Screening for Proteins Related to The Biosynthesis of Hispidin and Its Derivatives in Phellinus Igniarius Using iTRAQ Proteomic Analysis

Abstract

Background: Hispidin (HIP) and its derivatives, a class of natural fungal metabolites, possess extremely complex and interesting chemical structures with extensive pharmacological activities. Phellinus igniarius, which is the most common sources of HIP, can be used as both medicine and food. The biosynthetic pasthway of HIP in P. igniarius is not yet clear and hence effective regulatory mechanism of HIP is absent.The purpose of this paper was to illustrate a biosynthesis system for hispidin and its derivatives at the protein level. Results: We found that tricetolatone (TL) is a key biosynthetic precursor in the biosynthetic pathway of hispidin and that its addition led to increased production of hispidin and various hispidin derivatives. Based on the changes in the concentrations of precursors and intermediates, key timepoints in the biosynthetic process were identified. We used isobaric tags for relative and absolute quantification (iTRAQ) to study dynamic changes of related proteins in vitro. The 270 differentially expressed proteins were determined by GO enrichment analysis to be primarily related to energy metabolism, oxidative phosphorylation, and environmental stress responses after TL supplementation. The differentially expressed proteins were related to ATP synthase, NAD binding protein, oxidoreductase, and other elements associated with electron transfer and dehydrogenation reactions during the biosynthesis of hispidin and its derivatives. Multiple reaction monitoring (MRM) technology was used to selectively verify the iTRAQ results, leading us to screen 11 proteins that were predicted to be related to the biosynthesis pathways.Conclution: These findings help to clarify the molecular mechanism of biosynthesis of hispidin and its derivatives and may serve as a foundation for future strategies to identify new hispidin derivatives.