WO2005020721A1 - Beverage compositions comprising monatin and methods of making same - Google Patents

Abstract

The present invention relates to novel beverage compositions comprising monatin and methods for making such compositions. The present invention also relates to beverage compositions comprising specific monatin stereoisomers, specific blends of monatin stereoisomers, and/or monatin produced via a biosynthetic pathway in vivo (e.g., inside cells) or in vitro.

Description

BEVERAGE COMPOSITIONS COMPRISING MONATIN AND METHODS OF MAKING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application 60/497,627 filed August 25, 2003, the entire disclosure of which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to novel beverage compositions comprising monatin and methods for making such compositions. The present invention also relates to beverage compositions comprising specific monatin stereoisomers, specific blends of monatin stereoisomers, and/or monatin produced via a biosynthetic pathway in vivo (e.g., inside cells) or in vitro.

BACKGROUND

The use of non-caloric high intensity sweeteners is increasing due to health concerns raised over childhood obesity, type II diabetes, and related illnesses. Thus, a demand exists for sweeteners having a sweetness significantly higher than that in conventional sweeteners, such as granulated sugar (sucrose). Many high intensity sweeteners contain unpleasant off-flavors and/or unexpected and less-than-desirable sweetness profiles. In attempts to overcome these problems, the industry continues to conduct significant research into bitterness inhibitors, off- flavor masking technologies, and sweetener blends to achieve a sweetness profile similar to sucrose.

Monatin (2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid) is a naturally-occurring, high intensity sweetener isolated from the plant Sclerochiton ilicifolius, found in the Transvaal Region of South Africa. Monatin contains no carbohydrate or sugar, and nearly no calories, unlike sucrose or other nutritive sweeteners at equal sweetness. SUMMARY

The present invention relates to beverage compositions comprising monatin (2-hydroxy-2- (indol-3-ylmethyl)-4-aminoglutaric acid — also known as 4-amino-2-hydroxy-2-(lH-indol-3- ylmethyl)-pentanedioic acid, or alternatively, based on an alternate numbering system, 4- hydroxy-4-(3-indolylmethyl) glutamic acid), a compound having the formula:

Monatin is a naturally-occurring, high intensity sweetener. Monatin has four stereoisomeric forms: 2R, 4R (the "R,R stereoisomer" or "R,R monatin"), 2S, 4S (the "S,S stereoisomer" or "S,S monatin"), 2R, 4S (the "R,S stereoisomer" or "R,S monatin"), and 2S, 4R (the "S,R stereoisomer" or "S,R monatin"). As used herein, unless stated otherwise, "monatin" refers to all four stereoisomers of monatin, as well as any blends of any combination of monatin stereoisomers (e.g., a blend ofthe R,R and S,S, stereoisomers of monatin).

Monatin has an excellent sweetness quality. Monatin has a flavor profile that is as clean or cleaner that other known high intensity sweeteners. The dose response curve of monatin is more linear, and therefore more similar to sucrose than other high intensity sweeteners, such as saccharin. Monatin' s excellent sweetness profile makes monatin desirable for use in tabletop sweeteners, foods, beverages and other products.

Different stereoisomers of monatin, including the R,R and S,S stereoisomers, have potential in the sweetener industry, either as separate ingredients or in blends. Monatin has a desirable taste profile alone or when mixed with carbohydrates. Monatin, and blends of stereoisomers of monatin with other sweeteners, such as carbohydrates, are thought to have superior taste characteristics and/or physical qualities, as compared to other high intensity sweeteners. For example, monatin is more stable than aspartame (also known as "APM"), has a cleaner taste than saccharin, and one stereoisomer (R,R monatin) is more sweet than sucralose. Likewise, monatin sweeteners do not have the bitter aftertaste associated with saccharin, or the metallic, acidic, astringent or throat burning aftertastes of some other high potency sweeteners. In addition, monatin sweeteners do not exhibit the licorice aftertaste associated with certain natural sweeteners, such as stevioside and glycyrrhizin.

Furthermore, unlike aspartame sweeteners, monatin sweeteners do not require a phenylalanine warning for patients with phenylketonuria. Likewise, it is expected that monatin is not cariogenic (i.e., does not promote tooth decay) because it does not contain fermentable carbohydrates. It is also expected that monatin will not cause a drop below pH ~5.7 (which can be harmful to teeth) when mixed with saliva, as measured in a pH drop test. Because of its intense sweetness, the R,R stereoisomer in particular should be economically competitive compared to other high intensity sweeteners.

In one aspect, the present invention provides a beverage composition comprising monatin or salt thereof, such as R,R, S,S, R,S or S,R monatin or a blend of different stereoisomers. As used herein, "beverage composition" refers to a composition that is drinkable as is (i.e., does not need to be diluted, or is "ready-to-drink") or a concentrate that can be diluted or mixed with a liquid to form a drinkable beverage. For example, the beverage composition can be a dry beverage mix (e.g., chocolate beverage mix, fruit beverage mix, malted beverage, or lemonade mix) that can be mixed, for example, with water or milk, to form a drinkable beverage. As another example, the beverage composition can be a beverage syrup that can be diluted, e.g., with carbonated water to form a carbonated soft drink. As another example, a beverage syrup or mix can be diluted with water/ice and one or more other ingredients (e.g., tequila) to form an alcoholic drink (e.g., a margarita). As described herein, monatin can be substituted for other common bulk sweeteners without a noticeable difference in taste. Carbonated beverages containing monatin have an improved taste profile over cola-type carbonated soft drinks sweetened with aspartame. Monatin is more stable than aspartame under acidic soft drink conditions and it is expected that monatin has a longer shelf life. As used herein, the term "carbonated" means that the beverage contains both dissolved and dispersed carbon dioxide.

In some embodiments, beverage compositions include a blend of monatin and a sweetener (e.g., sucrose or high fructose corn syrup). In other embodiments, beverage compositions comprising monatin include a flavoring, caffeine and/or a bulk sweetener. Bulk sweeteners may be, for example, sugar sweeteners, sugarless sweeteners and lower glycemic carbohydrates (i.e., carbohydrates with a lower glycemic index than glucose). In other embodiments, monatin-containing beverage compositions include a high-intensity sweetener and/or a lower glycemic carbohydrate. In other embodiments, monatin-containing beverage compositions include a sweetness enhancer.

In some embodiments, the beverage compositions comprise monatin that consists essentially of S,S or R,R monatin. In other embodiments, the compositions contain predominantly S,S or R,R monatin. "Predominantly" means that of the monatin stereoisomers present in the composition, the monatin contains greater than 90% of a particular stereoisomer. In some embodiments, the compositions are substantially free of S,S or R,R monatin. "Substantially free" means that of the monatin stereoisomers present in the composition, the composition contains less than 2% of a particular stereoisomer. Additionally or alternatively, when used to describe monatin produced in a biosynthetic pathway, "substantially free" encompasses the amount of a stereoisomer (e.g., S,S monatin) produced as a by-product in a biosynthetic pathway involving chiral-specific enzymes (e.g., D-amino acid dehydrogenases or D-amino acid aminotransferases) and/or chiral-specific substrates (e.g., one having a carbon in the R- stereoconfiguration) to produce a different specific stereoisomer (e.g., R,R monatin)

In another aspect ofthe present invention, a beverage composition includes a stereoisomerically-enriched monatin mixture produced in a biosynthetic pathway. "Stereoisomerically-enriched monatin mixture" means that the mixture contains more than one monatin stereoisomer and at least 60% ofthe monatin stereoisomers in the mixture is a particular stereoisomer, such as R,R, S,S, S,R or R,S. In other embodiments, the mixture contains greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer. In another embodiment, a beverage composition comprises an stereoisomerically-enriched R,R or S,S monatin. "Stereoisomerically-enriched" R,R monatin means that the monatin comprises at least 60% R,R monatin. "Stereoisomerically-enriched" S,S monatin means that the monatin comprises at least 60% S,S monatin. In other embodiments, "stereoisomerically-enriched" monatin comprises greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R,R or S,S monatin. Monatin can be isolated from the bark ofthe roots ofthe plant Sclerochiton ilicifolius. For example, the bark can be ground and extracted with water, filtered and freeze dried to obtain a dark brown, amorphous mass. The mass can be re-dissolved in water and reacted with a cation resin in the acid form, e.g., "Biorad" AG50W x8 in the HCl form (Bio-Rad Laboratories, Richmond, CA). The resin can be washed with water and the compounds bound to the resin eluted using an aqueous ammonia solution. The eluate can be freeze dried and subjected to aqueous gel filtration. See, for example, U.S. PatentNo. 5,128,164. Alternatively, monatin can be chemically synthesized. See, for example, the methods of Holzapfel and Olivier, Synth. Commun. 23:2511 (1993); Holzapfel et al., Synth. Commun. 38:7025 (1994); U.S. PatentNo. 5,128,164; U.S. Patent No. 4,975,298; and U.S. PatentNo. 5,994,559. Monatin also can be recombinantly produced.

