Transposon engineering of Saccharomyces cerevisiae for allantoin-inducible cordycepin production.

Cordycepin is a high-value bioactive compound with significant therapeutic potential. In this study, we report a transposon-based genome engineering strategy for establishing an allantoin-inducible cordycepin production platform in Saccharomyces cerevisiae. We first optimized the allantoin-inducible system by refining the culture medium's components to improve cell growth and achieved an approximately 50-fold on/off ratio after reaching a plateau, thereby enhancing the robustness of engineered yeast strains. Next, a multiselection marker transposon system was implemented to achieve combinatorial integration of cordycepin biosynthetic genes (cns1/cns2) into the yeast genome. Engineered yeast strains obtained from the combinatorial library displayed varying cordycepin titers. Among them, one engineered strain produced up to 135 mg/L cordycepin, which is significantly higher than that achieved by the episomal plasmid-based strain. This work demonstrated a transposon-based workflow for combinatorial pathway optimization, which will be of particular interest for future microbial cell factory design.