Fatty acid photodecarboxylase is an ancient photoenzyme responsible for hydrocarbon formation in the thylakoid membranes of algae

Abstract

Fatty acid photodecarboxylase (FAP) is one of the three enzymes that require light for their catalytic cycle (photoenzymes). FAP has been first identified in the green microalga Chlorella variabilis NC64A and belongs an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from Chlorella and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activity, their activity and physiological function have not been studied in vivo. Besides, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a Chlamydomonas FAP knockout line (fap), we show that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon present in this alga. We further show that CrFAP is associated to the thylakoids and that 90% of 7-heptadecene is recovered in this cell fraction. In the fap mutant, photosynthesis activity was not affected under standard growth conditions but was reduced after cold acclimation. A phylogenetic analysis including sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was kept in photosynthetic secondary endosymbiosis lineages but absent in those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP activity is conserved in red and brown algae and is not limited to unicellular species. These results thus indicate that FAP has been conserved during evolution of most algal lineages when photosynthesis was kept and suggest that its function is linked to photosynthetic membranes.One sentence summaryFAP is present in thylakoids and conserved beyond green algae.

A Reconstructed Common Ancestor of the Fatty Acid Photo‐decarboxylase Clade Shows Photo‐decarboxylation Activity and Increased Thermostability

“…This assumption was later further confirmed by Moulin and coworkers that confirmed FAP activity for additional enzymes. [29] Two additional ancestors could not be characterized due to problems with the soluble production after expression of their genes in E. coli. ANC1 shows higher production of soluble protein in E. coli, which could be further increased by Nterminal truncation.…”

“…Giving the extremely rare frequency of photoenzymes, we decided to only include 12 sequences that were classified in the fatty acid photo-decarboxylases clade by Sorigue and co-workers where only two enzymes had confirmed photo-decarboxylation activity at the time of the reconstruction, lately during the experimental characterization more photo-decarboxylases were experimentally verified (Figure 1). [7,29] All sequences belong to the algal genus. As ancestral enzymes have shown before to have an improved soluble production in E. coli, the expectation was that the same could be achieved for the photo-decarboxylases.…”

“…Despite the differences, they all share the conserved amino acid positions that characterize FAPs: C432, R451 and A576 (numbering corresponding to CvFAP). [29,30] A576 is possibly one of the most striking difference between FAPs and other members of the GMC superfamily. At this position, most members of the GMC superfamily have a histidine.…”

Mechanism and dynamics of fatty acid photodecarboxylase

“…It allows the decarboxylation of C16-C18 free (non-esterified) fatty acids to the corresponding n-alka(e)nes (4). These hydrocarbon products are mostly located in chloroplast thylakoids but their exact role is still unknown (22,23). The initial spectroscopic characterization of FAP, based on monitoring the electronic state of the flavin following excitation by a laser flash, has led to a first model of the FAP photocycle (4).…”

Phylogeny and Structure of Fatty Acid Photodecarboxylases and Glucose-Methanol-Choline Oxidoreductases

“…Perhaps the most interesting GMC family members at the moment are fatty acid photodecarboxylases (FAP), a recently discovered class of enzymes initially identified in the green algae Chlorella variabilis [77], which convert long chain fatty acids to the corresponding C n-1 alkanes/alkenes in the presence of blue light. FAP activity was also confirmed for proteins from Chlamydomonas reinhardtii [77], Galdieria sulfuraria, Chondrus crispus, Nannochloropsis gaditana and Ectocarpus siliculosus [78]. Overall, FAP genes were identified in more than 30 algal species as well as in metagenomic datasets [78].…”