(12) United States Patent (10) Patent No.: US 9,518,097 B2 Hallen Et Al

USO09518097B2

(12) United States Patent (10) Patent No.: US 9,518,097 B2 Hallen et al. (45) Date of Patent: Dec. 13, 2016

(54) IDENTIFICATION AND USE OF GENES Walton et al. Ribosomal Biosynthesis of the Cyclic Peptide Toxins ENCOOING AMATOXIN AND of Amanita Mushrooms. Peptide Science (2010) 94(5): 659-664.* PHALLOTOXIN Machine translation of CN1341362 obtained on Dec. 21, 2010. 7 pages. (75) Inventors: Heather E. Hallen, Lincoln, NE (US); Trigueros et al. Biochimica et Biophysica Acta (2003) 1621: 292 Jonathan D. Walton, East Lansing, MI 298.* Rozen et al. Methods in Molecular Biology (2000) 132: 365-386.* (US); Hong Luo, Lansing, MI (US); Ahn, et al., “An extended physical map of the TOX2 locus of John S. Scott-Craig, East Lansing, MI Cochliobolus carbonum required for biosynthesis of HC-toxin.” (US) Fungal Genet Biol. 35(1):31-8 (2002). Alexandra, et al., “Highly Efficient Production of Laccase by the (73) Assignee: Board of Trustees of Michigan State Basidiomycete Pycnoporus cinnabarinus.” Appl Environ Microbiol. University, East Lansing, MI (US) 70(11):6379-638 (2004). Bamburg, “Proteins of the ADF/cofilin family: essential regulators (*) Notice: Subject to any disclaimer, the term of this of actin dynamics.” Annu. Rev. Cell Dev. Biol. 15:185-230 (1999). patent is extended or adjusted under 35 Benedict, et al., “Fermentative production of amanita toxins by a U.S.C. 154(b) by 0 days. strain of Galerina marginata.” J Bacteriol 91: 1380-1381 (1966). Beutler and der Marderosian, "Chemical variation in Amanita.” J. Nat. Prod. 44:422-431 (1981). (21) Appl. No.: 12/268,229 Brandt, et al., “Suggested functions for prolyl oligopeptidase: a puzzling paradox.” Clin Chim Acta. 377(1-2):50-61. Epub Oct. 10, (22) Filed: Nov. 10, 2008 2006 (2007). Bulaj, et al., “Efficient oxidative folding of conotoxins and the (65) Prior Publication Data radiation of venomous cone snails.” Proc Natl Acad Sci U S A. US 201OfO267O19 A1 Oct. 21, 2010 100(2): 14562-8. Epub Oct 22, 2003. Bushnell, et al., “Structural basis of transcription: alpha-amanitin Related U.S. Application Data RNA polymerase II cocrystal at 2.8 A resolution.” Proc. Natl. Acad. Sci. USA 99:1218-1222 (2002). (60) Provisional application No. 61/002,650, filed on Nov. Craik, et al., “The chemistry and biology of cyclotides.” Curr Opin 9, 2007. Drug Discov Devel 10:176-184 (2007). Craik, "Chemistry. Seamless proteins tie up their loose ends.” (51) Int. Cl. Science 311:1563-1564 (2006). C07K I4/375 (2006.01) Crooks, et al., “WebLogo: a sequence logo generator.” Genome Res. Jun. 14, 2004(6): 1188-90 (2004). C07K 7/64 (2006.01) Cunningham and O'Connor, “Proline specific peptidases.” Biochim CI2O I/68 (2006.01) Biophy's Acta 1343:160-186 (1997). (52) U.S. Cl. Duquesne, et al., “Two enzymes catalyze the maturation of a lasso CPC ...... C07K 14/375 (2013.01); C07K 7/64 peptide in Escherichia coli." Chem Biol. 14(7):793-803 (2007). (2013.01); C12O 1/6895 (2013.01); C12O Enjalbert, et al., “Treatment of Amatoxin Poisoning: 20-Year Ret 2600/142 (2013.01); C12O 2600/158 rospective Analysis, review of poisonings.' J. Toxicol. Clin. Toxicol. (2013.01) 40:715-757 (2002). (58) Field of Classification Search Escoubas, “Molecular diversification in spider venoms: a web of None combinatorial peptide libraries.” Mol Divers 10:545-554 (2006). See application file for complete search history. (Continued)

(56) References Cited Primary Examiner — Angela M. Bertagna U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Schwegman Lundberg & Woessner, P.A. 4,683, 195 A 7, 1987 Mullis et al...... 435/6.11 4,683.202 A 7, 1987 Mullis et al...... 435,912 4,965,188 A 10, 1990 Mullis et al...... 435/6.12 5,928,906 A 7, 1999 Koster et al...... 435,912 (57) ABSTRACT 9,273,100 B2 3/2016 Hallen-Adams et al. 2001/00 14445 A1 8, 2001 Urnovitz ...... 435/6 The present invention relates to compositions and methods 2004/0081978 A1* 4, 2004 Ziman et al. 435/6 comprising genes and peptides associated with cyclic pep 2005/0255486 A1* 11, 2005 Behlke et al. 435/6 tide toxins and toxin production in mushrooms. In particular,

2006/0141495 A1* 6, 2006 Wu ...... 435/6 2008/0076677 A1* 3, 2008 Yershov et al. ... 506,13 the present invention relates to using genes and proteins 2009/0093.620 A1* 4/2009 Kovalic et al...... 536,231 from Amanita species encoding Amanita peptides, specifi 2012/0276588 A1 11/2012 Hallen-Adams et al. cally relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods FOREIGN PATENT DOCUMENTS for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing CN 1341362. A * 3, 2002 Suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and moni OTHER PUBLICATIONS toring Suspected cases of mushroom poisoning in patients. Zhang et al. Production and characterization of Amanitin toxins from a pure culture of Amanita exitialis. FEMS Microbiology Letters (2005) 252: 223-228.* 9 Claims, 91 Drawing Sheets US 9,518,097 B2 Page 2

(56) References Cited Panaccione, “Multiple families of peptide synthetase genes from ergopeptine-producing fungi.” Mycological Research 100:429-436 (1996). OTHER PUBLICATIONS Panaro Fabrizio, et al., “Liver transplantation represents the optimal Finking and Marahiel, "Biosynthesis of Nonribosomal Peptides.” treatment for fulminant hepatic failure from amanita phalloides Annu Rey Microbiol 58:453-488 (2004). poisoning.” Transplant International 19(4):344-345 (2006). Frohman, et al., “Rapid production of full-length cDNAs from rare Polgar, “The prolyl oligopeptidase family.” Cell. Mol. Life Sci. transcripts: amplification using a single gene-specific oligonucle 59:349-362 (2002). otide primer.” Proc Natl AcadSci 85:8998-9002 (1988). Richter, et al., “cDNAs encoding D-Ala2 deltorphin precursors Garcia-Horsman, et al., “Deficient activity of mammalian prolyl oligopeptidase on the immunoactive peptide digestion in coeliac from skin of Phyllomedusa bicolor also contain genetic information disease.” Scand J Gastroenterol. 42(5):562-71 (2007). for three dermorphin-related opioid peptides.” Proc. Nat. Acad. Sci. Gardiner, et al., “The sirodesmin biosynthetic gene cluster of the USA 87:4836-4839 (1990). plant pathogenic fungus Leptosphaeria maculans.” Mol Microbiol. Rosengren, et al., “Microcin J25 has a threaded sidechain-to 53(5): 1307-18 (2004). backbone ring structure and not a head-to-tail cyclized backbone.” Gass and Khosla, “Prolyl endopeptidases.” Cell Mol Life Sci. JAm. Chem. Soc. 125:12464-12474 (2003). 64(3):345-55 (2007). Salamov and Solovyev, "Ab initio gene finding in Drosophila Hallen, et al., “Taxonomy and toxicity of Conocybe lactea and genomic DNA.” Genome Res. 10:516-522 (2000). related species.” Mycol. Res. 107:969-979 (2003). Saska, et al., “An asparaginyl endopeptidase mediates in vivo Hallen, Walton, 159. The utility of the incomplete genome: the proteinbackbone cyclization.” J Biol Chem. 282(40):29721-8. Epub Amanita bisporigera genome project. Mar. 15-20, 2005 Asilomar Aug. 13, 2007. Conference Center, Pacific Grove Calif. Fungal Genetics Newslet Schneider, "Mushroom in backyard kills curious puppy.” Lansing ter, vol. 52—Supplement XXIII Fungal Genetics Conference. State Journal, Sep. 30, 2008. Hallen, et al., “Gene expression shifts during perithecium develop Schuren, et al., “Highly-efficient transformation of the homobasidi ment in Gibberella Zea: (anamorph Fusarium graminearum), with omycete Schizophyllum commune to phleomycin resistance.” Curr: particular emphasis on ion transport proteins.” Fung. Genet. Biol., Genet. 26:179-183 (1994). 44; 1146-1156 (2007). Shan, et al., “Structural Basis for Gluten Intolerance in Celiac Hallen, et al., “Gene family encoding the major toxins of lethal Sprue.” Science 27 297 No. 5590 pp. 2275-2279 (2002). Amanita mushrooms.” Proc. Natl. Acad. Sci. USA 104: 19097 Shan, et al., “Identification and analysis of multivalent proteolyti 19101 (2007). cally resistant peptides from gluten: implications for celiac sprue.” Keller, et al., “Fungal secondary metabolism from biochemistry to J Proteome Res. 4(5):1732-41 (2005). genomics.” Nat Rey Microbiol. 3(12):937-47 (2005). Singh, et al., “The use of heparin as a simple cost-effective means Kroncke, et al., “alpha-Amanitin uptake into hepatocytes. Identifi of controlling background in nucleic acid hybridization proce cation of hepatic membrane transport systems used by amatoxins.” dures.” Nucl. Acids Res. 12:5627-5638 (1984). J. Biol. Chem., 261: 12562-12567 (1986). Szeltner, et al., “The noncatalytic beta-propeller domain of prolyl Kuo, Galerina marginata. Retrieved from the MushroomExpert. oligopeptidase enhances the catalytic capability of the peptidase Com Web site: http://www.mushroomexpert.com/galerina domain.” J Biol Chem. 275(20): 15000-5 (2000). marginata.html, (Aug. 2004). Szeltner, et al., “Substrate- and pH-dependent contribution of Le Quere, et al., “Size and complexity of the nuclear genome of the oxyanion binding site to the catalysis of prolyl oligopeptidase, a ectomycorrhizal fungus Paxillus involutus.” Fung. Genet. Biol. paradigm of the serine oligopeptidase family.” Protein Sci. 36:234-241 (2002). 9(2):353-60 (2000). Lengsfeld, et al., “Interaction of phalloidin with actin.” Proc. Natl. Trabi and Craik, “Circular proteins—no end in sight.” Trends Acad. Sci. USA, 71:2803-2807 (1974). Biochem Sci. 27(3): 132-8 (2002). Letschert, et al., “Molecular characterization and inhibition of Tudzynski and Holter, “Gibberellin biosynthetic pathway in Gib amanitin uptake into human hepatocytes.” Toxicol Sci. 91: 140-149 berella fujikuroi: evidence for a gene cluster.” Fungal Genet Biol. (2006). 25(3): 157-70 (1998). Lindell, et al., “Specific inhibition of nuclear RNA polymerase II by Tudzynski et al., “Characterization of the final two genes of the alpha-amanitin.” Science 170:447-449 (1970). gibberellin biosynthesis gene cluster of Gibberella fujikuroi: des Lugones, et al., “Introns are necessary for mRNA accumulation in and P450-3 encode GA4 desaturase and the 13-hydroxylase, respec Schizophyllum commune.” Mol. Microbiol. 32:681-700 (1999). tively.” J Biol Chem. 278(31):28635-43. Epub May 15, 2003. Malonek, et al., “Functional characterization of two cytochrome Tulloss, et al., “Amanita-beauty, danger, and diversity-almost every P450 monooxygenase genes, P450-1 and P450-4, of the gibberellic where.” Boll. Gr: micol. G. Bres. (n.s.) 43(2): 13-21 (2000). acid gene cluster in Fusarium proliferatum (Gibberella fujikuroi Tyler, et al., “Occurrence of Amanita toxins in American collections MP-D).” Appl Environ Microbiol. 71(3): 1462-72 (2005). of deadly amanitas.” J. Pharm. Sci. 55:590-593 (1966). Maniatis, et al., “Regulation of inducible and tissue specific gene Voss, et al., “The role of enhancers in the regulation of cell-type expression.” Science 236:1237-1245 (1987). specific transcriptional control.” Trends Biochem. Sci., 11:287-289 Margulies, et al., “Genome sequencing in microfabricated high (1986). density picolitre reactors.” Nature 437:376-380 (2005). Walton, "Horizontal gene transfer and the evolution of secondary May and Perrin, “Tryptathionine bridges in peptide synthesis.” metabolite gene clusters in fungi: an hypothesis.” Fungal Genet Biopolymers, 88(5):714-24 (2007). Biol. 30(3):167-71 (2000). Muraoka, et al., “Detection and identification of amanitins in the Welzel, et al., “Characterization of the ferrichrome A biosynthetic wood-rotting fungi Galerina fasciculata and Galerina helvoliceps.” gene cluster in the homobasidiomycete Omphalotus olearius.” Appl. Environ. Microbiol. 65:4207-4210 (1999). FEMS Microbiol Lett. 249(1): 157-63 (2005). Muraoka and Shinozawa, “Effective production of amanitins by WeiB, et al., “Molecular phylogenetic studies in the genus two-step cultivation of the basidiomycete, Galerina fasciculata Amanita.” Can J. Bot. 76: 1170-1180 (1998). GF-060.” J Biosci Bioeng 89:73-76 (2000). Williams, et al., “Loss of a prolyl oligopeptidase confers resistance Nikolskaya, et al., “Identification of peptide synthetase-encoding to lithium by elevation of inositol (14.5) trisphosphate.” EMBO.J. genes from filamentous fungi producing host-selective phytotoxins 18(10):2734-45 (1999). or analogs.” Gene 165:207-211 (1995). Williams, “Pharmacogenetics in model systems: defining a common Novick and Geisinger, “Quorum sensing in Staphylococci.” Annu mechanism of action for mood stabilisers.” Prog Rev Genet. 42:541-64 (2008) in the instant application. Neuropsychopharmacol Biol Psychiatry 29(6):1029-37 (2005). Olivera, "Conus peptides: biodiversity-based discovery and Woodward, et al., “Constant and hypervariable regions in conotoxin exogenomics.” J. Biol. Chem. 281:31173-31.177 (2006). propeptides.” EMBO.J. 9:1015-1020 (1990). US 9,518,097 B2 Page 3

