Limits...
Sugar Synthesis from CO 2 in Escherichia coli

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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, ). 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 (KM) and the maximal reaction rate (, where [E] is the enzyme concentration and  is the turnover number). For a steady-state concentration to be stable, the derivative of  with respect to R5P concentration has to be higher than the derivative of  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 ) 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 , a non-zero stable steady state exists if the enzymatic parameters of prs satisfy the relation  > .(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  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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4930481&req=5

fig6: 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, ). 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 (KM) and the maximal reaction rate (, where [E] is the enzyme concentration and is the turnover number). For a steady-state concentration to be stable, the derivative of with respect to R5P concentration has to be higher than the derivative of 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 ) 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 , a non-zero stable steady state exists if the enzymatic parameters of prs satisfy the relation > .(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 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.

Mentions: In contrast to linear metabolic pathways in which stable metabolic steady-state can be achieved, regardless of the specific kinetic properties, the stability of autocatalytic cycles (consisting of a set of metabolites which regenerate more of themselves with each turn of the cycle) is not guaranteed. The stability depends on the topology of the cycle and the kinetic parameters of the enzymes involved (Reznik and Segrè, 2010). This is demonstrated in the simplified model shown in Figure 6A, consisting of an autocatalytic effective carbon fixation reaction and a biomass generating reaction from which assimilated carbon is shunted toward biosynthesis. The stability of the steady state in this metabolic network depends on the kinetic properties of the enzymes, as these parameters govern the response to a perturbation in the intracellular concentration of the metabolite at the flux branchpoint. Explicitly, for a stable steady state to exist in the carbon fixation cycle, the Michaelis constant of prs must satisfy: (where and and are effective parameters that are a function of all the reactions composing the CBB cycle). This relation ensures that enough flux remains in the cycle to ensure autocatalysis (Figure 6B; Supplemental Experimental Procedures).


Sugar Synthesis from CO 2 in Escherichia coli
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, ). 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 (KM) and the maximal reaction rate (, where [E] is the enzyme concentration and  is the turnover number). For a steady-state concentration to be stable, the derivative of  with respect to R5P concentration has to be higher than the derivative of  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 ) 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 , a non-zero stable steady state exists if the enzymatic parameters of prs satisfy the relation  > .(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  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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930481&req=5

fig6: 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, ). 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 (KM) and the maximal reaction rate (, where [E] is the enzyme concentration and is the turnover number). For a steady-state concentration to be stable, the derivative of with respect to R5P concentration has to be higher than the derivative of 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 ) 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 , a non-zero stable steady state exists if the enzymatic parameters of prs satisfy the relation > .(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 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.
Mentions: In contrast to linear metabolic pathways in which stable metabolic steady-state can be achieved, regardless of the specific kinetic properties, the stability of autocatalytic cycles (consisting of a set of metabolites which regenerate more of themselves with each turn of the cycle) is not guaranteed. The stability depends on the topology of the cycle and the kinetic parameters of the enzymes involved (Reznik and Segrè, 2010). This is demonstrated in the simplified model shown in Figure 6A, consisting of an autocatalytic effective carbon fixation reaction and a biomass generating reaction from which assimilated carbon is shunted toward biosynthesis. The stability of the steady state in this metabolic network depends on the kinetic properties of the enzymes, as these parameters govern the response to a perturbation in the intracellular concentration of the metabolite at the flux branchpoint. Explicitly, for a stable steady state to exist in the carbon fixation cycle, the Michaelis constant of prs must satisfy: (where and and are effective parameters that are a function of all the reactions composing the CBB cycle). This relation ensures that enough flux remains in the cycle to ensure autocatalysis (Figure 6B; Supplemental Experimental Procedures).

View Article: PubMed Central - PubMed

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.

No MeSH data available.