biostudies-literatureDiao ZNational Natural Science Foundation of China (National Science Foundation of China)8769https://www.ebi.ac.uk/biostudies/studies/S-EPMC12514280biostudies-literatureUnknown16(1)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 overexNature communicationsAI-powered high-throughput digital colony picker platform for sorting microbial strains by multi-modal phenotypes.PMC1251428032030003321701033237009832470087Bao WKan LLi RXu JMa BDiao ZPeng QGao WWang XJi YLuo SYang SGe AfalseAI-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 overex2025-01-01T00:00:00Z2025 Oct2026-06-04T08:42:45.004Z2026-05-07T03:09:14.418ZS-EPMC125142804107341810.1038/s41467-025-63929-7