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Natural Products are molecular “special equipment” that impart survival benefits on their producers in nature. Due to their evolved functions to modulate biology these privileged metabolites are substantially represented in the drug market and are continuing to contribute to the discovery of innovative medicines such as the recently approved semi-synthetic derivative of the bacterial alkaloid staurosporin in oncology indications. The innovation of low molecular weight compounds in modern drug discovery is built on rapid progress in chemical, molecular biological, pharmacological and data sciences which together provide a rich understanding of disease-driving molecular interactions and how to modulate them. NPs investigated in these pharmaceutical research areas create new perspectives on their chemical and biological features and thereby new chances to advance medical research. New methods in analytical chemistry linked with searchable NP-databases solved the issue of reisolation and enabled targeted and efficient access to novel molecules from nature. Cheminformatics delivers high resolution descriptions of NPs and explores the substructures that systematically map NP-chemical space by sp3-enriched fragments. Whole genome sequencing has revealed the existence of collocated gene clusters that form larger functional entities together with proximate resistance factors thus avoiding self-inhibition of the encoded metabolites. The analysis of bacterial and fungal genes provides tantalizing glimpses of new compound-target pairs of therapeutic value. Furthermore, a dedicated investigation of structurally unique, selectively active NPs in chemical biology demonstrates their extraordinary power as shuttles between new biological target spaces of pharmaceutical relevance.

Access to the cyclic depsipeptide FR900359 (FR), a selective G

The depsipeptide FR900359 has been first described in literature in 1988 (Fujioka et al, 1988) to be isolated from a methanol extract of the whole plant of Ardisia crenata. FR900359 can be isolated from the leaves of A. crenata, but the very low quantities and the complex matrix prevent access to sufficient amounts of FR900359 to enable drug development efforts and potential commercial manufacturing. Almost two decades later, it has been discovered that FR900359 is in fact produced by a strictly obligate bacterial endosymbiont, Candidatus Burkholderia crenata, of the plant Ardisia crenata (Carlier et al, 2016). This study identified also the DNA sequence of the biosynthetic gene cluster (BGC) of FR900359. In order to identify alternative and scalable methods for production of FR900359, a genome mining effort on bacterial genomes from both public sequence databases and genome sequences generated from internal efforts at Novartis was initiated. Translated amino acid sequences of the FR900359‑BGC from Candidatus B. crenata were used as query sequence. While the query of public sequence databases did not return highly similar sequences, a gene cluster with very high homology in translated amino acid sequence and identical prediction of protein functions was discovered in the genome of Chromobacterium vaccinii DSM 25150, which had been sequenced internally at Novartis. Here we describe the genetic engineering of Chromobacterium vaccinii DSM 25150 resulting in mutants that exhibit improved production of FR900359 and improved characteristics concerning downstream processing and purification.

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.

The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway.

The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) multienzyme "assembly line" in the myxobacterium Chondromyces crocatus Cm c5. The most striking structural feature of these compounds is an isochromanone ring system; such an aromatic moiety is only known from two other complex polyketides, the electron transport inhibitor stigmatellin and the polyether lasalocid. The cyclization and aromatization reactions in the stigmatellin pathway are presumed to be catalyzed by a cyclase domain located at the end of the PKS, while the origin of the lasalocid benzenoid ring remains obscure. Notably, the ajudazol biosynthetic machinery does not incorporate a terminal cyclase, but instead a variant thioesterase (TE) domain. Here we present detailed phylogenetic and sequence analysis, coupled with experiments both in vitro and in vivo, that suggest that this TE promotes formation of the isochromanone ring, a novel reaction for this type of domain. As the ajudazol TE has homologues in several other secondary-metabolite pathways, these results are likely to be generalizable.

SummaryThe thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production.