Sugar Synthesis from CO2 in Escherichia coli

Abstract

Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PAPERCLIP.

Graphical abstract

Figure 1

Decoupling Energy Production and Carbon Fixation in E. coli to Achieve Hemiautotrophic Growth Two recombinant enzymes are needed to complete a carbon fixation cycle in E. coli: RuBisCO, the carboxylating enzyme, and the kinase prk. The remaining reactions required for the reduction and substrate regeneration phases of the cycle are endogenous to the metabolic network of the host, as part of gluconeogenesis and the pentose phosphate pathway. Deletion of the phosphoglycerate mutase genes (ΔgpmA and ΔgpmM) disrupts carbon flow in the glycolytic/gluconeogenic backbone and generates two disconnected sub-networks: (1) a carbon fixation module containing upper glycolysis, the pentose phosphate pathway, and the two foreign CBB enzymes and (2) an energy module, containing lower glycolysis and the TCA cycle, supplying reducing power and ATP. In a scenario in which an organic carbon source (e.g., pyruvate) is utilized by the energy module to supply the energetic demands of the carbon fixation cycle, the cellular building blocks derived from carbon fixation module metabolites (e.g., phospho sugars, such as ribose-P; see Figure S4 for details) can be synthesized from inorganic carbon using the non-native CBB cycle. The remainder of the biomass building blocks (those emanating from the energy module metabolites, e.g., organic acids for many of the amino acids), as well as the energy requirements of the cell, are supplied directly via the catabolism of the organic carbon source. In such a hemiautotrophic growth mode, CO₂ and energy carriers are the sole inputs for the production of biomass precursors in a carbon fixation cycle. See also Figures S1, S2, S3, and S4.

Figure 2

Chemostat Evolution Leads to a Hemiautotrophic Phenotype (A) The ancestor strain (left) containing gpmA and gpmM deletions was evolved in a xylose-limited chemostat supplied with an excess of pyruvate and CO₂. Additional deletions of pfkA, pfkB, and zwf resulted in RuBisCO-dependent (but not hemiautotrophic) catabolism of xylose in the initial heterotrophic growth (Figure S5). Propagation in a chemostat ensured xylose-limited growth, resulting in strong selective pressure toward increased carbon fixation flux. When mutations arise that create a fully functioning CBB cycle, they enable CO₂ to be the sole carbon input for the required biomass precursors. If carbon fixation by the CBB cycle could meet cellular demand for phospho-sugars, growth dependency on xylose would be alleviated and the hemiautotrophic strain (right) could take over the population. (B) Around day 50 of chemostat evolution, an increase in optical density (OD) (black diamonds) and a decrease in pyruvate concentration (purple circles) were observed, indicating a takeover by an evolved clone with a metabolically distinct phenotype. Culture samples from day ≈50 onward (shaded green) were able to grow in minimal media with pyruvate as the only organic carbon source in an elevated CO₂ atmosphere, both on liquid media and agar plates. Dashed lines are sigmoidal fits. See also Figure S5.

Figure 3

Growth without Xylose Is Dependent on CO₂ Availability In contrast to the ancestral strain, evolved clones isolated from all three chemostat experiments were able to grow in minimal media, supplemented solely with pyruvate (doubling time of ≈6 hr). In all cases, growth required elevated CO₂ conditions (pCO₂ = 0.1 atm) and no growth was detected under ambient atmosphere. Similarly, evolved clones, but not the ancestral strain, were able to form colonies on minimal media agar plates when supplemented with pyruvate under a high CO₂ atmosphere (inset). See also Figure S6.

Figure 4

Isotopic Labeling Experiments Demonstrate the Biosynthesis of Sugars and Other Biomass Components from CO₂ (A) Isotopic labeling analysis, in which evolved clones were grown with isotopically labeled ¹³CO₂ as an inorganic carbon source and non-labeled pyruvate as an energy source, showed almost full labeling of all CBB intermediates and the derived biomass building blocks. In marked contrast, TCA intermediates and biomass components originating from the energy module show low levels of labeling, as predicted for a hemiautotrophic growth mode. This indicates that the evolved strain synthesizes CBB module biomass precursors from CO₂ using the non-native CBB cycle, while the biomass precursors originating from the energy module are synthesized from the supplemented pyruvate. (B) In a reciprocal setup in which non-labeled CO₂ was supplied in addition to uniformly labeled pyruvate, the labeling pattern was again in agreement with hemiautotrophic growth expectations: CBB intermediates and the derived amino acids were mostly non-labeled (as the supplied CO₂), while metabolites in the energy module were mostly labeled. (C) Metabolically unperturbed BW25113 E. coli in which RuBisCO and prk were not recombinantly expressed. As expected when lacking the gpm metabolic cutoff and RuBisCO, when uniformly labeled ¹³C-pyruvate was supplied as a carbon source, the intermediates of glycolysis and the pentose-phosphate pathway were fully labeled. TCA cycle intermediates and the derived biomass components were not fully labeled due to the usage of non-labeled bicarbonate in anaplerotic reactions (denoted CO₂ for simplicity). The mean percentage (±SD) of three replicates is shown.

