Hydrogel compartmentalization enables flow cytometric selection of engineered yeast producing complex natural...

Saccharomyces cerevisiae is a powerful chassis for biosynthesis of plant-derived natural products, but optimizing multi-enzyme pathways remains bottlenecked by the lack of scalable screens for small molecules with weak to no fluorescence. Here we present a colony-level, fluorescence-activated cell sorting (FACS) workflow that converts weak metabolite fluorescence into a robust sorting signal via hydrogel compartmentalization. Single yeast cells are encapsulated into monodisperse, hollow PEG hydrogel microparticles ("PicoShells") that support clonal growth in suspension while permitting nutrient and gas exchange. Using the fluorescent properties of serpentine, a monoterpene indole alkaloid (MIA) natural product, we show while single-cell FACS cannot identify producers, PicoShell-encapsulated and grown yeast colonies generate a colony-amplified fluorescence signal suitable for standard FACS, reflecting total per-colony metabolite output. Time-resolved cytometry reveals delayed fluorescence accumulation consistent with production onset, and LC-MS (liquid chromatography-mass spectrometry) analysis of recovered cultures shows that stringent high-fluorescence gates enrich the producer phenotype from defined mixtures. In a spike-in experiment, we achieve up to ∼10-fold enrichment of producer strains by sorting the top 0.1-0.5% fluorescence fraction, highlighting the advantage of hydrogel encapsulation for metabolite-level screening. This approach enables scalable (>30 000 colonies per hour) enrichment of microbial producer phenotypes using a colony-based fluorescence signal without engineered reporter constructs and should generalize to other pathways where intrinsic or derivatized fluorescence can be measured at the colony level.