(56) References Cited OTHER PUBLICATIONS Yu, et al., “A draft sequence of the rice genome (Oryza sativa L. ssp. indica).” Science 296:79-92 (2002). Butera, R., et al., “Diagnostic accuracy of urinary amanitin in Suspected mushroom poisoning: a pilot study’. Clinical Toxicology, 42(6), (2004), 901-912. “U.S. Appl. No. 13/362,561, Response filed Oct. 16, 2013 to Restriction Requirement mailed Sep. 17, 2013', 7 pgs. “U.S. Appl. No. 13/362,561, Restriction Requirement mailed Sep. 17, 2013', 7 pgs. “U.S. Appl. No. 13/362,561, Response filed Apr. 8, 2014 to Non Final Office Action mailed Jan. 28, 2014', 8 pgs. “U.S. Appl. No. 13/362,561, Restriction Requirement mailed Jun. 3. 2014'. 6 pgs. “U.S. Appl. No. 13/362,561. Non Final Office Action mailed Jan. 28, 2014', 8 pgs. “U.S. Appl. No. 13/362.561, Response filed Aug. 4, 2014 to Restriction Requirement mailed Jun. 3, 2014, 14pgs. U.S. Appl. No. 13/362,561, PTO Response to Rule 312 Commu nication mailed Jan. 28, 2016, 2 pgs. U.S. Appl. No. 14/933.239, Preliminary Amendment filed Apr. 11, 2016, 9 pgs. * cited by examiner U.S. Patent Dec. 13, 2016 Sheet 1 of 91 US 9,518,097 B2

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Fig. 4A A:aii. &E : N, 55 a coc aac to C cat tog aac Cita act C & aga. CC. Clta aac C. & 3. at C : .

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A. f gcc ctt test taa attocc catccatttgtcc.gctgatatgacacgaagtag taggegat&Qaagttgttggacgt. tat caggattggg.ccgttgagcotgcatcggaaacaact tatgttcettcttitt.ttctgttt. toatttgttaaaatacagaacccatgttgatgatctgttgttgtag ticaätataaagttgtact gtgtttittgttcaaaaaaaaaaaaaaaaaaaaaa. Carats icieate tie positi is of three it'es. SEQ E: NO: 55 a coc aac too cat tog aac Cita act. ca. 3g a cit (ta 3 &c. citc. &ca. atc. cai atg tic. gaol atc: aat got acci gt cit co-c at (: Egg gg at gg. tiga aac ccg tge atc. g.gt gag gas gttc act aca. Ct c (tc act cgt. sigC gag gel C Clt tit. taaatt C.C.C.cat catttgttctg.cgct&ta Casjaa tatgggggata Caagttgttggettig titat aggottggcc.gttgagic{tgcatigga &3:Caact tatgttcett Cit.tttittctgttitt catttgttcaaaaaaagaacCCatgtc..gatge, Cistgttgtagt caatataaagttgat, gttcttgcaaaaaaaaaaa aaaaaaaaaaa. SEQ is NO. : SS (noncoding region of the propeptide atg tit gas at: 3 &t gg, acc 8:gt citt cl: at tigg gigt at ggt. Egg aac Cog tige at gSt gag gag to act aca, Ctc to acc cyt ggg gag gig citt g it U.S. Patent Dec. 13, 2016 Sheet 5 Of 91 US 9,518,097 B2

Fig. 4B SEC ID NO:57 Exemplary sequence of genomic DNA covering the amanitin gene. The nucleotides cncoding the anaraitin: peptide are undefined, CATCAAAACAAAACAC2CACTCGGCTASA GTCCATIAAGATGGGAGCGGAAGTC'? "GCCAAAAggi GACCA; CG'I''''''AAAAAIGAGICCGTGACCGACCATAAAGI AC:ApacTCAGOAT'GTAragg, CCCGAATATCATCCGCACA3CCGCCATEGICGGCGGCC AgggggiC3TAACGCCGCECAAACTA CAAACGTCGT3CACGTCCCACIA'''GGCIGT3 Citigats; Aggrg AAC'GACATCACGGACCiC3: CACITICCACC'GTCGAACAAG CARGCAACAAGA533CCGACGAAGCCGAAiGTACAACCEACA3GAGGCA; AGAIA ggigi ACGAACCACAARCTACECACAGGAACCACCCTGAACGGCCGSCGEAG GEACAGGGGCCAAEGEECCCT3AACGTAATGSCGCCCAACCGGGCAEGGAA TAGEEGA.C.G.A.GIGIGAATGACAACACCGCCGAAAAAGAAACAGCCC 3A.CGCAGCC 3C36AECCAGITCGACACCGCACCACGTCCCGTCACGT3: £3EACA CATAATGACCTACGT3AATGAAAAt TCGCTAGICGAAAAAGGAE3AA. C.C.G.A.ACACAAA;&CAAGAAGCEGACCGAAAACGGTAGACGCAGA CCACCGSA, CCAACTGGEGAATCGCCGICAGAAATCATACAA.CSCGCCGAGAAAIGAGEGE CAGAAAAACGCAGACCAC,CGCGA GAGASTCFAA "CAG." GAGCGCACAF;GCGA:ACGGGCCA2: GitaGGGAGAA SA:C:{SGETA CECGICGAGCCGGGCGGAAAAGAgACGGAAAAICEik AgCCASACECCCCGGAAAAACTGACEACA CAGASGTRACEiki IGIAEAG3CCOSAGGCCGTGCGCACAGC3GTCCATACCAGAAGECAGCGC (ACTIGGAEGEAGGGSCGCGCCGAGGGGCAAGGAAACGGACCAAEAA iCGAAACATCGACAAGCSEGSAACTGAACAEGAAGCCGITAACGT3A.G. (GAAAAAAAGGA, 38AA CCCASGSGA&CASASEGAGAAAA ICACAFCACCi AGAAAGAAACAGGCGGCAGGGAAGGGCEGGCGRGGEGACG AEGGGGGCSACCACCGACGSAGEGTCCCACCCCCGSCCAAGGGSCAAGC3, ACCASCGGGAGCCAAAAGATA3 ACGCEA.... EEGSACGEICAGCCACAATSGACCACAATAACACGTGT CAGEAC, CCGACAAGGGICTCTATAGICC" '''CECCiCA (CGAIGGCC: ACGCSE''{{CACAGACTEGECACCCCCAGGAATEACCAAATCGIGG AAAAAGEA ACGCCCGCACEACGGIE AACTGICCATICOCGCACA CICATCAITRGETICAGCCAATACAATTCTCCGACCAIAggATIGERTA, CAACE, A.C.G.A.A.A.F.S.AGASAOCGCSESAC Tasiaat (:CCACGAACCTEACCCAAGACCTCEAAACCC2C3AECCC.S.A.: CSACA CAA GCACCCG:CCCCA'ggiG: GAACGGCGCGGECACTCECA CRYCA&TOGI'gg C(AgCAAGCAC3ACTCTCCACAATGA:AGT3 CACA g'(EATCA:CCIGAAAICCCSICCATEGICG'K3CAGA (ACGA 3GAA CECACTCSSACGGGAAAAGGCAGOSCGCCAAGACCACAGGGGCG: AEAAGGCACACAGGGACCGECCGCACGGAEGAiGCECAAG TACA. GGGEAAASCACGAGAAAIGCT"'GCCASACEAACTTACTCECAT 'c''g'''CATEGAAAAAAAAAACCAGICGC2'C'g''g''CCAE; SAAGEA.G.G.E.''GTCAGCAGGA, GCATAACIGICAGAAACCiCACCCCC g;3TCGCCAGA CAEGCAAACAAACTGITAACS-CACAGCCACCTGACA AAAECOSAICG U.S. Patent Dec. 13, 2016 Sheet 6 of 91 US 9,518,097 B2

Fig. 5A assicis SEQ isis No.ss 619is s &: i , R. ga ict it to t3a 3.2 aca at ti &c. &c. &&t 3gs as: ct 3 gif . & E. y N & get tigg cti ta gat tic coa tage gte get Sac gate to aac git catc.

R. G. E. SS i. { s ctic &ct (St. gigg gag age: ct; tigg & a atti.etcateca tag teaag geaagttgttga caatgtcaggettgcc.gac.cgttgagectg categgaaacgacticacgttctittcticatct titctgattatcatttstaaacatataaaacce: 3K:gta&&tgat (Cgttgttgii.3tsgaatgcaatata.ittga&&3.838 a.a38,838 && 8888 & &: Carats indicate he positions of three iatrons. SE i & 3 g3 C getcat (istag itCi tasta atga { ai:3tigc at (a tistgegtC gas atgg. C &&.g3 82 gogat. &gic C C3tiac (tt.{g act; 535335i3 &f gt is tagSESSESSEESEggEEE333,35ESSESS as a tig taa K &tigaa&catastg??ategic gttgfig. tittstettigatetist 8tggaatgcaatatastigtgaaaasaa&aaaaaaaaaaaaa. titääacafestia?&c.ciis. 3& SE: NC : 83. coding sexiece of proprotein, & tig cit gig 8 ti aa gcc ace: g: ct ce: g: tsg, ett gas gat, tige: SE E3 gic gg gag at $3t a&C egt (its etc. &ist stgg & gag age

U.S. Patent Dec. 13, 2016 Sheet 9 Of 91 US 9,518,097 B2

U.S. Patent Dec. 13, 2016 Sheet 10 of 91 US 9,518,097 B2

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Fig. 7 SEQ E. NC ; 3.83 ECI vic?. WOAIX AS S ength = i. 1.5 TGGGigiCAGCGGATACCCA3 CECTCGAACTCGGAAAACCTTACC CASAAAACACIAE, AFCCCCGC C?ggi AAA

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SEC E NO: 2.88 SQPHETYSILYESIAB.SINEAR.p.g.a.Yge, gig

U.S. Patent Dec. 13, 2016 Sheet 16 of 91 US 9,518,097 B2

Fig. 7 (Coit.) SE NC: ; 2 (3 RY KLIGCEGWESEK. SNTVR.L. CosgravKYASISTFS: YAVNITAAAARITKVYFTWT. ''WHIHKERS G. HES-YHNERLEQDNMIDAEVPLNK- KKNHRRISKE, FASWVGGE, RFASAQISNRP