In one aspect ofthe present invention, a method of making a beverage composition comprising monatin is provided. The method includes biosynthetically producing monatin either in vivo or in vitro. A "biosynthetic pathway" comprises at least one biological conversion step. In some embodiments, the biosynthetic pathway is a multi-step process and at least one step is a biological conversion step. In other embodiments, the biosynthetic pathway is a multi-step process involving both biological and chemical conversion steps. In some embodiments, the monatin produced is a stereoisomerically-enriched monatin mixture.

In another aspect ofthe present invention, a beverage composition comprising a biosynthetically-produced monatin is provided. Although monatin can also be chemically synthesized, biosynthetically-produced monatin may provide advantages in beverage applications over chemically-synthesized monatin because the chemically-synthesized monatin can include undesirable contaminants.

In another aspect ofthe present invention, several biosynthetic pathways exist for making monatin from substrates chosen from glucose, tryptophan, indole-3 -lactic acid, as well as indole-3 -pyruvate and 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glutaric acid (also known as "the monatin precursor," "MP" or the alpha-keto form of monatin). Examples of biosynthetic pathways for producing or making monatin or its intermediates are disclosed in FIGS. 1-3 and 11-13, which show potential intermediate products and end products in boxes. For example, a conversion from one product to another, such as glucose to tryptophan, tryptophan to indole-3 -pyruvate, indole-3 -pyruvate to MP, MP to monatin, or indole-3 -lactic acid (indole-lactate) to indole-3 -pyruvate, occurs in these pathways.

These conversions within the biosynthetic pathways can be facilitated via chemical and/or biological conversions. The term "convert" refers to the use of either chemical means or at least one polypeptide in a reaction to change a first intermediate into a second intermediate. Conversions can take place in vivo or in vitro. The term "chemical conversion" refers to a reaction that is not actively facilitated by a polypeptide. The term "biological conversion" refers to a reaction that is actively facilitated by one or more polypeptides. When biological conversions are used, the polypeptides and/or cells can be immobilized on supports such as by chemical attachment on polymer supports. The conversion can be accomplished using any reactor known to one of ordinary skill in the art, for example in a batch or a continuous reactor.

Examples of polypeptides, and their coding sequences, that can be used to perform biological conversions are shown in FIGS. 1-3 and 11-13. Polypeptides having one or more point mutations that allow the substrate specificity and/or activity ofthe polypeptides to be modified, can be used to make monatin. Isolated and recombinant cells expressing such polypeptides can be used to produce monatin. These cells can be any cell, such as a plant, animal, bacterial, yeast, algal, archaeal, or fungal cell.

For example, monatin-producing cells can include one or more (such as two or more, or three or more) ofthe following activities: tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryptophan dehydrogenase (EC 1.4.1.19), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminase (EC 2.6.1.28), multiple substrate aminotransferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), L-amino acid oxidase (EC 1.4.3.2), tryptophan oxidase (no EC number, Ffadar et al., J Bacteriol 125:1096-1104, 1976 and Furuya et al., Biosci Biotechnol Biochem 64:1486-93, 2000), D-tryptophan aminotransferase (Kohiba and Mito, Proceedings ofthe 8^th International Symposium on

Vitamin B₆ and Carbonyl Catalysis, Osaka, Japan 1990), D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC 1.4.3.3), D-alanine aminotransferase (EC 2.6.1.21), synthase/lyase (EC 4.1.3.-), such as 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) or 4- hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17), and/or synthase/lyase (4.1.2.-).

In another example, cells can include one or more (such as two or more, or three or more) of the following activities: indolelactate dehydrogenase (EC 1.1.1.110), R-4- hydroxyphenyllactate dehydrogenase (EC 1.1.1.222), 3-(4)-hydroxyphenylpyruvate reductase (EC 1.1.1.237), lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol-5-yl) lactate dehydrogenase (EC 1.1.1.111), lactate oxidase (EC 1.1.3.-), synthase/lyase (4.1.3.-) such as 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) or 4-hydroxy-4-methyl-2- oxoglutarate aldolase (EC 4.1.3.17), synthase/lyase (4.1.2.-), tryptophan aminotransferase

(EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryptophan dehydrogenase (EC 1.4.1.19), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminase (EC 2.6.1.28), multiple substrate aminotransferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), D- tryptophan aminotransferase, D-amino acid dehydrogenase (EC 1.4.99.1), and/or D-alanine aminotransferase (EC 2.6.1.21).

In addition, the cells can include one or more (such as two or more, or three or more) ofthe following activities: tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryptophan dehydrogenase (EC 1.4.1.19), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminase (EC 2.6.1.28), multiple substrate aminotransferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), L-amino acid oxidase (EC 1.4.3.2), tryptophan oxidase, D-tryptophan aminotransferase, D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC 1.4.3.3), D-alanine aminotransferase (EC 2.6.1.21), indolelactate dehydrogenase (EC 1.1.1.110), R-4-hydroxyphenyllactate dehydrogenase (EC 1.1.1.222), 3-(4)-hydroxyphenylpyruvate reductase (EC 1.1.1.237), lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol-5-yl) lactate dehydrogenase (EC 1.1.1.111), lactate oxidase (EC 1.1.3.-), synthase/lyase (EC 4.1.3.-), such as 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16) or 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17), and/or synthase/lyase (4.1.2.-). As further example, the cells can include one or more ofthe following aldolase activities: KHG aldolase, ProA aldolase, KDPG aldolase and/or related polypeptides (KDPH), transcarboxybenzalpyruvate hydratase-aldolase, 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase, trans-O-hydroxybenzylidenepyruvate hydratase-aldolase, 3-hydroxyaspartate aldolase, benzoin aldolase, dihydroneopterin aldolase, L-threo-3-phenylserine benzaldehyde- lyase (phenylserine aldolase), 4-hydroxy-2-oxovalerate aldolase, 1,2- dihydroxybenzylpyruvate aldolase, and/or 2-hydroxybenzalpyruvate aldolase.

Monatin can be produced by methods that include contacting tryptophan and/or indole-3 - lactic acid with a first polypeptide, wherein the first polypeptide converts tryptophan and/or indole-3 -lactic acid to indole-3-pyruvate (either the D or the L form of tryptophan or indole- 3 -lactic acid can be used as the substrate that is converted to indole-3 -pyruvate; one of skill in the art will appreciate that the polypeptides chosen for this step ideally exhibit the appropriate specificity), contacting the resulting indole-3 -pyruvate with a second polypeptide, wherein the second polypeptide converts the indole-3 -pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4- keto glutaric acid (MP), and contacting the MP with a third polypeptide, wherein the third polypeptide converts MP to monatin. Exemplary polypeptides that can be used for these conversions are shown in FIGS. 2 and 3.

