Synthetic transactivator-promoter systems for exogenous gene expression in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, which is a unicellular photosynthetic eukaryote, has long served as a model microalga for fundamental biological research and biotechnological applications. Recently, it has attracted attention as a promising biological resource for the sustainable production of bio-oils and high-value biomolecules. To enhance the biotechnological utility of this species, various genetic engineering tools have been developed in recent years. In this study, the tetracycline repressor (TetR)-based transactivation system, which is widely used in mammalian and other eukaryotic cells, was repurposed to establish a synthetic transcriptional activation system for exogenous gene expression in Chlamydomonas. We first constructed a transient expression-based evaluation platform to screen for transcriptional activation domains (TADs) that are functional in Chlamydomonas. Among the analyzed TADs, VP192 (tandem repeat of 12 copies of the core VP16 domain) had the highest transcriptional activity when fused to either TetR or Gal4 DNA-binding domains. Furthermore, fusion proteins comprising VP192 and either TetR or reverse TetR enabled doxycycline-dependent regulation of transgene expression in a dose-dependent manner. Notably, transcriptional inducibility was maintained even when the tetracycline-responsive element (TRE) was fused to the HSP70A promoter. Combining TetR-VP192 and this synthetic chimeric promoter (TRE-P_HSP) yielded transgene expression levels that exceeded those resulting from the strong HSP70A/RbcS2 hybrid promoter by more than 14-fold. These findings suggest that artificial transcription factors and engineered promoters provide a versatile molecular toolkit for regulating exogenous gene expression in Chlamydomonas.