The present invention relates to transformed plants with an altered terpene content, preferably over-accumulating a mono- or sesqui-terpene. By transformation of plants with genes encoding terpene synthases (TS), and prenyl transferases (PRT), plants accumulating at least 1000 ng/per g of fresh leaf of a specific terpene were obtained. The present invention provides an advantageous system for production of terpenes in that any desired mono- or sesqui-terpene at the choice of the skilled person can be produced in plants. Preferably, the transformed plants contain at least one recombinant plastid targeted TS and PRT

Chappell, Joe

Clark, Anthony

Schalk, Michel

Wu, Shuiqin

English

University of Kentucky

University of KentuckyUKnowledgePlant and Soil Sciences Faculty Patents Plant and Soil Sciences9-13-2011Transformed Plants Accumulating TerpenesJoe ChappellUniversity of Kentucky, chappell@uky.eduShuiqin WuUniversity of KentuckyMichel SchalkAnthony ClarkRight click to open a feedback form in a new tab to let us know how this document benefits you.Follow this and additional works at: https://uknowledge.uky.edu/pss_patentsPart of the Plant Sciences CommonsThis Patent is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Plant and SoilSciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu.Recommended CitationChappell, Joe; Wu, Shuiqin; Schalk, Michel; and Clark, Anthony, "Transformed Plants Accumulating Terpenes" (2011). Plant and SoilSciences Faculty Patents. 3.https://uknowledge.uky.edu/pss_patents/3(12) United States Patent Chappell et a]. USOO8017835B2 US 8,017,835 B2 Sep. 13, 2011 (10) Patent N0.: (45) Date of Patent: (54) TRANSFORMED PLANTS ACCUMULATING TERPENES (75) Inventors: Joe Chappell, Lexington, KY (US); Shuiqin Wu, Lexington, KY (US); Michel Schalk, Collonges-Sous-Saleve (FR); Athony Clark, West Windsor, NJ (Us) (73) Assignee: University of Kentucky Research Foundation, Lexington, KY (US) ( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 681 days. (21) Appl. No.: 11/911,660 (22) PCT Filed: Apr. 18,2006 (86) PCT No.: PCT/IB2006/051198 § 371 (0X1)’ (2), (4) Date: Apr. 25, 2008 (87) PCT Pub. No.: WO2006/111924 PCT Pub. Date: Oct. 26, 2006 (65) Prior Publication Data US 2009/0123984 A1 May 14, 2009 (30) Foreign Application Priority Data Jun. 16, 2005 (EP) ................................... .. 05105281 (51) Int. Cl. C12N 15/29 (2006.01) C12N15/31 (2006.01) C12N15/52 (2006.01) C12N15/82 (2006.01) (52) US. Cl. .... .. 800/288; 800/298; 435/193; 435/320.1 (58) Field of Classi?cation Search ...................... .. None See application ?le for complete search history. (56) References Cited U.S. PATENT DOCUMENTS 7,091,027 B1 8/2006 WallaaIt et a1. .. .... .. 435/232 7,622,288 B2 * 11/2009 Schalk . . . . . . . . . . . . . . . . . .. 435/193 2004/0161819 A1 8/2004 Aharoni et al. ............ .. 435/691 FOREIGN PATENT DOCUMENTS EP 1 063 297 A1 12/2000 W0 WO 91/13078 9/1991 W0 WO 00/12725 3/2000 W0 WO 00/22150 4/2000 W0 WO 02/33060 A2 4/2002 W0 W0 02/064764 A2 8/2002 W0 WO 2004/031376 A2 4/2004 W0 WO 2005/052163 A2 6/2005 OTHER PUBLICATIONS Chen D. et al. Plant Science (2000) vol. 155; pp. 179-185.* Brodelius, M. et al. Eur. J. Biochem. (2000) vol. 269; pp. 3570 3577.* Aharoni, A. et al. Plant Cell (Dec. 2003); vol. 15; pp. 2866-2884.* Asaph Aharoni et al., XP002355234, “Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants”, Plant Cell, v01. 