US007033807B2 (12) United States Patent (10) Patent No.: US 7,033,807 B2 Bolten et al. (45) Date of Patent: Apr. 25, 2006

(54) ASPERGILLUS OCHRACEUS 11 ALPHA Arfin et al., Proc. Natl. Acad. Science (1995) 92:77147718. HYDROXYLASE AND OXDOREDUCTASE Armour et al., FEBS Lett. (1992) 307:113-115. (75) Inventors: Suzanne L. Bolten, Kirkwood, MO Baldwin et al., Gene Ther. (1997) 4:1142–1149. (US); Robert A. Clayton, Foristell, MO Barany, Proc. Natl Acad. Science (1991) 88:189-193. (US); Alan M. Easton, Maryland Zamechik et al. Proc. Natl. Acad. Sci. (1986) 83:4143–4146. Heights, MO (US); Leslie C. Engel, Baum et al., J. Hematother (1996) 5:323-329. Des Pere, MO (US); Dean M. Messing, Becker et al., EMBO J. (1989) 8:3685–3691. St. Louis, MO (US); John S. Ng, Ben-Bassat et al., J. Bacteriol. (1987) 169:751–757. Thousand Oaks, CA (US); Beverly Berkner, BioTechniques (1988) 6:616–629. Berkner, Current Top. Microbiol. Immunol. (1992) Reitz, Chesterfield, MO (US); Mark C. 158:39, 66. Walker, Chesterfield, MO (US); Ping Blobel and Dobberstein, J. Cell Biol. (1975) 67:835–851. T. Wang, Manchester, MO (US) Boris-Lawrie and Temin, Annal. New York Acad. Sci. (73) Assignee: Pharmacia Corporation, St. Louis, (1994) 716:59–71. Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. (1993) MO (US) 3:102 109. (*) Notice: Subject to any disclaimer, the term of this Bostian et al., Cell (1984) 36:741–751. patent is extended or adjusted under 35 Botstein et al., Ann. J. Hum. Genet. (1980) 32:314–331. U.S.C. 154(b) by 565 days. Bregni et al., Blood (1992) 80: 1418–1422. Breskvar Ket al., Biochem. Biophys. Res. Commun (1991) 178, 1078–1083. (21) Appl. No.: 10/021,425 Brody and Crystal, Annal. New York Acad. Sci. (1994) (22) Filed: Oct. 30, 2001 716:90 103. Capecchi, Cell (1980) 22:479–488. (65) Prior Publication Data Chen et al., Gene Ther. (1998) 5:50–58. US 2003/0148420 A1 Aug. 7, 2003 Clapp, Clin. Perinatol. (1993) 20:155–168. Collins, W.P. Alternative Immunoassays, (1985) Related U.S. Application Data 1-38,77–86,103-184. (60) Provisional application No. 60/244.300, filed on Oct. 30. Corbi and Lopez Rodriguez, Leuk. Lymphoma (1997) 2000. 25:415 425. (51) Int. Cl. (Continued) CI2N 9/02 (2006.01) Primary Examiner Charles L. Patterson, Jr. CI2N 15/53 (2006.01) (74) Attorney, Agent, or Firm—Christopher W. Slavinsky (52) U.S. Cl...... 435/189: 435/252.3; 435/320.1; (57) ABSTRACT 536/23.2 The present invention relates to a novel cytochrome P450 (58) Field of Classification Search ...... 435/189, like enzyme (Aspergillus ochraceus 11 alpha hydroxylase) 435/252.3, 25, 320.1; 536/23.2 and an oxidoreductase (Aspergillus och race us See application file for complete search history. oxidoreductase) isolated from cDNA library generated from the mRNA of Aspergillus ochraceus spores. When the (56) References Cited cDNA encoding the 11 alpha hydroxylase was co-expressed U.S. PATENT DOCUMENTS in Spodoptera frugiperda (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it 4.419,446 A 12/1983 Howley et al...... 435/68 successfully catalyzed the conversion of the steroid substrate 4,559,332 A 12, 1985 Grob et al...... 514,175 4,588,683 A 5/1986 Goodhue et al...... 435/59 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as 4,935,233 A 6, 1990 Bell et al...... 424,85.5 determined by HPLC analysis. The invention also relates to 5,348,886 A 9, 1994 Lee et al...... 435/320.1 nucleic acid molecules associated with or derived from these 5,384,253 A 1/1995 Krzyzek et al...... 435/1723 cDNAS including complements, homologues and fragments 5,422,262 A 6, 1995 Andersson et al...... 435.240.1 thereof, and methods of using these nucleic acid molecules, 5,679,521 A 10, 1997 Andersson et al...... 435/6 to generate, for example, polypeptides and fragments 5,869,283 A 2/1999 Slijkhuis et al. .... 435/69.1 thereof. The invention also relates to the generation of 6,046,023 A 4/2000 Wiersma et al...... 435/60 antibodies that recognizes the A. ochraceus 11 alpha hydroxylase and oxidoreductase and methods of using these FOREIGN PATENT DOCUMENTS antibodies to detect the presence of these native and recom WO O 528 906 B1 11, 1995 binant polypeptides within unmodified and transformed host WO WO98,25948 6, 1998 cells, respectively. The invention also provides methods of expressing the Aspergillus 11 alpha hydroxylase gene OTHER PUBLICATIONS separately, or in combination with human or Aspergillus Tudzynski, B., et al. (1998) Acc. No. GFP4501.* oxidoreductase, in heterologous host cells, to facilitate the Kennedy, J., et al. (1999) Acc. No. AF 141924.* bioconversion of steroid substrates to their 11 alpha Altschul SF, etal; J. Molec Biol. (Oct. 5, 1990) p. 403-410. hydroxy-counterparts. Anfossi et al., Proc. Natl. Acad. Science (1989) 86:3379. 3383. 15 Claims, 25 Drawing Sheets US 7,033,807 B2 Page 2

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Figure 1 - Nucleotide and protein sequence of Aspergillus ochraceus 11 alpha hydroxylase

tggaagtttt tacact tatt atgccggagc cgaaagattic tag togagg ggttggggaa 50 cala cactata agacctacala coacttggat ttggtgaatt tacacgggcai ttatcaaaac 120 agccacaa.gc tigacagctica ttatc atg cocc ttc tic act gigg Ctt ctg gCg 172 Met Pro Phe Phe Thr Gly Lieu Lieu Ala 1. 5 att tac cat ag, ctic atta coc grac aac Coca gtC cala acc ctg agc acc 220 Ile Tyr His Ser Leu Ile Lieu. Asp Asin Pro Wall Glin Thr Le Ser Thr 10 15 20 t 25 at gtC gta titg gcg gCa gCg tac tigg Ctic gca acg Ctic Cag ccg agc 258 Le Val Val Leu Ala Ala Ala Tyr Trip Leu Ala Thr reu Gin Pro Ser 30 3S AO gac citt CCt gag citg aat ccc gcc aaa coca t to gag to acc aat cqt 35 Asp Leu Pro Glu Lieu. Asn Pro Ala Lys Pro Phe Glu Phe Thr Asn Arg AS 50 55 cg Cgt gtt Cat gag titt gttgata aat agt aag age tig cit gct cgg 364 Arg Arg Val His Glu Phe Val Glu Asn Ser Lys Ser Leu Leu Ala Arg 60 65 70 ggg agg gaa ttg Caic ggg cac gag CC9 tact aga citc atg. tct gaa tigg 412 Gly Arg Glu Lieu. His Gly His Glu Pro Tyr Arg Lieu Met Ser Glu Trip 75 8O 85 gga toc ttg att gte citg ccc cca gag togc gcc gac "gag ctg cgc aac 460 Gly Ser Leu e Val Eleu Pro Pro Glu Cys Ala Asp Glu eu Arg Asn 9. 95 100 OS gae coca aga atg giac titt gag acg CCC alCC acc giac gac tocc CaC gga 508 Asp Pro Arg Met Asp Phe Glu Thr Pro Thr Thr Asp Asp Ser His Gly 110 15 120 tat atc cct ggc titc glac got Ctc. aac goal gaC cc.g. aac ctg act alala 556 Tyr Ile Pro Gly Phe Asp Ala Leu Asn Ala Asp Pro Asn Lieu. Thr Lys 25 130 35 gtg g to acc aag tac ctc aca aaa gcai ttg aac aag Ctt act gct CC 604 Val Val Thir Lys Tyr Lieu Thir Lys Ala .eu. Asn Lys Leu Thr Ala Pro 140 145 1SO atc. tcg cat gaa gog toc atc gcc atg aaa gog gtg ctg. g.g. t gale gat SS2 Ile Ser His Glu Ala Ser Ilie Ala Met Lys Ala Wall Le Gly Asp Asp 55 160 55 cca gat tig cit gag atc taC cca gcc aga gaC titg Ctic cag ctic to 700 Pro Asp Trp Arg Glu Ile Tyr Pro Ala Arg Asp Leu. Leu Gin Leu Vai 170 75 18O 185 gcc cgg atg tcg aca agai gtg tte ctic ggc gag gala atg to aat aac 748 Ala Arg Met Ser Thr Arg Val Phe Leu Gly Glu Glu Met Cys Asn Asn 190 1.9S 2OO cag gat tgg at C Caa a CC tea to a caa tac gig gcc ctit gcc tic gg. 796 Glin Asp Trp Ilie Glin Thr Ser Ser Glin Tyr Ala Ala Leu Ala Phe Gly 20S 210 25 gtc gg. t gac aag ctt aga ata tac CCg aga atg atc aga ccg ata gta 844 Val Giy Asp Llys Leu Arg Ile Tyr Pro Arg Met Ile Arg Pro Ile Val 220 225 230 cat tgg tte atg cca toc togt tgg gag ctg. Cgc cga tog Ctg cga cqc 892 His Trp Phe Met Pro Ser Cys Trp Glu Leu Arg Arg Ser Leu Arg Arg 235 240 25 tgc cg a cag att Ctc acg ccg tac att cac aaa cgc aag tec citg aag 940 Cys Arg Glin Ile Leu Thr Pro Tyr Ile His Lys Arg Lys Ser Leu Lys 250 255 250 265 U.S. Patent Apr. 25, 2006 Sheet 2 of 25 US 7,033,807 B2

Figure , continued

99. acc acg gac gag. cag grgc aag Cecc c atg t gat gat c ac 988 Gy Tr Thr Glu Gl Gly Lys Pro el et Phe Aso Asp Sea le 270 275 280

gag. tgg tec gag. cga. gas cg gg CCC c Cate gac cg gtC cg aag O36 Glu Trp Phe Glu Arg Gl Luel Gy Pro As His Asp Alia Wall Le Lys 285 290 29S

cag gtc acg cc tec aea get gct aea eas ca acg agt gac ca c O84. Gn Wa Thr Iue Se le Va. Ala le His Thr Thr Se Asp Le Le 300 3OS 30

ttg cag gCC atg agc gait ce gcg cag aac ccg. all gtg ca Ca gCa 32 e Gn Ala Me Sea As Le Ala Glin As Pro Iys Wall eu Glin Ala 35 32O 325

gtg cgc gag. gag. gtg gtic cga. gtg Ctg agic gag. 999 t agc aag 18O Wall Arg Glu Gu. Val Wall Arg Wall e Sea T Glu Gly el Ser Lys 330 335 340 345

gtic tog c aac agt tC aag c atg gac agc gcg ttg aag gaa agic 228 Wall Ser Lel s Sea Leu Lys eu Met Se Ala Leu Llys Glu Ser 350 355 360

cag cgt. ctC agg cc. acg citt ctic ggc tic titt cgt. Cgg cag gca acg 1276 Gr Arg Lel Arg el e Gly Se Phe Arg Gl Ala T 365 370 375

aat gac atc. aag ctg aag agc s t gtic atta tag 999 aga 1324. As Asp Ile Lys Leu Lys Ser Gly phe Wall le Lys Lys Gly Arg 380 35 390

gto gtg atc. gac agc YCC cate atg tgg at ccc. gag. ea teac gac 1372 Wall Wall e Asp Ser Thr His e Trp Asn Po Glu Tyr Asp 395 4OO 405 cost ctic cag tac gac tac cgc tac ttc C aag cgg Cag act cco 420 Pic eu Gin Tyr Asp Gly Arg Phe As Lys Arg Glth T Pro 40 45 20 25

sc gag. gac aag aac cg tte etc. gtc. agc agc gCC c Cac atg 1468 Gy G.u. Asp Iys Asn Ala e eu Wall Set Sea Ala As His Met AsO 435 440

gga tic gigt cac ggc gtt cac gcc. gt. cct go aga tic gcc. ecc 15.5 Gly Phe Gly His Gly Wall His Ala Cys Pro Gly Phe Ala Se 445 450

aac gag. ac aag att gcc ttg tgt. cate tec atc ta as tate ga egg 1564 Asn Glu Ile Lys Jille Alia Cys His e e Leul As Gill Trip 460 465 O

cgt. cCa gac ggs c aag c Cag c c C ac 999 atg act 1612 Arg e Pro As Gly Phe yS Pro Gin Po Le As le Gy et T 475 480 485

late citg gCg gat ccc. aat age atg Ctg atc. agg cca cgc aag gC9 1660 Tyr Leu Ala Also Pro As Arg Met el Ile Arg Pro Arg Lys Ala 49 495 SOO 505

gag. etc. gat atg gCg. agt act gtg tag gtcgaacacg aagttcCtgat 170 Gl le Asp Met Ala Se r Val 50 gaagttgttat tytcagtgg gttgaa.gcaa.g. tcgcagaa at tigaacaat titatalagaat 17 O aaaaa. U.S. Patent Apr. 25, 2006 Sheet 3 of 25 US 7,033,807 B2

Figure 2 - Nucleotide and protein sequence of human oxidoreductase

atg gga gac c Cac gtg gac Coc agc tec aCC gtg tcc gag. gcg gtg 48 Met Gy Asp Ser His Wall Asp T Se Sea Thr Wall St. Glu Ala Wall O 15

gcc. gaa gaa gta tott Ctte tetic agc atg acg gac atg att Ctg titt tog 95 Ala Glu Glu Val Ser Phe Ser Met Thr Asp Met Ile Lieu Phe Ser 20 25 30

ctic atc. gtg ggit ctic cite act ac tgg t ctic tetic aga aag --- s 44 Luel le Wall Gly eu. Trp Phe Lieu. Phe Arg Lys Lys Lys 35 40 AS

gaa gaa gto cCC gag. titc acc al att Cag AC ttg acc ticc tect gtC 192 Glu Glu Wall Pro Glu Phe Thr Lys Ile Glin Thr Leu Thr Ser Ser Wall 50 55 60 aga gag agc agc titt gtg gaa aag atg aag acg 9. ls aC atc 240 Arg Glu Se Ser Phe Wall Glu Lys Met ys Iys Thr Gly Arg As Ile 55 70 75 80 atc gtg titc. tac 9C tCc Cag acg 99 act gca 9a 99. titt gCC EC 288 le Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Giu Gu Phe Ala As 85 90 95

CgC Ctg toc aag gac gcc. CaC cgc taC 999 atg cga. ggc atg toa Cg 336 Arg Leu Se Lys Asp Ala His Arg Gy Met Gly Met Set Ala 100 105 1.0

gac cct gag. gag. tat gac ct CC gac Ctg agc agc Ctg cca gag atc 384 Asp Pro Glu Glu Asp Leu Ala Leu Ser Ser Lieu Pro Glu e 11S 120 12S

gac aac gcc Ctg gtg gtt tec tgc atg gotc sCC tac ggit gag. gga gac 432 Asp Asn Ala Leu Wall Wall Ple Cys Met Ala Thr Gly Glu Gy Asp 30 135 140

CCC acc gaic aat gcc cag gac titc. tac gac tgg Ctg Cag gag. C. gac 480 Pro Thr Asp As Ala Gn Asp Phe Tyr Asp Trp el Glin Glu Th Aso 145 150 55 160

gtg gat ce tect 999 gtc. aag titc gtg t 99t ct 999 aag 528 Wall Asp Lieu Se Gly Wall Lys Phe Wall Phe Gly Leu Gly Lys 1.65 170

CC tead gag CaC titc. aat gCC atg ggc alag tac gtg gac aag Ctg 576 Th Glu His Phe Asn Ala Met. Gly Lys Tyr Wall Asp Lys Leu 180 185 90

gag. cag ctic ggc gcc. cag CC atc t gag. Ctg 999 tg ggc gaC 624 Giu. Glin Lieu Gly Ala Glin Arg Ile Phe Gill Leu Gly Leul Gly Asp 19s 200 205

gat ggg s ttg gag gag. gac tC ac aCC tgg cga gag. cag ttc tgg 672 Asp Gly As el Glu Glui Asid Phe le Thr Trip Arg Glu Glin Phe Trp 210 215 220

cc.g gcc gtg gaa CaC t 999 gala gCC act 99C gag. 9a 9 CC 720 Pro Ala Wall Glu His Phe Gly Val Glu Ala Gly Gill Glu Sea 225 230 235 240

agc att cgc cag ac gag. citt gtg gto CC CC gac ata gat gcg gcc 768 Ser le Arg Glin Tyr Glu Lell Wall Wall His Th Asp Ile Asp Ala Ala 245 2SO 255 aag gtg taC atg 999 gag. atg ggC Cgg Ctg aag agc teac gag. aC cag 816 Lys Val Tyr Me Gly Glu Met Gly Arg Leu Lys Sea Tyr Glu As Glin 260 265 270

aag ccc Ccc titt gat gcc. aag aat ccg. t Ctg gct gca gtc. CC acc 864. Lys Pro Pro Phe Asp Ala Lys As Pro Phe Lieu Ala Ala Wall This h 275 280 28S

eaC cgg aag ctg LaC cag gga LCC CgC CaC ctic atg CLC Ctg gaa 912 Ast Arg Lys Leu As Glin Gly Th Arg His e Met His Leu Glu 290 295 300

ttg gac atc gac tecc la ac agg talt gaa tot ggg gac CaC 950

U.S. Patent Apr. 25, 2006 Sheet 5 Of 25 US 7,033,807 B2

Figure 2, continued

50 55 62 cac atc tact g to tgt ggg gat gca Cigg aac atg goc agg gat gtg cag 1920 His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Val Gln 525 630 535 640 aac acc titc. tac gaC atc gtg get gag Cto ggg gCC atg gag cac geg 1958 Asn Thr Phe Tyr Asp Ile Val Ala Glu Lieu Gly Ala Met Glu His Ala 645 650 555 Cag gog gtg gac tac atc aag aala Ctg atg acc aag ggc cgc tac tocc 2O6 Glin Ala Wall Asp Tyr le Lys Lys Leu Met Thr Lys Gy Arg Tyr Ser 560 565 670 Cg gaC gttg to agc 2O3 Leu Asp Val Trp Ser 6S U.S. Patent Apr. 25, 2006 Sheet 6 of 25 US 7,033,807 B2

Figure 3 - Nucleotide and protein sequence of Aspergillus ochraceus oxidoreductase

cittattitcgt. ttaggaagag caccggct to gigg tectic cetacecce accc. SO ct tctgactic cct ttttgtt attgatcgcc catcteggtg aacatttggg atatoctitcc 20 cc.cccc. ccc.gc.cccga cccticcittat ct tct citcC cgtocagat ttagotcgcc 80 atcgaattcg caatticct tc Ctcgg actC titCatcgctg agcgtCctica to atg gog 238 et Ala l

c etc. gat ace etc gat tg gtic gtC citg gtg ggs gic tg 9tg 99t. 286 Glin e Asp Thr Le As Le Wall Wall eu. Wall Ala Lieu eu Wall Gy 10 5

agc gtg gCC tact t ag ggc c tec tgg gcc gto gcc s gac 33A Sea Wall Ala Tyr Pe Thr Lys Gly Thr Tyr Ala Wall Ala Lys Asp 20 25 3.

c ta gCC tog got ggit ceg gog atg ae gga ggc gcc. aag goto gg 382 Po Tyr Ala Sea Ala Gly Pro Alia Met As Gly Gly Ala Lys Ala Gly 35 40 45 50

aag act cgic gac att gtt cag atg gac gaa act c --- --C tgtc. 430 Lys T Asp e Wall Glin Lys Me Glu Thr Gly ys As Cys 55 SO SS

gtg att c tac ggc tg ca ecc gst acc act as gac tac gcg tes 478 Wall Ile Phe Tyr Gly See Glin Th Gly Ala Glt Asp Ala Se O 75 BO

aga Ctg gcc. aag gaa ggc cc Cag cga. ttc gigt ctic aag acc atg gtg S25 Arg Le Lys Gl Gly See Gl Arg Ple Gy Lys et Wall 85 90 95

gcc. gat ctg ga gac ac gac ac gaa c Ctg gaa aag c cacc gag. 574 Ala Asid Lieu Gill Asp Tyr Asp Tyr Clu As e Glu Lys Phe Pro Glu 100 LOS 10

gac alas get gtt c gtt ctg gcc e t ggc gag. 99. gaa ecc 622 Asp Lys Wall Wall Phe Phe Wall Le Ala Tyr Gly Gu Gly Glu Pic 15 2O 25 30

acg gat aate gog gtt gaa tec ac Cag tec gtc. acg gic gaa gat gCt 670 Asp As Ala Val Gu Phe Gl Phe Wall r Gly Glu Asp Ala 135 O 145

gct tec gag. agc ggc gct se gCC gaC gat aag cct Ctg t tet cc. 718 Ala Phe Giu See Gly Ala T Ala Asp Asp Lys Pro Le Ser Sea e SO 55 is

aag late gtc. acg t gigt Ctg ggit s s ass te gag s tal ael 766 Lys Tyr Wall The Phe Gly Le Gly Asn As r Gu His 1.65 170 5

got atg gtt cgc aat gtg gac gotc. got cc. C aag ttic so gcc ca 84 Ala Met Wall Arg As Wall Asp Ala Ala Luell T Lys She Gly Ala G 8O 85 90

cgc. atte ggc tect gct ggt. gag. gigt gac gac ggc gct ggt as atg gaa 852 Arg le Gly Se Alia Gly Glu Gly Asp Asp Gly Ala Gly Thr et Gu 95 200 205 2O

gas gat tec ctg gcc. tgg ag gaa c atg tgg get gcc. t t gag. 90 Glu Asp Phe Leu Ala Trp Iys Glu Pro Me Trp Ala At a Ser Gu 25 220 225

c atg eae Ctg se cgc. gat c gt. t ag cc. gic etc 95 Ala et As Luel Glin Glu Arg As Ala Wall Glu Pro Wall Phe Asn 2O 235 240

gto ace sa gac gag. ecc Ctg agc cCc gaa gate ag ac gt teac ctic 1005 Wall Thr Gil Asp Glu. Se el See o Glu Asp Giu As Wall Teu 245 250 255

gget gas cc.c act Casa gigt cale cc Cala 9C s acc aag ggc ccg ac Gly Glu Pro Thr GT Gly His el GI) Gly Gl Pro Iys Gly Pro 25 265 270 U.S. Patent Apr. 25, 2006 Sheet 7 Of 25 US 7,033,807 B2

Figure 3, continued tot gig cac alac cog tec atc. get cocc atc. tcc gala tot cgt. gala cig O2 Ser Ala His Asn Pro Phe Ile Ala Pro le Ser Gil Ser Arg Gu Lieu 275 28O 285 290 tCc aac gttcaag gae cgc aac tet ctg. cac atg gaa atc agc atc gotc. 150 Phe Asn Val Lys Asp Arg Asn. Cys Lieu. His Met Glu. Ie Ser Ile Ala 295 300 305 ggit agc aac Ctc act tac Cag act ggit gaC cac atc gct gtt teg CCC 1.98 Gly Ser Asn Leu Thr Tyr Glin Thr Gly Asp His Ile Ala Val Trp Pro 3. 315 320 acc aac gcc gigt to gag gc gat c.g. t.t.c cteg Cag get titt gg. CC 1245 Ihr Asn Ala Gly Ser Glu Val Asp Arg Phe Leu Girl Alia Phe Gly Le 3.25 330 335 gaal gga aag cqc cac toc g to atc. aac att aag gg atc gat gtg acc 294 Glu Gly Lys Arg His Ser Val Ile Asin Ile Lys Gly Ile Asp Val Thr 30 35 350 gct aag get cog at coc act cot acg acc tat gaC gcc gca gtt cgc 1342 Ala Lys Val Pro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala Val Arg 35s 360 355 370 tac tac citg gaa gtC tgt gCC CCC gtt toc cgt cag titt g to tcg act 1390 Tyr Tyr Leu Giu Val Cys Ala Pro Wal Ser Arg Grh Phe Val Ser Thr 375 38O 385 ctic got gc titt gcc cct gat gala gcg acc aag gog gag atc gtt cgt. 138 Leu Ala Ala Phe Ala Pro Asp Glu Ala Thr Lys Ala Glu le Val Arg 390 39S 400 ttg ggit gge gac aag gac tat ttc cat gag aag att acc aac ca tige 185 Leu Gly Gly Asp Lys Asp Tyr Phe His Glu Lys Ile Thr Asn Arg Cys O5 410 415 ttc. alac atc gct cag got Ctc. Cag agc atc acg tec aag Cect titc acc 154 Phe Asn ille Alia. Glin Ala Leu Gln Ser Ile Thr Ser Lys Pro Phe Thr . 420 42S 430 gcc g. tc. Cog titc. tcc ct citt atc gala gigt atc acci aag citt Cag CCC 52 Ala Val Pro Phe Ser Leu Lieu. I e Glu Gly Ile Thr Lys Leu Gln Pro A35 440 45 450 cgt tac tac tog atc. tcc tog tot coc ctg. gtt cag aag gac aag att 1530 Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Glin Lys Asp Lys Ile 45S 50 46S agc att acc gcc gtt gtg gag tog et Cec titg coct get gag gaa cac 1578 ser Ile Thr Ala Val Val Glu Serval Arg. Leu Pro Gly Glu Glu His 47 475 480 att gttcaag get g to acc acg aac tat Ctt Cte cg Ctc aag gaa aag 125 Ile Wall Lys Gly Val Thr Thr Asn Tyr Leu Leu Ala Leu Lys Glu Lys 485 90 m 95 caa aac ggc gag cct tcc cct gac cog cac ggc ttg act tac tot atc 77 Gln Asn Gly Glu Pro Ser Pro Asp Pro His Gly Leu Thr Tyr Ser Ile 500 505 510 act giga CCC cit aac aag taic gat ggc atc cat gtic cocci gtte Cac gitc 1822 Thr Gly. Pro Arg Asn Lys Tyr Asp gly Ile His Wall Pro Wal His Wall 55 520 525 530 cgc. cac tog aac ttc aaattg CCC tog gat coc tog cga cot gtg atc 1870 Arg His Ser Asn Phe Lys Leu Pro Ser Asp Pro Ser Arg Pro Wall Ile 535 540 545 atg gtt gga ccc gg t act gg. t gtt gct cot tte cgt ggg ttt atc. cag 1918 Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Ile Gln SS Sss 560 gag cgt get gCC titg gCC gog aag ggc gag aag gtc. giga act acc ttg 1966 Glu Arg Ala Ala Leu Ala Ala Lys Gly Glu Lys Val Gly Thr Thr Lieu. 555 S70 575 Ct to tec C tec cit aag tec gac gala gat titc titg tact aag at 2014 Leu Phe Phe Gly Cys Arg Lys Ser Asp Glu Asp Phe Leu Tyr Lys Asp 580 585 59 gaa tug aag act tet Cag gag cag cit ggc gac tog citc. aag atc atc 2062 U.S. Patent Apr. 25, 2006 Sheet 8 of 25 US 7,033,807 B2

Figure 3, continued Glu Trp Llys Thr Phe Glin Glu Gln Leu Gly Asp Ser Leu Lys Elle Ile 595 600 SOS 610 act gcc tte tet cgt gaa teg got gag aaa g to tac g to cag cac agg 210 Thr Ala Phe Ser Arg Glu Ser Ala Glu Lys Val Tyr wall Gln His Arg 65 62O 625 Ctg cgt gag cat gcc gag Ctg g to agt gae ctg. citg aag cag aaa gcc 21.58 Leu Arg Glu His Ala Glu Leu Val Ser Asp Leu Leu Lys Gln Lys Ala 530 535 640 act to tat gtt tec got ac got gee alac atgy gcc cat gala gttcaac 2206 Thr Phe Tyr Val Cys Gly Asp Ala Alia. Asni Met Ala Arg Glu Val Asn 65 6SO 555 ctic gtg Ctt ggg Caa atc att gCC aag cag cc gg. t c to cct gcc gag 2254 Leu Val Lev Gly Glin Ile le Ala Lys Glin Arg, Gy Leu Pro Ala Glu 660 665 670 alag grge gag gag atg gtg aag cac atg cgc agic age gig C agc tact cag 2302 Lys Gly Glu Glu Met Wall Lys His Met Arg Ser Ser. Gly Ser Tyr Glin 675 880 585 690 gac gat gttc tgg toc taa aa 2322 Asp Asp Wall Trip Ser 59S

U.S. Patent Apr. 25, 2006 Sheet 11 of 25 US 7,033,807 B2

Figure 5 - Phylogenetic tree showing the relatedness of Aspergillus ochraceus II alpha hydroxylase to the top 10 BLAST hits from GenBank

pMON45624 CAA/5566 AAD 54-552 CAA767O 3 CAA/5567 CAA57874 CAA9 1268 CAB565O3 AAB 94-588 CAA/5565 CAB9 1316 U.S. Patent Apr. 25, 2006 Sheet 12 of 25 US 7,033,807 B2

Figure 6 - Percent homology of Aspergillus ochraceus 11 alpha hydroxylase to the top 10 BLAST hits from GenBank

Accession Number Species %. D to 11a. OH

CAB936 Neurospora crassa 40

CAA76565 Gibberellafujikuroi 37

CAA7SS66 Gibberellafiujikuroi 37

AAD34552 Aspergillus ferreus 29

CAA75567 Gibberellafiujikuroi 24

CAA57874 Fusarium auysporum 24

CAA76703 Gibberellafiujikurol 23

CAB56503 Catharanthus roseus 14

AAB94588 Glycinemax 4.

CAA9 1268 Caenorhabditis elegans 2 U.S. Patent Apr. 25, 2006 Sheet 13 Of 25 US 7,033,807 B2

Figure 7-Amino acid homology alignment of A. ochraceus and human oxidoreductase to NADPH cytochrome P450 reductases from A. niger, mouse, and S. cerevisiae

human EEWSLFSRTCS, FSLIV FE MON4S605se EiSEE pMON45632 niger yeast

PMON45605 6. SA's Risk sh Firuisi, Ul'EE: DL human 6. O O R rose 6. pMON45632 47 niger 47 yeast 42

PMON45605 i20 human 2O rose 20 pMON45632 lo niger O7 yeast 102

PMON4560S 69 . y REGG Twi. Fw Pi. hulla 69 W w File:Stapi. CSS 1.69 al pMon45632 1.65 niger 164: yeast A8

PMONS605 228 hurtlan 228 Olse 228 pMON45632 225 as 2 niger 224 AYPVFC.T.E. yeast 208 EAKFrsfoy rvLN---EITD

pMON 45 605 25 hunar 25 actise 25 pMON45632 273 T niger 272 O yeast 262 P . SSS F. Silpa

PMON45605 335 A- -WW-S NiSNK...??'" EE human 335 www.SN, FSNS itri O rose 335 i EESNFHFC LTi Yilil TelPR pMON45632 333 LE N AWi r OAAR wV niger 332 s 3 f 's r > yeast 322

PMON56OS 393 human 393 a m O O se 393 . E. S. V V AFPI. L. YPSLFPP DELELF pMON45632 391 a Gr niger 390 yeast 38 U.S. Patent Apr. 25, 2006 Sheet 14 of 25 US 7,033,807 B2

Figure 7, continued humanPMONASSOS aS45 f a E: EK----e.g., as -- Ssilis, Fr. FP-E -AGF-- cuse 45 ----gister pMON45632 448 EsvRLP---GEEivisitTilskEK. yeastniger 4.436 As SWR.P.--- s PMON45605 53 human 503 pMON45632Se 53505 PDPHG-TYSITs . PiH, KSFRLsittilisD'Ripyt4WCFGTav EYEYE APFRG. niger 503 SRPSR-DT, HHPR O FS, sia CE. E.g. yeast 495 AETN, YD E. is plvi riis-Crgsyap FRC FIRs

PMON4555 5-7 VGETIG human 5 47 RAF S.E. --VG3 rose 547 - pMON45632 564 niger 562 Ris yeast 555

PMON 5605 599 (, ). W.E. human 5.99 TWC, Divists rose 599 ) pMON45632 617 WC), niger 615 v. L. yeast 54 victs), ide. ,

MON45605 658 vil human 658 se 659 Av. pMON45632 676 niger 64 yeast 63 PMON45605 human Se nigerPMON45632 yeast U.S. Patent Apr. 25, 2006 Sheet 15 Of 25 US 7,033,807 B2

Figure 8-A mino acid homology alignment of A. ochraceus oxidoreductase to NADPH cytochrome P450 reductases from A. niger and S. cerevisiae ... niger ... ochraces FXGAWADP ... cerevisiae Snake MSc ... niger ... ochraceus ... cerevisiae ... niger 12 ... ochraceus 120 cerevisiae 113

niger 19 river CLSEEREAE ... ochraces 180 -VRS RIGSAGEGD3AGTEEDF.A.. EPMWAA SigEgg cerevisiae 63 -i-L3 bill:AG" TEDSiWis SILEVLKE lish KS ... niger 239 ... ochraceus 240 . cerevisiae 223 niger 290 YGD2 i.v. wiya C ... ochraces 291 CG.I.V.PTNAG . cerevisiae 280 ''S ... niger 350 i'i La’, Aw FY: W. v. PWSRF w."Lar ... ochraceus 35 OWAW CAAWR, WSROWSSI cerevisiae 338 sier PWSRFS ... niger 4.09 ... ochraceus 41 cerevisiae 398 niger 58 ochraceus a 70 cerevisiae AS8 ... niger S25 Plitv' PTG. PFR.Fi. ... ochraceus 527 PWW G.E. CE . cerevisiae 58 WWr c Hit"I'lly A PF - Gh R ... niger 579 ... ochraceus 581 ... cerevisiae 578 ... niger 638 ochraceus 64 O , cerevisiae 637 ... ochraceus, ONASS32 SEQ D NO: Os) niger (SEO ED NO: 38) . cerevisiae, yeast (SEQ ID NO : 37) U.S. Patent Apr. 25, 2006 Sheet 16 of 25 US 7,033,807 B2

Figure 9- Phylogenetic tree showing the relatedness of Aspergillus ochraceus and human oxidoreductase to reductases from A. niger, Jeast, and VS PMON456O5 hum On pMON45632 niger yed st

Y O US 6 U.S. Patent Apr. 25, 2006 Sheet 17 Of 25 US 7,033,807 B2

Figure 10- Percent homology between Aspergillus ochraceus axidoreductase to reductases from A. niger, yeast, and mouse and human.

