US00790671 OB2
(12) United States Patent (10) Patent No.: US 7,906,710 B2 Karunanandaa et al. (45) Date of Patent: *Mar. 15, 2011
(54) TRANSGENIC PLANTS CONTAINING FOREIGN PATENT DOCUMENTS ALTERED LEVELS OF STEROD EP 0486290 11, 1991 COMPOUNDS EP O480730 4f1992 JP O9121863 5, 1997 WO WO93,021.87 2, 1993 (75) Inventors: Balasulojini Karunanandaa, St. Louis, WO WO97/032O2 1, 1997 MO (US); Martha Post-Beittenmiller, WO WO97/34003 9, 1997 St. Louis, MO (US); Mylavarapu WO WO 98.45457 10, 1998 Venkatramesh, St. Louis, MO (US); WO WO99,04622 2, 1999 Ganesh M. Kishore, St. Louis, MO WO WOOOf 61771 10, 2000 (US); Gregory M. Thorne, St. Louis, WO WOO1/31027 3, 2001 MO (US); John R. LeDeaux, St. Louis, MO (US) OTHER PUBLICATIONS Bach et al., “Cloning of cDNAS or genes encoding enzymes of sterol (73) Assignee: Monsanto Company, St. Louis,MO biosynthesis from plants and other eukaryotes: heterologous expres (US) sion and complementation analysis of mutations for functional char acterization.” Progress in Lipid Research, 36(2/3): 197-226, 1997. (*) Notice: Subject to any disclaimer, the term of this Bak et al., “Cloning and expression in Escherichia coli of the patent is extended or adjusted under 35 obtusifoliol 14-alpha-demethylase of Sorghum bicolor (L.) Moench, U.S.C. 154(b) by 0 days. a cytochrome P450 orthologous to the sterol 14-alpha-demethylases This patent is Subject to a terminal dis (CYP51) from fungi and mammals.” Plant Journal, 11(2):191-201, claimer. 1997. Bak et al., “Cloning and expression in Escherichia coli of the obtusifoliol 14-alpha-demethylase of Sorghum bicolor (L.) Moench, (21) Appl. No.: 12/477,673 a cytochrome P450 orthologous to the sterol 14-alpha-demethylases (CYP51) from fungi and mammals.” EMBL Onlinel, Database (22) Filed: Jun. 3, 2009 Accession No. U74319, abstract, 1996. Bard et al., “Genetic and biochemical aspects of yeast sterol regula (65) Prior Publication Data tion involving 3-hydroxy-3-methylglutaryl coenzyme A reductase.” US 201O/OO37354 A1 Feb. 11, 2010 J. General Microbiology, 125:415-420, 1981. Basson et al., “Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the Related U.S. Application Data rate-limiting enzyme of sterol biosynthesis.” Molecular Cellular (62) Division of application No. 10/862,907, filedon Jun. 7, Biology, 8(9):3797-3808, 1998. 2004, now Pat. No. 7,544,863, which is a division of Broun et al., “Catalytic plasticity of fatty acid modification enzymes application No. 09/885,723, filed on Jun. 20, 2001, underlying chemical diversity of plant lipids.” Science, 282:1315 now Pat. No. 6,822,142. 1317, 1998. (60) Provisional application No. 60/260,114, filed on Jan. (Continued) 5, 2001. Primary Examiner — Russell Kallis (51) Int. Cl. CI2N 15/29 (2006.01) (74) Attorney, Agent, or Firm — Chunping Li, Esq.; SNR CI2N 15/52 (2006.01) Denton US LLP CI2N 15/82 (2006.01) AOIH 5/00 (2006.01) (57) ABSTRACT AOIH 5/10 (2006.01) (52) U.S. Cl...... 800/306; 800/312:800/314: 800/317; Disclosed are constructs comprising sequences encoding 800/320.1:536/23.1:536/23.2:536/23.6; 3-hydroxy-3 methylglutaryl-Coenzyme A reductase and at 435/320.1; 435/419 least one other Sterol synthesis pathway enzyme. Also dis (58) Field of Classification Search ...... None closed are methods for using Such constructs to alter sterol See application file for complete search history. production and content in cells, plants, seeds and storage organs of plants. Also provided are oils and compositions (56) References Cited containing altered sterol levels produced by use of the dis closed constructs. Novel nucleotide sequences useful in the U.S. PATENT DOCUMENTS alteration of sterol production are also provided. Also pro 5,306,862 A 4, 1994 Chappell et al...... 800,205 vided are cells, plants, seeds and storage organs of plants 5,349,126 A 9/1994 Chappell et al...... 800,205 comprising sequences encoding 3-hydroxy-3 methylglutaryl 5,365,017 A 11/1994 Chappell et al...... 800,205 Coenzyme A reductase, at least one other sterol synthesis 5,460,949 A 10, 1995 Saunders et al...... 435/55 5,480,805 A 1/1996 Wolfetal...... 435/320.1 pathway enzyme and at least one tocopherol synthesis 5,589,619 A 12/1996 Chappell et al...... 800,205 enzyme. 6,153,815 A 11/2000 Covello ...... 800,306 6,822, 142 B2 1 1/2004 Karunanandaa et al...... 800,298 7,544,863 B2 * 6/2009 Karunanandaa et al...... 800,306 12 Claims, 78 Drawing Sheets US 7,906.710 B2 Page 2
OTHER PUBLICATIONS Gill et al., “Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme. Cell. Cabello-Hurtado et al., “Cloning and functional expression in yeast 41:249-258, 1985. ofacDNA coding for an obtusifoliol 14-alpha-demethylase (CYP51) Gonzalez et al., Abstract of poster at Third Terpnet Meeting of the European Network on Plant Isoprenoids, May 29-30, Poiters, France, in wheat.” Biochemical and Biophysical Research Communications, 1997. 230(2):381-385, 1997. Jenkins et al., “Plant sterols, health claims and strategies to reduce Cabello-Hurtado et al., “Cloning and functional expression in yeast cardiovascular and strategies to reduce cardiovascular disease risk.” ofacDNA coding for an obtusifoliol 14-alpha-demethylase (CYP51) J. of the American College of Nutrition, 18:559-562, 1999. in wheat, EMBL Onlinel, Database Accession No.Y09291, abstract, Nakamura et al., “A large scale analysis if cDNA in Aradopsis 1996. thalian: generation of 12,028 non-redundant expressed sequence tags Chappell et al., “Is the reaction catalyzed by 3-hydroxy-3- from normalized size-selected cDNA libraries.” EMBL Onlinel, methylglutaryl coenzyme A reductase a rate-limiting step for Database Accession No. AV440215, 2000. Nguyen, “The cholesterol-lowering action of plant stanol esters. J. isoprenoid biosynthesis in plants?” Plant Physiology, 109(4): 1337 of Nutrition, 129:2109-2112, 1999. 1343, 1995. Register et al., “Structure and function of selectable and non-select Chin et al., “Nucleotide sequence of 3-hydroxy-3-methyl-glutaryl able transgenes in maize after introduction by particle bombard coenzyme A reductase, a glycoprotein of endloplasmic reticulum.' ment. Plant Mol. Biol., 25:951-961, 1994. Nature, 308 (5960):613-617, 1984. Schaeffer et al., “Plant sterol-C24-methyltransferases: different pro Colebatch et al., “Lotus faponicus root nodule ESTs: tools for func files of tobacco transformed with SMT1 or SMT2.” Lipids, 35:263 tional genomics.” EMBL Onlinel, Database Accession No. 269, 2000. 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 monooxygenase homologues in Arabidopsis Arabidopsis thaliana and Brassica napus.” Plant Molecular Biology, thaliana and Brassica napus," EMBL Onlinel, Database Accession 39(4):721-728, 1999. No. AJO05930, 1998. Schaller et al., “Expression of the Hevea brasiliensis (H.B.K.) mill. Dale et al., “Bacterial expression of the catalytic domain of Arg. 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 in 3-hydroxy-3-methylglutaryl-CoA reductase (isoform HMGR1) tobacco results in sterol overproduction.” Plant Physiol. 109:761 from Arabidopsis thaliana, and its inactivation by phosphorylation at 770, 1995. Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA Schaller et al., “Overexpression of an arabidopsis cDNA encoding a reductase kinase. Eur: J. Biochem., 233:506-513, 1995. sterol-C24(1)-methyltransferase in tobacco modifies the ration of Deavarenne, "Regulation of squalene synthase, a key enzyme of 24-methyl cholesterol to sitosterol and is associated with growth sterol biosynthesis, in tobacco.” Plant Physiology, 129:1095-1106, reduction.” Plant Physiol., 118:461–469, 1998. 2002. Shintani et al., “Elevating the vitamin E content of plants through Downing et al., “The isolation of two mutants of Saccharomyces metabolic engineering.” Science, 282(5396): 2098-2100, 1998. cerevisiae which demonstrate increased activity of 3-hydroxy-3- Tada et al., “Mechanism of photoregulated cartogenesis in methylglutaryl coenzyme a reductase. Biochemical and Biophysical Rhodoturul minuta v. photoinduction of 3-hydroxy-3-methyl Research Communications, 94(3):974-979, 1980. glutaryl coenzyme A reductase.” Plant and Cell Physiol. 23(4):615 Fourgoux-Nicol et al., “Isolation of rapeseed genes expressed early 621, 1982. and specifically during development of the male gametophyte. Plant Van der Hoeven et al., “Generation of ESTs retrieved from tomato Molecular Biology, 40:857-872, 1999. radicule tissue.” EMBL Onlinel, Database Accession No. GenBank Accession No. AJO05930, dated Apr. 20, 1999. AW625933, abstract, 2000. GenBank Accession No. AW.625933, dated Mar. 28, 2000. Yoder et al., “Transformation systems for generating marker-free GenBank Accession No. AW719774, dated Apr. 19, 2000. transgenic plants.” Bio/Technology, 12:263-267, 1994. GenBank Accession No. U74319, dated Mar. 18, 1997. GenBank Accession No. YO9291, dated Apr. 18, 2005. * cited by examiner U.S. Patent Mar. 15, 2011 Sheet 1 of 78 US 7,906,710 B2
squalene
epoxidase Sterol methyl transferase I -- HO W 24-methylene cycloartenol
HO cycloartenol Sterol C4 demethylase
obtusifoliol
HO obtusifoliol stigmasta-7-enol Cl4c-demethylase
campesterol HO HO
sterol methy sterol C5 transferase II desaturase 5.
HO / sitosterol
Sa
HO stigmasterol HO sitostanol
F.G. 1 U.S. Patent US 7,906,710 B2
(IO
Z*9IH
U.S. Patent Mar. 15, 2011 Sheet 3 Of 78 US 7,906,710 B2
Kpnl 8744 Sac 8738 EcoRI 8728 Stu 8724 Xbal 8716 Clai 8711 Bgll 8704 Ban.H. 7759 Sacl 8701 Xbal 7753 Xho 87OO Sal 7747 Kpni 8695 Pst 657 Not 669 Pst 7745 XhoI 678 Hind 7729 O Ora 7692 Pvt 1045 Drai 7615-N Pst 1235 Pyu 288 Pvu 736 PSt 7236 Nico 1614
Pvu 1882 Pvu. 6760 XhoI 2146 Cal 21 63 pMON29920 af-Pvu 2167 8746 bp Pst 6297 Pvu 2669 Oral 5937 Ora 2793 Oral 5918
Pvt 3336
Pyu 4752
Construct pMON29920
FG. 3 U.S. Patent Mar. 15, 2011 Sheet 4 of 78 US 7,906,710 B2
Kpril 12737 Sac 1273 Barnhi 12739 EcoRI 12721 Pst 657 Xbal 12472 Nott 669 Oral 12200 XhoI 678 Oral 12087 Pvui 1045 EcoRV 1923 \ Pst 1235 HindIII 1768 to El Pvui 288 NCO 1614 Pvul 11075 Pyu 1882 Sac 1 O52 Xho 246 Cal 2163 Pvt. 267
W Y Pst 99.78 Pvt. 2669 Pvu 9973 Dra 2793
Bani 9728 pMON43800 12739 bp Ca 969 Barn 952 Cal 909 ECORE 8728 Stu 8724 Right Border Xba 8716 Cia 871 Bgll 8704 Saci 8701 NII-9 Xho 87OO Dral 5918 Kpni 8695 Oral 5937 Ba?h 77.59 Pst 6297 Xbal 7753 Sai 7747 Pst 7745 Hind 7729 Drai 7692 Drai 765 Pvu 736 Pst 7236 Pyut 6760 Construct pMON43800 FIG. 4 U.S. Patent Mar. 15, 2011 Sheet 5 Of 78 US 7,906,710 B2
Hind 7204 EcoRV 8 EcoR 12 Pst 321 Bgll 321 Pst 538 Pvul 591 Drai 767 NCO 917 Oral 7090 BamH 1326
Pvu 6836 Pvul 1336 Clal 1585 Pvul 1589 EcoR 1593 Pvu 6235 Pst 1603 Srna 1607 Bam H. 1611 Xbal 623 Not 1630 Cal 1638 Dra 542 Pvu 1642 Oral 5393 pMON23616 7204 bp
Pvuil 2144 Oral 2268
Construct pMON23616
FIG.S U.S. Patent Mar. 15, 2011 Sheet 6 of 78 US 7,906,710 B2
XhoI 2478
pMON43818 12547 bp
Srna 6263
Construct pMON43818
F.G. 6 U.S. Patent Mar. 15, 2011 Sheet 7 Of 78 US 7,906,710 B2
Soy Alpha' Beta Conglycinin
pMON43052 10605 bp Arabidopsis HMGR catalytic domain
ori-W Left Border pot.polyA signal
Not 503 Construct pMON43052
FIG. 7 U.S. Patent Mar. 15, 2011 Sheet 8 of 78 US 7,906,710 B2
Not 7042 Sna 7007
pMON51850 7049 bp
Construct pMON51850
F.G. 8 U.S. Patent Mar. 15, 2011 Sheet 9 Of 78 US 7,906,710 B2
Not 10282 Pst 10270 Barth 96O4. Kpnl 9602 Sac 9596 ECOR 9586 Hird 10289 Sac. 9584 EcoRV 8 Kpnl 9578 EcoRI 12 Smal 9572 Bgll 321 Pst 9568 Pst 321 EcoR 95.58 Pst 538 EcoRV 93.35 Pvu S91 Pvut 9075 5. Pvul 9022 Ara SR Nicol 917 Dra 8953 Q Pvul 1185 EcoRV 8870 s KY Sac 8695 A
Cal 8068025 ?tAy is 53 Saci 7873 St Kpni 7867 Soy Alpha' Beta Conglycinin pMON43057 10289 bp Pvu 262O StulDaisy"9" 7307-W Richt Bord Ayf BaghOral Zog5976 (1va Ora 6899 Pyu 6645 Pst 652O Pvull 4036 PWL 6044 SNd Pst 558 Dra 522 Oral 52O2
Construct pMON43057
FIG. 9 U.S. Patent Mar. 15, 2011 Sheet 10 Of 78 US 7,906,710 B2
Cal 12867 Pvu 126 PSt 12592 Hind 12882 ECOR 12582 EcoRV 8 Pst 12262 ECOR 2 Bgll 1 1846 Pst 321 Sac 1694 DS 3' Bgll 321 Bgill 11556 in r-KS Pst 538 Sat 11428 LA Pyu 591 Bgill 11313 still /Ncol 97 ECOR 1 161 p- Pvu 85 Sac 11154 Kpnl 11148 s Pvu 1953 Dra 2077 Stu 10588 AY Oral 10386 A. Soy Alpha' Beta Conglycinin ori-V Pst 1 O27O linker-KS Pvu 262O
BamH 9604. Pl:MON43058 bp Kpnl 9602 Saci 9596 El EcoR 9586 8. ar G Sac 9584 W Arabidopsis HMGR catalytic domain Pvut 4036 Kpnl 9578 Wy Soy Alpha' Beta Conglycinin W Srna 9572 Q 2 E.A Right Border C2
EcoRV 93.35 "-yask Pvt 9075 s Oral 52O2 Pvu 9022 Dra 522 Ora 8953 Pst 5581 EcoRV 887O Pvu 6O44 Sac 8695 Cat 8064 Cal 8025 Saci 7873 Kpni 7867 Stu 7307 Oral 7 O5 Barn 7O13 Dra 6976 Oral 6899 Pyu 6645 Pst 652O Construct pMON43058 FIG. 10 U.S. Patent Mar. 15, 2011 Sheet 11 of 78 US 7,906,710 B2
s 2.4-Methy tens Cycloarta rol 70 GCycioarteriol Unknown 3 see Es s M SS3 Stigmasta-7-enol lso fucosterol so Sitos tano Sitosterol Obtusifoliol s Unknown 2 Stigmasterol O Campesterol 1000 - " ... w “. . . . n Jnknown Squalene
c S. Event nurther
FIG.
U.S. Patent Mar. 15, 2011 Sheet 13 Of 78 US 7,906,710 B2
Kpnl 10192 Sac O186 EcoR 10176 Pvul 1 OO86 Pvu OO33 Dra 9964 EcoRV 988 Barn H. O. 94 Sac 97O6 Pst 666 Cal 907S Not 678
Pvu 8848 Pvu 1056 Pvu 8800 Pst 1246 Cla 8800 Pvu .299 Stu 8414 Nicol 8395 Nico 1625 Bgll 8389 Pvu 1893 ECORV 8274. Xhoi 257 Cia 2174 Pst 77SO PV 21.78 Hind 774O Dra 77O3 Dra 7626 pMON53733 10194 bp PVU 268O Pvu 7372 Dra 2804 Pst 7247
Pvu 3347
Draf 5929 Pvu 4763 Construct pMON53733
F.G. 13 U.S. Patent Mar. 15, 2011 Sheet 14 of 78 US 7,906,710 B2
Kpnl 9826 Sac 982O EcoR 9810 Pvull 972O Pvu 9667 Dra 9598 EcoRV 9515 Saci 934O Barn 9828 Clal 8709 Cla. 8670 Pst 666 Pvu 84.82 Not 678 Pvul 8434 Xhol 687 Clal 8434 Pvu iO56 Pst 1246 NicolBgll 83898395 Pvul 1299 EcoRV 8274 Nico 1625 Pst 775O Pvu 1893 HindIII 774O e XhoI 257 Dral 77O3 \NS a Clal 2174 Drai 7626 - a-Pvul 2178 Pvul 7372 - pMON53734 E Pst 7247- 9828bp E Pvull 2680 Sf Drai 2804 Pvu. 677
PVU 3347 Pst 63O8
Dra 5948 Dra 5929 Pyut 4763 Construct pMON53734
FIG. 14 U.S. Patent Mar. 15, 2011 Sheet 15 Of 78 US 7,906,710 B2
Kpnl 9698 Saci 9692 EcoR 9682 Pvuil 9594 Pyu 9541 Banh 97OO Draf 9472 Pst 666 EcoRV 93.89 Not 678 Sac 9214 Cla. 8583 Cal 8544 Pvu 1056 Nicol 8395 Pstl 1246 EcoRV 8274 Pvul 1299 Pst 7750 Nicol 1625 HindIII 774O Pvul 1893 Dra 7703 XhoI 257 Ora 7626 Claf 2174 a Pvu 2178 Pvu 7372 Pst 7247 pMON53735 9700 bp
Pvu 268O Dra 2804
Pst 63O8 Dra 5948 - 7 Dra 5929
Pvt 4763 Construct pMON53735
FIG. 15 U.S. Patent Mar. 15, 2011 Sheet 16 of 78 US 7,906,710 B2
Kpnl 101.41 Sac 101.35 EcoRI 1.0125 Pyut OOS3 Sac 10O3O Ban 97.79 Banh O143 PSt 9429 Pst 666 PWU 94.24 Not 678 Barn H 93 Cal 8752 Pvu 1056 Barnh 8735 Pst 246 Clal 8674 v Pyu 299 NCO 8395 NCO 1625 Bgll 8389 PVU 1893 ECORV 8274 XhoI 257 Cla. 274 Pst 7750 a Pvt. 278 Hind 774O Dra 77O3 \S Drai 7626 pMON53736 101.43 bp P w ul 2 6 8 O Pvt 7372 Pst 7247
PSt 63O8
Dra. 5948 Oral 5929 Pvu 4763 Construct pMON53736
F.G. 16 U.S. Patent Mar. 15, 2011 Sheet 17 Of 78 US 7,906,710 B2
Kpnl 98.44 Saci 98.38 EcoR 98.28 PVU 9756 Sac 9733 Bar H 94-82 Barn 9846 Pst 9132 Pyu 927 Pst 666 Ban 904 Not! 678 Clal 8455 Ban 8438 Pvul 1056 Nicol 8395 Pst 1246 Bg7 8389 Pvt 1299 ECORV 8274 NCO 1625 Pst 77SO Pvul 1893 Hind 774O XhoI 2157 Da 7703 Cal 2 74. Ofa 7526 Pvul 2178 pMON53737 Pyut 7372 9846 bp Pst 7247 PVU 268O
Ora 2804
Construct pMON53737
F.G. 17 U.S. Patent Mar. 15, 2011 Sheet 18 Of 78 US 7,906,710 B2
Kpnl 9844 Sac 98.38 EcoR 9828 Pyu 9756 Saci 9733 Barn 9482 Pst 9132 Pvull 9127 Not 678 Ban 9014 XhoI 687 Cal 3455 Pvu 1056 Ban 84.38 Pst 1246 Nico 8395 Bgill 8389 PWU 299 EcoRV 8274 NCO 1625 Pst 7750 Pvu 1893 Hind 774O XhoI 257 Ora 7703 Cal 2174 Da 7626 Pvu 278 pMON53738 Pvt 7372 9846 bp Pstt 7247724 PVU 268O Dra 2804
Pst 63O8 Oral 5948 Oral 5929
PVul 4763
Construct pMON53738
FIG. 18 U.S. Patent Mar. 15, 2011 Sheet 19 Of 78 US 7,906,710 B2
Kpni 98.44 Saci 9838 EcoR 98.28 PvE 9756 Saci 9733 Barn 9482 Pst 9132 Pst 666 Pvu 927 Not 678 Earl 9014 Clal 8455 Pvu 1056 B. Pst 1246 Bgll 8389 PvuU 1299 EcoRV 8274 Nool 1625 Pst 7750 Pvt 1893 Hind 774O XhoI 257 Oral 7703 Clal 2174 Drai 7626 Pvu 21.78 pMON53739 Pvt 7372 9846 bp Pst 7247 Pvt. 268O
Dra 28O4.