Producing monatin in a biosynthetic pathway via one or more biological conversions provides certain advantages. For example, by using specific polypeptides and/or certain substrates in the biosynthetic pathway, one can produce a mixture enriched in a specific stereoisomer, and/or produce a monatin mixture that is substantially free of one or more stereoisomers.

A monatin composition may include impurities as a consequence ofthe method used for monatin synthesis. Monatin compositions produced by purely synthetic means (i.e., not involving at least one biological conversion) will contain different impurities than monatin compositions produced via a biosynthetic pathway. For example, based on raw materials used, monatin compositions produced by purely synthetic means may include petrochemical, toxic and/or other hazardous contaminants inappropriate for human consumption. Examples of such contaminants are hazardous chemicals, such as LDA, hydrogen-Pd/C, diazomethane, KCN, Grignard's reagent and Na/Hg. On the other hand, it is expected that a monatin composition produced via a biosynthetic pathway may contain edible or potable impurities, but will not contain petrochemical, toxic and/or other hazardous material.

It is expected that a method for producing monatin in a biosynthetic pathway via one or more biological conversions produces fewer toxic or hazardous contaminants and/or can provide a higher percentage of a particular stereoisomer, as compared to purely synthetic means. For example, it is expected that when making monatin using D-amino acid dehydrogenases, D- alanine (aspartate) aminotransferases, D-aromatic aminotransferases or D-methionine aminotranferases, one can obtain at least 60% R,R monatin and less than 40 % S,S, S,R and/or R,S monatin. It is also expected, for example, that when making monatin using the above-mentioned D-enzymes, as well as at least one substrate (e.g., the monatin precursor) having a carbon in the R-stereoconfiguration, one can obtain at least 95 % R,R monatin and less than 5% S,S, S,R and/or R,S monatin. In contrast, it is expected that when making monatin by purely synthetic means, one obtains about 25%-50% ofthe desired stereoisomer.

In one embodiment, a method for producing monatin via a biosynthetic pathway, for example, involving one or more biological conversion, produces no petrochemical, toxic or hazardous contaminants. "Petrochemical, toxic or hazardous contaminants" means any material that is petrochemical, toxic, hazardous and/or otherwise inappropriate for human consumption, including those contaminants provided as raw material or created when producing monatin via purely synthetic means. In another embodiment, a method for producing monatin via a biosynthetic pathway, for example, involving one or more biological conversion, produces only edible or potable material. "Edible or potable material" means one or more compounds or material that are fit for eating or drinking by humans, or otherwise safe for human consumption. Examples of edible or potable material include monatin, tryptophan, pyruvate, glutamate, other amino acids, as well as other compounds or material that are naturally present in the body.

In one embodiment, a beverage composition comprising monatin or salt thereof contains less calories and carbohydrates than the same amount ofthe beverage composition containing sucrose or high fructose corn syrup in place ofthe monatin or salt thereof at comparable sweetness. "A sweetness comparable" or "comparable sweetness" means that an experienced sensory evaluator, on average, will determine that the sweetness presented in a first composition is within a range of 80% to 120% ofthe sweetness presented a second composition.

In other embodiments, a beverage composition comprising monatin or salt thereof further comprises a citrus flavor, wherein the monatin or salt thereof is present in an amount that enhances the flavor provided by the citrus flavor. In another embodiment, the beverage composition further comprises a citrus flavor and a carbohydrate, and wherein the monatin or salt thereof and the carbohydrate are present in an amount that enhances the flavor provided by the citrus flavor. The carbohydrate may be chosen from, but is not limited to, erythritol, maltodextrin, sucrose and a combination thereof.

In one embodiment, a carbonated beverage comprises a syrup composition in an amount ranging from about 15% to about 25% by weight ofthe carbonated beverage, wherein the syrup composition comprises monatin or salt thereof.

In another embodiment, a beverage composition comprises from about 3 to about 10000 ppm monatin or salt thereof. In other embodiments, the beverage composition comprises from about 3 to less than about 30 ppm monatin, or from more than 2500 to about 10000 ppm monatin. In another embodiment, a beverage composition is a syrup or dry beverage mix, wherein the composition comprises from about 10 to about 10000 ppm monatin or salt thereof. For example, the beverage composition can be a syrup that is a concentrate adapted for dilution in a drink in a range of about 1 part syrup: 3 parts drink to about 1 part syrup: 5.5 drink. In one embodiment, the syrup comprises from about 600 to about 10000 ppm S,S monatin or salt thereof. In another embodiment, the syrup comprises from about 18 to about 300 ppm R,R monatin or salt thereof. Alternatively, the syrup comprises from about 0 to about 10000 ppm S,S monatin or salt thereof, and from 0 to about 300 ppm R,R monatin or salt thereof.

In another embodiment, a beverage composition is a dry beverage mix comprising from about 10 to about 10000 ppm monatin or salt thereof. In one embodiment, the dry beverage mix comprises from about 600 to about 10000 ppm S,S monatin or salt thereof. In another embodiment, the dry beverage mix comprises from about 10 to about 450 ppm R,R monatin or salt thereof. Alternatively, the dry beverage mix comprises from about 0 to about 10000 ppm S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

In other embodiments, a beverage composition comprises from about 3 to about 10000 ppm monatin or salt thereof, and the composition is substantially free of R,R monatin or salt thereof, or is substantially free of S,S monatin or salt thereof. In another embodiment, a beverage composition comprises from about 3 to about 450 ppm R,R monatin or salt thereof (e.g., from about 6 to about 225 ppm R,R monatin or salt thereof). In another embodiment, a beverage composition comprises from about 3 to about 10000 ppm S,S monatin or salt thereof (e.g., from about 60 to about 4600 ppm of S,S monatin or salt thereof). In another embodiment, a beverage composition comprises from about 0 to about 10000 ppm of S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

In one embodiment, a beverage composition is a ready-to-drink composition comprising from about 3 to about 2000 ppm monatin or salt thereof. In another embodiments, the ready-to- drink composition comprises from about 5 to about 50 ppm R,R monatin or salt thereof, or from about 60 to about 2000 ppm S,S monatin or salt thereof.

In another embodiment, a beverage composition comprises about 450 or less ppm R,R monatin or salt thereof, and is substantially free of S,S, S,R or R,S monatin or salt thereof.

Alternatively, a beverage composition comprises about 10000 or less ppm S,S monatin or salt thereof, and is substantially free of R,R, S,R or R,S monatin or salt thereof. In some embodiments, the monatin or salt thereof in a beverage composition consists essentially of R,R monatin or salt thereof, or consists essentially of S,S monatin or salt thereof. In other embodiments, the monatin or salt thereof in a beverage composition is a stereoisomerically- enriched R,R monatin or salt thereof, or is a stereoisomerically-enriched S,S monatin or salt thereof. In other embodiments, the monatin or salt thereof in a beverage composition comprises at least 95% R,R monatin or salt thereof, or at least 95% S,S monatin or salt thereof.

In one embodiment, a beverage composition comprises monatin or salt thereof that is produced in a biosynthetic pathway. In another embodiment, a beverage composition comprises a stereoisomerically-enriched monatin mixture, wherein the monatin mixture is produced via a biosynthetic pathway. In one embodiment, the biosynthetic pathway is a multi-step pathway and at least one step ofthe multi-step pathway is a chemical conversion. In other embodiments, the monatin mixture produced via a biosynthetic pathway is predominantly R,R monatin or salt thereof, or is predominantly S,S monatin or salt thereof.

In one embodiment, a beverage composition comprises a monatin composition produced in a biosynthetic pathway, wherein the monatin composition does not contain petrochemical, toxic or hazardous contaminants. In another embodiment, a beverage composition comprises monatin or salt thereof, wherein the monatin or salt thereof is produced in a biosynthetic pathway and isolated from a recombinant cell, and wherein the recombinant cell does not contain petrochemical, toxic or hazardous contaminants.

In one embodiment, a beverage composition comprising monatin or salt thereof is non- cariogenic. In other embodiments, a beverage composition comprising monatin or salt thereof further comprises erythritol, trehalose, a cyclamate, D-tagatose or combination thereof.