Accession number organism % id to A.oeh oxred CAasisso A. niger 84

BAAO2936 S. cerevisiae 37

BAA04496 Se 34

AAB284 human 33 U.S. Patent Apr. 25, 2006 Sheet 18 of 25 US 7,033,807 B2

Figure 11 - Amino acid homology alignment of human oxidoreductase with the top 4 hits from SwissProf

PMON455OS EAV, EEWSLFSt4T DigiLFSLI GLL.T. WFFF. KKKEEW PEFKITL human GDShiv SwS AFSS: Les Livist. Ywff Rikkk EEW PEFTTL rabbit a. ADBETAT Se DSESA'Ever ways VFSBL CITYFF & SEEK O'" pig

PON4 SSOs 'Gaie's SARY GRSSEE hurtar rabbit ra rose pig

PMONASSOs human rabbit rat Ilse pig 120 SSSLEEAA ADW DC via FG,Giv's

PMONS6OS 80 NAGWEKRLIGia, RE GODG EPA.V.A. human 18O rabbit 181 al 18O dise 18O pig 180

PON4S5OS 240 human 240 rabbit 24 Eas at 240 cuse 240 pig 240

PMON45605 300 human 3OO rabbit 301. at 3OO mouse 300 pig 3OO

PMON45605 360 PFCTSYRTAL'Y.DINpTN.W.E. ACYASSECELRRMASSSCE.G.L. LC. U.S. Patent Apr. 25, 2006 Sheet 19 Of 25 US 7,033,807 B2

PMON45.505 360 FIC". RTALTYDINPPR rif, Fafa S.PSDELL REaCSC Inar 350 rabbit 35 at 360 FPC PTK FAT Y.I.I.'s K.R.W.L. A.J.SS'SEE dise 350 pig 360

PMON4,5505 420 i. Riii. human 2O A v. E. S.; A. L. C.S. - ' ' ... it...... YYS ass-rvival vily rabbit 42 rat 420 420 420 was it a (Yes,’’

PMON45 605 480 human 48O rabbit 48. al 480 480 480 U.S. Patent Apr. 25, 2006 Sheet 20 of 25 US 7,033,807 B2

Figure 11, continued

PMONS5OS 540 hunar 50 rabbit S4 al 540 Se SAO pig 540

pMON4565 60 human 600 rabbit 60 EGGAEivOG Air NARDON" if at 6OO Olse 600 plg 600

PMON45 605 659 (SEQ D O3) human 659 (SEQ d 52) rabbit 661 WDYK. T. S. WIS (SEO O 53 ) at 660 visry's Grass (SEQ d 54 ) those 650 via F. S. WAS (SEQ ID 55) pig 650 di MI SRYS WS (SEQ d s 56) U.S. Patent Apr. 25, 2006 Sheet 21 of 25 US 7,033,807 B2

Figure 12- Phylogenetic tree showing the relatedness of human oxidoreductase (PI6435) with top 4 hits from Swiss Prof

PMON456O5 humon pig rot

Y O US 6 robbit U.S. Patent Apr. 25, 2006 Sheet 22 of 25 US 7,033,807 B2

Figure 13- Percent homology between human oxidoreductase and top 4 hits from Swiss Prot

Accession number Species % id to human oxred

POO388 at 92

P00389 rabbit 92

P37040 OS 92

PO475 pig 9. U.S. Patent Apr. 25, 2006 Sheet 23 Of 25 US 7,033,807 B2

Figure 14- Expression of Aspergillus ochraceus II alpha hydroxylase in transfected Sf9 insect cells Expression of 11-o-Hydroxylase in Transfected Sf9 Cells

U.S. Patent Apr. 25, 2006 Sheet 24 of 25 US 7,033,807 B2

Figure 15- Expression of Aspergillus ochraceus P450 oxidoreductase in transfected Sf9 insect cells Expression of Fungal P-450 Oxidoreductase in Transfected Sf9 Cells

D i. '',iii. 2O121 70 : 52.4 : ; :i : U.S. Patent Apr. 25, 2006 Sheet 25 Of 25 US 7,033,807 B2

Figure 16- Conversion of androstenedione to 11 alpha hydroxy androstenedione monitored by HPLC

60 androstenedione Y 40 20 11 a-hydroxyandrostenedione

O 60

40

20 B

O SO

40

O O 5 O 15 20 25 HPLC Retention Time (min.) US 7,033,807 B2 1. 2 ASPERGILLUS OCHRACEUS 11 ALPHA Econ. Microbiol., 5 (Microb. Enzymes Bioconvers.): HYDROXYLASE AND OXDOREDUCTASE 369–465, 1980). A variety of reactions have been characterized, including hydroxylation, epoxidation, PRIORITY oxidation, dehydrogenation, ring and side chain degradation, reduction, hydrolysis, and isomerization reactions. Many The present application claims priority under Title 35, types of microorganisms have also been used including United States Code, S 119 of U.S. Provisional Application species as diverse, for example, as Acremonium, Aspergillus, Ser. No. 60/244,300, filed Oct. 30, 2000. Rhizopus, Fusarium, Penic illium, Streptomyces, Actinomyces, Nocardia, Pseudomonas, Mycobacterium, FIELD OF THE INVENTION Arthrobacter and Bacillus. 10 The present invention relates to a novel cytochrome A variety of approaches have been used to facilitate the P450-like enzyme (Aspergillus ochraceus 11 alpha hydroxylation of intermediates used in the synthesis of hydroxylase) and an oxidoreductase (Aspergillus ochraceus commercially-important steroid compounds. U.S. Pat. No. oxidoreductase) isolated from cDNA library generated from 4.588,683, for example, describes a method of preparing 11 the mRNA of Aspergillus ochraceus spores. When the 15 beta, 17 alpha, 20, 21 tetrahydroxy steroids by incubating cDNA encoding the 11 alpha hydroxylase was co-expressed Substrate compounds in a medium comprising a fungal in Spodoptera frugiperda (Sf-9) insect cells with the cDNA culture of the genus Curvularia capable of effecting 11 beta encoding human oxidoreductase as an electron donor, it hydroxylation. Aspergillus ochraceus cultures and prepara successfully catalyzed the conversion of the steroid substrate tions of mycelia have also been used to convert progesterone 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as and other Steroids to their corresponding 11 alpha hydroxy determined by HPLC analysis. The invention also relates to forms (Tan, L. and Falardeau, P., 1970; Tan L., and nucleic acid molecules associated with or derived from these Falardeau P. J. Steroid Biochem. 1: 221–227, 1970; cDNAS including complements, homologues and fragments Samanta, T. B. et al., Biochem. J. 176, 593-594, 1978; thereof, and methods of using these nucleic acid molecules, Jayanthi, C. R. et al., Biochem. Biophys. Res. Commun. 106: to generate, for example, polypeptides and fragments 25 1262–1268, 1982). thereof. The invention also relates to the generation of The advent of new and expanded clinical uses of steroids antibodies that recognize the A. ochraceus 11 alpha for the treatment of a wide variety of disorders has created hydroxylase and oxidoreductase and methods of using these a need for improved methods for the production of steroid antibodies to detect the presence of these native and recom compounds and their intermediates on a commercial scale. binant polypeptides within unmodified and transformed host 30 U.S. Pat. No. 4,559,332, for example, describes a number of cells, respectively. The invention also provides methods of methods for the preparation of 20-spiroxane series of steroid expressing the Aspergillus 11 alpha hydroxylase gene compounds, including methods for the preparation of separately, or in combination with human or Aspergillus eplerenone methyl hydrogen 9,11C.-epoxy-17C.-hydroxy-3- oxidoreductase, in heterologous host cells, to facilitate the oxopregn-4-ene-7C,21-dicarboxylate, Y-lactone (also bioconversion of steroid substrates to their 11 alpha 35 referred to as eplerenone or epoxymexrenone) and related hydroxy-counterparts. compounds. WO 98/25948 and U.S. application Ser. No. 09/319,673 describe novel processes for the preparation of BACKGROUND OF THE INVENTION 9,11-epoxy steroid compounds, especially those of the 20-spiroxane series and their analogs, novel intermediates Microbial transformation or bioconversion reactions have 40 useful in the preparation of steroid compounds, and pro long been used to facilitate the chemical synthesis of a wide cesses for the preparation of such novel intermediates. U.S. variety of pharmaceutical products. Stereospecific reactions Pat. No. 6,046,023 discloses improved methods for the carried out under mild enzymatic conditions frequently offer microbial transformation of canrenone or estr-4-ene-3,17 advantages over comparable chemical processes which dione into its 11 O-hydroxy analogue using microorganisms result in undesireable side products. Microorganisms also 45 of the genus Aspergillus, Rhizopus, and Pestelotia, using have the ability to carry out simultaneous independent or steroid substrates having a purity of less than 97% and more sequential reactions on a substrate molecule, minimizing the than 90% at a concentration greater than 10 g/L. number of distinct steps in a synthesis and reducing the total Many modern, systematic approaches needed to optimize cost of the desired intermediate or end product. bioconversion of particular steroid intermediates are often General features of microbial systems used as biocatalysts 50 hindered by insufficient biochemical knowledge of the for the transformation of organic compounds has been enzymes involved in their synthesis and degradation. reviewed (See e.g., Goodhue, Charles T., Microb. Trans Eukaryotic cytochromes P450 appear to be associated with form. Bioact. Compa., 1: 9-44, 1982). Biotransformations the endoplasmic reticulum (ER) or mitochondrial mem can be carried out, for example, in continuous cultures or in branes. The electron donor for ER-associated cytochrome batch cultures. Enzymes secreted from the microorganism 55 P450 enzymes is often an FAD/FMN-dependent NADPH react with a substrate, and the product can be recovered from cytochrome P450 oxidoreductase. Electron transfer in the the medium. Intracellular enzymes can also react with a mitochondrial cytochromes P450 is usually mediated by an substrate if it is able to enter cells by an active or a passive NADPH-ferredoxin oxidoreductase and ferrodoxin. The diffusion process. Immobilized, dried, permeabilized, and specific electron donors known to be involved in mamma resting cells, and spores have also been used for microbial 60 lian steroidogenesis, are also called adrenodoxin reductase transformations. The use of cell extracts and purified and adrenodoxin, respectively. enzymes in solution, or immobilized on carriers, may even While fungal biotransformations are known to be medi tually offer significant cost or control advantages over ated by cytochrome P450 enzymes, many of these enzymes traditional fermentation methods. are extremely difficult to purify in an enzymatically-active Bioconversion reactions have been widely used in the 65 form (van den Brink et al., Fungal Genetics and Biology 23, field of steroids (Kieslich, K.; Sebek, O. K. Annu. Rep. 1–17, 1998). Many fungal P450 enzymes appear to be Ferment. Processes 3: 275-304, 1979; Kieslich, Klaus. associated with the endoplasmic reticulum (van den Brink et US 7,033,807 B2 3 4 al., Fungal Genetics and Biology 23, 1–17, 1998). Yeast facilitate the development of expression vectors and recom have an adrenodoxin reductase homologue which was binant host strains that can carry out more efficient biocon shown to couple with a mammalian 11 beta hydroxylase in versions of steroid intermediates and the synthesis of end vitro. (Lacour et al., Journal of Biological Chemistry 273, products on a commercial scale without the problems asso 23984–23992, 1998). In contrast, the electron donor which 5 ciated with partially-characterized host strains or an incom couples with Aspergillus ochraceus 11 alpha hydroxylase plete understanding of the enzymes involved in steroido was predicted to be an NADPH-cytochrome P450 oxi genesis. The present invention overcomes many of the doreductase (Samanta and Ghosh, J Steroid Biochem 28, limitations discussed above by identifying enzymes capable 327–32, 1987). The steroid 11 alpha hydroxylation complex of carrying out the 11 alpha hydroxylation of steroids. This in Rhizopus nigricans also appears to require an NADPH 10 approach not only greatly facilitates the use of 11 alpha cytochrome p450 oxidoreductase (Makovec and Breskvar, hydroxylation, but also permits the development of new Arch Biochem Biophys. 357, 310–6, 1998). Amplification of strategies for the identification of similar enzymes from cytochrome R. nigricans P450 and NADPH-cytochrome other fungi, the cloning of other enzymes involved in P450 reductase activities in preparations of progesterone steroidogenesis from Aspergillus ochraceus and other induced fungal mycelia may the facilitate biochemical char 15 microorganisms, and the development of improved host acterization of both enzymes (Makovec and Breskvar, strains or methods using free cells or immobilized cells or Pflugers Arch–Eur J. Physiol 439(Suppl): R111-R112, enzymes in bioconversion reactions. Similar approaches 2000). could also be developed to aid in the construction of Aspergillus ochraceus spores have been shown to cata expression vectors and recombinant host strains that are lyze the 11 alpha hydroxylation of steroid substrates such as more amenable to propagation and control than wild-type progesterone (Dutta T K. Datta J. Samanta T B, Biochem. microorganisms now commonly used for bioconversion in Biophys. Res. Commun. 192: 119–123, 1993). A. filmigatus is large scale bioreactors. also known to exhibit a steroid 11 alpha hydroxylase activity SUMMARY OF THE INVENTION (Smith et al., J Steroid Biochem Mol Biol 49: 93–100, 1994). The A. fumigatus enzyme is distinguished from the A. 25 In its broadest scope, the present invention provides a ochraceus enzyme, in that it appears to be a cytochrome method to clone enzymes involved in steroid metabolism P450 with dual site-specificity for 11 alpha and 15 beta and use of these enzymes to produce novel steroid interme hydroxylation and, unlike the A. ochraceus hydroxylase, diates and end-products. One aspect of the claimed inven appears to be non-inducible. tion is to provide a novel enzyme 11 alpha hydroxylase and 30 oxidoreductase, and their nucleic acids, proteins, peptides, Despite recent advances in sequencing technologies, fragments, and homologues. The invention also relates to detailed knowledge about the structural relationships of methods of identifying and cloning other enzymes involved fungal cytochrome P450s gleaned from nucleotide sequence in steroid metabolism. The invention also covers novel data remains primitive. Breskvar et al., (Biochem. Biophys. vectors and host cells, a novel method for making heterolo Res. Commun 1991; 178, 1078–1083, 1991) have described a genomic DNA sequence from Rhizopus nigricans for a 35 gous proteins by using the above vectors, and a method for putative P-450 encoding an 11 O-hydroxylase for progester identifying the Substrate specificity of the cloned enzymes. one. This sequence may not be complete, however, since the The invention provides a means for determining the predicted amino acid sequence lacks the canonical heme substrate specificity of the cloned 11 alpha hydroxylase, binding motif, FXXGXXXCXG, which is common to almost allelic variants, muteins, and fusion proteins thereof, per 40 mitting evaluation of a broad array of steroid Substrates all known cytochrome P-450 enzymes. (Nelson et al., Phar including 3 keto delta 4.5 steroids (3 keto delta 4 steroids); macogenetics 6: 1–42, 1996). 3 keto delta 4.5 delta 6.7 steroids (3 keto delta 4 delta 6 The cloning and characterization of the NADPH cyto steroids); 3 keto delta 6.7 steroids (3 keto delta 6 steroids); chrome P450 oxidoreductase (cprA) gene of Aspergillus or 3 keto delta 1.2 delta 4.5 steroids (3 keto delta 1 delta 4 niger has been described (van den Brink, J., et al., Genbank 45 steroids). Preferred substrates for testing include (a) can accession numbers Z26938, CAA81550, 1993, renone; (b) androstenedione; (c) aldona; (d) ADD (1.4 unpublished). The primary structure of Saccharomyces cer androstenedienedione) (e) mexrenone; (f) 6 beta mexrenone; evisiae NADPH-cytochrome P450 reductase has also been (g) 9 alpha mexrenone; (h) 12 beta mexrenone: (i) delta 12 deduced from the nucleotide sequence of its cloned gene meXrenone; () testosterone; (k) progesterone; (1) meXrenone (Yabusaki et al., J. Biochem. 103(6): 1004–1010, 1988). 50 6.7-bis-lactone; and (m) mexrenone 7.9-bislactone. Prefer Several other approaches have been used to facilitate the ably the cloned 11 alpha hydroxylase, allelic variants, cloning and analysis of steroid enzymes. U.S. Pat. Nos. muteins, and fusion proteins thereof do not also catalyze a 5,422,262, 5,679,521, and European patent EP 0 528 906 second hydroxylation selected from the group consisting of B1, for example, describes the expression cloning of steroid 15 alpha or beta hydroxylation, 6 alpha or beta 5 alpha reductase, type 2. U.S. Pat. No. 5,869,283, for 55 hydroxylation, 7alpha or beta hydroxylation, 9 alpha or beta example, describes an expression cassette comprising het hydroxylation, 12 alpha or beta hydroxylation, and 17 alpha erologous DNAS encoding two or more enzymes, each or beta hydroxylation of substrates selected from the group catalyzing an oxidation step involved conversion of choles consisting of 3 keto delta 4.5 steroids; 3 keto delta 4.5 delta terol into hydrocortisone, including the conversion of cho 6.7 steroids; or 3 keto delta 6.7 steroids. Most preferably the lesterol to pregnenolone; the conversion of pregnenolone to 60 cloned 11 alpha hydroxylase, allelic variants, muteins, and progesterone; the conversion of progesterone to 17 fusion proteins thereof do not catalyze the 15 beta hydroxy C.-hydroxy-progesterone; the conversion of 17 lation of Substrates selected from the group consisting of 3 C-hydroxyprogesterone to cortexolone; and the conversion keto delta 4.5 steroids; 3 keto delta 4.5 delta 6.7 steroids; or of cortexolone to hydrocortisone. 3 keto delta 6.7 steroids. The sequences of Aspergillus ochraceus 11 alpha 65 The invention provides an isolated and purified nucleic hydroxylase and A. Ochraceus oxidoreductase have not been acid, encoding Aspergillus ochraceus 11 alpha hydroxylase. reported. Knowledge about their sequence could greatly It also provides an isolated DNA, cDNA, gene, and an allele US 7,033,807 B2 5 6 of the gene encoding Aspergillus ochraceus 11 alpha Most preferably the hydroxylation is from canrenone to 11 hydroxylase. Preferably the isolated and purified nucleic alpha hydroxy canrenone. acid is as set forth in SEQID NO: 01. Preferably the isolated The invention also provides a method of expressing a DNA, cDNA, gene, and an allele of the gene is as set forth protein that can catalyze the 11 alpha hydroxylation of 3 in SEQ ID NO: 01. keto delta 4.5 steroids; 3 keto delta 4.5 delta 6, 7 steroids; 3 The invention provides an isolated protein having the keto delta 6, 7 steroids; or 3 keto delta 1, 2 delta 4, 5 steroids amino acid sequence of Aspergillus ochraceus 11 alpha comprising: (a) transforming or transfecting host cells with hydroxylase. It also provides an isolated variant of Aspergil an expression cassette comprising a promoter operably lus ochraceus 11 alpha hydroxylase, and a fusion protein linked to a nucleic acid that encodes said protein, and (b) comprising this hydroxylase. Preferably the protein is as set 10 expressing said protein in said host cells. The invention also forth in SEQ ID NO: 2. It also provides for variant of the provides for a method of producing the protein further protein set forth in SEQ ID NO. 2.; a polypeptide which comprising the step of recovering said protein. Preferably, comprises SEQ ID NO: 2 with at least one conservative this protein is Aspergillus ochraceus 11 alpha hydroxylase. amino acid substitution; polypeptides, with an amino acid More preferably, this method further comprises expressing sequence at least 99%. 95%, 90%, 75%, and 50% identical 15 an electron donor protein, wherein said electron donor to SEQ ID NO: 2. protein can donate electrons to said protein that can catalyze The invention provides an isolated and purified nucleic the 11 alpha hydroxylation of 3 keto delta 4, 5 steroids; 3 acid, encoding Aspergillus ochraceus 11 alpha oxidoreduc keto delta 4, 5 delta 6, 7 steroids; 3 keto delta 6, 7 steroids; tase. It also provides an isolated DNA, cDNA, gene, and or 3 keto delta 1, 62 delta 4, 5 steroids. Preferably, the allele of the gene encoding Aspergillus ochraceus oxi electron donor protein is selected from the group consisting doreductase. Preferably, the isolated and purified nucleic of human oxidoreductase and Aspergillus ochraceus oxi acid, wherein said nucleic acid sequence is as set forth in doreductase. More preferably the electron donor protein is SEQID NO: 5. It also provides for an isolated DNA, cDNA, Aspergillus ochraceus oxidoreductase. More preferably, the gene, and allele of the gene set forth in SEQ ID NO: 5. nucleic acid encoding said steroid 11 alpha hydroxylase and The invention provides an isolated protein having the 25 said electron donor protein are on separate expression cas amino acid sequence of Aspergillus ochraceus oxidoreduc settes. More preferably, the nucleic acid encoding said tase. It also provides an isolated variant of the protein having steroid 11 alpha hydroxylase and said electron donor protein the amino acid sequence of Aspergillus ochraceus are on the same expression cassettes. Even more preferably, oxidoreductase, and a fusion protein comprising the amino the steroid 11 alpha hydroxylase is Aspergillus ochraceus 11 acid sequence of Aspergillus ochraceus oxidoreductase. 30 alpha hydroxylase and said electron donor protein is human Preferably the isolated protein has the amino acid sequence oxidoreductase. Even more preferably, the steroid 11 alpha set forth in SEQ ID NO: 6. It also provides an isolated hydroxylase is Aspergillus ochraceus 11 alpha hydroxylase variant of a protein set forth in SEQ ID NO: 6. a purified and said electron donor protein is Aspergillus ochraceus polypeptide, the amino acid sequence of which comprises oxidoreductase. Preferably, the expression cassette is on an SEQ ID NO: 6 with at least one conservative amino acid 35 expression vector. More preferably, the expression vector is Substitution; and a polypeptides with an amino acid a baculovirus. Even more preferably, the baculovirus is a sequence at least 99%. 95%, 90%, 75%, and 50% identical nuclear polyhedrosis virus is selected from the group con to SEQ ID NO: 6. sisting of Autographa Californica nuclear polyhedrosis virus The invention provides an isolated and purified nucleic and Bombyx mori nuclear polyhedrosis virus. Most acid encoding an enzyme that can catalyze the 11 alpha 40 preferably, the nuclear polyhedrosis virus is Autographa hydroxylation of 3 keto delta 4.5 steroids (3 keto delta 4 californica nuclear polyhedrosis virus. Preferably, the host steroids); 3 keto delta 4.5 delta 6.7 steroids (3 keto delta 4 cells are insect cells. More preferably, the insect cells are delta 6 steroids); 3 keto delta 6.7 steroids (3 keto delta 6 Selected from the group consisting of Spodoptera steroids); or 3 keto delta 1.2 delta 4.5 steroids (3 keto delta frugiperda, Trichoplusia ni, Autographa Californica, and 1 delta 4 steroids). Preferably the enzyme does not catalyze 45 Manduca sexta cells. Most preferably the insect cells are the 15 beta hydroxylation of 3 keto delta 4.5 steroids; 3 keto Spodoptera frugiperda cells. The invention also provides a delta 4.5 delta 6.7 steroids; or 3 keto delta 6.7 steroids. More for a method of expressing a protein wherein the Aspergillus preferably, the hydroxylation is selected from the group ochraceus 11 alpha hydroxylase is SEQ ID NO: 2; the consisting of: (a) canrenone to 11 alpha hydroxy canrenone; human oxidoreductase is SEQID NO: 4; and the Aspergillus (b) androstenedione to 11 alpha hydroxy androstenedione; 50 ochraceus oxidoreductase is SEQ ID NO: 6. (c) aldona to 11 alpha hydroxy aldona; (d) ADD (1.4 The invention also provides for an isolated and purified androstenedienedione) to 11 alpha hydroxy ADD; (e) polypeptide that can catalyze the 11 alpha hydroxylation of mexrenone to 11 alpha hydroxy mexrenone; (f) 6 beta 3 keto delta 4.5 steroids (3 keto delta 4 steroids); 3 keto delta meXrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 4, 5 delta 6, 7 steroids (3 keto delta 4 delta 6 steroids); 3 keto alpha meXrenone to 11 alpha hydroxy 9 alpha mexrenone; 55 delta 6, 7 steroids (3 keto delta 6 steroids); or 3 keto delta (h) 12 beta mexrenone to 11 alpha hydroxy 12 beta 1, 2 delta 4, 5 steroids (3 keto delta 1 delta 4 steroids). meXrenone; (i) delta 12 meXrenone to 11 alpha hydroxy Preferably, the polypeptide does not catalyze the 15 beta delta 12 meXrenone; () testosterone to 11 alpha hydroxy hydroxylation of 3 keto delta 4.5 steroids; 3 keto delta 4, 5 testosterone; (k) progesterone to 11 alpha hydroxy proges delta 6, 7 steroids; or 3 keto delta 6, 7 steroids. More terone: (1) mexrenone 6.7-bis-lactone to 11 alpha hydroxy 60 preferably, the hydroxylation is selected from the group mex renone 6,7-bis-lactone; and (m) mexrenone 7,9- consisting of: (a) canrenone to 11 alpha hydroxy canrenone; bislactone to 11 alpha hydroxy mexrenone 7.9-bislactone. (b) androstenedione to 11 alpha hydroxy androstenedione; More preferably, the hydroxylation is selected from the (c) aldona to 11 alpha hydroxy aldona; (d) ADD (1.4 group consisting of: (a) canrenone to 11 alpha hydroxy androstenedienedione) to 11 alpha hydroxy ADD; (e) canrenone; (b) androstenedione to 11 alpha hydroxyandros 65 mexrenone to 11 alpha hydroxy mexrenone; (f) 6 beta tenedione; (c) aldona to 11 alpha hydroxy aldona; and (d) meXrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 ADD (1.4 androstenedienedione) to 11 alpha hydroxy ADD. alpha meXrenone to 11 alpha hydroxy 9 alpha mexrenone; US 7,033,807 B2 7 8 (h) 12 beta mexrenone to 11 alpha hydroxy 12 beta encoded enzymes in a recombinant host. Preferably the meXrenone; (i) delta 12 meXrenone to 11 alpha hydroxy heterologous DNA coding sequences in the expression cas delta 12 meXrenone; () testosterone to 11 alpha hydroxy sette are selected from the group consisting of the following testosterone; (k) progesterone to 11 alpha hydroxy proges genus and species: Aspergillus ochraceus, Aspergillus terone: (1) mexrenone 6.7-bis-lactone to 11 alpha hydroxy ochraceus, Aspergillus niger, Aspergillus nidulans, Rhizopus mex renone 6,7-bis-lactone; and (m) mexrenone 7,9- Oryzae, Rhizopus stolonifer, Streptomyces fradiae, Bacillus bislactone to 11 alpha hydroxy mexrenone 7.9-bislactone. megaterium, Pseudomonas cruciviae, Trichothecium More preferably, the hydroxylation is selected from the roseum, Fusarium oxysporum Rhizopus arrhizus, Absidia group consisting of: (a) canrenone to 11 alpha hydroxy coerula, Absidia glauca, Actinomucor elegans, Aspergillus canrenone; (b) androstenedione to 11 alpha hydroxyandros 10 flavipes, Aspergillus filmigatus, Beauveria bassiana, Botry tenedione; (c) aldona to 11 alpha hydroxy aldona; and (d) osphaeria obtusa, Calonectria decora, Chaetomium ADD (1.4 androstenedienedione) to 11 alpha hydroxy ADD. cochliodes, Corynespora cassicola, Cunninghamella Most preferably the hydroxylation is from can renone to 11 blakesleeana, Cunninghamella echinulata, Cunninghamella alpha hydroxy canrenone. elegans, Curvularia clavata, Curvularia lunata, Cylindro The invention also provides for an expression cassette 15 carpon radicicola, Epicoccum humicola, Gongronella comprising a promoter operably linked to an isolated and butleri, Hypomyces chrysospermus, Monosporium purified nucleic acid encoding a polypeptide that can cata Olivaceum, Mortierella isabellina, Mucor mucedo, Mucor lyze the 11 alpha hydroxylation of 3 keto delta 4.5 steroids griseocyanus, Myrothecium verrucaria, Nocardia Corallina, (3 keto delta 4 steroids); 3 keto delta 4.5 delta 6.7 steroids Paecilomyces carneus, Penicillum patulum, Pithomyces (3 keto delta 4 delta 6 steroids); 3 keto delta 6, 7 steroids (3 atroolivaceus, Pithomyces cynodontis, Pycnosporium sp., keto delta 6 steroids); or 3 keto delta 1, 2 delta 4, 5 steroids Saccharopolyspora erythrae, Sepedonium chrysospermum, (3 keto delta 1 delta 4 steroids). More preferably, the Stachylidium bicolor; Streptomyces hygroscopicus, Strepto hydroxylation is selected from the group consisting of: (a) myces purpurascens, Syncephalastrum racemosum, Tham canrenone to 11 alpha hydroxy canrenone; (b) androstene nostylum piriforme, Thielavia terricola, and Verticillium dione to 11 alpha hydroxy androstenedione; (c) aldona to 11 25 theobromae, Cephalosporium aphidicola, Cochliobolus alpha hydroxyaldona; (d) ADD (1.4 androstenedienedione) lunatas, Tieghemella Orchidis, Tieghemella hyalospora, to 11 alpha hydroxy ADD; (e) mexrenone to 11 alpha Monosporium Olivaceum, Aspergillus ustus, Fusarium hydroxy mexrenone; (f) 6 beta mexrenone to 11 alpha graminearum, Verticillium glaucum, and Rhizopus nigri hydroxy 6 beta mexrenone; (g) 9 alpha mexrenone to 11 cans. More preferably, the genus and species are selected alpha hydroxy 9 alpha mexrenone; (h) 12 beta mexrenone to 30 from the group consisting of Aspergillus ochraceus, 11 alpha hydroxy 12 beta mexrenone: (i) delta 12 mexrenone Aspergillus ochraceus, Aspergillus niger, Aspergillus to 11 alpha hydroxy delta 12 mexrenone; () testosterone to nidulans, Rhizopus Oryzae, Rhizopus stolonifer, Streptomy 11 alpha hydroxy testosterone; (k) progesterone to 11 alpha ces fradiae, Bacillus megaterium, Pseudomonas cruciviae, hydroxy progesterone; (1) meXrenone 6.7-bis-lactone to 11 Trichothecium roseum, Fusarium oxysporum, Rhizopus alpha hydroxy meXrenone 6.7-bis-lactone; and (m) 35 arrhizus, and Monosporium Olivaceum. Most preferably, mexrenone 7.9-bislactone to 11 alpha hydroxy mexrenone genus and species is Aspergillus Ochraceus. 7.9-bislactone. More preferably, the hydroxylation is Preferably, the recombinant host cell and progeny thereof selected from the group consisting of: (a) canrenone to 11 comprise at least one expression cassette. More preferably, alpha hydroxy canrenone; (b) androstenedione to 11 alpha the host is a microorganism. Most preferably, the host is a hydroxy androstenedione; (c) aldona to 11 alpha hydroxy 40 bacterium. The invention also provides for a process for aldona; and (d) ADD (1.4 androstenedienedione) to 11 alpha making one or more enzymes from the metabolic pathway hydroxy ADD. Most preferably the hydroxylation is from for the transformation of sitosterol to eplerenone comprising canrenone to 11 alpha hydroxy canrenone. incubating the recombinant host cell in a nutrient medium The invention also provides for an expression cassette under conditions where the one or more enzymes encoded comprising a promoter operably linked to an isolated and 45 by the heterologous DNA are expressed and accumulate. purified nucleic acid encoding Aspergillus ochraceus oxi More preferably the process comprises the steps of: (a) doreductase. Preferably the nucleic acid is SEQ ID NO: 6. incubating the compound to be oxidized in the presence the The invention also provides for an expression cassette recombinant host cells under conditions where the com comprising a heterologous DNA encoding an enzyme from pound is hydroxylated and the hydroxylated product the metabolic pathway for the synthesis of sitosterol to 50 accumulates, and (b) recovering the hydroxylated product. eplerenone wherein said enzyme catalyzes at least one Most preferably, the process comprises the steps of: (a) conversion selected from the group consisting of: (a) can incubating the compound to be oxidized in the presence of renone to 11 alpha hydroxy canrenone; (b) androstenedione the enzymes produced under conditions where the com to 11 alpha hydroxy androstenedione; (c) aldona to 11 alpha pound is hydroxylated and the hydroxylated product hydroxyaldona; (d) ADD (1.4 androstenedienedione) to 11 55 accumulates, and (b) recovering the hydroxylated product. alpha hydroxy ADD; (e) mexrenone to 11 alpha hydroxy The invention also provides for a host cells harboring an mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 beta expression cassette. More preferably the expression cassette meXrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 is integrated into the chromosome of said host cell. More alpha mexrenone; (h) 12 beta mexrenone to 11 alpha preferably, the expression cassette is integrated into an hydroxy 12 beta mexrenone: (i) delta 12 mexrenone to 11 60 expression vector. alpha hydroxy delta 12 meXrenone; () testosterone to 11 The invention also provides for a method of determining alpha hydroxy testosterone; and (k) progesterone to 11 alpha the specific activity of a cloned 11 alpha hydroxylase hydroxy progesterone; (1) meXrenone 6.7-bis-lactone to 11 comprising the steps of: (a) transforming host cells with an alpha hydroxy meXrenone 6.7-bis-lactone; and (m) expression vector comprising a nucleic acid that encodes mexrenone 7.9-bislactone to 11 alpha hydroxy mexrenone 65 said 11 alpha hydroxylase, (b) expressing said 11 alpha 7.9-bislactone and wherein the heterologous DNA is oper hydroxylase in said host cells; (c) preparing Subcellular ably linked to control sequences required to express the membrane fractions from said cells, (d) incubating said US 7,033,807 B2 10 subcellular membrane fractions with a steroid substrate, and amino acids SEQID NO: 24; and (e) amino acids SEQ ID (e) monitoring conversion of the steroid substrate to its 11 NO: 25. Preferably the antibody is purified on a peptide alpha hydroxy steroid counterpart. Preferably, the further column, wherein said peptide is selected from the group comprises transforming host cells with an expression vector consisting of: (a) the N-terminal amino acids 1–10 of SEQ nucleic acid that encodes an oxidoreductase, and expressing 5 ID NO: 2; (b) the last 10 C-terminal amino acids of SEQID said oxidoreductase in said host cells. More preferably, the NO: 2; (c) amino acids SEQ ID NO: 23; (d) amino acids oxidoreductase is human or Aspergillus ochraceus. Most SEQ ID NO: 24; and (e)amino acids SEQ ID NO: 25. preferably the oxidoreductase is human oxidoreductase. The invention also provides for a purified polypeptide, the Most preferably the oxidoreductase is Aspergillus Ochraceus amino acid sequence of which is selected from the group oxidoreductase. 10 consisting of SEQ ID NO: 26. The invention also provides for a purified immunogenic The invention also provides for a protein having SEQID polypeptide, the amino acid sequence of which comprises at NO: 2 and variants thereof that are at least 95% identical to least ten consecutive residues of SEQ ID NO: 6. SEQ ID NO: 2 and catalyze the 11 alpha hydroxylation of The invention also provides for an isolated and purified 3 keto delta 4, 5 steroids; 3 keto delta 4, 5 delta 6, 7 steroids; antibody having a binding specificity for 11 alpha hydroxy 3 keto delta 6, 7 steroids; or 3 keto delta 1, 2 delta 4, 5 15 lase having an amino acid sequence as shown in SEQ ID steroids, wherein said hydroxylation is selected from the NO: 6. Preferably the antibody binds to a protein region group consisting of: (a) canrenone to 11 alpha hydroxy selected from the group consisting of (a) the N-terminal canrenone; (b) androstenedione to 11 alpha hydroxyandros amino acids 1–10 of SEQ ID NO: 6; (b) the last 10 tenedione; (c) aldona to 11 alpha hydroxy aldona; (d) ADD C-terminal amino acids of SEQ ID NO: 6; and (c) amino (1.4 androstenedienedione) to 11 alpha hydroxy ADD; (e) acids SEQ ID NO: 26. More preferably, the antibody is mexrenone to 11 alpha hydroxy mexrenone; (f) 6 beta purified on a peptide column, wherein said peptide is meXrenone to 11 alpha hydroxy 6 beta mexrenone; (g) 9 selected from the group consisting of: (a) the N-terminal alpha meXrenone to 11 alpha hydroxy 9 alpha mexrenone; amino acids 1–10 of SEQ ID NO: 6; (b) the last 10 (h) 12 beta mexrenone to 11 alpha hydroxy 12 beta C-terminal amino acids of SEQ ID NO: 6; and (c) amino meXrenone; (i) delta 12 meXrenone to 11 alpha hydroxy 25 acids SEQ ID NO: 26. delta 12 meXrenone; () testosterone to 11 alpha hydroxy The invention also provides for a composition comprising testosterone; and (k) progesterone to 11 alpha hydroxy an antibody described above in an effective carrier, vehicle, progesterone. Preferably the enzyme does not catalyze the or auxiliary agent. It also provides for a composition com 15 beta hydroxylation of 3 keto delta 4.5 steroids; 3 keto prising Such an antibody and a solution. The antibody may delta 4, 5 delta 6, 7 steroids; or 3 keto delta 6, 7 steroids. 30 be a polyclonal antibody. The antibody may also be a The invention provides an isolated and purified nucleic monoclonal antibody. The antibody may be conjugated to an acid encoding an enzyme that can catalyze the 11 alpha immunoaffinity matrix. The invention also provides for a hydroxylation of 3 keto delta 4.5 steroids (3 keto delta 4 method of using an immunoaffinity matrix to purify a steroids); 3 keto delta 4.5 delta 6.7 steroids (3 keto delta 4 polypeptide from a biological fluid or cell lysate. Preferably delta 6 steroids); 3 keto delta 6, 7 steroids (3 keto delta 6 35 the immunoaffinity matrix is SEPHAROSE 4B. More pref steroids); or 3 keto delta 1.2 delta 4, 5 steroids (3 keto delta erably the method of using an immunoaffinity matrix to 1 delta 4 steroids) wherein the hydroxylation is selected purify a polypeptide from a biological fluid or cell lysate from the group consisting of: (a) canrenone to 11 alpha uses SEPHAROSE 4B as an immunoaffinity matrix. More hydroxy can renone; (b) androstenedione to 11 alpha preferably, the method of using an immunoaffinity matrix to hydroxy androstenedione; (c) aldona to 11 alpha hydroxy 40 purify a polypeptide from a biological fluid or cell lysate aldona; (d) ADD (1.4 androstenedienedione) to 11 alpha uses SEPHAROSE 4B as an immunoaffinity matrix. hydroxy ADD; (e) mexrenone to 11 alpha hydroxy The invention also provides for a method of using a mexrenone; (f) 6 beta mexrenone to 11 alpha hydroxy 6 beta peptide column to purify an antibody, wherein said peptide meXrenone; (g) 9 alpha mexrenone to 11 alpha hydroxy 9 45 is selected from the group consisting of: (a) the N-terminal alpha mexrenone; (h) 12 beta mexrenone to 11 alpha amino acids 1–10 of SEQ ID NO: 2; (b) the last 10 hydroxy 12 beta mexrenone: (i) delta 12 mexrenone to 11 C-terminal amino acids of SEQ ID NO: 2; (c) amino acids alpha hydroxy delta 12 meXrenone; () testosterone to 11 alpha hydroxy testosterone; and (k) progesterone to 11 alpha SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and (e) hydroxy progesterone. Preferably the enzyme does not cata amino acids SEQ ID NO: 25. 50 The invention also provides for a method of using a lyze the 15 beta hydroxylation of 3 keto delta 4, 5 steroids; peptide column to purify an antibody, wherein said peptide 3 keto delta 4, 5 delta 6.7 steroids; or 3 keto delta 6, 7 is selected from the group consisting of: (a) the N-terminal steroids. amino acids 1–10 of SEQ ID NO: 6; (b) the last 10 The invention also provides for a purified polypeptide, the C-terminal amino acids of SEQ ID NO: 6; and (c) amino amino acid sequence of which is selected from the group 55 acids SEQ ID NO: 26. consisting of SEQID NO: 23, SEQID NO: 24, SEQID NO: The invention also provides for a method of detecting a 25. first polypeptide in a biological fluid, wherein said first The invention provides for a purified immunogenic polypeptide is selected from the group consisting of 11 alpha polypeptide, the amino acid sequence of which comprises at hydroxylase and oxidoreductase, comprising the following least ten consecutive residues of SEQ ID NO: 2. 60 steps: (a) contacting said fluid with a second polypeptide, The invention provides for an isolated and purified anti having a binding specificity for said first polypeptide, and body having a binding specificity for 11 alpha hydroxylase (b) assaying the presence of said second polypeptide to having an amino acid sequence as shown in SEQID NO: 2. determine the level of said first polypeptide. Preferably, the Preferably the antibody binds to a protein region selected second polypeptide is an antibody. More preferably, the from the group consisting of (a) the N-terminal amino acids 65 second polypeptide is radiolabeled. 1–10 of SEQ ID NO: 2; (b) the last 10 C-terminal amino The invention also provides for a process for producing an acids of SEQID NO: 2; (c) amino acids SEQID NO:23; (d) isolated nucleic acid comprising hybridizing SEQID NO: 1 US 7,033,807 B2 11 12 to genomic DNA in 6xSSC and 65° C. and isolating the DEFINITIONS nucleic acid detected with SEQID NO: 1. The invention also provides for an isolated DNA nucleic acid prepared accord The following is a list of abbreviations and the corre ing to this process. sponding meanings as used interchangeably herein: The invention also provides for an isolated nucleic acid 11 alpha hydroxycan renone=11 alpha hydroxy-4- that specifically hybridizes under highly stringent conditions androstene-3,17-dione (CHO, MW 356.46) to the complement of the sequence set forth in SEQID NO: AcNPV=Autographa Californica nuclear polyhedrosis 1. virus, a member of the Baculoviridae family of insect The invention also provides for a process for producing an viruses isolated nucleic comprising hybridizing SEQ ID NO: 5 to 10 genomic DNA in 6xSSC and 65° C. and isolating the nucleic AD=androstenedione or 4-androstene-3,17-dione acid detected with SEQ ID NO: 5. The invention also (CH2O, MW 340.46) provides for an isolated DNA nucleic acid prepared accord aldadiene=canrenone ing to this process. Amp=ampicillin The invention also provides for an isolated nucleic acid 15 attTnT=attachment site for TnT (a preferential site for TnT. that specifically hybridizes under highly stringent conditions insertion into bacterial chromosomes) to the complement of the sequence set forth in SEQID NO: 5. bacmid=recombinant baculovirus shuttle vector isolated The invention also provides for a DNA construct which from E. coli alters the expression of a 11 alpha hydroxylase gene not Bluo-gal=halogenated indolyl-B-D-galactoside normally expressed in a cell when said DNA construct is bp=base pair(s) inserted into chromosomal DNA of the cell, said DNA Cam=chloramphenicol construct comprising: (a) a targeting sequence; (b) a regu cDNA=complementary DNA latory sequence; and (c) the structural gene for a steroid 11 alpha hydroxylase. The invention also provides for a host DMF=N,N-dimethylformamide cell harboring this DNA construct. 25 ds=double-stranded The invention also provides for a DNA construct which eplerenone or epoxymexrenone=methyl hydrogen 9,11C.- alters the expression of a 11 alpha hydroxylase gene not epoxy-17 C. -hydroxy-3-oxopregn-4-ene-7.C., 21 normally expressed in a cell when said DNA construct is dicarboxylate, Y-lactone (MW 414.5) inserted into chromosomal DNA of the cell, said DNA 30 g=gram(s) construct comprising: (a) a targeting sequence; (b) a regu Gen=gentamicin latory sequence; and (c) the structural gene for a steroid hoXr=human oxidoreductase oxidoreductase. The invention also provides for a host cell harboring this DNA construct. HPLC=high performance liquid chromatography hydroxy can re none = 11 alpha- or 11 beta The invention also provides for use of a host cell harbor 35 ing a cloned 11 alpha hydroxylase for the manufacture of a hydroxycan renone medicament for therapeutic application to treat heart IPTG=isopropyl-f-D-thiogalactopyranoside disease, inflammation, arthritis, or cancer. Kan=kanamycin The invention also provides for a composition comprising kb=kilobase(s), 1000 bp(s) from about 0.5-to about 500 g/L molasses, 0.5–50 g/L 40 mb=megabase(s) cornsteep liquid, 0.5–50 g/L KHPO, 2.5–250 g/L NaCl, 2.5-250 g/L glucose, and 0.04–4 g/L progesterone, pH Me=methyl 3.5–7. Preferably, this composition is comprised of from mg=milligram(s) about 10–250 g/L molasses, 1–25 g/L cornsteep liquid, 1-25 ml or mL=milliliter(s) g/L KHPO, 5–125 g/L NaCl, 5–125 g/L glucose, and 45 mm=millimeter 0.08–2 g/L progesterone, pH 4.5-6.5. More preferably, the composition is comprised of from about 25–100 g/L mM=millimolar molasses, 2.5–10 g/L cornsteep liquid, 2.5–10 g/L KHPO, NMR=nuclear magnetic resonance 12.5–50 g/L NaCl, 12.5–50 g/L glucose, and 0.2–0.8 g/L oXr=oxidoreductase progesterone, pH 5.5-6.0. Most preferably the composition 50 PCR=polymerase chain reaction comprises about 50 g/L molasses, 5 g/L cornsteep liquid, 5 r=resistant or resistance g/L KH2PO4, 25 g/L NaCl, 25 g/L glucose, 20 g/L agar, and RP-HPLC=reverse phase high performance liquid chro 0.4 g/L progesterone, pH 5.8. matography The invention also provides for a semisolid formulation of RT=room temperature any of the compositions described above, further comprising 55 from about 4-100 g/L agar. Preferably the agar is at a RT-PCR=reverse transcriptase polymerase chain reaction concentration of from about 10–40 g/L agar. More S=Sensitive preferably, the agar is about 20 g/L agar. SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel The invention also provides for the use of any of the electrophoresis compositions describe above to produce spores from the 60 Spc/Str=spectinomycin/streptomycin microorganism selected from the group consisting of Tet=tetracycline Aspergillus ochraceus, Aspergillus niger, Aspergillus Tn=transposon nidulans, Rhizopus Oryzae, Rhizopus stolonifer, and Tri chothecium roseum, Fusarium oxysporum Rhizopus ts=temperature-sensitive U=units arrhizus, Monosporium Olivaceum. Penic illum 65 chrysogenium, and Absidia coerula. Preferably, the compo ug or lug=microgram(s) sition is used to produce spores from Aspergillus ochraceus. ul or ul=microliter(s) US 7,033,807 B2 13 14 X-gal 5-bromo-3-chloro-indolyl-B-D-galactopyranoside The term “recombinant’ means any agent (e.g., DNA, X-gluc=5-bromo-3-chloro-indolyl-B-D-glucopyranoside peptide, etc.), that is, or results from, however indirectly, The following is a list definitions of various terms used human manipulation of a nucleic acid molecule. herein: The term “selectable or screenable marker genes' means The species “Aspergillus ochraceus NRRL 405' means genes whose expression can be detected by a probe as a the filamentous fungus Aspergillus ochraceus NRRL 405, means of identifying or selecting for transformed cells. accession number 18500, obtained from the American Type Culture Collection (ATCC). A. ochraceus NRRL 405 and A. The term “specifically bind' means that the binding of an ochraceus ATCC 18500 are the same strain, catalogued antibody or peptide is not competitively inhibited by the differently. 10 presence of non-related molecules. The term "amino acid(s) means all naturally occurring The term “specifically hybridizing” means that two L-amino acids, including norleucine, norvaline, nucleic acid molecules are capable of forming an anti homocysteine, and ornithine. parallel, double-stranded nucleic acid structure. The term “degenerate” means that two nucleic acid mol 15 The term “substantial complement’ means that a nucleic ecules encode for the same amino acid sequences but acid sequence shares at least 80% sequence identity with the comprise different nucleotide sequences. complement. The term "fragment’ means a nucleic acid molecule whose sequence is shorter than the target or identified The term "substantial fragment’ means a nucleic acid nucleic acid molecule and having the identical, the Substan fragment which comprises at least 100 nucleotides. tial complement, or the Substantial homologue of at least 10 The term “substantial homologue' means that a nucleic contiguous nucleotides of the target or identified nucleic acid molecule shares at least 80% sequence identity with acid molecule. another. The term “fusion protein’ means a protein or fragment thereof that comprises one or more additional peptide 25 The term “substantially hybridizing” means that two regions not derived from that protein. nucleic acid molecules can form an anti-parallel, double Stranded nucleic acid structure under conditions (e.g., salt The term “probe' means an agent that is utilized to and temperature) that permit hybridization of sequences that determine an attribute or feature (e.g. presence or absence, exhibit 90% sequence identity or greater with each other and location, correlation, etc.) of a molecule, cell, tissue, or 30 exhibit this identity for at least about a contiguous 50 organism. nucleotides of the nucleic acid molecules. The term “promoter is used in an expansive sense to refer to the regulatory sequence(s) that control mRNA production. The term “substantially-purified” means that one or more Such sequences include RNA polymerase binding sites, molecules that are or may be present in a naturally-occurring enhancers, etc. 35 preparation containing the target molecule will have been The term “protein fragment’ means a peptide or polypep removed or reduced in concentration. tide molecule whose amino acid sequence comprises a The following is a list of steroids, corresponding terms, Subset of the amino acid sequence of that protein. and their structures, as used interchangeably herein:

US 7,033,807 B2 25 26

ponu?uoo

QUITEN IZ US 7,033,807 B2 27 28 FIG. 1 Nucleotide and Protein Sequence of Aspergillus (its probable function, genus and species) for the top 10 ochraceus 11 Alpha Hydroxylase matches are as follows: CAA75565 (cytochrome P450 The nucleotide and protein sequences of Aspergillus monooxygenase Gibberellafiujikuroi CAB91316 (prob ochraceus 11 alpha hydroxylase (SEQ ID NO: 1, SEQ ID able cytochrome P450 monooxygenase (lov A) Neurospora NO: 2, respectively) are displayed. 5 crassa); CAB56503 (cytochrome P450 Catharanthus FIG. 2 Nucleotide and Protein Sequence of Human roseus); AAB94588 (CYP71D10p Glycine max): Oxidoreductase CAA75566 (cytochrome P450 monooxygenase Gibberella The nucleotide and protein sequences of human oxi fiujikuroi); AAD34552 (cytochrome P450 monooxygenase doreductase (SEQ ID NO:3, SEQ ID NO: 4, respectively) Aspergillus terreus); CAA75567 (cytochrome P450 are displayed. The predicted amino acid sequence of human 10 monooxygenase Gibberellafiujikuroi); CAA76703 (cyto oxidoreductase independently cloned from a cDNA library chrome P450 Gibberellafiujikuroil); CAA57874 (unnamed prepared by RT-PCR using the RNA from a human HepG2 protein product Fusarium oxysporum); CAA91268 (simi cells as a template, as disclosed in this specification, matches lar to cytochrome P450-cDNA ESTyk423b11.3 comes from that previous reported by three different laboratories. The this gene Caenorhabditis elegans ). GenBank accession numbers for these loci include A60557 15 References for these loci are as follows: CAA75565 (NADPH ferrihemoprotein reductase (EC 1.6.2.4)-hu Tudzynski, B. and Holter, K., Gibberellin biosynthetic man): AAG09798 (NADPH-cytochrome P450 reductase pathway in Gibberellafiujikuroi: evidence for a gene cluster. Homo sapiens), and P16435 (NADPH-CYTOCHROME Fungal Genet. Biol. 25 (3), 157-170 (1998); CAB91316 P450 REDUCTASE (CPR) (P450R)). Schulte, U., Aign, V., Hoheisel, J., Brandt, P., Fartmann, B., The amino acid sequence of AAB21814 (cytochrome Holland, R., Nyakatura, G. Mewes, H. W. and Mannhaupt, P450 reductase {EC 1.6.2.4 human, placenta, Peptide G., Unpublished; CAB56503 Schroeder, G., Unterbusch, Partial, 676 aa), differs from human oxidoreductase A60557 E., Kaltenbach, M., Schmidt, J., Strack, D. and Schroeder, J. and P16435 at 4 residues: A->V at 500, F->L at 518, V->W Light-induced cytochrome P450-dependent enzyme in at 537, and A->H at 538. The initial methionine is also indole alkaloid biosynthesis: tabersonine 16-hydroxylase missing from AAB21814. The cognate nucleic acid for 25 FEBS Lett. 458, 97-102 (1999): AAB94588 Siminszky, AA21814 (S90469 cytochrome P450 reductase human, B., Corbin, F.T., Ward, E. R., Fleischmann, T. J. and Dewey, placenta, mRNA Partial, 2403 nt) lacks the ATG codon for R. E. Expression of a soybean cytochrome P450 monooxy the initial methionine and includes a C->T change at 1496, genase cDNA in yeast and tobacco enhances the metabolism a C->A, change at 1551, and a frameshift due to a missing of phenylurea herbicides. Proc. Natl. Acad. Sci. U.S.A. 96 G at 1605 which is resolved by the addition of a T at 1616. 30 (4), 1750–1755 (1999); CAA75566 Tudzynski, B. and References for these loci are as follows: A60557 Ya Holter, K. Gibberellin biosynthetic pathway in Gibberella mano, S., Aoyama, T., McBride, O. W., Hardwick, J. P. fiujikuroi evidence for a gene cluster. Fungal Genet. Biol. 25 Gelboin, H. V. and Gonzalez, F. J. Human NADPH-P450 (3), 157–170 (1998); AAD34552 Kennedy, J., Auclair, K., oxidoreductase: complementary DNA cloning, sequence Kendrew, S. G., Park, C., Vederas, J. C. and Hutchinson, C. and vaccinia virus-mediated expression and localization of 35 R. Accessory Proteins Modulate Polyketide Synthase Activ the CYPOR gene to chromosome 7 Mol. Pharmacol. 36 (1), ity During Lovastatin Biosynthesis. Science (1999) In 83–88 (1989): AAG09798 Czerwinski, M., Sahni, M., press: CAA75567 Tudzynski, B. and Holter, K. Gibberel Madan, A. and Parkinson, A. Polymorphism of human lin biosynthetic pathway in Gibberellafiujikuroi evidence CYPOR: Expression of new allele. Unpublished, Direct for a gene cluster. Fungal Genet. Biol. 25 (3), 157-170 Submission), and P16435 Haniu, M., McManus, M. E., 40 (1998); CAA76703 Tudzynski, B. and Hoelter, K. Char Birkett, D. J. Lee, T. D. and Shively, J. E. Structural and acterization of P450 monooxygenase genes from Gibberella functional analysis of NADPH-cytochrome P-450 reductase fiujikuroi. Unpublished; CAA57874 Mouyna, I. and Bry from human liver: complete sequence of human enzyme and goo, Y. Disruption of a Fusarium oxysporum f.sp. elaeidis NADPH-binding sites. Biochemistry 28 (21), 8639–8645 cytochrome P450 gene by a repetitive sequence. Unpub (1989: AAB21814. Shephard, E. A., Palmer, C. N. Segall, 45 lished; and CAA91268 No Authors. Genome sequence of H. J. and Phillips, I. R. Quantification of cytochrome P450 the nematode C. elegans: a platform for investigating biol reductase gene expression in human tissues. Arch. Biochem. ogy. The C. elegans Sequencing Consortium. Science 282 Biophys. 294 (1), 168–172 (1992); S90469 Shephard, E. (5396), 2012–2018 (1998) Published errata appear in Sci A., Palmer, C. N. Segall, H. J. and Phillips, I. R. Quanti ence Jan. 1, 1999:283 (5398):35 and Mar. 26, fication of cytochrome P450 reductase gene expression in 50 1999:283(5410):2103 and Sep. 3, 1999:285(5433): 1493). human tissues. Arch. Biochem. Biophys. 294 (1), 168-172 FIG. 5 Phylogenetic Tree Showing the Relatedness of (1992)). Aspergillus ochraceus 11 Alpha Hydroxylase to the Top 10 FIG. 3 Nucleotide and Protein Sequence of Aspergillus BLAST Hits from GenBank ochraceus Oxidoreductase A phylogenetic tree displaying the genetic relatedness of The nucleotide and protein sequences of Aspergillus 55 Aspergillus Ochraceus Steroid 11 alpha hydroxylase, cloned ochraceus 11 oxidoreductase (SEQ ID NO: 5, SEQID NO: into plasmid pMON45624, was aligned with related 6, respectively) are displayed. enzymes found in GenBank. BLAST was used to find the FIG. 4-Amino Acid Homology Alignment of A. related enzymes within GenBank, and ClustalW was used ochraeeus 11 Alpha Hydroxylase with the Top 10 BLAST 60 generate the multiple sequence alignment and phylogenetic Hits from GenBank tree depicted in this figure. Descriptions of the GenBank Aspergillus ochraceus steroid 11 alpha hydroxylase (SEQ accession numbers used as labels in the figure are the same ID NO: 02), cloned into plasmid pMON45624 (SEQID NO: as that described above for the legend to FIG. 4. 01), was aligned with related enzymes found in GenBank FIG. 6 Percent Homology Between Aspergillus ochra using the BLASTP program that implements a heuristic 65 ceus 11 Alpha Hydroxylase and the Top 10 BLAST Hits matching algorithm (Altschul et al., J Mol Biol Oct from GenBank 5:215(3):403–10, 1990). The GenBank accession numbers The percent homology between Aspergillus ochraceus US 7,033,807 B2 29 30 steroid 11 alpha hydroxylase and the top 10 enzymes found The percent identity between Aspergillus ochraceus oxi in GenBank using BLAST was calculated using CLUSTAL doreductase and the oxidoreductases from A. niger, yeast, (Thompson et al., Comput. Appl. Biosci. 10:19–29, 1994). and mouse was calculated using Clustal W and Boxshade. FIG. 7—Amino Acid Homology Alignment of Aspergil FIG. 11—Alignment of Human Oxidoreductase with Top lus ochraceus and Human Oxidoreductase to NADPH Cyto 4 Hits from SwissProt The amino acid sequences of human steroid oxidoreduc chrome P450 Reductases from A. niger, Mouse, and S. tase (SEQ ID NO: 04), cloned into plasmid pMON45605 cerevisiae (SEQ ID NO: 03), which corresponds to the amino acid The amino acid sequences of Aspergillus ochraceus Ste sequence of the corrected sequence reported for P16435 roid oxidoreductase (SEQ ID NO: 06) cloned into plasmid 10 below, was aligned with the top 4 hits from the SWISSPROT pMON45632 (SEQID NO: 05), and human oxidoreductase protein sequence database, as described above. The SWIS (SEQ ID NO: 03), cloned into plasmid pMON45605 (SEQ SPROT accession numbers locus common name and ID NO: 04) were aligned with related enzymes from A. species) probable function) are as follows: P16435 niger, mouse, and S. cervisiase, as described above. The {NCPR HUMAN human NADPH-CYTOCHROME GenBank accession numbers (probable function, genus and 15 P450 REDUCTASE: P00389 (NCPR RABIT} rabbit) NADPH-CYTOCHROME P450 REDUCTASE: P00388 species) are as follows: BAAO2936 (NADPH-cytochrome {NCPR RAT} rat NADPH-CYTOCHROME P450 P450 reductase precursor Saccharomyces cerevisiae ); REDUCTASE: P37040 (NCPR MOUSE} (mouse) CAA81550 NADPH cytochrome P450 oxidoreductase As NADPH-CYTOCHROME P450 REDUCTASE; POs pergillus niger); P16435 (NADPH-CYTOCHROME P450 {NCPR PIG pig) (NADPH-CYTOCHROME P450 REDUCTASE (CPR) (P450R) human); BAA04496 REDUCTASE (NADPH-cytochrome P450 oxidoreductase References for these loci are as follows: P16435 Haniu, Mus musculus). M. McManus, M. E., Birkett, D. J. Lee, T. D. and Shively, References for these loci are as follows: BAAO2936 J. E. Structural and functional analysis of NADPH-cyto Yabusaki, Y. Murakami, H. and Ohkawa, H. Primary 25 chrome P-450 reductase from human liver: complete structure of Saccharomyces cerevisiae NADPH-cytochrome sequence of human enzyme and NADPH-binding sites. P450 reductase deduced from nucleotide sequence of its Biochemistry 28 (21), 8639–8645 (1989); P00389 Kata cloned gene. J. Biochem. 103 (6), 1004–1010 (1988): giri, M., Murakami, H., Yabusaki, Y., Sugiyama, T. Oka CAA81550 van den Brink, J., van Zeil, C. van den moto, M., Yamano, T. and Ohkawa, H. Molecular cloning Hondel, C. and Van Gorcom, R. Cloning and characteriza 30 and sequence analysis of full-length cDNA for rabbit liver tion of the NADPH cytochrome P450 oxidoreductase (cprA) NADPH-cytochrome P-450 reductase mRNA. J. Biochem. gene of Aspergillus niger. Unpublished; P16435 Haniu, 100 (4),945–954 (1986); P00388 Porter, T. D. and Kasper, M. McManus, M. E., Birkett, D. J. Lee, T. D. and Shively, C. B. Coding nucleotide sequence of rat NADPH-cyto J. E. Structural and functional analysis of NADPH-cyto chrome P-450 oxidoreductase cDNA and identification of chrome P-450 reductase from human liver: complete 35 flavin-binding domains. Proc. Natl. Acad. Sci. U.S.A. 82 (4), sequence of human enzyme and NADPH-binding sites Bio 973–977 (1985); P37040 Ohgiya, S., Shinriki, N., chemistry 28 (21), 8639–8645 (1989); BAAO4496Ohgiya, Kamataki, T. and Ishizaki, K. Mouse NADPH-cytochrome S., Shinriki, N., Kamataki, T. and Ishizaki, K. Mouse P-450 oxidoreductase: molecular cloning and functional NADPH-cytochrome P-450 oxidoreductase: molecular expression in yeast. Biochim. Biophys. Acta 1186 (1–2), cloning and functional expression in yeast. Biochim. Bio 40 137-141 (1994); PO7s Haniu, M., Iyanagi, T., Miller, P. phys. Acta 1186 (1–2), 137–141 (1994). Lee, T. D. and Shively, J. E. Complete amino acid sequence FIG. 8 Amino Acid Homology Alignment of A. ochra of NADPH-cytochrome P-450 reductase from porcine ceus Oxidoreductase to NADPH Cytochrome P450 Reduc hepatic microsomes. Biochemistry 25 (24), 7906–7911 tases from A niger, Mouse, and S. cerevisiae (1986)). The amino acid sequence of Aspergillus ochraceus steroid 45 FIG. 12 Phylogenetic Tree Showing the Relatedness of oxidoreductase (SEQ ID NO: 06) cloned into plasmid Human Oxidoreductases with Top 4 Hits from SwissProt pMON45632 (SEQ ID NO: 05), was aligned with related A phylogenetic tree displaying the genetic relatedness of fungal enzymes from A. niger and S. cervisiase, as described human oxidoreductase (SEQID NO: 04), cloned into plas above. Descriptions of the GenBank accession numbers mid pMON45604 (SEQ ID NO: 03), was aligned with used as labels in the figure are the same as that described 50 related enzymes found in SWISSPROT. BLAST was used to above for the legend to FIG. 7, above. find the related enzymes within SWISSPROT, and ClustalW FIG. 9 Phylogenetic Tree Showing the Relatedness of was used generate the multiple sequence alignment and Aspergillus ochraceus and Human Oxidoreductase to phylogenetic tree depicted in this figure. Descriptions of the SWISPROT accession numbers used as labels in the figure Reductases from A. niger, Yeast, and Mouse. 55 A phylogenetic tree displaying the genetic relatedness of are the same as that described above for the legend to FIG. Aspergillus ochraceus oxidoreductase (SEQ ID NO: 06). 11, above. cloned into plasmid pMON45632 (SEQ ID NO: 05), was FIG. 13—Percent Identity Between Human Oxidoreduc aligned with related enzymes. BLAST was used to find the tase and Top 4 Hits from SwissProt related enzymes within GenBank, and ClustalW was used 60 The percent identity between human oxidoreductase and generate the multiple sequence alignment and phylogenetic the top 4 hits found in SWISSPROT was calculated using tree depicted in this figure. Descriptions of the GenBank Clustal W and Boxshade. accession numbers used as labels in the figure are the same FIG. 14—Expression of Aspergillus ochraceus 11 Alpha as that described above for the legend to FIG. 7, above. Hydroxylase in Transfected Sf9 Insect Cells FIG. 10—Percent Identity Between Aspergillus ochra 65 Baculovirus-infected insect cells expressing Aspergillus ceus Oxidoreductase and Reductases from A. niger, Yeast, ochraceus 11 alpha hydroxylase were harvested at 25 and 48 and Mouse. hours post infection and microsomal membrane fractions US 7,033,807 B2 31 32 were prepared and separated by SDS-polyacrylamide gel and fragments of either, provides new and advantageous electrophoresis. The proteins in the gel were electrophoreti methods to convert steroid intermediates to their 11 alpha cally transferred to 0.2 um nitrocellulose membrane (Schle hydroxy counterparts. icher & Schuell Grimsehlstrasse 23 37574 Einbeck Ger The present invention also includes the DNA sequences many) and probed with antibodies GN-1187 and GN-1188 5 which code for the 11 alpha hydroxylases and prepared from peptide 11aOH peptide 2 CRQILTPYIH oxidoreductases, DNA sequences which are substantially KRKSLKGTTD (SEQ ID NO: 24). similar and perform Substantially the same function, and DNA sequences which differ from the DNAs encoding the FIG. 15 Expression of Aspergillus ochraceus P450 Oxi hydroxylases and oxidoreductases of the invention only due doreductase in Transfected Sf9 Insect Cells to the degeneracy of the genetic code. Also included in the Baculovirus-infected insect cells expressing Aspergillus 10 present invention are the oligonucleotide intermediates used Ochraceus 11 oxidoreductase were harvested at 25 and 48 to construct mutated versions of these DNAs and the hours post infection and microsomal membrane fractions polypeptides encoded by these oligonucleotides and mutant were prepared and separated by SDS-polyacrylamide gel DNAS. electrophoresis. The proteins in the gel were electrophoreti The present invention also includes antibodies which bind cally transferred to 0.2 um nitrocellulose membrane (Schle 15 specifically to A. Ochraceus 11 alpha hydroxylase or A. icher & Schuell Grimsehlstrasse 23 37574 Einbeck Ger Ochraceus oxidoreductase, including anti-peptide many) and probed with antibodies GN-2023 and GN-12024 antibodies, methods of using these anti-peptide antibodies to prepared from oxr peptide 1 CTYWAVAKDPYASAG purify these and other related polypeptides, methods of PAMNG (SEQ ID NO: 26). using the purified polypeptides to generate polyclonal or monoclonal antibodies to the full-length polypeptides, and FIG. 16 Conversion of Androstenedione to 11 Alpha methods of using antibodies to the full-length polypeptides Hydroxy Androstenedione Monitored by HPLC to assess the presence of the polypeptides in recombinant Microsomal and mitochondrial subcellular fractions were and non-recombinant host cells. The antibodies can be used prepared from insect cells co-infected with recombinant to identify related polypeptides in any of a variety of host baculoviruses expressing recombinant Aspergillus ochra- 2s organisms that possess the biological activities associated ceus 11 alpha hydroxylase and human oxidoreductase with these polypeptides. cloned from HepG2 cell RNA. The subcellular fractions Among the preferred organisms that can be used in this were incubated with 250 uMandrostenedione (AD) in the hydroxylation step are Aspergillus ochraceus NRRL 405, presence of an NADPH-generating system for 120 minutes, Aspergillus ochraceus ATCC 18500, Aspergillus niger and the resulting products were separated by HPLC and 30 ATCC 16888 and ATCC 26693, Aspergillus nidulans ATCC monitored by ultraviolet detection at 247 nm. Hydroxlase 11267, Rhizopus oryzae ATCC 11145, Rhizopus stolonifer activity was found in the microsomal fraction, as expected, ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillus but also appeared in the mitochondrial fraction. These megaterium ATCC 14945, Pseudomonas cruciviae ATCC results suggest that the 11 alpha hydroxylase may have a 13262, and Trichothecium roseum ATCC 12543. Other tendency to Stick to membranes in disrupted cells, or that the 35 preferred organisms include Fusarium oxysporum f. sp. separation of the Subcellular fractions in this experiment was cepae ATCC 11171 and Rhizopus arrhizus ATCC 11145. insufficient. Panel A illustrates a reaction carried out using Other organisms that have exhibited activity for this enzyme prepared from a mitochondrial fraction. The peak in reaction include Absidia coerula ATCC 6647, Absidia glauca ATCC 22752, Actinomucor elegans ATCC 6476, panel A that elutes after AD appears to be testosterone. When Aspergillus flavipes ATCC 1030, Aspergillus fumigatus a microsomal fraction was used, almost as much AD was 40 ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC converted to 11 alpha hydroxy AD, but relatively more 13144, Botryosphaeria obtusa IMI 038560, Calonectria testosterone was also produced. Panel B illustrates the same decora ATCC 14767, Chaetomium cochliodes ATCC 10195, reaction carried out for 120 minutes without a source of Corynespora cassicola ATCC 16718, Cunninghamella enzyme. Panel C illustrates an HPLC tracing with 11C.- blakesleeana ATCC 8688a, Cunninghamella echinulata hydroxyandrostenedione standard added to incubation 4s ATCC 3655, Cunninghamella elegans ATCC 9245, Curvu buffer. laria clavata ATCC 22921, Curvularia lunata ACTT 12071, Cylindrocarpon radicicola ATCC 1011, Epicoccum humi DETAILED DESCRIPTION OF THE cola ATCC 12722, Gongronella butleri ATCC 22822, Hypo INVENTION myces chrysospermus, Mortierella isabellina ATCC 42613, The present invention encompasses enzymes that facili- 50 Mucor mucedo ATCC 4605, Mucor griseocyanus ATCC tate the biosynthesis of steroid molecules, particularly 1207A, Myrothecium verrucaria ATCC 9095, Nocardia enzymes possessing cytochrome P450 or oxidoreductase corallina, Paecilomyces carneus ATCC 46579, Penicillum patulum ATCC 24550, Pithomyces atroolivaceus IFO 6651, activities. The present invention is directed, in part, to the Pithomyces cynodontis ATCC 26150, Pycnosporium sp. isolation of a nucleic acid encoding Aspergillus Ochraceus ATCC 12231, Saccharopolyspora erythrae ATCC 11635, 11 alpha hydroxylase, which exhibits sequence homology to 55 Sepedonium chrysospermum ATCC 13378, Stachylidium the highly conserved residues that correspond to cytochrome bicolor ATCC 12672, Streptomyces hygroscopicus ATCC P450 enzymes. It also directed to the isolation of nucleic 27438, Streptomyces purpurascens ATCC 25489, Synceph acids encoding human and Aspergillus ochraceus oxi alastrum racemosum ATCC 18192, Thamnostylum piri doreductase. Biological activities of the cloned hydroxylases forme ATCC 8992, Thielavia terricola ATCC 13807, and and oxidoreductases of the present invention can be deter- 60 Verticillium theobromae ATCC 12474. mined by a variety of assays, including incubation of steroid Additional organisms that may be expected to show Substrates in the presence of microsomes prepared from activity for the 11C. hydroxylation include Cephalosporium recombinant baculovirus-infected insect cells and monitor aphidicola (Phytochemistry (1996), 42(2), 411-415), ing the conversion to their 11 alpha hydroxy-counterparts by Cochliobolus lunatas (J. Biotechnol. (1995), 42(2), high pressure liquid chromatography (HPLC). The present 65 145–150), Tieghemella orchidis (Khim.-Farm. Zh. (1986), invention, comprising novel 11 alpha hydroxylase and oxi 20(7), 871-876), Tieghemella hyalospora (Khim.-Farm. Zh. doreductase nucleic acids, proteins, peptides, homologues, (1986), 2007), 871-876), Monosporium olivaceum (Acta US 7,033,807 B2 33 34 Microbiol. Poll., Ser. B. (1973), 5(2), 103-110), Aspergillus 1985) in which a portion of the coding sequence in a plasmid ustus (Acta Microbiol. Poll., Ser. B. (1973), 5(2), 103-110), is replaced with synthetic oligonucleotides that encode the Fusarium graminearum (Acta Microbiol. Pol. Ser. B. desired amino acid Substitutions in a portion of the gene (1973), 5(2), 103-110), Verticillium glaucum (Acta Micro between two restriction sites. biol. Poll., Ser. B. (1973), 5(2), 103-110), and Rhizopus Pairs of complementary synthetic oligonucleotides encod nigricans (J. Steroid Biochem. (1987), 28(2), 197–201). ing the desired gene can be made and annealed to each other. FIG. 1 sets forth the nucleotide and protein sequence of The DNA sequence of the oligonucleotide would encode Aspergillus ochraceus 11 alpha hydroxylase (SEQID NO: 1, sequence for amino acids of desired gene with the exception SEQ ID NO: 2, respectively). FIG. 2 sets forth the nucle of those substituted and/or deleted from the sequence. otide and protein sequence of human oxidoreductase (SEQ 10 Plasmid DNA can be treated with the chosen restriction ID NO:3, SEQID NO: 4, respectively). FIG.3 sets forth the endonucleases then ligated to the annealed oligonucleotides. nucleotide and protein sequence of Aspergillus ochraceus The ligated mixtures can be used to transform competent E. oxidoreductase (SEQ ID NO: 5, SEQ ID NO: 6, coli cells which will confer resistance to an appropriate respectively). antibiotic. Single colonies can be picked and the plasmid FIG. 4 sets forth an amino acid homology alignment of A. 15 DNA examined by restriction analysis or by DNA sequenc ochraceus 11 alpha hydroxylase cloned in pMON45624 and ing to identify plasmids with the desired genes. aligned with related enzymes found in GenBank using Cloning of DNA sequences encoding novel proteins and BLAST FIG. 5 is a phylogenetic tree showing the this fusion proteins may be accomplished by the use of inter relationship graphically. FIG. 6 shows the percent homology mediate vectors. Linkers and adapters can be used to join between Aspergillus ochraceus steroid 11 alpha hydroxylase DNA sequences, and to replace lost sequences, where a and the top 10 enzymes found in GenBank using BLAST, restriction site is internal to the region of interest. DNA calculated using Clustal W and Boxshade. encoding a single polypeptide or a fusion protein FIG. 7 sets forth the amino acid homology of Aspergillus (comprising a first polypeptide, a peptide linker, and a ochraceus and human oxidoreductase to NADPH cyto second polypeptide) is inserted into a Suitable expression chrome P450 reductases from A. niger, mouse, and S. 25 vector which is then transformed or transfected into appro cerevisiae (yeast). FIG. 8 sets forth the amino acid alignment priate bacterial, fungal, insect, or mammalian host cells. The for A. ochraceus, A. niger, and S. cerevisiae oxidoreduc transformed organism or host cell line is grown and the tases. FIG. 9 is a phylogenetic tree showing the relatedness recombinant protein isolated by standard techniques. of Aspergillus ochraceus and human oxidoreductase to Recombinant fusion proteins have all or a portion of a first reductases from A. niger, yeast, and mouse. FIG. 10 shows 30 protein joined by a linker region to a all or a portion of the percent homology between Aspergillus ochraceus ste second protein. roid 11 alpha hydroxylase and the oxidoreductases from A. Hybridization niger, yeast, and mouse, calculated using Clustal W and Nucleic acid molecules and fragment nucleic acid mol Boxshade. ecules encoding 11 alpha hydroxylases or oxidoreductases 35 can specifically hybridize with other nucleic acid molecules. FIG. 11—Alignment of human oxidoreductase with top 4 Two nucleic acid molecules are said to be capable of hits from SwissProt. FIG. 12 sets forth a phylogenetic tree specifically hybridizing to one another if the two molecules displaying the genetic relatedness of human oxidoreductase, are capable of forming an anti-parallel, double-stranded to these hits. FIG. 13 shows the percent identity between nucleic acid structure. A nucleic acid molecule is said to be human oxidoreductase and top 4 hits from SwissProt. 40 the “complement of another nucleic acid molecule, if they FIG. 14 sets forth an immunoblot illustrating expression exhibit complete complementarity. Molecules exhibit com of Aspergillus ochraceus P450 11 alpha hydroxylase in plete “complementarity” when every nucleotide of one of baculovirus-infected insect cells harvested at 25 and 48 the molecules is complementary to a nucleotide of the other. hours post infection. The nitrocellulose membrane was Two molecules are “minimally complementary’ if they can probed with a 1:1 mixture of antibodies prepared from two 45 hybridize to one another with sufficient stability to permit rabbits immunized with a conjugated synthetic peptide, them to remain annealed to one another under at least 11aOH peptide 2 (SEQ ID NO 24). conventional “low-stringency' conditions. Similarly, the FIG. 15 sets forth an immunoblot illustrating expression molecules are “complementary’ if they can hybridize to one of Aspergillus ochraceus P450 oxidoreductase in another with sufficient stability to permit them to remain baculovirus-infected insect cells harvested at 25 and 48 50 annealed to one another under conventional “high hours post infection. The nitrocellulose membrane was stringency' conditions. Conventional Stringency conditions probed with a 1:1 mixture of antibodies prepared two rabbits are described by Sambrook, et al., Molecular Cloning. A immunized with a conjugated synthetic peptide, OXr peptide Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, 1 (SEQ ID NO 26). Cold Spring Harbor, N.Y. (1989), and by Haymes, et al. FIG. 16 sets forth an HPLC tracing illustrating the con 55 Nucleic Acid Hybridization, A Practical Approach, IRL version of androstenedione (AD) to its 11 alpha hydroxy Press, Washington, D.C., 1985). Departures from complete counterpart after incubating AD with subcellular fractions complementarity are therefore permissible, as long as Such prepared from baculovirus-infected insect cells expressing departures do not completely preclude the capacity of the Aspergillus ochraceus 11 alpha hydroxylase and human molecules to form a double-stranded structure. oxidoreductase. 60 Appropriate Stringency conditions which promote DNA Cloning Techniques hybridization are well known to those skilled in the art, or Genetic engineering techniques now standard in the art can be found in Current Protocols in Molecular Biology, (U.S. Pat. No. 4,935,233 and Sambrook et al., “Molecular John Wiley & Sons, N.Y., 6.3.1–6.3.6. (1989). Basic con Cloning A Laboratory Manual, Cold Spring Harbor ditions would include, for example, 6xsodium saline citrate Laboratory, 1989) may be used in the construction of the 65 (SSC) at about 45° C., followed by a wash of 2xSSC at 50° DNA sequences of the present invention. One such method C. Stringency can be varied, for example, by altering the salt is cassette mutagenesis (Wells et al., Gene 34:315-323, concentration in the wash step from about 2xSSC at 50° C. US 7,033,807 B2 35 36 (moderately low stringency) to about 0.2xSSC at 50° C. myces cerevisiae. The proteins or fragments thereof of the (high Stringency). Stringency can also be altered by chang present invention can be expressed in S. cerevisiae by fusing ing the temperature in the wash step, from room it to the N-terminus of the URA3, CYC1 or ARG3 genes temperature, about 22° C. (low stringency conditions), to (Guarente and Ptashne, Proc. Natl. Acad. Sci. (U.S.A.) about 65° C. (high stringency conditions). Both temperature 78:2199–2203 (1981); Rose et al., Proc. Natl. Acad. Sci. and salt may be varied, or either the temperature or the salt (U.S.A.) 78:2460–2464 (1981); and Crabeel et al., EMBO.J. concentration may be held constant while the other variable 2:205–212 (1983)). Alternatively, proteins or fragments is changed. thereof of the present invention can be fused to either the Expression Vectors PGK or TRP1 genes (Tuite et al., EMBO J. 1:603-608 Another aspect of the present invention includes plasmid 10 (1982); and Dobson et al., Nucleic Acids. Res. 11:2287–2302 DNA vectors for use in the expression of these novel (1983)). More preferably, the protein or fragment thereof of hydroxylases and oxidoreductases. These vectors contain the present invention is expressed as a mature protein the novel DNA sequences described above which code for (Hitzeman et al., Nature 293:717–722 (1981); Valenzuela et the novel polypeptides of the invention. Appropriate vectors al., Nature 298:347–350 (1982); and Deryncket al., Nucleic which can transform microorganisms or cell lines capable of 15 Acids Res. 11:1819–1837 (1983)). expressing the hydroxylases and oxidoreductases include Native and engineered yeast promoters Suitable for use in expression vectors comprising nucleotide sequences coding the present invention have been reviewed by Romanos et al., for the hydroxylases and oxidoreductases joined to tran Yeast 8:423–488 (1992). Most preferably, the protein or Scriptional and translational regulatory sequences which are fragment thereof of the present invention is secreted by the selected according to the host cells used. yeast cell (Blobel and Dobberstein, J. Cell Biol. 67:835-851 Vectors incorporating modified sequences as described (1975); Kurjan and Herskowitz, Cell 30:933–943 (1982); above are included in the present invention and are useful in Bostian et al., Cell 36:741–751 (1984); Rothman and Orci, the production of the hydroxylases and oxidoreductases. The Nature 355:409–415 (1992); Julius et al., Cell 32:839-852 vector employed in the method also contains selected regu (1983); and Julius et al., Cell 36:309-318 (1984)). latory sequences in operative association with the DNA 25 Mammalian coding sequences of the invention and which are capable of General methods for expression of foreign genes in mam directing the replication and expression thereof in selected malian cells have been reviewed (Kaufman, R. J., 1987, host cells. “Genetic Engineering, Principles and Methods’. Vol. 9, J. K. Methods for producing the hydroxylases and oxidoreduc Setlow, editor, Plenum Press, New York; Colosimo et al., tases is another aspect of the present invention. The method 30 Biotechniques 29: 314-331, 2000). Recombinant proteins of the present invention involves culturing suitable cells or are generally targeted to their natural locations within the cell lines, which has been transformed with a vector con host cell (e.g., cytoplasm, nucleus, or various membrane taining a DNA sequence encoding novel hydroxylases and compartments), or are secreted, if a signal peptide is present. oxidoreductases. Suitable cells or cell lines may be bacterial An expression vector is constructed in which a strong cells. For example, various strains of E. coli are well-known 35 promoter capable of functioning in mammalian cells drives as host cells in the field of biotechnology. Examples of such transcription of a eukaryotic secretion signal peptide coding strains include E. coli strains DH5 alpha, DH10B and region, which is translationally joined to the coding region MON105 (Obukowicz et al., Applied Environmental Micro for the desired protein. For example, plasmids such as biology 58: 1511–1523, 1992). Also included in the present pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from invention is the expression of the hydroxylases and oxi 40 Invitrogen Corp., San Diego, Calif.) can be used. The doreductases utilizing a chromosomal expression vector for eukaryotic secretion signal peptide coding region can be E. coli based on the bacteriophage Mu (Weinberg et al., from the gene itself or it can be from another secreted Gene 126: 25–33, 1993). Various other strains of bacteria, mammalian protein (Bayne, M. L. et al., Proc. Natl. Acad. including the Enteric bacteria (e.g., Salmonella sp.) and B. Sci. USA 84: 2638-2642, 1987). After construction of the subtilis, may also be employed in this method. 45 vector containing the gene, the vector DNA is transfected When expressed in the E. coli cytoplasm, the gene encod into mammalian cells such as the COS7, HeLa, BHK, ing the proteins of the present invention may also be Chinese hamster ovary (CHO), or mouse L lines. The cells constructed Such that at the 5' end of the gene codons are can be cultured, for example, in DMEM media (JRH added to encode Met-Ala', Met-Ser', Met-Cys', or Scientific). The polypeptide secreted into the media can be Met' at the N-terminus of the protein. The N termini of 50 recovered by standard biochemical approaches following proteins made in the cytoplasm of E. coli are affected by transient expression for 24–72 hours after transfection of the post-translational processing by methionine aminopeptidase cells or after establishment of stable cell lines following (Ben Bassat et al., J. Bacteriol. 169:751–757, 1987), and selection for antibiotic resistance. The selection of suitable possibly by other peptidases, so that upon expression the mammalian host cells and methods for transformation, methionine is cleaved off the N-terminus. The proteins of the 55 culture, amplification, Screening and product production and present invention may include polypeptides having Met', purification are known in the art. See, e.g., Gething and Ala', Ser', Cys', Met-Ala', Met-Ser', or Met Sambrook, Nature, 293:620–625, 1981, or alternatively, Cys' at the N-terminus. These mutant proteins may also be Kaufman et al, Mol. Cell. Biol., 5(7): 1750–1759, 1985) or expressed in E. coli by fusing a secretion signal peptide to Howley et al., and U.S. Pat. No. 4,419,446. Other suitable the N-terminus. This signal peptide is cleaved from the 60 mammalian cell lines are the monkey COS-1 cell line and polypeptide as part of the secretion process. the CV-1 cell line. Yeast Mammalian cells can also be used to express the nucleic Many strains of yeast cells known to those skilled in the acid molecules of the present invention. The nucleic acid art are also available as host cells for expression of the molecules of the present invention can be cloned into a polypeptides of the present invention. Under another 65 suitable retroviral vector (see, e.g., Dunbar et al., Blood embodiment, the protein or fragment thereof of the present 85:3048–3057 (1995); Baum et al., J. Hematother 5: invention is expressed in a yeast cell, preferably Saccharo 323-329 (1996); Bregniet al., Blood 8.0:1418–1422 (1992); US 7,033,807 B2 37 38 Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102–109 Insect Cell Expression (1993); Boris-Lawrie and Temin, Annal. New York Acad. Insect cells may be used as host cells to express recom Sci. 716:59–71 (1994); Miller, Current Top. Microbiol. binant proteins of the present invention (See, e.g., Luckow, Immunol. 158:1–24 (1992)), adenovirus vector (Berkner, V. A., Protein Eng. J. L. Cleland. Wiley-Liss, New York, BioTechniques 6:616–629 (1988); Berkner, Current Top. N.Y.: 183–218, 1996, and references cited therein). General Microbiol. Immunol. 158:39–66 (1992); Brody and Crystal, methods for expression of foreign genes in insect cells using Annal. New York Acad. Sci. 716:90–103 (1994); Baldwin et baculovirus vectors have been described (O'Reilly, D. R. L. al., Gene Ther. 4:1142–1149 (1997)), RSV, MuSV, SSV. K. Miller et al. Baculovirus Expression Vectors: A Labora MuDV (Baum et al., J. Hematother. 5: 323-329 (1996)), tory Manual. New York, W. H. Freeman and Company, AAV (Chen et al., Gene Ther. 5:50–58 (1998); Hallek et al., 10 1992; and King, L. A. and R. D. Possee. The Baculovirus Cytokines Mol. Ther. 2: 69–79 (1996)), AEV, AMV, or CMV Expression System. A Laboratory Guide, London, Chapman (Griffiths et al., Biochem. J. 241: 313–324 (1987)). & Hall). Transformation and Transfection A baculovirus expression vector can be constructed by In another aspect, the invention provides a transformed inserting the desired gene (e.g., 11 alpha hydroxylase or cell having a nucleic acid molecule which comprises an 15 oxidoreductase) into abaculovirus transfer vector which can exogenous promoter region which functions in a cell to recombine into the baculovirus genome by homologous cause the production of an mRNA molecule which is linked recombination. Many transfer vectors use a strong baculovi to a structural nucleic acid molecule, wherein the structural rus promoter (such as the polyhedrin promoter) to drive nucleic acid molecule encodes an 11 alpha hydroxylase or transcription of the desired gene. Some vectors permit the oxidoreductase gene or fragment thereof. This nucleic acid expression of fusion proteins or direct the secretion of molecule is linked to a 3' non-translated sequence that proteins from the cell by fusing a eukaryotic secretion signal functions in a cell to cause termination of transcription and peptide coding region to the coding region of the desired addition of polyadenylated ribonucleotides to a 3' end of the gene. The plasmid pVL 1393 (obtained from Invitrogen mRNA molecule. Corp., San Diego, Calif.) can be used, for example, to direct Methods and compositions for transforming eukaryotic 25 transcription of nonfused foreign genes in baculovirus cells, bacteria and other microorganisms are known in the art infected insect cells. The baculovirus transfer vector con (see, for example, Sambrook et al., Molecular Cloning. A taining the desired gene is transfected into Spodoptera Laboratory Manual, Second Edition, Cold Spring Harbor frugiperda (Sf9) insect cells along with circular or linearized Laboratory Press, Cold Spring Harbor, N.Y., (1989); Colo genomic baculovirus DNA, and recombinant baculoviruses 30 purified and amplified after one or more plaque assays. simo et al., Biotechniques 29: 314-331, 2000). Recombinant baculoviruses can also be created using the Technology for introduction of DNA into cells is well baculovirus shuttle vector system (Luckow, V. A. et al., J. known to those of skill in the art. Four general methods for Virol. 67(8): 4566-4579, 1993; U.S. Pat. No. 5,348,886) delivering a gene into cells have been described: (1) chemi now marketed as the Bac-To-BacTM Expression System cal methods (Graham and van der Eb, Virology 54:536–539 35 (Life Technologies, Inc., Rockville, Md.). The desired genes (1973)); (2) physical methods such as microinjection are inserted downstream from the polyhedrin promoter in (Capecchi, Cell 22:479–488 (1980)), electroporation (Wong mini-Tm7 cassettes that are transposed in Vivo into a bacu and Neumann, Biochem. Biophys. Res. Commun. lovirus shuttle vector genome propagated in E. coli. Com 107:584-587 (1982); Fromm et al., Proc. Natl. Acad. Sci. posite viral DNAs are isolated from E. coli and transfected (U.S.A.) 82:5824–5828 (1985); U.S. Pat. No. 5,384,253); 40 into Sf9 cells and stocks of recombinant baculoviruses are and the gene gun (Johnston and Tang, Methods Cell Biol. rapidly prepared without the need for multiple rounds of 43:353–365 (1994); (3) viral vectors (Clapp, Clin. Perinatol. tedious plaque purification common to methods that rely on 20:155–168 (1993); Lu et al., J. Exp. Med. 178:2089. 2096 homologous recombination. (1993); Eglitis and Anderson, Biotechniques, 6:608–614 Recombinant baculoviruses can also created using the (1988)); and (4) receptor-mediated mechanisms (Curiel et 45 Gateway Recombinational Cloning System (Life al., Hum. Gen. Ther. 3:147–154 (1992), Wagner et al., Proc. Technologies) of shuttling genes from vector to vector using Natl. Acad. Sci. (U.S.A.) 89:6099–6103 (1992)). Other modified genetic elements (attachment sites) and modified methods well known in the art can also be used. proteins (e.g., int, IHF, xis) that are involved in the site Transformation can be achieved using methods based on specific integration and excision of bacteriophage lambda. calcium phosphate precipitation, polyethylene glycol 50 Pure recombinant baculoviruses carrying the 11 alpha treatment, electroporation, and combinations of these treat hydroxylase or oxidoreductase gene are used to infect cells ments (see for example Potrykus et al., Mol. Gen. Genet. cultured, for example, in Excell 401 serum-free medium 205:193–200 (1986); Lorz et al., Mol. Gen. Genet. 199: 178 (JRH Biosciences, Lenexa, Kans.) or Sf900-II (Life (1985): Fromm et al., Nature 319:791 (1986); Uchimiya et Technologies). Hydroxylases or oxidoreductases that are al., Mol. Gen. Genet. 204:204 (1986); Marcotte et al., Nature 55 localized to membranes can be prepared using standard 335:454 457 (1988)). protocols that fractionate and enrich for enzymes in mito Assays for gene expression based on the transient expres chondrial or microsomal fractions (Engel and White, Dev sion of cloned nucleic acid constructs have been developed Biol. 140: 196-208, 1990). Hydroxylases or oxidoreductases by introducing the nucleic acid molecules into cells by that are secreted or leak into the medium can also be polyethylene glycol treatment, electroporation, or particle 60 recovered by Standard biochemical approaches. bombardment (Marcotte et al., Nature 335: 454-457 (1988); Simultaneous expression of two or more recombinant McCarty et al., Cell 66: 895–905 (1991); Hattori et al., proteins in baculovirus-infected insect cells can be carried Genes Dev. 6: 609-618 (1992); Goffet al., EMBO J. 9: out by two general approaches. The simplest approach is to 2517–2522 (1990)). Transient expression systems may be coinfect insect cells with titered stocks of recombinant used to functionally dissect the regulatory and structural 65 baculoviruses harboring a single heterologous gene under features of expression cassettes comprising operably-linked the control of a strong baculovirus promoter, Such as the genetic elements. polyhedrin or the plC) promoter. These promoters are highly US 7,033,807 B2 39 40 transcribed during the late stages of infection when most Cloning, A Laboratory Manual, 2" edition, Cold Spring host cell protein synthesis has been shut down. Earlier Harbor Laboratory, 1989, and references cited therein, incor baculovirus promoters or other insect or eukaryotic cell porated herein by reference. General features and maps of a promoters can also be used to direct synthesis at other times, wide variety of cloning and expression vectors have been which generally result in lower expression levels. Varying 5 also been published (Gacesa, P. and Ramji. D. P. Vectors: the ratio of two or more recombinant viruses used in a Essential Data, John Wiley & Sons, 1994). General methods coinfection or selecting viruses that use different promoters for the cloning and expression of genes in mammalian cells to drive expression of the recombinant protein will permit are also found in Colosimo et al., Biotechniques 29: one skilled in the art to select conditions suitable for optimal 314-331, 2000. General and specific conditions and proce expression of the desired recombinant proteins. 10 dures for the construction, manipulation and isolation of Construction of dual- or multiple-expression vectors will polyclonal and monoclonal antibodies are well known in the also permit the expression of two or more recombinant art (See, for example, Harlow and Lane, Antibodies. A proteins in baculovirus-infected insect cells. Generally, Laboratory Manual, Cold Spring Harbor Press, Cold Spring these vectors permit the introduction two or more gene cassettes into a single locus in the baculovirus genome. The 15 Harbor, N.Y., 1988). structures of a variety of dual expression vectors have been Unless noted otherwise, all specialty chemicals were described (O'Reilly, D. R. L. K. Miller et al. Baculovirus obtained from Sigma (St. Louis, Mo.). Restriction endonu Expression Vectors: A Laboratory Manual. New York, W. H. cleases and T4 DNA ligase were obtained from Life Tech Freeman and Company, 1992; and King, L. A. and R. D. nologies (Rockville, Md.), New England Biolabs (Beverly, Possee. The Baculovirus Expression System: A Laboratory Mass.), Roche Molecular Biochemicals (Indianapolis, Ind.), Guide, London, Chapman & Hall). or Promega (Madison, Wis.). All parts are by weight and Materials and Methods temperatures are in degrees centigrade (C.), unless other General Methods wise indicated. General methods of cloning, expressing, and characteriz Strains, Plasmids, and Sequence Cross Listings ing proteins are found in T. Maniatis, et al., Molecular 25 The bacterial strains used in these studies are listed in Cloning, A Laboratory Manual, Cold Spring Harbor Table 1. Plasmids used or constructed for this study are listed Laboratory, 1982, and references cited therein, incorporated in Table 2. Brief descriptions of sequences of relevant herein by reference; and in J. Sambrook, et al., Molecular oligonucleotides, genes, or proteins are listed in Table 3.

