Here, we report the discovery and structural elucidation of myxoprincomide (1), a novel NRPS/PKS natural product from Myxococcus xanthus DK1622, by combining targeted mutagenesis, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), and statistical data evaluation (principal-component analysis, PCA). M. xanthus DK1622, a model organism for social motility and differentiation, was found to contain 18 biosynthetic gene clusters encoding NRPS, PKS, and hybrid systems, but only 5 compound classes had been characterized previously, with the remaining 13 pathways active at transcriptomic and proteomic levels but their products undetectable due to low abundance. We constructed a targeted mutant library of DK1622, analyzed replicate LC-HRMS data of wild-type and mutant extracts, and used PCA to identify molecular features absent in mutants. This revealed three new secondary metabolites (c506, c844, c329) assigned to their biosynthetic gene clusters. Compound c506, named myxoprincomide (1), is a product of the hybrid NRPS/PKS enzyme encoded by MXAN_3779 (mxp locus), the largest hybrid enzyme in DK1622. To isolate myxoprincomide, we screened 98 M. xanthus strains and identified A2 as a better producer, then engineered A2 with a T7A1 promoter upstream of the MXAN_3779 homologue to generate a strain producing 30-fold higher levels. Structural elucidation via 2D NMR and ESI-MS/MS showed myxoprincomide is an unusual linear peptide with nine amino acid residues, including atypical 2-oxo-b-leucine, g-OH-valine, and b-lysine. Based on structure and feeding experiments, a biosynthetic model for myxoprincomide by the giant NRPS/PKS hybrid assembly line Mxp was proposed. Our strategy, combining gene inactivation with comprehensive secondary metabolome mining, overcomes challenges of low-abundance compound detection, complements genome mining, and enabled discovery of myxoprincomide and two other new metabolites from DK1622. This approach has high potential for uncovering hidden natural products from myxobacteria and other microbes, particularly low-abundance ones.