fundamental aspects and practical applications in the production of non-toxic Citrus F. Bourgaud1, A. Dugrand1, A. Olry1, A. Hehn1, Y. Froehlicher2 1: Laboratoire Agronomie et Environnement - Université de Lorraine/INRA -Nancy (France) 2: Laboratoire AGAP – INRA/CIRAD – Montpellier/San Gugliano (France) R5 R5 R6 O O O O O O R8R6 Linear furanocoumarins Angular furanocoumarins Psoralen: R5, R8: H Angelicin: R5, R6: H Bergapten (5-MOP): R5: OMe Sphondin: R6: OMe Xanthotoxin (8-MOP): R8: OMe Pimpinellin: R5, R6: OMe Isopimpinellin (5-8-MOP): R5, R8: OMe Skin blisters and burns ! Giant Hogweed (Heracleum mantegazzianum) Blisters after contact with plants + UV/Sun exposure AFERP-STOLON 15-17 juillet 2015 – Lyon O + O O O O O Moraceae Rosids Fabaceae Ficus carica Rutaceae Cullen cinenea Core tricolpates Citrus bergamia Apiaceae Asterids Apiaceae Pastinaca sativa Ammi majus AFERP-STOLON 15-17 juillet 2015 – Lyon Ecological functions of furanocoumarins O • Phytoalexins O O •Allelopathy (germination inhibition) Fusarium sp. Genotoxicity (DNA crosslinks) Enzyme inhibition (MBI P450) Hordeum vulgare Papilio polyxenes •Anti-insect Grapefruit juice effect (effet pomélo) AFERP-STOLON 15-17 juillet 2015 – Lyon Cinnamic acid p-Coumaroyl CoA Umbelliferone OH HO OH HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin Multiple steps reaction HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins O O O O O O OH H Psoralen OH Angelicin O O O O O O OH Sphondin Xanthotoxol Bergaptol Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 P-Coumaroyl-CoA 2’Hydroxylase (C2’H) Dioxygenase 2-OGD-Dioxygenase Vialart et al., Plant J, 2012, 70(3), 460-470 P450 Cinnamic acid p-Coumaroyl CoA ? Dioxygenase OH HO OH Umbelliferone HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins O O O O O O OH H Psoralen OH Angelicin O O O O O O OH Sphondin Xanthotoxol Bergaptol Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Characterization of p-coumaroyl CoA 2’-hydroxylase MeO COSCoA COSCoA HO OH HO p-coumaroyl-CoA Feruloyl-CoA C2’H - Diox Arabidopsis thaliana Kai et al., F6’H - Diox Plant J 2008, 55(6), 989-999 Ruta graveolens MeO Vialart et al. Plant J, 2012, 70(3), 460-470 COSCoA COSCoA HO OH HO OH Spontaneous lactonization Spontaneous lactonization MeO Umbelliferone HO O O HO O O Scopoletin Ruta graveolens To furanocoumarins Vialart et al. Plant J, 2012, 70(3), 460-470 STOLON/AFERP Symposium, July 15-17th 2015 Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox Cinnamic acid - - - - p-Coumaric acid - - - - Ferulic acid - - - - Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - - In vitro activity / heterologous expression in E. coli *: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox Cinnamic acid - - - - p-Coumaric acid - - - - Diox 1 & 2: Ferulic acid - - - more - than 90% identity Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - - In vitro activity / heterologous expression in E. coli *: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Comparison of 2-OGD-dioxygenases specialized in the metabolization of cinnamic acids Arabidopsis Ruta Ruta Pastinaca compounds tested thaliana graveolens graveolens sativa for substrate F6'H1* Diox 1** Diox 2 Diox Cinnamic acid - - - - p-Coumaric acid - - - - Ferulic acid - - - - Caffeic acid - - - - Cinnamoyl CoA - - - - p-Coumaroyl CoA - + - + Feruloyl CoA + + + - Caffeoyl CoA - - - - In vitro activity / heterologous expression in E. coli *: Kai et al, Plant J 2008, 55(6), 989-999 **:Vialart et al., Plant J, 2012, 70(3), 460-470 AFERP-STOLON 15-17 juillet 2015 – Lyon Prenyltransferase Cinnamic acid p-Coumaroyl CoA C-Prenyltransferase Umbelliferone OH HO OH HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins Umbelliferone prenyltransferases O O O O O Karamat et al., Plant J, 2014, 77(4), 627-638 O OH Munakata et al., Plant Phys, 2014 H Psoralen OH Angelicin O O O O O O OH