Biosynthesis And Engineering Of Hybrid Polyketide/Nonribosomal Peptide Antibiotic Oxazolomycin

The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species. OZM, produced by Streptomyces sp. KBFP-2025 and Streptomyces albus JA3453, is structurally characterized by a unique spiro-linkedβ-lactone/γ-lactam moiety, a 5-substituted oxazole ring, an (E, E)-diene, and a (Z, Z, E)-triene chain. OZM exhibits potent antitumor, antiviral, but limited antibacterial activities. Using degenerated primers to clone genes HADH and ADH encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from S. albus JA3453 was cloned and localized to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned gene cluster in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment from the specific region of the chromosome to generate OZM non-producing mutants. Sequence analysis of this fragment revealed six genes likely responsible for methoxymalonyl-ACP biosynthesis. The involvement of the latter in OZM biosynthesis was proven directly by mutational and complementary analysis of selected genes, ozmF encoding a methyltransferase and ozmC encoding a ketoacyl synthase, within this locus.Chromosomal walking from the methoxymalonyl-ACP biosynthesis locus followed by extensive restriction endonuclease analysis resulted in the identification of the overlapping cosmids covering the 135-kb contiguous DNA region. A 127,755-bp DNA involved in oxazolomycin (OZM) biosynthesis was sequenced from Streptomyces albus JA3453 in South Center of National Human Genome, with an overall GC content of 73.8% characteristic of Streptomyces DNA. Bioinformatic analysis of the sequenced region revealed 55 open reading frames (ORFs), whose putative functions were individually assigned by comparing the proposed gene products with proteins of known functions in the databases as annotated. The boundaries of ozm gene cluster were preliminarily determined by gene inactivation. The ozm cluster covers about 79.5-kb genes encoding 20 genes: nonribosomal peptide synthetase (NRPS; OzmL and OzmO), polyketide synthetase (PKS; OzmJ, OzmK, OzmM, OzmN and OzmQ), hybrid NRPS-PKS (OzmH), resistance (OzmA and OzmS), regulatory (OzmR and OzmU) and tailoring enzymes (OzmBCDEFG).A model for OZM biosynthesis is proposed. Surprising of all, the only AT activity identified within the ozm gene cluster is a discrete trans AT (acyl carrier protein) encoded by ozmM whose deduced product is a protein of 1057 amino acids. Blast research suggested that OzmM contains three distinctive domains. N-terminal of OzmM (amino acid residues 1-332), designated as OzmM-AT1 and central of this protein (amino acid residues 333-631), designated as OzmM-AT2, both have similarity to most discovered modular or discrete acyltransferase. C-terminal of OzmM (amino acid residues 613-1057) encode a putative oxidoreductase domain show high homologous to trans AT LnmG C-terminus. Mutational analysis, complementary experiments and site-directed mutagenesis suggested that AT2 are essential for OZM biosynthesis but AT1 seems to be redundant. We propose that AT2 was recruited ten times required for transferring of malonyl-CoA and methoxymalonyl-ACP extender units onto all ACPs. Also, unusual features in polyketide synthase were characterized in OZM gene cluster, e.g. extra domain, domain missing and domain misposition. Above studies about OZM PKS and NRPS genetic organization will provide us new insights into"AT-less"PKS engineering to generate new"unnatural"natural compound by combinatorial chemistry.