Cytochrome P450S and Uses Thereof Joe Chappell University of Kentucky, [email protected]

Cytochrome P450S and Uses Thereof Joe Chappell University of Kentucky, Chappell@Uky.Edu

University of Kentucky UKnowledge

Plant and Soil Sciences Faculty Patents Plant and Soil Sciences

9-11-2012 Cytochrome P450S and Uses Thereof Joe Chappell University of Kentucky, [email protected]

Lyle F. Ralston Right click to open a feedback form in a new tab to let us know how this document benefits oy u.

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This Patent is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and Soil Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. US008263362B2

(12) United States Patent (10) Patent N0.: US 8,263,362 B2 Chappell et a]. (45) Date of Patent: *Sep. 11, 2012

(54) CYTOCHROME P450S AND USES THEREOF 2003/0166255 A1 9/2003 Chappell ...... 435/2523 2004/0078840 A1 4/2004 Chappell 6151. .. .. 800/278 2006/0218661 A1 9/2006 Chappell 6151. .. .. 800/278 (75) Inventors: Joseph Chappell, Lexington, KY (US); 2007/0231861 A1 10/2007 Millis et a1. . 435/69.l Lyle F. Ralston, Kirkwood, MO (US) 2007/0238157 A1 10/2007 Millis et a1. .. 435/166 2007/0238159 A1 10/2007 Millis et a1. 435/25233 (73) Assignee: University of Kentucky Research 2007/0238160 A1 10/2007 Millis et a1. 435/25233 Foundation, Lexington, KY (US) 2007/0254354 A1 11/2007 Millis et a1. 435/25233 2008/0178354 A1 7/2008 Chappell 6151. 800/298 2008/0233622 A1 9/2008 Julien et a1. .. 435/148 (*) Notice: Subject to any disclaimer, the term of this 2010/0035329 A1 2/2010 Millis et a1. .. 435/2542 patent is extended or adjusted under 35 2010/0151519 A1 6/2010 Julien et a1. 435/69.l U.S.C. 154(b) by 32 days. 2010/0151555 A1 6/2010 Julien et a1. .. 435/193 2010/0216186 A1 8/2010 Chappell 6151. .. . 435/69.l This patent is subject to a terminal dis 2011/0081703 A1 4/2011 Chappell 6151...... 435/193 claimer. FOREIGN PATENT DOCUMENTS (21) App1.No.: 12/182,000 JP 2000-511404 9/2000 wo 96/36697 11/1996 Filed: Jul. 29, 2008 wo 97/38571 10/1997 (22) wo 97/38703 10/1997 wo wo 97/37664 10/1997 (65) Prior Publication Data wo 00/17327 3/2000 US 2010/01201 10 A1 May 13, 2010 wo 02/072758 9/2002 wo 2010/019696 2/2010 Related US. Application Data OTHER PUBLICATIONS Walker & Croteau, “Molecular Cloning of a 10-deacetylbaccatin (63) Continuation of application No. 10/097,559, ?led on III-10-O-acetyl Transferase cDNA from Taxus and Functional Mar. 8, 2002, now Pat. No. 7,405,057. Expression in Escherichia coli,” Jan. 18, 2000, 97(2):583-587. Provisional application No. 60/274,421, ?led on Mar. Back et al., “Expression of a Plant Sesquiterpene Cyclase Gene in (60) Escherichia coli,” Archives in Biochemistry and Biophysics (1994) 9, 2001, provisional application No. 60/275,597, ?led 315:527-532. on Mar. 13, 2001. Mandujano-Chavez et al., “Differential Induction of Sesquiterpene Metabolism in Tobacco Cell Suspension Cultures by Methyl (51) Int. Cl. Jasmonate and Fungal Elicitor,” Archives in Biochemistry and Bio C12N1/00 (2006.01) physics (2000) 381:285-294. C12N1/06 (2006.01) Ralston et al.. “Cloning. Heterologous Expression, and Functional C12N 5/00 (2006.01) Characterization of 5-epi-arist010chene-1,3-Dihydr0xylase from Tobacco (Nicotiana tobacum), ” Archives in Biochemistry and Bio C12N 5/07 (2010.01) physics (2001) 393:222-235. (52) US. Cl. 435/69.1; 435/419; 435/348; 435/252.1; Zook et al., “Characterization of Novel Sesquiterpene Biosynthesis 435/252.2 in Tobacco Expressing Fungal Sesquiterpenoid Synthase,” Plant Field of Classi?cation Search ...... None Physiology (1996) 112:311-318. (58) Akiyoshi-Shibata et al., “Further Oxidation of Hydroxycalcidiol by See application ?le for complete search history. Calcidiol 24-Hydr0xylase. A Study with the Mature Enzyme Expressed in Escherichia coli,” Eur. J. Biochem. (1994) 224:335 (56) References Cited 343. Back & Chappell, “Cloning and Bacterial Expression of a U.S. PATENT DOCUMENTS Sesquiterpene Cyclase from Hyoscyamus muticus and its Molecular Comparison to Related Terpene Cyclases,” J. Biol. Chem. (1995) 5,589,619 A 12/1996 Chappellet a1...... 800/205 5,672,487 A 9/1997 Schweden et a1. 270:7375-7381. 5,741,674 A 4/1998 Schweden et a1. Back & Chappell, “Identifying Functional Domains within Terpene 5,766,911 A 6/1998 Koike et al...... 435/193 Cyclases Using a Domain-Swapping Strategy,” Proc. Natl. Acad. Sci. 5,824,774 A 10/1998 Chappellet a1. USA. (1996) 93:6841-6845. 5,981,843 A 11/1999 Chappellet a1...... 800/301 5,994,114 A 11/1999 Croteau et a1...... 435/232 (Continued) 6,072,045 A 6/2000 Chappellet a1. Primary Examiner * Medina A Ibrahim 6,100,451 A 8/2000 Chappellet a1...... 800/298 (74) Attorney, Agent, 0rFirm * McKenna Long &Aldridge 6,117,649 A 9/2000 Bellamine et a1. 6,194,185 B1 2/2001 Croteau et a1. LLP; Stephanie Seidman 6,331,660 B1 12/2001 Chomet etal. (57) ABSTRACT 6,368,837 B1 4/2002 Gatenby et a1. 6,468,772 B1 10/2002 Chappellet a1...... 435/183 The invention features isolated cytochrome P450 polypep 6,495,354 B2 12/2002 Chappellet a1. 435/183 tides and nucleic acid molecules, as well as expression vec 6,531,303 B1 3/2003 Millis et a1. 435/155 tors and transgenic plants containing these molecules. In 6,559,297 B2 5/2003 Chappellet a1. .. 536/231 addition, the invention features uses of such molecules in 6,569,656 B2 5/2003 Chappellet a1. 435/183 methods of increasing the level of resistance against a disease 6,645,762 B2 11/2003 Chappellet a1. 435/325 6,689,593 B2 2/2004 Millis et a1. 435/155 caused by a plant pathogen in a transgenic plant, in methods 6,890,752 B2 5/2005 Chappellet a1. 435/325 for producing altered compounds, for example, hydroxylated 7,186,891 B1 3/2007 Chappellet a1. 800/298 compounds, and in methods of producing isoprenoid com 7,405,057 B2* 7/2008 Chappellet a1. .. 435/69.1 pounds. 7,442,785 B2 10/2008 Chappellet a1. .. 536/236 7,622,614 B2 11/2009 Julien et a1...... 568/327 20 Claims, 11 Drawing Sheets US 8,263,362 B2 Page 2

