US 2011 O2O1059A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0201059 A1 Hall et al. (43) Pub. Date: Aug. 18, 2011

(54) COMPOSITIONS AND METHODS FOR CI2P 7/16 (2006.01) PRODUCING FERMENTABLE CI2P 7/02 (2006.01) CARBOHYDRATES CI2P 19/00 (2006.01) CI2P 19/14 (2006.01) (76) Inventors: Richard J. Hall, Durham, NC CI2P 19/18 (2006.01) (US); Simon Warner, Chapel Hill, CI2P 19/20 (2006.01) NC (US); Rogerio Prata, Chapel Hill, NC (US) (52) U.S. Cl...... 435/96; 435/98; 800/320.1; 800/320; 800/298; 435/160; 435/162:435/155; 435/72: (21) Appl. No.: 12/997,581 435/97; 435/99 (22) PCT Filed: Jun. 11, 2009 (57) ABSTRACT (86). PCT No.: PCT/US2O09/046968 Provided herein are methods for producing fermentable sugar obtained from a plant tissue. The methods include providing S371 (c)(1), transgenic plant material comprising one or more locked (2), (4) Date: Mar. 4, 2011 carbohydrates and contacting plant material with an enzyme capable of converting the locked carbohydrate into a ferment Related U.S. Application Data able Sugar. The methods are useful for providing Sugar or Sugar pre-cursors for several industrial purposes including (60) Provisional application No. 61/060,789, filed on Jun. ethanol production. The invention also encompasses plants 11, 2008. and plant parts that produce a lock enzyme to yield a locked O O carbohydrate, with the consequence of accumulating the Publication Classification locked carbohydrate in the plant. The invention also encom (51) Int. Cl. passes providing a key enzyme able to convert locked carbo CI2P 7/14 (2006.01) hydrates to fermentable Sugars. Key enzymes can be provided CI2P 9/6 (2006.01) by transgenic plants or plant parts, transgenic microbes, AOIH 5/00 (2006.01) transgenic yeast, microbes or yeast. US 2011/020 1 059 A1 Aug. 18, 2011

COMPOSITIONS AND METHODS FOR quence of increasing the total locked carbohydrate content in PRODUCING FERMENTABLE the plant. Further provided are hydrolytic enzymes (key CARBOHYDRATES enzymes) for converting the locked carbohydrate into a fer mentable Sugar. Fermentable Sugars are used for a variety of REFERENCE TO SEQUENCE LISTING industrial purposes including the production of ethanol. SUBMITTED ELECTRONICALLY 0001. The official copy of the sequence listing is submitted DETAILED DESCRIPTION OF THE INVENTION concurrently with the specification as a text file via EFS-Web, Overview in compliance with the American Standard Code for Infor mation Interchange (ASCII), with a file name of 0007 Plants accumulating large amounts of sugar are “71825USPSP2 sequence listing..txt, created Jun. 10, 2009, valuable as fermentation feedstocks for the downstream pro and a size of 313 KB. The sequence listing filed via EFS-Web duction of commercially-useful products. However, plants is part of the specification and is hereby incorporated in its have various mechanisms to regulate the flow of sugars, there entirety by reference herein. fore, Sugar accumulation is limited in many plants. Plants contain both internal receptors and membrane-bound exter FIELD OF THE INVENTION nal receptors for monitoring Sugar biosynthesis, transport, 0002 This invention relates to plant molecular biology, and uptake (reviewed in Lalonde et al. (1999) Plant Cell particularly to methods and compositions for improving 11:707-726). Intracellular receptors modulate metabolic pro plants for obtaining commercially desirable harvested plant cesses such as photosynthesis. Extracellular receptors sense external Sugar concentrations in order to control Sugar influx material, particularly for ethanol production. from the Surrounding environment. Thus, the plant cells are capable of maintaining Sufficient levels of Sucrose by regu BACKGROUND OF THE INVENTION lating metabolic processes and Sugar uptake. 0003 Plant biomass is comprised of sugars and represents 0008 Provided herein is a method for producing locked the greatest source of renewable hydrocarbon on earth. storage carbohydrates in plants so that they cannot be metabo Unlike other renewable energy sources, biomass can be con lized by the plant. The methods comprise introducing into the verted directly into liquid fuels. The two most common types plant or plant part one or more enzymes capable of converting of biofuels are ethanol (ethyl alcohol) and biodiesel. Ethanol an endogenous Sugar into a locked carbohydrate. By "endog is an alcohol, which can be produced by fermenting any enous Sugar or “native Sugar is intended a sugar that is biomass high in carbohydrates (starches, Sugars, or cellulo normally produced by a particular variety of plant. In con ses) once fermentable sugars have been obtained from the trast, a “locked carbohydrate' or a “locked sugar is one that biomass material. Sugars generated from degradation of plant is not produced under normal conditions of growth or devel biomass could provide plentiful, economically competitive opment of that variety of plant or in a particular plant part or feedstocks for fermentation to produce chemicals, plastics, plant organelle. Expression of an enzyme capable of convert and fuels or any other product of interest. ing the endogenous Sugar into a locked carbohydrate (which 0004 Fuel ethanol could be made from crops which con is herein referred to as a “lock enzyme’) in a plant will allow tain starch Such as feedgrains, food grains, and tubers, such as accumulation of the locked carbohydrates in the plant. potatoes and Sweet potatoes. Crops containing Sugar, Such as Because these locked carbohydrates are not metabolized in Sugar beets, Sugarcane, and Sweet Sorghum also could be used plants, they are unlikely to be subject to “futile cycles' of for the production of ethanol. Sugar, in the form of raw or degradation and synthesis in the mature storage tissues, refined Sugar, or as Sugar in molasses requires no pre-hydroly which have the potential to decrease storage efficiency and sis (unlike corn starch) prior to fermentation. Consequently, harvestable yield. Many of these oligosaccharides, polysac the process of producing ethanol from Sugar is simpler than charides, or monosaccharides will also evade the plant's car converting corn starch into ethanol. bohydrate detecting mechanisms, such as Sucrose sensing, 0005. The yield and concentration of desired carbohy Such that native and non-native carbohydrate synthesis may drates in plants are key determinants of the technical and occur to compensate for decreases in endogenous carbohy economic feasibility of downstream industrial processes. drates which have been diverted into the locked carbohydrate However, the metabolic networks of plants for biosynthesis of storage pathway. Sugars show Substantial internal buffering and redundancy, 0009 Recently, Wu and Birch, infra, have demonstrated with the consequence that alteration to a key gene in metabo that converting Sucrose to the non-metabolized Sucrose iso lism of a Sugar commonly results in no useful change to the mer isomalitulose allows accumulation of isomalitulose and harvestable yield of the sugar (Moore, Australian Journal of Sucrose providing combined Sugar production in Sugarcane. Plant Physiology 22: 661-679 (1995); Nguyen-Quoc and Isomalitulose is currently used to manufacture Sugar alcohols Foyer, J of Experimental Botany 52: 881-889 (2001); Fernie consumed as low-calorie Sweeteners (Schiweck et al. (1991) et al., Trends in Plant Science 7: 35-41 (2002)). In F. W. Lichtenthaler (ed.), Carbohydrates as organic raw materials. Wiley-VCH. Weinheim, Germany), and it is an SUMMARY OF THE INVENTION attractive renewable starting material for the manufacture of 0006 Provided herein are methods for producing locked biosurfactants and biocompatible polymers (Lichtenthaler carbohydrates in a plant tissue by providing one or more (2002) Accounts Chem. Res. 35:728-737). carbohydrate-metabolizing enzymed that catalyze the con 0010. The invention also comprises expressing hydrolytic version of an endogenous carbohydrate to a non-native car enzymes capable of hydrolyzing the locked carbohydrates bohydrate. The invention encompasses plants and plant parts into fermentable sugars. These enzymes are herein referred to that produce one or more carbohydrate-metabolizing as “key enzymes. These enzymes may be of plant, bacterial, enzymes to yield a locked carbohydrate, with the conse fungal, archeal, or other origin; may be provided exogenously US 2011/020 1 059 A1 Aug. 18, 2011

in an enzyme preparation, may be expressed in a separate line steps, but not the exclusion of any other element, integer or of plants or the same line of plants, or in yeast or other step, or group of elements, integers or steps. microbes, or may be provided in microbes that are used in a 0016 “Isolated' means altered “by the hand of man' from fermentative process converting fermentable Sugars, carbo its natural state; i.e., that, if it occurs in nature, it has been hydrates or di, tri, oligo or polymeric saccharides to useful changed or removed from its original environment, or both. fermentation products. Fermentable Sugars are carbohydrates For example, a naturally occurring polynucleotide or a which can be metabolized by conventional organisms such as polypeptide naturally present in a living animal in its natural yeast. Fermentation is the process of energy production in a state is not "isolated, but the same polynucleotide or cell and is not limited to the production of alcohols. Fermen polypeptide separated from the coexisting materials of its tation refers to the breakdown and re-assembly of biochemi natural state is "isolated’, as the term is employed herein. For cals for industry in either aerobic or anaerobic growth condi example, with respect to polynucleotides, the term isolated tions. It generally is the process of energy production in a cell means that it is separated from the chromosome and cell in and is not limited to the production of alcohols. Commonly which it naturally occurs. A sequence is also isolated if sepa known fermentable sugars include but are not limited to rated from the chromosome and cell in which it naturally Sucrose, glucose and fructose. occurs in but inserted into a genetic context, chromosome, or 0011 Commercial applications of the invention include cell in which it does not naturally occur. the production of Sugarcane, Sugar beet, or other plants capable of producing locked carbohydrates. In some embodi Locked Carbohydrates ments, accumulation of the normal storage carbohydrates 0017 Sucrose is the major intermediary in carbon flux (e.g., Sucrose) is not affected in these plants. These plants or between Source (photosynthetic) tissues and sink (growth and their extracts are then treated with enzyme preparations or storage) tissues within plants, and it is the primary storage with microbes or plant materials expressing key enzymes product in certain plants such as Sugarcane and Sugar beet. capable of hydrolyzing locked carbohydrates into ferment Plants have highly adapted sensors and transporters for able Sugar. These Sugars could then be used in fermentation Sucrose, but it is generally considered that these Sucrose sen for many purposes including ethanol production or any other sors and transporters are notable to respond in the same way product of interest. to locked carbohydrates (Loreti et al., Plant Physiol 123: 0012. Thus, the methods of the invention find particular 939-948 (2000); Sinha et al., Plant Physiol 128: 1480-1489 use in the integration of current practices for the cultivation of (2002)). In stark contrast with sucrose, plants are unable to crop plants for the purpose of obtaining a commercially metabolize these locked carbohydrates as a source of carbon desired plant material with increased accumulation of carbo and energy (Sinha et al., 2002). hydrates (locked or native) in a plant, and the use of the crop 0018 While not bound by any particular theory or mecha plant or plant part as a source of biomass for the production of nism, specific alterations to metabolism, involving the con fermentable Sugars, or for agricultural and/or human con version of a carbohydrate normally sensed by the plant into a Sumption. locked carbohydrate that is not perceived in an equivalent 0013 By a “crop plant' is intended any plant that is culti manner, can shift metabolism and result in the accumulation vated for the purpose of producing plant material that is of higher concentrations of locked carbohydrates or, in some sought after by man for either oral consumption, or for utili cases, accumulation of higher concentrations of total carbo Zation in an industrial, pharmaceutical, or commercial pro hydrates. cess. The invention may be applied to any of a variety of 0019. Thus, provided herein are methods for the expres plants, including, but not limited to maize, wheat, rice, barley, sion in a plant of an enzyme capable of converting an endog Soybean, cotton, Sorghum, oats, tobacco, strawberry, Mis enous Sugar into a locked Sugar. The endogenous Sugars pro canthus grass, Switch grass, trees, beans in general, rape/ duced by different plants may differ and as such an canola, alfalfa, flax, Sunflower, safflower, millet, rye, Sugar endogenous Sugar of one plant may be non-native to another. cane, Sugar beet, cocoa, tea, Brassica, cotton, coffee, Sweet Where the Sugar is non-native to a particular plant, that plant potato, flax, peanut, clover, vegetables Such as lettuce, is a candidate for production of a locked carbohydrate using tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, the methods of the invention. Also, a non-native carbohydrate cabbage, cauliflower, broccoli, Brussels sprouts, peppers, and may also refer to a carbohydrate that is not normally produced pineapple; tree fruits such as citrus, apples, pears, peaches, in a particular Subcellular compartment, or in a particular apricots, walnuts, avocado, banana, and coconut; and flowers plant part of the native plant. In this embodiment, the subcel Such as orchids, carnations and roses. lular compartment or the plant part would normally not be 0014. As used herein, the term “plant part or “plant tis capable of metabolizing or transporting out of the compart Sue' includes plant cells, plant protoplasts, plant cell tissue ment or plant part any non-native carbohydrate produced cultures from which plants can be regenerated, plant calli, therein. Thus, it is essential to determine which carbohydrates plant clumps, and plant cells that are intact in plants or parts are endogenously produced by a chosen plant or plant part to of plants such as embryos, pollen, ovules, seeds, leaves, flow thereby deduce which carbohydrates are non-native to the ers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, plant and the type of carbohydrate-metabolizing enzyme(s) root tips, anthers, and the like. that could be useful for producing a locked carbohydrate in 0015 The article “a” and “an are used herein to refer to the plant. one or more than one (i.e., to at least one) of the grammatical 0020 For example, amylose (i.e., a type of starch) is a object of the article. By way of example, “an element’ means polysaccharide consisting of glucosyl residues linked by one or more element. Throughout the specification the word alpha-(1-4) bonds and is the primary carbohydrate storage “comprising.” or variations such as "comprises' or “compris compound found in most plants. Producing starch in plants ing,” will be understood to imply the inclusion of a stated that use Sucrose as their primary carbohydrate storage com element, integer or step, or group of elements, integers or pound, Such as Sugarcane, may permit the accumulation of US 2011/020 1 059 A1 Aug. 18, 2011

starch which would behave as a "locked’ Sugar (i.e., Sugar lose-containing foods and beverages; 5) less hygroscopic; 6) that cannot be metabolized by the plant). simple conversion into Sugar alcohols with other useful prop 0021. The types of carbohydrates endogenously produced erties as foods. by plants can be determined using methods well known to 0025. Sucrose isomerases (E.C. 5.4.99.11) are enzymes persons of skill in the art. These methods include separation produced by organisms including various microbes, with the of Sugars or Sugar derivatives by electrophoresis or chroma capability to convert the disaccharide Sucrose into isomers tography (including paper chromatography, thin layer chro Such as isomalitulose (palatinose) or trehalulose. Sucrose matography, gas chromatography, gas-liquid chromatogra isomerases vary in their properties including the disaccharide phy and high-performance liquid chromatography) reaction products, the proportion of monosaccharides such as techniques. The separated components are typically identi glucose and fructose in the reaction products, the kinetic fied by comparison of separation profiles with standards of properties of the enzymes, the optimal reaction conditions, known identity, or by analytical techniques such as mass and the sensitivity of the enzyme to variations from the opti spectrometry and nuclear magnetic resonance spectroscopy. mal conditions (Veronese and Perlot, Enzyme. Microb. Tech See, for example, reference may be made to Robinson 1980, nol 24: 263-269 (1999)). An isolate of Pantoea dispersa des The Organic Constituents of Higher Plants, Cordus Press, ignated UQ68J is exceptionally efficient in sucrose isomerase North Amherst, USA; Adams et al. 1999, Anal. Biochem. activity (Wu and Birch (2004) J. Appl. Microbiol. 97:93 266: 77-84; Veronese and Perlot 1999, Enz. Microbial Tech. 103). Another exemplary sucrose isomerase has been isolated 24:263-269; Hendrix and Salvucci 2001, J. Insect Physiol. from Erwinia carotovora (GENBANK Accession No. 47:423-432;Thompson et al. 2001, Carbohydrate Res. 33.1: YP049947). 149-16.1; each of which is incorporated by reference herein 0026 Dextrans and Fructans for their teachings regarding analysis of Sugar content. 0027. This invention also comprises transforming plants 0022. The endogenous or the non-native carbohydrates with one or more genes involved in the synthesis of fructans may include monosaccharides, oligosaccharides, Sugar alco or dextrans. These genes may come from plant, bacterial, or hols, Sugar acids, amino Sugars or other variants such as fungal sources and should catalyze the formation of fructose deoxy Sugars, methyl Sugars and the like. Examples of and glucose polysaccharides or polysaccharides comprised of monosaccharides include compounds with formula (CH. Sub. mixed Sugars that are found in cane or Sugar beet, Sweet 2O). Sub.n where n=3 or more but suitably less than 10; Sorghum, mangel-Wurzel or other Sugar crops. The oligo—or including compounds comprising tetroses (e.g., erythrose, polysaccharides produced may also comprise mixed Sugar threose, erythrulose), pentoses (e.g., ribose, arabinose, monomers, for example glucose, fructose, mannose and Xylose, lyxose, ribulose, Xylulose), hexoses (e.g., allose, galactose. altrose, glucose, mannose, gulose, idose, galactose, talose, 0028 By producing these fructan, dextran and mixed fruc psicose, fructose, Sorbose, tagatose), and longer molecules tan and dextran carbohydrates in plants whose primary Stor Such as Sedoheptulose or mannoheptulose. Oligosaccharides, age carbohydrate is sucrose, such as Sugarcane and Sugarbeet, which are formed by linking together two or more monosac a method for sequestering carbohydrates is provided in a form charide units through glycosidic bonds, may be selected from that is non-metabolizable for the plant. Such compounds may disaccharides (e.g., maltose, lactose, gentibiose, melibiose, evade the Sucrose sensing mechanisms of the plant so that trehalose, Sophorose, primeverose, rutinose. Sucrose, isoma they can be accumulated for later enzymatic hydrolysis to litulose, trehalulose, turanose, maltulose, leucrose, 2-keto fermentable Sugars. Sucrose) and longer oligomers such as raffinose, melezitose, 0029 Dextran is a collective name for high-molecular isobemisiose or stachyose. Examples of Sugar alcohols weight polymers composed of D-glucose units connected include, but are not limited to, erythritol, ribitol, mannitol, with alpha-1,6 linkages and various amounts of side branches Sorbitol. Non-limiting examples of Sugar acids include glu linked with alpha-12, alpha-1,3, or alpha-14 to the main conic acid, glucaric acid, glucuronic acid. Non-limiting chains. The enzymes that synthesize these glucans from examples of amino Sugars include glucosamine, galac Sucrose are known by the generic term dextranslucrase (1.6- tosamine. Endogenous or non-native Sugars may also be alpha-D-glucan-6-alpha-glucosyltransferase, EC2.4.1.5.). selected from other variants such as deoxy Sugars and methyl The biosynthesis of dextran has been demonstrated in numer Sugars. Further encompassed are isobemisiose, tagatose, iso ous bacteria, especially in Streptococcus mutans, Leuconos maltotriose, dextrin, cyclodextrins, lactose, Verbascose, amy toc mesenteroides ssp. mesenteroides and Leuconostoc lose, and rhamnose. mesenteroides ssp. dextranicum. Leuconostoc produce the 0023 Isomaltulose and Trehalulose enzyme dextran Sucrase and secrete it into the culture medium 0024. In certain embodiments, the locked carbohydrate is in the presence of sucrose. This enzyme, dextran Sucrase, then an isomer of the endogenous carbohydrate. In one example of synthesizes dextran from the Sucrose Substrate. Dextran has this embodiment, the endogenous Sugar is Sucrose and the applications in several fields. It is used especially in biochem Sugar-metabolizing enzyme is a Sucrose isomerase, which istry as a Support for filtration chromatography on a gel of the converts the Sucrose by isomerization to a locked Sugar Sephadex type. Additionally, in the field of therapeutics, it is selected from isomalitulose and trehalulose. Isomalitulose al used as a substitute for blood plasma (Biochimie generale pha.-D-glucopyranosyl-1,6-D-fructofuranose (also called (General Biochemistry)—J. H. WEIL Masson, 6th edi palatinose) is a nutritive disaccharide, with Sweetness and tion—1990 p. 171). bulk similar to Sucrose. Several characteristics make isoma 0030 Exemplary dextranslucrase enzymes include (but are litulose advantageous over Sucrose for some applications in not limited to): the dextranslucrase from Streptococcus dow the food industry: 1) noncariogenic (not causing dental nei, gtfS gene (Gilmore et al. (1990) Infect. Immun. 58 (8), decay); 2) low glycemic index (useful for diabetics); 3) selec 2452-2458: GENBANK Accession No. P29336); the dex tive promotion of growth of beneficial bifidobacteria among translucrase from Streptococcus mutans, gtfI gene, produces a human intestinal microflora; 4) greater stability of isomaltu 1.3 glucose soluble dextrans (Shiroza et al. (1987) J. Bacte US 2011/020 1 059 A1 Aug. 18, 2011

riol. 169 (9), 4263-4270: GENBANK Accession No. in one glucose unit, which are linked to each other through P08987); and the dextranslucrase from Streptococcus mutans .beta.(2-1) fructosyl-fructose linkages. gtfl) gene, gtfS protein (Terao et al. (1998) FEMS Microbiol. 0036) Inulin molecules are synthesised by the concerted Lett. 161 (2), 331-336; GENBANKAccession No. P49331) action of two enzymes: Sucrose: Sucrose 1-fructosyltrans ferase (in short 1-SST enzyme or 1-SST, used interchange 0031. There is no common class of enzymes identified as ably) and fructan:fructan 1-fructosyltransferase (in short "Leucrose synthases.” Instead leucrose O-alpha-D-glucopy 1-FFT enzyme or 1-FFT, used interchangeably) (Koops and ranosyl-(1->5)-D-fructopyranoside is generally a byproduct Jonker, J of Experimental Botany 45: 1623-1631 (1994); and of dextranslucrase enzyme (EC 2.4.1.5) activity. These Koopos and Jonker, Plant Physiol 110: 1167-1175 (1996)). enzymes act as glucosyltransferases, and normally transfer a Both 1-SST and 1-FFT are active during the period of inulin glucose unit hydrolyzed from a Sucrose molecule to a grow synthesis and accumulation: 1-SST catalyses the initial reac ing dextran chain, or in the case of leucrose to a pyranosyl tion of inulin biosynthesis, the conversion of Sucrose into the fructose molecule yielding leucrose. Glucose can also serve smallest inulin molecule, the trisaccharide kestose (GFF). as an acceptor for the transglycosylase reaction resulting in 1-FFT catalyzes the redistribution of terminal fructosyl units isomaltose (O-C-D-glucopyranosyl-O. 1-6-O-D-glucopyra (-F) between inulin molecules, which results in a stepwise noside) production. Since the 1950's leucrose has been made increase in chain length. enzymatically typically using the Leuconostoc mesenteroides 0037 Amylose dextranslucrase (The Preparation, Properties and Structure of 0038. This invention further comprises transforming the Disaccharide Leucrose Journal of the American Chemical plants with one or more genes involved in the synthesis of Society, Stodolaet.al: (1956)78:2415) followed by chemical novel carbohydrates such as amylosucrase (E.C. 2.4.1.4) to purification. produce amylose in order to accumulate carbohydrates for 0032. Dextranslucrases can be mutated to produce more later fermentation into ethanol. Examples of enzymes that leucrose and or turanose. This has been shown for the dex may catalyze the desired conversions include isomerases, translucrase of Streptococcus oralis (Engineering the Glucan epimerases, mutases, kinases, aldolases, transferases, tran sucrase GTFREnzyme Reaction and Glycosidic Bond Speci sketolases, phosphatases, synthases, carboxylases, dehydro ficity: Toward Tailor-Made Polymer and Oligosaccharide genases and hydrolases. An exemplary amylosucrase Products, Biochemistry 2008, 47, 6678-6684, Hendrik Hell includes the enzyme produced by Neisseria polysacharea muth et. al). Since dextranslucrases can be mutated to produce (GENBANKAccession number Q97EU2), which catalyzes leucrose it is reasonable to assume that other related enzymes the conversion of Sucrose to a linear alpha-1,4-linked glucan. (e.g. amylosucrases EC 2.4.1.4) or unrelated enzymes that 0039. Alternan also produce Sucrose isomers could be mutated to produce 0040 Alternan is a polysaccharide consisting of glucosyl leucrose. Leucrose synthase activity is attributed to any residues linked by alternate alpha-(1-3)/alpha-(1-6) bonds. enzyme that produces leucrose by any mechanism, i.e. This polymer is highly soluble and has very low viscosity. isomerization, transglycosylation, hydrolysis, dehydrogena Accumulation of this polysaccharide in Sugarcane or other tion, reduction, etc. plants may allow the accumulation of excess carbohydrates. 0033. The production of leucrose can be assayed using 0041 Alternansucrase is an enzyme which catalyzes the HPAE chromatography with pulsed amperometric detection conversion of Sucrose to alternan. AlternanSucrase is encoded (PAD). This technique is widely accepted as a preferred by the ASr gene of Leuconostoc mesenteroides described in method for separating carbohydrates and is effective in sepa Jeannes et al. (1954) Am ChemSoc. 76:5041-5052. rating Sucrose isomers. Comparison of peak elution times with known standards is one method for determining the Key Enzymes presence of leucrose. Full verification of the bond arrange 0042. The invention also comprises expressing hydrolytic ments in the carbohydrate molecules can be determined either enzymes capable of hydrolyzing the locked carbohydrates by methylation and acetylation of leucrose followed by GC into fermentable sugars. These enzymes are herein referred to MS, or directly by NMR spectroscopy if the samples are of as “key enzymes. These enzymes may be of plant, bacterial, Sufficient quantity and purity. fungal, archeal, or other origin; may be provided exogenously 0034. Sucrose:sucrose fructosyltransferase (SST) (EC in an enzyme preparation, may be expressed in a separate line 2.4.1.99), 1, 2-B-fructan 1-fructosyltransferase (FFT) (EC of plants or the same line of plants, or in yeast or other 2.4.1.100), 2-3-fructan 1-fructosyltransferase (FFT) (EC 2.4. microbes, or may be provided in microbes that are used in a 1.100), glucan Sucrase, and levan Sucrase (EC 2.4.1.10) are fermentative process to convert the locked carbohydrates into enzymes within the larger class of fructosyltransferases. The fermentable sugars. Yeast or microbes used in the fermenta fructosyltransferase enzymes catalyze the formation of fruc tive process may also be identified or engineered to convert tans composed of fructose linked by B(2-> 1) and/or f3(2->6) locked carbohydrates to energy. Furthermore, the locked car glucoside bonds. Fructosyltransferases may be identified and bohydrates may be converted to a fermentable sugar by isolated from plant, bacterial, or fungal sources. These chemical methods, e.g., by one or more chemicals capable of enzymes may be expressed in plants to accumulate fructans as converting a locked carbohydrate into a fermentable Sugar. storage carbohydrates. Accumulation of this polysaccharide The chemical(s) can be added prior to fermentation, or during (fructan) in Sugarcane or other plants may allow the accumu the fermentation process. lation of excess carbohydrates. 0043 Key enzymes can be isolated from, produced by, 0035) Inulin is a fructan type carbohydrate polymer which provided by a wide range of sources. Recombinant organisms occurs as a polydisperse composition in many plants and can Such as plants, microbes or yeast, can be engineered to also be produced by certain bacteria and fungi. Inulin from express a key enzyme. The recombinant organism can be used plant origin consists of a polydisperse composition of mainly directly in a method of converting locked carbohydrates to linear chains composed of fructose units, mostly terminating fermentable sugars without further purification of the US 2011/020 1 059 A1 Aug. 18, 2011 enzyme. Alternatively, key enzymes may be isolated from Microbiology and Molecular Biology Reviews 2005:306 recombinant organisms for further use in the processing of 325, which is herein incorporated by reference as it describes locked carbohydrates. Native sources for key enzymes may and lists various dextran-hydrolyzing enzymes. also be used either directly (such as yeast or microbes which 0051 Fructanases are fructosydases which catalyze the express a key enzyme normally) or by further isolation of the hydrolysis of fructosidic linkages in fructans to break the key enzyme. A key enzyme may be provided by a source fructan down into simpler Sugar molecules. Fructans can be selected from the group consisting of transgenic plant hydrolyzed to fermentable Sugars through the catalytic activ expressing one or more key enzymes, recombinant microbe ity of fructanases. For example, the fructanase 2,1-B-D-fruc expressing one or more key enzymes, transgenic yeast tan fructanohydrolase IEC 3.2.1.7 can hydrolyze fructan expressing one or more key enzymes, microbe expressing one polymers into fructose monosaccharides which can be fer or more key enzymes, and yeast expressing one or more key mented to form ethanol. enzymes. 0.052 Inulin can be converted to a fermentable carbohy 0044) Isomalitulose and trehalulose can be hydrolyzed by drate using one or more inulase enzymes. Microbial inuli alpha-1,6-glucosidase enzymes. Exemplary glucosidase nases (2,1-3-D-fructan fructanohydrolase IEC 3.2.1.7) are enzymes are set forth in SEQ ID NO:1-6 herein. Additional usually inducible and exo-acting enzymes, which catalyze the sequences are described in U.S. Pat. No. 5,786,140, and in hydrolysis of inulin by splitting off terminal fructosyl units Börnke et al. (2001) Journal of Bacteriology 183(8):2425 2430, each of which is herein incorporated by reference in its (D-fructose). entirety. 0053 Alternans can be hydrolyzed to form fermentable 0045. Dextran-degrading enzymes form a diverse group of Sugars by the activity of a alpha-1,6-glucosidase or alpha-1, different carbohydrases and transferases. These enzymes 3-glucosidase. have often been classified as endo- and exodextranases based on the mode of action and commonly called dextranases and Methods include enzymes Such as dextranases (EC3.2.1.11), glucan 0054 Provided herein are methods for improving the yield 1,6-alpha-D-glucosidases (EC3.2.1.70), glucan-1,6-alpha of carbohydrate in plants by expressing an enzyme capable of isomaltosidases (EC3.2.1.94), dextran 1,6-alpha-isomaltotri converting endogenous carbohydrate into locked carbohy osidases (EC3.2.1.95), and branched-dextran exo-1,2-alpha drate. The locked carbohydrates accumulated in the plants glucosidases (EC3.2.1.115) described herein can be converted to fermentable carbohy 0046 Exodextranases, such as glucodextranase (EC3.2.1. drates using one or more of the key enzymes disclosed herein, 70; glucan 1,6-alpha-glucosidase), catalyze stepwise which can then be used as fermentation feedstocks for etha hydrolysis of the reducing terminus of dextran and derived nol, propanol, butanol or other fuel alcohol, ethanol-contain oligosaccharides to yield solely alpha-D-glucose; i.e., ing beverages (such as malted beverages and distilled spirits), hydrolysis is accompanied by inversion at carbon-1 in Such a and other fermentation products such as foods, nutraceuti way that new reducing ends are released only in the alpha cals, enzymes and industrial materials. The methods forfer configuration. Some bacteria and yeasts are known to pro mentation using plant-derived carbohydrate feedstocks are duce glucodextranases. Dextran-inducible extracellular glu well known to those skilled in the art, with established pro codextranase occurs in Arthrobacter globiformis strains I42 cesses for various fermentation products (see for example and T-3044 (Oguma and Kobayashi (1996).J. Appl. Glycosci. Vogel et al. 1996, Fermentation and Biochemical Engineer 43:73-78; Oguma et al. (1999) Biosci. Biotechnol. Biochem. ing Handbook: Principles, Process Design, and Equipment, 63:2174-2182). Noyes Publications, Park Ridge, N.J., USA and references 0047 Intracellular dextran glucosidases (EC3.2.1.) pro cited therein). Key enzyme proteins could also be incorpo ducing alpha-D-glucose from dextran exist in several strains rated into the ethanol production process downstream of the of Streptococcus mitis (Linder and Sund (1981) Caries Res. feedstock step. It is envisioned that locked carbohydrates 15:436-444; Walker and Pulkownik (1973) Carbohydr. Res. could be harvested and, in the process of making ethanol, the 29:1-14; Walker and Pulkownik (1974) Carbohydr. Res. key enzyme is added during the production process. Key 36:53-66). enzyme proteins could also be incorporated into the ferment 0048. The soil bacterium A. globiformis T6 isomaltodex able Sugar production process downstream of the feedstock tranase (EC3.2.1.94:1,6-alpha-D-glucan isomaltohydrolase) step. It is envisioned that locked carbohydrates could be har is an extracellular exoenzyme capable of hydrolyzing dextran Vested and, in the process of making fermentable Sugar, the by removing successive isomaltose units from the nonreduc key enzyme is added during the production process. ing ends of the dextran chains (Sawai and Yano (1974) J. 0055. In one embodiment, the use of the methods dis Biochem. 75:105-112: Sawai and Nawa (1976) Agric. Biol. closed herein results in a substrate that leads to higher ethanol Chem. 40:1246-1250). yields compared to the ethanol yield from plant material not 0049 Branched dextran exo-1,2-alpha-glucosidase (EC3. accumulating locked carbohydrates. The increase in ethanol 2.1.115) was found in the culture supernatant of the soil yield can be at least about 1%, at least about 2%, at least about bacterium Flavobacterium sp. strain M-73 by Mitsubishi et 3%, at least about 4%, at least about 5%, at least about 6%, at al. (1979) Agric. Biol. Chem. 43:2283-2290. The enzyme had least about 7%, at least about 8%, at least about 9%, at least a strict specificity for 1.2-alpha-D-glucosidic linkage at the about 10%, at least about 20%, at least about 20%, at least branch points of dextrans (containing 12 to 34% of 1.2-alpha about 30%, at least about 40%, at least about 50%, at least linkages) and related polysaccharides producing free D-glu about 60%, at least about 70%, at least about 80%, at least cose as the only reducing Sugar. about 90%, at least about 100%, at least about 2-fold, at least 0050. A list of additional exemplary microbial dextran about 3-fold, at least about 4-fold, at least about 5-fold, or hydrolyzing enzymes and their substrate specificities and greater. Even Small increases in ethanol yield will translate to hydrolysis products is provided in Khalikova et al. (2005) large Volumes of ethanol produced overtime in a commercial US 2011/020 1 059 A1 Aug. 18, 2011 scale fermentation process. Such improvements in ethanol 0061 A purified or semi-purified preparation of enzyme production could result in a significant increase in profit to the will contain at least one class of key enzyme, but may also ethanol producer. contain one or more additional enzymes of the same or dif 0056. In one embodiment, the use of the methods dis ferent class. The preparation may further comprise one or closed herein results in a substrate that leads to higher carbo more additional enzymes useful in the starch conversion hydrate yields compared to the carbohydrate yield from plant method. Such as amylase or glucoamylase. A "semi-purified’ material not accumulating locked carbohydrates. The enzyme preparation will contain one or more key enzymes, increase in carbohydrate yield can be at least about 1%, at one or more additional enzymes useful in the starch conver least about 2%, at least about 3%, at least about 4%, at least sion process, or may contain other buffers or stabilizing about 5%, at least about 6%, at least about 7%, at least about agents (e.g., glycerol). Furthermore, the semi-purified 8%, at least about 9%, at least about 10%, at least about 20%, enzyme preparation may also be culture Supernatant or crude at least about 20%, at least about 30%, at least about 40%, at extract collected from a cell population expressing and/or least about 50%, at least about 60%, at least about 70%, at secreting the enzyme. The preparation may also be a lyo least about 80%, at least about 90%, at least about 100%, at philized formulation of enzyme that is reconstituted upon least about 2-fold, at least about 3-fold, at least about 4-fold, addition to the locked carbohydrate-containing plant mate at least about 5-fold, or greater. Even Small increases in car rial. bohydrate yield will translate to large volumes of carbohy 0062. The various key enzymes discussed herein can be drate produced over time in a commercial-scale fermentation expressed in and isolated from any number of eukaryotic and process. The carbohydrate may be sucrose or a combination prokaryotic organisms. Appropriate expression cassettes, of Sucrose and a locked Sugar. vectors, transformation, and transfection techniques for a 0057. In another embodiment, the plants accumulating particular organism of interest will be evident to one of skill in locked carbohydrates can be used in various other down the art. stream products other than ethanol production. Locked car 0063. In one embodiment, bacterial cells, such as E. coli, bohydrates can be converted into fermentable sugars which Streptomyces, Bacillus subtilis; and various species within are used in many commercial fermentation processes includ the genera Escherichia, Pseudomonas, Serratia, Streptomy ing growing recombinant yeast which produce important ces, Corynebacterium, Brevibacterium, Bacillus, Microbac chemicals such as insulin, antibodies, or enzymes. Isomaltu terium, and Staphylococcus can be used as a host to express lose is currently used to manufacture Sugar alcohols con one or more classes of key enzymes encompassed herein. Sumed as low-calorie, non-cariogenic Sweeteners. Fructose Methods for transformation of bacterial hosts are described also has value as a Sweetener in high fructose syrups such as in, for example, U.S. Patent Publication No. 2003/0135885. high fructose corn syrup. Plants engineered to produce fruc 0064. In another embodiment, fungal hosts, such as fungal tans as a locked Sugar may be used as a source of fructans host cells belonging to the genera Aspergillus, Rhizopus, Tri which, after hydrolysis by a fructanase enzyme, produce a choderma, Neurospora, Mucor, Penicillium, etc., such as Solution with a high fructose concentration. In Such plants the yeast belonging to the genera Kluyveromyces, Saccharomy yield of fructan may be increased by expressing an additional ces, Schizosaccharomyces, TrichospOron, Schwanniomyces, enzyme (e.g., glucose isomerase) to catalyze the conversion etc. may be used. Transformation of fungus may be accom of glucose to fructose. The glucose isomerase (invertase) plished according to Gonnietal. Agric. Biol. Chem. 51:2549 could be expressed in maize endosperm, or expressed in (1987). microbes. The purified enzyme could be used to produce 0065. Another suitable host includes any number of fructans, glucans and alternans. eukaryotic cells, for example, insect cells such as Drosophila 0058 Sweeter plant products can be generated by express S2 and Spodoptera Sf9; animal cells such as CHO, COS or ing in plants a combination of enzymes that first allow for the Bowes melanoma, C127, 3T3, CHO, HeLa and BHK cell accumulation of fructans in the plant and then convert the lines. Any host can be used insofar as it can express the gene fructans directly or indirectly to fructose. Expressing inver of interest. The American Type Culture Collection (http:// tase (glucose isomerase) in plants accumulating fructans will www.atcc.org/) maintains cell lines from a wide variety of lead to a higher Sweetness index in the plant. Sources and many of these cultures can be used to generate a 0059. In another embodiment, plants accumulating locked transgenic cell line capable of expressing a heterologous carbohydrates as described herein are useful for providing enzyme. Transformation vectors appropriate for eukaryotic protection of the plant against disease. While not being bound cells are available commercially such as pXT1, pSG5 (Strat by any particular theory or mechanism, plants accumulating agene) pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). locked Sugars may be more tolerant or resistant to microbial Techniques for transformation and selection of transgenic infection due to the presence of carbohydrates other than eukaryotic cells are well known in the art. Exemplary meth Sucrose, since infection by some microbes depends upon the ods are also described elsewhere herein. content of Sucrose in the plant. 0066. In another embodiment, the key enzymes can be isolated from an organism that endogenously expresses the Enzyme Extracts for Key Enzyme enzyme, or the organism expressing the enzyme can be used 0060 Invarious embodiments of the present invention, the in one or more fermentation steps without the need for puri enzyme capable of converting the locked carbohydrate to a fication or isolation of the enzyme from the organism. fermentable carbohydrate (referred to herein as the “key' 0067. Additional methods for generating an enzyme enzyme) is provided as a purified or partially-purified prepa extract are described in, for example, Conrad et al. (1995) ration of the enzyme. The exogenously-added key enzyme Eur: J. Biochem. 230,481-490; Chiang et al. (1979) Starch 31 may be de novo synthesized, or may be isolated from an Nr.3, S.86–92: Schwardt, E. (1990) Food Biotechnology, organism expressing the enzyme prior to addition of the 4(1), 337-351; Morgan and Priest (1981) Journal of Applied enzyme to the locked carbohydrate-containing plant material. Bacteriology 50, 107-114; Laderman et al. (1993) Journal of US 2011/020 1 059 A1 Aug. 18, 2011

