Abstract Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO 2 . Here, we use iterative laboratory evolution to generate genetically diverse autotrophic strains. We identify that just three mutations are sufficient for E. coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (Rubisco, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis ( pgi ), central-carbon regulation ( crp ), and RNA transcription ( rpoB ). The pgi mutation reduces the enzyme activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. The other two mutations increase the ratio of NADH/NAD + - the cycle’s electron-donor. This study demonstrates the malleability of metabolism and evolution’s capacity to switch trophic modes on laboratory time-scales and could facilitate transforming other heterotrophic organisms into autotrophs.

Autotrophic growth ofE. coliis achieved by a small number of genetic changes