by a computational approach and compare them based on their specific activity and yield on methanol, formate, and CO₂/H₂ considering the kinetics and thermodynamics of the reactions. Besides pathways found in nature or implemented in the laboratory, this included two completely new cycles with favorable features: the reductive citramalyl-CoA cycle and the 2-hydroxyglutarate-reverse tricarboxylic acid cycle. A comprehensive kinetic data set was collected for all enzymes of all pathways, and missing kinetic data were sampled with the Parameter Balancing algorithm. Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway-specific activities. The specific activities of the reductive glycine pathway, the CETCH cycle, and the new reductive citramalyl-CoA cycle were predicted to match the best natural cycles with superior product-substrate yield. However, the CBB cycle performed better in terms of activity compared to the alternative pathways than previously thought. We make an argument that stoichiometric yield is likely not the most important design criterion of the CBB cycle. Still, alternative carbon fixation pathways were paretooptimal for specific activity and product-substrate yield in simulations with C1 substrates and CO₂/H₂ and therefore hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.

Figure 1

Schematic workflow that was applied to calculate the pathway-specific activities from the kinetic parameter set and the flux distributions of each pathway. Box with white-colored filling indicate input data(sources), blue boxes indicate processed data. $N$: stoichiometric matrix; $K_{\text{eq}}$: equilibrium constants; $K_m$: Michaelis constants; $k_{\text{cat}}$: forward rate constants; $M$: molecular weight.

Figure 2

Overview of natural occurring and artificial CO₂- and C1-fixing pathways that were integrated in the model. For simplification, reaction arrows can include multiple reactions and skip some metabolites. ADP, AMP, phosphate, water, and oxidized forms of electron carriers were also left out to improve clarity. Primary products of the pathways are underlined. Abbreviations: Ac-CoA: acetyl-CoA; Prop-CoA: propanoyl-CoA; β-MM-CoA: β-methylmalyl-CoA; CitMal-CoA: citramalyl-CoA; Pyr: pyruvate; MM-CoA: methylmalonyl-CoA; Succ-CoA: succinyl-CoA; Glyox: glyoxylate; 4HB-CoA: 4-hydroxybutyrate; AcAc-CoA: acetoacetyl-CoA; R15P: ribulose-1,5-bisphosphate; 3PG: 3-phosphoglycerate; 13BPG: 1,3-bisphophoglycerate; GAP: glyceraldehyde-3-phosphate; R5P: ribulose-5-phosphate; H6P: hexulose-6-phosphate; MSAld: malonate semialdehyde; Ala: alanine; β-Ala: β-alanine; Oxaloac: oxaloacetate; 6-PG: 6-phosphoglycerate; OH-Pyr: hydroxypyruvate; 2-PG: 2-phosphoglycerate; 10-CHO-THF: 10-formyltetrahydrofolate; CH2-THF: 5,10-methylenetetrahydrofolate; LP-S₂: [glycine-cleavage complex H protein]-N6-lipoyl-L-lysine; LP-S-CH₂NH₂: [glycine-cleavage complex H protein]-S-aminomethyl-N6-dihydrolipoyl-L-lysine; LP-SH: [glycine-cleavage complex H protein]-dihydrolipoyl-L-lysine.

Figure 3

Overview of (a) the reductive citramalyl-CoA cycle (rCCC) and (b) the 2-hydroxyglutarate-reverse TCA cycle (2-HG-rTCA cycle) that were designed in this work. For simplification, reaction arrows can include multiple reactions and skip some metabolites. ADP, AMP, phosphate, water, and oxidized forms of electron carriers were also left out to improve clarity. Abbreviations: Succ-CoA: succinyl-CoA; Succ: succinate.

