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.

Construction of a new integrating vector from actinophage ϕOZJ and its use in multiplex Streptomyces transformation.

Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets. Among these tools, vectors that enable combinatorial chromosomal manipulations are particularly desirable. Towards developing tools for facile multiplex engineering, we herein describe the development of new integrating vectors derived from BD1 subgroup actinophage OzzyJ (ϕOZJ). By demonstrating the transformation of several Streptomyces spp. using ϕOZJ-derived vectors, we reveal their potential for strain engineering. We further report the development of new ϕC31 and ϕBT1-based vectors having orthogonal resistance, replication and integration features for concomitant transformation with our ϕOZJ-derived vectors. Importantly, the resulting compatible vector panel enabled us to demonstrate the transfer of up to three plasmids each into Streptomyces venezuelae, Streptomyces roseosporus and Streptomyces pristinaespiralis during a single conjugation experiment. To our knowledge this is the first documentation of conjugation-mediated multiplex plasmid transformation, a useful approach for rapid combinatorial strain development.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c.

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; -19 mV, M81A; -69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these "release mutants."

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.