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A Method of Increasing the Total Or Soluble Carbohydrate Content Or (19) TZZ ¥___T (11) EP 2 348 116 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) C12N 9/90 (2006.01) 15.07.2015 Bulletin 2015/29 (21) Application number: 10184961.0 (22) Date of filing: 12.05.2004 (54) Amethod of increasing the total or soluble carbohydrate contentor sweetness of an endogenous carbohydrate by catalysing the conversion of an endogenous sugar to an alien sugar Verfahren zur Erhöhung des gesamten oder löslichen Kohlenhydratgehalts oder der Süße eines endogenen Kohlenhydrat durch die Katalysierung der Umwandlung eines endogenen Zuckers in einen Fremdzucker Procédépour augmenter la teneur totale ou soluble en hydrocarburesou le goût sucré d’unhydrocarbure endogène par catalyse de la conversion d’un saccharide endogène en un saccharide étranger (84) Designated Contracting States: US-A- 4 326 036 AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR • SEVENIER R ET AL: "HIGH LEVEL FRUCTAN ACCUMULATION IN A TRANSGENIC SUGAR (30) Priority: 12.05.2003 AU 2003902253 BEET", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. (43) Date of publication of application: 16, 1 January 1998 (1998-01-01), pages 843-846, 27.07.2011 Bulletin 2011/30 XP000877303, ISSN: 1087-0156, DOI: DOI: 10.1038/NBT0998-843 (62) Document number(s) of the earlier application(s) in • HAMERLIDENES ET AL:"Transgenic expression accordance with Art. 76 EPC: of trehalulose synthase results in high 04732254.0 / 1 623 031 concentrationsof the sucrose isomer trehalulose in mature stems of field-grown sugarcane", (73) Proprietor: THE UNIVERSITY OF QUEENSLAND PLANT BIOTECHNOLOGY JOURNAL, vol. 9, no. Brisbane, 1, January 2011 (2011-01), pages 32-37, Queensland 4067 (AU) XP002642324, ISSN: 1467-7644 • BORNKE F ET AL: "Tailoring plant metabolism (72) Inventors: forthe production of novel polymers and platform • Birch, Robert George chemicals", CURRENT OPINION IN PLANT Brisbane, Queensland 4074 (AU) BIOLOGY, QUADRANT SUBSCRIPTION • Wu, Luguang SERVICES, GB, vol. 13, no. 3, 1 June 2010 Brisbane, Queensland 4069 (AU) (2010-06-01), pages353-361, XP027081081, ISSN: 1369-5266, DOI: DOI:10.1016/J.PBI.2010.01.005 (74) Representative: Jones, Elizabeth Louise [retrieved on 2010-02-17] Dehns • VAN BEILEN J B: "Transgenic plant factories for St Bride’s House the production of biopolymers and platform 10 Salisbury Square chemicals", BIOFUELS, BIOPRODUCTS AND London BIOREFININGJOHN WILEY &SONS LTD.UK, vol. EC4Y 8JD (GB) 2, no. 3, May 2008 (2008-05), pages 215-228, ISSN: 1932-104X (56) References cited: EP-A1- 0 952 222 WO-A1-02/18603 WO-A1-95/13389 WO-A2-01/14595 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 2 348 116 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 348 116 B1 • GRESSEL ETAL: "Transgenics are imperativefor Remarks: biofuel crops", PLANT SCIENCE, ELSEVIER Thefile contains technical information submitted after IRELANDLTD, IE, vol. 174, no. 3, 3 December 2007 the application was filed and not included in this (2007-12-03), pages246-263, XP022489458, ISSN: specification 0168-9452, DOI: 10.1016/J.PLANTSCI. 2007.11.009 • MORANDINI ET AL: "Rethinking metabolic control", PLANT SCIENCE, ELSEVIER IRELAND LTD, IE, vol. 176, no. 4, 1 April 2009 (2009-04-01) , pages 441-451, XP025962006, ISSN: 0168-9452, DOI: 10.1016/J.PLANTSCI.2009.01.005 [retrieved on 2009-01-20] 2 EP 2 348 116 B1 Description FIELD OF THE INVENTION 5 [0001] THIS INVENTION relates generally to methods for increasing the total soluble carbohydrate content of a plant tissue by producing a sugar-metabolizing enzyme that catalyzes the conversion of an endogenous sugar, sucrose, (one that is normally produced in the plant) to an alien sugar (one that is not normally produced in the plant at the same developmental stage). The invention also relates to transgenic plants and plant parts (sink tissue) that produce a sugar- metabolizing enzyme to yield an alien sugar, with the consequence of an increased total soluble carbohydrate content, 10 and to the use of said plants and plant parts to produce fermentable carbohydrates and other products derived therefrom. [0002] Bibliographic details of the publications referred to in this specification are collected at the end of the description. BACKGROUND OF THE INVENTION 15 [0003] Plants are the primary source of renewable bioenergy, biomaterials and feedstocks for industrial biotransfor- mations. The yield and concentration of desired sugars in plants are key determinants of the technical and economic feasibility of downstream industrial processes. However, the metabolic networks of plants for biosynthesis of sugars show substantial internal buffering and redundancy, with the consequence that alteration to a key gene in metabolism of a sugar commonly results in no useful change to the harvestable yield of the sugar (Moore, 1995; Nguyen-Quoc and 20 Foyer, 2001; Fernie