Introduction of glycine synthase enables uptake of exogenous formate and strongly impacts the metabolism in Clostridium pasteurianum.

Autotrophic or mixotrophic use of one-carbon (C1) compounds is gaining importance for sustainable bioproduction. In an effort to integrate the reductive glycine pathway (rGP) as a highly promising pathway for the assimilation of CO and formate, genes coding for glycine synthase system from Gottschalkia acidurici were successfully introduced into Clostridium pasteurianum, a non-model host microorganism with industrial interests. The mutant harboring glycine synthase exhibited assimilation of exogenous formate and reduced CO formation. Further metabolic data clearly showed large impacts of expression of glycine synthase on the product metabolism of C. pasteurianum. In particular, 2-oxobutyrate (2-OB) was observed for the first time as a metabolic intermediate of C. pasteurianum and its secretion was solely triggered by the expression of glycine synthase. The perturbation of C1 metabolism is discussed regarding its interactions with pathways of the central metabolism, acidogenesis, solventogenesis, and amino acid metabolism. The secretion of 2-OB is considered as a consequence of metabolic and redox instabilities due to the activity of glycine synthase and may represent a common metabolic response of Clostridia in enhanced use of C1 compounds.