IGCKAGGGAFKQFESKIQSFRNLI,8. PFTSEAEPEPRESE.www.ewsworwewssex www.myrewerseyewee woHARDWSNELRATYY-FFINARY IDPLI. 'I HEGVAVSFQLQREEGSCTDLEVVVDTVVD iR.G. SiFiCEYEF: CGEA.G. ...I.E.Y. SEC E REO ; 2) 4 ceil. SC 38 it CC (AAEC, SC; ACGG ACCACTGACGGGAGAICICGIG-CATCCASG GAAACACAATCCAATCACGAGGAGCATGGGA, CGATEACCGI ACGECGGCTCACGCTGGCAGGAACGAAC CTTACCAAA CIGIAEA, ECG TAGGAA COAGA ACACCSCTCAGGIGSACGGAAGA CAiiAACC; ACGECACA (AEGACIA.C.C.C.C.C.O.G.C.A. TECAiiA CASEAGAAGAAAAGAAGEAEGi. EAAAACAIGCSEGASCAiiAAAGC3ACCGACAGCCAGC:" CAGCCEGGAECCAA. CA3CAGAGCCA CACEACA3 (AITECAAAGGSATAC (3:3CAGAAGAAACACAGECASECT; ACT CACTCCC:{CAG CGAGA, EAECE&G.A.G.CGCA ACACAGGGGGGAGASGCAEGAAGACA, CAAGAAA.S.C.O.G.G.C.E. G.A.A.G.A.G.A.''{{G.A.A. AIGAAEAgg-Gi AGA, ACGC:CG-CCAAGCACACGTCCCAGCCAGO CCCAGGCCCATTCCTAgTEGAGGCECGATCTATTG-CAAAAI'E'IGACAGCCEGCGTGGTA GGAAGASEAGACCACA32, AGACAEGEEEGECCAGCCAA EASCCA.A.A.G.A.GCGEACEAACGGAEGEEACT

SE E N C : 3 (5 K-ASLWRYTNTRSISGFDIWR3TCRISIEWSSSSIRR, SEALRASERNGPG.W.L.G

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Fig. 7 (Cont.) 3E D C : 27 L.E.G.A.R.S.E.S.PNSSEYNEYSE.S.E.A.R. 33s.C..Y S.E. E. N. ; 2 8 Cortig3 873 engths: 234 {CICACAGAACTACAGAACTTGACC CEO:AGFCTGACAICAAGC CAirgi'gccACT gaig CTCCAAECCCGICGGCGACACACGAG3 CCCSAGCCGECCAGGGGAGCCTAACAIGCAACGCGAAAAEGA, (CGGCGACA, CCTCCAAATACCCGGICAICAGCCGC (CTATSGCAG

3E I) NO : 2 OS SERYSSSE3 it is A. SYRIS3 - GSARSYN

SEC E N C : 2, g: . . . . CAA CGTGGGE EGGAAAAE3TGAAAACCEAAGAAAAAAATCAC ACT's CCCCCACAGACAAAAAACCACACTCCGAEG£3GCCAACGGCCAAGCC CAACGGAACAACTTGAAAAIAICAC TSCAAAACAACCACCEAACEG ECAiiA CAGSA GASSCEAGGCC&GCAC GAAC TEACCACCGA, ATA:::::ACACAGGT.A.GAL.GAAECCACCEGCAC {{TCAEgg Ac:GGTTCACG'C3'CECCASG CACGGAATCSG-3GACAARCCA GGCACAA.iii. GKCACA ACASACA Gigg IAAGGAGGCC Gigi CiA CAC CSS AGGAACGGGTG.A.GCCGCCGAC CTTACGCTCAGCCAAGGCCAAGCCAGGGGCALAGGGSAACEAA.A.G. CGCIECACTCGTCCC3TACAGCCAgQAAGCSGACCAGAGCC CCTAAGAASCCAAATCG EAEAEGGCCEAACGGAGA CIGTGICA&ACCSCGCGCGACFCCAACGCCEic. TCGGGTACAFG AESA (AGECCTA.A.A.A.A.A.GGCCGGACACACE.G.A.G.C.A.' GSGIGCTAACGCTA (GCGAGCCGGFG3CIAGAAAAAGGASCGGGCAGT GCiti'GAA.A.G.CGGGSCGECGIACSA, CE. ECCCCGAC SGTCCCGAAAAGAAECCCCCTCGAC (38CCACCCCCCACACCCA GCECACCACCGA-CA CASCAFCCCCCCGAGGAGCCACC GG2 CCGCACAAGACCGGEEGEEC.S.A, CGGCA ACAEACCC GCCCEGCAGCACCCGIGA CGACASECGGCE AGACEG. TCCAA.C.A.G.GTCCC, GCAAA CGC3, CGACACCCAAAATC CCCC (AGGCACECA-ACEAAGEACEACCECA, Gigi'i::gCACACCAAATCGCACCCCACAG CAAATTACA CECASECCAGECCA CASA CGG, CA. GGGCE.S.E.C.: CAC TGCCACAT. AACECGICTTCACGECCCGCAAGGCGCCTATCGG AEGEEAS GAAGCCAAAC&CCiCEACAAG AC&A.A. CTTC:GACACCACCACCAA CGAAGAAAAGECTCCAAGTCCCA {{CAAigCCAAACA's CgFC CIA's CGCCS's (GT CO; CACCiCGT C: CGCACAGACAGGACASACECCGGCGACCCCCCCACCiCGAGCCASGAEC TACAEGCCCCCCCCICCACAFTAATTAGGAC(ACGAC;

U.S. Patent Dec. 13, 2016 Sheet 19 Of 91 US 9,518,097 B2

Fig. 7 (Cat.) SE O NO; 228 {{GCACACAA3CCAC.S.C.C.C.CCACCCCAAGF. CACC, CCGGA, CGGCAAGACCiCAA. G.C.A.A.G.E. SE II NC: S FSSATRLSi F.E.L.E. CAGOWNEL.EKGEG

SEC I & 22. ACCACACAF,{{CCACCCCCCCXCAC; i.AEAGASCAFCC I'GCCGGTGACGE&GACAATTTGACCCTCACTAAAGGCGAAGGGTGA SEQ ID No: 18 iSOINATRiP FC.;3°CAGIVEN'T TKGEG.

SEQ II NO: 224 Beta-anaanit in from Arianita hailoides this sequence was found by PCR with diegnerate pairers AGCAGAAAACACCGCCCCATAGGGAAAGGCACCC {i. CGGAGACCACAACCCCCCIGGC3AG SE ED NO ; 223 SNAREGGCC CORGE: SEQ II NO: 226 Phalloidin from Amanita ocreata Ethis sequence was also four-d by SCR with siegenerate riters R.G.C.A.ACAAACCACCCCCCCXCIGGEECGCAC3 C3G CCCC G2GACCACCCTC.C.C.C.C.C.CGA SEC NO : 22: ASIDENAER.I.PAWACFCAGOVNP, I'RGE U.S. Patent Dec. 13, 2016 Sheet 20 of 91 US 9,518,097 B2

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( 33 A 13,254 bp lambda ciore red/underlined sequences (portions) are two copies of PHA encoding phaitacidia 5'-3' orientation

SEQ (NO:327 gaggggg. SAA: CGAEAAGTTCACATICAGCACACGCAGCCGAAGGGC33 (SAGE CCAGAGGAGCCAGCASAACCAGAAATAAGAA CGAEAAC:AiGAGGAAA33GAG ACGGCACAGGGGGGAGCAGAAATCCAAAAGAAAAAA; GAAGAAA Gigi'''''A.CC GAAAAAAAGGGECGAA CGGAACCACAGIGCAACCRCGSAACCAGCA CACCAAFC3G Aggi;AccGATc. ACCACAAGT CAAAS3GET''CCCTTACCAAEAATAACAATGGT figCACAATCCGIATA:CCTCGECACTCAGGCTATATCCAATTCCTCAAGC Ciga:CGCACA; CAGGACTEACAAGTCACGGAA''TATGETGAGC ACCAAACCCACCCaCE:GCCEEAAECGCCAAAAITGSTSATAAA CAEGCGEACAGACAA{EACAAAAACASCCACCTACCACCCAITGEAACCACAAC A.C.AGAAACAATGGEICACAAT3CCGAGAAAGACAAA'GOAT'GiGA is: CATCCAAECTGAAg3AAACAATAAG ACTTC:GA:AEGTGAf (ACCGCAGCCACCACATTGACAAGEACTTACA, CCAAACCATCGEACT ge:CGCCACACE:GCAAge:CCGSECCASGTG (TICACCACCAEACEAAGAGGCGAs ATCACACGTCACGTCCCEEGCGAAGGGACAFCATAAAAAGC&GGGCGAC CCCCCAACSTCAAGAATAACGEA ACAGCGGAAGATE"GGGS. TACA CATICCAAAgg'GGTCCAAGT3 (ICAAECACAFCCAAATCTCCGG CCGGCCGCATCGCGAA''CACA3AEGEECEAAAAAAAAAAiCGGGTAC ACT CACCA'GACCAG8 AGFACTCCAGCCAGCCTGGCACAAGE CGAAACAAggGACIGCGCTAGACA GGAGCCEGGAAGGGATGGACA (ACAGCCAAC2 gig. GCGS,,G,3C:{CAATAAC A&TGTEACCGAIGCCAECC: A. gGAAAGGCSEccGTTACGAGCTGA:"FCCCTGGATCGIGAAAGTGACTTAGCCGTGTGGCT AAFTA CAA.ACCACGTCCCAA CCGAiGACACCCACCATGGT ICACAF CAAATCCAAACCGAA.C.G.A.G.E.C.C.S.C.G.T.'GCECCACCG TACECAFARCGAAAACAGACCCCGSAACGiGi CG-CCCECGA (ACCAAGGEATCCGIACGA CAAAA'). CAAICCCGTOGAGACCCATCGCCG GT, CGC:CCAAAGSCCCCGAGCCAiiACCSCCGTCCCACCGAGCCAAGCiAAG CGigi'3:3GCTGSAEGAC.C.; AIACCGATCATAGA (333A GAAGGGACIGAAAICCGATGAACECGAGCTA TGIAAGGACA CCAAATGA-CACAFCCCGTGGGAECC GCS-CAAAAACC (AAECCAGCTAACGGAGAGAGCCACGTCGATACCGGCCAFEGACGACC ACCAETCECCASGAA GTAETAGGGAAECEGCGATCCSTC AGGCACAGGGGGCCSAGACGACAiiACTSGCAA CCGAC&CCGACG AGAGGAEACCACCESATA GGAGGACGSTCGC&ATC is: CACCSCTAAGAAAAAGGGGCA; AGECACACCCACCCAEA GACAAGCCAAGEGGAAAGCEAACAGAAAATC:{{IGGGGC CGASTCA CAGITATAGACCAGAASCAGGAAIGC's ACCEGA; 3G CGIAA; CC: TCCCAGGCGTGGAGCCA gCCAGEIGGSIGGCCAC{33G AcGigg AIFIGCTA 'CGCCEAGTGACAAGTi"TCTCAGCATIC32AGASATAGICTGACAGGA AC3ACAAGAATGAAACAC CTTCACAE.8GCCGGCCCCAATTGACIGCTGig

U.S. Patent Dec. 13, 2016 Sheet 61 of 91 US 9,518,097 B2

, FSERESH 2.5 Prezii citi or of potential geries in Castria as genotic tis, Tige isi it. . .2 : : f Sex && t&st sequence i. e.gth of 33 guage ; 3.25 4. is cit gredict ic gene S 6 if f : 3 in 2 i. - cali, ii 4. ifex, if pedit.eti et S is is + chai; 28 it -chair .3 Fositics of gredicted genes as Yd exons: Was art fre: Sacre: 29, i2937)

8. Sir S&E is Start Sic Scise R 8:

CSE 3 - 3 3 33 m. s CS, 835 - es 2 33 88 w 3 - 3 C3, 8 - 38 3 3S 8 . 883 3S - CSS, 3 s. 28 S. 8: 35 - 23 8. ; : Si, 2 -. 23 28 3.35 - 32. it & Si. : 3 ...... 4 a. 3 3.

