Conversion of Escherichia coli to Generate All Biomass Carbon from CO2

Abstract

Graphical Abstract Highlights d Conversion of obligate heterotroph to full autotrophy over laboratory timescales d Non-native Calvin cycle operation generates biomass carbon from CO 2 in E. coli d Formate is oxidized by heterologous formate dehydrogenase to provide reducing power d Chemostat-based directed evolution led to complete trophic mode change in z200 days In BriefMetabolic rewiring and directed evolution led to the emergence of E. coli clones that use CO 2 as their sole carbon source, while formate is oxidized to provide all the reducing power and energy demands. SUMMARYThe living world is largely divided into autotrophs that convert CO 2 into biomass and heterotrophs that consume organic compounds. In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO 2 as their sole carbon source has so far remained an outstanding challenge. Here, we report the achievement of this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all its biomass carbon from CO 2 . Reducing power and energy, but not carbon, are supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO 2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling.

Awakening a latent carbon fixation cycle inEscherichia coli

“…S5). A previous study has established the RuBP cycle in E. coli, demonstrating that this heterotrophic bacterium can be modified to grow autotrophically with CO2 as a sole carbon source 6,7 . However, to our knowledge, the current study is the first one in which the capacity for net carbon fixation was explored in vivo using only endogenous enzymes of a heterotrophic host, thus shedding light on the emergence of novel carbon fixation pathways.…”

“…The ΔPZF strain serves as an ideal starting point for such a future evolution experiment, as its growth is dependent on the activity of the GED shunt while it still harbors all necessary enzymes to run the GED cycle. The gradual evolution of autotrophic growth via the GED cycle would be achieved via the additional expression of formate dehydrogenase as an energy-supplying module and longterm cultivation with limiting amounts of xylose and saturating amounts of CO2 and formate 6,7 .…”

“…Engineering E. coli for autotrophic growth via the GED cycle would be a challenging task requiring considerable metabolic adaptation of the host; for example, establishing a delicate balance between the metabolic fluxes within the cycle and those diverging out of the cycle (as found in previous efforts to establish the RuBP cycle in E. coli 6,7 ). Hence, to check the feasibility of the cycle, we focused on establishing growth via the "GED shunt", representing a segment of the full cycle which consists of reductive carboxylation by Gnd and the subsequent ED pathway (blue reactions in Fig.…”

“…That is, growth on a pentose substrate is not possible due to the accumulation of fructose 6-phosphate that prevents further conversion of pentose phosphates into GAP (Fig. 4A) 7 . In contrast, flux in the opposite direction, as required for the GED cycle, can still occur, since fructose 1,6-bisphosphate can be dephosphorylated to fructose 6-phosphate which is then used to regenerate Ru5P.…”

“…Recent studies have made considerable progress in establishing carbon fixation pathways in heterotrophic organisms, with the longterm goal of achieving synthetic autotrophy, which could pave the way towards sustainable bioproduction schemes rooted in CO2 and renewable energy 4,5 . Most notably, overexpression of phosphoribulokinase and Rubisco, followed by long-term evolution, enabled the industrial hosts Escherichia coli 6,7 and Pichia pastoris 8 to synthesize all biomass from CO2 via the RuBP cycle. Also, overexpression of enzymes of the 3hydroxypropionate bicycle established the activity of different modules of this carbon fixation pathway in E. coli 9 .…”