AI-powered high-throughput digital colony picker platform for sorting microbial strains by multi-modal phenotypes.

Phenotype-based screening remains a major bottleneck in the development of microbial cell factories. Here, we present a Digital Colony Picker (DCP), an AI-powered platform for automated, high-throughput screening and export of microbial clones based on growth and metabolic phenotypes at single-cell resolution, without agar or physical contact. Using a microfluidic chip comprising 16,000 addressable picoliter-scale microchambers, individual cells are compartmentalized, dynamically monitored by AI-driven image analysis, and selectively exported via laser-induced bubble technique. Applied to Zymomonas mobilis, DCP enabled en masse screening and identified a mutant with 19.7% increased lactate production and 77.0% enhanced growth under 30 g/L lactate stress. This phenotype was linked to overexpression of ZMOp39x027, a canonical outer membrane autotransporter that promotes lactate transport and cell proliferation under stress. DCP provides a multi-modal phenotyping solution with spatiotemporal precision and scalable throughput, offering a generalizable strategy for accelerated strain engineering and functional gene discovery.