Si is a s 33 is - 858 8 CSi. 3 c. St. 2 a. S5 3 i Si. 3 - 388 . . . SS 3. CSS, 383 . 5 . F3 & S. E. s. & - Si 28 -e 383 25 - 8. i 3 CS, - { 2 or 269 . 3. S;

. . . 8. - 2. 2 - S. 28 O - 3.3 ..., S - 35. in CSi 38 A . 33 ... 33 3. - 3 CE. 338 - 33; 3 : 338 - 3i 3. 2 A CSi 3 . S. 5 32S - 33 83 r. S CS5. is - a 2, #2 it i; ; ; 3 2 r. is S. ; : - 33. , , & 1. : 33 38 2 : SSS 2 4s. E. : : . {2 & 8 f a S. S.S. 3 is . & i és 383 28 .. 3 CSi. £8, -. f:3. is 38 : - g3 - CSE 5 : - s 2: 36 S - & 2 - SS: 3. -2, 33

33 ... 3 3 r. S. 3 Si2 - 88 , , Si2 - 88 3 - 2 iS. S23 - 8.5 - ". . . 3 or SS 33 3 - 3 Si. 3 - 6339 8, 2 SS - 33 33 r CSS 3,3 - SS 22 3,3 .. r 5 : S. SS - is 558 - st - is Sf 53 - 8. r., E 8 - is 2. . SS 39 - , , 3.

& SS - S. 3 .. 8. ... : a 8: is 2. CSi 2: ... 7448 G. 9{ t; i. 3 CS -- : .. 8 is . . 2 3. i. CSi 3S - 332 : - S3 is U.S. Patent Dec. 13, 2016 Sheet 62 of 91 US 9,518,097 B2

Fi. 24 exist: i -- E. E.; i. is - 3 & 8 2s 32 8 : : -. 83 E3 3 : - S CBS; 8764 - 88: 2 8 : & 4 & 333 f 3 - i C. 83 & 3. SS 3 & 3 -. 33 8: & E.S. 3 (8 - 33 (3. SS 3 : - : 33 3 ; :- 3 CE;3. S: 3: - SS 8 . . . . S: 332 r. 58 ; : Cixis. SS - 3 33 SS - : ; s: - 3&S. E 33 *::: 3. 8s 3. 28, 3 : 3: Sas is Si. . S3 & 4 .. 5 3, . SS S{ i : 3.3 CE. S. S. 38 33 . . . . - 3.38 OS g 3 is is is ... 3. . .

r a. 3. & - C3. i. 823 - & 33 382:... -- SS s: : -. 3 CES ... 83 - 36 8 . . . 3.63: ... 38 SS 3 or S3 ... 8 ~. . . . .

8. -- Eks ... ; - 8 - E3 : 8 - 33.3 3 $3 82 - 3. 28 a r. S3 .3384 re. 3.

U.S. Patent Dec. 13, 2016 Sheet 64 of 91 US 9,518,097 B2

Fig. 25A (Cort, Fredicted protein(s): P450- (OF45) SEQ D NO. 597 FENES: , 3 sess {s} 33.5 - 26 (S. 523 as a chain - MVETSYSALVTERIEFFEXISAAAITYGLYR FRVIVE ISFIRELEGEANANIFL3 N: LK. WTOLSELYWTOQAES; LESR;YWYTKES TRRIGKLWGGLEPFVEGEDEKKRKI. WE, YFC.WWEEOCER NON AFGPLOSNAECS KGGECREVGCKSWDESSWFEO ARN KSFRY.E.I.S.S.RESSRWA: i:SQL:NLEEWRRFPLFPLRWETOL: ISKC3 RTSRiiNESEGSWRNN.ONSGS&LLSWREN SEVEIRRESIDEWEAOWSJAGR, SPEKEAWAFSIAKN REAKERRELLE, COTCINE SYSEDIVIRESSRWCA EP:AKERGN:3SS. KRGCWYESA. KKSINGEA. FRER.ECLEGVST EPGVWSEESFWGGPRSCIGF:RFAEAEviXA.I.FTLVRAI.EFAWAEOISVESSJR. IIT EIGRY FEEEYKARS U.S. Patent Dec. 13, 2016 Sheet 65 Of 91 US 9,518,097 B2

U.S. Patent Dec. 13, 2016 Sheet 66 of 91 US 9,518,097 B2

Fig. 25B (Coat.) * putative homolog show to have Hydroxylase activity: SEQ 13 NC: 598 OC3 AAT6823; 51, 4 aa in 233. & 2 - OCT-2 is DEFINITION cytochrotte P450 CYP739C. Tritican aestivat . ACCESSOR: AAé 323i WER3.ON A&6823 f {G : 4333 O 8 E33 SORCE accession AY6434 S. ORGANSA Triticum aestivum A HORS Kandel, S., Morant, M., Betiveliste, E., Blee, E. , Werck - Keichhart, D. and Pinot, F. E Cli Qing, Functional Expression, and Chaitacterization of CYP (SC), the First Sub tennial. Fiyodroxylase of Crg Chair. Fatty Acid in East ExCEOK 3Y CHEECAS AS MEEE YE ASME CUK.A. J. Bioi. Chera. 28) (43), 3588 - 35889 (205 3D ié120513 RESERENCE 2 (kesides J to 514) A HORS iviorarit, M . , Werck wreichhart, D, and Palett K. TE Birect. Subtilissio: iFEATURES Erocation/Qualifiers St. is . 53.4 A organigrass "Triticiata aestivuit." A cultivar : " Darilas' Anotese "Cytocicorne P450. Cytochrotte $450s are haetan thiciate proteins involved in the oxidative degradiation of various compounds. They are particularly well known for their role in the degr&ciation of environmental toxins and Eutagens; pfarra O0087 givwiavaaa wasawi as waf davylvwrp raitroglraq gvggggy:ff aginiaeiki & radis &gataid igdhdivory ghirk wipi hg, t, flywig akpticiady nwrvkgyisdir 2 ggypksign philariigkg witdgsidwk. thrkrrvipai nindki.kmintv titascicagsta 1.8 sewkakadkg gsveidis Sg feet advis htafgs systiq gkkwflagre lic fiafs twf 24 nwgilpsity, ptekrikiwi. J. dikewrtain ni ikgriat, ?ittagygnoll gift, eacape 33. digqnpils ind eii deck.tiff faghdits shi twtrff list pewtekire eviecgagi 363 ptgdrinkisi i v infllet x lyapy saic irkags devg gikytegtfi tipi, ati, hard 42 keywgeciank fkgrfergv tragkhpal isfssgprs iggrfari&& kavia'iligr 481 fsfsspiky v iiaprad vitr pkiglpai.R. sileir. U.S. Patent Dec. 13, 2016 Sheet 67 of 91 US 9,518,097 B2

Fig. 258 (Coat. : J Rio Cinsas. 2005 Oct 28, 280 (4.3 : 3538 - S. Egli 2035 Al3.g. 23. Clcaing, fu actioral expression, and characterization of CYF7030, the first Suk terminal hydroxylase of long chain fatty acici if plants. Induction by cherica is and methyl asimogate. Kandel S, Niorant. Y., Benvertiste I, Bié& E, Werck-Feichhart is, ict. . Départ emaint Régions e Métabolique à l'Environnement Biotique, IBMP-CNRS, UPR 2357, 28 Rue Goeths, F - 67083 Strasbourg Ceciex, France.

We cored and characterized CYP709C, a new plant cytochrome P450 belonging to the P450 family, that So far has no icientified farction except for listering with a fatty acid metabolizing cli&de of 45 enzyees. We showed here that CYF70383, is capable of hydroxylating fatty aci is at the oriega. -i arid onega-2 positions. is WKirk was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat ci. NA library in an engineered yeasts train. Investigation of substrate specificity indicates that CYP7 OSC3 metabolizes different fatty acids varying in their chain length (C.12 to C. 8) and arisatiration, CYP739C is the first i deitified part cytocircle :53 that car catalyze st-terminal. hydroxylation of C38 fatty acids, cis 9, 10 - Epoxy Stearic: acid is Yetabolized with the tigest efficiency, i.e. K{ {a} (app} } of 8 microV and W (max (app) } of 328 rama frain/raci. P450. This, together with the fact that wheat possesses a taicosocial peroxygenase attie to synthesize this compound firca oileic acid, strongly suggests that it is a girysiological shstrate. Hydroxylated fatty acids are implicated in paint defense events. We posta lated that YF709C1 could be involved in part defense by producing Such cottpour is . This receives support from the observation that {i} st-terraina. hygiroxylation of S, i.G. epoxy 8 tearic acici is iduced (1.5- fold after 3 h} ii. nicosories of wheat. Seedling 3 treated with the stress hormore methyl, a solate and iii. CY (93. is enhanced at the transcripticial level by this treatment. CYP7 (9:1 transcript also a scutthulated after U.S. Patent Dec. 13, 2016 Sheet 68 of 91 US 9,518,097 B2

Fig. 258 Coat.) treatient. With a continatio of the safe'ie aphthalic acid any dride &nd pherciabital. ii. 3 iiidiigates & possible detoxifying faction for CYP7 (9C that we discussed.

FG, 2.5i BEASTE of 3:45 - against Cop.ciis at Broad: tie to IC is contessipated to be sig; ificant . CC13 (92.6 g : iystéetical protei. & ). CC1G (3738: hypothetical grotein. 3. As - S CC3G (6332 : hypothetical protein E33 or 48 CC. (3068.4 : hypothetical protein. i8 48 x is CC16 02050: hypothetical protein 2er is CCG (0.121: hypothetical proteir ge - 39 CC3 (53.06 : hypothetical proteit. ST 3 - 39 CC18.12478 : hypothetical protein f 8ss w. 3 CC1G 04748: hypothetical proteir s Ser8 CCG 001.22 : hyottetical proteia 4. ge. 33 CCG 06334: predicted protein. 4 CC1305310 : hygiothetical groteir 3 is CC1G 1297 : predicted protein SS CCG (281.3 : predicted protein. U.S. Patent Dec. 13, 2016 Sheet 69 of 91 US 9,518,097 B2

FG 33 BEAS''2 of '45 against ...accaria. 3care Sequences proficing significant alignment.8 : its silius: N

* igil Lacbii. 31.5739 leu.2...bSSf999839.4230 25 e-23 to igil Lacbi.1315758 eu2. Ibsc f(0.08334,429 88 se 2.5 & 2.5 e - 39 8 igi.i.ackii. 324893 fgenesia3 sCl scaffold. 8033333. 3.55 ise - 93 igillagbill.i.232.344 le. Swall: 6.13.8.1. E. E. in 33 f SS se- 32 8 igiliacbilii.57726 igwill...s.lib.i. 25 e-S2 8 iii.acbiii 1782.07 estExt. Genewiss 1.W.2. E.C. 601.17 25 g -88 s

2 5 an 88 s igitacbi.1838.04 lest & 2. e - 88 s 3.

igi. Lacbill?2792 estExt genewisel. Atarian.C.surr-, 601.18 3. S & or 38 5 igidiagbill.315756 522. LibScf.000SS84400 2 . () & 8 - 8S 3.

igi. Lacbi. 324.807 fgeissa:3.29. C. Scaffold.6093 2 2. i 4. * Best accaria proteir hit :

SEQ (E) NO. 599 >igi Lacbi. 31.5739 ev.2, List: f(30&g (4230 GRC.S.A.A.G.W.YKAES3RY, CSKS3 F.3, E. EEEC'GSGIEWEKGRYKSGSSEY ARANHNSYEEKESR AATNI.GAGILVVEGIKKKQQRKIMNRAFGFAQIREITDAFVRKSQLi2OIWAE ECFKQCSGQ3RIEIES ALTWTT, DVIGAGFNYKFNALMRSKANE SEAFA' FC. AGSWNW-ES 3AEF3 PASE REAGIWEAKKASSTMSRIGKELESNSKAAVSC ESERINKSÍ). SEEWRANWA: TESRMLEVISCIEEWAGEEETSN2, ''TWAFAESQNDAKEN.E.I.' 'W3TNTMENA.PY AWWRETIKLIA WSSRWAMKai-ji. FSKGWEEERKCE, it is RKSGE AH8EREWESEAAS SPGWFGVLTFGSCIGY REALVEMKAFT, RSFEFELAVPASDIGKKAGIVERP22.I.SNPEGGSQM: FvKAYQRPEEA* U.S. Patent Dec. 13, 2016 Sheet 70 of 91 US 9,518,097 B2

U.S. Patent Dec. 13, 2016 Sheet 73 of 91 US 9,518,097 B2

- - - -, -, -, r. - its frk r- in- - - wn a 3 d 8 - is e i ga o -

U.S. Patent US 9,518,097 B2 U.S. Patent Dec. 13, 2016 Sheet 75 Of 91 US 9,518,097 B2

FE8, 26 335 st; against. 2...acfaria. E.gxi, its 3.4: rs i.atively weak capacec; to S45, ri Segaccess praigiri 53 g ifi : it 3 g {Ai} t S : is Scare 8. Wate 3 isi as: S. 62-28 33 is: 9 e-2f

s :-2. issa cais a 3 396 g (.333 3, 2; 25. 3&aii.5235,33883. 9. : ... 3. 8-24 age 1.34

U.S. Patent Dec. 13, 2016 Sheet 78 of 91 US 9,518,097 B2

N. : : r 3.

|1 U.S. Patent Dec. 13, 2016 Sheet 79 of 91 US 9,518,097 B2

Fig. 28C

BLAST: against Coprii as Scot & E Sequences producing significant. (bits Wallie alligratients: CC.G. (2050: hypothetical protein. 27 few 3 { CC1G (63.32 : hypothetical protein 262 m2 CC1G 992.6 (3 : hypothetical protein. 33 6 - 2 ) CCG 12478 : hypothetical protein 7. See - 5 CCG 04749; hypothetical protein ;3 2e-3 CCG 05306: hypothetical protein 62 58 - CCG 98334 predicted protein 63 86 - , CC1& 00:22; hypothetical protei: 6. 8 cer. CC13 (91.2 : hypothetical protein 6 : 28 - OS CCG (283.3 : predicted protein 3 is . S.

