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WO 2009/152285 Al (12) INTERNATIONALAPPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 17 December 2009 (17.12.2009) WO 2009/152285 Al (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, C12N 15/82 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/US2009/046968 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 11 June 2009 ( 11.06.2009) SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 61/060,789 11 June 2008 ( 11.06.2008) US ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, (71) Applicant (for all designated States except US): SYN- ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, GENTA PARTICIPATIONS AG [CH/CH]; 4058 MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR), Schwarzwaldallee 215, CH-4058 Basel (CH). OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (72) Inventors; and (75) Inventors/Applicants (for US only): HALL, Richard, J. Declarations under Rule 4.17: [US/US]; 3054 Cornwallis Road, Research Triangle Park, — as to applicant's entitlement to apply for and be granted NC 27709 (US). WARNER, Simon [GB/US]; 3054 a patent (Rule 4.1 7(U)) Cornwallis Road, Research Triangle Park, NC 27709 (US). PRATA, Rogerio [US/US]; 201 Cheaspeake Way, — as to the applicant's entitlement to claim the priority of Chapel Hill, NC 2751 6 (US). the earlier application (Rule 4.1 7(Hi)) (74) Agent: FRYE, Cathy; 3054 Cornwallis Road, Research Published: Triangle Park, NC 27709 (US). — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every — with sequence listing part of description (Rule 5.2(a)) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (54) Title: COMPOSITIONS AND METHODS FOR PRODUCING FERMENTABLE CARBOHYDRATES IN PLANTS (57) Abstract: Provided herein are methods for producing fermentable sugar obtained from a plant tissue. The methods include providing transgenic plant material comprising one or more locked carbohydrates and contacting plant material with an enzyme capable of converting the locked carbohydrate into a fermentable sugar. The methods are useful for providing sugar or sugar pre cursors for several industrial purposes including ethanol production. The invention also encompasses plants and plant parts that produce a lock enzyme to yield a locked carbohydrate, with the consequence of accumulating the locked carbohydrate in the plant. The inventon also encompasses providing a key enzyme able to convert locked carbohydrates to fermentable sugars. Key enzymes can be provided by transgenic plants or plant parts, transgenic microbes, transgenic yeast, microbes or yeast. COMPOSITIONS AND METHODS FOR PRODUCING FERMENTABLE CARBOHYDRATES IN PLANTS REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of "71 825USPSP2 sequence Hsting.txt, created June 10, 2009, and a size of 313KB. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein. FIELD OF THE INVENTION This invention relates to plant molecular biology, particularly to methods and compositions for improving plants for obtaining commercially desirable harvested plant material, particularly for ethanol production. BACKGROUND OF THE INVENTION Plant biomass is comprised of sugars and represents the greatest source of renewable hydrocarbon on earth. Unlike other renewable energy sources, biomass can be converted directly into liquid fuels. The two most common types of biofiiels are ethanol (ethyl alcohol) and biodiesel. Ethanol is an alcohol, which can be produced by fermenting any biomass high in carbohydrates (starches, sugars, or celluloses) once fermentable sugars have been obtained from the biomass material. Sugars generated from degradation of plant biomass could provide plentiful, economically competitive feedstocks for fermentation to produce chemicals, plastics, and fuels or any other product of interest. Fuel ethanol could be made from crops which contain starch such as feed grains, food grains, and tubers, such as potatoes and sweet potatoes. Crops containing sugar, such as sugar beets, sugarcane, and sweet sorghum also could be used for the production of ethanol. Sugar, in the form of raw or refined sugar, or as sugar in molasses requires no pre-hydrolysis (unlike corn starch) prior to fermentation. Consequently, the process of producing ethanol from sugar is simpler than converting corn starch into ethanol. The yield and concentration of desired carbohydrates 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, Australian Journal of Plant Physiology 22: 661- 679 (1995); Nguyen-Quoc and Foyer, J of Experimental Botany 52: 881-889 (2001); Fernie et al., Trends in Plant Science 7: 35-41 (2002)). SUMMARY OF THE INVENTION Provided herein are methods for producing locked carbohydrates in a plant tissue by providing one or more carbohydrate-metabolizing enzymed that catalyze the conversion of an endogenous carbohydrate to a non-native carbohydrate. The invention encompasses plants and plant parts that produce one or more carbohydrate-metabolizing enzymes to yield a locked carbohydrate, with the consequence of increasing the total locked carbohydrate content in the plant. Further provided are hydrolytic enzymes (key enzymes) for converting the locked carbohydrate into a fermentable sugar. Fermentable sugars are used for a variety of industrial purposes including the production of ethanol. DETAILED DESCRIPTION OF THE INVENTION Overview Plants accumulating large amounts of sugar are valuable as fermentation feedstocks for the downstream production of commercially-useful products. However, plants have various mechanisms to regulate die flow of sugars, therefore, sugar accumulation is limited in many plants. Plants contain both internal receptors and membrane-bound external receptors for monitoring sugar biosynthesis, transport, and uptake (reviewed in Lalonde et aJ. (1999) Plant Cell 11:707-726). Intracellular receptors modulate metabolic processes such as photosynthesis. Extracellular receptors sense external sugar concentrations in order to control sugar influx from the surrounding environment. Thus, the plant cells are capable of maintaining sufficient levels of sucrose by regulating metabolic processes and sugar uptake. Provided herein is a method for producing locked storage carbohydrates in plants so that they cannot be metabolized by the plant. The methods comprise introducing into the plant or plant part one or more enzymes capable of converting an endogenous sugar into a locked carbohydrate. By "endogenous sugar" or "native sugar" is intended a sugar that is normally produced by a particular variety of plant. In contrast, a "locked carbohydrate" or a "locked sugar" is one that is not produced under normal conditions of growth or development of that variety of plant or in a particular plant part or plant organelle. Expression of an enzyme capable of converting the endogenous sugar into a locked carbohydrate (which is herein referred to as a "lock enzyme") in plant will allow accumulation of the locked carbohydrates in the plant. Because these locked carbohydrates are not metabolized in plants, they are unlikely to be subject to "futile cycles" of degradation and synthesis in the mature storage tissues, which have the potential to decrease storage efficiency and harvestable yield. Many of these oligosaccharides, polysaccharides, or monosaccharides will also evade the plant's carbohydrate detecting mechanisms, such as sucrose sensing, such that native and non- native carbohydrate synthesis may occur to compensate for decreases in endogenous carbohydrates which have been diverted into the locked carbohydrate storage pathway. Recently, Wu and Birch, infra, have demonstrated that converting sucrose to the non-metabolized sucrose isomer isomaltulose allows accumulation of isomaltulose and sucrose providing combined sugar production in sugarcane. Isomaltulose is currently used to manufacture sugar alcohols consumed as low-calorie sweeteners (Schiweck et al. (1991) In F. W. Lichtenthaler (ed.), Carbohydrates as organic raw materials. Wiley-VCH, Weinheim, Germany), and it is an attractive renewable starting material for the manufacture of biosurfactants and biocompatible polymers (Lichtenthaler (2002)
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