Figure 5

The Genetic Basis Underlying the Hemiautotrophic Phenotype (A) Venn diagram summarizing the intersection among mutations accumulated in hemiautotrophic evolved strains isolated from three distinct chemostat experiments. A detailed description of the mutations can be found in Tables S1, S2, and S3. In the ancestral strain for the second chemostat experiment (exp. II) a ΔmutS deletion that induces hyper-mutability was introduced (only mutations occurring between chemostat inoculation and hemiautotrophic phenotype emergence are depicted). (B) Ribose-phosphate diphosphokinase (prs), the main flux branching enzyme of the CBB module by which ribose-phosphate is diverted toward biomass production, was the only gene in which mutations appeared in all of the chemostat evolution experiments. Structural analysis (based on the most closely available crystal structure, that of Bacillus subtilis showing ≈70% sequence similarity to the E.coli homolog) indicates that the mutations are located in catalytically active regions of the enzyme, either on the ribose-5-phosphate substrate binding loop (turquoise) or on a second loop containing the ATP binding site and an allosteric regulatory site (dark blue). See also Figure S7 and Tables S1, S2, and S3.

Figure 6

Stability Analysis of Autocatalytic Carbon Fixation Cycles (A) Simplified model for an autocatalytic carbon fixation cycle. We consider a two-reaction pathway in which a generalized carbon fixation reaction autocatalytically produces a metabolite (R5P) from an external supply of CO₂ (effectively performing five carboxylations, ${\nu }_{CBB}={\nu }_{RuBisCO}/5$). A second reaction consumes R5P for the production of biomass. For clarity, we assume irreversible Michaelis-Menten rate laws. Model parameters for each reaction are the Michaelis constant (K_M) and the maximal reaction rate ($V_{max}=\left[E\right]\cdot k_{cat}$, where [E] is the enzyme concentration and $k_{cat}$ is the turnover number). For a steady-state concentration to be stable, the derivative of ${\nu }_{prs}$ with respect to R5P concentration has to be higher than the derivative of ${\nu }_{CBB}$ at the steady-state point. This relation will imply that if R5P concentration deviates from its steady state, the flux through the reaction branching to biomass synthesis would stabilize the R5P concentration. In terms of metabolic control analysis, this is equivalent to requiring that the elasticity of the prs reaction is greater than the elasticity of the CBB reaction at the steady-state point. (B) Schematic of the stability analysis in the phase space of R5P concentration showing the net flux at the branchpoint. The steady-state concentration of R5P (inferred from setting ${\nu }_{CBB}={\nu }_{prs}$) and the stability of the steady state are determined by values of the parameters. Assuming the maximal rate of carbon fixation is lower than biomass synthesis $\left(V_{max}^{CBB}<V_{max}^{prs}\right)$, a non-zero stable steady state exists if the enzymatic parameters of prs satisfy the relation $\left(V_{max}^{CBB}/K_M^{CBB}\right)$ > $\left(V_{max}^{prs}/K_M^{prs}\right)$. (C) Experimental in vitro reaction rate measurements of purified prs enzymes, with either wild-type or mutated sequences. All of the mutated prs enzymes from the evolved strains show ≈2-fold decrease in their $k_{cat}/K_M$ values, as predicted by the stability analysis. The measured values in respect to the wild-type enzyme were 57% ± 2%, 65% ± 6%, and 38% ± 3% for the A95T, G226V, and R105_A110dup prs mutations, respectively. The mean percentage (±SD) of three replicates is shown.

