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Figure 1. 

Combinatorial integration of the cordycepin biosynthetic pathway for enhanced production in S. cerevisiae. (a) Schematic diagram of the cordycepin biosynthesis pathway. Phosphoribosyl pyrophosphate, PRPP; adenosine 5'-monophosphate, 5'-AMP, AMP; adenosine 3'-monophosphate, 3'-AMP. (b) Schematic illustration of multimarker combinatorial integration of the cns1/cns2 expression cassette. Cns1 and Cns2 were connected by a self-cleavable P2A peptide, enabling dual gene expression controlled by a single galactose-inducible promoter (GAL1) promoter.

Figure 2. 

Allantoin–GAL system for controlling EGFP expression in S. cerevisiae. (a) Schematic illustration of allantoin-inducible EGFP expression in yeast. (b) Growth comparison of JSA-URA-EGFP under different nutrient conditions. The growth curves of JSA-URA-EGFP were obtained in DO-supplemented YNBD medium (blue line), DO-supplemented YABD medium (red line), DO-free YNBD medium (green line) and DO-free YABD medium (orange line). (c) EGFP expression regulated by the allantoin-responsive GAL system in the JSA-URA-EGFP strain. The strain JSA-URA-EGFP was cultivated in YNBD, YABD, and a mixture of 10%YNBD + 90%YABD, and 100 μL of the cell culture was taken for measuring OD₆₀₀ and EGFP signals with a microplate reader (Biotek, Synergy H1). All experiments were conducted in triplicate, and data represent the mean values with standard deviations. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

Figure 3. 

Characterization of combinatorial library for cordycepin biosynthesis in S. cerevisiae. (a) Schematic illustration of combinatorial piggyBac transposon-mediated integration of the cns1/cns2 expression cassette using multiple auxotrophic selection markers. (b) Comparative analysis of cordycepin production in 20 strains (JSA-COR-S1~S20) constructed via single-marker piggyBac transposon integration. (c) Representative HPLC results of cordycepin production in engineered strains. (d) Comparative analysis of cordycepin production in 20 strains (JSA-COR-M1~M20) constructed via multimarker integration of the piggyBac transposon. The strain JSA-p426-COR indicates the multicopy episomal plasmid-based engineered strain used for cordycepin biosynthesis. Cordycepin production was screened under a 2-mL fermentation condition. All experiments were conducted in triplicate, and data represent the mean values with standard deviations.