Methods to study prFMN-UbiD mediated (de)carboxylation.

The microbial UbiX-UbiD system facilitates the reversible (de)carboxylation of alpha, beta-unsaturated carboxylic acids, including aromatic compounds. The direct C-H carboxylation presents an attractive method for functionalisation and carbon capture but is difficult to achieve under mild conditions. Hence, UbiD-mediated Csp2-H activation can serve as a versatile tool for developing new biocatalytic routes to transform aryl or alkene compounds and carbon dioxide into valuable commodity chemicals. UbiD activity is dependent on the prenylated flavin cofactor, prFMN, produced by UbiX. Oxidative maturation of the prFMNreduced UbiX product into the active prFMNiminium is a critical prerequisite for UbiD activity. However, efficiency of prFMN incorporation and oxidative maturation can vary considerably batch to batch and between distinct UbiD enzymes. Herein, we present detailed protocols for the production, reconstitution and characterisation of active UbiD enzymes, including UbiD-mediated (de)carboxylation.