Sugar Synthesis from CO2 in Escherichia coli

Abstract

SummaryCan 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

Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

“…The experimentally observed key evolutionary event enabling the functioning of the CBB cycle, was a mutation affecting the kinetic properties of the main branching reaction out of the CBB pathway, prs, weakening its affinity to its substrate, ribose-5p. The observed weakening of affinity of prs is directly in line with our predictions on the relationship between the affinity of branch reactions and the affinity of the corresponding cycle reactions (see Materials and methods section of Antonovsky et al, 2016).…”

“…The successful introduction of the genes did not suffice to make the cycle function, and further directed evolution was needed in order to achieve successful operation of the cycle. Strikingly, most evolutionary changes occurred in branch points from the cycle (Antonovsky et al, 2016). The change which was biochemically characterized in the evolutionary process was the decrease of the value of kcat KM of phosphoribosylpyrophosphate synthetase (prs), one of the enzymes responsible for flux out of the CBB cycle, corresponding to the branch reaction in our simple model.…”

“…An experimental demonstration of these principles in-vivo is the recent implementation of a functional CBB cycle in E. coli by introducing the two genes missing for its function (Antonovsky et al, 2016). The successful introduction of the genes did not suffice to make the cycle function, and further directed evolution was needed in order to achieve successful operation of the cycle.…”

“…This cycle has been recently introduced synthetically into E. coli and was shown to carry flux in it, given further metabolic engineering of central carbon metabolism (Antonovsky et al, 2016). The experimentally observed key evolutionary event enabling the functioning of the CBB cycle, was a mutation affecting the kinetic properties of the main branching reaction out of the CBB pathway, prs, weakening its affinity to its substrate, ribose-5p.…”

Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

“…In fact, recent developments such as the modification of the E. coli lifestyle to express the Calvin cycle [66] are close to our vision of the design of a modular chassis. In this vision, the chassis would be designed from first principles as a collection of genetic modules encoding the functions (structural, metabolic, etc.)…”