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Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, β-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, β-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. Key points: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases. [ABSTRACT FROM AUTHOR]

A new indole diterpene, 26-dihydroxyaflavininyl acetate (1), along with five known analogs (2–6) were isolated from the liquid fermentation of Aspergillus flavus GZWMJZ-288, an endophyte from Garcinia multiflora. The structures of these compounds were identified through NMR, MS, chemical reaction, and X-ray diffraction experiments. Enzyme inhibition activity screening found that compounds 1, 4, and 6 have a good binding affinity with NPC1L1, among which compound 6 exhibited a stronger binding ability than ezetimibe at a concentration of 10 µM. Moreover, compound 5 showed inhibitory activity against α-glucosidase with an IC50 value of 29.22 ± 0.83 µM, which is 13 times stronger than that of acarbose. The results suggest that these aflavinine analogs may serve as lead compounds for the development of drugs targeting NPC1L1 and α-glucosidase. The binding modes of the bioactive compounds with NPC1L1 and α-glucosidase were also performed through in silico docking studies. [ABSTRACT FROM AUTHOR]

A new indole diterpene, 26-dihydroxyaflavininyl acetate (1), along with five known analogs (2–6) were isolated from the liquid fermentation of Aspergillus flavus GZWMJZ-288, an endophyte from Garcinia multiflora. The structures of these compounds were identified through NMR, MS, chemical reaction, and X-ray diffraction experiments. Enzyme inhibition activity screening found that compounds 1, 4, and 6 have a good binding affinity with NPC1L1, among which compound 6 exhibited a stronger binding ability than ezetimibe at a concentration of 10 µM. Moreover, compound 5 showed inhibitory activity against α-glucosidase with an IC50 value of 29.22 ± 0.83 µM, which is 13 times stronger than that of acarbose. The results suggest that these aflavinine analogs may serve as lead compounds for the development of drugs targeting NPC1L1 and α-glucosidase. The binding modes of the bioactive compounds with NPC1L1 and α-glucosidase were also performed through in silico docking studies.

Perennial ryegrass (Lolium perenne L.), an economically important pasture and turf grass, is commonly infected with asexual Epichloë species endophytes. Endophytes provide enhanced bioprotection by producing alkaloids, and research often focusses on the negative impact on grazing animals. However, alkaloid distribution throughout the plant and their role in biocontrol of insect pests and diseases are less well understood. Additionally, intermediate compounds have not been investigated for their impacts on animal welfare and biological control in pasture-based scenarios. Here, a single liquid chromatography–mass spectrometry (LC-MS) method was used to measure seven alkaloids in different perennial ryegrass tissues infected with SE or NEA12 endophytes. High alkaloid recoveries and a clear plant matrix effect emphasize the importance of using matrix-matched standards for accurate quantitation. The method is sensitive, detecting alkaloids at low concentrations (nanogram levels), which is important for endophyte strains that produce compounds detrimental to livestock. Concentrations were generally highest in seeds, but distribution differed in the shoots/roots: peramine, terpendole E, terpendole C and lolitrem B were higher in shoots, whilst ergovaline, paxilline and epoxy-janthitrem I were more evenly distributed throughout the two tissues. Knowledge of alkaloid distribution may allow for concentrations to be predicted in roots based on concentrations in the shoots, thereby assisting future determinations of resistance to insects, especially subterranean root-feeding pests. [ABSTRACT FROM AUTHOR]

Indole diterpenes are a large class of secondary metabolites produced by fungi, possessing a cyclic diterpenoid backbone and an indole moiety. Novel structures and important biological activity have made indole diterpenes one of the focuses of synthetic chemists. Although the discovery, identification, structural diversity, biological activity and especially structure–activity relationship of indole diterpenes have been reported in some papers in recent years, they are absent of a systematic and comprehensive analysis, and there is no elucidation of enzymes related to this kind of natural product. Therefore, it is necessary to summarize the relevant reports to provide new perspectives for the following research. In this review, for the first time, the function of related synthases and the structure–activity relationship of indole diterpenes are expounded, and the recent research advances of them are emphasized. [ABSTRACT FROM AUTHOR]

