CRISPR-Enhanced β-Carotene Biosynthesis: Droplet Microfluidics-Driven Pathway Optimization and High-Yield Terpenoid Strain Screening.

Terpenoids are highly abundant and valuable natural products, yet high-throughput screening platforms for microbial producers remain scarce. To address this, we developed a microfluidic screening platform leveraging the intrinsic fluorescence of β-carotene. Using an engineered yeast chassis (YsL4) with a reinforced mevalonate pathway, we optimized cultivation conditions and achieved perfect (100%) phenotypic specificity in a 1:1 coencapsulation assay between producer (YsL4) and nonproducer (Ys011) strains. Screening a genome-wide CRISPRa/i library via multiround fluorescence-activated droplet sorting identified 15 target genes. Validation confirmed that 10 targets (5 for overexpression, 10 for knockout) significantly enhanced β-carotene production. A key mutant, YL19 (ΔHMO1), achieved a titer of 33.71 mg/L in shake-flasks─a 149.15% increase over the parent strain─with a concurrent lycopene reduction indicating redirected carbon flux toward β-carotene biosynthesis. This integrated platform enhances screening efficiency and provides a new paradigm for identifying critical terpenoid biosynthetic targets.