OTHER PUBLICATIONS Hutvagner et al., “Isolation and Sequence Analysis of a cDNA and a Back et al., “Cloning and Bacterial Expression of Sesquiterpene Related Gene for Cytochrome P450 Proteins from Solanum Cyclase, a Key Branch Point Enzyme for the Synthesis of chacoense,” Gene (1998) 188:247-252. Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Keller et al., “Sesquiterpene Cyclase Is not a Determining Factor for Capsicum annuum. ” Plant Cell Physiol. (1998) 39:899-904. Elicitor- and Pathogen-Induced Capsidiol Accumulation in Beckman et al., “Human 25-Hydroxyvitamin D3 -24-Hydroxylase, a Tobacco,” Planta (1998) 205:467-476. Multicatalytic Enzyme,” Biochemistry (1996) 35:8465-8472. Lupien et al., “Regiospeci?c Cytochrome P450 Limonene Boddupalli et al., “Fatty Acid Monooxygenation by P450BM-3: Hydroxylases from Mint (Mentha) Species: cDNA Isolation, Char Product Identi?cation and Proposed Mechanisms for the Sequential acterization, and Functional Expression of (—)-4S-Limonene-3 Hydroxylation Reactions,”Arch. Biochem. Biophys. (1992) 292:20 Hydroxylase and (—)-4S-Limonene-6-Hydroxylase.” Arch. 28. Biochem. Biophys. (1999) 368:181-192. Chappell et al., “Accumulation of Capsidiol in Tobacco Cell Cultures Mandujano-Chavez et al., “Differential Induction of Sesquiterpene Treated with Fungal Elicitor,” Phytochemisz (1987) 26:2259-2260. Chappell & Nable, “Induction of Sesquiterpenoid Biosynthesis in Metabolism in Tobacco Cell Suspension Cultures by Methyl Tobacco Cell Suspension Cultures by Fungal Elicitor,” Plant Physiol. Jasmonate and Fungal Elicitor,” Arch. Biochem. Biophys. (2000) (1987) 85:469-473. 381:285-294. Chappell, “Biochemistry and Molecular Biology of the Isoprenoid Mathis et al., “Pre-Steady-State Study of Recombinant Biosynthetic Pathway in Plants,” Annu. Rev. Plant Physiol. Plant Sesquiterpene Cyclases,” Biochemistry (1997) 36:8340-8348. Mol. Biol. (1995) 46:521-547. Maughan et al., “Expression of CYP71B7, a Cytochrome P450 Chapple, “Molecular-Genetic Analysis of Plant Cytochrome P450 Expressed Sequence Tag from Arabidopsis thaliana,” Arch. Dependent Monooxygenases.” Annu., Rev. Plant Physiol. Plant Mol. Biochem. Biophys. (1997) 341:104-111. Biol. (1998) 49:311-343. Milet et al., “Capsidiol and Ethylene Production by Tobacco Cells in Clark et al., “Spatially Distinct Expression of Two New Cytochrome Response to Cryptogein, an Elicitor from Phytophthora cryptogea,” P450s in Leaves of Nepeta racemosa: Identi?cation of a Trichome Phytochemistry (1991) 30:2171-2173. Speci?c Isoform,” Plant Mol. Biol. (1997) 33:875-885. Miller, “Structure of Genes Encoding Steriodogenic Enzymes,” J. Coolbaugh et al., “Studies on the Speci?city and Site ofAction ofa Steroid. Biochem. (1987) 27:759-766. alpha-Cyclopropyl-alpha-[p-Methoxphenyl]-5-Pyrimidine Methyl Molot et al., “Relations Between Capsidiol Concentration, Speed of Alcohol Amcymidol), a Plant Growth Regulator,” Plant Physiol. Fungal Invasion and Level of Induced Resistance in Cultivars of (1978) 62:571-576. Pepper (Capsicum annuum) Susceptible or Resistant to Cooper & Porter, “Mutagenicity of Nitrosamines in Phytophthora capsici,” Physiol. Plant Pathol. (1981) 18:379-389. Methyltransferase-De?cient Strains of Salmonella typhimurium Nedelkina et al., “Novel Characteristics and Regulation of a Diver Coexpressing Human Cytochrome P450 2E1 and Reductase,” Mutat. Res. (2000) 454:45-52. gent Cinnamate 4-Hydroxylase (CYP3A15) from French Bean: Diener et al., “Sterol Methyltransferase 1 Controls the Level of Cho Engineering Expression in Yeast,” Plant Mol. Biol. (1999) 39: 1079 lesterol in Plants,” Plant Cell (2000) 12:853-870. 1090. Dietz et al., “Nucleotide Sequences of Subunit E of the Vacuolar O’Donohue et al., “Chemical Synthesis, Expression and Proton-ATPase of Spinacia oleracea (Accession No. X96785) and Mutagenesis of a Gene Encoding beta-Cryptogein, an Elicitin Pro Arabidopsis thaliana (Accession No. X921117),” (Plant Gene Reg duced by Phytophthora cryptogea,” Plant Mol.Biol. (1995) 27:577 ister PGR 96-037) Plant Physiol. (1996) 111:652. 586. Dong & Porter, “Coexpression of Mammalian Cytochrome P450 and O’Keefe & Leto, “Cytochrome P-450 from the Mesocarp of Avocado Reductase in Escherichia coli,” Arch. Biochem. Biophys. (1996) (Persea americana),” Plant Physiol. (1989) 89: 1141-1 149. 327:254-259. Omura & Sato, “The Carbon Monoxide-Binding Pigment of Liver Facchini & Chappell, “Gene Family for an Elicitor-Induced Microsomes I. Evidence for Its Hemoprotein Nature,” J. Biol. Chem. Sesquiterpene Cyclase in Tobacco,” Proc. Natl. Acad. Sci. USA. (1964) 239:2370-2378. (1992) 89:11088-11092. Omura, “FortyYears of Cytochrome P450,” Biochem. Biophys. Res. Fahrendorf & Dixon, “Stress Responses in Alfalfa (Medicago sativa Commun. (1999) 266:690-698. L.) XVIII: Molecular Cloning and Expression of the Elicitor-Induc Pompon et al., “Yeast Expression of Animal and Plant P450s in ible Cinnamic Acid 4-Hydroxylase Cytochrome P450,” Arch. Optimized Redox Environments,” Methods Enzymol. (1996) Biochem. Biophys. (1993) 305:509-515. 272:5 1-64. GenBank Accession No. AAC39505, 1998. Porter & Chang, “Strategies to Enhance the Coexpression of GenBank Accession No. AAD44150, 2001. Cytochrome P450 2E1 and Reductase in Bacteria,” Drug Metab. Rev. GenBank Accession No. AAD44151, 2001 . (1999) 31:159-174. GenBank Accession No. AB015762, 2001 . Rademacher, “Growth Retardants: Effects on Gibberellin GenBank Accession No. CAA70575, 2004. Biosynthesis and Other Metabolic Pathways,” Annu. Rev. Plant GenBank Accession No. U48435, 1997. Physiol. Plant Mol. Biol. (2000) 51:501-531. GenBank Accession No. X96784, 1996. Ralston et al., “Cloning, Heterologous Expression, and Functional GenBank Accession No. Y09447. Characterization of 5-epi-Aristolochene-1,3-Dihydroxylase from Hallahan et al., “Cytochrome-P450-Catalysed Monoterpenoid Oxi Tobacco (Nicotiana t0bacum),” Arch. Biochem. Biophys. (2001) dation in Catmint (Nepeta racemosa) and Avocado (Persea 393:222-235. americana): Evidence for Related Enzymes with Different Activi Rising et al., “Demonstration of Germacrene A as an Intermediate in ties,” Biochim. Biophys. Acta. (1994) 1201:94-100. 5-epi-Aristolochene Synthesis Catalysis,” J. Am. Chem. Soc. (2000) Hallahan & West, “Cytochrome P450 in Plant/Insect Interactions: 122:1861-1866. Geraniol 10-Hydroxylase and the Biosynthesis of Iridoid Schalk & Croteau, “A Single Amino Acid Substitution (F3631) Con Monoterpenoids,” Drug Metabol. Drug Interact. (1995) 12:369-3 82. verts the Regiochemistry of the Spearmint (—)-Limonene Helliwell et al. “Cloning of the Arabidopsis ent-Kaurene Oxidase Hydroxylase from a C6- to a C3 -Hydroxylase,” Proc. Natl. Acad. Sci. Catalyzes Three Steps of Gibberellin Biosynthesis,” Plant Physiol. USA. (2000) 97: 1 1948-1 1953. (1999) 119:507-510. Schuler, “Plant Cytochrome P450 Monooxygenases,” Crit. Rev. Holton et al., “Cloning and Expression of Cytochrome P450 Genes Plant Sci. (1996) 15:235-284. Controlling Flower Colour,” Nature (1993) 366:276-279. Starks et al., “Structural Basis for Cyclic Terpene Biosynthesis by Ho shino et al., “5 -epi-Aristolochene 3-Hydroxylase from Green Pep Tobacco 5-epi-Aristolochene Synthase,” (1997) 277: 1815-1820. per,” Phytochemistry (1995) 38:609-613. Stolle et al., “Restricted Colonization by Peronospora tabacina and Humphreys & Chapple, “Molecular ‘Pharming’ with Plant P450s.” Phytoalexin Accumulation in Immunized Tobacco Leaves,” Trends in Plant Science (2000) 5:271-272. Phytopathology (1988) 78: 1 193-1 197. US 8,263,362 B2 Page 3