Biological Chemistry Vol. 268, No. 32, pp. 24394-24401, (Brassica oleracea), artichoke (Cynara Scolvinus), and saf each of which is herein incorporated by reference in its flower (Carthamus, e.g. tinctorius); fruits such as apple entirety. (Malus, e.g. domesticus), banana (Musa, e.g. acuminata), berries (such as the currant, Ribes, e.g. rubrum), cherries Transgenic Plants (such as the Sweet cherry, Prunus, e.g. avium), cucumber 0068. In one embodiment of the present invention, the (Cucumis, e.g. sativus), grape (Vitis, e.g. vinifera), lemon locked carbohydrate-containing plant material comprises (Citrus limon), melon (Cucumis melo), nuts (such as the plant parts derived from at least one variety of a transgenic Walnut, Juglans, e.g. regia; peanut, Arachis hypoaeae), plant expressing at least one polynucleotide encoding a lock orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica). enzyme. In another embodiment, the transgenic plant mate pear (Pyra, e.g. Communis), pepper (Solanum, e.g. capsicum), rial expresses more than one lock enzyme, resulting in the plum (Prunus, e.g. domestica), Strawberry (Fragaria, e.g. accumulation of more than one type of locked carbohydrate. moschata), tomato (Lycopersicon, e.g. esculentum); leafs, In yet another embodiment, both the lock and the key Such as alfalfa (Medicago, e.g. sativa), Sugar cane (Saccha enzymes are expressed in plant material. Where both the lock rum), cabbages (such as Brassica oleracea), endive and the key enzymes are provided as transgenic plant mate (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum), lettuce rial, each class of enzyme may be expressed in the same plant (Lactuca, e.g. sativa), spinach (Spinacia e.g. oleraceae), variety, or may be expressed in different plant varieties. tobacco (Nicotiana, e.g. tabacum); roots, such as arrowroot 0069. As used herein the term “transgenic’ refers to plants that include an exogenous polynucleotide (e.g., gene) that is (Maranta, e.g. arundinacea), beet (Beta, e.g. vulgaris), carrot stably maintained in the transformed plant and is stably inher (Daucus, e.g. Carota), cassava (Manihot, e.g. esculenta), tur ited by progeny in Successive generations. The term “trans nip (Brassica, e.g. rapa), radish (Raphanus, e.g. sativus) yam genic plant can refer either to the initially transformed plant (Dioscorea, e.g. esculenta), Sweet potato (Ipomoea batatas); or to the progeny of the initially transformed plant. Tech seeds, such as bean (Phaseolus, e.g. vulgaris), pea (Pisum, niques for transforming plants, plant cells or plant tissues can e.g. sativum), soybean (Glycine, e.g. max), wheat (Triticum, include, but are not limited to, transformation with DNA e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea, e.g. employing A. tumefaciens or A. rhizogenes as the transform mays), rice (Oryza, e.g. sativa); grasses, such as Miscanthus ing agent, electroporation, DNA injection, microprojectile grass (Miscanthus, e.g., giganteus) and Switchgrass (Pani bombardment, and particle acceleration. See, for example, cum, e.g. virgatum); trees such as poplar (Populus, e.g. EP 295959 and EP 138341. As used herein, the terms “plant tremula), pine (Pinus); shrubs, such as cotton (e.g., Gos material' or “plant part includes plant cells, plant proto sypium hirsutum); and tubers, such as kohlrabi (Brassica, e.g. plasts, plant cell tissue cultures from which plants can be oleraceae), potato (Solanum, e.g. tuberosum), and the like. regenerated, plant calli, plant clumps, and plant cells that are 0072 The locked carbohydrate-containing plant material intact in plants or parts of plants such as embryos, pollen, may also comprise one or more varieties of plants having ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, naturally-occurring genetic variability resulting in altered cobs, husks, stalks, roots, root tips, anthers, tubers, rhizomes starch metabolism. Many such plants carry mutations in and the like. genes encoding isoforms of starch synthesis or starch degra 0070. Where both the lock and the key enzymes are pro vided by transgenic plant material, it is not necessary for the dation enzymes. For example, plants have been identified plant material expressing the key enzyme to be 100% trans which are heterozygous or homozygous for one or more of the genic for the key enzyme. Rather, it is only necessary for the waxy (WX), amylose extender (ae), dull (du), horny (h), plant material to contain an amount of key enzyme that is shrunken (sh), brittle (bt), floury (fl), opaque (O), or Sugary Sufficient for the downstream use (e.g., for conversion of (su) mutant alleles. See, for example, U.S. Pat. Nos. 4,428, locked carbohydrates to fermentable Sugars). For example, 972; 4,767,849; 4,774,328; 4,789,738; 4,789,557; 4,790.997: for fermentation purposes, a Sufficient amount of the key 4,792.458; 4.798.735; and 4,801,470, herein incorporated by enzyme may be provided in the fermentation process by less reference. than 100% key enzyme-expressing plant material. For 0073 Dual Expression of Lock Enzymes example, a sufficient amount of key enzyme may be provided 0074 The invention also comprises the simultaneous to the fermentation process when only about 0.1% of the expression of two lock enzymes Such as two Sucrose locked carbohydrate-containing plant material expresses the isomerases, one that produces predominantly isomalitulose, key enzyme, or only about 1%, about 2%, about 3%, about and one that produces predominantly trehalulose, so that both 4%, about 5%, about 6%, about 7%, about 8%, about 9%, isomers of Sucrose may be accumulated in the same plant. about 10%, about 11%, about 12%, about 13%, about 14%, Sugarcane possesses an excess capacity for carbohydrate about 15%, about 16%, about 17%, about 18%, about 19%, or synthesis, however, there is a continuous “futile cycle” of about 20%, of the plant material. However, it is contemplated Sucrose synthesis and breakdown in Sugarcane. By diverting that the percentage of plant material expressing the key carbohydrates into a form that is not metabolized by the plant, enzyme could be as much as 100%, including, for example, these carbohydrates may be removed from that futile cycle, about 25%, about 30%, about 35%, about 40%, about 50%, and the plant may make up for the loss by producing more about 60%, about 65%, about 70%, about 80%, about 90%, sucrose. The fact that Wu and Birch have seen isomalitulose about 95%, or about 99% of the plant material. accumulate to the same level as Sucrose, without decreasing 0071. The methods of the invention are particularly useful the amount of Sucrose, Suggests that this excess capacity of in plants producing high amounts of Sugar, Such as (for Sugarcane for Sugar synthesis has not been exhausted. By example), Sugarcane, Sugar beet, and Sorghum. However, the genetically modifying Sugarcane with two or more lock plant material can be derived from any plant, including but not enzymes that produce more than one isomers of sucrose (iso limited to plants producing edible flowers such as cauliflower maltulose, trehalulose, leucrose, etc.) at equivalent levels it US 2011/020 1 059 A1 Aug. 18, 2011

may be possible to significantly increase the total Sugar con I0081 Locked Carbohydrates as Selectable Markers tent in Sugarcane, or to increase the level of locked Sugar in the I0082 Plant transformation requires the use of positive Sugarcane. selectable marker genes for identification and propagation of 0075. In one embodiment, the total carbohydrate content, transformed tissue and the elimination of non-transformed or the total locked carbohydrate content, or both, is increased tissue. One advantage of this system would be the ability to at least about 10%, at least about 20%, at least about 50%, at select and/or screen for expression and/or accumulation of least about 100%, at least about 125%, at least about 150%, at the key enzyme involved in the breakdown of the locked least about 2-fold, at least about 3-fold, at least about 4-foldor carbohydrates, from the very earliest stages of the plant trans greater when compared to the same variety of plant that does formation process. A transformation system using the desired not accumulate locked carbohydrate according to the meth enzyme end product as a means of initial selection would ods of the invention. permit early screening for position effects or genomic inser 0076 Sucrose isomerase enzymes producing predomi tion sites that lead to high level or constitutive expression of nantly isomalitulose include, for example, the P dispersa the transgene. Also, the use of the desired end product as the UQ68J enzyme described in U.S. Pat. No. 7,250,282, which selectable marker can reduce the number of genes that must is herein incorporated by reference in its entirety. Other be transferred into the plant. This will reduce the size of the enzymes producing predominantly trehalulose include, for T-DNA needed for transformation and be useful in the pro example, the whitefly enzyme characterized by Salvucci duction of "molecular stacks' in which multiple transgenes (2003) Comp. Biochem. Physiol. B 135:385-395. While not are desired in a single transgenic plant, i.e., eliminate the need to be limited by theory, the whitefly enzyme may be a repre for an extraneous selectable marker gene such as PMI, or sentative of the lock enzyme trehalulose synthase. antibiotic resistance genes that are necessary for production 0077 Subcellular Targeting of transgenic plants, but are no longer useful to the plant after 0078 For the purpose of producing starch in a transgenic transformation/selection. However, it is contemplated that plant, it may be advantageous to target the lockenzyme in the multiple selectable markers can be used in the methods of the plant to Subcellular compartments that have high concentra invention, including those used solely for selection. tions of Sucrose, such as the vacuole of Sugarcane. Another I0083. In one embodiment, an alpha-1,6-glucosidase target may be the vacuole of the maize endosperm. Targeting enzyme may be used to cleave the alpha-1,6-glucoside link an enzyme capable of synthesizing starch from Sucrose to the age between glucose and fructose in the disaccharide isoma vacuole of maize endosperm cells may permit the accumula litulose. This enzyme is desirable for converting isomalitulose tion of more starch in the maize endosperm as naturally produced by transgenic sugarcane plants into fermentable occurring enzymes do not produce starch in the vacuoles of Sugar or ethanol and may be useful as a novel selectable maize endosperm cells. Alternatively targeting to the apoplast marker for Sugarcane transformation. is another way to achieve conversion of Sucrose into locked Sugars such as starch or isomalitulose. In plants such as maize, Expression Cassettes Sucrose accumulates in the leaf and is transported to the ear I0084. A plant or plant part expressing a lock and/or key during grain filling which provides a carbon sink. enzyme can be obtained by introducing into the plant or plant 0079. In one embodiment, the lock enzyme is targeted to part a heterologous nucleic acid sequence encoding the the amyloplast, where locked carbohydrate can accumulate, enzyme. The heterologous nucleic acid sequences may be and the key enzyme (when expressed in the same plant) is present in DNA constructs or expression cassettes. "Expres targeted to the apoplast. The key enzyme can be targeted to sion cassette' as used herein means a nucleic acid molecule the apoplast using, for example, the maize Gamma Zein N-ter capable of directing expression of a particular nucleotide minal signal sequence, which confers apoplast-specific tar sequence in an appropriate host cell, comprising a promoter geting of proteins. The lock enzyme may be targeted to the operatively linked to the heterologous nucleotide sequence of amyloplast by, for example, fusion to the waxy amyloplast interest (i.e., lock and/or key enzyme) which is operatively targeting peptide (Klosgenet al., 1986) or to a starch granule. linked to termination signals. It also typically comprises For example, the polynucleotide encoding the lock enzyme sequences required for proper translation of the nucleotide may be operably linked to a chloroplast (amyloplast) transit sequence. The expression cassette comprising the lock and/or peptide (CTP) and a starch binding domain, e.g., from the key enzyme may be chimeric, meaning that at least one of its Waxy gene. components is heterologous with respect to at least one of its 0080 Directing the key enzyme to the apoplast will allow other components. The expression cassette may also be one the enzyme to be localized in a manner that it will not come that is naturally occurring but has been obtained in a recom into contact with the locked carbohydrate substrate. In this binant form useful for heterologous expression. Typically, manner the enzymatic action of the enzyme will not occur however, the expression cassette is heterologous with respect until the enzyme contacts its Substrate. The enzyme can be to the host. The expression of the nucleotide sequence in the contacted with its Substrate by the process of milling (physi expression cassette may be under the control of a constitutive cal disruption of the cell integrity), or heating the cells or plant promoter or of an inducible promoter that initiates transcrip tissues to disrupt the physical integrity of the plant cells or tion only when the host cell is exposed to Some particular organs that contain the enzyme. For example the key enzyme external stimulus. Additionally, the promoter can also be can be targeted to the apoplast or to the endoplasmic reticu specific to a particular tissue or organ or stage of develop lum so as not to come into contact with the locked carbohy ment. drate in the amyloplast. Milling of the grain will disrupt the I0085. The expression cassette may optionally comprise a integrity of the grain and the key enzyme will then contact the transcriptional and translational termination region (i.e. ter starch granules. In this manner the potential negative effects mination region) functional in plants. In some embodiments, of co-localization of an enzyme and the locked carbohydrate the expression cassette comprises a selectable marker gene to can be circumvented. allow for selection for stable transformants. Expression con US 2011/020 1 059 A1 Aug. 18, 2011 structs of the invention may also comprise a leader sequence disclosure. These include, for example, the rbcS promoter, and/or a sequence allowing for inducible expression of the specific for green tissue; the ocs, nos and Smas promoters lock and/or key enzyme. See, Guo et al. (2003) Plant J. which have higher activity in roots or wounded leaf tissue; a 34:383-92 and Chen et al. (2003) Plant 3.36:731-40 for truncated (-90 to +8) 35S promoter which directs enhanced examples of sequences allowing for inducible expression. expression in roots, an O-tubulin gene that directs expression I0086. The regulatory sequences of the expression con in roots and promoters derived from Zein storage protein struct are operably linked to the nucleic acid sequence encod genes which direct expression in endosperm. ing the lock and/or key enzyme. By “operably linked' is 0092 Tissue specific expression may be functionally intended a functional linkage between a first sequence and a accomplished by introducing a constitutively expressed gene second sequence for instance, the first sequence may be a (all tissues) in combination with an antisense gene that is promoter sequence which is operably linked to a second expressed only in those tissues where the gene product is not sequence wherein the promoter sequence initiates and medi desired. ates transcription of the DNA sequence corresponding to the 0093 Moreover, several tissue-specific regulated genes second sequence. Generally, operably linked means that the and/or promoters have been reported in plants. Some reported nucleotide sequences being linked are contiguous; however, tissue-specific genes include the genes encoding the seed the sequences may have linking sequences that join them storage proteins (such as napin, cruciferin, beta-conglycinin, together, thus the operably linked sequences may not be and phaseolin) Zein or oil body proteins (such as oleosin), or directly linked. genes involved in fatty acid biosynthesis (including acyl car 0087 Promoter rier protein, Stearoyl-ACP desaturase, and fatty acid desatu 0088 Any promoter capable of driving expression in the rases (fad 2-1)), and other genes expressed during embryo plant of interest may be used in the practice of the invention. development (such as Bce4, see, for example, EP255378 and The promoter may be native or analogous or foreign or het Kridlet al., Seed Science Research, 1:209 (1991)). Examples erologous to the plant host. The terms "heterologous' and of tissue-specific promoters, which have been described “exogenous” when used herein to refer to a nucleic acid include the lectin (Vodkin, Prog. Clin. Biol. Res., 138; 87 sequence (e.g. a DNA or RNA sequence) or a gene, refer to a (1983); Lindstrom et al., Der. Genet., 11:160 (1990)), corn sequence that originates from a source foreign to the particu alcohol dehydrogenase 1 (Vogel et al., EMBO J., 11:157 lar host cell or, if from the same source, is modified from its (1989): Dennis et al., Nucleic Acids Res., 12:3983 (1984)), original form. Thus, a heterologous gene in a host cell corn light harvesting complex (Simpson, 1986; Bansal et al., includes a gene that is endogenous to the particular host cell Proc. Natl. Acad. Sci. USA,89:3654 (1992)), cornheat shock but has been modified through, for example, the use of DNA protein (Odell et al., Nature, 313: 810 (1985)); pea small shuffling. The terms also include non-naturally occurring subunit RuBP carboxylase ((Poulsen et al., Mol. Gen. Genet. multiple copies of a naturally occurring DNA sequence. 205:193 (1986)); Ti plasmid mannopine synthase ((Lan Thus, the terms refer to a DNA segment that is foreign or gridge et al., Cell 34:1015 (1989)), Tiplasmid nopaline syn heterologous to the cell, or homologous to the cell but in a thase ((Langridge et al., Cell 34:1015 (1989)), petunia chal position within the host cell nucleic acid in which the element cone isomerase (vanTunen et al., EMBO J. 7: 1257 (1988)), is not ordinarily found. Exogenous DNA segments are bean glycine rich protein 1 (Keller et al., Genes Dev. 3:1639 expressed to yield exogenous polypeptides. (1989)), truncated CaMV 35S (Odellet al., Nature, 313:810 0089. The choice of promoters to be included depends (1985)), potato patatin (Wenzler et al., Plant Mol. Biol. upon several factors, including, but not limited to, efficiency, 13:347 (1989)), root cell (Yamamoto et al., Nucleic Acids selectability, inducibility, desired expression level, and cell Res., 18:7449 (1990)), maize Zein (Reina et al., Nucleic Acids or tissue-preferential expression. For example, where expres Res., 18:6425 (1990); Kriz et al., Mol. Gen. Genet. 207:90 sion in specific tissues or organs is desired, tissue-specific (1987); Wandelt et al., Nucleic Acids Res., 17:2354 (1989); promoters may be used. In contrast, where gene expression in Langridge et al., Cell, 34:1015 (1983); Reina et al., Nucleic response to a stimulus is desired, inducible promoters are the Acids Res., 18:7449 (1990)), globulin-1 (Belanger et al., regulatory elements of choice. Where continuous expression Genetics, 129:863 (1991)), C-tubulin, cab (Sullivan et al., is desired throughout the cells of a plant, constitutive promot Mol. Gen. Genet., 215:431 (1989)), PEPCase ((Hudspeth et ers are utilized. It is a routine matter for one of skill in the art al., Plant Mo. Bio., 12:579 (1989)), R gene complex-associ to modulate the expression of a sequence by appropriately ated promoters (Chandler et al., Plant Cell, 1:1175 (1989)), selecting and positioning promoters and other regulatory and chalcone synthase promoters (Franken et al., EMBO J. regions relative to that sequence. 10:2605 (1991)). Particularly useful for seed-specific expres 0090. A number of plant promoters have been described sion is the pea vicilin promoter (CZako et al., Mol. Gen. with various expression characteristics. Examples of some Genet., 235:33 (1992). (See also U.S. Pat. No. 5,625,136, constitutive promoters which have been described include the herein incorporated by reference.) Other useful promoters for rice actin 1 (Wang et al., Mol. Cell. Biol., 12:3399 (1992); expression in mature leaves are those that are Switched on at U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., Nature, the onset of senescence, Such as the SAG promoter from 313:810 (1985)), CaMV 19S (Lawton et al., 1987), nos (Ebert Arabidopsis (Gan et al., Science, 270: 1986 (1995). et al., 1987), Adh (Walker et al., 1987), sucrose synthase 0094. In various embodiments, the lock and/or key (Yang & Russell, 1990), and the ubiquitin promoters. enzyme is active in the fruit of the plant. A class of fruit 0091 Vectors for use in tissue-specific targeting of genes specific promoters expressed at or during antithesis through in transgenic plants will typically include tissue-specific pro fruit development, at least until the beginning of ripening, is moters and may also include other tissue-specific control discussed in U.S. Pat. No. 4,943,674, the disclosure of which elements such as enhancer sequences. Promoters which is hereby incorporated by reference. cDNA clones that are direct specific or enhanced expression in certain plant tissues preferentially expressed in cotton fiber have been isolated will be known to those of skill in the art in light of the present (John et al., Proc. Natl. Acad. Sci. USA, 89:5769 (1992). US 2011/020 1 059 A1 Aug. 18, 2011 cDNA clones from tomato displaying differential expression increased salinity, drought, pathogen and wounding. (Gra during fruit development have been isolated and character ham et al., J. Biol. Chem., 260:6555 (1985); Graham et al., J. ized (Mansson et al., Gen. Genet., 200:356 (1985), Slater et Biol. Chem., 260:6561 (1985), Smith et al., Planta, 168:94 al., Plant Mol. Biol. 5:137 (1985)). The promoter for polyga (1986)). Accumulation of metallocarboxypeptidase-inhibitor lacturonase gene is active in fruit ripening. The polygalactu protein has been reported in leaves of wounded potato plants ronase gene is described in U.S. Pat. No. 4,535,060, U.S. Pat. (Graham et al., Biochem. Biophys. Res. Comm., 101: 1164 No. 4,769,061, U.S. Pat. No. 4,801,590, and U.S. Pat. No. (1981)). Other plant genes have been reported to be induced 5,107,065, which disclosures are incorporated herein by ref by methyl jasmonate, elicitors, heat-shock, anaerobic stress, erence. The fruit specific E8 promoter is described in Deik or herbicide safeners. man et al. (1988, EMBO J. 2: 3315-3320) and DellaPenna et 0099 Preferably, in the case of a multicellular organism, al. (1989, Plant Cell 1:53-63). In another embodiment, pro the promoter can also be specific to a particular tissue, organ moters that selectively express coding sequences in Sucrose or stage of development. Examples of Such promoters storage tissues (such as the mature stems of Sugarcane and the include, but are not limited to, the Zea mays ADP-gpp and the tubers of Sugar beet) may be used. For example, promoters Zea mays Gamma Zein promoter and the Zea mays globulin specific for the mature stems of Sugarcane are described in promoter. International Publication WO 01/18211. 0100 Expression of a gene in a transgenic plant may be 0095. In another embodiment, the expression of the lock desired only in a certain time period during the development enzyme is under the control of a sink tissue-specific promoter. of the plant. Developmental timing is frequently correlated By "sink tissue-specific promoter' is meant a promoter that with tissue specific gene expression. Timing the expression of preferentially directs expression of an operably linked tran carbohydrate-metabolizing enzymes advantageously takes scribable sequence in the sink tissue of a plantas compared to into consideration the change in carbohydrate concentration expression in other tissues of the plant, including Source that occurs during plant development. The importance of a tissues (e.g., leaf). “Sink cell' and “sink tissue' as used carbohydrate within tissue may also change with time and, in herein, refer to cells, tissues or organs which at the time of this regard, sink tissue may undergo changes in Sucrose con harvest comprise organic carbon that has entered the cells by centrations during development. For example, Sucrose con net inflow in a form other than carbon dioxide. In plants, sink centration in certain fruits such as Sweet melons changes as tissues include all non-photosynthetic tissues, as well as pho the fruit matures. Hexose Sugars accumulate early in devel tosynthetic tissues with a net inflow of organic carbon fixed opment, followed by high levels of Sucrose at later stages by other photosynthetic cells or otherwise obtained from the (Schaffer et al., 1987, Phytochemistry 26: 1883-1887). In Surrounding medium or environment by means other than developing corn endosperm, Sucrose concentration increases direct fixation of carbon dioxide. from 8 to 12 days after pollination and then drops more than 0096. Other examples of tissue-specific promoters include tenfold 28 days after pollination (Tsai et al., 1970, Plant Phys. those that direct expression in leaf cells following damage to 46: 299-306). Additionally, sucrose concentration in soybean the leaf (for example, from chewing insects), in tubers (for seed changes significantly during development as raffinose example, patatin gene promoter), and in fiber cells (an saccharides content increases dramatically, 53 days after example of a developmentally-regulated fiber cell protein is anthesis (Amuti, 1977, Phytochemistry 16:529-532). In pea E6 (John et al., Proc. Natl. Acad. Sci. USA, 89:5769 (1992). seed, Sucrose content falls dramatically with continued devel The E6 gene is most active in fiber, although low levels of opment (Holl and Vose, Can. 1980, J. Plant Sci. 60: 1109 transcripts are found in leaf, ovule and flower. Other tissue 1114). These examples illustrate the desirability of promoter specific promoters can be isolated by one skilled in the art (see selection for specific expression of an enzyme gene timed to U.S. Pat. No. 5,589,379). take advantage of fluctuating Sucrose pools. Thus, in various 0097. Several inducible promoters have been reported. embodiments, the promoter is an inducible promoter which is Many are described in a review by Gatz, in Current Opinion capable of driving expression of the enzyme-encoding poly in Biotechnology, 7:168 (1996) and Gatz, C., Annu. Rev. nucleotide at an appropriate developmental stage of the plant. Plant Physiol. Plant Mol. Biol. 48:89 (1997), Examples In this embodiment, the transcriptional control element is include tetracycline repressor system, Lac repressor System, Suitably a developmentally regulated promoter to control the copper-inducible systems, Salicylate-inducible systems (such timing of expression. as the PR1a system), glucocorticoid-inducible (Aoyama T. et 0101 Localization Signals al., N—H Plant Journal, 11:605 (1997)) and ecdysone-induc 0102 The polynucleotide sequences encoding the lock ible systems. Other inducible promoters include ABA- and and/or key enzyme of the present invention may be operably turgor-inducible promoters, the promoter of the auxin-bind linked to polynucleotide sequences encoding localization sig ing protein gene (Schwob et al., Plant J., 4:423 (1993)), the nals or signal sequence (at the N- or C-terminus of a polypep UDPglucose flavonoid glycosyl-transferase gene promoter tide), e.g., to target the enzyme to a particular compartment (Ralstonet al., Genetics, 119:185 (1988)), the MPI proteinase within a plant. Examples of Such targets include, but are not inhibitor promoter (Cordero et al., Plant J., 6:141 (1994)), and limited to, the vacuole, endoplasmic reticulum, chloroplast, the glyceraldehyde-3-phosphate dehydrogenase gene pro amyloplast, starch granule, or cell wall, or to a particular moter (Kohler et al., Plant Mal. Biol., 29; 1293 (1995); Quig tissue, e.g., seed. The expression of a polynucleotide encod ley et al., J. Mol. Evol., 29:412 (1989); Martinez et al., J. Mol. ing a lock and/or key enzyme having a signal sequence in a Biol. 208:551 (1989)). Also included are the benzene sul plant, in particular, in conjunction with the use of a tissue phonamide-inducible (U.S. Pat. No. 5,364,780) and alcohol specific or inducible promoter, can yield high levels of local inducible (WO 97/06269 and WO 97/06268) systems and ized enzyme in the plant. Targeting or signal sequences can be glutathione S-transferase promoters. used to localize a lock or key enzyme Such that the enzyme 0098. Other studies have focused on genes inducibly regu does not come into contact with a specific Substrate during the lated in response to environmental stress or stimuli such as growth and development of the plant. For instance, key US 2011/020 1 059 A1 Aug. 18, 2011 enzymes expressed in plants that accumulate locked Sugars structs with the chloramphenicol acetyltransferase gene (Cal may be targeted away from the plant organelle or compart lis et al., Genes Develop. 1: 1183-1200 (1987)). In the same ment which contains the locked Sugar. At the time of harvest, experimental system, the intron from the maize bronze 1 gene the plant tissue may be physically disrupted in order to com had a similar effect in enhancing expression. Intron bine the key enzyme with the locked Sugar during the pro sequences have been routinely incorporated into plant trans cessing of the plant tissue. formation vectors, typically within the non-translated leader. 0103) Thus, vectors may be constructed and employed in 0110. A number of non-translated leader sequences the intracellular targeting of a specific gene product within the derived from viruses are also known to enhance expression. cells of a transgenic plant or in directing a protein to the Specifically, leader sequences from Tobacco Mosaic Virus extracellular environment. This will generally be achieved by (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus joining a DNA sequence encoding a transit or signal peptide (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown sequence to the coding sequence of a particular gene. The to be effective in enhancing expression (e.g. Gallie et al. Nucl. resultant transit, or signal, peptide will transport the protein to Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant a particular intracellular or extracellular destination, respec Molec. Biol. 15: 65-79 (1990)). Other leader sequences tively, and will then be post-translationally removed. Transit known in the art include but are not limited to: picornavirus or signal peptides act by facilitating the transport of proteins leaders, for example, EMCV leader (Encephalomyocarditis through intracellular membranes, e.g., vacuole, Vesicle, plas 5' noncoding region) (Elroy-Stein, O., Fuerst, T. R., and tid and mitochondrial membranes, whereas signal peptides Moss, B. PNAS USA 86:6126-6130 (1989)); potyvirus lead direct proteins through the extracellular membrane. ers, for example, TEV leader (Tobacco EtchVirus) (Allisonet 0104 Numerous signal sequences are known to influence al., Virology 154:9-20 (1986)); MDMV leader (Maize Dwarf the expression or targeting of a polynucleotide to a particular Mosaic Virus); Virology 154:9-20); human immunoglobulin compartment or outside a particular compartment. Suitable heavy-chain binding protein (BiP) leader, (Maceak, D. G., signal sequences and targeting promoters are known in the art and Samow, P., Nature 353:90-94 (1991); untranslated leader and include, but are not limited to, those provided herein. from the coat protein mRNA of alfalfa mosaic virus (AMV 0105. In one embodiment, the lock enzyme carbohydrate RNA 4), (Tobling, S.A., and Gehrke, L., Nature 325:622-625 can accumulate, and the key enzyme is targeted to the apo (1987); tobacco mosaic virus leader (TMV), (Gallie, D. R. et plast. The key enzyme can be targeted to the apoplast using, al., Molecular Biology of RNA, pages 237-256 (1989); and for example, the maize Gamma Zein N-terminal signal Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. sequence, which confers apoplast-specific targeting of pro A. et al., Virology 81:382-385 (1991). See also, Della-Cioppa teins. The lock enzyme may be targeted to the amyloplast by, et al., Plant Physiology 84:965-968 (1987). for example, fusion to the waxy amyloplast targeting peptide 0111 Regulatory Sequences (Klosgen et al., Mol Gen Genet. 203: 237-2441986) or to a 0112 The polynucleotides of the present invention, in starch granule. For example, the polynucleotide encoding the addition to processing signals, may further include other lock enzyme may be operably linked to a chloroplast (amy regulatory sequences, as is known in the art. "Regulatory loplast) transit peptide (CTP) and a starch binding domain, sequences” and "suitable regulatory sequences’ each refer to e.g., from the waxy gene. Alternatively, the maize Brittle 1 nucleotide sequences located upstream (5' non-coding transit peptide sequence (Bt1ts, Sullivan and Kaneko, Planta sequences), within, or downstream (3' non-coding sequences) 196: 477-484 (1995)) can be used for amyloplast targeting. In of a coding sequence, and which influence the transcription, other embodiments, the total carbohydrate content or sweet RNA processing or stability, or translation of the associated ness or the endogenous carbohydrate content of the sink coding sequence. Regulatory sequences include enhancers, tissue is increased by targeting the carbohydrate-metaboliz promoters, translation leader sequences, introns, and poly ing enzyme to a Sub-cellular compartment used for carbohy adenylation signal sequences. They include natural and Syn drate storage in the plant cells (e.g., vacuole or apoplasmic thetic sequences as well as sequences that are a combination space). of synthetic and natural sequences. 0106 A signal sequence Such as the maize Gamma Zein 0113. A variety of transcriptional terminators are available N-terminal signal sequence for targeting to the endoplasmic for use in expression cassettes. These are responsible for the reticulum and secretion into the apoplast may be operably termination of transcription beyond the transgene and correct linked to a polynucleotide encoding the key enzyme in accor mRNA polyadenylation. The termination region may be dance with the present invention (Torrent et al., Plant Mol. native with the transcriptional initiation region., may be Biol. 34:139 (1997)). Another signal sequence is the amino native with the operably linked DNA sequence of interest, acid sequence SEKDEL (SEQ ID NO:7) for retaining may be native with the plant host, or may be derived from polypeptides in the endoplasmic reticulum (Munro et al. Cell another source (i.e., foreign or heterologous to the promoter, 48:899 (1987)). the DNA sequence of interest, the plant host, or any combi 01.07 Enhancers nation thereof). Appropriate transcriptional terminators are 0108) Numerous sequences have been found to enhance those that are known to function in plants and include the gene expression from within the transcriptional unit and these CAMV 35S terminator, the timl terminator, the nopaline syn sequences can be used in conjunction with the genes of this thase terminator and the pea rbcs E9 terminator. These can be invention to increase their expression in transgenic plants. used in both monocotyledons and dicotyledons. In addition, a 0109 Various intron sequences have been shown to gene's native transcription terminator may be used. enhance expression. For example, the introns of the maize 0114 Selectable Markers Adhl gene have been found to significantly enhance the 0115 Generally, the expression cassette will comprise a expression of the wild-type gene under its cognate promoter selectable marker gene for the selection of transformed cells. when introduced into maize cells. Intron 1 was found to be Selectable marker genes are utilized for the selection of trans particularly effective and enhanced expression in fusion con formed cells or tissues. Selectable markers may also be used US 2011/020 1 059 A1 Aug. 18, 2011

in the present invention to allow for the selection of trans ture. However, any one of a variety of extensions and/or formed plants and plant tissue, as is well-known in the art. glycine-rich wall proteins (Keller et al., EMBO Journal, One may desire to employ a selectable or screenable marker 8:1309 (1989)) could be modified by the addition of an anti gene as, or in addition to, the expressible gene of interest. genic site to create a screenable marker. “Marker genes' are genes that impart a distinct phenotype to 0120 Possible selectable markers for use in connection cells expressing the marker gene and thus allow Such trans with the present invention include, but are not limited to, a neo formed cells to be distinguished from cells that do not have or nptII gene (Potrykus et al., Mol. Gen. Genet., 199:183 the marker. Such genes may encode either a selectable or (1985)) which codes for kanamycin resistance and can be screenable marker, depending on whether the marker confers selected for using kanamycin, G418, and the like; a bar gene a trait which one can select for by chemical means, i.e., which confers resistance to the herbicide phosphinothricin; a through the use of a selective agent (e.g., a herbicide, antibi gene which encodes an altered EPSP synthase protein otic, or the like), or whether it is simply a trait that one can (Hinchee et al., Biotech. 6:915 (1988)) thus conferring gly identify through observation or testing, i.e., by Screening phosate resistance; a nitrilase gene Such as bXn from Kleb (e.g., the R-locus trait). Of course, many examples of suitable siella Ozaenae which confers resistance to bromoxynil marker genes are known in the art and can be employed in the (Stalker et al., Science, 242:419 (1988)); a mutant acetolac practice of the invention. tate synthase gene (ALS) which confers resistance to imida 0116. In one embodiment, both the lock and the key Zolinone, sulfonylurea or other ALS-inhibiting chemicals enzymes are expressed in the same plant, and the expression (European Patent Application 154,204, 1985); a methotrex of the key enzyme is used as a selectable marker. In one ate-resistant DHFR gene (Thillet et al., J. Biol. Chem., 263: example, the selection system is based on the expression of 12500 (1988)); a dalapon dehalogenase gene that confers alpha-1,6-glucosidase in a plant accumulating isomalitulose. resistance to the herbicide dalapon; a phosphomannose In Such a system a means of breaking down isomalitulose into isomerase (PMI) gene; a mutated anthranilate synthase gene a Substrate for fermentation is necessary, and may be pro that confers resistance to 5-methyl tryptophan; the hph gene vided in the form of Sugarcane, Sugarbeet, etc. plants engi which confers resistance to the antibiotic hygromycin; or the neered to express an alpha-1,6-glucosidase (isomalitulase, mannose-6-phosphate isomerase gene (also referred to herein palatinase, etc.). Such a selectable marker system would be as the phosphomannose isomerase gene), which provides the useful in screening for high level expression of alpha-1,6- ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and glucosidase from the very earliest steps of plant transforma 5.994,629). One skilled in the art is capable of selecting a tion, this would be helpful in identifying integration events suitable selectable marker gene for use in the present inven that are stable, highly expressed, and resistant to gene silenc tion. ing. Also, this system could be used to select alpha-1,6- 0.121. An illustrative embodiment of a selectable marker glucosidases with improved activity and in selecting for vari gene capable of being used in Systems to select transformants ants that increase protein or mRNA stability, localization to are the genes that encode the enzyme phosphinothricin specific Subcellular locations etc. acetyltransferase, such as the bar gene from Streptomyces 0117. Also included within the terms selectable or screen hygroscopicus or the pat gene from Streptomyces viridochro able marker genes are also genes which encode a 'secretable mogenes. The enzyme phosphinothricin acetyl transferase marker whose secretion can be detected as a means of iden (PAT) inactivates the active ingredient in the herbicide biala tifying or selecting for transformed cells. Examples include phos, phosphinothricin (PPT). PPT inhibits glutamine syn markers which encode a secretable antigen that can be iden thetase, (Murakami et al., Mol. Gen. Genet. 205:42 (1986); tified by antibody interaction, or even secretable enzymes Twell et al., Plant Physiol. 91: 1270 (1989)) causing rapid which can be detected by their catalytic activity. Secretable accumulation of ammonia and cell death. The Success in proteins fall into a number of classes, including Small, diffus using this selective system in conjunction with monocots was ible proteins detectable, e.g., by ELISA; small active particularly Surprising because of the major difficulties which enzymes detectable in extracellular Solution (e.g., B-lacta have been reported in transformation of cereals (Potrykus, mase, phosphinothricin acetyltransferase); and proteins that Trends Biotech. 7:269 (1989)). are inserted or trapped in the cell wall (e.g., proteins that I0122) Where one desires to employ abialaphos resistance include a leader sequence Such as that found in the expression gene in the practice of the invention, aparticularly useful gene unit of extension or tobacco PR-S). for this purpose is the bar orpat genes obtainable from species 0118 With regard to selectable secretable markers, the use of Streptomyces (e.g., ATCC No. 21.705). The cloning of the of a gene that encodes a protein that becomes sequestered in bar gene has been described (Murakami et al., Mol. Gen. the cell wall, and which protein includes a unique epitope is Genet., 205:42 (1986); Thompson et al., EMBO Journal, also encompassed herein. Such a secreted antigen marker 6:2519 (1987)) as has the use of the bar gene in the context of would ideally employ an epitope sequence that would provide plants other than monocots (De Blocket al., EMBO Journal, low background in plant tissue, a promoter-leader sequence 6: 2513 (1987); De Block et al., Plant Physiol. 91:694 that would impart efficient expression and targeting across (1989)). the plasma membrane, and would produce protein that is I0123 Screenable markers that may be employed include, bound in the cell wall and yet accessible to antibodies. A but are not limited to, a B-glucuronidase or uidA gene (GUS) normally secreted wall protein modified to include a unique which encodes an enzyme for which various chromogenic epitope would satisfy all such requirements. Substrates are known; an R-locus gene, which encodes a 0119. One example of a protein suitable for modification product that regulates the production of anthocyanin pig in this manner is extension, or hydroxyproline rich glycopro ments (red color) in plant tissues (Dellaporta et al., in Chro tein (HPRG). For example, the maize HPRG (Steifel et al., mosome Structure and Function, pp. 263-282 (1988)); a The Plant Cell, 2:785 (1990)) molecule is well characterized |B-lactamase gene (Sutcliffe, PNAS USA, 75:3737 (1978)), in terms of molecular biology, expression and protein struc which encodes an enzyme for which various chromogenic US 2011/020 1 059 A1 Aug. 18, 2011

Substrates are known (e.g., PADAC, a chromogenic cepha 0127 Plant Transformation losporin); axylE gene (Zukowsky et al., PNAS USA, 80: 1101 0128. Once a nucleic acid sequence encoding the lock (1983)) which encodes a catechol dioxygenase that can con and/or key enzyme has been cloned into an expression sys Vert chromogenic catechols; a tyrosinase gene (Katz et al., J. tem, it is transformed into a plant cell. The word “plant” refers Gen. Microbiol., 129:2703 (1983)) which encodes an enzyme to any plant, particularly to seed plant, and “plant cell' is a capable of oxidizing tyrosine to DOPA and dopaquinone structural and physiological unit of the plant, which com which in turn condenses to form the easily detectable com prises a cell wall but may also refer to a protoplast. The plant pound melanin; a B-galactosidase gene, which encodes an cell may be inform of an isolated single cellor a cultured cell, enzyme for which there are chromogenic Substrates; a or as a part of higher organized unit Such as, for example, a luciferase (lux) gene (Ow et al., Science, 234:856 (1986)), plant tissue, or a plant organ. The term “transformation' which allows for bioluminescence detection; or an aequorin refers to the transfer of a nucleic acid fragment into the gene (Prasher et al., Biochem. Biophys. Res. Comm., 126: genome of a host cell, resulting in genetically stable inherit 1259 (1985)), which may be employed in calcium-sensitive ance. Host cells containing the transformed nucleic acid frag bioluminescence detection, or a green fluorescent protein ments are referred to as “transgenic cells, and organisms gene (Niedz et al., Plant Cell Reports, 14: 403 (1995)). comprising transgenic cells are referred to as “transgenic 0.124 Genes from the maize R gene complex are contem organisms.” plated to be particularly useful as screenable markers. The R I0129. Examples of methods of transformation of plants gene complex in maize encodes a protein that acts to regulate and plant cells include Agrobacterium-mediated transforma the production of anthocyanin pigments in most seed and tion (De Blaere et al., 1987) and particle bombardment tech plant tissue. A gene from the R gene complex is Suitable for nology (Klein et al. 1987; U.S. Pat. No. 4,945.050). Whole maize transformation, because the expression of this gene in plants may be regenerated from transgenic cells by methods transformed cells does not harm the cells. Thus, an R gene well known to the skilled artisan (see, for example, Fromm et introduced into such cells will cause the expression of a red al., 1990). pigment and, if stably incorporated, can be visually scored as 0.130. The expression cassettes of the present invention ared sector. If a maize line carries dominant allelles for genes can be introduced into the plant cell in a number of art encoding the enzymatic intermediates in the anthocyanin bio recognized ways. The term “introducing in the context of a synthetic pathway (C2, A1, A2, BZ1 and BZ2), but carries a polynucleotide, for example, a nucleotide encoding an recessive allele at the R locus, transformation of any cell from enzyme disclosed herein, is intended to mean presenting to that line with R will result in red pigment formation. Exem the plant the polynucleotide in Such a manner that the poly plary lines include Wisconsin 22 which contains the rg-Sta nucleotide gains access to the interior of a cell of the plant. dler allele and TR112, a K55 derivative which is r-g, b, P1. Where more than one polynucleotide is to be introduced, Alternatively any genotype of maize can be utilized if the C1 these polynucleotides can be assembled as part of a single and R alleles are introduced together. A further screenable nucleotide construct, or as separate nucleotide constructs, and marker contemplated for use in the present invention is firefly can be located on the same or different transformation vec luciferase, encoded by the lux gene. The presence of the lux tOrS. gene in transformed cells may be detected using, for example, I0131. Accordingly, these polynucleotides can be intro X-ray film, Scintillation counting, fluorescent spectropho duced into the host cell of interest in a single transformation tometry, low-light video cameras, photon counting cameras event, in separate transformation events, or, for example, in or multiwell luminometry. It is also envisioned that this sys plants, as part of a breeding protocol. The methods of the tem may be developed for populational screening for biolu invention do not depend on a particular method for introduc minescence, such as on tissue culture plates, or even for ing one or more polynucleotides into a plant, only that the whole plant screening. polynucleotide(s) gains access to the interior of at least one Additional Agronomic Traits cell of the plant. Methods for introducing polynucleotides 0125 into plants are known in the art including, but not limited to, 0126 The plants disclosed herein may further exhibit one transient transformation methods, stable transformation or more agronomic traits that primarily are of benefit to a seed methods, and virus-mediated methods. company, a grower, or a grain processor, for example, herbi I0132 “Transient transformation' in the context of a poly cide resistance, virus resistance, bacterial pathogen resis nucleotide is intended to mean that a polynucleotide is intro tance, insect resistance, nematode resistance, and fungal duced into the plant and does not integrate into the genome of resistance. See, e.g., U.S. Pat. Nos. 5,569,823: 5,304.730; the plant. 5,495,071; 6,329,504; and 6,337,431. Such trait may also be I0133. By “stably introducing” or “stably introduced” in one that increases plant vigor or yield (including traits that the context of a polynucleotide introduced into a plant is allow a plant to grow at different temperatures, Soil conditions intended the introduced polynucleotide is stably incorporated and levels of Sunlight and precipitation), or one that allows into the plant genome, and thus the plantis stably transformed identification of a plant exhibiting a trait of interest (e.g., with the polynucleotide. selectable marker gene, seed coat color, etc.). Various traits of I0134) “Stable transformation” or “stably transformed” is interest, as well as methods for introducing these traits into a intended to mean that a polynucleotide, for example, a nucle plant, are described, for example, in U.S. Pat. Nos. 5.569,823: otide cotext missing or illegible when filed plant inte 5,304,730; 5,495,071; 6,329,504; 6,337,431; 5,767,366; grates into the genome of the plant text missing or illeg 5,928,937; 4,761.373; 5,013,659: 4,975,374; 5,162,602; ible when filed progeny thereof, more particularly, by the 4,940,835; 4,769,061; 5,554,798; 5,879,903, 5,276.268; progeny of multiple successive generations. 5,561.236; 4,810,648; and 6,084, 155; in European applica 0.135 Numerous transformation vectors available for tion No. 0 242 246; in U.S. Patent Application No. plant transformation are known to those of ordinary skill in 20010016956; and on the worldwide web at wwww.lifesci. the plant transformation arts, and the genes pertinent to this Sussex.ac.uk/home/Neil Crickmore/Bt/. invention can be used in conjunction with any such vectors. US 2011/020 1 059 A1 Aug. 18, 2011

The selection of vector will depend upon the preferred trans 0141 Transformation of the target plant species by recom formation technique and the target species for transformation. binant Agrobacterium usually involves co-cultivation of the For certain target species, different antibiotic or herbicide Agrobacterium with explants from the plant and follows pro selection markers may be preferred as discussed elsewhere tocols well known in the art. Transformed tissue is regener herein. ated on selectable medium carrying the antibiotic or herbicide 0.136 Methods for regeneration of transformed plants are resistance marker present between the binary plasmid T-DNA well known in the art. For example, Tiplasmid vectors have borders. been utilized for the delivery of foreign DNA, as well as direct 0142. Another approach to transforming plant cells with a DNA uptake, liposomes, electroporation, microinjection, and gene involves propelling inert or biologically active particles microprojectiles. In addition, bacteria from the genus Agro at plant tissues and cells. This technique is disclosed in U.S. bacterium can be utilized to transform plant cells. Below are Pat. Nos. 4,945,050, 5,036,006, and 5,100,792. Generally, descriptions of representative techniques for transforming this procedure involves propelling inert or biologically active both dicotyledonous and monocotyledonous plants, as well particles at the cells under conditions effective to penetrate as a representative plastid transformation technique. the outer surface of the cell and afford incorporation within 0.137 Many vectors are available for transformation using the interior thereof. When inert particles are utilized, the Agrobacterium tumefaciens. These typically carry at least vector can be introduced into the cell by coating the particles one T-DNA border sequence and include vectors such as with the vector containing the desired gene. Alternatively, the pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construc target cell can be surrounded by the vector so that the vector tion of vectors useful in Agrobacterium transformation, see, is carried into the cell by the wake of the particle. Biologically for example, US Patent Application Publication No. 2006/ active particles (e.g., dried yeast cells, dried bacterium or a 0260011, herein incorporated by reference. bacteriophage, each containing DNA sought to be intro 0138 Transformation without the use of Agrobacterium duced) can also be propelled into plant cell tissue. tumefaciens circumvents the requirement for T-DNA 0.143 Transformation of most monocotyledon species has sequences in the chosen transformation vector and conse now also become routine. Preferred techniques include direct quently vectors lacking these sequences can also be utilized. gene transfer into protoplasts using PEG or electroporation Transformation techniques that do not rely on Agrobacterium techniques, and particle bombardment into callus tissue. include transformation via particle bombardment, protoplast Transformations can be undertaken with a single DNA spe uptake (e.g. PEG and electroporation) and microinjection. cies or multiple DNA species (i.e. co-transformation) and The choice of vector depends largely on the preferred selec both of these techniques are suitable for use with this inven tion for the species being transformed. For the construction of tion. Co-transformation may have the advantage of avoiding such vectors, see, for example, US Application No. complete vector construction and of generating transgenic 20060260011, herein incorporated by reference. plants with unlinked loci for the gene of interest and the 0139 Transformation techniques for dicotyledons are selectable marker, enabling the removal of the selectable well known in the art and include Agrobacterium-based tech marker in Subsequent generations, should this be regarded niques and techniques that do not require Agrobacterium. desirable. Non-Agrobacterium techniques involve the uptake of exog 0144) Patent Applications EP0292.435, EP0392 225, and enous genetic material directly by protoplasts or cells. This WO 93/07278 describe techniques for the preparation of cal method can be accomplished by PEG or electroporation lus and protoplasts from an elite inbred line of maize, trans mediated uptake, particle bombardment-mediated delivery, formation of protoplasts using PEG or electroporation, and or microinjection. Examples of these techniques are the regeneration of maize plants from transformed proto described by Paszkowski et al., EMBO J. 3: 2717-2722 plasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 and Fromm et al. (Biotechnology 8: 833-839 (1990)) have (1985), Reichet al., Biotechnology 4: 1001-1004 (1986), and published techniques for transformation of A188-derived Klein et al., Nature 327: 70-73 (1987). In each case the maize line using particle bombardment. Furthermore, WO transformed cells are regenerated to whole plants using stan 93/07278 and Koziel et al. (Biotechnology 11: 194-200 dard techniques known in the art. (1993)) describe techniques for the transformation of elite 0140 Agrobacterium-mediated transformation is a pre inbred lines of maize by particle bombardment. This tech ferred technique for transformation of dicotyledons because nique utilizes immature maize embryos of 1.5-2.5 mm length of its high efficiency of transformation and its broad utility excised from a maize ear 14-15 days after pollination and a with many different species. Agrobacterium transformation PDS-1000He Biolistics device for bombardment. typically involves the transfer of the binary vector carrying 0145 The plants obtained via transformation with a the foreign DNA of interest to an appropriate Agrobacterium nucleic acid sequence of the present invention can be any of a strain which may depend of the complement of vir genes wide variety of plant species, including those of monocots carried by the host Agrobacterium strain either on a co-resi and dicots; however, the plants used in the method of the dent Tiplasmid or chromosomally (Uknes et al. Plant Cell 5: invention are preferably selected from the list of agronomi 159-169 (1993)). The transfer of the recombinant binary vec cally important target crops set forth Supra. The expression of torto Agrobacterium is accomplished by a triparental mating a gene of the present invention in combination with other procedure using E. coli carrying the recombinant binary vec characteristics important for production and quality can be tor, a helper E. coli strain which carries a plasmid that is able incorporated into plant lines through breeding. Breeding to mobilize the recombinant binary vector to the target Agro approaches and techniques are known in the art. See, for bacterium strain. Alternatively, the recombinant binary vec example, Welsh J. R. Fundamentals of Plant Genetics and tor can be transferred to Agrobacterium by DNA transforma Breeding, John Wiley & Sons, NY (1981); Crop Breeding, tion (Hofgen & Willimitzer, Nucl. Acids Res. 16: 9877 Wood D. R. (Ed.) American Society of Agronomy Madison, (1988)). Wis. (1983); Mayo O. The Theory of Plant Breeding, Second US 2011/020 1 059 A1 Aug. 18, 2011