Pst 63O8
Oral 5948 Dra 5929
Pvt 4763 Construct pMON53739
FIG. 19 U.S. Patent Mar. 15, 2011 Sheet 20 Of 78 US 7,906,710 B2
Kpni 9685 Sac 96.79 EcoR 9669 Pyu 95.97 Sac 95.74. Barn 9687 Barn 9323 Pst 666 Pst 8973 Not 678 Pvu 8968 XhoI 687 Barr 8855 Pvu 1056 NCO 8395 Pst 1246 Bgill 8389 Pvu 1299 EcoRV 8274 Pst 7750 Nicol 1625 HindIII 7740 Pvul 1893 Oral 7703 V XhoI 257 Ora 7626 Clai 2.74 Pvul 7372 Pst 7247 pMON53740 96.87 bp
PVU 268O Drai 2804 Pvu 677
Pst 63O8
Oral 5948 Oral 5929
PV 4763 Construct pMON53740
FIG. 20 U.S. Patent Mar. 15, 2011 Sheet 21 of 78 US 7,906,710 B2
CYo Sir O CO. N. o O O. So so go so go go to St N.S N.is N.is N.5 N.S N.S N.S N.S O O LO to O O O LO Y Z Z Z Z Z Z Z 2 He O& O.> isO. O.is O.e O.> O.a. O.s. 2O o, o O O. S. O. O. C. O to a Y X 0 - 8
(515n). Jeluoo Oueeooko
U.S. Patent Mar. 15, 2011 Sheet 25 of 78 US 7,906.710 B2
N LO (O N OO O. O. 2 So So So So So so S NCrd N.to N. N.to koN. N. N.S N.S O cd O LO O O LO O. O. Y Z Z Z 2 2. 2 2 Z H O O O O. O. O. O. O. Z > d > si is is a s O O. O. O. C. C. C. C. C. O 0 a X X e --
a. -
C O O C O O Co O O O O O O O O O O O C O O O N- D D s c CN ens (f/6n) pue Loo ole SOS U.S. Patent
uosqueduuoogoIoue?SO??SSIÐA9"|u?oquefisueu],squela U.S. Patent US 7,906.710 B2
“?IHLZ
pees fu Jedi Oues 5u U.S. Patent Mar. 15, 2011 Sheet 28 Of 78 US 7,906,710 B2
1850INH?SeÐAu?S?oni?uoloH9LNHquelº ?ue?nÎN?nOX10oux!
er cN M. Aup le3 6used oues fit U.S. Patent Mar. 15, 2011 Sheet 29 Of 78 US 7,906,710 B2
Drai 571 Hind 808 Dral 815 Bgll 899 Xbal 74.79 Bgll 959 Hind 995 Kpnl 1126 Ora 1 178 Pst 187
W Oral 659 Pvu 5952 pMON43842 AC F-Pvul/Pvul 19231952 79 bp lac2. Kpni 2080 t NXhc 2334 Ora 5467 Sa 23-O Oral 5448 Cal 2350 Hindl 2355 EcoRV 2363 EcoFR 2367
Sac 238O Pvu 4513 E. xis Saci 4295 Bgill 2783 Hindll as 'ind 2807 Hind 3551 Barnh 3608 Bgll 3785 EcoRI 3795 Pst 3805 Small 38Og Barnh 3813 Xba 3825 Construct pMON43842
FIG. 29 U.S. Patent Mar. 15, 2011 Sheet 30 Of 78 US 7,906,710 B2
Pst 399 Xbal 80O8 EcoRV 605 p-AN Nico 622 s S Stu 853 S. URA3 Ssy KCQ 69 Q) GS A Draf 6688-g Pvu 1851 2- Pvul 1880 Pvul 6481 - AmpR pMON43843 i-Kpnl 2008
8439 bp AC Xhoi 2265 Sal 227 Ratif Gs "Noti 2282 a. W A. Oral 25O2 &Squalene epoxidase 1 (ATA506263) ^-EcorvC Dra 29s258 C & P-GPD s CO X. AC YY Drai 3180 Pvull 5042-7s s Kpril 3188 Sac 4824 thridlit S rtial 4516 Sall Hind 3768 Pst 4465 Sall 4094 Small 4101 EcoR 4104 Pst 414 Small 4118 Barnh 4122 Xbal 4134 Construct pMON438-43 F.G. 30 U.S. Patent Mar. 15, 2011 Sheet 31 Of 78 US 7,906,710 B2
Pst 399
Xbal 8217 EcoRV 605
Oral 6897 Pvul 1851 Pvu 880 Pvt 6690 Kpnl 2008
XhoI 2265 Oral 6205 Sail 2271 Drai 686 NotOt 2232223 Draf 2396 Hird 289
EcoRV 3 OO Pvu 5251 Kpni 3370 Sac SO33 Nico 3487 Sinal 4725 Salt 4303 Pst 4674 Srna 4310
Xbal 4343 EcoR 4313 Barnhil 4331 Pst 4323 Small 4327 Construct pMON43844
FG. 31 U.S. Patent Mar. 15, 2011 Sheet 32 of 78 US 7,906,710 B2
Plurality : 5. O0 Threshold: 4 AveWeight L. 00 AveMatch 2.91. AvMismatch -2 loo SO HMGR.clustalW (methanobach ...... is o o
a a s p o a r e.
u HMGRclustalWo e o up - a - e. yeast2} MSLPLKTIVH LVKPFACTAR FSARYPIHVI vivAvLLSAAA YLSVTQSYLN HMGRclustalW yeast MPPLFKGLKQ MAK?IAYVSR. FSAKRPIHII LFSLIISAA YLSVIQYYFN HMGRclustalW (phycomyces ...... HMGRclustalW ( fusarium) ...... w to as 4
w w w t w w is w
HMGRclustalW rice ......
HMGRclustalW { soybean) ......
HMGRclustal w{ tomato) ......
0 s a a
a a u- up o an - - -
w up is w w
4 y or a as a s
FIG. 32A U.S. Patent Mar. 15, 2011 Sheet 33 of 78 US 7,906,710 B2
o e s e s p a o e
HMGRclustalW rat) ...... HMGRclustalW { rabbit} ...... HMGRclustalW human} ...... HMGRclustalW { mouse) ...... HMGRclustalW { xenopus) ......
w w w w w w x HMGRclustalW (sea urchin} ...... HMGRclustalW ( cockroach) ......
us - a o
8 at 0 8 a 0
ow wh- w w
U.S. Patent Mar. 15, 2011 Sheet 35 of 78 US 7,906,710 B2
GLFWASPWE HMGRclustalW (syrianhamst ...... MLSRLFRMH GLFWASHPE HMGRclustalw rath ...... MLSRLFRMH GFWASHPWE HMGRclustalW rabbit} ...... MLSRFRMH GLEWASP.NE HMGRclustalW human ...... Y P A r u 8 s v. MSRFRM GFWASHPWE HMGRclustalw mouse ...... HMGRclustalW ( xenopus) ...... MSRLFRME GQFVASHPWE HMGRclustalW sea urchin} ...... MLSRLFLAC GRSCSSHPWE HMGRclustalW cockroach ...... MVGRFRAH GOFOASHPWE HMGRclustalW (drosophila) ...... MGPLFRA . OFCASHPWE HMGRclustalW (dictyostel ...... HMGRclustalW (schistosom} ...... HMGRclustalW (archaeoglo) ...... HMGRclustalW (pseudomoras) ......
Consensus ------MLSRFRME GWASPWE
FIG. 32D U.S. Patent Mar. 15, 2011 Sheet 36 of 78 US 7,906,710 B2
SO
w is 8 x + 8 w w
8 k & 8 × 8 v 8 a
8 8 4 p. 8 x d as HMGRclustalW yeast2 PSSYYLSTIS FQSKDNSTTL, PSLDDVIYSV DHTRYLLSEE PKPELVSE HMGRclustalW yeasti PHHYYLLNN FNSPNETDSI PELANTVsek DNTKYILCED SWSKESST HMGRclustalW (phycomyces ...... HMGRclustalW fusarium) EFLDLLKNAe TLDIVIMLLG YIAMHLTFVs LFLSMRKMGs KGCF HMGRclustalW { candida VDHYNVVPFQ FRRAGEYKEP VLSGIVELDE V KavySQSDA AEOWOQLTAE HMGRclustalW (dict/oste2} ...... w w & s &
?ix w w w w as it is w
4 - w w w w w
& N w w & 8 w; 4
w w x 8
Wik & 8 w w s w y & 0.
it 4
8 at t d X k d
& d & 8 8 8
as a s & 8
w X x so as v Y HMGRclustalW (rubbertre2} ...... *Y is 8 v is - Y - d - Y - - - - w a is as a HMGRclustalW (rubbertrel ...... w w w
a x & F.G. 32E U.S. Patent Mar. 15, 2011 Sheet 37 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} VIVGTVT.. L TICMMSMN. . MFTGNNK...... HMGRclustalW (syrianharnst VIVGTVT.. L TICMMSMN. . MFTGNNK...... HMGRclustalW ( rat} VIVGTVT..L TICMMSMN. . MFTGNNK...... AMGR.ciistalW ( rabbit} VIVGTVT. L. TICMMSMN.. METGNDK...... HMGReinstalW { human VIVGTVT.. L TICMMSMN. . MFTGNNK...... forciustalW ( mouse) ...... we w w w as w w w w - HMGR.ciistalw{ xenopus VIVCTVT . . L TICMMSMN. - MFTGNDK...... HMGR.ciistalw{sea urchin} VIVCTLT . . L TICMLSMN. . YFTGLPR...... HMGRciustalw cockroach} VIVATLt... t. TvCMLTVDQ. RPLGLP...... HMGR.ciistalW (drosophila} VIVALLT ... I TACMLNGGCE QYPGC2ORIG STAsAAAAG SGSGAGSGAS HMGRclustalW (dictyostel ......
HMGR.ciistalwischistosom} s a o 0 e o a a v s v e s - w is P - r w w - in a s - w ......
HMGRciustalW (archaeoglo} w W o - d e 8 a 0 to P - v - r v o 4 m w w a a d - - - - ......
HMGRciustalw (pseudomonas} w as a 4 W 4- 4 4 0 & w w w w w w w w a a ......
Consersus WWGVE- or TCMMISMN MEGNNK. ------
F.G. 32F U.S. Patent Mar. 15, 2011 Sheet 38 of 78 US 7,906,710 B2
2OO
Ad & 8 8 so
HMGRclustalW yeast2} NGTKWRLRNN SNFILDLENI YRNMVKQFSN KTSEFDQFDL FAAY HMGRclustalW yeast DGTKWRLRSD RKSLFDVKTL AYSLYDvir SE NVTOADPFDv MWAYLMM HMGRclustalW (phycomyces ...... & 8 w w & w w w - 4 x is as w
we w w w x x . . . . HMGRclustaiwi fusarium: SSVFAFLFGL VVTTKLGVPI SVILLSEGLP FLVVTIGFEK NVTRAVMS HMGRclustalW candida} DGTVWRSRAY HGKLGKYSDM AvGAFNKVLN LVRGAETFDI AVCAYAM HMGRclustalW (dictyoste2} ......
HMGRclustalW rice ......
8 s a w
HMGRclustalW soybean) ...... 8 8 v w a as 8 A
HMGRclustalW (rosyperiwi) ......
0 X & X w w - HMGRclustalW ( tomato ...... w w w w w is 48 -
4 - A 8 8
& a is 4 -
X w x s is
X 8 × 8 x X x 4 is
X x s as
X vs. a s HMGRclustalW (rubbertre2} ...... 8 v 8 8 s h $ 8 & 9 w & O is 8 s as a a s
w is us s- a s > BMGRclustalW (rubbertre1} ...... HMGRclustalW camptothec) ...... is se s > 0 is a Y As we a s 48 - 4 48 HMGRclustalW- 8 X is w a st (arabadops2} ......
o w HMGRclustalW (chineseham) ...... & - 0 CGWNYEO. PK FEEDWTSSD LTTRCA U.S. Patent Mar. 15, 2011 Sheet 39 Of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} ...... CGWNYEC. PK FEEDWSSD RCA HMGRclustalW (syrianhainst ...... CGWNYEC. PK FEEDWSSD TRCA HMGRclustalW rat ...... CGWNYEC. pK FEEDWSSD ILTTRCA HMGRclustalw rabbit} ...... CGWNYEC. PK FEEDWSSD TRCA HMGRclustalW human ...... CGWNYEC. PK FEEDWSSD TRCA HMGR.clustalW mouse ...... e - a a s HMGR.clustalW ( xenopus) ...... CGWNYAC. K. FEEDWSSD RCA HMGRclustalw{sea urchin} ...... I CGWNYECAPQ WKESSLSSDV VMCMRA HMGRclustalW cockroach ...... PGWGNC. EEYNAADM VMROVA HMGRclustalwdrosophila GTIPPSSMGG SATSSRHRPC HGWSQSC. DG LEAEYNAADV LMTVRCTA HMGRclustalw(dictyostel) ------...... HMGRclustalw(schistosom} ...... M. KLNVL, FOCFSTF VLFR HMGRclustalW (archaeoglo ...... HMGRclustalW (pseudomonas ......
Consensus ------CGWNYEC-K FEEWSSD TRCA
F.G. 32H
U.S. Patent Mar. 15, 2011 Sheet 42 of 78 US 7,906,710 B2
3OO HMGRclustalW (methanobac - - - - V 9 is as a a s ...... p r s o is a -
* * * r * * * v. W 8 a we h w w w is ...... a
v P. d w is a
P P P 8 a 9 P. B. P 0 v is a ......
up as or o e s s >
k a s we on w w no
a a y - e s sh HMGRclustalW ( yeast2 SLVIGLPFIV VIIG. FKHKV RIAAFSLQKF HRISIDKKIT WSNYSAMF HMGRclustalW ( yeastl TLFEGLPFIV VVVG. FKHKI KIAQYALEKF ERVGLSKRIT TDEIVFESVS HMGRclustalW (phycomyces} - as a st 4 a a W a w s a s w a w w HMGRclustalW fusarium) IVILVIGAAS GVOGGLOQFC FLAAWTLF.F DFILLFTFYr ASKRS HMGR.clustalW candida} SLSEGIPFFV AVVG. FNNK LLAEKVLQ.N. QLNAQSSKND APTVLYOALR HMGRclustalW (dictyoste2}
a w- 8 a. s. e. Ot
up v e s is o o HMGR.clustalW rice v h w w & a $ 8 of n e s a s p w w w W - v is s > ......
HMGRclustalW corn) on as w w x > as Y is w w w w w PPA
HMGRclustalW (wheat3} at w 8 w w w w w w w w w w 0 w is w w w w w w w w w w w w w w is w w w a 4 HMGRclustalW (wheat2} . . . 8 & 4 $ s p w w w w w w a w a n e w is ...... w 4 M W 0. As a w
soybean) O A to a P. t. a - - - - - P vs a s as a s is a s p as a - - - - d.
HMGRclustalW rubbertres) - P. P. P. a DSVRRRPP. K. HVRKDHDGE WLNSFSHG. HMGRclustal w{rosyperiwi) DSRRRSP. . . TWTAKAAAGE LPLAPHEGQ. HMGRclustalw tomato) DVRRRSEEPV YPSKVFAADE KPLKPHKKQQ HMGRclustalwwoodtobacc} OVRRRSEKPA YPTKEFAAGE KPKPEK. . . HMGRclustalW ( potato w DVRRRPVK, YTSKASAG. EPLKQQE. . . HMGRclustalW (radish) w w w w x 4 x w w 0 DRR. RPPK PPVNSN...... RFDNRS HMGRclustalW (arabadopsisl up k we we o 40 W DLRR. . RPPK PPVTNNNNSN GSFRSYQPRT HMGRclustalW (cucumismel) DRRRSLRPPR PNAVODADAT CTFRRDEQDA HMGRclustalW (rubbertre2) 8 O - as sp b d as 0 a O AP - O
HMGRclustalW (rubbertrel} so s vs. o e s - e. e. . . . . HR. . . . vo as a KHAT HMGRclustalW (camptothec) v is & 8 w HQIPSVGGTA PPMLKPROPT HMGRclustaWarabadops2} w w wr it EDLRRRFPTK KNGEEISN. . HMGRclustalW (chineseham FLDKELIGIN EALPS.LLI DLSRASALAK FALSSNSQDE WRENARGMA FIG. 32K U.S. Patent Mar. 15, 2011 Sheet 43 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} FDKETGLN EAPFFL. DLSRASALAK FALSSNSQoE WRENARGMA HMGRclustalW (syrianhamst FLOKETGN EAPFFL. DLSRASALAK FASSNSODE WRENARGMA HMGRclustalW rat) FOKEGN EAPFF DSRASALAK FALSSNSQDE WRENARGMA HMGRclustalW rabbit} FDKETGN EAFF, DSRASALAK FALSSNSQDE VRENARGMA HMGRclustal W{ human} Foke.TGN EAPFFLII DSRASTAK FALSSNSQDE WRENARGMA HMGRclustalw mouse HMGRclustalw( xenopus) FDKETGN EAPFFL. DSKASA-AK FALSSNSODE WRONARGMA HMGRclustalW (sea urchin) FGETGN EALPFFLI, OTKASALTK FALSSTTONE WWONARGMA HMGRclustalW cockroach) FLGSOVSDK AFFF.T. DSKAVAO FALSSRSQDE WKENARGA HMGRclustalW (drosophila) FGSDSEK DAFSFLIV DLSNSGRLRS GAMGSN. QAE VTONIARGLE HMGRclustalW (dictyostel) HMGRclustaiw (schistosom} SSVFLFS HMGRclustalW (archaeoglo
Consensus EALFF, a FALSSNSQDE
FIG.32L U.S. Patent Mar. 15, 2011 Sheet 44 of 78 US 7,906,710 B2
30
HMGRclustalW yeast2) QEGAYROY FYSSFIGC AYARELPG VNFCSTFM VFOLSAT HMGRciustalW ( yeast1) EEGGRLIQDH LLCIFAFIGC smYAHQLKTL NFCSAF FELLPT HMGRclustalW (phycomyces} HMGRclustalW fusarium WSSWMSCWW PLRMMASRRW ANWAKGDDE NRWRGDAP FGRKSSSPK HMGRclustalW candida} ECGP.L.R.H. LFMTAFLGC SFYASYLDG KNFCAA. LAFDLTSTHMGRclustalw{dictyoste2} HMGRclustalW (wheati) HMGRciustalW { rice) . . .NGAMVS HMGRclustalW corn PE. . . . PSRA ...... AA RVQAGAP . . .NEFSA. HMGRclustalW (wheat3 } HMGRclustalW (wheat2) HMGR.clustalW soybean HMGRclustalW (rubbertre3} ------...... P9 LKpSDYSLPL . . .NALWFSL HMGRclustalW rosyperiwi) - - NO O...... PS IPSSSDVPL PLY.A...... NGWFFT, HMGRclustalW { tomato QQ. . . . QEDK N...... T LIDASDALP PYLT ...... NGFFM BMGRclustalW (wood tobacc} QQ. . . . QECD N...... S. L.ASDAP PLYLT...... NGLFFM HMGRclustalw potato wn w w w w w w w w w w w w w w w w WS SPKASDAP, PLY.T. . . . . NGLFFM HMGRclustalW radish DD. . . . DDR.R. K. . . . . LTS PPKASDAP P.Y...... NAWFF HMGRclustalW (arabadopsis1} SO. . . . OR RR. ATTAP PPKASDAP, PLY.T. . . . . NAWFF HMGR.clustalW (cucumismel S.A. . . . ADHL. KR...... A SPKASDAP. PLYL. . . . . NTFF, HMGRclustal Wrubbertre2} HMGRclustalW (rubbertrel} PW. . . . DRS P...... T TPKASDAP, PLY...... NAWFF HMGRclustalW camptothec) KW. . . . DAVO ...... PID SPKASDAPL PLY.T...... NGVFFT, HMGRclustalW (arabadops2) ------WAV ...... PP RASDAP PLY.T...... NFFLS HMGRclustalW (chineseham) ILGPTFTLDA IV . . ECLVIG WGTMSGVRQL EMCCFGCMS WANYWFM FIG. 32M
U.S. Patent Mar. 15, 2011 Sheet 46 of 78 US 7,906,710 B2
4 OO
8 v set s > as a is us - d
4 & 8 d 8 w a s s a
u - 8 w & & w -
s is s a s s d e as HMGRclustalW yeast2} FYSALSMK, ENRSTW IROTL. . EED GVWPTTADI YEASEPH HMGRclustalw yeast1) FYSAALR MNVRST IKQTL. EED GVWPSTARI SKAEKSWSS HMGRclustalW (phycomyces) - - - Y - p & as 8 HMGRclustalW fusarium) FKVLMG VNVICS SNP SSM STRINASS GGWASWO HMGRclustalW candida} FSASK, EINOIHRSTL LREQL. . EDD GLTETTVODV LKSNSLAGTK HMGRclustalW (dictyoste2} 4 & w a w is wh
HMGRclustalW (wheati) & 4 w w 4 W & A w. HMGRclustalW{ rice VSSCD, WR CSRER. . . P...... GGREFA TVWCQLASVV HMGRclustalW corn FAASMR RNREKRSS PIHA...... WGAEM, AGWAS HMGRclustalW (wheat3}
we w w 8 × 8 W & &
soybean w x W, X - 8 w HMGR.clustal Wrubbertre3} FESWAYFILE RWREKRKST P.E...... VTEEA ALOWASW HMGRclustalW rosyperiwi) FFSWMYFLT RNREKRNA P.W...... VLSEA AASIIASW HMGRclustalw tomato) FFSWMYS RWREKRNS P.W...... WTLSELG AWSASW HMGRclustalW (woodtobacc} FFSWMYYLS RREERNIST PLEV...... WTFSEW AASLASW HMGRclustalW potato) FFSWMYFW RWREKRNS PIV ...... WISE. AMWSASW HMGRclustalW radish) FFSWAYYLE RWROKRYNT P.W...... WTVTEG AVALIASFI HMGRclustalW (arabadopsisi FFSWAYYLE RWROKRYNT PLEV...... VTEC AAASF HMGRclustalW (cucumismel) FSWAY Rors PW...... VSEA AVSAS HMGRclustalW rubbertre2) & a 8 & HMGRclustalW rubbertrel PLHI...... WLSEW AWSASF HMGRclustalW camptothec RWREKRNS P.W...... WSEA AIFTFIASFI HMGRclustalW (arabadops2} FFAWYFS RWREKRNS PW...... WoSEC AGFWASF HMGRclustalW (chineseham FFPACVSLV. ELSRESREGR PIWO. . . LSE FARVLEEEE. NKPNPVTORV FIG. 32O
