Oxazolomycin biosynthesis in Streptomyces albus JA3453 featuring an "acyltransferase-less" type I polyketide synthase that incorporates two distinct extender units

Abstract

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 peptide-polyketide hybrid compound containing a unique spiro-linked beta-lactone/gamma-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 non-ribosomal 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 malonyl-CoA (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.

FIGURE 1.

Chemical structure of oxazolomycin.

FIGURE 2.

Genetic organization of the ozm biosynthetic gene cluster. B, BamHI. Proposed functions for individual ORFs are summarized in Table 1. Numbers refer to genes outside ozm gene cluster. Letters refer to genes inside ozm gene cluster.

FIGURE 3.

A, proposed model for OZM biosynthesis. DH, dehydratase; ER, enoyl reductase; SAM, S-adenosylmethionine; ?, unknown. The upper section shows that OzmM consists of the cryptic AT1 and active AT2 domains that transfer two extender units (malonyl-CoA and methoxymalonyl-ACP) onto corresponding ACPs (OzmC is also involved). The bottom section indicates that the PKS-NRPS assembly line condenses various building blocks by step to biosynthesize oxazolomycin. B, proposed pathway for methoxymalonyl-ACP extender unit biosynthesis in ozm gene cluster.

FIGURE 4.

Determination of OzmH A domain substrate specificities. The ATP-PP_i exchange reactions were performed using amino acids Gly, Ala, and Ser as substrates and H₂O as a negative control (100% relative activity corresponds to 995,320 cpm).

FIGURE 5.

Deletion of ozmM and complementation of the ozmM mutant with either intact ozmM or ozmM-AT1 (S81G) or ozmM-AT2 (S402G) mutant and their effect on OZM biosynthesis. A, schematic representation of constructs for the generation of the ZH9 deletion mutant strain and its genetic complementation strains ZH10, ZH14, and ZH15. Details of site-specific mutagenesis with OzmM-AT1 and OzmM-AT2 are shown. B, HPLC analysis of OZM production in wild-type (I), ZH9 mutant (II), ZH 10 mutant (III), ZH14 mutant (IV), and ZH15 mutant (V) strains. ♦, OZM.