Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams

Abstract

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (alpha-methyl, alpha-alkyl, or beta-methyl) produced by the hit strains. H-1-N-15 HSQC-TOCSY NMR analysis of N-15-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported alpha-methyl macrolactam, and two Streptomyces strains producing new alpha-alkyl and beta-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an alpha-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a beta-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 mu M against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.