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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.


Related in: MedlinePlus

Growth without Xylose Is Dependent on CO2 AvailabilityIn 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.
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fig3: Growth without Xylose Is Dependent on CO2 AvailabilityIn 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.

Mentions: After inoculation, xylose concentration quickly fell below the detection limit (<1 mg/l) as expected for a sugar-limited chemostat regimen, while the concentration of pyruvate remained in considerable excess (≈2 g/l), as shown in Figure 2B. Due to severing gluconeogenesis by gpm deletion, carbon from pyruvate could not be used for sugar biosynthesis to compensate for the xylose limitation. However, excess pyruvate could potentially serve as a source of energy and reducing power to be utilized by the CBB module as it evolves to function as a xylose-independent CO2 fixation cycle. During the first 40 days of growth (≈100 chemostat generations), we observed no significant change in cell density and nutrient concentrations in the effluent media. Over the following 20 days, we noticed a gradual increase in cell density, accompanied by a steady decrease in pyruvate concentration. Finally, around day 60 (≈150 chemostat generations; Figure 2B), the concentration of pyruvate dropped to an undetectable level (<1 mg/l), suggesting that growth was no longer limited by xylose availability and that pyruvate became fully utilized. Importantly, in contrast to the ancestral strain, culture samples from day 50 onward (Figure 2B) were able to grow in minimal media when supplied with only pyruvate and elevated CO2 (doubling time of ≈6 hr; Figure 3). In ambient CO2, no growth was detected in either liquid media or agar plates.


Sugar Synthesis from CO 2 in Escherichia coli
Growth without Xylose Is Dependent on CO2 AvailabilityIn 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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig3: Growth without Xylose Is Dependent on CO2 AvailabilityIn 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.
Mentions: After inoculation, xylose concentration quickly fell below the detection limit (<1 mg/l) as expected for a sugar-limited chemostat regimen, while the concentration of pyruvate remained in considerable excess (≈2 g/l), as shown in Figure 2B. Due to severing gluconeogenesis by gpm deletion, carbon from pyruvate could not be used for sugar biosynthesis to compensate for the xylose limitation. However, excess pyruvate could potentially serve as a source of energy and reducing power to be utilized by the CBB module as it evolves to function as a xylose-independent CO2 fixation cycle. During the first 40 days of growth (≈100 chemostat generations), we observed no significant change in cell density and nutrient concentrations in the effluent media. Over the following 20 days, we noticed a gradual increase in cell density, accompanied by a steady decrease in pyruvate concentration. Finally, around day 60 (≈150 chemostat generations; Figure 2B), the concentration of pyruvate dropped to an undetectable level (<1 mg/l), suggesting that growth was no longer limited by xylose availability and that pyruvate became fully utilized. Importantly, in contrast to the ancestral strain, culture samples from day 50 onward (Figure 2B) were able to grow in minimal media when supplied with only pyruvate and elevated CO2 (doubling time of ≈6 hr; Figure 3). In ambient CO2, no growth was detected in either liquid media or agar plates.

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.&nbsp;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.


Related in: MedlinePlus