TABLE 1.

Strains Designation Description or Genotype Reference, Source DH5CTM F. phi80 dlaczdeltaM15, Life Technologies, Rockville, delta(laczYA-argF)U169, deoR, recA1, Maryland end A1, hsdR17 (rk, mk"), phoA, SupF44, lambda-, thi-1, gyra 96, relA1 DH1OBTM F. mcra D(mirr-hsdRMS-mcrBC) Life Technologies, Rockville, phi80 dlaczDM15 DlacX74 endA1 Maryland recA1 deoRD(ara, leu)7697 arad139 gally galKnupG rpsL DH1OBac TM DH 10B harboring the baculovirus Life Technologies, Rockville, shuttle vector bMON14272 (Kan.) Maryland; See also Luckow et al., J. and the helper plasmid pMON7124 Virol. 67: 4566 4579 (1993) (TetR)

TABLE 2

Plasmids Plasmid SEQ ID NO. Marker Description Source pFastBac1 Amp Baculovirus donor plasmid containing Life Technologies Gent multiple cloning site downstream Inc. (Rockville, of an AcNPV polyhedrin promoter MD): See also within a mini-Tn7 transposable Luckow et al., element capable of being transposed J. Virol. 67: to a baculovirus shuttle vector 4566 4579 (1993) pBluescript II Amp Multifunctional phagemid cloning Stratagene, La SK vector derived from plJC19. Jolla, CA pCRII-TOPO Amp Multifunctional cloning vector Invitrogen, Kan. for direct cloning of polymerase Carlsbad, CA chain reaction products using the T overhang pSport1 Amp Multifunctional cloning vector for Life Technologies, cloning and in vitro transcription Rockville, MD from either strand using SP6 or T7 promoters US 7,033,807 B2 41 42

TABLE 2-continued

Plasmids

Plasmid SEQ ID NO. Marker Description Source GEM-T Amp A derivative of pCEM-5Zf(+) with Promega, single 5 T overhangs at the Madison, WI insertion site to improve the efficiency of PCR product ligation MON45624 #1 Amp' pFastBac1 EcoRIXbal + PCR This work Gentr ragment EcoRIXbal encoding Aspergilius ochracetis 11 alpha hydroxylase MON45603 Amp' pBluescriptiISK BamHI/HincII + This work BamHI/HincII 5" segment of human oxidoreductase MON45604 Amp' pBluescriptiISK HincII/KpnI + This work HincII/KpnI 3 segment of human oxidoreductase MON4560S #3 Amp' pFastBac1 BamHI/KpnI + BamHI. This work Gent KpnI complete coding region of human oxidoreductase cDNA. MON456.30 Amp' pCRII-TOPO SalI/BamHI + SalI/ This work Kan. BamHI5 segment of A. ochraceus oxidoreductase cDNA MON45631 Amp' pCRII-TOPO BamHI/XhoI + This work Kan. BamHIXhoI 3 segment of A. ochraceus oxidoreductase cDNA which lacked the intron. MON45632 #5 Amp' pFastBac1 SalIXhoI + containing This work Gent assembled coding region of Aspergilius ochracetis oxidoreductase

TABLE 3 Table of Sequences SEQ ID NO Description Length/Sequence Type (SEQ NO O1) Nucleotide sequence of 1776 DNA Aspergillus ochraceus 11alphaOH gene from plMON45624

(SEQ NO 02)Aspergillus ochraceus 514 Protein 11alphaoH protein sequence from plMON45624

(SEQ NO O3) Nucleotide sequence of human 2031 DNA oxidoreductase gene from pMON45 605

(SEQ NO 04)Human oxidoreductase protein 677 Protein sequence from plMON45605

(SEQ NO 05)Nucleotide sequence of 2322 DNA Aspergillus ochraceus oxidoreductase gene from pMON45632

(SEQ ID NO 06)Aspergillus ochraceus 705 Protein oxidoreductase protein sequence from plMON45632

(SEQ ID NO 07)Primer H. oxred 1A gatcggat.ccaatATGG DNA GAGACTCCCACGTGGAC AC

(SEQ ID NO 08)Primer H. oxred 1B CAGCTGGTTGACGAGAG DNA CAGAG

(SEQ ID NO 09)Primer H. oxred 2A CTCTGCTCTCGTCAACC DNA AGCTG

(SEQ ID NO 10)Primer H. oxred 2B gatcgg taccttagcTC DNA CACACGTCCAGGGAGTA US 7,033,807 B2 43 44

TABLE 3-continued Table of Sequences

SEQ ID NO Description Length/Sequence Type

(SEQ ID NO : 1)Primer A. oxred-for 1 GACGGIGCIGGTACAAT DNA GCA

(SEQ ID NO : 2)Primer A. oxred-revil TTAIGACCAIACATCIT DNA CCTGGTAGC

(SEQ ID NO : 3)Primer pSport-for 1 CAAGCTCTAATACGACT DNA CACTATAGGGA

(SEQ ID NO : 4)Primer A. oxred-rev2 CAGGAACCGATCGACCT DNA CGGAA

(SEQ ID NO : 5)Primer A. oxred-rev3 GTCACCCT CACCAGCAG DNA AGCCAATG

(SEQ ID NO : 6)Primer A. oxred-rev4 CCACATTGCGAACCATA DNA GCGTTGTAGTG

(SEQ ID NO : 7) Primer pSport-for-2 GCCAAGCTCTAATACGA DNA CTCACTATAGGGAAAGC

(SEQ ID NO : 8)Primer A. oxred-for2 gtcgacATGGCGCAACT DNA CGATACTCTC

(SEQ ID NO : 9)Primer A. oxred-rev5 citcgagttaGGACCAGA DNA CATCGTCCTGGTAG

(SEQ ID NO : 20)Primer A. oxred-for 3 GGATCCCTCGCGACCTG DNA TGATCAT

(SEQ ID NO : 21)Primer A. oxred-for-4 CGAAGATTTCTTGTACA DNA AGGATGAATGGAAGACT TTTC

(SEQ ID NO : 22)Primer A. oxred-rev6 CTGAAAAGTCTTCCATT DNA CATCCTTGTACAAGAAA TC

(SEQ ID NO : 23) 11aoH peptide 1 AAAYWLATLQPSDLPEL Protein N

(SEQ ID NO : 24) 11aCH peptide 2 CRQILTPYIHKRKSLKG Protein TTDE

(SEQ ID NO : 25) 11aoH peptide 3 HMGFGHGWHACPGRFFA Protein SNEI

(SEQ ID NO : 26) oxir peptide 1 CTYWAWAKDPYASAGPA Protein MNG

(SEQ ID NO : 27)CAA75565; cytochrome P450 Protein monooxygenase Gibberella fujikuroi

(SEQ ID NO : 28) CAB91316; probable cytochrome Protein P450 monooxygenase (lova) Neurospora Crassa

(SEQ ID NO : 29) CAB56503; cytochrome P450 Protein Catharanthus roseus (SEQ ID NO : 30)AAB94588; CYP71D10p Glycine Protein max (SEQ ID NO : 31)CAA75566; cytochrome P450 Protein monooxygenase Gibberella fujikuroi

(SEQ ID NO : 32)AAD34552; cytochrome P450 Protein monooxygenase Aspergillus terreus US 7,033,807 B2 45 46

TABLE 3-continued Table of Sequences

SEQ ID NO Description Length/Sequence Type (SEQ ID NO : 33)CAA75567; cytochrome P450 Protein monooxygenase Gibberella fujikuroi

(SEQ ID NO : 34)CAA76703; cytochrome P450 Protein Gibberella fujikuroi (SEQ ID NO : 35) CAA57874; unnamed protein Protein product Fusarium oxysporum (SEQ ID NO : 36) CAA9 1268; similar to Protein cytochrome P450-cDNA EST yk423b11.3 comes from this gene; Caenorhabditis elegans (SEQ ID NO : 37)BAA02936 NADPH-cytochrome P450 Protein reductase precursor Saccharomyces cerevisiae (SEQ ID NO : 38)CAA81550 NADPH cytochrome P450 Protein oxidoreductase Aspergillus niger

(SEQ ID NO : 39)BAAO 4496 NADPH-cytochrome P450 Protein oxidoreductase Mus musculus (SEQ ID NO : 40) Universal bacteriophage M13 CAG GAA ACA GCT DNA reverse primer ATG AC (SEQ ID NO : 41) Universal bacteriophage T7 TAA TAC GAC TCA DNA promoter primer CTA TAG GG

(SEQ ID NO : 42)Aspergillus ochraceus Primer gatcgaatticATGCCCT DNA 11alphaOH- for TCTTCACTGGGCT

(SEQ ID NO : 43)Aspergillus ochraceus Primer gatctotagattacaca DNA 11alphaOH-rev gttaaacticgc.caTATC GAT

(SEQ ID NO : 44) pFastBac1 Primer Bacfwd CTGTTTTCGTAACAGTT DNA TTG

(SEQ ID NO : 45)pFastBac1 Primer PolyA CCTCTACAAATGTGGTA DNA TG

(SEQ ID NO : 46)Aspergillus ochraceus Primer GAGATCAAGATTGCCTT DNA 45 624-for1

(SEQ ID NO : 47)Aspergillus ochraceus Primer CTTCGACGCTCTCAA DNA 45 624-for2

(SEQ ID NO : 48).Aspergillus ochraceus Primer GCAATCTTGACTCGTT DNA 45 624-rev1.

(SEQ ID NO : 49) S90 469 human cytochrome P450 24 O3 DNA reductase placental mRNA Partial, 2403 nt. (SEQ ID NO : 50)AAB21814 human cytochrome P450 676 Protein reductase, placental partial (SEQ ID NO : 51) A60557 human NADPH 677 Protein ferrihemoprotein reductase (SEQ ID NO : 52)P16435 Human NADPH-cytochrome 677 Protein P450 reductase

(SEQ ID NO : 53)P00389 Rabbit NADPH-cytochrome 6.79 Protein P450 reductase

(SEQ ID NO : 54)P00388 Rat NADPH-cytochrome 678 Protein P450 reductase

(SEQ ID NO : 55)P37040 Mouse NADPH-cytochrome 678 Protein P450 reductase US 7,033,807 B2 47 48

TABLE 3-continued Table of Sequences SEQ ID NO Description Length/Sequence Type

(SEQ ID NO: 56)PO 4175 Pig NADPH-cytochrome 678 Protein P450 reductase (SEQ ID NO: 57 ) Universal bacteriophage SP6 gatttaggtgacact at DNA primer ag (SEQ ID NO: 58) NotI-poly-dT adapter 5'- pGACTAGT DNA TCTAGA TCGCGA GCGGCCGC CC (T) 15 -3' (SEQ ID NO: 59) Sall adapter, top strand 5'- DNA TCGACCCACGCGTCCG -3' (SEQ ID NO: 60) Sall adapter, bottom strand 3'- DNA GGGTGCGCAGGCp 5' (SEQ ID NO : 61)Primer oxred 1C GTGGACCACAAGCTCGT DNA ACTG (SEQ ID NO: 62)Primer oxred 2C CATCGACCACCTGTGTG DNA AGCTG (SEQ ID NO: 63)Primer oxred 2D GTACAGGTAGTCCTCAT DNA CCGAG (SEQ ID NO: 64)Aspergillus niger NADP CYP450 3710 DNA oxidoreductase Z26838

