US007214507B2 (12) United States Patent (10) Patent No.: US 7,214.507 B2 BOuWmeester et al. (45) Date of Patent: May 8, 2007 (54) PLANT ENZYMES FOR BIOCONVERSION (56) References Cited (75) Inventors: Hendrik Jan Bouwmeester, Renkum U.S. PATENT DOCUMENTS (NL); Jan-Willem de Kraker, Jena 6,200,786 B1* 3/2001 Huang et al. ............... 435,132 (DE); Marloes Schurink, Ede (NL); 6,451,576 B1* 9/2002 Croteau et al. ............. 435/232 Raoul John Bino, Wageningen (NL); Aede de Groot, Wageningen (NL); OTHER PUBLICATIONS Maurice Charles R. Franssen, Dedeyan B et al. 2000. Appl Env Microbiol 66: 925-929.* Wageningen (NL) Yoder OC et al. 2001. Curr Opin Plant Biol 4: 315-321.* Nielsen KAetal. 2005. Cytochrome P450s in plants. In Cytochrome (73) Assignee: Plant Research International B.V., P450: structure, mechanism, and biochemistry, 3' ed., Ortiz de Wageningen (NL) Montellano, ed., p. 553.* Bohlmann, Jorg, et al., “Plant terpenoid synthases: Molecular (*) Notice: Subject to any disclaimer, the term of this biology and phylogenetic analysis”. Proc. Natl. Acad. Sci. 1998, patent is extended or adjusted under 35 95:4126-4133. de Kraker, Jan-Willem, et al., “Biosynthesis of Germacrene A U.S.C. 154(b) by 0 days. Carboxylic Acid in Chicory Roots. Demonstration of a Cytochrome P450 (+)-Germacrene A Hydroxylase and NADP-Dependent (21) Appl. No.: 10/489,762 Sesquiterpenoid Dehydrogenase(s) Involved in Sesquiterpene Lactone Biosynthesis”. Plant Physiology 2001, 125:1930-1940. (22) PCT Filed: Sep. 17, 2002 de Kraker, Jan-Willem, et al., “Germacrenes from fresh costus roots”, Phytochemistry 2001, 58:481-487. (86). PCT No.: PCT/NLO2/OO591 de Kraker, Jan-Willem, et al., “(+)-Germacrene A Biosynthesis'. Plant Physiol. 1998, 117:1381-1392. S 371 (c)(1), Halkier, Barbara Ann, "Catalytic Reactivities and Structure/Func (2), (4) Date: Sep. 7, 2004 tion Relationships of Cytochrome P450 Enzymes'. Phytochemistry 1996, 43(1):1-21. (87) PCT Pub. No.: WO03/025193 Schuler, Mary A., “Plant Cytochrome P450 Monooxygenases”. Critical Reviews in Plant Sciences 1996, 15(3):235-284. PCT Pub. Date: Mar. 27, 2003 * cited by examiner (65) Prior Publication Data Primary Examiner Sandra E. Saucier Assistant Examiner Lora E. Barnhart US 2005/OO19882 A1 Jan. 27, 2005 (74) Attorney, Agent, or Firm Hoffmann & Baron, LLP (30) Foreign Application Priority Data (57) ABSTRACT Sep. 17, 2001 (EP) .................................. O1203519 The invention provides a method for converting sesquiter (51) Int. Cl. pene. The method includes reacting a sesquiterpene Sub CI2P I/00 (2006.01) stract with a sesquiterpene converting enzyme. The enzyme A6 IK 36/28 (2006.01) is from a species or organism containing sesquiterpenes. The (52) U.S. Cl. ........................ 435/41; 560/249; 424/736; sesquiterpene Substrate is not naturally present in the species 424/773; 424/725 or organism. (58) Field of Classification Search ..................... None See application file for complete search history. 5 Claims, 20 Drawing Sheets U.S. Patent May 8, 2007 Sheet 1 of 20 US 7,214.507 B2 Figure 1. actucin 8-deoxylactucin lactucopicrin guaianolides eldesmanoides genacranoides f R g R U.S. Patent May 8, 2007 Sheet 2 of 20 US 7,214.507 B2 Figure 2 Biopathway Cope rearrangement Cyclisation products products (+)-germacrene A (1) cation a 13 3 N 2 2. H Z *-OH 21 N 3 O 8. O 2 A.l C6-hydroxylationO : 9 OH (+)-costunolide(5) U.S. Patent May 8, 2007 Sheet 3 of 20 US 7,214.507 B2 Figure 3. NADPH oxygenated 2e product (GOE) +HO Ol SStateE. (RE (> S NH () SSM&RK.Cytochrone P-450 cytosol YS&YYYNY&CS."tunner NH U.S. Patent May 8, 2007 Sheet 4 of 20 US 7,214.507 B2 Figure 4 R B-terpineol (R-OH) ot-terpineol (R-OH) B-terpinyl acetate (R-OAc) ot-terpinyl acetate (R-OAc) U.S. Patent May 8, 2007 Sheet S of 20 US 7,214.507 B2 Figure 5 O R gH) O H3C H3C H H ou-Q Egg O OH O O OH H OH OH H (digitoxin) R = OH (digoxin) U.S. Patent May 8, 2007 Sheet 6 of 20 US 7,214.507 B2 Figure 6 (A - c. (-)-o-Cububene (-)-O-gurjunene gemacrone imonene U.S. Patent May 8, 2007 Sheet 7 of 20 US 7,214.507 B2 Figure 7. Glornerella 15 Cingulata Cichorium intybus OH U.S. Patent May 8, 2007 Sheet 8 of 20 US 7,214.507 B2 Figure 8 NADPH NADP 9. O A2 4H2O COOH 12 (+)-costunolide (5) U.S. Patent May 8, 2007 Sheet 9 of 20 US 7,214.507 B2 Figure 9. COOH COOH 15 6 U.S. Patent May 8, 2007 Sheet 10 of 20 US 7,214.507 B2 Figure 10 dHSauSSL 24.00 28.00 32.00 36.00 U.S. Patent May 8, 2007 Sheet 11 of 20 US 7,214.507 B2 Figure 11 COOH U.S. Patent May 8, 2007 Sheet 12 of 20 US 7,214.507 B2 Figure 12. s Leukodin se Dihydro s COStunoide Costunoide E Da N air N2 N2 CO CO dark dark light dark light U.S. Patent May 8, 2007 Sheet 13 of 20 US 7,214.507 B2 Figure 13. A Air B 1OO % 8 4. 