Analysis of Total Flavonoid Estimation

Flavonoids encompass a heterogeneous group of secondary metabolites with exceptional health benefits. Chrysin, a natural dihydroxyflavone, possesses numerous bioactive properties, such as anticancer, antioxidative, antidiabetic, anti-inflammatory, etc. However, using traditional sources of chrysin involves extracting honey from plants, which is non-scalable, unsustainable, and depends on several factors, including geography, climatic conditions, and the season, which limits its production at a larger scale. Recently, microbial production of desirable metabolites has garnered attention due to the cost-effectiveness, easy scale-up, sustainability, and low emission of waste. We previously reported for the first time the chrysin-producing marine endophytic fungus Chaetomium globosum, associated with a marine green alga. To extend our understanding of chrysin biosynthesis in C. globosum, in the present study, we have assessed the presence of flavonoid pathway intermediates in C. globosum extracts using LC-MS/MS. The presence of several key metabolites, such as dihydrokaempferol, chalcone, galangin, baicalein, chrysin, p-Coumaroyl-CoA, and p-Cinnamoyl-CoA, indicates the role of flavonoid biosynthesis machinery in the marine fungus. Further, we have aimed to enhance the production of chrysin with three different strategies: (1) optimizing the fermentation parameters, namely, growth medium, incubation time, pH, and temperature; (2) feeding key flavonoid pathway intermediates, i.e., phenylalanine and cinnamic acid; (3) elicitation with biotic elicitors, such as polysaccharide, yeast extract, and abiotic elicitors that include UV radiation, salinity, and metal stress. The combined effect of the optimized parameters resulted in a 97-fold increase in the chrysin yield, resulting in a fungal cell factory. This work reports the first approach for enhanced production of chrysin and can serve as a template for flavonoid production enhancement using marine endophytic fungi.