In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-l-isoasparagine or a β-methoxy-l-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound l-asparagine. Furthermore, CysQ O-methylates hydroxyl-l-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

Pentaminomycins C–E: Cyclic Pentapeptides as Autophagy Inducers from a Mealworm Beetle Gut Bacterium

Pentaminomycins C–E (1–3) were isolated from the culture of the Streptomyces sp. GG23 strain from the guts of the mealworm beetle, Tenebrio molitor. The structures of the pentaminomycins were determined to be cyclic pentapeptides containing a modified amino acid, N5-hydroxyarginine, based on 1D and 2D NMR and mass spectroscopic analyses. The absolute configurations of the amino acid residues were assigned using Marfey’s method and bioinformatics analysis of their nonribosomal peptide biosynthetic gene cluster (BGC). Detailed analysis of the BGC enabled us to propose that the structural variations in 1–3 originate from the low specificity of the adenylation domain in the nonribosomal peptide synthetase (NRPS) module 1, and indicate that macrocyclization can be catalyzed noncanonically by penicillin binding protein (PBP)-type TE. Furthermore, pentaminomycins C and D (1 and 2) showed significant autophagy-inducing activities and were cytoprotective against oxidative stress in vitro.

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 peptide-polyketide 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.