U.S. Patent Mar. 15, 2011 Sheet 48 of 78 US 7,906,710 B2
4 Ol 450 HMGR.clustalW (methanobach ......
HMGRciustalW (methanococ} w a v - r s a 10 - w w w w V w w - a ...... HMGRclustalW (halobacter) ...... HMGR.clustalW (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 ...... HMGRciustalW ( rice) YLLSLFAHPD APATTTGDDD ...... HMGRclustalW corn YLLSFFGIAF WQSIVSSGDD ...... HMGRclustalW (wheat}} ...... HMGRclustalW (wheat2) ...... HMGRclustal W soybean) ...... HMGRclustalW (rubbertre3} YLLGFFGIGF VHSFS. RAST ...... HMGRclustalW rosyperiwi YLVSFFGLDF voSLIYKPNN ...... HMGRclustalW tomato YLLGFFGIGF VQTFWSRGNN ...... HMGRclustalW (woodtobacc} YLLGFFGIGF voSFWSRDNN ...... HMGRclustalW potato YLLGFFGIGF VOSFVSRSNS ...... w a e. ge as a HMGRclustalW (radish YLLGFFGIDF voSFISRP...... a b to w w e - a w HMGRclustalW (arabadopsisi YLLGFFGIDF voSFISRASG ...... HMGRclustalW (cucumismel YLLGFFGIDF voSFIARSSP ...... HMGRclustalW (rubbertre2} ...... HMGRclustalW (rubbertrel YLLGFFGIDF vOSFIARASH ...... HMGRclustalW (camptothec YLLGFFGIGL VQPFTSRSSH ...... HMGRclustalW (arabadops2} YLLGFCGIDL IFRSS...SD...... HMGRclustalW (chineseham) KMIMSLGLVL WHAHSRWIAD PSPONST. . . TE.HSKVSLG OEDVSKRE FIG. 32O U.S. Patent Mar. 15, 2011 Sheet 49 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} KMIMSLGLVI, VHAHSRWIAD PSPQNST... TE.HSKVSLG DEOWSKRE HMGRclustalW (syrianhainst KMIMSLGLVL, WHAHSRWIAD PSPQNST... TEHSKVSLG OEOWSKRIE HMGRclustalW rat} KMIMSLGLVI, VHAHSRWIAD PSPONST. . . AE. OsKVSLG AEDWSKREE HMGRclustalW { rabbit} KMIMSIGLVL WHAHSRWAD PSPONST. . . AD. NSKVSLG ENWSKRIE HMGR.clustalw{ human}. KMIMSLGLVL, WHAHSRWIAD PSPQNST... AD. TSKVSLG OENWSKRE HMGRclustalw mouse HMGRclustalW ( xenopus) KMIMSLGLVI, VHAHSRWISE PSSQNST. . SSDHEWITM DDMMPKRVE HMGRclustalW (sea urchin} KMMRTGLV, WHAHSYWAS . . . .N.T. . . ELMSRMYd GNLDKKD HMGRclustalW ( cockroach) KVMSAGLM, WHAH.RWVRC ...... L. HMGRclustalW drosophila KMTTGLMA WYSREWSp . . AAT. . . TMVdKLTP SNWSNNR HMGRclustalW (dictyostel) KPLPYIPOHN QQQQQKCQPS ...... HMGRclustalW (schistosom} YGEQKKCLVS NKGVSSSTRK RRHSYSSGHS Y VEY RRMSVH NGYWVNPN HMGRclustalW (archaeoglo
Corises is YLL-FFG-VL W-A-SR-ISD PSPQNST------SKWSG LOEw
FIG. 32R U.S. Patent Mar. 15, 2011 Sheet 50 of 78 US 7,906,710 B2
5OO HMGRclustalW (methanobac
------E 6 - - - - to or ------w a e o se a ......
o or e o us a e s o o
o a o a si e a to e o a o O p 8 - - - - - e s a s a HMGRclustalW yeast2. SLPN FINYKD IGNLSNQVII SVLPKQYYTP LKKYHQIEDs WSVSN HMGRclustalw yeast SLPDFITSNA SENFKEQAIV SVTPLLYYKP IKSYQRIEDM WRNWSW HMGRclustalW (phycomyces) w - so 0 8 - w HMGR.clustalW fusarium) ASNPAYNDA FHHHFOGYGW GGRMWGGILK SLEDPVLSKW VAASVA HMGRclustalW candida} TLYDAVA.DQ IPIGSNGTLV TLFPTRFFLP EKLSTQIEAV WSFGST HMGRclustalW (dictyoste2}
rice - P. ------...... corn . . DEDFLWGS G...... e o e s is a o
soybean) HMGRclustalW rubbertre3} . . D. SWDVEE Y...... D ODNIIIKEDT R...... HMGR.clustal wrosyperiwi) . . E. GWEEE ...... EMVEDS RN...... HMGRclustalw tomato) - D. SDE...... N DEEEKDS RC...... HMGRclustalW (wood tobacc} . . DECNEED E...... N DEQFLLEEDS RR...... HMGRclustalw potato) . .D. SDIED E...... N AEOLEEDS RR...... HMGRclustalW radish) . .D. SGDSER ...... DFDDH R ------HMGRciustal Warabadopsis1} . .D. AWDAD. T...... I - - - - - OOOOH R ------HMGRclustalW (cucumismel} - -D. ADIED ...... EORT ...... HMGRclustalW rubbertre2} agiciustalW (rubbertrel} . . D. WWDLED T...... D 9. NY,DED R ------HMGRclustalW camptothec . . DDWWGWDD DE...... D WOEVLKEDT R...... HMGRclustalW (arabadops2} . . DDVWVNDG - ......
w Y is a w w w HMGR.clius talW (chineseham PSVSLWQFYI, SKMISMDIEQ VVTLSLAFLL AVKYIFFEQA E ESS FG. 32S
U.S. Patent Mar. 15, 2011 Sheet 52 of 78 US 7,906,710 B2
SSO HMGRclustalW (methanobac). a e s a e s ------o a o ...... up o o e o O - Y - w 0 - w - - - - - as a a e - e s a s e s a ......
s s a s HMGRclustalW (halobacter) - a a - P 8 p. e. e. ab a 0 to be a - 0 to w a a a a interciustalw(sulfolobus) HMGRclustalW yeast2} AIRDQFISKL LFFAFAVSIS INVYLLNAAK IHTGYMNFO. ... POSNKIDD HMGRclustalW yeastl) AIRDRFVSKL VLSALVCSAV INVYLLNAAR IHTSYTADQL WKTEVTKKSF BMGRclustalW (phycomyces} a s o e o e o so e e s - r s a e o e s 0 e o a - a s ...... HMGRciustalw{ fusarium) NGYLFNVAR WGIKDPNVPE ENIDRNEAR AREENDTGS. d P AT HMGRclustalW candida} AARDKYISKF ILFAFAVSAS INVYLLNWAR IHTTRLEDA, to o a up e IE HMGRclustalW (dictyoste2} - e a P - - w w b ------8 v e o e o o e o s s e s ...... HMGRclustalW (wheatl}
BMGRclustalW { rice s s to e - P - 8 a s a e o w w e 8 a. s. s. s. v. o. o. is a s s s ......
O BMGRclustalW { corn) s s p q r s a b & a s a d 4 to w e o e s a s o s a ......
HMGRclustalW (wheat3} a s p + 8 w or 8 & 8 8 an on 8 a. a w w w w is as s a
o a e. e. BMGRclustalW (wheat2} ------up o s s so a e e
HMGRclustalW soybean) s 8 m a o 0 t e s a si b (- - 8 e v - e o is 0 6 o o a a s is a a to 0 e s s s a w
8 - 0 a to w w 8 × 8 + 8 h 8 v 8 9 w a to w w as a ......
0 e o p is O 0. is a vo. us us o ------G- - - - -
P. D. o up de P a tomato) up a o as is ------...... G. . . . . as a s p O HMGRclustalW (woodtobacc} a e s a p is a o as s ------. . . . G. . . . .
HMGRclustalW potato - a - O O up set us s. s et a o o o a so a s o a o as a o a s s a - - - - G. . . . .
9 0 - 0 h p + v. v ar to 8 P - P - a. A h v 8 o b b g w w s s a
e - a e o o is a 0 e o up e o e s & 8 v 8 - w a e s w a ......
s a e s - a a s a o e o o a o e s p s s s a a - e s o o s a ......
• a e v - - - 8 e s a o 0 o 0 8 as e e s - - w w e a o e s a s a a
o s o w - o a s a w a s a e o a e s e s - - - - - a s is a ......
o a a o o o a s s a s s o a s a e e s - e o o e o e s a e o s a
w to o is o is as a
M is a HMGRclustalW (chineseham KN. . PITSPV VTPKKAPDNC CRREPLLVRR SEKLSSVEEE PGVSQDRKVE F.G. 32U
U.S. Patent Mar. 15, 2011 Sheet 54 of 78 US 7,906,710 B2
SS 600 HMGRclustalW (methanobac ...... HMGRclustalW (methanococ) ......
HMGRclustalW (halobacter) ...... 8 0 w a w w w to w s s s > w- b d 4 w s vs HMGRclustalW (sulfolobus) ...... HMGRclustalw yeast2} LVVQQKSATI EFSET...... RSMPA, SSGETWA KDIIISEEIQ HMGRclustalW yeastl). TAPVQKASTP VLTN...... KVS GSKVKSSSA OSSSSGPSSS HMGRclustalW (phycomyces) ...... HMGRclustalW fusarium) LPLGEYVPPT PMRTQ...... PSTPA TODEAEG. EMKARP EMGRclustalW candida LKKPKKKASK TAVSV...... PKAVW WKDSETTKSS ESSSESE HMGRclustalW dictyoste2} . . KGKSVNVE DLKDQ...... EIAL VDKGEOP. . . . .NETR HMGRclustalW (wheatl ...... HMGRclustalW { rice ...... GGGGSR...... s p n w w . . RA . . . . . A. PPEPAMHGH HMGR.clustalW corn ...... SSGS AAA...... PSROHAOA PACEGSP HMGRclustalW (wheat3} ...... 8 l k w w w w 4 a a A six u : HMGRclustalW wheat2) ...... HMGRclustalW soybean ...... HMGRclustalW (rubbertre3} ...... PTG AC...... AAPSLDCS SLTKEAP HMGR.clustalW rosyperiwi . .T. . NCTTL GC...... AWPPPSVP KAPVVPQQP HMGRclustalW tomato) . . P...ATTL GC...... AVPAPPAR QIAPMAPPQP HMGRclustalW (woodtobacc} . . P...ATTL GCT...... AWPPPA QIVPMVPPQP HMGRclustalW potato) . . PCAAATTL GC...... w to the s is to ... WWPPPPVR KTAPMVPQQp RMGRclustalW radish) . . . . . LWO PPP...... as o w w s a lo n ve . . PPP. . . . S QIVAAKLPNP HMGRclustalW (arabadopsis1} ...... LWTC SPP...... TP...... WSWAKPNP HMGRclustalW (cucumismel} ...... LIDN NRY ------AAPRSASA WALPSKWVDA HMGRclustalW (rubbertre2} ...... HMGRclustalW (rubbertrel ...... LWTC PPA...... NISTKTT AAPTKPTS HMGRclustalW camptothec...... TVP CAA...... APWDCPP PIKPKVVDPW HMGRclustal Warabadops2} ...... MIPC NQ...... SOCREW PKPNSWOpp HMGRclustalwchineseham VIKPLVVETE SAS...... - RATFWLG.A ... SCTSppVAA RTQELEELP U.S. Patent Mar. 15, 2011 Sheet 55 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2) VIKPLVVETE SAS...... RAFVGA SGSPVAA RTOELEIELP HMGR.clustalW (syrianhamst V(KPLVAETE STS...... RAVG. A - SGGCSVA, GTOEPEIELP HMGRclustalW rat VIKPLVAEAE TSG...... RATFVLG.A ..saas PPLAL GAQEPGIELP HMGRclustaiw rabbit) VIKPLVAETD SPH...... RAAFVVGGS . SFPDTSLVL ETKEPEELP HMGRclustalW human VIKPLVAETD TPN...... RATFVVGNS - SLLDTSSVL. WTQEPEIELP HMGRclustalW ( mouse) ...... w w w b or w w w w (8 W. W. w w HMGRclustalW { xenopus VIKPLPLETS P...... KARFWG OSSESP EDKNTMS HMGRclustalW (sea urchin} PVTASPRNSR SSSPVSSHSV KPARFTIGSS GSGSEDEEEE WREEEWEWV HMGRclustalW cockroach: ETRDELTTTR GMDG. . . . . W VEVSSPVEHK YVOTEOPSCS APEQPLEEPP HMGRclustalW (drosophila LFTIEDQSSA N...... ASTQTDLL PRHRWGP HMGRclustalW (dictyostel} . . . . . SGKeQ EO...... - - QCOCOCQ QQTPDITNQP HMGRclustalW (schistosom) PKIKETLISD QVKQSPVLPK FSKKLNDIPL. QSRKrIYCLH KOODYDRND HMGRclustalW (archaeoglo ......
Corsess WKPVAETE was So we w w an in wRATV-G-A SAPPPA- - - PEEE
FIG. 32X U.S. Patent Mar. 15, 2011 Sheet 56 of 78 US 7,906,710 B2
6SO HMGRclustalW (methanobac s a e s s as s as as s a 8 a 8 8 x - as MDME HMGRclustalW (methanococ s y s is a s NDEKMN HMGRclustalW (halobacter & w 's v i e s a Y - AASIADRVRE HMGR.clustalW (sulfolobus) a w w w w a 8x X x > b w IDEWWEKLVK HMGRclustalW { yeast2. NNE. CWYALS SODEPRP. L. NTEVSNLVVN HMGRclustalw yeast) SEEDDSRDE SDKKRP, . NEWAALWTH HMGR.cliustalW (phycomyces HMGRclustalW fusarium ANL...... PNRSN EELEK.I.S. . ENAREM DEEWSSMR HMGRclustalW candida} SEO...... SSR. EQVIELYK. . OGKKTLV DEVVSLWTA HMGR.clustalW (dictyoste2} PNN...... F QRAVHRR. . KLARDO KEHQRALHAQ HMGRclustalW wheati
HMGR.clustal W{ rice) G...... d 4 k V 4 + w w 8 . GMMEC DEEWAAVS HMGRclustalw{ corn) AA...... ExMPEO DEEWASWVA HMGRclustalW (wheat3} 8 w w & w is k 4 x X 0 &
HMGRclustalW (wheat.2) st we 8 A & 8 as a w up th k - ) is HMGRclustalW soybean)
s a a as a HMGRclustalW rubbertre3} s is a a Y A 9 8 × 8 w VSTT. . . . . TSSD DEQIIKSVVS HMGRclustalW rosyperiwi SK------MW, EKPA, . . . TPONSEE OEOKAWWA HMGRclustalw tomato S------MSM WEKPAP, SASSGE DEEIIKSVVQ HMGRclustalW woodtobacc} SEKA.W. PAASEE DEEKSVVO HMGRclustalW ( potato EKPSP. . . .MPASED DEEIIQSWVQ HMGRclustalW radish) w x w w w - w w wa 0 w w . . . QPPLPKE DEEWRSVD HMGRclustalwarabadopsis 1} w is p ESPEE EEWSVD HMGRclustalwcucumismel s is b : TPPEE OEVVKMVV EMGRclustalW (rubbertre2}
w ApVSEE BMGRclustal Wrubbertrel w w is as 8 EWNSWW HMGRclustalW (camptothec) as y as w is a 0 - SPPSSEE DEEKSVVE HMGRclustalW (arabadops2} OEEVKVo HMGRclustal w{chineseham SE...... x t w w PRP.N EECLQILE. . SAEKGAKFLS DAEEIOLVNA FIG. 32Y U.S. Patent Mar. 15, 2011 Sheet 57 Of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} SE...... PRPN EECLOILE. . SAEKGAKFLS DAEIOLVNA HMGRclustalW (syrianhamst) SE...... PRPN EECLOILE. . SAEKGAKFLS DAEIIQLVNA HMGR.clustalW { rat} SE...... PRPN EECLQILE. . SAEKGAKFLS DAEIIQLVNA HMGR.clius talW ( rabbit} KE------. . . . PRPN EECLQILG. . NAEKGAKFLS DAEIQLVNA HMGRclustalW ( human} RE...... PRPN EECLQILG. . NAEKGAKFLS DAE.IQLVNA HMGRclustal W{ mouse HMGRclustalW ( xenopus EE...... PRP. L. DECVRILK. . NPDKGAQYLT DAEWSVNA HMGR.clustal W(sea urchin} LET...... ELKAPRP.M PELE, ... NVGKGPNA DDEVOLVGA HMGRclustalW { cockroach} AS ...... NRS. I DECLSVC. . . KSDVGAQALS DCEVMALVTS HMGR.clustalW (drosophila KP...... PRPV QECONST EEGSGPAAS DEEWSWEA HMGRclustalW (dictyostel) TKTN...... KKPKELS NEELIKEK HMGRclustalW schistosom VLDLDMLTEK IKQGLGHELS DTEILQLLSH HMGRclustalW (archaeoglo) a Y O to a a dis P. p. a to a 0 e os .MQVLRLDRR HMGRclustalW (pseudomonas) • o 0 a s h e s s s 8 0 wo
CoSeSuS EECQ- - - -AEKGAKSS DEEKLVVA
FIG. 32Z. U.S. Patent Mar. 15, 2011 Sheet 58 Of 78 US 7,906,710 B2
55 700 HMGRclustalW methanobac. GR.. IKLYEI E.RHVPVDEA WRRREFE. . . . . RTCGWK LEEWSNYS HMGRclustalW (methanococ) GE.. IKPYQL. D. KMFGSKIA TERRKFIE...... KKWGIE ... FKHONYS HMGRclustalW (halobacter) GD.. LRLHEL. E.AHADADTA AEARRLLVE. . . . . SOSGAS DAWGNYC HMGRclustalW (sulfolobus GE.. ISFHEV D.NLLEANAA MWARRAE. . . . . KWGWG PSGSTV HMGRclustalW yeast2) G. . KLPLYSL, EKKLEDTTRA WLVRRKALST AESPLWS. . . . EKLPFRN HMGRclustalw yeastl G. . KLPLYAL, EKKLGDFTRA WAVRRKALS AEAPWAS. ORY KN HMGRclustalW (phycomyces ...... 48 Y is w 4 o HMGRclustalW fusarium) G. . KIPGYAL. EKTLGDFTRA NKAAADHS LDRSKLPYSN HMGRclustalW candida) G. . KLPLYAL. EKQLGDNLRA WARRKASD LADA PVRS. . . .NKPYLH HMGRclustalW (dictyoste2} A. . VVAAAEK AATSGEDPSS ICPVVPPTSN DFEGSTN. w us as a PWOH HMGRciustalW (wheatl ...... HMGRclustalW ( rice G. ...ALPSHRL ESRLGDCRRA RVTGRG ... WEGLPFDG HMGRclustalW corn, G. ... KVPSYAL EARLGDCRRA . . . RGRD . . IEGLPLOG HMGRclustalW (wheat3} ...... a a a
HMGR.clustalW (wheat2} ...... a v 0 is o w w q 0. HMGRclustalW soybean ...... HMGRclustalW (rubbertre3 G. . SIPSYSL. ESKLGNCKRA ALIRRETLQ. . . . . RMSGRS . . LEGPLOG HMGRclustalW rosyperiwi G. . KIPSYSL ESKLGDCKRA AGIRREALQ. . . . . RGKS . . LEGLPLEG HMGRclustalW ( tomato) G. . KIPSYSL ESKLGDCKRA ASIRKEVMQ. RGKS . . LEGPLEG EMGRclustalW (woodtobacc} G. . KMPSYSL ESKLGDCKRA ASIRK2ALO. . . . . RGKS . . LEGIPLEG HMGRclustalW ( potato) G. . KTPSYSL ESKLGDCMRA ASIRKEALO. . . . . RTGKS . . LEGPLEG HMGRclustalW radish G. .VVPSYSL ESRLGDCKRA ASIRREALQ. . . . . RGRS . . IEGPLOG HMGRclustalW (arabadopsis1} G. .VIPSYSL ESRLGDCKRA ASIRREALQ. . . . . RVGRS ... EGLPLDG HMGRclustalW (cucumismel} G..SVPSYSL ESKLGDPKRA ASIRREALQ. RGRS ... (EGPFEG HMGRclustal Wrubbertre2} ...... HMGRclustalW (rubbertrei} G..KIPSYSL ESKLGDCKRA RMRRS . EGLPVEG HMGRclustalW camptothec G..TTPSYAL ESKLGDSHRA RMTRKS . . AGIPLDG HMGRclustalW (arabadops2} G. .TIPSYSL, ETKLGDCKRA AAIRREAVO. . . . . RTGKS TGLPLEG HMGRclustalW (chineseham) K..HIPAYKL. ETLMETHERG WSIRRQLLST K. LPEPSS. . LOYLPYRD FIG. 32AA U.S. Patent Mar. 15, 2011 Sheet 59 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} R PAK WSIRROLIST K. PESS. ... LQYLPYRD HMGRclustal Wsyrianhamst K PAK ETMETERG WSIRRQLIST K..LPEPSS. ... LQYLPYRD HMGRclustalW rat) K. SAK, EMEERG WSRROLLSA K. .I.AEPSS. ... LQYLPYRD HMGRclustalW { rabbit} K. PAK EMETERG WSIRRQLLSK . ESS . LQYLPYRD HMGRclustalW ( human} K PAK ELMETERG WSIRRQLSK K. : SSS. . LOYLPYRD HMGRclustalw mouse HMGRclustalW ( xenopus E. HAK, EMMESPRE WARROMLSD .LPQRSA. . LQSLPYKN HMGRclustalW sea urchin) K. EPAYK ENONERG WAVRROSK . . . A EKYAS HMGRclustalW cockroach) G. ...EAGYQ, EKWVRNERG WGIRRQLTK ...A.K.A. ... , N.YKN HMGRclustalW (drosophila GGCK ESVODERG WRIRRCIIGS R. A.KMPWGR ow, HMGRclustalW (dictyostei) ...EWAYR ENELGOCSRA WERRMLLEK . . . . CLSKK. 0. ... EPG HMGRclusta Wschistosom} G. R.R.E. ESVWRNFRA WERLOS . . . . FNNP ERYKO HMGRclustalW (archaeoglo) YKSKIRRA MSSRGFK SWEEKW AEFAGSEE . VKAVLSCG HMGR.clustalW (pseudomonas w 8 . A MS OSRARN SPAARE GOLIGLSDD ... WSANAG
Consertists G SYS, ESKGCKrA. WSRREASK Kao arGSS EGYG
FIG.32BB U.S. Patent Mar. 15, 2011 Sheet 60 of 78 US 7,906,710 B2
70. 750 HMGRclustalW (methanobac IDMERASRRN IENPIGVVQI PLGVAGPLRV RGEHADGEYY WASEGA, HMGRclustalW (methanococ) IDEEMAMKKN IENMIGAIQI PLGFAGPLKI NGEYAKGEFY LAEGA, HMGRclustalW (halobacter FPAEAAES.A IENMVGSIQV PMGVAGPvsv DGGSvAGEKY AEGA, HMGRclustalW (sulfolobus) IDYSEIKNKN AENVIGAIQI PLGIVGPIRV NGDYAKGDFY WPMATTEGA, HMGRclustalw yeast2) YDYDRVFGAC CENVIGYMPI. PVGVIGPLII DGr. . . . SYH IPMATEGCL HMGRclustalW yeastl} YDYDRVFGAC CENVIGYMPL. PVGVIGPLVI D.G.T. . . . SYH
FIG. 32CC
U.S. Patent Mar. 15, 2011 Sheet 62 of 78 US 7,906,710 B2
WPMATTEGC, HMGRclustalW (sea urchin} YDYSFVSGAC CENVIGYMPV PWGWAGP DGQ. . . . EFO WPMATTEGO, HMGRclustalW cockroach} YoYLKVMGAC CENWGYMPV PWGWAGPLN, OGR. . . . LVH WPLATEGCL HMGRclustalW (drosophila) FDYRKVNAC CENVLGYWPI PWGYAGPL, OGE. TYY WRMATTEGA, HMGRclustalW (dictyostel) FDFAKVOGOC CENVIGYVPI PWGTAGPIQL NGO. . . . LWT PMATTEGCL BMGRclustal Wschistosom} YDYRLVYGOC CEEVIGYMPI PVGKGP, DGR, . . .SY PLATEGO, HMGRclustal Warchaeoglo PLOVAORM ENVGTFE, GANF, DGK. . . . DY. PMARESW HMGRclustalW (pseudomonas) ALPMDANGM IENVIGTFE, PYAVASNFQI NGR . . . . DVI VPLVVEEPSI
Corses S FOY-SVG-C CENVIGY - - PWGVAGPL OGK - - --EYS WPMATEGC, HMGCoA binding E.