Sphondin Xanthotoxol Bergaptol Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Parsley prenyltransferase C-Prenyltransferase Umbelliferone HO O O Osthenol Demethylsuberosin HO O O HO O O 100:5 To linear To angular furanocoumarins furanocoumarins O O O Umbelliferone prenyltransferase Karamat et al., Plant J, 2014, 77(4), 627-638 STOLON/AFERP Symposium, July 15-17th 2015 Parsley prenyltransferase C-Prenyltransferase Umbelliferone HO O O Osthenol Demethylsuberosin HO O O HO O O 100:5 To linear To angular furanocoumarins furanocoumarins O O O Ruta graveolens Umbelliferone prenyltransferase Karamat et al., Plant J, 2014, 77(4), 627-638 STOLON/AFERP Symposium, July 15-17th 2015 P450s P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Apiaceae : CYP71 AJ OH HO OH HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin HO O O HO O O Marmesin (+) Columbianetin Psoralen synthase Angelicin synthase CYP71AJ1, Ammi majus CYP71AJ4, Pastinaca sativa CYP71AJ2, Apium graveolens (celery) O O O O O O CYP71AJ3, Pastinaca sativa (parsnip) OH Larbat et al., JBC, (2009) celery Ammi majus H Larbat et al., JBC, 281, 542 (2007) OH Psoralen Larbat et al., JBC, 284, 4776 (2009) Angelicin Dueholm et al. BMC Evol Biol 15:122 (2015) O O O parsnip Thapsia O O O OH Sphondin Xanthotoxol Bergaptol Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Apiaceae : CYP71 AJ OH HO OH CYP71 xx Non apiaceae : CYP82 HO O O HOOC HOOC COSCoA Osthenol Demethylsuberosin HO O O HO O O Marmesin (+) Columbianetin O O O O O O Ammi majus OH H Psoralen OH Angelicin O O O parsnip O O O Psoralen 8-monooxygenase Sphondin Xanthotoxol Pimpinellin CYP71xx4, Pastinaca sativa (parsnip) OMe MeO MeO C.Krieger (PhD) O O O O O O OH O O O Xanthotoxin Xanthotoxin 5-monooxygenase CYP71xx1, Ammi majus Isopimpinellin CYP71xx6, Pastinaca sativa (parsnip) O O O OH OMe OMe C.Krieger (PhD), S. Rosselli (Post-doc) Biosynthetic routes CYP82 , Non apiaceae to furanocoumarins O O O O O O A. Dugrand (PhD) OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Crucial steps required to close the road ? P450 Cinnamic acid p-Coumaroyl CoA Umbelliferone Dioxygenase OH HO OH C-Prenyltransferase HO O O O-Methyltransferase HOOC Multiple steps HOOC COSCoA Osthenol reaction Demethylsuberosin HO O O HO O O Angular Linear Marmesin (+) Columbianetin furanocoumarins furanocoumarins O O O O O O OH H Psoralen OH Angelicin O O O O O O OH Sphondin Xanthotoxol Bergaptol Pimpinellin OMe MeO MeO O O O O O O O O O OH Bergapten O O O OMe Xanthotoxin Isopimpinellin O O O O O O OH OMe OMe Biosynthetic routes to furanocoumarins O O O O O O OMe OMe STOLON/AFERP Symposium, July 15-17th 2015 Demethylallyllated compounds Geranylated compounds 8-MOP-Derived 5-MOP-Derived Psoralen O OH HO O O O O O O O O O O O O O O O O O O OH OH HO O O O O O O O O O O Isoimperatorin Oxypeucedanin Oxypeucedanin Heraclenol Heraclenin Imperatorin O O O O O O hydrate OH Xanthotoxol Bergaptol O OH OH O O O OMe O O O O O O O O O O O O O O O O O O O 8-geranyloxypsoralen Bergamottin Epoxybergamottin 6’,7’-dihydroxy OMe Xanthotoxin Bergapten 8-MOP bergamottin 5-MOP OMe Citrus fruit O O O OMe Isopimpinellin 5-8-MOP 5-8-MOP-Derived C. paradisi C. maxima C. medica O O OMe OMe OMe O O O O O O O O O O O O O O O OMe O O O O OH HO C. aurantium C. aurantifolia C. limon O Cnidilin Cnidicin Phellopterin Byakangelicol Byakangelicin C. bergamia C. micrantha C. jambhiri STOLON/AFERP Symposium, July 15-17th 2015 Quantitative distribution of furanocoumarins in Citrus pulp C. Maxima C. Paradisi (pummelo) (grapefruit) C. Aurantifolia (lime) How to annihilate the Cinnamic acid p-Coumaroyl CoA biosynthesis of OH HO OH furanocoumarins in Umbelliferone Citrus ? HO O O HOOC HOOC COSCoA Demethylsuberosin HO O O Linear Marmesin furanocoumarins C. Paradisi (Grapefruit) O O O Gene editing strategy OH (CRISPR / Cas9) Psoralen O O O Xanthotoxol Bergaptol OH C.