Takemoto et al., “Molecular Cloning of a Defense-Response-Related Chappell et al., “Elicitor-inducible 3-hydroxy-3-methylglutaryl Cytochrome P450 Gene from Tobacco,” Plant Cell Physiol. (1999) coenzyme A reductase activity is required for sesquiterpene accumu 40: 1232-1242. lation in tobacco cell suspension cultures,” Plant Physiol. 97 :693 -698 Threlfall & Whitehead, “Co-Ordinated Inhibition of Squalene (1991). Synthetase and Induction of Enzymes of Sesquiterpenoid Chappell et al., “Is the reaction catalyzed by 3-hydroxy-3 Phytoalexin Biosynthesis in Cultures of Nicotiana tabacum,” methylglutaryl coenzyme A reductase a rate-limiting step for Phytochemistry (1988) 27:2567-2580. isoprenoid biosynthesis in plants,” Plant Physiol. 109:1337-1343 Urban et al., “Maximizing the Expression of Mammalian (1995). Cytochrome P-450 Monooxygenase Activities in Yeast Cells,” Chappell, “The biochemistry and molecular biology of isoprenoid Biochimie (1990) 72:463-472. metabolism,” Plant Physiol. 107:1-6 (1995). Urban et al., “Cloning, Yeast Expression, and Characterization of the Chen at al., “Cloning, expression and characterization of (+)-6 Coupling of Two Distantly Related Arabidopsis thaliana NADPH cadinene synthase: a catalyst for cotton phytoalexin biosynthesis,” Cytochrome P450 Reductases with P450 CYP73A5,” J. Biol. Chem. Arch. Biochem. Biophys. 324:255-266 (1995). (1997) 272:19176-19186. Chiu et al., “Engineered GFP as a vital reporter in plants,” Curr. Biol. Vogeli & Chappell, “Induction of Sesquiterpene Cyclase and Sup 6:325-330 (1996). pression of Squalene Synthetase Activities in Plant Cell Cultures Dekeyser et al., “Transient gene expression in intact and organized Treated with Fungal Elicitor,” Plant Physiol. (1988) 88: 1291-1296. rice tissues,” Plant Cell 2:591-602 (1990). Vogeli et al., “Puri?cation and Characterization of an Inducible Devarenne et al., “Molecular characterization of tobacco squalene Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspen synthase and regulation in response to fungal elicitor,” Arch. sion Cultures,” Plant Physiol. (1990) 93:182-187. Biochem. Biophys. 349:205-215 (1998). Walker & Croteau, “Molecular Cloning of a 10-Deacetylbaccatin Draper et al., “Ti plasmid homologous sequences present in tissues III-10-O-Acetyl Transferase cDNA from Taxus and Functional from Agrobacterium plasmid-transformed Petunia protoplasts,” Expression in Escherichia coli,” Proc. Natl. Acad. Sci. USA. (2000) Plant Cell Physiol. 23:451-458 (1982). 97:583-587. Fang et al., “Multiple cis regulatory elements for maximal expression Watson et al., “Sesquiterpenoid Stress Metabolites in Capsicums,” of the cauli?ower mosaic virus 35S promoter in transgenic plants,” Biochem. Soc. Trans. (1983) 11:589. Plant Cell 1:141-150 (1989). Werck-Reichhart et al., “Cytochromes P450 for Engineering Herbi Freeman et al., “A comparison of methods for plasmid delivery into cide Tolerance,” Trends in Plant Science (2000) 5:116-123. plant protoplasts,” Plant Cell Physiol. 25:1353-1365 (1984). Whitehead et al., “5 -epi-Aristolochene is a Common Precursor of the Frohman et al., “Rapid production of full-length cDNAs from rare Sesquiterpene Phytoalexins Capsidiol and Debneyol,” transcripts: ampli?cation using a single gene-speci?c Phytochemistry (1989) 28:775-779. oligonucleotide primer,” Proc. Natl. Acad. Sci. USA. 85:8998-9002 Whitehead et al., “Synthesis of (+)-5-epi-Aristolochene and (+)-1 (1988). Deoxycapsidiol from Capsidiol,” Phytochemistry (1990) From et al., “Stable transformation of maize after gene transfer by 29:479-482. electroporation,” Nature 319:791-793 (1986). Whitehead, “Cis-9,10-Dihydrocapsenone: A Possible Catabolite of From et al., “An octopine synthase enhancer element directs tissue Capsidiol from Cell Suspension Cultures of Capsicum annuum,” speci?c expression and binds ASF-1, a factor from tobacco nuclear Phytochemistry (1987) 26: 1367-1369. extracts,” Plant Cell 1:977-984 (1989). Wust et al, “Hydroxylation of Limonene Enantiomers and Analogs Gasser, C. and R. Fraley, “Genetically engineering plants for crop by Recombinant (-31 )-Limonene 3- and 6-Hydroxylases from Mint improvement,” Science 244:1293-1299 (1989). (Mentha) Species: Evidence for Catalysis within Sterically Con GenBank Accession No. CAC24711, Entry name: Cytochrome strained Active Sites,” Arch. Biochem. Biophys. (2001) 387:125 P450, updated last on Apr. 15, 2005, Version CAC24711.1 and 136. Retrieved from the Internet:

Kay et al., “Duplication of CaMV 35S promoter sequences creates a Tarshis et al., “Regulation of product chain length by isoprenyl strong enhancer for plant genes,” Science 236: 1299-1302 (1987). diphosphate synthases,” Proc. Natl. Acad. Sci. USA. 93:15018 Kindle, K., “High-frequency nuclear transformation of 15023 (1996). Chlamydomonas reinhardtii,” Proc. Natl. Acad. Sci. USA 87: 1228 Terada, R. and K. Shimamoto, “Expression of CaMV35S-GUS gene 1232 (1990). in transgenic rice plants,” Mol. Gen. Genet. 220:389-392 (1990). Koepp et al., “Cyclization of geranylgeranyl diphosphate to taxa Thai et al., “Farnesol is utilized for isoprenoid biosynthesis in plant 4(5),11(12)-diene is the committed step of taxol biosynthesis in cells via farnesyl pyrophosphate formed by successive Paci?c Yew,” J. Biol. Chem. 270:8686-8690 (1995). monophosphorylation reactions,” Proc. Natl. Acad. Sci. USA. Kuhlemeier et a1 ., “The pea rch-SA promoter mediates light respon 96:13080-13085 (1999). Thornburg et al., “Wound-inducible expression of a potato inhibitor siveness but not organ speci?city,” Plant Cell 1:471-478 (1989). II-chloramphenicol acetyltransferase gene fusion in transgenic Marcotte et al., “Abscisic acid-responsive sequences from the Em tobacco plants,” Proc. Natl. Acad. Sci. USA. 84:744-748 (1987). gene ofwheat,” Plant Cell 1:969-976 (1989). Trant, J ., “Functional expression of recombinant spiny dog?sh shark Nelson, D. and H. Strobel, “Evolution of cytochrome P-450 pro (Squalus acanthias)cytochrome P450c17 (17 alpha-hydroxylase/ teins,” Mol. Biol. Evol. 4(6):572-593 (1987). C17,20-lyase) in yeast (Pichia pastoris),” Arch. Biochem. Biophys. Newman et al., “Characterization of the TAC box, a cis-element 326(1):8-14 (1996). within a elicitor-inducible sesquiterpene cyclase promoter,” Plant J. Tudzynski, B., “Biosynthesis of gibberellins in Giberella fujikuroi: 16:1-12 (1998). biomolecular aspects,” Appl. Microbiol. Biotechnol. 52(3):298-310 Odell et a1 ., “Identi?cation of DNA sequences required for activity of (1999). the cauli?ower mosaic virus 25S promoter,” Nature 313:810-812 Umemoto et al., “cDNAs sequences encoding cytochrome P450 (1985). (CYP71 family) from eggplant seedlings,” FEBS 330(2): 169-173 Ohnuma et al., “A role of the amino acid residue located on the ?fth (1993). position before the ?rst aspartate-rich motif of farnesyl diphosphate UniProKB/Swiss-Prot entry P93530, Entry name: Cytochrome P450 sythase of determination of the ?nal product,” J. Biol. Chem. 71D6, updated last on Aug. 10, 2010, Version 53 and Retrieved from 271:30748-30754 (1996). the Internet:

Deguerry et al., “The diverse sesquiterpene pro?le of patchouli, Nunez, S. and J. Trant, “Isolation of the putative cDNA encoding Pogostemon cablin, is correlated With a limited number of cholesterol side chain cleavage cytochrome P450 (CYP11A) of the sesquiterpene synthases,” Arch. Biochem. Biophys. 454: 123-136 southern stingray (Dasyatis americana),” Gene 187(1): 123-129 (2006). (1997). Devarenne et al., “Regulation of squalene synthase, a key enzyme of Ralston et al., “Biochemical and molecular characterization of 5-epi aristolochene 3-hydroxylase, a putative regulatory enzyme in the sterol biosynthesis, in tobacco,” Plant Physiol. 129: 1095-1 106 biosynthesis of sesquiterpene phytoalexins in tobacco,” Plant Inter (2002). actions With Other Organisms. Annual Meeting of the American Greenhagen et al., “Probing sesquiterpene hydroxylase activities in a Society of Plant Physiologists. Madison, WI., Jun. 27-Jul. 1, 1998, coupled assay With terpene synthases,” Arch. Biochem. Biophys. Session 47:Abstract #737 (Poster Presentation) 2 pages. 409:385-394 (2003). Trant, J ., “Isolation and characterization of the cDNA encoding the Greenhagen et al., “Identifying and manipulating structural determi spiny dog?sh shark (Squalus acanthias) form of cytochrome nates linking catalytic speci?cities in terpene synthases,” Proc. Natl. P450c17,” J. Exp. Zool. 272(1):25-33 (1995). Acad. Sci. USA. 103:9826-9831 (2006). Wu et al., “Expression cloning and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal enzyme O’Maille et al., “Biosynthetic potential of sesquiterpene synthases: possessing 20 alpha-hydroxysteroid dehydrogenase activity,” J. Biol. alternative products of tobacco 5-epi-aristolochene synthase,” Arch. Chem. 268(17): 12964-12969 (1993). Biochem. Biophys. 448:73-82 (2006). Decision to Grant, issued May 19, 2011, in connection With corre Schenk et al., “Stereochemistry and deuterium isotope effects asso sponding European Patent Application No. 027097971, 1 page. ciated With the cyclization-rearrangements catalyzed by tobacco Appeal Decision, issued Oct. 21, 2011, in connection With corre epiaristolochene and hyoscyamus premnaspirodiene synthases, and sponding Japanese Patent Application No. 2002-571814, 18 pages. the chimeric CH4 hybrid cyclase,” Arch. Biochem. Biophys. 448 :3 1 - Allylix, “Protein engineering and chembiosynthesis to produce novel 44 (2006). sesquiterpenoids,” Presentation at BIO World Congress on Industrial Takahashi et al., “Kinetic and molecular analysis of Biotechnology & Bioprocessing, Washington, DC, held on Jun. 28, 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme 2010, 19 pages. GenBank Accession No. AB015762, (2000). catalyzing successive hydroxylations of sesquiterpenes,” J. Biol. GenBank Accession No. A35867, (1996). Chem. 280:3686-3696 (2005). GenBank Accession No. CAA70575, (2004). Takahashi et al., “Functional characterization of premnaspirodiene GenBank Accession No. U48435, (1997). oxygenase, a cytochrome P450 catalyzing regio- and stereo-speci?c GenBank Accession No. X96784, (2006). hydroxylations of diverse sesquiterpene substrates,” J. Biol. Chem. GenBank Accession No. Y09447, ( 1999). 43:31744-31754 (2007). Gonzalez & Korzekwa, “Cytochromes P450 Expression Systems,” Takahasi et al., “Metabolic engineering of sesquiterpene metabolism Annu. Rev. Pharmacol. Toxicol. (1995) 35:369-390. in yeast,” Biotechnol. Bioeng. 97: 170-181 (2007). Gotoh, “Substrate Recognition Sites in Cytochrome P450 Family 2 Wu et al., “Surrogate splicing for functional analysis of sesquiterpene (CYP2) Proteins Inferred From Comparative Analyses of Amino synthase genes,” Plant Physiol. 138:1322-1333 (2005). Acid and Coding Nucleotide Sequences,” J. Biol. Chem. (1992) Wu et a1 ., “Redirection of cyto solic or plastidic isoprenoid precursors 267:83-90. elevates terpene production in plants,” Nat. Biotechnol. 24:1441 Guarente et al., “A GAL 10-CYC1 Hybrid Yeast Promoter Identi?es 1447 (2006). the GAL4 Regulatory Region as an Upstream Site,” Proc. Natl. Acad. Zhao et al., “Eremophilane sesquiterpenes from capsidiol,” J. Org. Sci. USA. (1982) 79:7410-7414. Chem. 69:7428-7435 (2004). Hahn, “Microbial Elicitors and their Receptors in Plants,”Annu. Rev. US. Appl. No. 13/199,349, ?ling date Aug. 26, 2011. Phytopathol. (1996) 34:387-412. Andersson, S., and N. Moghrabi, “Physiology and molecular genet Hallahan et al., “Cytochrome-P450-Catalysed Monoterpenoid Oxi ics of 17 beta-hydroxysteroid dehydrogenases,” Steroids 62(1): 143 dation in Catmint (Nepeta racemosa) and Avocado (Persea 147 (1997). americana); Evidence for Related Enzymes With Different Activi Nomura et al., “The cDNA cloning and transient expression of a ties,” Biochim. Biophys. Acta. (1994) 1201:94-100. chicken gene encoding cytochrome P-450scc,” Gene 185(2):217-222 (1997). * cited by examiner US. Patent Sep. 11,2012 Sheet 1 0f 11 US 8,263,362 B2

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famesyi diphosphateOPP 5-» epi-aristolocheneg ' ‘

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+02 "in 1-deoxycapsidioi B-Eeoxycapsid i0} (3hydroxy-S?pi-aristoiochene} (1-hydroxy-5~epi-arisiofochene) US. Patent Sep. 11,2012 Sheet 2 0f 11 US 8,263,362 B2

FIG. 2

100

Enzymeofactivity(%maximum) 50

0 3 6 9121518212427 Time after elicitation (h) US. Patent Sep. 11,2012 Sheet 3 0f 11 US 8,263,362 B2

FIG. 3A

125

100 ? Enzymeactivity(%ofmaximum) 75 50

U I 1 i i 0 25 50 75 ion Inhibitor concentration (,uM) FIG. 3B

125

Enzymeactivity(%maximum)of 50

0 i l i * U 25 50 75 190 Inhibitor concentration (pM)

US. Patent Sep. 11,2012 Sheet 5 0f 11 US 8,263,362 B2

FIG.5

control eiicitor-treated {J 24 .515 3 I 9 12 18 24

CYP71D

CYP73A

CYPBZE

CYP92A

Loading control US. Patent Sep. 11,2012 Sheet 6 0f 11 US 8,263,362 B2

FIG. 6A

0.006

Absorbance 41005

-0.018

l 400 450 500 Wavelength (nm) FIG. 65

0.006

-0.006

Absorbance 41012

{3.018

-0.024 | 1 r 400 450 509 Waveiength (nm) US. Patent Sep. 11,2012 Sheet 7 0f 11 US 8,263,362 B2

FIG. 7A é E E % 71D71 D AA+ ig

FIG. 78 empty A+ empty A

FIG. 7C

71D D+ 71D!)

FIG. 7D empty D+ empty D~

'21: Hz"? 81 1 14 Time (min)

US. Patent Sep. 11,2012 Sheet 10 0f 11 US 8,263,362 B2

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MY US. Patent Sep.11,2012 Sheetllrofll IJS 8 9 263,362 B2