Figure 7

Acquired Mutations Focus on Flux Branchpoints and Carbon Metabolism Regulators A fourth evolution experiment was initialed with a modified ancestral strain, containing additional mutations in prs (R105_A110dup, underlined) and ppsR (knockout, underlined). Emergence of the hemiautotrophic phenotype was detected in <100 chemostat generations. Whole-genome sequencing of isolated clones revealed that six mutations, in addition to prs and ppsR, were acquired in the evolutionary transition toward hemiautotrophic growth (red). Out of a total of eight mutations appearing in this hemiautotrophic strain, four are found in flux branchpoints, shunting cycle intermediates toward biomass (serA, pgi, and glmU in addition to prs), an additional three are in regulators of carbon metabolism (crp, malT, and ppsR), and one (xylA) is in the catabolism of the surrogate xylose sugar not used by the final evolved phenotype. A detailed description of the mutations can be found in Table S4. See also Table S4.

Figure S1

Endogenous Pentose Phosphate Pathway Enzymes of E. coli Can Regenerate Pentoses in the Non-native CBB Cycle, Related to Figure 1 (A) Pentose regeneration in the CBB cycle is achieved in most photosynthetic organisms by utilizing a designated sedoheptulose-bisphosphate phosphatase (SBPase) to dephosphorylate sedoheptulose-bisphosphate (SBP). Alternatively, pentose regeneration can occur using a bifunctional FBPase as was found to be the case in cyanobacteria. (B) While the E.coli genome does not contain bona fide SBPase, pentose-phosphates regeneration can be accomplished in an SBP-independent manner using the native enzymes of the pentose phosphate pathway (PPP). In Figure 1 we depict option (B) but E. coli can also operate option (A) using promiscuous activity of the endogenous FBPase.

Figure S2

Systematic Identification of Metabolic Configurations in Which Pathway Activity Is Coupled to Cellular Growth, Related to Figure 1 (A) Metabolic toy model containing three endogenous reactions (V₁-V₃) and one target recombinant reaction (V_target). Flux through the target reaction is not essential for biomass generation on either of two considered carbon sources (turquoise, purple). (B) Computational frameworks for identifying gene deletions leading to the overproduction of a pre-specified metabolite (such as OptKnock) ensure maximal flux through the target pathway at the point of maximal biomass production (red background). However, maximal biomass production may not be the prevailing growth condition so global coupling between target flux and biomass production (red line) is not ensured. We performed an exhaustive search for combinations of reaction knockouts in which V_target is globally essential for growth (blue background). For example, eliminating reactions V₁ and V₃ results in making V_target essential for growth on either of the two carbon sources. (C) While metabolizing carbon source A, the selection slope, defined as V_target/V_BM at the origin, is 0.5. (D) When carbon source B is metabolized, the selection slope increases, now requiring 1 unit of target flux per biomass flux, and thus a tighter coupling. (E) Flux dependency space depicting the feasible fluxes for each of the metabolic configurations previously described. While the unmodified network allows biomass to be generated independently from the target flux (gray line), by eliminating reactions V₁ and V₃ the flux space is reduced only to flux modes in which biomass generation is coupled to the target flux; either with weak or strong coupling slope (purple and turquoise lines, respectively). (F) If several alternatives lead to coupling, choosing a combination of knockouts that yields variable selection slopes on different carbon sources (red circle) is preferred, thus giving the experimentalist options to increase the selection stringency in steps. The software implementation is detailed in the Supplemental Experimental Procedures.