(SEQ ID NO: 65)Aspergillus niger NADP CYP450 693 Protein oxidoreductase CAA81550

Specific Methods micrograms/mL, ug/ml) when selecting for amplicillin Transformation of E. coli Strains resistant transformants, or spectinomycin (75 ug/ml) when E. coli strains such as DH5 alpha and DH 10B (Life selecting for spectinomycin-resistant transformants. The Technologies, Rockville, Md.) are routinely used for trans 40 plates are incubated overnight at 37° C. Colonies are picked formation of ligation reactions and are the hosts used to and inoculated into LB plus appropriate antibiotic (100 prepare plasmid DNA for transfecting mammalian cells. E. ug/ml amplicillin or 75 ug/ml spectinomycin) and are grown coli strains, such as DH10B and MON105 (Obukowicz, et at 37° C. while shaking. al., Appl. and Envir. Micr., 58: 1511–1523, 1992) can be DNA Isolation and Characterization used for expressing the proteins of the present invention in 45 Plasmid DNA can be isolated by a number of different the cytoplasm or periplasmic space. methods and using commercially available kits known to DH10B and DH5alpha subcloning efficiency cells are those skilled in the art. Plasmid DNA is isolated using the purchased as competent cells and are ready for transforma Promega WizardTM Miniprep kit (Madison, Wis.), the tion using the manufacturer's protocol. Other E. coli strains Qiagen QIAwell Plasmid isolation kits (Chatsworth, Calif.) are rendered competent to take up DNA using a CaCl2 50 or Qiagen Plasmid Midi or Mini kit. These kits follow the method. Typically, 20 to 50 mL of cells are grown in LB same general procedure for plasmid DNA isolation. Briefly, medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 150 cells are pelleted by centrifugation (5000xg), the plasmid mMNaCl) to a density of approximately 1.0 absorbance unit DNA released with sequential NaOH/acid treatment, and at 600 nanometers (OD600) as measured by a Baush & cellular debris is removed by centrifugation (10000xg). The Lomb Spectronic spectrophotometer (Rochester, N.Y.). The 55 Supernatant (containing the plasmid DNA) is loaded onto a cells are collected by centrifugation and resuspended in column containing a DNA-binding resin, the column is one-fifth culture volume of CaCl, solution 50 mM CaCl, washed, and plasmid DNA eluted. Mter screening for the 10 mM Tris-Cl ((10 mM 2-amino-2-(hydroxymethyl) 1,3- colonies with the plasmid of interest, the E. coli cells are propanediol hydrochloride, pH 7.4 and are held at 4°C. for inoculated into 50–100 ml of LB plus appropriate antibiotic 30 minutes. The cells are again collected by centrifugation 60 for overnight growth at 37° C. in an air incubator while and resuspended in one-tenth culture Volume of CaCl shaking. The purified plasmid DNA is used for DNA solution. Ligated DNA is added to 0.1 ml of these cells, and sequencing, further restriction enzyme digestion, additional the samples are held at 4°C. for 30–60 minutes. The samples Subcloning of DNA fragments and transfection into E. coli, are shifted to 42° C. for 45 seconds and 1.0 ml of LB is mammalian cells, or other cell types. added prior to shaking the samples at 37° C. for one hour. 65 DNA Sequencing Protocols Cells from these samples are spread on plates (LB medium Purified plasmid DNA is resuspended in dHO and its plus 1.5% Bacto-agar) containing either amplicillin (100 concentration is determined by measuring the absorbance at US 7,033,807 B2 49 50 260/280 nm in a Baush and Lomb Spectronic 601 UV which share only isolated regions of similarity (Altschulet spectrometer. DNA samples are sequenced using ABI al., J. Mol. Biol. 215: 403-410, 1990). PRISMTM DyeDeoxyTM terminator sequencing chemistry Two parameters can be varied which alter the sensitivity (Applied Biosystems Division of PerkinElmer Corporation, and quantity of BLAST search results. Parameter B (with a Lincoln City, Calif.) kits (Part Number 401388 or 402078) default value of 10) regulates the number of high-scoring according to the manufacturers suggested protocol. segment pairs (alignments) reported in the results. Parameter Occasionally, 5% DMSO is added to the mixture in repeat V (with a default value of 10) is the maximum number of experiments, to facilitate the sequencing of difficult tem database sequences (hits) for which one-line descriptions plates. will be reported. Matches are based on high-scoring segment Sequencing reactions are performed in a DNA thermal 10 pairs (HSPs). Two sequences may share more than one HSP. cycler (Perkin Elmer Corporation, Norwalk, Conn.) follow if the HSPs are separated by gaps. The BLAST algorithm is ing the recommended amplification conditions. Typically, sensitive to ambiguities in the sequence and is not well DNA samples were prepared containing 500 ng of template Suited for sequences that contain many gaps. DNA and 100 ng of primer of choice in thin-walled 0.2 mL The program blastp compares an amino acid query PCR tubes that have been brought to 12 uL with Millipore 15 sequence against a protein sequence database. blastin com milli-Q (mCP)-quality water. 2 ul of 2 mM Mg" was added pares a nucleotide query sequence against a nucleotide to each tube. Tubes were denatured for 5 minutes at 96° C. sequence database. blastX compares a nucleotide query in a Perkin-Elmer System 9700 thermal cycler. After sequence translated in all reading frames against a protein denaturation, the tubes were chilled to a temperature of 4°C. sequence database. You could use this option to find poten by the thermal cycler. 6 ul of ABI Prism Big Dye Terminator tial translation products of an unknown nucleotide sequence. Cycle Sequencing Ready Reaction Kit was added to each thlastin compares a protein query sequence against a nucle tube. The samples were returned to the thermal cycler and otide sequence database dynamically translated in all read cycle-sequenced using the following program: (1) 96° C. for ing frames. thlastX compares the six-frame translations of a 30 sec; (2) 50° C. for 5 sec; (3) 60° C. for 4 min, followed nucleotide query sequence against the six-frame translations by step (1) for 24 additional cycles and then held at 4°C. 25 of a nucleotide sequence database (See http:// Cycle sequencing was complete after about 2.5 hours. www.ncbi.nlm.nih.gov/Education/BLASTinfo/ for more Samples are purified to remove excess dye terminators information on BLAST, related programs, and pattern with using Centri-SepTM spin columns (Princeton matching algorithms). Separations, Adelphia, N.J.) or purified through a Millipore MAHV N45 50 Multiscreen-HV filtration plate which had 30 Nucleotides searches performed with BLAST, score= been filled with 25 uL Sephadex G-50 superfine resin and 98-557, word length 514 letters, were used to obtain nucle 300 uL mO water. Before loading samples onto filtration otide sequences homologous to nucleic acid molecules of plates, the plate was prespun in a centrifuge at 750xg for 2 the present invention. Protein searches are performed with minto remove excess water. The samples were loaded onto BLASTP. score=50, word length=3 to obtain amino acid the resin and the plate spun again at 750xg for 4 min. The 35 sequences homologous to a reference polypeptide (e.g., SEQ purified sample was collected into a 96-well plate that was ID NO: 2). placed directly underneath the Sephadex-filled plate during Clustal W version 1.74, which implements a different the spin. The liquid in the 96-well plate was dried at room algorithm for alignment of multiple DNA or protein temperature in a Speed Vac. After 45–60 min the DNA was sequences, was also used to prepare alignments and to assign dried and pelleted at the bottom of the plate. Samples were 40 percent identities between different sequences. This program resuspended in 3 ul of a formamide/blue Dextran loading improves the sensitivity of progressive multiple sequence dye and were heated for 2 minutes (see p.33 of Perkin-Elmer alignment through sequence weighting, position specific gap Big Dye manual for loading buffer recipe). Samples were penalties and weight matrix choice (Thompson et al., loaded onto 48 cm well-to-read length 4.5% acrylamide gels Nucleic Acids Research, 22(22):4673-4680, 1994). The and sequenced for 7 hr using ABI automated DNA sequenc 45 default parameters for version 1.74 were used facilitate ers (typically run module Seq Run 48E-1200 and dye set DT. alignments and to assign percent identities between two Program BD, Set Any-Primer). sequences. The input consisted of sequences in FASTA Overlapping DNA sequence fragments are analyzed and format and the output is the alignment shown in the figures. assembled into master DNA contigs using Sequencher DNA For nucleic acid sequences, the iub DNA weight matrix was Analysis software (Gene Codes Corporation, Ann Arbor, 50 used. For amino acid sequences, the blosum protein weight Mich.) or the Perkin-Elmer Data Collection and Sequence matrix was used (See http://www.ncbi.nlm.nih.gov/ Analysis programs to assign bases to the data collected. Education/BLASTinfo/ for more information on BLAST, BLAST, ClustalW, and Boxshade Homology Alignment related programs, and pattern matching algorithms. Tools Boxshade V 3.31 is a public domain program for creating A variety of programs can be used to align nucleotide or 55 nicely formatted printouts from muliple-aligned protein or peptide sequences to each other and to facilitate homology DNA sequences. Boxshade, by itself, does not create searches in large sequence databases. BLAST (Basic Local alignments, but applies shading or coloring to files that were Alignment Search Tool), which implements the statistical previously prepared by other sequence alignment programs. matching theory by Karlin and Altschul (Proc. Natl. Acad. The inputs to Boxshade are the alignments created by Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 60 ClustalW and the threashold values for the residues to be 90: 5873–5877, 1993), is a widely used program for rapidly colored or shaded. In most cases, except where specified, a detecting ungapped nucleotide or peptide Subsequences that 50% identity value was used. With this setting, if a position match a given query sequence (Available from the National has greater than or equal to half of the sequences sharing an Center for Biotechnology Information, http:// identical residue, then it is shaded. Boxshade is available by www.ncbi.nlm.nih.gov). BLAST uses a heuristic algorithm 65 ftp from ftp. or by e-mail from Kay Hofmann which seeks local as opposed to global alignments and is (khofmann(a)isrec-sun1-unil.ch or Michael D. Baron therefore able to detect relationships among sequences (michael.baron(abbSrc.ac.uk). US 7,033,807 B2 51 52 Protein Purification and Characterization dried over anhydrous magnesium sulfate, filtered, and Protein purification can be accomplished using any of a evaporated to dryness. The residue was dissolved in 8 ml variety of chromatographic methods such as: ion exchange, methanol so that the final concentration of canrenone was gel filtration or hydrophobic chromatography or reversed approximately 15 mM (assuming quantitative recovery). phase HPLC. In some cases, proteins which are properly 5 The media extract was diluted 10- to 15-fold into 50% folded can be affinity-purified using affinity reagents, such as methanol for HPLC analysis. Stock solutions of canrenone monoclonal antibodies or receptor subunits attached to a and 11 O-hydroxy canrenone were prepared in methanol. suitable matrix. These and other protein purification meth Standards for HPLC analysis were prepared from these ods are described in detail in Methods in Enzymology, stock solutions by diluting to a final concentration of 750 uM Volume 182 “Guide to Protein Purification” edited by Mur 10 with 50% methanol. Media extract and standards were ray Deutscher, Academic Press, San Diego, Calif., 1990. chromatographed over a C-4 reverse phase HPLC column. The purified protein can be analyzed by RP-HPLC, elec The media exhibited a component with the same retention trospray mass spectrometry, and SDS-PAGE. Protein quan time as the 11 O-hydroxy canrenone standard, as monitored titation is done by amino acid composition, RP-HPLC, at 254 nm (data not shown). and/or Bradford protein dye-binding assays. In some cases, 15 tryptic peptide mapping is performed in conjunction with Example 3 electrospray mass spectrometry to confirm the identity of the protein. Growth of A. ochraceus Mycelia for RNA Extraction EXAMPLES Liquid cultures of Aspergillus ochraceus mycelia were The following examples will illustrate the invention in grown in 10 g/L peptone, 10 g/L yeast extract and 10 g/L greater detail, although it will be understood that the inven glucose containing 20 g/L canrenone for 24 to 72 hours at tion is not limited to these specific examples. Various other 28°C. in a volume of 160 ml. Ten ml samples of cells were examples will be apparent to the person skilled in the art filtered, washed with cold water, frozen, and stored at -80° after reading the present disclosure without departing from 25 C. the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the Example 4 appended claims. Extraction of Total RNA from Induced Spores 30 Example 1 Approximately 0.4 g spores were disrupted in 40 ml Preparation of A. ochraceus Spores for RNA Trizol reagent (Life Technologies, Rockville, Md.) using a Mini-BeadbeaterTM model 3110 (Biospec Products, Extraction Bartlesville, Okla.). Briefly, spore-Trizol mixture was sub Aspergillus ochraceus ATCC 18500 stock culture (50 ul) 35 jected to four 30 second pulses at low speed. Between was grown for 3–4 days on plates containing sporulation pulses, tubes containing spores were chilled on ice. Visual medium: 50 g(L molasses, 5 g/L cornsteep liquid, 5 g/L inspection with the aid of a microscope indicated that the KHPO, 25 g/L NaCl, 25 g/L glucose, 20 g/L agar, and 0.4 majority of the spores were disrupted by this treatment. The g/L progesterone, pH 5.8. Progesterone was included in the debris was pelleted by low-speed centrifugation and the total media to induce the steroid 11 O-hydroxylase. Spores were 40 RNA in the supernatant was extracted following the manu scraped from the plates into 5 to 7 ml saline, washed in facturer's recommended protocols for use with Trizol. saline, collected by centrifugation, and Suspended in saline Briefly, 2 ml chloroform was added for each 10 ml Trizol in containing 15% glycerol. The spores were frozen on dry ice 11 ml polypropylene centrifuge tubes. Following a 3 minute and stored at -80° C. Approximately 0.8 g. spores were extraction of proteins, phase separation was done by cen incubated at 30° C. in a 1 liter flask containing 400 ml 1% 45 trifugation and the aqueous phase containing the RNA was glucose, 50 mM. KHPO and 0.1 g canrenone, pH 7.0. This transferred to a clean tube for precipitation with an equal treatment prior to spore disruption has three benefits: (1) to volume of isopropanol. The precipitated RNA was recov induce the steroid 11 O-hydroxylase by incubation with ered by centrifugation and washed with 70% ethanol. The canrenone; (2) to determine whether the spores were cata RNA was resuspended in 10 ml water, re-extracted with lyzing the 11 O-hydroxylation of canrenone; (3) and to 50 chloroform and precipitated with ethanol overnight at -20° soften the spore wall. After approximately 26 hours of C. Total RNA (3 mg) was recovered by centrifugation and incubating with shaking at 30° C. to provide better aeration, rehydrated in 2 ml water, and precipitated on ice by adding the spores were collected by centrifugation. Visual inspec an equal volume of cold 4 M lithium chloride. This precipi tion with the aid of a microscope indicated that very few had tation was done to remove DNA, carbohydrates, heme, and started to germinate. The spore pellets were flash frozen in 55 other impurities which can carry over from guanidine meth liquid nitrogen and stored at -80° C. The media was ods. The RNA was recovered by a 25 minute centrifugation. analyzed for presence of 11 alpha hydroxy canrenone by HPLC to determine whether spores used for library con Example 5 struction demonstrated the desired activity. 60 Extraction of Total RNA from Induced Mycelia Example 2 Approximately 0.5 g wet weight cells were pulverized to a fine powder under liquid nitrogen with a mortar and pestle A. ochraceus Spores Catalyze 11 C-Hydroxylation pre-chilled in dry ice. The powder was added to 10 ml Trizol of Canrenone Reagent (Life Technologies) and homogenized with a Kine Approximately 160 ml of media from the spore induction 65 matica polytron (Kinematica AG, Lucerne, Switzerland) at was extracted three times with 70 ml ethyl acetate to collect setting #4. Cellular debris was removed by centrifugation the Steroid Substrate and products. The organic phase was prior to chloroform extraction. The aqueous phase contain US 7,033,807 B2 53 54 ing nucleic acids was precipitated with isopropanol for 10 minutes at room temperature. The precipitate was collected by centrifugation and washed with 70% ethanol. The RNA oligo-dT-NotI primer- adapter (SEQ ID NO:58) was rehydrated in water and re-extracted with chloroform to 5'-pCACTAGT TCTAGA TCGCGA GCGGCCGC CC (T) 15-3' remove any residual proteins. The aqueous phase was pre Spel Xbal NruI Not cipitated at -20°C. with /10 volume of 3 M sodium acetate and 2.5 volumes absolute ethanol. The final yield was 424 A radiolabeled tracer (O-PdCTP) was not added. The ug. Approximately 4 ug and 16 ug of total RNA were second strand of cDNA was synthesized in a reaction separated by electrophoresis through a 1.2% agarose gel and volume of 150 ul. The final composition of this mixture visualized by staining in ethidium bromide. Chromosomal 10 including the first strand reaction was 25 mM Tris-HCl, pH DNA was present as a minor contaminant. 7.5, 100 mM KC1, 5 mM (NHA)SO, 0.15 mM B-NAD", Example 6 250 uM each dATP, dCTP, dGTP and dTTP 1.2 mM DTT, 65 units/ml E. coli DNA ligase, 250 units/ml E. coli DNA Extraction of Total RNA from HepG2 Cells 15 polymerase 1 and 13 units/ml E. coli Rnase H. After a 2 hour incubation at 16° C., 10 units of T4 DNA polymerase was Hepatocellular human liver carcinoma cells (HepG2), added, and incubated 5 minutes at 16°C. The reaction was ATCC HB-8065, were maintained in DMEM high glucose stopped with 10 ul 0.5 M EDTA and the cDNA was media supplemented with Penstrep, glutamate and 10% fetal separated from cDNAs smaller than 300 base pairs, primer bovine serum (Life Technologies, Rockville, Md.). Cells adaptors and deoxynucleotides with GENECLEAN II (BIO were induced overnight with 0.05% ethanol and harvested for RNA extraction by trypsinization. Briefly, the cell pellet 101 Inc. La Jolla, Calif.). Annealed Sal I adaptors (Life was resuspended in >10x volumes of 4 M guanidine Technologies) that were phosphorylated at their 5' blunt end isothiocyanate, 50 mM Tris-HCl, pH 7.5, 25 mM EDTA were ligated to the cDNA overnight at 16°C. (solution D. Life Technologies) and then vortexed. Water 25 and sodium acetate, pH 4.1, were added such that the final Sal I adapter concentration of sodium acetate was 0.1 M. The RNA 5'-TCGACCCACGCGTCCG-3' (SEQ ID NO: 59) solution was extracted with one half volume of chloroform and placed on ice for 15 minutes. The aqueous phase was 3'-GGGTGCGCAGGCp-5' (SEQ ID NO: 60) re-extracted with chloroform and precipitated overnight 30 with isopropanol. Total RNA was resuspended in solution D and re-precipitated with isopropanol, followed by two pre GENECLEAN II was used to remove the adaptors. The cipitations in water containing 0.3 M sodium acetate pH 5.5 cDNA was then digested with Not. QIAquick columns and 2.5 volumes of ethanol. PolyA selection was performed (QIAGEN, Valencia, Calif.) were used to remove small twice as described below. 35 DNA fragments from the cDNA, which was ethanol pre cipitated. Example 7 Example 9 PolyA" Selection of mRNA 40 Size Fractionation of cDNA PolyA" RNA was selected from total RNA with an Eppendorf 5Prime, Inc. kit (Boulder Colo.). Briefly, each 1 The cDNA was enriched for species approximately 1.5 kb mg of total RNA was selected twice over a column contain and larger by gel electrophoresis through 0.8% Sea-Plaque ing oligo dT cellulose. The column slurry was packed by agarose (FMC BioProducts, Rockland Me.) in TAE buffer. gentle centrifugation and equilbrated with 0.5 MNaCl. RNA 45 The preparative gel had a lane of DNA size markers which was allowed to bind to the dT cellulose for 15 minutes at was excised from the gel after electrophoresis and stained room temperature. The columns were washed once with 0.5 with ethidum bromide for visualization under ultraviolet M. NaCl, and twice with 0.1 M NaCl. PolyA" RNA was light next to a ruler so that the appropriate region of the eluted in 0.5 ml 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. The cDNA could be recovered from the gel. GENECLEAN II selection by oligo dT cellulose was performed twice. The 50 mRNA was precipitated at -20° C. with 0.3 M sodium was used to extract the cDNA, which was eluted in 20 ul acetate in 50% ethanol, with glycogen added as carrier. Water. Example 8 Example 10 55 Library Construction in Vector pSport1 and cDNA Synthesis and Library Construction Electroporation into E. coli The SuperscriptTM Plasmid System for cDNA Synthesis An aliquot of the size-selected cDNA was ligated over and Plasmid Cloning kit (Life Technologies) was used for night at 4° C. with pSport 1 (Life Technologies, Inc., cDNA systhesis and library construction. Superscript II 60 Rockville, Md.) predigested with NotI and SalI in a 20 ul reverse transcriptase catalyzed the first strand of cDNA in a reaction containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl, 20 ul reaction for 1 hour at 42°C. The final composition was 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT, 2.5 lug/ml 50 mM Tris-HCl, pH 8.3, 75 mM KC1, 3 mM MgCl, 10 mM pSport1, approximately 0.5 ug/ml cDNA, and 50 units/ml DTT, 50 uM each dATP, dCTP, dGTP and dTTP, 50 ug/ml T4 DNA ligase. The ligation mixture was precipitated by the oligo-dT-NotI primer-adaptors that were phosphorylated at 65 addition of 12.5 ul 7.5 M ammonium acetate, 5 ul yeast their 5' end (Life Technologies) and 50,000 units/ml Super tRNA carrier and 70 ul absolute ethanol. The ligated cDNA Script II reverse transcriptase. was recovered by centrifugation at room temperature for 20 US 7,033,807 B2 55 56 minutes and rehydrated in 5 ul sterile water. One ul of the Construction of pMON45624 ligated cDNA was introduced into ElectroMAX DH10B E. The amplified fragments described above were purified coli (Life Technologies) by electroporation. Cells were through a QIAquick column (Qiagen, Valencia Calif.) and allowed to recover in 1 ml SOC medium (Life Technologies) digested with EcoRI and Xbal prior to ligation into pFast for 1 hour at 37°C., before plating an aliquot on LB with 100 Bac1 cleaved with EcoRI and Xbal. The resulting plasmid ug/ml amplicillin. The titer of the Aspergillus ochraceus was designated pMON45624 and the DNA sequence veri spore library (designated LIB3025) was determined by fied using primers based on the vector sequence and internal preparing serial dilutions of the cell suspension in SOC. The primers based on the 11 alpha hydroxylase sequence (shown equivalent of 1 ul, 0.1 ul and 0.01 ul samples of the cell below). Suspension were plated, and the resulting titer was calcu 10 lated to be 1.75x10°/ml colony forming units. Primer Bacfwd: CTGTTTTCGTAACAGTTTTG (SEQ ID NO:44) Example 11 Primer PolyA: CCTCTACAAATGTGGTATG (SEQ ID NO: 45) Identification of Clones Encoding Cytochrome 15 Primer GAGATCAAGATTGCCTT (SEQ ID NO:46) P450 Enzymes by DNA Eequence Analysis and 45 624-for1: Construction of Plasmid pMON45624 Encoding Aspergillus ochraceus 11 Alpha Hydroxylase Primer CTTCGACGCTCCAA (SEQ ID NO: 47) Cloning of 11 Alpha Hydroxylase from Aspergillus Ochra 45 624-for2: CaS Primer GCAATCTTGACCGTT (SEQ ID NO : 48) Approximately 2,000 colonies were selected on LB agar 45 624-rev1 : plates containing 100 ug/ml amplicillin and miniprep plas mid DNA samples were prepared for sequencing. Unidirec The nucleotide and predicted amino acid sequences of the tional sequencing was performed from the 3' end of the cloned 11 alpha hydroxylase are displayed in FIG. 1 as SEQ expressed sequence tags (ESTs) beginning at the Not site 25 ID NO: 1 and SEQ ID NO: 2, respectively. encompassing part of the poly dT primer used for cDNA FIG. 4 sets forth an amino acid homology alignment of A. synthesis. Two universal primers were used to facilitate the ochraceus 11 alpha hydroxylase cloned in pMON45624 and sequencing: aligned with related enzymes found in GenBank using BLAST FIG. 5 is a phylogenetic tree showing the this 30 relationship graphically. FIG. 6 shows the percent homology M13 reverse: between Aspergillus ochraceus steroid 11 alpha hydroxylase CAG GAA ACA GCT. ATG AC (SEQ ID NO: 40) and the top 10 enzymes found in GenBank using BLAST, T7 promoter: calculated using Clustal W and Boxshade. TAA TAC GAC. TCA CTA TAG GG (SEQ ID NO: 41) Example 12 Most known cytochrome p450s contain a conserved heme 35 binding region approximately 50 amino acid residues (150 nucleotides) upstream of the stop codon (Nelson et al. Amplification of cDNA Encoding Human NADPH Pharmacogenetics 6: 1–42, 1996). The 2,000 ESTs were Cytochrome P450 Reductase and Cloning Into screened for sequences encoding the canonical heme Plasmids pMON45603, pMON45604, and 40 pMON45605 binding motif (FXXGXXXCXG, where “X” is any amino acid) in the appropriate region using BLASTX and visual Gene Amplification of Human Oxidoreductase inspection of the sequences scored as hydroxylases for the Approximately 1 ug polyA mRNA from HepG2 cells canonical heme-binding motif. Only fifteen ESTs had the was heated to 65° C. for 10 minutes with 100 ng random heme-binding motif. One EST was unique and the other hexamers (Invitrogen, Carlsbad, Calif.) in an 11 ul reaction. fourteen appeared to be overlapping sequences. The cDNA 45 The mixture was chilled on ice, then incubated at 42°C. for inserts from seven clones encoding putative cytochrome 75 minutes in a 20 ul reaction containing 1 ul RNase p450 enzymes were then sequenced to completion. All seven inhibitor (Promega, Madison, Wis.), 0.01 M DTT, 5 mM encoded the same enzyme. dNTPs, 50 mM Tris-HCl, pH 8.3, 75 mMKC1, 3 mM MgCl, Gene Amplification of Aspergillus ochraceus 11 Alpha and 1 ul SuperScriptiI enzyme (Life Technologies). The Hydroxylase 50 reverse transcriptase was inactivated by heating to 95°C. for The coding region of the 11 alpha hydroxylase was 2 minutes. First strand cDNA was stored at -20° C. Forward amplified by PCR using a unique clone from the A. ochra and reverse primers were based on the nucleotide sequence ceus cDNA spore library (LIB3025) as a template. The of accession number S90469 (human placental partial primers included recognition sites for EcoRI (forward) and mRNA encoding cytochrome P450 reductase (SEQ ID NO: Xbal (reverse) for directional cloning into pFastbac1. 55 49)). The accession number of the corresponding protein Amplification was carried out for 32 cycles using a PCR sequence is AAB21814 (SEQ ID NO: 50). The human core kit (Roche) and 50 pmol of each primer. One cycle oxidoreductase was cloned in two pieces which were consisted of a denaturation step at 94° C. for 45 seconds, an assembled in pFastEac1 (Life Technologies) by ligation at annealing step at 60° C. for 45 seconds, and an elongation an internal HincII site. The primers included restriction sites step at 72° C. for 60 seconds. for directional subcloning into pFastEac1.

Primer 11alphaOH- for: gatcgaatticATGCCCTTCTTCACTGGGCT (SEQ ID NO: 42)

Primer 11alphaOH-rev: gatctotagaTTACACAGTTAAACTCGCCATATCGAT (SEQ ID NO: 43) US 7,033,807 B2 57 58

Primer H. oxred 1A: gatcggatccaatATGGGAGACTCCCACGTGGACAC (SEQ ID NO: 07) Primer H. oxred 1B: CAGCTGGTTGACGAGAGCAGAG (SEQ ID NO: 08) Primer H. oxred 2A CTCTGCTCTCGTCAACCAGCTG (SEQ ID NO: 09) Primer H. oxred 2B: gatcggtaccttaGCTCCACACGTCCAGGGAGTAG (SEQ ID NO: 10)

The second strand was synthesized using 400 uM dNTP and a partial clNA clone 804561639F1 from Aspergillus fumi 167nM of each primer set per 150 ul reaction. Amplification 10 gatus (PathoSeq Database, Incyte Pharmaceuticals) was was performed with Deep Vent polymerase (New England visually scanned to select regions of high homology for the Biolabs, Beverly, Mass.). The reaction for segment 2 (the 3' half of the oxidoreductase cDNA) was adjusted to 5% design of primers for PCR. A primer set was selected which DMSO. The amplification included an initial cycle of dena spanned the coding region of the cprA gene product from amino acids 203 to 693. turation at 94° C. for 90 seconds, followed by annealing at 15 62° C. for 2 minutes and elongation at 72°C. for 2 minutes. Primers were selected from the 5' most region of overlap This was followed by 30 cycles, consisting of a 45 second denaturation step, a 45 second annealing step, and a 60 where the amino acid sequence was identical between both second elongation step. The elongation step was extended to and the nucleic acid sequence differed by 2 positions in the 5 minutes for the final cycle. 3rd codon position. For the 3' primer, the nucleic acid Construction ofpMON45603, pMON45604, pMON45605 encoding the stop codon, last 7 amino acid residues and 2 The PCR fragments for the 5' half of the oxidoreductase additional bases corresponding to second and third positions cDNA were digested with BamHI and HincII. The PCR in the codon of the amino acid residue 8 positions from the fragments for the 3' half of the oxidoreductase cDNA were stop codon encodes ARG in A. niger and SER in A. fumigatis digested with HincII and Kipni and ligated into pBluescript (CGC vs. AGC). Inosines replaced the third base in codons II (Stratagene, La Jolla, Calif.) for sequencing. The resulting 25 when there was a discrepancy between the A. niger and A. plasmids were designated pMON45603 (5' segment) and fumigatus sequence. pMON45604 (3' segment). The BamHI/HincII fragment from pMON45603 and the HincII/KpnI fragment from pMON45604 were ligated into pFastbac1 cut with BamHI Primer A. oxred-for1: and KpnI, to generate pMON45605. GACGGIGCIGGTACAATGGA (SEQ ID NO:11) Sequencing primers were based on the sequence of Gen 30 Bank accession number S90469 (SEQ ID NO 49), a cDNA Primer A. oxred-revil: encoding cytochrome P450 reductase human, placenta, TTAIGACCAIACATCITCCTGGTAGC (SEQ ID NO:12) mRNA Partial, 2403 nt). The cognate protein sequence is: (where I=Inosine) AAB21814 (SEQ ID NO 50) cytochrome P450 reductase A partial clNA clone was amplified from approximately 5 {EC 1.6.2.4} human, placenta, Peptide Partial, 676 aa 35 Homo sapiens). The cDNA insert of pMON45603 was ug of total RNA extracted from A. ochraceus mycelia. sequenced using primer OXred 1C, and the cDNA insert of Before the first strand synthesis, the RNA was heated to 65° pMON45604 was sequenced using primer oxred 2C and 2D. C. for 10 minutes with 100 ng random hexamers (Promega Universal T7 (SEQ ID NO: 41) and M13 reverse (SEQ ID Madison Wis.) in an 11 ul reaction mixture. The mixture was NO: 40) primers, which annealed to vector sequences flank 40 chilled on ice, then incubated at 42° C. for 75 minutes in a ing the cDNA inserts were also used for sequencing. 20 ul reaction containing 1 ul RNase inhibitor (Promega), 0.01 M DTT, 5 mM dNTPs, 50 mM Tris-HCl, pH 8.3), 75 Primer oxred 1C: mM KC1, 3 mM MgCl, and 1 ul SuperScriptiI (LTI). The GTGGACCACAAGCTCGTACTG (SEQ ID NO : 61) reverse transcriptase was inactivated by heating to 95°C. for 2 minutes. The first strand cDNA was stored at -20°C. The Primer oxred 2C: 45 second strand was synthesized using 5 ul of the first strand CATCGACCACCTGTGTGAGCTG (SEQ ID NO: 62) as template. The reaction included 500 nM primers, 200 uM Primer oxred 2D : each dNTP and Taq polymerase and buffer as supplied in GTACAGGTAGTCCTCATCCGAG (SEQ ID NO: 63) PCR core kit (Roche Molecular Biochemicals, Indianapolis, Ind.). Amplification was performed using 32 cycles of a 30 The nucleotide and predicted amino acid sequences of the 50 cloned human oxidoreductase are displayed in FIG. 2 as second denaturation step at 94° C., a 30 second annealing SEQID NO:3 and SEQID NO: 4, respectively. FIG. 11 sets step at 60° C. and a 60 second elongation step at 72°C. The forth an alignment of human oxidoreductase with top 4 hits amplified DNA products were cloned into pGEM-T from SwissProt. FIG. 12 sets forth a phylogenetic tree (Promega, Madison, Wis.) and sequenced using universal T7 displaying the genetic relatedness of human oxidoreductase, 55 (SEQ ID NO: 41) and SP6 (SEQ ID NO. 57) primers. to these hits. FIG. 13 shows the percent identity between Primer SP6 GATTTAGGTGACACTATAG (SEQ ID NO: human oxidoreductase and top 4 hits from SwissProt. 57) Alignment of the sequences with the A. niger cprA gene Example 13 revealed that the A. Ochraceus clones had an intron in the Amplification of cDNA Encoding NADPH 60 same position as the intron in the A. niger gene. This Cytochrome P450 Reductase from A ochraceus and indicated that the A. ochraceus PCR products might have Cloning into Plasmids pMON45630, pMON45631, been amplified from a genomic DNA contaminant of the and pMON45632. total RNA. A reverse primer based on the A. ochraceus Gene Amplification of Aspergillus ochraceus Oxidoreduc sequence was designed to amplify the approximately 600 tase 65 missing bp including the initial methionine. The A. ochra An alignment of sequences from the Aspergillus niger ceus cDNA library was then used as a template for PCR. The cprA gene accession number Z26938 (SEQ ID NO: 65) and forward primer was based on the reverse complement of US 7,033,807 B2 59 60 vector pSport1 (Life Technologies) bases 299 to 326. The Primers A.oxred-for-4 and rev6 were complementary and other primer, A.OXred-rev2 was bases on the A. Ochraceus flanked the intron. The first PCR reaction used an A.oxred sequence encoding residues 326–333. clone linearized at the internal BamHI site as template. Polymerase and buffers were supplied by the PCR core kit (Roche Molecular Biochemicals, Indianapolis, Ind.). Primer Primer pSport-for1: and dNTP concentrations were 500 nM and 200 uM, respec CAAGCTCTAATACGACTCACTATAGGGA (SEQ ID NO : 13) tively. Two reactions were performed, using a combination of A.oxred-for3 with A.OXred-rev6, and A.OXred-for-4 with Primer A. oxred-rev 2: A.oxred-reviš. Following a 2 minute initial denaturation, 28 CAGGAACCGATCGACCTCGGAA (SEQ ID NO: 14) cycles of PCR amplification were performed. One cycle The A. ochraceus spore library size made from gel-purified 10 included a 45 second denaturation at 94° C., a 45 second fragments >1.5 kb in size was then used as a template for denaturation step at 62° C. and a 45 second elongation step amplifying the final 200 bases of coding region. Two new at 72°C. One ul of each reaction served as template for the reverse primers were designed from the A.OXred sequence, second PCR amplification with primers A.oxred-for3 and and a new forward primer based on pSport1 (bases 295–328) A.OXred-revs using Elongase enzyme and buffers. Amplifi was also used. cation consisted of 30 cycles with a 30 second denaturation

Primer A. oxred-rev3: GTCACCCTCACCAGCAGAGCCAATG (SEQ ID NO: 15) Primer A. oxred-rev4 : CCACATTGCGAACCATAGCGTTGTAGTG (SEQ ID NO: 16) Primer pSport-for2: GCCAAGCTCTAATACGACTCACTATAGGGAAAGC (SEQ ID NO: 17)

Amplification was performed using an Elongase polymerase 25 step at 94° C., a 30 second annealing step at 62° C., and a kit (Life Technologies, Rockville Md.) for 35 cycles con 5 minute elongation step at 68°C. The PCR products were sisting of a denaturation step at 94° C. for 30 seconds, an directly cloned into pCR11-TOPO. DNA sequencing dem onstrated that the intron had been removed. This clone was annealing step at 63° C. for 30 seconds, and an elongation designated pMON45631. step at 68°C. for 5 minutes. The PCR products were cloned Plasmid pMON45632 was constructed in a three-way directly into pCR11 TOPO (Invitrogen). Twelve clones were 30 ligation by ligating the SalI/BamHI fragment from sequenced, and the composite sequence, extended for 232 pMON45630 with the BamHI/XhoI fragment from bases upstream of the initial methionine, and included 2 pMON45631 and vector pFastEac1, which had been cut in-frame stop codons (Data not shown). with SalI and XhoI and de-phosphorylated to enhance the Primers incorporating the complete coding region of recovery of vectors with the desired inserts. A.oxred were designed with a 5' SalI site and a 3' XhoI site 35 The nucleotide and amino acid sequences of the cloned for ligation into expression vector pFastEac1. Aspergillus ochraceus 11 oxidoreductase are displayed in FIG.3 as SEQ ID NO: 5 and SEQ ID NO: 6, respectively. FIG. 7 sets forth the amino acid homology of Aspergillus Primer A. oxred-for2: ochraceus and human oxidoreductase to NADPH cyto gtogacATGGCGCAACTCGATACTCTC (SEQ ID NO: 18) 40 chrome P450 reductases from A. niger, mouse, and S. Primer A. oxred-rev5: cerevisiae. FIG. 8 sets forth the amino acid alignment for A. citcgagittaGGACCAGACATCGTCCTGGTAG (SEQ ID NO : 19) ochraceus, A. niger, and S. cerevisiae oxidoreductases. FIG. 9 is a phylogenetic tree showing the relatedness of Aspergil A. ochraceus total RNA was used as a template for PCR with lus ochraceus and human oxidoreductase to reductases from these primers and the Elongase kit. Amplification consisted 45 of 35 cycles with a 30 second denaturation step at 94° C., a A. niger, yeast, and mouse. FIG. 10 shows the percent 30 second annealing step at 64° C., and a 5 minute elonga homology between Aspergillus ochraceus steroid 11 alpha tion step at 68°C. An aliquot of the cDNA from reaction ran hydroxylase and the oxidoreductases from A. niger, yeast, as a single band of approximately 2.1 kb. and mouse, calculated using Clustal W and Boxshade. Construction of pMON45630 50 Example 15 The PCR products were cloned directly into pCR11 Generation of Polyclonal Antibodies Recognizing TOPO (Invitrogen, Carlsbad, Calif.). All clones contained Aspergillus Ochraceus 11 Alpha Hydroxylase and the internal intron noted earlier. One clone was designated Aspergillus ochraceus NADPH Cytochrome p450 pMON45630. Reductase Construction ofpMON45631 and pMON45632 55 Generation of Anti-11-C-Hydroxylase Antibodies A strategy based on two step PCR from an internal BamHI Polyclonal antibodies against Aspergillus ochraceus 11 site approximately 170 bp upstream of the 5' splice site was alpha hydroxylase and NADPH cytochrome p450 reductase employed to generate clones lacking the intron. were raised in rabbits against Synthetic peptides (prepared

Primer A. oxred-for3: GGATCCCTCGCGACCTGTGATCAT (SEQ ID NO: 20) Primer A. oxred-for 4: CGAAGATTTCTTGTACAAGGATGAATGGAAGACTTTTC (SEQ ID NO: 21) Primer A. oxred-rev6: CTGAAAAGTCTTCCATTCATCCTTGTACAAGAAATC (SEQ ID NO: 22) US 7,033,807 B2 61 62 by Sigma/Genosis, The Woodlands, TX) corresponding to for Spodoptera frugigperda (Sf9) cells. Cells were seeded in several regions of the following predicted protein sequences: 6-well tissue culture plates at 9x10 cells per well in SF-900 serum-free medium (Life Technologies) and allowed to attach for at least one hour. The transfection mixtures were 11aOH peptide 1: made following the addition of 5 ul miniprep DNA and 5ul AAAYWLATLQPSDLPELN (SEQ ID NO: 23) Cellfectin to polystyrene tubes that contained 200 ul SF-900 11aOH peptide 2: medium. The mixtures were allowed to incubate for 15–30 CRQILTPYIHKRKSLKGTTD (SEQ ID NO: 24) minutes at room temperature. Prior to transfection, 800 ul SF-900 medium was added to each tube. The cells were 11aoH peptide 3: HMGFGHGWHACPGRFFASNEI (SEQ ID NO: 25) 10 washed one time with 2 ml SF-900 medium, and the DNA mixtures were added to the cells. The cultures were allowed oxir peptide 1: to incubate for 5 hours at 27° C. Following the 5 hr CTYWAWAKDPYASAGPAMING (SEQ ID NO: 26) incubation period, the transfection mixture was removed and The 11aOH peptide 2 (SEQ ID NO: 24) corresponds to the the cultures were replenished with 3 ml per well IPL-41 G helix, G/H loop, and H helix region present in an 15 medium (Life Technologies) supplemented with 10% fetal alignment of the amino acid sequence of 11 alpha hydroxy bovine serum. Following a three day incubation period, the lase with the corresponding sequence of CYP3A4 described cells were harvested, centrifuged, and the Supernatant that by Wang and Lu, (Drug Metab. Dispos. 25(6), 762–767, contained recombinant virus (designated as passage 1 or P1 1997). The 11aOH peptide 3 (SEQID NO:25) corresponded stock) was removed and stored at 4°C. A larger viral stock to the peptide fragment from the heme-binding domain. was made by infecting 100 ml fresh Sf9 cells at 5x10 cells Immunological grade peptides were monitored for purity per ml with 0.5 ml of the P1 medium. This larger (P2) stock using reverse phase high performance liquid chromatogra was then titered using a plaque assay protocol (O'Reilly et phy (HPLC). Each peptide was conjugated to keyhole limpet al., 1992), and used for production of the 11 alpha hydroxy hemacyanin (KLH) and suspended in Complete Freund's lase or oxidoreductase enzymes, separately or in combina Adjuvant. The conjugated peptide was then injected Subcu 25 tion with each other. taneously at multiple sites into rabbits. Each conjugated FIG. 14 sets forth an immunoblot illustrating expression peptide was injected into two rabbits. All Subsequent immu of Aspergillus ochraceus P450 11 alpha hydroxylase in nizations were given in incomplete Freund's Adjuvant. In baculovirus-infected insect cells harvested at 25 and 48 general, five Subsequent injections were given at two-week hours post infection. The nitrocellulose membrane was intervals following the initial immunization. IgG fractions 30 probed with a 1:1 mixture of antibodies prepared two rabbits were affinity-purified using a Sepharose-Protein A column. immunized with a conjugated synthetic peptide 11aOH Fractions from the two rabbits injected with each peptide peptide 2 (SEQ ID NO 24). were combined at a 1:1 ratio. The pooled anti-11 alpha FIG. 15 sets forth an immunoblot illustrating expression hydroxylase (rabbits GN 1187/1188) was 0.34 mg/ml IgG. of Aspergillus ochraceus P450 oxidoreductase in The pooled anti-oxred (rabbits GN 2023/2024) was 0.26 35 baculovirus-infected insect cells harvested at 25 and 48 mg/ml IgG. The combined IgGs were each diluted 1:10, hours post infection. The nitrocellulose membrane was 1:100 and 1:1,000 for a pilot experiment to determine which probed with a 1:1 mixture of antibodies prepared two rabbits was dilution was optimal for probing Western blots. The immunized with a conjugated synthetic peptide oXr peptide 1:10 dilution gave the best results and was used for probing 1 (SEQ ID NO 26). subsequent Westerns. 40 Example 17 Example 16 Insect Cell Infection and Heterologous Expression Co-Infection Baculoviruses Expressing of Aspergillus Ochraceus 11 Alpha Hydroxylase and Proteins were expressed in Sf9 insect cells using bacu 45 Human Oxidoreductase lovirus shuttle vectors (Luckow et al., J. Virol. 67: 4566-4579, 1993). The baculovirus shuttle vector (bacmid) Sf9 cells were co-infected with virus particles that con contains a mini-F replicon for expression in bacterial cells, tained the steroid 11 alpha hydroxylase cDNA and a separate a kanamycin resistance marker for selection, and attTnT (the virus containing a human NADPH P450-oxidoreductase. target site for the bacterial TnT transposon) within the lacZa 50 Both viruses were added at a multiplicity of infection (MOI) sequence. Each of these elements is inserted into the poly ratio of 0.1:0.01 (11 aOH to oxr). One day after infection, hedrin locus of the Autographa Californica nuclear polyhe 0.9 ug/ml hemin chloride was added to the culture. The cells drosis virus (AcNPV, the native baculovirus) genome. A were harvested by centrifugation three days after infection donor plasmid (pFastEac1, Life Technologies) was used to (unless specified differently), and the washed cell pellets deliver the gene to be expressed and was inserted into the 55 were frozen until processed for sub-cellular fractions. bacmid via the bacterial Tn7 transposition elements. pFast Bac1 contains the TnT. left and right ends flanking the Example 18 polyhedrin promoter, a polylinker cloning sequence, the Co-Infection Baculoviruses Expressing of SV40 polyA transcription termination sequence, and the Aspergillus Ochraeeus 11 Alpha Hydroxylase and gentamicin resistance gene for selection. Recombinant 60 viruses were generated following transformation of the Aspergillus ochraceus Oxidoreductase pFastEac1 plasmid, which contained a single 11 alpha Sf9 cells are co-infected with virus particles that contain hydroxylase or oxidoreductase cDNA, into DH1 OBac E. coli the steroid 11 alpha hydroxylase cDNA and a separate virus cells (Life Technologies) that contained the bacmid and containing A. ochraceus NADPH P450-oxidoreductase. helper plasmid. 65 Both viruses are added at a multiplicity of infection (MOI) Transfections were performed using CellFectinTM reagent ratio of 0.1:0.01 (11 aOH to oxr). One day after infection, (Life Technologies) following the manufacturer's protocol 0.9 g/ml hemin chloride is added to the culture. The cells US 7,033,807 B2 63 64 are harvested by centrifugation three days after infection (Hewlett-Packard, Naperville, Ill.), and a Model TC-50 (unless specified differently), and the washed cell pellets are temperature controller and Model CH-30 column heater frozen until needed in Subsequent experiments that require (both Eppendorf, Madison, Wis.). processing into for Sub-cellular fractions. Cell membrane fractions derived from insect cells trans fected with recombinant baculoviruses expressing Example 19 11-hydroxylase and complementary electron transport pro Preparation of Subcellular Fractions from teins were analyzed for 11-hydroxylase activity in a reaction Baculovirus-infected Insect Cells mixture containing 80 mM phosphate buffer, pH 7.4, 8 mM MgCl, and 0.9 mM NADP in a final volume of 200 ul. In One half gram of the cell pastes from infected Sf9 cells 10 order to insure an adequate source of reducing equivalents, and uninfected control cells were thawed and Suspended in an NADPH regenerating system was provided by adding 40 ml of 0.25 M sucrose with 10 mM. KHPO4, adjusted to glucose-6-phosphate dehydrogenase (1.5 U/ml) and 8 mM pH 7.4. The Suspensions were homogenized using a Fisher glucose-6-phosphate. Steroid Sub Strate (e.g., Sonic Dismembrator, model 300 probe sonicator (Fisher androstenedione) was provided at a final concentration of Scientific, St. Louis, Mo.). The samples were transferred to 15 0.3 mM. Reaction mixtures were incubated at 37° C. for 30 a conical centrifuge tube (Corning Costar Corporation, min. The reactions were terminated by the addition of 200 ul Cambridge, Mass.) and subjected to centrifugation at 500xg methanol and then placed on ice. Samples were pelleted by at 5°C. for 15 minutes. The pellets were resuspended in the centrifugation to remove precipitated protein. same volume of fresh buffer and viewed under a microscope On one occasion, the incubation was carried out in a to confirm complete lysis. Few or no whole cells were volume of 0.5 ml in siliconized polypropylene 1.5 ml observed. The Supernatants were then subjected to centrifu microcentrifuge tubes at 37° C. for 120 minutes. The gation at 10,000xg for 30 minutes at 5° C. to collect enzyme, prepared from microsomal or mitochondrial mitochondria, Golgi and other Subcellular organelles. The fractions, was added and the Substrate added at a concen pellets were resuspended in fresh buffer and subjected to tration of 250 uM (e.g., 25 mM methanol stock solution of centrifugation at 7,800xg for 30 minutes at 5° C. to collect 25 AD). The cofactor buffer was 100 mM potassium phosphate, mitochondria. pH 7.4, 7.5 mM MgCl2, 7.5 mM glucose-6-phosphate, 0.80 The mitochondrial pellets were resuspended in buffer as mM NADP, and 1.0 units/mL glucose-6-phosphate dehy described about and the centrifugation was repeated. The drogenase. HPLC samples were prepared by terminating the mitochondrial pellets were resuspended in 2 ml buffered 0.5 ml reaction mixture by addition of 0.3 ml methanol, sucrose solution and stored at -80° C. in 100 ul aliquots. 30 Vortexing three times for 2 seconds and storing on ice. The The Supernatants from the original mitochondrial frac tubes were spun for 5 minutes at -20,000xg in a microcen tionation were subjected to centrifugation at 200,000xg for trifuge and the samples transferred to autosampler vials and 1 hour at 5°C. The microsomal pellets were resuspended in capped. 2 ml buffered sucrose solution and stored at -80° C. in 100 Steroid components present in reaction mixtures and ul aliquots. 35 media extract were separated and analyzed by reverse phase Microsomal Incubations HPLC using a 250 mmx4 mm Vydac analytical C-4 column. Incubation mixtures consisted of Sf9 microsomes (1.0 mg Chromatograms were developed using a solvent gradient of protein/mL final concentration), an NADPH-generating from 40% to 100% methanol over a ten minute time period system and 250 uM substrate (AD) in 100 mM potassium and holding at 100% methanol for 5 minutes before phosphate buffer, pH 7.4 or 150 mM HEPES buffer, pH 7.4. 40 re-equilibration to initial conditions. The column effluent The NADPH-generating system was composed of the fol was monitored for UV absorbance at both 254 and 220 nm. lowing at the indicated final concentrations: MgCl, (7.5 Androstenedione, testosterone and monohydroxylated mM), D-glucose-6-phosphate (7.5 mM), NADP (0.80 mM), androstenedione metabolites were resolved on a Nova-pak and glucose-6-phosphate dehydrogenase (1.0 units/mL). C 18 column, 4 micron, 3.9 x 150 mm (Waters Incubations were carried out for the indicated times at 37° C. 45 Chromatography, Milford, Mass.) equipped with a 0.22 in a water bath. Following incubation, reactions were ter micron Rheodyne precolumn filter at 40° C. and 1.0 ml minated by the addition of 0.3 ml methanol. The samples mobile phase/min. A stepped gradient was utilized with were Vortexed three times for two seconds and placed on ice, water as mobile phase solvent A and methanol as solvent B. or stored at -70° C. for later analysis. The initial concentration of solvent B was 42% for 6 min. Example 20 50 The percentage of B was increased linearly to 45% over 4 minutes and then held for 3 minutes. The percentage of B HPLC Assays to Measure Conversion of Steroid was then increased linearly to 80% over 10 minutes and held Substrates to Their Hydroxylated Counterparts there for an additional 2 minutes for a total run time of 25 High Performance Liquid Chromatography (HPLC) minutes. The ultraviolet detection wavelength was 247 nm The HPLC method used to separate hydroxylated steroid 55 and the injection volume was 200 ul. compounds from Steroid substrates, such as 11 C.- Both the “mitochondria” sample and the “microsomal hydroxyandrostenedione from androstenedione, is a modi sample produced peaks matching the HPLC retention time fied version of the testosterone hydroxylase assay, described of the 11C.-hydroxyandrostenedione standard, while other by Sonderfan et al., Arch. Biochem. Biophys. 255: 27-41, fractions did not. These “mitochondria' and “microsomal' 1987). The standards for androstenedione and 11-beta 60 peaks were 3.2 and 2.3%, respectively, of the total peak area hydroxyandrostenedione were obtained from Sigma. quantitated at 247 nm. The 11C.-hydroxyandrostenedione 11-alpha-hydroxyandrostenedione (89.5% pure, with the standard was also spiked into a blank microsomal incubation major impurity being androstenedione) was provided by sample at a concentration of 5.0 g/mL. The concentration Searle Medicinal Chemistry. HPLC grade water and metha of the “mitochondria” and “microsomal 1 1 C.- nol were obtained from Burdick & Jackson. 65 hydroxyandrostenedione peaks were 1.75 and 1.31 ug/mL, The HPLC system consisted of a Model 1050 series after correcting for the purity of the standard (89.5%). These pump, autoinjector and variable wavelength detector concentrations represent 2.3 and 1.7% of substrate converted US 7,033,807 B2 65 66 to 11 C-hydroxyandrostenedione, using a substrate concen sequence of the genome. Analysis of the open reading tration of 250 uM. frames, will reveal regions which are homologous to the FIG. 16 sets forth an HPLC tracing illustrating the con steroid hydroxylase and oxidoreductase genes of the present version of androstenedione (AD) to its 11 alpha hydroxy invention, and regions of the translated open reading frames counterpart after incubating AD with subcellular fractions which are homologous to these enzymes using programs prepared from baculovirus-infected insect cells expressing designed to facilitate multiple sequence alignments of nucle Aspergillus ochraceus 11 alpha hydroxylase and human otide and protein sequence data such as BLAST, CLUSTAL oxidoreductase. W, and BoxShade. Genes which encode these proteins are obtained from the artificial chromosomes and recloned into Example 21 10 expression vectors such as pFastEac1, transformed into appropriate host cells, which are assayed for the presence of Recognition of Aspergillus ochraceus 11 Alpha enzymes capable of carrying out the conversion of steroid Hydroxylase and Aspergillus ochraceus NADPH substrates to their oxidized counterparts. Cytochrome p450 Reductase by Immunoblotting It is intended that the scope of the present invention be Using Polyclonal Antibodies Generated Against 15 determined by reference to the appended claims. It is rec Synthetic Peptides ognized that a number of variations can be made to this Proteins from Sf9 cell lysates (obtained from uninfected invention as it is currently described but which do not depart and recombinant baculovirus-infected cells) were loaded from the Scope and spirit of the invention without compro onto lanes of a 10% gradient acrylamide minigel (BioRad, mising any of its advantages. These include isolation of Hercules, Calif.) at equal concentrations (10 ug per well). homologous genes from microorganisms known to carry out The proteins were separated by electrophoresis at 16 11 alpha hydroxylation of steroid substrates, preferably mAmps constant current for approximatley 1 hr in a Tris fungi and bacteria. This invention is also directed to any glycine buffer containing 0.1% SDS (Sigma, St. Louis, Substitution of analogous components. This includes, but is Mo.). The proteins were transferred to nitrocellulose not restricted to use of these techniques to isolate other (Schleicher & Schuell, Keene, NH) for 40 min at 70 mAmp 25 P450s which are involved in steroidogenesis, including constant current. Primary antibodies were diluted 1:10 (from hydroxylases that act at other positions in the core molecule, stock concentrations of 0.34 mg/ml IgG for anti-11 alpha and use of these enzymes to facilitate bioconversion of hydroxylase (antibodies GN-1187 and GN-1188 prepared steroid intermediates in modified host microorganisms. from peptide 11a(OH peptide 2 CRQILTPYIHKRK All references, patents, or applications cited herein are SLKGTTD (SEQ ID NO: 24)), and 0.26 mg/ml IgG for 30 incorporated by reference in their entirety, as if written anti-oxred (antibodies GN-2023 and GN-12024 prepared herein. from oxr peptide 1 CTYWAVAKDPYASAGPAMNG (SEQ References ID NO: 26)) and used to probe the nitrocellulose membrane. The antigens were detected using anti-rabbit horseradish Altschul S F: Gish W: Miller W: Myers E. W.; Lipman DJ peroxidase (HRP)-linked secondary antibody as recom 35 Basic local alignment search tool. J. Molec. Biol. (Oct. 5, mended by the manufacturer (New England Biolabs, 1990), 215(3), 403–10. Beverly, Mass.). Chemilumiescence was detected using Anfossi et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:3379–3383 luminol and peroxide reagents (New England Biolabs, (1989) Beverly, Mass.) following the protocol provided by the Arfin et al. Proc. Natl. Acad. Sci. (U.S.A.) 92:77147718 vendor. Light emission was recorded using X-OMAT AR 40 (1995) film (Eastman Kodak Company, Rochester, N.Y.). Images Armour, et al., FEBS Lett. 307: 113-115 (1992) were recorded using a Minolta Dimage V digital camera Baldwin et al., Gene Ther. 4:1142–1149 (1997) (Minolta Corporation, Ramsey, N.J.). Barany, Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193 (1991) Bassat et al., J. Bacteriol. 169:751–757 (1987) Example 22 45 Baum et al., J. Hematother. 5: 323-329 (1996) Becker et al., EMBO J. 8:3685–3691 (1989) Characterization of the Aspergillus ochraceus Ben-Bassat et al., J. Bacteriol. 169:751–757 (1987) Genomic DNA Encoding 11 Alpha Hydroxylase Berkner, BioTechniques 6:616–629 (1988) and Oxidoreductase Berkner, Current Top. Microbiol. Immunol. 158:39–66 50 (1992) The approaches described above can be used to facilitate Blobel and Dobberstein, J. Cell Biol. 67:835–851 (1975) the identification of genes encoding steroid hydroxylases Boris-Lawrie and Temin, Annal. New York Acad. Sci. and oxidoreductases within the genome of Aspergillus 716:59.71 (1994) ochraceus and closely related microorganisms, including Boris-Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102–109 Aspergillus niger and Aspergillus nidulans. Other preferred 55 (1993) organisms are Rhizopus Oryzae, Rhizopus stolonifer; Strep Bostian et al., Cell 36:741–751 (1984) tomyces fradiae, Bacillus megaterium, Pseudomonas Botstein et al., Ann. J. Hum. Genet. 32:314–331 (1980) cruciviae, Trichothecium roseum, Fusarium oxysporum f.sp. Bregni et al., Blood 8.0:1418–1422 (1992) Cepae, Rhizopus arrhizus, and Monosporium Olivaceum. Breskvar K, Cresnar B. Plaper A, Hudnik-Plevnik T. Local Other preferred organisms that are known to have steroid 11 60 ization of the Gene Encoding Steroid Hydroxylase Cyto alpha hydroxylase activity are described in the detailed chrome P-450 from Rhizopus Nigricans inside a HindIII description of the invention, above. Fragment of Genomic DNA. Biochem. Biophys. Res. Briefly, genomic DNA is prepared and shotgun cloned Commun 1991; 178, 1078–1083. into low copy artificial chromosomes propagated in bacteria. Brody and Crystal, Annal. New York Acad. Sci. 716:90–103 A large number of clones are sequenced to ensure statistical 65 (1994) representation of the entire genome, and the sequences of Capecchi, Cell 22:479–488 (1980) overlapping clones merged to produce the final map and Chen et al., Gene Ther. 5:50–58 (1998) US 7,033,807 B2 67 68 Clapp, Clin. Perinatol. 20:155–168 (1993) Harley and Reynolds, Nucleic Acids Res. 15:2343-2361 Collins, In: Alternative Immunoassays, John Wiley & Sons, (1987) NY (1985) Harlow and Lane. In Antibodies. A Laboratory Manual, Corbi and Lopez-Rodriguez, Leuk. Lymphoma 25:415–425 Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1997) (1988) Crabeel et al., EMBO J. 2:205 212 (1983) Harms and Splitter, Hum. Gene Ther. 6:1291–1297 (1995) Curiel et al., Hum. Gen. Ther. 3:147–154 (1992) Current Protocols in Molecular Biology, John Wiley & Sons, Hasan et al., Gene 56:141–151 (1987) N.Y. (1989) Hattori et al., Genes Dev. 6: 609-618 (1992) Czerwinski, M., Sahni, M., Madan, A. and Parkinson, A. 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SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 65 <210> SEQ ID NO 1 &2 11s LENGTH 1776 &212> TYPE DNA <213> ORGANISM: Aspergillus ochraceus &22O > FEATURE <221 NAME/KEY: CDS <222> LOCATION: (146) . . . (1690) <223> OTHER INFORMATION: Aspergillus ochraceus 11 alpha hydroxylase <400 SEQUENCE: 1 tggaagttitt tacacttatt at gcc.ggagc cqaaagattic toagtc gagg ggttggggaa 60 caa.cactata agacctacaa coactitggat ttggtgaatt tacacgggca ttatcaaaac 120 agccacaagc togacagotca ttatc atg ccc titc titc act ggg citt citg gcg 172 Met Pro Phe Phe Thr Gly Leu Leu Ala 1 5

att tac cat agt citc at a citc gac aac cca gtc. caa acc ctd agc acc 220 Ile Tyr His Ser Leu Ile Leu Asp Asn Pro Val Glin Thr Leu Ser Thr 10 15 20 25

att gtc gta ttg gog go a gog tac togg citc goa acg ctic cag ccg agc 268 Ile Val Val Leu Ala Ala Ala Tyr Trp Leu Ala Thr Leu Gln Pro Ser 30 35 40

gac citt cott gag citg aat coc goc aaa cca titc gag titc acc aat cqt 316 Asp Leu Pro Glu Lieu. Asn Pro Ala Lys Pro Phe Glu Phe Thr Asn Arg

US 7,033,807 B2 75 76

-continued Glin Arg Lieu Arg Pro Thr Lieu Lieu Gly Ser Phe Arg Arg Glin Ala Thr 365 370 375 aat gac atc aag citg aag agc ggg titt gtc. ata aag aaa ggg act aga 324 Asn Asp Ile Lys Lieu Lys Ser Gly Phe Val Ile Lys Lys Gly. Thir Arg 38O 385 390 gto gtg atc gac agc acc cat at g tog aat coc gag tat tac act gac 372 Val Val Ile Asp Ser Thr His Met Trp Asin Pro Glu Tyr Tyr Thr Asp 395 400 405 cct citc cag tac gac ggg tac cqc tac titc aac aag cqg cag aca coc 420 Pro Leu Gln Tyr Asp Gly Tyr Arg Tyr Phe Asn Lys Arg Glin Thr Pro 410 415 420 425 ggc gag gac aag aac gog titg citc gtc agc aca agc gcc aac cac atg 468 Gly Glu Asp Lys Asn Ala Leu Lieu Val Ser Thr Ser Ala Asn His Met 430 435 4 40 gga titc ggt cac ggc gtt cac goc tot cott ggc aga titc ttic goc to c 516 Gly Phe Gly His Gly Val His Ala Cys Pro Gly Arg Phe Phe Ala Ser 445 450 455 aac gag atc aag att gcc titg tot cat atc atc tta aat tat gag togg 564 Asn Glu Ile Lys Ile Ala Lieu. Cys His Ile Ile Lieu. Asn Tyr Glu Trip 460 465 470 cgt citt coa gac ggc titc aag coc cag cott citc aac atc ggg at g act 612 Arg Lieu Pro Asp Gly Phe Lys Pro Glin Pro Leu Asn. Ile Gly Met Thr 475 480 485 tat citg gog gat coc aat acc agg at g citg atc agg cca cqc aag gog 660 Tyr Lieu Ala Asp Pro Asn. Thir Arg Met Lieu. Ile Arg Pro Arg Lys Ala 490 495 5 OO 505 gag atc gat at g g cq agt tta act gtg tag gtogaacacg aagttcCtgat 710 Glu Ile Asp Met Ala Ser Leu Thr Val * 510 gaagttgttat tdgtoagtgg gtgaa.gcaag togcagaaat gtgtaacaat ttataagaat 770 aaaaaa. 776

<210> SEQ ID NO 2 &2 11s LENGTH 514 &212> TYPE PRT <213> ORGANISM: Aspergillus ochraceus <400 SEQUENCE: 2 Met Pro Phe Phe Thr Gly Leu Leu Ala Ile Tyr His Ser Leu Ile Leu 1 5 10 15 Asp Asin Pro Val Glin Thr Leu Ser Thr Ile Val Val Leu Ala Ala Ala 2O 25 30 Tyr Trp Lieu Ala Thr Lieu Gln Pro Ser Asp Leu Pro Glu Lieu. Asn Pro 35 40 45 Ala Lys Pro Phe Glu Phe Thr Asn Arg Arg Arg Val His Glu Phe Val 50 55 60 Glu Asn. Ser Lys Ser Lieu Lleu Ala Arg Gly Arg Glu Lieu. His Gly. His 65 70 75 8O Glu Pro Tyr Arg Leu Met Ser Glu Trp Gly Ser Leu Ile Val Leu Pro 85 90 95 Pro Glu Cys Ala Asp Glu Lieu Arg Asn Asp Pro Arg Met Asp Phe Glu 100 105 110 Thr Pro Thr Thr Asp Asp Ser His Gly Tyr Ile Pro Gly Phe Asp Ala 115 120 125 Lieu. Asn Ala Asp Pro Asn Lieu. Thir Lys Val Val Thr Lys Tyr Lieu. Thr 130 135 1 4 0 Lys Ala Lieu. Asn Lys Lieu. Thir Ala Pro Ile Ser His Glu Ala Ser Ile US 7,033,807 B2 77 78

-continued

145 15 O 155 160

Ala Met Lys Ala Val Lieu Gly Asp Pro Asp Trp Arg Glu Ile 1.65 170 175

Pro Ala Arg Asp Leu Lleu Glin Telu Wall Ala Arg Met Ser Thr Arg Wall 18O 185 19 O

Phe Telu Gly Glu Glu Met Cys Asn Asn Glin Asp Trp Ile Glin Thr Ser 195 200

Ser Glin Tyr Ala Ala Leu Ala Phe Gly Wall Gly Asp Telu Arg Ile 210 215 220

Tyr Pro Met Ile Arg Pro Ile Wall His Trp Phe Met Pro Ser Cys 225 230 235 240

Trp Glu Telu Arg Ser Lieu Cys Arg Glin Ile Telu Thr Pro 245 250 255

Ile His Lys Arg Lys Ser Telu Lys Gly Thr Thr Asp Glu Glin Gly 260 265 27 O

Pro Telu Met Phe Asp Asp Ser Ile Glu Trp Phe Glu Arg Glu Telu 275 280 285

Gly Pro Asn His Asp Ala Wal Telu Lys Glin Wall Thr Teu Ser Ile Wall 29 O 295

Ala Ile His Thr Thr Ser Asp Telu Telu Telu Glin Ala Met Ser Asp Telu 305 310 315 320

Ala Glin Asn Pro Lys Val Lieu Glin Ala Wall Arg Glu Glu Wall Wall Arg 325 330 335

Wall Telu Ser Thr Glu Gly Leu Ser Lys Wall Ser Teu His Ser Telu 340 345 35 O

Teu Met Asp Ser Ala Lieu Lys Glu Ser Glin Arg Teu Arg Pro Thr Telu 355 360 365

Teu Gly Ser Phe Ala Thr Asn Asp Ile Telu Ser 370 375

Gly Phe Wall Ile Lys Lys Gly Thr Wall Wall Ile Ser Thr His 385 390 395 400

Met Trp Asn Pro Glu Tyr Tyr Thr Pro Teu Glin Asp Gly Tyr 405 410 415

Arg Phe Asn Lys Arg Glin Thr Pro Gly Glu Asp Asn Ala Telu 420 425 43 O

Teu Wall Ser Thr Ser Ala Asn His Met Gly Phe Gly His Gly Wall His 435 4 40 4 45

Ala Cys Pro Gly Arg Phe Phe Ala Ser Asn Glu Ile Ile Ala Telu 450 455 460

Cys His Ile Ile Lieu. Asn Tyr Glu Trp Teu Pro Asp Gly Phe Lys 465 470 475 480

Pro Glin Pro Telu Asn. Ile Gly Met Thr Tyr Teu Ala Asp Pro Asn Thr 485 490 495

Arg Met Telu Ile Arg Pro Arg Lys Ala Glu Ile Asp Met Ala Ser Telu 5 OO 505 51O.

Thr Wall

SEQ ID NO 3 LENGTH 2031 TYPE DNA ORGANISM: human FEATURE: NAME/KEY: CDS LOCATION: (1) . . . (2031) OTHER INFORMATION: human oxidoreductase

US 7,033,807 B2 83 84

-continued cac atc tac gtc tgt ggg gat gCa cgg aac atg gcc agg gat gtg Cag 1920 His Ile Wall Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Wall Glin 625 630 635 640 aac acc titc. tac gac atc gtg gct gag citc. ggg gcc atg gag. cac gCg 1968 Asn Thr Phe Tyr Asp Ile Wall Ala Glu Telu Gly Ala Met Glu His Ala 645 650 655

Cag gCg gtg gac tac atc aag a.a.a. citg atg acc aag ggC cgc tac to c 2016 Glin Ala Wall Asp Tyr Ile Lys Lys Telu Met Thr Lys Gly Arg Ser 660 665 67 O citg gac gtg tgg agC 2031 Teu Asp Wall Trp Ser 675

SEQ ID NO 4 LENGTH 677 TYPE PRT ORGANISM: human

<400 SEQUENCE: 4

Met Gly Asp Ser His Wall Asp Thr Ser Ser Thr Wall Ser Glu Ala Wall 1 5 10 15

Ala Glu Glu Wall Ser Teu Phe Ser Met Thr Asp Met Ile Telu Phe Ser 2O 25 30

Teu Ile Wall Gly Teu Teu Thr Tyr Trp Phe Teu Phe Arg 35 40 45

Glu Glu Wall Pro Glu Phe Thr Lys Ile Glin Thr Teu Thr Ser Ser Wall 50 55 60

Arg Glu Ser Ser Phe Wall Glu Lys Met Lys Lys Thr Gly Arg Asn Ile 65 70 75

Ile Wall Phe Gly Ser Glin Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95

Arg Telu Ser Lys Asp Ala His Tyr Gly Met Arg Gly Met Ser Ala 100 105 110

Asp Pro Glu Glu Tyr Teu Ala Asp Telu Ser Ser Teu Pro Glu Ile 115 120 125

Asp Asn Ala Telu Wall Wall Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 1 4 0

Pro Thr Asp Asn Ala Glin Phe Tyr Trp Teu Glin Glu Thr Asp 145 15 O 155 160

Wall Asp Telu Ser Gly Wall Lys Phe Ala Wall Phe Gly Teu Gly Asn 1.65 170 175

Thr Glu His Phe Asn Ala Met Gly Lys Wall Asp Lys Arg Telu 18O 185 19 O

Glu Glin Telu Gly Ala Glin Ile Phe Glu Teu Gly Teu Gly Asp Asp 195 200

Asp Gly Asn Telu Glu Glu Asp Phe Ile Thr Trp Arg Glu Glin Phe Trp 210 215 220

Pro Ala Wall Glu His Phe Gly Wall Glu Ala Thr Gly Glu Glu Ser 225 230 235 240

Ser Ile Arg Glin Tyr Glu Teu Wall Wall His Thr Asp Ile Asp Ala Ala 245 250 255

Wall Met Gly Glu Met Gly Arg Telu Ser Glu Asn Glin 260 265 27 O

Pro Pro Phe Ala Lys Asn Pro Phe Teu Ala Ala Wall Thr Thr 275 280 285 US 7,033,807 B2 85

-continued Asn Arg Lys Lieu. Asn Glin Gly Thr Glu Arg His Leu Met His Leu Glu 29 O 295 3OO Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Ala Lieu Val Asn Gln Leu Gly Lys 325 330 335 Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Lieu. Asn. Asn Lieu. Asp 340 345 35 O Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 38O Val Leu Tyr Glu Leu Ala Glin Tyr Ala Ser Glu Pro Ser Glu Glin Glu 385 390 395 400 Leu Lieu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Lieu. Tyr 405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Glin 420 425 43 O Asp Cys Pro Ser Leu Arg Pro Pro Ile Asp His Lieu. Cys Glu Lieu Lieu 435 4 40 4 45 Pro Arg Lieu Glin Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Wal 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465 470 475 480 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr Asn Trp Lieu Arg Ala 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu Val Pro Met Phe 5 OO 505 51O. Val Arg Lys Ser Glin Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro Val 515 52O 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly Phe Ile 530 535 540 Glin Glu Arg Ala Trp Lieu Arg Glin Glin Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Lleu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr Lieu. Tyr Arg 565 570 575 Glu Glu Lieu Ala Glin Phe His Arg Asp Gly Ala Lieu. Thr Glin Lieu. Asn 58O 585 59 O Val Ala Phe Ser Arg Glu Gln Ser His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys Glin Asp Arg Glu His Leu Trp Llys Lieu. Ile Glu Gly Gly Ala 610 615 62O His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Val Glin 625 630 635 640 Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly Ala Met Glu His Ala 645 650 655 Glin Ala Val Asp Tyr Ile Lys Lys Lieu Met Thr Lys Gly Arg Tyr Ser 660 665 67 O Leu Asp Val Trp Ser 675

<210 SEQ ID NO 5 <211& LENGTH 2.322 &212> TYPE DNA

US 7,033,807 B2 89 90

-continued

Wall Thr Glu Asp Glu Ser Teu Ser Pro Glu Asp Glu Asn Wall Tyr Telu 245 25 O 255 ggit gag coc act Cala ggit cat citc. Cala ggC gag cco aag ggc cc.g tac Gly Glu Pro Thr Glin Gly His Telu Glin Gly Glu Pro Lys Gly Pro 260 265 270 tot gCg cac aac cc.g titc. atc gct coc atc too tot cgt. gaa citg 102 Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ser Ser Arg Glu Telu 275 280 285 290 titc. aac gtc aag gac cgc aac tgt citg cac atg agc atc gcc 15 O Phe Asn Wall Lys Asp Arg Asn Cys Telu His Met Ser Ile Ala 295 3OO 305 ggit agc aac citc. act tac Cag act ggit gac cac gtt tgg coc 198 Gly Ser Asn Telu Thr Glin Thr Gly Asp His Wall Trp Pro 310 315 32O acc aac gcc ggit too gag gto gat cgg titc. citg titt ggit citc. 246 Thr Asn Ala Gly Ser Glu Wall Asp Arg Phe Teu Phe Gly Telu 325 330 gaa gga aag cgc cac too gto atc aac att aag gat gtg acc 2.94 Glu Gly Lys Arg His Ser Wall Ile Asn Ile Lys Asp Wall Thr 34 O 345 350 gct aag gtt cc.g att cco act cost acg acc tat gac gca gtt cgc 342 Ala Lys Wall Pro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Wall Arg 355 360 365 370 tac tac citg gaa gto tgt gcc coc gtt to c cgt. Cag titt gtc tog act 39 O. Telu Glu Wall Cys Ala Pro Wall Ser Arg Glin Phe Wall Ser Thr 375 38O 385 citc. gct gCC titt gcc cost gat gaa gC9 acc aag gCg gag atc. gtt cgt. 4.38 Teu Ala Ala Phe Ala Pro Asp Glu Ala Thr Lys Ala Glu Ile Wall Arg 390 395 4 OO ttg ggit ggC gac aag gac tat titc. cat gag aag att acc aac cga. tgc 486 Teu Gly Gly Asp Lys Asp Tyr Phe His Glu Lys Ile Thr Asn Arg Cys 405 410 415 titc. aac atc gct Cag gct citc. Cag agc atc acg too aag cost titc. acc 534 Phe Asn Ile Ala Glin Ala Teu Glin Ser Ile Thr Ser Lys Pro Phe Thr 420 4.25 430 gcc gtc cc.g titc. too citg citt atc gaa ggit atc. acc aag citt Cag coc 582 Ala Wall Pro Phe Ser Teu Teu Ile Glu Gly Ile Thr Lys Telu Glin Pro 435 4 40 4 45 450 cgt. tac tac tog atc too tog tot to c citg gtt Cag aag gac aag att 630 Arg Ser Ile Ser Ser Ser Ser Telu Wall Glin Lys Asp Lys Ile 455 460 465 agC att acc gcc gtt gtg gag tog gtt cgc ttg cost ggit gag. gaa cac 678 Ser Ile Thr Ala Wall Wall Glu Ser Wall Arg Teu Pro Gly Glu Glu His 470 475 48O att gtc aag ggit gtg acc acg aac tat citt citc. gCg citc. aag gaa aag 726 Ile Wall Lys Gly Wall Thr Thr Asn Telu Teu Ala Teu Lys Glu Lys 485 490 495

Cala aac ggC gag cost too cost gac cc.g cac ggC ttg act tac tot atc 774 Glin Asn Gly Glu Pro Ser Pro Asp Pro His Gly Teu Thr Ser Ile 5 OO 505 510 act gga coc cgt. aac aag tac gat ggC atc cat gto cco gtt cac gtc 822 Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Wall Pro Wall His Wall 515 52O 525 530

cac tog aac titc. a.a.a. ttg coc tog gat cco tog Cga cost gtg atc 870 Arg His Ser Asn Phe Teu Pro Ser Asp Pro Ser Arg Pro Wall Ile 535 540 545 atg gtt gga coc ggit act ggit gtt gct cost titc. cgt. ggg titt atc Cag 918 Met Wall Gly Pro Gly Thr Gly Wall Ala Pro Phe Arg Gly Phe Ile Glin 550 555 560 US 7,033,807 B2 91 92

