Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes

Abstract

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO₂. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for <i>Escherichia coli</i> 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 (<i>pgi</i>), central-carbon regulation (<i>crp</i>), and RNA transcription (<i>rpoB</i>). The <i>pgi</i> mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (<i>rpiB</i>) and the energy module (<i>fdoGH</i>), as well as an increased ratio of NADH/NAD⁺ - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.