The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptidepolyketide hybrid compound containing a unique spiro-linked ␤-lactone/␥-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid nonribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonylCoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.

Zorbamycin (1, ZBM) is a glycopeptide antitumor antibiotic first reported in 1971. The partial structures of 1 were speculated on the basis of its acid hydrolysis products, but the structure of the intact molecule has never been established. The low titer of 1 from the wild-type strain, combined with its acid-instability, has so far hampered its isolation. By random mutagenesis of Streptomyces flavoviridis ATCC21892, a wild-type producer of 1, with UV irradiation, two high-producing strains of 1, S. flavoviridis SB9000 and SB9001, were isolated. Under the optimized fermentation conditions, these two strains produced about 10 mg/L of 1, which was about 10-fold higher than the wild-type ATCC21892 strain, as estimated by HPLC analysis. Finally, 1 was isolated as both a 1-Cu complex and Cu-free molecule, and the intact structure of 1 was established on the basis of a combination of mass spectrometry and 1H and 13C NMR spectroscopic analyses.

The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.