Figure S3

Systematic Approach for Identifying Metabolic Configurations in Which Carbon Fixation Activity Is Essential for Cell Growth, Related to Figure 1 (A) Each block (see 3x3 yellow grid, top left) refers to a combination of two knockouts in metabolic reactions within central carbon metabolism of E. coli (knockout genes marked on axes). We computationally analyzed the coupling of growth (biomass formation) to RuBisCO target flux in each combination of reaction knockouts for nine different carbon sources (Supplemental Experimental Procedures). The coupling between target flux and biomass production is shown using a color code. Feasible fluxes are calculated for the perturbed metabolic network (Figure S2; Supplemental Experimental Procedures) when grown on each of the nine carbon sources as inputs (in addition to CO₂). Metabolic configurations (a specific combination of knockout mutations and carbon source) in which carbon fixation activity is essential for cell growth are highlighted in shades of green. The coupling slope is defined as the minimal carbon fixation flux, required per unit of biomass produced. Gray cells refer to configurations in which carbon fixation is not essential for growth, while black cells are configurations where growth is not possible. (B) The flux requirements for biomass formation of four metabolic configurations. A mutant strain which lacks the enzymatic activities of phosphofructokinase (pfkA and pfkB, denoted for simplicity as Δpfk) and glucose-6-phosphate dehydrogenase (Δzwf) is predicted to grow on pyruvate even without RuBisCO flux, and it thus contains points along the x axis and is colored gray. In contrast, the same set of knockouts when using pentoses (e.g., xylose) requires flux through RuBisCO (and prk). This result stems from the stoichiometry of the pentose phosphate pathway which converts three pentose sugars into two molecules of fructose-6-P (F6P) and a glyceraldehyde 3-phosphate triose. Downstream glycolysis of F6P requires pfk activity to generate fructose-1,6-P and feed the glycolytic pathway. Therefore, pfk deficient mutant is predicted not to grow on pentose sugars as a single carbon source without carbon fixation. Recombinant expression of RuBisCO and prk creates a pfk independent route from pentoses to trioses through the RuBisCO-dependent carboxylation of RuBP to 3-phosphoglycerate, hence rescuing growth. In contrast to pentose metabolism, three-carbon inputs to glycolysis (e.g., pyruvate) do not require pfk activity for catabolism, nor for the gluconeogenic biosynthesis of hexose phosphates which takes place via fructose 1,6-bisphosphatase (fbp). An interesting scenario is the combined knockout of zwf and phosphoglycerate mutase (gpmA and gpmM, denoted for simplicity as Δgpm). In this strain, expression of CBB enzymes allows growth on carbon sources which enter central carbon metabolism through lower glycolysis (e.g., pyruvate, bottom left). The metabolic cutoff prevents gluconeogenic carbon flow and therefore sugar synthesis from the organic carbon source. Since sugars (e.g., glucose-6-phosphate, ribose-5-phosphate) are essential components for biomass synthesis, heterotrophic growth is not feasible. However, pyruvate can be readily metabolized in the TCA cycle to generate ATP and reducing power, hence supplying the energy to allow carbon fixation using a non-native CBB cycle. We term such growth hemiautotrophic. The RuBisCO-dependent assimilation of CO₂ into biomass, supplies the demand for biomass precursors, such as sugars, which cannot be synthesized from organic carbon in this Δgpm Δzwf strain. Uptake rate was set to 10 mmol gCDW^-1 h^-1 for xylose, and 16.7 for pyruvate. (C) The RuBisCO dependency of a Δpfk Δzwf strain was experimentally validated. Gene deletions were performed using iterative P1 transductions (Supplemental Methods) and growth was tested on permissive (i.e., glycerol or pyruvate) versus restrictive carbon sources (i.e., xylose) under elevated CO₂ conditions (pCO₂ = 0.1 atm). As predicted from the stoichiometric model, growth on xylose was observed only in pCBB transformants, expressing RuBisCO and prk. (D) E. coli BW25133, with an unperturbed metabolic network, can grow heterotrophically on minimal media supplied with a single carbon source. In contrast, a metabolically perturbed Δgpm Δzwf strain requires a minimum of two carbon sources for heterotrophic growth, each feeding into one of the two disconnected sub networks formed by the metabolic cutoff. When two carbon sources are co-supplied (e.g., xylose and pyruvate), lack of either one would limit growth, even in excess of the other. As the experimental results show, per given concentration of supplied pyruvate (feeding the energy module), maximal biomass formation is initially proportional to the amount of supplied xylose. An excess of xylose beyond the point in which the ratio of carbon sources matches the consumption uptake ratio, does not contribute to the formation of additional biomass since growth becomes limited by the availability of pyruvate. Knowledge of these different domains can be used to ensure a feeding ratio in the chemostat that will ensure xylose limited conditions with excess pyruvate ready to be used for making energy for carbon fixation. In a hemiautotrophic growth mode (in the presence of a fully functional CBB cycle), growth can be obtained using only pyruvate and CO₂.

Figure S4

Schematic Representation of the Main Sinks for Biomass Precursor Metabolites in Central Carbon Metabolism, Related to Figure 1 12 precursor metabolites in glycolysis, the PPP and the TCA cycle serve as metabolic branchpoints, in which flux branches out from central carbon metabolism to build the components of the cell’s biomass. We detail the percentage (by mass) contributed to the total of cellular carbon derived from each of these key metabolites (as calculated based on Neidhardt, 1987). This serves as a basis for predicting the expected total labeled carbon fraction in the hemiautotrophic strain. In mutant E. coli lacking the phosphoglycerate mutase activity (gpmA, gpmM) central carbon metabolism is divided into two sub-networks disconnected in terms of carbon flow between them. Due to the cutoff, heterotrophic growth requires a minimum of two carbon sources: one feeding lower glycolysis and the TCA cycle (energy module, blue), while the other feeding upper glycolysis and the pentose phosphate pathway (CBB module, green). The relative percentage of the biomass emanating from precursor metabolites in the CBB module sums to approximately 30% of total biomass in the cell (or almost equivalently, carbon biomass), while the metabolites derived from the energy module contribute the remaining 70%. In evolved cells showing a hemiautotrophic phenotype, CO₂ is the sole carbon input into the CBB module, hence about 30% of the cell’s carbon is expected to be supplied from RuBisCO-dependent assimilation of inorganic carbon.

