Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning the Oxazolomycin Biosynthetic Gene Cluster from Streptomyces albus JA3453

Abstract

Oxazolomycin (OZM), a hybrid peptide-polyketide antibiotic, exhibits potent antitumor and antiviral activities. Using degenerate primers to clone genes encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from Streptomyces albus JA3453 was cloned and found to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned genes in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment containing six genes for methoxymalonyl-ACP biosynthesis from the specific region of the chromosome, as well as deletion of the ozmC gene within this region, to generate OZM-nonproducing mutants.

Oxazolomycin Biosynthesis in Streptomyces albus JA3453 Featuring an “Acyltransferase-less” Type I Polyketide Synthase That Incorporates Two Distinct Extender Units

“…Sequencing and in Vivo Analysis of the ozm Gene Cluster-DNA sequencing was performed using a set of five cosmids covering the whole oxazolomycin biosynthetic gene cluster: pJTU1059, pJTU1060, pJTU1061, pJTU1062, and pJTU1063 (8). The insert of each cosmid DNA was cut out with DraI and purified using a Plasmid Maxi kit (Qiagen) and sonicated with a 550 Sonic Dismembrator (Fisher).…”

“…The resultant PCR product was recovered as a 3300-bp NdeI-EcoRI fragment and ligated into the same sites of pBluescript SK to verify PCR fidelity by sequencing and yielding pJTU1088. The ozmM gene was then recovered as a NdeI-EcoRI fragment from pJTU1088 and ligated into the same sites of pJTU1351 (8) to generate pJTU1078 in which the expression of ozmM is under the control of the ErmE* promoter. Introduction of pJTU1078 into S. albus ZH9 by conjugation with apramycin selection afforded the complemented strain named S. albus ZH10 (see Fig.…”

“…All of these steps were monitored by nanospray Fourier transform ion cyclotron resonance mass spectrometry. The exact role of OzmC remained unclear, although gene inactivation and complementation experiments have suggested that it is absolutely required for OZM biosynthesis (8).…”

“…Its involvement in oxazolomycin biosynthesis was confirmed by gene inactivation to generate non-OZM-producing mutants. Sequence analysis of a 12-kb DNA fragment from this region identified six enzymes (OzmB, OzmC, OzmD, OzmE, OzmF, and OzmG) involved in the biosynthesis of the methoxymalonyl-ACP for oxazolomycin production (8). Another related study showed that OzmB serves as a bifunctional glyceryl transferase/phosphatase belonging to the HAD superfamily, which first diverts the primary metabolite 1,3-bisphosphoglycerate into a phosphoglyceryl-S-OzmB intermediate, then removes the phosphate group to afford the 3-glyceryl-S-OzmB (acting as a phosphatase), and finally transfers the glyceryl group to an ACP to set the stage for polyketide biosynthesis (acting as a glyceryl transferase) (10).…”

“…The oxazolomycins (OZMs), represented by oxazolomycin A, B, C, 16-methyl-oxazolomycin, KSM-2690B, KSM-2690C, triedimycin A, B, neooxazolomycin, inthomycins A, B, C and lajollamycin, are a growing family of antibiotics produced by several Streptomyces species that show diverse antibacterial, antitumor, and anti-HIV activity (7,8). Oxazolomycin A is a peptide-polyketide hybrid compound containing a unique spiro-linked ␤-lactone/␥-lactam, a 5-substituted oxazole ring, and (E,E)-diene and (Z,Z,E)-triene moieties (Fig.…”