Identification of Enzymes Involved in Sesterterpene Biosynthesis in Marine Fungi.

Diverse and bioactive terpene compounds are produced by marine fungi. The biosynthesis of sesterterpene molecules containing a C25 skeleton is catalyzed by chimeric enzymes that carry out both chain elongation and terpene cyclization reactions. These bifunctional characteristics facilitate using different chain length polyisoprenoid diphosphates as precursors to yield the geranylfarnesyl diphosphate intermediate, which is then converted to a sesterterpene in one step. In this chapter, we describe the identification of sesterterpene synthase enzymes, together with other related enzymes such as diterpene and farnesyl diphosphate synthases, in a single fungal strain. The processes are based on genome sequencing, in silico analysis of terpene synthase, in vivo gene (cluster) deletion and complementation, and in vitro protein function verification, together with the methods of detecting terpenes using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).

A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites-an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Analysis of the lithiated leucosceptroids from Leucosceptrum canum to facilitate their identification and differentiation by electrospray ionization tandem mass spectrometry.

RATIONALE: Leucosceptroids, isolated from Leucosceptrum canum (L. canum), are sesterterpenoids with novel molecular scaffolds which possess potent antifeedant activities. Their molecular scaffolds comprise a 5,6,5-framework with a great diversity due to different hydroxylation and substituent positions. The biological activities of leucosceptroids are affected by the subtle structural differences. The structural characterization and differentiation of the leucosceptroid isomers are of great importance. METHODS: Firstly, different kinds of cation adducts of leucosceptroids were examined by adding alkali metal ions to the solution, and the lithiated adducts of leucosceptroid were found to be readily formed and yielded characteristic fragment ions under collision-induced dissociation (CID). Then, a systematic mass spectrometric investigation of the [M + Li](+) ions was carried out to clarify their characteristic fragment pathways by electrospray ionization quadrupole time-of-flight-type tandem mass spectrometry (ESI-QTOF-MS/MS). The proposed fragmentation pathways were confirmed through ion trap ESI-MS(n) (n ≥ 3) spectra. Finally, the proposed MS/MS method was applied to investigate the extracts of L. canum. RESULTS: A specific fragmentation pathway of the lithiated adduct, which leads to the production of diagnostic ions of leucosceptroids, was observed. This fragmentation is initiated by cleavage of the C-ring and leads to formation of two types of ions by further dissociation. Both pathways could yield characteristic fragment ions, which could be used to define the substituents at the skeletal structure and at the C-ring. The representative characteristic fragmentations of [M + Li](+) ions and the proposed fragmentation pathways were successfully adopted to investigate the L. canum extracts, and a total of eleven leucosceptroids were identified or tentatively characterized. CONCLUSIONS: The characteristic fragmentation pathways of [M + Li](+) species of leucosceptroid isomers were proposed. Three types of leucosceptroid isomers were successfully differentiated. Eleven leucosceptroids were characterized from L. canum extracts. The fragmentation knowledge will facilitate the analysis of leucosceptroids and other sesterterpenoids in future research. Copyright © 2016 John Wiley & Sons, Ltd.

Coleifolides A and B, Two New Sesterterpenoids from the Aerial Parts of Scutellaria coleifolia H.Lév.

Coleifolides A and B (1 and 2), two new sesterterpenoids with a ß-methyl-α,ß-unsaturated-γ-lactone moiety, were isolated from the aerial parts of Scutellaria coleifolia H.Lév. (Lamiaceae), together with three known compounds. Their structures were elucidated by NMR and MS examinations. Coleifolides A and B were concluded to be partially racemic compounds by the HPLC analysis using a chiral column or introduction of chiral derivatizing agents. The absolute configuration of the major isomer was determined by analyses of the CD spectrum as well as NMR data of (R)- and (S)-2-NMA derivatives. Coleifolides A and B are structurally similar to manoalide derivatives, previously isolated from marine sponges, and appear to be the first examples of this type of compounds being isolated from higher plants.

A fast and efficient LC-MS/MS method for detection, identification and quantitative analysis of bioactive sesterterpenes in Salvia dominica crude extracts.

Sesterterpenes are a small group of terpenoids showing a number of interesting pharmacological properties, including cytotoxicity, anti-inflammatory, anti-microbial and anti-angiogenic activities and platelet aggregation inhibition. Recently, some sesterterpene lactones isolated from Salvia dominica have been shown to modulate enzymatic activity of tubulin tyrosine ligase (TTL), a promising target for new anticancer therapeutic strategies. However, to allow a direct use of S. dominica extracts as a source of TTL inhibitors, analytical method aimed to their fast qualitative and quantitative characterization is required. Despite the structural features and diverse biological activities of sesterterpenoids, actually no analytical method for their quantization into complex mixtures has been published. Here we describe an LC-MS/MS method aimed to qualitative and quantitative analysis of sesterterpenes lactones in the crude extracts obtained from different parts of S. dominica. This approach allowed us to characterize all the sesterterpenes by a single step analysis and also to identify two unknown compounds. Moreover, a quantitative comparison of the composition in sesterterpenes of extracts obtained from S. dominica leaves, roots and leaf galls was performed, leading to the definition of both leaves and leaf galls as suitable sources of TTL inhibitors.

Antifouling sesterterpenes from the New Zealand marine sponge Semitaspongia bactriana.

Three antifouling (AF) sesterterpenes have been isolated from the New Zealand marine sponge Semitaspongia bactriana with toxicity against the diatom Nitzschia closterium and bryozoan Bugula neritina. The three metabolites have been characterised by spectroscopic techniques as 7E,12E,20Z-variabilin (1), cavernosolide (2) and lintenolide A (3) (also called spongianolide C) and have low micromolar activity against our two test species. The gamma-hydroxybutenolide containing sesterterpenes (2 and 3) show the most promise, with relative stability and suitable lipophilicity for incorporation of either these metabolites, or synthetic analogues, as biocides to produce paints or plastics with AF properties.