A Modular T7 RNAP Expression Architecture for Orthogonal Multigene Expression in Yeast

Orthogonal gene expression systems based on bacteriophage-derived T7 RNA polymerase (T7 RNAP) offer a promising strategy for decoupling engineered transcription from host regulatory networks. Although recent advances have enabled productive T7 RNAP-mediated expression in Saccharomyces cerevisiae , strategies for independently controlling multiple genes within a shared T7 transcriptional framework remain limited. Here, we developed a modular pYTK-compatible T7 RNAP expression architecture that separates overall transcriptional capacity from gene-specific control of protein output. We established interchangeable T7 promoter, terminator, polyadenylation signal, and 5’ untranslated region (UTR) parts for combinatorial assembly. Analysis of expression cassette dosage showed that transcript abundance and expression output scale with cassette copy number, with high-copy constructs producing transcript levels comparable to those driven by the GAL1 promoter. To enable gene-specific tuning, we constructed a library of fourteen 18 nt UTR elements that generated a ∼38-fold range of protein output from a common T7 promoter. Application of this library to a three-gene β-carotene biosynthesis pathway enabled ∼20-fold variation in product titer through UTR assignment alone, without altering promoter identity or gene copy number. Together, these results establish a modular T7 RNAP expression framework in which cassette dosage defines overall transcriptional capacity while interchangeable UTR elements enable gene-specific tuning of expression output, providing a practical strategy for multigene expression control in yeast.