Perennial ryegrass (Lolium perenne L.), an economically important pasture and turf grass, is commonly infected with asexual Epichloë species endophytes. Endophytes provide enhanced bioprotection by producing alkaloids, and research often focusses on the negative impact on grazing animals. However, alkaloid distribution throughout the plant and their role in biocontrol of insect pests and diseases are less well understood. Additionally, intermediate compounds have not been investigated for their impacts on animal welfare and biological control in pasture-based scenarios. Here, a single liquid chromatography–mass spectrometry (LC-MS) method was used to measure seven alkaloids in different perennial ryegrass tissues infected with SE or NEA12 endophytes. High alkaloid recoveries and a clear plant matrix effect emphasize the importance of using matrix-matched standards for accurate quantitation. The method is sensitive, detecting alkaloids at low concentrations (nanogram levels), which is important for endophyte strains that produce compounds detrimental to livestock. Concentrations were generally highest in seeds, but distribution differed in the shoots/roots: peramine, terpendole E, terpendole C and lolitrem B were higher in shoots, whilst ergovaline, paxilline and epoxy-janthitrem I were more evenly distributed throughout the two tissues. Knowledge of alkaloid distribution may allow for concentrations to be predicted in roots based on concentrations in the shoots, thereby assisting future determinations of resistance to insects, especially subterranean root-feeding pests.

Analytical scale chemical/cultivation profiling prioritized the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339. Subsequent investigation permitted isolation of noonindoles A–F (5–10) and detection of eight minor analogues (i–viii) as new examples of a rare class of indole diterpene (IDT) amino acid conjugate, indicative of an acyl amino acid transferase capable of incorporating a diverse range of amino acid residues. Structures for 5–10 were assigned by detailed spectroscopic and X-ray crystallographic analysis. The metabolites 5–14 exhibited no antibacterial properties against G-ve and G+ve bacteria or the fungus Candida albicans, with the exception of 5 which exhibited moderate antifungal activity.

Indole diterpenes are a large class of secondary metabolites produced by fungi, possessing a cyclic diterpenoid backbone and an indole moiety. Novel structures and important biological activity have made indole diterpenes one of the focuses of synthetic chemists. Although the discovery, identification, structural diversity, biological activity and especially structure–activity relationship of indole diterpenes have been reported in some papers in recent years, they are absent of a systematic and comprehensive analysis, and there is no elucidation of enzymes related to this kind of natural product. Therefore, it is necessary to summarize the relevant reports to provide new perspectives for the following research. In this review, for the first time, the function of related synthases and the structure–activity relationship of indole diterpenes are expounded, and the recent research advances of them are emphasized.

A new indole diterpene, named penicindopene A (1), together with seven known compounds (2 – 8), was isolated from the deep-sea fungus Penicillium sp. YPCMAC1. The structure of penicindopene A was elucidated by extensive spectroscopic analyses (1 D and 2 D NMR, and HRESIMS data), in addition to the ECD calculations for the assignments of its absolute configuration. Penicindopene A represented the first example of indole diterpenes possessing a 3-hydroxyl-2-indolone moiety, and it exhibited moderate cytotoxicities against A549 and HeLa cell lines with IC50 values of 15.2 and 20.5 µM, respectively. [ABSTRACT FROM AUTHOR]

