Comparative characterization and physiological function of putative fatty acid photodecarboxylases

Light-driven fatty acid photodecarboxylases (FAPs) are unique and promising biocatalysts for chemical synthesis and biomanufacturing. However, the exploitation of FAP enzymes is still in its early stage. To date, although it has been proposed that FAPs are conserved in a group of the glucose-methanol-choline (GMC) oxidoreductases, the prior studies have been limited to CvFAP (from Chlorella variabilis NC64A) and CrFAP (from Chlamydomonas reinhardtii 137C). In this study, a comparative characterization of several enzymes classified in the FAPs clade was performed to elucidate their physiological functions. A library of recombinant enzymes including 15 putative FAPs, CvFAP, and CrFAP were prepared. Their catalytic performance was investigated by the photoenzymatic decarboxylation of six fatty acids and seven dicarboxylic acids yielding C1- and C2-shortening alkanes, respectively. In addition, identification of the flavin prosthetic group provided experimental evidence that the cofactor flavin adenine dinucleotide (FAD) was present in FAPs. Five new enzymes with superior FAP photochemical activity to CvFAP/CrFAP in terms of overproduction in Escherichia coli, activity/substrate specificity and thermostability were obtained. This work expands the state-of-the-art of photodecarboxylases in sustainable fuel production and organic synthesis.