Edition, Clarendon Press, Oxford (1987); Singh, D. P. Breed coli isolates containing the modified pET24b expression vec ing for Resistance to Diseases and Insect Pests, Springer tor were selected on standard LB agar containing 50 ug/mL Verlag, NY (1986); and Wricke and Weber, Quantitative kanamycin. Genetics and Selection Plant Breeding, Walter deGruyter and 0152 Recombinant E. coli isolates were grown with shak Co., Berlin (1986). ing at 37 degrees C. for 8 hours to overnight in 20 mL of LB 0146 The genetic properties engineered into the trans media containing 50 ug/mL, kanamycin. The 20 mL of E. coli genic seeds and plants described above are passed on by culture was transferred to 1 L of autoinduction media (9.57g sexual reproduction or vegetative growth and can thus be trypton, 4.8 g yeast extract, 2 ml of 1 M MgSO4, 1 mL of maintained and propagated in progeny plants. Generally, 1000x trace metals, 20 ml of 50x5052, 20 mL of 50xM) maintenance and propagation make use of known agricultural (1000x trace metals: 36 mL sterile water, 50 mL of 0.1M methods developed to fit specific purposes such as tilling, FeC13 in 0.12M HCl, 2 mL of 1M CaCl2, 1 mL of 1MMnCl2 Sowing or harvesting. 4 H20, 1 mL of 1M ZnSO47 H20, 1 mL of 0.2M CoCl2 6 0147 The lock and/or key enzymes disclosed herein may H20, 2 mL of 0.1MCuCl22 H20, 1 mL of 0.2M NiC12 6 H20, also be incorporated into or maintained in plant lines through 2 mL of 0.1M Na2MoCl42 H20, 2 mL of 0.1M H3BO3) breeding or through common genetic engineering technolo (50x5052: 25 g glycerol, 73 mL H20, 2.5 g glucose 10 g gies. Breeding approaches and techniques are known in the alpha-lactose monohydrate) (50xM: 80 mL H20, 17.75 g art. See, for example, Welsh J. R., Fundamentals of Plant Na2HPO4, 17.0 g KH2PO4, 13.4 g NH4C1, 3.55g Na2SO4) Genetics and Breeding, John Wiley & Sons, NY (1981); Crop with 25 ug/mL, kanamycin and grown with shaking at 28 Breeding, Wood D. R. (Ed.) American Society of Agronomy degrees C. overnight. The E. coli cells were harvested out of Madison, Wis. (1983); Mayo O., The Theory of Plant Breed the autoinduction media by centrifugation at 10,000xg for 15 ing, Second Edition, Clarendon Press, Oxford (1987); Singh, minutes and the collected cells were frozen at -80 degrees C. D. P. Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quan 1B: Sucrose Isomerase (E.C. 5.4.99.11) titative Genetics and Selection Plant Breeding, Walter de 0153. The amino acid sequence for a sucrose isomerase Gruyter and Co., Berlin (1986). expressed by Erwinia carotovora has been listed in 0148. The relevant techniques are well known in the art GeneBank under the accession number YPO49947 (SEQ ID and include but are not limited to hybridization, inbreeding, NO: 14). The amino acid sequence of this Sucrose isomerase backcross breeding, multi-line breeding, dihaploid inbreed was back translated into a polynucleotide coding sequence ing, variety blend, interspecific hybridization, aneuploid using the codon preference of E. coli. The polynucleotide techniques, etc. Hybridization techniques also include the sequence was generated by gene synthesis (GeneArt) and sterilization of plants to yield male or femalesterile plants by cloned into the expression vector pET24b (Novagen) using mechanical, genetic (including transgenic), chemical, or bio restriction sites that place the coding sequence in-frame chemical means. downstream of an inducible T71ac promoter. This expression 014.9 The following examples are offered by way of illus plasmid was introduced into an E. coli expression strain, tration and not by way of limitation. BL21, harboring DE3 lysogen. After growing for 3 hours in LB media containing 50 microgram/microliter kanamycin, EXPERIMENTAL the cells were induced to produce the E. carotovora Sucrose isomerase enzyme with IPTG at a final concentration of 1 0150 Standard recombinant DNA and molecular cloning mM. The E. coli cells were harvested 3 hours after induction techniques used here are well known in the art and are by centrifugation at 10,000xg for 10 min and the supernatant described by J. Sambrook, E. F. Fritsch and T. Maniatis, was removed. Cells were lysed by resuspending the cell pellet Molecular Cloning: A Laboratory manual, Cold Spring Har in BugBuster reagent (Novagen) containing lysozyme bor laboratory, Cold Spring Harbor, N.Y. (1989) and by T.J. (1 KU/1 mL BugBuster) and benzonase (25 units/1 mL Bug Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Buster) followed by incubation for 10 mM on a shaking Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring platform. Insoluble debris was removed by centrifugation at Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current 16,000xg for 20 min at 4 degrees C. Supernatant containing Protocols in Molecular Biology, pub. by Greene Publishing total soluble protein and the recombinant enzyme was trans Assoc. and Wiley-Interscience (1987). ferred to a fresh 1.5 mL Eppendorf tube and aliquots were stored at 4 degrees C. and -20 degrees C. for further charac Example 1 terization. 0154 Sucrose isomerase enzyme activity was assayed by Enzymes that can Produce Locked Sugars combining the enzyme with the Substrate, Sucrose, and mea Suring the production of isomalitulose and trehalulose. The 1A: Bacterial Expression System of His-Tagged Enzymes total soluble protein extract from the recombinant E. coli was assayed for Sucrose isomerase activity by incubating 10 0151. Selected genes coding for specific enzymes were microliters of Supernatant E. coli lysate, as described above, cloned into an Escherichia coli expression vector, pFT24b with 90 microliters of 292 mM sucrose 50 mM sodium phos (Novagen), using restriction sites that place the coding phate buffer (pH 6.0) at 30 degrees C. for 20 hours. The sequence in-frame downstream of an inducible T71ac pro reaction product was screened for the presence of isomaltu moter. Polynucleotide sequences coding for specific enzymes lose and trehalulose by thin layer chromatography (TLC) and were generated by back translating the polypeptide sequence high pressure liquid chromatography (HPLC). of the enzyme using the codon preference for E. coli. The 0155 TLC was performed by spotting 3 microliters of the expression plasmids were introduced into an E. coli expres Supernatants of the growth media onto AL SIL G silica gel sion strain, BL21 Star (DE3) (Invitrogen). Recombinant E. plates (Whatman) and developed twice in a solvent consisting US 2011/020 1 059 A1 Aug. 18, 2011

of 3 parts ethylacetate: 3 parts acetic acid: 1 part distilled equilibrated with extraction buffer. The clarified lysate was water. After drying, the plates were sprayed with a dye mix loaded onto the equilibrated column at 5 mL/min. Bound ture consisting of 4 milliliters aniline, 4 g diphenylamine, 200 his-tagged Sucrose isomerase was eluted in a linear imidazole milliliters acetone, and 30 milliliters 80% phosphoric acid. gradient from 50 mM sodium phosphate, 500 mM NaCl, 10 Isomaltulose and trehalulose were distinguished from other mM Imidazole, pH 8 to 50 mM sodium phosphate, 500 mM Sugars, such as Sucrose, by their relative mobility and by the NaCl, 200 mMImidazole over 100 mL. Fractions containing distinct colors produced when they reacted with aniline dye. the enzyme were collected and diluted in 50 mM Tris-HCl, Greenish yellow indicates the presence of isomalitulose, red pH 8. Diluted sample was loaded onto a 5 mL HiTrap QHP indicates the presence of trehalulose, and brown/black indi cates the presence of Sucrose. The monosaccharides, glucose anion exchange column (GE Healthcare). Bound proteins and fructose, produced by hydrolysis of sucrose were blue or were eluted from the column by running a linear NaCl gra red-orange respectively. dient from 50 mM Tris-HCl, pH 8 to 50 mM Tris-HCl, 500 0156 Identification of the sugars present in each lane of mM NaCl, pH 8 over 100 mL. Active sucrose isomerase was the developed TLC plate was possible by comparing both the detected in the flow through and fractions that eluted at relative mobility of the Sugars present in the samples and the approximately 100 mM NaCl. These fractions were pooled staining color with aniline dye to the relative mobility and and concentrated to a final protein concentration of 0.8 staining color of Sugar standards. The reaction product of mg/mL. Samples were aliquoted and stored at -80 degrees C. Sucrose isomerase incubated with Sucrose as described above 0159. Sucrose isomerase enzyme activity was measured in was three colored bands. The highest mobility band had a the samples by combining 6 ug/mL his-tagged Sucrose purple color and migrated with the same mobility as both isomerase, 70 mM 0.1 M Citrate-phosphate buffer, pH 6 and glucose and fructose standards blue and red colored respec 584 mM sucrose at 30 degrees C. for 2 hours. Sample was tively and is therefore interpreted to be a mixture of co migrat analyzed by Dionex essentially as described in Example 1G. ing glucose and fructose released by hydrolysis of one of the Table 1 outlines the sucrose isomerase activity detected in disaccharides: Sucrose, isomalitulose, or trehalulose. The recombinant E. coli cells expressing Sucrose isomerase (SEQ middle band corresponded with the isomalitulose standard in ID NO: 14). Activity is demonstrated by the accumulation of both coloration and relative mobility and is therefore identi the locked Sugars trehalulose and isomalitulose. fied as isomalitulose. The slowest migrating band had a red coloration and migrated slower than either the isomalitulose, TABLE 1 or sucrose standards. The relative mobility of this sugar band corresponds well with published reports on the migration of Sucrose isomerase (SEQID NO: 14) activity measured trehalulose in similar TLC assays (Cho et al. Biotechnology using Sucrose as the Substrate after 2 hr. Letters (2007) 29:453-458; an isomalitulose-producing Glucose Fructose Sucrose Trehallulose Isomalitulose microorganism isolated from traditional Korean food.) Time (mM) (mM) (mM) (mM) (mM) Therefore this sugar band was concluded to be trehalulose. No trehalulose standard was available at the time of the TLC Sucrose S.98 4.97 O.61 227.96 248.45 isomerase assay, however, subsequent HPLC (Dionex) analysis of Negative O O 512 O O Sucrose isomerase reaction products and standards obtained control later indicate that this band was definitely trehalulose. Also, it is important to note that the reaction product 6 did not contain any Sucrose which has a higher relative mobility than isoma litulose and trehalulose and slower mobility than the monosac 1C: Dextranslucrase Enzyme (E.C. 2.4.1.5) charides glucose and fructose. The absence of Sucrose was 0160 Dextranslucrases (E.C. 2.4.1.5) are glucosyl trans expected due to the complete conversion of Sucrose into iso ferase enzymes capable of transferring glucose from a maltulose and trehalulose due to the activity of the sucrose Sucrose molecule to form glucose homopolymers known as isomerase enzyme. dextrans. This type of enzymatic reaction is an example of 0157 Alternatively, supernatants were scretext miss transglycosylation. The dextran is composed of mainly 1.6 ing or illegible when filed separate sucrose isomerase alpha D glucose linkages of varying length. The dextran can reaction products followed by a linear gradient from 10 to 40 also contain a variety of 1.4 alpha D glucose linkages which min using 200 mM NaOH at 1 ml/min on a Dionex DX-600 form branch points in the dextran molecule. These branching system with ED50 electrochemical detector (Dionex Co.). points have a direct impact on the physiochemical properties (such as solubility) of the dextran molecules. The polynucle His-Tagged Sucrose Isomerase (SEQID NO: 14) otide sequence coding for a dextranslucrase enzyme will be 0158 Recombinant BL21 DE3 cell pellets expressing generated that uses the codon preference for E. coli. This his-tagged sucrose isomerase (SEQID NO: 14) were gener polynucleotide sequence will be synthesized, cloned into an ated essentially as described in Example 1A. The recombi expression vector and expressed in E. coli as described in nant BL21 cell pellets were brought up to a volume of 40 mL Example 1A. in extraction buffer (50 mM sodium phosphate, 500 mM 0.161 Dextranslucrase enzyme activity will be monitored NaCl, 10 mMImidazole, pH 8 containing protease inhibitors using a colorimetric assay to detect the rate of fructose release (Roche Complete EDTA-free protease inhibitor tablets)). from sucrose (Kobayashi, Metal. (1980) Biochimica et Bio Cells were lysed by 2 passages through a FRENCH Press physica Acta Vol 614, pp. 46–62). Dextran accumulation will (Thermo IEC). Cell lysate was centrifuged for 30 minutes at be monitored using methods similar to those described in 10,000xg at 4 degrees C. Supernatant was filtered using 0.45 Zhang, S., et al. (2007) Transgenic Res. 16:467-478 in com micron vacuum filter devices (Millipore) to generate a clari bination with HPLC techniques such as size exclusion chro fied lysate. A HisTrap FF 5 ml column (GE Healthcare) was matography. Dextranslucrase enzyme activity assays will be US 2011/020 1 059 A1 Aug. 18, 2011 validated by comparing dextranslucrase activity recovered except for elution from HisTrap was in 50 mM sodium phos from recombinant E. coli with commercially available dex phate, 500 mMNaCl, containing 500 mMImidazole, pH 7.2. translucrase enzyme. 0166 His-tagged dextranslucrase with leucrose synthase 0162 Dextranslucrase activity will be measured using sug activity was diluted to 0.1 mg/mL in 40 mM HEPES, 40 mM arcane juice as the source of Sucrose. Selected E. coli expressed dextranslucrases will be incubated in a similar fash NaCl, 10% glycerol, pH 7.2-100 uI reactions were set up for ion as described above, however sucrose will be replaced with the leucrose synthase and the negative control with the fol Sugarcane juice as the Substrate. These experiments will be lowing conditions: designed to test the ability of the expressed enzymes to pro duce dextrans from Sucrose in the presence of other proteins and unknown compounds found in Sugarcane juice. #1 #2 0163 A mutant dextranslucrase has been characterized by Sample (0.1 mg/ml) 10 10 Hellmuth et al. Biochemistry 47: 6678-6684 (2008) which Buffer (200 mM Sorensen's Buffer + 500 60.8 60.8 alters the activity of the enzyme such that it can catalyze the mM CaCl2, pH 7) conversion of Sucrose to isomalitulose or leucrose. This dex 2M Sucrose 14.6 14.6 translucrase variant has leucrose synthase activity due to the 2MFructose O 14.6 ability of the variant enzyme to catalyze the conversion of Water 14.6 O Sucrose to leucrose. Total Reaction Volume 1OO 100 Analysis of His-Tagged Dextranslucrase with Leucrose Syn thase Activity (SEQID NO: 29). Wolumes in column 1 and H2 are in microliters 0164 Recombinant BL21 DE3 cell expressing a His tagged dextranslucrase with leucrose synthase activity (SEQ 0.167 Table 2 outlines data demonstrating that his-tagged ID NO: 29) was generated essentially as described in dextranslucrase (SEQ ID NO: 29) with leucrose synthase Example 1A. Frozen cell pellets were brought up to a volume activity is enzymatically active and converts Sucrose to leu of 30-40 mL in extraction buffer (50 mM sodium phosphate, crose and isomaltose. Dextranslucrase enzymes catalyze the 500 mMNaCl, 10 mMImidazole, pH 7.2 containing protease conversion of Sucrose to locked Sugars through a transglyco inhibitors (Roche Complete EDTA-free protease inhibitor Sylation reaction. Table 2, comparing sample 1 and sample 2. tablets)). Cells were lysed by 2 passages through a FRENCH demonstrates that dextranslucrase with leucrose synthase Press (Thermo IEC). Cell lysates were centrifuged for 30 activity has altered specificity toward producing leucrose ver minutes at 10,000xg at 4 degrees C. Supernatants were fil sus isomaltose dependent on the addition of fructose as a tered using 0.45 micron vacuum filter devices (Millipore). A secondary Substrate.

TABLE 2 Dionex analysis of carbohydrate products from microbially expressed His-tagged dextranslucrase with leucrose synthase activity. Enzyme activity indicated by the change in percent Sugar determined by comparing samples collected at time 0 and time 24 hours. Glucose Fructose Sucrose Isomaltose Isomalitulose Leucrose Sample (% total (% total (% total (% total (% total (% total set up Sugar) Sugar) Sugar) Sugar) Sugar) Sugar) 1 8.99 20.55 -37.46 3.16 O.66 4.09 2 140 -0.29 -6.57 O.12 0.57 4.77 1 O.O8 O.14 -0.22 O O O (Negative control) 2 -0.01 O.63 -0.62 O O O (Negative control) Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, Negative control contains bacterial fractions collected as described in Example 1A from cells containing an empty pHT24 vector,

HisTrap FF 5 ml column (GE Healthcare) was equilibrated 1D: Levan Sucrase, Fructosyl Transferase (E.C. 2.4.1.10, with extraction buffer and the clarified lysates were loaded at E.C. 2.4.1.99, E.C. 2.4.1.100) 5 mL/min. Bound his-tagged enzymes were eluted in 50 mM sodium phosphate, 500 mM NaCl, containing 300 mM Imi 0168 Sucrose:sucrose fructosyltransferase (SST) (EC dazole, pH 7.2. All samples were buffer exchanged into 50 2.4.1.99), 1.2-?3-fructan 1-fructosyltransferase (FFT) (EC mM HEPES, 50 mMNaCl, pH 7 using a HiPrep 26/10 desalt 2.4.1.100), and levan sucrase (EC 2.4.1.10) are enzymes ing column (GE Healthcare). 50% Glycerol was added to within the larger class of fructosyltransferases. The fructosyl such that the final buffer was 40 mM HEPES, 40 mM. NaCl, transferase enzymes catalyze the formation of fructans com 10% glycerol, pH 7. Protein concentrations were estimated posed of fructose linked by B(2-> 1) and/or f3(2->6) glucoside by Bradford assay. Samples were stored at -80 degrees C. bonds. Fructosyl transferases may be identified and isolated 0.165. As a negative control, BL21 DE3 cell pellets from plant, bacterial, or fungal sources. These enzymes may expressing the empty pET24b vector were processed as above be expressed in plants to accumulate fructans as storage car US 2011/020 1 059 A1 Aug. 18, 2011 bohydrates. Accumulation of this polysaccharide (fructan) in the activity of a alpha-1,6-glucosidase or alpha-1,3-glucosi Sugarcane or other plants may allow the accumulation of dase or a combination of the two enzymes. excess carbohydrates. 0169. The polynucleotide sequence coding for a fructosyl 1F: Amylosucrase (E.G. 2.4.1.4) transferase enzyme will be generated that uses the codon 0177 Amylose or starch, is a polysaccharide consisting of preference for E. coli. This polynucleotide sequence will be glucosyl residues linked by alpha-(1-4) bonds and is the pri synthesized, cloned into an expression vector and expressed mary carbohydrate storage compound found in most plants. in E. coli essentially as described in Example 1A. Producing starch in plants that use Sucrose as their primary 0170 Fructosyl transferase activity will be estimated by carbohydrate storage compound. Such as Sugarcane, may per TLC and HPLC similar to the procedures described above for Sucrose isomerase and the Dionex analysis described in mit the accumulation of starch which would behave as a Example 1B. Modifications to the protocol in order to locked Sugar. increase the sensitivity for fructans may include development 0.178 Neisseria polysacharea produces an amylosucrase in a solution of propanol:butanol: water (12:3:4) and the use of enzyme (GenBank Accession number Q97EU2) which cata a urea-phosphoric acid dye mixture (Wise et al., 1955, Anal lyzes the conversion of Sucrose to a linear alpha-1,4-linked Chem 27:33-36). Long polymers of fructose have low mobil glucan. For the purpose of producing starch in a transgenic ity in the TLC assay and will remain in the location where plant, it may be advantageous to target the amylosucrase they are spotted on the silica gel plate. Hydrolysis of fructans enzyme in the plant to Subcellular compartments that have to fructose by HCl solution will allow specific identification high concentrations of Sucrose, such as the vacuole of Sugar of fructose using the aniline dye described above. Alterna cane. Another target may be the vacuole of the maize tively a fructanase enzyme may be used to hydrolyze fructans endosperm. Targeting an enzyme capable of synthesizing to fructose. This technique will be useful in determining that starch from Sucrose to the vacuole of maize endosperm cells large polymers are indeed fructans as only fructans would be may permit the accumulation of more starch in the maize hydrolyzed by a fructanase enzyme. endosperm as naturally occurring enzymes do not produce 0171 Fructose, as the sweetest naturally occurring sugar, starch in the vacuoles of maize endosperms cells. Targeting also has value as a Sweetener in high fructose syrups such as Such an enzyme to endosperm vacuoles may be expected to high fructose corn syrup. Plants engineered to produce fruc create up to 10% more starch because of starch accumulation tans as a locked Sugar may be used as a source of fructans in a Subcellular compartment that normally does not accumu which, after hydrolysis by a fructanase enzyme, produce a late starch. Alternatively targeting to the apoplast is another Solution with a high fructose concentration. In Such plants the way to achieve conversion of sucrose into locked sugars such yield of fructan may be increased by expressing an additional as starch or isomaltulose. In plants such as maize, Sucrose enzyme glucose isomerase to catalyze the conversion of glu accumulates in the leaf and is transported to the ear during cose to fructose. The glucose isomerase (invertase) could be grain filling which provides a carbon sink. Table 3 outlines the expressed in maize endosperm, or expressed in microbes. The Sugar content of maize tissue with and without removal of the purified enzyme could be used to produce fructans, glucans ear. Note that when the ear is removed, excess Sugar accumu and alternans. lates in the leaf tissue. 0172 Sweeter plant products can be generated by express ing in plants a combination of enzymes that first allow for the TABLE 3 accumulation of fructans in the plant and then convert the fructans directly or indirectly to fructose. Expressing inver Sugar content of maize with and without ears. tase (glucose isomerase) in plants accumulating fructans will Sugar, mg/mL Earless maize Maize with Ear lead to a higher Sweetness index in the plant. Sucrose 7.42 2.6 0173 Endogenous sucrose synthase activity in the Glucose 1.34 1.OS endosperm will create additional Sucrose which may be used Fructose 1.32 O.9S as a substrate for further fructan synthesis. Total, mg/mL. 10.08 4.6 1E: Alternanslucrase 0174 Alternan is a polysaccharide consisting of glucosyl 0179 A codon optimized polynucleotide sequence coding residues linked by alternate alpha-(1-3)/alpha-(1-6) bonds. for the N. polysacharea amylosucrase enzyme may be Syn This polymer is highly soluble and has very low viscosity. thesized, cloned into an expression vector and expressed in E. Accumulation of this polysaccharide in Sugarcane or other coli essentially as described in Example 1A. plants may allow the accumulation of excess carbohydrates. His-Tagged AmyloSucrase Alternanslucrase is an enzyme which catalyzes the conversion of Sucrose to alternan. 0180 Recombinant BL21 cells expressing an amylosu 0175 Alternansucrase is encoded by the Asr gene of Leu crase will be generated essentially as described in Example conostoc mesenteroides NRRL B-1355, 1498, and 1501 1A. Frozen BL21 DE3 cell pellets expressing amylosucrase (Jeannes et al. Am Chem Soc 76:5041-5052, 1954). The Asr will be recovered from a 30 mL overnight culture in autoin gene may be synthesized, cloned into an expression vector duction media and will be resuspended in 3 mL BugBuster and expressed in E. coli essentially as described in Example HT (Novagen) containing Complete EDTA-free protease 1A. inhibitors (Roche). Samples will be incubated at room tem 0176 Alternansucrase activity may be detected by perature for 10 minutes with occasional mixing to lyse cells. enzyme-linked immunosorbent assay (ELISA) as described Cell lysate will be centrifuged at 10,000xg for 10 minutes at by Kok-Jacor et al. J. Plant Physiol 160: 765-777 (2005) 4 degrees C. 10 uI of supernatant will be incubated in a 500 Alternans can be hydrolyzed to form fermentable sugars by uL reaction containing 1xPBS and 100 mM sucrose overnight US 2011/020 1 059 A1 Aug. 18, 2011

at 30 degrees C. The presence of a visible white precipitate fructan by the fructanase will release the monosaccharide indicates amylosucrase activity. Determination that this pre fructose which may be detected by TLC or HPLC as cipitate is starch can be done by washing the precipitate in described above for sucrose isomerase (Example 1B). 80% ethanol several times, followed by solubilization in DMSO and gel permeation chromatography. Susceptibility to digestion by amylase enzyme would further demonstrate the 2B: Glucosidase precipitate is composed of starch. 0185. Gene sequences for alpha-1,6-glucositext miss ing or illegible when filed search the NCBI database for 1G: Dionex HPAEC Analysis of Carbohydrates genes homologtext missing or illegible when filed 0181 Carbohydrate separation and detection was ana polypeptide sequences (SEQID NOs: 1-6) were back trans lyzed utilizing a Dionex IC3000 system with a Dionex AS lated (using Vector NTI program) into polynucleotide autosampler, a Dionex DC detection compartment (pulsed sequences using the codon preference of E. coli. The E. coli amperometric detection (PAD) using a disposable Dionex codon optimized polynucleotide sequences were synthesized carbohydrate certified gold surface electrode), and a Dionex by GeneArt and expressed in E. coli essentially as described SP pump system. For high resolution separation, one Carbo in Example 1B. pac PA1 4x50mMGuard Column followed by two Carbopac 0186 Alpha-1,6-glucosidase activity was assayed by PA1 4x250 mManalytical columns were used for all analysis. measuring the production of glucose from hydrolysis of the The electrode potentials were set to the carbohydrates stan alpha-1,6-glucoside bond of isomalitulose. 13 microliters of dard quad with AgCl reference electrode as specified by crude E. coli extract was added to 37 microliters of isomaltu Dionex Corporation. The eluent system utilized an isocratic mobile phase consisting of 100 mMNaOH and 2 mMNaOAc lose reaction buffer (100 mM isomalitulose and 30 mM with a 38 min run time. Peak identification was based on HEPES (pH 7.5)) at 30 degrees, 40 degrees, 50 degrees, 60 standard retention times of glucose, fructose, Sucrose degrees, 70 degrees, or 80 degrees C. depending on the (Sigma), leucrose (Carbosynth), isomalitulose (Fischer) and enzyme; for 10 minutes, 20 minutes, 30 minutes, or 40 min trehalulose. Peak analysis utilized Chromeleon version 6.80 utes. 20 microliters of the reaction product was added to a 96 software (Dionex Corp., Sunnyvale, Calif.). well microplate, then 250 microliters of glucose oxidase reagent (Pointe Scientific) was added and the mixture was Example 2 incubated at 37 degrees C. for 10 minutes. After this incuba Enzymes that Unlock Locked Sugars tion, the Absorbance at 500 nm was read using a SpectraMax plus 384. Sample absorbance was compared with the absor 2A: Fructanase (EC 3.2.1.80, E.C.3.2.1.7) bance at 500 nm of controls which were 13 microliters each of 0182 Fructanases are fructosydases which catalyze the a set of glucose standards that were also allowed to react with hydrolysis of fructosidic linkages in fructans to break the the glucose oxidase reagent. A standard curve was created fructan down into simpler Sugar molecules. Fructans can be from the controls and the production of glucose from the hydrolyzed to fermentable Sugars through the catalytic activ hydrolysis of isomalitulose by the samples was estimated by ity of fructanases. For Example, the fructanase 2,1-B-D-fruc tan fructanohydrolase IEC 3.2.1.7 can hydrolyze fructan comparing the absorbance at 500 nm for the samples to the polymers into fructose monosaccharides which can be fer standard curve. mented to form ethanol. 0187 Using this method, the alpha-1,6-glucosidase 0183. A codon optimized polynucleotide sequence coding enzymes described by SEQID NOs: 1-6 were screened and for a fructanase enzyme may be synthesized, cloned into an found to have activities at temperatures ranging from 30 expression vector and expressed in E. coli essentially as degrees C. to 80 degrees C. Table 4 describes the alpha-1,6- described in Example 1A. glucosidase activity measured in total cell lysate of an E. coli 0184 Fructanase activity may be estimated by incubating strain expressing the Bacillus thermoamyloliquefaciens a fructanase enzyme with a solution of fructan. Hydrolysis of enzyme (SEQID NO:5). US 2011/0201059 A1 Aug. 18, 2011 20 Table 4: Alpha-1,6-glucosidase (SEQ ID NO. 5) activity measured using sucrose as the substrate.

1 2

s O s

2 8 lsomalitulose elsomaltotriose 6 Dextran t E 4.

50 °C Temperature US 2011/020 1 059 A1 Aug. 18, 2011

His Tagged Enzyme Recovery from Recombinant E. coli G. thermoglucosidasius alpha-1,6-glucosidase (SEQID NO: 0188 Recombinant BL21 E. coli cells expressing an 1): alpha-1,6-glucosidase (SEQ ID NOs: 1, 3, 5 and 6) were 0194 His-tagged G. thermoglucosidasius alpha-1,6-glu generated essentially as described in Example 1A. The frozen cosidase (SEQ ID NO: 1) was recovered from recombinant cell pellets expressing the his-tagged alpha-1,6-glucosidase BL21 E. coli cells essentially as described above (Example 2B“His tagged enzyme recovery from recombinant E. coli). key enzymes were brought up to a Volume of 40 mL in Fractions containing his-tagged enzyme were pooled and extraction buffer (50 mM sodium phosphate, 500 mM NaCl, diluted in 50 mM Tris-HCl, pH 7. Sample was applied to a 5 10 mM Imidazole, pH 7.2-8 containing protease inhibitors mL HiTrap QHP column (GE Healthcare). Bound proteins (Roche Complete EDTA-free protease inhibitor tablets)). were eluted by washing the column with a linear NaCl gra Cells were lysed by 2 passages through a FRENCH Press dient over 100 mL from 50 mM HEPES, 10 mM. NaCl, pH7 (Thermo IEC). Cell lysates were centrifuged for 30 minutes at to 50 mM HEPES, 1 MNaCl, pH 7. The fractions containing 10,000xg at 4 degrees C. Supernatants were collected and the enzyme were pooled and concentrated to 1 mL Centri filtered using 0.45 micron vacuum filter device (Millipore). prep YM-30 concentrator device (Amicon). Sample was (0189 A His Trap FF column was used to recover the applied to a HiPrep 26/60 S-100 HR size exclusion column his-tagged enzymes from the Supernatant. A HisTrap FF 5 mL and eluted with 20 mM Tris-HCl, 250 mM. NaCl, pH 7. column (GE Healthcare) was equilibrated with extraction Fractions containing the enzyme were pooled and diluted in buffer. The clarified lysates were loaded at 5 mL/min. Bound 1.5 M Ammonium Sulfate, 50 mMSodium phosphate, pH7. his-tagged enzymes were eluted in 50 mM sodium phosphate, Sample was applied to a 5 mL HiTrap Phenyl HP column (GE 500 mMNaCl, containing 150-500 mMImidazole, pH 7.2-8. Healthcare). Bound proteins were eluted by washing the col umn with a linear ammonium sulfate gradient over 100 mL (0190. The negative control was BL21 DE3) cell pellets from 50 mM Sodium phosphate, 1.5 Mammonium sulfate, transformed with empty pET24b vector essentially as pH 7 to 50 mM sodium phosphate buffer pH 7 containing no described in Example 1A. Negative control cell pellets were ammonium Sulfate. Fractions containing the enzyme were extracted essentially as described above for the his-tagged pooled. alpha-1,6-glucosidase enzymes; however, the extraction B. thermoamyloliquefaciens alpha-1,6-glucosidase (SEQID buffer and elution buffers were at pH 7.2. NO. 5): 0191 All samples collected from the HisTrap FF column 0.195 His-tagged B. thermoamyloliquefaciens alpha-1,6- were bufferexchanged into 50 mM HEPES, 50 mMNaCl, pH glucosidase (SEQ ID NO. 5) was recovered from recombi 7 using either Bio-Rad Econo-Pac 10-DG desalting column nant BL21 E. coli cells essentially as described above (Ex or HiPrep 26/10 desalting column (GE Healthcare). 50% ample 2B “His tagged enzyme recovery from recombinant E. Glycerol was added such that the final buffer was 40 mM coli). Fractions containing his-tagged enzyme were pooled HEPES, 40 mM. NaCl, 10% glycerol, pH 7. Protein concen and diluted in 20 mM Tris-HCl, pH 7. Sample was applied to trations were estimated by Bradford assay. Samples were a 5 mL HiTrap Q HP column (GE Healthcare). Bound pro stored at -80 degrees C. teins were eluted by washing the column with a linear NaCl T. ethanolicus alpha-1,6-glucosidase (SEQID NO: 6): gradient over 100 mL from 20 mM Tris-HCl, 50 mM. NaCl, pH 7 to 50mMHEPES, 1 MNaCl, pH 7. Fractions containing 0.192 His-tagged T. ethanolicus alpha-1,6-glucosidase the enzyme were pooled and concentrated to 1 mL Centri (SEQ ID NO: 6) was recovered from recombinant BL21 E. prep YM-30 concentrator device (Amicon). Sample was coli cells essentially as described above (Example 2B “His applied to a HiPrep 26/60 S-100 HR size exclusion column tagged enzyme recovery from recombinant E. coli). Frozen and eluted with 50 mM HEPES, 50 mM. NaCl, pH 7.4. Frac samples derived from the HisTrapFF column were combined tions containing the enzyme were pooled in 1.5 M Ammo with 3 Mammonium sulfate, 50 mMammonium phosphate, nium Sulfate, 50 mM Sodium phosphate, pH7. Sample was pH 7 to a final ammonium sulfate concentration of 1 M. This applied to a 5 mL HiTrap Phenyl HP column (GE Health sample was applied to a 5 mL HiTrap Phenyl HP column (GE care). Bound proteins were eluted by washing the column Healthcare). Bound proteins were eluted from the column by with a linear ammonium sulfate gradient over 100 mL from washing the column with a linear ammonium sulfate gradient 50 mMSodium phosphate, 1.5 Mammonium sulfate, pH 7 to over 100 ml from 50 mM Sodium phosphate, 1.5 Mammo 50 mM sodium phosphate buffer pH 7 containing no ammo nium sulfate, pH 7 to 50 mM sodium phosphate buffer pH 7 nium sulfate. Fractions containing the enzyme were pooled. containing no ammonium sulfate. Fractions containing the Activity of His-Tagged alpha-1,6-glucosidase Key Enzymes enzyme were pooled and concentrated using Centri-prep 0196. The enzyme activity of the alpha-1,6-glucosidase YM-30 concentrator device (Amicon). enzymes (SEQID NOs: 1, 3, 5 and 6) recovered from recom B. thurgiensis alpha-1,6-glucosidase (SEQID NO:3): binant BL21 E. coli cells was measured. Samples collected 0193 His-tagged B. thurgiensis alpha-1,6-glucosidase from the purification schemes described above (Example 2B) (SEQ ID NO:3) was recovered from recombinant BL21 E. were diluted to 0.2 mg/mL in 50 mM HEPES, 50 mM NaCl, coli cells essentially as described above (Example 28 "His pH 7. Reactions were initiated by mixing samples with an tagged enzyme recovery from recombinant E. coli). Frac equal volume of 100 mM HEPES, 4 mM EDTA, 0.04% tions containing his-tagged enzyme were pooled and diluted Tween-20, 200 mM Isomalitulose, pH 7. For buffer controls, in 50 mM HEPES, pH 6. Sample was applied to a 5 mL 100 mM HEPES, 4 mM EDTA, 0.04% Tween-20, pH 7 was HiTrap QHP column (GE Healthcare). Bound proteins were combined with an equal volume of 200 mM Isomalitulose. eluted by washing the column with a linear NaCl gradient Reactions were incubated at optimal temperature for the over 100 mL from 50 mM HEPES, pH 6 to 50 mM HEPES, enzyme (37, 45, or 60 degrees C.) for 40 minutes in a Biorad 1 MNaCl, pH 6. The fractions containing the enzyme were Tetrad 2 thermocycler for the appropriate time. Reactions pooled. were terminated by heating samples at 95 degrees C. for 5 US 2011/020 1 059 A1 Aug. 18, 2011 22 minutes. Glucose concentrations in reactions were estimated mg/mL in 40 mM HEPES, 40 mMNaCl, 10% glycerol, pH 7. using the GOPOD assay. Enzyme activity is detected as the Enzyme activity assasys were initiated by mixing samples conversion of isomalitulose to glucose. with an equal volume of 100 mM HEPES, 4 mM EDTA, (0197) The GOPODassay was performed by combining 20 0.04% Tween-20, 200 mM leucrose (for alpha-1,5-glucosi uL aliquots of reaction samples, or glucose standards of dase key enzymes (SEQID NOs: 30-33)) or 135 mM treha known concentrations, with 250 uL GlucoseCox Reagent lulose/67 mM isomalitulose mixture (for alpha-1,1-glucosi (Pointe Scientific) in a 96-well assay plate (Costar 3370) and dase key enzyme (SEQ ID NO. 34)), pH 7. Reactions were incubated for 10 minutes at 37 degrees C. Absorbance at incubated at optimal temperature (70 degrees C. for alpha-1, wavelength of 500 nm was measured using SpectraMax 384 5-glucosidase enzymes and 80 degrees C. for alpha-1,1-glu Plus plate reader. Absorbance values of sample reactions cosidase key enzyme) for 40 minutes in a text missing or were converted to glucose concentrations using the equation illegible when filediate time. Reactions were terminated from a glucose standard curve generated by plotting the by text missing or illegible when fileds. Key enzyme activity was demonstrated by the conversion of a locked sub absorbance value versus the known glucose standard concen strate (leucrose or trehalulose and/or isomalitulose) to glu tration. The activity of the various alpha-1,6-glucosidase cose. Glucose concentrations in reactions were estimated enzymes is described in Table 5. using GOPOD assay essentially as described above. Table 6 outlines data which demonstrates that his-tagged alpha-1,5- TABLE 5 glucosidase enzymes and alpha-1,1-glucosidase enzyme are active and convert locked Sugar Substrates to fermentable Activity data for alpha-1,6-glucosidase enzymes Sugar. Reaction temperature in Sample (SEQ ID NO) Glucose (mM) degrees C. TABLE 6 T. ethanolicus (6) 19.72 60 Conversion of locked Sugars to glucose by his-tagged key enzymes. G. thermoglucosidasius (1) 29.16 60 Negative control O.O7 60 Sample name GK24 N- GK24 HB27 HB8 Negative Buffer only negative control O.O3 60 (SEQID NO:) del (30) (31) (32) (33) Control B. thungiensis (3) 23.35 37 Negative control O.O7 37 Glucose O.94 1.01 O42 1.56 O.OS Buffer only negative control O.O1 37 Conc. (mM) B. thermoamyloiquefaciens 1.17 45 Sample name SAM1606 Negative (5) (SEQID NO: (34) control Negative control O.09 45 Glucose 8.67 O46 Buffer only negative control O.O1 45 concentration (mM) Purification of His-Tagged alpha-1,5-glucosidase and alpha 1,1-glucosidase Key Enzymes. 2C: Dextranase (E.C.3.2.1.11) (0198 Recombinant BL21 DE3) cell pellets expressing 0201 Dextranases are glycosidases which catalyze the His-tagged alpha-1,5-glucosidase and alpha-1,1-glucosidase exo or endohydrolysis of 1.6 alpha D glucosidic linkages in key enzymes were generated essentially as described in dextrans thus converting the dextran to Smaller Sugar mol Example 1A. Frozen cell pellets were brought up to a volume ecules. A codon optimized polynucleotide sequence coding of 30-40 mL in extraction buffer (50 mM sodium phosphate, for a dextranase enzyme may be synthesized, cloned into an 500 mMNaCl, 10 mMImidazole, pH 7.2 containing protease expression vector and expressed in E. coli essentially as inhibitors (Roche Complete EDTA-free protease inhibitor described in Example 1A. tablets)). Cells were lysed by 2 passages through a FRENCH 0202 Dextranase enzyme activity assays will monitor the Press (Thermo EC). Cell lysates were centrifuged for 30 rate of isomaltose released from a dextran molecule during a minutes at 10,000x g at 4 degrees C. Supernatants were hydrolysis reaction. HPLC size exclusion chromatography filtered using 0.45 micron vacuum filter devices (Millipore). will also be employed to determine the level of dextran A HisTrap FF 5 ml column (GE Healthcare) equilibrated with hydrolysis achieved by measuring the release of individual extraction buffer was used to clarify the lysates which were Sugars. loaded at 5 mL/min. Bound his-tagged enzymes were eluted 0203 Assays will be validated using a commercially in 50 mM sodium phosphate, 500 mM NaCl, containing 300 available dextranase from Penicillium sp I.U.B.: 3.2.1.11 mM Imidazole, pH 7.2. All samples were buffer exchanged (Worthington Biochemical Corporation, N.J. 08701). The into 50 mM HEPES, 50 mMNaCl, pH 7 using a HiPrep 26/10 dextran hydrolysis can be measured by incubating 0.1 mL of desalting column (GE Healthcare). 50% Glycerol was added 5-20 micrograms/mL of dextranase with 1.9 mL of commer to such that the final buffer was 40 mMHEPES, 40 mMNaCl, cially available dextran solution (substrate). Thermostability 10% glycerol, pH 7. Protein concentrations were estimated of dextranases will be tested in experiments performed at 60 by Bradford assay. Samples were stored at -80 degrees C. to 70 degrees C. which are temperatures relevant to sugar mill (0199. As a negative control, BL21 DE3) cell pellets Sugarcane juice processing. Validated assays will be further expressing the empty pET24b vector were processed as optimized for detection of functional dextranases cloned and described above except for elution from HisTrap was in 50 expressed in E. coli. mM sodium phosphate, 500 mM NaCl, containing 500 mM Example 3 Imidazole, pH 7.2. Activity Analysis of His-tagged alpha-1,5-glucosidase and Transgenic Plants alpha-1,1-glucosidase Key Enzymes 3A. Transgenic Sugarcane 0200 Extracts of his-tagged enzymes were generated 0204 Embryogenic callus was produced from the imma essentially as described above and were diluted to 0.08 ture leaf tissue of Sugarcane. In greenhouse, cane was har US 2011/020 1 059 A1 Aug. 18, 2011