FG, 28 BLASTP agiast Laccaria: Score E. Sequences producing significant alignments : (bits Value N

igifilacsil. 3:34-3 leu.2. Libs.cf.00&ig9Q300 f3 is . ." 2 jgill.3 chil 2554iii.e. geWi. 61.27.3. f3 46 - 2 . isi. Eacbi. 240704 leg whi.6.30. T3 : & a 2 it 48. f3 4 e- 2 . igi. Eacbill.339:14 iglhi. 61.38.i. 73 4 & 2 . igiliacbil 334243 fgenesh.3.g3. C. Scaffold 5000021. f 2e . . igi Läckii. 334242.Égenesb3 gg, C. Scaffold Sio9992.0 & 8 26 - ... 3 igittagbii. 34.522 estExt. Eger&sh;3. Rg. c. 620912 3 22- ... 3 igiliacbill 39974 Oleta. Libs.cf.09%lg0500 42 7& 3 igittacbili 31819, 232. Libs.cf.09883 02839 4 & - 9 2 U.S. Patent Dec. 13, 2016 Sheet 80 Of 91 US 9,518,097 B2

FE8. A $3 E II) N. : 606 > FGENESH: iRNA is 2 exon (; 1638 - 1786 3.5 oil, cair. - Accra CACAAggs, CCG'CCCCCTEGCGEAGAGCCC2, GCEC3G AGAGCA ACCGICTCCACCGTCCC; i3AGCCTTGEAA SEQ hio; FGENESH: 5 2 exor {s} i. 626 - i. 786 (60 nt) 34 aa, chai... r MSDINATRLPAWI.VDC2CVGDDVNRLLTRGESLC No identity hits in any of the genotes of the rotoxic mushroots. This is a sequence encoding PHA. 2. SE) is N.; SOS SE: I NC: Gene 5 re: : ; OUS ETASCAAAGGCTCTCGCCACAGT3AG3AGACG3'3ACAICGTCA, CGACGCA33GCAAIC TACAAGCCAAGCC AAACGGGTGGCCSAGCEC,

3 SE: I NC : 699 > FGENESH; mRNA. S. i. exor {s} i. 2372 - 1,3 .28 258 p, chain - AGCCAAAACAAAACCGCCAA.C.G.C.EAAAECGGCEGCGAGCCGEGG, EAGGGGGECCCCCAAAACTTGAGCCGGGACGCECA.A.A.C.A.A.G CGECGACCAGGGGTACCAAGGCECA.A.C.E.C.C.C.C.C.C.G.A.A. TACACCGTECGAGTCGGAAGAAAC33SGACAATAAGCECIC CCGIGCSEG stost No. xF3ENESH: 6 exon (s) 12872 - .312935 aa, chain - 3. MWCSKSETW, KAWWEASRGYWWWSSEYMCWSLGIAQYK-MAVAIASGY...WR HSFPIP3 YHRE GREYESEAEASE

Ro icientity its in any of the strict gencies. U.S. Patent Dec. 13, 2016 Sheet 81 of 91 US 9,518,097 B2

'E A.G&ESS O& Pg 50 GENES 2 ARE; GBAA;3969.1 cy:GCEROME P450, PUTATIVE (CRYPTOCOCCUS REOF, .. 232 28-59 E.B.E.238.31 FYPOTHETICAL PROPEEN (NBF3489 CRY20COCCUS, .. 229 2e-58 REFKE, SO338, HYPOTHETICRE, FROTEIN US0489. STIL&G0 3:... 79 3e-43 REXES349,x 582. Hyoristica.HYPOTE: CA, RoeiRGE EN 133262:Mi380 itsSTEREO ago. 3...3, , , 5; 3e38ie-35 REENES 13)2), PTATIVE CY''{CHROME P450 OR2A S.A. Ya JA, , , 49 3e-34 g8A2882.92.SBAR33. CEOCHROMECYOCHRO8E 2.592450 CYP789CERTICPAESSE) (TRITICUP ARSTYYi} 45is ie-333e-33 REFRB 389.24, P:FEAFIVE CYPOCHROME P450 ORY2A SAINA JA... 8e-33 RS:X2477.684, PUTATIVE CY2008ROME P450 ORY2A SATIVA JA, , , 43 2e-32 HYPOTHETICAL PROTEIN MG849. MAGNAPORPH3... 42 28-32 aYPOTHETICAL PROTEIN EG 363. GIS3ERELIA 2, , , 3S 38-39 PUTAEYE CYTOCHROSE P458 (ORY2A SATYA JA, , , 32 38-29

g3A3843.969, CYPOCERORE P450, PYESTIVE CRYP2000CCGS NEOF, , . .38 Se-3: g3 EA,289. HYEORECA. PROTEIN CRSF3408 CRYPE(COCCS , , , 13. 98-3 REXP. 25.827. HYPOTHETICAL PROTEIN 38.980. USILAGO 2,..., 8 68-25 REFX269,336. HYPOTHETICA, PROFSIR IMG 4189. (SEELAG) 8,... 106 28-2 G8 EAA728, HYPOTHSTICA, FROTEIN G6967, GIESERELA 2, .. 8 Be-19 SEEA82 iii. CYPOCHROME P450 MONOOXYGENASE, PULATIVE ASP,..., 98 le-8 33 EAAS3: 84.1 BYPOTHETICAL FROTEN F301:48, G88EREEik 2,..., 8: e-it . SEEAS G. CY20CEROSE P450, PUTATIVE ASPERGILESS FMJG, , , 93 3e-2 REFXP.3263. ENSANGP900000282) A30PHELES GAMBIAE SER,..., 93 38-37 gai AK3295. CYOCSR03E P450 ANOPELES GAMBIAE 92 e REFENS1824, PURATIV8 CYYOCHROME 430 ORY2A SAVA. J.A., '92 Ge DB RAB8935, CYPOCSROME 2459-LIKE PROTEIN ARAB130PSIS ... 92 Se G8 EAA522.39. HYPOTHETICAL PROTEIN MG04911, MASNAPOREBE , , , 92 Se-27 ES: HYPOTHETICAL PROTEIN EG9388, GE888REELA 2, 9 lei6

CONSERVED BYPOTHETICAL PROTEIN CRYPTOCOCCUS, .. 8s 3e-5 HYPOTHE ICAL PROTEIN C-880620 CRYPTOCOCC-IS,..., 79 se 13 CYTOCHRO8E P450, PUTATIVE CRYPOCOCCQS REOF, , . 12 5e-1} HYPOTHETICAL PROPEN CNBF3400 CRYPTOCGCCUS, , , , 58-1} FUTATIVE 8732-LIKE (Y20CEROME P450 GINK:0 B., , ) 28-1} HYPOTHETICAE PROTEIN MAE (338c: ;YCOBACER..., 53. 48-18 REsi Ye. 3899, CYFOCHROME P450 MON308 GENASE (NOCARDIA FA... 5S 68-0 Girassia.FX873822 i :Purari YPOTHERICAL VE CYPOCRRO8E PROTEIN P45080386. MycoBAceRION USILAGQ VAN3., 8, , , 5538 68-69e-99 REENE 331846, CYTCCHR088 P450, FAMILY 3, SSBFAMILY A, PO, , , 65 8e-09 SSE2635. GA i39-Pi B808088 IEA FSESD(OSSCURA 33 85-39 23. BAB33854, it cYEOCHROME P450 MUSCA D08ESTICA) 63 8e-03 REFX2 3205). ENSANGP03090816957 ANOPHELES GAMBIAE SER, , , 6 ie-88 FG. 3OA U.S. Patent Dec. 13, 2016 Sheet 82 of 91 US 9,518,097 B2