Figure S5

Metabolic Perturbations Allow Selections for RuBisCO Activity during Chemostat Evolution toward Hemiautotrophic Growth, Related to Figure 2 (A) In contrast to our stoichiometric model prediction, a Δgpm Δzwf strain expressing the pCBB plasmid failed to grow hemiautotrophically in an elevated CO₂ atmosphere while provided with pyruvate as a single organic carbon source. To harness natural selection toward establishing hemiautotrophic growth we analyzed the effect of further deleting phosphofructokinase (pfkA and pfkB) in the context of a xylose limited (pyruvate and CO₂ enriched) chemostat evolution experiment. As indicated in the phenotypic phase space resulting from the computational analysis (Supplemental Methods), pfk deletion does not affect the feasibility of hemiautotrophic growth (using solely pyruvate as an organic carbon source), as phosphofructokinase is not required to operate the CBB cycle. However, this additional deletion couples carbon fixation by RuBisCO to cellular growth even when a second organic carbon source feeding directly to the CBB module (e.g., xylose) is supplied in addition to pyruvate (Figure S3), as occurs during xylose limited chemostat evolution. Usefully for the adaptation, when xylose is supplied (in addition to pyruvate and CO₂), the minimal RuBisCO activity required to support growth is significantly lower than the activity required for hemiautotrophic growth. Furthermore, under these conditions, while xylose utilization is dependent on the activity of RuBisCO, the CBB cycle is not required to operate in an autocatalytic manner, as required in the hemiautotrophic growth mode (Figure 1). Uptake rate was set to 10 mmol gCDW^-1 h^-1 for xylose, 16.7 for pyruvate and 6.2 mmol gCDW^-1 h^-1 for each of the two carbon sources when combined. (B) We constructed the Δgpm Δzwf Δpfk strain using iterative P1 transductions and transformed it with the pCBB plasmid (Experimental Procedures). In contrast to the predicted feasibility of hemiautotrophic growth for this strain, growth on minimal media was observed only when xylose (0.1 g/L) was supplemented in addition to pyruvate (5 g/l) and CO₂ (pCO₂ = 0.1 atm), and not on pyruvate and CO₂ alone. (C) As shown in a ¹³CO₂ labeling experiment, expression of RuBisCO and prk in a Δgpm Δzwf Δpfk strain supported growth on minimal media supplemented with pyruvate, xylose and isotopically labeled CO₂ (p¹³CO₂ = 0.1 atm). Labeling of intermediates of upper glycolysis and the pentose phosphate pathway is due to the flux from pentose-phosphates to 3PG through RuBisCO, in which ¹³CO₂ is fixed. Intermediates of lower glycolysis and the TCA cycle are synthesized from pyruvate and therefore are mostly unlabeled. Low levels of labeling in TCA intermediates results from inorganic carbon fixation in anaplerotic reactions. The mean percentage (±SD) of labeled carbon in intracellular metabolites and hydrolyzed amino acids was calculated according to the mass isotopologues distributions obtained using LC-MS (n = 3).