Abstract Ipomoea asarifolia has been associated with a tremorgenic syndrome in livestock. Recently indole diterpene compounds were identified in I. asarifolia , some of which have been shown to cause a tremorgenic syndrome. In this study, the tremorgenic nature of I. asarifolia was assessed using a mouse model. Adult mice were fed rodent chow containing 10, 15, 20 and 25% endophyte infected (E+), or 25% endophyte free (E-), I. asarifolia for 14 days. The mice fed E+ chow developed a tremorgenic syndrome as characterized by visually observed muscle tremors and an inability to traverse a balance beam, whereas the mice fed E- chow did not develop tremors and had similar muscle coordination to control mice. A lactating mouse model was also used to determine if the compounds can be transferred to nursing pups via the milk. Nursing pups were exposed via their mother's milk for 21 days, from post-natal day 0–21. The pups from dams exposed to E+ chow developed a similar tremorgenic syndrome. Data presented in this study demonstrate that the tremorgenic compounds in I. asarifolia are endophyte derived. Additionally, both adult mice and nursing pups are good models for studying the tremorgenic nature of I. asarifolia and related plants. Highlights • Indole diterpenes are the primary tremorgenic principles in I. asarifolia. • Mice can be used as a small animal model to study the tremorgenic nature of I. asarifolia. • I. asarifolia causes a dose-dependent physiological response that results in visually observable muscle tremors. • The tremorgenic compounds in I. asarifolia are transferred via milk to nursing young causing muscle tremors in the pups. • The muscle tremors caused by I. asarifolia are transient in nature, as they appear to resolve quickly once exposure stops. [ABSTRACT FROM AUTHOR]

[Display omitted] Oxidative modifications are key biosynthetic processes responsible for expanding the structural diversity of indole diterpenes (IDTs). In this study, we focused on the function of prenyltransferases and flavoprotein oxidases for the synthesis of nodulisporic acid-type and shearinine-type bicyclic rings found on the indole moiety. An in vitro enzymatic reaction of a prenyltransferase JanD, which mediates diprenylation at the C5- and C6-positions, revealed that JanD recognizes a C13‑hydroxy group on the diterpene moiety. Subsequent enzymatic reactions with flavoprotein oxidases, JanO, and NodO, indicated that JanO can accommodate penta/hexacyclic IDTs, while NodO exhibits strict substrate specificity toward pentacyclic IDTs. The broad substrate tolerance of JanD and JanO provides opportunities for the synthesis of unnatural IDTs based on artificial reconstitution in a heterologous host. [ABSTRACT FROM AUTHOR]

The article discusses research on synthesis of (±)-terpendole E, kinesin Eg5 inhibitor, is a biosynthetic intermediate of indole-diterpenes in the fungus Chaunopycnis alba. It mentions synthesis of the racemate of terpendole E from tricyclic dihydroxy ketone by a 13-step sequence that involves diastereoselective installation of its C3 quaternary stereocenter via a cyclopropyl ketone intermediate and Palladium (Pd)-mediated two-step construction of the indole ring moiety .

The article discusses a research on enzymatic activity of prenyltransferase which catalyzes the di-prenylation of alkaloid paxilline. Topics discussed include expression of recombinant prenyltransferase genes in Escherichia coli, identification of indole diterpene biosynthetic gene clusters, and cloning of genes which controls biosynthesis of fungal organic compound fusicoccin A.

Analytical scale chemical/cultivation profiling prioritized the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339. Subsequent investigation permitted isolation of noonindoles A-F ( 5 - 10 ) and detection of eight minor analogues ( i - viii ) as new examples of a rare class of indole diterpene (IDT) amino acid conjugate, indicative of an acyl amino acid transferase capable of incorporating a diverse range of amino acid residues. Structures for 5 - 10 were assigned by detailed spectroscopic and X-ray crystallographic analysis. The metabolites 5-14 exhibited no antibacterial properties against G-ve and G+ve bacteria or the fungus Candida albicans , with the exception of 5 which exhibited moderate antifungal activity., Competing Interests: The authors declare no conflict of interest.

Epichloë festucae var. lolii and Epichloë sp. Lp TG-3 are filamentous fungal endophytes of perennial ryegrass ( Lolium perenne ) that have a substantial impact on New Zealand's agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry., Competing Interests: The authors declare that this study received funding from Grasslanz Technology Ltd and PGG Wrightson Seeds Ltd. The funders were not involved in the study design, collection, analysis, interpretation of dat or the writing of this article. They were however involved in the decision to submit it for publication., (Copyright © 2022 Miller, Hudson, Johnson, Singh, Mace, Forester, Maclean, Voisey and Johnson.)

