Awakening a latent carbon fixation cycle in Escherichia coli

Abstract

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

Fig. 1. In silico identification of latent carbon fixation pathways.

a The GED cycle, the simplest carbon fixation pathway that can be generated from E. coli endogenous enzymes. The pathway is based on the reductive carboxylation of ribulose 5-phosphate (Ru5P) to 6-phosphogluconate (6PG) by Gnd, followed by the activity of the Entner–Doudoroff pathway (enzymes Edd and Eda) to produce pyruvate and glyceraldehyde-3-phosphate (GAP). Pyruvate is converted to GAP via gluconeogenesis and the intermediates phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PG). To close the cycle, GAP is recycled to Ru5P via the pentose phosphate pathway. The GED cycle mirrors the ribulose bisphosphate cycle (i.e., Calvin–Benson cycle), which is shown in gray. b, c Two other general archetypes of carbon fixation pathways that were computationally uncovered. Both are based on integrated cycles, which together reduce CO₂ to formate and then assimilate formate into pyruvate. AcCoA corresponds to acetyl-CoA and PrCoA corresponds to propionyl-CoA. CH₂-THF corresponds to methylene-THF. d A phylogenetic tree of bacteria, showing the three phyla that harbor all key enzymes of the GED cycle (shown in color). Numbers in parentheses correspond to the number of species in which the key pathway enzymes were found.

Fig. 2. Activity of the GED shunt in a ∆rpe strain.

a Design of the ∆rpe selection scheme. Ribose can be assimilated only via the activity of the GED shunt, where the biosynthesis of almost all biomass building blocks is dependent on the pathway (marked in yellow). Growth on gluconate (violet) is not dependent on reductive carboxylation via Gnd and thus serves as a positive control. Reaction directionalities are shown as predicted by flux balance analysis. b Growth of a ∆rpe strain on ribose (20 mM) as a sole carbon source is dependent on elevated CO₂ concentration (20%, i.e., 200 mbar) and overexpression of gnd, edd, and eda (pGED). Overexpression of only gnd (pG) or only edd and eda (pED) failed to establish growth (less than two doublings). Cultivation at ambient CO₂ also failed to achieve growth. Values in parentheses indicate doubling times. Curves represent the average of technical duplicates, which differ from each other by <5%. Growth experiments were repeated independently three times to ensure reproducibility. c Cultivation on ¹³CO₂ confirms the operation of the GED shunt. On the left, a prediction of the labeling pattern of key amino acids is shown. The observed labeling fits the prediction and differs from the WT control cultivated under the same conditions. Labeling of amino acids in the WT strain stems from the natural occurrence of ¹³C as well as from reactions that exchange cellular carbon with CO₂, e.g., the glycine cleavage system and anaplerotic/cataplerotic cycling. Values represent averages of two independent cultures that differ from each other by <10%. 3PG 3-phosphoglycerate, ALA Alanine, GAP glyceraldehyde-3-phosphate, GLY Glycine, HIS Histidine, PYR pyruvate, SER Serine, VAL Valine. Source data underlying b and c are provided as a Source Data file.

Fig. 3. Activity of the GED shunt in a ∆tktAB strain.

a Design of the ∆tktAB selection scheme. Xylose can be assimilated only via the GED shunt. E4P supplements are provided as the ∆tktAB strain cannot synthesize erythrose 4-phosphate. Growth on gluconate is not dependent on reductive carboxylation by Gnd and thus serves as a positive control. Reaction directionalities are shown as predicted by flux balance analysis. b Growth on xylose upon overexpression of gnd, edd, and eda (pGED) was achieved only after mutation and was dependent on elevated CO₂ concentration. Values in parentheses indicate doubling times. Curves represent the average of technical duplicates, which differ from each other by <5%. Growth experiments were repeated independently three times to ensure reproducibility. c Expression analysis by quantitative RT-PCR revealed that the transcript level of pntA increased ~3-fold in the mutated strain. Bars correspond to the average of two independent experiments, which are shown as circles. Gluconate and xylose indicate carbon sources used. d Genomic overexpression of pntAB using medium (M) or strong (S) promoter, but not weak (W) promoter, supported growth of a ∆tktAB pGED strain on xylose (legend to the left). e Deletion of glucose 6-phosphate dehydrogenase (∆zwf) supported the growth of a ∆tktAB pGED strain on xylose (legend to the left). f ¹³C-labeling experiments confirm the operation of the GED shunt. Cells were cultivated with xylose (1-¹³C) and ¹³CO₂. Observed labeling fits the expected pattern and differs from that of a WT strain cultured under the same conditions. Results from additional labeling experiments are shown in Supplementary Fig. 2. 3PG 3-phospho-glycerate, ALA Alanine, GAP glyceraldehyde-3-phosphate, GLY Glycine, HIS Histidine, PYR pyruvate, SER Serine, VAL Valine. Source data underlying b–f are provided as a Source Data file.

Fig. 4. Activity of the GED shunt in a ∆PZF strain that enables a smooth transition into a GED cycle.

a Design of the ∆PZF (∆pfkAB ∆zwf ∆fsaAB ∆fruK) selection scheme. Xylose can be assimilated only via the activity of the GED shunt, where the biosynthesis of most biomass building blocks is dependent on the pathway (marked in yellow). Growth on gluconate (violet) is not dependent on reductive carboxylation via Gnd and thus serves as a positive control. Reaction directionalities are shown as predicted by flux balance analysis. b Growth on xylose upon overexpression of gnd, edd, and eda (pGED) was achieved only after adaptive evolution and was dependent on elevated CO₂ concentration. Values in parentheses indicate doubling times. Curves represent the average of technical quadruplicates, which differ from each other by <5%. Growth experiments were repeated independently three times to ensure reproducibility. c ¹³C-labeling experiments confirm the operation of the GED shunt (mutant ‘C’). Cells were cultivated with xylose (1-¹³C) and ¹³CO₂. Observed labeling fits the expected pattern and differs from that of a WT strain cultured under the same conditions. Results from additional labeling experiments are shown in Supplementary Fig. 3. 3PG 3-phosphoglycerate, ALA Alanine, GAP glyceraldehyde-3-phosphate, GLY Glycine, HIS Histidine, PYR pyruvate, SER Serine, VAL Valine. Source data underlying b and c are provided as a Source Data file.

Fig. 5. Rerouting sugar fermentation via the GED shunt can increase product yields.

In this example, we chose xylose as the sugar substrate, due to its growing relevance as a renewable feedstock derived from lignocellulosic wastes. An additional analysis with glucose as the fermentation feedstock is shown in Supplementary Fig. 4. a An overview comparing xylose utilization routes: Canonical utilization (gray) via the pentose phosphate pathway and glycolysis; the GED shunt (blue); and a linear pathway based on carboxylation by Rubisco (RuBP shunt, orange). b Maximal theoretical yields of 15 fermentation products are shown relative to the WT reference, as calculated by flux balance analysis. Presented values are normalized to the product yield from canonical sugar utilization (pentose phosphate pathway and glycolysis). Most products are predicted to require the secretion of other organic compounds (e.g., acetate, formate) to achieve a balancing of reducing equivalents or support ATP biosynthesis. Of the products shown in the figure, only butyrate is predicted to be produced without byproducts. Source data underlying b are provided as a Source Data file.