A growth-coupled progesterone-responsive biosensor for high-throughput microfluidic screening in Saccharomyces cerevisiae

Poor aqueous solubility of steroid precursors, such as pregnenolone and progesterone, limits microbial biotransformation and high-throughput strain screening, representing a bottleneck for strain improvement and potential industrial applications. To address this, we developed a growth-coupled progesterone-responsive biosensor in <i>Saccharomyces cerevisiae</i>, integrated with a hydroxypropyl-β-cyclodextrin (HP-β-CD) system to enhance intracellular steroid availability. The biosensor links progesterone formation to cell growth and fluorescence, with selection stringency finely tuned via an IPTG-inducible lac operator and 3-aminotriazole (3-AT) to suppress low-producing cells. Coupled with atmospheric and room temperature plasma (ARTP) mutagenesis, the growth-coupled biosensor-FADS platform identified five yeast variants capable of improved conversion of pregnenolone to progesterone while expressing 3β-hydroxysteroid dehydrogenase (3β-HSD) without altering the enzyme itself. The progesterone production of these selected variants was subsequently validated using 1 mM pregnenolone as the substrate, showing 2.0-3.37-fold higher titers than the wild-type strain, demonstrating proof-of-concept. Microfluidic droplet encapsulation allowed clear separation of high-producers, highlighting the platform's selectivity, robustness, and scalability. This synthetic biology-driven system integration platform provides a practical, modular, and high-throughput strategy for screening poorly water-soluble steroid-producing yeast. It is adaptable to other bioactive molecules, can support future enzyme evolution, and demonstrates potential for broader biotechnological applications.