USOO7544863B2
(12) United States Patent (10) Patent No.: US 7,544,863 B2 Karunanandaa et al. (45) Date of Patent: Jun. 9, 2009
(54) TRANSGENIC PLANTS CONTAINING WO WO97/032O2 1, 1997 ALTERED LEVELS OF STEROD WO WO97/34003 9, 1997 COMPOUNDS WO WO 98.45457 10, 1998 (75) Inventors: Balasulojini Karunanandaa, St. Louis, WO WO99,04622 2, 1999 MO (US); Martha Post-Beittenmiller, WO WOOOf 61771 10, 2000 St. Louis, MO (US); Mylavarapu WO WOO1/31027 3, 2001 Venkatramesh, St. Louis, MO (US); Ganesh M. Kishore, St. Louis, MO (US); Gregory M. Thorne, St. Louis, MO (US); John R. LeDeaux, St. Louis, OTHER PUBLICATIONS MO (US) Chappell J. et al. Plant Physiology; 1995, vol. 109, pp. 1337-1343.* (73) Assignee: Monsanto Technology LLC, St. Louis, Schaeffer A. et al. Lipids, 2000; vol. 35, No. 3, pp. 263-269.* MO (US) Bach and Benveniste, “Cloning of cDNAS or genes encoding enzymes of sterol biosynthesis from plants and other eukaryotes: (*) Notice: Subject to any disclaimer, the term of this heterologous expression and complementation analysis of mutations patent is extended or adjusted under 35 for functional characterization.” Progress in Lipid Research, 36(2/3): U.S.C. 154(b) by 529 days. 197-226, 1997. Bak et al., “Cloning and expression in Escherichia coli of the (21) Appl. No.: 10/862,907 obtusifoliol 14-alpha-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14-alpha-demethylases (22) Filed: Jun. 7, 2004 (CYP51) from fungi and mammals.” Plant Journal, 11(2):191-201, 1997. (65) Prior Publication Data Bak et al., “Cloning and expression in Escherichia coli of the obtusifoliol 14-alpha-demethylase of Sorghum bicolor (L.) Moench, US 2005/OO86713 A1 Apr. 21, 2005 a cytochrome E. E. to the E. ERS (CYP51) from fungi and mammals.” EMBL Onlinel, Database Related U.S. Application Data Accession No. U74319, abstract, 1996. (62) Division of application No. 09/885,723, filed on Jun. Bard et al., “Genetic and biochemical aspects of yeast sterol regula 20, 2001, now Pat. No. 6,822,142. tion involving 3-hydroxy-3-methylglutaryl coenzyme A reductase.” J. General Microbiology, 125:415-420, 1981. (60) Provisional application No. 60/260,114, filed on Jan. Basson et al., “Structural and functional conservation between yeast 5, 2001. and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.” Molecular Cellular (51) Int. Cl. Biology, 8(9):3797-3808, 1998. CI2N 15/29 (2006.01) Broun et al., “Catalytic plasticity of fatty acid modification enzymes CI2N 15/52 (2006.01) underlying chemical diversity of plant lipids.” Science, 282:1315 CI2N 5/82 (2006.01) 1317, 1998. AOIH 5/00 (2006.01) AOIH 5/10 (2006.01) (Continued) (52) U.S. Cl...... 800/306; 800/312 800/314: Primary Examiner Russell Kallis 800/317; 800/320.1536/23.1:536/23.2: (74) Attorney, Agent, or Firm—Chunping Li, Esq.; 536/23.6:435/320.1; 435/419 Sonnenschein Nath & Rosenthal LLP (58) Field of Classification Search ...... 536/23.1, 536/23.2, 23.5, 23.6, 23.7; 435/419,320.1; (57) ABSTRACT 800/287, 288, 298,306, 312, 314, 317.2, 800/317.3, 320.1, 322 See application file for complete search history. Disclosed are constructs comprising sequences encoding 3-hydroxy-3 methylglutaryl-Coenzyme A reductase and at (56) References Cited least one other Sterol synthesis pathway enzyme. Also dis U.S. PATENT DOCUMENTS closed are methods for using Such constructs to alter sterol 5.306,862 A 4, 1994 Chappelletal production and content in cells, plants, seeds and storage 5,349,126- WW A 9, 1994 NEHa etca. al. organs of plants. Also provided are oils and compositions 5,365,017 A 1 1/1994 Chappell et al. containing altered sterol levels produced by use of the dis 5,460,949 A 10/1995 Saunders et al. closed constructs. Novel nucleotide sequences useful in the 5,480,805 A 1, 1996 Wolfetal. alteration of sterol production are also provided. Also pro 5,589,619 A 12/1996 Chappell et al. vided are cells, plants, seeds and storage organs of plants 6,153,815 A 1 1/2000 Covello ...... 800,306 comprising sequences encoding 3-hydroxy-3 methylglutaryl Coenzyme A reductase, at least one other sterol synthesis FOREIGN PATENT DOCUMENTS pathway enzyme and at least one tocopherol synthesis EP O480730 4f1992 enzyme. JP O9121863 5, 1997 WO WO93,021.87 2, 1993 25 Claims, 78 Drawing Sheets US 7,544.863 B2 Page 2
OTHER PUBLICATIONS Gonzalez et al., Abstract of poster at Third Terpnet Meeting of the European Network on Plant Isoprenoids, May 29-30, Poiters, France, Cabello-Hurtado et al., “Cloning and functional expression in yeast 1997. ofacDNA coding for an obtusifoliol 14-alpha-demethylase (CYP51) Jenkins et al., “Plant sterols, health claims and strategies to reduce in wheat.” Biochemical and Biophysical Research Communications, cardiovascular and strategies to reduce cardiovascular disease risk.” 230(2):381-385, 1997. J. of the American College of Nutrition, 18:559-562, 1999. Cabello-Hurtado et al., “Cloning and functional expression in yeast Nakamura et al., “A large scale analysis if cDNA in Aradopsis ofacDNA coding for an obtusifoliol 14-alpha-demethylase (CYP51) thalian: generation of 12,028 non-redundant expressed sequence tags in wheat, EMBL Onlinel, Database Accession No.Y09291, abstract, from normalized size-selected cDNA libraries.” EMBL Onlinel, 1996. Database Accession No. AV440215, 2000. Chin et al., “Nucleotide sequence of 3-hydroxy-3-methyl-glutaryl Nguyen, “The cholesterol-lowering action of plant stanol esters. J. coenzyme A reductase, a glycoprotein of endloplasmic reticulum.' of Nutrition, 129:2109-2112, 1999. Nature, 308 (5960):613-617, 1984. Register et al., “Structure and function of selectable and non-select Colebatch et al., “Lotus faponicus root nodule ESTs: tools for func able transgenes in maize after introduction by particle bombard tional genomics.” EMBL Onlinel, Database Accession No. ment. Plant Mol. Biol., 25:951-961, 1994. AW719774, abstract, 2000. Schafer et al., “An example of intron junctional sliding in the gene Covello, “An example of intronjunctional sliding in the gene families families encoding Sqalene monooxygenase homologues in encoding squalene monoxygenase homologues in Arabidopsis Arabidopsis thaliana and Brassica napus. Plant Molecular Biology, 39(4):721-728, 1999. thaliana and Brassica napus.” EMBL Onlinel, Database Accession Schaller et al., “Overexpression of an arabidopsis cDNA encoding a No. AJO05930, 1998. sterol-C24(1)-methyltransferase in tobacco modifies the ration of Dale et al., “Bacterial expression of the catalytic domain of 24-methyl cholesterol to sitosterol and is associated with growth 3-hydroxy-3-methylglutaryl-CoA reductase (isoform HMGR1) reduction.” Plant Physiol., 118:461–469, 1998. from arabidopsis thaliana, and its inactivation by phosphorylation at Schaller et al., “Expression of the hevea brasiliensis (H.B.K.) mill. Ser577 by brassica oleracea 3-hydroxy-3-methylglutaryl-CoA Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 in reductase kinase. Eur: J. Biochem.; 233:506-513, 1995. tobacco results in sterol overproduction.” Plant Physiol. 109:761 Deavarenne, "Regulation of squalene synthase, a key enzyme of 770, 1995. sterol biosynthesis, in tobacco.” Plant Physiology, 129:1095-1106, Shintani et al., “Elevating the vitamin E content of plants through 2002. metabolic engineering.” Science, 282(5396): 2098-2100, 1998. Downing et al., “The isolation of two mutants of Saccharomyces Tada and Shiroishi, “Mechanism of photoregulated cartogenesis in cerevisiae which demonstrate increased activity of 3-hydroxy-3- Rhodoturul minuta v. photoinduction of 3-hydroxy-3-methyl methylglutaryl coenzyme a reductase. Biochemical and Biophysical glutaryl coenzyme A reductase.” Plant and Cell Physiol. 23(4):615 Research Communications, 94(3):974-979, 1980. 621, 1982. Fourgoux-Nicol et al., "Isolation of rapeseed genes expressed early Van der Hoeven et al., “Generation of ESTs retrieved from tomato and specifically during development of the male gametophyte. Plant radicule tissue.” EMBL Onlinel, Database Accession No. Molecular Biology, 40:857-872, 1999. AW625933, abstract, 2000. Gill et al., “Membrane-bound domain of HMG CoA reductase is Yoder et al., “Transformation systems for generating marker-free required for sterol-enhanced degradation of the enzyme. Cell, transgenic plants.” Bio/Technology, 12:263-267, 1994. 41:249-258, 1985. * cited by examiner U.S. Patent Jun. 9, 2009 Sheet 1 of 78 US 7,544,863 B2
squalene
epoxidase Sterol methyl transferase I -- HO 24-methylene cycloartenol
cycloartenol sterol C4 dennethylase
obtusifoliol obtusifoliol stigmasta-7-enol Cl4c-dennethylase HO HO c5. sterol methy sterol C5- transferase II desaturase
HO / sitosterol
N
HO stigmasterol HO sitostanol
F.G. 1 U.S. Patent Jun. 9, 2009 Sheet 2 of 78 US 7,544,863 B2
(IC)
Zº?IH IJn](150)ANH
O O
U.S. Patent Jun. 9, 2009 Sheet 3 Of 78 US 7,544,863 B2
Kpnl 8744 Sac 8738 EcoRI 8728 Stu 8724 Xbal 8716 Clai 8711 H 7759 Bgll 8704 Ban.H 77 Sac. 8701 XbalSal 77537747 xholKpni 86958700 famil 976,S Pst 7745 Nott 669 Hind 7729 Drai 7692 Pvt 1045 Drai 7615 - Pst 1235 Pyu 288 PVU 736 Pst 7236 Nicol 1614
Pvu 1882 XhoI 2146 Pyut 676O NOS 3' Cal 263 pMON29920 F-Pvu 2167 8746 bp Left Border Pst 6297
PVU 2669 Oral 5937 Ora 2793 Oral 5918
Pvt 3336
Pvu 4752
Construct pMON29920
FG. 3 U.S. Patent Jun. 9, 2009 Sheet 4 of 78 US 7,544,863 B2
Kpnl 12737 Sac 12731 Barnh 12739 EcoRI 12721 Pst 657 Xbal 12472 Not 669 Oral 122OO Xhol 678 Oral 12087 Pvui 1045 EcoRV 11923 \ Pst 1235 Hind 1768 ad-lifting Pvui 288 NCO 1614 Pvul 11075 PVu 1882 Sac 1052 J Xhol 2146 & Clal 2163 Q1-Pvul 2167 Y pi 99.78 l PVU 2669 Vul 9973 Dra 2793
Bani 9728 pMON43800
12739 bp Pvul 3336 Cla. 9169 Ban 9152 Cla. 909 { ECOR 8728 69 Stu 8724 Right Border 6 Xbai 8716 59 l s7 PVU 4752 E. SNCC Int Kpni 8695 Dral 5918 BamH 7759. Dra 5.937 Xbal 7753 Pst 6297 Sal 7747 Pst 7745 Hind 7729 Dra 7692 Oral 7615 Pyu 736 Pst 7236 Pyut 676O Construct pMON43800 FIG. 4 U.S. Patent Jun. 9, 2009 Sheet 5 Of 78 US 7,544,863 B2
Hind 7204 EcoRV 8 EcoR 12 Pst 321 Bgill 321 Pst 538 Pvul 591 Drai 7167 Nool 917 Oral 7O90 Bam H. 1326 Pvul 6836 Pvul 1336
Pst 671 Cal 1585 Pvu 1589 EcoR 593 Pst 1603 Sma? 16O7 BamH 1611 Xbal 1623 Not 1630 Cal 1638 Ea pMON23616 Pvu 1642 7204 bp
Pvull 2144 Oral 2268
Construct pMON23616
F.G. 5 U.S. Patent Jun. 9, 2009 Sheet 6 of 78 US 7,544,863 B2
XhoI 2478
pMON43818 12547 bp
Sma 6253
Construct pMON43818
F.G. 6 U.S. Patent Jun. 9, 2009 Sheet 7 Of 78 US 7,544,863 B2
Soy Alpha' Beta Conglycinin
pMON43052 10605 bp Arabidopsis HMGR catalytic domain
Ori-V left Border pot.polyA signal
Not 5031 Construct pMON43052
F.G. 7 U.S. Patent Jun. 9, 2009 Sheet 8 of 78 US 7,544,863 B2
Not 7042 Small 7007
pMON51850 7049 bp
Construct pMON51850
FIG. 8 U.S. Patent Jun. 9, 2009 Sheet 9 Of 78 US 7,544,863 B2
Not 0282 Pst 10270 Barn- 9604. Kpnl 9602 Sac 9596 ECOR 9586 Sac 9584 Hind 10289 EcoRV 8 Kpni 9578 ECOR 12 SmallAs 9572g568 8gilt 321 EcoR 9558 Ps EcoRV 93.35 St. 538 Pvull 9075 Pyut 591 Pvu 9022 Dra 895.3 EcoRV 8870 Sac 8695
Cal 8064 Cal 8025 Saci 7873 Kpnl 7867 Soy Alpha' Beta Conglycinin pMON43057 10289 bp
Stu 73O7 Dra 705 Ban 7013 Da 6976 Ora 6899 Pyu 6645 Pst 652O PVul 4036
Pst 558 Dra 522 Oral 52O2
Construct pMON43057
FIG. 9 U.S. Patent Jun. 9, 2009 Sheet 10 Of 78 US 7,544,863 B2
Cal 12867 Pvu 126 Pst 12592 Hind 2882 ECOR 12582 ECORV 8 Pst 2262 ECOR 2 Bgll 11846 Pst 321 Sac 1694 DS 3' Bgll 321 Bgll 11556 in r-KS Pst 538 Sat 11428 PVU 591 Bgill 11313 NCO 917 ECOR 1116 Pvu 1.185 Sac 1154. Kpnl 11148 as C Stu O588 A. Oral 10386 C Soy Alpha' Beta Conglycinin ori-V Pst 1027O linker-KS
pMON43058 BamHE 9604 12882 bp Kpni 9602 Sac 9596 ECOR 9586 Sac 9584 WA Arabidopsis HMGR catalytic domain Kpnl 9578 Soy Alpha' Beta Conglycinin Pvut 4036 W.KY Sna 9572 Q PSt 9568 KY ECOR 9558 EcoRV 93.35 Oral 52O2 t E. Nort Draf 522 Oral 8953 Pst 5581 EcoRV 887O Pvul 6O44 Sac 8695 Clal 8064 Cal 8025 Saci 7873 Kpni 7867 Stu 73O7 Oral 705 Ban 7013 Ora 6976 Drai 6899 Pvu 6645 Pst 652O Construct pMON43058 F.G. 10 U.S. Patent Jun. 9, 2009 Sheet 11 of 78 US 7,544.863 B2
8000 24-Methylene Cycloartanol GCycloartenol OUnknown 3 6000 SS ga S553 SA 2. Stigmasta-7-enol
s Sooo. lso fucosterol Sit ". . . . - O Si to stano s E. F. . . . . E - o 4000 . . ili - f s Sitosterol g - Obtusifoliol 3000 s Unknown 2
200 Stigmasterol OCampesteroi looo : . . . . . - . Unknown Squalene O ofSS EY ent number
FIG. 11 U.S. Patent Jun. 9, 2009 Sheet 12 of 78 US 7,544,863 B2
Cycloartenol uglg OUnknown 3 v g/g Stigma sta 7 enouglg sofucosterol uglg O Sitos tanol ug/g Sitosteroiugig
Obtusifoliol uglg
Unknown 2 uglg Stigmasterol ug/g Campesterol uglg unknown ugg Squalene ug/g
Ewert Nuner
FIG. 12 U.S. Patent Jun. 9, 2009 Sheet 13 Of 78 US 7,544,863 B2
Kpnl 10192 Sac O186 EcoRI 1 of 76. Pvul 1 OO86 PVU OO33 Oral 9964 EcoRV 988 Bann H. O. 94. Sac 97O6 Pst 666 Cal 9075 Not 678 Call 9036 Xho 687 Pvu 8848 Pvu 1056 PWu 8800 Pst 1246 Cla 8800 Pvu .299 Stu 8414 NCO 8395 Nico 1625 Bglli 8389 PVU 1893 EcoRV 8274 XhoI 2157 Cla. 2174 Pst 7750 Pvt 21.78 Hind 774O Dra 7703 Dra 7626 pMON53733 10194 bp Pvu 268O Pvt 7372 Dra 2804 Pst 7247
PWU 3347
C ori-322 rop Pst 6308 SSQ
Da 5948 Nith Draf 5929 PvE 4763 Construct pMON53733
F.G. 13 U.S. Patent Jun. 9, 2009 Sheet 14 of 78 US 7,544,863 B2
Kpnl 9826 Saci 982O EcoR 9810 Pvt 972O Pvu 9667 Dra 9598 EcoRV 9515 Saci 934O Barnh 9828 Cal 3709 Pst 666 Cla. 8670 Not 678 Pvu 84.82 XhoI 687 Pvu 8434 Pvul 1056 Clal 8434 Nool 8395 Pst 1246 Bglli 8389 Pvul 1299 EcoRV 8274 NCO 1625 Pst 7750 Pvu 1893 Hind 774O XhoI 257 Oral 7703 Cla. 274 Ora 7626 2- Pvu 2178 Pvul 7372 pMON53734 Pst 7247 9828bp
PWu 268O Oral 2804
Construct pMON53734
F.G. 14 U.S. Patent Jun. 9, 2009 Sheet 15 Of 78 US 7,544,863 B2
Kpnl 9698 Saci 9692 EcoRI 9682 Pvu 9594 PVul 9541 Barnh 97OO Oral 9472 P st 666 EcoRV 93.89 Not 678 Sac 9214 Cal 8583 Pvul 1056 NCO 8395 EcoRV 8274 Pvul 1299 Pst 7750 NCol 1625 Hind 774O Pvul 1893 Dra 7703 Xho 2157 Ora 7626 Cal 2174 st-Pvu 2178 t 2 pMON53735 St 9700 bp
Pvu 268O Dra 2804. Pyu 677
Pst 63O8 PVU 3347
Da 5948 Da 5929 Pvul 4763 Construct pMON53735
F.G. 15 U.S. Patent Jun. 9, 2009 Sheet 16 of 78 US 7,544,863 B2
Kpnl 101.41 Sac 101 35 EcoRI 1.0125 Pvt 10053 Sac 1 OO3O Barnhi 101.43 Bar H 97.79 Pst 666 Pst 9.429 Not 678 PVU 94.24 No Barn HE 93 Clal 8752 Pvu 1056 Barnh 8735 Pst 246 Cla. 8674 Pvt 1299 Nico 8.395 NCO 1625 Bgll 8389 Pvu 1893 ECORV 8274 XhoI 257 Cla. 2174 Pst 7750 PVU 278 Hind 774O Ora 77O3 N. Dra 7626 pMON53736 101.43 bp Pvu 268O Pyu 7372 Oral 28O4. Pst 7247
Pvu. 6771 Pvu 3347
Pst 63O8 Dra. 5948 Oral 5929 Pvull 4763 Construct pMON53736
F.G. 16 U.S. Patent Jun. 9, 2009 Sheet 17 Of 78 US 7,544,863 B2
Kpnl 98.44 Saci 9838 EcoR 98.28 PVul 9756 Sac 9733 Bar 94-82 BamH 9846 Pst 9132 Pst 666 Pyut 927 Ban 904 Not 678 Clal 8455 Pvt. O56 Ban 8438 Pst 246 Nicol 8395 Pvul 1299 Bgll 8389 ECORV 8274 NCO 1625 Pst 775O Pyut 1893 Hind 774O XhoI 257 Da 77O3 Cla. 274 Ora 7626 PV 21.78 pMON53737 Pyut 7372 9846 bp Pst 7247 PVUI 268O
Ora 2804
Pst 63O8
Oral S948 Drat 5929
Construct pMON53737
F.G. 17 U.S. Patent Jun. 9, 2009 Sheet 18 Of 78 US 7,544,863 B2
Kpnd 9844 Sach 9838 ECOR 9828 Pvull 9756 Saci 9733 BamHt 9482 Pst 9132 Pst 666 PWU 927 Not 678 Barnhl 9014 Xho. 687 Cal 8455 Ban.H. 84.38 PV U 1056 Pst 1246 NCO 8395 PWU 299 Bgll 8389 ECORV 8274 NCO 1625 Pst 7750 Pvul 1893 Hind 774O XhoI 257 Da 7703 Cal 2174 Da 7626 Pvul 21.78
Pst 7247 9846 bp PVU 268O Ora 2804 Pyut 6771
Pvt 3347 Pst 63O8
Oral 5948 Oral 5929
PVU 4763
Construct pMON53738
FIG. 18 U.S. Patent Jun. 9, 2009 Sheet 19 Of 78 US 7,544,863 B2
Kpni 98.44 Sac 98.38 ECOR 9828 Pvu 9756 Sac 9733 Barm 94.82 Barn 9846 Pst 9132 PSt 666 PVU 927 Not 678 Ban 9014 Xhol 687 Clal 8455 PVU 1056 Barnh 84.38 Pst 1246 NCO 8395 Bgll 8389 PvulU 1299 EcoRV 8274 NCO 1625 Pst 775O Pvu 893 Hind 774.O XhoI 257 Oral 77O3 Clal 2174 Drai 7626 Pvu 21.78 PVUE 7372 pMON53739 Pst 7247 9846 bp
PWu 268O Dra 2804
Pyut 677
Pvu 3347 Pst 63O8 Oral 5948 Dra. 5929
PVU 4763 Construct pMON53739
FIG. 19 U.S. Patent Jun. 9, 2009 Sheet 20 Of 78 US 7,544,863 B2
Kpnl 9685 Sac 96.79 EcoR 9669 Pyu 95.97 Sac 95.74. Barnh 93.23 Barnh 96.87 Pst 8973 Pst 666 Pvull 8968 Not 678 Barnh 8855 Pvul 1056 Nico 8395 Pst 1246 Bgill 8389 Pvul 1299 EcoRV 8274 Pst 7750 Nco 1625 Hind 7740 Pvul 1893 Oral 7703 Xho 2157 Oral 7626 A Cal 2174 a-Pvu 21.78 PvulA. E.737 pMON53740 left Border 96.87 bp
Pvu 268O Pyu 677. Dra 2804
Pst 6308 Pyu 3347 Dral 5948 Oral 5929 Pyu 4763 Construct pMON53740
F.G. 20
U.S. Patent Jun. 9, 2009 Sheet 24 of 78 US 7,544,863 B2
98/99NOWNdx /9/99NOWd* 8€/CGNOWd• 6C/CGNOWd+ 0?7/9GNOWd- |OJ?u00-.
S?ueldo?uefisuelLu?SIÐAÐTIoue,seduueogouosqueduuoºo
008 00/ 009 00|| 0 (fffin),ueuod ouese ULeC U.S. Patent Jun. 9, 2009 Sheet 25 of 78 US 7,544.863 B2
S LO CO. N. CO. O. C. s? So So So So Sp Sp N Nc? N.O N.C N.is N.to N. N. N. O co O O. O. O. O. Y Z Z Z Z Z Z Z Z H O O. O. O. O. O. O. O. Z is is is is is is is is O o, os o do o os o ( ) 0 : X % B --
C O O C O O O O O O O O O O O O O O C O C O N- C O r c CN -- (6/6n) pue Loo ole SOS U.S. Patent Jun. 9, 2009 Sheet 26 of 78 US 7,544,863 B2 £8/19 9€/19 6€/9 9
NOWd•| NOWNd 9€/89NOWdx NOWdx 8€/9GNOWd• NOWId+ 0?7/€GNOWd- | TOHL?OO-
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?
O C 009Z O ()007 0090 000€ 000Z Y (fyf5n), ue Joo ou2SOS U.S. Patent Jun. 9, 2009 Sheet 27 of 78 US 7.544,863 B2
499×]©WH) SOIH 999XJSOWHì
00! ·|09 ·|09 paeS fu Jed DJeS 5u U.S. Patent US 7,544,863 B2
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·9 M. Aup leg 6u Jed oues fif U.S. Patent Jun. 9, 2009 Sheet 29 Of 78 US 7,544,863 B2
Dral 57 Hindi 808 Dral 815 Bglli 899 Xbal 7479 Bgill 959 Hind 995 Kpnl 1126 Ora 1 178 Pst 187
O Oral 61.59
Pvul 5952 pMON43842 ACTF-Pvul/Pvul 19231952
7911 bp T-Sc.CycaC2 E-Kpni 2080 Lacz-alpha RXho 2334 Ora 54.67 Sa 23-0 Dra 5448 Cla. 2350 obtus. C. EcoRVHind 23552363 EcoR 2367 so Sac 238O Pvul 4513 Shi Hitti X Ellis Sac. 4295 Bgil 2783 Hindll as 'Hindi 2807 Hind 3551 'BamH 3608 Bgll 3785 EcoRI 3795 Pst 3805 Small 38Og Barnh 3813 Xball 3825 Construct pMON43842
FG. 29 U.S. Patent Jun. 9, 2009 Sheet 30 Of 78 US 7,544,863 B2
Pst 399 Xbai 8008 EcoRV 605
A. Oral 6688 t Pvu 1851 Pvu 648 E. -Pyull 1880 AmpR pMON43843 CYC Kpnl 2008
8439 bp AC Xhoi 2265 lacz O d
Draf 5996 Sal 227 Oral 5977 W. A Noti 2282 W 97 Oral 2502 & Squalene epoxidase 1 (ATA506263) XY Oral 2518 & & YEcoRV 2918
QS P-GPD SS Pvu SO42 & fS s x’ KpniOra 318O388 Sac 4824 Sittitt Small 4516 Hind 3768 Pst 4465 Sall 4O94 Small 4101 EcoRI 4104 Pst 414 Small 4118 Ban HF 422 Xbal 4134 Construct py(ON438-43
FG. 30 U.S. Patent Jun. 9, 2009 Sheet 31 Of 78 US 7,544,863 B2
Pst 399
Xbal 827 EcoRV 605
Oral 6897 Pv 1851 Pvu 188O Pvu 6690 Kpnl 2008
Xhof 2265 Oral 6205 NotSal 227123: Drai 618.6 Dra 2396
Hird: 289
EcoRV 3 OO
Pvu 525 Kpni 3370 Sac SO33 NCO 3487 Sna 4725 Salt 4303 Pst 4674 Srna 4310 Xbal 4343 EcoR 433 Bar H 4331 Pst 4323 Small 4327 Consuruct pMON438-44
FG. 31 U.S. Patent Jun. 9, 2009 Sheet 32 of 78 US 7,544,863 B2
Plurality: 5. OO Threshold: 4 AveWeight 1.00 AveMatch 2.91. AvMismatch -2 loo SO
a e s
a p r u p a s a
d a HMGRclustaiwP - b. O O 0 (sulfolobus ...... HMGRclustalW yeast2). MSLPLKTIVE LVKPFACTAR FSARYPIHVI wVAVLLSAAA YLSVTQSYLN HMGRclustalW yeast MP9LFKGLKQ MAK2IAYVSR FSAKRPIHII LFSLIISASA YLSVIQYYFN HMGRclus talW (phycomyces ...... HMGR.clustalW ( fusarium ...... o is o s to a
HMGRclustalW ( rice ......
