Gene purA, a Key Upstream Target for Cordycepin Overproduction in Cordyceps militaris

ABSTRACT Background Cordycepin, a bioactive compound derived from Cordyceps militaris with diverse pharmacological properties, is predominantly produced using this fungus for industrial applications. However, current strategies for cordycepin yield improvement primarily rely on random mutagenesis and omics analysis, with limited application of systematic metabolic engineering approaches in this biosynthesis system. Results A ternary approach was adopted to systematically evaluate the effect of gene purA on cordycepin overproduction: (1) computational biology analysis implied that gene CCM_06768 coded PurA and it was essential for cell survival; (2) replacing the coding sequence of purA with the fusion module of purA‐mScarlet ‐I to show its low expression level; and (3) regulating purA expression using different promoters and resulting a 27.3% increase on cordycepin production. Conclusion This study first characterized the low native expression level of purA in C. militaris using an advanced reporter and demonstrated its development potential. After systematically fine‐tuning its expression using a unique promoter engineering toolkit, the yield of cordycepin was increased with the enhancement of the precursor pathway. These efforts not only lay out a foundation for multiplex genome editing to build a versatile C. militaris cell factory but also provide a novel research paradigm for the numerous kinds of unexplored mushrooms.