Evolution of a methanol-essential Escherichia coli

Methanol represents an attractive substrate for biotechnological applications. Utilisation of reduced one-carbon compounds for growth is limited to methylotrophic organisms, and engineering synthetic methylotrophy remains a major challenge. Here we applied apply an in silico-guided multiple knockout approach to engineer a methanol-essential Escherichia coli strain, which contains the ribulose monophosphate cycle for methanol assimilation. Methanol conversion to biomass was is stoichiometrically coupled to the metabolisation of gluconate and the designed strain was is subjected to laboratory evolution experiments. Evolved strains incorporated up to 24% methanol into core metabolites under a co-consumption regime and utilized methanol at rates comparable to natural methylotrophs. Genome sequencing revealed mutations in genes coding for glutathione-dependent formaldehyde oxidation (frmA), NAD(H) homeostasis/biosynthesis (nadR), phosphopentomutase (deoB), and gluconate metabolism (gntR). This study demonstrates for the first time a successful metabolic re-routing linked to a heterologous pathway to achieve methanol-dependent growth and represents a crucial step in generating a fully synthetic methylotrophic organism.