vested by cutting off immature shoots at or above ground level tured to fresh selection media every 2 weeks until they were and outer leaves and leaf sheaths were stripped. Basal nodes large enough for analysis. Typically, 2 to 3 rounds of Subcul and emergent leaves were trimmed. In the laboratory (laminar ture were required. flow cabinet), excess leaf sheaths were unfurled, nodes were Regeneration of Plants from Transgenic Callus Lines trimmed and cane was sterilized (sprayed with 70% ethanol 0210. After 4-5 weeks on mannose selection media, Sur or immersed in 20% bleach for 20 minutes). Additional outer viving embryogenic callus colonies are selectively isolated leaf sheaths were removed to expose inner 4-6 leaf rolls and from original cultures and transferred onto regeneration leaf roll was cut to manageable size (12-15 mm in length). media (MS salts and B5 vitamins, 30 g/L sucrose, supple Remaining basal nodes and internodes were removed to mented with 3-6 g/L mannose and 2 mg/L BAP) at 28 degrees expose the leafroll region just above the apical meristem. C. in dark in Flambeau boxes. 0211 One week later, the cultures are transferred to a light 0205 Transverse sections of the leafroll were cut to form room for shoot development under 16 hours light at 28 discs 0.5-1.0 mm in thickness, using not more than a 3.0 cm degrees C. After 3-4 weeks in the regeneration media, the length of the leafroll material. Leafroll discs were plated onto visible green buds or shoots are sub-cultured on elongation MS media containing 2-3 mg/L of 2,4-D and cultured in the media (MS basal salts and B5 vitamins, sucrose 30 g/L with dark for 3-4 weeks. Leafroll discs were cutor split apart at the hormone-free). time of initiation or 2 weeks following initiation and the 0212 Regenerated shoots are rooted in the text missing resulting pieces spread across media to promote a more con or illegible when filedi rooting cultures are kept at 28 sistent and prolific embryogenic/proto-embryogenic culture degrees C. under light for another 2 weeks before transfer to response. After 3-4 weeks of culture, embryogenic callus was the greenhouse and soil. Any of the genes described in selectively excised from leaf disc rolls and sub-cultured on Example 1. Example 2 or Example 12 can be transformed into same (MS+2,4-D) media. Further selective subcultures were Sugarcane to generate transgenic plants using the above performed every 2-3 weeks, dependent upon growth and described protocol. Agrobacterium mediated genetic trans development to produce additional cultures, until cultures formation is also possible and methods are described in the literature such as Arencibia, Ariel D. and Carmona, Elva R. reach 8-10 weeks of age. Sugarcane (Saccharum spp.) Methods in Molecular Biology Gene Delivery using the Biolistics PDS 2000 Particle Deliv (Totowa, N.J., United States) (2006), 344 (Agrobacterium ery Device for Sugarcane Transformation Protocols (2nd Edition), Volume 2), 227-235 0206 Target embryogenic cultures were prepared for gene delivery by selecting high quality target tissue pieces and 3B: Transgenic Sugarcane Expressing Dextranslucrase Activ preculturing them for 3-6 days on fresh media before gene ity delivery. 0213 Selected dextranslucrases are sequence optimized 0207. At 2-5 hours prior to gene delivery, target tissues based upon the codon preference for Sugarcane. The Sugar were arranged in a target pattern on high osmotic potential cane codon optimized sequence is cloned into transformation media containing MS basal salts and B5 Vitamins supple vectors for Sugarcane transformation. One of skill in the art is mented with sucrose 30 g/L and 0.2 M sorbitol and 0.2 M able to select the appropriate promoter and terminator for the mannitol plus 2 mg/l 2,4-D. dextranslucrase gene as well as select an appropriate select 0208. To prepare DNA for bombardment, gold particles able marker for Sugarcane transformation. Targeting (0.6 micrometer size, Bio-Rad) were re-suspended in 50% sequences are incorporated into the expression construct for sterile glycerol by Vortexing. An aliquot of the glycerol— dextranslucrases to target the enzyme to the vacuolar compart gold particle Suspension was combined by gentle mixing with ment of parenchyma cells where Sucrose is stored. 2x10" mol DNA of the gene encoding the selectable marker 0214 Transgenic Sugarcane plants are generated as (PMI) and genes of interest outlined in Table 29 of Example described in Example 3A. Transformed plants are analyzed 12. The mixture was combined with 2.5M CaCl2 and cold 1M using routine methods for DNA analysis of transgenic plants spermidine to precipitate the DNA onto the gold particles. in order to determine if the expression construct has been The gold particles with precipitated DNA were washed with incorporated into the nuclear DNA of the Sugarcane plant. ethanol. The gold particles were repeatedly re-suspended in 0215 Transgenic Sugarcane plants are evaluated for dex ethanol and aliquots of DNA/particle Suspension were placed translucrase enzyme activity. Mature plant tissue is crushed evenly onto the center of individual macrocarrier membrane and the juice will be collected and chilled prior to assaying for disks and allowed to dry. The macrocarrier was loaded into dextran accumulation using the detection methods described the gene gun above the stopping screen. Bombardment of in Example 1C.. Enzyme assay methods described in Example embryos was performed with a PDS—1000 Helium gene 1C are used to determine the functionality of the expressed gun. A rupture disc of 1300 psi was used and the distance from enzyme in transgenic plants. the rupture disc and the macrocarrier was set at 8 mm with a stopping screen at 10 mm. The distance between the stopping 3C: Generation of Transgenic Plants Expressing Dextranase screen and the embryos was about 7 cm. The pressure on the Activity. helium tank was set at about 1400 psi. Target tissues (embryo 0216 Selected dextranases are codon optimized for genic cultures) were bombarded with 2 shots before being expression in Sugarcane using the codon preference for Sug transferred to the dark at 28 degrees C. for about 12 hours. arcane. The Sugarcane optimized gene sequence is cloned 0209. After recovery, the bombarded cultures were trans into a transformation vector designed for Sugarcane transfor ferred to maintenance medium and cultured at 28 degrees C. mation. One of skill in the art is able to select the appropriate in the dark. After 7 days, the bombarded cultures were trans promoter and terminator for the dextranase as well as select ferred to fresh selection medium containing mannose (7-9 an appropriate selectable marker for Sugarcane transforma grams/L), 5 g/L Sucrose plus 2 mg/L 2,4-D and incubated for tion. The dextranase enzyme is targeted to the ER subcellular 4-5 weeks in dark. Growing callus pieces were then Subcul compartment of parenchyma cells using the appropriate tar US 2011/020 1 059 A1 Aug. 18, 2011 24 geting sequences. The dextranase enzyme is targeted away containing an origin of replication from BCTV, beet curly top from the Sucrose and dextran storage compartment of the virus (SEQID NO: 8). The binary vectors without the BCTV sugarcane plant.text missing or illegible when filed origin of replication were transferred into Agrobacterium Transgenic plants are generated as described in Example 3A. Enzyme activity is evaluated in mature plant tissue by crush tumefaciens strain LBA4404 using the freeze-thaw method ing and extracting juice from the transgenic plant and per (An et al., Binary vector. In: Gelvin SB, Schilproot RA (eds), forming the assays for dextranase activity as described in Plant molecular biology manual. Kluwar Academic Publish Example 2C. Enzyme assay methods described in Example ers, Dordrecht, pp A31-19 (1988)). The BCTV containing 2C are used to determine the functionality of the expressed binary vectors were transferred into Agrobacterium tumefa enzyme in Sugarcane juice 3D: Transient expression in ciens strain LBA4404 containing a helperplasmid containing tobacco and Sugar beet leaves a BCTV replicase sequence (SEQID NO:9) using the freeze 0217 Expression cassettes described in Example 12 were cloned into either a binary vector or a binary vector also thaw method (An et al., Binary vector. In: Gelvin SB, Schil containing an origin of replication from BCTV, beet curly top proot R A (eds), Plant molecular biology manual. Kluwar virus, (SEQID NO:8). The binary vectors without the origin Academic Publishers, Dordrecht, pp A31-19 (1988)). of replication from BCTV were transferred into Agrobacte 0221) Leaves from sugar beet or tobacco were used for rium tumefaciens strain LBA4404 using the freeze-thaw transient expression of enzymes. Transgenic TEV-B tobacco method (An et al., Binary vector. In: Gelvin SB, Schilproot R plants (made in the tobacco cultivar Xanthi) containing a A (eds), Plant molecular biology manual. Kluwar Academic mutated Pl/HC-Pro gene from TEV that suppresses post Publishers, Dordrecht, pp A31-19 (1988)). The binary vec transcriptional gene silencing (Mallory et al., Nat Biotechnol tors containing the origin of replication from BCTV (BCTV 20:622 (2002)) were used for transient expression of selected binary vectors) were transferred into Agrobacterium tumefa enzymes in tobacco leaves. Preparation of Agrobacterium ciens strain LBA4404 containing a helperplasmid containing cultures and infiltration of tobacco or Sugar beet plants was a replicase sequence from BCTV (SEQID NO: 9) using the carried out as described by Azhakanandam et al., Plant Mol. freeze-thaw method (An et al., Binary vector. In: Gelvin SB, Biol. 63: 393-404 (2007). In brief, the genetically modified Schilproot. RA (eds), Plant molecular biology manual. Klu agrobacteria were grown overnighttext missing or illeg war Academic Publishers, Dordrecht, pp A31-19 (1988)). ible when filed acetosyringone and 10 uMMES (pH 5.6), 0218 Leaves from sugar beet or tobacco were used for the and subsequently were pelleted by text missing or illeg transient expression of enzymes in plant tissue. Tobacco ible when filedcentrifugation at 4000xg for 10 min. The pellets were resuspended in the infection medium Murashige leaves from transgenic TEV-B tobacco plants (made in the and Skoog salts with vitamins, 2% sucrose, 500 uMMES (pH tobacco cultivar Xanthi) containing a mutated Pl/HC-Pro 5.6), 10 uM MgSO, and 100 uM acetosyringone to gene from TEV that Suppresses post-transcriptional gene ODoo-1.0 and subsequently held at 28 degrees C. for 3 silencing (Mallory et al., Nat Biotechnol 20:622 (2002)) were hours. Infiltration of individual leaves was carried out on used for transient expression of selected enzymes. Prepara sugar beet (about 3 weeks old) and TEV-B tobacco plants tion of Agrobacterium cultures and infiltration of tobacco or (about 4 weeks old) using a 5 mL Syringe by pressing the tip Sugar beet leaves was carried out as described by AZhakanan of the Syringe (without a needle) against the abaxial Surface of dam et al., Plant Mol. Biol. 63: 393-404 (2007). In brief, the the leaf. Infiltrated plants were maintained at 22-25 degrees genetically modified agrobacteria were grown overnight in C. with a photoperiod of 16 hours light and 8 hours dark. Plant 50 mL of LB medium containing 100 uMacetosyringone and tissue was harvested after 5 days post infiltration for subse 10 uMMES (pH 5.6), and subsequently were pelleted by quent analysis. centrifugation at 4000xg for 10 min. The pellets were resus pended in the infection medium Murashige and Skoog salts 3E. Maize Transient Expression System with vitamins, 2% sucrose, 500 uMMES (pH 5.6), 10 uM 0222 Expression cassettes described in Example 12 were MgSO, and 100 uMacetosyringone to ODoo-1.0 and sub cloned into a binary vector. The constructs were transferred sequently held at 28 degrees C. for 3 hours. Infiltration of into Agrobacterium tumefaciens strain LBA4404 containing individual leaves was carried out on sugar beet (about 3 weeks helper plasmid (pSBI) using a freeze-thaw method (An et al., old) and TEV-B tobacco plants (about 4 weeks old) using a 5 Binary vector. In: Gelvin S B, Schilproot R A (eds), Plant mL Syringe by pressing the tip of the Syringe (without a molecular biology manual. Kluwar Academic Publishers, needle) against the abaxial surface of the leaf. Infiltrated Dordrecht, pp A31-19 (1988)). plants were maintained at 22-25 degrees C. with a photope 0223) The maize transient expression system was estab riod of 16 hours light and 8 hours dark. Plant tissue was lished using young maize seedlings (5-12 dold). Preparation harvested after 5 days post infiltration for subsequent analy of Agrobacterium cultures and infiltration of maize leaves S1S. was carried out as described by AZhakanandam et al., Plant 0219. To ensure that enzyme activity measured was due to Mal. Biol. 63:393-404 (2007). In brief, the genetically modi plant expression of the enzymes, the expression constructs fied agrobacteria were grown overnight in 50 mL of LB also incorporated an intron in the polynucleotide sequence medium containing 100 uMacetosyringone and 10 uMMES coding for the enzyme. The presence of the intron ensures that (pH 5.6), and subsequently were pelleted by centrifugation at expression of the enzyme is due to plant expression (able to 4000xg for 10 min. The pellets were resuspended in the process out the intron and therefore express a fully processed infection medium (Murashige and Skoog salts with Vitamins, enzyme) versus agrobacterium expression (unable to process 2% sucrose, 500 uMMES (pH 5.6), 10uMMgSO, and 100 the intron and thus notable to express a functional enzyme). uMacetosyringone) to ODoo 1.0 and Subsequently held at 28 degrees C. for 3 hours. Infiltration of individual leaves was 3D: Transient Expression in Tobacco and Sugar Beet Leaves carried out on maize seedlings using a 5 mL Syringe, without 0220 Expression cassettes described in Example 12 were a needle, by pressing the tip of the Syringe against the abaxial cloned into either a binary vector or a binary vector also surface of the leaf. Infiltrated plants were maintained at 22-25 US 2011/020 1 059 A1 Aug. 18, 2011 degrees C. with a photoperiod of 16 hours light and 8 hours light for another week prior to transplanting to Soil for the dark. Plant tissue was harvested after 5-7 days post infiltration remainder of the maize growing cycle. for Subsequent analysis. 0224. To ensure that enzyme activity measured was due to 3G: Analysis of Key Enzymes in Plant Tissue plant expression of the enzymes, the expression constructs 0230 Whole leaves from tobacco or sugar beet transiently also incorporated an intron in the polynucleotide sequence expressing an enzyme were frozen at -80 degrees C. in coding for the enzyme. The presence of the intron ensures that 24-well blocks containing 3/16" chrome ball bearings. The expression of the enzyme is due to plant expression (able to frozen material was shaken at setting 9 for 2 min in a Kleco process out the intron and therefore express a fully processed Titer plate/Microtube Grinding Mill creating a powder. enzyme) versus agrobacterium expression (unable to process Buffer (50 mM HEPES, 2 mM EDTA, 0.02% Tween-20, 100 the intron and thus notable to express a functional enzyme). mM locked Sugar (isomaltulose, leucrose, or trehalulose depending upon the enzyme), pH 7) was added to the pow 3F. Transgenic Maize Callus and Plants dered samples to give a thick slurry. Samples were incubated in a Glas-Col rotator at 80% speed for 30 min. Samples were 0225. Transformation of maize callus was performed transferred by wide-bore P200 pipet to PCR tubes at 100 uL using a biolistic transformation method. Maize embryos were per tube and incubated at the appropriate temperature for the collected from maize kernels about 8 to 11 days after polli enzyme (50, 60, 70, 80 degrees C. depending on enzyme) in nation. The ears were collected and sterilized in 20% Germi a Biorad Tetrad 2 thermocycler. The sample was transferred to eithera Millipore Biomax 5 KDMW membrane spin filter cidal Clorox for 20 minutes on an orbital shaker set at 120 rpm and centrifuged at 12,000xg for 20 minor a Millipore Mul followed by extensive rinsing of the ear in sterile water. tiscreen-HV filter plate and filtered at 20 text missing or Embryos were collected from the kernels and kept on culture illegible when filed were diluted in Milli-Q water as nec media in the dark for 3 to 7 days. essary artext missing or illegible when filed vials with 0226 To prepare DNA for bombardment, gold particles split caps for carbohydrate analysis by Dionex HPAEC. (0.6 to 1 micrometer size, Bio-Rad) were resuspended in 50% sterile glycerol by Vortexing. An aliquot of the glycerol— 3H: Analysis of Locking Enzymes in Plant Tissue gold particle Suspension was combined by gentle mixing with 0231 Whole leaves from tobacco, sugar beet, or maize 2x10" mol DNA of the gene encoding the selectable marker were rolled and placed into filtration baskets (DNA IQ Spin (PM) and gene of interests outlined in. Table 29 of Example Basket) and the filled filtration baskets placed into 1.5 mL 12. The mixture was combined with 2.5M CaCl2 and cold 1M eppendorf tubes. The filled filtration baskets and eppendorf spermidine to precipitate the DNA onto the gold particles. tubes were frozen on dry ice for 5-8 min (or until frozen) The gold particles with precipitated DNA were washed in followed by thawing on ice for 5-8 min (or until thawed). The ethanol. The washed gold particles were re-suspended in thawed filled filtration baskets and eppendorf tubes were then ethanol and aliquots of DNA suspension were placed evenly centrifuged at 10,000xg for 15 min at 4 degrees C. and the onto the center of individual macrocarrier membrane disks filtrate collected. and allowed to dry. The macrocarrier was loaded into the gene 0232. The filtrate was boiled at 100 degrees C. for 5 min gun above the stopping screen. Bombardment of embryos followed by centrifugation at 16,000xg for 20 min. The was performed with a PDS Helium—1000 gene gun. A rup boiled filtrate was further filtered by transferring the boiled ture disc in the range of 650-1800 psi was used and the filtrate to either a Millipore Biomax 5 KD MW membrane distance from the rupture disc and the macrocarrier was set at spin filter and centrifuged at 12,000xg for 20 minor a Milli 8 mm with a stopping screen at 10 mm. The distance between pore Multiscreen-HV filter plate and filtered at 20 InHg. The the stopping screen and the embryos was about 7 cm. The filtrate was collected and diluted in Milli-Q water as neces pressure on the helium tank was set at about 1200 psi. Target sary and placed into either 0.3 or 1.5 mL sample vials with tissues (embryos) were bombarded 3 times before being split caps for analysis. transferred to the dark at 28 degrees C. to recover for 3 days. Example 4 0227. After recovery, the bombarded embryos were trans ferred to maintenance medium and cultured at 28 degrees C. Plant Expressed Sucrose Isomerase Enzyme in the dark. After 3 days, the bombarded embryo tissue was transferred to fresh callus induction medium and incubated 4A: Transient Expression of Sucrose Isomerase in Sugar Beet for 1 week to induce callus formation. The calli were then and Tobacco Leaves transferred to selection medium containing mannose for three 0233. The transformation vector 17588, as described in weeks at 28 degrees C. in the dark. Example 12, was used to transiently expressing enzymes in 0228 Selection of transgenic calli was performed by tobacco or Sugar beet leaves essentially as described in transferring living callus tissue to selection medium and cul Example 3D. Tobacco or sugar beet leaves transiently tured at 28 degrees C. in the dark for 3 weeks. Surviving calli expressing a Sucrose isomerase were generated using the were transferred to fresh selection medium and cultured an vector 17588 which contains a dicot optimized polynucle additional 2 weeks at 28 degrees C. in the dark. Surviving calli otide sequence encoding a sucrose isomerase (SEQ ID NO: were then transferred to regeneration medium and cultured at 16). Leaves transiently expressing Sucrose isomerase were 28 degrees C. in the dark for 2 weeks. harvested and extracted essentially as described in Example 0229 Callus tissues will be incubated under 16 hours of 3H and analyzed by Dionex for carbohydrates essentially as light at 24 degrees C. to encourage shoot development. Once described in Example 1G. shoot development starts, callus with shoots will be trans 0234 Dionex HPAE chromatography utilized pure sugar ferred to rooting medium and cultured at 24 degrees C. with standards as a reference for retention time and standard curve US 2011/020 1 059 A1 Aug. 18, 2011 26 production for determining Sugar concentrations. Sugar con centrations were based on the total Sugar consisting of glu TABLE 9 cose, fructose, Sucrose, trehalulose and isomalitulose when Carbohydrate analysis (HPAEC) of Sugar beet leaves transiently present. These five sugars represent >98% of the total peak expressing sucrose isomerase (SEQ ID NO: 16). area of the chromatograms with the remainder coming from Sucrose Trehallulose Isomalitulose Total minor unknown peaks from the biological extraction milieu Sample (mM) (mM) (mM) disaccharide (mM) of the leaf. 17588 8.5 9.9 3.1 21.5 0235 Sucrose isomerase activity in transiently infiltrated 17588 16.6 0.7 O.1 17.3 leaves was directly detected by the formation of the two major 17588 15.1 2.5 1.3 18.9 17588 31.8 O.S O.3 32.6 products of the enzymatic conversion of sucrose to the locked Negative 1O.O O.O O.O 1O.O sugars, trehalulose and isomalitulose. Neither of the locked control sugars were present in control leaves. Tables 7-10 summarize Negative 15.3 O.O O.O 15.3 control the analysis of tobacco and Sugar beet transiently expressing Negative 17.6 O.O O.O 17.6 a sucrose isomerase (vector 17588) and demonstrate that control tobacco and Sugar beet plants are able to express an active Negative 7.8 O.O O.O 7.8 Sucrose isomerase which catalyzes the conversion of Sucrose control to the locked Sugars isomalitulose and trehalulose and accu mulate the locked Sugars in the leaves. TABLE 10 TABLE 7 Carbohydrate analysis (HPAEC) of Sugar beet leaves transiently expressing sucrose isomerase (SEQ ID NO: 16). Carbohydrate analysis (HPAEC) of tobacco leaves Glucose + Sucrose Trehallulose Isomalitulose expressing a sucrose isomerase (SEQID NO: 16). fructose (% total (% total (% total (% total Sample Sugar) Sugar) Sugar) Sugar)

Sucrose Trehallulose Isomalitulose Total 17588 28.2 28.5 33.1 10.2 sample (mM) (mM) (mM) Disaccharide (mM) 17588 43.2 54.2 2.3 O.3 17588 56.5 34.7 5.8 3.0 17588 42.4 56.1 O.9 O.6 17588 3.6 17.7 6.4 27.7 Negative SO4 49.6 O.O O.O 17588 6.8 34.3 14.1 55.2 control 17588 4.2 23.9 8.1 36.2 Negative 42.9 57.1 O.O O.O control 17588 14.7 33.1 13.8 61.6 Negative 39.8 6O2 O.O O.O Negative 11.9 O.O O.O 11.9 control Negative 744 25.6 O.O O.O control control Negative 11.8 O.O O.O 11.8 control Negative 6.3 O.O O.O 6.3 4B: Transient Expression of Enzymes in Maize Leaves control Negative 4.2 O.O O.O 4.2 0236 Transient expression of enzymes in maize leaves was performed essentially as described in Example 3E using control the binary vectorpEB47 (described in Example 12) compris ing a monocot optimized polynucleotide sequence encoding a sucrose isomerase (SEQ ID NO: 24). Maize leaves were TABLE 8 harvested and analyzed for the presence of isomalitulose and trehalulose (products of Sucrose isomerase activity within the Carbohydrate analysis (HPAEC) of tobacco leaves maize leaf) essentially as described above for tobacco and transiently expressing Sucrose isomerase. Sugar beet leaves transiently expressing Sucrose isomerase. Glucose + Sucrose Trehallulose Isomalitulose Table 11 outlines data that demonstrates Sucrose isomerase is Fructose (% total (% total (% total (% total actively expressed in maize leaves transiently expressing sample Sugar) Sugar) Sugar) Sugar) Sucrose isomerase and leads to the accumulation of the locked 17588 39.2 7.9 38.8 14.1 Sugars, isomalitulose and trehalulose within the maize leaf. 17588 514 6.O 30.2 12.4 17588 47.9 6.O 34.4 11.7 TABLE 11 17588 51.7 11.5 26.0 10.8 Negative 40.6 59.4 O.O O.O Carbohydrate analysis (HPAEC) of maize leaves transiently control expressing Sucrose isomerase (SEQ ID NO. 24). Negative 58.5 41.5 O.O O.O control Glucose + Sucrose Trehallulose Isomalitulose Negative 45.7 S4.3 O.O O.O fructose (% total (% total (% total (% total control Sample Sugar) Sugar) Sugar) Sugar) Negative 53.3 46.7 O.O O.O control 47-6 (pEB47) 78.9 17.2 2.4 1.5 47-7 (pEB47) 63.7 33.3 2.1 O.9 US 2011/020 1 059 A1 Aug. 18, 2011 27

strates that transgenic Sugarcane callus which expresses TABLE 11-continued Sucrose isomerase accumulated the locked Sugars trehalulose and isomalitulose. Carbohydrate analysis (HPAEC) of maize leaves transiently expressing sucrose isomerase (SEQ ID NO. 24). TABLE 13 Glucose + Sucrose Trehallulose Isomalitulose fructose (% total (% total (% total (% total Carbohydrate analysis (HPAEC) of transgenic Sugarcane Sample Sugar) Sugar) Sugar) Sugar) callus tissue expressing Sucrose isomerase. 47-8 (pEB47) 73.1 16.O 7.3 3.6 Glucose + Sucrose Trehallulose Isomalitulose Negative 694 30.6 O.O O.O Fructose % total % total % total % total control (GUS Sample Sugar Sugar Sugar Sugar containing construct) 1 pEB38 44.13 37.70 8.87 9.30 Negative 58.2 4.1.8 O.O O.O Negative 34.61 65.39 O.O O.O control leaf control tissue Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, Negative control is transgenic sugarcane callus generated by bombardment with a polynucleotide sequence encoding the selectable markerPMI. 4C: Transgenic Maize Callus Expressing Sucrose Isomerase 0237 Transgenic maize callus expressing Sucrose 4E: Transgenic Sugar Beet Expressing Sucrose Isomerase isomerase was generated by bombarding maize embryos with (SEQID NO: 16) linear polynucleotide sequence. The method of embryo trans 0239 Transgenic Sugar beet plants containing the expres formation and generation of callus was essentially as sion cassette 17588 (described in Example 12) were gener described in Example 3F; however, two polynucleotide ated essentially as described in patent application WO02/ sequences were bombarded at the same time. One of the 14523 which is a multiple shoot method of transformation. polynucleotide sequences contained the selectable marker, The transgenic Sugar beet callus was selected using mannose PMI, which allows for selection of transgenic maize cells by selection (the selectable marker gene was PMI) which was growth on mannose. The second polynucleotide sequence, performed essentially as described in patent application pEB38, contained a maize optimized polynucleotide WO9472O627. sequence encoding a sucrose isomerase (SEQ ID NO: 20). 0240. The transgenic sugar beet plants were analyzed by The sucrose isomerase was targeted to the vacuole. Table 12 PCR to determine if the selectable marker (PMI) and the outlines data which demonstrates that transgenic maize callus sucrose isomerase gene (SEQID NO: 16) were present in the which expresses Sucrose isomerase accumulated the locked plant. In addition, the transgenic Sugar beet plants were ana Sugars trehalulose and isomalitulose. lyzed for the accumulation of locked Sugars. 0241 To analyze the Sugar content of the transgenic Sugar TABLE 12 beet plants, leaves from the transgenic Sugar beet plants were sampled into a Costar 96-well box. The box was placed on ice Carbohydrate analysis (HPAEC) of transgenic maize during the sampling procedure. After filling the box with callus tissue expressing Sucrose isomerase. glass balls the leaf samples were placed into the wells and 100 Glucose + Sucrose Trehallulose Isomalitulose uL sterileddH0 was added. The wells were closed using strip Fructose % total % total % total % total caps or a lock and the box shaken in a Tissue laser (25-30s, 30 Sample Sugar Sugar Sugar Sugar Hz.) to pulverize the tissue in the water. The locks covering 1 pEB38 14.8 O.9S 38.2 46.0 the wells were pierced and the samples were boiled on a water 2 pEB38 2SO O.69 35.3 39.0 bath for 10 min. After boiling, an additional 100 uL sterile 3 pEB38 32.O S.13 34.8 28.1 ddH0 was added followed by centrifugation (10 min, 3000 Negative 7O.O 3O.O O.O O.O rpm). The supernatants were transferred to Millipore spin control filter and centrifuged at 12000 rpm, 5 min. The filtered super Total sugar = total amount of identifiable sugars in sample based on retention times of pure natants were stored at -20 degrees C. or in 4 degrees C. if the sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, The negative control is transgenic maize callus generated by bombardment with the analysis was performed directly. polynucleotide sequence encoding PMI only, 0242. The samples were diluted 100 times with distilled water prior to analysis using the Dionex HPAE-system. The 4D: Transgenic Sugarcane Callus Expressing Sucrose Dionex HPAE-system, ICS-3000 was used to separate the Isomerase carbohydrates. The instrument was equipped with a tempera ture regulated auto sampler, CarboPacPA203x30mm guard 0238 Transgenic Sugarcane callus expressing Sucrose column, CarboPac PA20 3x15 mm analytical column and isomerase was generated essentially as described in Example pulsedamperometric detector (PAD). The mobile phase used 3A; however, two polynucleotide sequences were bombarded was 200 mM NaOH solution and water in following gradient at the same time. One of the polynucleotide sequences con program: 8 min/16% NaOHsolution/2 min 16-100% tained the selectable marker, PMI, which allows for selection NaOHSolution//3 min 100% NaOHSolution//2 min 100 of transgenic Sugarcane cells by growth on mannose. The 16%//7 min 16% NaOHsolution. The column temperature second polynucleotide sequence, pEB38, contained a mono was set at 30 degrees C. and the flow 0.43 mL/min. The cot optimized polynucleotide sequence encoding a Sucrose approximate retention times were glucose 7.7 min, fructose isomerase (SEQ ID NO: 20). The sucrose isomerase was 9.3 min, sucrose 11.0 min, trehalulose 13.1 min and isoma targeted to the vacuole. Table 13 outlines data which demon litulose 14.5 min. The peaks were identified using the standard US 2011/020 1 059 A1 Aug. 18, 2011 28 solutions. Table 14 outlines data which demonstrates trans NO: 35). Transiently expressing leaves were harvested and genic Sugar beet plants expressing a Sucrose isomerase extracted essentially as described in Example 3H and ana enzyme and the Subsequent accumulation of the locked Sug lyzed by Dionex for carbohydrates essentially as described in ars, isomalitulose and trehalulose. Locked Sugars are detected Example 1G. in transgenic Sugar beet plants expressing Sucrose isomerase 0244. Dionex HPAE chromatography utilized pure sugar indicating that the enzyme is both expressed and is able to standards as a reference for retention time and standard curve perform the enzymatic activity which converts Sucrose to production for determining Sugar concentrations. Sugar con isomalitulose and trehalulose. centrations were based on the total Sugar consisting of glu cose, fructose, Sucrose, and locked Sugars when present. TABLE 1.4 These sugars represent >98% of the total peak area of the chromatograms with the remainder coining from minor Transgenic Sugar beet plants expressing sucrose isomerase. unknown peaks from the biological extraction milieu of the PCR PCR Dionex- Dionex leaf. Even PMI GOI isomalitulose trehallulose 0245. Dextranslucrase with leucrose synthase activity tran siently expressed in leaves was directly detected by the for O851B:1 A ------biennia mation of the locked Sugar leucrose. Leucrose was not present O851B:2A ------in control leaves. Table 15 summarizes the analysis of biennia tobacco leaves transiently expressing a dextranslucrase with O851F:2A ------biennia leucrose synthase activity (vector 902195) and demonstrates O851I:1 B ------that tobacco leaves are able to express an active dextransu biennia crase which catalyzes the conversion of sucrose to the locked O851K:2A ------Sugar leucrose which accumulates in the leaf. biennia O851 Ki:2B -- biennia 5B: Transient Expression of Dextranslucrase (SEQ ID NO: O851 Ki:2C -- 24) in Maize Leaves. biennia 0246 Maize leaves transiently expressing dextranslucrase O851K:4A ------biennia with leucrose synthase activity were generated essentially as O851N:1 A -- -- described in Example 3E using the vector pBB47 (described biennia in Example 12) comprising a monocot optimized polynucle O851O:1 A ------biennia otide sequence encoding a dextransurase (SEQID NO: 47). O851O:2A ------Maize leaves were harvested and extracted essentially as biennia described in Example 3H. The extract was analyzed for car O851O:3A ------bohydrate content essentially as described in Example 1G. biennia Table 15 outlines data that demonstrates dextranase is O851O:4A ------biennia actively expressed in maize leaves and leads to the accumu O851O.S.A ------lation of the locked sugar leucrose within the maize leaf. biennia O903B:SA ------5C: Transgenic Sugarcane Callus Expressing Dextranslucrase annual O903B:7 A ------(SEQ ID NO:37) annual 0247 Transgenic Sugarcane callus expressing dextransu O903D:1 A ------annual crase with leucrose synthase activity (SEQ ID NO: 37) was O903F:1 B ------generated essentially as described in Example 3A; however, annual two polynucleotide sequences were bombarded at the same O903F:1 C ------time. One of the polynucleotide sequences contained the annual selectable marker, PMI, which allows for selection of trans O903G:1 A ------annual genic Sugarcane cells by growth on mannose. The second O903I:1 A ------polynucleotide sequence, pEB28, contained a monocot opti annual mized polynucleotide sequence encoding a dextranslucrase (SEQ ID NO: 37). The dextranslucrase was targeted to the vacuole. Table 15 outlines data which demonstrates that Example 5 transgenic Sugarcane callus which expresses Sucrose isomerase accumulated the locked Sugar leucrose. Transgenic Plants Expressing Dextranslucrase with Leucrose Synthase Activity TABLE 1.5 5A: Transient Expression of Dextranslucrase (SEQ ID NO: Plant tissue expressing dextranslucrase 35) in Tobacco Leaves accumulates leucrose and/or isomaltose. 0243 The transformation vector 902195, as described in tobacco maize Sugar cane Example 12, was used to generate tobacco leaves transiently dextranslucrase Leucrose Leucrose Leucrose and expressing dextranslucrase essentially as described in isomaltose Example 3D. Transient expression of dextranslucrase in Negative control tobacco leaves was performed using the vector 902195 which Leucrose synthase activity is determined by the accumulation of leucrose above 10x signal: contains a dicot optimized polynucleotide sequence encoding noise on a Dionex IC, a dextranslucrase with leucrose synthase activity (SEQ ID US 2011/020 1 059 A1 Aug. 18, 2011 29

Example 6 be transferred to a 50 degree C. water bath where 4 mL of Transgenic Plants Expressing Amylosucrase NaOAc buffer pH-4.5 and 0.1 mL of amyloglucosidase (MegaZyme) will be added and then incubated for 30 minutes 6A: Total Starch Analysis of Amylosucrase-Expressing at 50 degree C. After incubation, all samples should be Maize and Sugarcane Callus brought to 10 mL with water, vortexed, and centrifuged for 10 minutes at 3000 rpm. This supernatant contains the solubi 0248. The effectiveness of the amylosucrase gene, when lized glucose monomers that remain from the digestion of the expressed in either maize or Sugar cane callus, can be evalu carbohydrate polymers that were extracted from the lyo ated by comparing the total starch content of the amylosu philized tissue samples. To enumerate the glucose in this crase expressing calli to control calli that have not been trans mixture, 2 mL should be added in duplicate to glass test tubes, formed with the gene. The total starch content of any plant mixed with 3 mL of GOPOD reagent, and incubated for 20 tissue of interest can be measured using a protocol similar to minutes at 50 degree C. Once cooled to room temperature, the that of the MegaZyme Total Starch Assay kit. In this assay, the optical density of the samples can be read at 510 nm. Based on starch contained in a plant sample is broken down into glu the ODreading of the samples and its comparison to a known cose monomers through digestion by both an alpha-amylase standard, the amount of glucose, and therefore starch, in the and an amyloglucosidase. The resulting solution of glucose original dry weight sample can be calculated. can be enumerated by a glucose oxidase-peroxidase (GO 0251 Upon completion of total starch content analysis, it POD) reaction essentially as is described in Example 2B. In is expected that calli expressing the amyloSucrase gene will this reaction, the glucose oxidase enzymes break down glu show an increased level of total starch over the negative cose to hydrogen peroxide which the peroxidase then digests, control calli due to the additional production of carbohydrate releasing oxygen which reacts with the 4-aminoantipyrine in polymers by the enzyme. Additionally, targeted expression of Solution to evolve a pink color. The pink color can be mea the amylosucrase enzyme to the vacuole or apoplast of trans Sured with a spectrophotometerand, when compared with the genic plant cells would serve to isolate the de novo starch absorbance of a glucose standard, can give a measure of the from the endogenous starch metabolizing enzymes allowing amount of glucose and therefore, the amount of starch in a given sample. for accumulation of a locked carbohydrate. Therefore, when 0249. To accurately measure the production of carbohy the calli are depleted of transient starch after growth on sor drate polymers by the amylosucrase gene in callus, several bitol media, the total starch content would be expected to fall controls and conditions will need to be established. For every slightly, but remain at an increased level over the negative calli that is transformed with the amylosucrase gene, a dupli controls. cate calli should be transformed with an empty vector that can act as a control sample. Both transformed and control calli 6B: Starch Structure: Amylose/Amylopectin Differentiation should initially be grown on Sucrose media to provide amy by Iodine Binding losucrase with its natural substrate and raise the overall starch 0252. The structure of the carbohydrate polymers pro content in the calli. After sufficient growth, some calli (both duced by the amylosucrase enzyme can potentially be iden AMS and control) should be transferred to sorbitol media tified by developing a method to enumerate the proportions of where the natural metabolism of the tissue will lower the amylose and amylopectin in plant material. The comparison background of transient starch and, theoretically, leave the of control samples with samples expressing the amylosucrase amylosucrase produced carbohydrate polymer. In tissue cul gene could identify structural composition changes that may ture, sorbitol is assimilated and metabolized by plants to a be present in the polymers produced by amylosucrase much lesser degree than sucrose. With sorbitol as a carbon expressing events, suggesting that a carbohydrate polymer Source, plant cells are expected to deplete transient and stor lock is being produced. One possible method for accomplish age starch reserves leaving an amylosucrase derived starch to ing this is through an iodine binding assay. In this assay, the accumulate. plant produced carbohydrate polymers are solubilized from 0250 Once the calli are harvested from the media, similar the tissue and then stained with iodine. The resulting iodine events can be pooled into wells of a 24-well block to bulk up starch complexes will absorb at different wavelengths the amount of tissue and lyophilized so that calculations can depending on the proportions of amylose and amylopectin be made on a dry weight basis. Lyophilized tissue can be present in the extract. Through comparison with known stan easily ground in the 24-well blocks using a Kleco. As men dards and mixtures of amylose and amylopectin, both the tioned previously, the MegaZyme total starch protocol can be total amount of starch present and the proportions of amylose used to effectively measure the total starch content of tissue and amylopectin present in the starch produced in the tissue samples. The following is an example of a slightly modified can be calculated. protocol that could be employed to analyze lyophilized callus 0253) The following is an example of a starch extraction material. Approximately 30-70 mg of the ground tissue and iodine staining procedure that could be used to analyze should be washed with 5 mL of 80-90% ethanol for 30-60 lyophilized, ground tissue samples. Approximately 100-200 minutes and centrifuged for 5 minutes at 3000 rpm to wash mg of ground, lyophilized tissue should be washed with 5 mL away any soluble Sugars or other soluble compounds. Addi of 90% ethanol, incubated for 15 minutes in a 100 degree C. tional ethanol washes may be added as necessary, as long as water bath, and centrifuged for 5 minutes at 3000 rpm to all samples are treated identically. The pelleted material remove the supernatant. This wash step should be repeated at should then be washed in 5 mL of cold water and centrifuged least two more times to ensure sufficient removal of soluble again for 5 minutes at 3000 rpm to remove any remaining Sugars and other potential iodine binding compounds from ethanol. At this stage, the pellet should be completely resus the samples. To the sample material, 5 mL of 100% ethanol pended in 3 mL of a 1:30 dilution of alpha-amylase (Mega should be added and incubated again for 15 minutes at 100 Zyme) in 50 mM MOPS buffer pH-7 and incubated for 6 degree C. Prior to centrifuging the sample, 5 mL of acetone minutes in a 100 degree C. water bath. Samples should then should be added to the mixture. The pellet should then be US 2011/020 1 059 A1 Aug. 18, 2011 30

Suspended once more in 5 mL of acetone to ensure the com pressed key enzyme (alpha-amylase). Locked Sugars pro plete removal of any residual ethanol, centrifuged for 5 min duced in tobacco or another plant system by the amylosucrase utes at 3000 rpm, and the pellet allowed to dry overnight. To gene can be extracted in boiling water from lyophilized plant material after washing with 80-90% ethanol to remove any solubilize the starch from the dried pellet, 5 mL of 0.5MKOH soluble sugars or compounds (Spoehr and Milner J. Biol. should be added and incubated for 2-3 hours at 100 degree C. Chem. 111 (3): 679-687. (1935)). The alpha-amylase will not Debris may be pelleted by centrifugation for 10 min at 3000 yield strictly glucose in its digest, the amount of glucose rpm. For the staining of the solubilized carbohydrate poly produced should be sufficient to be detected by the GOPOD mers, 1 mL of the KOH extract should first be neutralized reaction assay when compared to a control sample of the with 5 mL of 0.1M HCl, then 0.5 mL of Lugol's Iodine undigested locked Sugar. It is expected that a difference in solution should be added and diluted to between 25 and 50 glucose levels would be detected in this type of digestion mL with water to bring the absorbance into an appropriate assay, Verifying that plant expressed key enzymes are, indeed, range. The color should be allowed to develop for about 15 capable of digesting plant produced locks. 0256 Additionally, in the process of performing HPSEC minutes and then samples can be added to a microtiter plate on debranched amylosucrose polymer mixture, sample frac for measuring the optical density along with pure amylose tions could be collected, and a plant expressed alpha amylase and pure amylopectin stained standards. The spectra of the or glucoamylase key enzyme could be used to hydrolyze the samples and standards should be measured first to determine starch in the collected fractions to glucose. A GOPOD reac at which wavelength the maximum absorbance occurs for tion assay could be used to detect the glucose liberated from each sample, since this is indicative of the proportions of the amyloSucrose locked-carbohydrate fraction. amylose and amylopectin in the samples. To analyze the 6D: Detection of Amylosucrase Activity in Stably Trans sample spectra, a system of equations will be set up using formed Plants or Plants Transiently Expressing Amylosu Beer's law based on the absorbance values at 6 different CaSC. wavelengths. Measurements of the absorbance will be 0257 Amylosucrase may be expressed either transiently recorded at 504 nm, the wavelength of greatest difference or through stable transformation of maize, cane, beets, between the amylose and amylopectin peaks where amy tobacco or other plants with a promoter that drives expression lopectin’s absorbance is greater than amylase's absorbance; in the appropriate target tissue (leaf, endosperm, embryo, 548 nm, the wavelength of the pure amylopectin peak; 630 etc.) and with targeting sequences that direct the amylosu nm, the wavelength of the pure amylose peak; 700 nm, the crase to the desired subcellular location (vacuole, chloroplast, wavelength of greatest difference between the amylose and cytoplasm, apoplast, etc.). A variety of techniques may be amylopectin peaks where amylase's absorbance is greater used to detect the activity of the amylosucrase gene in plants. 0258 For instance, plant tissue samples expressing the than amylopectin’s absorbance: 800 nm, the wavelength of amylosucrase polypeptide may be incubated in the dark for greatest absorbance due to amylase where amylopectin’s 24 to 48 hours in order for transient starch produced in the absorbance approaches Zero; and the wavelength determined chloroplast to be broken down by the plant. Leaf or other to be the location of the sample spectra's maximum (Jarvis tissue may be excised from the plant and dipped into boiling and Walker J. Sci. Food Agric. 63: 53-57 (1993)). The results water for one minute to heat kill the tissue. After heat killing of this system of equations will give a concentration value of plant tissue samples may be incubated in hot ethanol to the amount of amylose and the amount of amylopectin remove the chlorophyll, repeated washing with hot ethanol present in the sample extract, from which a ratio of the two may be necessary to remove all the chlorophyll. Once the starch forms can be determined. chlorophyll has been removed, the tissue can be rinsed with cold water and placed on a petri dish. Lugol's Solution (5 g 0254. Upon successful completion of the iodine binding iodine (I) and 10g potassium iodide (KI) mixed with 85 ml assay, it is expected that the assay data will Support the total distilled water), may then be poured over the sample an starch assay data in showing an overall starch increase in the allowed to incubate at room temperature. Control samples samples expressing the amylosucrase gene. In addition, it is that have been in the dark for 24 hours should contain no expected that the amylosucrase expressing events will pro starch and should not stain black in Lugols solution. Samples duce a carbohydrate polymer that is more closely related to expressing the amylosucrase gene should stain black due to amylose than amylopectin, therefore a larger proportion of starch that is produced in the vacuole or other organelles amylose when compared to control samples should be targeted for expression of the Amylosucrase enzyme. 0259 Leaves contain a variety of unique cell types such as observed. This shift in composition of the starch produced in the pavement cells that are highly specialized cells making up amylosucrase expressing events will also support the Success the majority of the leaf surface. These are easily identified by ful production of a locked Substrate in plant tissue. their puzzle piece shapes (in dicots) and are only found at the 6C: Digestion of Plant Produced Carbohydrate Polymers leaf Surface. They contain no chloroplasts or amyloplasts, so with Plant-Expressed Enzymes if pavement cells are found to have what appeared to be dark 0255. The ability of a plant produced key enzyme to digest staining "amyloplasts' and these are not observed in pave a plant produced locked Substrate can be exemplified using ment cells from “vector only” controls, this would be good the principle underlying the glucose oxidase-peroxidase evidence that the construct is working and that starch is being (GOPOD) reaction. If the plant purified key enzyme acts on produced. the plant produced locked Sugar, glucose monomers should 6E: Analysis of Locked Amylosucrose Carbohydrates by be liberated from the locked sugar which can be enumerated HPSEC by the GOPOD reaction. In order to complete this digestion, 0260 Another means of analyzing structural composition however, an appropriate plant expressedky enzyme must be changes that may be present in the polymers produced by purified and a carbohydrate polymer produced by the amylo amylosucrase expressing events is by the use of High-perfor Sucrase enzyme must be solubilized in an appropriate buffer. mance size exclusion chromatography, HPSEC. Using Alpha-amylase can be collected from transgenic maize plants HPSEC, a locked amylosucrase carbohydrate polymer could expressing alpha-amylase in the seed through laboratory be identified and characterized based on its molecular weight established FPLC methods yielding a purified plant-ex or chain length distribution. US 2011/020 1 059 A1 Aug. 18, 2011

0261 The extraction of starch from plant material for generate a standard curve. In this way, the chain length of an analysis by HPSEC could be carried out essentially as amylosucrose polymer may be determined and characterized. described by Santacruz et al J. Agric. Food Chem. 2004, 52 (7): 1985-1989. Starch could be extracted from plant material Example 7 Such as leaf or callus by lyophilizing and grinding plant material. Powdered lyophilized plant tissue could be mixed Transgenic Plants Expressing Key Enzymes with 90% ethanol (v/v) and placed in a boiling water bath for 15 minutes. After centrifugation at 1000 g for 10 minutes, the 0264 7A: Transient Transgenic Tobacco and Sugar Beet pellet could be washed three more times with hot 90% etha Expressing alpha-1,6-glucosidase nol. The pellet can be washed again with 100% ethanol, 0265 Tobacco and sugar beet leaves transiently express boiled for 15 minutes. After centrifugation, the supernatant ing an alpha-1,6-glucosidase enzyme were generated essen can be discarded and the pellet washed further with acetone, tially as described in Example 3D. Leaves transiently centrifuged and Supernatant discarded. The pellet can be expressing alpha-1,6-glucosidase were generated using the dried overnight at room temperature. The dried plant material binary vector 902525 or the BCTV binary vector 902526. can be further extracted by addition of 0.2% EDTA to the Both of the binary vectors contain expression cassettes dried residual pellet and mixed overnight with shaking at encoding an alpha-1,6-glucosidase (SEQID NO: 11) which room temperature. After centrifugation, the resulting starch has been targeted through the ER and is expected to accumu pellet can be further extracted by addition of 90% ethanol and late in the apoplast. Infiltrated tobacco and Sugar beet leaves boiled for 30 minutes. After centrifugation, the supernatant were harvested, extracted and enzyme activity assayed essen can be saved and the pellet extracted again with 90% ethanol. tially as described in Example 3G. The key enzyme, alpha-1, The supernatants can be combined and mixed with 100% 6-glucosidase, catalyzes the conversion of isomalitulose to the ethanol in a ratio of 1 part DMSO to 9 parts ethanol. The fermentable Sugars fructose and glucose and was assayed at Solution can be incubated at room temperature for 15 minutes, 60 degrees C. Carbohydrate analysis of the final filtrate was centrifuged to obtain a starchpellet. The starch pellet can then performed using the Dionex system essentially as described be solubilized in 90% DMSO with boiling for 15 minutes. in Example 1G. Tables 16-17 outline data demonstrating The starch could be done debranched for GPC analysis essen transient expression of an alpha-1,6-glucosidase in tobacco tially as described by Yao et al Carbo. Research. 2005, 340: and Sugar beet leaves. 701-710. Debranching of starch can be carried out in a 50 mM Sodium Acetate, pH 4.0 buffer which has been warmed to TABLE 16 42-SOC. A reaction which combines 880 ul of warm NaAc Carbohydrate analysis of tobacco leaves transiently expressing buffer, 120 ul of the DMSO solubilized starch pellet can be an alpha-1,6-glucosidase enzyme (SEQID NO: 11). Enzyme activity prepared. To keep the starch solubilized, the reaction can be is indicated by the change in abundance of each Sugar as a percentage heated to 100 C for 10 minutes and then cooled to 22-42 C of the total sugars over a 24 hour period. before addition of 1 U/ml of isoamylase (MegaZyme Inc., Glucose Fructose Isomalitulose Ireland.) The digestion reaction can be incubated at 37-42 C sample (% total Sugar) (% total Sugar) (% total Sugar) with constant agitation for 16-24 hours. After digestion, the 902525 11.97 1246 -24.43 debranching reaction can be heated in a boiling water bath for binary 10 minutes. The starch dispersion can then be concentrated in 902526 22.66 26.95 -49.61 a Speed-Vac Vacuum evaporator. BCTV Gel permeation chromatography or HPSEC could be carried Negative -1.67 3.75 -2.08 out on this concentrated Starch sample to characterize the control starch structure of the locked amylosucrose carbohydrate. Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample Starch samples can be diluted up to 30 fold in DMSO in analysis, The negative control is tobacco leaves transiently expressing a binary vector preparation for analysis by the HPSEC system. containing an origin of replication from beet curly top, 0262. Using an HPSEC system such as a Waters Breeze 717 system. 50 ul of debranched starch polymer could be TABLE 17 injected into a Ultrahydrogel-6x40 mm Guard column (WAT 011565) and Ultrahydrogel 250 A-7.8x300 mm column HPAEC analysis of carbohydrate products from Sugar beet leaves transiently expressing an alpha-1,6-glucosidase (WAT01 1525) with Waters 1515 isocratic HPLC pump and a enzyme (SEQID NO: 11). Enzyme activity is indicated differential refractometer such as Waters Model 410 for by the change in abundance of each Sugar as a percentage detection. A flow rate of 0.5 mL/min at a column, column of the total Sugars Over a 24 hour period. temperature of 35C and detector temperature of 40 C may be used. The molecular weight standards for column calibration Glucose Fructose Isomalitulose could be maltotriose (Sigma), maltohepatose (Sigma), and sample (% total Sugar) (% total Sugar) (% total Sugar) pullulan standards (P-5, MW 5800; P-10, MW 12,200; P-20, 902525 19.73 1910 -38.83 binary MW 23,700; P-50, MW48,000, from Shodex, Japan). On the 902526 14.05 11.91 -25.96 chromatogram the differential refractive index (DRI) value BCTV on the y-axis will be the mass response to the carbohydrate at Negative 6.14 6.61 -12.74 a particular retention time (RT). control 0263. Within the separation range of the HPSEC media, Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample the RT on the x-axis will be approximately proportional to the analysis, The negative control is sugar beet leaves transiently expressing a binary vector logarithm of the molecular weight (or chain length), and containing an origin of replication from beet curly top, using standards the precise relationship may be determined to US 2011/020 1 059 A1 Aug. 18, 2011 32

