Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams

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
(α-methyl, α-alkyl, or β-methyl) produced by the
hit strains. 1H–15N HSQC-TOCSY NMR analysis
of 15N-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 α-methyl
macrolactam, and two Streptomyces strains producing
new α-alkyl and β-methyl macrolactams. Subsequent purification
and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl
macrolactam with diene amide and tetraene chromophores, and concolactam
(3), a β-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 μM 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.