Identification of a Napsamycin Biosynthesis Gene Cluster by Genome Mining

Abstract

Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N-methyl diaminobutyric acid, an ureido group, a methionine and two non-proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5'-amino-3'-deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non-ribosomal peptide synthetase (NRPS)-like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N-methyl diaminobutyric acid, to the generation of m-tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC-ESI-MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.

tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics

“…Pacidamycins are of interest due to their unusual peptidyl-nucleoside structure features, and for the development of next generation antibacterial drugs inhibiting the clinically underexplored cell wall enzyme target MraY. The biosynthetic gene clusters for pacidamycins and closely related napsamycins were identified in parallel recently by three research groups via genome mining (5)(6)(7). Bioinformatic analysis revealed the building blocks in pacidamycins are assembled using a nonribosomal peptide synthetase (NRPS) thiotemplate logic, whereas the encoded NRPS modules and domains are highly dissociated and predicted to work with each other in trans.…”

SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

“…Recently, the biosynthetic gene clusters for pacidamycins (7,8) and napsamycins (9) were identified and characterized, suggesting a general biosynthetic pathway to the uridyl peptide antibiotics. Based on the bioinformatic analysis and in vivo and in vitro experiments, the assembly of pacidamycin is catalyzed by nonribosomal peptide synthetases (NRPSs) with highly dissociated modules (7,10).…”

“…Based on the bioinformatic analysis and in vivo and in vitro experiments, the assembly of pacidamycin is catalyzed by nonribosomal peptide synthetases (NRPSs) with highly dissociated modules (7,10). The clusters also contain genes responsible for the biosynthesis of the DABA nonproteinogenic amino acid, the modification of uridine nucleoside, the export of the antibiotic, and other tailoring reactions (7)(8)(9). Generally, at least one transcriptional regulatory gene lies within an antibiotic biosynthetic gene cluster, controlling biosynthesis of the respective antibiotic.…”

“…However, early bioinformatic analyses did not propose a candidate regulator for pacidamycin biosynthesis (7,8). In the napsamycin gene cluster, npsA was deduced to encode a putative regulator homologous to ArsR-type transcriptional repressors (9), but no biological experiments were conducted to verify its involvement in the production of napsamycins. Streptomycetes are well known for their ability to produce various secondary metabolites, including clinically used antibiotics, antitumor agents, immunosuppressants, etc.…”

Draft Genome Sequence of Streptomyces sp. Strain SS, Which Produces a Series of Uridyl Peptide Antibiotic Sansanmycins

“…The genetic organization of the sansanmycin biosynthetic gene cluster resembles the recently characterized napsamycin gene cluster of Streptomyces sp. DSM5940 (2). In contrast to the pacidamycin gene cluster, ssaX (homologues of pacX), encoding a phenylalanine hydroxylase, was not in a digene cassette but was located in the defined gene cluster downstream of ssaT.…”