-continued gag cgt. gct gcc ttg gcc gCg aag ggC gag aag gto gga act acc ttg 1966 Glu Arg Ala Ala Teu Ala Ala Lys Gly Glu Lys Wall Gly Thr Thr Telu 565 570 575 citt titc. titc. ggC tgc cgt. aag to c gac gaa gat titc. tac aag gat 2014 Teu Phe Phe Gly Cys Arg Lys Ser Asp Glu Asp Phe Teu Lys Asp 58O 585 590 gaa tgg aag act titt Cag gag Cag citt ggC gac tog citc. aag atc atc Glu Trp Lys Thr Phe Glu Glin Telu Gly Asp Ser Teu Lys Ile Ile 595 605 610 act gcc titc. tot cgt. tog gct gag a.a.a. gto tac gto Cag cac agg 2110 Thr Ala Phe Ser Arg Ser Ala Glu Lys Wall Wall Glin His Arg 615 62O 625 citg cgt. gag cat gcc citg gtc agt gac citg citg aag Cag a.a.a. gcc 215.8 Teu Arg Glu His Ala Teu Wall Ser Asp Teu Teu Lys Glin Ala 630 635 64 O act titc. tat gtt tgc gac gct gcc aac atg gcc cgt. gaa gtc aac 22O6 Thr Phe Tyr Wall Cys Asp Ala Ala Asn Met Ala Arg Glu Wall Asn 645 650 655 citc. gtg citt ggg Cala att gcc aag Cag cgc ggit citc. cost gcc gag 2254 Teu Wall Telu Gly Glin Ile Ala Lys Glin Arg Gly Teu Pro Ala Glu 660 665 670 aag ggC gag gag atg gtg aag cac atg cgc agC agC ggC agc tac Cag Lys Gly Glu Glu Met Wall Lys His Met Arg Ser Ser Gly Ser Glin 675 680 685 690 gac gat gtc tgg too taa a.a. 2322 Asp Asp Wall Trp Ser 695

SEQ ID NO 6 LENGTH 695 TYPE PRT ORGANISM: Aspergillus ochraceus

<400 SEQUENCE: 6

Met Ala Glin Leu Asp Thr Teu Asp Telu Wall Wall Teu Wall Ala Telu Telu 1 5 10 15

Wall Gly Ser Wall Ala Tyr Phe Thr Lys Gly Thr Trp Ala Wall Ala 25 30

Asp Pro Tyr Ala Ser Ala Gly Pro Ala Met Asn Gly Gly Ala 35 40 45

Ala Gly Lys Thr Asp Ile Wall Glin Lys Met Asp Glu Thr Gly Lys 50 55 60

Asn Cys Wall Ile Phe Tyr Gly Ser Glin Thr Gly Thr Ala Glu Asp Tyr 65 70 75

Ala Ser Arg Telu Ala Lys Glu Gly Ser Glin Arg Phe Gly Telu Lys Thr 85 90 95

Met Wall Ala Asp Teu Glu Tyr Asp Tyr Glu Asn Teu Glu Phe 100 105 110

Pro Glu Asp Lys Wall Wall Phe Phe Wall Telu Ala Thr Tyr Gly Glu Gly 115 120 125

Glu Pro Thr Asn Ala Wall Glu Phe Tyr Glin Phe Wall Thr Gly Glu 130 135 1 4 0

Asp Ala Ala Phe Glu Ser Gly Ala Thr Ala Asp Asp Pro Telu Ser 145 15 O 155 160

Ser Telu Tyr Wall Thr Phe Gly Telu Gly Asn Asn Thr Glu His 1.65 170 175

Asn Ala Met Wall Arg Asn Wall Asp Ala Ala Teu Thr Lys Phe Gly 18O 185 19 O US 7,033,807 B2 93 94

-continued

Ala Glin Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr 195 200 2O5

Met Glu Glu Asp Phe Teu Ala Trp Lys Glu Pro Met Trp Ala Ala Telu 210 215 220

Ser Glu Ala Met Asn Teu Glin Glu Arg Asp Ala Wall Glu Pro Wall 225 230 235 240

Phe Asn Wall Thr Glu Asp Glu Ser Telu Ser Pro Glu Asp Glu Asn Wall 245 250 255

Telu Gly Glu Pro Thr Glin Gly His Telu Glin Gly Glu Pro Gly 260 265 27 O

Pro Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ser Glu Ser Arg 275 280 285

Glu Telu Phe Asn Wall Asp Arg Asn Teu His Met Glu Ile Ser 29 O 295

Ile Ala Gly Ser Asn Teu Thr Tyr Glin Thr Gly Asp His Ile Ala Wall 305 310 315 320

Trp Pro Thr Asn Ala Gly Ser Glu Wall Asp Arg Phe Teu Glin Ala Phe 325 330 335

Gly Telu Glu Gly Lys Arg His Ser Wall Ile Asn Ile Gly Ile Asp 340 345 35 O

Wall Thr Ala Wall Pro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala 355 360 365

Wall Arg Teu Glu Wall Cys Ala Pro Wall Ser Arg Glin Phe Wall 370 375

Ser Thr Telu Ala Ala Phe Ala Pro Asp Glu Ala Thr Ala Glu Ile 385 390 395 400

Wall Arg Telu Gly Gly Asp Lys Tyr Phe His Glu Ile Thr Asn 405 410 415

Arg Phe Asn Ile Ala Glin Ala Telu Glin Ser Ile Thr Ser Pro 420 425 43 O

Phe Thr Ala Wall Pro Phe Ser Telu Telu Ile Glu Gly Ile Thr Telu 435 4 40 4 45

Glin Pro Arg Tyr Ser Ile Ser Ser Ser Ser Teu Wall Glin Asp 450 455 460

Lys Ile Ser Ile Thr Ala Wall Wall Glu Ser Wall Arg Teu Pro Gly Glu 465 470 475 480

Glu His Ile Wall Lys Gly Wall Thr Thr Asn Teu Teu Ala Telu 485 490 495

Glu Glin Asn Gly Glu Pro Ser Pro Asp Pro His Gly Telu Thr 5 OO 505

Ser Ile Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Wall Pro Wall 515 525

His Wall Arg His Ser Asn Phe Lys Telu Pro Ser Asp Pro Ser Arg Pro 530 535 540

Wall Ile Met Wall Gly Pro Gly Thr Gly Wall Ala Pro Phe Arg Gly Phe 545 550 555 560

Ile Glin Glu Arg Ala Ala Teu Ala Ala Lys Gly Glu Wall Gly Thr 565 570 575

Thr Telu Telu Phe Phe Gly Cys Lys Ser Asp Glu Asp Phe Telu 585 59 O

Asp Glu Trp Lys Thr Phe Glin Glu Glin Teu Gly Asp Ser Telu 595 600 605 US 7,033,807 B2 95 96

-continued Ile Ile Thr Ala Phe Ser Arg Glu Ser Ala Glu Lys Wall Tyr Val Glin 610 615 62O

His Arg Lieu Arg Glu His Ala Glu Lieu Val Ser Asp Teu Lieu Lys Glin 625 630 635 640

Lys Ala Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu 645 650 655

Val Asn Lieu Val Lieu Gly Glin Ile Ile Ala Lys Glin Arg Gly Lieu Pro 660 665 67 O

Ala Glu Lys Gly Glu Glu Met Wall Lys His Met Arg Ser Ser Gly Ser 675 680 685 Tyr Glin Asp Asp Val Trp Ser 69 O. 695

<210 SEQ ID NO 7 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: human primer H. oxred 1A SEQUENCE: 7 gatcggat.cc aatatgg gag acticccacgt ggacac 36

SEQ ID NO 8 LENGTH 36 TYPE DNA ORGANISM: human primer H. oxred 1B <400 SEQUENCE: 8 gatcggat.cc aatatgg gag acticccacgt ggacac 36

SEQ ID NO 9 LENGTH 22 TYPE DNA ORGANISM: human primer H. oxred 2A <400 SEQUENCE: 9 citctgctdtc gtcaaccago to 22

SEQ ID NO 10 LENGTH 35 TYPE DNA ORGANISM: human primer H. oxred 2B <400 SEQUENCE: 10 gatcggtacc ttagcticcac acgtocaggg agtag 35

SEQ ID NO 11 LENGTH 2.0 TYPE DNA ORGANISM: Aspergillus primer A. oxred-for 1 FEATURE: NAME/KEY: modified base LOCATION: (6) . . . (6) OTHER INFORMATION: I - Inosine FEATURE: NAME/KEY: modified base LOCATION: (9) . . . (9) OTHER INFORMATION: I - Inosine FEATURE: NAME/KEY: misc feature LOCATION: (1) . . . (20) OTHER INFORMATION: n = Inosine

<400 SEQUENCE: 11 gacgging Cng gtacaatgga 20 US 7,033,807 B2 97 98

-continued

<210> SEQ ID NO 12 &2 11s LENGTH 26 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-revil &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (4) . . . (4) <223> OTHER INFORMATION: I - Inosine &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (10 ) . . . (10) <223> OTHER INFORMATION: I - Inosine &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (16) . . . (16) <223> OTHER INFORMATION: I - Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (1) . . . (26) <223> OTHER INFORMATION: n = Inosine

<400 SEQUENCE: 12 ttan gaccan acatcntcct ggtagc 26

<210> SEQ ID NO 13 &2 11s LENGTH 2.8 &212> TYPE DNA <213> ORGANISM: E. coli Primer pSport-for 1 <400 SEQUENCE: 13

Caag Ctctaa tacgacitcac tataggga 28

<210> SEQ ID NO 14 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-rev2 <400 SEQUENCE: 14 caggaaccga to gaccitcgg aa 22

<210 SEQ ID NO 15 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-rev3 <400 SEQUENCE: 15 gtoaccctica ccagoaga.gc caatg 25

<210> SEQ ID NO 16 &2 11s LENGTH 2.8 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-rev4 <400 SEQUENCE: 16 ccacattgcg aaccatagog ttgtagtg 28

<210 SEQ ID NO 17 &2 11s LENGTH 34 &212> TYPE DNA <213> ORGANISM: E. coli Primer pSport-for2 <400 SEQUENCE: 17 gccaagcticit aatacgactic actataggga aagc 34

<210> SEQ ID NO 18 US 7,033,807 B2 99 100

-continued

&2 11s LENGTH 27 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-for2 <400 SEQUENCE: 18 gtogacatgg cqcaactcga tactcitc 27

<210 SEQ ID NO 19 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-rev5 <400 SEQUENCE: 19 citcgagittag gaccagacat cqtcc togta g 31

<210> SEQ ID NO 20 <211& LENGTH 24 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-for3 <400 SEQUENCE: 20 ggat.ccct cq cqacct gtga totat 24

<210> SEQ ID NO 21 &2 11s LENGTH 38 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-for 4 <400 SEQUENCE: 21 cgaagattitc ttgtacaagg atgaatggaa gacttittc 38

<210> SEQ ID NO 22 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Aspergillus Primer A. oxred-rev6 <400 SEQUENCE: 22 citgaaaagttc titccattcat cottgtacaa gaaatc 36

<210> SEQ ID NO 23 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: Aspergillus 11aoH peptide 1 <400 SEQUENCE: 23 Ala Ala Ala Tyr Trp Lieu Ala Thr Lieu Glin Pro Ser Asp Leu Pro Glu 1 5 10 15

Teu Asn

<210> SEQ ID NO 24 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: Aspergillus 11aoH peptide 2: <400 SEQUENCE: 24 Cys Arg Glin Ile Leu Thr Pro Tyr Ile His Lys Arg Lys Ser Lieu Lys 1 5 10 15 Gly. Thir Thr Asp 2O

<210> SEQ ID NO 25 <211& LENGTH 21 &212> TYPE PRT US 7,033,807 B2 101 102

-continued <213> ORGANISM: Aspergillus 11aoH peptide 3 <400 SEQUENCE: 25 His Met Gly Phe Gly His Gly Val His Ala Cys Pro Gly Arg Phe Phe 1 5 10 15

Ala Ser Asn. Glu Ile

SEQ ID NO 26 LENGTH 2.0 TYPE PRT ORGANISM: Human oxir peptide 1 <400 SEQUENCE: 26 Cys Thr Tyr Trp Ala Val Ala Lys Asp Pro Tyr Ala Ser Ala Gly Pro 1 5 10 15 Ala Met Asn Gly

SEQ ID NO 27 LENGTH 52 6 TYPE PRT ORGANISM: Gibberella fujikuroi CAA755 65

<400 SEQUENCE: 27

Met Ala Asn His Ser Ser Ser Tyr Tyr His Glu Phe Lys Asp His 1 5 10 15

Ser His Thr Wall Teu Thr Teu Met Ser Glu Pro Wall Ile Telu Pro 25 30

Ser Telu Ile Telu Gly Thr Cys Ala Wall Telu Teu Ile Glin Trp Telu 35 40 45

Pro Glin Pro Teu Ile Met Wall Asn Gly Arg Lys Phe Gly Glu Telu 50 55 60

Ser Asn Wall Arg Ala Lys Arg Asp Phe Thr Phe Gly Ala Glin Telu 65 70 75

Teu Glu Gly Teu Met Ser Pro Asp Pro Phe Ile Met 85 90 95

Gly Asp Wall Gly Glu Lel His Ile Telu Pro Pro Ala Glu 100 105 110

Wall Arg Asn Asn Glu Teu Ser Phe Thr Met Ala Ala Phe Trp 115 120 125

Phe Tyr Ala His Teu Gly Phe Glu Gly Phe Arg Glu Gly Thr Asn 130 135 1 4 0

Glu Ser His Ile Met Lys Teu Wall Ala Arg His Glin Teu Thr His Glin 145 15 O 155 160

Teu Thr Telu Wall Thr Gly Ala Wall Ser Glu Glu Ala Telu Wall Telu 1.65 170 175

Asp Wall Tyr Thr Asp Ser Pro Glu Trp His Asp Ile Thr Ala Lys 18O 185 19 O

Asp Ala Asn Met Lys Teu Met Ala Arg Ile Thr Ser Arg Wall Phe Telu 195 200

Gly Lys Glu Met Cys Arg Asn Pro Glin Trp Teu Arg Ile Thr Ser Thr 210 215 220

Tyr Ala Wall Ile Ala Phe Ala Wall Glu Glu Teu Arg Telu Trp Pro 225 230 235 240

Ser Trp Telu Pro Wall Wall Glin Trp Phe Met Pro His Thr Glin 245 250 255 US 7,033,807 B2 103 104

-continued

Ser Arg Ala Telu Wall Glu Ala Arg Asp Teu Ile Asn Pro Telu Telu 260 265 27 O

Glu Arg Arg Arg Glu Ala Glu Ala Glu Arg Thr Gly Glu 275 280 285

Wall Thr Asn Asp Wall Glu Trp Telu Asp Asp Teu Ala Arg Glu 29 O 295

Lys Gly Wall Gly Tyr Pro Ala Cys Ala Glin Teu Ser Telu Ser Wall 305 315 320

Ala Ala Telu His Ser Thr Phe Phe Thr Glin Wall Met Phe Asp 325 330 335

Ile Ala Glin Asn Pro Teu Ile Glu Pro Teu Arg Glu Glu Ile Ile 340 345 35 O

Ala Wall Telu Gly Lys Gly Trp Ser Asn Ser Teu Asn Telu 355 360 365

Telu Met Asp Ser Wall Teu Lys Glu Ser Glin Arg Teu Pro Ile 370 375

Ala Ile Ala Ser Met Arg Arg Phe Thr Thr His Asn Wall Telu Ser 385 390 395 400

Asp Gly Wall Ile Teu Pro Asn Lys Telu Thr Teu Wall Ser Ala His 405 410 415

Glin His Trp Asp Pro Glu Tyr Lys Asp Pro Teu Phe Asp Gly 420 425 43 O

Arg Phe Phe Asn Met Arg Arg Glu Pro Gly Glu Ser Ala 435 4 40 4 45

Glin Telu Wall Ser Ala Thr Pro Asp His Met Gly Phe Gly Gly Telu 450 455 460

His Ala Pro Gly Arg Phe Phe Ala Ser Glu Glu Ile Ile Ala 465 470 475 480

Teu Ser His Ile Teu Teu Tyr Phe Pro Wall Glu Gly Ser 485 490 495

Ser Met Glu Pro Arg Lys Gly Telu Asn Met Asn Ala Asn Pro Thr 5 OO 505 51O.

Ala Telu Ser Wall Arg Arg Arg Lys Glu Glu Ile Ala Ile 515 52O 525

SEQ ID NO 28 LENGTH 514 TYPE PRT ORGANISM: Neurospora crassa CAB91316

<400 SEQUENCE: 28

Met Glu Arg Lieu Asp Ile Lys Ser Ile Thr Asp Pro Ser Ala Thr Pro 1 5 10 15

Phe Ser Tyr Telu Wall Thr Ala Phe Telu Telu Ala Wall Wall Wall Ser 25 30

Teu Glin Gly Pro Phe Pro Lys Asn Ile His Teu Asn Pro 35 40 45

Gly Pro Telu Glu Phe Ser Asp Thr Pro Lys Glu Phe Wall 50 55 60

Gly Ser Arg Glin Met Teu Ala Asn Trp Phe Lys Ala Asn Pro Asn 65 70 75

Pro Arg Wall Ile Ser Asp Phe Gly Glu Ala Ile Wall Telu Pro Pro 85 90 95

Arg Met Ala Asn Glu Ile Lys Asn Asp Asp Arg Teu Ser Phe Thr Arg US 7,033,807 B2 105 106

-continued

100 105 110

Trp Thr Tyr Lys Ala Phe His Gly His Telu Pro Gly Phe Glu Gly Phe 115 120 125

Gly Glu Ala Ser Glu Ser His Ile Wall Glin Glu Wall Ile Met Arg 130 135 1 4 0

Asp Telu Thr Tyr Teu Asn Wall Thr Glu Pro Teu Ala Glin Glu 145 15 O 155 160

Thr Ser Met Ala Met Glu Ala Asn Telu Pro Ala Ala Asn Gly Glu 1.65 170 175

Trp Ser Thr Ile Asn Teu Arg Ser Lys Ile Teu Pro Ile Wall Ala Arg 18O 185 19 O

Ile Ser Ser Arg Wall Phe Teu Gly Glu Glu Teu Arg Asn Glu Glu 195 200

Trp Telu Wall Thr Glin Glin Thr Ile Asp Gly Phe Gly Ala Ala 210 215 220

Glu Asp Telu Arg Teu Trp Pro Ala Ala Telu Arg Pro Ile Wall His Trp 225 230 235 240

Phe Telu Pro Ser Cys Glin Arg Ala Arg Ala Asp Wall Arg Wall Ala Arg 245 250 255

Ser Ile Telu Asp Pro Wall Teu Lys Arg Arg Glin Glu Lys Ala Ala 260 265 27 O

Asn Gly Gly Lys Ala Glu His Asp Asp Ala Ile Glu Trp Phe Glu Arg 275 280 285

Thr Ala Gly Lys Tyr Asp Pro Ala Wall Ala Glin Telu Wall Telu 29 O 295 3OO

Ser Telu Wall Ala Ile His Thr Thr Ser Asp Teu Thr Glin Wall Met 305 310 315 320

Thr Asn Telu Met Glin Asn Pro Glu Phe Ile Ala Pro Teu Arg Glu Glu 325 330 335

Met Ile Glin Wall Teu Ser Glu Gly Gly Trp Thr Ser Telu 340 345 35 O

Asn Met Lys Telu Teu Asp Ser Wall Ile Glu Ser Glin Arg Wall 355 360 365

Pro Thr Gly Wall Ala Ser Met Arg Ala Glu Asp Wall Thr 370 375

Teu Ser Asp Gly Thr Phe Ile Pro Lys Gly Gly Phe Wall Ala Wall Ser 385 390 395 400

Ala His Asp Met Trp Asn Ser Glu Wall Tyr Glu Glin Ala Glu Lys Trp 405 410 415

Asp Gly Arg Phe Teu Arg Met Arg Glu Thr Pro Gly Ala Gly 420 425 43 O

Glu Asn Wall Ala Glin Teu Wall Ser Thr Ala Pro Glu His Telu Gly Phe 435 4 40 4 45

Gly His Gly Glin His Ala Cys Pro Gly Phe Phe Ala Ala Asn Glu 450 455 460

Ile Ile Ala Teu Wall His Telu Telu Telu Asn Glu Trp Arg Telu 465 470 475 480

Pro Glu Gly Ser Asp Pro Lys Ile Arg Thr Phe Gly Phe Ser Met Gly 485 490 495

Wall Asp Pro Ser Teu Lys Wall Glu Tyr Gly Arg Glin Pro Glu Ile 5 OO 505 51O.

Glu Telu US 7,033,807 B2 107 108

-continued

SEQ ID NO 29 LENGTH 495 TYPE ORGANISM: Catharanthus roseus CAB56503

<400 SEQUENCE: 29

Leu Lleu Phe Cys Phe Ile Teu Ser Lys Thr Thr Phe Gly Glin 1 5 10 15

Asn Ser Glin Tyr Ser Asn His Asp Glu Telu Pro Pro Gly Pro Pro Glin 2O 25 30

Ile Pro Ile Telu Gly Asn Ala His Glin Telu Ser Gly Gly His Thr His 35 40 45

His Ile Telu Arg Asp Teu Ala Lys Gly Pro Teu Met His Telu 50 55 60

Lys Ile Gly Glu Wall Ser Thr Ile Wall Ala Ser Ser Pro Glin Ile Ala 65 70 75

Glu Glu Ile Phe Thr His Asp Ile Telu Phe Ala Asp Arg Pro Ser 85 90 95

Asn Telu Glu Ser Phe Lys Ile Wall Ser Tyr Asp Phe Ser Asp Met Wall 100 105 110

Wall Ser Pro Tyr Gly Asn Trp Arg Glin Teu Arg Lys Ile Ser Met 115 120 125

Met Glu Telu Telu Ser Glin Lys Ser Wall Glin Ser Phe Arg Ser Ile Arg 130 135 1 4 0

Glu Glu Glu Wall Teu Asn Phe Ile Ser Ile Gly Ser Glu Gly 145 15 O 155 160

Thr Arg Ile Asn Teu Ser Lys Glu Ile Ser Teu Teu Ile Gly Ile 1.65 170 175

Thr Thr Ala Ala Phe Gly Glu Lys Asn Lys Asn Thr Glu Glu Phe 18O 185 19 O

Ile Arg Telu Telu Asp Glin Teu Thr Ala Wall Ala Glu Pro Asn Ile 195 200

Ala Asp Met Phe Pro Ser Teu Lys Telu Glin Teu Ile Ser Thr Ser 210 215 220

Lys Lys Ile Glu Lys Ile His Glin Phe Asp Wall Ile Wall Glu 225 230 235 240

Thr Ile Telu Lys Gly His Lys Glu Ile Asn Pro Telu Ser Glin 245 250 255

Glu Asn Gly Glu Lys Glu Lel Wall Asp Wall Teu Telu Asn Ile 260 265 27 O

Glin Arg Arg Asn Asp Phe Glu Ala Telu Gly Asp Lys Asn Ile 275 280 285

Ala Ile Ile Phe Asn Ile Phe Ser Ala Gly Thr Glu Thr Ser Ser Thr 29 O 295

Thr Wall Asp Trp Ala Met Cys Glu Met Ile Lys Asn Pro Thr Wall Met 305 310 315 320

Ala Glin Glu Glu Wall Arg Lys Wall Phe Asn Glu Glu Gly Asn 325 330 335

Asp Glu Thr Lys Teu His Glin Telu Lys Teu Glin Ala Wall Ile 340 345 35 O

Glu Thr Telu Teu His Pro Pro Wall Pro Teu Teu Telu Pro Arg 355 360 365

Glu Cys Arg Glu Glin Cys Lys Ile Lys Gly Tyr Thr Ile Pro Ser 370 375 38O US 7,033,807 B2 109 110

-continued

Ser Arg Val Ile Val Asn Ala Trp Ala Ile Gly Arg Asp Pro Asn Tyr 385 390 395 400

Trp Ile Glu Pro Glu Lys Phe Asn Pro Asp Arg Phe Teu Glu Ser 405 410 415

Val Asp Phe Lys Gly Asn Ser Phe Glu Tyr Teu Pro Phe Gly Gly Gly 420 425 43 O

Arg Arg Ile Cys Pro Gly Ile Thr Phe Ala Teu Ala Asn Ile Glu Telu 435 4 40 4 45

Pro Leu Ala Glin Leu Lleu Phe His Phe Asp Trp Glin Ser Asn Thr Glu 450 455 460

Lys Lieu. Asn Met Lys Glu Ser Arg Gly Val Thr Wall Arg Arg Glu Asp 465 470 475 480

Asp Leu Tyr Leu Thr Pro Val Asin Phe Ser Ser Ser Ser Pro Ala 485 490 495

SEQ ID NO 30 LENGTH 510 TYPE PRT ORGANISM: Glycine max AAB94588 <400 SEQUENCE: 30

Met Wal Met Glu Lieu. His Asn His Thr Pro Phe Ser Ile Phe Ile 1 5 10 15

Thir Ser Ile Leu Phe Ile Phe Phe Wall Phe Phe Teu Wall Glin Arg 2O 25 30

Ser Asp Ser Lys Thr Ser Ser Thr Cys Lys Teu Pro Pro Gly Pro Arg 35 40 45

Thr Leu Pro Leu Ile Gly Asn Ile His Glin Ile Wall Gly Ser Telu Pro 50 55 60

Val His Tyr Tyr Lieu Lys Asn Lieu Ala Asp Lys Gly Pro Telu Met 65 70 75

His Lieu Lys Lieu Gly Glu Val Ser Asn. Ile Ile Wall Thr Ser Pro Glu 85 90 95

Met Ala Glin Glu Ile Met Lys Thr His Asp Teu Asn Phe Ser Asp Arg 100 105 110

Pro Asp Phe Val Leu Ser Arg Ile Val Ser Tyr Asn Gly Ser Gly Ile 115 120 125

Val Phe Ser Gln His Gly Asp Tyr Trp Arg Glin Teu Arg Ile 130 135 1 4 0

Thr Val Glu Leu Leu Thr Ala Lys Arg Val Glin Ser Phe Arg Ser Ile 145 15 O 155 160

Arg Glu Glu Glu Val Ala Glu Lieu Val Lys Ile Ala Ala Thr Ala 1.65 170 175

Ser Glu Glu Gly Gly Ser Ile Phe Asn Lieu Thr Glin Ser Ile Ser 18O 185 19 O

Met Thr Phe Gly Ile Ala Ala Arg Ala Ala Phe Gly Lys Ser Arg 195 200

Tyr Glin Glin Val Phe Ile Ser Asn Met His Glin Teu Met Telu Telu 210 215 220

Gly Gly Phe Ser Val Ala Asp Leu Tyr Pro Ser Ser Arg Wall Phe Glin 225 230 235 240

Met Met Gly Ala Thr Gly Lys Lieu Glu Lys Wall His Arg Wall Thr Asp 245 250 255

Arg Val Lieu Glin Asp Ile Ile Asp Glu His Asn Arg Asn Arg Ser US 7,033,807 B2 111 112

-continued

260 265 27 O

Ser Glu Arg Glu Ala Wall Glu Asp Telu Wall Asp Wall Telu Telu 275 280 285

Phe Lys Glu Ser Glu Phe Telu Thr Asp Asp Asn Ile Ala 295 3OO

Wall Glin Asp Ile Phe Ile Gly Gly Gly Glu Thr Ser Ser Ser Wall 305 310 315 320

Wall Trp Gly Met Ser Glu Telu Ile Arg Asn Pro Arg Wall Met Glu 325 330 335

Glu Glin Ala Glu Wall Arg Wall Tyr Asp Ser Gly Wall 340 345 35 O

Asp Thr Glu Teu His Glin Telu Ile Tyr Teu Ser Ile Ile 355 360 365

Glu Met Arg Teu His Pro Pro Wall Pro Teu Teu Wall Pro Arg Wall 370 375

Ser Arg Glu Arg Cys Glin Ile Asn Gly Tyr Glu Ile Pro Ser Thr 385 390 395 400

Arg Ile Ile Ile Asn Ala Trp Ala Ile Gly Arg Asn Pro Tyr Trp 405 410 415

Gly Glu Thr Glu Ser Phe Pro Glu Arg Phe Teu Asn Ser Ser Ile 420 425 43 O

Asp Phe Arg Gly Thr Phe Glu Phe Ile Pro Phe Gly Ala Gly 435 4 40 4 45

Arg Ile Pro Gly Ile Thr Phe Ala Ile Pro Asn Ile Glu Telu Pro 450 455 460

Teu Ala Glin Telu Teu Tyr His Phe Trp Lys Teu Pro Asn Met 465 470 475 480

Asn Glu Glu Teu Asp Met Thr Glu Ser Asn Gly Ile Thr Telu Arg 485 490 495

Arg Glin Asn Asp Teu Cys Teu Ile Pro Ile Thr Arg Teu Pro 5 OO 505 51O.

SEQ ID NO 31 LENGTH 524 TYPE PRT ORGANISM: Gibberella fujikuroi CAA755 66

<400 SEQUENCE: 31

Met Ser Ile Phe Asn Met Ile Thr Ser Tyr Ala Gly Ser Glin Telu Telu 1 5 10 15

Pro Phe Tyr Ile Ala Ile Phe Wall Phe Thr Teu Wall Pro Trp Ala Ile 2O 25 30

Arg Phe Ser Trp Teu Glu Teu Arg Lys Gly Ser Wall Wall Pro Telu Ala 35 40 45

Asn Pro Pro Asp Ser Teu Phe Gly Thr Gly Lys Thr Arg Arg Ser Phe 50 55 60

Wall Telu Ser Glu Ile Telu Ala Lys Ala Arg Ser Telu Phe Pro 65 70 75

Asn Glu Pro Phe Teu Ile Thr Trp Gly Glu Wall Telu Ile Telu 90 95

Pro Pro Asp Phe Ala Glu Ile Arg Asn Asp Pro Arg Telu Ser Phe 100 105 110

Ser Ala Ala Met Glin Asn His Ala Gly Ile Pro Gly Phe Glu 115 120 125 US 7,033,807 B2 113 114

-continued

Thr Wall Ala Telu Wall Gly Arg Glu Asp Glin Teu Ile Glin Lys Wall Ala 130 135 1 4 0

Arg Glin Telu Thr Lys His Telu Ser Ala Wall Ile Glu Pro Telu Ser 145 15 O 155 160

Arg Glu Ser Thr Teu Ala Wall Ser Telu Asn Phe Gly Glu Thr Thr Glu 1.65 170 175

Trp Arg Ala Ile Arg Teu Lys Pro Ala Ile Teu Asp Ile Ile Ala Arg 18O 185 19 O

Ile Ser Ser Arg Ile Tyr Teu Gly Asp Glin Teu Arg Asn Glu Ala 195 200

Trp Telu Ile Thr Lys Thr Tyr Thr Thr Asn Phe Thr Ala Ser 210 215 220

Thr Asn Telu Arg Met Phe Pro Ser Ile Arg Pro Teu Ala His Trp 225 230 235 240

Phe Telu Pro Glu Cys Arg Lys Telu Glin Glu Arg Asp Ala Ile 245 250 255

Gly Ile Ile Thr Pro Teu Ile Glu Arg Arg Arg Glu Teu Arg Arg Ala 260 265 27 O

Ala Ile Ala Ala Gly Glin Pro Telu Pro Wall Phe His Asp Ala Ile Asp 275 280 285

Trp Ser Glu Glin Glu Ala Glu Ala Ala Gly Thr Gly Ala Ser Phe Asp 29 O 295

Pro Wall Ile Phe Glin Teu Thr Telu Ser Telu Teu Ala Ile His Thr Thr 305 310 315 320

Asp Telu Telu Glin Glin Thr Met Ile Asp Teu Gly Arg His Pro Glu 325 330 335

Ile Glu Pro Teu Glin Glu Wall Wall Glin Teu Teu Arg Glu Glu 340 345 35 O

Gly Trp Lys Thr Thr Teu Phe Lys Met Teu Teu Asp Ser Ala 355 360 365

Ile Lys Glu Ser Glin Arg Met Lys Pro Gly Ser Ile Wall Thr Met Arg 370 375

Arg Wall Thr Glu Asp Ile Thr Telu Ser Ser Gly Teu Thr Telu Lys 385 390 395 400

Thr Arg Teu Asn Wall Asp Asn Arg Arg Teu Asp Asp Pro 405 410 415

Ile Asp Asn Pro Glu Wall Tyr Asn Pro Arg Phe Tyr Asp Met 420 425 43 O

Arg Ser Glu Ala Gly Lys His Gly Ala Glin Teu Wall Ser Thr Gly 435 4 40 4 45

Ser Asn His Met Gly Phe Gly His Gly Glin His Ser Pro Gly Arg 450 455 460

Phe Phe Ala Ala Asn Glu Ile Lys Wall Ala Teu His Ile Telu Wall 465 470 475 480

Asp Trp Lys Teu Cys Pro Asp Thr Glu Thr Pro Asp Thr 485 490 495

Arg Met Ile Ala Lys Ser Ser Pro Wall Thr Asp Ile Telu Ile 5 OO 505 51O.

