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Abstract

C-C bonds are the basis for virtually all organic molecules on Earth. In nature, hundreds of different enzymes catalyze reactions in which C-C bonds are formed. A major task of these enzymes is the fixation of carbon, i.e. a C-C bond formation with at least one-carbon (C1) molecule. Most of these enzymes utilize electrophilic C1 species to fix carbon, while only very few use nucleophilic or radical C1 species. In this work the repertoire of enzymatic C-C bond formation was expanded by 4 new examples, 3 of which are based on a C1 nucleophile and one on a C1 radical. The thiamine diphosphate (ThDP)-dependent enzyme oxalyl-CoA decarboxylase (OXC) generates a highly reactive carbanion/enamine intermediate that is protonated and released as formyl-CoA. Here it was shown that this intermediate can also undergo C-C bond formation with an electrophilic carbonyl center. This insight allowed to establish three novel C-C bond formation reactions. First, it was demonstrated that benzaldehyde serves as an excellent electrophile, giving rise to mandelyl-CoA. In combination with oxalyl-CoA synthetase and a thioesterase this enabled the one-pot synthesis of aromatic (S)-α-hydroxy acids with enantiomeric excess up to 99%. Second, it was found that OXC can also generate the carbanion/enamine intermediate from formyl-CoA. By coupling to exergonic reactions at high CO2 concentrations, OXC was shown to be reversible, that is, it can carboxylate formyl-CoA to oxalyl-CoA. Third, OXC was engineered to accept the C1 molecule formaldehyde as substrate, producing glycolyl-CoA. Through directed evolution the catalytic efficiency was improved by a factor of ~200 and the resulting variant was successfully employed in a whole-cell biocatalyst for the production of glycolate from formaldehyde. The glycyl radical enzyme pyruvate formate-lyase (PFL) can abstract a hydrogen atom from formate, thereby generating a highly reactive formyl radical that undergoes C-C bond formation with an acetyl moiety stemming from acetyl-CoA. Here it was shown that PFL exhibits promiscuous activity with glycolyl-CoA. Based on this activity, a pathway was established in vitro that converts glycolate and formate to glycerate. These additions to the toolbox of enzymatic C-C bond formation could contribute to achieve synthetic carbon fixation pathways in the future. Such pathways are thought to be instrumental in achieving a carbon neutral economy.