FIG. 32EE
U.S. Patent Mar. 15, 2011 Sheet 64 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} WASNRGCRA GGGGASSR VLADGMT. RG PVVRPRACO SAVKAWE HMGRclustalW (syrianhamst) VASTNRGCRA GLGGGASSR WLADGMT. RG PVVRLPRACD SASVKAWE HMGRclustalW { rat) VASTNRGCRA CSLGGGASSR VADGM.S. RG PVVRPRACO SAEVKSWET HMGRclustalW ( rabbit} WASNRGCSA CLGGGASSR VAOGMT. RG PVVRPRAC SAEVKAWE HMGRclustalw human} WASNRGCRA GGGGASSR VLADGMT. RG PVVRLPRACO SAEWKAWLET HMGRclustalW { mouse HMGRclustalW ( xenopus) VASTNRGCSA IMLGGGAKSR VADGMT. RG PWVRLPTACO AAVKANDS HMGRclustalW (sea urchin} WASTNRGCSA RSAGGHSW LIGOGMT. RG PLVRLPSAQE AGAIKQWLEV HMGRclustalW cockroach VASTNRGMRA MRCGVTSR VAOGMT. RG PVVRFPNIDR ASEAMLWMOV HMGRclustalW (drosophila) VASTNRGCKA SVRG. VRSW VEDVGMT. RA PCVRFPSVAR AAEAKSWEN HMGRclustalW (dictyostel) VASTHRGCKA TESGGAKCT TSRGMTRA PWVRFSDIVK ASEFWSWIND HMGRclustalW (schistosom} VASTNRGCRA FAGGKSW VYRDQMT.RA PVVWFPSIID SVKCAWIDS HMGRclustalW (archaeoglo) VAAASNAARM ARESGGTTD YTGSLMIGQI OWTKLLN 9NA AKFEVLRQKD HMGRclustalW (pseudomonas) WAAASYMA 3. ARANGGFS SSAPLMHAQV QVGIQDPLN ARLSRRKD Consensus WASTNRGCKA -SGGATSV WADGM-RA PVWRFPSAKR AAELKSWED
FIG. 32GG
U.S. Patent Mar. 15, 2011 Sheet 66 of 78 US 7,906,710 B2
HMGR.clustalW (chineseha2} PEGFAWIKIDA, FDSTSRFARL, QKLH.W. . . . . TMAGRNLYR FQSKTGDAMG BMGRclustalW syrianhamst) PEGFAWKDA. FOSTSRFARL QKLHV. . . . . TMAGRNLYIR FQSKTGDAMG HMGRclustalW { rat PEGFAVWKEA. FOSISRFAR. QKLHV. . . . . TLAGRNLYIR LOSKTGDAMG HMGRclustalW { rabbit) PEGFAVIKEA FDSTSRFARL, QKLHI . . . . . SMAGRNLYER FQSRTGDAMG HMGRclustalW human} SEGFAVIKEA FTDSTSRFARL QKLHT. . . . . SAGRNLYER FQSRSGDAMG HMGRclustalW mouse) HMGRclustalW ( xenopus) ASEGFKVIKDA FDSTSRFARL GRLQN. . . . . CWAGRNLYIR FQSKTGDAMG HMGRclustalW (sea urchin) PENFAAIKER FESTSRFAKL. KSIQT. . . . . AAGRYMFLR SKATGDAMG HMGRclustalW ( cockroach) PYNFEQIKKN FDSTSRFARL SKIHI. . . . . RVAGRHLFR FATTGAMG HMGRclustalW (drosophila) DENYSVVKT FDSTSRFGRL, KIDCir. . . . . AMDGPQLYIR FWAITGDRMG HMGRclustalW (dictyostel CNYOAKAW FOSTSRFARL SAIKC. . . . . TAGRSVYR FKCOTGDAMG HMGR.clustalW (schistosom) EGFOT.KSA FKTSAHVNL, LSVFA. . . . . CPAGRY HISR FAARTGDAMG HMGRclustalw{archaeoglo) ErANSC PMLVNLGCGC Kidear. VID TMGKMLVH WDVKOAMG HMGRclustalW (pseudomonas EIIELANRKD QLINSLGGGC RDIEVETFAD TRGPMLVAH WDWRDAMG Consensus PSENFETLK-A FNSTSRFARL. QSIQC - - - - - AAGRNLYR FSCSTGDAMG NADH binding domain i (continued)
FIG. 32
U.S. Patent Mar. 15, 2011 Sheet 68 of 78 US 7,906,710 B2
HMGRclustalW (chineseha2} MNMISKGTEK ALLKLQEF. . . . FPEMQILA WSGNYCDKK PAAN WIEGR EMGRclustalW (syrianhamst} MNMISKGTEK. ALVKLQEF. . . . FPEMQILA VSGNCTDKK PAAVNWEGR HMGRclustalW ( rat) MNMISKGTEK ALLKLQEG. . . . VPELOILA WSGNYCOKK PAANWEGR HMGRclustalw rabbit MNMISKGTEK ALSKLHEY. . . . FPEMOILA WSGNYCTDKK PAAWNEGR HMGRclustalW human} MNMISKGTEK ALSKLHEY. . . . FPEMOILA WSGNYCTDKK PAAN WIEGR HMGRclustalW mouse) us 8 ... . . EK ALLKOEF. . . . FPDMOILA WSGNYCTDKK PAANNEGR HMGRclustalW xenopus MNMISKVTEQ ALARLQEE. . . . FPDLHVLA VSeNYCTDKK PAANNEGR HMGRclustalW (sea urchin) MNMISKGTEQ ALHALOTM. . . . FPNEIMS LSGNYCTOKK WAANEGR HMGRclustalW cockroach) MNMLSKGTEV ALAYVOQV. . . . YPDMEILS LSGNFCTDKK PAVNWEGR HMGRclustalW drosophila) MNMVSKALRW PFAEFTLH. . . . FPDMQIIS SGNFCCDKX AANGR HMGRclustalW dictyostel) MNMWSKGWEA WLEHL.K. . . . FDOMTLLS SGNMCTDKK SSNWTEGR HMGRclustalW schistosom} MNMVSKAIDS ALHCLKKY. . . . FSNMOVIS SGMCDKK PANTIGR HMGRclustal Warchaeoglo ANAVNTMCEK VAPFIERITG . GKVYLRIIS NLAAYRLARA KAVFOKOVG EMGRclustalW pseudomonas ANTVNTMAEA VAPLMEAITG . GOVRLRILS NLADRARA QVRITPQQLE Corsensus MNMVSKGVEN VL--LQED- - - GFPDMDVIS SCNYCDKK PAAVNWEGR NADH binding domain l (concluded)
FIG. 32KK
U.S. Patent Mar. 15, 2011 Sheet 71 of 78 US 7,906,710 B2
OOO HMGRclustalW methanobac AHANGA FLATGODEAH IVEGSGVT . . . . . GOLYF HMGRclustalW (methanococ) AEYANIIGA FLAGODEAH IVEGSLGITM - - - - - OGYF HMGRclustalW (halobacter) AEWANWWAAM FLATOQDEAQ WWEGANATE . . . . . GO.YV HMGRclustalW (sulfolobus) AHFANVA FIATGQDVAQ VSSSGYTW - - - - - EDLY HMGR.clustalW ( yeast2} AEAAN.W.A. FLALGODPAO NVESSNCITI, . . . . . GDLR HMGRclustalw yeastill AHAANLVTAV FLA LGQDPAQ GDR HMGRclustalW (phycomyces} HMGRclustalW fusarium) AHAANVAA FLATCQDPAQ VVSANCT - - - - - GALQ HMGRclustalW candida} ACAANMVTAV YLALGODPAQ NVeSSNCTL . . . . . GOL&W HMGRclustalW (dictyoste2} AHASNVAL YIATGQDPAQ NVESSNCTL MESINCG. . . - - - - KDLY HMGRclustalW (wheatl AHASNATAL FATGQDPAQ NVESSQCITM LEAVNEG. . . w w KOL HMGR.cliustaiw rice AHASN.W.A. FIATGQDPAQ NVESSQCITM EEVNOG... . w h ODH HMGRclustalW corn ABASN.W.A. FLATGQDPAQ NVESSCTM EPVNAG...... RDLI HMGRclustalW wheat3} AEASNWA FIATGQDPAQ NVESSOCIAM LEAVNOG... KCLE HMGRclustalW (wheat2} ABASNVSA FIATGQDPAQ NVESSOCITM LEAVNGG...... RDLHI HMGRclustalW ( soybean) AEASNVSA FIATGQDPAQ NVESSHCTM MEANOG... . . - RDLH HMGRclustalW rubbertre3} AASNMVTAV YIATGQDPAQ NWSSSHCTM MEAVNDG...... KDLHI HMGRclustalW (rosyperiwi AHASNWSA FIATGQDPAQ NVESSQCTM MEAVNDG. . . ------ROH HMGRclustalW tomato) AASNWSAV FIATGQDPAQ NESSHCTM MEAVNDG...... KDLi HMGRclustalW (woodtobacc} AEASNIWSAW YIATGQDPAQ NESSECTM MEAVNOG... . . - - - - - KOW HMGRclustalW potato AASNWSAW YLATGODPAC NVESSRCTM MEAVNG...... KOLEV HMGRclustalW (radish) RASNWSAV FLATGQDPAQ NVESSQCTM MEANDG. . . Kids HMGRclustalW (arabadopsisl} AEASNWSAV FIATGQDPAQ NVESSQCITM MEANOG, ...... Kid HMGRclustalW (cucumismel} AHSSNWSA FLATGQDPAQ NVESSHCTM MEWNNG. - - - - RDLE HMGRclustalW rubbertre2} AHAGNWSA FIATGQDPAQ NVESSECTM MEAVNOG...... KDLHI HMGRclustalW rubbertrel ABAGNWSA FIATGQDPAQ NVESSECTM MEAVNG...... KOL HMGRclustalW camptothec APASNWSAV YLATGQDPAQ NVESSHCTM MEANG...... KDIEW HMGR.clustalwarabadops2} AESSNVSAW FATGQDPAQ NVESSECMT. LPG) . . . .
w w w m w 0 HMGRclustal Wohinesehaim) APEAANVTA YACGQDAAQ NVGSSNCITT, MEASGPTN. . FIG. 32NN U.S. Patent Mar. 15, 2011 Sheet 72 Of 78 US 7,906,710 B2
acticals (chineseha2) AEAANIVA YACGODAAQ NVGSSNCITI, MEASGPIN. . . 'icials (syrianhanss) AAANVTA YACGQDAAQ NVGSSNCITI, MEASGPIN. 'Eustalw rat}. LHAANIVTAI YIACGQDAAQ NVGSSNCITL MEASGPTN. . igitals rabbit}. AHAANYVTAI YIACGQDAAQ NVGSSNCITL MEASGPPN. . EGiant human). ARAANIVTAI YIACGQDAAQ NVGSSNCITL MEASGPTN.. 'Eicialist mouse AHAANIVTAI YIACGQDAAQ NVGSSNCITL MEASGPTN .. d itals ( xenopus) AHAANIVTAI YIACGQDAAQ NVGSSNCITI MEATGPTY.. o acticals ( sea urchin). AHAANIVTAI YIATGQDAAQ NIASSNCMTL METRGPKG. . Agitals ( cockroach) AAANIVTAI FIATGQDPAQ NVGSSNCMTL MEPWGEDG.. it tals (arosophila) AAANMVTAV FLATGQDPAO NWTSSNCSTA MENAENS - . icitals (dictyostel) AHASNIVTAI FLATGQDCAQ NVESSNCETO MEACNDG. . . indicals (schistoson) AEAANIAGM FAATGODLAQ VVDSSSCE.TO LEVLSD . . .
ideals (archaeoglo) A - 4 ------ATGNOFRA EAGASYAA GGYKTT YEWDRKGW HMGRclustalW (pseudomonas) ...... WAGNWRA WEAGAHAYAC RSGHYGSTT WEKONNGLV Consensus AEAANVTA FIATGQDPAO NVESSNCTM MEAVNDGN------KOLE
FIG. 32OO U.S. Patent Mar. 15, 2011 Sheet 73 of 78 US 7,906,710 B2
OO. OSO HMGRclustalW (methanobac AVNLPDVPLA TWGGGTGLET ASSCLDIMGW RGGG ...... RVAFAEWG HMGRclustalW (methanococ) SVTLPDVPIG TWGGGTRVET QKECLEMGC YGDN ...... KALKFAEVG HMGR.clusta Whalobacter SVSIASLEVG VGGGTOLp OSSGLDLGV SGGGDP, AGS NADALAECIA HMGRclustalW sulfolobus) SVTLPSLEVG TWGGGTRLPT QKSALSIMGW YGSGNP PGS NAKKAEA HMGRclustalW yeast2) SVSMPSIEVG TIGGGVLEP OGAMOLLGV RGHTEPGA NARCARA HMGRclustalW yeast SVSMPSIEVG TIGGGVLEP OGAMLDLLGV RGPHATAPGT NAROARWA HMGRclustalW (phycomyces ...... & 8 & 4 x 8 s HMGRclustalW fusarium) SVSMPSLEVG TLGGGTLE OGAMLDILGV RGSHPTNPGD NARRARG HMGR.cliustalW candida} SVSMPSIEVG TIGGGTILDP QGSMLELLGV RG PAWGIE NARQL.AKVA HMGRclustalW (dictyoste2} SVTMPSIEVG TWGGGTA OSACOLLK RGANERGA NSEOLARVVA RMGRclustalW (wheat SVTMPPEV. HMGRclustalW rice SVTMPSIEVG QAACN.LGV KGSNGSPGA NAGRATWA HMGRclustalW corn SVTMPSIEVG OSACLDLLGV RGASRDRPGS NARAWWA HMGRclustalW wheat3 SVTMPPIEV.
HMGR.clius talW (wheat2 SVTMPPIEv. a s > s 8 - - x 4 as 8 HMGR.clustalW { soybean SVTMPSIEVG OSAC NLLGV KGASKESPGS NSRLATWA HMGRclustalW rubbertres) SVSMPSIELG TVGGGTOLAS OSACLNLLGV KGASKSPGS NSRATIVA HMGRclustalW (rosyperiwi svTMPSIEVG TWGGGTQLAS OSAC NLLGV KGASKOSGA NSRLATIVA HMGRclustalW ( tomato SVTMPSIEVG TWGGGTOLAS QSACLNLLGV KGANREAPGS NARAVVA HMGRclustalW wood tobacc} SVTMPSIEVG TVGGGTOLAS OSACLNLLGV KGAN REVPGS NARATWA HMGRclustalW potato SVTMPSIEVG TVGGGTQLAS QSACLNLLGV KGANSDAPGS NARATWA RMGRclustalW radish SVTMPSIEVG TWGGGTOLAS OSACEN.GW KGASKESPG NSRRATWA HMGRciustal Warabadopsis) SVTMPSIEVG TWGGGTQIAS QSACENLLGW GASESPG NARRATWA HMGRclustalW (cucumistinel SVTMPSIEvg TWGGGTQLAs QSACLNLLGV KGASKESPGA NSRLAWA BMGRclustal Wrubbertre2} SVTLPSIEVG TWGGGTQLAS QSACLNLLGV MGACKESPGS YSRATWA HMGRclustalW (rubbertrel SVTMPSIEVG TVGGGTOAS OSACLNLLGV KGANKESPGS NSRLIAAVA HMGRclustalW camptothec) SVTMPSIEVG TVGGGTOLAS QSACLNLLGV KGASKEAPGS NARATWA HMGR.clustalW farabadops2} SVSMPCIEVG TWGGGTQLAS QAACLNLLGV KGSNNEKGS NAQQARIVA HMGRclustalW (chineseham SCTMPSIEIG TWGGGN, QGACKDNPGE NARQE.ARIWC FIG. 32PP
U.S. Patent Mar. 15, 2011 Sheet 75 Of 78 US 7,906,710 B2
OS OO HMGR.clustalW (methanobac GAVAGES IGrciustalw(methanococ)e s p at a AAVAGES 8 HMGRciustalW& W 3 - 0 (halobacter WGSAGES w agiciustalw4 & sulfolobus SWSELN, EMGR.clustalw yeast2. CAVAGELS, QSHMTHNRK. .NKANEL OPS...... HMGRclustalw yeasti CAVAGES. QSEMTHNRKP AEKNNL ATDI...... Grciustalw{phycomyces a 8 HMGRclustalW eusarium) AAVLAGELSI CSALAAGHLW RAEMCHNRSA SHARTGHD HMGRclustalW ( candida} SWSGESL WSAIAAGEW OSEMOENRAA HMGRclustal w{dictyoste? AAVSGELS, MSALAAGELV RSLKNRK SMTHNLPHSDHMGRclustalW (wheatl rice GSWWAGRA, HMGR.clustalW corn) GGVLAGELSL p. HMGR.clustalW (wheat) - is HerclustalW (wheat2}
& w y 4 HMGR.clustalW { GSWAGES NSMNRSS KOW...... w w Mercistalw{w w x rubbertre) GSWAGES NSMKNRSA KDWSK. . . . . HMGRclustalw{rogyperii) CSVAGES MSASAGOW RSE MKNRSS KON. . . . . HMGRclustalW tomato) GSWAGES MSASSGOLV SMKYNRST KDVR . . . HMGRciustalW (woodtobacc} GSWAGES MSAISAGOLV KSKYNRST KCWK. . . . . HMGRclustalW potato GSWAGELS MSASAGQLW KSMKNS KS......
8 w w w HMGR.clustalW (radish) GAVLAGELSL MSAAAGOLV RSMKSNRSS RDISG. . . . . Hercistalw(arabadopsis)w is s GAVAGEISI. SAAAGOV RSMKYNRSS RDSG. . . . . 8 w Herciustalww w w & 8 (cucumismel) GSWAGES MSAAAGQLV RSEKNRSS ROWSK. . . . . HMGR.clustalw rubbertre?} SVAGES MSAAAGOLY KSMY Ness KVSK . . . HMGRciustalw(rubbertrel) SWAGES, SAAAGOV KSMYRSS KMSK. . . . . IMGrcistalW (camptothec) GSWAGS, MSAAAGOV NSMENRSN KDWK. . . . .