7B: Transgenic Maize Callus Expressing alpha-1,6-glucosi contained a polynucleotide sequence encoding an alpha-1,6- dase glucosidase (SEQ ID NO. 56). The alpha-1,6-glucosidase 0266 Transgenic maize callus expressing an alpha-1,6- was targeted to the chloroplast. glucosidase enzyme was generated by bombarding maize embryos with linear polynucleotide sequence. The method of 0269 Sugarcane calli expressing the alpha-1,6-glucosi embryo transformation and generation of callus was essen dase were collected 1 callus per well in 96-well 2 mL plates tially as described in Example 3F; however, two polynucle (Whatman) containing one 3/16" chrome ball bearing per well. otide sequences were bombarded at the same time. One of the The plate was shaken at setting 9 for 2 min in a Kleco Titer polynucleotide sequences contained the selectable marker, plate/Microtube Grinding Mill creating a powder. Buffer PMI, which allows for selection of transgenic maize cells by (100 mM HEPES, 4 mM EDTA, 0.04% Tween-20, pH 7) was growth on mannose. The second polynucleotide sequence, added to the powdered samples to give a thick slurry. Samples 902435 or 902425, contained a maize optimized polynucle otide sequence encoding an alpha-1,6-glucosidase (SEQ ID were incubated in a Glas-Col rotator at 80% speed for 30 min. NO: 54 or SEQID NO:56). The alpha-1,6-glucosidase was Samples were transferred by wide-bore P200 pipet to a 96 targeted to the endoplasmic reticulum (902435) or to the well PCR at 100 uL per well and incubated at 60 degrees C. chloroplast (902425). for 20 minutes. Extracts were centrifuged at 1770xg for 30 0267 Analysis of alpha-1,6-glucosidase enzyme activity mins to pellet denatured proteins in Samples. Equal Volumes in transgenic maize calli was performed by extracting the of clarified extract and 271 mM trehalulose? 134 mM isoma enzyme from the transgenic calli and incubating the extract litulose were combined and incubated at 60 degrees C. in with isomalitulose. If alpha-1,6-glucosidase enzyme activity BioRad Tetrad 2 thermocycler. Samples were collected at is present, the isomalitulose is converted to glucose and fruc times 0 and 24 hours. Collected samples were incubated at 95 tose. Essentially, maize calli expressing the alpha-1,6-glu degrees C. for 5 minutes before freezing at -20 degrees C. cosidase were collected 8 calliper well in Slicprep 96 device. Samples were analyzed by HPAE chromatography essen Samples were frozen at -80 degrees C. and thawed at room tially as described in Example 1G. Table 19 demonstrates that temperature. Thawed samples were centrifuged at 1770xg Sugarcane callus expresses an active alpha-1,6-glucosidase and flow-through extract collected. Extracts were heated at 60 that also shows alpha-1,1-glucosidase activity. degrees C. for 10 minutes. Extracts were centrifuged at 1770xg 30 minutes at 4 degrees C. to pellet denatured pro TABLE 19 teins in Samples. Equal Volumes of clarified extract and reac tion buffer (200 mM Isomalitulose, 100 mM HEPES, 0.04% Carbohydrate analysis (HPAE chromatography) of products from transformed sugarcane callus tissue expressing an Tween-20, 4 mMEDTA, 40 mMNaOH, 2x protease inhibitor alpha-1,6-glucosidase enzyme. Enzyme activity is indicated Roche Complete EDTA-free) were combined and incu by the change in abundance of each Sugar as a percentage bated at 60 degrees C. in. BioRad Tetrad 2 thermocycler. of the total sugars over a 24 hour period. Samples were collected at times 0 and 24 hours. Collected Glucose Fructose Isomalitulose Trehallulose samples were incubated at 95 degrees C. for 5 minutes before (% total (% total (% total (% total freezing at -20 degrees C. Samples were analyzed by Dionex. Sample Sugar) Sugar) Sugar) Sugar) Table 18 outlines data which demonstrates that transgenic 902425 8.98 9.59 -6.86 -9.60 maize callus expresses an active alpha-1,6-glucosidase (plastid) enzyme. Negative 2.53 3.70 -2.82 -2.15 control TABLE 18 Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample HPAEC analysis of carbohydrate products from transformed maize analysis, Negative control is wildtype sugarcane callus, callus tissue expressing alpha-1,6-glucosidase enzymes. Enzyme activity is indicated by the change in abundance of each Sugar 7D: Transient Expression of alpha-1,1-glucosidase (SEQ ID as a percentage of the total Sugars over a 24 hour period. NO: 27) Enzyme in Sugar Beet or Tobacco Leaves Glucose Fructose Isomalitulose 0270 Tobacco and sugar beet leaves transiently express Sample (% total Sugar) (% total Sugar) (% total Sugar) ing an alpha-1,1-glucosidase (SEQID NO: 27) enzyme were 902435 1428 18.03 -32.31 generated essentially as described in Example 3D. The vector ER for transient expression was 901612 or 902522 which are 902425 7.24 9.26 -16.50 (plastid) described in Example 12. The binary vector 901612 contains Negative O.49 -0.18 -0.31 an expression cassette encoding an alpha-1,1-glucosidase control (SEQ ID NO: 27) targeted to the chloroplast. The binary vector 902522 contains an expression cassette encoding an Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample alpha-1,1-glucosidase (SEQ ID NO: 27) targeted to pass analysis. Negative control is maize callus transformed with a vector that contains the PMI through the endoplasmic reticulum and accumulate in the selectable marker only, apoplast. Infiltrated tobacco and Sugar beet leaves were har 7C: Transgenic Sugarcane Callus Expressing alpha-1,6-glu Vested, extracted and enzyme activity assayed essentially as cosidase described in Example 3G. The key enzyme, alpha-1,1-glu 0268 Transgenic Sugarcane callus expressing an alpha-1, cosidase, catalyzes the conversion of isomaltulose or treha 6-glucosidase enzyme was generated essentially as described lulose to the fermentable Sugars fructose and glucose and was in Example 3A; however, two polynucleotide sequences were assayed at 70 degrees C. Carbohydrate analysis of the final bombarded at the same time. One of the polynucleotide filtrate was performed using the Dionex system essentially as sequences contained the selectable marker, PMI, which described in Example 1G. Tables 20-21 outline data demon allows for selection of transgenic Sugarcane cells by growth strating transient expression of an alpha-1,1-glucosidase in on mannose. The second polynucleotide sequence, 902425, tobacco and Sugar beet leaves. US 2011/020 1 059 A1 Aug. 18, 2011 33

times 0 and 24 hours. Collected samples were incubated at 95 TABLE 20 degrees C. for 5 minutes before freezing at -20 degrees C. Samples were analyzed by Dionex essentially as described in HPAEC analysis of carbohydrate products from tobacco leaves transiently expressing an alpha-1,1-glucosidase enzyme. Enzyme activity is Example 1G. Table 22 demonstrates that maize callus indicated by the change in abundance of each Sugar as a percentage expresses an active alpha-1,1-glucosidase. of the total Sugars Over a 24 hour period. TABLE 22 Glucose Fructose Trehallulose Isomalitulose (% total (% total (% total (% total HPAEC analysis of carbohydrate products from transformed maize Sample Sugar) Sugar) Sugar) Sugar) callus tissue expressing an alpha-1,1-glucosidase enzyme. Enzyme 9.01612 21.61 23.38 -22.57 -22.41 activity is indicated by the change in abundance of each Sugar Negative 1.47 1.55 1.93 -4.95 as a percentage of the total Sugars over a 24 hour period. control Glucose Fructose Trehallulose Total sugar = total amount of identifiable sugars in sample based on retention times of pure Sample (% total Sugar) (% total Sugar) (% total Sugar) sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, The negative control is tobacco leaves transiently expressing empty binary vector, 902429 1O.O2 11.32 -6.47 Negative 3.51 3.46 1...SO control TABLE 21 Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, The negative control was transgenic maize callus generated by transformation with HPAEC analysis of carbohydrate products from Sugar beet leaves the binary vector expressing the selectable marker (PMI) only, transiently expressing alpha-1,1-glucosidase enzymes. Enzyme activity is indicated by the change in abundance of each Sugar 7F: Transient Expression of alpha-1,5-glucosidase by as a percentage of the total Sugars over a 24 hour period. Tobacco Leaves Glucose Fructose Trehallulose 0273 Tobacco leaves transiently expressing an alpha-1,5- sample (% total Sugar) (% total Sugar) (% total Sugar) glucosidase (SEQID NO: 46) enzyme were generated essen 9.01612 1248 13.70 -13.59 tially as described in Example 3D. The vector for transient chloroplast expression was BCTV binary vector 902550 which is 902522 1873 19.51 -22.46 described in Example 12. BCTV binary vector 902550 con apoplast tains an expression cassette encoding an alpha-1,5-glucosi Negative 6.94 7.45 -5.49 control dase (SEQID NO: 46) which is targeted to the chloroplast. Infiltrated tobacco and sugar beet leaves were harvested, Total sugar = total amount of identifiable sugars in sample based on retention times of pure extracted and enzyme activity assayed essentially as sugar standards, Extraneous peaks in samples are indeterminate and not included in sample analysis, The negative control is sugar beetleavestransiently expressing empty binary vector described in Example 3G. The key enzyme, alpha-1,5-glu cosidase, catalyzes the conversion of leucrose to the ferment 7E: Transgenic Maize Callus Expressing alpha-1,1-glucosi able Sugars glucose and fructose and was assayed at 80 degrees C. Table 23 outlines data demonstrating tobacco dase leaves transiently expressed the alpha-1,5-glucosidase 0271 Transgenic maize callus expressing alpha-1,1-glu cosidase enzyme was generated by bombarding maize enzyme. embryos with two binary vectors. The method of embryo transformation and generation of callus was essentially as TABLE 23 described in Example 3F; however, two polynucleotide HPAEC analysis of carbohydrate products from tobacco leaves transiently sequences were bombarded at the same time. One of the expressing an alpha-1,5-glucosidase enzyme. Enzyme activity is polynucleotide sequences contained the selectable marker, indicated by the change in abundance of each Sugar as a percentage PMI, which allows for selection of transgenic maize cells by of the total sugars over a 24 hour period. growth on mannose. The second polynucleotide sequence, Glucose Fructose Leucrose 902429, contained a maize optimized polynucleotide sample (% total Sugar) (% total Sugar) (% total Sugar) sequence encoding an alpha-1,1-glucosidase (SEQ ID NO: 902550 18.07 20.36 -38.43 49). The alpha-1,1-glucosidase was targeted to be retained by Negative 3.30 1...SO -4.80 the endoplasmic reticulum. control 0272. Maize calli expressing the alpha-1,1-glucosidase Total sugar = total amount of identifiable sugars in sample based on retention times of pure was collected 1 callus per well in 96-well 2 mL plates (What sugar standards, Extraneous peaks in samples are indeterminate and not included in sample man) containing one 3/16" chrome ball bearing per well. The analysis, The negative control is tobacco leaves transiently expressing empty BCTV vector, plate was shaken at setting 9 for 2 min in a Kleco Titer plate/Microtube Grinding Mill. Sets of 4 pulverized callus 7G: Transgenic Maize Callus Expressing alpha-1,5-glucosi tissue samples were combined and transferred to microfuge dase (SEQID NO: 43) tubes. The samples were centrifuged at 20,000xg30 minutes 0274 Transgenic maize callus expressing alpha-1,5-glu at 4 degrees C. The Supernatants containing protein extract cosidase enzyme was generated by bombarding maize were transferred to new tubes and extracts with volumes <20 embryos with two binary vectors. The method of embryo uL were pooled such that all samples were >30 ul involume. transformation and generation of callus was essentially as Equal volume of extract and reaction buffer (-185 mM tre described in Example 3F; however, two polynucleotide halulose, 93 mM isomalitulose, 100 mM HEPES, 0.04% sequences were bombarded at the same time. One of the Tween-20, 4 mM EDTA, 40 mM NaOH, Roche protease polynucleotide sequences contained the selectable marker, inhibitors) were combined and incubated at 70 degrees C. in PMI, which allows for selection of transgenic maize cells by BioRad Tetrad 2 thermocycler. Samples were collected at growth on mannose. The second polynucleotide sequence, US 2011/020 1 059 A1 Aug. 18, 2011 34

902423, contained a maize optimized polynucleotide alpha-1,6-glucosidase (SEQID NO: 11). Both binary vectors sequence encoding an alpha-1,5-glucosidase (SEQ ID NO: were infiltrated into the same tobacco leaf. 43). The alpha-1,5-glucosidase was targeted to the chloro 0277 Essentially as described in Example 3D, whole plast. leaves from tobacco were co-infiltrated with both binary vec 0275 Maize calli expressing an alpha-1,5-glucosidase tors 17588 and 092526. Co-infiltration was performed essen (SEQID NO: 43) was collected 1 callus per well in 96-well 2 tially as described in Example 3D except that two strains of mL plates (Whatman) containing one 3/16" chrome ball bear Agrobacterium, each containing one of the two vectors, were ing per well. Samples were frozen at -80 degrees C. The infiltrated into the tobacco leaf. Infiltrated leaves were col frozen material was shaken at setting 9 for 4 min in a Kleco Titer plate/Microtube Grinding Mill. 200 uI of extraction lected and frozen at -80 degrees C. in 24-well blocks con buffer (100 mM HEPES, 4 mM EDTA, 0.04% Tween-20, pH taining 3/16" chrome ball bearings. The frozen material was 7) was added to each sample. Extracts were incubated in a shaken at setting 9 for 2 min in a Kleco Titer Plate/Microtube Glas-Col rotator at 80% speed for 10 min. Extract was cen Grinding Mill creating a powder. Powder samples were trans trifuged at 1770xg for 10 minutes at 4 degrees C. in Eppen ferred to 30 mL centrifuge tubes and centrifuged at 20,000xg dorf 581OR Swing bucket centrifuge. Extract was frozen at for 20 minutes at 4 degrees C. The Supernatants were trans -80 degrees C. Extract was later thawed and transferred to a ferred to new tubes and adjusted to 50 mM HEPES, 0.02% 96-well PCR plate (Thermo Sci). Samples were heated at 80 Tween-20, 2 mM EDTA and 20 mM NaOH resulting in a degrees C. for 15 minutes in BioRad Tetrad 2 thermocycler. mixture with pH between 7 and 8. Samples were then trans Plates were again centrifuged at 1770xg for 10 minutes at 4 ferred to PCR tubes and incubated at 60 degrees C. in a Biorad degrees C. in Eppendorf 581OR Swing bucket centrifuge. Tetrad 2 thermocycler. Samples were collected from the ther Supernatants were filtered using a Millipore Multiscreen-HV mocycler at times 0, 18, and 48 hours and heated at 95 degrees filter plate. Filtered extracts of 8 callus samples were com C. before freezing at -20 degrees C. The sugar contents of the bined. Combined samples were concentrated from ~1.6 mL samples thawed after the -20 degree C. freeze were analyzed to 100-500 uL using Microcon concentrators with MWCO3 by Dionex. k membrane filters (Amicon). An equal volume of 200 mM 0278 Table 25 demonstrates that plants transiently leucrose and extract was added to 96-well PCR plate and expressing both Sucrose isomerase and alpha-1,6-glucosidase incubated at 80 degrees C. in the thermocycler. Samples were expressed an active Sucrose isomerase. Sucrose isomerase collected at times 0 and 24 hours. Collected samples were activity was demonstrated by the accumulation of trehalulose incubated at 95 degrees C. 5 minutes before freezing at -20 and isomalitulose in both the negative control (17588) and the degrees C. Samples were analyzed by Dionex essentially as sample (17588 and 902526). It is noted that the sample described in Example 1G. Alpha-1,5-glucosidase activity (17588 and 902526) accumulated less trehalulose and isoma was confirmed by measuring the conversion of the locked litulose than the negative control (17588). While not to be Sugar, leucrose, to the fermentable Sugars glucose and fruc limited by theory, this observation Suggests that the alpha-1, tose. Table 24 demonstrates that maize callus expressed an 6-glucosidase enzyme is active in the sample (17588 and active alpha-1,5-glucosidase enzyme. 902526) and thus leads to the conversion of the trehalulose and isomalitulose to fermentable Sugars. TABLE 24 0279 Tables 25-26 demonstrate that plants transiently HPAEC analysis of carbohydrate products from transformed maize expressing both Sucrose isomerase and alpha-1,6-glucosidase callus tissue expressing an alpha-1,5-glucosidase enzyme. Enzyme expressed active enzymes. Alpha-1,6-glucosidase activity activity is indicated by the change in abundance of each Sugar was demonstrated by comparing time 0 samples with Samples as a percentage of the total Sugars over a 24 hour period. collected at 48 hours which demonstrated the conversion of Glucose Fructose Leucrose the locked Sugars, trehalulose and isomalitulose, to the fer sample (% total Sugar) (% total Sugar) (% total Sugar) mentable Sugars, glucose and fructose. 902423 6.86 12.71 -19.57 0280 Data outlined in Table 25-26 demonstrates the co Negative O.48 0.73 -1.21 expression of a locking enzyme (sucrose isomerase) and an control key enzyme (alpha-1,6-glucosidase) in a plant. Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample TABLE 25 analysis, Negative control consisted of maize callus transformed with the binary vector containing the selectable marker (PMI) only, HPAEC analysis of carbohydrate products from tobacco leaves transiently expressing both Sucrose isomerase and an alpha Example 8 1,6-glucosidase enzyme. Accumulation of Sucrose isomers in a plant co-expressing both lock and key enzymes before incubating Combining Plant Expressed Locking and Key for key activity. (T. ethanolicus Enzymes Glucose + Sucrose Trehallulose Isomalitulose Fructose % total % total % total % total 0276 Tobacco leaves transiently expressing enzymes sample Sugar Sugar Sugar Sugar were generated essentially as described in Example 3D. 17588 and 75.88 O 15.91 8.21 Leaves were generated by transiently expressing two binary 902526 vectors simultaneously. One of the binary vectors was 17588 Negative 80.99 19.01 O O (described in Example 12) which contains a polynucleotide control Total sugar = total amount of identifiable sugars in sample based on retention times of pure sequence encoding a sucrose isomerase (SEQ ID NO: 16). sugar standards, Extraneous peaks in samples are indeterminate and not included in sample The second binary vector was 902526 (described in Example analysis, Negative control consisted of non-infiltrated tobacco leaves, 12) which contains a polynucleotide sequence encoding an US 2011/020 1 059 A1 Aug. 18, 2011 35

Strains were grown in a media containing 10 g yeast extract, TABLE 26 and 20 g peptone per liter of media. This media was Supple HPAEC analysis of carbohydrate products from tobacco leaves mented with glucose or isomalitulose to the appropriate final transiently expressing both Sucrose isomerase and an alpha concentration. 1,6-glucosidase enzyme. Table 254 convers hydrolysis of the lock Sugars by key activity after incubation. Enzyme activity 0284. Single yeast colonies were inoculated into 5 mL 2% is indicated by the change in abundance of each Sugar as a glucose media and incubated for 24 hours at 30 degrees C. percentage of the total sugars over a 24 hour period. cells were centrifuged at 3000xg for 5 minutes, supernatant Glucose Fructose Isomalitulose Trehallulose was discarded, cells were washed by resuspending the cells in (% total (% total (% total (% total 5 mLs of distilled water, washed cells were centrifuged at sample Sugar) Sugar) Sugar) Sugar) 3000xg for 5 minutes, supernatant was discarded, cells were 17588 and O.15 10.34 -4.20 -6.30 resuspend in 5 mLS of yeast media containing 1% isomaltu 902526 lose media and incubated for 12 hours at 30 degrees C. After Negative -8.18 3.58 1.19 3.41 12 hours cells were centrifuged at 3000xg for 5 minutes, control Supernatant was discarded, cells were washed by resuspend Total sugar = total amount of identifiable sugars in sample based on retention times of pure sugar standards, Extraneous peaks in samples are indeterminate and not included in sample ing in 5 mLs of distilled water, washed cells were centrifuged analysis, Negative control consisted tobacco leaves transiently expressing sucrose isomerase at 3000xg for 5 minutes, supernatant was discarded, cells and an empty control vector, were resuspend in 5 mLs of 4% isomalitulose media or 4% Example 9 glucose media for fermentation. Samples for ethanol and Sugar analysis were removed every hour for six hours and Production of Fermentable Sugars and/or Ethanol stored at -20 degrees C. After all samples were collected they 9A: Glucose Production Using Both Dextranslucrase and were thawed and filtered in 0.45 Micron nylon SpinX col Dextranase umns by centrifugation at 7000 rpm for 5 minutes. Filtered 0281 Dextranslucrase and dextranase form a pair of solution was then subjected to HPLC to determine the con enzymes that are a lock and key combination. The dextran Sucrase catalyzes the formation of dextrans which are a centration of ethanol and the Sugar composition of the solu locked form of Sugar or carbohydrate. The dextranase is a key tion which is shown in table27. The graph below outlines the enzyme which can be used to convert the dextran back to a ethanol produced by various yeast Strains grown in the pres fermentable form of Sugar. ence of glucose or isomaltulose over time. 0282. The dextranslucrase is expressed in transgenic Sug arcane plants such that dextrans accumulate in the Sugarcane TABLE 27 plant. Dextrans produced from dextranslucrase reactions in Ethanol yield from yeast strains grown Sugarcane juice (Example 1C) or dextrans produced by trans with isomalitulose or glucose genic plants expressing dextranslucrases (Example 3B) are harvested. These dextrans are used as substrate for dextranase Percentage Ethanol Percentage of activity assays to demonstrate the ability of the selected dex Yeast Strain Sugar Yield Theoretical Yield tranases to convert the dextrans back into glucose, maltose B Glucose 2.1 80.1 and other Small reducing Sugars. The dextranase is provided B Isomalitulose 1.49 57.4 C Glucose 2.14 82.O as either transgenic plant produced enzyme (Example 3C) or C Isomalitulose O3S 13.6 as microbially produced enzyme (Example 2C). A. Glucose 1.9 72.4 9B: Isomalitulose Fermented to Produce Ethanol A. Isomatlulose O 0283 Yeast, Saccharomyces cerevisiae, strains were screened for the ability to ferment isomalitulose into ethanol. US 2011/020 1 059 A1 Aug. 18, 2011 36

EtOH yield from isomal tulose and Glucose

2.5

-- Conrnercial yeast with IM -o-Commercial yeast with Glic

-- Strain B With M

ens Strain B with Gltic

alue Strain F with M

-- Strain F With Giuc US 2011/020 1 059 A1 Aug. 18, 2011 37

Example 10 0290 An empty-vector control consisting of the pEB68 backbone but lacking any gene behind the TP1 promoter was Transfer of Ethanol Producing Genes Between Yeast made by cutting pEB68 with XhoI/Xbal, purification of the Strains backbone, blunting the ends, and self-ligation. This vector 0285. Not all yeast strains, including commercial yeast was named pEB70. strains used in the ethanol industry, possess the capacity for 0291 Saccharomyces cerevisiae strain X1049-9C (ATCC isomalitulose fermentation. Genes needed for isomalitulose number 204802) was transformed with the vectors pEB68, fermentation can be introduced into commercial Strains by pEB77, and pEB70. Yeast competent cells were made and mating, mutagenesis or transformation. These genes may transformed using the S. c. EasyCompTM Transformation kit include an alpha glucosidase enzyme in addition to a receptor (Invitrogen). Transformed yeast cells were recovered by holding them at 30 degreesC for 4-5 hours after transforma which senses the presence of isomalitulose and induces the tion and then plated on YPD medium containing 200 ug/mL expression of an alpha-glucoside transporter which transports of G418. isomalitulose and other alpha glucosides into the cell. Genes 0292 Glucosidase enzyme activity associated with vector involved with these functions occurat the melezitose locus in pEB69 was measured in transformed yeast cells by selected S. cerevisiae and may be introduced into other strains of yeast three yeast clones expressing DEX4-Sc SAM1606 fusion by mating techniques known to skilled practitioners in the art protein and three untransformed yeast clones which were (Hwang & Lindegren Nature Vol 203 no 4946, pp 791-792 inoculated on 5 mL of YPD with G418 (untransformed yeast (1964)). Alternatively, the coding sequence of a highly effi was inoculated in YPD without selection). After 24 hours of growth, cells were pelleted and the media was separated and cient alpha-1,6-glucosidase enzyme may be introduced into used for enzyme analyses. yeast in place of the alpha glucosidase gene at the melezitose 0293 Sc-SAM1606 activity was measured at 70 degreesC locus by homologous recombination or they may be inserted for 16 hours by combining 10 uI of yeast media, 25 uL of elsewhere in the genome. By replacing the endogenous alpha buffer (100 mM Hepes, 4 mM EDTA, 0.04% Tween-20, pH glucosidase gene with a gene that more efficiently hydrolyzes 7.0), and 15 uL of a sugar solution containing 280 mM tre isomalitulose or other locked Sugars it may be possible to halulose, 100 mM isomalitulose, 70 mM citrate. Enzyme improve the rate of fermentation of these Sugars. Similarly, activity was estimated by measuring the amount of glucose genes for alpha-glucoside transporters and receptors may be released from the conversion of locked Sugar (trehalulose and overexpressed or altered by site directed mutagenesis in order isomalitulose) to glucose using a GO-POD assay essentially as described in Example 2B. Table 27 outlines data demon to increase the rate of isomalitulose uptake by yeast strains to strating the transformed yeast expressed an active glucosi improve the efficiency of isomalitulose fermentation. Another dase enzyme. approach may be to identify strains which constitutively 0294 Glucosidase enzyme activity associated with vector express the genes necessary for isomaltulose fermentation or pEB77 was demonstrated by isolating two clones of each to mutagenize or engineer yeast strains so that they constitu transformation (pEB77 and pPB70) and inoculated into tively express the genes necessary for isomalitulose fermen medium containing 10 g yeast extract, 20 g peptone, 4 g. tation. The techniques necessary for these approaches are isomalitulose, and 0.5% glucose per liter of medium. Cultures widely known to skilled practitioners of the art. were grown until glucose was exhausted (24 hours). After 24 hours, the cells were spun and 1 mL of medium was saved for 10A: Transgenic Yeast Expressing Key Enzymes enzyme activity. To evaluate glucosidase activity on isoma litulose the following reaction was set up: 25 ul of 2x Buffer 0286 A yeast codon optimized gene for Bacillus (100 mM Hepes pH: 7.0, 4 mM EDTA, 0.04% Tween-20, SAM1606 (Sc. SAM1606) glucosidase (GeneBank Acces protease inhibitors), 10 ul isomalitulose (500 mM), and 15 ul sion CAA54266) was cloned into the XhoI/Xbal sites of medium obtained as described above. The 50 uL reaction was pGEM30 (ATCC 53345), which contains an N-terminus incubated overnight at 37 degrees C. 20 uL of the above DEX4 secretion signal. This created a DEX4-Sc. SAM1606 reaction were added to 250 uL of Glucose oxidase reagent glucosidase fusion protein. (GOPOD assay essentially as described in Example 2B) and incubated at 37 degrees C. for 10 minutes. The reactions (0287. The URA3 marker was replaced with the kanMX consisted of three technical replicates. The glucose concen locus, which confers resistance to the antibiotic Geneticin tration measured was termed GlucoseA. To account for any (G418) (Wachet al. Yeast 10: 1793-1808 (1994)). The URA3 glucose left in the medium after 24 hours of yeast growth, the cassette was excised with SmaI and Clal and the backbone same GOPOD assay was conducted by diluting 15 uL of was gel-purified. The kanMX cassette was amplified from a medium with 35 uL of water (no isomalitulose) and using 20 uL of this dilution to the Glucose oxidase reagent. All the yeast insertional library (ATCC number GSA-7) using Phu glucose measured this way is considered background noise sion High Fidelity DNA polymerase (Finnzymes) with prim and must come from the medium. This was termed GlucoseB. ers bearing 30 bp of homology to the ends of the SmaI/ClaI 0295 The amount of glucose produced by hydrolysis of backbone fragment. isomalitulose was calculated as Glucose A minus GlucoseB 0288 The SmaI/Clal backbone fragment and the kanMX and correspond to the values shown in Table 29. cassette were recombined using SLIC recombination (Li and Elledge, Nature Methods 4: 251-256 (2007)). Briefly, both TABLE 28 fragments were treated with T4 DNA polymerase at room Glucose Conc of samples (mM): Transformed raw temperature to create single stranded DNA, the reaction was data from yeast expressing glucosidase using stopped after 15 minutes with dCTP, and the fragments were equation from glucose standard curve. co-transformed into E. coli TOP10 competent cells (Invitro gen). Plasmids isolated from recombinant E. coli cells were Sample # sequenced and analyzed by restriction enzymes. The result Sample Negative ing vector was named pBB68. Replicate pEB68 pEB68 pEB68 pEB68 control 0289. A second yeast vector containing the Bacillus thur ingiensis alpha-1,6-glucosidase gene was generated by clon A. 4.74 7.19 4.21 4.73 1.49 ing a yeast codon optimized polynucleotide sequence encod B 4.81 3.86 4.26 4.59 1.65 ing the alpha-1,6-glucosidase into the pEB68 backbone by C 4.83 4...SO 4.47 4.90 1.63 SLIC recombination to create pEB77. US 2011/020 1 059 A1 Aug. 18, 2011 38 Table 29: Transgenic yeast containing plasmid pEB77 demonstrate glucosidase activity.

| 802_77_1 802772 802.701 802. 702 ------Clone number and vector US 2011/020 1 059 A1 Aug. 18, 2011 39

Example 11 optimized polynucleotide sequence encoding a Sucrose isomerase (SEQID NO: 24); a NOS terminator. Improvement of Molecules to Increase Activity, 0301 The vector pEB38 contains an expression cassette Thermostability, and Catalytic Efficiency and Prod with the following components operatively linked together in uct Specificity this order: maize ubiquitin promoter (SEQID NO: 18); maize 0296 Improvement of sucrose isomerase enzymes can be gamma Zein signal sequence (SEQID NO: 19) which targets achieved through rational design of the enzyme. For example, the polypeptide encoded by the Sucrose isomerase polynucle the product of the pall gene (GenBank accession number otide sequence to the endoplasmic reticulum; sporamin vacu AY040843) contains a product specificity domain olar targeting sequence (SEQ ID NO: 15) which directs the *RLDRD’ which influences the proportion of trehalulose polypeptide encoded by the Sucrose isomerase polynucle or isomalitulose produced by the enzyme. By mutating these otide sequence from the endoplasmic reticulum to the vacu four charged amino acid residues (Arg325, Arg328, Asp327 ole; monocot optimized polynucleotide sequence encoding and Asp329) trehalulose formation can be increased by sucrose isomerse (SEQID NO: 20); and the NOS terminator. 17-61% and formation of isomalitulose can be decreased by 0302) The binary vector 902525 contains an expression 26-67% (Zhanget al. FEBS Letters 534 (2003) 151-155). An cassette with the following components operatively linked aromatic clamp formed by Phe 256 and Phe280 has also been together in this order: Arabidopsis ubiquitin promoter (SEQ identified as important in Substrate recognition and product ID NO: 7); GY 1 ER targeting sequence (SEQ ID NO: 13), specificity. (Ravaudet al. The Journal of Biological Chemis which targets the polypeptide encoded by the Sucrose isomerase coding region through the endoplasmic reticulum; try VOL. 282, NO. 38, pp. 28126-28136, Sep. 21, 2007). dicot optimized polynucleotide sequence encoding Sucrose isomerase polypeptide (SEQ ID NO: 11); NOS terminator. Example 12 The Sucrose isomerase enzyme expressed by this expression Constructs for Transient Expression cassette is expected to accumulate in the apoplast of the transgenic plant cell comprising the expression cassette. 0297 Table 1 outlines expression constructs used forgen (0303. The BCTV binary vector 902526 contains an eration of stable, transgenic plants as well as for the expres expression cassette with the following components opera sion of enzymes transiently in plant tissues. The DNA tively linked together in this order: Agrobacterium NOS pro sequences encoding proteins were codon optimized for the moter (SEQ ID NO: 10); GY 1 ER targeting sequence (SEQ appropriate host; for example, expression constructs ID NO: 13), which targets the polypeptide encoded by the designed for tobacco and Sugarbeet transient and stable trans Sucrose isomerase coding region through the endoplasmic genic plant expression were codon optimized for dicots while reticulum; dicot optimized polynucleotide sequence encod expression constructs designed for Sugarcane or maize tran ing sucrose isomerase polypeptide (SEQ ID NO: 11); NOS sient and stable transgenic plant expression were codon opti terminator. The Sucrose isomerase enzyme expressed by this mized for monocots. Codon optimization tables are available expression cassette is expected to accumulate in the apoplast through commercial Software applications such as Vector of the transgenic plant cell comprising the expression cas NTI9.O. Sette. 0298 Standard cloning techniques such as restriction 0304. The binary vector 901612 contains an expression enzyme digestion, gel electrophoresis and Subsequence frag cassette with the following components operatively linked ment purification, DNA ligation, bacterial cell transformation together in this order: Arabidopsis ubiquitin promoter (SEQ and selection, and the like were used to generate the vectors ID NO: 7); FNR plastid targeting sequence (SEQID NO: 26) described in Table 29. Some of the components of the expres which directs the alpha-1,1-glucosidase polypeptide to the sion vectors described in Table 1 were synthesized by Gene chloroplast, dicot optimized polynucleotide sequence encod Art (Germany), additionally, some of the vectors were cloned ing alpha-1,1-glucosidase (SEQ ID NO: 27); NOS termina by GeneArt (Germany). tor. The alpha-1,1-glucosidase enzyme expressed by this 0299 The binary vector 17588 contains an expression cas expression cassette is expected to accumulate in the chloro sette with the following components operatively linked plast of the transgenic plant cell comprising the expression together in this order: the Arabidopsis ubiquitin promoter CaSSette. (SEQ ID NO: 7); GY 1 ER targeting sequence (SEQID NO: (0305. The binary vector 902195 contains an expression 13), which targets the polypeptide encoded by the sucrose cassette with the following components operatively linked isomerase coding region through the endoplasmic reticulum; together in this order: Agrobacterium NOS promoter (SEQ the sporamin vacuolar targeting sequence (SEQ ID NO 15) ID NO: 10): GY 1 ER targeting sequence (SEQ ID NO: 13) which directs the Sucrose isomerase polypeptide from the which targets the dextranslucrase polypeptide to the endoplas endoplasmic reticulum to the vacuole; a dicot optimized poly mic reticulum; sporamin vacuolar targeting sequence (SEQ nucleotide sequence encoding a Sucrose isomerase (SEQID ID NO: 15) which directs the polypeptide encoded by the NO: 16); and a NOS termination sequence. dextranslucrase polynucleotide sequence from the endoplas 0300. The binary vector pBB47 contains an expression mic reticulum to the vacuole; dicot optimized polynucleotide cassette with the following components operatively linked sequence encoding a dextranslucrase with leucrose synthase together in this order: an FMV enhancer (SEQID NO: 22); a activity (SEQID NO:35): NOS terminator. 35S enhancer (SEQID NO. 23); a maize ubiquitin promoter 0306 The vector pEB28 contains an expression cassette (SEQID NO: 18); a maize gamm-Zein ER targeting sequence with the following components operatively linked together in (SEQ ID NO: 19) which directs the sucrose isomerase this order: maize ubiquitin promoter (SEQID NO: 18); maize polypeptide to the ER; a sporamin vacuolar targeting gamma Zein signal sequence (SEQID NO: 19) which targets sequence (SEQ ID NO: 15) which directs the sucrose the polypeptide encoded by the dextranslucrase polynucle isomerase polypeptide from the ER to the vacuole; a maize otide sequence to the endoplasmic reticulum; sporamin vacu US 2011/020 1 059 A1 Aug. 18, 2011 40 olar targeting sequence (SEQ ID NO: 15) which directs the together in this order: Arabidopsis ubiquitin promoter (SEQ polypeptide encoded by the dextranslucrase polynucleotide ID NO: 7); GY 1 ER targeting sequence (SEQ ID NO: 13) sequence from the endoplasmic reticulum to the vacuole; which targets the alpha-1,1-glucosidase polypeptide to the monocot optimized polynucleotide sequence encoding a dex endoplasmic reticulum; dicot optimized polynucleotide translucrase with leucrose synthase activity (SEQID NO: 37): sequence encoding an alpha-1,1-glucosidase (SEQ ID NO: NOS terminator. 52); NOS terminator. The expectation is that the alpha-1,1- 0307. The binary vector 902550 contains an expression glucosidase polypeptide will be processed through the endo cassette with the following components operatively linked plasmic reticulum and accumulate in the apoplast. together in this order: Arabidopsis ubiquitin promoter (SEQ 0310. The vector 902435 contains an expression cassette ID NO: 7); chloroplast targeting sequence (SEQID NO: 42): with the following components operatively linked together in dicot optimized polynucleotide sequence encoding an alpha this order: maize ubiquitin promoter (SEQID NO: 29); TMV 1.5-glucosidase (SEQID NO: 46): NOS terminator. enhancer sequence (SEQID NO: 40); maize optimized poly 0308 The vector 902423 contains an expression cassette nucleotide sequence encoding an alpha-1,6-glucosidase with the following components operatively linked together in (SEQ ID NO. 54); ER retention sequence (SEQID NO:51); this order: maize ubiquitin promoter (SEQID NO:39); TMV maize ubiquitin termination sequence (SEQID NO: 45). enhancer (SEQ ID NO: 40); chloroplast targeting sequence 0311. The vector 902425 contains an expression, cassette (SEQ ID NO: 41) which directs the alpha-1,5-glucosidase with the following components operatively linked together in polypeptide encoded by the polynucleotide sequence (SEQ this order: maize ubiquitin promoter (SEQID NO: 29); TMV ID NO: 43) to the chloroplast; maize optimized polynucle enhancer sequence (SEQ ID NO: 40); chloroplast targeting otide sequence encoding alpha-1,5-glucosidase (SEQID NO: sequence (SEQID NO: 26); monocot optimized polynucle 43); terminator from maize ubiquitin (SEQ ID NO: 45). otide sequence encoding an alpha-1,6-glucosidase (SEQ ID 0309 The binary vector 90522 contains an expression cas NO: 56); maize ubiquitin termination sequence (SEQID NO: sette with the following components operatively linked 45).

TABLE 29 Expression constructs

Vector number Promoter Regulatory elements Enzyme crop 17588 Arabidopsis GY1 ER targeting sequence Sucrose Sugar beet (binary ubiquitin (SEQID NO: 13); sporamin isomerase and tobacco vector) promoter vacuolar targeting sequence (SEQ ID NO: (SEQ ID (SEQID NO: 15) 16) NO: 7) pEB47 maize FMV enhancer (SEQ ID Sucrose Maize and (binary ubiquitin NO: 22); 35S enhancer isomerase Sugarcane vector) promoter (SEQID NO: 23); Maize? (SEQ ID NO: (SEQ ID gamma Zein ER targeting 24) NO: 18) sequence (SEQID NO: 19); sporamin vacuolar targeting sequence (SEQID NO: 15) pEB38 maize Maize gamma Zein ER Sucrose Maize and ubiquitin targeting sequence (SEQID isomerase Sugarcane promoter NO: 19); sporamin vacuolar (SEQ ID NO: (SEQ ID targeting sequence (SEQID 20) NO: 18) NO: 15) 902525 Arabidopsis GY1 ER targeting sequence T. ethanolicus Sugar beet binary ubiquitin (SEQID NO: 13) alpha-1,6- and tobacco promoter glucosidase (SEQ ID (SEQ ID NO: NO: 7) 11) 902526 NOS GY1 ER targeting sequence T. ethanolicus Sugar beet (BCTV promoter (SEQID NO: 13) alpha-1,6- and tobacco binary) (SEQ ID glucosidase NO: 10) (SEQ ID NO: 11) 902195 NOS GY1 ER targeting sequence Dextranslucrase Tobacco and promoter (SEQID NO: 13); sporamin (SEQID NO: Sugarbeet (SEQ ID vacuolar targeting sequence 35) NO: 10) (SEQID NO: 15) pEB28 maize Maize gamma Zein ER Dextranslucrase Maize and ubiquitin targeting sequence (SEQID (SEQID NO: Sugarcane promoter NO: 19); sporamin vacuolar 37) (SEQ ID targeting sequence (SEQID NO: 18) NO: 15) 902435 maize ER retention sequence (51); Alpha-1,6- Maize and ubiquitin maize ubiquitin terminator glucosidase Sugarcane promoter (SEQID NO: 45); TMV (SEQ ID NO: (SEQ ID enhancer (SEQ ID NO:40) 54) NO:39) US 2011/020 1 059 A1 Aug. 18, 2011

TABLE 29-continued Expression constructs

Vector number Promoter Regulatory elements Enzyme crop 902425 maize TMV enhancer (SEQID Alpha-1,6- Maize and ubiquitin NO: 40); FNR chloroplast glucosidase Sugarcane promoter targeting sequence (SEQID (SEQID NO: (SEQ ID NO: 41); maize ubiquitin 56) NO:39) terminator (SEQ ID NO: 45) 9.01612 Arabidopsis Plastid targeting sequence Bacilius alpha Sugar beet ubiquitin FNR (SEQID NO: 26) 1,1- and tobacco promoter glucosidase (SEQ ID (SEQID NO: NO: 7) 27) 902522 Arabidopsis GY1 ER targeting sequence Alpha-1,1- Sugar beet ubiquitin (SEQID NO: 13) glucosidase and tobacco promoter (SEQID NO: (SEQ ID 52) NO: 7) 902429 maize TMV enhancer (SEQID Alpha-1,1- Maize and ubiquitin NO: 40); ER targeting glucosidase Sugarcane promoter sequence (SEQID NO: 48); (SEQID NO: (SEQ ID ER retention sequence (51); 49) NO:39) maize ubiquitin terminator (SEQID NO: 45) 902550 Arabidopsis Plastid targeting sequence Alpha-1,5- Sugarbeet ubiquitin FNR (SEQID NO: 26) glucosidase and tobacco promoter (SEQID NO: (SEQ ID 46) NO: 7) 902423 maize TMV enhancer (SEQID Alpha-1,5- Maize and ubiquitin NO: 40): FNR chloroplast glucosidase Sugarcane promoter targeting sequence (SEQID (SEQID NO: (SEQ ID NO: 41); maize ubiquitin 43) NO:39) terminator (SEQ ID NO: 45)

The following embodiments are encompassed by the present 0315 b) contacting said transgenic plant material with invention: one or more key enzymes wherein said contacting is 1. A method for producing fermentable Sugar comprising: under conditions sufficient for conversion of said locked 0312 a) providing transgenic plant material comprising carbohydrate to fermentable Sugar. one or more locked carbohydrates; and 7. The method of claim 6, wherein the one or more locked 0313 b) contacting said transgenic plant material with carbohydrate is selected from the group consisting of isoma one or more key enzymes wherein said contacting is litulose, trehalulose, leucrose, starch, dextran, fructan, maltu under conditions sufficient for conversion of said locked lose, turanose and isomaltose. carbohydrate to fermentable Sugar. 8. The method of claim 6, wherein the one or more lock 2. The method of claim 1, wherein the one or more locked enzymes is selected from the group consisting of dextransu carbohydrate is selected from the group consisting of isoma crase, levan Sucrose, alternanslucrase, Sucrose isomerase and litulose, trehalulose, leucrose, starch, dextrans, fructans, amylosucrase. maltulose, turanose and isomaltose. 9. The method of claim 6, wherein the one or more key 3. The method of claim 1, wherein the one or more key enzymes is selected from the group consisting of dextranase, enzyme is selected from the group consisting of dextranase, alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha 1,1-glucosidase and alpha-1,6-glucosidase. 1,1-glucosidase and alpha-1,6-glucosidase. 10. The method of claim 6, wherein the one or more key 4. The method of claim 1, wherein the one or more key enzymes is provided by a source selected from the group enzyme is provided by a source selected from the group consisting of transgenic plant material expressing a key consisting of transgenic plant material expressing a key enzyme, recombinant microbe expressing a key enzyme, enzyme, recombinant microbe expressing a key enzyme, transgenic yeast expressing a key enzyme, microbe express transgenic yeast expressing a key enzyme, microbe express ing a key enzyme and yeast expressing a key enzyme. ing a key enzyme and yeast expressing a key enzyme. 11. The method of claim 6, wherein the transgenic plant is 5. The method of claim 1, wherein the transgenic plant is selected from the group consisting of maize, Sugar beet, Sor selected from the group consisting of maize, Sugar beet, Sor ghum and Sugarcane. ghum and Sugarcane. 12. A method for producing alcohol comprising: 6. A method for producing fermentable Sugar comprising: 0316 a) providing transgenic plant material comprising 0314 a) providing transgenic plant material comprising one or more locked carbohydrates; one or more lock enzymes and one or more locked 0317 b) contacting said transgenic plant material with carbohydrates; and one or more key enzymes wherein said contacting is US 2011/020 1 059 A1 Aug. 18, 2011 42

under conditions sufficient for conversion of said one or 26. The method of claim 25, wherein the one or more key more locked carbohydrates to fermentable Sugar, and enzymes is targeted away from the one or more locked car 0318 c) fermenting said fermentable sugar to form bohydrates. alcohol. 27. The method of claim 25, wherein the one or more key 13. The method of claim 12, wherein the locked carbohydrate enzymes is targeted to an organelle selected from the group is selected from the group consisting of isomalitulose, treha consisting of chloroplast, vacuole, cytoplasm, apoplast and lulose, leucrose, starch, dextran, fructan, maltulose, turanose endoplasmic reticulum. and isomaltose. 28. The method of claim 25, wherein the one or more locked 14. The method of claim 12, wherein the one or more key carbohydrates is selected from the group consisting of isoma enzyme is selected from the group consisting of dextranase, alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha litulose, trehalulose, leucrose, starch, dextran, fructan, maltu 1,1-glucosidase and alpha-1,6-glucosidase. lose, turanose and isomaltose. 15. The method of claim 12, wherein the one or more key 29. The method of claim 25, wherein the one or more key enzyme is provided by a source selected from the group enzymes is selected from the group consisting of dextranase, consisting of transgenic plant material expressing a key alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha enzyme, recombinant microbe expressing a key enzyme, 1,1-glucosidase and alpha-1,6-glucosidase. transgenic yeast expressing a key enzyme, microbe express 30. The method of claim 25, wherein the one or more key ing a key enzyme and yeast expressing a key enzyme. enzymes is provided by a source selected from the group 16. The method of claim 12, wherein the alcohol is selected consisting of transgenic plant material expressing a key from the group consisting of ethanol and butanol. enzyme, recombinant microbe expressing a key enzyme, 17. The method of claim 12, wherein the transgenic plant is transgenic yeast expressing a key enzyme, microbe express selected from the group consisting of maize, Sugar beet, Sor ing a key enzyme and yeast expressing a key enzyme. ghum and Sugarcane. 31. The method of claim 25, wherein the transgenic plant is 18. A method for producing alcohol comprising: selected from the group consisting of maize, Sugar beet, Sor 0319 a) providing transgenic plant material comprising ghum and Sugarcane. one or more lock enzymes and one or more locked 32. A method for producing fermentable Sugar comprising: carbohydrates: 0324 a) providing transgenic plant material comprising 0320 b) contacting said transgenic plant material with one or more lock enzymes, one or more locked carbo one or more key enzymes wherein said contacting is hydrates and one or more key enzymes; and under conditions sufficient for conversion of said one or 0325 b) processing said transgenic plant material under more locked carbohydrates to fermentable Sugar, and conditions Sufficient for said one or more key enzymes 0321 c) fermenting said fermentable sugar to form to convert said one or more locked carbohydrates to alcohol. fermentable Sugar. 19. The method of claim 18, wherein the one or more locked 33. The method of claim 32, wherein the one or more lock carbohydrates is selected from the group consisting of isoma enzymes is selected from the group consisting of dextransu litulose, trehalulose, leucrose, starch, dextran, fructan, maltu crase, levan Sucrose, alternanslucrase, Sucrose isomerase and lose, turanose and isomaltose. amylosucrase. 20. The method of claim 18, wherein the one or more lock 34. The method of claim 32, wherein the one or more key enzymes is selected from the group consisting of dextransu enzymes is targeted away from the one or more locked car crase, levan Sucrose, alternanslucrase, Sucrose isomerase and bohydrates. amylosucrase. 35. The method of claim 32, wherein the one or more key 21. The method of claim 18, wherein the one or more key enzymes is targeted to an organelle selected from the group enzymes is selected from the group consisting of dextranase, alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha consisting of chloroplast, vacuole, cytoplasm, apoplast and 1,1-glucosidase and alpha-1,6-glucosidase. endoplasmic reticulum. 22. The method of claim 18, wherein the one or more key 36. The method of claim 32, wherein the one or more locked enzymes is provided by a source selected from the group carbohydrates is selected from the group consisting of isoma consisting of transgenic plant material expressing a key litulose, trehalulose, leucrose, starch, dextran, fructan, maltu enzyme, recombinant microbe expressing a key enzyme, lose, turanose and isomaltose. transgenic yeast expressing a key enzyme, microbe express 37. The method of claim 32, wherein the one or more key ing a key enzyme and yeast expressing a key enzyme. enzymes is selected from the group consisting of dextranase, 23. The method of claim 18, wherein the alcohol is selected alpha-amylase, glucoamylase, alpha-1,5-glucosidase, alpha from the group consisting of ethanol and butanol. 1,1-glucosidase and alpha-1,6-glucosidase. 24. The method of claim 18, wherein the transgenic plant is 38. The method of claim 32, wherein the one or more key selected from the group consisting of maize, Sugar beet, Sor enzymes is provided by a source selected from the group ghum and Sugarcane. consisting of transgenic plant material expressing a key 25. A method for producing fermentable Sugar comprising: enzyme, recombinant microbe expressing a key enzyme, 0322 a) providing transgenic plant material comprising transgenic yeast expressing a key enzyme, microbe express one or more locked carbohydrates and one or more key ing a key enzyme and yeast expressing a key enzyme. enzymes; and 39. The method of claim 32, wherein the transgenic plant is 0323 b) processing said transgenic plant material under selected from the group consisting of maize, Sugar beet, Sor conditions Sufficient for one or more key enzymes to ghum and Sugarcane. convert one or more locked carbohydrates to ferment 40. A transgenic plant comprising one or more heterologous able Sugar. lock enzymes and one or more heterologous key enzymes. US 2011/020 1 059 A1 Aug. 18, 2011

