Genetic and biocatalytic basis of formate dependent growth of Escherichia coli strains evolved in continuous culture

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO₂ as sole carbon sources. Labeling with ¹³C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO₂, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.