4 w HMGRciustalw arabadops2 GSWAGELS SAAAGQV ROGP. . . . . fidistalw(chineseham) GWMAGES MAAAAGPW RSMVNRSK NOD. . . . . FIG. 32RR U.S. Patent Mar. 15, 2011 Sheet 76 of 78 US 7,906,710 B2
{ w X - 8 8 HMGRclustalW (chineseha2} GTVMAGELSL, MAALAAGHLV RSHMVHNRSK INLQD. . . . . is v 8 × 8 HMGRclustalW (syrianhainst GTVMAGELSL. MAAAAGHLV RSEMVHNRSK INLOD. . . . . fierciustalW rat} GTVMAGELSL MAAAAGHLV RSEMVHNRSK INLQD. . . . . GRciustalw{ rabbit} GTVMAGELSL MAALAAGHLV KSHMIHNRSK INLQD. . . . . iMGRciustalW human GTVMAGELSL MAALAAGHLV KSEMIHNRSK INLQD. . . . . Aigreinstalw{ mouse) GTVMAGELSL MAALAAGHLV RSEMVHNRSK INLQD. . . . . s AMGRciustalw Xeroes SIVMAGES MAAAAGHV KSMVENRSK NLQD. . . . .
8 w w x is w 4 w HMGRclustalW (sea urchin ATVMAGELSL, MSALAAGHLV KSEMKENRSA INIASPLPSI EVARRSK HMGRclustalW cockroach) GTVLAGELSL, MSALAAGHLV KSEMRENRSS VSTSG. . . . .
HMGRclustalW (dictyostel SAVMAGELSL, MSALSAGHLM KSHLQYNRAK TN...... HMGRclustalW (schistosom GTVLAAESL. MAALDTDDLV KAHMHFNRAK CSTNSHSCSH STDNNDN EMGRclustalW (archaeoglo ...GLAQNFAA LRALATEGIQ RGEMELEARN LAIMAGATGD EVORVVEIMW HMGRclustalW pseudomonas . . GLAQNLGA MRALATEGIQ RGHMALEARN IAV VAGARGD EVDWVARQLV Corsess GVAGES, MSAAAGHLW KSMK-NrSS KVSK------
FIG. 32.SS U.S. Patent Mar. 15, 2011 Sheet 77 of 78 US 7,906,710 B2
O S2 HMGRclustalW (methanobach ...... 0 w w w
HMGRciustalW (methanococ} w w w w w 4 op 4 w w w 4 w 4 & 9 w
HMGRciustalW (halobacter} o a o a w as a ex as e o e o a a s a as a s a as a s - a as a to s a -s as a o a
HMGRciustalW (sulfolobus) o a a - a se a s o is e a o o o o O p e s up a e o a o 0 e o e s e. e. e. e s o HMGRclustalw{ yeast2) ...... NKGPPCKT SALL...... HMGRciustalW ( yeast1 ...... NRLKIDGSW TCKS......
o HMGRciustalW (phycomyces) a o - w w do o a e o o ve or w P 0 - w w w or up o up w w w O e A 8 - 8 s : o o BMGRciustalw{ fusarium) QCPRALSVNN VDERRRYSEV KAIDE...... HMGRciustalW ( candida} ......
HMGRciustalW (dictyoste2} - w a w s a a P o 0 w 0 as a s as a e o u q w as a
we v a v HMGR.clustalW (wheat1} us s is v or 0 p - - p. v. s. s. s is u w o n - a s a s s e o up
HMGRciustalw{ rice} ...... A . . . . . AS...... e a w as a so e o a w w w as HMGR.clustalW corn ...... T . . . . . TATEK TRQREVDV......
w w w HMGR.clustalW (wheat3} 0 w w w w w w - 0 + w w w w w w w w x w w w w
w HMGR.clustalw{wheat2} w w p s w w e a so w w w w w w w is & we w e w a w a a a 4s e o HMGRciustalw{ soybean) ...... I . . . . . S......
HMGRciustalW (rubbertre3} o a I - - - - - TF. ------...... HMGR.ciistalW (rosyperiwi) w I - - - - - ASSQL ESDS ...... HMGRciustalw{ tomato) ...... A . . . . . SS. ------
HMGR.ciistalw{woodtobacc} us p a o o us s. s. s. A - - - - - SS. ------...... HMGRciustalW potato) ...... t 8 HMGRclustalw{radish} ...... A . . . . .TTTT......
iMGRciustalW (arabadopsis1} 0 w w up we so A - - - - - ......
HMGR.ciistalW (cucumismel to 0 w to L...... ES......
fiercistalw{rubbertre2} ...... A .....As...... 4 P V P a d is 8 + 8 + 9
HMGRciustalw{rubbertrel} a w w w do - - - - A . . . . . AS...... w w w w w w w w & 8 w w g
fiercistalw{camptothec} w a o a o w w . A . . . . . SS. ------
HMGRciustalW (arabadops2} to w to - we S . . . . . SOVNR ...... we
Big Reinstalw{chineseham) we w 40 w w w w 0 . . . LOGTCTK KSA...... FIG. 32TT
US 7,906,710 B2 1. 2 TRANSGENC PLANTS CONTAINING rols in yeast by increasing HMGR in yeast having decreased ALTERED LEVELS OF STEROD erg5 and erg6 activity—Sc and hamster HMGR). COMPOUNDS WO9845457 (SMTI, Erg6 from A.t., corn, yeast; trans genic plants with altered sterol levels using DNA encoding This application is a divisional of U.S. application Ser. No. an enzyme binding a first sterol and producing a second 10/862,907, filed Jun. 7, 2004, now U.S. Pat. No. 7,544,863 sterol—altered carotenoid, tocopherol, modified FA levels— that is a divisional application of U.S. patent application Ser. HMGR, 5C.-reductase, geranylgeranyl pyrophosphate Syn No. 09/885,723, filed Jun. 20, 2001, now U.S. Pat. No. 6,822, thase, phytoene synthase, phytoene desaturase, isopentenyl 142, which application claims benefit of priority from U.S. diphosphate isomerase). Provisional Application Ser. No. 60/260,114, filed Jan. 5, 10 Acetate is the metabolic precursor of a vast array of com 2001, each of the disclosures of which are incorporated herein pounds vital for cell and organism viability. Acetyl coenzyme by reference in their entirety. A (CoA) reacts with acetoacetyl CoA to form 3-hydroxy-3 methylglutaryl CoA (HMG-CoA). HMG-CoA is reduced to TECHNICAL FIELD mevalonate in an irreversible reaction catalyzed by the 15 enzyme HMG-CoA reductase. Mevalonate is phosphorylated The present invention relates to biotechnology with an and decarboxylated to isopentenyl-pyrophosphate (IPP). emphasis on plant biotechnology, and particularly biotech Through the sequential steps of isomerization, condensation nology affecting the biosynthesis of Steroid compounds. and dehydrogenation, IPP is converted to geranyl pyrophos phate (GPP). GPP combines with IPP to form farnesyl pyro BACKGROUND phosphate (FPP), two molecules of which are reductively 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 the agricultural community. Several approaches involve 25 HMGR). Schaller et al. (Plant Physiol. 109: 761-770, 1995) manipulation of already present cellular biosynthetic path found that over-expression of rubber HMGR (hmg1) 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. esters in lipid bodies. HMGR activity was increased by 4- to Several related U.S. patents address increasing sterol accu 30 8-fold. mulation in higher plants. Those patents include U.S. Pat. No. 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) and a terminal cyclopentane ring (D) having the formula (I) and U.S. Pat. No. 5,306,862 “Method and composition for 35 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 tance—in tobacco, tomato, corn, carrot, soybean, cotton, bar (I) ley, arabidopsis, guayule and petunia; seeds with elevated 40 sterol/cycloartenol. 7S promoter and CaMV promoters), U.S. Pat. No. 5.365,017 “Method and composition for increasing 19 ) sterol accumulation in higher plants” (DNA construct with 9 HMGR-CaMV 35S, transgenic plants, hybrid plants, corn, soy, barley, tomato, Arabidopsis), U.S. Pat. No. 5,349,126 45 “Process and composition for increasing squalene and sterol accumulation in higher plants' (increase in squalene and sterol accumulation by increasing HMGR activity in trans where R is an 8 to 10 carbon-atom side chain. genic tobacco, cotton, Soybean, tomato, alfalfa, Arabidopsis, In plants, squalene is converted to squalene epoxide, which corn, barley, carrot and guayule plants), and EP 486290 (en 50 is then cyclized to form cycloartenol (4.4,14C.-trimethyl-93, hancement of squalene and specific sterol squalene Zymos 19-cyclo-5C.-cholest-24-en-3?-ol). Cycloartenol has two terol, cholest-7,24-dienol, cholest-5.7.24-trienol accumula methyl groups at position 4, a methyl group at position 14, a tion in yeast by increasing HMGR activity in yeast deficient methylene bridge between the carbon atoms at positions 9 in enzymes that convert squalene to ergosterol). and 19 that forms a disubstituted cyclopropyl group at those In those patents, the amount of a protein exhibiting 3-hy 55 positions, and includes an 8-carbon sidechain of the formula: droxy-3-methylglutaryl Coenzyme-A reductase (HMGR) CHCH(CH)CH=C(CH). Squalene epoxide can alter activity is typically increased. HMGR widens a “bottleneck” natively be converted into pentacyclic sterols, containing five near the beginning of a biosynthetic path to steroid produc instead of four rings. Exemplary pentacyclic sterols include tion, permitting a higher carbon flux through steroid biosyn the phytoalexins and Saponins. thetic pathways and resulting in increased sterol accumula 60 Being one of the first sterols in the higher plant biosynthetic tion. pathway, cycloartenol serves as a precursor for the production U.S. Pat. No. 5,480,805 “Composition for modulating ste of numerous other sterols. In normal plants, cycloartenol is rols in yeast” (enhancement of delta 8-7 isomerase activity converted to predominantly 24-methylene cycloartenol (4.4, ERG2 enhances accumulation of specific sterols in yeast). 14C.-dimethyl-9B. 19-cyclo-22.23-dihydro-ergosta-24(28)- U.S. Pat. No. 5,460.949 “Method and composition for 65 en-3-3-ol), cycloeucalenol, (4,14C-trimethyl-9B. 19 cyclo increasing the accumulation of squalene and specific sterols 5C-ergosta-24(28)-en-3?-ol), isoflucosterol (5C.-Stigmasta-5- in yeast' (increasing squalene, Zymosterol and specific Ste 24(28)-dien-3?-ol), sitosterol (5C.-stigmasta-5-en-3?-ol), US 7,906,710 B2 3 4 Stigmasterol-(stigmasta-5,-22-dien-33-ol), campesterol (5C.- cholesta-7.24-dienol and cholest-5,724-trenol accumulation ergosta-5-en-3?-ol), and cholesterol (5C.-cholesta-5-en-3? in yeast with ERG5 and ERG6 mutants. ol). These transformations are illustrated in FIG. 1. EP 480730“Plant-sterol accumulation and pest resistance Although sterols produced by plants, and particularly by increasing copy number of 3-hydroxy-3-methyl glutaryl higher (vascular) plants, can be grouped by the presence or 5 coenzyme-A reductase gene in tobacco, tomato and corn absence of one or more of several functionalities, plant sterols WO 9913086 “Human Delta 7-sterol reductase polypep are classified into two general groups herein; i.e., those con tide-useful for diagnosis or treatment of genetic defects e.g. taining a double bond between the carbon atoms at positions hereditary Smith-Lemli-Opitz syndrome' teaches making 5 and 6 (delta-5 or A5 sterols) and those not containing a and using the recombinant polypeptide with humans. double bond between the carbon atoms at positions 5 and 6 10 Chappellet al. U.S. Pat. No. 5,589,619 teaches that trans (non-delta-5 sterols). formation of higher plants with truncated HMG-CoA reduc Exemplary naturally-occurring delta-5 plant sterols are tase enhanced the production of squalene, cycloartenol and isoflucosterol, sitosterol, Stigmasterol, campesterol, choles certain sterols, particularly compounds having unsaturations terol, and dihydrobrassicasterol. Exemplary naturally occur at the 5-position. Several intermediate sterols as are shown in ring non-delta-5 plant sterols are cycloartenol, 24-methylene 15 FIG. 1 were also produced. It would be beneficial if the cycloartenol, cycloeucalenol, and obtusifoliol. The most production of sitosterol and Stigmasterol could be enhanced abundant sterols of vascular plants are campesterol, sito while lessening the accumulation of the intermediate sterols. sterol, and stigmasterol, all of which contain a double bond The present invention provides avenues for enhancing pro between the carbonatoms at positions 5 and 6 are classified as duction of sitosterol and stigmasterol and lessening the accu delta-5 sterols. mulation of the intermediate sterols. The HMG-CoA reductase enzymes of animals and yeasts Gonzalez et al. (Abstract of poster at Third Terpnet Meet are integral membrane glycoproteins of the endoplasmic ing of the European Network on Plant Isoprenoids, May reticulum. The intact enzyme comprises three regions: a cata 29-30, 1997, Poitiers, France) over-expressed the Arabidop lytic region containing the active site of the enzyme; a mem sis HMGR cDNA (hmg1 and himg2) and found sterol over brane binding region anchoring the enzyme to the endoplas 25 production with himgil only. They used two forms ofthehmg1 mic reticulum; and a linker region joining the catalytic and gene, a full-length form and a truncated form containing only membrane binding regions of the enzymes. The membrane the catalytic domain. HMGRs have three domains, an N-ter binding region occupies the amino-terminal (N-terminal) minal membrane spanning domain, a short linker domain, portion of the intact protein, whereas the catalytic region and a C-terminal catalytic domain. In this case the transgenic occupies the carboxy-terminal (C-terminal) portion of the 30 plants were also Arabidopsis. The difference between the protein, with the linker region constituting the remaining full-length and truncated forms was a greater accumulation of portion. M. E. Basson et al., Mol. Cell Biol., 8(9):3797-3808 pathway intermediates in the case of the truncated form. More (1988). importantly, the intermediates demonstrated as accumulating The activity of HMG-CoA reductase in animals and yeasts were cycloartenol, 24-methylenecycloartanol and obtusifo is known to be subject to feedback inhibition by sterols. Such 35 liol. feedback inhibition requires the presence of the membrane Finally, U.S. Pat. Nos. 5,365,017 and 5,306,862, both binding region of the enzyme. See, e.g., G. Gil et al. Cell, assigned to Amoco Corp., disclose a method for increasing 41:249-258 (1985); M. Bard and J. F. Downing, J. Gen. sterol accumulation in plants by increasing the copy number Microbiol., 124:415-420 (1981). of a gene having HMG-CoA reductase activity. These inven Given that mevalonate is the precursor for sterols and other 40 tions disclose a method usinghamster truncated HMGR that isoprenoids, it might be expected that increases in the amount consisted of the catalytic domain and the linker domain. or activity of HMG-CoA reductase would lead to increases in According to the claims the linker domain was essential for the accumulation of both sterols and other isoprenoids. activity. They also demonstrated a greater accumulation of In mutant strains of the yeast Saccharomyces cerevisiae (S. pathway intermediates Such as cycloartenol. cerevisiae) having abnormally high levels of HMG-CoA 45 reductase activity, the production of two sterols, 4, 14-dim BRIEF SUMMARY ethylzymosterol and 14-methylfucosterol is markedly increased above normal. Downing, et al., Biochem. Biophys. The present invention relates to transgenic plants and their Res. Comm., 94(3): 874-979 (1980). progeny having improved nutritional characteristics. More When HMG-CoA reductase activity was increased by illu 50 particularly, the present invention relates to transgenic plants mination in non-photosynthetic microorganisms, isoprenoid and their progeny, the storage organs (e.g. seed, fruit and (carotenoid), but not sterol (ergosterol), synthesis was vegetable parts) of which contain modified levels of steroid enhanced. Tada, et al., Plant and Cell Physiology, 23(4):615 compounds, such as (i) elevated levels of beneficial phytoster 621 (1982). ols (e.g., sitosterol), phytostanols (e.g., sitostanol), and esters WO 9703202 discloses a method for identifying agents 55 thereof, relative to an otherwise identical plant transformed modulating sterol biosynthesis using a yeast acetoacetyl CoA only with a truncated HMG-CoA reductase gene or a wild thiolase (ERG10) gene linked to a reporter system to evaluate type plant, and (ii) reduced levels of steroid pathway inter compounds, such as lovastatin and other HMG-CoA synthase mediate compounds (e.g. one or more of squalene, inhibitors, that affect cholesterol biosynthesis. cycloartenol, 24-methylene cycloartenol, obtusifoliol, Stig U.S. Pat. No. 5,668,001 teaches a recombinant avian 60 masta-7-enol and campesterol) in their storage organs relative HMG-CoA synthase preparation useful for evaluating drugs to an otherwise identical transgenic plant transformed only that inhibit cholesterol biosythesis. with a truncated HMG-CoA reductase gene. Nucleic acid JP 09 121863 discloses a plant with increased 3-hydroxy sequences encoding enzymes that affect the biosynthesis and 3-methylglutaryl coenzyme A reductase (HMGR) activity as accumulation of steroid compounds in plants (HMG-CoA a result of increasing the expression of a mutant protein 65 reductase and a steroid pathway enzyme), and methods for kinase gene that regulates expression of the HMGR gene. The using these sequences to produce Such transgenic plants, are increased HMGR activity increases squalene, Zymosterol, also provided. These methods comprise, for example, intro US 7,906,710 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 has 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 7,906,710 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 dase, and an Arabidopsis enzyme having nucleic acid simi These and other features, aspects, and advantages of the larity to an obtusifoliol C14C.-demethylase enzyme. Thus, the present invention will become better understood with regard invention contemplates an isolated DNA molecule having a to the following description, appended claims and accompa 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 similar 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 Hevea brasilien 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 spectinomycin transformed host cell as compared to an otherwise identical 50 and 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 reductase 1 (HMGR1) in sense orientation driven by the US 7,906,710 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 pea rbcs. 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 overexpressed (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 reductase 1 (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 E. for Agrobacterium mediated transformation of soybean, car 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 reduc origin of replication in E. coli: Spc/Str. coding region for TnT. tase1 (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 7,906,710 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 reductase1 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 reductase1 (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 E. tance to neomycin and kanamycin; NOS3': 3' termination end 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 reductase1 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 reductase1 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 E. of replication in E. coli: Spc/Str: coding region for TnTade 45 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: clNA 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 reductase1 (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 reductase 1 (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 7,906,710 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: clNA 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 Cyc1-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-cytochrome c 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 comparison 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 Cyc1-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 dimensional structure sis putative obtusifoliol C14C-demethylase; P-Sc.Gall: a of Pseudomonas mevalonii HMGR (Lawrence et al., 1995, promoter from Gall-galactokinase of Saccharomyces cerevi Science 268: 1758): boxed-HMGCoA binding domain, light siae to direct expression with galactose induction; Lac Z shade-NAD(H) binding domain, underlined consensus-do alpha: partial coding sequence for beta-d-galactosidase or 65 mains 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 t and US 7,906,710 B2 15 16 boldface, and are located at the C-terminal region of the egies it is possible to convert all of the squalene and other protein, adjacent to a highly conserved arginine, marked with intermediates to end sterols such as sitosterol, Stigmasterol tand boldface. Also indicated are the conserved Glu (E), Lys and campesterol. Thus, Sterol level in soybean oils can be (K), and Asp (D) residues, marked by E, K, and D, respec elevated from 0.3% up to 3.5%. Expression of the full-length tively. These residues are thought to be critical in catalysis, rubber HMGR in soybeans results in a sterol level increase up based on the crystal structure (Tabernero et al., 1999; PNAS to 2.7%. 96(13):7167-71). Appendices A through C show SEQID Nos: 1 through 3, Enhancement of 3-hydroxy-3-methylglutaryl-CoA reduc respectively. Appendices D through G show SEQID Nos 20 tase (HMG Co-A reductase) activity in certain cells results in thorough 23, respectively. 10 increased sterol biosynthesis. See, e.g. Chappell, U.S. Pat. No. 5,589,619. The present discovery further contemplates an DETAILED DESCRIPTION increase of steroid pathway end products Such as A5 sterols and their stanol counterparts with a decreased accumulation The following detailed description is provided to aid those of certain steroid pathway intermediates by also enhancing skilled in the art in practicing the present invention. Even So, 15 various specific steroid pathway enzyme activities, such that this detailed description should not be construed to unduly more of the steroid pathway intermediate compounds are limit the present invention as modifications and variations in converted to steroid pathway end products. the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or DNA sequences encoding squalene epoxidases are useful Scope of the present inventive discovery. for removal of squalene accumulation, genes encoding sterol All publications, patents, patent applications, databases methyl transferase I enzymes are useful for removal of and other references cited in this application are herein incor cycloartenol accumulation, genes encoding Sterol C4-dem porated by reference in their entirety as if each individual ethylase are useful for removal of 24-methylene cycloartenol publication, patent, patent application, database or other ref accumulation, genes encoding obtusifoliol C14C-demethy erence were specifically and individually indicated to be 25 lases are useful for removal of accumulation of obtusifoliol, incorporated by reference. genes encoding sterol C5-desaturases are useful for removal We have expressed the full-length forms of the rubber and of stigmasta-7-enol accumulation, and genes encoding sterol Arabidopsis HMGRS driven by seed-specific promoters in methyl transferase II enzymes are useful for the reduction of transgenic canola and Soybean. We have demonstrated Sterol 30 accumulated campesterol and concomitant increase of sito over-production up to 2-4 fold higher in seeds from these sterol. transgenic plants. We also demonstrated a higher accumula Levels of sitostanol and sitostanol esters can be elevated tion of pathway intermediates in Soybean than canola. These further by approximately 2- to 40-fold over the transgenic results were disclosed in PCT publication WO 00/61771. plants of the art having only added genes for HMG CoA However, we have expressed a truncated form of the Arabi 35 reductase by introducing additional genes encoding one or dopsis himgil without the linker and membrane spanning more of the following sterol pathway enzymes: a squalene domains in Arabidopsis and Soybean. The results in Arabi epoxidase, a sterol methyltransferase I, a sterol C4-demethy dopsis were similar to that demonstrated by Gonzalez et al. lase, an obtusifoliol C14C-demethylase, a sterol C5-desatu (1997) and we compared the sterol profiles of our transgenic rase, a sterol methyl transferase II. plants with those produced by Gonzalez et al., using our 40 As used herein, the term "structural coding sequence' methods to show they are comparable. We found the same means a DNA sequence which encodes for a peptide, types of pathway intermediates accumulating. However, in polypeptide, or protein which may be made by a cell follow Soybean seeds we have demonstrated the accumulation of ing transcription of the DNA to mRNA, followed by transla squalene to a very high level (~3 mg/g seed which is around 45 tion to the desired peptide, polypeptide, or protein. 100-fold higher than in nontransgenic controls). This is an The term “sterol as applied to plants refers to any chiral unexpected result not disclosed or Suggested in the prior art. tetracyclic isopentenoid which may be formed by cyclization Squalene is a precursor for sterols and in Soybean it appears of squalene oxide through the transition state possessing Ste that there is a “bottleneck” in the further conversion of this reochemistry similar to the trans-syn-trans-anti-trans-anti precursor to sterols. Thus, it appears that there could be addi 50 configuration, i.e., protosteroid cation, and which retains a tional ways of over-producing sterols in soybean to levels polar group at C-3 (hydroxyl or keto), an all-trans-anti Stere 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. 55 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 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): 60 As used herein, the term “sterol refers to unsaturated addition of oxygen to squalene which is a 30-carbon linear hydroxyl group-containing derivatives of a fused, reduced isoprenoid chain thus allowing for cyclization to form ring system, cyclopenta C-phenanthrene, comprising three cycloartenol. Additional enzymes downstream that can be fused cyclohexane rings (A, B and C) in a phenanthrene also be manipulated are sterol methyltransferase 1, C-4 dem arrangement, and a terminal cyclopentane ring (D). The ethylase, C-14 demethylase, Sterol methyltransferase 2, and 65 exemplary steroid below (FORMULAII) illustrates the num C-5 desaturase that would all deplete other pathway interme bering system employed herein in describing the location of diates shown to accumulate in Soybeans. By using Such strat groups and Substituents. US 7,906,710 B2 18 monomers) form of nucleotides of any length, either ribo
