Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

Bioinformatic and Functional Evaluation of Actinobacterial Piperazate Metabolism.

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration. The in vitro characterization of Kutzneria enzymes KtzI and KtzT recently led to a biosynthetic pathway for Piz. However, Piz anabolism in vivo has remained completely uncharacterized. Herein, we describe the systematic interrogation of actinobacterial Piz metabolism using a combination of bioinformatics, genetics, and select biochemistry. Following studies in Streptomyces flaveolus, Streptomyces lividans, and several environmental Streptomyces isolates, our data suggest that KtzI-type enzymes are conditionally dispensable for Piz production. We also demonstrate the feasibility of Piz monomer production using engineered actinobacteria for the first time. Finally, we show that some actinobacteria employ fused KtzI-KtzT chimeric enzymes to produce Piz. Our findings have implications for future piperazyl drug discovery, pathway engineering, and fine chemical bioproduction.

Draft Genome Sequence of Streptomyces sp. Strain JV178, a Producer of Clifednamide-Type Polycyclic Tetramate Macrolactams.

Here, we report the draft genome sequence of Streptomyces sp. JV178, a strain originating from Connecticut (USA) garden soil. This strain produces the polycyclic tetramate macrolactam compounds clifednamides A and B. The draft genome contains 10.65 Mb, 9,045 predicted protein coding sequences, and several natural product biosynthetic loci.