41. The transgenic plant of claim 40, wherein the one or more 0330 b) processing said transgenic plant material under lock enzymes is selected from the group consisting of dex conditions Sufficient for said one or more key enzymes translucrase, levan Sucrose, alternanSucrase, Sucrose to convert said one or more locked carbohydrates to isomerase and amylosucrase. fermentable Sugar. 42. The transgenic plant of claim 40, wherein the one or more 54. A transgenic plant comprising: key enzymes is targeted away from the locked carbohydrate. 0331 a) one or more lock enzymes wherein said one or 43. The transgenic plant of claim 40, wherein the one or more more lockenzymes is selected from the group consisting key enzymes is targeted to an organelle selected from the of dextranslucrase, levan Sucrose, alternansucrase, group consisting of chloroplast, vacuole, cytoplasm, apoplast Sucrose isomerase and amylosucrase, and endoplasmic reticulum. 0332 b) one or more locked carbohydrates wherein said 44. The transgenic plant of claim 40, wherein the locked one or more locked carbohydrates is selected from the carbohydrate is selected from the group consisting of isoma group consisting of isomaltulose, trehalulose, leucrose, litulose, trehalulose, leucrose, starch, dextran, fructan, mal starch, dextrans, fructans, maltose, turanose and isoma tose, turanose and isomaltose. ltose, 45. The transgenic plant of claim 40, wherein the one or more 0333 c) one or more key enzymes wherein said one or key enzyme is selected from the group consisting of dextra more key enzymes is selected from the group consisting nase, alpha-amylase, glucoamylase, alpha-1,5-glucosidase, of dextranase, alpha-amylase, glucoamylase, alpha-1,5- alpha-1,1-glucosidase and alpha-1,6-glucosidase. glucosidase, alpha-1,1-glucosidase and alpha-1,6-glu 46. The transgenic plant of claim 40, wherein the transgenic cosidase; and wherein said one or more key enzymes is plant is selected from the group consisting of maize, Sugar targeted away from the one or more locked carbohy beet, Sorghum and Sugarcane. drates, and 0334 d) wherein said transgenic plant is selected from 47. A transgenic plant comprising one or more locked carbo the group consisting of maize, Sugar beet, Sorghum and hydrates and one or more key enzymes. Sugarcane. 48. The transgenic plant of claim 47, wherein the one or more 55. A method for producing fermentable sugar derived from a key enzymes is targeted away from the one or more locked plant comprising: carbohydrates. 0335) a) providing plant material comprising locked 49. The transgenic plant of claim 47, wherein the key enzyme carbohydrate; and, is targeted to an organelle selected from the group consisting 0336 b) contacting said plant material with one or more of chloroplast, vacuole, cytoplasm, apoplast and endoplasmic enzymes capable of converting the locked carbohydrate reticulum. into fermentable Sugar (key enzyme), wherein said con 50. The transgenic plant of claim 47, wherein the one or more tacting is under conditions Sufficient for said conversion. locked carbohydrates is selected from the group consisting of 56. The method of embodiment 55, wherein said plant mate isomalitulose, trehalulose, leucrose, starch, dextran, fructan, rial comprising locked carbohydrate is derived from a trans maltose, turanose and isomaltose. genic plant expressing one or more enzymes capable of con 51. The transgenic plant of claim 47, wherein the one or more Verting an endogenous carbohydrate of said transgenic plant key enzyme is selected from the group consisting of dextra into said locked carbohydrate (lock enzyme). nase, alpha-amylase, glucoamylase, alpha-1,5-glucosidase, 57. The method of embodiment 55 or 56, wherein the key alpha-1,1-glucosidase and alpha-1,6-glucosidase. enzyme is provided as a purified or semi-purified enzyme 52. The transgenic plant of claim 47, wherein the transgenic preparation. plant is selected from the group consisting of maize, Sugar 58. The method of embodiment 55 or 56, wherein at least one beet, Sorghum and Sugarcane. of the key enzymes is provided as plant material derived from 53. A method for producing fermentable Sugar comprising: a plant expressing said key enzyme. 0326 a) providing transgenic plant material wherein 59. The method of embodiment 58, wherein at least one of the said transgenic plant material is selected from the group key enzymes is expressed in the same plant as the plant consisting of Sugar beet, Sorghum, maize, and Sugar comprising the locked carbohydrate. cane, and wherein said transgenic plant material com 60. The method of embodiment 55, wherein the locked car prises: bohydrate is selected from the group consisting of isomaltu 0327 i) one or more lock enzymes wherein said one lose, trehalulose, dextran, fructan, amylose, leucrose and or more lock enzymes is selected from the group alternan. consisting of dextranslucrase, levan Sucrose, alternan 61. The method of embodiment 56, wherein the transgenic Sucrase. Sucrose isomerase and amylosucrase, plant expresses at least two Sucrose isomerase enzymes, 0328 ii) one or more locked carbohydrates wherein wherein at least the first Sucrose isomerase enzyme catalyzes said one or more locked carbohydrates is selected the conversion of Sucrose primarily into isomalitulose, and from the group consisting of isomalitulose, trehalu wherein at least the second Sucrose isomerase enzyme cata lose, leucrose, starch, dextrans, fructans, maltose, lyzes the conversion of Sucrose primarily into trehalulose. turanose and isomaltose, 62. The method of embodiment 55, wherein said plant mate 0329 iii) one or more key enzymes wherein said one rial comprising the locked carbohydrate is derived from a or more key enzymes is selected from the group con plant selected from the group consisting of maize, wheat, rice, sisting of dextranase, alpha-amylase, glucoamylase, barley, soybean, cotton, Sorghum, oats, tobacco, Miscanthus alpha-1,5-glucosidase, alpha-1,1-glucosidase and grass, Switch grass, trees, beans, rape/canola, alfalfa, flax, alpha-1,6-glucosidase; and wherein said one or more Sunflower, safflower, millet, rye, Sugarcane, Sugar beet, key enzymes is targeted away from said one or more cocoa, tea, Brassica, cotton, coffee, Sweet potato, flax, pea locked carbohydrates; and nut, clover, vegetables such as lettuce, tomato, cucurbits, US 2011/020 1 059 A1 Aug. 18, 2011 44 cassava, potato, carrot, radish, pea, lentils, cabbage, cauli 0343 b) a nucleotide sequence encoding an enzyme flower, broccoli, Brussels sprouts, peppers, and pineapple; capable of converting the locked carbohydrate into a tree fruits such as citrus, apples, pears, peaches, apricots, fermentable Sugar. walnuts, avocado, banana, and coconut; and flowers such as 70. The plant of embodiment 69, wherein the locked carbo orchids, carnations and roses. hydrate is selected from the group consisting of isomalitulose, 63. The method of embodiment 62, wherein said plant mate rial comprising the locked carbohydrate is derived from Sug trehalulose, dextran, fructan, amylose, leucrose and alternan. arcane, Sugar beet, or Sweet Sorghum. 71. The plant of embodiment 70, wherein the transgenic plant 64. The method of embodiment 55, wherein the key enzyme expresses at least two Sucrose isomerase enzymes, wherein at is derived from a microorganism. least the first Sucrose isomerase enzyme catalyzes the conver 65. The method of embodiment 64, wherein the key enzyme sion of Sucrose primarily into isomaltulose, and wherein at is endogenous to said microorganism. least the second Sucrose isomerase enzyme catalyzes the con 66. The method of embodiment 64, wherein the key enzyme version of Sucrose primarily into trehalulose. is a recombinant enzyme expressed in the microorganism. 72. The transgenic plant of embodiment 69 selected from the 67. The method of embodiment 65, wherein the microorgan group consisting of maize, wheat, rice, barley, soybean, cot ism is a Saccharomyces strain capable of fermenting isoma ton, Sorghum, oats, tobacco, Miscanthus grass, Switch grass, litulose. 68. A method of selecting a transformed plant comprising: trees, beans, rape/canola, alfalfa, flax, Sunflower, safflower, 0337 a) introducing into said plant or part thereof: millet, rye, Sugarcane, Sugar beet, cocoa, tea, Brassica, cot 0338 i) an expression cassette comprising a nucle ton, coffee, Sweet potato, flax, peanut, clover, vegetables Such otide sequence encoding an enzyme capable of con as lettuce, tomato, cucurbits, cassava, potato, carrot, radish, Verting an endogenous Sugar in said plant to a locked pea, lentils, cabbage, cauliflower, broccoli, Brussels sprouts, carbohydrate; and, peppers, and pineapple; tree fruits such as citrus, apples, 0339 ii) an expression cassette comprising a nucle pears, peaches, apricots, walnuts, avocado, banana, and coco otide sequence encoding an enzyme capable of con nut; and flowers such as orchids, carnations and roses. verting the locked carbohydrate into a fermentable 73. The plant of embodiment 62, wherein said plant is sugar Sugar, cane, Sugar beet, or Sorghum. 0340 b) maintaining said plant or part thereof under 0344 All publications and patent applications mentioned conditions sufficient for the expression of the lock in the specification are indicative of the level of skill of those enzyme and the key enzyme; and, skilled in the art to which this invention pertains. All publi 0341 c) evaluating the sugar profile of said plant; cations and patent applications are herein incorporated by wherein the presence of one or more of the fermentable sugars reference to the same extent as if each individual publication produced by said key enzyme is indicative of a transformed or patent application was specifically and individually indi plant. cated to be incorporated by reference. 69. A transgenic plant useful for the production of ethanol, 0345 Although the foregoing invention has been wherein said plant comprises: described in some detail by way of illustration and example 0342 a) a nucleotide sequence encoding an enzyme for purposes of clarity of understanding, it will be obvious capable of converting an endogenous Sugar in said plant that certain changes and modifications may be practiced to said locked carbohydrate; and, within the scope of the appended claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 58

SEO ID NO 1 LENGTH: 562 TYPE PRT &213s ORGANISM: (Geo) Bacillus thermoglucosidasius KP1 OO6 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (562) <223> OTHER INFORMATION: alpha-1,6-glucosidase

<4 OOs SEQUENCE: 1

Met Glu Arg Val Trp Trp Llys Glu Ala Val Val Tyr Glin Ile Tyr Pro 1. 5 1O 15

Arg Ser Phe Tyr Asp Ser Asn Gly Asp Gly Ile Gly Asp Ile Arg Gly 2O 25 3 O

Ile Ile Ala Lys Lieu. Asp Tyr Lell Lys Glu Lieu. Gly Wall Asp Val Val 35 4 O 45

Trp Leu Ser Pro Val Tyr Lys Ser Pro Asn Asp Asp Asn Gly Tyr Asp US 2011/020 1 059 A1 Aug. 18, 2011 45

- Continued

SO 55 6 O

Ile Ser Asp Asp Ile Met Asp Glu Phe Gly Thir Met Ala Asp 65 70

Trp Thir Met Lell Glu Glu Met His Lys Arg Gly Ile Luell Wall 85 90 95

Met Asp Luell Wall Wall Asn His Thir Ser Asp Glu His Pro Trp Phe Ile 1OO 105 11 O

Glu Ser Arg Ser Asp Asn Pro Arg Asp Tyr Ile Trp 115 12 O 125

Arg Pro Gly Asn Gly Lys Glu Pro Asn ASn Trp Glu Ser Wall Phe 13 O 135 14 O

Ser Gly Ser Ala Trp Glu Asp Glu Met Thir Gly Glu Luell 145 150 155 160

His Luell Phe Ser Lys Glin Pro Asp Luell ASn Trp Glu Asn Pro Lys 1.65 17O 17s

Wall Arg Arg Glu Wall Glu Met Met Phe Trp Lell Asp Gly 18O 185 19 O

Wall Asp Gly Phe Met Asp Wall Ile Asn Met Ile Ser Wall Pro 195

Glu Luell Pro Asp Gly Glu Pro Glin Ser Gly Lys Ala Ser Gly 21 O 215 22O

Ser Arg Met Asn Gly Pro Arg Wall His Glu Phe Luell Glin Glu 225 23 O 235 24 O

Met Asn Arg Glu Wall Lell Ser Asp Ile Met Thir Wall Gly Glu 245 250 255

Thir Pro Gly Wall Thir Pro Glu Gly Ile Luell Thir Asp Pro Ser 26 O 265 27 O

Arg Arg Glu Luell Asn Met Wall Phe Glin Phe Glu His Met Asp Luell Asp 28O 285

Ser Gly Pro Gly Gly Trp Asp Ile Arg Pro Trp Ser Luell Ala Asp 29 O 295 3 OO

Lell Thir Met Thir Trp Glin Glu Lell Glu Gly Gly 3. OS 310 315

Trp Asn Ser Luell Tyr Lell Asn Asn His Asp Glin Pro Arg Ala Wall Ser 3.25 330 335

Arg Phe Gly Asp Asp Gly Arg Wall Glu Ser Ala Lys Met Luell 34 O 345 35. O

Ala Thir Phe Luell His Met Met Glin Gly Thir Pro Ile Glin Gly 355 360 365

Glu Glu Ile Gly Met Thir Asn Wall Arg Phe Pro Ser Ile Glu Asp Tyr 37 O 375 38O

Arg Asp Ile Glu Thir Lell Asn Met Glu Arg Wall Glu Glu Tyr 385 390 395 4 OO

Gly Glu Asp Pro Glin Glu Wall Met Glu Lys Ile Gly Arg 4 OS 41O 415

Asp Asn Ala Arg Thir Pro Met Glin Trp Asp Asp Ser Glu Asn Ala Gly 425 43 O

Phe Thir Ala Gly Thir Pro Trp Ile Pro Wall ASn Pro Asn Glu 435 44 O 445

Ile Asn Wall Lys Ala Ala Lell Glu Asp Pro ASn Ser Wall Phe His Tyr 450 45.5 460 US 2011/020 1 059 A1 Aug. 18, 2011 46

- Continued

Tyr Lys Llys Lieu. Ile Glin Lieu. Arg Lys Gln His Asp Ile Ile Val Tyr 465 470 47s 48O Gly. Thir Tyr Asp Lieu. Ile Lieu. Glu Asp Asp Pro Tyr Ile Tyr Arg Tyr 485 490 495 Thr Arg Thr Lieu. Gly Asn Glu Gln Lieu. Ile Val Ile Thr Asin Phe Ser SOO 505 51O Glu Lys Thr Pro Val Phe Arg Lieu Pro Asp His Ile Ile Tyr Lys Thr 515 52O 525 Lys Glu Lieu. Lieu. Ile Ser Asn Tyr Asp Wall Asp Glu Ala Glu Glu Lieu. 53 O 535 54 O Lys Glu Ile Arg Lieu. Arg Pro Trp Glu Ala Arg Val Tyr Lys Ile Arg 5.45 550 555 560

Leul Pro

<210s, SEQ ID NO 2 &211s LENGTH: 551 212. TYPE: PRT <213> ORGANISM: Erwinia rhapontici DSM 4484 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (551) <223> OTHER INFORMATION: alpha-1,6-glucosidase Genebank AAK28.737

<4 OOs, SEQUENCE: 2 Met Arg Ser Thr Pro His Trp Lys Glu Ala Val Val Tyr Glin Val Tyr 1. 5 1O 15 Pro Arg Ser Phe Met Asp Ser Asn Gly Asp Gly Thr Gly Asp Lieu. Asn 2O 25 3O Gly Ile Ile Ser Llys Lieu. Asp Tyr Lieu. Glin Glin Lieu. Gly Ile Thr Lieu. 35 4 O 45 Lieu. Trp Leu Ser Pro Val Tyr Arg Ser Pro Met Asp Asp Asin Gly Tyr SO 55 6 O Asp Ile Ser Asp Tyr Glu Glu Ile Ala Asp Ile Phe Gly Ser Met Ser 65 70 7s 8O Asp Met Glu Arg Lieu. Ile Ala Glu Ala Lys Ala Arg Asp Ile Gly Ile 85 90 95 Lieu Met Asp Leu Val Val Asn His Thr Ser Asp Glu. His Pro Trp Phe 1OO 105 11 O Ile Asp Ala Lieu. Ser Ser Lys Asn. Ser Ala Tyr Arg Asp Phe Tyr Ile 115 12 O 125 Trp Arg Ala Pro Ala Ala Asp Gly Gly Pro Pro Asp Asp Ser Arg Ser 13 O 135 14 O Asn Phe Gly Gly Ser Ala Trp Thir Lieu. Asp Glu Ala Ser Gly Glu Tyr 145 150 155 160 Tyr Lieu. His Glin Phe Ser Thr Arg Gln Pro Asp Lieu. Asn Trp Glu Asn 1.65 17O 17s Pro Arg Val Arg Glu Ala Ile His Ala Met Met Asn Arg Trp Lieu. Asp 18O 185 19 O Lys Gly Ile Gly Gly Phe Arg Met Asp Val Ile Asp Lieu. Ile Gly Lys 195 2OO 2O5 Glu Val Asp Pro Glin Ile Met Ala Asn Gly Arg His Pro His Lieu. Tyr 21 O 215 22O Lieu. Glin Gln Met Asn Arg Ala Thr Phe Gly Pro Arg Gly Ser Val Thr US 2011/020 1 059 A1 Aug. 18, 2011 47

- Continued

225 23 O 235 24 O Val Gly Glu Thir Trp Ser Ala Thr Pro Glu Asp Ala Lieu. Leu Tyr Ser 245 250 255 Ala Glu Glu Arg Glin Glu Arg Glin Glu Lieu. Thir Met Val Phe Glin Phe 26 O 265 27 O Glu. His Ile Llys Lieu. Phe Trp Asp Glu Glin Tyr Gly Llys Trp Cys Asn 27s 28O 285 Glin Pro Phe Asp Lieu. Lieu. Arg Phe Lys Ala Val Ile Asp Llys Trp Glin 29 O 295 3 OO Thir Ala Lieu Ala Asp His Gly Trp Asn. Ser Lieu. Phe Trp Ser Asn His 3. OS 310 315 32O Asp Lieu Pro Arg Ala Val Ser Llys Phe Gly Asp Asp Gly Glu Tyr Arg 3.25 330 335 Val Val Ser Ala Lys Met Lieu Ala Thir Ala Lieu. His Cys Lieu Lys Gly 34 O 345 35. O Thr Pro Tyr Ile Tyr Glin Gly Glu Glu Ile Gly Met Thr Asn Val Asn 355 360 365 Phe Ala Asp Ile Asp Asp Tyr Arg Asp Ile Glu Ser Lieu. Asn Lieu. Tyr 37 O 375 38O Glin Glu Arg Ile Ala Glu Gly Met Ser His Glu Ala Met Met Arg Gly 385 390 395 4 OO Ile His Ala Asn Gly Pro Asp Asn Ala Arg Thr Pro Met Gln Trp Thr 4 OS 41O 415 Ala Val His Met Pro Gly Lieu Pro Pro Val Ser Pro Gly Lieu. Arg Lieu. 42O 425 43 O Ile Lieu. Thir Ser Gly Glin Trp Asin Val Ala Ala Ala Lieu. Asp Asp Pro 435 44 O 445 Asp Ser Val Phe Tyr His Tyr Glin Llys Lieu Val Ala Lieu. Arg Lys Glin 450 45.5 460 Lieu Pro Lieu. Lieu Val His Gly Asp Phe Arg Glin Ile Val Val Glu. His 465 470 47s 48O Pro Glin Val Phe Ala Trp Lieu. Arg Thr Lieu. Gly Glu Gln Thr Lieu Val 485 490 495 Val Ile Asn. Asn. Phe Thr Arg Asp Ala Wal Met Lieu Ala Ile Pro Asp SOO 505 51O Asn Lieu. Glin Ser Glin Glin Gly Arg Cys Lieu. Ile Asn. Asn Tyr Ala Pro 515 52O 525 Arg Glu Gln Leu Glu Pro Ile Met Glu Lieu Gln Pro Tyr Glu Ser Phe 53 O 535 54 O Ala Lieu. Lieu. Ile Glu Arg Lieu 5.45 550

<210s, SEQ ID NO 3 &211s LENGTH: 564 212. TYPE: PRT <213> ORGANISM: Bacillus thuringiens is str. Al Hakam 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (564) <223> OTHER INFORMATION: alpha-1,6-glucosidase

<4 OOs, SEQUENCE: 3 Met Lys Trp Gly Ser Ile Met Glu Lys Glin Trp Trp Lys Glu Ser Val 1. 5 1O 15 US 2011/020 1 059 A1 Aug. 18, 2011 48

- Continued

Val Tyr Glin Ile Tyr Pro Arg Ser Phe Met Asp Ser Asn Gly Asp Gly 2O 25 3O Ile Gly Asp Lieu. Arg Gly Ile Ile Ser Lys Lieu. Asp Tyr Lieu Lys Glu 35 4 O 45 Lieu. Gly Ile Asp Val Ile Trp Leu Ser Pro Val Tyr Glu Ser Pro Asn SO 55 6 O Asp Asp Asin Gly Tyr Asp Ile Ser Asp Tyr Cys Lys Ile Met Asn. Glu 65 70 7s 8O Phe Gly Thr Met Glu Asp Trp Asp Glu Lieu. Lieu. His Glu Met His Glu 85 90 95 Arg Asn Met Lys Lieu Met Met Asp Lieu Val Val Asn His Thir Ser Asp 1OO 105 11 O Glu. His Asn Trp Phe Ile Glu Ser Arg Llys Ser Lys Asp Asn Llys Tyr 115 12 O 125 Arg Asp Tyr Tyr Ile Trp Arg Pro Gly Lys Glu Gly Lys Glu Pro Asn 13 O 135 14 O Asn Trp Gly Ala Ala Phe Ser Gly Ser Ala Trp Glin Tyr Asp Glu Met 145 150 155 160 Thir Asp Glu Tyr Tyr Lieu. His Lieu. Phe Ser Lys Lys Glin Pro Asp Lieu. 1.65 17O 17s Asn Trp Asp Asn. Glu Lys Val Arg Glin Asp Val Tyr Glu Met Met Lys 18O 185 19 O Phe Trp Lieu. Glu Lys Gly Ile Asp Gly Phe Arg Met Asp Val Ile Asn 195 2OO 2O5 Phe Ile Ser Lys Glu Glu Gly Lieu Pro Thr Val Glu Thr Glu Glu Glu 21 O 215 22O Gly Tyr Val Ser Gly His Llys His Phe Met Asn Gly Pro Asn Ile His 225 23 O 235 24 O Llys Tyr Lieu. His Glu Met Asn. Glu Glu Val Lieu. Ser His Tyr Asp Ile 245 250 255 Met Thr Val Gly Glu Met Pro Gly Val Thir Thr Glu Glu Ala Lys Lieu. 26 O 265 27 O Tyr Thr Gly Glu Glu Arg Lys Glu Lieu Gln Met Val Phe Glin Phe Glu 27s 28O 285 His Met Asp Lieu. Asp Ser Gly Glu Gly Gly Lys Trp Asp Wall Lys Pro 29 O 295 3 OO Cys Ser Lieu. Lieu. Thir Lieu Lys Glu Asn Lieu. Thir Lys Trp Gln Lys Ala 3. OS 310 315 32O Lieu. Glu. His Thr Gly Trp Asn. Ser Lieu. Tyr Trp Asn. Asn His Asp Glin 3.25 330 335 Pro Arg Val Val Ser Arg Phe Gly Asn Asp Gly Met Tyr Arg Ile Glu 34 O 345 35. O Ser Ala Lys Met Leu Ala Thr Val Lieu. His Met Met Lys Gly Thr Pro 355 360 365 Tyr Ile Tyr Glin Gly Glu Glu Ile Gly Met Thr Asn Val Arg Phe Glu 37 O 375 38O Ser Ile Asp Glu Tyr Arg Asp Ile Glu Thir Lieu. Asn Met Tyr Lys Glu 385 390 395 4 OO Llys Val Met Glu Arg Gly Glu Asp Ile Glu Lys Val Met Glin Ser Ile 4 OS 41O 415 US 2011/020 1 059 A1 Aug. 18, 2011 49

- Continued Tyr Ile Lys Gly Arg Asp Asn Ala Arg Thr Pro Met Gln Trp Asp Asp 42O 425 43 O Gln Asn His Ala Gly Phe Thir Thr Gly Glu Pro Trp Ile Thr Val Asn 435 44 O 445 Pro Asn Tyr Lys Glu Ile Asn. Wall Lys Glin Ala Ile Glin Asn Lys Asp 450 45.5 460 Ser Ile Phe Tyr Tyr Tyr Llys Llys Lieu. Ile Glu Lieu. Arg Lys Asn. Asn 465 470 47s 48O Glu Ile Val Val Tyr Gly Ser Tyr Asp Lieu. Ile Lieu. Glu Asn. Asn Pro 485 490 495 Ser Ile Phe Ala Tyr Val Arg Thr Tyr Gly Val Glu Lys Lieu. Leu Val SOO 505 51O Ile Ala Asn. Phe Thr Ala Glu Glu. Cys Ile Phe Glu Lieu Pro Glu Asp 515 52O 525 Ile Ser Tyr Ser Glu Val Glu Lieu. Lieu. Ile His Asn Tyr Asp Val Glu 53 O 535 54 O Asn Gly Pro Ile Glu Asn Ile Thr Lieu. Arg Pro Tyr Glu Ala Met Val 5.45 550 555 560 Phe Llys Lieu Lys

<210s, SEQ ID NO 4 &211s LENGTH: 576 212. TYPE: PRT <213> ORGANISM: Arthrobacter globiformis 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (576.) <223> OTHER INFORMATION: alpha-1,6-glucosidase Genebank AB113246

<4 OOs, SEQUENCE: 4 Met Thr Ile Glu Glu Thr Glu Glu Glu Ala Thr Tyr Arg Ala Gly Arg 1. 5 1O 15 Glu Trp Phe Llys Ser Ala Val Val Tyr Glin Ile Tyr Pro Arg Ser Phe 2O 25 3O Ala Asp Ser Asp Gly Asp Gly Val Gly Asp Lieu. Arg Gly Ile Ile Gly 35 4 O 45 Llys Lieu. Asp Tyr Lieu. Glin Llys Lieu. Gly Val Asp Val Val Trp Lieu. Ser SO 55 6 O Pro Val Tyr Arg Ser Pro Glin Asp Asp Asn Gly Tyr Asp Ile Ser Asp 65 70 7s 8O Tyr Arg Glu Ile Asp Pro Val Phe Gly Gly Lieu. Glu Thir Lieu. Asp Glu 85 90 95 Lieu. Lieu. Asp Gly Lieu. His Ala Arg Gly Met Lys Lieu Val Met Asp Lieu. 1OO 105 11 O Val Val Asn His Thr Ser Asp Glu. His Pro Trp Phe Val Glu Ser Arg 115 12 O 125 Ser Ser Lys Asp Ser Pro Lys Arg Asp Trp Tyr Trp Trp Arg Pro Ala 13 O 135 14 O Arg Glu Gly Ala Glu Pro Gly Thr Ala Gly Ala Glu Pro Asn. Asn Trp 145 150 155 160 Gly Ser Ala Phe Ser Gly Pro Ala Trp Glu Tyr Asp Ala Ala Thr Gly 1.65 17O 17s Glu Tyr Tyr Lieu. His Lieu. Phe Ser Arg Lys Glin Pro Asp Lieu. Asn Trp 18O 185 19 O US 2011/020 1 059 A1 Aug. 18, 2011 50

- Continued

Glu Asn Pro Glu Val Arg Ala Ala Val Tyr Asp Met Met Asn Trp Trp 195 2OO 2O5 Lieu. Asp Arg Gly Val Asp Gly Phe Arg Met Asp Val Ile Asin Phe Ile 21 O 215 22O Ser Lys Asp Glin Thr Lieu Pro Asp Gly Pro Arg Ala Asp Gly Met Lieu. 225 23 O 235 24 O Phe Gly Asp Gly Gly Pro His Tyr Ile Cys Gly Pro Arg Ile His Glu 245 250 255 Phe Lieu. Glin Glu Met His Glin Glu Val Phe Ala Gly Arg Asp Lys Asp 26 O 265 27 O Lieu. Lieu. Thr Val Gly Glu Met Pro Gly Val Thr Val Asp Glu Ala Val 27s 28O 285 Lieu. Phe Thr Asp Pro Gly Arg Arg Glu Val Asp Met Val Phe Glin Phe 29 O 295 3 OO Glu. His Val Ala Lieu. Asp Glin Glu Gly Gly Asn Llys Trp Arg Pro Llys 3. OS 310 315 32O Llys Lieu. Lieu. Lieu. Thir Asp Lieu Lys Llys Ser Lieu. Gly Arg Trp Glin Glu 3.25 330 335 Ala Lieu. Gly Glu Arg Gly Trp Asn. Ser Lieu. Tyr Trp Gly Asn His Asp 34 O 345 35. O Glin Ala Arg Ala Val Ser Arg Phe Gly Asp Asp Gly Glu Tyr Arg Glu 355 360 365 Glin Ser Ala Lys Met Lieu Ala Ala Val Lieu. His Lieu. His Arg Gly. Thir 37 O 375 38O Pro Tyr Val Tyr Glin Gly Glu Glu Lieu. Gly Met Thr Asn Met Ala Phe 385 390 395 4 OO Gly Ala Ile Ser Asp Tyr Arg Asp Ile Glu Val Lieu. Asn His His Arg 4 OS 41O 415 Glu Ala Thir Thr His Lieu. Gly His Thr Asp Ala Glu Val Lieu Ala Ala 42O 425 43 O Lieu Ala Pro Lieu. Asn Arg Asp Asn Ala Arg Thr Pro Val Glin Trp Asp 435 44 O 445 Ala Ser Arg His Gly Gly Phe Thr Thr Gly Ala Pro Trp Ile Ala Val 450 45.5 460 Asn Pro Asn Ala Asn. Thir Ile Asn Ala Ala Ala Glin Val Asp Asp Pro 465 470 47s 48O Asp Ser Val Phe Ser Phe Tyr Arg Arg Val Ile Ala Lieu. Arg His Ala 485 490 495 Asp Pro Val Val Ala Tyr Gly Asp Phe Thr Met Lieu Lleu Pro Asp Asp SOO 505 51O Glu. His Val Tyr Ala Phe Arg Arg Ser Lieu Pro Asp Ala Glu Lieu. Lieu. 515 52O 525 Val Lieu. Gly Asn. Phe Ser Gly Thr Gly Glin Ser Ala Gly Val Asp Gly 53 O 535 54 O Ser Trp Gly Asp Ala Glu Lieu Val Lieu. Gly Asn Tyr Pro Ala Ala Pro 5.45 550 555 560 Gly Lieu. Gly Lieu. Arg Pro Trp Glu Val Llys Val Phe Arg Arg Asn Lieu. 565 st O sts

<210s, SEQ ID NO 5 211 LENGTH: 787 US 2011/020 1 059 A1 Aug. 18, 2011 51

- Continued

212. TYPE: PRT <213> ORGANISM: Bacillus thermoamyloliquefaciens 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (787) <223> OTHER INFORMATION: alpha-1,6-glucosidase Q9F234 <4 OOs, SEQUENCE: 5 Met Lieu. Glu Asp Thir Ser Phe Ala Ile Glin Pro Glu Glin Asp Asp Llys 1. 5 1O 15 Thr Glin Glu Thr His Arg Ile Asp Ile Gly Asn Met His Thr Phe Ser 2O 25 3O His Thr Glu. His Val Phe Ser Phe His Cys Asp Thr Gly Ile Val Lys 35 4 O 45 Ile Arg Phe Tyr Arg Glu Asp Ile Val Arg Ile Ala Phe ASn Pro Phe SO 55 6 O Gly Glu Thir Ser Leu Ser Thr Ser Val Ala Val Val Lys Glu Pro Glu 65 70 7s 8O Llys Val Asp Ala Ser Val His Glu Thr Glu Glu Glu Val Thr Lieu. Thr 85 90 95 Ser Ala Lys Glin Thr Val Val Lieu. Glin Lys Arg Pro Phe Arg Val Arg 1OO 105 11 O Ile Tyr Asp Asn His Gly Arg Lieu. Lieu Val Ala Glu Gly Lys Lys Gly 115 12 O 125 Met Ala Phe Thr Tyr Glin Gly Glu Val Cys Cys Phe Lys Met Met Asp 13 O 135 14 O Glu Ala Asp His Phe Tyr Gly Phe Gly Glu Lys Thr Gly Phe Lieu. Asp 145 150 155 160 Lys Arg Gly Glu Thr Met Thr Met Trp Asn Thr Asp Val Tyr Ala Pro 1.65 17O 17s His Asn Pro Glu Thr Asp Pro Leu Tyr Glin Ser His Pro Tyr Phe Met 18O 185 19 O Thr Val Arg Asn Gly Ser Ala His Gly Ile Phe Phe Asp Asn Thr Tyr 195 2OO 2O5 Lys. Thir Thr Phe Asp Phe Glin Thr Ala Thr Asp Glu Tyr Cys Phe Ser 21 O 215 22O Ala Glu Gly Gly Ala Ile Asp Tyr Tyr Val Phe Ala Gly Pro Thr Pro 225 23 O 235 24 O Lys Asp Val Lieu. Glu Glin Tyr Thr Asp Lieu. Thr Gly Arg Met Pro Lieu. 245 250 255 Pro Pro Llys Trp Ala Lieu. Gly Tyr His Glin Ser Arg Tyr Ser Tyr Glu 26 O 265 27 O Thr Glu Glin Glu Val Arg Glu Ile Ala Glin Thr Phe Ile Glu Lys Asp 27s 28O 285 Ile Pro Leu Asp Val Ile Tyr Lieu. Asp Ile His Tyr Met Asn Gly Tyr 29 O 295 3 OO Arg Val Phe Thr Phe Asp Arg Asn Arg Phe Pro Asn Lieu Lys Glin Lieu 3. OS 310 315 32O Ile Ala Asp Lieu Lys Glin Lys Gly Ile Arg Val Val Pro Ile Val Asp 3.25 330 335 Pro Gly Val Lys Glu Asp Pro Glu Tyr Val Ile Tyr Glin Glu Gly Ile 34 O 345 35. O Arg His Asp Tyr Phe Cys Llys Tyr Ile Glu Gly Asn Val Tyr Phe Gly US 2011/020 1 059 A1 Aug. 18, 2011 52

- Continued

355 360 365 Glu Val Trp Pro Gly Lys Ser Ala Phe Pro Asp Phe Thr Asn Llys Lys 37 O 375 38O Val Arg Llys Trp Trp Gly Glu Lys His Glin Phe Tyr Thr Asp Lieu. Gly 385 390 395 4 OO Ile Glu Gly Ile Trp Asn Asp Met Asn Glu Pro Ser Val Phe Asin Glu 4 OS 41O 415 Thir Lys Thr Met Asp Val Llys Val Ile His Asp Asn Asp Gly Asp Pro 42O 425 43 O Lys Thr His Arg Glu Lieu. His Asn Val Tyr Gly Phe Met Met Gly Glu 435 44 O 445 Ala Thr Tyr Lys Gly Met Lys Llys Lieu. Lieu. Asn Gly Lys Arg Pro Phe 450 45.5 460 Lieu. Lieu. Thir Arg Ala Gly Phe Ser Gly Ile Glin Arg Tyr Ala Ala Val 465 470 47s 48O Trp Thr Gly Asp Asn Arg Ser Phe Trp Glu. His Leu Gln Met Ser Lieu. 485 490 495 Pro Met Cys Met Asn Lieu. Gly Lieu Ser Gly Val Ala Phe Cys Gly Pro SOO 505 51O Asp Val Gly Gly Phe Ala His Asn. Thir Asn Gly Glu Lieu. Lieu. Thir Arg 515 52O 525 Trp Met Glin Val Gly Ala Phe Thr Pro Tyr Phe Arg Asn His Cys Ala 53 O 535 54 O Ile Gly Phe Arg Arg Glin Glu Pro Trp Ala Phe Gly Glu Lys Tyr Glu 5.45 550 555 560 Arg Ile Ile Llys Llys Tyr Ile Arg Lieu. Arg Tyr Glin Trp Lieu Pro His 565 st O sts Lieu. Tyr Thr Lieu Phe Ala Glu Ala His Glu Thr Gly Ala Pro Val Met 58O 585 59 O Arg Pro Leu Phe Phe Glu Tyr Pro Asp Asp Glu Asn Thr Tyr Asn Lieu. 595 6OO 605 Tyr Asp Glu Phe Lieu Val Gly Ala Asn Val Lieu. Ile Ala Pro Ile Met 610 615 62O Thr Pro Ser Thr Thr Arg Arg Val Ala Tyr Phe Pro Lys Gly Asn Trp 625 630 635 64 O Val Asp Tyr Trp Thr Gly Glu Val Lieu. Glu Gly Gly Glin Tyr His Leu 645 650 655 Ile Ser Ala Asp Lieu. Glu Thir Lieu Pro Ile Phe Ile Lys Glin Gly Ser 660 665 67 O Ala Ile Ala Lieu. Gly Asp Wall Lys Arg Ser Thr Glu Met Pro Asp Glu 675 68O 685 His Arg Thr Val His Ile Tyr Lys Ala Asn Gly Gly Lys Ala Thr Tyr 69 O. 695 7 OO Val Lieu. Tyr Asp Asp Asp Gly Glin Thr Phe Ser Tyr Glu Lys Gly Asp 7 Os 71O 71s 72O Tyr Lieu. Arg Met Tyr Ile Glu Val Glu Tyr Gly Glu Asn Ser Val His 72 73 O 73 Ile Val Thr Lys Ser Glu Gly Thr Tyr Glin Pro Ser Trp Llys Lieu Ser 740 74. 7 O Phe Ala Ile His His Ala Thr Glu Gln Thr Llys Val Thr Ile Asp Gly 7ss 760 765 US 2011/020 1 059 A1 Aug. 18, 2011 53

- Continued

Asn Glu Glin Asn Ala Ile Phe Asp Pro His Glin Arg Ile Lieu. Lieu. Ile 770 775 78O

Glin Ser Glu 78s

<210s, SEQ ID NO 6 &211s LENGTH: 752 212. TYPE: PRT <213> ORGANISM: Thermoanaerobacter ethanolicus 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (752) <223> OTHER INFORMATION: alpha-1,6-glucosidase ABR2623 O <4 OOs, SEQUENCE: 6 Met Tyr Glin Llys Thir Ser Glu Lys Ile Val Val Arg Asn. Glu Gly Lys 1. 5 1O 15 Llys Lieu. Glu Lieu. Arg Val Lieu. Gly Asp Llys Ile Ile Asn Val Phe Val 2O 25 3O Ser Asn Lys Glu Glu Lys Arg Lys Asp Thir Ile Ala Ile Glu Arg Llys 35 4 O 45 Glu Tyr Asp Thr Pro Glu Phe Ser Ile Ser Asp Glu Lieu. Glu Ser Ile SO 55 6 O Lieu. Ile Glu Thir Asn. Ser Lieu Lys Val Lys Ile Asn Lys Asn Asp Lieu. 65 70 7s 8O Ser Val Ser Phe Lieu. Asp Lys Asn Gly Asn. Ile Ile Asn. Glu Asp Tyr 85 90 95 Asn Gly Gly Ala Lys Phe Asn. Glu Thir Asp Val Arg Cys Tyr Lys Llys 1OO 105 11 O Lieu. Arg Glu Asp His Phe Tyr Gly Phe Gly Glu Lys Ala Gly Tyr Lieu. 115 12 O 125 Asp Llys Lys Gly Glu Arg Lieu. Glu Met Trp Asn. Thir Asp Glu Phe Met 13 O 135 14 O Thr His Asn Gln Thr Thr Lys Lieu. Leu Tyr Glu Ser Tyr Pro Phe Phe 145 150 155 160 Ile Gly Met Asn Asp Tyr His Thr Tyr Gly Ile Phe Lieu. Asp Asn Ser 1.65 70 17s Phe Arg Ser Phe Phe Asp Met Gly Glin Glu Ser Glin Glu Tyr Tyr Phe 18O 185 19 O Phe Gly Ala Tyr Gly Gly Gln Met Asn Tyr Tyr Phe Ile Tyr Gly Glu 195 2OO 2O5 Asp Ile Lys Glu Val Val Glu Asn Tyr Thr Tyr Lieu. Thr Gly Arg Ile 21 O 215 22O Ser Leu Pro Pro Leu Trp Val Lieu. Gly Asin Glin Glin Ser Arg Tyr Ser 225 23 O 235 24 O Tyr Thr Pro Glin Glu Arg Val Lieu. Glu Val Ala Lys Thr Phe Arg Glu 245 250 255 Lys Asp Ile Pro Cys Asp Val Ile Tyr Lieu. Asp Ile Asp Tyr Met Glu 26 O 265 27 O Gly Tyr Arg Val Phe Thr Trp Asn Lys Glu Thir Phe Lys Asn His Lys 27s 28O 285 Glu Met Leu Lys Gln Leu Lys Glu Met Gly Phe Llys Val Val Thir Ile 29 O 295 3 OO US 2011/020 1 059 A1 Aug. 18, 2011 54

- Continued Val Asp Pro Gly Val Lys Arg Asp Tyr Asp Tyr His Val Tyr Arg Glu 3. OS 310 315 32O Gly Ile Glu Lys Gly Tyr Phe Val Lys Asp Llys Tyr Gly Ile Thr Tyr 3.25 330 335 Val Gly Llys Val Trp Pro Gly Glu Ala Cys Phe Pro Asp Phe Leu Gln 34 O 345 35. O Glu Glu Val Arg Tyr Trp Trp Gly Glu Lys His Arg Glu Phe Ile Asn 355 360 365 Asp Gly Ile Asp Gly Ile Trp Asn Asp Met Asn. Glu Pro Ala Val Phe 37 O 375 38O Glu Thr Pro Thr Lys Thr Met Pro Glu Asp Asn Ile His Ile Leu Asp 385 390 395 4 OO Gly Glu Lys Val Lieu. His Lys Glu Ala His Asn Val Tyr Ala Asn Tyr 4 OS 41O 415 Met Ala Met Ala Thr Arg Asp Gly Phe Lieu. Arg Ile Arg Pro Asn. Glu 42O 425 43 O Arg Pro Phe Val Lieu. Thr Arg Ala Ala Phe Ser Gly Ile Glin Arg Tyr 435 44 O 445 Ala Ala Met Trp Thr Gly Asp Asn Arg Ser Lieu. Tyr Glu. His Lieu. Lieu 450 45.5 460 Met Met Met Pro Met Leu Met Asn Ile Gly Leu Ser Gly Glin Pro Phe 465 470 47s 48O Val Gly Ala Asp Val Gly Gly Phe Glu Gly Asp Cys His Glu Glu Lieu 485 490 495 Phe Ile Arg Trp Ile Glu Ala Ala Val Phe Thr Pro Phe Lieu. Arg Val SOO 505 51O His Ser Ala Ile Gly Thr Lys Asp Glin Glu Pro Trp Ser Phe Gly Lys 515 52O 525 Arg Ala Glu Asp Ile Ser Arg Llys Tyr Ile Llys Met Arg Tyr Glu Lieu 53 O 535 54 O Lieu Pro Tyr Lieu. Tyr Asp Leu Phe Tyr Ile Ala Ser Gln Lys Gly Tyr 5.45 550 555 560 Pro Ile Met Arg Pro Leu Val Phe Glu Tyr Gln Lys Asp Glu Asn Thr 565 st O sts His Lys Ile Tyr Asp Glu Phe Met Phe Gly Glu Gly Lieu. Lieu Val Ala 58O 585 59 O Pro Val Tyr Lieu Pro Ser Lys Glu Arg Arg Glu Val Tyr Lieu. Pro Glu 595 6OO 605 Gly Ile Trp Tyr Asp Tyr Trp Thr Gly Lys Gly Phe Lys Gly Lys Asn 610 615 62O Tyr Tyr Lieu Val Asp Ala Pro Ile Glu Val Ile Pro Leu Phe Val Lys 625 630 635 64 O Glu Gly Gly Ile Lieu Lleu Lys Glin Glin Pro Glin Ser Phe Ile Gly Glu 645 650 655 Llys Llys Lieu. Glu Glu Lieu. Thr Val Glu Ile Tyr Lys Gly Lys Glu Gly 660 665 67 O His Tyr Lieu. His Tyr Glu Asp Asp Gly Lys Ser Phe Asp Tyr Thr Lys 675 68O 685 Gly Val Tyr Asn Lieu. Phe Asp Ile Ser Phe Cys Tyr Lys Glu Gly Arg 69 O. 695 7 OO Met Asp Ile Llys Phe Asp Llys Ile His Phe Gly Tyr Asp Llys Gly Val US 2011/020 1 059 A1 Aug. 18, 2011 55

- Continued

Llys Llys Tyr Llys Phe Ile Phe Lys Asn. Phe Asp Asp Ile Lys Glu Ile 72 73 O 73 Lys Ile Asin Gly Glu Lys Val Glu Lys Glu Ser Cys Glu Ile Glu Lieu. 740 74. 7 O

