Ribosomal Route to Small-Molecule Diversity

The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli. These studies reveal the potential of RPs for the rational synthesis of complex, new small molecules over multiple-step biosynthetic pathways using simple genetic engineering

Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Schematic representation of the draft mitochondrial genome of <i>L. patella</i> animal L2 (top, to scale).

<p>The hive plots <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone.0095850-Krzywinski1" target="_blank">[61]</a> at the bottom of the figure show that the L2 assembly is syntenic with contigs assembled of the mitochondrial genomes of L5 and L6. In these plots, protein-coding genes are represented as circles, and contig boundaries are represented as zigzag lines. Homologous genes are joined by curved lines colored according to the sequence identity of the gene relevant gene pair. Abbreviations: COX, cytochrome <i>c</i> oxidase; NADH, nicotinamide adenine dinucleotide dehydrogenase.</p

Collection locations, phylogeny and divergence of <i>Lissoclinum patella</i> individuals collected across areas of the Southeastern Pacific between 2002 and 2011.

<p>(a) Collection sites, with a portion of the phylogenetic tree based on mitochondrial cytochrome c oxidase 1 (COXI) genes overlaid, with individuals colored by clades that diverge by 5% or more in their nucleotide sequence, as shown in (b).</p

Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.

<p>Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.</p

Heatmap and hierarchical clustering of select secondary metabolite peak volumes in LCMS runs on <i>L. patella</i> extracts (top left).

<p>Clustering based on secondary metabolites that are produced by the symbiotic bacteria <i>Prochloron didemni</i> and <i>Ca.</i> Endolissoclinum faulkneri closely follows the hosts' phylogeny as determined by COXI sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g002" target="_blank">Fig. 2</a>). The <i>P. didemni</i> compounds shown are all cyanobactins produced either by a patellamide-type pathway (red), or a trunkamide-type pathway (blue). These two types of are closely related ribosomal pathways that are highly tolerant to changes in the precursor peptide sequence. The patellazoles (magenta) are produced by another symbiont, <i>Candidatus</i> Endolissoclinum faulkneri, by a polyketide synthase pathway.</p

Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from Didemnidae animals not included in Figure S3.

<p>The COXI sequences for <i>L. patella</i> amimals L2, L5 and L6 used to make this tree were obtained from the respective mitochondrial genome assembly from Illumina sequencing data. The inset identity matrix shows the pairwise nucleotide identities of sequences in this clade, indicating that <i>L. patella</i> sequence AB602781.1 likely is a group B animal.</p

Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona savignyi</i> acting as the outgroup.

<p>Note: the <i>Didemnum vexillum</i> clade is collapsed for space. The Didemnidae COXI genes found in the NCBI database cover two non-overlapping regions of the gene (see Main Text), and therefore two separate trees were constructed (for the other tree, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g004" target="_blank">Figure 4</a>).</p

Genome-Directed Lead Discovery: Biosynthesis, Structure Elucidation, and Biological Evaluation of Two Families of Polyene Macrolactams against <i>Trypanosoma brucei</i>

Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A–C from a marine actinobacterium, <i>Micromonospora</i> sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite <i>Trypanosoma brucei</i> (lobosamide A IC₅₀ = 0.8 μM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a “molecules-to-genes-to-molecules” approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from <i>Actinosynnema mirum</i>. These additional analogs have allowed us to identify specific structure–activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets