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Plant and Soil Sciences Faculty Patents Plant and Soil Sciences
5-13-2014 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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Recommended Citation Chappell, Joe and Ralston, Lyle F., "Cytochrome P450S and Uses Thereof" (2014). Plant and Soil Sciences Faculty Patents. 16. https://uknowledge.uky.edu/pss_patents/16
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(12) United States Patent (10) Patent N0.2 US 8,722,363 B2 Chappell et a]. (45) Date of Patent: *May 13, 2014
(54) CYTOCHROME P450S AND USES THEREOF 6,531,303 B1 3/2003 Millis et a1...... 435/155 6,559,297 B2 5/2003 Chappellet a1. .. . 536/23.1 ~ . ~ 6,569,656 B2 5/2003 Chappellet a1. .. .. 435/183 (n)A”mmtu%ER$mm&Mmm@m QMWQB21MW3Qm?MAH “Mw? . 6,689,593 B2 2/2004 Millis et al. .. 435/155 (72) Inventors: Joseph Chappell,~Lex1ngton, KY (US); 6,890,752 B2 500% chappenet 31‘ u N 435625 Lyle F. Ralston, Sao Paulo (BR) 7,186,891 B1 3/2007 Chappell et a1. .. .. 800/298 7,405,057 B2* 7/2008 Chappellet a1. .. . 435/69.1 (73) Assignee: University of Kentucky Research 9141313611? 31~ ~~ ~ 5536655332? - - , , u 1en e a ...... Foundatlon’ Lexmgton’ KY (Us) 8,106,260 B2 1/2012 Chappelletal. .. .. 800/298 8,124,811 B2 2/2012 J l' t l...... 568/367 ( * ) Notice: Subject to any disclaimer, the term ofthis 8,192,950 B2 @2012 6111;123:112 31‘ n 435/41 patent 1s extended or adjusted under 35 8,263,362 B2* 9/2012 Chappellet a1. .. . 435/69.1 Lrsx3.154(h)hy()days. 8354504 B2 02013 (happenetm... n 530679 _ _ _ _ _ 8362309 B2 02013 Juhenetal.n ..583B60 Th1s patent 1s 511131601 to a term1nal d1s- 8,445,231 B2* 5/2013 Chappellet a1. . 435/69.1 Claimer_ 8,481,286 B2 7/2013 Julien etal. 435/69.1 2003/0166255 A1 9/2003 Chappell ...... 435/252.3 _ 2004/0078840 A1 4/2004 Chappellet a1. .. .. 800/278 (21) APPI'NO" 13/986’446 2006/0218661 A1 9/2006 Chappelletal. .. .. 800/278 _ 2007/0231861 A1 10/2007 Millis et al. . 435/69.1 Q3 Fm?l WhylZM3 mmmmmwim 10mm Mmmmmnu “m4unm 2007/0238159 A1 10/2007 Millis et al. 435/252.33 (65) Prior Publication Data 2007/0238160 A1 10/2007 Millis et al. 435/252.33 2007/0254354 A1 11/2007 Millis et al...... 435/252.33 US 2013/0330793 A1 Dec. 12, 2013 . (Cont1nued)
(63) Continuation of application No. 13/ 199,349, ?led on / Aug. 26, 2011, HOW Pat. No. 8,445,231, which is a {50 Zooggs/éégg‘; I? continuation of application No. 12/ 182,000, ?led on I Jul. 29, 2008, HOW Pat. No. 8,263,362, which is a (Commued) continuation of application No. 10/097,559, ?led on OTHER PUBLICATIONS Mar. 8, 2002, now Pat. No. 7,405,057. International Search Report, issued Dec. 17, 2002, in connection With (60) Provisional application No. 60/274,421, ?led on Mar. 9, 2001, provisional application No. 60/275,597, ?led corresponding International Patent Application No. PCT/US02/ on Mar. 13,2001. 06912, 4 pages. International Preliminary Examination Report, issued Apr. 24, 2003, (51) Int. Cl. in connection With corresponding International Patent Application No. PCT/US02/06912, 4 pages. C12N1/00 (2006.01) Partial European Search Report, issued Jun. 16, 2004, in connection C12N1/06 (2006.01) With corresponding European Patent Application No. 027097971, 7 C12N 5/00 (2006.01) pages. C12N 5/07 (2010.01) Of?ce Action, issued Jul. 1, 2004, in connection With corresponding (52) US. Cl. U.S. Appl. No. 10/097,559, 10 pages. USPC ...... 435/69.1; 435/468; 435/348; 435/252.1; Supplemental European Search Report, issued Sep. 8, 2004, in con 435/252.2; 435/155; 435/419 nection With corresponding European Patent Application No. (58) Field of Classi?cation Search 027097971, 6 pages. None See application ?le for complete search history. (Continued) (56) References Cited Primary Examiner * Medina A Ibrahim (74) Attorney, Agent, 0rFirm * McKenna Long &Aldridge U.S. PATENT DOCUMENTS LLP; Stephanie Seidman 5,589,619 A 12/1996 Chappell et a1...... 800/205 (57) ABSTRACT 5,672,487 A 9/1997 Schweden et a1. .. .. 435/69.1 5,741,674 A 4/1998 Schweden et a1. .. .. 435/69.1 The invention features isolated cytochrome P450 polypep 5,766,911 A 6/1998 Koike et a1. 435/193 tides and nucleic acid molecules, as well as expression vec 5,824,774 A 10/1998 Chappell et a1. 530/350 tors and transgenic plants containing these molecules. In 5,981,843 A 11/1999 Chappell et a1. 800/301 5,994,114 A 11/1999 Croteau et a1. .. 435/232 addition, the invention features uses of such molecules in 6,072,045 A 6/2000 Chappell et a1. .. 536/23.1 methods of increasing the level of resistance against a disease 6,100,451 A 8/2000 Chappell et a1. 800/298 caused by a plant pathogen in a transgenic plant, in methods 6,117,649 A 9/2000 Bellamine et a1...... 435/25 for producing altered compounds, for example, hydroxylated 6,194,185 B1 2/2001 Croteau et a1...... 435/189 compounds, and in methods of producing isoprenoid com 6,331,660 B1 12/2001 Chomet et a1. .. 800/278 6,368,837 B1 4/2002 Gatenby et a1. . 435/146 pounds. 6,468,772 B1 10/2002 Chappell et a1. 435/183 6,495,354 B2 12/2002 Chappell et a1...... 435/183 29 Claims, 11 Drawing Sheets US 8,722,363 B2 Page 2
(56) References Cited Examination Report, issued Nov. 20, 2012, and received Nov. 23, 2012, in connection With corresponding Canadian Patent Application U.S. PATENT DOCUMENTS No. 2,440,278, 7 pages. Akiyoshi-Shibata et al., “Further oxidation of hydroxycalcidiol by 2008/0178354 A1 7/2008 Chappell et al...... 800/298 calcidiol 24 -hydroxylase. A study With the mature enzyme expressed 2008/0233622 A1 9/2008 Julien et al. 435/148 in Escherichia coli,” Eur. J. 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Patent May 13, 2014 Sheet 1 0f 11 US 8,722,363 B2 FIG. 1 1 \ EAS 2 / \ 3 . farnesyldiphosphateOPP 5~ep1~ansto|ochene. \ I" OH “\ +NADPH HOW“ +02 1-deoxycapsidiol 3-deoxycapsidiol (3-hydroxy-5-epi-aristolochene) (1-hydroxy-5-epi-a?stolochene) \ \ \ OH Oapsidiol US. Patent May 13, 2014 Sheet 2 0f 11 US 8,722,363 B2 FIG. 2 100 75 Enzymeactivity(%ofmaximum) 50 25 I I 6 9 12 15 1 8 2 12427 Time after elicitation (h) US. Patent May 13, 2014 Sheet 3 0f 11 US 8,722,363 B2 FIG. 3A 125 100 Enzymeactivity(%ofmaximum) 01\i OU! I N 01 O I _| I I O 25 50 75 100 inhibitor concentration (pM) FIG. 3B 125 100 Enzymeactivity(%ofmaximum) (It\I O01 N 01 O l I I 0 25 50 75 1 00 Inhibitor concentration (1.1M) U S. Patent May 13, 2014 Sheet 5 0f 11 US 8,722,363 B2 km, g imading mmimi US. Patent May 13, 2014 Sheet 6 0f 11 US 8,722,363 B2 FIG. 6A 0.006 -0.006 I Absorbance -0.012 I -0.018 400 450 500 Wavelength (nm) FIG. GB 0.006 -0.006 Absorbance -0.012 ~0.018 -0.024 | 1 I 400 450 500 Wavelength (nm) US. Patent May 13, 2014 Sheet 7 0f 11 US 8,722,363 B2 FIG. 7A 71 D A+ 1-deoxycapsidiot- 'QDSM'IO!- 71DA , --»’-/'--...... -,...... lill'lllllll FIG. 7B empty A+ empty A iIlljltllllll FIG. 7C 71D D+ 71D D FIG. 7D empty D+ empty D Time (min) US. Patent May 13, 2014 Sheet 8 0f 11 US 8,722,363 B2 US. Patent May 13, 2014 Sheet 9 0f 11 US 8,722,363 B2 S H D A US. Patent May 13, 2014 Sheet 10 0f 11 US 8,722,363 B2 324 321 332 327 295 339 363 360 371 366 334 377 400 397 409 404 372 415 439 436 448 443 411 454 KETLRLH PFGAGRR E Figure8C PERF mRVVGKKAKVE US. Patent May 13, 2014 Sheet 11 or 11 US 8,722,363 B2 478 475 487 482 450 489 Figure8D US 8,722,363 B2 1 2 CYTOCHROME P450S AND USES THEREOF to elicitor-induced cell cultures but not control cultures. Whitehead et al. (Phytochemistry 28:775-779, 1989) there RELATED APPLICATIONS fore concluded that the 3-hydroxylase, responsible for hydroxylation of 5-epi-aristolochene at C3 to generate This application is a continuation of US. patent applica 1-deoxycapsidiol, was pathogen/elicitor inducible, while the tion Ser. No. 13/199,349, ?led Aug. 26, 2011 (now issued 1-hydroxylase, responsible for hydroxylating 1-deoxycap US. Pat. No. 8,445,231), which is a continuation of US. sidiol at the C1 to generate capsidiol, was constitutive. patent application Ser. No. 12/182,000, ?led Jul. 29, 2008 Hoshino et al. (Phytochemistry 38:609-613, 1995) added to (now issued US. Pat. No. 8,263,362), which is a continuation the observations of Whitehead et al. (Phytochemistry 28:775 of US. patent application Ser. No. 10/097,559, ?led Mar. 8, 779, 1989) by directly measuring 3-hydroxylase-activity in 2002 (now issued US. Pat. No. 7,405,057), which claims the microsomal preparations of arachidonic acid-elicited Capsi bene?t of US. Provisional Application Nos. 60/274,421 and cum annuum fruits and seedlings. These assays consisted of 60/275,597, ?led on Mar. 9, 2001 and Mar. 13, 2001, respec incubating 5-epi-aristolochene with microsome preparations tively, all of which are hereby incorporated by reference. and subsequently determining the amount of 1 -deoxycapsid iol generated by a combination of thin-layer chromatography FIELD OF THE INVENTION (TLC) separations and gas chromatography (GC). Their evi dence demonstrated that the conversion of 5-epi-aris This invention relates to cytochrome P450s and uses tolochene to 1-deoxycapsidiol was dependent on both thereof. NADPH and O2, and that 1-deoxycapsidiol accumulation in 20 vitro was arrested by the P450 antagonists carbon monoxide BACKGROUND OF THE INVENTION (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964), ancymidol (Coolbaugh et al., Plant Physiol. 62:571-576, Cytochrome P450s encompass a superfamily of oxidases 1978), and ketoconazole (Rademacher, Annu. Rev. Plant responsible for the oxidation of numerous endobiotics and Physiol. Plant Mol. Biol. 51:501-531, 2000). thousands of xenobiotics. In addition, in plants, cytochrome 25 Recent results suggest that the hydroxylation of 5-epi P450s play important roles in wound healing, pest resistance, aristolochene is an important regulated step in capsidiol bio signaling, and anti-microbial and anti-fungal activity. synthesis. In studies to evaluate the effectiveness of methyl Capsidiol is a bicyclic, dihydroxylated sesquiterpene pro jasmonate as an inducer of capsidiol biosynthesis in tobacco duced by many Solanaceous species in response to a variety cell cultures, Mandujano-Chavez et al. (Arch. Biochem. Bio of environmental stimuli, including exposure to UV (Back et 30 phys. 381 :285-294, 2000), reported that the modest accumu al., Plant Cell. Physiol. 389:899-904, 1998) and infection by lation of this phytoalexin was accompanied by a strong induc microorganisms (Molot et al., Physiol. Plant Pathol. 379-389, tion of EAS. This result implied that steps before or after the 1981; Stolle et al., Phytopathology 78:1193-1197, 1988; sesquiterpene cyclase reaction were limiting. Using an in vivo Keller et al., Planta. 205:467-476, 1998). It is the primary assay measuring the conversion rate of radiolabeled 5-epi antibiotic or phytoalexin produced in tobacco in response to 35 aristolochene to capsidiol, a very limited induction of the fungal elicitation, and it is derived from the isoprenoid path hydroxylase activity was observed in cells treated with way via its hydrocarbon precursor, 5-epi-aristolochene (FIG. methyl jasmonate relative to that in fungal elicitor-treated 1). Several of the biosynthetic enzymes leading up to 5-epi cells. This result pointed to the hydroxylase reactions as a aristolochene formation have been studied (Chappell, Annu. potentially limiting step in capsidiol biosynthesis. Rev. Plant Physiol. Plant Mol. Biol. 46:521-547, 1995), espe 40 cially 5-epi-aristolochene synthase (BAS) (Vogeli and Chap SUMMARY OF THE INVENTION pell, Plant Physiol. 88: 1291 -1296, 1988; Back and Chappell, Proc. Natl. Acad. Sci. USA. 93:6841-6845, 1996; Mathis et In one aspect, the invention features several isolated cyto al., Biochemistry 36:8340-8348, 1997; Starks et al., Science chrome P450 polypeptides (such as CYP71D20, CYP71D21, 277: 1815-1820, 1997). BAS commits carbon to sesquiter 45 CYP73A27, CYP73A28, and CYP92A5, and P450s having pene metabolism by catalyzing the cyclization of famesyl substantial identity to these polypeptides), as well as isolated diphosphate (FPP) to 5-epi-aristolochene. However, until the nucleic acid molecules that encode these P450s. present invention, the enzyme(s) responsible for the conver In related aspects, the invention features a vector (such as sion of 5-epi-aristolochene to capsidiol has yet to be fully an expression vector) including an isolated nucleic acid mol identi?ed and characterized. 50 ecule of the invention and a cell (for example, a prokaryotic Biochemical evidence from previous studies in tobacco cell, such as Agrobaclerium or E. coli, or a eukaryotic cell, (Whitehead et al., Phytochemistry 28:775-779, 1989) and such as a mammalian, insect, yeast, or plant cell) including green pepper (Hoshino et al., Phytochemistry 38:609-613, the isolated nucleic acid molecule or vector. 1995) have suggested that the oxidation of 5-epi-aris In yet another aspect, the invention features a transgenic tolochene to capsidiol occurs in a two step process with one of 55 plant or transgenic plant component including a nucleic acid the hydroxylation steps being constitutive and the other being molecule of the invention, wherein the nucleic acid molecule mediated by an elicitor-inducible cytochrome P450 (FIG. 1). is expressed in the transgenic plant or the transgenic plant Because 1-deoxycapsidiol had been isolated from natural component. Preferably, the transgenic plant or transgenic sources (Watson et al., Biochem. Soc. Trans. 11:589, 1983), plant component is an angiosperm (for example, a monocot or Whitehead et al. (Phytochemistry 28:775-779, 1989), sur 60 dicot). In preferred embodiments, the transgenic plant or mised that perhaps the biosynthesis of this intermediate was transgenic plant component is a solanaceous, maize, rice, or due to pathogen induction of a corresponding hydroxylase. cruciferous plant or a component thereof. The invention fur They therefore prepared synthetic 1-deoxycapsidiol and ther includes a seed produced by the transgenic plant or reported a modest conversion of this compound to capsidiol transgenic plant component, or progeny thereof. when fed to control or unelicited tobacco cell cultures. This 65 In another aspect, the invention features a method of pro was further supported by their observation that radiolabeled viding an increased level of resistance against a disease 5-epi-aristolochene was only converted to capsidiol when fed caused by a plant pathogen in a transgenic plant. The method US 8,722,363 B2 3 4 involves: (a) producing a transgenic plant cell including the or nucleic acid sequence (for example, the nucleic acid nucleic acid molecule of the invention integrated into the sequences shown in SEQ ID NOS12, 4, 6, 8 and 12, respec genome of the transgenic plant cell and positioned for expres tively). Forpolypeptides, the length of comparison sequences sion in the plant cell; and (b) growing a transgenic plant from will generally be at least 16 amino acids, preferably at least 20 the plant cell wherein the nucleic acid molecule is expressed amino acids, more preferably at least 25 amino acids, and in the transgenic plant and the transgenic plant is thereby most preferably 35 amino acids. For nucleic acids, the length 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 5 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 20 cine; aspartic acid, glutamic acid, asparagine, glutamine; the sequences described in SEQ ID N012 (CYP71D20), SEQ serine, threonine; lysine, arginine; and phenylalanine, ID N014 (CYP71D21), SEQ ID N016 (CYP73A27), SEQ ID tyrosine. N018 (CYP73A28), or SEQ ID N0112 (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 N011), CYP71D21 polypeptide. 25 (SEQ ID N013), CYP73A27 (SEQ ID N015), CYP73A28 In another aspect, the invention features a host cell express (SEQ ID N017), or CYP92A5 (SEQ ID N011 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 30 occurring organic molecules with which it is naturally asso transferase. In other preferred embodiments, the host 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 35 source (for example, a plant cell); by expression of a recom producing an isoprenoid compound, the method including the 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 40 analysis. an isoprenoid compound not normally produced by the cell; 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 45 By “isolated nucleic acid molecule” is meant a nucleic acid ments, the host cell expresses an endogenous or recombinant 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 50 term therefore includes, for example, a recombinant DNA methods. 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 C0 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. 55 duced by PCR or restriction endonuclease digestion) inde Such P450s may also include, without limitation, hydroxy 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 sequences. 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, 60 sequence is capable of hybridizing to a DNA sequence at least regardless of length or post-translational modi?cation (for 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 80 or 85%, preferably 90%, about 50% formamide, 0.1 mg/mL sheared salmon sperm more preferably 95%, and most preferably 97%, or even 98% 65 DNA, 1% SDS, 2>