(II) 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 and RNA. It also includes known types of modifications, for example, labels, methylation, "caps'. Substitution of one or 10 more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phos photriesters, phosphoamidates, carbamates, etc.), those con Sterols may or may not contain a C-5 to C-6 double bond, taining pendant moieties, such as, for example, proteins (in as this is a feature introduced late in the biosynthetic pathway 15 cluding for e.g., nucleases, toxins, antibodies, signal (note Scheme 1, below). Sterols containa Cs-Co side chain at peptides, poly-L-lysine, etc.), those with intercalators (e.g., the C-17 position, as shown above. acridine, psoralen, etc.), those containing chelators (e.g., met The term “phytosterol, which applies to sterols found als, radioactive metals, boron, oxidative metals, etc.), those uniquely in plants, refers to a sterol containing a C-5, and in containing alkylators, those with modified linkages (e.g., some cases a C-22, double bond. Phytosterols are further alpha anomeric nucleic acids, etc.), as well as unmodified characterized by alkylation of the C-17 side-chain with a forms of the polynucleotide. Polynucleotides include both methyl or ethyl substituent at the C-24 position. Major phy sense and antisense Strands. tosterols include, but are not limited to, Sitosterol, Stigmas The alternative nucleotide sequences described above are terol, campesterol, brassicasterol, etc. Cholesterol, which considered to possess Substantially similar enzymatic activity lacks a C-24 methyl or ethyl side chain, is found in plants but 25 to that of the polypeptide-encoding polynucleotide sequences is not unique thereto, and is not a “phytosterol of the present invention if they encode polypeptides having “Phytostanols are saturated forms of phytosterols wherein enzymatic activity differing from that of any of the polypep the C-5 and, when present, C-22 double bond(s) is(are) tides encoded by the polynucleotide sequences of the present reduced, and include, but are not limited to, sitostanol, invention by about 30% or less, preferably by about 20% or campestanol, and 22-dihydrobrassicastanol. 30 less, and more preferably by about 10% or less when assayed “Phytosterol esters' and “phytostanol esters' are further by Standard enzymatic assays. characterized by the presence of a fatty acid or phenolic acid As used herein “effective amount” is intended to qualify moiety rather than a hydroxyl group at the C-3 position. the amount of an agent which will achieve the goal of a The term “steroid compounds' includes sterols, phytoster lessening in the severity and/or the frequency of incidence of ols, phytosterol esters, phytostanols, and phytostanol esters. 35 a disease condition or disorder, over no treatment. The term “phytosterol compound” refers to at least one The phrase “steroid pathway products” refers to the prod phytosterol, at least one phytosterol ester, or a mixture ucts of steroid biosynthesis produced by the action of one or thereof. 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 40 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 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.” 45 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-methylglutaryl-Coenzyme A reductase exclusive activity of these promoters in these organs or 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 50 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. 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 55 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 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 60 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 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. 65 Sources of nucleic acid sequences encoding these enzymes. As used herein “polynucleotide' and "oligonucleotide' are Various steps in the steroid compound biosynthetic path used interchangeably and refer to a polymeric (2 or more way in plants are shown in Scheme 1, below. The numbers US 7,906,710 B2 19 20 over the arrows refer to plant sterol compound biosynthetic acetoacetyl CoA to yield HMGCoA. HMGCoA synthase has pathway enzymes and genes as indicated in Table 1. been purified from yeast. A plant HMGCoA synthase cDNA has also been isolated from Arabidopsis thaliana (Montamat TABLE 1. et al., Gene 167: 197-201 (1995)). 5 HMGCoA reductase, also referred to as 3-hydroxy-3-me Plant Sterol Compound Pathway Enzymes and Genes thylglutaryl-coenzyme A (EC 1.1.1.34), catalyzes the reduc Step in GenBank tive conversion of HMGCoA to mevalonic acid (MVA). This Enzyme Pathway Gene ID reaction is reported to play a role in controlling plant iso Acetoacetyl-CoA thiolase 1 X78116 prenoid biosynthesis (Gray, Adv. Bot. Res. 14:25-91 (1987); HMG-CoA synthase 2 X83882 10 Bachet al., Lipids 26: 637-648 (1991); Stermeret al., J. Lipid HMG-CoA reductase 3 X15032 Res. 35: 1133-1140 (1994). Plant HMGCoA reductase genes L19262 are often encoded by multigene families. The number of Mevalonate kinase 4 X77793 genes comprising each multigene family varies, depending Phosphomevalonate kinase 5 Not available on the species, ranging from two in Arabidopsis thaliana to at Mevalonate pyrophosphate 6 Y14325 15 least seven in potato. Overexpression of plant HMGCoA decarboxylase reductase genes in transgenic tobacco plants has been Isopentenyl diphosphate 7 U492.59 reported to result in the overproduction of phytosterols isomerase U47324 Farnesyl pyrophosphate 8 X75789 (Schaller et al., Plant Physiol. 109: 761-770 (1995)). synthase Mevalonate kinase (EC 2.7.1.36) catalyzes the phosphory Squalene synthase 9 AFOO4S60 lation of mevalonate to produce mevalonate 5-phosphate. It Squalene epoxidase 10 ABO16883 Squalene cyclase 11 U87266 has been reported that mevalonate kinase plays a role in the Sterol C-24 12, 18 U714OO control of isoprenoid biosynthesis (Lalitha et al., Indian. J. methyltransferase Biochem. Biophys. 23:249-253 (1986)). A mevalonate kinase Sterol C-4 demethylase 13, 19 Not gene from Arabidopsis thaliana has been cloned (GeneBank available 25 Cycloeucalenol- 14 Not accession number X77793; Riou et al., Gene 148: 293-297 obtusifoliol isomerase available (1994)). Sterol C-14 demethylase 15 U74319 Phosphomevalonate kinase (EC 2.7.4.2) (MVAP kinase) is Sterol C-14 reductase 16 PCTWO an enzyme associated with isoprene and ergosterol biosyn 97,48793 Sterol C-8 isomerase 17 AFO3O357 thesis that converts mevalonate-5-phosphate to mevalonate Sterol C-5 desaturase 2O X90454 30 5-pyrophosphate utilizing ATP (Tsay et al., Mol. Cell. Biol. Sterol C-7 reductase 21 U49398 11: 620-631 (1991)). Sterol C-24 isomerase 22 Klahre Mevalonate pyrophosphate decarboxylase (“MVAPP et al. decarboxylase') (EC 4.1.1.33) catalyzes the conversion of (1998) Plant Cell mevalonate pyrophosphate to isopentenyl diphosphate 10: 1677-1690 35 (“IPP). The reaction is reported to be a decarboxylation/ Sterol C-24 reductase 23 Same dehydration reaction which hydrolyzes ATP and requires as 22 Sterol C-22 desaturase 24 Not Mg". A cDNA encoding Arabidopsis thaliana MVAPP available decarboxylase has been isolated (Toth et al., J. Biol. Chem. Sterol C-5 reductase 25 WO 271: 7895-7898 (1996)). An isolated Arabidopsis thaliana OO61771 40 MVAPP decarboxylase gene was reported to be able to complement the yeast MVAPP decarboxylase. The plant sterol compound biosynthesis pathway has two Isopentenyl diphosphate isomerase (“IPP:DMAPP) (EC distinct components. The early pathway reactions, leading 5.3.3.2) catalyzes the formation of dimethylallyl pyrophos from acetyl-CoA to squalene via mevalonic acid, are common phate (DMAPP) from isopentenyl pyrophosphate (IPP). to other isoprenoids. The later pathway reactions, leading 45 Plant IPP:DMAPP isomerase gene sequences have been from squalene to the major plant sterol compounds such as 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 50 Farnesyl pyrophosphate synthase (EC 2.5.1.1) is a prenyl and Parish, Eds. CRC Press, 85-99 (1997); Newman and 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)). 55 DMAPP with IPP to yield geranyl pyrophosphate (“GPP”). Acetoacetyl CoA thiolase (EC 2.3.1.9) catalyzes the first 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 60 ways leading to the synthesis of quinones or sesquiterpenes. been isolated by functional complementation in Saccharomy 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)). 65 phate. The enzyme is Subject to sterol demand regulation HMGCoA synthase (EC 4.1.3.5) catalyzes the production 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 US 7,906,710 B2 21 22 effect on the incorporation of FPP into sterol and other com thereby forming a A-sterol (Parks et al., Lipids 30: 227-230 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 10 sterol biosynthesis of animals, fungi, and plants. flavoprotein. The A-desaturase is reported to be a mono The later pathway of sterol compound biosynthetic steps oxygenase that utilizes electrons derived from a reduced pyri starts with the cyclization of squalene epoxide and ends with dine nucleotide via cytochrome bs. the formation of A5-24-alkyl sterols in plants. Sterol C-7 reductase catalyzes the reduction of a A-double 2,3-oxidosqualene cycloartenol cyclase (EC 5.4.99.8) 15 bond in A7-sterols to generate the corresponding A-sterol. (also called cycloartenol synthase) is the first step in the sterol It has been reported that the mechanism involves, like many compound pathway that is plant-specific. The cyclization of other sterol enzymes, the formation of a carbocationic inter 2.3-oxidosqualene leads to lanosterol in animals and fungi, mediate via electrophilic “attack” by a proton. while in plants the product is cycloartenol. Cycloartenol con Sterol C-24(28) isomerase catalyzes the reduction of a tains a 9, 19-cyclopropyl ring. The cyclization is reported to A*-A, a conversion that modifies the side chain. The proceed from the epoxy end in a chair-boat-chair-boat product is a A-24-alkyl sterol. Sterol C-24 reductase sequence that is mediated by a transient C-20 carbocationic catalyzes the reduction of the D'' double bond at C-24, intermediate. which produces sitosterol. Recently, Klahre et al. (1998) S-adenosyl-L-methionine:sterol C-24 methyl transferase Plant Cell 10:1677-1690) discovered that both the isomeriza (“SMT1') (EC 2.1.1.41) catalyzes the transfer of a methyl 25 tion and reduction steps are catalyzed by an enzyme coded by group from S-adenosyl-L-methionine to the C-24 center of the same gene, i.e., DIM/DWF1. the sterol side chain (Nes et al. (1991).J. Biol. Chem. 266(23): Sterol C-22 desaturase (EC 2.7.3.9) catalyzes the forma 15202-15212). This is the first of two methyl transfer reac tion of a double bond at C-22 on the side chain. This forma tions that have been reported to be an obligatory and rate tion of a double bond at C-22 on the side chain marks the end limiting step of the sterol compound-producing pathway in 30 of the sterol compound biosynthetic pathway, and results in plants. The second methyltransfer reaction occurs later in the the formation of stigmasterol (Benveniste (1986) Annu. Rev. pathway after the A7 isomerase. The enzyme responsible for Plant Physiol. 37:275-308). The C-22 desaturase in yeast, the second methyl transfer reaction is named SMTII (Bou which is the reported final step in the biosynthesis of ergos vier-Nave, P. et al., (1997) Eur: J. Biochem., 246:518-529). terol in that organism, requires NADPH and molecular oxy An isoform, SMTII, catalyzes the conversion of cycloartenol 35 gen. In addition, the reaction is also reported to involve a to a A-24-alkyl sterol, cyclosadol (Guo et al. (1996) cytochrome P450 that is distinct from a cytochrome P450 Tetrahed. Lett. 37(38):6823-6826). participating in demethylation reactions (Lees et al. (1995) Sterol C-4 demethylase catalyzes the first of several dem Lipids 30: 221-226). ethylation reactions, which results in the removal of the two Phytosterols are biogenetic precursors of brassinosteroids, methyl groups at C-4. While in animals and fungi the removal 40 steroid alkaloids, steroid sapogenins, ecdysteroids, and Ste of the two C-4 methyl groups occurs consecutively, in plants roid hormones. This precursor role of phytosterols is often it has been reported that there are other steps between the first described as a “metabolic function. A common transforma and second C-4 demethylations. The C-4 demethylation is tion of free sterols in tissues of Vascular plants is the conju catalyzed by a complex of microsomal enzymes consisting of gation at the 3-hydroxy group of sterols with long-chain fatty a monooxygenase, an NAD-dependent sterol 4-decarboxy 45 acids to form steryl esters, or with a Sugar, usually with a lase, and an NADPH-dependent 3-ketosteroid reductase. single molecule of B-D-glucose, to form steryl glycosides. Cycloeucalenol-obtusifoliol isomerase (“COI) catalyzes Some of the sterylglycosides are additionally esterified, at the the opening of the cyclopropyl ring at C-9. The opening of the 6-hydroxy group of the Sugar moiety, with long-chain fatty cyclopropyl ring at C-9 creates a double bond at C-8. acids to form acylated steryl glycosides. Sterol C-14 demethylase catalyzes demethylation at C-14, 50 The existence of several enzymes that are specifically asso which removes the methyl group at C-14 and creates a double ciated with the synthesis and breakdown of conjugated Sterols bond at that position. In both fungi and animals, this is the first has been reported (Wojciechowski, Physiology and Biochem step in the sterol synthesis pathway. Sterol 14-demethylation istry of Sterols, eds. Patterson, Nes, AOCS Press, 361 (1991)). is mediated by a cytochrome P-450 complex. Enzymes involved in this process include: UDPGlc:Sterol Sterol C-14 reductase catalyzes a C-14 demethylation that 55 glucosyltransferase, phospho(galacto)glyceride sterylgluco results in the formation of a double bond at C-14 (Ellis et al., side acyltransferase, and sterylglycoside and sterylester Gen. Microbiol. 137:2627-2630 (1991)). This double bond is hydrolases. removed by a A' reductase. The normal substrate is 4.C.-me UDPGlc:sterol glucosyltransferase (EC 2.4.1.173) cata thyl-8, 14.24 (24)-trien-3 (3-ol. NADPH is the normal reduc lyzes glucosylation of phytosterols by glucose transfer from tant. 60 UDP-glucose (“UDPGl'). The formation of sterylglycosides Sterol C-8 isomerase catalyzes a reaction that involves can be measured using UDP-''Clglucose as the substrate. further modification of the tetracyclic rings or the side chain Despite certain differences in their specificity patterns, all (Duratti et al., Biochem. Pharmacol. 34: 2765-2777 (1985)). reported UDPGlc:sterol glucosyltransferases preferentially The kinetics of the sterol isomerase-catalyzed reaction favor glycosylate only sterols or sterol-like molecules that contain a A 6A isomerase reaction that produces a A' group. 65 a C-3 hydroxy group, a B-configuration, and which exhibit a Sterol C-5 desaturase catalyzes the insertion of the planar ring. It has been reported that UDPGlc:sterol gluco A-double bond that normally occurs at the A-sterol level, Syltransferases are localized in the microsomes. US 7,906,710 B2 23 24 Phospho(galacto)glyceride steryl glucoside acyltrans of a corresponding plant comprising no introduced DNA ferase catalyzes the formation of acylated steryl glycosides encoding a polypeptide or protein that affects the biosynthe 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. 5 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 10 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, 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 15 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 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 25 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 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 30 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 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 35 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 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 40 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 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 45 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 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 50 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 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 55 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, 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 60 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. 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, 65 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 are specifically discussed). US 7,906,710 B2 25 26 A non-transgenic plant that serves as the Source of the plant formed) to native (untransformed) showing transformation. cell that is transformed, i.e. the precursor cell, is referred to Higher relative activity ratios such as about 15:1 have also herein as a “native, non-transgenic' plant. The native, non been observed. transgenic plant is of the same strain as the formed transgenic Sterol accumulation can also be used to distinguish plant. between native, non-transgenic and transgenic plants. A Sterol production in a transgenic plant of the present inven transgenic plant has at least about twice the total sterol con tion is increased by increasing the activity of the enzyme tent as a native, non-transgenic plant where a single added HMG-CoA reductase, which enzyme catalyzes the conver gene is present. Greater differences up to about forty-fold sion of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG have also been observed. CoA) to mevalonate and the activity of at least one other 10 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 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 15 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 of campesterol, campestanol, and their esters can be reduced enhances the activity of that enzyme. as these enzymes shunt Sterol intermediates away from The amount of the expressed gene is increased by trans campesterol, and toward sitosterol and sitostanol. forming a plant cell with a recombinant DNA molecule com III. DNA Encoding Useful Polypeptides prising a vector operatively linked to a DNA segment that The present invention contemplates a recombinant con encodes a polypeptide having HMG-CoA reductase activity, structor a recombinant vector that contains a DNA sequence and a promoter Suitable for driving the expression of that encoding a polypeptide exhibiting 3-hydroxy-3-methylglu polypeptide in that plant cell, and culturing the transformed taryl-Coenzyme A (HMG-CoA) reductase activity and a plant cell into a transgenic plant. Such a polypeptide includes 25 DNA sequence encoding a polypeptide exhibiting the activity intact as well as a catalytically active, truncated HMG-CoA of a steroid pathway enzyme. Each polypeptide-encoding reductase proteins. DNA sequence is operably linked in the 5' to 3" direction Thus, a transformed plant cell and a transgenic plant have independent of the other sequence. Each DNA sequence in one or more added, exogenous genes that encode a polypep the 5' to 3' direction comprises a promoter, then the DNA tide having HMG-CoA reductase activity and at least one 30 sequence encoding the polypeptide then a transcription ter other steroid pathway enzyme activity relative to a native, mination signal sequence. The steroid pathway enzyme is a non-transgenic plant or untransformed plant cell of the same squalene epoxidase enzyme, a sterol methyl transferase I type. As such, a transformed plant cell or transgenic plant can enzyme, a sterol C4-demethylase enzyme, a obtusifoliol be distinguished from an untransformed plant cell or native, C14C-demethylase enzyme, a sterol C5-desaturase enzyme, nontransgenic plant by standard technology Such as agarose 35 or a sterol methyl transferase II enzyme. It is contemplated separation of DNA fragments or mRNAs followed by transfer that HMG-CoA reductase and steroid pathway enzyme and appropriate blotting with DNA or RNA, e.g., Southern or activities come from a mutant or truncated form of those Northern blotting, or by use of polymerase chain reaction enzymes, such as a truncated HMG-CoA reductase lacking technology, as are well known. Relative HMG-CoA reduc the transmembrane region while retaining a functional cata tase activity of the transformed cell or transgenic plant with 40 lytic domain. Several HMG CoA reductase sequences are untransformed cells and native, non-transgenic plants or cell known in the art. An amino acid alignment for these is shown cultures therefrom can also be compared, with a relative in FIG. 32. The sources of the sequences used in building the activity for that enzyme of about 1.5:1 for transgenic (trans multiple alignment are listed in Table 5. TABLE 5 Sources of Sequences Used In Building The Multiple Alignment methanobac Swissprot: Begin: End: 397 O26.662 methanobacterium thermoautotrophicum. 