Enhanced Adenosine Biosynthesis in Paecilomyces hepiali Through Strain Evolution and Transcriptomic Insights.

Adenosine is a major bioactive nucleoside and quality marker in cordyceps-derived fungal products, but its efficient biosynthesis in Paecilomyces hepiali remains limited by insufficient strain performance and incomplete understanding of metabolic regulation. Here, we developed an integrated strategy combining protoplast-based strain evolution, medium optimization, and transcriptomic analysis to enhance adenosine biosynthesis in P. hepiali. Efficient protoplast preparation was achieved using 48 h seed cultures digested with 1% driselase and 1% yatalase at 28°C for 7 h, and 40 s ultraviolet irradiation was selected for mutant library construction. A stable mutant, P. hepiali A3, produced 65.32 mg/L adenosine, representing a 31.62% increase over the parental strain. Subsequent response surface optimization identified maltose, peptone, and aspartic acid as key nutritional factors, increasing the adenosine titer to 170.41 mg/L in shake flasks and 191.36 mg/L in a 5-L bioreactor. Comparative transcriptomic analysis revealed extensive metabolic remodeling involving central carbon metabolism, ribose precursor supply, purine nucleotide metabolism, and sterol biosynthesis. Upregulation of ribose-5-phosphate isomerase RPIB and downregulation of ADA related to purine degradation were associated with enhanced purine nucleoside accumulation. These results provide transcriptomic insights into adenosine biosynthesis and establish a practical framework for improving fungal nucleoside cell factories.