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Ep 2257630 B1 (19) TZZ ¥Z_T (11) EP 2 257 630 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/82 (2006.01) 28.09.2016 Bulletin 2016/39 (86) International application number: (21) Application number: 09713735.0 PCT/IB2009/000500 (22) Date of filing: 27.02.2009 (87) International publication number: WO 2009/106985 (03.09.2009 Gazette 2009/36) (54) BIOSYNTHETIC ENGINEERING OF GLUCOSINOLATES BIOSYNTHETISCHE KONSTRUKTION VON GLUCOSINOLATEN MISE AU POINT PAR BIOSYNTHÈSE DE GLUCOSINOLATES (84) Designated Contracting States: • ZANGYUN-XIANG ET AL: "Metabolic engineering AT BE BG CH CY CZ DE DK EE ES FI FR GB GR of aliphatic glucosinolates in Chinese cabbage HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL plants expressing Arabidopsis MAM1, CYP79F1, PT RO SE SI SK TR and CYP83A1." BMB REPORTS 30 JUN 2008, vol. 41, no. 6, 11 February 2008 (2008-02-11), pages (30) Priority: 27.02.2008 US 31914 472-478, XP002545714 ISSN: 1976-6696 • MIKKELSEN MICHAEL DALGAARD ET AL: (43) Date of publication of application: "Metabolic engineering of valine- and 08.12.2010 Bulletin 2010/49 isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from (73) Proprietor: University of Copenhagen cassava." PLANT PHYSIOLOGY (ROCKVILLE), 1017 Copenhagen K (DK) vol. 131, no. 2, February 2003 (2003-02), pages 773-779, XP002545715 ISSN: 0032-0889 (72) Inventors: • CHEN S ET AL: "UPDATE ON GLUCOSINOLATE • GEU FLORES, Fernando METABOLISM AND TRANSPORT" PLANT 01239 Cambridge, MA (US) PHYSIOLOGY AND BIOCHEMISTRY, • NIELSEN, Morten, Thrane GAUTHIER-VILLARS, PARIS, FR, vol. 39, no. 9, 1 DK-1871 Copenhagen (DK) September 2001 (2001-09-01), pages 743-758, • MIKKELSEN, Michael, Dalgaard XP001085172 ISSN: 0981-9428 DK-1871 Copenhagen (DK) • MAJSE NAFISI ET AL: "Cytochromes P450 in the • HALKIER, Barbara, Ann biosynthesis of glucosinolates and indole DK-1871 Copenhagen (DK) alkaloids" PHYTOCHEMISTRY REVIEWS, • MØLDRUP, Morten, Emil KLUWER ACADEMIC PUBLISHERS, DO, vol. 5, DK-1871 Copenhagen (DK) no. 2-3, 28 October 2006 (2006-10-28), pages 331-346, XP019453402 ISSN: 1572-980X (74) Representative: Kremer, Simon Mark et al • GEU-FLORES FERNANDO ET AL: "Towards Mewburn Ellis LLP engineering glucosinolates into non-cruciferous City Tower plants" PLANTA (BERLIN), vol. 229, no. 2, 40 Basinghall Street January 2009 (2009-01), pages 261-270, London EC2V 5DE (GB) XP002545716 ISSN: 0032-0935 • GEU-FLORESFERNANDO ET AL: "Glucosinolate (56) References cited: engineering identifies gamma-glutamyl peptidase"NATURE CHEMICALBIOLOGY, vol. 5, no. 8, August 2009 (2009-08), pages 575-577, XP002545717 ISSN: 1552-4450(print) 1552-4469(ele Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 257 630 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 257 630 B1 Description Technical field 5 [0001] The present invention relates generally to genes and polypeptides which have utility in reconstituting or modifying glucosinolate (GSL) production or hydrolysis in host cells. The invention further relates to systems, methods and products employing the same. Background art 10 Biosynthesis of GSLs [0002] Glucosinolates are amino acid-derived secondary metabolites present in the Brassicales order, including the agriculturally important cruciferous vegetables, e.g. broccoli, and oilseed rape. 15 [0003] The molecule consists of a ’GSL skeleton’ and a variable side chain derived from an amino acid. In the majority of Capparalean families, GSLs have phenolic side chains derived from phenylalanine (PHE) and branched aliphatic side chains, derived from valine and leucine. However, the predominant GSLs in the Brassicaceae possess side chains derived from chain elongated forms of methionine (MET) and PHE. Lower amounts of GSLs with indolylic side chains derived from tryptophan (TRP) also occur. The MET derived (’aliphatic’) GSLs exhibit considerable variation in the length 20 and structure of the side chain. [0004] The biosynthesis of aliphatic GSLs can be considered in three parts: • Firstly, the development of chain elongation homologues of MET. 25 • Secondly the synthesis of the GSL skeleton. This is called ’core biosynthesis’, and includes at least five different enzymatic steps. The enzymes involved include two cytochromes P450 enzymes from the CYP79 and CYP83 families, which respectively catalyze the conversion of precursor amino acids to the corresponding oximes, followed by oxidation of the oximes to reactive compounds. In a sulfur donation step, which may be either non-enzymatic or involve a glutathione-S-transferase-type of protein, S-alkyl thiohydroximates are formed. The