Pacidamycin Biosynthesis: Identification and Heterologous Expression of the First Uridyl Peptide Antibiotic Gene Cluster

Abstract

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3'-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics

“…In summary, the in vivo gene disruption experiments unambiguously verified that the identified gene cluster from S. coeruleorubidus genome is directly involved in the biosynthesis of pacidamycins. Independently, Rackham et al have shown that heterologous expression of this gene cluster in Streptomyces lividans yielded pacidamycin D and a related variant, providing a parallel in vivo identification of the cluster, but they did not report any in vitro studies (18). In contrast, we studied the activity of seven encoded enzymes that allowed the assignment of their functions in the biosynthesis of pacidamycins.…”

“…The identity of the cluster has been confirmed by both genetic and biochemical characterizations. After completion of this study, a parallel effort in S. coeruleorubidus genome sequencing has independently identified the same 22 gene cluster (18).…”

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides

“…Two new series of peptidyl-uridines whose structures are based upon mureidomycin A and tunicamycin have been synthesised as inhibitors of C. jejuni PglC, and selected compounds show IC50 values in the range 40-250 µM [35]. The biosynthetic pathway to both the pacidamycin [36][37][38] and caprazamycin [39] antibiotics has been elucidated, hence the use of biosynthetic engineering offers the possibility of generating modified uridyl peptide antibiotics via fermentation in the future. shown that mureidomycin A and liposidomycin B are both slow-binding inhibitors, while tunicamycin is a reversible inhibitor [40,41].…”

Exploiting the genome sequence of Streptomyces nodosus for enhanced antibiotic production

“…Some cryptic gene clusters have been activated by changing growth conditions or by overproduction of transcriptional activators (Sherlach and Hertweck, 2009;Laureti et al, 2011). Genome sequences are also valuable for unravelling new biosynthetic pathways (Rackham et al, 2010), and for metabolic engineering to increase yields of specific secondary metabolites.…”