A functional betalain-producing dioxygenase in Neolecta irregularis reveals an expanded evolutionary origin of the...

Betalains are nitrogen-containing pigments with important biological, ecological, and biotechnological roles, yet their biosynthesis has been described almost exclusively in plants of the order Caryophyllales and in a few fungal and bacterial lineages. The early-diverging ascomycete Neolecta irregularis, considered a morphological and genomic "living fossil", offers a unique opportunity to explore the evolutionary origins of this pathway. Here, we combine pigment analysis, gene expression profiling, and biochemical characterization to investigate the betalain-forming potential of this species. Although no betalains were detected in fruiting bodies-whose yellow coloration was instead attributed to riboflavin-RNA-seq data revealed expression of a putative 4,5-DOPA-extradiol-dioxygenase (DODA), the key enzyme in the formation of betalamic acid. We cloned, expressed, and purified the N. irregularis dioxygenase (NiDODA) in Escherichia coli, demonstrating that it is a monomeric, iron-dependent enzyme capable of producing betalamic acid, muscaflavin, and dopaxanthin from L-DOPA. NiDODA displayed the highest affinity for L-DOPA reported for any DODA to date, and exhibited both 4,5- and 2,3-extradiol-dioxygenase activities. Phylogenetic analysis positioned NiDODA within a distinct fungal branch, expanding the diversity of known betalain-producing enzymes.