The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin-a diterpene produced by Clitopilus passeckerianus-being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics.

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

Biosynthesis of ubiquinones requires the intramembrane UbiA enzyme, an archetypal member of a superfamily of prenyltransferases that generates lipophilic aromatic compounds. Mutations in eukaryotic superfamily members have been linked to cardiovascular degeneration and Parkinson's disease. To understand how quinones are produced within membranes, we report the crystal structures of an archaeal UbiA in its apo and substrate-bound states at 3.3 and 3.6 angstrom resolution, respectively. The structures reveal nine transmembrane helices and an extramembrane cap domain that surround a large central cavity containing the active site. To facilitate the catalysis inside membranes, UbiA has an unusual active site that opens laterally to the lipid bilayer. Our studies illuminate general mechanisms for substrate recognition and catalysis in the UbiA superfamily and rationalize disease-related mutations in humans.

The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function.

Tiamulin, a water-soluble and highly effective semisynthetic derivative of pleuromutilin leads to the formation of physiologically inactive polypeptide chain initiation complexes which readily decompose and do not enter the phase of peptide chain elongation. Once elongation has begun it continues even in the presence of tiamulin as has been shown by measuring the formation of N-acetylphenylalanine-poly(phenylalanine). The formation of abortive initiation complexes was observed regardless of whether AcPhe-tRNA of fMet-tRNA was used as an initiator or whether artificial messengers or a natural messenger, like R17 bacteriophage RNA, was used. When this drug was acting on whole cells, it led to the disappearance of polysomes. The only structures which could be detected were of the monosome size. Therefore, polysomes seem to elongate the polypeptide chains in whole cells in the presence of this antibiotic, but since effective reinitiation is blocked, the polysome pool of the cell soon becomes depleted.

Tobacco (Nicotiana sylvestris) glandular trichomes make an attractive target for isoprenoid metabolic engineering because they produce large amounts of one type of diterpenoids, alpha- and beta-cembratrien-diols. This article describes the establishment of tools for metabolic engineering of tobacco trichomes, namely a transgenic line with strongly reduced levels of diterpenoids in the exudate and the characterization of a trichome specific promoter. The diterpene-free tobacco line was generated by silencing the major tobacco diterpene synthases, which were found to be encoded by a family of four highly similar genes (NsCBTS-2a, NsCBTS-2b, NsCBTS-3 and NsCBTS-4), one of which is a pseudogene. The promoter regions of all four CBTS genes were sequenced and found to share over 95% identity between them. Transgenic plants expressing uidA under the control of the NsCBTS-2a promoter displayed a specific pattern of GUS expression restricted exclusively to the glandular cells of the tall secretory trichomes. A series of sequential and internal deletions of the NsCBTS-2a promoter led to the identification of two cis-acting regions. The first, located between positions -589 to -479 from the transcription initiation site, conferred a broad transcriptional activation, not only in the glandular cells, but also in cells of the trichome stalk, as well as in the leaf epidermis and the root. The second region, located between positions -279 to -119, had broad repressor activity except in trichome glandular cells and is mainly responsible for the specific expression pattern of the NsCBTS-2a gene. These results establish the basis for the identification of trans-regulators required for the expression of the CBTS genes restricted to the secretory cells of the glandular trichomes.

As the largest class of natural products, terpenes have a variety of roles in mediating antagonistic and beneficial interactions among organisms. They defend many species of plants, animals and microorganisms against predators, pathogens and competitors, and they are involved in conveying messages to conspecifics and mutualists regarding the presence of food, mates and enemies. Despite the diversity of terpenes known, it is striking how phylogenetically distant organisms have come to use similar structures for common purposes. New natural roles undoubtedly remain to be discovered for this large class of compounds, given that such a small percentage of terpenes has been investigated so far.

In the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (1-3) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A-C (7-9); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isoprenoids are synthesized by consecutive condensations of their five-carbon precursor, isopentenyl diphosphate, to its isomer, dimethylallyl diphosphate. Two pathways for these precursors are known. One is the mevalonate pathway, which operates in eucaryotes, archaebacteria, and cytosols of higher plants. The other is a recently discovered pathway, the nonmevalonate pathway, which is used by many eubacteria, green algae, and chloroplasts of higher plants. To date, five reaction steps in this new pathway and their corresponding enzymes have been identified. EC numbers of these enzymes have been assigned by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) and are available at http://www.chem.qmw.ac.uk/iubmb/enzyme/reaction/terp/nonMVA.html.

The UbiA superfamily of intramembrane prenyltransferases catalyzes a key biosynthetic step in the production of ubiquinones, menaquinones, plastoquinones, hemes, chlorophylls, vitamin E, and structural lipids. These lipophilic compounds serve as electron and proton carriers for cellular respiration and photosynthesis, as antioxidants to reduce cell damage, and as structural components of microbial cell walls and membranes. This article reviews the biological functions and enzymatic activities of representative members of the superfamily, focusing on the remarkable recent research progress revealing that the UbiA superfamily is centrally implicated in several important physiological processes and human diseases. Because prenyltransferases in this superfamily have distinctive substrate preferences, two recent crystal structures are compared to illuminate the general mechanism for substrate recognition.Copyright © 2016 Elsevier Ltd. All rights reserved.