HMGRclustalw soybean) ------......
w up o
& a 0 is e e
a b x . . . . .
b u a a s is a
8 w w up a a - a s
- - up o r s s FIG. 32A U.S. Patent Jun. 9, 2009 Sheet 33 of 78 US 7,544,863 B2
0 X 4.
HMGRclustalW ( rat ...... HMGRclustalW { rabbit} ------...... HMGRclustalW ( human ------...... HMGRclustalW ( mouse ...... BMGRclustalW ( xenopus ...... HMGRclustalW (sea urchin} ......
HMGRclustalW ( cockroach) ...... to e ------e a o u e o a s - ......
d is .
e up o e o a s as a
w e s a e
opp de v - U.S. Patent Jun. 9, 2009 Sheet 34 of 78 US 7,544,863 B2
OO HMGRclustalW (methanobach ......
s s a - a s r. s. s.
d a 0
P - - 4 · · · a
is & X • d HMGRclustalW yeast2: EWKLDSN.OY STYLSIKPDE LFEKCTHY: R SPvSDTWKLL SSKEAADYT HMGRclustalW yeastl) GWQLDSNSVF ETAPNKDSNT LFOECSHYYr DSSLDG visi AEEASEPA HMGRclustalW (phycomyces ...... HMGRclustalW fusarium) ...... MCH EGCOGCH pCO CCQWVSNAWS HMGRclustalW ( candida ...... MF GASANQHWA WDDLSKWPWD HMGRclustalW (dictyoste2} ...... HMGRclustalW wheatl ...... HMGRclustalW rice ...... HMGRclustal ( corr} ......
HMGRclustalW soybean) ......
HMGRclustalW (wcodtobacc} ...... a > 0 t is is us is us is s - us is & s as & HMGRclustalw Potato ...... 8 w w w HMGRclustalW radish...... Y -
BMGRclustalW (rubbertre2} ...... as a se -- ......
HMGRclustalW (rubbertrel} ...... 0 up to 8 d e a s is a 8 so t e is HMGRclustalW- - - - - (camptothec) ...... HMGRclustalW (arabadops2} ...... HMGRclustalW (chineseham) ...... MLSRLFRMH GFWASPWE HMGRclustalW (chineseha2 ...... MLSRLFRMHE U.S. Patent Jun. 9, 2009 Sheet 35 of 78 US 7,544,863 B2
GLFWASH9WE HMGRclustalW (syrianhamst} ...... MLSRLFRMH GLFWASHPWE HMGRclustalW rat ...... MLSRLFRMH GWASPWE HMGRclustalW rabbit} ...... MLSRFRMH GFWASPWS HMGRclustalW human) ...... 9 - p - P - vr e e i s m o p a e s s ... MLSRLFRM GFWASHPWE RMGRclus talW mouse ...... w 8 - w as ......
HMGRclustalW ( xenopus) ...... on to a so e s so e us us o a on a s a MSRFRME GQFVASHPWE HMGRclustalW (sea urchin} ...... to a s o e o a to o a u e s is s is a ... MLSRLFLAC GRSCSSHPWE HMGRclustalW cockroach) ...... MVGRFRAH GOFOASHPWE HMGRclustalW (drosophila) ...... MGPLFRA QFCASHPWE HMGRclustal W{dictyostel ......
HMGRclustalW (schistosom} ...... 8 - - - w w w - d e to v at - -- w a a e o a p r or w s- a w HMGRclustalW (archaeoglio) ...... HMGRclustalW (pseudemoras) ......
Consensus ------MESRFRNR GVASHPWE
FIG. 32D U.S. Patent Jun. 9, 2009 Sheet 36 of 78 US 7,544,863 B2
O SO HMGRclustalW (methanobac) ...... HMGRclustalw{methanococ) ...... HMGRclustalW (halobacter ...... a a P · F a a s HMGRclustalW (sulfolobus) ...... HMGR.clius talW yeast2} PFYYLSTS FORSKCNSTTL, PSLDDVISV DHTRYLLSEE PKPTELVSE HMGRclustalW { yeasti PHHYYLLNN FNSPNETDSI PELANTV Sex DNTKYILCED SWSKESST HMGRclustalW (phycomyces ...... p q a a s is a
- is - e s is HMGRclustalW fusarium. EFLDLLKNAE TLDIVIMLLG YIAMHLTFVs LFLSMRKMGs KWGCTLF HMGR.clius talW { candida} VDHYNVVPFQ FRRAGEYKEP VLSGIVELDE V KFVySQSDA AEQWOQLTAE HMGRclustalW (dicta oste2} ......
w
At - as - d -
HMGRclustalw tomato ...... 4 b e 8 a. s. s is is a w s as s- s is a s HMGRclustalW (Woodtobacc} ...... HMGRclustalW ( Potato ...... fit to th a to es e s as a s
4& . . . . is s is .
8 s - as x s
w - - Y - P - - - - HMGRclustalW (rubbertre2} ...... a ed - e s - HMGRclustalW rubbertrel ...... as s as as - to 9 - 8 e o e s is a s is
us - e s p + · · HMGRclustalW camptothec) ...... a - 8 & & is s s a s is s a a > b BMGRclustalWis a (arabadops2} ...... HMGRclustalwchineseham VIVGTVT. . . TICMMSMN. . MFTGNNK...... k - 4 - s - d. F.G. 32E U.S. Patent Jun. 9, 2009 Sheet 37 Of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} VIVGTVT. L., TICMMSMN. . MFIGNNK...... HMGR.ciistalW (syrianhamst} WIWGTWT. . L TICMMSMN. . MFTGNNK...... increiustalW ( rat} VIVGTVT..L TICMMSMN. . MFTGNNK...... AMGRciustalw{ rabbit VLVGTVT..L TICMMSMN. . MFTGNDK...... HMGRcilistalW { human} WIWGTWT. . L TICMMSMN. . MFTGNNK...... HMGRciustalw{ mouse ------...... HMGReiustalw{ xenopus. WIWGTVT.. L TICMMSMN. . MFTGNDK...... -- HMGRciustalW sea urchin} VIVCTLT ... L TICMLSMN. . YFTGLpR...... HMGRciustalW ( cockroach} VIVATL.T., L TvCMLTVDQ. RPLGLP...... HMGR.ciistalW (drosophila} VIVALLT.. I TACMLNGGQE QYPGCEQRIG HSTASAAAAG SGSGAGSGAS HMGRclustalW (dictyostel ......
AIGrciustalw{schistoscm) > si e s e s p s a s a a s a w- r s > 0 & e s > s is a ......
b HMGRciustalW (archaeoglo} w 8 - 8 a e e s e o 0- 8 w w 8 - 0 & 8 & a is a ......
HMGR.ciistal w (pseudomonas} w is 8 v 8 8 v 8 8 v - & 8 w 8 e s a s is a s
Cosensus WVGTW- - TOMMSMN- a MFGNNK------was a
FG. 32F U.S. Patent Jun. 9, 2009 Sheet 38 of 78 US 7,544,863 B2
S. 2 OO HMGRclustai W(methanobac) ...... is a s is us us as
HMGRclustalW (methanococ) ...... e s ed a as as a s vs a is s : s &
HMGRclustalW (halobacter ...... 9 - 8 s ...... HMGRclustalW (sulfolobus) ...... to as is s s e s is s HMGRclustalW yeast2} NGTKWRLRNN SNFILDLENI YRNMVKQFSN KTSEFDQFDL FAAYLL HMGRclustalW yeastill DGTKWRLRSD RKSLFDVKTL AYSLYDVFSE NVTOADPFDv MVTAYLMM HMGRclustalW (phycomyces ...... - d - - - b > b is a a a s s HMGRclustalW fusarium: SSVFAFLFGL VVTTKLGVPI SVILLSEGLP FLVVTIGFEK NVTRAVMS HMGRclustalW { candida} DGTVWRSRAY HGKLGKYSDM AVGAF NKVLN LVRGAETFDI AVCAYAM HMGRclustalW (dictyoste2} ......
w Y- as a s us d > 4
8
a as as as as w w -
a b d x as
- s - as as as e
is is a w w is a
8 X 8 × 8 wid was ap is
X > Y as
8 v X HMGRclustalW rubbertre2} ...... & 9 ep a b d w a 4 to es e s is s s - ¥ 8
8 - Y. g. 4- - - - - HMGRclustal Wrubbertrel) ------as a > X ye to - a - BMGRclustalW camptothec) ------P p - Y - - - - ) > 0 - Y HMGRclustalW (arabadops2} ...... HMGRclustalW (chineseham) . . . . . P 8 CGWNYEC. PK FEEDVLSSD ILTTRCIA U.S. Patent Jun. 9, 2009 Sheet 39 Of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} CGWNYEC. K. FEEDWSSD LTTRCA HMGRclustalW (syrianhainst CGWNYECPK FEEDWSSD LTTRCA HMGRclustalW ( rat} CGWNYEC.PK FEEDWSSD ILTTRCA HMGRclustalw rabbit} 4 × & & CGWNYEC. PK FEEDVLSSD LTTRCA HMGRclustalW { human CGWNYEC. PK FEEDVLSSD LTTRCA HMGRclustalW mouse as s is a s s is a s w d or 8 HMGRclustalW xenopus) CGWNYAC. PK FEEDWSSD LTTRCA HMGRclustalW sea urchin} CGWNYECAPQ WKESSLSSOV VMCMRTA HMGRclustalW cockroach. PGWGHNC, TLEEYNAADM VMTRCWA HMGRclustalW (drosophila HGWSOSC. DG LEAEYNAAOV LMTIVRCTA HMGRclustalW dictyoste1)
HMGRclustalW schistosom} F - WLYLFTR, HMGRclustalW (archaeoglio)
s as s > - p > a is as a s a a s or
s s
Consensus - are a re- a a -a ------are a se - CGWNYEC-PK FEEDWSSD LITRCA
FG. 32H U.S. Patent Jun. 9, 2009 Sheet 40 of 78 US 7,544,863 B2
25 O
as s - 't
e s s
- HMGRclustalW yeast2 FYTLCCLFND MRKIGSKFWL, SFSALSNSAC ALYLSLYTTH SLKKPASLE HMGRclustalW yeast1} FYTIFGLFND MRKTGSNFWL, SASTVVNSAS SLFLALYVTQ CGKEWSA HMGRclustalW (phycomyces) ...... HMGRclustalW fusarium) HAIEHRRIQA QNSKSGKRSP DGSTQNMIQY AVOAAIKEKG FEROYAE HMGRclustal W candida} FYTLFNLFAR MRAVGSKVWL, GLSTLVSSFF AFLFALYITT RVOSPFL HMGR.clustalW (dictyoste2} ......
a
& Ad 8 P. HMGRclustalW rice ......
- s s s at HMGR.clustalW corn ......
as a P
w- - as a
4 · · · · a -
to X d w
. s we
8 ......
d as to 8 g » to 8
- X & -
X { us s
8 × 8 is is 8 -
0 was
q p s- as a as so
FIG. 32
U.S. Patent Jun. 9, 2009 Sheet 42 of 78 US 7,544,863 B2
3 OO o no o a o p is so e o - as a s us as w w o a s a
o o is e s- e P
o e o o a r u se e o o o o as a e s e up to so o e s -
p - up to do o is a 9
a o a to o 0 & - 0
- 0 v o 0.
- or o 0- vs - HMGRclustalW { yeast2. SLVIGLPFIV VIIG. FKHKV RIAAFSLOKF BRISDKKT WSNIYEAMF HMGRclustalW ( yeast1) TLFEGLPFIV VVVG. FKHKI KIAQYALEKF ERVGISKRT TDEVFESVS HMGRclustalW (phycomyces P - P - P - HMGRclustal W{ fusarium) IVILVIGAAS GVQGGLQQFC FAAWTLFF DFILFFYT ALSIKLRST HMGRclustalW candida SLSEGIPFFV AVVG. FNNK. LAEKVLO. N. QLNAQSSKND APTVLYQALR HMGRclustalW (dictyoste2}
e o 0.
- O wo P vs. P W - O - - - - a 0 o w a a wo rice! ...... HMGRclustalW { corn - - - - - H99A HMGRclustalW (wheat3}
o o e e g e P. p. soybean
A is HMGRclustalW rubbertres) . . . DEVRRRPP. K. HIVRKDDGE WLNSFSHG. HMGRclustal wrosyperiwi) OSRRRSP. . . TWTAKAAAGE LPLAPHEGO. HMGRclustalW { tomato) ...... DVRRRSEEPW YPSKVFAADE KPLKPHKKQQ HMGRclustalW (wood tobacc} DVRRRSEKPA YPTKEFAAGE KPKPK. . . HMGRclustalW potato ...... OVRRRVKP. YSKDASAG EPLKOOE. . . HMGRclustalW (radish DRRRPPK PPWNSN. . . . RFDNRS HMGRclustalW (arabadopsis l; ORRRPPK PPWTNNNNSN GSFRSYQPRT HMGRclustalW (cucumismel} DRRRSRPPR PNAVODADAT CTFRRDEQDA HMGRclustalW (rubbertre2} . . . v o X ed vp p P 0 0 HMGRclustalW (rubbertrel} ...... •M DTTG. R.H. ... -HR...... KHAT HMGRclustalW (camptothec) ...... M DVRRRSNS HQIPSVGGTA PPMLKPKQPT HMGRclustalW (arabadops2} ep a a w e s - v EDLRRRFPTK KNGEESN HMGRclustalW (chineseham FLDKELTGLN EALPSFL.LLI DLSRASALAK FALSSNSQDE WRENARGMA FIG. 32K U.S. Patent Jun. 9, 2009 Sheet 43 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2) FDKELTGN EALPFFL DLSRASALAK FALSSNSODE WRENARGMA HMGRclustalW (syrianhamst) FLOKETGLN EALPFFLE DLSRASALAK FALSSNSODE WRENARGMA HMGRclustalW ( rat) FLOKEGN EALPFFL DLSRASALAK FALSSNSODE WRENARGMA HMGRclustalW ( rabbit) FOKEGIN EALPFFL, DSRASALAK FALSSNSODE WRENARGMA HMGRclustalW human} FDKELTGN EAPFFLEL DSRASTLAK FALSSNSODE WRENARGMA HMGRclustalw mouse) HMGRclustalW ( xenopus FDKELTGIN APFF OLSKASALAK FALSSNSODE WRONARGMA HMGRclustalW (sea urchin} FGLETGN EALPFFL.L.L DITKASALTK FALSSTTONE WVDNARGMA HMGRclustalW cockroach) FLGSDWSDK OAFFFL.L. DLSKATVLAQ FALSSRSQDE WKENARGA HMGRclustalW drosophila FLGSDISELK DAFSLIV DLSNSGR LRS GAMGSN. QAE WONIARGLE HMGRclustalW (dictyostel) HMGRclustaiW (schistosom} LSTSWFLFS HMGRclustalW (archaeoglo as as a 4 4 - is a 4& × 8 + 9 s is is us is us is is as us & 4 & w is is k
a ap a- - - so
4 4 & 4- - - - d. FDKETGN EALPFFL.L.L. - DL-RASALAK FALSSNSOE
F.G. 32. U.S. Patent Jun. 9, 2009 Sheet 44 of 78 US 7.544,863 B2
3 SO HMGRclustalW (methanobac e p r - as so o o e o a o s - a e - e s p - s - d - 4- a v- w -
P o as up - e o a o o us do e s - is
a e o no e a a HMGRclustal Whalobacter a b o us s. s s as s o up us so is
HMGRclustalW (sulfolobus C- 8 P. F. - P - o HMGRciustalw{ yeast2. QEGAYE,ERDY LFYISSFIGC WNFCISTFM LWFTDLLSAT HMGRclustalW yeast. EEGGRLEQDH CIFAFGC TNFCISAF LIFELILTPTHMGRclustalW (phycomyces) HMGRclustal W. fusarium) VSSVMSCVW ASNWAKGDDE LNRWRGDAP, FGRKSSSPK HMGRclustal W{ candida) ECGPLLLRDH SFYASYLDG, KNFC, AAL LAFDILTTSTHMGRclustalW dictyoste2}
s a e. e. to e or HMGRclustalW (wheatl - a u s w r a r a a
HMGRclustalW { rice) - a . . . NGLAMVS HMGRclustalW { corn . . . NIFSA HMGRclustalW (wheat3.
8 v or p ...... d is ...... to s r
HMGRclustalW soybean A a P - 4 4 - a up a a HMGRclustalW rubbertres } KSDYSLP . . . NALWFSL HMGRclustal Wrosyperiwi) PRSSOWPL PLYL.A...... NGVFFT, HMGRclustalW tomato LOASDALP, PYLTT. . . . NGFFTM HMGRclustalW (woodtobacc} L. ASDALP, PTY LT...... NGLFFM PLYIT. . . . . HMGRclustalW potato) s so o or a e s is a SPKASDAIP . . .NGLFFTM HMGR.clius talW radish DO. . . . DDRR PPKASDAP PLY LT...... NAWFFT, HMGRclustalW (arabadopsis1 SD. . . . DOHR PPKASDALP PLYIT...... NAWFFT, EMGRclustalW (cucumismel S.A. . . . ADHL. SPKASDAP, PLYLT...... NTFFL EMGRclustal Wrubbertre2} e u un a n o e a r is HMGRclustalW (rubbertrel PKASDAP PLY ...... NAVFFT EMGRclustalW camptothec) SPKASDAP, PLYIT...... NGVFFT, HMGRclus tail Warabadops2) RKASDAIPL PLY.L.T...... NTFFLSL, HMGRclustalW (chineseham) LGPTFTDA LW. . ECLVG WGTMSGVRQL. EMCCFGCMS WANYFWFMT FG. 32M
U.S. Patent Jun. 9, 2009 Sheet 46 of 78 US 7.544,863 B2
4 OO HMGR.clustalW methanobac). * V P - or et d is - 8 s as a a as
a w g + w a w
e a e s up s r. w o e or w a
is is s a & P w re r w w & a s v w as wr HMGRclustalW yeast2. FSASMK ENHRSTV ROTL. . EED GVWPTTAD YODETASEPH HMGRclustalW yeast) FYSAALRI. EMNWRRST IKQTL. . . EED GWV PSTARI SKAEKKSWSS HMGR.clustalW (phycomyces) is or - up u or -n is a s- a s us HMGRclustal W fusarium) FKVLMELGF FWNIWNICS PFRNP. SSM STRTWASS, GGWAPSWD HMGRciustalW candida FLSAILSOL EINQIHRSTL LREQL., EDD GLTETTVODV LKSNSLAGTK HMGRclustalW (dictyoste2} u- - - as a s- a .
- p r 8 a HMGRclustalW rice WSSCDWR CSORSER. . . P...... GGREFA TVWCQLASVV HMGRclustalW { corn FAASLAYMR RWRSKRSST PIHA...... WGLAEML. AFGWAS, HMGRclustalW (wheat 3
0 P v ar - a w
soybean) w e o w to a w HMGRclustalW rubbertres) FFSWAYFILF RWREKRKST P.E.I ...... WTFPEA ALCLWASW HMGRclustalW rosyperiwi FFSWMYSLT RWRCKRNAT F.W...... WTLSEA ALASLASW HMGRclustalW tomato) FFSWMYFLS RWRSKIRNIST PLE.W...... VSELG AVSASW HMGR.clius talW wood tobacc} FFSWMYYS RWREKRNST PLHW...... WTFSEW AAS,ASW HMGRclustalW potato) FFSWMYFW RWREKRNS PLHV...... WTSELL AMWSLASW HMGRclustalW radish) FFSWAYYLE RWROKRYNT PLRV...... WTWTELG AVALASF HMGRclustalW (arabadopsis FFSWAYYLH RWROKRYNT PH.W...... WTEG AIALASF HMGRclustalW (cucumismel) FFSWAYY RWROKRNIST PLHW ...... WISEIA AVSMASF HMGRclustal Wrubbertre2} 8 w in HMGRclustalW rubbertrel} RWROKRNST PLHI...... WTLSEW AVSLASF HMGRclustalW (carptothec) RWREKRNST PLBW...... WTSEA AIRTFWASFI HMGRcius talW (arabadops2) FFATVYES RWREKIRNST PLEV...... WOLSEIC AGFWASF HMGRclustal Wichineseham) FFPACWSLV. ESRESREGR PIWQ. . . LSHE FARVLEEEE. FG. 32O
U.S. Patent Jun. 9, 2009 Sheet 48 of 78 US 7,544,863 B2
40. 4SO HMGR.clustalW (methanobach ...... - 0 p q r a g g o a HMGRclustalW (methanococ) ...... HMGRclustalW (halobacter) ...... HMGRclustalW (sulfolobus ...... HMGRclustalw yeast2} FLRSNVAIIL. GKASVIGLLL LINLY VF. . . . TDKLNATIL NTVYFDSTIY HMGRclustalW { yeastl FLNLSVVVII MKLSVILLFV FINFYNF. . . GANwVN. DAF NSYFDKERV HMGRclustalW (phycomyces ...... HMGRclustalW fusarium PFKVASNGLD AILPTAKSNN RPTLVTV... LTPIKYELEY PSEYAGSA HMGRclustalW candida} TFTDAPSTLV TVAKVAGVSV FFGLHFY. . . GFGSAWLSDL SAGNETNOTF HMGRclustalW (dictyoste2} ...... HMGRclustalW (wheatl ...... HMGRclustalW rice) YLLSLFAHPD APATTTGDDD ...... HMGRclustalW corn YLLSFFGIAF WQSIVSSGDD ...... HMGRclustalW (wheat} ...... HMGRclustalW (wheat2) ...... HMGRclustalW ( soybean) ------...... HMGRclustalW (rubbertre3} YLLGFFGIGF VHSFS. RAST ...... HMGRclustalW rosyperiwi YLVSFFGLDF VOSLIYKPNN ...... HMGRclustalW ( tomato YLLGFFGIGF VQTFVSRGNN ...... HMGRclustalW (wood tobacc} YLLGFFGIGF VOSFVSRDNN ...... HMGRclustalW potato YLLGFFGIGF voSFVSRSNS ...... HMGR.clustalW (radish YLLGFFGIDF VQSFISRP...... BMGRclustalW (arabadopsisi YLLGFFGIDF voSFISRASG ...... HMGRclustalW (cucumismel YLLGFFGIDF VOSFIARSSP ...... HMGRclustal Wrubbertre2} ...... HMGRclustalW (rubbertrel YLLGFFGIDF VOSFIARASH ...... HMGRclustalW (camptothec YLLGFFGIGL VQPFTSRSSH ...... HMGRclustalW (arabadops2} YLLGFCGIDL IFRSS...SD...... HMGRclustalW (chineseham) KMMSLGV WHAESRWAD PSPONST. . . E. BSKWSG DEDWSKRIE FIG. 32O U.S. Patent Jun. 9, 2009 Sheet 49 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2) KMIMSGLV, WEAHSRWAD PSPONST. . . TE. BSKVSLG DEOWSKRE HMGRclustalW (syrianhamst KMMSGV WHABSRWAD PSPQNST. . . TE. HSKVSLG DEOWSKRE HMGRclustalW ( rat) KMMSLGV VHAHSRWAD PSPONST. . . AE. QSKVSLG ADVSKRE HMGRclustalW { rabbit) KMMSGLV, VHABSRWAD PSPONST. . . AD. NSKVSLG DENWSKRE HMGRclustalW human) KMMSGV VHABSRWAD PSPONST. . . AD. TSKVSLG CENWSKRE HMGRclustalW mouse HMGRclustalW ( xenopus KMMSGV WHAHSRWISE PSSQNST. . . SISDHEVTTM LDDMMPKRVE HMGRclustalW (sea urchin) KMMRTGV WHAHSYWAS . . . NDT. . . ELMSRDMYD GNTDKKID HMGRclustalW ( cockroach) KVMSAGLM, VHAHRWWRC HMGRclustalW (drosophila KMTTGMA WHIYSREVSP SNWSNNRT HMGRclustalW (dictyostel) KPLPYIPOHN OQQQQKCOPS HMGRclustalW (schistosom NXGVSSSTRK NLIGYWVNPN HMGRclustalW (archaeoglo so to s r. s. O. O. O. O O. W. P. p.
as as
w w wo
Cosensus Y-FFG-V W-A-SR-SD PSPQNST------SKWSLG DE
FIG. 32R U.S. Patent Jun. 9, 2009 Sheet 50 of 78 US 7,544,863 B2
SOO HMGRclustalW (methanobac as as a so o o s p s o e o a p up e- as as a - r u e o s p s s ap a o o v- - -
HMGRciustalW (methanococ} p - a e o or a
HMGRclustalW (halobacter) up a a as as a 6 p.
HMGRclustalW (sulfolobus) a p s a- as - - up to e g a P p a so as o - HMGRclustalW yeast2. SLPNFINYKD IGNLSNQVII SVLPKQYYTP LKKYHQIEDs WLLDSVSN HMGRclustalW yeastl SLPDFITSNA SENFKEQAIV SVTPLLYYKP IKSYQRIEDM WILLRNWSW HMGRclustalW (phycomyces a o ------O. P. s. B O O. P. HMGRclustalW fusarium) ASNPAYN. DA FHHHFOGYGV GGRMVGGILK SLEDPVLSKW WIAALSWA HMGRclustalW candida} TLYDAVA. Do IPIGSNGTLV TLFPTRFFLP EKLSTOIEAV VLSFIGLIST HMGRclustalW (dictyoste2}
HMGRclustalW (wheatl a no do o o
HMGRclustalW rice e o go a s a up o - - e o
HMGRclustalW corn w in Y a p- - a o
HMGRclustalW (wheat3} wn ------e o O HMGRclustalW (wheat2}
HMGRclustalW soybean) o P to p r s e s - a O a s h g HMGRclustalW rubbertre3} . . O. SWDVEE Y...... D ODNIKEDT R ------HMGRclustalW rosyperiwi) . . E. GWEIEE o O so up so s as s RN...... HMGRclustalW tomato) . . D. SWDE. . to o v vs is RC...... HMGRclustalW (wood tobacc} . . DECWOEED 0 0 d N DEQFLLEEDS RR...... HMGRclustalW potato) . . DSWDED is 0 d e o up a N AEQLIEEDS RR...... HMGRclustalW (radish w w- a - e s p so R...... HMGRclustalW (arabadopsis R...... HMGRclustalW (cucumismel} o e o a so go go e e to P - P - O...... HMGRclustalW rubbertre2} 48 - 8 is 8 HMGRclustalW rubbertrel) . O. WWDED as a . e. e. up D P. NY, DED R...... HMGRclustalW camptothec) DDWWGWDD DE...... ------O D WDEVLKEDT R...... HMGRclustalwarabadops2} . . DOVWVNDG e o us as as as a d di uo e - a HMGRclustalW (chineseham SKMISMDIEO WVTLSLAFL.L., AWKYIFFEQA E.T. . ESTLSL FIG. 32S U.S. Patent Jun. 9, 2009 Sheet 51 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} PSVSLWOFY, SKMISMDIEO VVTLS,AFL, AVKYIFFEOA S. EST.S. HMGRclustalW (syrianhamst) PSVSLWOFYL SKMISMDIEQ WWTSAF, AVKIFFEQA St. ESTLSL HMGRclustalW rat} PSVSLWQFYL SKMISMDIEQ WITLSLALLL AVKYIFFEQA E. ESS HMGRclustalW rabbit) PSVSLWQFYL SKMISMDIEQ WITLSLALL, AVKYIFFEQA ETESS HMGRclustalW human} PSVSLWOFYL SKMISMDIEO WTLSLALLL AVKYIFFEQT. E. ESTLSL HMGRclustalW mouse HMGRclustalW xenopus) PSMPLWQFYL ITLGALLE AVKYIFFEQT ETESTFSM HMGRclustalW sea urchin) PTMPLWEFYA TAAV, ASHYIFFSO AYPEKRVS HMGRclustalW cockroach} SAWDT FCTHCDTGVS YSRWSFASEG EE P1VK, HMGRclustalW (drosophila ESGEAD IVISIWLA, VVKFCFDNR DP. . . LPDO HMGRclustalW (dictyostel} . . QDYICQPQ . . N. . . . DNN IN...... HMGRclustalW (schistosom} CHYKCWSTTF SFKHNSSENE WFPVLYHITA HMGRclustalW (archaeoglo) 0 p up p as a o o o
o O o a o p a s
O
Cosensus PSDSWOFY SKMISMDIEO WVSA AVKYFFED- RT - -
FIG. 32T U.S. Patent Jun. 9, 2009 Sheet 52 of 78 US 7,544,863 B2
SSO HMGRclustalW (methanobac). a 0 w a • b ------up a s ...... O to o O - P - r - - - P - P - r - a ------e - e s ......
s s o o s r. e. e. HMGRclustalW (halobacter) - - - - P - P - d 4 a to a a e s - up to o e s is a s a ......
HMGRclustalW (sulfolobus) ------8 to - P ------e o s vs. s o ...... g g g e P HMGRclustalW yeast2) AIRDQFISKL LFFAFAVSIS INVYLLNAAK IHTGYMNFQ. . . POSNKDD HMGR.clustalW { yeastl) AERDRFWSKL VLSALVCSAV INVYLLNAAR IHTSYTADQL WKTEVTKKSF HMGRclustalW (phycomyces) HMGRclustalW ( fusarium) LNGYLFNWAR WGIKDPNVPE ENIDRNELAR AREFNDTGS. 'iGR&itals candida} AARDKYISKF ILFAFAVSAS INVYLLNWAR IHTTRLEDA,
'iReitals (dictyoste?) te e o O or a 0 o- e s p r t t t e - P - t e s a e s e s ......
X O P. HMGR.clustalW (wheatl} to a do h 8 v 8 & 8 as a a r s a o a w s s r. s - is ......
BMGRciustalW { rice - s h P - P - 8 as a - a 0 w w - 8 o w is s s a ...... vo e on a o e o so s e o up - r a te e o o a w w w w e o re e s p r s se g e o s
e o a to a s
a a w p is a 9 b - 0 to a e s a 0 to o e s p a a s
v v as ul
------w w a a - - 0 or 0 & - w 0 a w bi e s p s is o a a . . . . .
te e o a as a so
t t t t t e s - a to t t P R P Y P - Y P 9 w w e s a
as a
8 to h is us V & 4 is us a b d e s 8 8 is 0 - as as as s a s
us as up us as - O - a up o us e o e o up o o e - e - a o o o us e s as a a - - - - G. . . . .
0. - P - o P. O. P. e. 9 v s a up to w w - - - - G. . . . .
s p is s s up a di b - d - d. 4- 4 s - d - is a s as . . .G. . . . .
to o O p up o o o s- a - O - P - P - op e P - p. p up up a as a e o a to e s a- as a . . . .G. . . . .
r an e
- 0 - 0 oh p is v Q b P × b is to u to e a 8 a p us w is
+ k is d is ------. . . .
- P. P. P. - p a o e
to a or or a e e o a P. P. P. P. v - - - - - w w - - r s s a a
P a s h g X 4 - 8 to 8 s is w is a a e o e o a s is as . . . . .
O 0 as o o o a - e. s so o e o o a s e o a e o o o a e o O p s - r s is a ------......
1 P - to - e O o es o or o e o a c e s w ------e e s a
. . s. s e s a o a s a e s ------V ------a s ......
e - P - - - - - a e HMGRclustalW (chineseham KN. . PITSPV VTPKKAPDNC CRREPLLVRR SEKLSSVEEE PGVSQDRKVE F.G. 32U U.S. Patent Jun. 9, 2009 Sheet 53 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2) KN PTSPW WTPKKAPDNC CRREPLLVRR SEKLSSWEEE PGVSQDRKVE HMGRclustalW (syrianhamst} KN. PTSPW ATPKKAPDNC CRREPVSRR NEKLSSVEEE PGVNQDRKVE HMGRclustalW rat KN. PTSPW WTPKKAQDNC CRREPLLVRR NOKLSSWEED PGVNQDRKVE HMGRclustalW rabbit KN. . PTSPW WTOKKVPDSC CRREPVVVRN NOKFCSVEEE AGMSQDRKVE BMGRclustalW human KN. PTSPW WTQKKVPDNC CRREPMLVRN NQKCDSVEEE GNRERKVE HMGR.cliustalW ( mouse HMGRciustalW ( xenopus KN PSPV AVOKKQISSC CRREPSQ.EK TVHVSTTEA S. SKEEEA RMGRclustalW (sea urchin} MEGHEWWNPG SDEEDASEWE TIGTLSSSPS TSWRWESM TSRTOACQITD RMGRclustalW cockroach) WTGOSVWNSN STDDAQLHYY MRW.T.W. S AdW, AAW KFWFF HMGR.clustalW drosophila RQ. . . . SGPW AIEAKASQTT PDE3HVE. or a QEKD TENSAAVRT, HMGRclustalW (dictyostel o o O a so p a EMGRclustalW (schistosom} NSQLRNLN HMGRclustal Warchaecglo) a 0 as a
w a - P - p. 8
b s- or us up a as a s h
s to , a n e o Consensus KN--PTSPV --K-SSWEEE. --G-
FIG. 32V U.S. Patent Jun. 9, 2009 Sheet 54 of 78 US 7,544,863 B2
6OO HMGRclustalW (methanobach ...... HMGRclustal W(methanococ) ...... HMGRclustalW (halobacter) ...... HMGRclustalW (sulfolobus) ...... HMGRclustalW yeast2} LVVQQKSATI EFSET...... RSMPA SSGLETPVTA KDIISEEIO HMGRclustalW { yeasti). TAPVQKASTP VLTN...... KTVS GSKWKSLSSA OSSSSGPSSS HMGRclustalW (phycomyces) ...... HMGRclustalW fusarium) LPLGEYVPPT PMRTQ...... PSTPA TODEAEG. . EMKARP HMGRclustalW ( candida) LKKPKKKASK TAVSV...... PKAVV VKDSETTKSS EHSSSESE HMGRclustalW {dictyoste2} ... KGKSVNVE DLKDQ...... EIIAL VDKGEOP. . ENER HMGRclustalW (wheatl ...... HMGRclustalW ( rice) ...... GCG GSR...... RA . . . . . A PPEPAMHG HMGRclustalW corn) ...... SSGS AAA ...... PSROHAOA PA9CEGSP HMGRclustalW (wheat3} ...... w w w th a
HMGRclustalW wheat2) ...... as a a Q- a to P. O. as as a s as up o e s as a s a e s HMGRclustalW soybean) ------HMGRclustalW (rubbertre3} ...... PTG AC...... AAPSLDCS SPTKIEAP HMGRclustalW rosyperiwi . .T. . NCTTL. GC...... AWPPPSVP KAPVVPQQP HMGRclustalW tomato. . . P. . . ATTL GC...... AVPAPPAR CAPMAPPQP HMGRclustalW (woodtobacc} . . P. . . ATTL GCT...... AVP999A QIVPMVPPQP HMGRclustalW potato) . . PCAAATTL GC...... WWPPPPVR KTAPMVPOOP HMGRclustalW (radish ...... LVO 999...... ------. . . PPP. . . . S QIVAAKIPNP HMGRclustalW (arabadopsis1} . . . . . LWTC SPP ...... us o O to to - - - TP...... WSWAKOPNP HMGRclustalW (cucumismel} ...... IDN NRY...... as as as a do as . . . . AAPRSASA WALPSKVWDA HMGRclustalW (rubbertre2} ...... HMGRclustalW (rubbertrel) ...... LWTC PPA...... NISTKTI AAPTKPTS HMGRclustalW (camptothec) ...... TVP CAA...... APVDCPLP PKPKWWDPW HMGRclustalW (arabadops2} ...... MIPC N.O...... SDCREVL PKPNSWOPP HMGRclustalW (chineseham) WKPLWETE SAS...... RATFWLG.A ... SGTSppVAA RTQELEELP FIG. 32W U.S. Patent Jun. 9, 2009 Sheet 55 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} WKPVVER SAS...... RATEWLGA SGTSPPVAA RTOELEIELP HMGRclustalW (syrianhamst) WKPLWAETR STS...... RAFVGA SGGCSPVA GTQEPSIELP HMGRclustalW rat} WIKPLWARAE TSG...... RATFWG. A SAASPPLAL GAQEPGEP HMGRclustalW rabbit WKPWAED S...... RAAFWWGGS SFP'SW ETKEPEELP HMGR.clustalw human WKPWASD TN...... RAFVVGNS SLTSSW WTQEPEIELP HMGRclustalW mouse HMGR.clustalW xenopus) WIKIPPLETS P...... KAKFVG DSSPESP EDKNTMEOP HMGRclustalW sea urchia PWTAS25NSR SSSPWSSHSV KPARFTIGSS GSGSaoEEEE WKEEEVIEWW HMGRclustalW cockroach} ERDETTR GMDG. . . . . W WEWSSpVEHK YVOTEQPSCS APEQPLEEPP HMGRclustalW (drosophila) LFTIEDOSSA N...... ASTCTD.L. PRERIWGP HMGR.clustalW dictyostel} H P o SGKEQ EQ...... QQQQQQQQ OOTPDITNOP HMGRclustalW schistosom} PKIKETLISD QWKQSPWLPK FSKKLNDIPL. QSRKSRIYCLH Kotor HMGR.clustai Warchaeoglio
Cols ess PPEEP
FIG. 32X U.S. Patent Jun. 9, 2009 Sheet 56 of 78 US 7,544,863 B2
6SO HMGRclustalW (methanobac) e - is MODME HMGRclustalW (methanococ X is us - A NDEKMLN HMGRclustalW (halobacter > X > s is a as d b - 8 to s s a AASLADRVRE HMGRclustalW (sulfolobus IDEWWEKLVK HMGRclustalW ( yeast2. NNE. CWYALS SODEPIRP.L. SNLVEME . . KEOLKNMN NTEVSNVVN HMGRclustalW yeasti) SEEDOSRDE SDKKRP. EELEALIS . . SGNTKQLK NKEWAALVIH HMGRclustalW (phycomyces} HMGRclustalW fusarium ANL...... PNRS, N EEEKLL.S. . . . ENALREMT DEEWISISMR HMGRclustalW candida} SEQ ------. . . . SSRP.L. EQVIELYK.. DGKKTLV ODEVWSLWTA HMGRclustalW dictyoste2} PNN...... F QRAWHIRR. . . . K.L.ARIOLO KEHQRALHAQ HMGRclustalW (wheati) HMGRclustalW rice) G------...... DEEWAAVAS HMGRclustalW corn) A ------...... A. . DEEWASWVA HMGRclustalW (wheat:3} HMGRclustalW (wheat2) HMGRclustalW ( soybean)
AB is ...... HMGRclustal wrubbertres - a WSTTT. . . . . SSOD DEQIIKSVVS HMGRclustalW rosy periwi SK...... MW. EKPAP, . . .TPQNSEE OEOKAVVA HMGR.clustalW tomato) S------MS. M. WEKPAP, . . . SASSGE DEEIIKSVVO HMGRclustalW (wood tobacc} SKV...... AAM SEKPAPW. . . . PAASEE DEEIKSVVO HMGRclustalW ( potato) AKV ------A.S.O. TEKPSP. . . . MPALSED DEEIIOSVWQ HMGRclustalW radish) E. : ------. . . . . QPPLPKE OEEWKSWO HMGRclustalW (arabadopsis1} EP------W. . . . ESPEE DEEVKSVID HMGRclustalW (cucumismel} EA------I.N. . PLPEE DEEWWKMVVD EMGRclustalW rubbertre2} w- Y - - HMGRclustal Wrubbertrel EP------II. . . . APLWSEE OEMVNSVVD HMGRclustalW (camptothec) P------I. . DEEKSWWE HMGRclustalW (arabadops2 RE...... DEEWKLVD HMGRclustalW (chineseham) SE------...... PRP.N EECLOILE - SAEKGAKFLS DAEIIQLVNA FIG. 32Y U.S. Patent Jun. 9, 2009 Sheet 57 Of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} S.E...... PRP.N EECLOILE. SAEKGAKFLS OAEOLVNA HMGRclustalW (syrian hamst) SE------PRP.N EECLOLE. SAEKGAKFLS DAEIOLVNA HMGRclustalW { rat} SE...... PRP.N EECOIL.E. . SAEKGAKFLS DAEIQLVNA HMGRclustalw rabbit: KE...... PRP.N EECLOILG. ... NAEKGAKFLS DAEIOLVNA HMGRclustalW human} RE...... PRP.N EECLQLG . . NAEKGAKFLS DAEQLVNA HMGRclustalw mouse HMGRclustalW ( xenopus EE------PRP. L. DECVRIL.K. . NPDKGAQYLT AEWSVNA HMGRclustalW sea urchin} LE...... ELKAPRP. M. PELE. . . NWGKGPNALT ODEWOLLWGA HMGRclustalW cockroach} AS - - - - - * as 8 s NRS. I DECLSWC. . . KSDWGAOALS DCEWMALVTS HMGRclustalW (drosophila KP...... PRP. W OECDILNST EEGSGPAAS DEEWSWEA HMGRclustalW (dictyostei) TKTN ------... KKPKES NEEILIKEK HMGRclustal Wschistosom} DTELOLISH HMGRclustalW (archaeoglo MOVERLDRR HMGRclus talW (pseudomonas)
P - P -
Consensus SE------PRP-N EECQL- - - -AEKGAKSLS DEEKLWWA
F. G. 32Z. U.S. Patent Jun. 9, 2009 Sheet 58 Of 78 US 7,544,863 B2
55 7 OO HMGRclustalW (methanobac). GR. . IKLYEI E.RHVPVDEA WRIRREFIE. . . . . RTCGVK LEBVSNYS HMGRclustalW (methanococ) GE. . IKPYQL. D. KMFGSKIA TERRKFE . . . . KKWGIE FKHCNYS HMGRclustalW (halobacter) GD.. LRLHEL, E.AHADADTA AEARRILLVE . . . . SOSGAS DAWGNYG HMGRclustalW (sulfolobus GE. . ISFHEV D. NLLEANAA MWARRA.E. . . . . KWGWG PSIGSTW HMGR.clustalW ( yeast2) G. . KLPLYSL EKKLEDTTRA WLVRRKALST AESPLWS. . . . EKLPFRN HMGRclustalW yeastl. G. . KLPLYAL EKKLGDTTRA WAVRRKALSI LAEAPVAS. . . . DREPYKN HMGRclustalW (phycomyces ...... a HMGRclustalW fusarium G. . KIPGYAL. EKTLGDFTRA WKRRSAR NKAAADTHS LDRSK, PYEN HMGRclustalW candida G. . KLPLYAL. EKQLGDNLRA WARRKASD LADA PVRS. NKPYLH HMGRclustalW (dictyoste2 A. . VVAAAEK AATSGEDPSS QPVVPPTSN LDFEGSTN. P. P. PVOE HMGRclustalW (wheatl) ...... HMGRclustalW ( rice G. . ALPSHRL ESRLGDCRRA . VEGLPFDG HMGRclustalW corn) G. ... KVPSYAL EARLGDCRRA . EGLPLOG HMGRclustaiw (wheat3} ......
HMGRclustalW (wheat2} ...... 0 v P ab o o O p q >
HMGRclustalW { soybean ...... W a a o e s is a so o P. P. P. HMGRclustalW (rubbertre3 G. . SIPSYSL. ESKLGNCKRA ALIRRETLQ. . . LEGLPLOG HMGRclustalW rosyperiwi. G. . KIPSYSL ESKLGDCKRA AGIRREALQ. . . LEGLPLEG HMGRclustalW ( tomato) G. . KIPSYSL ESKLGDCKRA ASIRKEVMO. . . LEGLPLEG HMGRclustalW woodtobacc} G. . KMPSYSL ESKLGDCKRA ASIRKEALO. . . . . RTGKS . . LEGLPLEG HMGRclustalw potato) G. . KTPSYSL ESKLGDCMRA ASIRKEALO. . . . . RTGKS . . LEGL?LEG HMGRclustalW (radish) G. . VVPSYSL ESRLGDCKRA ASRREALQ. . . . . RIGRS . . IEGPLDG HMGRclustalW (arabadopsis 1} G. . VIPSYSL ESRLGDCKRA ASIRREALQ. . . . . RVTGRS . . EGLPLDG HMGRclustalW (cucumismel) G. . SVPSYSL ESKLGDPKRA ASRREALQ. . . . . RTGRS IHGPFEG HMGRclustalW (rubbertre2} ...... ea is e o o vo o HMGRclustalW (rubbertrel G. . KIPSYSL. ESKLGDCKRA . . .EGIPVEG HMGRclustalW camptothec. G. ..TTPSYAL ESKLGDSHRA . . AGIPLDG HMGRclustal Warabadops2} G. .TIPSYSL ETKLGDCKRA AAIRREAVO. . . . . RTGKS . . LTGLPLEG HMGRclustalW (chineseham K. . HIPAYKI, ETLMETHERG VSIRRQLIST K. . LPEPSS. ... LQYLPYRD FIG. 32AA U.S. Patent Jun. 9, 2009 Sheet 59 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2 K. HIPAYK., ETLMETHERG WSIRROLIST K. LPEPSS. . . LOYLPYRD HMGRclustalW (syrianhamst} K. PAYE, ETMETHERG WSIRRQLST. K. . LPEPSS. . . LORYPYRD HMGRclustalw rat) K. PAYO, ETMETHERG WSIRROLISA. K. .I.AEPSS. . . LOYLPYRD HMGRclustalW rabbit K. PAK, MEEERG WSRRQLSK K. LPEPSS. . . LOYLPYRD HMGRclustalW human} K. HIPAK ETLMETHERG WSIRROLSK K. LSEPSS. . . LOYLPYRD EMGRclustalW mouse HMGR.cliustalW ( xenopus) K. HIPAYK, ETMMESPREG WAIRRQMLSD. K. . LPQRSA. . . LQSLPY KN HMGRclustal W{sea urchin) EPAYKL ENIONPERG WAVRRQSK L. L.PITDA. - LEKIPYAS HMGRclustalW cockroach) G., HIAGYQL EKVWRNPERG WGRRQTK T. , ADIKOA. ONLYKN HMGRclustalW (drosophila GGHCPEKE ESWOOPERG WRIRROGS R. . AKMPWGR OWEH HMGRclustalW (dictyostel) G. ... EWLAYRL ENELGOCSRA WEIRRMLLEK . . . .QLSKK. EPPEG HMGRclustai Wschistosom} G. RLKTRE, ESWWRNPFRA WSLRRLDS. . . . . TENNP HERIPYKD HMGRclustalW (archaeoglo) YKSGKIRRA MSSRGFYK LSWEERLKKV AFAGSEE . . WKAWLSQG HMGR.clius talW (pseudomonas MS OSRPAFRN LSPAARLDH GQLIGLSDD WSIANAG
Consensis G- - - PSYST, ESKGDKRA WSRSEALSK K--LRTGSS - - LEGLYEG
FIG. 32BB U.S. Patent Jun. 9, 2009 Sheet 60 of 78 US 7,544,863 B2
70. 750 HMGRclustalW (methanobac IDMERASRRN IENPIGVVQI PLGVAGPLRV RGEHADGEYY WPASEGA HMGR.clustalW (methanococ) IDEEMAMKKN IENMIGAIQI PLGFAGPLKI NGEYAKGEFY PLATTEGA, HMGR.clustaw (halobacter FPAEAAES-A IENMVGSIQV PMGVAGPWSV DGGSVAGEKY PIATEGA. HMGRclustalw sulfolobus IDYSEIKNKN AENVIGAIQI PLGIWGPIRV NGDYAKGDFY WPMATTEGA, HMGRclustalW yeast2} YDYDRVFGAC CENVIGYMPI. PVGVIGPLII DGT. . . . SYH PMATTEGC, HMGRclustalW yeastl YDYDRVFGAC CENVIGYMPL. PVGVIGPLVI D.G.T. . . . SYH
FIG. 32CC
U.S. Patent Jun. 9, 2009 Sheet 62 of 78 US 7,544,863 B2
WPMATTEGO, HMGRclustalW (sea urchin YDYSFVSGAC CENVIGYMPV PvoVAGPL, LL DGQ. . . . EFQ WPMATTEXGC HMGRclustalW cockroach YDYLKVMGAC CENVIGYMPV PVGVAGPLNL DGR. . . .LVH WPATTEGC HMGRclustalW (drosophila) FDYRKVLNAC CENVLGYVPI. PVGYAGPLLL DGE. . . . TYY WPMATEGA HMGRclustalW (dictyostel) FDFAKVOGQC CENVIGYVPI. PVGTAGPIQL NGO. . . . LVT PMAEGC, BMGRclustalW (schistosom) YDYRLVYGQC CEEVIGYMPI. PVGKIGPLLL DGR, . . . SHY PAEGCL HMGRclustal Warchaeoglo) . LPLDVADRM IENVIGTFEL, PLGIATNFLI DGK....DYL IPMAREPSW HMGRclustalW (pseudomonas ALPMDIANGM IENVIGTFEL PYAVASNFQI NGR. . . . DVL. VPLVVEEPSI
Consensus FDY-SWLG-C CENVIGY-- PWGVAGPLLL OGK- - - -EYS WPMATTEGC, HMGCoA binding E.
FIG. 32EE U.S. Patent Jun. 9, 2009 Sheet 63 of 78 US 7,544,863 B2
75 BOO HMGRclustalW (methanobac) VASVNRGCSV TRAGGATVR WTGDSMTRA PWIRTGSVVE ALQLREWIYE HMGR.clius talW (methanococ) VASVNRGCSI ITKCGGATVR WDOKMTRA PCLKTKSVVD AKVRDWIRE HMGRclustalW (halobacter) LASVNRGCSV NSAGGAAR WKSGMRA PWFRVADVAE AEALWSWTRD HMGRclustalW sulfolobus) IASVNRGIKA WTSGGWRAK WLKOEMT, RA PVFKFDSIEO PNFLKFEE HMGRclustalw yeast2} VASAMRGCKA NAGGGATIV LTKOGMT. RG PWVRFTLR SGACKWLDS HMGRclustalW { yeastl). VASAMRGCKA NAGGGAW TKOGMT. RG PVVRFTLCR SGACKWLDS HMGRclustalW (phycomyces VASTARGCKA NAGGGAST WIADGMT. RG PCVEFPTILR AAACKLWEN HMGRclustalW fusarium VASASRGCKA NSGGGAW LTADGMT. RG PCVAFETLER AGAAKLWOS HMGRclustalW candida} VASAMRGCKA NLGGGVW TKOGMT. RG PCVKPSLKR AGOCKLWLDS HMGRclustalW dictyoste2} VASTHRGAKA TKSGGAKTW LLOSGMT. RA PVCRPSSR AGELKQWIEN HMGRclustalW (wheatl ...... HMGRclustalW rice VASVNRRVQG HLWSGGASW PAWKPCPMR AAELKAFAEA HMGRclustalW corn. VASTNRGCKA AESG-GASW PWARFOTARR AAEKAFILEO HMGRclustalW (wheat3} ...... HMGRclustalW (wheat2) ...... HMGRclustalW ( soybean) ...... HMGRclustalW (rubbertre3) VASANRGCKA YASGGATSV LLRDGMT. RA PVVRFPTAKR AADKFFMEO HMGR.clustalW rosyperiwi VASTNRGCKA LASGGANSW IRDGMT. RA PVVRFGTAKR AAEKFYMED HMGRclustalW tomato VASTNRGCKA YASGGACE LROGMT. RA PCVRFGTAKR AAEKFVED HMGRclustalW (wood tobacc} VASTNRGCKA YASGGASW LLRCGMT. RA PCVRSGTAFOR AAELKFFVED HMGRclustalW potato VASTNRGCKA FWSGGADSW LLROGMT. R.A. PVV RFTAKR AAEKFFVED HMGRclustalW (radish) VASTNRGCKA MVSGGATST WLKOGMT. RA PWVREASARR ASELKFFLES HMGRclustal Warabadopsis...} VASTNRGCKA MFSGGATST WLKOGMT. R.A. PWWRFASARR ASKFFEN HMGRclustalW (cucumismel. VASTNRGCKA YASGGATSM LKOGMRA PVV RFGSAKR ASEKFFLED EMGRclustalW (rubbertre2} ...... GRclustalW (rubbertrel VASTNRGCKA YSGGATSV LKOGM. RA PWVRFASATR AAEROFFLEO BMGRclustalW camptothec) VASTNRGCKA FACGGATSV RDAMTRA PWRFGSAKR AADKFFLEN HMGRclustal Warabadops2} VASTNRGFKA EHLSGGAFSV WKDAMTRA PWWRFPSARR AALVMFYLOD HMGRclustalW (chineseham VASTNRGCRA GGGGASSR WADGMT. RG PVVRLPRACD SAEWKAWLET FIG. 32FF U.S. Patent Jun. 9, 2009 Sheet 64 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} WASTNRGCRA GLGGGASSR VLADGMT. RG PVVRPRACD SAVAWE HMGRclustalW (syrianhamst) WASTNRGCRA GLGGGASSR VLADGMT. RG PVVRPRACD SASVKAWET HMGRclustalW rat} WASTNRGCRA SLGGGASSR VLADGMS. RG PVVRPRACO SAEVKSWET HMGRclustalW rabbit} VASTNRGCRA CLGGGASSR VLADGMT. RG PVVR,2RAC SAEWKAWE HMGRclustalW human} GLGGGASSR WADGMT RG PWVRPRACD SAEWKAWLET HMGRclustalW mouse a 4- 8 C. P. HMGRclustalW ( xenopus WASTNRGCRA IMLGGGAKSR WEADGMT. RG PWVRLPTACO AAVKAWLDS HMGRclustalW (sea urchin) WASTNRGCRA LRSAGGIHSW LIGDGMT. RG PLVRLPSAQE AGAIKOWLEV HMGRclustalW cockroach WASTNRGMRA MRCG. WTSR VAOGMT. RG PWVRFPNIDR ASEAMLWMOV HMGRclustalW (drosophila) WASNRGCKA SWRGWRSW VEDVGMT. RA PCVRFPSWAR AAEAKSWIEN HMGRclustalW (dictyostel) WASTHRGCKA ITESGGAKCT ITSRGMT. RA pVWRFSDIVK ASEFWSWIND HMGRclustalW (schistosom} WASTNRGCSRA FAGGKSW WYRDQMT. RA PVWWFPSID SVKCIAWDS HMGRclustalW (archaeoglo WAAASNAARM ARESGGTTD YTGSLMIGQI QVTKLLN 9NA AKFEVLRQKD HMGRclustalW (pseudomonas) WAAASYMAKL ARANGGFS SSAPLMHAQV QIVGIQDPLN ARSLRRKD
Consen SS WASTNRGCKA --SGCASW WADGM-RA PWVRFPSAKR AAEKSWED
F.G. 32GG U.S. Patent Jun. 9, 2009 Sheet 65 of 78 US 7,544,863 B2
8O 8 SO HMGRclustalW (methanobac). NM. . DALREE AESTTRHGKL WKDP. . . . WAGSYWYPR FWYTTGDSMG HMGRclustalW (methanococ NF. . ERIKEv AESTTRHGKL KIEP. . . . WGRNLY PR FVFKTGDAMG HMGR.clustaw (halobacter NF. . AALKEA AEETTNHGEL. DWT. . . . . YWWGNSWYR FRYOTRDAMG HMGRclustalW sulfolobus NL. . EKIRNI ANSTSHHGKL RSITP . . . . . FVLGNNWWLR FSFETGDAMG HMGRclustalW yeast2} EEGQNSIKKA FNSTSRFARf. QHIOT. . . . . CAGDLFMR FRTTTGDAMG HMGR.clus talW yeast EEGONAIKKA FNSTSRFARL QHEIOT. . . . . CLAGDLLFMR FRTTGDAMG HMGRclustalW (phycomyces E.G. NDLVTNA FNSTSRFARL RK.K. . . . . AAGKVSR. FSTITGDAMG HMGRclustalW fusarium EAGQDMMKKA FNSTSRFARL QSMKT. . . . . ALAGTNLYIR FKTTGDAMG HMGRclustaW candida} DEGQEEMKKA FNSTSRFARL OH.QT. . . . . AAGDLFIR FRTWTGDAMG HMGRclustalW (dictyoste2 QENFYOVASA FNSTSRFARL KSKW...... WAGRVR KSSTGOAMG HMGRclustal (wheatl ...... a to e GOAMG HMGRclustalW rice PANFELLAAV FNRSSRFGRL QDRC. . . . . ALAGRNMR FSCITGDAMG HMGRclustalW corn) PANFDTLSVV FNRSSRFARL OGVOC. . . . . AMAGRNLYMR FSCSTGDAMG HMGRclus talW (wheat3} . . . . . is a up a s or p is a p up o F 8 or GDAMG HMGRclustalW (wheat2 ...... - GDAMG EMGRclustalW soybean ...... HMGRclustal wrubbertres }. PDNFDTIAVV FN KSSRFARL QSVOC. . . . . AAGKNYMR FSCSTGOAMG RMGRclustal Wrosyperiwi. TQNFETISVV FNKSSRFAKI. QS WQC. . . . . AIAGKNLYIR FSCSTGDAMG HMGRclustalW tomato PIKFESLANV FNQSSRFARL QRIOC. . . . . AAGKNLYMR CCSTGDAMG BMGRclustalW (wood tobacc). PVKFETLAAV FNQSSRFARL OROC. . . . . AAGKNLYMR FWCSTGDAMG HMGRclustalw potato) PLNFETLSLM FNKSSRFARL OGIQ.C. . . . . AACKNLYT FSCSTGDAMG HMGRclustalW radish PENFETLAVW FNRSSRFARL. OSVMC. . . . . TLAGKNAYVR FSCSTGDAMG HMGRclustalW (arabadopsisi PENFDTLAVV FNRSSRFARL QSVKC - . . . . TIAGKNAYVR FCOSTGDAMG HMGRcustaw (cucumismell PSNFDTLAVV FNRSSRFARI. OSRC. . . . . SAGKNYVR FCCSTGDAMG HMGR.clustal w{rubbertre2} ...... P. e. HMGRclustal w (rubbertrel} PDNFDTLAVV FN KSSRFARL OGIKC. . . . . SIAGKNYIR FSCSTGDAMG HMGRclustalW camptothec PLNFETLAAV FNSSSRFGKL QNIKC. . . . . AAGKNLYMR YSCSTGOAMG HMGRclustalwarabadops2. PSNFERLSLI FN KSSRFARL CSTC. . . . . TAGRNLYPR FACSTGDAMG HMGRclustalwchineseham PEGFAVIKDA FDSTSRFARL QKLHW. . . . . TMAGRNLYR FQSKTGDAMG F.G. 32.HH U.S. Patent Jun. 9, 2009 Sheet 66 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2) PEGFAWIKIDA FDSTSRFARL QKLHV. . . . . TMAGRNLYIR FQSKTGDAMG HMGRclustalW (syrianhamst) PEGFAWKDA FOSTSRFAR OKLHV. . . . . MAGRN.YISR FQSKTGDAMG HMGRclustalW rat PEGFAWWKEA FDSTSRFARL OKLHV. . . . . TLAGRN.YIR LQSKTGDAMG HMGRclustalW rabbit) PSEGFAWKGA FDSTSRFAR OKLH. . . . . SMAGRNLYR FOSRTGDAMG HMGRclustalW human) SEGFAWIKEA FDSTSRFAR QKHT. . . . . SEAGRNLYR FQSRSGDAMG HMGRclustalW mouse) HMGRclustalW xenopus ASGFKWKDA FDSTSRFARL GRLQN. . . . . CVAGRNLYIR FQSKTGDAMG HMGRcius talW (sea urchin) PSNFAAIKER FESTSRFAK, KSIOT. . . . . ALAGRYMFIR FKATGDAMG HMGRclustalW cockroach) PYNFEQIKKN FDSTSRFARL SKIHI. . . . . RVAGRHLFIR FIATTGOAMG HMGRclustalW (drosophila) DENYRVVKTE FOSSRFGRL KOCH . . . . . AMDGPOLYIR FWATGDRMG HMGRclustalW (dictyostel TDNYOALKAV FSTSRFARL SAIKC. . . . . TAGRSVYIR FKCOTGDAMG HMGRclustalW (schistosom} EcGOTLKSA FDKTSAHVNL, LSVFA. . . . . CPAGRY HIS FAARTGDAMG HMGRclustalW (archaeoglo ESRANSCD PMLVNLGGGC KDEAR: WID TIMGKMLIVH VDWKOAMG HMGRclustalW (pseudomonas EIELANRKD OLLNSLGGGC RDEVETFAO PRGPMWA WDWRDAMG Consensus PSNFELK-A FNSTSRFAR QSIQC - - - - - AAGRNLYR FSCSTGDAMG NADH binding domain li (continued)
FIG. 32 U.S. Patent Jun. 9, 2009 Sheet 67 of 78 US 7.544,863 B2
8S 9 OO HMGRclustalW (methanobac) MNMVTIATER ALELLT ... R. ETGAHV.. IA LSGNLCTDKK PAAVNLEGR HMGRclustalW (methanococ MNMVTIATEK ACNFIEGELK KEGIFVKTVA VSGNACVDKK PSGMNLNGR HMGRclustalW (halobacter) MNMATIATEA VOGVVE. . . A ETAASL. . VA LSGNLCSDKK PAANAVEGR HMGRclustalW (sulfolobus MNMVTIAVEK VCEFIE. . . . ENFPSADCLA vs GNMcSDKK QTNVNSLFGR HMGRclustalW ( yeast2} MNMISKGVEY SLKQMVEEY. . GWEDMEVVS VSGNYCTDKK 9AANWIEGR HMGRclustalW { yeastl). MNMISKGVEY SLKQMVEEY. . GWEDMEVVS VSGNYCTDKK PAAIN WIEGR HMGRclustalW (phycomyces MNM...... HMGRclustalW fusarium) MNMISKGVEH ALSVMANDG. . GFDDMQIIS VSGNYCTDKK AAALNWOGR HMGRclustalW candida} MNMISKGVEY ALKQMTEVF. . GWDDMMWVs vsgNYCTDKK PAAVNWINGR HMGRclustalW (dictyoste2} MNMVSKGVEK ALEVITEY. . . . FPEMEVLS LSGNVCTDKK PSSNWLEGR HMGRclustalW (wheatl. MNMVSKGVEN VLGYIRNN. . . . FPDMDVIS ISGNYCSDKK ATAVN WIDGR HMGRclustalW ( rice MNMVSKGVEN VLGYLQNV. . . . FPDMDVIS VSGNYCSDKK PTAVN WIEGR HMGRclustalW corn MNMVSKGVON VLDFLQDD. . . . FHDMDVIS ISGNFCSDKK PSAVNWIEGR HMGRclustalW (wheat3} MNMISKGVQN VLDYLQDD. . . . FPDMDVIS ISGNFCSDKK PAAVN WIEGR HMGRclustalW wheat 2} MNMISKGVQH VLDYLEED. . . . FPDMDVVS ISGNFCSDKK SAAVN WIEGR HMGRclustalW scybean ...... HMGRclustalW (rubbertre3} MNMVSKAVQN VIDYLQND.. . FPDMDVIG LTGNFCADKK AAAVN WIEGR HMGRclustalW rosy periwi) MNMVSKGVQN VLEFLQTD. . . . YPDMDVLG ISGNFCADKK PAAVNWEGRHMGRclustalW tomato) MNMVSKGVON VLDYLONE. . . . YPDMDVIG ISGNFCSDKKs PAAVNWEGR HMGRclustalW (woodtobacc} MNMVSKGVQN VLDYLQNE. . . . YPDMDVIG IsGNFCSDKK PAAVNWEGR HMGRclustalW potato) MNMVSKGVON VLDYLQSE. . . .YPDMDVIG ISGNFCSDKK PAAVNWIEGR HMGRclustalW radish) MNMVSKGVQN VLEFLTED. . . . FPDMDVIG ISGNFCSDKK PAAVNWEGR HMGRclustalW (arabadopsis l MNMVSKGVON VLEYLTDD. . . . FPDMDVIG ISGNFCSDKK PAAVNWEGR HMGRclustalW (cucumismel} MNMVSKGVQN VLEFLQHD. . . . FSDMEVIG ISGNFCADKK PAAWNWIEGR HMGRclustalW (rubbertre2} ...... LESD. . . . FADMDVIG ISGNFCSDKK PAAVN WIEGR HMGRclustalW rubbertrel} MNMVSKGVON VLEFLQSD. . . . FSDMDVIG ISGNFCSDKK PAAVNWEGR HMGRclustalW camptothec MNMISKGVQN VLDFLQDD. . FPDMDVG SGNYCSDKK PAAVNWIEGRHMGRclustalwarabadops2} MNMVSKGVON VLDFVKSE. . . . FPDMDVIG ISGNYCSDKK ASAVNWIEGR HMGRclustalW (chineseham) MNMISKGTEK ALLKLQEF. . . . FPEMQILA VSGNYCTDKK PAAIN WIEGR FIG. 32JJ U.S. Patent Jun. 9, 2009 Sheet 68 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} MNMISKGTEK ALLKLQEF. . . . FPEMQILA WSGNYCTDKK PAAN WIEGR HMGRclustalW (syrianhamst) MNMISKGTEK ALVKLQEF. . . . FPEMOILA vs GNYCTDKK PAAWNWIEGR HMGRclustalW ( rat) MNMISKGTEK ALLKLQEG. . . .VPELOILA WSGNYCTDKK PAAN WIEGR HMGRclustalW ( rabbit) MNMISKGTEK ALSKLHEY. . . . FPEMOILA WSGNYCTDKK AAWNWEGR HMGRclustalw human} MNMISKGTEK ASKLHEY. . . . FPEMOILA WSGNYCTDKK PAAN WIEGR HMGRclustalW mouse) is us 4 - - - EK ALLKOEF. . . . FPDMOILA WSGNYCTDKK PAAN WEGR HMGRclustalW xenopus MNMISKVTEO ALARLQEE. . . . FPDLHVLA VSGNYCTDKK PAANWEGR HMGRclustalW (sea urchin} MNMISKGEQ ALHALOT.M. . . . FPNIEIMS LSGNYCTDKK WAANWEGR HMGRclustalW cockroach MNMLSKGTEV ALAYVQQV. . . . YPDMEILS LSGNFCTDKK PAAWNWEGR HMGRclustalW (drosophila) MNMVSKALRW PFAEFTL.H. . . . FPDMQIIS LSGNFCCDKK PAAINWKGR HMGRclustalW (dictyostel) MNMVSKGWEA WLEHL.K. . . . FDDMTLLS ISGNMCTOKX PSSINWTEGR HMGRclustalW (schistosom} MNMVSKATDS ALHCLKKY. . . . FSNMOVIS LSGNMCTDKK PATENTILGR HMGRclustalW (archaeoglo ANAVNTMCEK VAPFIERITG GKVYLRS NLAAYRLARA KAVFOKDVG HMGRclustalW (pseudomonas ANVNMAEA WAPLMEATG . GOVRLRILS NLA DRLARA QVRITPQQLE Cosensus MNMVSKGWEN VL--LQED- - - GFPDMDVIS SGNYCTDKK PAAVNWEGR NADH binding domain l (concluded)
FIG. 32KK
U.S. Patent Jun. 9, 2009 Sheet 71 of 78 US 7,544,863 B2
OOO HMGRclustalW (methariobac). AHYANIIGAI FLATGQDEAH IVEGSEGVT - 8. GOLYF HMGRclustalW (methanococ) AHYANIIGAI FLATGODEAH IVEGSLGITM OGLYF HMGRclustalW (halobacter) AHVANVVAAM FLATGQDEAQ WWEGANAITT e s to up GDIYV HMGRclustalW (sulfolobus) AHFANIVTAI FIATGQDVAQ IVESSSGYTW O to O O. O. EDLY HMGRclustalW ( yeast2. AHAANLVTAL FLALGQDPAQ NVESSNCIT, . . . . . GDR HMGRclustalW ( yeast 1 AHAANLVTAV FLALGQDPAO NVESSNCT 0 d as GDLR
HMGRclustalW (phycomyces ...... vP 4 w P. P. E. O. P. HMGRclustalW fusarium. AHAANIVAAI FLATGQDPAQ VVESANCIT s s as GALOI HMGRclustalW candida} AQAANMVTAV YALGODPAO NVESSNCITL MTSETEO. . . . o GOLOW HMGRclustalW (dictyoste2 AHASNIVTAL YIATGQDPAQ NVESSNCTL MESNGG . . . KDLY HMGRclustalW (wheatl. AHASNIATAL FATGQDPAQ NVESSQCITM LEAVNEG. . . to do KLH HMGR.clustalW rice AHASNIVTAL FIATGQDPAQ NVESSQCITM EEVNOG... . . O X - 8 DOH HMGRclustalW corn AHASNIVTAI FIATGQDPAQ NVESSHCITM EPWNAG . . P RDLH HMGRclustalW (wheat3. AHASNIVTAI FIATGQDPAQ NVESSQCIAM LEAVNDG. . . 4 KOLE HMGRclustalW (wheat2} AHASNIVSAI FIATGQDPAQ NVESSQCITM LEAVNGG. . . a ROH HMGRclustalW ( soybean AHASNIVSAI FIATGQDPAQ NVESSHCTM MEANDG. . . up o do ROH HMGRclustalW (rubbertre3 AHASNMVTAV YIATGQDPAQ NVESSHCTM MEAVNDG. . . a KDH HMGRclustalW (rosyperiwi AHASNIVSAI FIATGQDPAQ NVESSQCTM MEAVNOG O HMGRclustalW { tomato. AHASNIVSAV FIATGQDPAQ NESSECTM MEAVNDG. . . P KD HMGRclustalwwoodtobacc} AHASNIVSAV YIATGQDPAO N5SSHCITM MEAVNDG. . . O P 0 O KDEV BMGRclustalW potato AFIASNIVSAV YLATGQDPAQ NVESSHCTM MEAVNDG. . . p KDLHV HMGRclustalW (radish RHASNIVSAV FLATOQDPAO NVESSCCITM MEANDG. . . B is KDS HMGRclustalW (arabadopsisi AHASNIVSAV FIATGQDPAQ NVESSQCITM MEANOG O KDE BMGRclustalW (cucumismel. AHSSNIVSAI FLATCODPAQ NVESSECTM MEPWNNG. RDLE HMGRclustalW rubbertre2 AHAGNIVSAI FIATGQDPAQ NVESSHCTM MEAVNOG... . . do 0 - P KDLH HMGRclustalW (rubbertrel} AHAGNIVSAI FIATGQDPAO NVESSRICITM MEAVNDG. . . KOE HMGRclustalW (camptothec. AHASNIVSAV YLATGODPAQ NVESSHCTM MEANOG o v as a KDLHW HMGRclustalW (arabadops2 AHSSNIVSAV FATGQDPAQ NVESSHCMTM LPCGD . . . . o o or o a DLHI HMGRclustalW (chineseham) AHAANIVTAI YACGODAAQ NVGSSNCT, MEASGPTN. . FIG. 32NN U.S. Patent Jun. 9, 2009 Sheet 72 Of 78 US 7,544,863 B2
. . . . . EDIYI HMGRclustalW (chineseha2) AHAANIVTAI YIACGQDAAQ NVGSSNCITE, MEASGPTN . . . . . EDLYI HMGRclustalW (syrianhamst) AHAANIVTAI YIACGQDAAQ NVGSSNCITE, MEASGPTN. EDLY HMGRclustalW rat) LHAANIVTAI YIACGQDAAQ NVGSSNCIT, MEASG9N. s p - as EDLY HMGRclustalW ( rabbit. AHAANYVTAI YIACGQDAAQ NVGSSNCTL MEASGPPN. 0 EOY HMGRclustalW human AHAANIVTAI YIACGQDAAQ NVGSSNCTL MEASGPTN . . - EDLY HMGRclustalW mouse AHAANIVTAI YIACGQDAAQ NVGSSNCIT, MEASGTN . w w - as EOY HMGRclustalW xenopus) AHAANIVTAI YIACGQDAAQ NVGSSNCIT MEATGPTY. P - EDLY HMGRclustalW (sea urchin} AHAANIVTAI YIATGQDAAQ NIASSNCMT, METRGKG - - GO.Y. HMGRclustalW cockroach). AEAANIVTAI FIATGQDPAQ NVGSSNCAMT, MEPWGEOG d p. 8 KOLYW HMGRclustalW (drosophila AHAANMVTAV FLATGQDPAQ NVSSNCSTA MECAENS...... EOYM HMGRclustalW (dictyostel. AHASNIVTAI FLATGQDCAQ NVESSNCTO MEACNDG. QDLY HMGRclustalW (schistosom AHAANIIAGM FAATGQDLAQ VVDSSSCLTQ LEVOLSD. . . 8X DSLWA HMGR.cliustalW (archaeoglo ...... ATGNOFrA IEAGASYAA GGYKPLTT YEWDRKGNLV HMGRclustalW (pseudomonas) ...... WAGNOWRA WEAGAHAYAC RSGHYGSTT WEKONNGHV
Consensus AEAANVTAI FIATGQDPAO NVESSNCTM MEAVNOGN------KDE
D
F.G. 32OO U.S. Patent Jun. 9, 2009 Sheet 73 of 78 US 7,544,863 B2
OSO HMGR.clustalW methanobac AVNLPDVPLA TWGGGTGLET ASECLDIMGV RGGG...... RWEAFAEWG HMGRclustal wraethanococ SVTLPDVBIG TVGGGTRVET OKECLEMGC YGON...... KAKFAEWG HMGRclustai Whalobacter SVSIASLEVG TWGGGTKLPT OSEGOILGW SGGGDP. AGS NADALAECIA HMGRclustalW (sulfolobus) SVTLPSLEVG TVGGGTRLPT QKEALSIMGW YGSGNP. PGS NAKKLAEIA HMGRclustalW { yeast2) SVSMPSIEVG TIGGGTVLEP (CGAM.DLLGV RGPHPTEPGA NAROLAREA HMGRclustalW ( yeast. SwsMPSIEvG TIGGGTVLEP OGAM.DLLGV RGPHATAPGT NARCARIVA HMGRclustalW (phycomyces ...... p s h a a - do a HMGRclustalW fusarium) SVSMPSLEVG TLGGGTILEP CGAMLDILGW RGSHPTNPGD NARRARIG HMGRclustalW candida} SVSMPSIEVG TIGGGTILDP QGSMELLGV RG 9ADWPGE NARQ,AKVA HMGRclustalW dictyoste2} SVTMPSIEVG TVGGGTHLPA OSACfDLLKI RGANERPGA NSEQLARVVA HMGRclustalW wheati) SVTMPPIEV...... HMGRclustalw rice SVTMPSIEVG TIGGGTCLAS CAACLNLLGV KGSNHGSPGA NAGRATIVA HMGRclustalW corn SVTMPSIEvG TWGGGTQLAS OSACOLLGV RGASRORPGS NARLLATWVA HMGRclustalW (wheat3 SVTMPPIEV......
HMGRclustai W (wheat2} SVTMPPIEV...... a - us - HMGRclustalw soybean SVTMPSISVG TVGGGTQLAS QSACTNLLGV KGASKESPGS NSRLLATIVA HMGRclustalW rubbertres } SVSMPSIELG TVGGGTQEAS OSACTNLLGV KGASKCSPGS NSRLLATIWA BMGRclustalW rosyperiwi) SVTMPSIEVG TVGGGTQLAS OSACNLLGW KGASKDSPGA NSRLLATIVA HMGR.clustalW { tomato SVTMPSIEVG TWGGGTQLAS QSACLNLLGV KGANREAPGS NARAWWA HMGRclustalW (wood tobacc} SVTMPSISVG TVGGGTQLAS QSACLNLLGV KGAN REVGS NARATWA HMGRclustalW potato swimpsIEvG TVGGGTOLAS OSAC.NLLGV KGANSDAPGS NARATIVA HMGRclustalW radish SVTMPSIEVG TVGGGTQLAS QSACLNLLGW KGASKESPGM NSRRAWA HMGRclustal Warabadopsisi SVTMPSIEVG TVGGGTQLAS QSACNILGW KGASTESPGM NARRATIWA HMGRclustalW (cucumismel SVTMPSISVG TVGGGTQLAS QSACLNLLGV KGASKESPGA NSRLATIVA HMGRclustalW rubbertre2} SVTLPSIEVG TWGGGTQIAS OSACLNLLGV MGACKESGS YSRATWA HMGRclustalW rubbertrel SVTMPSIEVG TVGGGTQLAS OSACLNILGW KGANKESPGS NSRLLAAIWA HMGRclustalW camptothec) SVTMPSIEVG TVGGGTQLAS QSACLNLLGV KGASKEAPGS NARATWA HMGRclustalW (arabadops2} SVSMPCIEVG TWGGGTQLAS QAACLNLLGV KGSNNEKPGS NACQARIWA HMGRclustalwchineseham SCTMPSIEIG TVGGGTNLLP QQACLCMLGV OGACKDNPGE NARQL.ARIWC F.G. 32PP
U.S. Patent Jun. 9, 2009 Sheet 75 Of 78 US 7,544,863 B2
OO HMGRclustaw (methanobac GAVAGEISL MGALAAGHLA HMGRciustal w (methanococ) AAWAGES WGSAGES,
HMGRcustalW sulfolobus STWLSGELNL EMGRclustalW yeast2. CAVAGELSI OPS...... HMGRclustalW ( yeastl CAVLAGEISL ATDI...... Grciustalw{phycomyces} & is 8 + · · · HMGRclustalW eusarium) AAVAGES CSAAAGHLW RAMCHNRSA SHOARTGH HMGRcustalw{ candida} SWSGESL WSAAAGHLW OSEMOENRAA F we - - - AAVSGESL MSAAAGHW RSLKENRK SMHNLPESO HMGR.clustalW (wheatl as as a
is ...... 4- 8 HMGRclustalW ( GSWWAGRALL AAASGHIV GGVAGELS LSALAAGOLV
vs as w- HMGR.clustalW (wheat) as a v is 4 - d.
------HMGR.clustalW (wheat2) & q as we w a d - d 8 HMGR.clustalW { soybean) GSWAGES MSAIAAGOLV NSMKYNRSS HMGR.clustal w{ rubbertre GSWAGELS MSAIAAGQLV NSE MKYNRSA HMGRciustalw{ rosyperii) GSWAGES MSAESAGQLV RSMKYNRSS OTN. . . . . s- HMGRclustalWas a s s as e. ( tomato) GSWAGES MSASSGOLV NSMKYNRST KDWK. . . . . HMGRciustalw(woodtobacc) GSWAGES. MSAISAGOLV KSEMKYNRST KDWTK. . . . . HMGRclustalW ( potato) GSWAGELS MSAISAGQLV KSEMKYNRS DSK as GrclustalW (radish) GAVLAG2LSL MSAAAGOV RSE MKNRSS RDSG. . . . . HMGRclustalwcarabadopsis)- - GAVIAGELSI MSAAAGOLV RSMKYNRSS RDISG. . . . . Grciustalw (cucumismel) GSVLAGELS MSAIAAGQLV RSE MKYNRSS RDVSK. . . . . HMGR.clustalW (rubbertre2} GSWLAGELS MSAAAGOLV KSEMKNRSS KDVSK. . . . . HMGRciustalw(rubbertrel) GSVLAGES MSAAAGOLW KSEMKYNRSS KOMSK. . . . . HMGRciustalw (camptothec) GSWAGES. MSAAAGOLV NSMKYNRSN GSVLAGELS MSAAAGOLV KSEMKYNRSS
Gristalw(chineseham) GVMAGES MAALAAGHLV RSMVNRSK FG. 32RR U.S. Patent Jun. 9, 2009 Sheet 76 of 78 US 7,544,863 B2
4 8 8 d 4 HMGRclustalW (chineseha2} GTVMAGELSL MAALAAGHLV RSHMVENRSK INLOD. . . . . HMGRclustalW (syrianhamst} GTVMAGELSL. MAALAAGHLV RSEMVENRSK INLOD. . . . . X X w. 8 ty HMGRclustalW rat} GTVMAGELSL MAALAAGHLV RSHMVHNRSK INLQD. . . . . HMGRclustalW rabbit} GTVMAGELSL MAALAAGHLV KSEMIHNRSK (NLOD. . . . .
s p as 0 8 HMGRclustalW human GTVMAGELSL MAALAAGHLV KSEMIHNRSK INLQD. . . . . HMGRclustalW ( mouse GTVMAGELSL MAALAAGHLV RSEMVHNRSK INLQD. . . . .
8 8 X d - EMGRclustalW XeOS STVMAGELSL MAALAAGHLV KSEMVENRSK INLOD. . . . . HMGRclustalW sea urchin ATVMAGELSL, MSALAAGHLV KSEMKENRSA INIASPLPSI DEWARRSK HMGRclustalW cockroach) GTVLAGELSL, MSALAAGHLV KSEMRHNRSS VSTSG. . . . .
HMGRclustalW (schistosom GTVLAAESL. MAALDTDDLV KAHMHFNRAK QSTNSHSCSH STTTDNNDNI EMGRclustalW (archaeoglo) . . GLAQNFAA LRALATEGIQ RGEMELEARN LA IMAGATGD EVORVVEIMV HMGRclustalW (pseudomonas . . GLAQNLGA MRALATEGIQ RGHMALEARN IAVVAGARGD EVDWVARQLV Consensus GVAGES, MSAAAGHLV KSMK-NRSS KDWSK------
FIG. 32.SS U.S. Patent Jun. 9, 2009 Sheet 77 of 78 US 7,544,863 B2
52 HMGRclustalW (methanobach ......
HMGRciustalW (methanococ} a o o o o v e o so as o 0 - F. p. p a r s p so a e e s o O e o as s
HMGR.clustalW (halobacter) o a o o e s e e a o O 9 to - d - - a p e g o a a s
HMGRciustalW (sulfolobus) - - e o a ta HMGRciustalW yeast2} ...... NKGPPCKT SALL...... BMGRciustalW Yeastl} ...... NRLKDGSW TCIKS ......
AMGRciustalW (phycomyces) s p q r u up up e s is a sp. o a a . . . . .
HMGRciustalw{ fusarium) QCPRALSVNN VDERRRYSEV KAIDE. . . . . is a u o is n e o O a HMGRciustalW ( candida} ...... HMGRciustalw{dictyoste2} HMGR.clustalW (wheat1) BMGRciustalw{ rice} ...... A . . . . . AS ------d to o HMGR.clustalw{ corn ...... T . . . . . TATEK TRQREVDV......
e us as HMGRclustalW (wheat3} as s 8 s - O to b to 8 o o up to a so es us . . . . s. v. s ld 4 HMGR.clustalW (wheat2} BMGRciustalw{ soybean) ...... I - - - - - S. ------...... HMGR.ciistalW (rubbertre3} ...... I - - - - - TF...... HMGR.ciistalw{rosype=iwi) w I . . . . . ASSQL ESDS ...... HMGRciustalwt tomato ...... A . . . . . SS------
HMGReinstalw{woodtobacc} - P - P - vo e - e. A . . . . . SS------...... figreinstalw( potato) ...... to 8 O b HMGRclustalw{radish} ...... A . . . . .T.T.T......
fierciustalW (arabadopsis1} to up or O - to a s a A . . . . . T.T......
EMGRciustalW (cucumismel} a as a - - - - - ES------......
HMGR.ciistalw(rubbertre2) ------A - - - - - AS......
HMGR.ciistalw(rubbertrel} o O p p. A . . . . . AS...... - - - -
fierciustalW (camptothec} o e o so to e s s A . . . . . SS. ------HMGRciustalw{arabadops2} O th S - - - - - SOVNR ......
Big ReinstalW (chineseham) 0 O to U. S. . . . LOGTCTK KSA......
e - so e o so a o or a FIG. 32TT U.S. Patent Jun. 9, 2009 Sheet 78 of 78 US 7,544,863 B2
HMGRclustalW (chineseha2} ...... LOGTCTK KSA......
timerciustalW (syrianhamst} - Y ------. . . LQGTCTK KAA ...... HMGRciustalW { rat ...... LOGTCTK KAA...... HMGRciustalW ( rabbit} ...... LEGACTK KAA...... AMGRciustalW { human} ...... ICGACTK KTA ...... HMGRciustalW { mouse ...... LQGTCTK KAA...... HMGRciustalw{ xenopus) ...... LPGTCTK KAA...... HMGRciustalW (sea urchin SVDFSAL:GeS SAAAPGTCTA NAS...... HMGRciustalw{ cockroach ...... S . . . EPSTPAC KS......
BMGR.ciistalW (drosophila} ------NP NVVSSCS. S......
s HMGRciustalW (dictyostel} 8 as a a w w w s a a e o 8 - 8 - e s a s a e s is s s s s s s a HMGRclustalW schistosom} SNYNENVA LSSKPVTON SdRESVHS, HIVKFPWXSD SWNPESHY TM HMGRclustalW (archaeoglo) RDGKIRLYA KeVLERLRs...... EMGRclustalW (pseudomonas EYHDVRADRA VALLKQKRGQ ......
Cosensus ------A - - - OGTOTK KAA ------
F.G. 32UU US 7,544,863 B2 1. 2 TRANSGENC PLANTS CONTAINING an enzyme binding a first sterol and producing a second ALTERED LEVELS OF STEROD sterol—altered carotenoid, tocopherol, modified FA levels— COMPOUNDS HMGR, 5C.-reductase, geranylgeranyl pyrophosphate Syn thase, phytoene synthase, phytoene desaturase, isopentenyl The present application is a divisional application of U.S. 5 diphosphate isomerase). patent application Ser. No. 09/885,723, filed Jun. 20, 2001, Acetate is the metabolic precursor of a vast array of com now U.S. Pat. No. 6,822,142, which application claims ben pounds vital for cell and organism viability. Acetyl coenzyme efit of priority from U.S. Provisional Application Ser. No. A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3 60/260,114, filed Jan. 5, 2001. methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to 10 mevalonate in an irreversible reaction catalyzed by the TECHNICAL FIELD enzyme HMG-CoA reductase. Mevalonate is phosphorylated and decarboxylated to isopentenyl-pyrophosphate (IPP). The present invention relates to biotechnology with an Through the sequential steps of isomerization, condensation emphasis on plant biotechnology, and particularly biotech and dehydrogenation, IPP is converted to geranyl pyrophos nology affecting the biosynthesis of Steroid compounds. 15 phate (GPP). GPP combines with IPP to form farnesyl pyro phosphate (FPP), two molecules of which are reductively BACKGROUND condensed to form squalene, a 30-carbon precursor of sterols. Enhancement of the nutritional or health benefits of oils A key enzyme in sterol biosynthesis is 3-hydroxy-3-meth through genetic engineering is being addressed throughout ylglutaryl-Coenzyme A reductase (HMG-COA reductase or HMGR). Schaller et al. (Plant Physiol. 109: 761-770, 1995) the agricultural community. Several approaches involve found that over-expression of rubber HMGR (hmg1) manipulation of already present cellular biosynthetic path genomic DNA in tobacco leads to the overproduction of sterol ways. Steroid biosynthetic pathways are of current interest, end-products (sitosterol, campesterol and stigmasterol) up to particularly for the enhancement of health benefits from food 6-fold in leaves. Further, the excess sterol was stored as steryl oils. 25 Several related U.S. patents address increasing sterol accu esters in lipid bodies. HMGR activity was increased by 4- to mulation in higher plants. Those patents include U.S. Pat. No. 8-fold. Sterols are derivatives of a fused, reduced ring system, 5,589,619 “Process and Composition for increasing squalene cyclopenta-a-phenanthrene, comprising three fused cyclo and sterol accumulation in higher plants” (accumulation of hexane rings (A, B, and C) in a phenanthrene arrangement, squalene in transgenic plants by increasing HMGR activity) 30 and U.S. Pat. No. 5,306,862 “Method and composition for and a terminal cyclopentane ring (D) having the formula (I) increasing sterol accumulation in higher plants' (increasing and carbon atom position numbering shown below: HMGR activity to increase plant sterol accumulation in cluding sterol and cycloartenol, which affects insect resis (I) tance—in tobacco, tomato, corn, carrot, soybean, cotton, bar 35 ley, arabidopsis, guayule and petunia; seeds with elevated sterol/cycloartenol. 7S promoter and CaMV promoters), U.S. Pat. No. 5.365,017 “Method and composition for increasing sterol accumulation in higher plants” (DNA construct with (C) HMGR-CaMV 35S, transgenic plants, hybrid plants, corn, 40 soy, barley, tomato, Arabidopsis), U.S. Pat. No. 5,349,126 “Process and composition for increasing squalene and sterol accumulation in higher plants' (increase in squalene and sterol accumulation by increasing HMGR activity in trans genic tobacco, cotton, soybean, tomato, alfalfa, Arabidopsis, 45 where R is an 8 to 10 carbon-atom side chain. corn, barley, carrot and guayule plants), and EP 486290 (en In plants, squalene is converted to squalene epoxide, which hancement of squalene and specific sterol.squalene Zymos is then cyclized to form cycloartenol (4.4,14C.-trimethyl-93, terol, cholest-7,24-dienol, cholest-5.7.24-trienol accumula 19-cyclo-5C.-cholest-24-en-3?-ol). Cycloartenol has two tion in yeast by increasing HMGR activity in yeast deficient methyl groups at position 4, a methyl group at position 14, a in enzymes that convert squalene to ergosterol). 50 methylene bridge between the carbon atoms at positions 9 In those patents, the amount of a protein exhibiting 3-hy and 19 that forms a disubstituted cyclopropyl group at those droxy-3-methylglutaryl Coenzyme-A reductase (HMGR) positions, and includes an 8-carbon sidechain of the formula: activity is typically increased. HMGR widens a “bottleneck” CHCH(CH)CH=C(CH). Squalene epoxide can alter near the beginning of a biosynthetic path to steroid produc natively be converted into pentacyclic sterols, containing five tion, permitting a higher carbon flux through steroid biosyn 55 instead of four rings. Exemplary pentacyclic sterols include thetic pathways and resulting in increased sterol accumula the phytoalexins and Saponins. tion. Being one of the first sterols in the higher plant biosynthetic U.S. Pat. No. 5,480,805 “Composition for modulating ste pathway, cycloartenol serves as a precursor for the production rols in yeast” (enhancement of delta 8-7 isomerase activity of numerous other sterols. In normal plants, cycloartenol is ERG2 enhances accumulation of specific sterols in yeast). 60 converted to predominantly 24-methylene cycloartenol (4.4, U.S. Pat. No. 5,460.949 “Method and composition for 14C.-dimethyl-9B. 19-cyclo-22.23-dihydro-ergosta-24(28)- increasing the accumulation of squalene and specific sterols en-3-3-ol), cycloeucalenol, (4,14C-trimethyl-9B. 19 cyclo in yeast' (increasing squalene, Zymosterol and specific Ste 5C-ergosta-24(28)-en-3?-ol), isoflucosterol (5C.-Stigmasta-5- rols in yeast by increasing HMGR in yeast having decreased 24(28)-dien-3?-ol), sitosterol (5C.-stigmasta-5-en-3?-ol), erg5 and erg6 activity—Sc and hamster HMGR). 65 Stigmasterol-(stigmasta-5,-22-dien-33-ol), campesterol (5C.- WO9845457 (SMTI, Erg6 from A.t., corn, yeast; trans ergosta-5-en-3?-ol), and cholesterol (5C.-cholesta-5-en-3? genic plants with altered sterol levels using DNA encoding ol). These transformations are illustrated in FIG. 1. US 7,544,863 B2 3 4 Although sterols produced by plants, and particularly EP 480730“Plant-sterol accumulation and pest resistance higher (vascular) plants, can be grouped by the presence or by increasing copy number of 3-hydroxy-3-methyl glutaryl absence of one or more of several functionalities, plant sterols coenzyme-A reductase gene in tobacco, tomato and corn are classified into two general groups herein; i.e., those con WO 9913086 “Human Delta 7-sterol reductase polypep taining a double bond between the carbon atoms at positions tide-useful for diagnosis or treatment of genetic defects e.g. 5 and 6 (delta-5 or A5 sterols) and those not containing a hereditary Smith-Lemli-Opitz syndrome' teaches making double bond between the carbon atoms at positions 5 and 6 (non-delta-5 sterols). and using the recombinant polypeptide with humans. Exemplary naturally-occurring delta-5 plant sterols are Chappellet al. U.S. Pat. No. 5,589,619 teaches that trans isoflucosterol, sitosterol, Stigmasterol, campesterol, choles 10 formation of higher plants with truncated HMG-CoA reduc terol, and dihydrobrassicasterol. Exemplary naturally occur tase enhanced the production of squalene, cycloartenol and ring non-delta-5 plant sterols are cycloartenol, 24-methylene certain sterols, particularly compounds having unsaturations cycloartenol, cycloeucalenol, and obtusifoliol. The most at the 5-position. Several intermediate sterols as are shown in abundant sterols of vascular plants are campesterol, sito FIG. 1 were also produced. It would be beneficial if the sterol, and stigmasterol, all of which contain a double bond 15 production of sitosterol and Stigmasterol could be enhanced between the carbonatoms at positions 5 and 6 are classified as while lessening the accumulation of the intermediate sterols. delta-5 sterols. The present invention provides avenues for enhancing pro The HMG-CoA reductase enzymes of animals and yeasts duction of sitosterol and stigmasterol and lessening the accu are integral membrane glycoproteins of the endoplasmic mulation of the intermediate sterols. reticulum. The intact enzyme comprises three regions: a cata Gonzalez et al. (Abstract of poster at Third Terpnet Meet lytic region containing the active site of the enzyme; a mem ing of the European Network on Plant Isoprenoids, May brane binding region anchoring the enzyme to the endoplas 29-30, 1997, Poitiers, France) over-expressed the Arabidop mic reticulum; and a linker region joining the catalytic and sis HMGR CDNA (hmg1 and himg2) and found sterol over membrane binding regions of the enzymes. The membrane production with himgil only. They used two forms ofthehmg1 binding region occupies the amino-terminal (N-terminal) 25 gene, a full-length form and a truncated form containing only portion of the intact protein, whereas the catalytic region the catalytic domain. HMGRs have three domains, an N-ter occupies the carboxy-terminal (C-terminal) portion of the minal membrane spanning domain, a short linker domain, protein, with the linker region constituting the remaining and a C-terminal catalytic domain. In this case the transgenic portion. M. E. Basson et al., Mol. Cell Biol. 8(9):3797-3808 plants were also Arabidopsis. The difference between the (1988). 30 full-length and truncated forms was a greater accumulation of The activity of HMG-CoA reductase in animals and yeasts pathway intermediates in the case of the truncated form. More is known to be subject to feedback inhibition by sterols. Such importantly, the intermediates demonstrated as accumulating feedback inhibition requires the presence of the membrane were cycloartenol, 24-methylenecycloartanol and obtusifo binding region of the enzyme. See, e.g., G. Gil et al. Cell. liol. 35 41:249-258 (1985); M. Bard and J. F. Downing, J. Gen. Finally, U.S. Pat. Nos. 5,365,017 and 5,306,862, both Microbiol., 124:415-420 (1981). assigned to Amoco Corp., disclose a method for increasing Given that mevalonate is the precursor for sterols and other sterol accumulation in plants by increasing the copy number isoprenoids, it might be expected that increases in the amount of a gene having HMG-CoA reductase activity. These inven or activity of HMG-COA reductase would lead to increases in 40 tions disclose a method usinghamster truncated HMGR that the accumulation of both sterols and other isoprenoids. consisted of the catalytic domain and the linker domain. In mutant strains of the yeast Saccharomyces cerevisiae (S. According to the claims the linker domain was essential for cerevisiae) having abnormally high levels of HMG-COA activity. They also demonstrated a greater accumulation of reductase activity, the production of two sterols, 4, 14-dim pathway intermediates Such as cycloartenol. ethylzymosterol and 14-methylfucosterol is markedly 45 increased above normal. Downing, et al., Biochem. Biophys. BRIEF SUMMARY Res. Comm., 94(3): 874-979 (1980). When HMG-COA reductase activity was increased by illu The present invention relates to transgenic plants and their mination in non-photosynthetic microorganisms, isoprenoid progeny having improved nutritional characteristics. More (carotenoid), but not sterol (ergosterol), synthesis was 50 particularly, the present invention relates to transgenic plants enhanced. Tada, et al., Plant and Cell Physiology, 23(4):615 and their progeny, the storage organs (e.g. seed, fruit and 621 (1982). vegetable parts) of which contain modified levels of steroid WO 9703202 discloses a method for identifying agents compounds, such as (i) elevated levels of beneficial phytoster modulating sterol biosynthesis using a yeast acetoacetyl CoA ols (e.g., sitosterol), phytostanols (e.g., sitostanol), and esters thiolase (ERG10) gene linked to a reporter system to evaluate 55 thereof, relative to an otherwise identical plant transformed compounds, such as lovastatin and other HMG-CoA synthase only with a truncated HMG-COA reductase gene or a wild inhibitors, that affect cholesterol biosynthesis. type plant, and (ii) reduced levels of steroid pathway inter U.S. Pat. No. 5,668,001 teaches a recombinant avian mediate compounds (e.g. one or more of squalene, HMG-COA synthase preparation useful for evaluating drugs cycloartenol, 24-methylene cycloartenol, obtusifoliol, Stig that inhibit cholesterol biosythesis. 60 masta-7-enol and campesterol) in their storage organs relative JP 09 121863 discloses a plant with increased 3-hydroxy to an otherwise identical transgenic plant transformed only 3-methylglutaryl coenzyme A reductase (HMGR) activity as with a truncated HMG-CoA reductase gene. Nucleic acid a result of increasing the expression of a mutant protein sequences encoding enzymes that affect the biosynthesis and kinase gene that regulates expression of the HMGR gene. The accumulation of steroid compounds in plants (HMG-CoA increased HMGR activity increases squalene, Zymosterol, 65 reductase and a steroid pathway enzyme), and methods for cholesta-7.24-dienol and cholest-5,724-trenol accumulation using these sequences to produce Such transgenic plants, are in yeast with ERG5 and ERG6 mutants. also provided. These methods comprise, for example, intro US 7,544,863 B2 5 6 ducing into cells nucleic acid sequences encoding enzymes and introduced DNA encoding a steroid pathway enzyme that that affect the levels of accumulated steroid pathway end is a squalene epoxidase enzyme, a sterol methyl transferase I products. enzyme, a sterol C4-demethylase enzyme, a obtusifoliol The present invention contemplates a recombinant con C14C-demethylase enzyme, a sterol C5-desaturase enzyme, struct or a recombinant vector that contains 2 DNA or a sterol methyl transferase II enzyme. The storage organs sequences. The first encodes a polypeptide exhibiting 3-hy of such a plant contain an elevated level of total accumulated droxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reduc sterol, compared to storage organs of an otherwise identical tase activity. The second DNA sequence encodes a polypep plant, the genome of which does not comprise said introduced tide exhibiting the activity of another steroid pathway DNA. Further, the storage organs of the plant contain a enzyme. Each polypeptide-encoding DNA sequence is oper 10 reduced level of squalene, cycloartenol, 24-methylene ably linked in the 5' to 3’ direction to a promoter and a cycloartenol, obtusifoliol, Stigmasta-7-enol, or campesterol transcription termination signal sequence independent of the compared to the seeds of an otherwise identical plant or a other sequence. The promoter is located upstream and the plant comprising an introduced DNA encoding an HMG termination sequence downstream of each polypeptide-en CoA reductase enzyme. coding DNA sequence. The second DNA sequence encoding 15 The invention contemplates a method of producing a plant a steroid pathway enzyme can code for a squalene epoxidase that accumulates an elevated level of sterol pathway products enzyme, a sterol methyl transferase I enzyme, a sterol compared to a corresponding plant comprising no introduced C4-demethylase enzyme, a obtusifoliol C14C-demethylase DNA encoding a peptide, polypeptide, or protein that affects enzyme, a sterol C5-desaturase enzyme, or a sterol methyl the biosynthesis and accumulation of a sterol pathway prod transferase II enzyme. It is contemplated that HMG-CoA uct, comprising sexually crossing plants to arrive at a plant reductase and the steroid pathway enzyme activity comes comprising nucleic acid encoding an HMG CoA reductase from a mutant or truncated form of those enzymes, such as a and a steroid pathway enzyme, including crosses with a nurse truncated HMG-CoA reductase lacking the transmembrane cultivar. The plants, including apomicitic plants, uniform region while retaining a functional catalytic domain. populations of the plants and their seeds and parts other than Examples of such preferred HMG CoA reductases include 25 seeds are contemplated. the truncated rubber and Arabidopsis HMG CoA reductases Another aspect of the invention is oils containing at least disclosed herein. one sterol pathway product, extracted from the seeds of a Preferably, the regulatory function of a promoter is sub contemplated plant. Preferably sitosterol, at least one sito stantially unaffected by cellular levels of squalene such as the sterol ester, or mixtures thereof, comprise at least about 57% CaMV 35S promoter. In one aspect, a promoter is seed 30 by weight of the total sterol compounds of a contemplated oil. specific. In another aspect, a promoter is derived from a Preferably sitosterol, that at least one sitosterol ester, or mix species in a different order from a host cell. In another aspect, tures thereof, comprise at least about 0.08% of the dry weight the HMG-CoA reductase or steroid pathway enzymes is from of a contemplated seed. Preferably, the oil as a reduced a species in a different order from the order that of the host amount of squalene, cycloartenol, 24-methylene cell. The invention contemplates a construct or recombinant 35 cycloartenol, obtusifoliol, Stigmasta-7-enol, campesterol, or vector having more than one DNA sequence encoding a ste combinations thereof, compared to oil from a corresponding roid pathway enzyme that do not have to be under the control transgenic plant that does not contain introduced DNA encod of the same promoter. Preferably, a recombinant vector is a ing a squalene epoxidase enzyme, a sterol methyl transferase plant expression vector. I enzyme, a sterol C4-demethylase enzyme, a obtusifoliol In another aspect of the invention, a transformed host cell 40 C14C-demethylase enzyme, a sterol C5-desaturase enzyme, comprises a recombinant construct or vector as described a sterol methyl transferase II enzyme, or mixture thereof; above. Preferably, a host cell is plant cell, preferably that plant wherein the reduction is in the range of from about 10% to cell is from canola, soybean, corn, maize, tobacco, cotton, about 100%. rape, tomato oralfalfa. The invention contemplates a host cell Sitosterol ester compositions derived from transgenic in a cell culture, plants derived from transformed host cells, 45 plants of the present invention, their progeny or their seeds are and storage organs (seeds, fruits and vegetable parts) from also contemplated, preferably wherein an esterifying fatty transgenic plants. acid has 2 to 22 carbon atoms in the main chain. In addition to contemplating transgenic plants and seeds, A further aspect of the invention is cholesterol-lowering the invention contemplates transgenic plant seeds capable of compositions comprising contemplated oils and sitosterol germinating into a transgenic plant and mutants, recombi 50 ester compositions. Another further aspect of the invention is nants, genetically engineered derivatives thereof and hybrids foods, food ingredients, or food compositions comprising derived therefrom. The plant over-accumulates steroid path contemplated oils. way products relative to a native, non-transgenic plant of the Still further, the invention contemplates pharmaceutical same strain, wherein said mutants, recombinants, genetically compositions comprising a cholesterol-lowering effective engineered derivatives thereof and hybrids derived therefrom 55 amount of a contemplated oil, and a pharmaceutically accept maintain the ability to overaccumulate steroid pathway prod able carrier, excipient, or diluent. uctS. A method of lowering the plasma concentration of low The invention contemplates a process of increasing the density lipoprotein cholesterol is contemplated, comprising formation of steroid pathway products in a transformed host orally administering to a human oranimal Subject an effective cell as compared to an otherwise identical non-transformed 60 amount of an above composition. Also contemplated is a host cell. Contemplated processes use the described recom method of treating or preventing an elevated plasma concen binant constructs and vectors to transform host cells, then tration of low-density lipoprotein cholesterol, comprising growing the host cells or regenerating transgenic plants there orally administering to a human oranimal Subject an effective from. amount of a contemplated composition. In one aspect of the invention, the genome of a contem 65 A related aspect of the invention is a method of making a plated plant, its progeny, seeds or cell culture, comprises food additive composition, comprising obtaining oil contain introduced DNA encoding an HMG-CoA reductase activity ing a sterol pathway product compound from seed of a con US 7,544,863 B2 7 8 templated transgenic plant and mixing the oil with an edible synthesis pathway enzyme, and a transcription termination solubilizing agent, an effective amount of a dispersant, and sequence; and (3) regenerating said transformed plant cell optionally, an effective amount of an antioxidant. into said transgenic plant. Novel forms of two sterol pathway enzymes and the nucleic acids that encode them are disclosed: an Arabidopsis BRIEF DESCRIPTION OF THE DRAWINGS enzyme having nucleic acid similarity to a squalene epoxi These and other features, aspects, and advantages of the dase, and an Arabidopsis enzyme having nucleic acid simi present invention will become better understood with regard larity to an obtusifoliol C14C.-demethylase enzyme. Thus, the to the following description, appended claims and accompa invention contemplates an isolated DNA molecule having a 10 nying figures where: nucleotide sequence of disclosure SEQID NO: 4, 6, 8, 10, 14. FIG. 1 is an abbreviated version of a plant steroid com 15, 17 or the complements thereof. Also contemplated is a pound biosynthetic pathway that shows the enzymes affect nucleotide sequence that hybridizes to the nucleotide ing steroid compound biosynthesis and accumulation. These sequence of SEQ ID NO;4, 6, 8, 10, 14, 15, 17 or their include: HMG-CoA reductase, squalene epoxidase, sterol complements under a wash stringency equivalent to 0.5xSSC 15 methyl transferase I, sterol C4-demethylase, obtusifoliol to 2xSSC, 0.1% SDS, at 55-65° C., and that encode a C14C.-demethylase, sterol C5 desaturase and sterol methyl polypeptide having squalene epoxidase or obtusifoliol C14C.- transferase II. demthylase enzymatic activity. Preferably, that enzymatic FIG. 2 depicts the forms of Arabidopsis and rubber activity is Substantially similar to that of a disclosed squalene HMGR1 tested in Arabidopsis and yeast to compare expres epoxidase or obtusifoliol C14C-demethylase, respectively. Sion, activity and sterol production. By Substantially Smiliar is meant having enzymatic activity FIG. 3 is a map showing the structure of construct differing from that of the disclosed enzymes by about 30% or pMON29920.pMON29920 is a binary transformation vector less, preferably by about 20% or less, and more preferably by with P-7S/E93'cassette and the KAN gene flanked by the two about 10% or less when assayed by standard enzymatic borders where P-7S is the promoter of alpha'beta conglycinin assays. Also contemplated is a nucleotide sequence encoding 25 protein from soybean, E93' is the 3' end of pea rbc E9 gene the same genetic information as said nucleotide sequence of and KAN is the coding sequence for NPTII that confers SEQ ID NO: 4, 6, 8, 10, 14, 15, 17 or their complements or resistance to kanamycin. The NPTII gene is driven by the 35S that hybridize as described above, but which is degenerate in promoter from cauliflower mosaic virus. Spc. Stris the coding accordance with the degeneracy of the genetic code. Recom region for TnT adenylyltransferase conferring resistance to binant constructs, vectors and transformed host cells com 30 spectinomycin and streptomycin; ori-V: the vegetative origin prising the novel isolated and purified nucleic acid sequences of replication; rop: coding region for repressor of primer; are also contemplated. In one embodiment, the vector is a ori-322: minimum known sequence required for a functional plant vector and the host cell is a plant cell. Methods of origin of replication: NOS 3': the 3' termination end of nopa producing the disclosed squalene epoxidase or obtusifoliol line synthase coding region. C14C-demethylase enzymes are also contemplated compris 35 FIG. 4 is a map showing the structure of construct ing culturing a transformed host cell for a time and under pMON43800. pMON43800 is a recombinant binary vector conditions conductive to the production of the squalene for Agrobacterium-mediated transformation, carrying the epoxidase or obtusifoliol C14C-demethylase enzyme, and rubber HMGR1 gene cassette. The HMGR1 gene is driven by recovering the produced squalene epoxidase or obtusifoliol the 7S alpha' beta conglycinin promoter from soybean. P-7S: C14C-demethylase enzyme. 40 7S promoter, rubber HMGR1 gene: coding sequence for Yet another aspect provides any of the above described 3-hydroxy-3-methylglutaryl reductase from Heveabrasilien transformed host cells, further comprising a recombinant sis; E93': 3' end of pea rbcS E9 gene: P-35S: 35S promoter construct or expression vector encoding a tocopheral synthe from cauliflower mosaic virus; KAN: coding region for sis pathway enzyme, and in particular, S-adenosylmethion NPTII gene conferring resistance for kanamycin; NOS 3': 3 ine-dependent C-tocopherol methyltransferase. Also 45 termination end of nopaline synthase coding region: Left included are plants, seeds and storage organs comprising the Border: Octapine left border from Octapine Ti plasmid transformed host cells. pTiA6; ori-V: the vegetative origin of replication; rop: coding Another aspect provides, a process of increasing the for region for repressor of primer; Spc/Str; coding region for TnT. mation of Steroid pathway products and tocopherols in a adenylyltransferase conferring resistance to spectinomyci transformed host cell as compared to an otherwise identical 50 nand streptomycin. non-transformed host cell comprising (1)transforming a host FIG. 5 is a map showing the structure of construct cell with a recombinant vector comprising (a) as operably pMON23616. pMON23616 is a plant expression vector con linked components in the 5' to 3’ direction, a promoter, a DNA taining P-NOS/ORF-7/KAN/NOS-3' cassette. P-NOS: NOS sequence encoding a first polypeptide having 3-hydroxy-3- promoter from Agrobacterium tumefaciens pTiT37; ORF-7: methylglutaryl-Coenzyme A reductase enzyme activity, and a 55 a short open reading frame that attenuates expression of KAN transcription termination signal sequence; and (b) as operably in plants; KAN: coding sequence of NPTII gene that confers linked components in the 5' to 3’ direction, a promoter, a DNA resistance to kanamycin and neomycin; ble: confers resis sequence encoding at least one polypeptide having steroid tance to bleomycin; NOS 3': 3' termination end of nopaline pathway enzyme activity selected from the group consisting synthase coding region: Left Border: Octapine left border of squalene epoxidase enzyme activity, Sterol methyl trans 60 from Octapine Tiplasmid pTiA6; ori-V: the vegetative origin ferase I enzyme activity, Sterol C4-demethylase enzyme of replication; rop: coding region for repressor of primer; activity, obtusifoliol C14C-demethylase enzyme activity, ste Spc/Str: coding region for TnT adenylyltransferase confer rol C5-desaturase enzyme activity, and sterol methyl trans ring resistance to spectinomycin and streptomycin. ferase II enzyme activity, and a transcription termination FIG. 6 is a map showing the structure of construct signal sequence; (2) transforming the host cell of (1) with a 65 pMON43818. pMON43818 is a recombinant binary vector recombinant vector comprising as operably linked compo carrying the gene encoding rubber hydroxymethyl glutaryl nents, a promoter, a DNA sequence encoding a tocopherol CoA reductasel (HMGR1) in sense orientation driven by the US 7,544,863 B2 10 soybean alpha' beta conglycinin promoter. P-NOS: nopaline rium: Soy Alpha' Beta Conglycinin: soybean 7S alpha' beta synthase gene promoter, kan: coding region for neomycin conglycinin gene promoter, Arabidopsis HMGR catalytic phospho transferase protein to confer resistance to kanamy domain: coding sequence for Arabidopsis HMGR1 catalytic cin; NOS 3': 3' termination end of nopaline synthase coding domain; E93': 3' end of pea rbcS E9 gene. region; Soy Alpha' Beta Conglycinin: 7S alpha' beta congly FIG. 10 is a map showing the structure of construct cinin gene promoter from soybean; Rubber HMGR1 gene: pMON43058, p MON43058 is a recombinant binary vector coding sequence for HMGR1 gene from Hevea brasiliensis; for Agrobacterium-mediated Soybean transformation, carry E93': 3' end of pea rbcS E9 gene: Left border: octopine left ing gene expression cassettes for catalytic domain of border, sequence essential for transfer of T-DNA into Agro HMGR1 from Arabidopsis thaliana and SMTII from Arabi bacterium; ori-V: plasmid origin of replication in Agrobacte 10 dopsis thaliana. P-NOS: nopaline synthase gene promoter; rium; rop: coding sequence for repressor of primer; Ori-322: kan: coding region for neomycin phosphotransferase protein origin of replication in E.coli: Spc/Str: coding region for TnT. to confer resistance to kanamycin; NOS 3': 3' termination end adenylyltransferase (AAD(3")) conferring resistance to spec of nopaline synthase coding region; Left border: octopine left tinomycin and streptomycin; Right Border: right border border sequence essential for transfer of T-DNA into Agro sequence of T-DNA essential for integration into Agrobacte 15 bacterium; ori-V: plasmid origin of replication in Agrobacte Filii. rium; rop: coding sequence for repressor of primer; ori-322: FIG. 7 is a map showing the structure of construct origin of replication in E.coli: Spc/Str: coding region for TnT. pMON43052. p MON43052 is a recombinant shuttle vector, adenylyltransferase (AAD(3")) conferring resistance to spec carrying the cDNA fragment encoding the catalytic domain tinomycin and streptomycin; Right Border: right border of Arabidopsis HMGR1 in sense orientation driven by the sequence essential for transfer of T-DNA into Agrobacte soybean alpha' beta conglycinin promoter. P-NOS: nopaline rium: Soy Alpha' Beta Conglycinin: 7S alpha' beta conglyci synthase gene promoter, kan: coding region for neomycin nin gene promoter from soybean; Arabidopsis HMGR cata phosphotransferase protein to confer resistance to kanamy lytic domain: Sequence encoding the catalytic domain of cin; NOS 3': 3' termination end of nopaline synthase coding Arabidopsis HMGR1; E93': 3' end of pearbcS E9 gene: Soy region; Soy Alpha' Beta Conglycinin: 7S alpha' beta congly 25 Alpha' Beta Conglycinin: soybean 7S alpha'beta conglycinin ciningene promoter from soybean; Arabidopsis HMGR cata gene promoter; Arabidopsis SMT2: cDNA encoding sterol lytic domain: coding sequence for the catalytic domain of methyl transferase II enzyme from Arabidopsis thaliana (ac Arabidopsis HMGR1 protein; E93': 3' end of pea rbcs E9 cession no: X89867): NOS 3': 3' termination end of nopaline gene; Left border: octopine left border, sequence essential for Synthase coding region. transfer of T-DNA into Agrobacterium; ori-V: plasmid origin 30 FIG. 11 is profile (histogram) of the sterol composition of of replication in Agrobacterium; rop: coding sequence for R1 transgenic Soybean seeds when Arabidopsis truncated repressor of primer: Ori-322: origin of replication in E.coli: HMGR (catalytic domain without linker) was overexpressed Spc/Str: coding region for TnT adenylyltransferase (AAD using seed-specific 7S promoter (data from pMON43057: (3")) conferring resistance to spectinomycin and streptomy p7S: At HMGR truncated). cin; Right Border: right border sequence of T-DNA essential 35 FIG. 12 is a profile (histogram) of the sterol composition of for integration into Agrobacterium. R1 transgenic Soybean seeds when Arabidopsis truncated FIG. 8 is a map showing the structure of construct HMGR (catalytic domain without linker) and Arabidopsis pMON51850. pMON51850 is a binary vector for Agrobac SMTII were overexpresed (data from pMON43058:p7S::At terium mediated transformation of soybean. P-NOS: nopa HMGR truncated and p7S::At SMTII). The expression of the line synthase gene promoter, kan: coding region for neomy 40 genes is controlled by the seed-specific 7S promoter. cin phosphotransferase protein to confer resistance to FIG. 13 is a map showing the structure of construct kanamycin; NOS 3': 3' termination end of nopaline synthase pMON53733. p MON53733 is a recombinant binary vector coding region; Left border: octopine left border sequence carrying the cDNA encoding full-length form of Arabidopsis essential for transfer of T-DNA into Agrobacterium; ori-V: hydroxymethyl glutaryl CoA reductasel (HMGR1) in sense plasmid origin of replication in Agrobacterium; rop: coding 45 orientation driven by the enhanced cauliflower mosaic virus sequence for repressor of primer; ori-322: origin of replica 35S promoter. P-35S: 35S promoter from cauliflower mosaic tion in E.coli: Spc/Str; coding region for TnT adenylyltrans virus; kan: confers resistance to neomycin and kanamycin; ferase (AAD(3")) conferring resistance to spectinomycin and NOS 3': 3' termination end of nopaline synthase coding streptomycin; Right Border: right border sequence of T-DNA region, Left border: Octopine left border, sequence essential essential for integration into Agrobacterium. 50 for transfer of T-DNA into Agrobacterium; ori-V: plasmid FIG. 9 is a map showing the structure of construct origin of replication in Agrobacterium; rop: coding sequence pMON43057.pMON43057 is a recombinant binary vector for repressor of primer; ori-322: origin of replication in for Agrobacterium mediated transformation of soybean, car E.coli: Spc/Str: coding region. for TnT adenylyltransferase rying the gene cassette for expressing catalytic domain of (AAD(3")) conferring resistance to spectinomycin and strep HMGR1 from Arabidopsis thaliana. The catalytic domain of 55 tomycin; Right Border: right border sequence of T-DNA the HMGR1 cDNA is driven by soybean 7S alpha' beta con essential for integration into Agrobacterium; P-e35S: glycinin promoter. P-NOS: nopaline synthase gene promoter; enhanced cauliflower mosaic virus promoter, Arabidopsis kan: coding region for neomycin phosphotransferase protein HMGR1: cloNA sequence encoding full-length form of Ara to confer resistance to kanamycin; NOS 3': 3' termination end bidopsis HMGR1; E93': 3' end of pea rbcs. E9 gene. of nopaline synthase coding region; Left border: octopine left 60 FIG. 14 is a map showing the structure of construct border sequence essential for transfer of T-DNA into Agro pMON53734. p MON53734 is a recombinant binary vector bacterium; ori-V: plasmid origin of replication in Agrobacte carrying the cDNA encoding catalytic domain with linker rium; rop: coding sequence for repressor of primer; ori-322: region of Arabidopsis hydroxymethyl glutaryl CoA reducta origin of replication in E.coli: Spc/Str: coding region for TnT. sel (HMGR1) in sense orientation driven by the enhanced adenylyltransferase (AAD(3")) conferring resistance to spec 65 cauliflower mosaic virus 35S promoter. P-35S: 35S promoter tinomycin and streptomycin; Right Border: right border from cauliflower mosaic virus; kan: confers resistance to sequence essential for transfer of T-DNA into Agrobacte neomycin and kanamycin; NOS 3': 3' termination end of US 7,544,863 B2 11 12 nopaline synthase coding region; Left border: octopine left rium; P-e35S: enhanced cauliflower mosaic virus promoter; border, sequence essential for transfer of T-DNA into Agro rubbertHMGR1: cloNA sequence encoding catalytic domain bacterium; ori-V: plasmid origin of replication in Agrobacte with linker region of rubber HMGR1; E93':3' endofpearbcS rium; rop: coding sequence for repressor of primer; ori-322: E9 gene. origin of replication in E.coli: Spc/Str: coding region for TnT. FIG. 18 is a map showing the structure of construct adenylyltransferase (AAD(3")) conferring resistance to spec pMON53738. pMON53738 is a recombinant binary vector tinomycin and streptomycin; Right Border: right border carrying the cDNA encoding mutant form of rubber (Hevea sequence of T-DNA essential for integration into Agrobacte brasiliensis) hydroxymethyl glutaryl CoA reductasel rium; P-e35S: enhanced cauliflower mosaic virus promoter; (HMGR1) in sense orientation driven by the enhanced cauli Arabidopsis thMGR1: cloNA sequence encoding catalytic 10 flower mosaic virus 35S promoter. In the mutant rubber domain with linker region of Arabidopsis HMGR1; E93': 3' HMGR1 the putative phosphorylation site, the serine amino end of pea rbcS E9 gene. acid residue at position 566 is changed to alanine amino acid FIG. 15 is a map showing the structure of construct residue (SEQID 23). P-35S: 35S promoter from cauliflower pMON53735. p MON53735 is a recombinant binary vector mosaic virus; kan: confers resistance to neomycin and kana carrying the CDNA encoding catalytic domain without the 15 mycin; NOS 3': 3' termination end of nopaline synthase cod linker region of Arabidopsis hydroxymethyl glutaryl CoA ing region; Left border: octopine left border, sequence essen reductasel (HMGR1) in sense orientation driven by the tial for transfer of T-DNA into Agrobacterium; ori-V: plasmid enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S origin of replication in Agrobacterium; rop: coding sequence promoter from cauliflower mosaic virus; kan: confers resis for repressor of primer; ori-322: origin of replication in tance to neomycin and kanamycin; NOS3': 3' termination end E.coli: Spc/Str; coding region for TnT adenylyltransferase of nopaline synthase coding region; Left border: octopine left (AAD(3")) conferring resistance to spectinomycin and strep border, sequence essential for transfer of T-DNA into Agro tomycin; Right Border: right border sequence of T-DNA bacterium; ori-V: plasmid origin of replication in Agrobacte essential for integration into Agrobacterium; P-e35S: rium; rop: coding sequence for repressor of primer; ori-322: enhanced cauliflower mosaic virus promoter; rubber origin of replication in E.coli: Spc/Str: coding region for TnT. 25 thMGR1 Ala 566: clNA sequence encoding catalytic adenylyltransferase (AAD(3")) conferring resistance to spec domain with linker region of rubber HMGR1 in which serine tinomycin and streptomycin; Right Border: right border amino acid residue at position 566 is changed to alanine sequence of T-DNA essential for integration into Agrobacte amino acid residue using site directed mutagenesis; E93': 3' rium; P-e35S: enhanced cauliflower mosaic virus promoter; end of pea rbcS E9 gene. Arabidopsis cFIMGR1: clNA sequence encoding catalytic 30 FIG. 19 is a map showing the structure of construct domain without the linker region of Arabidopsis HMGR1; E9 pMON53739. p MON53739 is a recombinant binary vector 3': 3' end of pea rbcS E9 gene. carrying the cDNA encoding mutant form of rubber (Hevea FIG. 16 is a map showing the structure of construct brasiliensis) hydroxymethyl glutaryl CoA reductasel pMON53736. pMON53736 is a recombinant binary vector (HMGR1) in sense orientation driven by the enhanced cauli carrying the cDNA encoding full-length form of rubber (He 35 flower mosaic virus 35S promoter. In the mutant rubber vea brasiliensis) hydroxymethyl glutaryl CoA reductasel HMGR1 the putative phosphorylation site, the serine amino (HMGR1) in sense orientation driven by the enhanced cauli acid residue at position 567 is changed to alanine amino acid flowermosaic virus 35S promoter. P-35S: 35S promoter from residue (SEQID 24). P-35S: 35S promoter from cauliflower cauliflowermosaic virus; kan: confers resistance to neomycin mosaic virus; kan: confers resistance to neomycin and kana and kanamycin; NOS 3': 3' termination end of nopaline syn 40 mycin; NOS 3': 3' termination end of nopaline synthase cod thase coding region; Left border: octopine left border, ing region; Left border: octopine left border, sequence essen sequence essential for transfer of T-DNA into Agrobacte tial for transfer of T-DNA into Agrobacterium; ori-V: plasmid rium; ori-V: plasmid origin of replication in Agrobacterium; origin of replication in Agrobacterium; rop: coding sequence rop: coding sequence for repressor of primer, ori-322: origin for repressor of primer; ori-322: origin of replication in of replication in E.coli: Spc/Str; coding region for TnTade 45 E.coli: Spc/Str; coding region for TnT adenylyltransferase nylyltransferase (AAD(3")) conferring resistance to specti (AAD(3")) conferring resistance to spectinomycin and strep nomycin and streptomycin; Right Border: right border tomycin; Right Border: right border sequence of T-DNA sequence of T-DNA essential for integration into Agrobacte essential for integration into Agrobacterium; P-e35S: rium; P-e35S: enhanced cauliflower mosaic virus promoter; enhanced cauliflower mosaic virus promoter; rubber Hevea HMGR1: cloNA sequence encoding full-length form 50 thMGR1 Ala 567: clNA sequence encoding catalytic of rubber HMGR1; E93': 3' end of pea rbcs E9 gene. domain with linker region of rubber HMGR1 in which serine FIG. 17 is a map showing the structures of construct amino acid residue at position 567 is changed to alanine pMON53737. pMON53737 is a recombinant binary vector amino acid residue using site directed mutagenesis; E93': 3' carrying the cDNA encoding catalytic domain with linker end of pea rbcS E9 gene. region of rubber (Hevea brasiliensis) hydroxymethylglutaryl 55 FIG. 20 is a map showing the structure of construct CoA reductasel (HMGR1) in sense orientation driven by the pMON53740. pMON53740 is a recombinant binary vector enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S carrying the cDNA encoding catalytic domain without linker promoter from cauliflower mosaic virus; kan: confers resis region of rubber (Hevea brasiliensis) hydroxymethylglutaryl tance to neomycin and kanamycin; NOS3': 3' termination end CoA reductasel (HMGR1) in sense orientation driven by the of nopaline synthase coding region; Left border: octopine left 60 enhanced cauliflower mosaic virus 35S promoter. P-35S: 35S border, sequence essential for transfer of T-DNA into Agro promoter from cauliflower mosaic virus; kan: confers resis bacterium; ori-V: plasmid origin of replication in Agrobacte tance to neomycin and kanamycin; NOS3': 3' termination end rium; rop: coding sequence for repressor of primer; ori-322: of nopaline synthase coding region; Left border: octopine left origin of replication in E.coli: Spc/Str: coding region for TnT. border, sequence essential for transfer of T-DNA into Agro adenylyltransferase (AAD(3")) conferring resistance to spec 65 bacterium; ori-V: plasmid origin of replication in Agrobacte tinomycin and streptomycin; Right Border: right border rium; rop: coding sequence for repressor of primer; ori-322: sequence of T-DNA essential for integration into Agrobacte origin of replication in E.coli: Spc/Str: coding region for TnT. US 7,544,863 B2 13 14 adenylyltransferase (AAD(3")) conferring resistance to spec region is known to affect copy number when expressed in tinomycin and streptomycin; Right Border: right border bacteria; AMP: contains the P3 promoter and the beta-lacta sequence of T-DNA essential for integration into Agrobacte mase coding sequence, conferring resistance to ampicillin, rium; P-e35S: enhanced cauliflower mosaic virus promoter; penicillin, and carbenicillin, Sc.2 micron: 2 micron origin of rubber chMGR1: CDNA sequence encoding catalytic 5 replication. domain without linker region of rubber HMGR1; E93': 3' end FIG. 30 is a map showing the structure of construct of pea rbcs. E9 gene. pMON43843. pMON43843 is a yeast expression vector car FIG. 21 is a graph comparing the cycloartenol content in rying cDNA encoding Arabidopsis putative squalene epoxi micrograms of steroid compound per gram of seeds analyzed dase 1 (ATA506263) in sense orientation driven by the in transgenic Arabidopsis plants transformed with 10 p423Gall promoter. Sc. His3: HIS3 region from Saccharomy pMON53733 through pMON53740 compared to control ces cerevisiae encoding imidazoleglycerol-phosphate dehy plants. dratase for histidine synthesis: Ori-fl: bacteriophage fl ori FIG. 22 is a graph comparing the 24-methylene gin of replication; LAC: contains partial laci coding cycloartenol content in micrograms of steroid compound per sequence, promoter Plac, promoter Pt7, promoter Pt3, KS gram of seeds analyzed in transgenic Arabidopsis plants 15 polylinker, and partial lacZ coding sequence; lac7: partial transformed with pMON53733 through pMON53740 com coding sequence for beta-d-galactosidase or lacZ protein; pared to control plants. T-Sc.Cycl: a terminator from Cycl- iso-1-cytochrome c from FIG. 23 is a graph comparing the obtusifoliol content in Saccharomyces cerevisiae to terminates transcription; micrograms of steroid compound per gram of seeds analyzed Squalene epoxidase 1 (ATA506263): clNA encoding Arabi in transgenic Arabidopsis plants transformed with dopsis putative squalene epoxidase 1 (ATA506263); P-Sc pMON53733 through pMON53740 compared to control Gall: a promoter from Gall-galactokinase of Saccharomy plants. ces cerevisiae to direct expression with galactose induction; FIG. 24 is a graph comparing the campesterol content in Lac Z-alpha: partial coding sequence for beta-d-galactosidase micrograms of steroid compound per gram of seeds analyzed or lacZ protein; Ori-pUC: minimum sequence required for a in transgenic Arabidopsis plants transformed with 25 functional origin of replication, sequence downstream of this pMON53733 through pMON53740 compared to control region is known to affect copy number when expressed in plants. bacteria; AMP: contains the P3 promoter and the beta-lacta FIG. 25 is a graph comparing the sitosterol content in mase coding sequence, conferring resistance to ampicillin, micrograms of steroid compound per gram of seeds analyzed penicillin, and carbenicillin, Sc.2 micron: 2 micron origin of in transgenic Arabidopsis plants transformed with 30 replication. pMON53733 through pMON53740 compared to control FIG. 31 is a map showing the structure of construct plants. pMON43844. p MON43844 is a yeast expression vector car FIG. 26 is a graph comparing the sitostanol content in rying cDNA encoding Arabidopsis putative squalene epoxi micrograms of steroid compound per gram of seeds analyzed dase 1 (ATA304243) in sense orientation driven by the in transgenic Arabidopsis plants transformed with 35 p423Gall promoter. Sc. His3: HIS3 region from Saccharomy pMON53733 through pMON53740 compared to control ces cerevisiae encoding imidazoleglycerol-phosphate dehy plants. dratase for histidine synthesis: Ori-fl: bacteriophage fl ori FIG. 27 is a sterol profile (histogram) of transgenic Arabi gin of replication; LAC: contains partial lacI coding dopsis harboring different forms of rubber HMGR. sequence, promoter Plac, promoter Pt7, promoter Pt3, KS FIG. 28 is a graph of the squalene, Zymosterol and erogos 40 polylinker, and partial lacZ coding sequence; lac7: partial terol content in micrograms of sterol per milligram of cell dry coding sequence for beta-d-galactosidase or lacZ protein; weight from HMGR constructs in yeast HMGR1 knockout T-Sc.Cycl: a terminator from Cyc1- iso-1-cytochromec from mutants for constructs having full length and truncated HMG Saccharomyces cerevisiae to terminate transcription; Arab. CoA reductase (HMGR) sequences. The truncated sequences squalene epoxidase 1 (ATA304243): cDNA encoding Arabi contain Substantial portions of the catalytic region but lack the 45 dopsis putative squalene epoxidase 1 (ATA304243); P-Sc linker region and the transmembrane region of HMGR. These Gall: a promoter from Gall-galactokinase of Saccharomy sequences are derived from Arabidopsis and rubber plants. ces cerevisiae to direct expression with galactose induction; FIG. 29 is a map showing the structure of construct Lac Z-alpha: partial coding sequence for beta-d-galactosidase pMON43842. p MON43842 is a yeast expression vector car or lacZ protein; Ori-pUC: minimum sequence required for a rying cDNA encoding Arabidopsis putative obtusifoliol 50 functional origin of replication, sequence downstream of this C14C.-demethylase (AC002329) in sense orientation driven region is known to affect copy number when expressed in by the p423Gall promoter. Sc. His3: HIS3 region from Sac bacteria; AMP: contains the P3 promoter and the beta-lacta charomyces cerevisiae encoding imidazoleglycerol-phos mase coding sequence, conferring resistance to ampicillin, phate dehydratase for histidine synthesis: Ori-fl: bacterioph penicillin, and carbenicillin, Sc.2 micron: 2 micron origin of age fl origin of replication; LAC: contains partial lacI coding 55 replication. sequence, promoter Plac, promoter Pt7, promoter Pt3, KS FIG. 32 is a comparision of known HMG CoA reductase polylinker, and partial lacZ coding sequence; lac7: partial amino acid sequences. ClustalW alignment of forty-three coding sequence for beta-d-galactosidase or lacZ protein; non-redundant HMG-CoA reductase sequences to represent T-Sc.Cycl: a terminator from Cycl- iso-1-cytochrome c from archaebacterial, eubacterial, fungal, plant and animal groups. Saccharomyces cerevisiae to terminate transcription; obtus. 60 The putative functional domains in the alignment marked as C14C.demethylase (AC002329): cDNA encoding Arabidop described below are based on the three dimnensional struc sis putative obtusifoliol C14C-demethylase; P-Sc.Gall: a ture of Pseudomonas mevalonii HMGR (Lawrence et al., promoter from Gall-galactokinase of Saccharomyces cerevi 1995, Science 268:1758): boxed-HMGCoA binding domain, siae to direct expression with galactose induction; Lac Z light shade-NAD(H) binding domain, underlined consensus alpha: partial coding sequence for beta-d-galactosidase or 65 domains involved in catalysis, * underneath consensus and lacZ protein; Ori-pUC: minimum sequence required for a boldface-key histidine residue involved in catalysis. The functional origin of replication, sequence downstream of this putative phosphorylation site residues are marked with and US 7,544,863 B2 15 16 boldface, and are located at the C-terminal region of the elevated from 0.3% up to 3.5%. Expression of the full-length protein, adjacent to a highly conserved arginine, marked rubber HMGR in soybeans results in a sterol level increase up withi and boldface. Also indicated are the conserved Glu (E), to 2.7%. Lys (K), and Asp (D) residues, marked by E, K, and D, respectively. These residues are thought to be critical in Enhancement of 3-hydroxy-3-methylglutaryl-CoA reduc catalysis, based on the crystal structure (Tabernero et al., tase (HMG Co-A reductase) activity in certain cells results in 1999; PNAS 96(13):7167-71). increased sterol biosynthesis. See, e.g. Chappell, U.S. Pat. Appendices A through C show SEQID Nos: 1 through 3, No. 5,589,619. The present discovery further contemplates an respectively. Appendices D through G show SEQID Nos 20 increase of steroid pathway end products Such as A5 sterols thorough 23, respectively. 10 and their stanol counterparts with a decreased accumulation of certain steroid pathway intermediates by also enhancing DETAILED DESCRIPTION various specific steroid pathway enzyme activities, such that more of the steroid pathway intermediate compounds are The following detailed description is provided to aid those converted to steroid pathway end products. skilled in the art in practicing the present invention. Even So, 15 DNA sequences encoding squalene epoxidases are useful this detailed description should not be construed to unduly for removal of squalene accumulation, genes encoding sterol limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of methyl transferase I enzymes are useful for removal of cyl ordinary skill in the art without departing from the spirit or coartenol accumulation, genes encoding sterol C4-demethy Scope of the present inventive discovery. lase are useful for removal of 24-methylene cycloartenol All publications, patents, patent applications, databases accumulation, genes encoding obtusifoliol C14C-demethy and other references cited in this application are herein incor lases are useful for removal of accumulation of obtusifoliol, porated by reference in their entirety as if each individual genes encoding sterol C5-desaturases are useful for removal publication, patent, patent application, database or other ref of stigmasta-7-enol accumulation, and genes encoding sterol erence were specifically and individually indicated to be 25 methyl transferase II enzymes are useful for the reduction of incorporated by reference. accumulated campesterol and concomitant increase of sito We have expressed the full-length forms of the rubber and sterol. Arabidopsis HMGRS driven by seed-specific promoters in Levels of sitostanol and sitostanol esters can be elevated transgenic canola and Soybean. We have demonstrated Sterol 30 over-production up to 2-4 fold higher in seeds from these further by approximately 2- to 40-fold over the transgenic transgenic plants. We also demonstrated a higher accumula plants of the art having only added genes for HMG CoA tion of pathway intermediates in Soybean than canola. These reductase by introducing additional genes encoding one or results were disclosed in PCT publication WO 00/61771. more of the following sterol pathway enzymes: a squalene However, we have expressed a truncated form of the Arabi 35 epoxidase, a sterol methyltransferase I, a sterol C4-demethy dopsis himgil without the linker and membrane spanning lase, an obtusifoliol C14C-demethylase, a sterol C5-desatu domains in Arabidopsis and Soybean. The results in Arabi rase, a sterol methyl transferase II. dopsis were similar to that demonstrated by Gonzalez et al. As used herein, the term "structural coding sequence' (1997) and we compared the sterol profiles of our transgenic means a DNA sequence which encodes for a peptide, plants with those produced by Gonzalez et al., using our 40 methods to show they are comparable. We found the same polypeptide, or protein which may be made by a cell follow types of pathway intermediates accumulating. However, in ing transcription of the DNA to mRNA, followed by transla Soybean seeds we have demonstrated the accumulation of tion to the desired peptide, polypeptide, or protein. squalene to a very high level (~3 mg/g seed which is around The term “sterol as applied to plants refers to any chiral 100-fold higher than in nontransgenic controls). This is an 45 tetracyclic isopentenoid which may be formed by cyclization unexpected result not disclosed or Suggested in the prior art. of squalene oxide through the transition state possessing Ste Squalene is a precursor for sterols and in Soybean it appears reochemistry similar to the trans-syn-trans-anti-trans-anti that there is a “bottleneck” in the further conversion of this configuration, i.e., protosteroid cation, and which retains a precursor to sterols. Thus, it appears that there could be addi polar group at C-3 (hydroxyl or keto), an all-trans-anti Stere tional ways of over-producing sterols in soybean to levels 50 greater than 10-fold which would include combining a trun ochemistry in the ring system, and a side-chain 20R-configu cated form of HMGR with other genes coding for enzymes ration (Parker et al. (1992) In Nes et al., Eds. Regulation of down-stream of squalene. Isopentenoid Metabolism, ACS Symposium Series No. 497, This opens the potential to combine other genes such as p. 110; American Chemical Society, Washington, D.C.). The squalene epoxidase for further enhancing the levels of desir 55 numbering of the carbon atoms of a representative sterol able sterols. Such a combination has not been disclosed or (cholesterol) is shown in the following structure (FORMULA Suggested in the prior art. Squalene expoxidase catalyzes the II): addition of oxygen to squalene which is a 30-carbon linear As used herein, the term “sterol refers to unsaturated isoprenoid chain thus allowing for cyclization to form hydroxyl group-containing derivatives of a fused, reduced cycloartenol. Additional enzymes downstream that can be 60 also be manipulated are sterol methyltransferase 1, C-4 dem ring system, cyclopenta C-phenanthrene, comprising three ethylase, C-14 demethylase, Sterol methytransferase 2, and fused cyclohexane rings (A, B and C) in a phenanthrene C-5 desaturase that would all deplete other pathway interme arrangement, and a terminal cyclopentane ring (D). The diates shown to accumulate in Soybeans. By using Such strat exemplary steroid below (FORMULAII) illustrates the num egies it is possible to convert all of the squalene and other 65 bering system employed herein in describing the location of intermediates to end sterols such as sitosterol, Stigmasterol groups and Substituents. and campesterol. Thus, Sterol level in Soybean oils can be US 7,544,863 B2 18 As used herein “polynucleotide' and "oligonucleotide' are used interchangeably and refer to a polymeric (2 or more
