Fengan Yu

A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.

In many macroorganisms, the ultimate source of potent biologically active natural products has remained elusive due to an inability to identify and culture the producing symbiotic microorganisms. As a model system for developing a meta-omic approach to identify and characterize natural product pathways from invertebrate-derived microbial consortia, we chose to investigate the ET-743 (Yondelis) biosynthetic pathway. This molecule is an approved anticancer agent obtained in low abundance (10(-4)-10(-5) % w/w) from the tunicate Ecteinascidia turbinata and is generated in suitable quantities for clinical use by a lengthy semisynthetic process. On the basis of structural similarities to three bacterial secondary metabolites, we hypothesized that ET-743 is the product of a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the tunicate/microbial consortium, we targeted and assembled a 35 kb contig containing 25 genes that comprise the core of the NRPS biosynthetic pathway for this valuable anticancer agent. Rigorous sequence analysis based on codon usage of two large unlinked contigs suggests that Candidatus Endoecteinascidia frumentensis produces the ET-743 metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. Moreover, the predicted activity of an enzyme for assembly of the tetrahydroisoquinoline core of ET-743 was verified in vitro. This work provides a foundation for direct production of the drug and new analogues through metabolic engineering. We expect that the interdisciplinary approach described is applicable to diverse host-symbiont systems that generate valuable natural products for drug discovery and development.

Covering: up to February 2017 Various fungi of the genera Aspergillus, Penicillium, and Malbranchea produce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system. After the discovery of distinct enantiomers of the natural alkaloids stephacidin A and notoamide B, from A. protuberus MF297-2 and A. amoenus NRRL 35660, another fungi, A. taichungensis, was found to produce their diastereomers, 6-epi-stephacidin A and versicolamide B, as major metabolites. Distinct enantiomers of stephacidin A and 6-epi-stephacidin A may be derived from a common precursor, notoamide S, by enzymes that form a bicyclo[2.2.2]diazaoctane core via a putative intramolecular hetero-Diels-Alder cycloaddition. This review provides our current understanding of the structural and stereochemical homologies and disparities of these alkaloids. Through the deployment of biomimetic syntheses, whole-genome sequencing, and biochemical studies, a unified biogenesis of both the dioxopiperazine and the monooxopiperazine families of prenylated indole alkaloids constituted of bicyclo[2.2.2]diazaoctane ring systems is presented.

Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A-C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 μM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 μM).