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The UbiX/UbiD system is widespread in microbes and responsible for the reversible decarboxylation of unsaturated carboxylic acids. The UbiD enzyme catalyzes this unusual reaction using a prenylated flavin (prFMN) as cofactor, the latter formed by the flavin prenyltransferase UbiX. A detailed picture of the biochemistry of flavin prenylation, oxidative maturation, and covalent catalysis underpinning reversible decarboxylation is emerging. This reveals the prFMN cofactor can undergo a wide range of transformations, complemented by considerable UbiD-variability. These provide a blueprint for biotechnological applications aimed at producing hydrocarbons or aromatic C-H activation through carboxylation.

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effects in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis. A class of nepetalactol-related short-chain dehydrogenase/reductases (NEPS) captures a reactive enol intermediate produced by iridoid synthase for cyclization and subsequent oxidation into nepetalactones, the active ingredients in catnip.

The direct C–H carboxylation of aromatic compounds is an attractive route to the corresponding carboxylic acids, but remains challenging under mild conditions. It has been proposed that the first step in anaerobic microbial degradation of recalcitrant aromatic compounds is a UbiD-mediated carboxylation. In this study, we use the UbiD enzyme ferulic acid decarboxylase (Fdc) in combination with a carboxylic acid reductase to create aromatic degradation-inspired cascade reactions, leading to efficient functionalization of styrene through CO2 fixation. We reveal that rational structure-guided laboratory evolution can expand the substrate scope of Fdc, resulting in activity on a range of mono- and bicyclic aromatic compounds through a single mutation. Selected variants demonstrated 150-fold improvement in the conversion of coumarillic acid to benzofuran + CO2 and unlocked reactivity towards naphthoic acid. Our data demonstrate that UbiD-mediated C–H activation is a versatile tool for the transformation of aryl/alkene compounds and CO2 into commodity chemicals. Biocatalytic cascade reactions using engineered variants of ferulic acid decarboxylase coupled to carboxylic acid reductase utilize carbon dioxide fixation to enable the carboxylation and functionalization of styrene and other aromatic compounds.