A FULLY FUNCTIONAL CALVIN CYCLE IN E. COLI

Can an obligatory heterotrophic organism be evolved to synthesize biomass directly from CO2? A positive answer will affect our understanding of metabolic plasticity and stimulate exciting avenues for increasing agricultural productivity and the sustainable production of chemicals. While in-vivo activity of recombinant carbon fixing enzymes have been previously demonstrated in heterotrophic microorganisms, a fully functional, auto-catalytic, carbon fixation cycle in which biomass precursors are synthesized solely from CO2 remained an elusive grand challenge. Here we demonstrate how a combination of rational metabolic rewiring and laboratory evolution leads to the emergence of a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation via the non-native CBB cycle solely provides all carbons for major biomass building blocks (e.g., serine, histidine, pentose phosphates) while reducing power, energy and the rest of the biomass precursors are obtained by metabolizing a supplied organic compound (e.g., pyruvate). Using whole-genome whole-population sequencing we describe the evolutionary dynamics and identify essential mutations for this novel, synthetically semiautotrophic growth mode. We analyze the effect of these mutations on carbon and energy metabolism of the host using a kinetic model, and elucidate their role in the emergence of the semiautotrophic growth. The success in evolving a non-native carbon fixation pathway in an obligatory heterotrophic host provides a striking demonstration of the capacity for rapid trophic-mode evolution in metabolism with relevance to biotechnology.