Arg Glu Ser Wall Glu Teu Asp Telu Glu Ala Ile 515 52O

<210> SEQ ID NO 32 &2 11s LENGTH 528 &212> TYPE PRT US 7,033,807 B2 115 116

-continued <213> ORGANISM: Aspergillus terreus AAD34552 <400 SEQUENCE: 32

Met Thr Val Asp Ala Teu Thr Glin Pro His His Teu Teu Ser Telu Ala 1 5 10 15

Trp Asn Asp Thr Glin Glin His Gly Ser Trp Phe Ala Pro Telu Wall Thr 25 30

Thr Ser Ala Gly Teu Teu Cys Telu Telu Telu Teu Cys Ser Ser Gly 35 40 45

Arg Ser Asp Teu Pro Wall Phe Asn Pro Thr Trp Trp Glu Telu 50 55 60

Thr Thr Met Arg Ala Lys Arg Asp Phe Asp Ala Asn Ala Pro Ser Trp 65 70 75

Ile Glu Ser Trp Phe Ser Glin Asn Asp Lys Pro Ile Arg Phe Ile Wall 85 90 95

Asp Ser Gly Tyr Cys Thr Ile Telu Pro Ser Ser Met Ala Asp Glu Phe 100 105 110

Arg Lys Met Glu Teu Cys Met Tyr Lys Phe Teu Gly Thr Asp Phe 115 120 125

His Ser His Telu Pro Gly Phe Gly Phe Glu Wall Thr Arg Asp 130 135 1 4 0

Ala His Telu Ile Thr Lys Wall Wall Met Asn Glin Phe Glin Thr Glin Ala 145 15 O 155 160

Pro Lys Wall Lys Pro Teu Ala Asn Glu Ala Ser Gly Ile Ile Thr 1.65 170 175

Asp Ile Phe Gly Asp Ser Asn Glu Trp His Thr Wall Pro Wall Asn 18O 185 19 O

Glin Cys Telu Asp Teu Wall Thr Arg Thr Wall Thr Phe Ile Met Wall Gly 195 200

Ser Lys Telu Ala His Asn Glu Glu Trp Telu Asp Ile Ala His His 210 215 220

Ala Wall Thr Met Ala Ile Glin Ala Arg Glin Teu Arg Teu Trp Pro Wall 225 230 235 240

Ile Telu Arg Pro Teu Wall His Trp Telu Glu Pro Glin Gly Ala Lys Telu 245 250 255

Arg Ala Glin Wall Arg Arg Ala Glin Telu Teu Asp Pro Ile Ile Glin 260 265 27 O

Glu Arg Ala Glu Arg Asp Ala Cys Arg Ala Gly Ile Glu Pro 275 280 285

Pro Arg Wall Asp Ser Ile Glin Trp Phe Glu Asp Thr Ala Gly 29 O 295 3OO

Lys Trp Asp Ala Ala Gly Ala Glin Telu Ala Met Asp Phe Ala Gly 305 310 315 320

Ile Tyr Gly Thr Ser Asp Teu Telu Ile Gly Gly Teu Wall Asp Ile Wall 325 330 335

Arg His Pro His Teu Teu Glu Pro Telu Asp Glu Ile Arg Thr Wall 340 345 35 O

Ile Gly Glin Gly Gly Trp Thr Pro Ala Ser Teu Lys Telu Telu 355 360 365

Teu Asp Ser Teu Lys Glu Ser Glin Arg Wall Lys Pro Wall Glu 370 375 38O

Ala Thr Met Arg Ser Tyr Ala Telu Glin Asp Wall Thr Phe Ser Asn Gly 385 390 395 400 US 7,033,807 B2 117 118

-continued

Thr Phe Ile Pro Lys Gly Glu Telu Wall Ala Wall Ala Ala Asp Arg Met 405 410 415

Ser Asn Pro Glu Wall Trp Pro Glu Pro Ala Lys Tyr Asp Pro Tyr Arg 420 425 43 O

Met Arg Telu Glu Pro Ala Lys Ala Phe Ser Ala Glin Telu 435 4 40 4 45

Glu Asn Thr Asn Gly His Ile Gly Phe Gly Trp His Pro Arg Ala 450 455 460

Cys Pro Gly Arg Phe Phe Ala Ser Lys Glu Ile Met Met Telu Ala 465 470 475 480

Telu Telu Ile Arg Tyr Trp Lys Wall Wall Pro Asp Glu Pro Telu 485 490 495

Glin Arg His Ser Phe Ser Wall Arg Ile His Pro Thr Thr 5 OO 505

Teu Met Met Arg Asp Glu Asp Ile Arg Teu Pro Gly Ser Telu 515 52O 525

SEQ ID NO 33 LENGTH 388 TYPE PRT ORGANISM: Gibberella fujikuroi CAA755 67

<400 SEQUENCE: 33

Met Lys Tyr Thr Thr Cys Glin Met Asn Ile Phe Pro Ser Telu Trp Ser 1 5 10 15

Met Lys Thr Ser Phe Arg Trp Pro Arg Thr Ser Trp Ser Ser Wall 2O 25 30

Ser Telu Tyr Asp Met Met Teu Arg Thr Wall Ala Teu Teu Ser Gly 35 40 45

Ala Phe Wall Gly Teu Pro Teu Cys Asp Glu Gly Trp Telu Glin Ala 50 55 60

Ser Ile Gly Tyr Thr Wall Glin Cys Wall Ser Ile Arg Asp Glin Telu Phe 65 70 75

Thr Trp Ser Pro Wall Teu Pro Ile Ile Gly Pro Phe Telu Pro Ser 85 90 95

Wall Arg Ser Wall Arg His Telu Arg Phe Ala Ala Glu Ile Met Ala 100 105 110

Pro Telu Ile Ser Glin Ala Teu Glin Asp Glu Glin His Arg Ala Asp 115 120 125

Thr Telu Telu Ala Glin Thr Glu Gly Arg Gly Thr Phe Ile Ser Trp 130 135 1 4 0

Teu Telu Arg His Teu Pro Glu Glu Telu Thr Pro Glu Glin Wall Gly 145 15 O 155 160

Teu Asp Glin Met Teu Wall Ser Phe Ala Ala Ile His Thr Thr Thr Met 1.65 170 175

Ala Telu Thr Lys Wall Wall Trp Glu Telu Wall Arg Pro Glu Ile 18O 185 19 O

Glu Pro Telu Arg Thr Glu Met Glin Asp Wall Phe Gly Pro Asp Ala Wall 195 200

Ser Pro Asp Ile Cys Ile Asn Lys Glu Ala Teu Ser Arg Telu His 210 215 220

Teu Asp Ser Phe Ile Arg Glu Wall Glin Arg Trp Pro Ser Thr Phe 225 230 235 240

Wall Thr Pro Ser Arg Arg Wall Met Lys Ser Met Thr Teu Ser Asn Gly 245 250 255 US 7,033,807 B2 119 120

-continued

Ile Lys Leu Glin Arg Gly Thr Ser Ile Ala Phe Pro Ala His Ala Ile 260 265 27 O

His Met Ser Glu Glu Thr Pro Thr Phe Ser Pro Asp Phe Ser Ser Asp 275 280 285

Phe Glu Asn Pro Ser Pro Arg Ile Phe Asp Gly Phe Arg Telu Asn 29 O 295 3OO

Leu Arg Ser Ile Lys Gly Glin Gly Ser Gln His Glin Ala Thr Thr 305 310 315 320

Gly Pro Asp Tyr Lieu. Ile Phe Asn His Gly Lys His Ala Pro Gly 325 330 335

Arg Phe Phe Ala Ile Ser Glu Ile Lys Met Ile Leu Ile Glu Telu Telu 340 345 35 O

Ala Lys Tyr Asp Phe Arg Lieu Glu Asp Gly Lys Pro Gly Pro Glu Telu 355 360 365

Met Arg Val Gly Thr Glu Thir Arg Leu Asp Thr Lys Ala Gly Telu Glu 370 375 38O Met Arg Arg Arg 385

SEQ ID NO 34 LENGTH 525 TYPE PRT ORGANISM: Gibberella fujikuroi CAA76703 <400 SEQUENCE: 34

Met Ser Lys Ser Asn Ser Met Asn Ser Thr Ser His Glu Thr Telu Phe 1 5 10 15

Glin Glin Lieu Val Lieu Gly Lieu. Asp Arg Met Pro Leu Met Asp Wall His 2O 25 30

Trp Leu Ile Tyr Val Ala Phe Gly Ala Trp Leu Cys Ser Wall Ile 35 40 45

His Val Leu Ser Ser Ser Ser Thr Val Lys Val Pro Wall Wall Gly Tyr 50 55 60

Arg Ser Val Phe Glu Pro Thir Trp Leu Leu Arg Leu Arg Phe Wall Trp 65 70 75

Glu Gly Gly Ser Ile Ile Gly Glin Gly Tyr Asn Lys Phe Asp Ser 85 90 95

Ile Phe Glin Val Arg Lys Lieu Gly Thr Asp Ile Val Ile Ile Pro Pro 100 105 110

Asn Tyr Ile Asp Glu Val Arg Lys Lieu Ser Glin Asp Lys Thr Arg Ser 115 120 125

Val Glu Pro Phe Ile Asn Asp Phe Ala Gly Glin Tyr Thr Arg Gly Met 130 135 1 4 0

Val Phe Leu Glin Ser Asp Leu Glin Asn Arg Val Ile Glin Glin Arg Telu 145 15 O 155 160

Thr Pro Llys Leu Val Ser Leu Thr Lys Val Met Lys Glu Glu Telu Asp 1.65 170 175

Tyr Ala Lieu. Thir Lys Glu Met Pro Asp Met Lys Asn Asp Glu Trp Wall 18O 185 19 O

Glu Val Asp Ile Ser Ser Ile Met Val Arg Leu Ile Ser Arg Ile Ser 195 200

Ala Arg Val Phe Leu Gly Pro Glu His Cys Arg Asn Glin Glu Trp Telu 210 215 220 Thir Thr Thr Ala Glu Tyr Ser Glu Ser Leu Phe Ile Thr Gly Phe Ile US 7,033,807 B2 121 122

-continued

225 230 235 240

Teu Wall Wall Pro His Ile Telu Pro Phe Ile Ala Pro Telu Telu 245 250 255

Pro Ser Arg Thr Teu Teu Asn Wall Ser Ser Gly Arg Arg Wall 260 265 27 O

Ile Gly Asp Ile Ile Ser Glin Glin Gly Asp Gly Asn Glu Asp Ile 275 280 285

Teu Ser Trp Met Arg Asp Ala Ala Thr Gly Glu Glu Glin Ile Asp 29 O 295

Asn Ile Ala Glin Met Teu Ile Telu Ser Teu Ala Ser Ile His Thr 305 310 315 320

Thr Ala Met Thr Met Thr His Ala Met Tyr Asp Teu Ala Cys Pro 325 330 335

Glu Tyr Ile Glu Pro Teu Asp Glu Wall Ser Wall Wall Gly Ala 340 345 35 O

Ser Gly Trp Asp Lys Thr Ala Telu Asn Phe His Lys Telu Asp Ser 355 360 365

Phe Telu Glu Ser Glin Arg Phe Asn Pro Wall Phe Teu Telu Thr Phe 370 375

Asn Ile Tyr His Glin Ser Met Thr Telu Ser Asp Gly Thr Asn Ile 385 390 395 400

Pro Ser Gly Thr Arg Ile Ala Wall Pro Ser His Ala Met Telu Glin Asp 405 410 415

Ser Ala His Wall Pro Gly Pro Thr Pro Pro Thr Glu Phe Asp Gly Phe 420 425 43 O

Arg Tyr Ser Lys Ile Ser Asp Ser Asn Ala Glin Telu 435 4 40 4 45

Phe Ser Met Thr Ser Ser Asn Met Ala Phe Gly Tyr Gly Lys 450 455 460

Ala Cys Pro Gly Phe Tyr Ala Ser Asn Glu Met Telu Thr Telu 465 470 475 480

Ala Ile Telu Telu Teu Glin Phe Glu Phe Lys Teu Pro Asp Gly Lys Gly 485 490 495

Arg Pro Arg Asn Ile Thr Ile Ser Asp Met Ile Pro Asp Pro Arg 5 OO 505 51O.

Ala Telu Cys Wall Arg Lys Arg Ser Telu Arg Asp Glu 515 52O 525

SEQ ID NO 35 LENGTH 2.94 TYPE PRT ORGANISM: Fusarium oxysporum CAA57874 <400 SEQUENCE: 35

Met Ala Pro Met Leu Arg Pro Telu Wall Tyr Arg Phe Ile Pro Glu Arg 1 5 10 15

Ala Arg Ile Lys Asp Glin Trp Thr Lys Gly Arg Arg Wall Met Ala 2O 25 30

Ser Met Arg Glu Arg Glin Glu Lys Gly Gly Asn Teu Glu Asp Pro Pro 35 40 45

Thr Met Telu Asp His Teu Ser Asn Gly Arg Asn Glu His Ile Ala Asp 50 55 60

Asp Wall Glu Telu Glin Teu Teu His Glin Met Thr Teu Ile Ala Wall 65 70 75 US 7,033,807 B2 123 124

-continued

Thr Wall Thr Thr Phe Ser Ser Thr Thr Glin Ala Ile Tyr Asp Telu Wall 85 90 95

Ala His Pro Glu Tyr Ile Thr Ile Telu Glu Glu Wall Glu Ser Wall 100 105 110

Pro Arg Asp Pro Asn Gly Asn Phe Thr Lys Asp Ser Thr Wall Ala Met 115 120 125

Asp Lys Telu Asp Ser Phe Lel Lys Glu Ser Glin Arg Phe Asn Ser Pro 130 135 1 4 0

Asp Telu Ser Met Ser Asn Lel Lys Asn Tyr Lys Teu Glu Ser Telu 145 15 O 155 160

Thr Gly His Ser Asn Teu Thr Thr Ile Ala Asp Met Lys Telu 1.65 170 175

Pro Asp Gly Thr Phe Wall Lys Gly Thr Teu Glu Ile Asn Thr 18O 185 19 O

Ser Ile His Lys Asp His Lys Telu Tyr Glu Asn Pro Glu Glin Phe 195 200

Asp Gly Telu Arg Phe His Lys Trp Lys Ala Pro Gly Glu 210 215 220

Arg Met Ser Ser Ser Gly Thr Asp Teu Ser Trp Gly Phe 225 230 235 240

Gly Arg His Ala Cys Pro Gly Tyr Telu Ser Ala Ile Asn Ile 245 250 255

Teu Ile Met Ala Glu Teu Teu Met Asn Tyr Asp Ile Telu Pro Asp 260 265 27 O

Gly Telu Ser Arg Pro Asn Ile Glu Phe Glu Wall Teu Ala Ser Telu 275 280 285

Asn Ala Ala Asn Ala 29 O

SEQ ID NO 36 LENGTH 510 TYPE PRT ORGANISM: Caenorhabditis elegans CAA9 1268 <400 SEQUENCE: 36

Met Ala Leu Lieu Ile Teu Ser Ser Telu Wall Ile Ser Ile Phe Thr Phe 1 5 10 15

Phe Ile Tyr Ile Ile Teu Ala Arg Arg Glu Arg Phe Telu Arg Glu 25 30

Ile Gly Telu Ser Gly Pro Glu Pro His Trp Phe Teu Gly Asn Telu 35 40 45

Glin Thr Ala Arg Lys Glu Telu Gly Tyr Asp Asp Ala Asn 50 55 60

Trp Phe Asn Teu His Glu Glin Gly Glu Thr Phe Gly Ile 70 75

Gly Ser Met Asn Ile Wall Ile Ser Asn Glu Asp Ile 90 95

Glu Wall Phe Asn Phe Ser Asn Phe Ser Asp Arg Ser Wall 100 105 110

Pro Ser Ile Tyr Ala Asn Glin Telu Thr Ala Ser Teu Telu Met Asn 115 120 125

Ser Tyr Ser Ser Trp Lys His Thr Arg Ser Ala Ile Ala Pro Ile 130 135 1 4 0

Phe Ser Thr Gly Lys Met Lys Ala Met Glin Glu Thr Ile Asn Ser Lys 145 15 O 155 160 US 7,033,807 B2 125 126

-continued

Wall Asp Telu Phe Teu Asp Ile Telu Glu Lys Ala Ser Ser Gly Glin 1.65 170 175

Trp Asp Ile Tyr Asp Asp Phe Glin Gly Teu Thr Teu Asp Wall Ile 18O 185 19 O

Gly Lys Cys Ala Phe Ala Ile Asp Ser Asn Glin Arg Asp Arg Asn 195 200

Asp Wall Phe His Pro Wall Thr Wall Lys Ile Thr Ile Asn Asn Phe 210 215 220

Thr Phe His Ser Ser Ser Pro Gly Thr Phe His Phe Telu Glu Ser 225 230 235 240

Thr Telu Glin Ile His Thr Thr Gly Arg Cys Arg Asn Ser Thr Cys Arg 245 250 255

Arg Thr Wall Lys Cys Wall Gly Phe Arg Glin Asp Ala Lys Phe 260 265 27 O

Ser Asp Tyr Glu Gly Gly Glu Gly Ser Asp Ser Wall Asp 275 280 285

Teu Telu Telu Teu Teu Asn Glu Asp Asp Lys Ser Pro Met 29 O 295

Thr Glin Glu Wall Ile Glu Asn Cys Phe Ala Phe Teu Telu Ala Gly 305 310 315 320

Thr Thr Ser Thr Ala Met Thr Tyr Ser Telu Telu Ser 325 330 335

Pro Asn Wall Glin Glin Lys Telu Tyr Glu Glu Ile Met Glu Ala 340 345 35 O

Glu Asn Gly Gly Teu Thr Tyr Asp Ser Ile His Asn Met Lys 355 360 365

Teu Asp Wall Tyr Lys Glu Thr Telu Phe Tyr Pro Pro His Phe 370 375

Ser Phe Ile Arg Teu Cys Glu Asp Ile Thr Ile Arg Gly Glin 385 390 395 400

Phe Pro Gly Ala Ile Wall Wall Cys Teu Pro His Thr Wall His 405 410 415

Arg Asn Pro Glu Asn Trp Asp Ser Pro Glu Glu Phe His Pro Glu Arg 420 425 43 O

Phe Glu Asn Trp Glu Glu Lys Ser Ser Ser Teu Trp Ile Pro Phe 435 4 40 4 45

Gly Wall Gly Pro Tyr Cys Wall Gly Met Arg Phe Ala Glu Met Glu 450 455 460

Phe Thr Thr Ile Wall Lys Telu Telu Asp Thr Phe Glu Telu Lys Glin 465 470 475 480

Phe Glu Gly Glu Ala Asp Teu Ile Pro Asp Asn Gly Wall Ile Met 485 490 495

Arg Pro Asn Asp Pro Wall Arg Telu His Telu Pro Arg Asn 5 OO 505 51O.

SEQ ID NO 37 LENGTH 691 TYPE PRT ORGANISM: yeast P450 reductase <400 SEQUENCE: 37 Met Pro Phe Gly Ile Asp Asn Thr Asp Phe Thr Val Leu Ala Gly Leu 1 5 10 15 Val Lieu Ala Val Lieu Lleu Tyr Val Lys Arg Asn. Ser Ile Lys Glu Lieu US 7,033,807 B2 127 128

-continued

25 30

Teu Met Ser Asp Asp Gly Ile Thr Ala Wall Ser Ser Gly Asn Arg 35 40 45

Asp Ile Ala Glin Wall Wall Thr Glu Asn Asn Asn Telu Wall Telu 50 55 60

Tyr Ala Ser Glin Thr Gly Thr Ala Glu Asp Tyr Ala Phe Ser 65 70 75

Glu Telu Wall Ala Lys Phe Asn Telu Asn Wall Met Ala Asp Wall 85 90 95

Glu Asn Asp Phe Glu Ser Telu Asn Asp Wall Pro Wall Ile Wall Ser 100 105 110

Ile Phe Ile Ser Thr Tyr Gly Glu Gly Asp Phe Pro Asp Gly Ala Wall 115 120 125

Asn Phe Glu Asp Phe Ile Cys Asn Ala Glu Ala Gly Ala Telu Ser Asn 130 135 1 4 0

Teu Arg Asn Met Phe Gly Telu Gly Asn Ser Thr Glu Phe Phe 145 15 O 155 160

Asn Gly Ala Ala Lys Lys Ala Glu Lys His Teu Ser Ala Ala Gly Ala 1.65 170 175

Ile Arg Telu Gly Lys Teu Gly Glu Ala Asp Asp Gly Ala Gly Thr Thr 18O 185 19 O

Asp Glu Asp Tyr Met Ala Trp Lys Asp Ser Ile Teu Glu Wall Telu 195 200

Asp Glu Telu His Teu Asp Glu Glin Glu Ala Phe Thr Ser Glin Phe 210 215 220

Glin Thr Wall Teu Asn Glu Ile Thr Asp Ser Met Ser Telu Gly Glu 225 230 235 240

Pro Ser Ala His Tyr Teu Pro Ser His Glin Teu Asn Arg Asn Ala Asp 245 250 255

Gly Ile Glin Telu Gly Pro Phe Telu Ser Glin Pro Ile Ala Pro 260 265 27 O

Ile Wall Lys Ser Glu Teu Phe Ser Ser Asn Asp Arg Asn Ile 275 280 285

His Ser Glu Phe Teu Ser Gly Ser Asn Ile Lys Ser Thr Gly 29 O 295

Asp His Telu Ala Wall Trp Pro Ser Asn Pro Teu Glu Wall Glu Glin 305 310 315 320

Phe Telu Ser Ile Phe Asn Teu Asp Pro Glu Thr Ile Phe Asp Telu 325 330 335

Pro Telu Asp Pro Thr Wall Lys Wall Pro Phe Pro Thr Pro Thr Thr Ile 340 345 35 O

Gly Ala Ala Ile Lys His Tyr Telu Glu Ile Thr Gly Pro Wall Ser Arg 355 360 365

Glin Telu Phe Ser Ser Teu Ile Glin Phe Ala Pro Asn Ala Asp Wall 370 375

Glu Telu Thr Teu Teu Ser Lys Asp Asp Glin Phe Ala Wall Glu 385 390 395 400

Ile Thr Ser Lys Tyr Phe Asn Ile Ala Asp Ala Teu Telu Ser 405 410 415

Asp Gly Ala Lys Trp Asp Asn Wall Pro Met Glin Phe Teu Wall Glu Ser 420 425 43 O

Wall Pro Glin Met Thr Pro Tyr Tyr Ser Ile Ser Ser Ser Ser Telu 435 4 40 4 45 US 7,033,807 B2 129 130

-continued

Ser Glu Lys Glin Thr Val His Val Thr Ser Ile Val Glu Asn Phe Pro 45 O 455 460

Asn Pro Glu Leu Pro Asp Ala Pro Pro Gly Val Gly Val Thr Thr Asn 465 470 475 480

Leu Lieu Arg Asn. Ile Glin Leu Ala Glin Asn. Asn. Wall Asn Ile Ala Glu 485 490 495

Thr Asn Lieu Pro Wal His Tyr Asp Lieu. Asn Gly Pro Arg Lys Telu Phe 5 OO 505

Ala Asn Tyr Lys Lieu Pro Wal His Val Arg Arg Ser Asn Phe Arg Telu 515 52O 525 Pro Ser Asn Pro Ser Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly 53 O 535 540

Val Ala Pro Phe Arg Gly Phe Ile Arg Glu Arg Val Ala Phe Telu Glu 545 550 555 560

Ser Glin Lys Lys Gly Gly Asn. Asn. Wal Ser Lieu Gly Lys His Ile Telu 565 570 575

Phe Tyr Gly Ser Arg Asn. Thir Asp Asp Phe Leu Tyr Glin Asp Glu Trp 58O 585 59 O

Pro Glu Tyr Ala Lys Lys Lieu. Asp Gly Ser Phe Glu Met Wall Wall Ala 595 600 605

His Ser Arg Leu Pro Asn Thr Lys Llys Val Tyr Val Glin Asp Telu 61 O 615 62O

Lys As p Tyr Glu Asp Glin Val Phe Glu Met Ile Asn. Asn Gly Ala Phe 625 630 635 640

Ile Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Lys Gly Wall Ser Thr 645 650 655

Ala Lieu Val Gly Ile Leu Ser Arg Gly Lys Ser Ile Thr Thr Asp Glu 660 665 67 O

Ala Thr Glu Lieu. Ile Lys Met Leu Lys Thr Ser Gly Arg Glin Glu 675 680 685 Asp Val Trp 69 O

SEQ ID NO 38 LENGTH 693 TYPE PRT ORGANISM: Aspergillus niger P450 reductase <400 SEQUENCE: 38

Met Al a Glin Lieu. Asp Thr Lieu. Asp Leu Val Val Lieu Ala Wall Telu Telu 1 5 10 15

Wall Gl y Ser Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp Ala Wall Ala 2O 25 30

Lys Thr Arg Met Pro Leu Pro Ala Pro Arg Met Asn Gly Ala Ala 35 40 45

Ala Gl y Lys Thr Arg Asn. Ile Ile Glu Lys Met Glu Glu Thr Gly 50 55 60

Asn Cys Val Ile Phe Tyr Gly Ser Glin Thr Gly. Thr Ala Glu Asp Tyr 65 70 75 8O

Ala Ser Arg Lieu Ala Lys Glu Gly Ser Glin Arg Phe Gly Telu Lys Thr 85 90 95

Met Val Ala Asp Leu Glu Glu Tyr Asp Tyr Glu Asn Lieu Asp Glin Phe 100 105 110

Pro Gl u Asp Llys Val Ala Phe Phe Val Leu Ala Thr Tyr Gly Glu Gly US 7,033,807 B2 131 132

-continued

115 120 125

Glu Pro Thr Asp Asn Ala Wall Glu Phe Tyr Glin Phe Phe Thr Gly Asp 130 135 1 4 0

Asp Wall Ala Phe Glu Ser Ala Ser Asp Glu Pro Telu Ser Lys 145 15 O 155 160

Teu Lys Wall Ala Phe Gly Telu Asn Asn Thr Glu His 1.65 170 175

Asn Ala Met Wall Arg Glin Wall Asp Ala Phe Glin Telu Gly Pro 18O 19 O

Glin Ile Gly Ser Glu Asp Asp Gly Ala Gly Thr Met 195 200

Glu Glu Asp Phe Teu Lys Pro Met Trp Ala Ala Telu Ser 210 215 220

Glu Ser Met Asp Teu Glu Glu Ala Wall Glu Pro Wall Phe 225 235 240

Wall Thr Glu Asn Glu Ser Telu Ser Pro Glu Asp Glu Thr Wall 245 250 255

Teu Gly Glu Pro Thr Gn. Ser His Telu Glin Gly Thr Pro Lys Gly Pro 260 265 27 O

Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ala Glu Ser Arg Glu 275 280 285

Teu Phe Thr Wall Lys Asn Cys Telu His Met Glu Ile Ser Ile 29 O 295

Ala Gly Ser Asn Teu Ser Tyr Glin Thr Gly Asp His Ile Ala Wall Trp 305 310 315 320

Pro Thr Asn Ala Gly Ala Glu Wall Asp Arg Phe Teu Glin Wall Phe Gly 325 330 335

Teu Glu Gly Lys Arg Asp Ser Wall Ile Asn Ile Gly Ile Asp Wall 340 345 35 O

Thr Ala Lys Wall Pro Ile Pro Thr Pro Thr Thr Asp Ala Ala Wall 355 360 365

Arg Tyr Tyr Met Glu Val Cys Ala Pro Wall Ser Arg Glin Phe Wall Ala 370 375

Thr Telu Ala Ala Phe Ala Pro Met Arg Lys Ala Arg Glin Arg Telu Cys 385 390 395 400

Wall Trp Wall Ala Glin Gly Lieu Phe Pro Arg Glu Gly His Glin Pro Met 405 410 415

Teu Glin His Ala Glin Ala Lieu Glin Ser Ile Thr Ser Pro Phe Ser 420 425 43 O

Ala Wall Pro Phe Ser Teu Teu Ile Glu Gly Ile Thr Lys Telu Glin Pro 435 4 40 4 45

Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Telu Wall Glin Asp Ile 450 455 460

Ser Ile Thr Ala Wall Wall Glu Ser Wall Arg Teu Pro Gly Ala Ser His 465 470 475 480

Met Wall Gly Wall Thir Thr Asn Tyr Telu Teu Ala Teu Glin 485 490 495

Glin Asn Gly Arg Ser Teu Ser Pro Ser Arg Teu Asp Telu Telu His 5 OO 505 51O.

His Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Wall Pro Wall His Wall 515 52O 525

Arg His Ser Asn Phe Lys Lieu Pro Ser Asp Pro Ser Arg Pro Ile Ile 530 535 540 US 7,033,807 B2 133 134

-continued

Met Wall Gly Pro Gly Thr Gly Wall Ala Pro Phe Arg Gly Phe Ile Glin 545 550 555 560

Glu Arg Ala Ala Teu Ala Ala Lys Gly Glu Wall Gly Pro Thr Wall 565 570 575

Teu Phe Phe Gly Cys Lys Ser Asp Glu Asp Phe Teu Tyr Asp 585 59 O

Glu Trp Lys Thr Tyr Glin Glin Telu Gly Asp Asn Teu Ile Ile 595 600 605

Thr Ala Phe Ser Glu Gly Pro Glin Lys Wall Tyr Wall Glin His Arg 610 615

Teu Arg Glu His Ser Glu Teu Wall Ser Asp Teu Teu Glin Ala 625 630 635 640

Thr Phe Tyr Wall Cys Gly Ala Ala Asn Met Ala Arg Glu Wall Asn 645 650 655

Teu Wall Telu Gly Glin Ile Ile Ala Ala Glin Arg Gly Teu Pro Ala Glu 660 665 67 O

Gly Glu Glu Met Wall Lys His Met Arg Arg Arg Gly Arg Glin 675 680 685

Glu Asp Wall Trp Ser 69 O.

SEQ ID NO 39 LENGTH 678 TYPE PRT ORGANISM mouse

<400 SEQUENCE: 39

Met Gly Asp Ser His Glu Thr Ser Ala Thr Wall Pro Glu Ala Wall 1 5 10 15

Ala Glu Glu Wall Ser Teu Phe Ser Thr Thr Asp Ile Wall Telu Phe Ser 2O 25 30

Teu Ile Wall Gly Wall Teu Thr Tyr Trp Phe Ile Phe Lys 35 40 45

Glu Glu Ile Pro Glu Phe Ser Lys Ile Glin Thr Thr Ala Pro Pro Wall 50 55 60

Lys Glu Ser Ser Phe Wall Glu Lys Met Lys Lys Thr Gly Asn Ile 65 70 75

Ile Wall Phe Gly Ser Glin Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95

Arg Telu Ser Lys Asp Ala His Tyr Gly Met Arg Gly Met Ser Ala 100 105 110

Asp Pro Glu Glu Tyr Teu Ala Asp Telu Ser Ser Teu Pro Glu Ile 115 120 125

Asp Lys Ser Telu Wall Wall Phe Cys Met Ala Thr Tyr Gly Gly Asp 130 135 1 4 0

Pro Thr Asp Asn Ala Glin Asp Phe Tyr Trp Teu Glin Thr Asp 145 15 O 155 160

Wall Asp Telu Thr Gly Wall Lys Phe Ala Wall Phe Gly Teu Asn 1.65 170 175

Thr Glu His Phe Asn Ala Met Gly Lys Wall Asp Arg Telu 18O 185

Glu Glin Telu Gly Ala Glin Ile Phe Glu Teu Gly Teu Asp Asp 195 200

Asp Gly Asn Telu Glu Glu Phe Ile Thr Trp Arg Glu Phe Trp US 7,033,807 B2 135 136

-continued

210 215 220

Pro Ala Wall Cys Glu Phe Phe Gly Wall Glu Ala Thr Gly Glu Glu Ser 225 230 235 240

Ser Ile Glin Tyr Glu Teu Wall Wall His Glu Asp Met Asp Thr Ala 245 250 255

Wall Thr Gly Glu Met Gly Arg Telu Ser Glu Asn Glin 260 265 27 O

Pro Pro Phe Ala Lys Asn Pro Phe Teu Ala Ala Wall Thr Thr 275 280 285

Asn Arg Lys Telu Asn Glin Gly Thr Glu Arg His Teu Met His Telu Glu 29 O 295

Teu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Wall 305 310 315 320

Ala Wall Pro Ala Asn Asp Ser Thr Telu Wall Asn Glin Ile Gly Glu 325 330 335

Ile Telu Gly Ala Asp Teu Asp Wall Ile Met Ser Teu Asn Asn Telu Asp 340 345 35 O

Glu Glu Ser Asn Lys His Pro Phe Pro Pro Thr Thr Arg 355 360 365

Thr Ala Telu Thr Tyr Tyr Teu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375

Wall Telu Tyr Glu Teu Ala Glin Tyr Ala Ser Glu Pro Ser Glu Glin Glu 385 390 395 400

His Telu His Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Telu 405 410 415

Teu Ser Trp Wall Wall Glu Ala Arg His Ile Teu Ala Ile Telu Glin 420 425 43 O

Asp Tyr Pro Ser Teu Pro Pro Ile Asp His Teu Cys Glu Telu Telu 435 4 40 4 45

Pro Arg Telu Glin Ala Tyr Tyr Ser Ile Ala Ser Ser Ser Wall 450 455 460

His Pro Asn Ser Wall His Ile Cys Ala Wall Ala Wall Glu Glu Ala 465 470 475 480

Ser Gly Wall Asn Lys Gly Wall Ala Thr Ser Trp Telu Arg Thr 485 490 495

Glu Pro Ala Gly Glu Asn Gly Arg Arg Ala Teu Wall Pro Met Phe 5 OO 505 51O.

Wall Arg Lys Ser Glin Phe Telu Pro Phe Pro Thr Thr Pro Wall 515 52O 525

Ile Met Wall Gly Pro Gly Thr Gly Wall Ala Pro Phe Met Gly Phe Ile 530 535 540

Glin Glu Ala Trp Teu Arg Glu Glin Gly Lys Glu Wall Gly Glu Thr 545 550 555 560

Teu Telu Tyr Gly Cys Ser Asp Glu Asp Telu Tyr Arg 565 570 575

Glu Glu Telu Ala Arg Phe His Lys Asp Gly Ala Teu Thr Glin Telu Asn 58O 585 59 O

Wall Ala Phe Ser Glu Glin Ala His Lys Wall Wall Glin His Telu 595 600 605

Teu Lys Arg Asp Lys Glu His Telu Trp Lys Teu Ile His Glu Gly Gly 610 615 62O

Ala His Ile Wall Cys Gly Ala Arg Asn Met Ala Asp Wall 625 630 635 640 US 7,033,807 B2 137 138

-continued

Gln Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly Pro Met Glu His 645 650 655 Thr Glin Ala Val Asp Tyr Val Lys Lys Lieu Met Thr Lys Gly Arg Tyr 660 665 67 O Ser Lieu. Asp Val Trp Ser 675

<210> SEQ ID NO 40 &2 11s LENGTH 17 &212> TYPE DNA <213> ORGANISM: baceriophage M13 reverse primer <400 SEQUENCE: 40 caggaalacag citatgac 17

EQ ID NO 41 ENGTH 2.0 YPE DNA RGANISM: bacteriophage T7 promoter primer <400 SEQUENCE: 41 taatacgact cactataggg 20

EQ ID NO 42 ENGTH 30 YPE DNA RGANISM: Aspergillus ochraceus Primer 11alphaOH-for <400 SEQUENCE: 42 gatc gaattic atgcc.cittct tcactgggct 30

<210> SEQ ID NO 43 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Aspergillus ochraceus Primer 11alphaOH-rev <400 SEQUENCE: 43 gatctotaga ttacacagtt aaacticgc.ca tatcg at 37

<210> SEQ ID NO 44 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: pFastBacI Primer Bacfwd <400 SEQUENCE: 44 citgtttitcgt aacagttittg 20

<210> SEQ ID NO 45 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: pFastBacI Primer PolyA <400 SEQUENCE: 45 ccitctacaaa totgg tatg 19

<210> SEQ ID NO 46 &2 11s LENGTH 17 &212> TYPE DNA <213> ORGANISM: Aspergillus ochraceus Primer 45624-for 1 <400 SEQUENCE: 46 gagatcaaga ttgccitt 17

US 7,033,807 B2 143 144

-continued

Gly Asn Telu Glu Glu Phe Ile Thr Trp Arg Glu Glin Phe Trp Pro 210 215 220

Ala Wall Glu His Phe Gly Wall Glu Ala Thr Gly Glu Glu Ser Ser 225 230 235 240

Ile Arg Glin Tyr Glu Teu Wall Wall His Thr Asp Ile Asp Ala Ala 245 250 255

Wall Met Gly Glu Met Gly Telu Lys Ser Glu Asn Glin 260 265 27 O

Pro Pro Phe Asp Ala Lys Asn Pro Phe Telu Ala Ala Wall Thr Thr Asn 275 280 285

Arg Lys Telu Asn Glin Gly Thr Glu Arg His Teu Met His Telu Glu Telu 29 O 295

Asp Ile Ser Asp Ser Lys Ile Tyr Glu Ser Gly Asp His Wall Ala 305 310 315 320

Wall Pro Ala Asn Asp Ser Ala Telu Wall Asn Glin Teu Gly Lys Ile 325 330 335

Teu Gly Ala Asp Teu Asp Wall Wall Met Ser Teu Asn Asn Telu Asp Glu 340 345 35 O

Glu Ser Asn Lys His Pro Phe Pro Pro Thr Ser Arg Thr 355 360 365

Ala Telu Thr Tyr Teu Asp Ile Thr Asn Pro Pro Arg Thr Asn Wall 370 375

Teu Glu Telu Ala Glin Ala Ser Glu Pro Ser Glu Glin Glu Telu 385 390 395 400

Teu Arg Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Telu Tyr Telu 405 410 415

Ser Trp Wall Wall Glu Ala Arg His Ile Teu Ala Ile Telu Glin Asp 420 425 43 O

Pro Ser Telu Pro Pro Ile Asp His Teu Glu Telu Telu Pro 435 4 40 4 45

Arg Telu Glin Ala Tyr Tyr Ser Ile Ala Ser Ser Ser Wall His 450 455 460

Pro Asn Ser Wall His Ile Ala Wall Wall Wall Glu Glu Thr Lys 465 470 475 480

Ala Gly Ile Asn Lys Gly Wall Ala Thr Asn Trp Teu Arg Ala 485 490 495

Glu Pro Wall Gly Glu Asn Gly Gly Arg Ala Teu Wall Pro Met Phe Wall 5 OO 505

Arg Ser Glin Teu Teu Pro Phe Ala Thr Thr Pro Wall Ile 515 525

Met Wall Gly Pro Gly Thr Gly Trp His Pro Phe Ile Gly Phe Ile Glin 530 535 540

Glu Arg Ala Trp Teu Arg Glin Glin Gly Lys Glu Wall Gly Glu Thr Telu 545 550 555 560

Teu Gly Cys Arg Arg Ser Asp Glu Asp Teu Arg Glu 565 570 575

Glu Telu Ala Glin Phe His Arg Asp Gly Ala Teu Thr Glin Telu Asn Wall 585 59 O

Ala Phe Ser Arg Glu Glin Ser His Lys Wall Wall Glin His Telu Telu 595 600 605

Glin Asp Arg Glu His Teu Trp Lys Telu Ile Glu Gly Gly Ala His 610 615

Ile Wall Cys Gly Ala Asn Met Ala Arg Asp Wall Glin Asn