Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype–phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey’s analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

[1]  H. Brückner,et al.  Sequences of Tolypins, Insecticidal Efrapeptin‐Type Peptaibiotics from Species of the Fungal Genus Tolypocladium , 2020, Chemistry & biodiversity.

[2]  Christine M. Aceves,et al.  Reproducible molecular networking of untargeted mass spectrometry data using GNPS , 2019, Nature Protocols.

[3]  M. Kawada,et al.  Leucinostatin Y: A Peptaibiotic Produced by the Entomoparasitic Fungus Purpureocillium lilacinum 40-H-28. , 2019, Journal of natural products.

[4]  R. Cichewicz,et al.  Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides , 2017, Proceedings of the National Academy of Sciences.

[5]  Y. Di,et al.  Differential Expression of Genes Involved in Host Recognition, Attachment, and Degradation in the Mycoparasite Tolypocladium ophioglossoides , 2016, G3: Genes, Genomes, Genetics.

[6]  J. Spatafora,et al.  The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics , 2015, BMC Genomics.

[7]  Kai Blin,et al.  antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters , 2015, Nucleic Acids Res..

[8]  A. Mujic.  Symbiosis in the Pacific Ring of Fire : evolutionary-biology of Rhizopogon subgenus Villosuli as mutualists of Pseudotsuga , 2015 .

[9]  R. Gazis,et al.  Novel endophytic lineages of Tolypocladium provide new insights into the ecology and evolution of Cordyceps-like fungi , 2014, Mycologia.

[10]  Shuifang Zhu,et al.  Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads , 2014, BMC Bioinformatics.

[11]  Jason S. Cumbie,et al.  The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster , 2013, PLoS genetics.

[12]  Xiaoxun Ma,et al.  Solubility, dissolution enthalpy and entropy of l-glutamine in mixed solvents of ethanol + water and acetone + water , 2013 .

[13]  S. Baker,et al.  The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea , 2012, Chemistry & biodiversity.

[14]  B. Horwitz,et al.  Secondary metabolism in Trichoderma--a genomic perspective. , 2012, Microbiology.

[15]  Ya Cao,et al.  Isolation and characterization of aphidicolin and chlamydosporol derivatives from Tolypocladium inflatum. , 2011, Journal of natural products.

[16]  R. Singhal,et al.  Cyclosporin A--a review on fermentative production, downstream processing and pharmacological applications. , 2011, Biotechnology advances.

[17]  N. Friedman,et al.  Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data , 2011, Nature Biotechnology.

[18]  Robert D. Finn,et al.  HMMER web server: interactive sequence similarity searching , 2011, Nucleic Acids Res..

[19]  M. Sulyok,et al.  Production of fumonisins B2 and B4 in Tolypocladium species , 2011, Journal of Industrial Microbiology & Biotechnology.

[20]  D. Haft,et al.  SMURF: Genomic mapping of fungal secondary metabolite clusters. , 2010, Fungal genetics and biology : FG & B.

[21]  Sean R Eddy,et al.  A new generation of homology search tools based on probabilistic inference. , 2009, Genome informatics. International Conference on Genome Informatics.

[22]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[23]  Sofia M. C. Robb,et al.  MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. , 2007, Genome research.

[24]  J. Spatafora,et al.  Phylogenetic classification of Cordyceps and the clavicipitaceous fungi , 2007, Studies in mycology.

[25]  Alexandros Stamatakis,et al.  RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models , 2006, Bioinform..

[26]  Burkhard Morgenstern,et al.  AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints , 2005, Nucleic Acids Res..

[27]  Pavel A. Pevzner,et al.  De novo identification of repeat families in large genomes , 2005, ISMB.

[28]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[29]  B. Schlegel,et al.  The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions , 2003, Journal of peptide science : an official publication of the European Peptide Society.

[30]  C. Toniolo,et al.  Lipopeptaibol metabolites of tolypocladium geodes: total synthesis, preferred conformation, and membrane activity. , 2003, Chemistry.

[31]  Kazuo Tachibana,et al.  Stereochemical Determination of Acyclic Structures Based on Carbon-Proton Spin-Coupling Constants. A Method of Configuration Analysis for Natural Products. , 1999, The Journal of organic chemistry.

[32]  K. Harada,et al.  A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Elucidation of Limitations of Marfey's Method and of Its Separation Mechanism , 1997 .

[33]  Y. Tsantrizos,et al.  Structural Assignment of the Peptide Antibiotic LP237-F8, a Metabolite of Tolypocladium geodes , 1996 .

[34]  Y. Tsantrizos,et al.  Peptaibol metabolites of Tolypocladium geodes , 1996 .

[35]  J. Clardy,et al.  Structure of Efrapeptins From the Fungus Tolypocladium Niveum: Peptide Inhibitors of Mitochondrial ATPase , 1992 .

[36]  J. Clardy,et al.  Structures of the efrapeptins : potent inhibitors of mitochondrial ATPase from the fungus Tolypocladium niveum , 1991 .

[37]  H. Loosli,et al.  Neue Cyclosporine aus Tolypocladium inflatum. Die Cyclosporine K–Z† , 1987 .

[38]  M. Suzuki,et al.  Studies on peptide antibiotics, leucinostatins. I. Separation, physico-chemical properties and biological activities of leucinostatins A and B. , 1983, The Journal of antibiotics.

[39]  P. Henderson,et al.  The isolation and purification of the elvapeptins , 1982 .

[40]  H. Weber,et al.  Crystal and molecular structure of an iodo-derivative of the cyclic undecapeptide cyclosporin A. , 1976, Helvetica chimica acta.

[41]  H. Loosli,et al.  [Cyclosporin A, a Peptide Metabolite from Trichoderma polysporum (Link ex Pers.) Rifai, with a remarkable immunosuppressive activity]. , 1976, Helvetica chimica acta.