<210s, SEQ ID NO 7 &211s LENGTH: 1717 &212s. TYPE: DNA <213> ORGANISM: Arabidopsis 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (1) . . (1717 <223> OTHER INFORMATION: UBQ promoter <4 OO > SEQUENCE: 7 ggagcCaagt ct cataaacg C cattgttgga agaaagt citt gagttggtgg taatgitalaca 6 O gagtag taag alacagagaag agagagagtg tagata cat gaattgtcgg gcaacaaaaa 12 O t cctgaac at Ctt attittag caaagaga aa gagttc.cgag tictgtag cag aagagtgagg 18O agaaatttaa gct cittggac ttgttgaattig titcc.gc.ctct togaat acttic ttcaatcctic 24 O atatatt citt cittctatott acctgaaaac cqgcatttaa tot cqcgggit ttatt coggit 3OO t caa.catttt ttttgttittg agittattatc tdggcttaat aacgcaggcc tdaaataaat 360 tdaaggcc.ca actgttttitt tttittaagaa gttgctgtta aaaaaaaaaa aagggaatta 42O acaacaacaa caaaaaaaga taaagaaaat aataacaatt actittaattig tag actaaaa 48O aaacatagat tittat catga aaaaaagaga aaagaaataa aaacttggat caaaaaaaaa 54 O acatacagat cittctaatta tta acttitt c ttaaaaatta ggit cotttitt cocaacaatt 6OO aggtttagag titttggaatt aaaccaaaaa gattgttcta aaaaatactic aaatttggta 660 gataagtttc cittattittaa ttagt caatig gtagatactt ttttitt ctitt tottt attag 72 O agtagattag aatcttittat gccaagtatt gataaattaa atcaagaaga taalactato a 78O taatcaa.cat gaaattaaaa gaaaaatcto atatatagta ttag tatt ct ctatatatat 84 O tatgattgct tatt cittaat giggttgggitt aaccalagaca tagt cittaat ggaaagaatc 9 OO tttitttgaac tttitt.cctta ttgattaa at t cittctatag aaaagaaaga aattatttga 96.O ggaaaagt at atacaaaaag aaaaatagala aaatgtcagt galagcagatg taatggatga O2O cctaatccaa ccaccaccat aggatgtttc tacttgagtic ggit cittittaa aaacgcacgg O8O tggaaaatat gacacgitatic atatgattico titcctittagt titcgtgataa taatcct caa 14 O citgatat citt cottttitttgttittggctaa agatattitta ttct cattaa tagaaaagac 2OO ggttittgggc titttggitttg catataaag aagacct tcg tdtggaagat aataatt cat 26 O c ctitt.cgt.ct ttittctgact cittcaatc to tcc caaagcc taaag.cgatc. tctgcaaatc 32O t citcgcgact citct ctitt ca aggtatattt totgatt citt tttgtttittg attcgitat ct 38O gatcto caat ttttgttatg toggattattgaat ctitttgt ataaattgct tittgacaata 44 O ttgttcgttt cqt caatcca gctitctaaat tttgtcc tiga t tact aagat atcgatt cqt SOO agtgtttaca totgtgtaat ttcttgcttg attgttgaaat taggattitt c aaggacgatc 560 tatt caattt ttgttgttitt c tttgttcg at t ct citctgtt ttaggitttct tatgtttaga 62O tcc.gtttctic tittggtgttg ttittgatttic ticttacggct tittgatttgg tatatgttcg 68O

US 2011/020 1 059 A1 Aug. 18, 2011 60

- Continued

<210s, SEQ ID NO 12 &211s LENGTH: 752 212. TYPE: PRT <213> ORGANISM: T. ethanolicus

<4 OOs, SEQUENCE: 12 Met Tyr Glin Llys Thir Ser Glu Lys Ile Val Val Arg Asn. Glu Gly Lys 1. 5 1O 15 Llys Lieu. Glu Lieu. Arg Val Lieu. Gly Asp Llys Ile Ile Asn Val Phe Val 2O 25 3O Ser Asn Lys Glu Glu Lys Arg Lys Asp Thir Ile Ala Ile Glu Arg Llys 35 4 O 45 Glu Tyr Asp Thr Pro Glu Phe Ser Ile Ser Asp Glu Lieu. Glu Ser Ile SO 55 6 O Lieu. Ile Glu Thir Asn. Ser Lieu Lys Val Lys Ile Asn Lys Asn Asp Lieu. 65 70 7s 8O Ser Val Ser Phe Lieu. Asp Lys Asn Gly Asn. Ile Ile Asn. Glu Asp Tyr 85 90 95 Asn Gly Gly Ala Lys Phe Asn. Glu Thir Asp Val Arg Cys Tyr Lys Llys 1OO 105 11 O Lieu. Arg Glu Asp His Phe Tyr Gly Phe Gly Glu Lys Ala Gly Tyr Lieu. 115 12 O 125 Asp Llys Lys Gly Glu Arg Lieu. Glu Met Trp Asn. Thir Asp Glu Phe Met 13 O 135 14 O Thr His Asn Gln Thr Thr Lys Lieu. Leu Tyr Glu Ser Tyr Pro Phe Phe 145 150 155 160 Ile Gly Met Asn Asp Tyr His Thr Tyr Gly Ile Phe Lieu. Asp Asn Ser 1.65 17O 17s Phe Arg Ser Phe Phe Asp Met Gly Glin Glu Ser Glin Glu Tyr Tyr Phe 18O 185 19 O Phe Gly Ala Tyr Gly Gly Gln Met Asn Tyr Tyr Phe Ile Tyr Gly Glu 195 2OO 2O5 Asp Ile Lys Glu Val Val Glu Asn Tyr Thr Tyr Lieu. Thr Gly Arg Ile 21 O 215 22O Ser Leu Pro Pro Leu Trp Val Lieu. Gly Asin Glin Glin Ser Arg Tyr Ser 225 23 O 235 24 O Tyr Thr Pro Glin Glu Arg Val Lieu. Glu Val Ala Lys Thr Phe Arg Glu 245 250 255 Lys Asp Ile Pro Cys Asp Val Ile Tyr Lieu. Asp Ile Asp Tyr Met Glu 26 O 265 27 O Gly Tyr Arg Val Phe Thr Trp Asn Lys Glu Thir Phe Lys Asn His Lys 27s 28O 285 Glu Met Leu Lys Gln Leu Lys Glu Met Gly Phe Llys Val Val Thir Ile 29 O 295 3 OO Val Asp Pro Gly Val Lys Arg Asp Tyr Asp Tyr His Val Tyr Arg Glu 3. OS 310 315 32O Gly Ile Glu Lys Gly Tyr Phe Val Lys Asp Llys Tyr Gly Ile Thr Tyr 3.25 330 335 Val Gly Llys Val Trp Pro Gly Glu Ala Cys Phe Pro Asp Phe Leu Gln 34 O 345 35. O Glu Glu Val Arg Tyr Trp Trp Gly Glu Lys His Arg Glu Phe Ile Asn US 2011/020 1 059 A1 Aug. 18, 2011 61

- Continued

355 360 365 Asp Gly Ile Asp Gly Ile Trp Asn Asp Met Asn. Glu Pro Ala Val Phe 37 O 375 38O Glu Thr Pro Thr Lys Thr Met Pro Glu Asp Asn Ile His Ile Leu Asp 385 390 395 4 OO Gly Glu Lys Val Lieu. His Lys Glu Ala His Asn Val Tyr Ala Asn Tyr 4 OS 41O 415 Met Ala Met Ala Thr Arg Asp Gly Phe Lieu. Arg Ile Arg Pro Asn. Glu 42O 425 43 O Arg Pro Phe Val Lieu. Thr Arg Ala Ala Phe Ser Gly Ile Glin Arg Tyr 435 44 O 445 Ala Ala Met Trp Thr Gly Asp Asn Arg Ser Lieu. Tyr Glu. His Lieu. Lieu 450 45.5 460 Met Met Met Pro Met Leu Met Asn Ile Gly Leu Ser Gly Glin Pro Phe 465 470 47s 48O Val Gly Ala Asp Val Gly Gly Phe Glu Gly Asp Cys His Glu Glu Lieu 485 490 495 Phe Ile Arg Trp Ile Glu Ala Ala Val Phe Thr Pro Phe Lieu. Arg Val SOO 505 51O His Ser Ala Ile Gly Thr Lys Asp Glin Glu Pro Trp Ser Phe Gly Lys 515 52O 525 Arg Ala Glu Asp Ile Ser Arg Llys Tyr Ile Llys Met Arg Tyr Glu Lieu. 53 O 535 54 O Lieu Pro Tyr Lieu. Tyr Asp Leu Phe Tyr Ile Ala Ser Gln Lys Gly Tyr 5.45 550 555 560 Pro Ile Met Arg Pro Leu Val Phe Glu Tyr Gln Lys Asp Glu Asn Thr 565 st O sts His Lys Ile Tyr Asp Glu Phe Met Phe Gly Glu Gly Lieu. Lieu Val Ala 58O 585 59 O Pro Val Tyr Lieu Pro Ser Lys Glu Arg Arg Glu Val Tyr Lieu. Pro Glu 595 6OO 605 Gly Ile Trp Tyr Asp Tyr Trp Thr Gly Lys Gly Phe Lys Gly Lys Asn 610 615 62O Tyr Tyr Lieu Val Asp Ala Pro Ile Glu Val Ile Pro Leu Phe Val Lys 625 630 635 64 O Glu Gly Gly Ile Lieu Lleu Lys Glin Glin Pro Glin Ser Phe Ile Gly Glu 645 650 655 Llys Llys Lieu. Glu Glu Lieu. Thr Val Glu Ile Tyr Lys Gly Lys Glu Gly 660 665 67 O His Tyr Lieu. His Tyr Glu Asp Asp Gly Lys Ser Phe Asp Tyr Thr Lys 675 68O 685 Gly Val Tyr Asn Lieu. Phe Asp Ile Ser Phe Cys Tyr Lys Glu Gly Arg 69 O. 695 7 OO Met Asp Ile Llys Phe Asp Llys Ile His Phe Gly Tyr Asp Llys Gly Val 7 Os 71O 71s 72O Llys Llys Tyr Llys Phe Ile Phe Lys Asn. Phe Asp Asp Ile Lys Glu Ile 72 73 O 73 Lys Ile Asin Gly Glu Lys Val Glu Lys Glu Ser Cys Glu Ile Glu Lieu. 740 74. 7 O

<210s, SEQ ID NO 13 US 2011/020 1 059 A1 Aug. 18, 2011 62

- Continued

&211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM; soybean 22 Os. FEATURE: <221> NAME/KEY: targeting <222s. LOCATION: (1) . . (19 <223> OTHER INFORMATION: ER targeting sequence

<4 OOs, SEQUENCE: 13 Met Ala Lys Lieu Val Phe Ser Lieu. Cys Phe Leu Lleu Phe Ser Gly Cys 1. 5 1O 15 Cys Phe Ala

<210s, SEQ ID NO 14 &211s LENGTH: 566 212. TYPE: PRT <213> ORGANISM: Erwinia carotovora 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (566) <223> OTHER INFORMATION: Sucrose isomerase YP O4994.7

<4 OOs, SEQUENCE: 14 Met Val Ala Val Asn Asp Gly Val Ser Ala His Pro Val Trp Trp Llys 1. 5 1O 15 Glu Ala Val Phe Tyr Glin Val Tyr Pro Arg Ser Phe Lys Asp Ser Asp 2O 25 3O Gly Asp Gly Ile Gly Asp Lieu Lys Gly Lieu. Thr Glu Lys Lieu. Asp Tyr 35 4 O 45 Lieu Lys Ala Lieu. Gly Ile Asn Ala Ile Trp Ile ASn Pro His Tyr Asp SO 55 6 O Ser Pro Asn. Thir Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys Ile 65 70 7s 8O Met Lys Glu Tyr Gly Thr Met Asp Asp Phe Asp Arg Lieu. Ile Ala Glu 85 90 95 Met Lys Lys Arg Asp Met Arg Lieu Met Ile Asp Val Val Val Asn His 1OO 105 11 O Thir Ser Asp Glu. His Glu Trp Phe Val Glu Ser Llys Llys Ser Lys Asp 115 12 O 125 Asn Pro Tyr Arg Asp Tyr Tyr Ile Trp Arg Asp Gly Lys Asp Gly Thr 13 O 135 14 O Gln Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glin Lys 145 150 155 160 Asp Asn Ala Thr Glin Glin Tyr Tyr Lieu. His Tyr Phe Gly Val Glin Glin 1.65 17O 17s Pro Asp Lieu. Asn Trp Asp Asn Pro Llys Val Arg Glu Glu Val Tyr Asp 18O 185 19 O Met Lieu. Arg Phe Trp Ile Asp Llys Gly Val Ser Gly Lieu. Arg Met Asp 195 2OO 2O5 Thr Val Ala Thr Phe Ser Lys Asn Pro Ala Phe Pro Asp Lieu. Thr Pro 21 O 215 22O Lys Gln Leu Glin Asn Phe Ala Tyr Thr Tyr Thr Glin Gly Pro Asn Lieu. 225 23 O 235 24 O His Arg Tyr Ile Glin Glu Met His Glin Llys Val Lieu Ala Lys Tyr Asp 245 250 255 Val Val Ser Ala Gly Glu Ile Phe Gly Val Pro Lieu. Glu Glu Ala Ala US 2011/020 1 059 A1 Aug. 18, 2011 63

- Continued

26 O 265 27 O Pro Phe Ile Asp Glin Arg Arg Lys Glu Lieu. Asp Met Ala Phe Ser Phe 27s 28O 285 Asp Lieu. Ile Arg Lieu. Asp Arg Ala Val Glu Glu Arg Trp Arg Arg Asn 29 O 295 3 OO Asp Trp Thir Lieu. Ser Glin Phe Arg Glin Ile Asn. Asn Arg Lieu Val Asp 3. OS 310 315 32O Met Ala Gly Gln His Gly Trp Asn Thr Phe Phe Leu Ser Asn His Asp 3.25 330 335 Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Glu Trp Arg 34 O 345 35. O Thir Arg Ser Ala Lys Ala Lieu Ala Thr Lieu Ala Lieu. Thr Glin Arg Ala 355 360 365 Thr Pro Phe Ile Tyr Glin Gly Asp Glu Lieu. Gly Met Thr Asn Tyr Pro 37 O 375 38O Phe Thir Ser Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp 385 390 395 4 OO Glin Asp Phe Val Glu Thr Gly Llys Val Llys Pro Asp Val Phe Lieu. Glu 4 OS 41O 415 Asn Val Lys Glin Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Glin Trp 42O 425 43 O Ser Asn Thr Ala Glin Ala Gly Phe Thr Thr Gly Thr Pro Trp Phe Arg 435 44 O 445 Ile Asin Pro Asn Tyr Lys Asn. Ile Asn Ala Glu Glu Glin Thr Glin Asn 450 45.5 460 Pro Asp Ser Ile Phe His Phe Tyr Arg Glin Lieu. Ile Glu Lieu. Arg His 465 470 47s 48O Ala Thr Pro Ala Phe Thr Tyr Gly Thr Tyr Glin Asp Lieu. Asp Pro Asn 485 490 495 Asn Asn. Glu Val Lieu Ala Tyr Thr Arg Glu Lieu. Asn Glin Glin Arg Tyr SOO 505 51O Lieu Val Val Val Asn Phe Lys Glu Lys Pro Val His Tyr Val Lieu Pro 515 52O 525 Llys Thr Lieu. Ser Ile Lys Glin Ser Lieu. Lieu. Glu Ser Gly Glin Lys Asp 53 O 535 54 O Llys Val Glu Pro Asn Ala Thr Thr Lieu. Glu Lieu Gln Pro Trp Glin Ser 5.45 550 555 560 Gly Ile Tyr Glin Lieu. Asn 565

<210s, SEQ ID NO 15 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: sweet potato 22 Os. FEATURE: <221 > NAMEAKEY: SIGNAL <222s. LOCATION: (1) . . (16 <223> OTHER INFORMATION: sporamin vacuolar targeting sequence

<4 OOs, SEQUENCE: 15 His Ser Arg Phe Asn Pro Ile Arg Lieu Pro Thir Thr His Glu Pro Ala 1. 5 1O 15

<210s, SEQ ID NO 16 US 2011/020 1 059 A1 Aug. 18, 2011 64

- Continued

&211s LENGTH: 1701 &212s. TYPE: DNA <213s ORGANISM: unknown 22 Os. FEATURE: <223> OTHER INFORMATION: artificial sequence 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1701 <223> OTHER INFORMATION: sucrose isomerase dicot optimized <4 OOs, SEQUENCE: 16 atg gtt gct gtt aac gat ggt gtt tot gct cat cca gtt tdg tog aaa 48 Met Val Ala Val Asn Asp Gly Val Ser Ala His Pro Val Trp Trp Llys 1. 5 1O 15 gag got gtt titc tac caa gtt tac cca cqt tot ttcaag gat tcc gat 96 Glu Ala Val Phe Tyr Glin Val Tyr Pro Arg Ser Phe Lys Asp Ser Asp 2O 25 3O ggt gat gga att gga gat Ctc aag gga citt act gag aag ct c gat tac 144 Gly Asp Gly Ile Gly Asp Lieu Lys Gly Lieu. Thr Glu Lys Lieu. Asp Tyr 35 4 O 45 citt aag got ct c got att aac got atc tigg atc aac cca cac tac gat 192 Lieu Lys Ala Lieu. Gly Ile Asn Ala Ile Trp Ile ASn Pro His Tyr Asp SO 55 6 O t ct coa aac act gat aac giga tac gat at c agg gat tac cqt aaa atc 24 O Ser Pro Asn. Thir Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys Ile 65 70 7s 8O atg aag gaa tac gga act atg gat gat tt c gat agg Ctt at C gCt gala 288 Met Lys Glu Tyr Gly Thr Met Asp Asp Phe Asp Arg Lieu. Ile Ala Glu 85 90 95 atg aag aaa agg gat atg agg Ct c atg att gat gtt gtg gtgaac cac 336 Met Lys Lys Arg Asp Met Arg Lieu Met Ile Asp Val Val Val Asn His 1OO 105 11 O act tct gat gag cat gag td tt C gtt gag tot aag aag to C aag gat 384 Thir Ser Asp Glu. His Glu Trp Phe Val Glu Ser Llys Llys Ser Lys Asp 115 12 O 125 aac cca tac cqt gat tac tac at C tog Cdt gat gga aag gat gga act 432 Asn Pro Tyr Arg Asp Tyr Tyr Ile Trp Arg Asp Gly Lys Asp Gly Thr 13 O 135 14 O cag cca aat aac tac cca tot titc titc ggit gga t cit gct togg caa aag 48O Gln Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glin Lys 145 150 155 160 gat aat gct act cag cag tac tac citt cac tac titc gga gtt caa cag 528 Asp Asn Ala Thr Glin Glin Tyr Tyr Lieu. His Tyr Phe Gly Val Glin Glin 1.65 17O 17s cca gat Ct c aat it gat aac cca aaa gtt agg gaa gag gtg tac gat 576 Pro Asp Lieu. Asn Trp Asp Asn Pro Llys Val Arg Glu Glu Val Tyr Asp 18O 185 19 O atg Ctt agg tt C tig atc gat aag ggt gtt agt gga ctic aga atg gat 624 Met Lieu. Arg Phe Trp Ile Asp Llys Gly Val Ser Gly Lieu. Arg Met Asp 195 2OO 2O5 act gtg gct act titc. tct aag aat coa got titc cca gat citt act coa 672 Thr Val Ala Thr Phe Ser Lys Asn Pro Ala Phe Pro Asp Lieu. Thr Pro 21 O 215 22O aag cag citt cag aac titc gct tac act tac act cag giga cca aat citt 72 O Lys Gln Leu Glin Asn Phe Ala Tyr Thr Tyr Thr Glin Gly Pro Asn Lieu. 225 23 O 235 24 O cac cqt tac at c caa gag atg cac caa aag gtt citc gct aag tac gat 768 His Arg Tyr Ile Glin Glu Met His Glin Llys Val Lieu Ala Lys Tyr Asp 245 250 255

US 2011/020 1 059 A1 Aug. 18, 2011 66

- Continued gga atc tac cag ctic aac tga 1701 Gly Ile Tyr Glin Lieu. Asn 565

SEO ID NO 17 LENGTH: 566 TYPE : PRT ORGANISM: unknown FEATURE: OTHER INFORMATION: Synthetic Construct

<4 OOs, SEQUENCE: 17

Met Wall Ala Wall Asn Asp Gly Wall Ser Ala His Pro Wall Trp Trp 1. 5 15

Glu Ala Wall Phe Tyr Glin Wall Pro Arg Ser Phe Asp Ser 25

Gly Asp Gly Ile Gly Asp Lell Lys Gly Luell Thir Glu Lys Luell Asp 35 4 O 45

Lell Lys Ala Luell Gly Ile Asn Ala Ile Trp Ile Asn Pro His Asp SO 55 6 O

Ser Pro Asn Thir Asp Asn Gly Tyr Asp Ile Arg Asp Arg Ile 65 70

Met Glu Tyr Gly Thir Met Asp Asp Phe Asp Arg Lell Ile Ala Glu 85 90 95

Met Arg Asp Met Arg Luell Met Ile Asp Wall Wall Wall Asn His 105 11 O

Thir Ser Asp Glu His Glu Trp Phe Wall Glu Ser Lys Ser Asp 115 12 O 125

Asn Pro Arg Asp Tyr Ile Trp Arg Asp Gly Asp Gly Thir 13 O 135 14 O

Glin Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glin Lys 145 150 155 160

Asp Asn Ala Thir Glin Glin Luell His Phe Gly Wall Glin Glin 1.65 17s

Pro Asp Luell Asn Trp Asp Asn Pro Lys Wall Arg Glu Glu Wall Asp 18O 185 19 O

Met Luell Arg Phe Trp Ile Asp Lys Gly Wall Ser Gly Lell Arg Met Asp 195 2OO

Thir Wall Ala Thir Phe Ser Lys Asn Pro Ala Phe Pro Asp Luell Thir Pro 21 O 215 22O

Lys Glin Luell Glin Asn Phe Ala Thir Tyr Thir Glin Gly Pro Asn Luell 225 23 O 235 24 O

His Arg Ile Glin Glu Met His Glin Lys Wall Lell Ala Tyr Asp 245 250 255

Wall Wall Ser Ala Gly Glu Ile Phe Gly Wall Pro Lell Glu Glu Ala Ala 26 O 265 27 O

Pro Phe Ile Asp Glin Arg Arg Lys Glu Luell Asp Met Ala Phe Ser Phe 285

Asp Luell Ile Arg Lell Asp Arg Ala Wall Glu Glu Arg Trp Arg Arg Asn 29 O 295 3 OO

Asp Trp Thir Luell Ser Glin Phe Arg Glin Ile ASn Asn Arg Luell Wall Asp 3. OS 310 315

Met Ala Gly Glin His Gly Trp Asn Thir Phe Phe Lell Ser Asn His Asp 3.25 330 335 US 2011/020 1 059 A1 Aug. 18, 2011 67

- Continued

Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Glu Trp Arg 34 O 345 35. O Thir Arg Ser Ala Lys Ala Lieu Ala Thr Lieu Ala Lieu. Thr Glin Arg Ala 355 360 365 Thr Pro Phe Ile Tyr Glin Gly Asp Glu Lieu. Gly Met Thr Asn Tyr Pro 37 O 375 38O Phe Thir Ser Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp 385 390 395 4 OO Glin Asp Phe Val Glu Thr Gly Llys Val Llys Pro Asp Val Phe Lieu. Glu 4 OS 41O 415 Asn Val Lys Glin Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Glin Trp 42O 425 43 O Ser Asn Thr Ala Glin Ala Gly Phe Thr Thr Gly Thr Pro Trp Phe Arg 435 44 O 445 Ile Asin Pro Asn Tyr Lys Asn. Ile Asn Ala Glu Glu Glin Thr Glin Asn 450 45.5 460 Pro Asp Ser Ile Phe His Phe Tyr Arg Glin Lieu. Ile Glu Lieu. Arg His 465 470 47s 48O Ala Thr Pro Ala Phe Thr Tyr Gly Thr Tyr Glin Asp Lieu. Asp Pro Asn 485 490 495 Asn Asn. Glu Val Lieu Ala Tyr Thr Arg Glu Lieu. Asn Glin Glin Arg Tyr SOO 505 51O Lieu Val Val Val Asn Phe Lys Glu Lys Pro Val His Tyr Val Lieu Pro 515 52O 525 Llys Thr Lieu. Ser Ile Lys Glin Ser Lieu. Lieu. Glu Ser Gly Glin Lys Asp 53 O 535 54 O Llys Val Glu Pro Asn Ala Thr Thr Lieu. Glu Lieu Gln Pro Trp Glin Ser 5.45 550 555 560 Gly Ile Tyr Glin Lieu. Asn 565

<210s, SEQ ID NO 18 &211s LENGTH: 1993 &212s. TYPE: DNA <213> ORGANISM: Zea maize 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (1) . . (1993) <223> OTHER INFORMATION: maize UBQ promoter <4 OOs, SEQUENCE: 18 Ctgcagtgca gcgtgacccg gtcgtgcc cc tict ctagaga taatgagcat tdatgtcta 6 O agittataaaa aattaccaca tatttitttitt gtcacacttig tittgaagtgc agitttatcta 12 O t ctittataca tatatttaaa citt tacticta cqaataatat aatctatagt act acaataa 18O tat cagtgtt ttagagaatc atataaatga acagttagac atggit ctaaa gogacaattga 24 O gtattittgac aac aggactic tacagttitta t ctittittagt gtgcatgtgt tot cotttitt 3OO ttittgcaaat agctt cacct atataatact tcatc cattt tattagtaca to catttagg 360 gtttagggitt aatggtttitt atagactaat tttitt tagta catct attitt attct attitt 42O agcct ctaaa ttaagaaaac taaaacticta ttt tagttitt tittatttaat aatttagata 48O taaaatagaa taaaataaag tdactaaaaa ttaaacaaat accctittaag aaattaaaaa 54 O

US 2011/020 1 059 A1 Aug. 18, 2011 71

- Continued

&211s LENGTH: 565 212. TYPE: PRT <213s ORGANISM: unknown 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Construct <4 OOs, SEQUENCE: 21 Val Ala Val Asn Asp Gly Val Ser Ala His Pro Val Trp Trp Llys Glu 1. 5 1O 15 Ala Val Phe Tyr Glin Val Tyr Pro Arg Ser Phe Lys Asp Ser Asp Gly 2O 25 3O Asp Gly Ile Gly Asp Lieu Lys Gly Lieu. Thr Glu Lys Lieu. Asp Tyr Lieu 35 4 O 45 Lys Ala Lieu. Gly Ile Asn Ala Ile Trp Ile Asn. Pro His Tyr Asp Ser SO 55 6 O Pro Asn. Thir Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys Ile Met 65 70 7s 8O Lys Glu Tyr Gly Thr Met Asp Asp Phe Asp Arg Lieu. Ile Ala Glu Met 85 90 95 Llys Lys Arg Asp Met Arg Lieu Met Ile Asp Val Val Val Asn His Thr 1OO 105 11 O Ser Asp Glu. His Glu Trp Phe Val Glu Ser Lys Llys Ser Lys Asp Asn 115 12 O 125 Pro Tyr Arg Asp Tyr Tyr Ile Trp Arg Asp Gly Lys Asp Gly Thr Glin 13 O 135 14 O Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glin Lys Asp 145 150 155 160 Asn Ala Thr Glin Glin Tyr Tyr Lieu. His Tyr Phe Gly Val Glin Glin Pro 1.65 17O 17s Asp Lieu. Asn Trp Asp Asn Pro Llys Val Arg Glu Glu Val Tyr Asp Met 18O 185 19 O Lieu. Arg Phe Trp Ile Asp Llys Gly Val Ser Gly Lieu. Arg Met Asp Thr 195 2OO 2O5 Val Ala Thr Phe Ser Lys Asn Pro Ala Phe Pro Asp Lieu. Thr Pro Llys 21 O 215 22O Gln Leu Glin Asn Phe Ala Tyr Thr Tyr Thr Glin Gly Pro Asn Lieu. His 225 23 O 235 24 O Arg Tyr Ile Glin Glu Met His Glin Llys Val Lieu Ala Lys Tyr Asp Wall 245 250 255 Val Ser Ala Gly Glu Ile Phe Gly Val Pro Lieu. Glu Glu Ala Ala Pro 26 O 265 27 O Phe Ile Asp Glin Arg Arg Lys Glu Lieu. Asp Met Ala Phe Ser Phe Asp 27s 28O 285 Lieu. Ile Arg Lieu. Asp Arg Ala Val Glu Glu Arg Trp Arg Arg Asn Asp 29 O 295 3 OO Trp. Thir Lieu. Ser Glin Phe Arg Glin Ile Asn. Asn Arg Lieu Val Asp Met 3. OS 310 315 32O Ala Gly Gln His Gly Trp Asn. Thir Phe Phe Lieu. Ser Asn His Asp Asn 3.25 330 335 Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Glu Trp Arg Thr 34 O 345 35. O Arg Ser Ala Lys Ala Lieu Ala Thr Lieu Ala Lieu. Thr Glin Arg Ala Thr 355 360 365 US 2011/020 1 059 A1 Aug. 18, 2011 72

- Continued

Pro Phe Ile Tyr Glin Gly Asp Glu Lieu. Gly Met Thr Asn Tyr Pro Phe 37 O 375 38O Thir Ser Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Glin 385 390 395 4 OO Asp Phe Val Glu Thr Gly Llys Val Llys Pro Asp Val Phe Lieu. Glu Asn 4 OS 41O 415 Val Lys Glin Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln Trp Ser 42O 425 43 O Asn Thr Ala Glin Ala Gly Phe Thr Thr Gly Thr Pro Trp Phe Arg Ile 435 44 O 445 Asn Pro Asn Tyr Lys Asn. Ile Asn Ala Glu Glu Gln Thr Glin Asn Pro 450 45.5 460 Asp Ser Ile Phe His Phe Tyr Arg Glin Lieu. Ile Glu Lieu. Arg His Ala 465 470 47s 48O Thr Pro Ala Phe Thr Tyr Gly Thr Tyr Glin Asp Lieu. Asp Pro Asn Asn 485 490 495 Asn Glu Val Lieu Ala Tyr Thr Arg Glu Lieu. Asn Glin Glin Arg Tyr Lieu SOO 505 51O Val Val Val Asin Phe Lys Glu Lys Pro Val His Tyr Val Lieu Pro Llys 515 52O 525 Thir Lieu. Ser Ile Lys Glin Ser Lieu. Lieu. Glu Ser Gly Glin Lys Asp Llys 53 O 535 54 O Val Glu Pro Asn Ala Thr Thr Lieu. Glu Lieu Gln Pro Trp Glin Ser Gly 5.45 550 555 560 Ile Tyr Glin Lieu. Asn 565

<210s, SEQ ID NO 22 &211s LENGTH: 194 &212s. TYPE: DNA <213> ORGANISM: Fig wort mosaic virus 22 Os. FEATURE: <221 > NAMEAKEY: enhancer <222s. LOCATION: (1) . . (194)

<4 OOs, SEQUENCE: 22 agctgcttgt ggggaccaga caaaaaagga atggtgcaga attgttaggc gcaccitacca 6 O aaag catctt togc ctittatt gcaaagataa agcagatt.cc tictag tacaa gtggggalaca 12 O aaataacgtg gaaaagagct gtc.ctgacag cccact cact aatgcgt atg acgaacgcag 18O tgacgaccac aaaa 194

<210s, SEQ ID NO 23 &211s LENGTH: 293 &212s. TYPE: DNA <213> ORGANISM: 35S virus 22 Os. FEATURE: <221 > NAMEAKEY: enhancer <222s. LOCATION: (1) ... (293)

<4 OOs, SEQUENCE: 23 acttittcaac aaaggg taat atc.cggaaac ctic ct cqgat tcc attgcc c agctatotgt 6 O cactitt attg talagatagt ggaaaaggaa ggtggct cot acaaatgcca t cattgcgat 12 O aaaggaaagg ct atcgttga agatgcct ct gcc.gacagtg gtc.ccaaaga tiggacccc.ca 18O

US 2011/020 1 059 A1 Aug. 18, 2011 75

- Continued

53 O 535 54 O aag gt C gag cc.g aac gcc acc acc Ct c gag ctt cag C cc tig cag agc 168O Lys Wall Glu Pro Asn Ala Thir Thr Lieu. Glu Lieu Gln Pro Trp Glin Ser 5.45 550 555 560 ggc at C tat cag Ctg aac tga 1701 Gly Ile Glin Lell Asn 565

SEO ID NO 25 LENGTH: 566 TYPE : PRT ORGANISM: unknown FEATURE: OTHER INFORMATION: Synthetic Construct

<4 OOs, SEQUENCE: 25

Met Wall Ala Wall Asn Asp Gly Val Ser Ala His Pro Val Trp Trp Llys 1. 5 1O 15

Glu Ala Wall Phe Tyr Glin Val Tyr Pro Arg Ser Phe Lys Asp Ser Asp 25 3O

Gly Asp Gly Ile Gly Asp Lieu Lys Gly Lieu. Thr Glu Lys Lieu. Asp Tyr 35 4 O 45

Lell Lys Ala Luell Gly Ile Asn Ala Ile Trp Ile Asn Pro His Tyr Asp SO 55 6 O

Ser Pro Asn Thir Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys Ile 65 70 7s 8O

Met Glu Tyr Gly Thir Met Asp Asp Phe Asp Arg Lieu. Ile Ala Glu 85 90 95

Met Arg Asp Met Arg Lieu Met Ile Asp Val Val Val Asn His 105 11 O

Thir Ser Asp Glu His Glu Trp Phe Val Glu Ser Llys Llys Ser Lys Asp 115 12 O 125

Asn Pro Arg Asp Tyr Ile Trp Arg Asp Gly Lys Asp Gly. Thir 13 O 135 14 O

Glin Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln Lys 145 150 155 160

Asp Asn Ala Thir Glin Glin Tyr Tyr Lieu. His Tyr Phe Gly Val Glin Glin 1.65 17O 17s

Pro Asp Luell Asn Trp Asp Asn Pro Llys Val Arg Glu Glu Val Tyr Asp 18O 185 19 O

Met Luell Arg Phe Trp Ile Asp Llys Gly Val Ser Gly Lieu. Arg Met Asp 195 2OO 2O5

Thir Wall Ala Thir Phe Ser Lys Asn Pro Ala Phe Pro Asp Lieu. Thr Pro 21 O 215 22O

Lys Glin Luell Glin Asn Phe Ala Tyr Thr Tyr Thr Glin Gly Pro Asn Lieu 225 23 O 235 24 O

His Arg Ile Glin Glu Met His Glin Llys Val Lieu Ala Lys Tyr Asp 245 250 255

Wall Wall Ser Ala Gly Glu Ile Phe Gly Val Pro Lieu. Glu Glu Ala Ala 26 O 265 27 O

Pro Phe Ile Asp Glin Arg Arg Lys Glu Lieu. Asp Met Ala Phe Ser Phe 27s 28O 285

Asp Luell Ile Arg Lell Asp Arg Ala Val Glu Glu Arg Trp Arg Arg Asn 29 O 295 3 OO US 2011/020 1 059 A1 Aug. 18, 2011 76

- Continued

Asp Trp Thir Lieu. Ser Glin Phe Arg Glin Ile Asn. Asn Arg Lieu Val Asp 3. OS 310 315 32O Met Ala Gly Gln His Gly Trp Asn Thr Phe Phe Leu Ser Asn His Asp 3.25 330 335 Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Glu Trp Arg 34 O 345 35. O Thir Arg Ser Ala Lys Ala Lieu Ala Thr Lieu Ala Lieu. Thr Glin Arg Ala 355 360 365 Thr Pro Phe Ile Tyr Glin Gly Asp Glu Lieu. Gly Met Thr Asn Tyr Pro 37 O 375 38O Phe Thir Ser Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp 385 390 395 4 OO Glin Asp Phe Val Glu Thr Gly Llys Val Llys Pro Asp Val Phe Lieu. Glu 4 OS 41O 415 Asn Val Lys Glin Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Glin Trp 42O 425 43 O Ser Asn Thr Ala Glin Ala Gly Phe Thr Thr Gly Thr Pro Trp Phe Arg 435 44 O 445 Ile Asin Pro Asn Tyr Lys Asn. Ile Asn Ala Glu Glu Glin Thr Glin Asn 450 45.5 460 Pro Asp Ser Ile Phe His Phe Tyr Arg Glin Lieu. Ile Glu Lieu. Arg His 465 470 47s 48O Ala Thr Pro Ala Phe Thr Tyr Gly Thr Tyr Glin Asp Lieu. Asp Pro Asn 485 490 495 Asn Asn. Glu Val Lieu Ala Tyr Thr Arg Glu Lieu. Asn Glin Glin Arg Tyr SOO 505 51O Lieu Val Val Val Asn Phe Lys Glu Lys Pro Val His Tyr Val Lieu Pro 515 52O 525 Llys Thr Lieu. Ser Ile Lys Glin Ser Lieu. Lieu. Glu Ser Gly Glin Lys Asp 53 O 535 54 O Llys Val Glu Pro Asn Ala Thr Thr Lieu. Glu Lieu Gln Pro Trp Glin Ser 5.45 550 555 560 Gly Ile Tyr Glin Lieu. Asn 565

<210s, SEQ ID NO 26 &211s LENGTH: 65 212. TYPE: PRT <213> ORGANISM: Cyanophora paradoxa 22 Os. FEATURE: <221> NAME/KEY: signal <222s. LOCATION: (1) . . (65 <223> OTHER INFORMATION: FNR plastid targeting sequence

<4 OOs, SEQUENCE: 26 Met Ala Phe Val Ala Ser Val Pro Val Phe Ala Asn Ala Ser Gly Lieu. 1. 5 1O 15 Llys Thr Glu Ala Lys Val Cys Glin Llys Pro Ala Lieu Lys Asn. Ser Phe 2O 25 3O Phe Arg Gly Glu Glu Val Thr Ser Arg Ser Phe Phe Ala Ser Glin Ala 35 4 O 45 Val Ser Ala Lys Pro Ala Thir Thr Gly Glu Val Asp Thr Thr Ile Arg SO 55 6 O

US 2011/020 1 059 A1 Aug. 18, 2011 79

- Continued Ala Glu Lieu Val Ile Ala Asn Tyr Pro Val Asp Asp Ser Glu Ala Gly 5.45 550 555 560 gga cca gct gct gcc ggit gct cot cat agg ttt agg Ctt agg cca tat 1728 Gly Pro Ala Ala Ala Gly Ala Pro His Arg Phe Arg Lieu. Arg Pro Tyr 565 st O sts gag tit cqt gtt tac Clt Ctt ttg gga tigg cac taa 1764 Glu Cys Arg Val Tyr Arg Lieu. Lieu. Gly Trp His 58O 585

<210s, SEQ ID NO 28 &211s LENGTH: 587 212. TYPE: PRT <213> ORGANISM; Bacillus

<4 OOs, SEQUENCE: 28

Met Ser Thir Ala Lieu. Thir Glin. Thir Ser Thir Asn. Ser Glin Glin Ser Pro 1. 5 1O 15 Ile Arg Arg Ala Trp Trp Llys Glu Ala Val Val Tyr Glin Ile Tyr Pro 2O 25 3O Arg Ser Phe Met Asp Ser Asn Gly Asp Gly Ile Gly Asp Lieu. Arg Gly 35 4 O 45 Ile Lieu. Ser Lys Lieu. Asp Tyr Lieu Lys Lieu. Lieu. Gly Val Asp Val Lieu. SO 55 6 O Trp Lieu. Asn Pro Ile Tyr Asp Ser Pro Asn Asp Asp Met Gly Tyr Asp 65 70 7s 8O Ile Arg Asp Tyr Tyr Lys Ile Met Glu Glu Phe Gly Thr Met Glu Asp 85 90 95 Phe Glu Glu Lieu. Lieu. Arg Glu Val His Ala Arg Gly Met Lys Lieu Val 1OO 105 11 O Met Asp Leu Val Ala Asn His Thr Ser Asp Glu. His Pro Trp Phe Ile 115 12 O 125 Glu Ser Arg Ser Ser Arg Asp Asn Pro Tyr Arg Asp Trp Tyr Ile Trp 13 O 135 14 O Arg Asp Pro Lys Asp Gly Arg Glu Pro Asn. Asn Trp Lieu. Ser Tyr Phe 145 150 155 160 Ser Gly Ser Ala Trp Glu Tyr Asp Glu Arg Thr Gly Glin Tyr Tyr Lieu. 1.65 17O 17s His Lieu. Phe Ser Arg Arg Glin Pro Asp Lieu. Asn Trp Glu Asn Pro Llys 18O 185 19 O Val Arg Glu Ala Ile Phe Glu Met Met Arg Phe Trp Lieu. Asp Llys Gly 195 2OO 2O5 Ile Asp Gly Phe Arg Met Asp Val Ile Asn Ala Ile Ala Lys Ala Glu 21 O 215 22O Gly Lieu Pro Asp Ala Pro Ala Arg Pro Gly Glu Arg Tyr Ala Trp Gly 225 23 O 235 24 O Gly Glin Tyr Phe Lieu. Asn Gln Pro Llys Val His Glu Tyr Lieu. Arg Glu 245 250 255 Met Tyr Asp Llys Val Lieu Ser His Tyr Asp Ile Met Thr Val Gly Glu 26 O 265 27 O Thr Gly Gly Val Thir Thr Lys Asp Ala Lieu. Lieu. Phe Ala Gly Glu Asp 27s 28O 285 Arg Arg Glu Lieu. Asn Met Val Phe Glin Phe Glu. His Met Asp Ile Asp 29 O 295 3 OO US 2011/020 1 059 A1 Aug. 18, 2011 80

- Continued

Ala Thir Asp Gly Asp Llys Trp Arg Pro Arg Pro Trp Arg Lieu. Thr Glu 3. OS 310 315 32O

Lell Thir Ile Met Thir Arg Trp Glin Asn Asp Lieu. Tyr Gly Lys Ala 3.25 330 335

Trp Asn Ser Luell Tyr Trp Thir Asn His Asp Glin Pro Arg Ala Wall Ser 34 O 345 35. O

Arg Phe Gly Asn Asp Gly Pro Tyr Arg Wall Glu Ser Ala Met Lieu. 355 360 365

Ala Thir Wall Lieu. His Met Met Gln Gly Thr Pro Tyr Ile Glin Gly 37 O 375

Glu Glu Ile Gly Met Thir Asn Pro Phe Asp Ser Ile Asp Glu Tyr 385 390 395 4 OO

Arg Asp Wall Glu Ile His Asn Lieu. Trp Arg His Arg Wall Met Glu Gly 4 OS 415

Gly Glin Asp Pro Ala Glu Wall Lieu. Arg Wall Ile Glin Lieu Lys Gly Arg 425 43 O

Asp Asn Ala Arg Thir Pro Met Glin Trp Asp Asp Ser Pro Asn Ala Gly 435 44 O 445

Phe Thir Thr Gly Thr Pro Trp Ile Wall ASn Pro Asn Arg Glu 450 45.5 460

Ile Asn Wall Glin Ala Lieu Ala Pro ASn Ser Ile Phe His Tyr 465 470 48O

Arg Arg Lieu. Ile Glin Lieu. Arg Glin His Pro Ile Wall Wall Tyr 485 490 495 Gly Asp Lieu. Ile Lieu Pro Asp His Glu Glu Ile Trp Ala Tyr SOO 505

Thir Arg Thir Lieu. Gly Asp Glu Arg Trp Luell Ile Wall Ala Asn. Phe Phe 515 525

Gly Gly Thir Pro Glu Phe Glu Leul Pro Pro Glu Wall Arg Glu Gly 53 O 535 54 O

Ala Glu Luell Wall Ile Ala Asn Pro Wall Asp Asp Ser Glu Ala Gly 5.45 550 555 560

Gly Pro Ala Ala Ala Gly Ala Pro His Arg Phe Arg Lieu. Arg Pro Tyr 565 st O sts

Glu Arg Wall Tyr Arg Lieu. Lieu. Gly Trp His 58O 585

SEQ ID NO 29 LENGTH: 1541 TYPE PRT ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: synthetic gene FEATURE: NAME/KEY: enzyme LOCATION: (1) ... (1541) OTHER INFORMATION: dextranslucrase with leucrose synthase activity

SEQUENCE: 29 Met Lieu. Glu Ser Gly Val Val His Ala Asp Asp Wall Lys Glin Val Val 1. 15

Wall Glin Glu Pro Ala Thir Ala Glin. Thir Ser Gly Pro Gly Glin Gln Thr 2O 25 3O Pro Ala Glin Ala Lys Ile Ala Ser Glu Glin Glu Ala Glu Lys Val Thr 35 4 O 45 US 2011/020 1 059 A1 Aug. 18, 2011 81

- Continued

Pro Ala Asp Llys Val Thr Asp Asp Val Ala Ala Ser Glu Lys Pro Ala SO 55 6 O Llys Pro Ala Glu Asn Thr Glu Ala Thr Val Glin Thr Asn Ala Glin Glu 65 70 7s 8O Pro Ala Lys Pro Ala Asp Thir Lys Glu Ala Ser Thr Glu Lys Ala Ala 85 90 95 Val Ala Glu Glu Val Lys Ala Ala Asn Ala Ile Thr Glu Ile Pro Llys 1OO 105 11 O Thr Glu Val Ala Asp Glin Asn Lys Glin Ala Arg Pro Thir Thir Ala Glin 115 12 O 125 Asp Glin Glu Gly Asp Lys Arg Glu Lys Thr Ala Val Glu Asp Llys Ile 13 O 135 14 O Val Ala Asn. Pro Llys Val Ala Lys Lys Asp Arg Lieu Pro Glu Pro Gly 145 150 155 160 Ser Lys Glin Gly Ala Ile Ala Glu Arg Met Val Ala Asp Glin Ala Glin 1.65 17O 17s Pro Ala Pro Val Asn Ala Asp His Asp Asp Asp Val Lieu. Ser His Ile 18O 185 19 O Llys Thir Ile Asp Gly Lys Asn Tyr Tyr Val Glin Asp Asp Gly Thr Val 195 2OO 2O5 Llys Lys Asn. Phe Ala Val Glu Lieu. Asn Gly Arg Ile Lieu. Tyr Phe Asp 21 O 215 22O Ala Glu Thr Gly Ala Lieu Val Asp Ser Asn. Glu Tyr Glin Phe Glin Glin 225 23 O 235 24 O Gly. Thir Ser Ser Lieu. Asn Asn Glu Phe Ser Gln Lys Asn Ala Phe Tyr 245 250 255 Gly. Thir Thr Asp Lys Asp Ile Glu Thr Val Asp Gly Tyr Lieu. Thir Ala 26 O 265 27 O Asp Ser Trp Tyr Arg Pro Llys Phe Ile Lieu Lys Asp Gly Lys Thir Trip 27s 28O 285 Thr Ala Ser Thr Glu Thir Asp Leu Arg Pro Leu Lleu Met Ala Trp Trp 29 O 295 3 OO Pro Asp Lys Arg Thr Glin Ile Asn Tyr Lieu. Asn Tyr Met Asin Glin Glin 3. OS 310 315 32O Gly Lieu. Gly Ala Gly Ala Phe Glu Asn Llys Val Glu Glin Ala Lieu. Lieu. 3.25 330 335 Thr Gly Ala Ser Glin Glin Val Glin Arg Lys Ile Glu Glu Lys Ile Gly 34 O 345 35. O Lys Glu Gly Asp Thir Lys Trp Lieu. Arg Thr Lieu Met Gly Ala Phe Val 355 360 365 Lys Thr Glin Pro Asn Trp Asn Ile Llys Thr Glu Ser Glu Thir Thr Gly 37 O 375 38O Thir Lys Lys Asp His Lieu. Glin Gly Gly Ala Lieu. Lieu. Tyr Thir Asn. Asn 385 390 395 4 OO Glu Lys Ser Pro His Ala Asp Ser Llys Phe Arg Lieu. Lieu. Asn Arg Thr 4 OS 41O 415 Pro Thir Ser Glin Thr Gly Thr Pro Llys Tyr Phe Ile Asp Llys Ser Asn 42O 425 43 O Gly Gly Tyr Glu Phe Lieu. Lieu Ala Asn Asp Phe Asp Asn. Ser Asn Pro 435 44 O 445 US 2011/020 1 059 A1 Aug. 18, 2011 82