53 Oxygen-18 79 57 S3 11 123 49 29 Allisi,t . H Alth E. E. 40 SO 80 12 140 so 180 20 220 m2 C (+)-Costunolide U.S. Patent May 8, 2007 Sheet 14 of 20 US 7,214.507 B2 Figure 14. A O09 24S Airo 41 5 s s 81 15 173 1351.45 is 29 159 y 231 |HER B AHHhHull,15 213 , , 29, , , Oil use west Hill:- B toos, 2 Oxygen-18 4. 55 s s B 175 1gs 221 109 149 is 235 R NIAA ... in 1919. 297 O t A. R I hul B.E., all 40 80 100 2O 14 SD 80 200 220 240 m2 C 100% 246 Leucodin U.S. Patent May 8, 2007 Sheet 15 of 20 US 7,214.507 B2 Figure 15. NADPH NADP GS,too CS)tei () H "to 4 H- (+)-costunolide (5) U.S. Patent May 8, 2007 Sheet 16 of 20 US 7,214.507 B2 Figure 16. -Sesquiterpene Lactones y O Sesquiterpene actones 11 (S). 13-dihydro-8-deoxylactucin U.S. Patent May 8, 2007 Sheet 17 of 20 US 7,214.507 B2 Figure 17. Wal NKatone 24.00 27.00 30.00 U.S. Patent May 8, 2007 Sheet 18 of 20 US 7,214.507 B2 Figure 18. NADP+ n, NAD(P) (+)-valencene o-nootkatol U.S. Patent May 8, 2007 Sheet 19 of 20 US 7,214.507 B2 trans,trans-farneso farnesal farthesoic acid U.S. Patent May 8, 2007 Sheet 20 of 20 US 7,214.507 B2 (+)-costunolide (+)-valenceme rootkatone US 7,214,507 B2 1. 2 PLANT ENZYMES FOR BOCONVERSION the structural aspects and biological activities of these type of compounds (e.g. Picman, 1986; Harborne et al., 1999; This application is the U.S. National Phase of Interna Seigler, 1995). tional Application Number PCT/NL02/00591 filed on 17 Sesquiterpene lactones may constitute up to 5% of the dry Sep. 2002, which is incorporated herein by reference. weight of the plant and occur mainly in members of the Asteraceae (in about 450 species), the largest of all plant BACKGROUND OF THE INVENTION families, but also occur in other higher plant families Such as the Apiaceae (12 species) and lower plants such as The invention relates to the field of bioconversion, using liverworts, for examnple the genus Frullania (Harborne et plant enzymes for the production of flavor, fragrance, phar 10 al., 1999). Sesquiterpene lactones may occur throughout a maceutical or bio-control agents from less valuable Sub plant, but are most commonly associated with leaves, flower strates. More specifically it relates to a process for produc parts and taproots. tion of terpenoid compounds by the use of plant enzymes. The Asteraceae contain 1317 genera and 21000 species. They are mainly herbaceous plants, sometimes shrubs or Modern chemistry strongly depends on the use of cata 15 trees, usually with a taproot, sometimes with tubers. Many lysts in order to have efficient and clean reactions with a of the Asteraceae contain (essential) oil bearing organs. Such minimum of waste. Especially the use of biocatalysts (en as ducts or trichomes, some of them contain latex. The Zymes, cells) is strongly increasing, also in industry. The Asteraceae contain many economic and ornamental plants, most important features of biocatalysts are (Faber, 2000): such as Sunflower (Helianthus annuus; for the production of 1. they are very efficient; seeds and sunflower oil), Jerusalem-Artichoke (Helianthus 2. they are environmentally acceptable; tuberosus; of which the tubers are eaten and that is also used 3. they operate under mild conditions; for the fructan production), and Artemisia annua (for the production of the effective anti-malarial artemisinin. Also 4. they are selective. used as food are the roots of Salsify (Tragopogon porrifo Up to now, the large majority of enzymes used in bio 25 lius) and ScorZonera (Scoizomera hispanica), the young conversion employing biocatalysis for industrial and labo flower-heads of Globe Artichoke (Cynara scolymus), and ratory applications is obtained from microbial Sources. A the leaves of Lettuce (Lactuca sativa), Endive (Cichorium minor fraction of enzymes is obtained from plant sources endiva) and Radicchio and Brussels Endive (Cichorium (Faber, 2000). Nevertheless, the plant kingdom is an impor inty bus). In addition, there are many ornamental Asteraceae, tant source for the chemist and the biotechnologist because 30 for example in the genera Dahlia, Doronlicum, Heleniumn, plants produce a unique variety of chemicals (Franssen and Tagetes, Helianthus, Aster; Centaurea, Gerbera, etc.