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NJ; , mw US 8,263,362 B2 1 2 CYTOCHROME P450S AND USES THEREOF tutive. Hoshino et al. (Phytochemistry 38:609-613, 1995) added to the observations of Whitehead et al. (Phytochemis RELATED APPLICATIONS try 28:775-779, 1989) by directly measuring 3-hydroxylase activity in microsomal preparations of arachidonic acid-elic This application claims the bene?t of the priority date of ited Capsicum annuum fruits and seedlings. These assays US. patent application Ser. No. 10/097,559 ?led on Mar. 8, consisted of incubating 5-epi-aristolochene with microsome 2002 (now issued US. Pat. No. 7,405,057), which claims the preparations and subsequently determining the amount of bene?t of US. Provisional Application Nos. 60/274,421 and 1-deoxycapsidiol generated by a combination of thin-layer 60/275,597, ?led on Mar. 9, 2001 and Mar. 13, 2001, respec chromatography (TLC) separations and gas chromatography tively, all of which are hereby incorporated by reference. (GC). Their evidence demonstrated that the conversion of 5-epi-aristolochene to 1-deoxycapsidiol was dependent on FIELD OF THE INVENTION both NADPH and OZ, and that 1-deoxycapsidiol accumula tion in vitro was arrested by the P450 antagonists carbon This invention relates to cytochrome P450s and uses thereof. monoxide (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964), ancymidol (Coolbaugh et al., Plant Physiol. 62:571 BACKGROUND OF THE INVENTION 576, 1978), and ketoconazole (Rademacher, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51:501-531, 2000). Cytochrome P450s encompass a superfamily of oxidases Recent results suggest that the hydroxylation of 5-epi responsible for the oxidation of numerous endobiotics and 20 aristolochene is an important regulated step in capsidiol bio thousands of xenobiotics. In addition, in plants, cytochrome synthesis. In studies to evaluate the effectiveness of methyl P450s play important roles in wound healing, pest resistance, jasmonate as an inducer of capsidiol biosynthesis in tobacco signaling, and anti-microbial and anti-fungal activity. cell cultures, Mandujano-ChaveZ et al. (Arch. Biochem. Bio Capsidiol is a bicyclic, dihydroxylated sesquiterpene pro phys. 381 :285-294, 2000), reported that the modest accumu duced by many Solanaceous species in response to a variety 25 lation of this phytoalexin was accompanied by a strong induc of environmental stimuli, including exposure to UV (Back et tion of EAS. This result implied that steps before or after the al., Plant Cell. Physiol. 389:899-904, 1998) and infection by sesquiterpene cyclase reaction were limiting. Using an in vivo microorganisms (Molot et al., Physiol. Plant Pathol. 379-389, assay measuring the conversion rate of radiolabeled 5-epi 1981; Stolle et al., Phytopathology 78:1193-1197, 1988; aristolochene to capsidiol, a very limited induction of the Keller et al., Planta. 205:467-476, 1998). It is the primary 30 hydroxylase activities was observed in cells treated with antibiotic or phytoalexin produced in tobacco in response to methyl jasmonate relative to that in fungal elicitor-treated fungal elicitation, and it is derived from the isoprenoid path cells. This result pointed to the hydroxylase reactions as a way via its hydrocarbon precursor, 5-epi-aristolochene (FIG. potentially limiting step in capsidiol biosynthesis. 1). Several of the biosynthetic enzymes leading up to 5-epi aristolochene formation have been studied (Chappell, Annu. 35 SUMMARY OF THE INVENTION Rev. Plant Physiol. Plant Mol. Biol. 46:521-547, 1995), espe cially 5-epi-aristolochene synthase (BAS) (Vogeli and Chap In one aspect, the invention features several isolated cyto pell, Plant Physiol. 88: 1291 -1296, 1988; Back and Chappell, chrome P450 polypeptides (such as CYP71D20, CYP71D21, Proc. Natl. Acad. Sci. U.S.A. 93:6841-6845, 1996; Mathis et CYP73A27, CYP73A28, and CYP92A5, and P450s having al., Biochemistry 36:8340-8348, 1997; Starks et al., Science 40 substantial identity to these polypeptides), as well as isolated 277: 1815-1820, 1997). BAS commits carbon to sesquiter nucleic acid molecules that encode these P450s. pene metabolism by catalyZing the cyclization of famesyl In related aspects, the invention features a vector (such as diphosphate (FPP) to 5-epi-aristolochene. However, until the an expression vector) including an isolated nucleic acid mol present invention, the Biochemical evidence from previous ecule of the invention and a cell (for example, a prokaryotic studies in tobacco (Whitehead et al., Phytochemistry 28:775 45 cell, such as Agrobaclerium or E. coli, or a eukaryotic cell, 779, 1989) and green pepper (Hoshino et al., Phytochemistry such as a mammalian, insect, yeast, or plant cell) including 38:609-613, 1995) have suggested that the oxidation of 5-epi the isolated nucleic acid molecule or vector. aristolochene to capsidiol occurs in a two step process with In yet another aspect, the invention features a transgenic one of the hydroxylation steps being constitutive and the plant or transgenic plant component including a nucleic acid other being mediated by an elicitor-inducible cytochrome 50 molecule of the invention, wherein the nucleic acid molecule P450 (FIG. 1). Because 1-deoxycapsidiol had been isolated is expressed in the transgenic plant or the transgenic plant from natural sources (Watson et al., Biochem. Soc. Trans. component. Preferably, the transgenic plant or transgenic 11:589, 1983), Whitehead et al. (Phytochemistry 28:775-779, plant component is an angiosperm (for example, a monocot or 1989), surmised that perhaps the biosynthesis of this inter dicot). In preferred embodiments, the transgenic plant or mediate was due to pathogen induction of a corresponding 55 transgenic plant component is a solanaceous, maize, rice, or hydroxylase. They therefore prepared synthetic 1-deoxycap cruciferous plant or a component thereof. The invention fur sidiol and reported a modest conversion of this compound to ther includes a seed produced by the transgenic plant or capsidiol when fed to control or unelicited tobacco cell cul transgenic plant component, or progeny thereof. tures. This was further supported by their observation that In another aspect, the invention features a method of pro radiolabeled 5-epi-aristolochene was only converted to cap 60 viding an increased level of resistance against a disease sidiol when fed to elicitor-induced cell cultures but not con caused by a plant pathogen in a transgenic plant. The method trol cultures. Whitehead et al. (Phytochemistry 28:775-779, involves: (a) producing a transgenic plant cell including the 1989) therefore concluded that the 3-hydroxylase, respon nucleic acid molecule of the invention integrated into the sible for hydroxylation of 5-epi-aristolochene at C3 to gen genome of the transgenic