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Hispidin (HIP) and its derivatives, a class of natural fungal metabolites, possess complex chemical structures with extensive pharmacological activities. Phellinus igniarius, the most common source of HIP, can be used as both medicine and food. However, the biosynthetic pathway of HIP in P. igniarius remains unclear and we have a limited understanding of the regulatory mechanisms related to HIP. In this work, we sought to illustrate a biosynthesis system for hispidin and its derivatives at the protein level. We found that tricetolatone (