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LNOO808SO4-: U.S. Patent Dec. 13, 2016 Sheet 89 of 91 US 9,518,097 B2 U.S. Patent Dec. 13, 2016 Sheet 90 Of 91 US 9,518,097 B2 U.S. Patent Dec. 13, 2016 Sheet 91. Of 91 US 9,518,097 B2 US 9,518,097 B2 1. 2 IDENTIFICATION AND USE OF GENES ingestion of an Amanita mushroom may notherald the need ENCOOING AMATOXIN AND for the extreme medical treatment necessary to save a PHALLOTOXN patient. Even experienced mycologists may not be able to distin This application claims priority to U.S. Provisional Appli 5 guish edible from poisonous mushrooms even with micro cation Ser. No. 61/002,650, filed on Nov. 9, 2007. scopic examination (EMedicine webmdat hypertext transfer protocol site: emedicine.com/ and hypertext transfer proto GOVERNMENT INTERESTS col site: emedicine.com/emerg/topic874.htm. Thus physicians and veterinarians need to be able to This invention was made with government Support under 10 directly and quickly confirm whether a patient or an animal DE-FG02-91 ER20021 awarded by the U.S. Department of showing gastrointestinal symptoms of unknown origin, or Energy. The government has certain rights in the invention. who has accidentally eaten an unknown mushroom, is in danger of serious illness or death from eating a deadly FIELD OF THE INVENTION poisonous mushroom containing amatoxins. 15 The present invention relates to compositions and meth SUMMARY OF THE INVENTION ods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In par The present invention relates to compositions and meth ticular, the present invention relates to using genes and ods comprising genes and peptides associated with cyclic proteins from Amanita species encoding Amanita peptides, peptide toxins and toxin production in mushrooms. In par specifically relating to amatoxins and phallotoxins. In a ticular, the present invention relates to using genes and preferred embodiment, the present invention also relates to proteins from Amanita species encoding Amanita peptides, methods for detecting Amanita peptide toxin genes for specifically relating to amatoxins and phallotoxins. In a identifying Amanita peptide-producing mushrooms and for preferred embodiment, the present invention also relates to diagnosing Suspected cases of mushroom poisoning. Fur 25 methods for detecting Amanita peptide toxin genes for ther, the present inventions relate to providing kits for identifying Amanita peptide-producing mushrooms and for diagnosing and monitoring Suspected cases of mushroom diagnosing Suspected cases of mushroom poisoning. Fur poisoning in patients. ther, the present inventions relate to providing kits for diagnosing and monitoring Suspected cases of mushroom BACKGROUND 30 poisoning in patients. The present invention provides an isolated nucleic acid More than 90% of human deaths resulting from mush sequence comprising at least one sequence set forth in SEQ room poisoning are due to peptide toxins found in Amanita ID NOs: 1-4, 55-56, 79-81, 85-86, and 95-96. In one species of mushrooms, such as A. phalloides, A. bisporigera, embodiment, the nucleic acid encodes a polypeptide com A. ocreata, and A. virosa. Animals, especially dogs, are 35 prising at least one sequence set forth in SEQ ID NOs: 50. frequent victims of poisoning by Amanita mushrooms. 113, 118, 121-132, and 135. In one embodiment, the nucleic Recently, two dogs died after eating toxin containing mush acid sequence comprises a sequence at least 50% identical to rooms in Michigan in the last few months, See Schneider: any sequence set forth in SEQ ID NOs: 82-87. In one Mushroom in backyard kills curious puppy, Lansing State embodiment, the nucleic acid sequence encodes a peptide Journal, Sep. 30, 2008 (at lansingStatejournal.com.apps/ 40 set forth in any one of SEQID NOs: 136-151 and 80. In one pbcs.dll/article?AID=/20080930/COLUMNISTS09/809300 embodiment, the nucleic acid sequence comprises SEQ ID 321. NOs: 86-87. In one embodiment, the polypeptide is selected High concentrations of peptide toxins are found in the from the group consisting of IWGIGCNP (SEQ ID NO: 50) above ground mushroom portion (otherwise known as car and AWLVDCP (SEQ ID NO: 69). In one embodiment, the pophores or fruiting bodies) of the toxin producing Amanita 45 invention provides a polypeptide encoded by the nucleic species. These toxins include two major families of com acid sequences SEQ ID NOs: 55-56, 79-81, and 85-86. pounds called amatoxins (for example, C.-amanitin, FIG. The present invention provides a composition comprising 1A) and phallotoxins (for example, phalloidin, phallacidin, a nucleic acid sequence, wherein said nucleic acid sequence FIG. 1B). Both classes of compounds are bicyclic peptides comprises at least one sequence set forth in SEQ ID NOs: with a Cys-Trp cross-bridge. In general, amatoxins are 8 50 1-4, 55-56, 79-81, 85-86, and 95-96. amino acids in length while phallotoxins are 7 amino acids The present invention provides a composition comprising in length. Although phallotoxins are toxic when injected, a polypeptide, wherein said polypeptide is encoded by a phallotoxins do not Survive the human intestinal tract and nucleic acid sequence comprising at least one sequence set therefore are usually not responsible for deadly mushroom forth in SEQ ID NOs: 55-56, 79-81, and 85-86. poisonings in humans and animals. On the other hand, 55 The present invention provides a set of at least two amatoxins do Survive cooking and remain intact in the polymerase chain reaction primer sequences, wherein said intestinal tract where they are absorbed into the body where primers are capable of amplifying a mushroom nucleic acid large doses irreversibly damage the liver. Liver failure due sequence associated with encoding an Amanita toxin. In one to poisoning by amatoxins can be “cured only with a liver embodiment, the two polymerase chain reaction primer transplant (Enjalbert et al., (2002) J. Toxicol. Clin. Toxicol. 60 sequences are selected from the group SEQ ID NOs: 1-4, 40:715; herein incorporated by reference). 95-96. There are an estimated 900-1000 species of Amanita, of The present invention provides a method of identifying a which the majority do not produce amatoxins or phallotox toxin producing mushroom, comprising, a) providing, i) a ins and some are actually safe for humans to eat (Bas, (1969) sample, ii) a set of at least two polymerase chain reaction Persoonia 5:285: Tulloss et al., (2000) Micologico G. Bres 65 primers, wherein said primers are capable of amplifying a adola, 43:13; Wei?Bet al., (1998) Can J. Bot. 76:1170; all of mushroom nucleic acid sequence associated with encoding which are herein incorporated by reference). Thus mere a toxin, and iii) a polymerase chain reaction, b) mixing said US 9,518,097 B2 3 4 sample with said set of polymerase chain reaction primers, A composition, comprising a polypeptide, wherein said c) completing a polymerase chain reaction under conditions polypeptide is encoded by a sequence derived from a fungal capable of amplifying a mushroom nucleic acid sequence species. associated with encoding a toxin, and d) testing for an A method, comprising a polypeptide, wherein said poly amplified toxin associated sequence for identifying a toxin peptide is encoded by a sequence derived from a fungal producing mushroom. In one embodiment, the testing com species. prises detecting the presence or absence of an amplified The present invention provides an antibody having speci mushroom nucleic acid sequence. In one embodiment, the ficity for a preproprotein comprising a toxin sequence, sample is selected from the group consisting of a raw wherein said preproprotein is encoded by a nucleotide sample, a cooked sample, and a digested sample. In one 10 embodiment, the sample comprises a mushroom sample. In sequence derived from a fungal species. In one embodiment, one embodiment, the sample is obtained from a subject. The the preproprotein includes but is not limited to SEQID NOs: Subject may be any mammal, e.g., the Subject may be a 50, 110, 113, 118, 121-132, 135, 249, 303-306, 308-318. In human. In one embodiment, the set of polymerase chain one embodiment, the toxin includes but is not limited to a reaction primer sequences may identify any Amanita pep 15 cyclic toxin, a linear amino acid sequence of a cyclic toxin, tide. In one embodiment, the set of polymerase chain a portion of a linear amino acid sequence of a cyclic toxin. reaction primer sequences may identify an amanitin peptide. In one embodiment, the toxin includes but is not limited to In one embodiment, the set of polymerase chain reaction an amatoxin and phallotoxin. In one embodiment, the toxin primer sequences are selected from the group consisting of includes but is not limited to an amanitin. In one embodi SEQ ID NOs: 1-4, 95-96. ment, the toxin includes but is not limited to an alpha, beta, The present invention provides a diagnostic kit for iden gamma, etc., amanitin. In one embodiment, the toxin tifying a poisonous mushroom, providing, comprising, a set includes but is not limited to 110, 246, 303-306, 308-117. of at least two polymerase chain reaction primers, wherein A composition, comprising an antibody having specificity said primers are capable of amplifying a mushroom nucleic for a preproprotein comprising a toxin sequence, wherein acid sequence associated with producing a toxin. In one 25 said preproprotein is encoded by a nucleotide sequence embodiment, the two polymerase chain reaction primer derived from a fungal species. sequences are selected from the group consisting of SEQID A method, comprising an antibody having specificity for NOs: 1-4, 95-96. In one embodiment, the kit further com a preproprotein comprising a toxin sequence, wherein said prises a nucleic acid sequence associated with producing a preproprotein is encoded by a nucleotide sequence derived mushroom toxin, wherein said nucleic acid sequence is 30 from a fungal species. capable of being amplified by said polymerase chain reac The present invention provides an antibody having speci tion primers. In one embodiment, the kit further comprises ficity for a toxin encoded by a nucleotide sequence derived instructions for amplifying said mushroom nucleic acid from a fungal species. In one embodiment, the toxin sequence. In one embodiment, the kit further comprises includes but is not limited to a cyclic toxin, a linear amino instructions for detecting the presence or absence of an 35 acid sequence of a cyclic toxin, a portion of a linear amino amplified mushroom nucleic acid sequence. In one embodi acid sequence of a cyclic toxin. In one embodiment, the ment, the kit further comprises instructions for identifying toxin includes but is not limited to an amanitin and a the species of an amplified mushroom nucleic acid phallatoxin. In one embodiment, the toxin includes but is not Sequence. limited to an alpha, beta, gamma, etc., amanitin. In one The present invention provides a polypeptide, wherein 40 embodiment, the toxin includes but is not limited to SEQID said polypeptide is encoded by a sequence derived from a NOS. 110, 303-306, 308-317. In one embodiment, the anti fungal species. In one embodiment, the polypeptide is an body includes but is not limited to a polyclonal antibody and isolated polypeptide. In one embodiment, the isolated poly a monoclonal antibody. In one embodiment, the antibody peptide is isolated from a cell. In one embodiment, the cell includes but is not limited to a rat, rabbit, mouse, chicken includes but is not limited to a fungal cell and a bacterial 45 antibody. cell. In one embodiment, the isolated polypeptide is a A composition, comprising an antibody having specificity synthetic polypeptide. It is not meant to limit the sequence for a toxin encoded by a nucleotide sequence derived from of the polypeptide. In one embodiment, the polypeptide a fungal species. includes but is not limited to a polypeptide comprising a A method, comprising an antibody having specificity for toxin sequence. In one embodiment, the polypeptide com 50 a toxin encoded by a nucleotide sequence derived from a prises at least one preproprotein sequence set forth in SEQ fungal species. ID NOs: 50, 110, 113, 118, 121-132, 135, 249, 303-306, The present invention provides an isolated prolyloligo 308-318. In one embodiment, the polypeptide is a MDIN peptidase protein, wherein said prolyloligopeptidase protein amino acid sequence. In one embodiment, the polypeptide is encoded by nucleic acid sequence derived from a fungal comprises a toxin amino acid sequence. In one embodiment, 55 species. In one embodiment, the prolyloligopeptidase the polypeptide comprises IWGIGCNP (SEQ ID NO: 50) includes but is not limited to a prolyloligopeptidase, pro and AWLVDCP (SEQ ID NO: 69). In one embodiment, the lyloligopeptidase A, prolyloligopeptidase B, and fragments polypeptide comprises at least one sequence set forth in SEQ thereof. In one embodiment, the prolyloligopeptidase A ID NOs: 249, and 318. In one embodiment, the polypeptide comprises any one sequence set forth in SEQ ID NOS. 69, is linear. In one embodiment, the polypeptide is cyclic. In 60 250-256, 258-276. In a preferred embodiment, the prolylo one embodiment, the polypeptide comprises at least one ligopeptidase B comprises any one sequence set forth in sequence set forth in 110, 303-306, 308-317. In one embodi SEQ ID NOs. 54, 252, 256, 261,267, 270, 271, 273, 276, ment, the polypeptide includes but is not limited to a 280-282, 286, 288-293, 296-297, 302. polypeptide comprising a prolyloligopeptidase sequence. In A composition, comprising an isolated prolyloligopepti one embodiment, the prolyloligopeptidase sequence com 65 dase protein, wherein said prolyloligopeptidase protein is prises at least one sequence set forth in SEQID NOs: 54, 69. encoded by nucleic acid sequence derived from a fungal 236, 237, 250-256, 258-276. species. US 9,518,097 B2 5 6 A method, comprising an isolated prolyloligopeptidase FIG. 7 shows exemplary fragment DNA sequences from protein, wherein said