Efficient production of cordycepin in engineered Saccharomyces cerevisiae

Cordycepin (COR) is an important bioactive nucleoside with antitumor and immunomodulatory activities, but its conventional production via Cordyceps militaris fermentation is limited by long cultivation periods and low titers. Therefore, microbial heterologous synthesis represents an attractive alternative. In this study, the COR biosynthetic pathway was introduced into Saccharomyces cerevisiae CEN.PK530-1C, resulting in a COR titer of 870.7 mg/L. To further enhance COR biosynthesis, different fusion constructs were evaluated to relocalize the key enzymes into membraneless organelles in CEN.PK530-1C, which markedly increased COR production to 1209.5 mg/L. Metabolic engineering strategies were then applied to prevent COR degradation through deletion of the adenosine deaminase gene (YJL070C) and to enhance ATP and NADPH supply via overexpression of pgk1 and pos5, resulting in strain SCC7 with a COR titer of 1515.5 mg/L. Furthermore, metabolic effector molecules were screened and used to improve COR production to 1676.5 mg/L. In fed-batch fermentation, SCC7 achieved 6972.8 mg/L COR in a 6 L bioreactor, representing the highest COR titer reported in S. cerevisiae to date. In conclusion, this study establishes an efficient yeast platform for COR production and provides a useful reference for biosynthesis of other bioactive nucleosides.