last three enzymatic 30 steps include a C-S lyase, which converts S-alkylthiohydroxamates to thiohydroxamates, a glucosyltransferase, which glucosylates thiohydroxamates to yield desulfoglucosinolates, and a sulfotransferase, which adds a sulfate moiety to desulfoglucosinolates to produce GSLs. Each of the mentioned enzymatic steps is catalyzed by different enzymes of a gene family, with the possible exception of the C-S lyase step, which may have functional homologues although only the SUR1 gene has been characterized (see e.g. Mikkelsen et al (2004) Plant J.; Mikkelsen MD, Naur 35 P, and Halkier BA (2004) 37, 770-777) • Thirdly side chain modifications. [0005] Taking the glucosinolate 4-methyl-sulfinyl-butyl glucosinolate (4-MSB) as an example, 4-MSB is derived from 40 dihomomethionine (DHM), which is produced from MET through chain-elongation mechanisms similar to those of the branched-chain amino acid biosynthesis (Fig. 10). This procedure requires five distinct activities, of which three (MAM1, BCAT4 and BCAT3) have been characterized (Schuster et al., 2006; Kroymann et al., 2001: Knill et al., 2007). Secondly, DHM is then converted to 4-methylthiobutyl glucosinolate (4-MTB). This requires a minimum of five distinct activities, which have been characterized previously (Fig. 11; Hansen et al., 2001; Bak and Feyereisen, 2001; Mikkelsen et al., 45 2004; Piotrowski et al., 2004). Finally, 4-MTB is converted to 4-MSB through one of several flavin-containing monoox- ygenases (Hansen et al., 2007; prior filed unpublished PCT/IB2007/002588). [0006] As an example of a glucosinolate that does not undergo amino acid chain elongation or secondary modifications in its biosynthesis, benzylglucosinolate (BGSL) is directly synthesized from PHE via the core structure pathway. 50 GSLs and their economic and biological importance [0007] Aliphatic GSLs in cruciferous crops are of economic and biological importance, largely as a result of hydrolytic products released upon tissue disruption. GSLs and their breakdown products are often collectively referred to as ’mustard oils’. GSLs are degraded by endogenous myrosinases, or by microbial organisms in the gut. Isothiocyanates 55 derived from methylsulfinylalkyl GSLs via the activity of myrosinases are associated with protection from carcinogens (Zhang et al. (1992). Proc. Natl. Acad. Sci. USA 89, 2399-2403). In particular, 4-methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding GSL 4-MSB (glucoraphanin), has previously been found to be a potent inducer of "phase 2" detoxifying enzymes, which have a role in detoxification of exogenous compounds (Zhang et al. 2 EP 2 257 630 B1 (1992) The Plant Cell 18: 1524-1536; Juge et al., 2007). The corresponding heptyl- and octyl- GSLs have also been found to hold cancer preventive properties - for example 7-methylthioheptyl glucosinolate (7-MSH) and 8-methylthiooctyl glucosinolate (8-MSO) are potent cancer-preventive agents (Rose et al (2000). Carcinogenesis 21. 1983-1988). [0008] Furthermore, sulphoraphane has been found to have an effect on bacteria that cause ulcers and stomach 5 cancer (Fahey et al. (2002) PNAS 99. 7610-7615). [0009] Moreover, many aliphatic GSLs have been implicated in mediating plant-herbivore interactions (Giamoustaris A & Mithen, R.F. (1995) Ann Appl Biol. 126. 347-363). [0010] Additionally. GSLs and plants containing them have a role in biofumigation, wherein (for example) hydrolysis of glucosinolates in Brassica green manure or rotation crops leads to the release of biocidal compounds into the soil 10 and the suppression of soil-bome pests and pathogens (J. A. Kirkegaard and M. Sawar, Plant and Soil, 201, 71-89.1998). [0011] Although glucosinolate-derived isothiocyanates have been known for decades as antibacterial compounds, interest in their use as therapeutic agents has become evident following identification of several human pathogens as effective targets, among them Escherichia coli. Staphylococcus aureus. and more recently Helicobacter pylori. Since isothiocyanates are unstable compounds, production of glucosinolates as their stable precursors is an attractive alter- 15 native to their direct production. Glucosinolates can be purified from natural sources, but their purification is complicated by the fact that different glucosinolates generally co-occur in a single plant tissue. [0012] Unfortunately commercially important GSLs are found in only relatively small number of commonly grown species - for example 4-MSB has been found in a limited number of species in only five families of the
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