plant cell and positioned for expres erate 1-deoxycapsidiol, was pathogen/elicitor inducible, 65 sion in the plant cell; and (b) growing a transgenic plant from while the 1-hydroxylase, responsible for hydroxylating the plant cell wherein the nucleic acid molecule is expressed 1-deoxycapsidiol at the C1 to generate capsidiol, was consti in the transgenic plant and the transgenic plant is thereby US 8,263,362 B2 3 4 provided with an increased level of resistance against a dis of comparison sequences will generally be at least 50 nucle ease caused by a plant pathogen. otides, preferably at least 60 nucleotides, more preferably at In another aspect, the invention features a method for pro least 75 nucleotides, and most preferably 110 nucleotides. ducing an altered compound, the method including the steps Sequence identity is typically measured using sequence of contacting the compound with one or more of the isolated analysis software (for example, Sequence Analysis Software polypeptides disclosed herein under conditions allowing for Package of the Genetics Computer Group, University of Wis the hydroxylation, oxidation, demethylation, or methylation consin Biotechnology Center, 1710 University Avenue, of the compound and recovering the altered compound. Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX In still another aspect, the invention features a hydroxylat programs). Such software matches identical or similar ing agent including any of the isolated polypeptides disclosed sequences by assigning degrees of homology to various sub herein. stitutions, deletions, and/or other modi?cations. Conserva In yet another embodiment, the invention features an iso tive substitutions typically include substitutions within the lated nucleic acid molecule that speci?cally hybridizes under following groups: glycine alanine; valine, isoleucine, leu highly stringent conditions to the complement of any one of cine; aspartic acid, glutamic acid, asparagine, glutamine; the sequences described in SEQ ID NO:2 (CYP71D20), SEQ serine, threonine; lysine, arginine; and phenylalanine, ID NO:4 (CYP71D21), SEQ ID N016 (CYP73A27), SEQ ID tyrosine. N018 (CYP73A28), or SEQ ID NO: 12 (CYP92A5), wherein By an “isolated polypeptide” is meant a P450 polypeptide such a nucleic acid molecule encodes a cytochrome P450 (for example, a CYP71D20 (SEQ ID NO: 1), CYP71D21 polypeptide. (SEQ ID NO:3), CYP73A27 (SEQ ID NO:5), CYP73A28 In another aspect, the invention features a host cell express 20 (SEQ ID NO:7), or CYP92A5 (SEQ ID NO:1 1) polypeptide) ing a recombinant isoprenoid synthase and a recombinant that has been separated from components that naturally cytochrome P450. In preferred embodiments, the host cell accompany it. Typically, the polypeptide is isolated when it is further expresses, independently or in combination, a recom at least 60%, by weight, free from the proteins and naturally binant acetyltransferase, methyltransferase, or fatty acyl occurring organic molecules with which it is naturally asso transferase. In other preferred embodiments, the host 25 ciated. Preferably, the preparation is at least 75%, more pref expresses an endogenous or recombinant cytochrome reduc erably at least 90%, and most preferably at least 99%, by tase. Preferably, the host cell is a yeast cell, a bacterial cell, an weight, a P450 polypeptide. An isolated P450 polypeptide insect cell, or a plant cell. may be obtained, for example, by extraction from a natural In a related aspect, the invention features a method for source (for example, a plant cell); by expression of a recom producing an isoprenoid compound, the method including the 30 binant nucleic acid encoding a P450 polypeptide; or by steps of: (a) culturing a cell that expresses a recombinant chemically synthesizing the protein. Purity can be measured isoprenoid synthase and a recombinant cytochrome P450 by any appropriate method, for example, column chromatog under conditions wherein the isoprenoid synthase and the raphy, polyacrylamide gel electrophoresis, or by HPLC cytochrome P450 are expressed and catalyze the formation of analysis. an isoprenoid compound not normally produced by the cell; 35 By “derived from” or “obtained from” is meant isolated and (b) recovering the isoprenoid compound. In preferred from or having the sequence of a naturally-occurring embodiments, the host cell further expresses a recombinant sequence (e.g., cDNA, genomic DNA, synthetic, or combi acetyltransferase, a recombinant methyltransferase, or a nation thereof). recombinant fatty acyltransferase. In other preferred embodi By “isolated nucleic acid molecule” is meant a nucleic acid ments, the host cell expresses an endogenous or recombinant 40 molecule, e.g., a DNA molecule, that is free of the nucleic cytochrome reductase. Preferably, the host cell is a yeast cell, acid sequence(s) which, in the naturally-occurring genome of a bacterial cell, an insect cell, or a plant cell. the organism from which the nucleic acid molecule of the In yet another aspect, the invention features an isoprenoid invention is derived, ?ank the nucleic acid molecule. The compound produced according to the above-mentioned term therefore includes, for example, a recombinant DNA methods. 45 that is incorporated into a vector; into an autonomously rep By “P450 polypeptide,” “cytochrome P450,” or “P450” is licating plasmid or virus; or into the genomic DNA of a meant a polypeptide that contains a heme-binding domain prokaryote or eukaryote; or that exists as a separate molecule and shows a CO absorption spectra peak at 450 nm according (for example, a cDNA or a genomic or cDNA fragment pro to standard methods, for example, those described herein. duced by PCR or restriction endonuclease digestion) inde Such P450s may also include, without limitation, hydroxy 50 pendent of other sequences. The term “isolated nucleic acid lase activity, dual hydroxylase activity, demethylase activity, molecule” also includes a recombinant DNA which is part of or oxidase activity. Such enzymatic activities are determined a hybrid gene encoding additional polypeptide sequence. using methods well known in the art. By “speci?cally hybridizes” is meant that a nucleic acid By “polypeptide” is meant any chain of amino acids, sequence is capable of hybridizing to a DNA sequence at least regardless of length or post-translational modi?cation (for 55 under low stringency conditions, and preferably under high example, glycosylation or phosphorylation). stringency conditions. For example, high stringency condi By “substantially identical” is meant a polypeptide or tions may include hybridization at approximately 42° C. in nucleic acid exhibiting at least 800r 85%, preferably 90%, about 50% formamide, 0.1 mg/ml sheared salmon sperm more preferably 95%, and most preferably 97%, or even 98% DNA, 1% SDS, 2>