3 hmdh metth hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). December 1998 methanococ Swissprot: Begin: End: 405 Q58116 methanococcus jannaschii. 3-hydroxy-3- hmdh meta methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). July 1998 halobacter Swissprot: Begin: End: 403 Q59468 halobacterium volcanii (haloferax hmdh halvo volcanii). 3-hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). July 1998 sulfolobus Swissprot: Begin: End: 409 O08424 sulfolobus solfatanicus. 3-hydroxy-3- hmdh Sulso methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). December 1998 yeast2 gp pln1: Begin: End: 1045 M22255 Saccharomyces cerevisiae Yeast HMG-CoA ySchmgcr2 1 reductase (HGM2) gene, complete cols; 3-hydroxy 3-methyl glutaryl coenzyme A reductase. April 1993 yeast1 gp pln1: Begin: End: 1054 M22002 Saccharomyces cerevisiae Yeast HMG-CoA ySchmgcr1 1 reductase (HGM1) gene, complete cols; 3-hydroxy 3-methyl-glutaryl coenzyme A reductase. April 1993 phycomyces Swissprot: Begin: End: 105 Q12649 phycomyces blakesleeanus. 3-hydroxy-3- hmdh phybl methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase) (fragment). November 1997 US 7,906,710 B2 27 28 TABLE 5-continued Sources of Sequences Used In Building The Multiple Alignment fitsarium Swissprot: Begin: : 976 Q12577 fissarium moniiforme (gibberella hmdh fuSmo filiikiiroi). 3-hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). November 1997 candida gp pln1: Begin: : 934 AB012603 Candida utilis Candida utilis HMG mRNA abO12603 1 or HMG-CoA reductase, complete cols. July 1998 dictyoste2 Swissprot: Begin: : 481 P34136 dictyostelium discoideum (slime mold). 3 hmd2 dicci hydroxy-3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg-coa reductase 2) (fragment). 35735 wheat1 pir2: Begin: : 150 hydroxymethylglutaryl-CoA reductase (NADPH) (EC pq0761 .1.1.34) (HMGR 10) - wheat (fragment) rice Swissprot: Begin: : 509 P48019 oryza sativa (rice). 3-hydroxy-3- hmdh orysa methylglutaryl-coenzyme a reductase (ec .1.1.34) (hmg-coa reductase) (fragment). February 1996 COil sp plant: Begin: : 579 O24594 zea mays (maize). 3-hydroxy 3 o24594 methylglutaryl coenzyme a reductase (ec .1.1.88). May 1999 wheat bir2: Begin: : 150 hydroxymethylglutaryl-CoA reductase (NADPH) (EC paO763 .1.1.34) (HMGR23) - wheat (fragment) wheat2 bir2: Begin: : 150 hydroxymethylglutaryl-CoA reductase (NADPH) (EC paO762 .1.1.34) (HMGR 18) - wheat (fragment) Soybean gmtX6: Begin: 101 : 259 rom proprietary soy sequence database 30820 1rS9f1 rubbertre3 Swissprot: Begin: : 586 Q00583 hevea brasiliensis (para rubber tree). 3 hmd3 hewbr hydroxy-3-methylglutaryl-coenzyme a reductase 3 (ec 1.1.1.34) (hmg-coa reductase 3). July 1998 rosyperiwi Swissprot: Begin: Q03163 catharanthus roseus (rosy periwinkle) hmdh catro (madagascar periwinkle). 3-hydroxy-3- methylglutaryl-coenzyme a reductase (ec .1.1.34) (hmg-coa reductase). July 1998 tomato Swissprot: Begin: : 602 P48022 lycopersicon esculentum (tomato). 3 hmd2 lyces hydroxy-3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg-coa reductase 2). July 1998 woodtobacc Swissprot: Begin: : 604 Q01559 nicotiana Sylvestris (wood tobacco). 3 hmdh nicsy hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). July 1998 potato gp pln1: Begin: : 596 LO1400 Soianum tuberosum Potato pothmgiri 1 hydroxymethylglutaryl coenzyme A reductase (hmgr) mRNA, complete cols; putative. April 1996 radish sp plant: Begin: : 573 Q43826 raphanus sativus (radish). q43826 hydroxymethylglutaryl-coa reductase (ec .1.1.34) (hydroxymethylglutaryl-coa reductase (nadph)) (3-hydroxy-3-methylglutaryl-coenzyme a red arabadopsis1 gp pln1: Begin: : 592 L19261 Arabidopsis thaliana Arabidopsis thaliana athhmgcoar 1 HMG-cCA reductase gene, complete cods. April 1994 cucumismel gp pln1: Begin: : 587 AB021862 Cucumis meio Cucumis meio mRNA for HMG abO21862. 1 CoA reductase, complete cols; putative. January 1999 rubbertre2 Swissprot: Begin: :210 P29058 hevea brasiliensis (para rubber tree). 3 hmd2 hewbr hydroxy-3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg-coa reductase 2) (fragment). 35735 rubbertre1 Swissprot: Begin: : 575 P29057 hevea brasiliensis (para rubber tree). 3 hmdli hewbr hydroxy-3-methylglutaryl-coenzyme a reductase 1 (ec 1.1.1.34) (hmg-coa reductase 1). July 1998 camptothec Swissprot: Begin: : 593 P48021 camptotheca acuminata. 3-hydroxy-3- hmdh camac methylglutaryl-coenzyme a reductase (ec .1.1.34) (hmg-coa reductase). November 1997 arabadops2 Swissprot: Begin: : S62 P43256 arabidopsis thaliana (mouse-ear cress). hmd2 arath 3-hydroxy-3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg-coa reductase 2) (hmgr2). uly 1998 chineseham Swissprot: Begin: : 887 P00347 cricetulus griseus (chinese hamster). 3 hmdh crigr hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). November 1997 chineseha2 gp rod: Begin: : 887 L00183 Cricetulus sp. Hamster 3-hydroxy-3- cruhmg14 1 methylglutaryl coenzyme A (HMG CoA) reductase gene, exons 19 and 20:3-hydroxy-3- methylglutaryl coenzyme A (HMG CoA). April 1993 Syrian hamst gp rod: Begin: : 887 M12705 Mesocricetus auratus Syrian hamster 3 hamhmgcob 1 hydroxy-3-methylglutaral coenzyme A reductase (HMG-CoA reductase) mRNA, complete cols; 3 hydroxy-3-methylglutaral coenzyme A red rat Swissprot: Begin: : 887 P51639 rattus norvegicus (rat). 3-hydroxy-3- hmdh rat methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase). December 1998 US 7,906,710 B2 29 30 TABLE 5-continued Sources of Sequences Used In Building The Multiple Alignment rabbit Swissprot: Begin: End: 888 Q295 2 Oryctoliagi is clinicatius (rabbit). 3 hmdh rabit hydroxy-3-methylglutaryl-coenzyme a reductase (ec 1. .1.34) (hmg-coa reductase). July 1999 human gp pri2: Begin: End: 888 M11058 Homo sapiens Human 3-hydroxy-3- humhmgcoa. 1 methylglutaryl coenzyme A reductase mRNA, complete cols; 3-hydroxy-3-methylglutaryl coenzyme A reductase. November 1994 OUSE gp rod: Begin: End: 224 M62766 Mus musculius Mouse HMG-CoA reductase mushmgcoa 1 mRNA, 3' end. April 993 avenopus Swissprot: Begin: End: 883 P2O7 5 xenopus laevis (african clawed frog). 3 hmdh Xenla hydroxy-3-methylg utaryl-coenzyme a reductase (ec 1. .1.34) (hmg-coa reductase). November 1997 seaurchin Swissprot: Begin: End: 932 P16393 Strongylocentrottis purpuratus (purple seaurchin). hmdn Surpu 3-hydroxy-3-methy glutaryl-coenzyme a reductase (ec .1.1.34) (hmg-coa reductase). November 199 cockroach Swissprot: Begin: End: 856 P54960 blatteila germanica (german cockroach). hmdh blage 3-hydroxy-3-methy glutaryl-coenzyme a reductase (ec 1 .1.1.34) (hmg-coa reductase). November 1997 drosophila Swissprot: Begin: End: 916 P14773 drosophila melanogaster (fruit fly). 3 hmdh drome hydroxy-3-methylg utaryl-coenzyme a reductase (ec 1. .1.34) (hmg-coa reductase). December 1998 dictyoste1 Swissprot: Begin: End: SS2 P34135 dictyostelium discoideum (slime mold). 3 hmdli dicci hydroxy-3-methylg utaryl-coenzyme a reductase 1 (ec 1. .1.34) (hmg-coa reductase 1). November 1997 Schistosom Swissprot: Begin: End: 948 P16237 schistosoma mansoni (blood fluke). 3 hmdh Schma hydroxy-3-methylg utaryl-coenzyme a reductase (ec 1. .1.34) (hmg-coa reductase). July 1998 archaeoglo Swissprot: Begin: End: 436 O28538 archaeoglobus fulgidus. 3-hydroxy-3- hmdh arcfu methylglutary -coenzyme a reductase (ec .1.1.34) (hmg-coa reductase). December 1998 pseudomonas gp bct1: Begin: End: 428 M24015 Pseudomonas mevaioni P. mevalonii HMG-CoA psehmgcoa. 1 reductase (mvaA) gene, complete cols; HMG-CoA reductase (EC 1.1.1.88). April 1993
These sequences, and their truncated counterparts, are use present invention contemplates allelic variants of structural ful in the present invention. Examples of such preferred HMG 35 genes encoding a polypeptide having HMG-CoA reductase CoA reductases include the truncated rubber and Arabidopsis activity. HMG CoA reductases disclosed herein. The previously described DNA segments are noted as hav Other enzyme-encoding DNAs can be introduced into ing a minimal length, as well as total overall length. That plants to elevate even further the levels of desirable A5 sterols minimal length defines the length of a DNA segment having and their reduced Stanol counterparts as well as other phy 40 a sequence that encodes a particular polypeptide having tosterols and tocopherols. Thus, the DNA sequences contem HMG-CoA reductase activity. As is well known in the art, as plated for use in the present invention, which can be used long as the required DNA sequence is present (including start alone or in various combinations as discussed below, include, and stop signals), additional base pairs can be present at either but are not limited to, those encoding the following enzymes: end of the segment and that segment can still be utilized to 3-hydroxysteroid oxidases; steroid 5-reductases; sterol meth 45 express the protein. This, of course, presumes the absence in yltransferases; Sterol acyltransferases; and S-adenosylme the segment of an operatively linked DNA sequence that thionine-dependent C.-tocopherol methyltransferases. represses expression, expresses a further product that con In each case, the sequences encoding these enzymes can Sumes the enzyme desired to be expressed, expresses a prod comprise an expression cassette comprising, operably linked uct other than the desired enzyme or otherwise interferes with in the 5' to 3'direction, a seed-specific promoter, the enzyme 50 the structural gene of the DNA segment. coding sequence, and a transcriptional termination signal Thus, as long as the DNA segment is free of such interfer sequence functional in a plant cell such that the enzyme is ing DNA sequences, a DNA segment of the invention can be successfully expressed. For use in the methods disclosed up to 15,000 base pairs in length. The maximum size of a herein, the recombinant constructs or expression cassettes recombinant DNA molecule, particularly a plant integrating can be incorporated in a vector, for example a plant expres 55 vector, is governed mostly by convenience and the vector size sion vector. Such vectors can be transformed into host cells that can be accommodated by a host cell, once all of the Such as bacterial cells, for example during the preparation or minimal DNA sequences required for replication and expres modification of the recombinant constructs, and plant cells. sion, when desired, are present. Minimal vector sizes are well Thus, the invention encompasses plants and seeds comprising known. Such transformed plant cells. 60 Also encompassed by the present invention are nucleotide It will be apparent to those of skill in the art that the nucleic sequences biologically functionally equivalent to those dis acid sequences set forth herein, either explicitly, as in the case closed herein, that encode conservative amino acid changes of the sequences set forth above, or implicitly with respect to within the amino acid sequences of the presently disclosed nucleic acid sequences generally known and not present enzymes, producing 'silent changes therein. Such nucle herein, can be modified due to the built-in redundancy of the 65 otide sequences contain corresponding base Substitutions genetic code and noncritical areas of the polypeptide that are based upon the genetic code compared to the nucleotide Subject to modification and alteration. In this regard, the sequences encoding the presently disclosed enzymes. Substi US 7,906,710 B2 31 32 tutes for an amino acid within the enzyme sequences dis The disclosures of Chappell, et al., U.S. Pat. No. 5,349,126, closed herein is selected from other members of the class to are incorporated in full herein by reference. The mammalian which the naturally occurring amino acid belongs. Amino genome contains a single gene encoding HMG-CoA reduc acids can be divided into the following four groups: (1) acidic tase. The nucleotide sequence of the hamster and human gene amino acids; (2) basic amino acids; (3) neutral polar amino 5 for HMG-CoA reductase have been described in Chappellet acids; and (4) neutral non-polar amino acids. Representative al. A composite nucleotide sequence of DNA corresponds to amino acids within these various groups include, but are not the mRNA SEQID NO:1 of Chappell et al., as well as the limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) derived amino acid residue sequence SEQID NO:2 of Chap amino acids such as arginine, histidine, and lysine; (3) neutral 10 pell et al., for hamster HMG-CoA reductase is provided in polaramino acids such as glycine, serine, threonine, cysteine, FIG. 2 of Chappell et al, reprinted from Chin et al., Nature, cystine, tyrosine, asparagine, and glutamine; and (4) neutral 308:613 (1984). The composite nucleotide sequence of FIG. nonpolar (hydrophobic) amino acids Such as alanine, leucine, 2, SEQID NO:1 of Chappell et al., comprising about 4768 isoleucine, Valine, proline, phenylalanine, tryptophan, and base pairs, includes the nucleotide sequence encoding the methionine. 15 intact hamster HMG-CoA reductase enzyme. 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, 20 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 25 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 30 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 35 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- 40 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 amino acid residues and comprises about fifty percent of the 45 by base pairs from about nucleotide position 1180 to about NH2-terminal portion of intact HMG-CoA reductase nucleotideposition 1283 or from about position 1180 to about enzyme. position 1540, respectively of SEQID NO:1 of Chappellet al. The linker region connects the catalytic and membrane The structural gene encoding the linker region is operatively binding regions, and constitutes the remaining about ten per linked to the structural gene encoding the catalytic region. cent of the intact enzyme. 50 In one particularly preferred embodiment, a structural gene As discussed in greater detail below, only the catalytic encoding a catalytically active, truncated HMG-CoA reduc region of HMG-CoA reductase is needed herein to provide tase enzyme can optionally contain base pairs encoding a the desired enzyme activity. Thus, an exogenous structural Small portion of the membrane region of the enzyme. gene that encodes a polypeptide corresponding to that cata A structural gene encoding a polypeptide comprising a lytic region is the minimal HMG Co. A reductase gene 55 catalytically active, truncated or intact HMG-CoA reductase required for transforming plant cells in addition to one of the enzyme from other organisms such as yeast can also be used steroid pathway enzymes discussed below. The present in accordance with the present invention. invention therefore contemplates use of both intact and trun Yeast cells contain two genes encoding HMG-CoA reduc cated structural genes that encode a polypeptide having tase. The two yeast genes, designated HMG1 and HMG2. HMG-CoA reductase activity. 60 encode two distinct forms of HMG-CoA reductase, desig A structural gene encoding a polypeptide having HMG nated HMG-CoA reductase 1 SEQID NO:3 of Chappellet al. CoA reductase activity can be obtained or constructed from a are presented in FIG. 3 of Chappell et al., are taken from variety of sources and by a variety of methodologies. See, e.g. Basson et al. Mol. Cell. Biol., 8(9):3797 (1988). The nucle Carlson et al., Cell, 28:145 (1982); Rine et al., Proc. Natl. otide base sequences of HMG2 SEQID NO:5 of Chappellet Acad. Sci. USA, 80:6750 (1983). Exemplary of such struc- 65 al. as well as the amino acid residue sequence of HMG-CoA tural genes are the mammalian and yeast genes encoding reductase 2 SEQ ID NO:6 of Chappell et al. are set forth HMG-CoA reductase or the catalytic region thereof. therein in the Sequence Listing. US 7,906,710 B2 33 34 The entire HMG1 gene comprises about 3360 base pairs M22002 Saccharomyces cerevisiae Yeast HMG-CoA SEQID NO:3 of Chappellet al. Intact HMG-CoA reductase reductase (HGM1) gene, complete cds: 3-hydroxy-3-methyl 1 comprises an amino acid sequence of about 1054 amino glutaryl coenzyme A reductase. 4/1993 acid residues SEQ ID NO:4 of Chappell et al. Thus, the Q12649 phycomyces blakesleeanus. 3-hydroxy-3-methyl minimal portion of the HMG1 gene that encodes an intact glutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc enzyme comprises base pairs from about nucleotide position tase) (fragment). 11/1997 121 to about position 3282 of FIG. 3, SEQ ID NO:3 of Q12577 fitsarium moniliforme (gibberellafiujikuroi).3-hy Chappell et al. droxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) The entire HMG2 gene comprises about 3348 base pairs (hmg-coa reductase). 11/1997 SEQID NO:5 of Chappellet al. Intact HMG-CoA reductase 10 ABO12603 Candida utilis Candida utilis HMG mRNA for 2 comprises about 1045 amino acid residues SEQID NO:6 of HMG-CoA reductase, complete cds. 7/1998 Chappellet al. Thus, the minimal portion of HMG2 gene that P34136 dictyostelium discoideum (slime mold). 3-hy encodes intact HMG-CoA reductase 2 comprises base pairs droxy-3-methylglutaryl-coenzyme a reductase 2 (ec from about nucleotide position 121 to about position 3255 of 15 1.1.1.34) (hmg-coa reductase 2) (fragment).35735 SEQ ID NO:5 of Chappellet al. PQ0761 hydroxymethylglutaryl-CoA reductase By analogy to the truncated hamster structural gene, struc (NADPH) (EC 1.1.1.34) (HMGR 10) wheat (fragment) tural genes encoding polypeptides comprising catalytically P48019 Oryza sativa (rice). 3-hydroxy-3-methylglutaryl active, truncated HMG-CoA reductase enzymes from yeast coenzyme a reductase (ec 1.1.1.34) (hmg-coa reductase) can also be used in accordance with the present invention. (fragment). 2/1996 The catalytic region of HMG-CoA reductase 1 comprises O24594 Zea mays (maize). 3-hydroxy-3-methylglutaryl amino acid residues from about residue 618 to about reside coenzyme a reductase (ec 1.1.1.88).5/1999 1054: i.e., the COOH-terminus. A structural gene that PQ0763 hydroxymethylglutaryl-CoA reductase encodes the catalytic region comprises base pairs from about (NADPH) (EC 1.1.1.34) (HMGR 23) wheat (fragment) nucleotide position 1974 to about position 3282 of FIG. 3 of 25 PQ0762 hydroxymethylglutaryl-CoA reductase Chappell et al. (NADPH) (EC 1.1.1.34) (HMGR 18) wheat (fragment) The linker region of HMG-CoA reductase 1 comprises an from proprietary soy sequence database amino acid sequence from about residue 525 to about residue Q00583 hevea brasiliensis (para rubber tree). 3-hydroxy 617. A structural gene that encodes the linker region com 3-methylglutaryl-coenzyme a reductase 3 (ec 1.1.1.34) (hmg prises nucleotides from about position 1695 to about position 30 1973 of FIG.3 of Chappellet al. A structural gene encoding coa reductase 3). 7/1998 the linker region of the enzyme operatively linked to the Q03163 catharanthus roseus (rosy periwinkle) (madagas structural gene encoding the catalytic region of the enzyme. car periwinkle). 3-hydroxy-3-methylglutaryl-coenzyme a Also by analogy to the truncated hamster gene, a truncated reductase (ec 1.1.1.34) (hmg-coa reductase). 7/1998 HMG1 gene can optionally contain nucleotide base pair 35 P48022 lycopersicon esculentum (tomato). 3-hydroxy-3- sequences encoding a small portion of the membrane-binding methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg region of the enzyme. Such a structural gene preferably com coa reductase 2). 7/1998 prises base pairs from about nucleotide position 121 to about Q01559 nicotiana Sylvestris (wood tobacco). 3-hydroxy position 147 and from about position 1695 to about position 3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg 3282 of FIG.3 of Chappellet al. 40 coa reductase). 7/1998 A construct similar to those above from an analogous por LO1400 Solanum tuberosum Potato hydroxymethylglu tion of yeast HMG-CoA reductase 2 can also be utilized. taryl coenzyme A reductase (hmgr) mRNA, complete cds; A nucleic acid sequence encoding HMG-CoA reductase putative. 4/1996 from Hevea brasiliensis has been disclosed by Chye et al. Q43826 raphanus sativus (radish). hydroxymethylglu (1991) Plant Mol. Biol. 16:567-577. A nucleic acid sequence 45 taryl-coa reductase (ec 1.1.1.34) (hydroxymethylglutaryl-coa encoding an Arabidopsis thaliana HMG-CoA reductase has reductase (nadph)) (3-hydroxy-3-methylglutaryl-coenzyme been published by Caelles et al. (1989) Plant Mol. Biol. 13: a red 627-638, and is also available as GenBank accession number L19261 Arabidopsis thaliana Arabidopsis thaliana HMG L19261. U.S. Pat. Nos. 5,306,862 and 5,365,017 disclose cCA reductase gene, complete cds. 4/1994 additional DNA sequences encoding HMG-CoA reductases. 50 AB021862 Cucumis melo Cucumis melo mRNA for The following sequences are listed by Genbank Accession HMG-CoA reductase, complete cds; putative. 1/1999 numbers: P29058 hevea brasiliensis (para rubber tree). 3-hydroxy O26.662 methanobacterium thermoautotrophicum. 3-hy 3-methylglutaryl-coenzyme a reductase 2 (ec 1.1.1.34) (hmg droxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) coa reductase 2) (fragment).35735 (hmg-coa reductase). 12/1998 55 Q58116 methanococcus jannaschii. 