Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

Abstract

Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptidepolyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for L-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis.The five-member heterocycles of thiazole or oxazole or their reduced structures of thiazoline and thiazolidine or oxazoline and oxazolidine are signature pharmacophores common to many clinically important natural products (44), such as bacitracin (26) and pristinamycin II B (4) (antibacterial) and bleomycin (12) and epothilone (35, 49) (anticancer) (Fig. 1A). They are also common structural motifs for many microbial pathogenesis factors (10), such as pyochelin from Pseudomonas aeruginosa (9, 39), vibriobactin from Vibrio cholerae (23, 52), yersiniabactin from Yersinia pestis (17, 40), mycobactin from Mycobacterium tuberculosis (41), and microcin B17 from Escherichia coli (24, 29) (Fig. 1B).Biosynthetically these heterocycles result from cyclization (Cy) of the cysteine (Cys), serine, or threonine side chain onto the proceeding carbonyl group of the peptide substrates, and two mechanisms are known for this process. One is exemplified by the maturation of microcin B17, where the heterocycleforming steps occur posttranslationally, catalyzed by the microcin B17 synthetase (29). The other is observed for nonribosomal peptide biosynthesis where the peptide elongation and heterocycle-forming steps proceed processively, catalyzed by nonribosomal peptide synthetases (NRPS). The latter enzymes are characterized by unique catalytic domains, such as the Cy, oxidization (Ox), and reduction domains, which act on the nascent peptidyl-S-NRPS intermediate to furnish the heterocycles into the resultant natural products (11,27,44,51).Leinamycin (LNM) is a novel thiazole-containing natural product produced by several Streptomyces atroolivaceus species (18,20,36). Its structure was established by spectroscopic (18,...

Biosynthesis and function of polyacetylenes and allied natural products

“…A series of three consecutive acyl carrier protein domains may provide a conduit of way stations through which side-chain modification intermediates are shuttled. This ACP cluster is an arrangement previously observed in gene clusters for pksX, mupirocin, and leinamycin [227][228][229]. The timing and mechanism of both the alkene chlorination and alkyne bromination steps have not yet been defined.…”

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

“…DNA sequencing, bioinformatic analyses of the sequenced 135,638-bp contiguous DNA, and functional assignments of the deduced gene products were summarized in GenBank (accession no. AF484556) (17). The boundaries of the lnm gene cluster were identified by gene replacement of orf(Ϫ13), orf(Ϫ11), orf(Ϫ2), orf(Ϫ1), and lnmA for the upstream boundary and of orfZЈ, orf(ϩ1), orf(ϩ2), orf(ϩ3), orf(ϩ4), and orf(ϩ6) for the downstream boundary, respectively.…”

“…1) is a hybrid peptide-polyketide natural product that showed potent antitumor activity, most significantly against tumor cell lines that were resistant to clinically important anticancer drugs (13-16). We recently cloned and localized the lnm biosynthesis gene cluster to a 172-kb DNA region from Streptomyces atroolivaceus S-140, and DNA sequence analysis of the four overlapping cosmids (pBS3004, pBS3005, pBS3006, and pBS3007) revealed 72 ORFs (17). Sequential inactivation of ORFs from both ends of the sequenced region led to the assignment of the lnm gene cluster to consist of 27 ORFs, of which two (lnmQ and lnmP) encode nonribosomal peptide synthetase (NRPS), one (lnmI) encodes a hybrid NRPS-PKS enzyme, and two (lnmJ and lnmG) encode PKS.…”

Less than the sum of its parts, a leinamycin precursor has superior properties

“…Rather, the researchers have been working to characterize the biosynthetic apparatus responsible for LNM, and thus identified a gene cluster responsible for LNM synthesis composed of 27 ORFs (8,9). Of these ORFs, they have characterized the roles of 9 encoded proteins, leaving up to 18 potential proteins with functions that cannot be easily predicted.…”