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Abstract

Carbon fixation is one of the most important biochemical processes. Most
natural carbon fixation pathways are thought to have emerged from
enzymes that originally performed other metabolic tasks. 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. We identify the GED
(Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate
with high thermodynamic driving force. 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 demonstrate the in vivo feasibility of this new-to-nature
pathway by constructing E. 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. Several metabolic
adaptations, most importantly the increased production of NADPH, assist
in establishing sufficiently high flux to sustain this growth. Our study
exemplifies a trajectory for the emergence of carbon fixation in a
heterotrophic organism and demonstrates a synthetic pathway of
biotechnological interest. Current efforts to establish synthetic carbon
fixation in model heterotrophs rely on expression of heterologous
enzymes. Here, the authors explore the presence and activity of a latent
CO2-assimilation pathway in E. coli based only on endogenous enzymes and
a reversible decarboxylase.