Figure S6

Hemiautotrophic Growth Is Dependent on Co-expression of RuBisCO and prk, Related to Figure 3 (A) Evolved hemiautotrophic cells that were cured from their pCBB plasmid could not grow on M9 minimal media agar plate supplemented only with pyruvate and elevated CO₂ (pCO₂ = 0.1 atm). Growth was restored only upon re-transformation with a plasmid containing all three genes (cbbM coding for RuBisCO, prk and CA). Cured cells re-transformed with modified plasmids, in which either cbbM, prkA or CA was deleted, did not grow hemiautotrophically. The reason for the apparent lack of growth with pCBB without CA awaits further research. (B) In a control experiment, agar plates supplemented with glycerol (0.2%) in addition to pyruvate support growth which is independent of CBB activity (as glycerol can be used as carbon source for sugar synthesis; Figure S3). As expected, with glycerol, plasmid cured cells formed colonies independently from the expression of cbbM, prk or CA. (C) Re-transformation of cured cells with a modified plasmid in which RuBisCO and a bicistronic operon of prk and CA were placed under the control of inducible promoters (Ptet and Para promoters, respectively) restores the ability to grow hemiautotrophically only when both inducers (aTc and arabinose) were added. Cells were incubated for ∼96 hr in elevated CO₂ atmosphere (pCO₂ = 0.1 atm) at 37°C. Notably, the BW 25113 strain cannot utilize arabinose as a carbon source due to ara operon deletion. (D) Cells were propagated in M9 minimal media supplemented with non-labeled pyruvate (5 g/L) and isotopically labeled ¹³CO₂ atmosphere (p¹³CO₂ = 0.1 atm). Growth of the ancestor strain required the addition of xylose (non-labeled, 0.2 g/L). Isotopic carbon composition in total cell biomass was measured (mean ± SD) by analyzing lyophilized cells pellet using an elemental mass analyzer (n = 3; Supplemental Experimental Procedures). In the evolved hemiautotrophic strain over one third of cellular carbon originated from fixed inorganic carbon. This value is in line with the expected fraction of biomass precursors originating from the CBB module in the literature (Figure S4). Labeled carbon in the ancestor strain results from the RuBisCO dependent catabolism of xylose (due to the pfkA, pfkB, and zwf deletions, as detailed in Figures S3 and S5) and from the assimilation of inorganic carbon via anaplerotic reactions. Similarly, labeling in the metabolically unperturbed E. coli BW25113 (lacking the RuBisCO and prk encoding plasmid) is due to anaplerotic reactions.

Figure S7

Hemiautotrophic Growth Evolved in Three Independent Chemostat Experiments and in a Fourth Experiment in Which prs and ppsR Mutations Were Introduced to the Ancestor, Related to Figure 5 (A) Ancestral strain was cultured in xylose limited chemostat with a dilution rate of 0.08 h^-1. Xylose concentration in the feed media was kept constant at 100 mg/L. Pyruvate was provided in excess and its concentration in the feed media was 5 g/L. Residual concentrations and cell density inside the chemostat were measured by routine sampling. Once steady-state was reached, residual xylose concentration fell below detection limit while cell density remained relatively constant for the next 40 days. From this point onward, cell density increased while the residual pyruvate levels decreased. In addition, culture samples from day 50 onward were able to grow on liquid media, as well as on agar plates, in elevated CO₂ conditions while supplemented solely with pyruvate. The number of colonies formed increased from day 50, reaching saturation at approximately day 70. Green shading denotes the emergence of the hemiautotrophic phenotype on pyruvate plates. (B) For the second experiment we further modified the ancestor strain by knocking out a component of the mismatch repair system (mutS) known to increase the mutation rate. In addition, we slowed the dilution rate to 0.035 h^-1 and decreased the xylose concentration in the feed to 25 mg/L. We further decreased the xylose concentration in the feed media whenever residual pyruvate concentration in the culture fell below detection limit, thus avoiding the possibility that pyruvate becomes the limiting nutrient instead of xylose. Clones displaying the hemiautotrophic phenotype were isolated from day 55 onward. (C) In the third experiment, the ancestor strain was propagated under the same conditions as in the first experiment but the xylose concentration in the feed media was reduced whenever pyruvate concentration approached the detection limit as in described for the second experiment. Clones with hemiautotrophic phenotype were first isolated on day 130. Xylose was completely omitted from the feed media from day 150 onward, validating the ability of cells to grow in the complete absence of an organic carbon source feeding into the CBB module. (D) Based on the genomic changes observed in the first three experiments, the ancestral strain in the fourth xylose limited chemostat evolution experiment contained a mutation in the prs gene (R105_A110dup) in addition to a knockout of the ppsR gene encoding the PEP synthase regulatory protein. The dilution rate was set to 0.035 h^-1. The xylose concentration in the feed media was kept constant at 25 mg/L. Pyruvate was provided in excess and its concentration in the feed media was 5 g/L. Random mutagenesis was not performed prior to the inoculation into the chemostat. As early as day 30 onward an increase in biomass optical density was observed along with a decrease in the residual concentration of pyruvate. Culture samples from day 50 onward (shaded green) were able to grow in minimal media with pyruvate as the only organic carbon source in an elevated CO₂ incubator, both on liquid media and agar plates. Sigmoid fits are added as guides to the eye. Note that the novel phenotype appears on a faster time-scale than in previous experiments.