Abstract: Lolitrem B is an indole-diterpenoid neurotoxin which is the main causative agent of ryegrass staggers, an animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance calcium-activated potassium (BK) channel activity (IC50 =4 nM). Furthermore, we have recently shown that the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the toxin a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B and seven structurally-related lolitrems on BK channel activity has been measured. Concentration–responses and conductance–voltage (G–V) relationships were determined for each compound and related to the different structure types. This study has identified seven new BK channel inhibitors and has allowed the identification of two key structural features required for high potency BK channel activity by lolitrems. [Copyright &y& Elsevier]

Abstract: The effects of the mycotoxin lolitrem B on the function of hSlo large conductance calcium-activated potassium channels expressed in HEK293 cells have been investigated using inside-out membrane patches. Lolitrem B potently inhibited hSlo potassium currents activated by depolarising voltage pulses in the presence of 10μM free calcium. At a concentration of 100nM, lolitrem B rapidly and completely inhibited outward potassium currents. The concentration that produced half-maximal inhibition was 3.7nM, indicating a high apparent affinity for hSlo channels. This is the first time that a molecular site of action has been identified for a compound of the lolitrem structural class of indole diterpene and identifies a novel BK channel blocker. [Copyright &y& Elsevier]

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed. [ABSTRACT FROM AUTHOR]

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n = 30) for indole diterpene alkaloids, representing four genera, Argyreia , Ipomoea , Stictocardia , and Turbina , that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species. • Convolvulaceous species (n = 30) were screened for fungal symbiont derived compounds. • Ergot alkaloids were detected in 18 species representing four genera. • Indole diterpenes were detected in two Argyreia species and eight Ipomoea species. • Swainsonine was detected in two Ipomoea species. [ABSTRACT FROM AUTHOR]

Abstract: Diastereoselective construction of a DEF ring model of terpendole E, a M phase-specific antimitotic natural product, has been achieved from a known bicyclic keto ester by a 10-step (or 12-step) sequence that features an efficient one-pot intramolecular etherification of an olefinic alcohol with mCPBA. The synthesis proceeded in an excellent overall yield of 36% through 10 steps or in 56% via 12 steps which involved a highly diastereoselective reduction of a ketone derived from an undesired diastereomeric alcohol intermediate. [Copyright &y& Elsevier]

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

High conductance, calcium-activated potassium (BK) channels (KCa1.1) are important in regulating physiologic responses in many types of tissues and, as such, present opportunities for development of new therapeutic agents. Both channel agonists and inhibitors could have therapeutic utility, depending on medical application under consideration. However, characterization of molecular pharmacology of BK channels is incomplete and has been difficult to accomplish because of paucity of chemical leads that are acceptable templates for Medicinal Chemistry investigation. Only through continued prosecution of new high-throughput screening campaigns can this situation be rectified. Examples are presented of BK channel agonist and inhibitor discovery paradigms which will be useful for progressing BK channel future drug discovery strategies., (© 2016 Elsevier Inc. All rights reserved.)

The first synthesis of the racemate of terpendole E, a specific inhibitor of the mitotic kinesin Eg5, has been achieved from a known tricyclic dihydroxy ketone by a 13-step sequence that involves diastereoselective installation of its C3 quaternary stereocenter via a cyclopropyl ketone intermediate and Pd-mediated two-step construction of the indole ring moiety as the key transformations.

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A putative indole diterpene biosynthetic gene cluster composed of eight genes was identified in a genome database of Phomopsis amygdali, and from it, biosynthetic genes of fusicoccin A were cloned and characterized. The six genes showed significant similarities to pax genes, which are essential to paxilline biosynthesis in Penicillium paxilli. Recombinants of the three putative prenyltransferase genes in the cluster were overexpressed in Escherichia coli and characterized by means of in vitro experiments. AmyG is perhaps a GGDP synthase. AmyC and AmyD were confirmed to be prenyltransferases catalyzing the transfer of GGDP to IGP and a regular di-prenylation at positions 20 and 21 of paxilline, respectively. AmyD is the first know example of an enzyme with this function. The Km values for AmyD were calculated to be 7.6 ± 0.5 μM for paxilline and 17.9 ± 1.7 μM for DMAPP at a kcat of 0.12 ± 0.003/s.