15, N0. 12, pp. 2866-2884 (2003). Andrea Hemmerlin et al., XP002407595 “Enzymes Encoded by the Farnesyl Diphosphate Synthase Gene Family in the Big Sagebrush Artemisia Tridentata Ssp.Spicif0rmis.” Journal of Biological Chem istry, vol. 278, N0. 34, pp. 32132-32140 (2003). H. Nakashita et al., XP009056668, “Introduction of Bacterial Metabolism Into Higher Plants by Polycistronic Transgene Expres sion”, Bioscience Biotechnology Biochemistry, v01. 65, N0. 7, pp. 1688-1691 (2003). Maria Brodelius et al., XP002407596, “Fusion of Farnesyldiphosphate Synthase and Epi-Aristolochene Synthase, A Sesquiterpene Cyclase Involved in Capsidiol Biosynthesis in Nicotiana Tabacum” European Journal of Biochemistry, vol. 269, N0. 14, pp. 3570-3577 (2002). Da-Hua Chen et al., XP002305638, “Expression of a Chimeric Farnesyl Diphosphate Synthase Gene in Artemisia Annua L. Transgenic Plants Via Agrobacterium T umefaciens-Mediated Trans formation”, Plant Science, vol. 155, N0. 2, pp. 179-185 (2000). Asaph Aharoni et al., XP002355235, “Gain and Loss of Fruit Flavor Compounds Produced by Wild and Cultivated Strawberry Species” Plant Cell, vol. 16, N0. 11, pp. 3110-3131 (2004). R. Bock, XP001084937, “Transgenic Plastids in Basic Research and Plant Biotechnology” Journal ofMolecular Biology, v01. 3, N0. 312, pp. 425-438 (2001). * cited by examiner Primary Examiner * Russell Kallis (74) Attorney, Agent, or Firm * Winston & Strawn LLP (57) ABSTRACT The present invention relates to transformed plants with an altered terpene content, preferably over-accumulating a mono- or sesqui-terpene. By transformation of plants with genes encoding terpene synthases (TS), and prenyl trans ferases (PRT), plants accumulating at least 1000 ng/per g of fresh leaf of a speci?c terpene were obtained. The present invention provides an advantageous system for production of terpenes in that any desired mono- or sesqui-terpene at the choice of the skilled person can be produced in plants. Pref erably, the transformed plants contain at least one recombi nant plastid targeted TS and PRT. 10 Claims, 20 Drawing Sheets US. Patent Sep. 13, 2011 Sheet 1 0120 US 8,017,835 B2 Figure 1 (A and B) SUUUUUDCP A 12 45000000— .1 .1 40000000— .1 35000000— 0) 250000005 20000000: Remive Abundant 15000000l j 5 1O l 3 10000000 _ 6 50000001 ~ 7 8 9 : MM 10000000 B 3 \NT 5000000 A _J\_ )1 [IIIIIIIIIUHIllllllrnllrll\[lrrl?lirfnerlrrTU[IWFIIH—[IIIITHITrlrrl-T 1U 11 12 13 14 15 15 'IT 18 19 20 21 22 23 24 25 Time (min) US. Patent Sep. 13, 2011 Sheet 2 0f 20 US 8,017,835 B2 Figure 1 C 55.0 100: 2 4'1 U 90% 55? 410 m o 1 so .A 0 138.1 ulln mm 981 rag 2223 9&1 Relative Abundanc 01 D 1251 931 108.1 | 1M m l M 1: 1111 :m f“ :2 0 1 11160.1] US. Patent Sep. 13, 2011 Sheet 3 0f 20 US 8,017,835 B2 Figure 1 D BSD - 43.0 1331 69.0 070') am am US 01 93,9 55.0 79 ,9 10m 125.1 91,0 222.2 W ‘ 1611 Law mm mm M 01 m D 132.1 15“ 1792 207-2 1233.1, I 1 31,1“ i | x [ 220 240 US. Patent Sep. 13, 2011 Sheet 4 0f 20 US 8,017,835 B2 Figure 2 * Amorpha aaaunnesmi . 4,1 1-di€H€ ADS transgenlc line mmaasmI Retaiive Abundammammasa ‘ . , , . , . . , , '11,? Retention iime t‘m‘im { Cubenol i , Retention time {min} US. Patent Sep. 13, 2011 Sheet 5 0f 20 US 8,017,835 B2 Figure 3 35000 30000 ' 25000 ' 20000 - 15000 - 10000 - Patchoulol (ng/ gW)5000 - 5 6121412 4 5111213196 71415129333413 813 hsPTS FPS-PTS PTS +FPP tpPTS thPS-PTS tpPTS + thPP Cytosol target Plastid target US 8,017,835 B2 Sheet80f20 Sep.13,2011 US. Patent kxwm 36mm <>2 @mooim-62-1 10 o: w upswE US. Patent Figure 7 b ands3. 4 Ram Sep. 13, 2011 Sheet 9 0f 20 US 8,017,835 B2 i US. Patent Sep. 13, 2011 Sheet 10 0f 20 US 8,017,835 B2 Figure 9 A Ndel EcoRl Xhol Ncol attB1 Pcv TNOS attB pTIVION Figure 9 B S SEICI attB1 Pcv Tnos Pca Tnos attB2 pTDUAL U-S- Patent Sep. 13, 2011 Sheet 11 0f 20 Us 8317,835 B2 Figure 10 A Pnos HPT PTS HIS Pcv P n O S H PT Pcv Pnos HPT FPS Poa D PTS Pcv Figure 10 B PTS tp Pcv Pnos HPT \l > PTS FPS tp Pcv / Pnos HPT FPS tp Pea ‘I > / Pno HPT ADS TP Pcv Pca US. Patent Sep. 13, 2011 Sheet 12 0120 US 8,017,835 B2 Figure 11 4”, No Xba -HP -CaUTR (15) Sph Sst P-NOSI-IKanHHT-NOSIIMCS HGUS | T-NOS R No Xba pB|101 (invitroqen, Carlsbad, CA) HPT 16 Sph ¢ No Xb ( ) K P 5,011 No i (13) DTRBP 4% Sph SST ( ) R | ¢ (19) Sph N0 Xba 8011 V”) > SD attp attp US. Patent Sep. 13, 2011 Sheet 13 0f 20 US 8,017,835 B2 Figure 12 22 ( ) NdeI EcoRl NdeI EcoRI ' $51M ‘ attB1 Pcv (23) pGPcv (24) Ndeli ECORI NdelEcoRl Xhol Q Ndel EcoRl Xhol | | Q Gene1 Gene1 pECVS —> attB1 Pcv (25) pEPCVS (26) i NdeI/Xho|,fragment isolation Xho' 300' Ndel EcoRl Xhol Tnos §\a1tt52 Gene1' attB1 PCV (27) NdeP<h0| Ncol (28) Ndel EcoRl Xhol Ncol Gene1 PCV pTMON US 8,017,835 B2 Sheet140f20 Sep.13,2011 US. Patent PO mmrmmock mom $2 Nmtm mockOQIQFQ Away ‘|||||||| moma _ mmtw ockj 6% =62 Lwagx _oww CQx any  xmozkog mmtw ockFL Anny ‘| a mom OQIPQ 960 E as/ mom wa Ame M tan 2aswiUS. Patent Sep. 13, 2011 Sheet 15 0120 US 8,017,835 B2 Figure 13 part II Xb M S p 61 KpnI SacI ,\ lam; \ Pca attBZ (37) 4' nTPHPOT SphI EcoRI XhoI XbaI l (39) Gene1 H KpnI SacI XbaI peI Q ¢ §\\\\ Pca attBZ thl ECORI XhoI Xbal SacI Gene 1 attBl Pcv Tnos DTPCVST (38) (4o) SQfI ECORI XbaI Sad Gene1 [any x attBl Pcv Tnos Pea Tnos attB2 pTDUAL US. Patent Sep. 13, 2011 Sheet 16 0120 US 8,017,835 B2 Figure 14 PBDON ~ Pnos HPT Tca AscI EcoRI XhOI pTMON § § attB1 Pcv“ 6XHis PTS Tnos 31th A501 EcoRI XhoI Spel attBl Pcv F6XHis PTS Tnos Pod FPS (43) US 8,017,835 B2 Sheet 17 0f 20 Sep. 13, 2011 US. Patent <w§+m2~13 n23 Pam<3“EQCL %§ .5313I§ A_ ._cam <9;2;232? 525ng Egmzawuozw \Mamangg?amz wuozw“1‘ Mam HEEBWEEEEQM mi&\\\1_ EOE :35 Q 2%Ex 5.5+ “wigsm? =5<62HEEL aim iv AV1513 2&ng EEEEE Ha?mamuozm ‘Arse , .52: £55%: w :6Eaéioowm _6%: 18cmqu 9mk+cbm+<w§+mzmdm _mam Fan<3“6mgaitg 23:25 :02 mtgEcomw2% Ex 3meng QEEEBZ2353:2me $2 _ § :3ng ram6% 28m:25 gem.BGQZEw 2% Ex 25:22 :avmmaw 3502350* Egéi?gx ram3% 28M Ema miaswUS. Patent Sep. 13, 2011 Sheet 18 0120 US 8,017,835 B2 Figure 15 part II ngI Smal SmalenI HindIIIStuI Xbal SphI ' ' ' -- ii - ' Tatp lacI Prrn Trbcl aadA Prrn attP pLattP+aadA+1acI ngI Smal SmaIKpnI HindIIIStuI Xbal SphI Trbcl Tatp lacI Trbcl aadA [51111 attP pLattP+aadA+1acI+Tfocl ‘\ :\ \ Prrnlac ngI SmaI SmaIKpnI HindIIIStuI XbaI SphI P11111210 Trbcl Tatp lacI Prrn Trbcl aadA attP / vaec US 8,017,835 B2 Sheet 19 0f 20 Sep. 13, 2011 US. Patent ms“ PE<2.$5962 @aeEngEmw HEX B Edamne62mrrm mood chm <©§3ongmH02 QUEHUQHEEgg“? :5 EEEMwem?emH62mam mrwm chm UQHP 95 3mmm 95%wmm A? @m<2.“Bm8%\ 629g H 5EYE2% Ex 52:35ng?am idemmDU mD U aim$3 5 :2 2:me

Transformed Plants Accumulating Terpenes

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Transformed Plants Accumulating Terpenes

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