Figure 4

Overview of the phosphatase-less Calvin-Benson-Bassham cycle variant that was designed in this work. Dashed lines represent the phosphotransferase reactions that were added which replace the phosphatase reaction and transfer 2 phosphate groups to ribulose-5-phosphate (R5P). For simplification, reaction arrows can include multiple reactions and skip some metabolites. ADP, AMP, phosphate, water, and oxidized forms of electron carriers were also left out to improve clarity. Abbreviations: R15P: ribulose-1,5-bisphosphate; 3PG: 3-phosphoglycerate; 13BPG: 1,3-bisphosphoglycerate; GAP: glyceraldehyde-3-phosphate; FBP: fructose-1,6-bisphosphate; F6P: fructose-6-phosphate; S17BP: seduheptulose-1,7-bisphosphate; PP_i: pyrophosphate; S7P: seduheptulose-7-phosphate; R5P: ribulose-5-phosphate.

Figure 5

Design of a pathway to convert acetyl-CoA (Ac-CoA) to pyruvate (Pyr) using parts of the reductive citramalyl-CoA cycle (blue) and the ethylmalonyl-CoA pathway (grey). The dashed arrow denotes the possible recycling of acetyl-CoA for the first reaction of the ethylmalonyl-CoA pathway. Abbreviations: EM-CoA: ethylmalonyl-CoA; Mesac-CoA: mesaconyl-CoA; CitMal-CoA: citramalyl-CoA; Ac-CoA: acetyl-CoA; Pyr: pyruvate; AcAc-CoA: acetoacetyl-CoA; 3-HB-CoA: 3-hydroxybutyryl-CoA.

Figure 6

Comparison of the specific activity of natural and artificial carbon fixation pathways with the Enzyme Cost Minimization algorithm. The final product of choice was glyceraldehyde-3-phosphate in this case.. The concentrations of CO₂ and HCO₃^- were assumed to not exceed 10 μM and 100 μM, respectively, corresponding approximately to air saturation. Pathway-specific activities with standard deviations and the full name of the main pathways and the connecting modules to transform their primary product to glyceraldehyde-3-phosphate. Pathway abbreviations: CBB(PTS): phosphatase-free CBB cycle; HP-bicycle(glyox): glyoxylate-producing subcycle of the 3-HP bicycle; PyrC: pyruvate carboxylase; OAAtoGAP: gluconeogenesis; b-OH-AspCycle: β-hydroxyaspartate cycle; AcCoAtoOAA(HP): acetyl-CoA to oxaloacetate-converting module derived from the 3-HP/4-HB cycle using either ADP- or AMP-producing CoA-ester synthases; rCC-AcCoAtoPyr: acetyl-CoA to pyruvate-converting module derived from the rCCC; GlyoxToOAA: glyoxylate- to oxaloacetate-converting module based on the Serine cycle; GlyoxToGAP(GCL/GDH): glyoxylate conversion by glyoxylate carboligase and glycerate dehydrogenase. The overall stoichiometries of all pathways are listed in supplementary Table S5.

Figure 7

Comparison of carbon fixation pathways using methanol as a substrate for the production of pyruvate with the Enzyme Cost Minimization algorithm. “(w/o SHMT)” indicates that the cost of the serine hydroxymethyltransferase are not included in the respective pathway’s activity. The concentration of CO₂ was assumed to be 1 mM and the concentration of HCO₃^- to be 10 mM. (a) Pathway-specific activities with standard deviations and the full name of the main pathways and the connecting modules to transform their primary product to pyruvate. Pathway abbreviations: CBB(PTS): phosphatase-free CBB cycle; HP-bicycle(glyox): glyoxylate producing subcycle of the 3-HP bicycle; GAPtoPyr: glycolysis; FCL: formaldehyde:NADP+ oxidoreductase (formyl-CoA-forming); GlyoxToPyr: GCL/GDH route; b-OH-AspCycle: β-hydroxyaspartate cycle; AcCoAtoOAA(HP): acetyl-CoA to oxaloacetate-converting module derived from the 3-HP/4-HB cycle using either ADP- or AMP-producing CoA-ester synthases; rCC-AcCoAtoPyr: acetyl-CoA to pyruvate-converting module derived from the rCCC; GlyoxToOAA: glyoxylate- to oxaloacetate-converting module based on the Serine cycle. (b) Pathway-specific activities compared to product-substrate yield. The overall stoichiometries of all pathways and modules are listed in Table 1 and supplementary Table S4 and S5. The small letters label each pathway and correspond to the labels and the respective pathway combinations in (a).