In other embodiments, a beverage composition comprising monatin or salt thereof further comprises a bulk sweetener, a high-intensity sweetener, a lower glycemic carbohydrate, a flavoring, an antioxidant, caffeine, a sweetness enhancer or a combination thereof. For example, the flavoring may be chosen from a cola flavor, a citrus flavor and a combination thereof. For example, the bulk sweetener may be chosen from corn sweeteners, sucrose, dextrose, invert sugar, maltose, dextrin, maltodextrin, fructose, levulose, high fructose corn syrup, corn syrup solids, levulose, galactose, trehalose, isomaltulose, fructo-oligosaccharides and a combination thereof. For example, the high-intensity sweetener may be chosen from sucralose, aspartame, saccharin, acesulfame K, alitame, thaumatin, dihydrochalcones, neotame, cyclamates, stevioside, mogroside, glycyrrhizin, phyllodulcin, monellin, mabinlin, brazzein, circulin, pentadin and a combination thereof. For example, the lower glycemic carbohydrate may be chosen from D-tagatose, sorbitol, mannitol, xylitol, lactitol, erythritol, maltitol, hydrogenated starch hydrolysates, isomalt, D-psicose, 1,5 anhydro D-fructose and a combination thereof. For example, the sweetness enhancer may be chosen from curculin, miraculin, cynarin, chlorogenic acid, caffeic acid, strogins, arabinogalactan, maltol, dihyroxybenzoic acids and a combination thereof. In another embodiment, a beverage composition comprises monatin or salt thereof that is a blend of R,R and S,S, monatin or salt thereof. In addition, a beverage composition may comprises a blend of monatin or salt thereof and a non-monatin sweetener. Non-monatin sweetener may be chosen from, for example, sucrose and high fructose corn syrup.

In some embodiments, methods for making a beverage composition comprising monatin or salt thereof comprise producing monatin or salt thereof from at least one substrate chosen from glucose, tryptophan, indole-3 -lactic acid, indole-3 -pyruvate and the monatin precursor. The methods may further comprise combining the monatin or salt thereof with at least one other ingredient that is not monatin or salt thereof (e.g., erythritol, trehalose, a cyclamate, D- tagatose, maltodextrin or combination thereof). In some embodiments, the other ingredient may be chosen from, for example, bulking agents, bulk sweeteners, liquid sweeteners, lower glycemic carbohydrates, high intensity sweeteners, thickeners, fats, oils, emulsifiers, antioxidants, sweetness enhancers, colorants, flavorings, caffeine, acids, powders, flow agents, buffers, protein sources, flavor enhancers, flavor stabilizers and a combination thereof. The bulk sweeteners may be chosen from, for example, sugar sweeteners, sugarless sweeteners, lower glycemic carbohydrates and a combination thereof. In other embodiments, beverage compositions made by the methods comprise from about 0 to about 10000 ppm of S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

In other embodiments, methods for making a beverage composition comprising monatin or salt thereof comprise producing monatin or salt thereof through a biosynthetic pathway. In some embodiments, methods for making a beverage composition comprising monatin or salt thereof comprise producing monatin or salt thereof using at least one biological conversion, or using only biological conversions. In another embodiment, a method for making a beverage composition comprising a monatin composition comprises: (a) producing monatin or salt thereof in a biosynthetic pathway in a recombinant cell; (b) isolating the monatin composition from the recombinant cell, wherein the monatin composition consists of monatin or salt thereof and other edible or potable material.

In other embodiments, a method for making a beverage composition comprising a monatin composition comprises producing the monatin composition in a biosynthetic pathway, wherein the monatin composition does not contain petrochemical, toxic or hazardous contaminants. In other embodiments, a method for making a beverage composition comprising a monatin composition comprises producing the monatin composition from a substrate in a multi-step pathway, wherein one or more steps in the multi-step pathway is a biological conversion, and wherein the monatin composition does not contain petrochemical, toxic or hazardous contaminants.

In other embodiment, a method for making a beverage composition comprising a monatin composition comprises producing the monatin composition in a biosynthetic pathway, wherein the monatin composition consists of monatin or salt thereof and other edible or potable material. In another embodiment, a method for making a beverage composition comprising a monatin composition comprises producing the monatin composition from a substrate in a multi-step pathway, wherein one or more steps in the multi-step pathway is a biological conversion, and wherein the monatin composition consists of monatin or salt thereof and other edible or potable material.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

It will be apparent to one of ordinary skill in the art from the teachings herein that specific embodiments ofthe present invention may be directed to one or a combination ofthe above- indicated aspects, as well as other aspects. Other features and advantages ofthe invention will be apparent from the following detailed description. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows biosynthetic pathways used to produce monatin and/or indole-3-pyruvate. One pathway produces indole-3 -pyruvate via tryptophan, while another produces indole-3- pyruvate via indole-3 -lactic acid. Monatin is subsequently produced via a MP intermediate.

Compounds shown in boxes are substrates and products produced in the biosynthetic pathways. Compositions adjacent to the arrows are cofactors, or reactants that can be used during the conversion of a substrate to a product. The cofactor or reactant used will depend upon the polypeptide used for the particular step ofthe biosynthetic pathway. The cofactor PLP (pyridoxal 5' - phosphate) can catalyze reactions independent of a polypeptide, and therefore, merely providing PLP can allow for the progression from substrate to product.

FIG. 2 is a more detailed diagram ofthe biosynthetic pathway that utilizes the MP intermediate. The substrates for each step in the pathways are shown in boxes. The polypeptides allowing for the conversion between substrates are listed adjacent to the arrows between the substrates. Each polypeptide is described by its common name and an enzymatic class (EC) number.

FIG. 3 shows a more detailed diagram ofthe biosynthetic pathway ofthe conversion of indole-3 -lactic acid to indole-3 -pyruvate. The substrates are shown in boxes, and the polypeptides allowing for the conversion between the substrates are listed adjacent to the arrow between the substrates. Each polypeptide is described by its common name and an EC number.

FIG. 4 shows one possible reaction for making MP via chemical means.

FIGS. 5A and 5B are chromatograms showing the LC/MS identification of monatin produced enzymatically.

FIG. 6 is an electrospray mass spectrum of enzymatically synthesized monatin. FIGS. 7 A and 7B are chromatograms ofthe LC/MS/MS daughter ion analyses of monatin produced in an enzymatic mixture.

FIG. 8 is a chromatogram showing the high-resolution mass measurement of monatin produced enzymatically.

FIGS. 9A-9C are chromatograms showing the chiral separation of (A) R-tryptophan, (B) S- tryptophan, and (C) monatin produced enzymatically.

FIG. 10 is a bar graph showing the relative amount of monatin produced in bacterial cells following IPTG induction. The (-) indicates a lack of substrate addition (no tryptophan or pyruvate was added).

FIGS. 11-12 are schematic diagrams showing pathways used to increase the yield of monatin produced from tryptophan or indole-3 -pyruvate.

FIG. 13 is a schematic diagram showing a pathway that can be used to increase the yield of monatin produced from tryptophan or indole-3 -pyruvate.

FIG. 14 presents a dose response curve obtained with an R,R, stereoisomer of monatin.

FIG. 15 presents a dose response curve obtained with an R,R/S,S stereoisomer mixture of monatin.

FIG. 16 compares the dose response curve obtained with an R,R/S,S stereoisomer mixture of monatin to a dose response curve obtained with saccharin.

FIG. 17 shows reversed phase chromatography of standards of synthetically produced monatin.