- Continued Ala Val Glin Ala Glu Gln Lieu. Asn Trp Lieu. His Tyr Met Met Asin Phe 450 45.5 460 Gly Ser Ile Val Ala Asn Asp Pro Thr Ala Asn. Phe Asp Gly Val Arg 465 470 47s 48O Val Asp Ala Val Asp Asn Val Asn Ala Asp Lieu. Lieu. Glin Ile Ala Ser 485 490 495 Asp Tyr Phe Llys Ser Arg Tyr Llys Val Gly Glu Ser Glu Glu Glu Ala SOO 505 51O Ile Llys His Lieu. Ser Ile Lieu. Glu Ala Trp Ser Asp Asn Asp Pro Asp 515 52O 525 Tyr Asn Lys Asp Thir Lys Gly Ala Glin Lieu Ala Ile Asp Asn Llys Lieu. 53 O 535 54 O Arg Lieu. Ser Lieu. Lieu. Tyr Ser Phe Met Arg Asn Lieu. Ser Ile Arg Ser 5.45 550 555 560 Gly Val Glu Pro Thr Ile Thr Asn Ser Lieu. Asn Asp Arg Ser Ser Glu 565 st O sts Llys Lys Asn Gly Glu Arg Met Ala Asn Tyr Ile Phe Val Arg Ala His 58O 585 59 O Asp Asp Glu Val Glin Thr Val Ile Ala Asp Ile Ile Arg Glu Asn. Ile 595 6OO 605 Asn Pro Asn Thr Asp Gly Lieu. Thr Phe Thr Met Asp Glu Lieu Lys Glin 610 615 62O Ala Phe Lys Ile Tyr Asn. Glu Asp Met Arg Lys Ala Asp Llys Llys Tyr 625 630 635 64 O Thr Glin Phe Asin Ile Pro Thr Ala His Ala Leu Met Leu Ser Asn Lys 645 650 655 Asp Ser Ile Thr Arg Val Tyr Tyr Gly Asp Lieu. Tyr Thr Asp Asp Gly 660 665 67 O Glin Tyr Met Glu Lys Llys Ser Pro Tyr His Asp Ala Ile Asp Ala Lieu. 675 68O 685 Lieu. Arg Ala Arg Ile Llys Tyr Val Ala Gly Gly Glin Asp Met Llys Val 69 O. 695 7 OO Thr Tyr Met Gly Val Pro Arg Glu Ala Asp Llys Trp Ser Tyr Asn Gly 7 Os 71O 71s 72O Ile Lieu. Thir Ser Val Arg Tyr Gly Thr Gly Ala Asn. Glu Ala Thr Asp 72 73 O 73 Glu Gly Thr Ala Glu Thr Arg Thr Glin Gly Met Ala Val Ile Ala Ser 740 74. 7 O Asn Asn Pro Asn Lieu Lys Lieu. Asn. Glu Trp Asp Llys Lieu. Glin Val Asn 7ss 760 765 Met Gly Ala Ala His Lys Asn. Glin Tyr Tyr Arg Pro Val Lieu. Lieu. Thir 770 775 78O Thir Lys Asp Gly Ile Ser Arg Tyr Lieu. Thir Asp Glu Glu Val Pro Glin 78s 79 O 79. 8OO Ser Lieu. Trp Llys Llys Thr Asp Ala Asn Gly Ile Lieu. Thir Phe Asp Met 805 810 815 Asn Asp Ile Ala Gly Tyr Ser Asn Val Glin Val Ser Gly Tyr Lieu Ala 82O 825 83 O Val Trp Val Pro Val Gly Ala Lys Ala Asp Glin Asp Ala Arg Thir Thr 835 84 O 845 Ala Ser Lys Llys Lys Asn Ala Ser Gly Glin Val Tyr Glu Ser Ser Ala US 2011/020 1 059 A1 Aug. 18, 2011 83

- Continued

850 855 860 Ala Lieu. Asp Ser Glin Lieu. Ile Tyr Glu Gly Phe Ser Asn. Phe Glin Asp 865 87O 87s 88O Phe Ala Thr Arg Asp Asp Glin Tyr Thr Asn Llys Val Ile Ala Lys Asn 885 890 895 Val Asn Lieu Phe Lys Glu Trp Gly Val Thr Ser Phe Glu Lieu Pro Pro 9 OO 905 91 O Gln Tyr Val Ser Ser Glin Asp Gly Thr Phe Lieu. Asp Ser Ile Ile Glin 915 92 O 925 Asn Gly Tyr Ala Phe Glu Asp Arg Tyr Asp Met Ala Met Ser Lys Asn 93 O 935 94 O Asn Llys Tyr Gly Ser Lieu Lys Asp Lieu. Lieu. Asn Ala Lieu. Arg Ala Lieu 945 950 955 96.O His Ser Val Asn. Ile Glin Ala Ile Ala Asp Trp Val Pro Asp Glin Ile 965 97O 97. Tyr Asn Lieu Pro Gly Lys Glu Val Val Thir Ala Thr Arg Val Asn. Asn 98O 985 99 O Tyr Gly Thr Tyr Arg Glu Gly Ala Glu Ile Lys Glu Lys Lieu. Tyr Val 995 1OOO 1005 Ala Asn. Ser Llys Thir Asn. Glu Thir Asp Phe Glin Gly Lys Tyr Gly O1O O15 O2O Gly Ala Phe Lieu. Asp Glu Lieu Lys Ala Lys Tyr Pro Glu. Ile Phe O25 O3 O O35 Glu Arg Val Glin Ile Ser Asn Gly Gln Lys Met Thr Thr Asp Glu O4 O O45 OSO Lys Ile Thr Lys Trp Ser Ala Lys Tyr Phe Asn Gly Thr Asn Ile O55 O6 O O65 Lieu. Gly Arg Gly Ala Tyr Tyr Val Lieu Lys Asp Trp Ala Ser Asn Of O O7 O8O Asp Tyr Lieu. Thir Asn Arg Asn Gly Glu Ile Val Lieu Pro Llys Glin

Lieu Val Asn Lys Asn. Ser Tyr Thr Gly Phe Val Ser Asp Ala Asn OO O5 10 Gly Thr Lys Phe Tyr Ser Thr Ser Gly Tyr Glin Ala Lys Asn Ser

Phe e Glin Asp Glu Asn Gly Asn Trp Tyr Tyr Phe Asp Lys Arg

Gly Tyr Lieu Val Thr Gly Ala His Glu Ile Asp Gly Llys His Val

Tyr Phe Lieu Lys Asn Gly e Glin Lieu. Arg Asp Ser Ile Arg Glu

Asp Glu Asn Gly Asn. Glin Tyr Tyr Tyr Asp Glin Thr Gly Ala Glin

Val Lieu. Asn Arg Tyr Tyr Thir Thr Asp Gly Glin Asn Trp Arg Tyr 90 95 2OO Phe Asp Ala Lys Gly Wal Met Ala Arg Gly Lieu Val Lys Ile Gly 2O5 21 O 215 Asp Gly Glin Glin Phe Phe Asp Glu Asn Gly Tyr Glin Val Lys Gly 22O 225 23 O Lys Ile Val Ser Ala Lys Asp Gly Lys Lieu. Arg Tyr Phe Asp Llys 235 24 O 245 US 2011/020 1 059 A1 Aug. 18, 2011 84

- Continued

Asp Ser Gly Asn Ala Val Ile Asin Arg Phe Ala Glin Gly Asp Asn 250 255 26 O Pro Ser Asp Trp Tyr Tyr Phe Gly Val Glu Phe Ala Lys Lieu. Thr 265 27 O 27s Gly Lieu Gln Lys Ile Gly Glin Glin Thr Lieu. Tyr Phe Asp Glin Asp 28O 285 29 O Gly Lys Glin Val Lys Gly Lys Ile Val Thir Lieu. Ser Asp Llys Ser 295 3OO 305 Ile Arg Tyr Phe Asp Ala Asn. Ser Gly Glu Met Ala Val Gly Lys 310 315 32O Phe Ala Glu Gly Ala Lys Asn. Glu Trip Tyr Tyr Phe Asp Llys Thr 3.25 33 O 335 Gly Lys Ala Val Thr Gly Lieu Gln Lys Ile Gly Lys Glin Thr Lieu. 34 O 345 350 Tyr Phe Asp Glin Asp Gly Lys Glin Val Lys Gly Llys Val Val Thr

Lieu Ala Asp Llys Ser Ile Arg Tyr Phe Asp Ala Asp Ser Gly Glu

Met Ala Val Gly Llys Phe Ala Glu Gly Ala Lys Asn. Glu Trp Tyr 385 390 395 Tyr Phe Asp Glin Thr Gly Lys Ala Val Thr Gly Lieu Gln Lys Ile 4 OO 405 41 O Asp Llys Glin Thr Lieu. Tyr Phe Asp Glin Asp Gly Lys Glin Val Lys 415 42O 425 Gly Lys Ile Val Thir Lieu. Ser Asp Llys Ser Ile Arg Tyr Phe Asp 43 O 435 44 O Ala Asn. Ser Gly Glu Met Ala Thr Asn Llys Phe Val Glu Gly Ser 445 450 45.5 Glin Asn. Glu Trp Tyr Tyr Phe Asp Glin Ala Gly Lys Ala Val Thr 460 465 47 O Gly Leu Gln Glin Val Gly Glin Gln Thr Lieu. Tyr Phe Thr Glin Asp 47s 48O 485 Gly Lys Glin Val Lys Gly Llys Val Val Asp Wall Asn Gly Val Ser 490 495 SOO Arg Tyr Phe Asp Ala Asn. Ser Gly Asp Met Ala Arg Ser Llys Trp 5 OS 510 515 Ile Glin Lieu. Glu Asp Gly Ser Trp Met Tyr Phe Asp Arg Asp Gly 52O 525 53 O Arg Gly Glin Asn. Phe Gly Arg Asn 535 54 O

<210s, SEQ ID NO 3 O &211s LENGTH: 529 212. TYPE: PRT <213> ORGANISM: Thermus thermophilus 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (508) <223> OTHER INFORMATION: deletion of signal sequence GK24, alpha-1, 5, -glucosidase

<4 OOs, SEQUENCE: 30 Met Ser Trp Trp Glin Arg Ala Val Ile Tyr Glin Val Tyr Pro Arg Ser 1. 5 1O 15 US 2011/020 1 059 A1 Aug. 18, 2011 85

- Continued

Phe Glin Asp Thir Asn Gly Asp Gly Val Gly Asp Lieu. Glu Gly Ile Arg 2O 25 3O Arg Arg Lieu Pro Tyr Phe Llys Ser Lieu. Gly Val Asp Ala Phe Trp Lieu 35 4 O 45 Ser Pro Phe Tyr Lys Ser Pro Met Lys Asp Phe Gly Tyr Asp Val Ala SO 55 6 O Asp Tyr Cys Asp Val Asp Pro Val Phe Gly Thr Lieu. Glin Asp Phe Asp 65 70 7s 8O Arg Lieu. Lieu. Glu Glu Ala His Ala Lieu. Gly Lieu Lys Val Lieu Val Asp 85 90 95 Lieu Val Pro Asn His Thir Ser Ser Glu. His Pro Trp Phe Lieu. Glu Ser 1OO 105 11 O Arg Ala Ser Arg Asn. Ser Pro Lys Arg Asp Trp Tyr Val Trp Lys Asp 115 12 O 125 Pro Ala Pro Asp Gly Gly Pro Pro Asn Asn Trp Glin Ser Phe Phe Gly 13 O 135 14 O Gly Pro Ala Trp Thr Lieu. Asp Glu Ala Thr Gly Glin Tyr Tyr Lieu. His 145 150 155 160 Lieu. Phe Lieu Pro Glu Glin Pro Asp Lieu. Asn Trp Asp ASn Pro Glu Val 1.65 17O 17s Arg Glu Ala Ile Lys Glu Val Met Arg Phe Trp Lieu. Arg Arg Gly Val 18O 185 19 O Asp Gly Phe Arg Val Asp Val Lieu. Trp Lieu. Lieu. Gly Lys Asp Pro Lieu 195 2OO 2O5 Phe Arg Asp Glu Pro Gly Ser Pro Lieu. Trp Arg Pro Gly Lieu Pro Asp 21 O 215 22O Arg Ala Arg His Glu. His Lieu. Tyr Thr Glu Asp Gln Pro Glu. Thr Tyr 225 23 O 235 24 O Ala Tyr Val Arg Glu Met Arg Glin Val Lieu. Asp Glu Phe Ser Glu Pro 245 250 255 Gly Arg Glu Arg Val Met Val Gly Glu Ile Tyr Lieu Pro Lieu Pro Arg 26 O 265 27 O Lieu Val Arg Tyr Tyr Ala Ala Gly Cys His Leu Pro Phe Asin Phe Ser 27s 28O 285 Lieu Val Thr Glu Gly Lieu. Ser Asp Trp Arg Pro Glu Asn Lieu Ala Arg 29 O 295 3 OO Ile Val Glu Thr Tyr Glu Gly Lieu. Lieu. Thir Arg Trp Asp Trp Pro Asn 3. OS 310 315 32O Trp Val Lieu. Gly Asn His Asp Glin Pro Arg Lieu Ala Ser Arg Lieu. Gly 3.25 330 335 Glu Pro Glin Ala Arg Val Ala Ala Met Lieu. Lieu. Phe Thr Lieu. Arg Gly 34 O 345 35. O Thr Pro Thir Trp Tyr Tyr Gly Asp Glu Lieu Ala Leu Pro Asn Gly Lieu. 355 360 365 Ile Pro Pro Glu Lys Val Glin Asp Pro Ala Ala Lieu. Arg Glin Arg Asp 37 O 375 38O Arg Glu Pro Thr Ala Tyr His Thr Lieu. Gly Arg Asp Pro Glu Arg Thr 385 390 395 4 OO Pro Met Pro Trp Asp Ala Ser Pro Tyr Gly Gly Phe Ser Thr Val Glu 4 OS 41O 415 US 2011/020 1 059 A1 Aug. 18, 2011 86

- Continued Pro Trp Lieu Pro Lieu. ASn Pro Asp Tyr Lys Thr Arg Asn Val Ala Ala 42O 425 43 O Glin Glu Lys Asp Pro Arg Ser Met Lieu. His Lieu Val Lys Arg Lieu. Ile 435 44 O 445 Ala Lieu. Arg Lys Asp Pro Gly Lieu. Lieu. Tyr Gly Ala Tyr Arg Thr Tyr 450 45.5 460 Arg Ala Arg Glu Gly Val Tyr Ala Tyr Lieu. Arg Gly Glu Gly Trp Lieu 465 470 47s 48O Val Ala Lieu. Asn Lieu. Thr Glu Lys Glu Lys Ala Lieu. Glu Lieu Pro Arg 485 490 495 Gly Gly Arg Val Val Lieu. Ser Thr His Lieu. Asp Arg Glu Glu Arg Val SOO 505 51O Gly Glu Arg Lieu. Phe Lieu. Arg Pro Asp Glu Gly Val Ala Val Arg Lieu. 515 52O 525 Asp

<210s, SEQ ID NO 31 211 LENGTH: 575 212. TYPE: PRT <213> ORGANISM: Thermus thermophilus 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (554) <223> OTHER INFORMATION: alpha-1, 5-glucosidase

<4 OOs, SEQUENCE: 31 Met Val Asp Gly Glu Gly Arg Lieu. Lieu. Gly Ile Val Thr Arg Gly Arg 1. 5 1O 15 Lieu. Lieu Ala Ala Lieu Ala Gly Arg Tyr Thr Pro Glu Val Pro Glin Ser 2O 25 3O Gly Val Asp Ser Gly Pro Glin Ser Gly Val Asp Ser Gly Ser Met Ser 35 4 O 45 Trp Trp Glin Arg Ala Val Ile Tyr Glin Val Tyr Pro Arg Ser Phe Glin SO 55 6 O Asp Thr Asn Gly Asp Gly Val Gly Asp Lieu. Glu Gly Ile Arg Arg Arg 65 70 7s 8O Lieu Pro Tyr Phe Lys Ser Lieu. Gly Val Asp Ala Phe Trp Leu Ser Pro 85 90 95 Phe Tyr Lys Ser Pro Met Lys Asp Phe Gly Tyr Asp Val Ala Asp Tyr 1OO 105 11 O Cys Asp Wall Asp Pro Val Phe Gly. Thir Lieu. Glin Asp Phe Asp Arg Lieu. 115 12 O 125 Lieu. Glu Glu Ala His Ala Lieu. Gly Lieu Lys Val Lieu Val Asp Lieu Val 13 O 135 14 O Pro Asn His Thr Ser Ser Glu. His Pro Trp Phe Lieu. Glu Ser Arg Ala 145 150 155 160 Ser Arg Asn. Ser Pro Lys Arg Asp Trp Tyr Val Trp Lys Asp Pro Ala 1.65 17O 17s Pro Asp Gly Gly Pro Pro Asn Asn Trp Glin Ser Phe Phe Gly Gly Pro 18O 185 19 O Ala Trp Thr Lieu. Asp Glu Ala Thr Gly Glin Tyr Tyr Lieu. His Leu Phe 195 2OO 2O5 Lieu Pro Glu Gln Pro Asp Lieu. Asn Trp Asp Asn. Pro Glu Val Arg Glu 21 O 215 22O US 2011/020 1 059 A1 Aug. 18, 2011 87

- Continued

Ala Ile Lys Glu Val Met Arg Phe Trp Lieu. Arg Arg Gly Val Asp Gly 225 23 O 235 24 O Phe Arg Val Asp Val Lieu. Trp Lieu. Lieu. Gly Lys Asp Pro Lieu. Phe Arg 245 250 255 Asp Glu Pro Gly Ser Pro Lieu. Trp Arg Pro Gly Lieu Pro Asp Arg Ala 26 O 265 27 O Arg His Glu. His Lieu. Tyr Thr Glu Asp Gln Pro Glu Thr Tyr Ala Tyr 27s 28O 285 Val Arg Glu Met Arg Glin Val Lieu. Asp Glu Phe Ser Glu Pro Gly Arg 29 O 295 3 OO Glu Arg Val Met Val Gly Glu Ile Tyr Lieu Pro Lieu Pro Arg Lieu Val 3. OS 310 315 32O Arg Tyr Tyr Ala Ala Gly Cys His Leu Pro Phe Asn Phe Ser Leu Val 3.25 330 335 Thr Glu Gly Lieu. Ser Asp Trp Arg Pro Glu Asn Lieu Ala Arg Ile Val 34 O 345 35. O Glu Thr Tyr Glu Gly Lieu Lleu. Thr Arg Trp Asp Trp Pro Asn Trp Val 355 360 365 Lieu. Gly Asn His Asp Glin Pro Arg Lieu Ala Ser Arg Lieu. Gly Glu Pro 37 O 375 38O Glin Ala Arg Val Ala Ala Met Lieu. Lieu. Phe Thr Lieu. Arg Gly Thr Pro 385 390 395 4 OO Thir Trp Tyr Tyr Gly Asp Glu Lieu Ala Lieu Pro Asn Gly Lieu. Ile Pro 4 OS 41O 415 Pro Glu Lys Val Glin Asp Pro Ala Ala Lieu. Arg Glin Arg Asp Arg Glu 42O 425 43 O Pro Thr Ala Tyr His Thr Lieu. Gly Arg Asp Pro Glu Arg Thr Pro Met 435 44 O 445 Pro Trp Asp Ala Ser Pro Tyr Gly Gly Phe Ser Thr Val Glu Pro Trp 450 45.5 460 Lieu Pro Lieu. Asn. Pro Asp Tyr Lys Thr Arg Asn Val Ala Ala Glin Glu 465 470 47s 48O Lys Asp Pro Arg Ser Met Lieu. His Lieu Val Lys Arg Lieu. Ile Ala Lieu. 485 490 495 Arg Lys Asp Pro Gly Lieu. Lieu. Tyr Gly Ala Tyr Arg Thr Tyr Arg Ala SOO 505 51O Arg Glu Gly Val Tyr Ala Tyr Lieu. Arg Gly Glu Gly Trp Lieu Val Ala 515 52O 525 Lieu. Asn Lieu. Thr Glu Lys Glu Lys Ala Lieu. Glu Lieu Pro Arg Gly Gly 53 O 535 54 O Arg Val Val Lieu. Ser Thr His Lieu. Asp Arg Glu Glu Arg Val Gly Glu 5.45 550 555 560 Arg Lieu. Phe Lieu. Arg Pro Asp Glu Gly Val Ala Val Arg Lieu. Asp 565 st O sts

<210s, SEQ ID NO 32 &211s LENGTH: 528 212. TYPE: PRT <213> ORGANISM: Thermus thermophilus 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (507) <223> OTHER INFORMATION: alpha-1, 5-glucosidase US 2011/020 1 059 A1 Aug. 18, 2011 88

- Continued

<4 OOs, SEQUENCE: 32 Met Trp Trp Lys Glu Ala Val Ile Tyr Glin Val Tyr Pro Arg Ser Phe 1. 5 1O 15 Glin Asp Thir Asn Gly Asp Gly Val Gly Asp Lieu. Glu Gly Val Arg Arg 2O 25 3O Arg Lieu Pro Tyr Lieu Lys Ser Lieu. Gly Val Asp Ala Lieu. Trp Lieu. Ser 35 4 O 45 Pro Phe Tyr Lys Ser Pro Met Lys Asp Phe Gly Tyr Asp Val Ala Asp SO 55 6 O Tyr Cys Asp Wall Asp Pro Val Phe Gly. Thir Lieu. Glin Asp Phe Asp Arg 65 70 7s 8O Lieu. Lieu. Glu Glu Ala His Ala Lieu. Gly Lieu Lys Val Lieu Val Asp Lieu. 85 90 95 Val Pro Asn His Thr Ser Ser Glu. His Pro Trp Phe Lieu. Glu Ser Arg 1OO 105 11 O Ala Ser Arg Asn. Ser Pro Lys Arg Asp Trip Tyr Ile Trp Lys Asp Pro 115 12 O 125 Ala Pro Asp Gly Gly Pro Pro Asn Asn Trp Glin Ser Phe Phe Gly Gly 13 O 135 14 O Pro Ala Trp Thr Lieu. Asp Glu Ala Thr Gly Glin Tyr Tyr Lieu. His Glin 145 150 155 160 Phe Lieu Pro Glu Glin Pro Asp Lieu. Asn Trp Arg ASn Pro Glu Val Arg 1.65 17O 17s Glu Ala Ile Tyr Glu Wal Met Arg Phe Trp Lieu. Arg Arg Gly Val Asp 18O 185 19 O Gly Phe Arg Val Asp Val Lieu. Trp Lieu. Lieu Ala Glu Asp Lieu. Lieu. Phe 195 2OO 2O5 Arg Asp Glu Pro Gly Asn Pro Asp Trp Arg Pro Gly Met Trp Asp Arg 21 O 215 22O Gly Arg His Lieu. His Ile Phe Thr Glu Asp Gln Pro Glu Thr Tyr Ala 225 23 O 235 24 O Tyr Val Arg Glu Met Arg Glin Val Lieu. Asp Glu Phe Ser Glu Pro Gly 245 250 255 Arg Glu Arg Val Met Val Gly Glu Ile Tyr Lieu Pro Tyr Pro Glin Leu 26 O 265 27 O Val Arg Tyr Tyr Glin Ala Gly Cys His Leu Pro Phe Asn Phe His Leu 27s 28O 285 Ile Phe Arg Gly Lieu Pro Asp Trp Arg Pro Glu Asn Lieu Ala Arg Ile 29 O 295 3 OO Val Glu Glu Tyr Glu Ser Lieu. Lieu. Thir Arg Trp Asp Trp Pro Asn Trp 3. OS 310 315 32O Val Lieu. Gly Asn His Asp Glin Pro Arg Lieu Ala Ser Arg Lieu. Gly Glu 3.25 330 335 Ala Glin Ala Arg Val Ala Ala Met Lieu. Lieu. Phe Thr Lieu. Arg Gly Thr 34 O 345 35. O Pro Thir Trp Tyr Tyr Gly Asp Glu Ile Gly Met Lys Asn Gly Glu Ile 355 360 365 Pro Pro Glu Lys Val Glin Asp Pro Ala Ala Lieu. Arg Gln Lys Asp Arg 37 O 375 38O Lieu. Gly Glu. His Asn Lieu Pro Pro Gly Arg Asp Pro Glu Arg Thr Pro US 2011/020 1 059 A1 Aug. 18, 2011 89

- Continued

385 390 395 4 OO Met Glin Trp Asp Asp Thr Pro Phe Ala Gly Phe Ser Thr Val Glu Pro 4 OS 41O 415 Trp Lieu Pro Val Asn Pro Asp Tyr Lys Thr Arg Asn Val Ala Ala Glin 42O 425 43 O Glu Glin Asp Pro Arg Ser Met Lieu. His Lieu Val Arg Arg Lieu. Ile Ala 435 44 O 445 Lieu. Arg Lys Asp Pro Asp Lieu. Lieu. Tyr Gly Ala Tyr Arg Thr Tyr Arg 450 45.5 460 Ala Arg Glu Gly Val Tyr Ala Tyr Lieu. Arg Gly Glu Gly Trp Lieu Val 465 470 47s 48O Ala Lieu. Asn Lieu. Thr Glu Lys Glu Lys Ala Lieu. Glu Lieu Pro Arg Gly 485 490 495 Gly Arg Val Val Lieu. Ser Thr His Lieu. Asp Arg Glu Glu Arg Val Gly SOO 505 51O Glu Arg Lieu. Phe Lieu. Arg Pro Asp Glu Gly Val Ala Val Arg Lieu. Asp 515 52O 525

<210s, SEQ ID NO 33 &211s LENGTH: 529 212. TYPE: PRT <213> ORGANISM: Thermus thermophilus 22 Os. FEATURE: <221 > NAME/KEY: enzyme <222s. LOCATION: (1) . . (508) <223> OTHER INFORMATION: alpha-1, 5-glucosidase

<4 OOs, SEQUENCE: 33 Met Ser Trp Trp Glin Arg Ala Val Ile Tyr Glin Val Tyr Pro Arg Ser 1. 5 1O 15 Phe Glin Asp Thir Asn Gly Asp Gly Val Gly Asp Lieu. Glu Gly Ile Arg 2O 25 3O Arg Arg Lieu Pro Tyr Lieu Lys Ser Lieu. Gly Val Asp Ala Lieu. Trp Lieu 35 4 O 45 Ser Pro Phe Tyr Lys Ser Pro Met Lys Asp Phe Gly Tyr Asp Val Ala SO 55 6 O Asp Tyr Cys Asp Val Asp Pro Val Phe Gly Thr Lieu. Glin Asp Phe Asp 65 70 7s 8O Arg Lieu. Lieu. Glu Glu Ala His Ala Lieu. Gly Lieu Lys Val Lieu Val Asp 85 90 95 Lieu Val Pro Asn His Thir Ser Ser Glu. His Pro Trp Phe Lieu. Glu Ser 1OO 105 11 O Arg Ala Ser Arg Asn. Ser Pro Lys Arg Asp Trp Tyr Ile Trp Lys Asp 115 12 O 125 Pro Ala Pro Asp Gly Gly Pro Pro Asn Asn Trp Glin Ser Phe Phe Gly 13 O 135 14 O Gly Pro Ala Trp Thr Lieu. Asp Glu Ala Thr Gly Glin Tyr Tyr Lieu. His 145 150 155 160 Lieu. Phe Lieu Pro Glu Glin Pro Asp Lieu. Asn Trp Arg ASn Pro Glu Val 1.65 17O 17s Arg Glu Ala Ile Lys Glu Val Met Arg Phe Trp Lieu. Arg Arg Gly Val 18O 185 19 O Asp Gly Phe Arg Val Asp Val Lieu. Trp Lieu. Lieu. Gly Lys Asp Pro Lieu 195 2OO 2O5 US 2011/020 1 059 A1 Aug. 18, 2011 90

- Continued

Phe Arg Asp Glu Pro Gly Ser Pro Lieu. Trp Arg Pro Gly Lieu Pro Asp 21 O 215 22O Arg Ala Arg His Glu. His Lieu. Tyr Thr Glu Asp Gln Pro Glu. Thr Tyr 225 23 O 235 24 O Ala Tyr Val Arg Glu Met Arg Glin Val Lieu. Asp Glu Phe Ser Glu Pro 245 250 255 Gly Arg Glu Arg Val Met Val Gly Glu Ile Tyr Lieu Pro Lieu Pro Arg 26 O 265 27 O Lieu Val Arg Tyr Tyr Ala Ala Gly Cys His Leu Pro Phe Asin Phe Ser 27s 28O 285 Lieu Val Thr Glu Gly Lieu. Ser Asp Trp Arg Pro Glu Asn Lieu Ala Arg 29 O 295 3 OO Ile Val Glu Thir Tyr Glu Gly Lieu. Lieu. Ser Arg Trp Asp Trp Pro Asn 3. OS 310 315 32O Trp Val Lieu. Gly Asn His Asp Glin Pro Arg Lieu Ala Ser Arg Lieu. Gly 3.25 330 335 Glu Pro Glin Ala Arg Val Ala Ala Met Lieu. Lieu. Phe Thr Lieu. Arg Gly 34 O 345 35. O Thr Pro Thir Trp Tyr Tyr Gly Asp Glu Lieu Ala Leu Pro Asn Gly Lieu. 355 360 365 Ile Pro Pro Glu Lys Val Glin Asp Pro Ala Ala Lieu. Arg Glin Arg Asp 37 O 375 38O Arg Glu Pro Thr Ala Tyr His Thr Lieu. Gly Arg Asp Pro Glu Arg Thr 385 390 395 4 OO Pro Met Pro Trp Asp Ala Ser Pro Tyr Gly Gly Phe Ser Thr Val Glu 4 OS 41O 415 Pro Trp Lieu Pro Lieu. ASn Pro Asp Tyr Arg Thr Arg Asn Val Ala Ala 42O 425 43 O Glin Glu Lys Asp Pro Arg Ser Met Lieu. His Lieu Val Lys Arg Lieu. Ile 435 44 O 445 Ala Lieu. Arg Lys Asp Pro Asp Lieu. Lieu. Tyr Gly Ala Tyr Arg Thr Tyr 450 45.5 460 Arg Ala Arg Glu Gly Val Tyr Ala Tyr Lieu. Arg Gly Glu Gly Trp Lieu 465 470 47s 48O Val Ala Lieu. Asn Lieu. Thr Glu Lys Glu Lys Ala Lieu. Glu Lieu Pro Arg 485 490 495 Gly Gly Arg Val Val Lieu. Ser Thr His Lieu. Asp Arg Glu Glu Arg Val SOO 505 51O Gly Glu Arg Lieu. Phe Lieu. Arg Pro Asp Glu Gly Val Ala Val Arg Lieu. 515 52O 525 Asp

<210s, SEQ ID NO 34 &211s LENGTH: 587 212. TYPE: PRT <213> ORGANISM; Bacillus 22 Os. FEATURE: <221> NAME/KEY: enzyme <222s. LOCATION: (1) . . (587) <223> OTHER INFORMATION: alpha-1,1-glucosidase

<4 OOs, SEQUENCE: 34

Met Ser Thir Ala Lieu. Thir Glin. Thir Ser Thir Asn. Ser Glin Glin Ser Pro US 2011/020 1 059 A1 Aug. 18, 2011 91

- Continued

1O 15

Ile Arg Arg Ala Trp Trp Glu Ala Wall Wall Glin Ile Tyr Pro 2O 25

Arg Ser Phe Met Asp Ser Asn Gly Asp Gly Ile Gly Asp Luell Arg Gly 35 4 O 45

Ile Luell Ser Lell Asp Tyr Luell Luell Luell Gly Wall Asp Wall Luell SO 55 6 O

Trp Luell Asn Pro Ile Tyr Asp Ser Pro Asn Asp Asp Met Gly Tyr Asp 65 70

Ile Arg Asp Tyr Ile Met Glu Glu Phe Gly Thir Met Glu Asp 85 90 95

Phe Glu Glu Luell Lell Arg Glu Wall His Ala Arg Gly Met Lys Luell Wall 1OO 105 11 O

Met Asp Luell Wall Ala Asn His Thir Ser Asp Glu His Pro Trp Phe Ile 115 12 O 125

Glu Ser Arg Ser Ser Arg Asp Asn Pro Tyr Arg Asp Trp Ile Trp 13 O 135 14 O

Arg Asp Pro Asp Gly Arg Glu Pro Asn ASn Trp Lell Ser Phe 145 150 155 160

Ser Gly Ser Ala Trp Glu Tyr Asp Glu Arg Thir Gly Glin Tyr Tyr Luell 1.65 17s

His Luell Phe Ser Arg Arg Glin Pro Asp Luell ASn Trp Glu Asn Pro 18O 185 19 O

Wall Arg Glu Ala Ile Phe Glu Met Met Arg Phe Trp Lell Asp Gly 195

Ile Asp Gly Phe Met Asp Wall Ile Asn Ala Ile Ala Ala Glu 21 O 215

Gly Luell Pro Asp Ala Pro Ala Arg Pro Gly Glu Arg Ala Trp Gly 225 23 O 235 24 O

Gly Glin Tyr Phe Lell Asn Glin Pro Wall His Glu Tyr Luell Arg Glu 245 250 255

Met Asp Lys Wall Lell Ser His Tyr Asp Ile Met Thir Wall Gly Glu 26 O 265 27 O

Thir Gly Gly Wall Thir Thir Asp Ala Luell Luell Phe Ala Gly Glu Asp 285

Arg Arg Glu Luell Asn Met Wall Phe Glin Phe Glu His Met Asp Ile Asp 29 O 295 3 OO

Ala Thir Asp Gly Asp Lys Trp Arg Pro Arg Pro Trp Arg Luell Thir Glu 3. OS 310 315

Lell Thir Ile Met Thir Arg Trp Glin Asn Asp Lell Tyr Gly Lys Ala 3.25 330 335

Trp Asn Ser Luell Tyr Trp Thir Asn His Asp Glin Pro Arg Ala Wall Ser 34 O 345 35. O

Arg Phe Gly Asn Asp Gly Pro Tyr Arg Wall Glu Ser Ala Met Luell 355 360 365

Ala Thir Wall Luell His Met Met Glin Gly Thir Pro Tyr Ile Glin Gly 37 O 375

Glu Glu Ile Gly Met Thir Asn Pro Phe Asp Ser Ile Asp Glu Tyr 385 390 395 4 OO

Arg Asp Wall Glu Ile His Asn Luell Trp Arg His Arg Wall Met Glu Gly 4 OS 41O 415 US 2011/020 1 059 A1 Aug. 18, 2011 92

- Continued

Gly Glin Asp Pro Ala Glu Val Lieu. Arg Val Ile Glin Lieu Lys Gly Arg 42O 425 43 O Asp Asn Ala Arg Thr Pro Met Gln Trp Asp Asp Ser Pro Asn Ala Gly 435 44 O 445 Phe Thir Thr Gly Thr Pro Trp Ile Llys Val Asn Pro Asn Tyr Arg Glu 450 45.5 460 Ile Asin Val Lys Glin Ala Lieu Ala Asp Pro Asn. Ser Ile Phe His Tyr 465 470 47s 48O Tyr Arg Arg Lieu. Ile Glin Lieu. Arg Lys Gln His Pro Ile Val Val Tyr 485 490 495 Gly Lys Tyr Asp Lieu. Ile Lieu Pro Asp His Glu Glu Ile Trp Ala Tyr SOO 505 51O Thir Arg Thr Lieu. Gly Asp Glu Arg Trp Lieu. Ile Val Ala Asn. Phe Phe 515 52O 525 Gly Gly Thr Pro Glu Phe Glu Lieu Pro Pro Glu Val Arg Cys Glu Gly 53 O 535 54 O Ala Glu Lieu Val Ile Ala Asn Tyr Pro Val Asp Asp Ser Glu Ala Gly 5.45 550 555 560 Gly Pro Ala Ala Ala Gly Ala Pro His Arg Phe Arg Lieu. Arg Pro Tyr 565 st O sts Glu Cys Arg Val Tyr Arg Lieu. Lieu. Gly Trp His 58O 585

<210s, SEQ ID NO 35 &211s LENGTH: 4626 &212s. TYPE: DNA <213s ORGANISM: unknown 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic gene 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) ... (4626) <223> OTHER INFORMATION: dicot optimized dextranslucrase with leucrose synthase activity

<4 OOs, SEQUENCE: 35 atg Ctt gag tot ggt gtt gtt cac gct gat gat gtt aag caa gtg gtt 48 Met Lieu. Glu Ser Gly Val Val His Ala Asp Asp Wall Lys Glin Val Val 1. 5 1O 15 gtt caa gaa cca gct act gct caa act tct gga cca gga caa caa act 96 Val Glin Glu Pro Ala Thr Ala Glin Thr Ser Gly Pro Gly Glin Glin Thr 2O 25 3O cca gct cag gCt aag att gct tct gala caa gag gct gag aaa gtt act 144 Pro Ala Glin Ala Lys Ile Ala Ser Glu Glin Glu Ala Glu Lys Val Thr 35 4 O 45 cca gct gat aag gtg aca gat gat gtt gct gct tct gala aag cca gct 192 Pro Ala Asp Llys Val Thr Asp Asp Val Ala Ala Ser Glu Lys Pro Ala SO 55 6 O aaa cca gct gag aat act gag gct act gtt cag act aat gct cala gag 24 O Llys Pro Ala Glu Asn Thr Glu Ala Thr Val Glin Thr Asn Ala Glin Glu 65 70 7s 8O cca gca aag cct gct gat aca aaa gala gct tcc act gag aag gCt gct 288 Pro Ala Lys Pro Ala Asp Thir Lys Glu Ala Ser Thr Glu Lys Ala Ala 85 90 95 gtt gct gala gala gtt aag gct gct aac gct att act gag at C cca aag 336 Val Ala Glu Glu Val Lys Ala Ala Asn Ala Ile Thr Glu Ile Pro Llys 1OO 105 11 O

US 2011/020 1 059 A1 Aug. 18, 2011 98

- Continued

1. 5 1O 15 Val Glin Glu Pro Ala Thr Ala Glin Thr Ser Gly Pro Gly Glin Glin Thr 2O 25 3O Pro Ala Glin Ala Lys Ile Ala Ser Glu Glin Glu Ala Glu Lys Val Thr 35 4 O 45 Pro Ala Asp Llys Val Thr Asp Asp Val Ala Ala Ser Glu Lys Pro Ala SO 55 6 O Llys Pro Ala Glu Asn Thr Glu Ala Thr Val Glin Thr Asn Ala Glin Glu 65 70 7s 8O Pro Ala Lys Pro Ala Asp Thir Lys Glu Ala Ser Thr Glu Lys Ala Ala 85 90 95 Val Ala Glu Glu Val Lys Ala Ala Asn Ala Ile Thr Glu Ile Pro Llys 1OO 105 11 O Thr Glu Val Ala Asp Glin Asn Lys Glin Ala Arg Pro Thir Thir Ala Glin 115 12 O 125 Asp Glin Glu Gly Asp Lys Arg Glu Lys Thr Ala Val Glu Asp Llys Ile 13 O 135 14 O Val Ala Asn. Pro Llys Val Ala Lys Lys Asp Arg Lieu Pro Glu Pro Gly 145 150 155 160 Ser Lys Glin Gly Ala Ile Ala Glu Arg Met Val Ala Asp Glin Ala Glin 1.65 17O 17s Pro Ala Pro Val Asn Ala Asp His Asp Asp Asp Val Lieu. Ser His Ile 18O 185 19 O Llys Thir Ile Asp Gly Lys Asn Tyr Tyr Val Glin Asp Asp Gly Thr Val 195 2OO 2O5 Llys Lys Asn. Phe Ala Val Glu Lieu. Asn Gly Arg Ile Lieu. Tyr Phe Asp 21 O 215 22O Ala Glu Thr Gly Ala Lieu Val Asp Ser Asn. Glu Tyr Glin Phe Glin Glin 225 23 O 235 24 O Gly. Thir Ser Ser Lieu. Asn Asn Glu Phe Ser Gln Lys Asn Ala Phe Tyr 245 250 255 Gly. Thir Thr Asp Lys Asp Ile Glu Thr Val Asp Gly Tyr Lieu. Thir Ala 26 O 265 27 O Asp Ser Trp Tyr Arg Pro Llys Phe Ile Lieu Lys Asp Gly Lys Thir Trip 27s 28O 285 Thr Ala Ser Thr Glu Thir Asp Leu Arg Pro Leu Lleu Met Ala Trp Trp 29 O 295 3 OO Pro Asp Lys Arg Thr Glin Ile Asn Tyr Lieu. Asn Tyr Met Asin Glin Glin 3. OS 310 315 32O Gly Lieu. Gly Ala Gly Ala Phe Glu Asn Llys Val Glu Glin Ala Lieu. Lieu. 3.25 330 335 Thr Gly Ala Ser Glin Glin Val Glin Arg Lys Ile Glu Glu Lys Ile Gly 34 O 345 35. O Lys Glu Gly Asp Thir Lys Trp Lieu. Arg Thr Lieu Met Gly Ala Phe Val 355 360 365 Lys Thr Glin Pro Asn Trp Asn Ile Llys Thr Glu Ser Glu Thir Thr Gly 37 O 375 38O Thir Lys Lys Asp His Lieu. Glin Gly Gly Ala Lieu. Lieu. Tyr Thir Asn. Asn 385 390 395 4 OO Glu Lys Ser Pro His Ala Asp Ser Llys Phe Arg Lieu. Lieu. Asn Arg Thr 4 OS 41O 415 US 2011/020 1 059 A1 Aug. 18, 2011 99

- Continued

Pro Thir Ser Glin Thr Gly Thr Pro Llys Tyr Phe Ile Asp Llys Ser Asn 42O 425 43 O Gly Gly Tyr Glu Phe Lieu. Lieu Ala Asn Asp Phe Asp Asn. Ser Asn Pro 435 44 O 445 Ala Val Glin Ala Glu Gln Lieu. Asn Trp Lieu. His Tyr Met Met Asin Phe 450 45.5 460 Gly Ser Ile Val Ala Asn Asp Pro Thr Ala Asn. Phe Asp Gly Val Arg 465 470 47s 48O Val Asp Ala Val Asp Asn Val Asn Ala Asp Lieu. Lieu. Glin Ile Ala Ser 485 490 495 Asp Tyr Phe Llys Ser Arg Tyr Llys Val Gly Glu Ser Glu Glu Glu Ala SOO 505 51O Ile Llys His Lieu. Ser Ile Lieu. Glu Ala Trp Ser Asp Asn Asp Pro Asp 515 52O 525 Tyr Asn Lys Asp Thir Lys Gly Ala Glin Lieu Ala Ile Asp Asn Llys Lieu. 53 O 535 54 O Arg Lieu. Ser Lieu. Lieu. Tyr Ser Phe Met Arg Asn Lieu. Ser Ile Arg Ser 5.45 550 555 560 Gly Val Glu Pro Thr Ile Thr Asn Ser Lieu. Asn Asp Arg Ser Ser Glu 565 st O sts Llys Lys Asn Gly Glu Arg Met Ala Asn Tyr Ile Phe Val Arg Ala His 58O 585 59 O Asp Asp Glu Val Glin Thr Val Ile Ala Asp Ile Ile Arg Glu Asn. Ile 595 6OO 605 Asn Pro Asn Thr Asp Gly Lieu. Thr Phe Thr Met Asp Glu Lieu Lys Glin 610 615 62O Ala Phe Lys Ile Tyr Asn. Glu Asp Met Arg Lys Ala Asp Llys Llys Tyr 625 630 635 64 O Thr Glin Phe Asin Ile Pro Thr Ala His Ala Leu Met Leu Ser Asn Lys 645 650 655 Asp Ser Ile Thr Arg Val Tyr Tyr Gly Asp Lieu. Tyr Thr Asp Asp Gly 660 665 67 O Glin Tyr Met Glu Lys Llys Ser Pro Tyr His Asp Ala Ile Asp Ala Lieu. 675 68O 685 Lieu. Arg Ala Arg Ile Llys Tyr Val Ala Gly Gly Glin Asp Met Llys Val 69 O. 695 7 OO Thr Tyr Met Gly Val Pro Arg Glu Ala Asp Llys Trp Ser Tyr Asn Gly 7 Os 71O 71s 72O Ile Lieu. Thir Ser Val Arg Tyr Gly Thr Gly Ala Asn. Glu Ala Thr Asp 72 73 O 73 Glu Gly Thr Ala Glu Thr Arg Thr Glin Gly Met Ala Val Ile Ala Ser 740 74. 7 O Asn Asn Pro Asn Lieu Lys Lieu. Asn. Glu Trp Asp Llys Lieu. Glin Val Asn 7ss 760 765 Met Gly Ala Ala His Lys Asn. Glin Tyr Tyr Arg Pro Val Lieu. Lieu. Thir 770 775 78O Thir Lys Asp Gly Ile Ser Arg Tyr Lieu. Thir Asp Glu Glu Val Pro Glin 78s 79 O 79. 8OO Ser Lieu. Trp Llys Llys Thr Asp Ala Asn Gly Ile Lieu. Thir Phe Asp Met 805 810 815