CRISPR/Cas9-mediated disruption of Cmpks1 reveals its role as a key regulator of carotenoid biosynthesis and metabolic adaptation in Cordyceps militaris

Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the ΔCmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.