FIG. 18 shows chiral chromatography of monatin standards. DETAILED DESCRIPTION

Overview of Biosynthetic pathways for Monatin Production

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice ofthe present disclosure. As used herein, "including" means "comprising." In addition, the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. The term "about" encompasses the range of experimental error that occurs in any measurement. Unless otherwise stated, all measurement numbers are presumed to have the word "about" in front of them even if the word "about" is not expressly used. The term "% wt/vol" or "% w/v" refers to percentage weight per volume, where 100% wt/vol is 1 g/mL. Thus, for example, 1 g/100 mL is 1% wt/vol (in liquid compositions). The term "ppm" refers to parts per million. Eighty ppm of monatin, for example, means 80 grams (g) of monatin in a million grams. Likewise, 1 ppm = 0.0001 % w/w or, for aqueous solutions, = 1 mg/L = 1 μg/mL = 0.0001 % wt/vol.

As shown in FIGS. 1-3 and 11-13, many biosynthetic pathways can be used to produce monatin or its intermediates such as indole-3 -pyruvate or MP. For the conversion of each substrate (e.g., glucose, tryptophan, indole-3 -lactic acid, indole-3 -pyruvate, and MP) to each product (e.g., tryptophan, indole-3 -pyruvate, MP and monatin), several different polypeptides can be used. Moreover, these reactions can be carried out in vivo, in vitro, or through a combination of in vivo reactions and in vitro reactions, such as in vitro reactions that include non-enzymatic chemical reactions. Therefore, FIGS. 1-3 and 11-13 are exemplary, and show multiple different pathways that can be used to obtain desired products.

Glucose to Tryptophan

Many organisms can synthesize tryptophan from glucose. The construct(s) containing the gene(s) necessary to produce monatin, MP, and/or indole-3 -pyruvate from glucose and/or tryptophan can be cloned into such organisms. It is shown herein that tryptophan can be converted into monatin. In other examples, an organism can be engineered using known polypeptides to produce tryptophan, or overproduce tryptophan. For example, U.S. Patent No. 4,371,614 describes an E. coli strain transformed with a plasmid containing a wild type tryptophan operon.

Maximum titers of tryptophan disclosed in U.S. Patent No. 4,371,614 are about 230 ppm. Similarly, WO 8701130 describes an E. coli strain that has been genetically engineered to produce tryptophan and discusses increasing fermentative production of L-tryptophan. Those skilled in the art will recognize that organisms capable of producing tryptophan from glucose are also capable of utilizing other carbon and energy sources that can be converted to glucose or fructose-6-phosphate, with similar results. Exemplary carbon and energy sources include, but are not limited to, sucrose, fructose, starch, cellulose, or glycerol.

Tryptophan to Indole-3-pyruvate

Several polypeptides can be used to convert tryptophan to indole-3 -pyruvate. Exemplary polypeptides include, without limitation, members ofthe enzyme classes (EC) 2.6.1.27, 1.4.1.19, 1.4.99.1, 2.6.1.28, 1.4.3.2, 1.4.3.3, 2.6.1.5, 2.6.1.-, 2.6.1.1, and 2.6.1.21. These classes include, without limitation, polypeptides termed tryptophan aminotransferase (also termed L-phenylalanine-2-oxoglutarate aminotransferase, tryptophan transaminase, 5- hydroxytryptophan-ketoglutaric transaminase, hydroxytryptophan aminotransferase, L- tryptophan aminotransferase, L-tryptophan transaminase, and L-tryptophan:2-oxoglutarate aminotransferase) which converts L-tryptophan and 2-oxoglutarate to indole-3 -pyruvate and L-glutamate; D-tryptophan aminotransferase which converts D-tryptophan and a 2-oxo acid to indole-3 -pyruvate and an amino acid; tryptophan dehydrogenase (also termed NAD(P)-L- tryptophan dehydrogenase, L-tryptophan dehydrogenase, L-Trp-dehydrogenase, TDH and L- tryptophan:NAD(P) oxidoreductase (deaminating)) which converts L-tryptophan and

NAD(P) to indole-3-pyruvate and NH₃ and NAD(P)H; D-amino acid dehydrogenase, which converts D-amino acids and FAD to indole-3 -pyruvate and NH₃ and FADH₂; tryptophan- phenylpyruvate transaminase (also termed L-tryptophan-α-ketoisocaproate aminotransferase and L-tryptophan:phenylpyruvate aminotransferase) which converts L-tryptophan and phenylpyruvate to indole-3 -pyruvate and L-phenylalanine; L-amino acid oxidase (also termed ophio-amino-acid oxidase and L-amino-acid: oxygen oxidoreductase (deaminating)) which converts an L-amino acid and H₂O and O₂ to a 2-oxo acid and NH₃ and H₂O₂; D-amino acid oxidase (also termed ophio-amino-acid oxidase and D-amino-acid:oxygen oxidoreductase (deaminating)) which converts a D-amino acid and H₂O and O to a 2-oxo acid and NH₃ and H₂O ; and tryptophan oxidase which converts L-tryptophan and H O and O₂ to indole-3- pyruvate and NH₃ and H₂O₂. These classes also contain tyrosine (aromatic) aminotransferase, aspartate aminotransferase, D-amino acid (or D-alanine) aminotransferase, and broad (multiple substrate) aminotransferase which have multiple aminotransferase activities, some of which can convert tryptophan and a 2-oxo acid to indole-3 -pyruvate and an amino acid.

Eleven members ofthe aminotransferase class that have such activity are described below in Example 1, including a novel aminotransferase shown in SEQ ID NOS: 11 and 12.

Therefore, this disclosure provides isolated nucleic acid and amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or even at least 99% sequence identity to the sequences set forth in SEQ ID NOS: 11 and 12, respectively. Also encompassed by this disclosure are fragments and fusions ofthe sequences set forth in SEQ ID NOS: 11 and 12 that encode a polypeptide having aminotransferase activity or retaining aminotransferase activity. Exemplary fragments include, but are not limited to, at least 10, 12, 15, 20, 25, 50, 100, 200, 500, or 1000 contiguous nucleotides of SEQ ID NO: 11 or at least 6, 10, 15, 20, 25, 50, 75, 100, 200, 300 or 350 contiguous amino acids of SEQ ID NO: 12. The disclosed sequences (and variants, fragments, and fusions thereof) can be part of a vector. The vector can be used to transform host cells, thereby producing recombinant cells which can produce indole-3 -pyruvate from tryptophan, and in some examples can further produce MP and/or monatin.

L-amino acid oxidases (1.4.3.2) are known, and sequences can be isolated from several different sources, such as Vipera lebetine (sp P81375), Ophiophagus hannah (sp P81383), Agkistrodon rhodostoma (spP81382), Crotalus atrox (sp P56742), Burkholderia cepacia, Arάbidopsis thaliana, Caulobacter cresentus, Chlamydomonas reinhardtii, Mus musculus, Pseudomonas syringae, and Rhodococcus str. In addition, tryptophan oxidases are described in the literature and can be isolated, for example, from Coprinus sp. SF-1, Chinese cabbage with club root disease, Arabidopsis thaliana, and mammalian liver. One member ofthe L- amino acid oxidase class that can convert tryptophan to indole-3 -pyruvate is discussed below in Example 3, as well as alternative sources for molecular cloning. Many D-amino acid oxidase genes are available in databases for molecular cloning. Tryptophan dehydrogenases are known, and can be isolated, for example, from spinach, Pisum sativum, Prosopis juliflora, pea, mesquite, wheat, maize, tomato, tobacco, Chromobacterium violaceum, and Lactobacilli. Many D-amino acid dehydrogenase gene sequences are known.

As shown in FIGS. 11-13, if an amino acid oxidase, such as tryptophan oxidase, is used to convert tryptophan to indole-3-pyruvate, catalase can be added to reduce or even eliminate the presence of hydrogen peroxide.

Indole-3 -lactate to Indole-3 -pyruvate

The reaction that converts indole-3 -lactate to indole-3 -pyruvate can be catalyzed by a variety of polypeptides, such as members of the 1.1.1.110, 1.1.1.27, 1.1.1.28, 1.1.2.3, 1.1.1.222, 1.1.1.237, 1.1.3.-, or 1.1.1.111 classes of polypeptides. The 1.1.1.110 class of polypeptides includes indolelactate dehydrogenases (also termed indolelactic acid: NAD⁺ oxidoreductase). The 1.1.1.27, 1.1.1.28, and 1.1.2.3 classes include lactate dehydrogenases (also termed lactic acid dehydrogenases, lactate: NAD⁺ oxidoreductase). The 1.1.1.222 class contains (R)-4- hydroxyphenyllactate dehydrogenase (also termed D-aromatic lactate dehydrogenase, R- aromatic lactate dehydrogenase, and R-3-(4-hydroxyphenyl)lactate:NAD(P)⁺2- oxidoreductase) and the 1.1.1.237 class contains 3-(4-hydroxyphenylpyruvate) reductase (also termed hydroxyphenylpyruvate reductase and 4-hydroxyphenyllactate: NAD⁺ oxidoreductase). The 1.1.3.- class contains lactate oxidases, and the 1.1.1.111 class contains (3-imidazol-5-yl) lactate dehydrogenases (also termed (S)-3-(imidazol-5-yl)lactate:NAD(P)⁺ oxidoreductase). It is likely that several ofthe polypeptides in these classes allow for the production of indole-3 -pyruvate from indole-3 -lactic acid. Examples of this conversion are provided in Example 2.

Chemical reactions can also be used to convert indole-3 -lactic acid to indole-3 -pyruvate. Such chemical reactions include an oxidation step that can be accomplished using several methods, for example: air oxidation using a B2 catalyst (China Chemical Reporter, vol. 13, no. 28, pg. 18(1), 2002), dilute permanganate and perchlorate, or hydrogen peroxide in the presence of metal catalysts. Indole-3 -pyruvate to 2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid (MP) Several known polypeptides can be used to convert indole-3 -pyruvate to MP. Exemplary polypeptide classes include 4.1.3.-, 4.1.3.16, 4.1.3.17, and 4.1.2.-. These classes include carbon-carbon synthases/lyases, such as aldolases that catalyze the condensation of two carboxylic acid substrates. Polypeptide class EC 4.1.3.- are synthases/lyases that form carbon-carbon bonds utilizing oxo-acid substrates (such as indole-3 -pyruvate) as the electrophile, while EC 4.1.2.- are synthases/lyases that form carbon-carbon bonds utilizing aldehyde substrates (such as benzaldehyde) as the electrophile.

For example, the polypeptide described in EP 1045-029 (EC 4.1.3.16, 4-hydroxy-2- oxoglutarate glyoxylate-lyase also termed 4-hydroxy-2-oxoglutarate aldolase, 2-oxo-4- hydroxyglutarate aldolase or KHG aldolase) converts glyoxylic acid and pyruvate to 4- hydroxy-2-keto glutaric acid, and the polypeptide 4-hydroxy-4-methyl-2-oxoglutarate aldolase (EC 4.1.3.17, also termed 4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase or ProA aldolase), condenses two keto-acids such as two pyruvates to 4-hydroxy-4-methyl-2- oxoglutarate. Reactions utilizing these lyases are described herein.

FIGS. 1-2 and 11-13 show schematic diagrams of these reactions in which a 3 -carbon (C3) molecule is combined with indole-3-pyruvate. Many members of EC 4.1.2.- and 4.1.3.-, particularly PLP-utilizing polypeptides, can utilize C3 molecules that are amino acids such as serine, cysteine, and alanine, or derivatives thereof. Aldol condensations catalyzed by representatives of EC 4.1.2.- and 4.1.3.- require the three carbon molecule of this pathway to be pyruvate or a derivative of pyruvate. However, other compounds can serve as a C3 carbon source and be converted to pyruvate. Alanine can be transaminated by many PLP-utilizing transaminases, including many of those mentioned above, to yield pyruvate. Pyruvate and ammonia can be obtained by beta-elimination reactions (such as those catalyzed by tryptophanase or β-tyrosinase) of L-serine, L-cysteine, and derivatives of serine and cysteine with sufficient leaving groups, such as O-methyl-L-serine, O-benzyl-L-serine, S- methylcysteine, S-benzylcysteine, S-alkyl-L-cysteine, O-acyl-L-serine, and 3-chloro-L- alanine. Aspartate can serve as a source of pyruvate in PLP-mediated beta-lyase reactions such as those catalyzed by tryptophanase (EC 4.1.99.1) and/or β-tyrosinase (EC 4.1.99.2, also termed tyrosine-phenol lyase). The rate of beta-lyase reactions can be increased by performing site-directed mutagenesis on the (4.1.99.1-2) polypeptides as described by Mouratou et al (J. Biol. Chem 274:1320-5, 1999) and in Example 8. These modifications allow the polypeptides to accept dicarboxylic amino acid substrates. Lactate can also serve as a source of pyruvate, and is oxidized to pyruvate by the addition of lactate dehydrogenase and an oxidized cofactor or lactate oxidase and oxygen. Examples of these reactions are described below. For example, as shown in FIG. 2 and FIGS. 11-13, ProA aldolase can be contacted with indole-3 -pyruvate when pyruvate is used as the C3 molecule.

The MP can also be generated using chemical reactions, such as the aldol condensations provided in Example 5.

MP to Monatin

Conversion of MP to monatin can be catalyzed by one or more of: tryptophan aminotransferases (2.6.1.27), tryptophan dehydrogenases (1.4.1.19), D-amino acid dehydrogenases (1.4.99.1), glutamate dehydrogenases (1.4.1.2-4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminases (2.6.1.28), or more generally members ofthe aminotransferase family (2.6.1.-) such as aspartate aminotransferase (EC 2.6.1.1), tyrosine (aromatic) aminotransferase (2.6.1.5), D-tryptophan aminotransferase, or D-alanine (2.6.1.21) aminotransferase (FIG. 2). Eleven members ofthe aminotransferase class are described below (Example 1), including a novel member ofthe class shown in SEQ ID NOS: 11 and 12, and reactions demonstrating the activity of aminotransferase and dehydrogenase enzymes are provided in Example 7.

This reaction can also be performed using chemical reactions. Amination ofthe keto acid (MP) is performed by reductive amination using ammonia and sodium cyanoborohydride.

FIGS. 11-13 show additional polypeptides that can be used to convert MP to monatin, as well as providing increased yields of monatin from indole-3 -pyruvate or tryptophan. For example, if aspartate is used as the amino donor, aspartate aminotransferase can be used to convert the aspartate to oxaloacetate (FIG. 11). The oxaloacetate is converted to pyruvate and carbon dioxide by a decarboxylase, such as oxaloacetate decarboxylase (FIG. 11). In addition, if lysine is used as the amino donor, lysine epsilon aminotransferase can be used to convert the lysine to allysine (FIG. 12). The allysine is spontaneously converted to 1-piperideine 6- carboxylate (FIG. 12). If a polypeptide capable of catalyzing reductive amination reactions (e.g., glutamate dehydrogenase) is used to convert MP to monatin, a polypeptide that can recycle NAD(P)H and/or produce a volatile product (FIG. 13) can be used, such as formate dehydrogenase.

Additional Considerations in the Design ofthe Biosynthetic Pathways

Depending on which polypeptides are used to generate indole-3 -pyruvate, MP, and/or monatin, cofactors, substrates, and/or additional polypeptides can be provided to the production cell to enhance product formation. In addition, genetic modification can be designed to enhance production ofproducts such as indole-3 -pyruvate, MP, and/or monatin. Similarly, a host cell used for monatin production can be optimized.

Removal of Hydrogen Peroxide

Hydrogen peroxide (H₂O₂) is a product that, if generated, can be damaging to production cells, polypeptides or products (e.g., intermediates) produced. The L-amino acid oxidase described above generates H₂O₂ as a product. Therefore, if L-amino acid oxidase is used, the resulting H₂O₂ can be removed or its levels decreased to reduce potential injury to the cell or product.