Diterpenoid is a huge group of nature products isolated from plants and fungi. Diterpene cyclase, which is responsible for the diterpene carbon skeleton formation from geranylgeranyl diphosphate (GGPP), is a key enzyme in the biosynthetic pathway of diterpene. The specificity of diterpene cyclase in different species results in structural diversity and bioactivity variety of diterpenoid. Isolation and characterization of the diterpene cyclase in various species will facilitate studies on the biosynthesis and regulation of diterpenoid in future. Compared to plant diterpenoids, few fungal diterpenoid and diterpene cyclase were studied. This article reviews the research advancement of fungal diterpene cyclase in recent years, especially describes the biosynthesis pathway of diterpenoid, the characteristics and cloning strategies of fungal diterpene cyclase, and the metabolic engineering of diterpenoid.

BC-3781 is a novel semisynthetic pleuromutilin antibiotic inhibiting bacterial protein synthesis. BC-3781 has completed a phase 2 clinical trial in acute bacterial skin and skin structure infections (ABSSSI). Its antibacterial spectrum additionally covers the predominant pathogens causing community-acquired bacterial pneumonia (CABP). In this study, the antibacterial activity of BC-3781 was evaluated against a contemporary collection of 10,035 bacterial isolates predominately causing ABSSSI and CABP, among other infections, collected within the SENTRY Antimicrobial Surveillance Program worldwide in 2010. BC-3781 exhibited potent activity against organisms commonly isolated from ABSSSI such as Staphylococcus aureus (MIC50/90, 0.12/0.12 μg/ml; 99.8% inhibited at ≤0.5 μg/ml), beta-hemolytic streptococci (MIC50/90, 0.03/0.03 μg/ml; 99.3% inhibited at ≤0.5 μg/ml), and coagulase-negative staphylococci (CoNS; MIC50/90, 0.06/0.12 μg/ml; 97.8% inhibited at ≤1 μg/ml). BC-3781 displayed similar MIC distributions among methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) S. aureus strains. BC-3781 was also active against Enterococcus faecium, with 76.3% of vancomycin-susceptible and 97.0% of vancomycin-resistant isolates being inhibited at BC-3781 concentrations of ≤1 μg/ml. Beta-hemolytic and viridans group streptococci were highly susceptible to BC-3781, with 99.3% and 96.7% of isolates inhibited at ≤0.5 μg/ml, respectively. Further, activity of BC-3781 against Streptococcus pneumoniae (MIC50/90, 0.12/0.25 μg/ml), Haemophilus influenzae (MIC50/90, 1/2 μg/ml), and Moraxella catarrhalis (MIC50/90, 0.12/0.25 μg/ml) was not negatively influenced by β-lactamase production or resistance to other antimicrobial classes tested. In all, BC-3781 displayed a very potent antibacterial profile including the most prevalent bacterial pathogens causing ABSSSI and CABP, thus warranting further clinical development of this antibiotic in these and possibly other indications.

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of trans-humulyl cation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids.

Plants and fungi are seemingly inexhaustible sources of interesting natural products with remarkable structural and biological diversity. One of the most important groups is the terpenes, ubiquitous natural products that are generated by 2 now well-established biosynthetic pathways: the older mevalonate and the more recently discovered 1-deoxyxylulose-5-phosphate. Among the diterpenes, the pimarane diterpenes are a very representative subgroup with several and interesting biological activities resulting from different functional group modifications. In this review, we outline the method of their structure determination, mainly spectroscopic results, their absolute configuration, and structure-activity relationships, were reported, as well as the mode of action for selected examples from plants, marine organisms, and fungi. The pimarane, isopimarane, and ent-pimarane diterpenes covered in this review have a wide range of biological activities including antimicrobial, antifungal, antiviral, phytotoxic, phytoalexin, cytotoxicity, and antispasmodic and relaxant effects.© 2018 Wiley Periodicals, Inc.

Basidiomycota fungi have received little attention for applications in biocatalysis and biotechnology and remain greatly understudied despite their importance for carbon recycling, ecosystem functioning and medicinal properties. The steady influx of genome data has facilitated detailed studies aimed at understanding the evolution and function of fungal lignocellulose degradation. These studies and recent explorations into the secondary metabolomes have uncovered large portfolios of enzymes useful for biocatalysis and biosynthesis. This review will provide an overview of the biocatalytic repertoires of Basidiomycota characterized to date with the hope of motivation more research into the chemical toolkits of this diverse group of fungi.Copyright © 2016 Elsevier Ltd. All rights reserved.

Chorismate pyruvate-lyase activity was detected in extracts of Escherichia coli. 4-Hydroxybenzoate was identified as the product of the enzymatic reaction by chemical derivatization and GC-MS analysis. The ubiC gene, coding for the chorismate pyruvate-lyase, was cloned and sequenced. The molecular weight of the gene product was calculated as 18,776 Da and confirmed by expression of the protein in E. coli minicells. The ubiA gene, coding for the 4-hydroxybenzoate octaprenyl transferase, was identified by sequence homology and complementation of a ubiA- strain. It is located directly downstream of ubiC in a typical operon structure.

The diterpene pleuromutilin is a ribosome-targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo-pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane-related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5-6-8-tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Retapamulin is a semisynthetic pleuromutilin derivative being developed as a topical antibiotic for treating bacterial infections of the skin. It is potent in vitro against susceptible and multidrug-resistant organisms commonly associated with bacterial skin infections. We report detailed mode of action studies demonstrating that retapamulin binds to the bacterial ribosome with high affinity, inhibits ribosomal peptidyl transferase activity, and partially inhibits the binding of the initiator tRNA substrate to the ribosomal P-site. Taken together, these data distinguish the mode of action of retapamulin from that of other classes of antibiotics. This unique mode of action may explain the lack of clinically relevant, target-specific cross-resistance of retapamulin with antibacterials in current use.