This synthetic route combines reductive carboxylation of ribulose 5-phosphate (Ru5P) with the Entner–Doudoroff (ED) pathway, gluconeogenesis, and the pentose phosphate pathway. We identify a promising candidate route-the GED cycle-that is expected to enable carbon fixation with minimal reactions and with a high thermodynamic driving force. Here, we use a computational approach to comprehensively search for all thermodynamically feasible carbon fixation pathways that rely solely on endogenous E. To understand this process better we aimed to recreate it in a heterotrophic bacterium. The limited number of natural carbon fixation pathways indicates that the recruitment of endogenous enzymes to support carbon fixation is a rather exceptional event. Similarly, acetyl-CoA carboxylase likely originated as a key component of fatty acid biosynthesis before being recruited into carbon fixation pathways in several prokaryotic lineages 2, 3. For example, Rubisco-the carboxylating enzyme of the RuBP cycle-probably evolved from a non-CO 2-fixing ancestral enzyme, thus emerging in a non-autotrophic context 12. While such studies help us to gain a deeper understanding of the physiological changes required to adapt a heterotrophic organism to autotrophic growth, they do not, however, shed light on the origin of the carbon fixation pathways themselves.īesides the reductive acetyl-CoA pathway and the reductive TCA cycle-both of which are believed to have originated early in the evolution of metabolism 11-the other carbon fixation routes are thought to have evolved by recruiting enzymes from other metabolic pathways. Also, overexpression of enzymes of the 3-hydroxypropionate bicycle established the activity of different modules of this carbon fixation pathway in E. Most notably, overexpression of phosphoribulokinase and Rubisco, followed by long-term evolution, enabled the industrial hosts Escherichia coli 7, 8 and Pichia pastoris 9 to synthesize all biomass from CO 2 via the RuBP cycle. Recent studies have made considerable progress in establishing carbon fixation pathways in heterotrophic organisms, with the long-term goal of achieving synthetic autotrophy, which could pave the way towards sustainable bioproduction schemes rooted in CO 2 and renewable energy 5, 6. Six other carbon fixation pathways are known to operate in various bacterial and archaeal lineages 2, 3, 4 and also contribute to primary production 1. Most primary production occurs via the ribulose bisphosphate (RuBP) cycle-better known as the Calvin–Benson cycle-used in bacteria, algae, and plants 1. The ability to assimilate inorganic carbon into biomass sets a clear distinction between autotrophic primary producers and the heterotrophs depending on them for the supply of organic carbon. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner–Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We identify the GED (Gnd–Entner–Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Carbon fixation is one of the most important biochemical processes.
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