Catalases can be used to reduce the level of H O₂ in the cell (FIGS. 11-13). The production cell can express a gene or cDNA sequence that encodes a catalase (EC 1.11.1.6), which catalyzes the decomposition of hydrogen peroxide into water and oxygen gas. For example, a catalase can be expressed from a vector transfected into the production cell. Examples of catalases that can be used include, but are not limited to: tr|Q9EV50 (Staphylococcus xylosus), tr|Q9KBE8 (Bacillus hάlodurans), tr|Q9URJ7 (Candida άlbicans), tr|P77948 (Streptomyces coelicolor), tr|Q9RBJ5 (Xanthomonas campestris) (SwissProt Accession Nos.). Biocatalytic reactors utilizing L-amino acid oxidase, D-amino acid oxidase, or tryptophan oxidase can also contain a catalase polypeptide.

Modulation ofpyridoxal-5 '-phosphate (PLP) Availability As shown in FIG. 1, PLP can be utilized in one or more ofthe biosynthetic steps described herein. The concentration of PLP can be supplemented so that PLP does not become a limitation on the overall efficiency ofthe reaction. The biosynthetic pathway for vitamin B₆ (the precursor of PLP) has been thoroughly studied in E. coli, and some ofthe proteins have been crystallized (Laber et α , FEBS Letters, 449:45-8, 1999). Two ofthe genes (epd or gαpB and serC) are required in other metabolic pathways, while three genes (pdxA,pdxB, andpdxJ) are unique to pyridoxal phosphate biosynthesis. One ofthe starting materials in the E. coli pathway is l-deoxy-D-xylulose-5- phosphate (DXP). Synthesis of this precursor from common 2 and 3 carbon central metabolites is catalyzed by the polypeptide l-deoxy-D-xylulose-5-phosphate synthase (DXS). The other precursor is a threonine derivative formed from the 4-carbon sugar, D- erythrose 4-phosphate. The genes required for the conversion to phospho-4-hydroxyl-L threonine (HTP) are epd, pdxB, and serC. The last reaction for the formation of PLP is a complex intramolecular condensation and ring-closure reaction between DXP and HTP, catalyzed by the gene products oϊpdxA andpdxJ.

If PLP becomes a limiting nutrient during the fermentation to produce monatin, increased expression of one or more of the pathway genes in a production host cell can be used to increase the yield of monatin. A host organism can contain multiple copies of its native pathway genes or copies of non-native pathway genes can be incorporated into the organism's genome. Additionally, multiple copies ofthe salvage pathway genes can be cloned into the host organism.

One salvage pathway that is conserved in all organisms recycles the various derivatives of vitamin B₆ to the active PLP form. The polypeptides involved in this pathway axepdxK kinase, pdxH oxidase, and pdxY kinase. Over-expression of one or more of these genes can increase PLP availability.

Vitamin B₆ levels can be elevated by elimination or repression ofthe metabolic regulation of the native biosynthetic pathway genes in the host organism. PLP represses polypeptides involved in the biosynthesis ofthe precursor threonine derivative in the bacterium Flαvobαcterium sp. strain 238-7. This bacterial strain, freed of metabolic control, overproduces pyridoxal derivatives and can excrete up to 20 mg/L of PLP. Genetic manipulation ofthe host organism producing monatin in a similar fashion will allow the increased production PLP without over-expression ofthe biosynthetic pathway genes. Ammonium Utilization

Tryptophanase reactions can be driven toward the synthetic direction (production of tryptophan from indole) by making ammonia more available or by removal of water. Reductive amination reactions, such as those catalyzed by glutamate dehydrogenase, can also be driven forward by an excess of ammonium.

Ammonia can be made available as an ammonium carbonate or ammonium phosphate salt in a carbonate or phosphate buffered system. Ammonia can also be provided as ammonium pyruvate or ammonium formate. Alternatively, ammonia can be supplied if the reaction is coupled with a reaction that generates ammonia, such as glutamate dehydrogenase or tryptophan dehydrogenase. Ammonia can be generated by addition ofthe natural substrates of EC 4.1.99.- (tyrosine or tryptophan), which will be hydrolyzed to phenol or indole, pyruvate and NH₃. This also allows for an increased yield of synthetic product over the normal equilibrium amount by allowing the enzyme to hydrolyze its preferred substrate.

Removal ofproducts and byproducts

The conversion of tryptophan to indole-3 -pyruvate via a tryptophan aminotransferase can adversely affect the production rate of indole-3 -pyruvate because the reaction produces glutamate and requires the co-substrate 2-oxoglutarate (α-ketoglutarate). Glutamate can cause inhibition ofthe aminotransferase, and the reaction can consume large amounts ofthe co-substrate. Moreover, high glutamate concentrations can be detrimental to downstream separation processes.

The polypeptide glutamate dehydrogenase (GLDH) converts glutamate to 2-oxoglutarate, thereby recycling the co-substrate in the reaction catalyzed by tryptophan aminotransferase. GLDH also generates reducing equivalents (NADH or NADPH) that can be used to generate energy for the cell (ATP) under aerobic conditions. The utilization of glutamate by GLDH also reduces byproduct formation. Additionally, the reaction generates ammonia, which can serve as a nitrogen source for the cell or as a substrate in a reductive amination for the final step shown in FIG. 1. Therefore, a production cell that over-expresses a GLDH polypeptide can be used to increase the yield and reduce the cost of media and/or separation processes. In the tryptophan to monatin pathway, the amino donor of step three (e.g., glutamate or aspartate) can be converted back to the amino acceptor required for step 1 (e.g., 2-oxoglutarate or oxaloacetate), if an aminotransferase from the appropriate enzyme classes is used. Utilization of two separate transaminases for this pathway, in which the substrate of one transaminase does not competitively inhibit the activity ofthe other transaminase, can increase the efficiency of this pathway.

Many ofthe reactions in the described pathways are reversible and can, therefore, reach an equilibrium between substrates and products. The yield ofthe pathway can be increased by continuous removal ofthe products from the polypeptides. For example, secretion of monatin into the fermentation broth using a permease or other transport protein, or selective crystallization of monatin from a biocatalytic reactor stream with concomitant recycle of substrates will increase the reaction yield.

Removal of byproducts via additional enzymatic reactions or via substitution of amino donor groups is another way to increase the reaction yield. Several examples are discussed in Example 13 and shown in FIGS. 11-13. For example, a byproduct can be produced that is unavailable to react in the reverse direction, either by phase change (evaporation) or by spontaneous conversion to an unreactive end product, such as carbon dioxide.

Modulation ofthe Substrate Pools

The indole pool can be modulated by increasing production of tryptophan precursors and/or altering catabolic pathways involving indole-3 -pyruvate and/or tryptophan. For example, the production of indole-3 -acetic acid from indole-3 -pyruvate can be reduced or eliminated by functionally deleting the gene coding for EC 4.1.1.74 in the host cell. Production of indole from tryptophan can be reduced or eliminated by functionally deleting the gene coding for EC 4.1.99.1 in the host cell. Alternatively, an excess of indole can be utilized as a substrate in an in vitro or in vivo process in combination with increased amounts ofthe gene coding for EC 4.1.99.1 (Kawasaki et al, J. Ferm. and Bioeng., 82:604-6, 1996). In addition, genetic modifications can be made to increase the level of intermediates such as D-erythrose-4- phosphate and chorismate. Tryptophan production is regulated in most organisms. One mechanism is via feedback inhibition of certain enzymes in the pathway; as tryptophan levels increase, the production rate of tryptophan decreases. Thus, when using a host cell engineered to produce monatin via a tryptophan intermediate, an organism can be used that is not sensitive to tryptophan concentrations. For example, a strain of Catharanthus roseus that is resistant to growth inhibition by various tryptophan analogs was selected by repeated exposure to high concentrations of 5-methyltryptophan (Schallenberg and Berlin, Z Naturforsch 34:541-5, 1979). The resulting tryptophan synthase activity ofthe strain was less effected by product inhibition, likely due to mutations in the gene. Similarly, a host cell used for monatin production can be optimized.