prolyloligopeptidase protein is A. bisporigera that contain conserved motifs of the amanitin encoded by nucleic acid sequence derived from a fungal and phallacidin genes. Each DNA sequence is followed by species. the translation of the presumed correct reading frame. Con The present invention provides an antibody having speci- 5 served upstream and downstream amino acid sequences with ficity to a prolyloligopeptidase protein, wherein said pro known and putative toxin sequences underlined. lyloligopeptidase protein is encoded by a nucleotide FIG.8 shows exemplary DNA blots of different species of sequence derived from a fungal species. In one embodiment, Amanita. (A) Probed with AMA1 cDNA. (B) Probed with the prolyloligopeptidase includes but is not limited to a PHA1 cDNA. (C) Probed with a fragment of the B-tubulin prolyloligopeptidase, prolyloligopeptidase A prolyloligo- 10 gene isolated from A. bisporigera (19). (D) Ethidium peptidase B, and fragments thereof. In one embodiment, the prolyloligopeptidase A comprises any one sequence set forth stained gel showing relative lane loading. Markers are in SEQ ID NOS. 69, 250-256, 258-276. In a preferred lambda cut with BstEII. Species and provenances: Lane 1, A. embodiment, the prolyloligopeptidase B comprises any one aff. Suballiacea (Ingham County, Michigan); lane 2, A. sequence set forth in SEQ ID NOS. 54, 252, 256, 261, 267, 15 bisporigera (Ingham County); lane 3, A. phalloides 270, 271, 273, 276, 280-282, 286, 288-293, 296-297, 302. (Alameda County, California); lane 4, A. ocreata (Sonoma A composition, comprising a mushroom P450 protein. County, California); lane 5, A. movinupta (Sonoma County); A method, comprising a mushroom P450 protein. lane 6, A. franchetii (Mendocino County, California); lane 7, A. porphyria (Sonoma County); lane 8, a second isolate of DESCRIPTION OF THE FIGURES 2O A. franchetii (Sonoma County); lane 9, A. muscaria (Monterey County, California); lane 10, A. gemmata (Men FIG. 1 shows exemplary structures of (A) amatoxins and docino County); lane 11, A. hemibapha (Mendocino (B) phallotoxins. Exemplary amino acids have the L con County); lane 12, A. velosa (Napa County, California); lane figuration except hydroxy Asp in phallacidin and Thr in 13, A. sect. Vaginatae (Mendocino County). Mushrooms phalloidin. 25 represent sect. Phalloideae (his 1-4), sect. Validae (it's 5-8), FIG. 2 shows exemplary fungi of the genus Amanita. A. sect. Amanita(ii's 9-10), sect. Caesarea (#11), sect. Vagina A. bisporigera (collected in Oakland County, Michigan). B: tae (Hs 12-13). Four separate gels were run; the lanes are in A. phalloides (Alameda County, California). C: Non-deadly the same order on each gel and approximately the same species of Amanita. From left to right: three specimens of A. amount of DNA was loaded per lane. A and B are to the same gemmata, A. muscaria, and two specimens of A. franchetii 30 scale, and C and D are to the same scale. (Mendocino County, California). FIG. 9 shows an exemplary schematic of a WebLogo FIG. 3 shows an exemplary hypothetical peptide syn (Crooks et al., 2004, herein incorporated by reference) thetase showing conserved motifs found in many NRPS showing a representation of amino acid frequency within at proteins that served as the basis for the design of PCR least 15 predicted “MSDIN sequences from DNA primers (see, Table 4). 35 sequences of Amanita species. FIG. 4 shows exemplary amanitin (an amatoxin) cDNA FIG. 10 shows an exemplary correlation of toxin genes sequences, genomic DNA sequences, prepropolypeptide and expression with toxin producing species of mushrooms sequences, and polypeptide sequences coding for peptide in addition to a schematic of types of genes discovered near toxins, A) shows exemplary cDNA sequences of the toxin producing genes in at least one lambda clone from a C.-amanitin gene and predicted amino acid sequence, where 40 toxin producing mushroom. A) and B) Southern blot of 5' and 3' ends were determined by Rapid amplification of DNA from species of Amanita that do (A. bisporigera and A. cDNA ends (RACE). * indicates a stop codon. The string of phalloides) or do not (A. gemmata, A. muscaria, A. flavo As at the end are a contemplated poly-A tail. The amatoxin conia, A. section Vaginatae, and A. hemibapha) make ama peptide sequence is underlined. B) shows an exemplary toxin (probe used in A) and phallotoxin (probe used in B); sequence of genomic DNA covering the amanitin gene 45 C) PCR amplification of the gene for O-amanitin. Primers based on inverse PCR. The nucleotides encoding the aman were based on the sequences in FIG. 4. A. gemmata and A. itin peptide are underlined. muscaria are species of Amanita that do not make amatoxins FIG. 5 shows exemplary phallacidin cDNA, genomic (or phallotoxins). A. bisporigera is 1-3 are three different DNA, propolypeptide, and polypeptide sequences encoding isolates of A. bisporigera; and D) Exemplary Schematic phallacidin peptide toxin. A) shows exemplary cDNA 50 Map of Amanita bisporigera genes in a lambda clone (13.4 sequences and predicted amino acid sequence, where 5' and kb) isolated using PHA1 as probe; showing two copies of 3' ends were determined by RACE, * indicates the stop PHA1 clustered with each other and with three P450 genes, codon. The string of A's at the end are the poly-A tail and NOTE: p450 genes were predicted using the Coprinus were not found encoded within the genomic DNA, and B) model however Coprinus doesn’t have a PHA1 gene. shows an exemplary genomic nucleic acid coding regions 55 FIG. 11 shows exemplary sequences found in genomic for phallacidin sequence #1, 1893 bp. SacI and phallacidin sequencing of Galerina (G. marginata, Gm) A) Nucleic sequence #2, 1613 nt. PVul where the nucleotides encoding Acid Sequences (GmAM1) and B) Amino acid sequences a phallacidin peptide were underlined. These two genomic deduced from sequences in A (GmAM1). (-nonsense sequences encoding a phallacidin peptide were obtained by codon) inverse PCR and confirmed by sequencing both strands. 60 FIG. 12 shows exemplary Galerina marginata amanitin FIG. 6 shows an exemplary alignment of a (A) clNA (GmAM1) preproprotein amino acid sequence alignment nucleotide and (B) predicted amino acid sequences of exem between Galerina and Amanita including A) alpha-amanitin plary coding regions of AMA1 and PHA1 proproteins, the toxins alpha-amanitin/gamma-amanitin from Amanita com mature toxin sequences were underlined, and (C) shows pared to alpha-amanitin/gamma-amanitin from Galerina homologous regions in nucleic acids from other species to 65 and B) a Southern blot of Galerina (G.) marginata (m) (Gm) coding regions of AMA1 and PHA1 proproteins (BLAST DNA probed with GmAM1 under high stringency condi results). tions. US 9,518,097 B2 7 8 FIG. 13 shows an exemplary RNA blot of the Galerina FIG. 20 shows exemplary A) HPLC analysis of an enzy marginata amanitin gene (GmAMAl). The results show that matic reaction of POPB with a boiled isolate of POPB the gene is expressed in two known amanitin-producing showing no cleavage product at the vertical arrow where a species of Galerina (G. marginata and G. badipes) but not AWLVDCP (SEQ ID NO: 69) should be found and B) in a species that is a nonproducer of toxin (G. hybrida). 5 cleavage of a synthetic phallacidin precursor by purified Induction of gene expression was triggered by low carbon Conocybe albipes POPB enzyme (see, FIG. 19) showing a growth conditions. Lane 1. G. hybrida, high carbon. Lane 2. cleavage product matching AWLVDCP (SEQID NO: 69) at G. hybrida, low carbon. Lane 3. G. marginata, high carbon. the vertical arrow. The results show that purified POPB cuts Lane 4. G. marginata, low carbon. Lane 5. G. badipes, high a synthetic amanatin peptide precisely at the expected flank ing Pro residues. carbon. Lane 6. G. badipes, low carbon. The probe was G. FIGS. 21A-21B shows exemplary expression of POPB in marginata AMA1 gene (GmAMA1) predicted to encode E. coli and production of anti-POPB antibodies. FIG. 21A alpha-amanitin (FIG. 4). Each lane was loaded with 15 ug shows Lanes 1-3: stained with Coomassie Blue. FIG. 21B total RNA. Fungi were grown in liquid culture for 30 d on shows Lanes 4-5 with antibody binding visualized by 0.5% glucose (high carbon) then switched to fresh culture of 15 enhanced chemiluminescence. Lane 1: Markers. Lane 2: 0.5% glucose or 0.1% glucose (low carbon) for 10 d before POPB purified from inclusion bodies. Lane 3: Soluble harvest. The major band in lanes 3-6 is ~300 bp. The high extract of Amanita bisporigera. Lane 4: immunoblot of MW signal in lane 1 is spurious. POPB inclusion body. Lane 5: immunoblot of Amanita FIG. 14 shows exemplary Galerina marginata amanitin extract. Crude antiserum was used at 1:5000 dilution. sequences (GmAMA1) found in genomic sequencing of FIG.22 shows exemplary Galerina POP sequences iden Galerina (G. marginata, Gm). A) Nucleic Acid Sequences tified using Amanita bisporigera A) POPA and B) POPB as (GmAM1), B) Amino acid sequences deduced from query sequences for searching a library of Galerina sequences in A (GmAM1). (.. nonsense codon), and C) sequences created by the inventors for their use during the amino acid sequence alignment of two Galerina amanitins development of the present inventions. The higher scoring (GaAMA1 above SEQ ID NO.244, GmaMA2 below, SEQ 25 hits were strong evidence that the Galerina genome contains ID NO.248). at least two POP genes. FIG. 15 shows exemplary BLASTP results using human FIG. 23 shows exemplary sequences found in the prolyloligopeptidase (POP) as query against fungi in Gen genomic schematic sequence of FIG. 10D inserted into a Bank. The results indicate that an ortholog of human POP lambda clone; 13,254 bp lambda clone red/underlined exists in at least some Homobasidiomycetes (Coprinus) and 30 sequences (portions) are two copies of PHA1 encoding Heterobasidiomycetes (Ustilago and Cryptococcus) and few phallacidin 5'-3' orientation, (SEQ ID NO: 327). other fungal species showing various levels of significant FIG. 24 shows an exemplary FGENESH 2.5 Prediction of identity and where scores and e-values of the two Aspergil potential genes in Coprinus genomic DNA of SEQ ID lus fungal sequences were considered statistically insignifi NOXX. Cant. 35 FIG. 25 shows an exemplary contemplated P450 gene FIG. 16 shows exemplary prolyloligopeptidase (POP)- sequence, A) P450-1 (OP451) and putative encoded amino like homologs in fungi with strong amino acid sequence acid sequences, B) blastp results of Predicted protein(s): similarity to human prolyloligopeptidase (gi:41349456). P450-1 (OP451), C). BLASTP of OP45-1 against Coprinus Shown are the DNA sequences and the alignments of the sequences at Broad, D) BLASTP of OP451 against Laccaria human protein (query) with each predicted translation prod 40 genomic sequences and E), OP451 as a query sequence for uct from A. bisporigera (Subject). a BLASTP against nr., showing an excellent hit against a FIG. 17 shows A) two exemplary prolyloligopeptidase Coprinus protein. (POP)-like A. bisporigera genome sequences POPA and FIG. 26 shows an exemplary contemplated P450 gene POPB, B) two exemplary cDNA sequences for POPA and sequence, A) P450-2 (OP452) and putative encoded amino POPB, and C) two exemplary amino acid sequences for 45 acid sequences, B) blastp results of Predicted protein(s): POPA and POPB. P450-2 (OP452), C). BLASTP of P450-2 (OP452) against FIG. 18 shows exemplary Southern blot of different Coprinus at Broad, and D) BLASTP of P450-2 (OP452) Amanita species probed with (A) POPA or (B) POPB of A. against Laccaria genomic sequences. bisporigera. DNA was from the same species of mushroom FIG. 27 shows an exemplary FGENESH mRNA and in lanes of the same order as FIG. 8, herein, and FIG. 5 in 50 protein 3 resulting in no hits to any of the BLAST searches. Hallen et al., 2007, Proc. Natl. Acad. Sci. USA 104: 19097 This region overlaps with PHA1-1, which is on + strand 19101, herein incorporated by reference. Lanes 1-4 are (gene 3 is on - Strand). Amanita species in sect. Phalloideae and the others are toxin FIG. 28 shows an exemplary contemplated P450 gene non-producers. Note the presence of POPA and absence of sequence, A) P450-3 (OP453) and putative encoded amino POPB in sect. Validae (lanes 5-8), the sister group to sect. 55 acid sequences, B) blastp results of Predicted protein(s): Phalloideae (lanes 1-4). the weaker hybridization of POPA P450-3 (OP453), C). BLASTP of P450-3 (OP453) against to the Amanita species outside sect. Phalloideae (lanes 5-13) Coprinus at Broad, and D) BLASTP of P450-3 (OP453) to lower DNA loading and/or lower sequence identity due to against Laccaria genomic sequences. taxonomic divergence (cf. FIG. 5 in Hallen et al., 2007, FIG. 29 shows exemplary A) PHA1-2 as described herein Proc. Natl. Acad. Sci. USA 104: 19097-19101, herein incor 60 (5th identified sequence) and B) a 6" identified sequence porated by reference). POPB does not hybridize to any FIG. 30 shows exemplary A) alignments of P450 genes species outside sect. Phalloideae even after prolonged auto 1.2.4 corresponding to OP451, OP452 and OP453 and B) radiographic exposure. exemplary sequences from the entire lambda clone reverse FIG. 19 shows exemplary purified POPB protein isolated complement (3'-5') and C) FGENESH of reverse comple from Conocybe albipes separated by standard SDS-PAGE 65 ment showing a different gene 4, which is gene 3 in the gel electrophoresis and Coomassie dye stained to show the reverse complement, resulting in D) a new set of exemplary location of protein. gene identities. US 9,518,097 B2 9 10 DEFINITIONS a 'sac fungus' group. Of the Ascomycota, a class “Asco mycetes’ includes Candida albicans, unicellular yeast, To facilitate an understanding of the present invention, a Morchella esculentum, the morel, and Neurospora crassa. number of terms and phrases as used herein are defined Some ascomycetes cause disease, for example, Candida below: albicans causes thrush and vaginal infections; or produce The use of the article “a or “an is intended to include chemical toxins associated with diseases, for example, Ole O. O. Aspergillus flavus produces a contaminant of nuts and stored As used herein, terms defined in the singular are intended grain called aflatoxin, that acts both as a toxin and a deadly to include those terms defined in the plural and vice versa. natural carcinogen. As used herein the term “microorganism” refers to micro 10 As used herein, the term “toxin' in reference to a poison scopic organisms and taxonomically related macroscopic refers to any substance (for example, alkaloids, cyclopep organisms within the categories of algae, bacteria, fungi tides, coumarins, and the like) that is detrimental (i.e., (including lichens), protozoa, viruses, and Subviral agents. poisonous) to cells and/or organisms, in particular a human The terms “eukaryotic' and “eukaryote” are used in the organism. In particularly preferred embodiments of the broadest sense. It includes, but is not limited to, any organ 15 present inventions, the term “toxin' encompasses toxins, isms containing membrane bound nuclei and membrane Suspected toxins, and pharmaceutically active peptides pro bound organelles. Examples of eukaryotes include but are duced by various fungal species, including, but not limited not limited to animals, plants, algae, diatoms, and fungi. to, a cyclic peptide toxin Such as an amanitin, that provides The terms “prokaryote” and “prokaryotic' are used in the toxic activity towards cells and humans. However, it is not broadest sense. It includes, but is not limited to, any organ intended that the present invention be limited to any par isms without a distinct nucleus. Examples of prokaryotes ticular fungal toxin or fungal species. Indeed, it is intended include but are not limited to bacteria, blue-green algae that the term encompass fungal toxins produced by any (cyanobacteria), archaebacteria, actinomycetes and myco organism. As used herein, a toxin encompasses linear plasma. In some embodiments, a host cell is any microor sequences of cyclic pharmaceutically active peptides and ganism. 