3-hydroxy-3-methyl P29057 hevea brasiliensis (para rubber tree). 3-hydroxy glutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc 3-methylglutaryl-coenzyme a reductase 1 (ec 1.1.1.34) (hmg tase).7/1998 coa reductase 1). 7/1998 Q59468 halobacterium volcanii (haloferax volcanii). P48021 camptotheca acuminata. 3-hydroxy-3-methylglu 3-hydroxy-3-methylglutaryl-coenzyme a reductase (ec 60 taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc 1.1.1.34) (hmg-coa reductase). 7/1998 tase). 11/1997 O008424 sulfolobus solfataricus. 3-hydroxy-3-methylglu P43256 arabidopsis thaliana (mouse-ear cress). 3-hy taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc droxy-3-methylglutaryl-coenzyme a reductase 2 (ec tase). 12/1998 1.1.1.34) (hmg-coa reductase 2) (hmgr2). 7/1998 M22255 Saccharomyces cerevisiae Yeast HMG-CoA 65 P00347 cricetulus griseus (chinese hamster). 3-hydroxy reductase (HGM2) gene, complete cds: 3-hydroxy-3-methyl 3-methylglutaryl-coenzyme A reductase (ec 1.1.1.34) (hmg glutaryl coenzyme A reductase. 4/1993 coA reductase). 11/1997 US 7,906,710 B2 35 36 L00183 Cricetulus sp. Hamster 3-hydroxy-3-methylglu to date there are no reports on functional characterization of taryl coenzyme A (HMGCoA) reductase gene, exons 19 and any plant squalene epoxidase gene or enzyme. 20: 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA). Squalene Epoxidase, also known as squalene monooxyge 4f1993 nase is enzyme reference number 1.14.99.7, Enzyme Nomen M12705 Mesocricetus auratus Syrian hamster 3-hydroxy clature 1992, p. 146. 3-methylglutaral coenzyme A reductase (HMG-CoA reduc Several squalene epoxidase enzymes are known to the art. tase) mRNA, complete cds; 3-hydroxy-3-methylglutaral These include Arabidopsis squalene epoxidase protein coenzyme A red sequence Accession No. AC004786 (SEQID NO:1), Arabi P51639 rattus norvegicus (rat). 3-hydroxy-3-methylglu dopsis squalene epoxidase Accession No. N64916 (SEQ ID taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc 10 NO:2), and Arabidopsis squalene epoxidase Accession No. tase). 12/1998 T44667 (SEQ ID NO:3). Japanese patent application No. Q295 12 Oryctolagus cuniculus (rabbit). 3-hydroxy-3-me 07194381 A discloses a DNA encoding a mammalian thylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa squalene epoxidase. reductase). 7/1999 15 In order to facilitate the modifications to sterol biosynthe M11058 Homo sapiens Human 3-hydroxy-3-methylglu sis and accumulation described herein, the present invention taryl coenzyme A reductase mRNA, complete cds; 3-hy also provides an isolated DNA molecule, comprising a nucle droxy-3-methylglutaryl coenzyme A reductase. 11/1994 otide sequence selected from the group consisting of: M62766 Mus musculus Mouse HMG-CoA reductase (a) Arabidopsis squalene epoxidase from clone ID mRNA, 3' end. 4/1993 ATA506263 disclosure SEQID NO:4, clone IDATA304243 P20715 xenopus laevis (african clawed frog). 3-hydroxy disclosure SEQ ID NO:6, clone ID ATA102071 disclosure 3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg SEQID NO: 8, clone ATA504158 disclosure SEQID NO:10, coa reductase). 11/1997 or the complement thereof; P16393 strongylocentrotus purpuratus (purple sea urchin). (b) a nucleotide sequence that hybridizes to said nucleotide 3-hydroxy-3-methylglutaryl-coenzyme a reductase (ec 25 sequence of (a) under a wash stringency equivalent to 0.5x 1.1.1.34) (hmg-coa reductase). 11/1997 SSC to 2xSSC, 0.1% SDS, at 55-65° C., and which encodes P54960 blattella germanica (german cockroach). 3-hy a polypeptide having squalene epoxidase enzymatic activity droxy-3-methylglutaryl-coenzyme a reductase (ec 1.1.1.34) Substantially similar to that of the disclosed squalene epoxi (hmg-coa reductase). 11/1997 dase; P14773 drosophila melanogaster (fruit fly). 3-hydroxy-3- 30 methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa (c) a nucleotide sequence encoding the same genetic infor reductase). 12/1998 mation as said nucleotide sequence of (a), but which is degen P34135 dictyostelium discoideum (slime mold). 3-hy erate in accordance with the degeneracy of the genetic code: droxy-3-methylglutaryl-coenzyme a reductase 1 (ec and 1.1.1.34) (hmg-coa reductase 1). 11/1997 35 (d) a nucleotide sequence encoding the same genetic infor P16237 schistosoma mansoni (blood fluke). 3-hydroxy-3- mation as said nucleotide sequence of(b), but which is degen methylglutaryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa erate in accordance with the degeneracy of the genetic code. reductase). 7/1998 An additional aspect of the invention is the recombinant O28538 archaeoglobus filgidus. 3-hydroxy-3-methylglu constructs and vectors (pMON48343, FIG.30; p MON43844, taryl-coenzyme a reductase (ec 1.1.1.34) (hmg-coa reduc 40 FIG. 31) comprising nucleic acid sequences encoding the tase). 12/1998 novel squalene epoxidase, as well as a method of producing M24015 Pseudomonas mevalonii P. mevalonii HMG-CoA the novel squalene epoxidase, comprising culturing a host reductase (mvaA) gene, complete cas; HMG-CoA reductase cell transformed with the novel constructs or vectors for a (EC 1.1.1.88). 4/1993 time and under conditions conductive to the production of the B. Steroid Pathway Enzymes 45 squalene epoxidase, and recovering the squalene epoxidase The present invention contemplates nucleic acid sequences produced thereby. encoding polypeptides having the enzyme activity of the Ste ii. Sterol Methyl Transferase I roid pathway enzymes squalene epoxidase, Sterol methyl S-adenosyl-L-methionine:sterol C-24 methyl transferases transferase I, sterol C4-demethylase, obtusifoliol C14C.-dem (SMT1 and SMT2) catalyze the transfer of a methyl group ethylase, sterol C5-desaturase and sterol methyl transferase 50 from a cofactor, S-adenosyl-L-methionine, to the C-24 center II. of the sterol side chain (Bach, T.J. and Benveniste, P. (1997), i. Squalene Epoxidase Prog. Lipid Res. 36: 197-226). SMT in higher plant cells are Squalene epoxidase (also called squalene monooxyge responsible for their capability to produce a mixture of nase) catalyzes the conversion of squalene to squalene 24-methyl and 24-ethyl sterols (Schaffer, A., Bouvier-Navé, epoxide (2,3-oxidosqualene), a precursor to the initial sterol 55 Benveniste, P., Schaller, H. (2000) Lipids 35:263-269). Func molecule in phytosterol biosynthetic pathway, cycloartenol. tional characterization of the SMT using a yeasterg6 expres This is the first reported step in the pathway where oxygen is sion system demonstrated unambiguously that an SMT1 required for activity. The formation of squalene epoxide is sequence encodes a cycloartenol-C24-methyltransferase and also the last common reported Step in sterol biosynthesis of a SMT2 sequence encodes a 24-methylene lophenol-C24 animals, fungi and plants. Recently, several homologues of 60 methyltransferase in a given plant species (Bouvier-Navé, P. Arabidopsis and Brassica squalene epoxidase genes were Husselstein, T., and Benveniste, P. (1998), Eur. J. Biochem. reported (Schafer, U.A., Reed, D. W., Hunter, D.G.Yao, K., 246: 518-529). Several plant genes coding for SMT1 and Weninger, A.M., Tsang, E.W., Reaney, M.J., MacKenzie, S. SMT2 have been reported and reviewed (Schaffer, A., Bou L., and Covello, P. S. (1999). Plant Mol. Biol. 39(4): 721 vier-Navé, Benveniste, P., Schaller, H. (2000) Lipids 35:263 728). The same authors also have a PCT application disclos 65 269). Transgenic plants expressing homologues of either ing the use of antisense technology with squalene epoxidase SMT1 or SMT2 have been studied (Schaffer, A., Bouvier to elevate squalene levels in plants (WO97/34003). However, Navé, Benveniste, P., Schaller, H. (2000) Lipids 35:263-269). US 7,906,710 B2 37 38 The use of these genes to modify plant sterol composition are (b) a nucleotide sequence that hybridizes to said nucleotide also covered by two Monsanto patent applications (WO sequence of (a) under a wash stringency equivalent to 0.5x 98/45457 and WO 00/61771). SSC to 2xSSC, 0.1% SDS, at 55-65° C., and which encodes Sterol methyl transferase I enzymes known in the art are a polypeptide having obtusifoliol C14C-demethylase enzy useful in the present invention. Examplary sequences include matic activity substantially similar to that of the disclosed the known Arabidopsis sterol methyl transferase I protein obtusifoliol C14C-demethylase; sequence Accession No. U71400 (disclosure SEQ ID (c) a nucleotide sequence encoding the same genetic infor NO:20), the known tobacco sterol methyl transferase I pro mation as said nucleotide sequence of (a), but which is degen tein sequence Accession No. U81312 (disclosure SEQ ID erate in accordance with the degeneracy of the genetic code: NO:21) and Ricinus communes sterol-C-methyltransferase, 10 and Eur: J. Biochem., 246(2), 518-529 (1997). (Complete cds, (d) a nucleotide sequence encoding the same genetic infor Accession No.g.2246457). mation as said nucleotide sequence of(b), but which is degen S-Adenosyl-L-Methionine-Sterol-C24-Methyltrans erate in accordance with the degeneracy of the genetic code. ferase—A nucleic acid sequence encoding an Arabidopsis An additional aspect of the invention is the recombinant thaliana S-adenosyl-L-methionine-sterol-C24-methyltrans 15 constructs and vectors (pMON43842, FIG. 29) comprising ferase has been published by Husselstein et al. (1996) FEBS nucleic acid sequences encoding the novel obtusifoliol C14C.- Letters 381: 87-92. A -sterol C-methyltransferase is enzyme demethylase, as well as a method of producing the novel number 2.1.1.41, Enzyme Nomenclature 1992, page 160. obtusifoliol C14C-demethylase, comprising culturing a host iii. Sterol C4-Demethylase cell transformed with the novel constructs or vectors for a Sterol C-4 demethylase catalyses the first of several dem time and under conditions conductive to the production of the ethylation reactions, which results in the removal of the two obtusifoliol C14C.-demethylase, and recovering the obtusifo methyl groups at C-4. While in animals and fungi the removal liol C14C-demethylase produced thereby. of the two C-4 methyl groups occurs consecutively, in plants V. Sterol C5-Desaturase it has been reported that there are other steps between the first Sterol C-5 desaturase catalyzes the insertion of the and second C-4 demethylations (Bach, T.J. and Benveniste, P. 25 A-double bond that normally occurs at the A-sterol level, (1997), Prog. Lipid Res. 36: 197-226). The C-4 demethyla thereby forming a A 7-sterol (Parks et al., Lipids 30:227-230 tion is catalyzed by a complex of microsomal enzymes con (1995)). The reaction has been reported to involve the ste sisting of a monooxygenase, an NAD"-dependent sterol 4-de reospecific removal of the 5C, and 6C. hydrogen atoms, bio carboxylase and an NADPH-dependent 3-ketosteroid synthetically derived from the 4 pro-Rand 5 pro-Shydrogens reductase. 30 of the (+) and (-) R-mevalonic acid, respectively (Goodwin, iv. Obtusifoliol C14C-Demethylase T. W. (1979) Annu. Rev. Plant Physiol. 30: 369-404). The Sterol C-14 demethylase catalyzes demethylation at C-14 reaction is obligatorily aerobic and requires NADPH or which removes the methyl group at C-14 and creates a double NADH. The desaturase has been reported to be a multien bond at that position. In both fungi and animals, this is the first Zyme complex present in microsomes. It consists of the step in the sterol synthesis pathway. However, in higher 35 desaturase itself, cytochromebs and a pyridine nucleotide plants, the 14C.-methyl is removed after one C-4 methyl has dependent flavoprotein. The A-desaturase is reported to be a disappeared. Thus, while lanosterol is the substrate for C-14 mono-oxygenase that utilizes electrons derived from a demethylase in animal and fungal cells, the plants enzyme reduced pyridine nucleotide via cytochrome, (Taton, M., and uses obtusifoliol as substrate. Sterol 14-demethylation is Rahier, A. (1996) Arch. Biochem. Biophys. 325: 279-288). mediated by a cytochrome P-450 complex. The mechanism 40 An Arabidopsis thaliana cDNA encoding a sterol-C5-desatu of 14C.-methyl removal involves two oxidation steps leading rase was cloned by functional complementation of a yeast to an alcohol, then an aldehyde at C-29 and a further oxidative mutant, erg3 defective in ERG3, the gene encoding the sterol step involving a deformylation leading to formic acid and the C5-desaturase required for ergosterol biosynthesis (Gachotte sterol product with a typical 8, 14-diene (Aoyama, Y. D., Husselstein, T., Bard, M., Lacroute F., and Benveniste, P. Yoshida, Y., Sonoda, Y., and Sato, Y. (1989) J. Biol. Chem. 45 (1996) The Plant Journal 9(3): 391-398). Known sterol 264: 18502-18505). Obtusifoliol 14C-demethylase from Sor C5-desaturase enzymes are useful in the present invention, ghum bicolor (L) Moench has been cloned using a gene including Arabidopsis sterol C5-desaturase protein sequence specific probe generated using PCR primers designed from an Accession No. X90454, disclosure SEQ ID NO:23, and the internal 14 amino acid sequence and was functionally Arabidopsis thaliana mRNA for sterol-C5-desaturase expressed in E. coli (Bak, S. Kahn, R. A., Olsen, C. E. and 50 described in Plant J. 9(3):391-398 (1996) (complete cds Halkier, B. A. (1997) The Plant Journal 11(2): 191-201). Accession No.g. 1061037). Also, Saccharomyces cerevisiae CYP51A1 encoding lanos The NCBI (National Center for Biotechnology Informa terol-14-demethylase was functionally expressed in tobacco tion) database shows 37 sequences for sterol desaturase that (Grausem, B., Chaubet, N., Gigot, C., Loper, J. C., and Ben are useful in the present invention. The following are exem veniste, P. (1995)The Plant Journal 7(5): 761-770). 55 plary of Such sequences. From yeast: C5 sterol desaturase Sterol C-14 demethylase enzymes and sequences are NP 013157 (Saccharomyces cerevisiae); hypothetical C5 known in the art. For example Sorghum bicolor obtusifoliol sterol desaturase-fission T40027 (Schizosaccharomyces 14C-demethylase CYP51 mRNA, described in Plant J., pombe); C5 sterol desaturase-fission T37759 (Schizosaccha 11(2):191-201 (1997) (complete cds Accession No. romyces pombe); C5 sterol desaturase JQ1146 (Saccharomy U74319). In order to facilitate the modifications to sterol 60 ces cerevisiae): C5 sterol desaturase BAA21457 (schizosac biosynthesis and accumulation described herein, the present charomyces pombe); C5 sterol desaturase CAA22610 invention also provides an isolated DNA molecule, having a (Schizosaccharomyces pombe); putative C5 Sterol desaturase nucleotide sequence selected from the group consisting of: CAA16898 (Schizosaccharomyces pombe); probable C5 ste (a) obtusifoliol C14C.-demethylase from clone ID: rol desaturase 013.666 (erg3 schpo); C5 sterol desaturase ATA101105 disclosure SEQID NO:14, clone IDATA202967 65 P50860 (Erg3 canga); C5 sterol desaturase P32353 disclosure SEQID NO:15, clone IDATA403931 disclosure (erg3 yeast); C5.6 desaturase AAC99343 (Candida albi SEQ ID NO:17, or the complement thereof; cans); C5 sterol desaturase BAA20292 (Saccharomyces cer US 7,906,710 B2 39 40 evisiae): C5 sterol desaturase AAB39844 (Saccharomyces and/or steroid pathway enzymes is(are) from a species in a cerevisiae): C5 sterol desaturase AAB29844 (Saccharomyces different order from the order that of the host cell. cerevisiae): C5 sterol desaturase CAA64303 (Saccharomy It is contemplated that a construct comprises more than one ces cerevisiae): C5 sterol desaturase AAA34595 (Saccharo of the DNA sequences encoding a steroid pathway enzyme. myces cerevisiae): C5 sterol desaturase AAA34594 (Saccha The invention also contemplates a recombinant vector romyces cerevisiae). From plants: C5 Sterol desaturase comprising the above-described recombinant construct, S71251 (Arabidopsis thaliana); putative sterol-C5-desatu wherein that vector is preferably a plant expression vector. rase AAF32466 (Arabidopsis thaliana); sterol-C5-desaturase A recombinant DNA molecule of the present invention can AAF32465 (Arabidopsis thaliana); putatuve sterol desatu be produced by operatively linking a vector to a useful DNA rase AAF22921 (Arabidopsis thaliana); delta7 sterol C5 10 segment discussed herein to form a plasmid. A vector capable desaturase (Arabidopsis thaliana); sterol C5(6) desaturase of directing the expression of a polypeptide having HMG homolog AAD20458 (Nicotiana tabacum); sterol C5 desatu CoA reductase activity is referred to herein as an HMG-CoA rase AAD12944 (Arabidopsis thaliana); sterol C5.6 desatu reductase “plant integrating vector. rase AAD04034 (Nicotiana tabacum); sterol C5 desaturase Such plant integrating vectors contain control elements CAA62079 (Arabidopsis thaliana). From mammals: sterol 15 that direct and regulate expression, including a promoter, a C5-desaturase (Mus musculus) BAA33730; sterol-C5-de marker, a terminator and insertion sequence (e.g. FIG. 5). The saturase BAA33729 (Homo sapiens); lathosterol oxidase polypeptide coding genes are operatively linked to the plant CAB65928 (Leishmania major); lathosterol oxidase (lathos integrating vector to allow the promoter sequence to direct terol 5-desaturase) O88822 (Mus musculus); lathosterol 5-de RNA polymerase binding and expression of the desired saturase O75845 (Homo sapiens); delta7 sterol C5 desaturase polypeptide coding gene. AAF00544 (Homo sapiens). Others: fungal sterol C5 desatu Useful in expressing the polypeptide coding gene are pro rase homolog BAA 18970 (Homo sapiens). moters that are inducible, viral, synthetic, constitutive as For DNA sequences encoding a sterol-C5-desaturase use described by Poszkowski et al., EMBO.J., 3:2719 (1989) and ful in the present invention, the NCBI nucleotide search for Odell et al., Nature, 313:810 (1985), and temporally regu “sterol desaturase' came up with 110 sequences. The follow 25 lated, spatially regulated and spatiotemporally regulated as ing are exemplary of such sequences. NC 001139 (Saccha given in Chau et al., Science, 244:174-181 (1989). The pro romyces cerevisiae): NC 001145 (Saccharomyces cerevi moterpreferably comprises a promoter sequence whose func siae): NC 001144 (Saccharomyces cerevisiae): AW700015 tion in regulating expression of the structural gene is Substan (Physcomitrella patens); AB004539 (Schizosaccharomyces tially unaffected by the amount of sterol or squalene in the pombe); and AW596303 (Glycine max); AC012188 (Arabi 30 cell. As used herein, the term “substantially unaffected dopsis thaliana). means that the promoter is not responsive to direct feedback vi. Sterol Methyl Transferase II control by the sterols or squalene accumulated in transformed The combination of introduction of an HMG-CoA reduc cells or transgenic plants. tase gene along with a sterol methyl transferase II gene into a A promoter is also selected for its ability to direct the cell serves to reducesteroid pathway intermediate compound 35 transformed plant cells or transgenic plants transcriptional accumulation in addition to reducing the accumulation of activity to the structural gene encoding a polypeptide having 24-methyl sterols such as campesterol. HMG-CoA reductase activity. Structural genes can be driven Known sterol methyl transferase II enzymes are useful in by a variety of promoters in plant tissues. Promoters can be the present invention, including Arabidopsis sterol methyl near-constitutive, such as the CaMV 35S promoter, or tissue transferase II protein sequence (complete mRNA cols from 40 specific or developmentally specific promoters affecting FEBS Lett. 381 (12):87-92 (1996) Accession No. X89867), dicots or monocots. disclosure SEQID NO:22. As exemplified and discussed in detail herein, where the Recombinant constructs encoding any of the forgoing near-constitutive promoter CaMV 35S is used to transform enzymes affecting the steroid biosynthetic pathway can be tobacco plants, increases in total sterol and squalene accumu incorporated into recombinant vectors comprising the recom 45 lation are found in a variety of transformed plant tissues (e.g. binant constructs comprising the isolated DNA molecules. callus, leaf, seed and root). Alternatively, the effects of trans Such vectors can be bacterial or plant expression vectors. formation (e.g. increased amount of a gene coding for HMG IV. Recombinant Constructs and Vectors CoA reductase, increased total sterol accumulation and The present invention contemplates a recombinant con increased squalene accumulation) can be directed to specific struct that contains a DNA sequence encoding a polypeptide 50 plant tissues by using plant integrating vectors containing a exhibiting 3-hydroxy-3-methylfluaryl-Coenzyme A (HMG tissue-specific promoter. CoA) reductase activity and a DNA sequence encoding a An exemplary tissue-specific promoter is the Lectin pro polypeptide exhibiting the activity of a steroid pathway moter, which is specific for seed tissue. The Lectin protein in enzyme. Each polypeptide-encoding DNA sequence is oper Soybean seeds is encoded by a single gene (Lel) that is only ably linked in the 5' to 3" direction independent of the other 55 expressed during seed maturation and accounts for about 2 to sequence. Each DNA sequence in the 5' to 3’ direction com about 5 percent of total seed mRNA. The Lectin gene and prises a promoter, then the DNA sequence encoding the seed-specific promoter have been fully characterized and polypeptide then a transcription termination signal sequence. used to direct seed specific expression in transgenic tobacco The steroid pathway enzyme is a squalene epoxidase enzyme, plants. See, e.g., Vodkin et al., Cell, 34:1023 (1983) and a sterol methyltransferase I enzyme, a sterol C4-demethylase 60 Lindstrom et al., Developmental Genetics, 11:160 (1990). enzyme, a obtusifoliol C14C-demethylase enzyme, a sterol A plant integrating vector containing a structural gene cod C5-desaturase enzyme, or a sterol methyl transferase II ing for a polypeptide having HMG-CoA reductase activity is enzyme. engineered to be under control of the Lectin promoter and that Preferably, the promoters in the recombinant construct are vector is introduced into Soybean plants using a protoplast seed-specific promoters. In one embodiment, the promoter is 65 transformation method. E. G. Dhir et al., Plant Cell Reports, derived from a species in a different order from the host cell. 10:97 (1991). The expression of the polypeptide having In other embodiments, the encoded HMG CoA reductase HMG-CoA reductase activity is directed specifically to the