The emergence of multidrug-resistant Gram-negative pathogens poses a serious threat to global health. Gram-negative bacteria have become increasingly recognized as underexplored sources of Gram-negative-active cationic lipopeptide (CLP) antibiotics. We systematically screened 8982 sequenced genomes from 42 underexplored Gram-negative bacterial genera and identified eight potential CLP biosynthetic gene clusters. Their predicted products were rapidly accessed by solid-phase total synthesis, which led to the novel antibiotic chospeptin with good activities against clinically isolated colistin-resistant Gram-negative pathogens.