(II) monomers) form of nucleotides of any length, either ribo nucleotides or deoxyribonucleotides. Although nucleotides are usually joined by phosphodiester linkages, the term also includes polymeric nucleotides containing neutral amide backbone linkages composed of aminoethyl glycine units. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA 10 and RNA. It also includes known types of modifications, for example, labels, methylation, "caps'. Substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phos Sterols may or may not contain a C-5 to C-6 double bond, 15 photriesters, phosphoamidates, carbamates, etc.), those con as this is a feature introduced late in the biosynthetic pathway taining pendant moieties, such as, for example, proteins (in (note Scheme 1, below). Sterols contain a C-C side chain at cluding for e.g., nucleases, toxins, antibodies, signal the C-17 position, as shown above. peptides, poly-L-lysine, etc.), those with intercalators (e.g., The term “phytosterol, which applies to sterols found acridine, psoralen, etc.), those containing chelators (e.g., met uniquely in plants, refers to a sterol containing a C-5, and in als, radioactive metals, boron, oxidative metals, etc.), those some cases a C-22, double bond. Phytosterols are further containing alkylators, those with modified linkages (e.g., characterized by alkylation of the C-17 side-chain with a alpha anomeric nucleic acids, etc.), as well as unmodified methyl or ethyl substituent at the C-24 position. Major phy forms of the polynucleotide. Polynucleotides include both tosterols include, but are not limited to, Sitosterol, Stigmas sense and antisense Strands. terol, campesterol, brassicasterol, etc. Cholesterol, which 25 The alternative nucleotide sequences described above are lacks a C-24 methyl or ethyl side chain, is found in plants but considered to possess Substantially similar enzymatic activity is not unique thereto, and is not a “phytosterol to that of the polypeptide-encoding polynucleotide sequences “Phytostanols are saturated forms of phytosterols wherein of the present invention if they encode polypeptides having the C-5 and, when present, C-22 double bond(s) is(are) enzymatic activity differing from that of any of the polypep reduced, and include, but are not limited to, sitostanol, 30 tides encoded by the polynucleotide sequences of the present campestanol, and 22-dihydrobrassicastanol. invention by about 30% or less, preferably by about 20% or “Phytosterol esters’ and “phytostanol esters' are further less, and more preferably by about 10% or less when assayed characterized by the presence of a fatty acid or phenolic acid by Standard enzymatic assays. moiety rather than a hydroxyl group at the C-3 position. As used herein “effective amount” is intended to qualify The term “steroid compounds' includes sterols, phytoster 35 the amount of an agent which will achieve the goal of a ols, phytosterol esters, phytostanols, and phytostanol esters. lessening in the severity and/or the frequency of incidence of The term “phytosterol compound” refers to at least one a disease condition or disorder, over no treatment. phytosterol, at least one phytosterol ester, or a mixture The phrase “steroid pathway products” refers to the prod thereof. ucts of steroid biosynthesis produced by the action of one or 40 more of squalene epoxidase enzyme, sterol methyl trans The term “phytostanol compound” refers to at least one ferase I enzyme, sterol C4-demethylase enzyme, obtusifoliol phytostanol, at least one phytostanol ester, or a mixture C14C-demethylase enzyme, sterol C5-desaturase enzyme, thereof. and sterol methyltransferase II enzyme. Specific examples of The term “constitutive promoter” refers to a promoter that steroid pathway products include, but are not limited to, sito operates continuously in a cell, and which is not subject to 45 sterol, Sitostanol, Stigmasterol and Stigmastanol. quantitative regulation. The gene with which Such a promoter In the context of the present disclosure, a “non-trans is associated is always “turned on.” formed plant or cell refers to a plant or cells which does not The terms “seed-specific,” “fruit-specific,” “plastid-spe comprise introduced polynucleotides encoding a polypeptide cific, etc., as they apply to promoters refer to preferential or having 3-hydroxy-3-methyulglutaryl-Coenzyme A reductase exclusive activity of these promoters in these organs or 50 enzyme activity and at least one polypeptide having squalene organelles, respectively. “Preferential expression” refers to epoxidase enzyme activity, Sterol methyl transferase I promoteractivity greater in the indicated organs or organelles enzyme activity, Sterol C4-demethylase enzyme activity, than elsewhere in the plant. “Seed-specific’ comprehends obtusifoliol C14C-demethylase enzyme activity, sterol expression in the aleurone layer, endosperm, and/or embryo C5-desaturase enzyme activity, or sterol methyltransferase II of the seed. 55 enzyme activity. Thus, a plant or cell that contains introduced As used herein "isolated polynucleotide' means a poly polynucleotide sequences other than those above, would still nucleotide that is free of one or both of the nucleotide be considered “non-transformed.” sequences which flank the polynucleotide in the naturally As used herein, "peptide' and “protein’ are used inter occurring genome of the organism from which the polynucle changeably and mean a compound that consists of two or otide is derived. The term includes, for example, a polynucle 60 more amino acids that are linked by means of peptide bonds. otide or fragment thereof that is incorporated into a vector or I. Plant Steroid Biosynthesis expression cassette; into an autonomously replicating plas To aid the reader in understanding the present invention, mid or virus; into the genomic DNA of a prokaryote or descriptions of the Sterol compound biosynthetic pathway are eukaryote; or that exists as a separate molecule independent presented below. These descriptions identify enzymes useful of other polynucleotides. It also includes a recombinant poly 65 in achieving the modifications to the biosynthesis and accu nucleotide that is part of a hybrid polynucleotide, for mulation of sterol compounds described herein, and identify example, one encoding a polypeptide sequence. Sources of nucleic acid sequences encoding these enzymes. US 7,544,863 B2 19 20 Various steps in the steroid compound biosynthetic path- over the arrows refer to plant sterol compound biosynthetic way in plants are shown in Scheme 1, below. The numbers pathway enzymes and genes as indicated in Table 1. US 7,544, 863 B2 21 22
*HOHO
HO Hooo,)
IGIWEIHOS OHO Hooo,)
Z
O ||
I
O | US 7,544,863 B2 23 24
ponunuop
Ioue,sous
US 7,544,863 B2 27 28 been purified from yeast. A plant HMGCoA synthase cDNA TABLE 1. has also been isolated from Arabidopsis thaliana (Montamat et al., Gene 167: 197-201 (1995)). Plant Sterol Compound Pathway Enzymes and Genes HMGCoA reductase, also referred to as 3-hydroxy-3-me Step in GenBank thyglutaryl-coenzyme A (EC 1.1.1.34), catalyzes the reduc Enzyme Pathway Gene ID tive conversion of HMGCoA to mevalonic acid (MVA). This reaction is reported to play a role in controlling plant iso Acetoacetyl-CoA thiolase 1 X78116 HMG-CoA synthase 2 X83882 prenoid biosynthesis (Gray, Adv. Bot. Res. 14:25-91 (1987); HMG-CoA reductase 3 X15032 Bachet al., Lipids 26: 637-648 (1991); Stermeret al., J. Lipid L19262 10 Res. 35: 1133-1140 (1994). Plant HMGCOA reductase genes Mevalonate kinase 4 X77793 Phosphomevalonate kinase 5 Not are often encoded by multigene families. The number of available genes comprising each multigene family varies, depending Mevalonate pyrophosphate 6 Y14325 on the species, ranging from two in Arabidopsis thaliana to at decarboxylase least seven in potato. Overexpression of plant HMGCoA Isopentenyl diphosphate 7 U492.59 15 reductase genes in transgenic tobacco plants has been isomerase U47324 Farnesyl pyrophosphate 8 X75789 reported to result in the overproduction of phytosterols synthase (Schaller et al., Plant Physiol. 109: 761-770 (1995)). Squalene synthase 9 AFOO4S60 Mevalonate kinase (EC 2.7.1.36) catalyzes the phosphory Squalene epoxidase 10 ABO16883 Squalene cyclase 11 U87266 lation of mevalonate to produce mevalonate 5-phosphate. It Sterol C-24 12, 18 U714OO has been reported that mevalonate kinase plays a role in the methyltransferase control of isoprenoid biosynthesis (Lalitha et al., Indian. J. Sterol C-4 demethylase 13, 19 Not Biochem. Biophys. 23:249-253 (1986)). A mevalonate kinase available Cycloeucalenol- 14 Not gene from Arabidopsis thaliana has been cloned (GeneBank obtusifoliol isomerase available accession number X77793; Riou et al., Gene 148: 293-297 Sterol C-14 demethylase 15 U74319 25 (1994)). Sterol C-14 reductase 16 PCTWO Phosphomevalonate kinase (EC 2.7.4.2) (MVAP kinase) is 97,48793 an enzyme associated with isoprene and ergosterol biosyn Sterol C-8 isomerase 17 AFO3O357 Sterol C-5 desaturase 2O X90454 thesis that converts mevalonate-5-phosphate to mevalonate Sterol C-7 reductase 21 U49398 5-pyrophosphate utilizing ATP (Tsay et al., Mol. Cell. Biol. Sterol C-24 isomerase 22 Klahre 30 11: 620-631 (1991)). et al. Mevalonate pyrophosphate decarboxylase (“MVAPP (1998) Plant Cell decarboxylase') (EC 4.1.1.33) catalyzes the conversion of 10: 1677-1690 mevalonate pyrophosphate to isopentenyl diphosphate Sterol C-24 reductase 23 Same (“IPP). The reaction is reported to be a decarboxylation/ as 22 35 dehydration reaction which hydrolyzes ATP and requires Sterol C-22 desaturase 24 Not available Mg". A cDNA encoding Arabidopsis thaliana MVAPP Sterol C-5 reductase 25 WO decarboxylase has been isolated (Toth et al., J. Biol. Chem. OO61771 271: 7895-7898 (1996)). An isolated Arabidopsis thaliana MVAPP decarboxylase gene was reported to be able to The plant sterol compound biosynthesis pathway has two 40 complement the yeast MVAPP decarboxylase. distinct components. The early pathway reactions, leading Isopentenyl diphosphate isomerase (“IPP:DMAPP) (EC from acetyl-CoA to squalene via mevalonic acid, are common 5.3.3.2) catalyzes the formation of dimethylallyl pyrophos to other isoprenoids. The later pathway reactions, leading phate (DMAPP) from isopentenyl pyrophosphate (IPP). from squalene to the major plant sterol compounds such as Plant IPP:DMAPP isomerase gene sequences have been 45 reported for this enzyme. It has also been reported that IPP: sitosterol, campesterol and stigmasterol, are committed bio DMAPP isomerase is involved in rubber biosynthesis in a synthetic reactions. latex extract from Hevea (Tangpakdee et al., Phytochemistry The early pathway reactions have been studied in fungi and 45:261-267 (1997). plants (Lees et al., Biochemistry and Function of Sterols, Nes Farnesyl pyrophosphate synthase (EC 2.5.1.1) is a prenyl and Parish, Eds. CRC Press, 85-99 (1997); Newman and 50 transferase which has been reported to play a role in providing Chappell, Biochemistry and Function of Sterols, Nes and polyisoprenoids for sterol compound biosynthesis as well as Parish, Eds. CRC Press, 123-134 (1997); Bach et al., Bio a number of other pathways (Li et al., Gene 17: 193-196 chemistry and Function of Sterols, Nes and Parish, Eds. CRC (1996)). Farnesyl pyrophosphate synthase combines Press, 135-150 (1997)). DMAPP with IPP to yield geranyl pyrophosphate (“GPP”). Acetoacetyl CoA thiolase (EC 2.3.1.9) catalyzes the first 55 The same enzyme condenses GPP with a second molecule of reported reaction, which consists of the formation of IPP to produce farnesyl pyrophosphate (“FPP). FPP is a acetoacetyl CoA from two molecules of acetyl CoA (Dixon et molecule that can proceed down the pathway to sterol com al., J. Steroid Biochem. Mol. Biol. 62: 165-171 (1997)). This pound synthesis, or that can be shuttled through other path enzyme has been purified from radish. A radish cDNA has ways leading to the synthesis of quinones or sesquiterpenes. been isolated by functional complementation in Saccharomy 60 Squalene synthase (EC 2.5.1.21) reductively condenses ces cerevisiae (GeneBank Accession if X78116). A radish two molecules of FPP in the presence of Mg" and NADPH to cDNA has also been screened against a cDNA library of form squalene. The reaction involves ahead-to-head conden Arabidopsis thaliana (Vollack and Bach, Plant Physiology sation, and forms a stable intermediate, presqualene diphos 111: 1097-1107 (1996)). phate. The enzyme is Subject to sterol demand regulation HMGCOA synthase (EC 4.1.3.5) catalyzes the production 65 similar to that of HMGCOA reductase. The activity of of HMGCoA. This reaction condenses acetyl CoA with squalene synthase has been reported to have a regulatory acetoacetyl CoA to yield HMGCoA. HMGCoA synthase has effect on the incorporation of FPP into sterol and other com US 7,544,863 B2 29 30 pounds for which it serves as a precursor (Devarenne et al., (1995)). The reaction has been reported to involve the ste Arch. Biochem. Biophys. 349: 205-215 (1998)). reospecific removal of the 5C, and 6C. hydrogen atoms, bio Squalene epoxidase (EC 1.14.99.7) (also called squalene synthetically derived from the 4 pro-Rand 5 pro-Shydrogens monooxygenase) catalyzes the conversion of squalene to of the (+) and (-) R-mevalonic acid, respectively. The reac squalene epoxide (2,3-oxidosqualene), a precursor to the ini tion is obligatorily aerobic, and requires NADPH or NADH. tial sterol molecule in the sterol compound biosynthetic path The desaturase has been reported to be a multienzyme com way, cycloartenol. This is the first reported step in the path plex present in microsomes. It consists of the desaturase way where oxygen is required for activity. The formation of itself, cytochrome bs, and a pyridine nucleotide-dependent squalene epoxide is also the last common reported step in flavoprotein. The A-desaturase is reported to be a mono sterol biosynthesis of animals, fungi, and plants. 10 oxygenase that utilizes electrons derived from a reduced pyri The later pathway of sterol compound biosynthetic steps dine nucleotide via cytochrome bs. starts with the cyclization of squalene epoxide and ends with Sterol C-7 reductase catalyzes the reduction of a A-double the formation of 5-24-alkyl sterols in plants. bond in A'-sterols to generate the corresponding A-sterol. 2,3-oxidosqualene cycloartenol cyclase (EC 5.4.99.8) It has been reported that the mechanism involves, like many (also called cycloartenol synthase) is the first step in the sterol 15 other sterol enzymes, the formation of a carbocationic inter compound pathway that is plant-specific. The cyclization of mediate via electrophilic “attack” by a proton. 2.3-oxidosqualene leads to lanosterol in animals and fungi, Sterol C-24(28) isomerase catalyzes the reduction of a while in plants the product is cycloartenol. Cycloartenol con A*-A, a conversion that modifies the side chain. The tains a 9, 19-cyclopropyl ring. The cyclization is reported to product is a A-24-alkyl sterol. Sterol C-24 reductase proceed from the epoxy end in a chair-boat-chair-boat catalyzes the reduction of the “double bond at C-24, sequence that is mediated by a transient C-20 carbocationic which produces sitosterol. Recently, Klahre et al. (1998) intermediate. Plant Cell 10:1677-1690) discovered that both the isomeriza S-adenosyl-L-methionine:sterol C-24 methyl transferase tion and reduction steps are catalyzed by an enzyme coded by (“SMT1') (EC 2.1.1.41) catalyzes the transfer of a methyl the same gene, i.e., DIM/DWF1. group from S-adenosyl-L-methionine to the C-24 center of 25 Sterol C-22 desaturase (EC 2.7.3.9) catalyzes the forma the sterol side chain (Nes et al. (1991).J. Biol. Chem. 266(23): tion of a double bond at C-22 on the side chain. This forma 15202-15212). This is the first of two methyl transfer reac tion of a double bond at C-22 on the side chain marks the end tions that have been reported to be an obligatory and rate of the sterol compound biosynthetic pathway, and results in limiting step of the sterol compound-producing pathway in the formation of stigmasterol (Benveniste (1986) Annu. Rev. plants. The second methyltransfer reaction occurs later in the 30 Plant Physiol. 37:275-308). The C-22 desaturase in yeast, pathway after the A7 isomerase. The enzyme responsible for which is the reported final step in the biosynthesis of ergos the second methyl transfer reaction is named SMTII (Bou terol in that organism, requires NADPH and molecular oxy vier-Nave, P. et al., (1997) Eur: J. Biochem. 246:518-529). gen. In addition, the reaction is also reported to involve a An isoform, SMTII, catalyzes the conversion of cycloartenol cytochrome P450 that is distinct from a cytochrome P450 to a A-24-alkyl sterol, cyclosadol (Guo et al. (1996) 35 participating in demethylation reactions (Lees et al. (1995) Tetrahed. Lett. 37(38):6823-6826). Lipids 30: 221-226). Sterol C-4 demethylase catalyzes the first of several dem Phytosterols are biogenetic precursors of brassinosteroids, ethylation reactions, which results in the removal of the two steroid alkaloids, steroid sapogenins, ecdysteroids, and Ste methyl groups at C-4. While in animals and fungi the removal roid hormones. This precursor role of phytosterols is often of the two C-4 methyl groups occurs consecutively, in plants 40 described as a “metabolic function. A common transforma it has been reported that there are other steps between the first tion of free sterols in tissues of Vascular plants is the conju and second C-4 demethylations. The C-4 demethylation is gation at the 3-hydroxy group of sterols with long-chain fatty catalyzed by a complex of microsomal enzymes consisting of acids to form steryl esters, or with a Sugar, usually with a a monooxygenase, an NAD-dependent sterol 4-decarboxy single molecule of B-D-glucose, to form steryl glycosides. lase, and an NADPH-dependent 3-ketosteroid reductase. 45 Some of the sterylglycosides are additionally esterified, at the Cycloeucalenol-obtusifoliol isomerase (“COI) catalyzes 6-hydroxy group of the Sugar moiety, with long-chain fatty the opening of the cyclopropyl ring at C-9. The opening of the acids to form acylated steryl glycosides. cyclopropyl ring at C-9 creates a double bond at C-8. The existence of several enzymes that are specifically asso Sterol C-14 demethylase catalyzes demethylation at C-14, ciated with the synthesis and breakdown of conjugated Sterols which removes the methyl group at C-14 and creates a double 50 has been reported (Wojciechowski, Physiology and Biochem bond at that position. In both fungi and animals, this is the first istry of Sterols, eds. Patterson, Nes, AOCS Press, 361 (1991)). step in the sterol synthesis pathway. Sterol 14-demethylation Enzymes involved in this process include: UDPGlc:Sterol is mediated by a cytochrome P-450 complex. glucosyltransferase, phospho(galacto)glyceride sterylgluco Sterol C-14 reductase catalyzes a C-14 demethylation that side acyltransferase, and sterylglycoside and sterylester results in the formation of a double bond at C-14 (Ellis et al., 55 hydrolases. Gen. Microbiol. 137:2627-2630 (1991)). This double bond is UDPGlc:sterol glucosyltransferase (EC 2.4.1.173) cata removed by a A' reductase. The normal substrate is 4-me lyzes glucosylation of phytosterols by glucose transfer from thyl-8, 14.24 (24)-trien-33-ol. NADPH is the normal reduc UDP-glucose (“UDPGl'). The formation of sterylglycosides tant. can be measured using UDP-''Clglucose as the substrate. Sterol C-8 isomerase catalyzes a reaction that involves 60 Despite certain differences in their specificity patterns, all further modification of the tetracyclic rings or the side chain reported UDPGlc:sterol glucosyltransferases preferentially (Duratti et al., Biochem. Pharmacol. 34: 2765-2777 (1985)). glucosylate only sterols or sterol-like molecules that contain The kinetics of the sterol isomerase-catalyzed reaction favor a C-3 hydroxy group, a B-configuration, and which exhibit a a A 6A isomerase reaction that produces a A' group. planar ring. It has been reported that UDPGlc:sterol gluco Sterol C-5 desaturase catalyzes the insertion of the 65 Syltransferases are localized in the microsomes. A-double bond that normally occurs at the A-sterol level, Phospho(galacto)glyceride steryl glucoside acyltrans thereby forming a A-sterol (Parks et al., Lipids 30: 227-230 ferase catalyzes the formation of acylated steryl glycosides US 7,544,863 B2 31 32 from the Substrate sterylglycoside by transfer of acyl groups sis of sterols, phytosterols, phytosterol esters, phytostanols, from Some membranous polar acyllipids to steryl glycoside phytostanol esters, or combinations thereof, comprising molecules. sexually crossing a transgenic plant of the present invention Acylglycerol:sterol acyltransferase (EC 2.3.1.26) cata with Such a corresponding plant. The latter can be a non lyzes the reaction wherein certain acylglycerols act as acyl transgenic plant, or a transgenic plant containing introduced donors in a phytosterol esterification. In plants, the activity of DNA encoding a trait other than one affecting sterol, phy acylglycerol: Sterol acyltransferase is reported to be associ tosterol, etc., biosynthesis. For example, such trait may be ated with membranous fractions. A pronounced specificity insector herbicide resistance. Plants produced by this method for shorter chain unsaturated fatty acids was reported for all also form part of the present invention. acyltransferase preparations studied in plants. For example, 10 Also included are plants that accumulate an elevated level acylglycerol: Sterol acyltransferases from spinach leaves and of sitosterol, at least one sitosterol ester, sitostanol, at least mustard roots can esterify a number of phytosterols. one sitostanol ester, or mixtures thereof, in seeds thereof Sterylglycoside and sterylester hydrolases (“SG-hydro compared to seeds of a corresponding plant comprising no lases) catalyze the enzymatic hydrolysis of sterylglycosides introduced DNA encoding a polypeptide or protein that to form free sterols. The SG-hydrolase activity is not found in 15 affects the biosynthesis of sterols, phytosterols, phytosterol mature, ungerminated seeds, is reported to emerge only after esters, phytostanols, phytostanol esters, or combinations the third day of germination, and is found mainly in the thereof, which are apomictic. cotyledons. It has been reported that phospho(galacto)glyc A process of increasing the formation of steroid pathway eride:SG acyltranaferase may catalyze a reversible reaction. products in a transformed host cell as compared to an other Enzymatic hydrolysis of sterylesters in germinating seeds of wise identical non-transformed host cell comprising the fol mustard, barley and corn is reported to be low in dormant lowing steps. A host cell is transformed with a recombinant seeds, but increases during the first ten days of germination. vector comprising (a) as operably linked components in the 5' This activity is consistent with a decrease in sterylesters and to 3' direction, a promoter, a DNA sequence encoding a an increase in free sterols over the same temporal period. polypeptide exhibiting 3-hydroxy-3-methylglutaryl-Coen II. Processes for Modifying Steroid Compound 25 Zyme A reductase enzyme activity, and a transcription termi Biosynthesis and Accumulation nation signal sequence; and (b) as operably linked compo In order to obtain seed producing oil containing elevated nents in the 5' to 3’ direction, a promoter, a DNA sequence levels of phytostanols and phytostanol esters such as sito encoding a steroid pathway enzyme, and a transcription ter stanol and sitostanol esters, these recombinant constructs or mination signal sequence. The steroid pathway enzyme is a expression cassettes can be introduced into plant cells by any 30 squalene epoxidase enzyme, a sterol methyl transferase I number of conventional means known in the art and regener enzyme, a sterol C4-demethylase enzyme, a obtusifoliol ated into fertile transgenic plants. The genome of such plants C14C.-demethylase enzyme, a sterol C5-desaturase enzyme, can then comprise introduced DNA encoding various steroid and a sterol methyl transferase II enzyme. The transformed pathway enzymes, alone or in combination, that achieves the plant cell is regenerated into a transgenic plant. desirable effect of enhancing the levels of phytostanols, phy 35 A plant contemplated by this invention is a vascular, mul tostanol esters, mixtures thereof in the oil of seed thereof. ticellular higher plant. Such higher plants will hereinafter by Preferably, the genome can comprise introduced DNA usually referred to simply as “plants'. Such “plants' include encoding a HMG CoA reductase enzyme and an introduced both complete entities having leaves, stems, seeds, roots and DNA encoding one or more of a squalene epoxidase, a sterol the like as well as callus and cell cultures that are monocoty methyl transferase I, a sterol C4-demethylase, an obtusifoliol 40 ledonous and dicotyledonous. Dicotyledonous plants are a C14C.-demethylase, a sterol C5-desaturase, a sterol methyl preferred embodiment of the present invention. transferase II. In each case, the foregoing introduced DNAS Preferred plants are members of the Solanaceae, Legumi can be operatively linked to regulatory signals that cause nosae, Ammiaceae, Brassicaceae, Gramineae, Carduaceae seed-specific expression thereof. and Malvaceae families. Exemplary plant members of those The present invention encompasses not only such trans 45 families are tobacco, petunia and tomato (Solanaceae), Soy genic plants, but also transformed plant cells, including cells bean and alfalfa (Leguminosae), carrot (Ammiaceae), corn, and seed of such plants, as well as progeny of such plants, for maize and barley (Gramineae), Arabidopsis (Brassicaceae), example produced from the seed. Transformed plant cells and guayule (Carduaceae), and cotton (Malvaceae). A preferred cells of the transgenic plants encompassed herein can be plant is tobacco of the strain Nicotiana tabacum (N. grown in culture for a time and under appropriate conditions 50 Tabacum), cotton of the strain Coker line 3 12-5A, soybean of to produce oil containing elevated levels of phytosterols and/ the strain Glycine max, alfalfa of the strain RYSI or tomato of or phytostanols and their corresponding esters. Alternatively, the strain Lycopersicon esculentium. Other plants include the phytosterols, phytostanols, and their corresponding esters canola, maize and rape. can be isolated directly from the cultures. A transgenic plant contemplated by this invention is pro In addition, of course, seed obtained from the transgenic, 55 duced by transforming a plant cell or protoplast with an progeny, hybrid, etc., plants disclosed herein can be used in added, exogenous structural gene that encodes a polypeptide methods for obtaining oil containing phytosterols, phy having HMG-CoA reductase activity and an exogenous struc tosterol esters, phytostanols, phytostanol esters, or mixtures tural gene that encodes at least one polypeptide have steroid thereof employing extraction and processing procedures pathway enzyme activity to produce a transformed plant cell, known in the art. Note, in this regard, Kochhar (1983) Prog. 60 and regenerating a transgenic plant form the transformed Lipid Res. 22:161-188. plant cell. The encoded polypeptide is expressed both in the The present invention also encompasses a method of pro transformed plant cell or protoplast and the resulting trans ducing a plant that accumulates an elevated level of sitosterol, genic plant. (The phrase “plant cell' will hereinafter be used at least one sitosterol ester, sitostanol, at least one sitostanol to include a plant protoplast, except where plant protoplasts ester, or mixtures thereof, in seeds thereof compared to seeds 65 are specifically discussed). of a corresponding plant comprising no introduced DNA A non-transgenic plant that serves as the source of the plant encoding a polypeptide or protein that affects the biosynthe cell that is transformed, i.e. the precursor cell, is referred to US 7,544,863 B2 33 34 herein as a “native, non-transgenic' plant. The native, non Sterol accumulation can also be used to distinguish transgenic plant is of the same strain as the formed transgenic between native, non-transgenic and transgenic plants. A plant. transgenic plant has at least about twice the total sterol con Sterol production in a transgenic plant of the present inven tent as a native, non-transgenic plant where a single added tion is increased by increasing the activity of the enzyme gene is present. Greater differences up to about forty-fold HMG-CoA reductase, which enzyme catalyzes the conver sion of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG have also been observed. CoA) to mevalonate and the activity of at least one other Sitostanol, sitostanol ester, and tocopherol biosynthesis steroid pathway enzyme. As used herein, the term “specific and accumulation in plants can be modified in accordance activity” means the activity normalized to cellular protein 10 with the present invention by variously expressing the nucleic COntent. acid coding sequences discussed above, alone or in combina HMG-COA reductase activity is increased by increasing tion, as described herein. The expression of sequences encod the amount (copy number) of a gene encoding a polypeptide ing Sterol methyltransferase II enzymes facilitates the pro having HMG-CoA reductase catalytic activity. Expression of duction of plants in which the biosynthesis and accumulation the increased amount of that encoded structural gene 15 enhances the activity of that enzyme. of campesterol, campestanol, and their esters can be reduced The amount of the expressed gene is increased by trans as these enzymes shunt Sterol intermediates away from forming a plant cell with a recombinant DNA molecule com campesterol, and toward sitosterol and sitostanol. prising a vector operatively linked to a DNA segment that III. DNA Encoding Useful Polypeptides encodes a polypeptide having HMG-CoA reductase activitiy, The present invention contemplates a recombinant con and a promoter Suitable for driving the expression of that polypeptide in that plant cell, and culturing the transformed structor a recombinant vector that contains a DNA sequence plant cell into a transgenic plant. Such a polypeptide includes encoding a polypeptide exhibiting 3-hydroxy-3-methylglu intact as well as a catalytically active, truncated HMG-CoA taryl-Coenzyme A (HMG-CoA) reductase activity and a reductase proteins. 25 DNA sequence encoding a polypeptide exhibiting the activity Thus, a transformed plant cell and a transgenic plant have of a steroid pathway enzyme. Each polypeptide-encoding one or more added, exogenous genes that encode a polypep DNA sequence is operably linked in the 5' to 3" direction tide having HMG-CoA reductase activity and at least one independent of the other sequence. Each DNA sequence in other steroid pathway enzyme activity relative to a native, the 5' to 3' direction comprises a promoter, then the DNA non-transgenic plant or untransformed plant cell of the same 30 sequence encoding the polypeptide then a transcription ter type. As such, a transformed plant cell or transgenic plant can mination signal sequence. The steroid pathway enzyme is a be distinguished from an untransformed plant cell or native, squalene epoxidase enzyme, a sterol methyl transferase I nontransgenic plant by standard technology Such as agarose enzyme, a sterol C4-demethylase enzyme, a obtusifoliol separation of DNA fragments or mRNAs followed by transfer C14C-demethylase enzyme, a sterol C5-desaturase enzyme, and appropriate blotting with DNA or RNA, e.g., Southern or 35 Northern blotting, or by use of polymerase chain reaction or a sterol methyl transferase II enzyme. It is contemplated technology, as are well known. Relative HMG-CoA reduc that HMG-CoA reductase and steroid pathway enzyme tase activity of the transformed cell or transgenic plant with activities come from a mutant or truncated form of those untransformed cells and native, non-transgenic plants or cell enzymes, such as a truncated HMG-CoA reductase lacking cultures therefrom can also be compared, with a relative 40 the transmembrane region while retaining a functional cata activity for that enzyme of about 1.5:1 for transgenic (trans lytic domain. Several HMG CoA reductase sequences are formed) to native (untransformed) showing transformation. known in the art. An amino acid alignment for these is shown Higher relative activity ratios such as about 15:1 have also in FIG. 32. The sources of the sequences used in building the been observed. multiple alignment are listed in Table 5.