25 linear sequences showing identity to known toxins regard As used herein, the term “fungi' is used in reference to less of whether these sequences are known to be toxic. eukaryotic organisms such as mushrooms, rusts, molds and As used herein, the term "Amanita peptide' or “Amanita yeasts, including dimorphic fungi. "Fungus' or “fungi also toxin' or “Amanita peptide toxin refers to any linear or refers to a group of lower organisms lacking chlorophyll and cyclic peptide produced by a mushroom, including but not dependent upon other organisms for Source of nutrients. 30 limited to species of Lepiota, Conocybe, Galerina, and the As used herein, "mushroom” refers to the fruiting body of like. However, it is not intended that the present invention be a fungus. limited to a toxin or a peptide produced by an Amanita As used herein, “fruiting body” refers to a reproductive mushroom and includes similar peptides and toxins pro structure of a fungus which produces spores, typically duced by other fungi. In particular, an Amanita peptide toxin comprising the whole reproductive structure of a mushroom 35 resembles any of the amatoxins and phallotoxins, such as including cap, gills and stem, for example, a prominent similarity of amino acid sequences, matching toxin motifs as fruiting body produced by species of Ascomycota and shown herein, encoded between the conserved regions (A Basidiomycota, examples of fruiting bodies are "mush and B) of their proproteins, encoded by hypervariable rooms,” “carpophores,” “toadstools.” “puffballs’, and the regions of their proproteins (P), and the like. For example, like. 40 an exemplary Amanatin peptide toxin is 7-11 amino acids in As used herein, “fruiting body cell refers to a cell of a length. cap or stem which may be isolated or part of the structure. As used herein, the term “toxic' refers to any detrimental As used herein, 'spore' refers to a microscopic reproduc or harmful effects on a cell or tissue. tive cell or cells. As used herein, "Amanita' refer to a genus of fungus As used herein, "mycelium” refers to a mass of fungus 45 whose members comprise poisonous mushrooms, e.g., hyphae, otherwise known as a vegetative portion of a Amanita(A.) bisporigera, A. virosa, A. ocreata, A. suballia fungus. cea, and A. tenuifolia which are collectively referred to as As used herein, “Basidiomycota' in reference to a Phy “death angels' or “Destroying Angels” and Amanita phal lum or Division refers to a group of fungi whose sexual loides' or “A. phalloides var.alba” or “A. phalloides var. reproduction involves fruiting bodies comprising basidi 50 verna” or “A. verna, referred to as “death cap. The toxins ospores formed on club-shaped cells known as basidia. of these mushrooms frequently cause death through liver As used herein, “Basidiomycetes’ in reference to a class and kidney failure in humans. Not all species of this genus of Phylum Basidiomycota refers to a group of fungi. Basidi are deadly, for example, Amanita muscaria, the fly agaric, omycetes include mushrooms, of which some are rich in induces gastrointestinal distress and/or hallucinations while cyclopeptides and/or toxins, and includes certain types of 55 others do not induce detectable symptoms. yeasts, rust and Smut fungi, gilled-mushrooms, puffballs, As used herein, “amatoxin' generally refers to a family of polypores, jelly fungi, brackets, coral, mushrooms, boletes, peptide compounds, related to and including the amanitins. puffballs, Stinkhorns, etc. For the purposes of the present inventions, an amatoxin As used herein, “Homobasidiomycetes’ in reference to refers to any Small peptide, linear and cyclic, comprising an fungi refers to a recent classification of fungi, including 60 exemplary chemical structure as shown in FIG. 1 or encoded Amanita spp. and all other gilled fungi (commonly known as by nucleic acid sequence of the present invention, wherein mushrooms), based upon cladistics rather than morphology. the nucleic acid sequence and/or proprotein has a higher As used herein, “Heterobasidiomycetes’ in reference to sequence homology to AMA1 than to an analogous fungi refers to those basidiomycete fungi that are not sequence of PHA1. Homobasidiomycetes. 65 As used herein, “phallotoxin' generally refers to a family As used herein, “Ascomycota’ or “ascomycetes’ in ref of peptide compounds, related to and including phallacidin erence to members of a fungal Phylum or Division refers to and phalloidin. For the purposes of the present inventions, a US 9,518,097 B2 11 12 phallotoxin refers to any Small peptide encoded by nucleic transferred or introduced into the host cell. For example, a acid sequences where the nucleic acid sequence and/or host cell may be located in a transgenic mushroom. A proprotein has a higher sequence homology to PHA1 than to transformed cell includes the primary subject cell and its an analogous sequence of AMA1. progeny. As used herein, nonribosomal peptide synthetase (NRPS) As used herein, "host fungus cell refers to any fungal is an enzyme that catalyzes the biosynthesis of a small (20 cell, for example, a yeast cell, a mold cell, and a mushroom or fewer amino acids) peptide or depsipeptide, linear or cell (such as Neurospora crassa, Aspergillus nidulans, circular, and is composed of one or more domains (modules) Cochliobolus carbonum, Coprinus cinereus, and the like). typical of this class of enzyme. Each domain is responsible As used herein, the term "Fungal expression system' for aminoacyl adenylation of one component amino acid. 10 refers to a system using fungi to produce (express) enzymes NRPSs can also contain auxiliary domains catalyzing, e.g., and other proteins. Examples of filamentous fungi which are N-methylation and amino acid epimerization (Walton, et al., currently used or proposed for use in Such processes are in Advances in Fungal Biotechnology for Industry, Agricul Neurospora crassa, Acremonium chrysogenium, Tolypocla ture, and Medicine, et al., Eds. (Kluwer Academic/Plenum, dium geodes, Mucor circinelloides, Trichoderma reesei, N.Y., 2004, pp. 127-162: Finking, et al., (2004) Annu Rev 15 Aspergillus nidulans, Aspergillus niger; Coprinus cinereus, Microbiol 58:453-488, all of which are herein incorporated Aspergillus Oryzae, etc. Further examples include an expres by reference). Examples are gramicidin synthetase, HC sion system for basidiomycete genes (for example, Gola, et toxin synthetase, cyclosporin synthetase, and enniatin Syn al., (2003) J Basic Microbiol. 43(2):104-12; herein incor thetase. porated by reference) and fungal expression systems using, As used herein, “prolyl oligopeptidase' or “POP” or for example, a monokaryotic laccase-deficient Pycnoporus “prolyloligopeptidase' refers to a member of a family of cinnabarinus strain BRFM 44 (Banque de Resources enzymes classified and referred to as EC 3.4.21.26-enzymes Fongiques de Marseille, Marseille, France), and Schizophyl that are capable of cleaving a peptide sequence, Such that lum commune, (for example, Alexandra, et al., (2004) Appl hydrolysis of Pro-I-Xaa->Ala-I-Xaa in oligopeptides, also Environ Microbiol. 70(11):6379-638; Lugones, et al., (1999) referred to as any one of “post-proline cleaving enzyme.” 25 Mol. Microbiol. 32:681-700; Schuren, et al., (1994) Curr. “proline-specific endopeptidase.” “post-proline endopepti Genet. 26:179-183; all of which are herein incorporated by dase.” “proline endopeptidase,” “endoprolylpeptidase.” reference). “prolyl endopeptidase.” “post-proline cleaving enzyme.” The term “transgene' as used herein refers to a foreign “post-proline endopeptidase, and “prolyl endopeptidase.” gene. Such as a heterologous gene, that is placed into an A POPA of the present inventions refers to a mushroom 30 organism by, for example, introducing the foreign gene into sequne found in the majority of mushrooms. A POPB of the cells or primordial tissue. The term “foreign gene' refers to present inventions refers to a sequence which in one any nucleic acid (e.g., gene sequence) that is introduced into embodiment has approximately a 55% amino acid homology the genome of a host cell by experimental manipulations and to POPA, wherein said POPB sequence is primarily found in may include gene sequences found in that cell so long as the toxin producing mushroom species. 35 introduced gene does not reside in the same location as does As used herein, the terms “cell,” “cell line,” and “cell the naturally-occurring gene. culture' may be used interchangeably. All of these terms As used herein, the term “vector” is used in reference to also include their progeny, which are any and all Subsequent nucleic acid molecules that transfer DNA segment(s) from generations. It is understood that all progeny may not be one cell to another. The term "vehicle' is sometimes used identical due to deliberate or inadvertent mutations. In the 40 interchangeably with “vector.” A vector “backbone' com context of expressing a heterologous nucleic acid sequence, prises those parts of the vector which mediate its mainte “host cell refers to a prokaryotic or eukaryotic cell, and it nance and enable its intended use (e.g., the vector backbone includes any transformable organism that is capable of may contain sequences necessary for replication, genes replicating a vector and/or expressing a heterologous gene imparting drug or antibiotic resistance, a multiple cloning encoded by a vector. Several types of fungi and cultures are 45 site, and possibly operably linked promoter and/or enhancer available for use as a host cell, such as those described for elements which enable the expression of a cloned nucleic use in fungal expression systems, described below. Prokary acid). The cloned nucleic acid (e.g., Such as a cDNA coding otes include but are not limited to gram negative or positive sequence, or an amplified PCR product) is inserted into the bacterial cells. Numerous cell lines and cultures are avail vector backbone using common molecular biology tech able for use as a host cell, and they can be obtained through 50 niques. the American Type Culture Collection (ATCC), an organi A “recombinant vector indicates that the nucleotide Zation that serves as an archive for living cultures and sequence or arrangement of its parts is not a native configu genetic materials (worldwide web.atcc.org). An appropriate ration, and has been manipulated by molecular biological host can be determined by one of skill in the art based on the techniques. The term implies that the vector is comprised of vector nucleic acid sequence and the desired result. A 55 segments of DNA that have been artificially joined. plasmid or cosmid, for example, can be introduced into a The terms “expression vector” and “expression cassette' prokaryote host cell for replication of many vectors. Bacte refer to a recombinant DNA molecule containing a desired rial cells used as host cells for expression vector replication coding sequence and appropriate nucleic acid sequences and/or expression include, among those listed elsewhere necessary for the expression of the operably linked coding herein, DH5O, JM109, and KC8, as well as a number of 60 sequence in a particular host organism. Nucleic acid commercially available bacterial hosts such as SURETM sequences necessary for expression in prokaryotes usually Competent Cells and SOLOPACKTM Gold Cells (Strata include a promoter, an operator (optional), and a ribosome gene, La Jolla). Alternatively, bacterial cells such as E. coli binding site, often along with other sequences. Eukaryotic LE392 can be used as host cells for phage viruses. In some cells are known to utilize promoters, enhancers, and termi embodiments, a host cell is used as a recipient for vectors. 65 nation and polyadenylation signals. A host cell may be “transfected' or “transformed, which As used herein, “recombinant nucleic acid' or “recombi refers to a process by which exogenous nucleic acid is nant gene' or “recombinant DNA molecule' indicates that