Heterologous Expression and Functional Analysis of NADPH-Dependent FMN Reductase and Hydrolase Enzymes Involved in Chlorflavonin Biosynthesis in Aspergillus candidus

Flavonoids are a class of secondary metabolites that have antioxidant, anti-microbial, anti-inflammatory, anti-cancer properties, and are also used in pharmaceuticals. Certain fungi, like Aspergillus candidus, are also known to produce flavonoid compounds like chlorflavonin and dechlorflavonin. However, their biosynthetic pathways are not fully characterized. In this study, two important genes from the chlorflavonin biosynthetic cluster, Cfv7 and Cfv4, were heterologously expressed in Escherichia coli and then purified. These genes encode for hydrolase and NADPH dependent flavin mononucleotide (FMN) reductase respectively. Cfv7 catalyzes the conversion of naringenin chalcone to naringenin, and Cfv4 spontaneously converts naringenin into apigenin via a dehydrogenation reaction. Their enzyme activities were validated via in-vitro assays, which confirmed their roles in the chlorflavonin biosynthesis.