TABLE 5 Sources of Sequences Used In Building The Multiple Alignment methanobac Swissprot:hmdh metth Begin: End:397 O26.662 methanococ Swissprot:hmdh meta Begin: End:405 Q58116 halobacter Swissprot:hmdh halvo Begin: End:403 Q59468 Sulfolobus Swissprot:hmdh Sulso Begin: End:409 OO8424 yeast2 gp pln1:ySchmgcr2 1 Begin: End: 1045 M222.SS yeast1 gp pln1:ySchmgcrl. 1 Begin: End:1054 M22002 phycomyces Swissprot:hmdh phybl Begin: End: 105 Q12649 fusarium Swissprot:hmdh fuSmo Begin: End:976 Q12577 candida gp pln1:ab012603 1 Begin: End:934 ABO12603 dictyoste2 Swissprot:hmd2 dicoli Begin: End:481 P34136 wheat1 pir2:pg.0761 Begin: End:150 hydroxymethylglutaryl-CoA reductase (NADPH) rice Swissprot:hmdh orysa Begin: End:SO9 P4.8019 CO sp plant:o24594 Begin: End:579 O24594 wheat pir2:pg.0763 Begin: End:150 hydroxymethylglutaryl-CoA reductase (NADPH) wheat2 pir2:pg.0762 Begin: End:150 hydroxymethylglutaryl-CoA reductase (NADPH) Soybean gmtx6:30820 1rS9f1 Begin:101 End:259 from proprietary soy sequence database rubbertre3 Swissprot:hmd3 hewbr Begin: End:586 Q00583 rosyperiwi Swissprot:hmdh catro Begin: End:601 Q03163 tomato Swissprot:hmd2 lyces Begin: End:6O2 P4.8022 woodtobacc Swissprot:hmdh nicsy Begin: End:604 Q01559 potato gp pln1:pothmgri 1 Begin: End:596 LO1400 US 7,544,863 B2 35 36
TABLE 5-continued Sources of Sequences Used In Building The Multiple Alignment radish sp plant:q43826 Begin: End:573 Q43826 arabadopsis1 gp pln1:athhmgcoar 1 Begin: End:592 L19261 cucumismel gp pln1:ab021862 1 Begin: End:587 AB021862 rubbertre2 Swissprot:hmd2 hewbr Begin: End:210 P29058 rubbertre1 Swissprot:hmd1 hewbr Begin: End:575 P29057 camptothec Swissprot:hmdh camac Begin: End:593 P48021 arabadops2 Swissprot:hmd2 arath Begin: End:562 P432S6 chineseham Swissprot:hmdh crigr Begin: End:887 POO347 chineseha2 gp rod:cruhmg14 1 Begin: End:887 LOO183 Syrian hamst gp rod:hamhmgcob 1 Begin: End:887 M127OS rat Swissprot:hmidh rat Begin: End:887 P51639 rabbit Swissprot:hmidh rabit Begin: End:888 Q29512 human gp pri2:humhmgcoa. 1 Begin: End:888 M11058 OUSE gp rod:mushmgcoa. 1 Begin: End:224 M62766 Xenopus Swissprot:hmdh Xenla Begin: End:883 P2O715 seaurchin Swissprot:hmdh Strpu Begin: End:932 P16393 cockroach Swissprot:hmdh blage Begin: End:856 PS4960 drosophila Swissprot:hmdh drome Begin: End:916 P14773 dictyoste1 Swissprot:hmd1 dicci Begin: End:SS2 P34135 Schistosom Swissprot:hmdh Schma. Begin: End:948 P16237 archaeoglo Swissprot:hmdh arcfu Begin: End:436 O28.538 pseudomonas gp bct1:psehmgcoa. 1 Begin: End:428 M24O15
These sequences, and their truncated counterparts, are use 25 long as the required DNA sequence is present (including start ful in the present invention. Examples of such preferred HMG and stop signals), additional base pairs can be present at either CoA reductases include the truncated rubber and Arabidopsis end of the segment and that segment can still be utilized to HMG CoA reductases disclosed herein. express the protein. This, of course, presumes the absence in Other enzyme-encoding DNAs can be introduced into the segment of an operatively linked DNA sequence that plants to elevate even further the levels of desirable A5 sterols 30 represses expression, expresses a further product that con and their reduced stanol counterparts as well as other phy Sumes the enzyme desired to be expressed, expresses a prod tosterols and tocopherols. Thus, the DNA sequences contem uct other than the desired enzyme or otherwise interferes with plated for use in the present invention, which can be used the structural gene of the DNA segment. alone or in various combinations as discussed below, include, but are not limited to, those encoding the following enzymes: 35 Thus, as long as the DNA segment is free of such interfer 3-hydroxysteroid oxidases; steroid 5reductases; sterol meth ing DNA sequences, a DNA segment of the invention can be yltransferases; Sterol acyltransferases; and S-adenosylme up to 15,000 base pairs in length. The maximum size of a thionine-dependent C.-tocopherol methyltransferases. recombinant DNA molecule, particularly a plant integrating In each case, the sequences encoding these enzymes can vector, is governed mostly by convenience and the vector size comprise an expression cassette comprising, operably linked 40 that can be accommodated by a host cell, once all of the in the 5' to 3" direction, a seed-specific promoter, the enzyme minimal DNA sequences required for replication and expres coding sequence, and a transcriptional termination signal sion, when desired, are present. Minimal vector sizes are well sequence functional in a plant cell such that the enzyme is known. successfully expressed. For use in the methods disclosed Also encompassed by the present invention are nucleotide herein, the recombinant constructs or expression cassettes 45 sequences biologically functionally equivalent to those dis can be incorporated in a vector, for example a plant expres closed herein, that encode conservative amino acid changes sion vector. Such vectors can be transformed into host cells within the amino acid sequences of the presently disclosed Such as bacterial cells, for example during the preparation or enzymes, producing 'silent changes therein. Such nucle modification of the recombinant constructs, and plant cells. 50 otide sequences contain corresponding base Substitutions Thus, the invention encompasses plants and seeds comprising based upon the genetic code compared to the nucleotide Such transformed plant cells. sequences encoding the presently disclosed enzymes. Substi It will be apparent to those of skill in the art that the nucleic tutes for an amino acid within the enzyme sequences dis acid sequences set forth herein, either explicitly, as in the case closed herein is selected from other members of the class to of the sequences set forth above, or implicitly with respect to 55 which the naturally occurring amino acid belongs. Amino nucleic acid sequences generally known and not present acids can be divided into the following four groups: (1) acidic herein, can be modified due to the built-in redundancy of the amino acids; (2) basic amino acids; (3) neutral polar amino genetic code and noncritical areas of the polypeptide that are acids; and (4) neutral non-polar amino acids. Representative Subject to modification and alteration. In this regard, the amino acids within these various groups include, but are not present invention contemplates allelic variants of structural 60 limited to: (1) acidic (negatively charged) amino acids such as genes encoding a polypeptide having HMG-CoA reductase aspartic.acid and glutamic acid; (2) basic (positively charged) activity. amino acids such as arginine, histidine, and lysine; (3) neutral The previously described DNA segments are noted as hav polar amino acids such as glycine, serine, threonine, cysteine, ing a minimal length, as well as total overall length. That cystine, tyrosine, asparagine, and glutamine; and (4) neutral minimal length defines the length of a DNA segment having 65 nonpolar (hydrophobic) amino acids Such as alanine, leucine, a sequence that encodes a particular polypeptide having isoleucine, Valine, proline, phenylalanine, tryptophan, and HMG-CoA reductase activity. As is well known in the art, as methionine. US 7,544,863 B2 37 38 A. HMG-CoA Reductase Intact hamster HMG-CoA reductase comprises about 887 The introduction of an HMG CoA reductase gene into a amino acid residues (SEQ ID NO:2 of Chappell et al.). A cell results in a higher carbon throughput through the steroid structural gene encoding an intact hamster HMG-CoA reduc synthesis pathway. The introduction of a truncated HMG tase enzyme of 887 amino acid residues comprises base pairs CoA reductase gene (lacking the transmembrane region, from about nucleotide position 164 to about nucleotide posi resulting in a soluble HMG CoA reductase enzyme) provides tion 2824 of SEQID NO:1 of Chappellet al. higher HMG CoA reductase activity and thus increased A preferred structural gene is one that encodes a polypep delta-5 steroid compound production over the same case with tide corresponding to only the catalytic region of the enzyme. an introduced full-length HMG CoA reductase gene. A useful Two catalytically active segments of hamster HMG-CoA truncated HMG CoA reductase nucleic acid encodes at least 10 reductase have been defined. Liscum et al., J. Biol. Chem. the catalytic domain. 260(1):522 (1985). One segment containing a catalytic region Hydroxymethylglutaryl-CoA reductase is enzyme number has an apparent molecular weight of 62 kDa and comprises 1.1.1.88, using the recommended nomenclature of the Inter amino acid residues from about position 373 to about position national Union of Biochemistry and Molecular Biology on 887. A second segment containing a catalytic region has an the Nomenclature and Classification of Enzymes, Enzyme 15 apparent molecular weight of 53 kDa segment and comprises Nomenclature 1992, Edwin C. Webb, ed., Academic Press, amino acid residues from about position 460 to about position Inc. (San Diego, Calif: 1992), page 35. 887. The 62 kDa catalytically active segment is encoded by The present invention contemplates transforming a plant base pairs from about nucleotide position 1280 to about cell with a structural gene that encodes a polypeptide having nucleotide position 2824 of SEQID NO:1 of Chappellet al. HMG-CoA reductase activity. The HMG-CoA reductase The 53 kDa catalytically active segment is encoded by base enzymes of both animal and yeast cells comprise three dis pairs from about nucleotide position 1541 to about nucleotide tinct amino acid residue sequence regions, which regions are position 2824 of SEQID NO:1 of Chappellet al. designated the catalytic region, the membrane-binding region In a preferred embodiment, the utilized structural gene and the linker region. encodes the catalytic region and at least a portion of the linker The catalytic region contains the active site of the HMG 25 region of HMG-CoA reductase. The linker region of hamster CoA reductase enzyme and comprises about forty percent of HMG-CoA reductase comprises amino acid residues from the COOH-terminal portion of intact HMG-CoA reductase about position 340 to about position 373 or from about posi enzyme. tion 340 to about position 460, depending upon how the The membrane-binding region contains hydrophobic catalytic region is defined. These linker regions are encoded 30 by base pairs from about nucleotide position 1180 to about amino acid residues and comprises about fifty percent of the nucleotideposition 1283 or from about position 1180 to about NH-terminal portion of intact HMG-CoA reductase position 1540, respectively of SEQIDNO:1 of Chappellet al. enzyme. The structural gene encoding the linker region is operatively The linker region connects the catalytic and membrane linked to the structural gene encoding the catalytic region. binding regions, and constitutes the remaining about ten per 35 In one particularly preferred embodiment, a structural gene cent of the intact enzyme. encoding a catalytically active, truncated HMG-CoA reduc As discussed in greater detail below, only the catalytic tase enzyme can optionally contain base pairs encoding a region of HMG-CoA reductase is needed herein to provide Small portion of the membrane region of the enzyme. the desired enzyme activity. Thus, an exogenous structural A structural gene encoding a polypeptide comprising a gene that encodes a polypeptide corresponding to that cata 40 catalytically active, truncated or intact HMG-CoA reductase lytic region is the minimal HMG Co. A reductase gene enzyme from other organisms such as yeast can also be used required for transforming plant cells in addition to one of the in accordance with the present invention. steroid pathway enzymes discussed below. The present Yeast cells contain two genes encoding HMG-CoA reduc invention therefore contemplates use of both intact and trun tase. The two yeast genes, designated HMG1 and HMG2. cated structural genes that encode a polypeptide having 45 encode two distinct forms of HMG-CoA reductase, desig HMG-CoA reductase activity. nated HMG-CoA reductase 1 SEQID NO:3 of Chappellet al. A structural gene encoding a polypeptide having HMG are presented in FIG. 3 of Chappell et al., are taken from CoA reductase activity can be obtained or constructed from a Basson et al. Mol. Cell Biol. 8(9):3797 (1988). The nucle variety of sources and by a variety of methodologies. See, e.g. otide base sequences of HMG2 SEQID NO:5 of Chappellet Carlson et al., Cell, 28:145 (1982); Rine et al., Proc. Natl. 50 al. as well as the amino acid residue sequence of HMG-CoA Acad. Sci. USA, 80:6750 (1983). Exemplary of such struc reductase 2 SEQ ID NO:6 of Chappell et al. are set forth tural genes are the mammalian and yeast genes encoding therein in the Sequence Listing. HMG-CoA reductase or the catalytic region thereof. The entire HMG1 gene comprises about 3360 base pairs The disclosures of Chappell, et al., U.S. Pat. No. 5,349,126, SEQID NO:3 of Chappellet al. Intact HMG-CoA reductase are incorporated in full herein by reference. The mammalian 55 1 comprises an amino acid sequence of about 1054 amino genome contains a single gene encoding HMG-CoA reduc acid residues SEQ ID NO:4 of Chappell et al. Thus, the tase. The nucleotide sequence of the hamster and human gene minimal portion of the HMG1 gene that encodes an intact for HMG-CoA reductase have been described in Chappellet enzyme comprises base pairs from about nucleotide position al. A composite nucleotide sequence of DNA corresponds to 121 to about position 3282 of FIG. 3, SEQ ID NO:3 of the mRNA SEQ ID NO:1 of Chappell et al., as well as the 60 Chappell et al. derived amino acid residue sequence SEQID NO:2 of Chap The entire HMG2 gene comprises about 3348 base pairs pell et al., for hamster HMG-CoA reductase is provided in SEQID NO:5 of Chappellet al. Intact HMG-CoA reductase FIG. 2 of Chappell et al, reprinted from Chin et al., Nature, 2 comprises about 1045 amino acid residues SEQID NO:6 of 308:613 (1984). The composite nucleotide sequence of FIG. Chappellet al. Thus, the minimal portion of HMG2 gene that 2, SEQID NO:1 of Chappell et al., comprising about 4768 65 encodes intact HMG-CoA reductase 2 comprises base pairs base pairs, includes the nucleotide sequence encoding the from about nucleotide position 121 to about position 3255 of intact hamster HMG-CoA reductase enzyme. SEQ ID NO:5 of Chappellet al. US 7,544,863 B2 39 40 By analogy to the truncated hamster structural gene, struc P48019 Oryza sativa (rice). 3-hydroxy-3-methylglutaryl tural genes encoding polypeptides comprising catalytically coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase) active, truncated HMG-CoA reductase enzymes from yeast (fragment). 2/1996 can also be used in accordance with the present invention. O24594 Zea mays (maize). 3-hydroxy-3-methylglutaryl The catalytic region of HMG-CoA reductase 1 comprises coenzyme a reductase (ec 1.1.1.88).5/1999 amino acid residues from about residue 618 to about reside PQ0763 hydroxymethylglutaryl-CoA reductase 1054: i.e., the COOH-terminus. A structural gene that (NADPH) (EC 1.1.1.34) (HMGR 23) wheat (fragment) encodes the catalytic region comprises base pairs from about PQ0762 hydroxymethylglutaryl-CoA reductase nucleotide position 1974 to about position 3282 of FIG. 3 of (NADPH) (EC 1.1.1.34) (HMGR 18) wheat (fragment) Chappell et al. 10 from proprietary soy sequence database The linker region of HMG-CoA reductase 1 comprises an Q00583 hevea brasiliensis (para rubber tree). 3-hydroxy amino acid sequence from about residue 525 to about residue 3-methylglutaryl-coenzyme a reductase 3 (ec 1.1.1.34) (hmg 617. A structural gene that encodes the linker region com coa reductase 3). 7/1998 prises nucleotides from about position 1695 to about position Q03163 catharanthus roseus (rosy periwinkle) (madagas 1973 of FIG.3 of Chappellet al. A structural gene encoding 15 car periwinkle). 3-hydroxy-3-methylglutaryl-coenzyme a the linker region of the enzyme operatively linked to the reductase (ec 1.1.1.34) (hmg-coa reductase). 7/1998 structural gene encoding the catalytic region of the enzyme. P48022 lycopersicon esculentum (tomato). 3-hydroxy-3- Also by analogy to the truncated hamster gene, a truncated methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg HMG1 gene can optionally contain nucleotide base pair coa reductase 2). 7/1998 sequences encoding a small portion of the membrane-binding Q01559 nicotiana Sylvestris (wood tobacco). 3-hydroxy region of the enzyme. Such a structural gene preferably com 3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg prises base pairs from about nucleotide position 121 to about coa reductase). 7/1998 position 147 and from about position 1695 to about position LO1400 Solanum tuberosum Potato hydroxymethylglu 3282 of FIG.3 of Chappellet al. taryl coenzyme A reductase (hmgr) mRNA, complete cds; A construct similar to those above from an analogous por 25 putative. 4/1996 tion of yeast HMG-CoA reductase 2 can also be utilized. Q43826 raphanus sativus (radish). hydroxymethylglu A nucleic acid sequence encoding HMG-CoA reductase taryl-coa reductase (ec 1.1.1.34) (hydroxymethylglutaryl-coa from Hevea brasiliensis has been disclosed by Chye et al. reductase (nadph)) (3-hydroxy-3-methylglutaryl-coenzyme (1991) Plant Mol. Biol. 16:567-577. A nucleic acid sequence a red encoding an Arabidopsis thaliana HMG-CoA reductase has 30 L19261 Arabidopsis thaliana Arabidopsis thaliana HMG been published by Caelles et al. (1989) Plant Mol. Biol. 13: cCA reductase gene, complete cds. 4/1994 627-638, and is also available as GenBank accession number AB021862 Cucumis melo Cucumis melo mRNA for L19261. U.S. Pat. Nos. 5,306,862 and 5,365,017 disclose HMG-CoA reductase, complete cds; putative. 1/1999 additional DNA sequences encoding HMG-CoA reductases. P29058 hevea brasiliensis (para rubber tree). 3-hydroxy The following sequences are listed by Genbank Accession 35 3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg numbers: coa reductase 2) (fragment).35735 026.662 methanobacterium thermoautotrophicum. 3-hy P29057 hevea brasiliensis (para rubber tree). 3-hydroxy droxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) 3-methylglutaryl-coenzyme a reductase 1 (ec 1.1.1.34) (hmg (hmg-coa reductase). 12/1998 coa reductase 1). 7/1998 Q58116 methanococcus jannaschii. 3-hydroxy-3-methyl 40 P48021 camptotheca acuminata. 3-hydroxy-3-methylglu glutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc tase).7/1998 tase). 11/1997 Q59468 halobacterium volcanii (haloferax volcanii). P43256 arabidopsis thaliana (mouse-ear cress). 3-hy 3-hydroxy-3-methylglutaryl-coenzyme a reductase (ec droxy-3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg-coa reductase). 7/1998 45 1.1.1.34) (hmg-coa reductase 2) (hmgr2). 7/1998 O08424 sulfolobus solfataricus. 3-hydroxy-3-methylglu P00347 cricetulus griseus (chinese hamster). 3-hydroxy taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc 3-methylglutaryl-coenzyme A reductase (ec 1.1.1.34) (hmg tase). 12/1998 coA reductase). 11/1997 M22255 Saccharomyces cerevisiae Yeast HMG-CoA L00183 Cricetulus sp. Hamster 3-hydroxy-3-methylglu reductase (HGM2) gene, complete cds: 3-hydroxy-3-methyl 50 taryl coenzyme A (HMG CoA) reductase gene, exons 19 and glutaryl coenzyme A reductase. 4/1993 20: 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA). M22002 Saccharomyces cerevisiae Yeast HMG-CoA 4f1993 reductase (HGM1) gene, complete cds: 3-hydroxy-3-methyl M12705 Mesocricetus auratus Syrian hamster 3-hydroxy glutaryl coenzyme A reductase. 4/1993 3-methylglutaral coenzyme A reductase (HMG-CoA reduc Q12649 phycomyces blakesleeanus. 3-hydroxy-3-methyl 55 tase) mRNA, complete cds; 3-hydroxy-3-methylglutaral glutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc coenzyme A red tase) (fragment). 11/1997 P51639 rattus norvegicus (rat). 3-hydroxy-3-methylglu Q12577 fitsarium moniliforme (gibberellafujikuroi).3-hy taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc droxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) tase). 12/1998 (hmg-coa reductase). 11/1997 60 Q295 12 oryctolagus cuniculus (rabbit). 3-hydroxy-3-me AB012603 Candida utilis Candida utilis HMG mRNA for thylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa HMG-CoA reductase, complete cds. 7/1998 reductase).7/1999 P34136 dictyostelium discoideum (slime mold). 3-hy M11058 Homo sapiens Human 3-hydroxy-3-methylglu droxy-3-methylglutaryl-coenzyme a reductase 2 (ec taryl coenzyme A reductase mRNA, complete cds; 3-hy 1.1.1.34) (hmg-coa reductase 2) (fragment).35735 65 droxy-3-methylglutaryl coenzyme A reductase.11/1994 PQ0761 hydroxymethylglutaryl-CoA reductase M62766 Mus musculus Mouse HMG-CoA reductase (NADPH) (EC 1.1.1.34) (HMGR 10) wheat (fragment) mRNA, 3' end. 4/1993