Figure 8

Comparison carbon fixation pathways using formate as a substrate for the production of pyruvate with the Enzyme Cost Minimization algorithm. “(w/o SHMT)” indicates that the cost of the serine hydroxymethyltransferase is not included in the respective pathway’s activity. The concentration of CO₂ was assumed to be 1 mM and the concentration of HCO₃^- to be 10 mM. (a) Pathway-specific activities with standard deviations and the full name of the main pathways and the connecting modules to transform their primary product to pyruvate. Pathway abbreviations: CBB(PTS): phosphatase-free CBB cycle; HP-bicycle(glyox): glyoxylate producing subcycle of the 3-HP bicycle; GAPtoPyr: glycolysis; FCL: formaldehyde:NADP+ oxidoreductase (formyl-CoA-forming); GlyoxToPyr: GCL/GDH route; b-OH-AspCycle: β-hydroxyaspartate cycle; AcCoAtoOAA(HP): acetyl-CoA- to oxaloacetate-converting module derived from the 3-HP/4-HB cycle using either ADP- or AMP-producing CoA-ester synthases; rCC-AcCoAtoPyr: acetyl-CoA- to pyruvate-converting module derived from the rCCC; GlyoxToOAA: glyoxylate to oxaloacetate converting module based on the Serine cycle. (b) Pathway-specific activities compared to product-substrate yield. The overall stoichiometries of all pathways and modules are listed in Table 1 and supplementary Table S4 and S5. The small letters label each pathway and correspond to the labels and the respective pathway combinations in (a).

Figure 9

Enzyme demands to sustain a total pathway activity of 1 mmol product per second. Contribution of the capacity, the reversibility, and the saturation with substrates or products of each reaction to the demand of enzymes for the CBB cycle, the rCCC, and the 2-HG-rTCA cycle. The figure follows the wording of Noor et al. [29]. “Capacity”: demand of enzyme caused by a limitation by the catalytic rate constant; “Reversibility”: extra amount of enzyme needed because of a backward flux; “Saturation”: additional enzyme necessary because of undersaturation with a substrate or oversaturation with a product. The values present the optimized state as predicted by the ECM algorithm assuming a CO₂ concentration of 10 μM and HCO₃^- concentration of 100 μM. Glyceraldehyde-3-phosphate was chosen as a product for the pathways. Reaction names correspond to their identifiers in the SBtab model file (Supplementary file Reactions_Composite22_model.tsv (available here)).

Figure 10

Comparison carbon fixation pathways using H₂/CO₂ as a substrate for the production of pyruvate with the Enzyme Cost Minimization algorithm. “(w/o SHMT)” indicates that the cost of the serine hydroxymethyltransferase is not included in the respective pathway’s activity. The concentration of CO₂ was assumed to be 1 mM and the concentration of HCO₃^- to be 10 mM. (a) Pathway-specific activities with standard deviations and the full name of the main pathways and the connecting modules to transform their primary product to pyruvate. Pathway abbreviations: CBB(PTS): phosphatase-free CBB cycle; HP-bicycle(glyox): glyoxylate-producing subcycle of the 3-HP bicycle; GAPtoPyr: glycolysis; FCL: formaldehyde:NADP+ oxidoreductase (formyl-CoA-forming); GlyoxToPyr: GCL/GDH route; b-OH-AspCycle: β-hydroxyaspartate cycle; AcCoAtoOAA(HP): acetyl-CoA- to oxaloacetate-converting module derived from the 3-HP/4-HB cycle using either ADP- or AMP-producing CoA-ester synthases; rCC-AcCoAtoPyr: acetyl-CoA- to pyruvate-converting module derived from the rCCC; GlyoxToOAA: glyoxylate- to oxaloacetate-converting module based on the Serine cycle. (b) Pathway-specific activities compared to product-substrate yield. The overall stoichiometries of all pathways and modules are listed in Table 1 and supplementary Table S4 and S5. The small letters label each pathway and correspond to the labels and the respective pathway combinations in (a).