DESIGN AND EVOLUTION OF A FULLY FUNCTIONAL CALVIN CYCLE IN E. COLI

The Calvin-Benson-Bassham (CBB) cycle is the gateway to the organic world being the main pathway for turning CO2 into biomass and storing energy in the biosphere. Heterotrophic organisms are dependent on supply of organic carbon fixed by autotrophs. How difficult is it to evolve from one trophic mode of growth to another? Moreover, can an obligatory heterotrophic organism be evolved to synthesize biomass from CO2? A positive answer will affect our understanding of metabolic plasticity and stimulate exciting avenues towards agricultural productivity as well as sustainable production of chemicals.

We explored such shift of growth modes by a combination of rational metabolic rewiring and laboratory evolution under selective conditions that lead to the emergence of a fully functional CBB cycle in E.coli.

We rewired the metabolic network of an E.coli host by introducing two enzymes (RuBisCO and prk) and severing the glycolysis pathway to decouple carbon-fixation from energy production. After several months under selective conditions in a chemostat, the bacteria evolved to semiautotrophic growth, in which carbon fixation via the CBB cycle solely provides all carbons for major biomass building blocks (e.g. serine, histidine, ribose-5P). Reducing power, energy and the rest of the biomass precursors are obtained by metabolizing a supplied organic compound (e.g. pyruvate).

We characterized the evolutionary process by sequencing whole-population samples throughout the experiment. Various genetic events such as mutational sweeps, clonal interferences, durable co-existence of several lineages and mutational cohorts can be clearly observed in the data. The success in evolving a non-native carbon fixation pathway in a heterotrophic host provides a striking demonstration of the capacity for rapid trophic-mode evolution in metabolism with relevance to biotechnology.