The genetic basis for the adaptation of E. coli to sugar synthesis from CO2.

Understanding the evolution of a new metabolic capability in full mechanistic detail is a standing grand challenge. Causative mutations underlying the phenotypic transition may be masked by non-essential mutations accumulated during the evolutionary trajectory. Here we reveal the genetic basis underlying an extreme metabolic transition by E. coli to utilize CO2 fixation for sugar synthesis, a novel phenotype that emerged following adaptive laboratory evolution of a rationally engineered ancestor strain. By screening different subsets of mutations for the desired phenotype we find that five mutations are sufficient to enable robust growth when a non-native Calvin-Benson-Bassham cycle provides all cellular demand for sugars. These mutations are found either in enzymes that affect the efflux of intermediates from the autocatalytic CO2 fixation cycle towards biomass (prs, serA, pgi), or in key regulators of carbon metabolism (crp, ppsR). We further characterize the functional role of each mutation by using suppressor analysis to identify distinct genetic variants that reproduce the phenotype. Specifically, we show that a decrease in catalytic capacity is a common feature of all mutations found in enzymes. These findings highlight the contribution of proper carbon balance to the metabolic stability of autocatalytic cycles which is applicable to future efforts in constructing non-native carbon fixation pathways.