et al., 2002). For example, Botha et al. (2001) have shown that 70% reduction in activity of a key enzyme in sucrose breakdown (acid invertase) results in no significant change in sucrose yield or purity in transgenic sugarcane. [0004] Sucrose isomerases are enzymes produced by organisms including various microbes, with the capability to convert the disaccharide sucrose into isomers such as isomaltulose (palatinose) or trehalulose. Sucrose isomerases 25 vary in their properties including the disaccharide reaction products, the proportion of monosaccharides such as glucose and fructose in the reaction products, the kinetic properties of the enzymes, the optimal reaction conditions, and the sensitivity of the enzyme to variations from the optimal conditions (Veronese and Perlot, 1999). [0005] Sucrose is the major intermediary in carbon flux between source (photosynthetic) tissues and sink (growth and storage) tissues within plants, and it is the primary storage product in certain plants such as sugarcane and sugarbeet. 30 Several reports indicate that expression of introduced sucrose isomerase genes in plants can result in the conversion of sucrose to an isomer such as isomaltulose, and it has been envisaged that this conversion could be beneficial for the industrial production of the isomer. The emphasis has been towards the high-level or complete conversion of sucrose to the isomer as a desired industrial product (Birch and Wu, 2002; Börnke et al., 2002b), or as a precursor for in planta conversion to derived industrial materials (Kunz et al., 2002). 35 [0006] It has also been envisaged that the conversion could be lethal to plant cells by conversion of an essential carbon and energy reserve to an unavailable form, with applications in cell ablation for purposes such as engineered male sterility (Bornke and Sonnewald, 2001). Indeed, several reports indicate that sucrose isomerase transgene expression is harmful to plant development and yield, causing severe growth abnormalities, reduced starch content, and reduced soluble carbohydrate content (Börnke et al., 2002a, b). 40 [0007] In tobacco, there were severe and damaging effects on plant development due to constitutive expression from the CaMV35S promoter of a sucrose isomerase gene fused to the potato proteinase inhibitor II signal peptide to direct the sucrose isomerase enzyme to the extracellular (apoplasmic) space. Low isomaltulose levels (0.3 to 0.6 mM m-2 of leaf tissue) were detected, equivalent to about 20% to 44% of the normal carbohydrate levels in leaf starch or 10 to 45 times the normal low transitory sucrose levels in this source tissue. Effects on stored carbohydrates in sink tissues were 45 not reported. Growth of leaves and other organs was severely disrupted, and the plants were unable to reproduce (Börnke et al., 2002a). [0008] In potato, expression of the same apoplasm-targeted sucrose isomerase from the tuber-specific patatin B33 promoter resulted in plants without apparent adverse effects on growth and development. Isomaltulose yields were again low (10-15 mmol g-1 fresh weight of tuber tissue, equivalent to about 4% to 5% of the normal stored carbohydrate levels 50 in tuber starch), and slightly below the usual sucrose levels in this starch-storing sink tissue. Furthermore, because of accompanying decreases in contents of sucrose, hexoses and starch, yields of total soluble carbohydrate (excluding starch) and total fermentable carbohydrate (including starch) were decreased in the modified lines (Börnke et al., 2002b). [0009] To overcome these problems, the present inventors conceived a novel approach based upon combining (i) a sucrose isomerase enzyme, especially a highly efficient sucrose isomerase such as UQ68J; (ii) use of a plant species 55 such as sugarcane that accumulates sucrose as the stored carbohydrate; andiii) ( targeting the introduced sucrose isomerase to the sucrose storage compartment, for example the large vacuole of sucrose storage parenchyma within the mature culm in the case of sugarcane. Because isomaltulose is not metabolized in plants, the present inventors hypothesized that, in contrast to sucrose, it might not be subject to ’futile cycles’ of degradation and synthesis in the 3 EP 2 348 116 B1 mature storage tissues, which have the potential to decrease storage efficiency and harvestable yield. Therefore, the inventors’ approach was designed to achieve higher yields of soluble carbohydrates and fermentable carbohydrates in the modified plants, in contrast with the reduction of these yields reported from previous approaches. Consistent with this hypothesis, it was found that isomaltulose concentrations above 500 mM in juice can be achieved in sugarcane, by 5 expressing a sucrose isomerase (e.g., the highly efficient sucrose isomerase UQ68J), targeted to the sucrose storage vacuoles in mature stem parenchyma.
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