Tryptophan production can be optimized through the use of directed evolution to evolve polypeptides that are less sensitive to product inhibition. For example, screening can be performed on plates containing no tryptophan in the medium, but with high levels of non- metabolizable tryptophan analogs. U.S. Pat. Nos. 5,756,345; 4,742,007; and 4,371,614 describe methods used to increase tryptophan productivity in a fermentation organism. The last step of tryptophan biosynthesis is the addition of serine to indole; therefore the availability of serine can be increased to increase tryptophan production.

The amount of monatin produced by a fermentation organism can be increased by increasing the amount of pyruvate produced by the host organism. Certain yeasts, such as Trichosporon cutaneum (Wang et al., Lett. Appl. Microbiol. 35:338-42, 2002) and Torulopsis glabrata (Li et al, Appl Microbiol. Biotechnol. 57:451-9, 2001) overproduce pyruvate and can be used to practice the methods disclosed herein. In addition, genetic modifications can be made to organisms to promote pyruvic acid production, such as those in E. coli strain W1485/ p2 (Kawasaki et al., J. Ferm. and Bioeng. 82:604-6, 1996).

Controlling Chirality

The taste profile of monatin can be altered by controlling its stereochemistry (chirality). For example, different monatin stereoisomers may be desired in different blends of concentrations for different food systems. Chirality can be controlled via a combination of pH and polypeptides.

Racemization at the C-4 position of monatin (see numbered molecule above) can occur by deprotonation and reprotonation ofthe alpha carbon, which can occur by a shift in pH or by reaction with the cofactor PLP bound to an enzyme such as a racemase or free in solution. In a microorganism, the pH is unlikely to shift enough to cause the racemization, but PLP is abundant. Methods to control the chirality with polypeptides depend upon the biosynthetic route utilized for monatin production.

When monatin is formed using the pathway shown in FIG. 2, the following can be considered. In a biocatalytic reaction, the chirality of carbon-2 can be determined by an enzyme that converts indole-3 -pyruvate to MP. Multiple enzymes (e.g., from EC 4.1.2.-, 4.1.3.-) can convert indole-3 -pyruvate to MP, thus, the enzyme that forms the desired stereoisomer can be chosen. Alternatively, the enantiospecificity ofthe enzyme that converts indole-3 -pyruvate to MP can be modified through the use of directed evolution, or catalytic antibodies can be engineered to catalyze the desired reaction. Once MP is produced (either enzymatically or by chemical condensation), the amino group can be added stereospecifically using a transaminase, such as those described herein. Either the R or S configuration of carbon-4 can be generated depending on whether a D- or L- aromatic acid aminotransferase is used. Most aminotransferases are specific for the L-stereoisomer; however, D-tryptophan aminotransferases exist in certain plants (Kohiba and Mito, Proceedings ofthe 8th International Symposium on Vitamin B₆ and Carbonyl Catalysis, Osaka, Japan 1990). Moreover, D-alanine aminotransferases (2.6.1.21), D-methionine-pyruvate aminotransferases (2.6.1.41), and both (R)-3-amino-2-methylρropanoate aminotransferase (2.6.1.61) and (S)-3- amino-2-methylpropanoate aminotransferase (2.6.1.22) have been identified. Certain aminotransferases may only accept the substrate for this reaction with a particular configuration at the C2 carbon. Therefore, even if the conversion to MP is not stereospecific, the stereochemistry ofthe final product can be controlled through the appropriate selection of a transaminase. Since the reactions are reversible, the unreacted MP (undesired stereoisomer) can be recycled back to its constituents, and a racemic mixture of MP can be reformed.

Activating Substrates

Phosphorylated substrates, such as phosphoenolpyruvate (PEP), can be used in the reactions disclosed herein. Phosphorylated substrates can be more energetically favorable and, therefore, can be used to increase the reaction rates and/or yields. In aldol condensations, the addition of a phosphate group stabilizes the enol tautomer of the nucleophilic substrate, making it more reactive. In other reactions, a phosphorylated substrate can provide a better leaving group. Similarly, substrates can be activated by conversion to CoA derivatives or pyrophosphate derivatives.

Use of monatin in a beverage composition

The S,S stereoisomer of monatin is approximately 50-200 times sweeter than sucrose by weight. The R,R stereoisomer of monatin is approximately 2000-2400 times sweeter than sucrose by weight. The sweetness ofthe monatin is calculated using experienced sensory evaluators in a sweetness comparison procedure, where a test sweetener solution is matched for sweetness intensity against one of a series of reference solutions. The solutions may be prepared, for example, using a buffer comprising 0.16% (w/v) citric acid and 0.02% (w/v) sodium citrate at ~pH 3.0.

Specifically, one may assess sweetness of a sweetener relative to sucrose by using a panel of trained sensory evaluators experienced in the sweetness estimation procedure. All samples (in same buffers) are served in duplicate at a temperature of 22°C ± 1°C. Sample solutions may be prepared, for example, using a buffer comprising 0.16% (w/v) citric acid and 0.02% (w/v) sodium citrate at ~pH 3.0. Test solutions, coded with 3 digit random number codes, are presented individually to panelists, in random order. Sucrose reference standards, ranging from 2.0 - 10.0% (w/v) sucrose, increasing in steps of 0.5%o (w/v) sucrose are also provided. Panelists are asked to estimate sweetness by comparing the sweetness ofthe test solution to the sucrose standards. This is carried out by taking 3 sips ofthe test solution, followed by a sip of water, followed by 3 sips of sucrose standard followed by a sip of water, etc. Panelists estimate the sweetness to one decimal place, e.g., 6.8, 8.5. A five minute rest period is imposed between evaluating the test solutions. Panelists are also asked to rinse well and eat a cracker to reduce any potential carry over effects.

Sucrose equivalent value (SEV) (e.g., % sucrose), determined by the panel of trained sensory evaluators, is plotted as a function of monatin concentration to obtain a dose response curve. A polynomial curve fit is applied to the dose response curve and used to calculate the sweetness intensity or potency at a particular point, e.g., 8% SEV, by dividing the sucrose equivalent value (SEV) by the monatin concentration (e.g., % monatin). See e.g., FIG. 15 (R,R/S,S monatin dose response curve); FIG. 14 (R,R monatin dose response curve). The above-mentioned sweetness intensities for S,S and R,R monatin (i.e., approximately 50-200 times sweeter and approximately 2000-2400 times sweeter than sucrose by weight, respectively) were determined at approximately 8% SEV.

Monatin is soluble in aqueous solutions in concentrations that are appropriate for consumption. Various blends of monatin stereoisomers may be qualitatively better in certain matrices, or in blending with other sweeteners. Blends of monatin with other sweeteners may be used to maximize the sweetness intensity and/or profile, and minimize cost. Monatin may be used in combination with other sweeteners and/or other ingredients to generate a temporal profile similar to sucrose, or for other benefits.

For example, monatin may be blended with other nutritive and nonnutritive sweeteners to achieve particular flavor profiles or calorie targets. Thus, sweetener compositions can include combinations of monatin with one or more ofthe following sweetener types: (1) sugar alcohols (such as erythritol, sorbitol, maltitol, mannitol, lactitol, xylitol, isomalt, low glycemic syrups, etc.); (2) other high intensity sweeteners (such as aspartame, sucralose, saccharin, acesulfame-K, stevioside, cyclamate, neotame, thaumatin, alitame, dihydrochalcone, monellin, glycyrrihizin, mogroside, phyllodulcin, mabinlin, brazzein, circulin, pentadin, etc.) and (3) nutritive sweeteners (such as sucrose, D-tagatose, invert sugar, fructose, corn syrup, high fructose corn syrup (HFCS), glucose/dextrose, trehalose, isomaltulose, etc.). Monatin may be used in such blends as a taste modifier to suppress aftertaste, enhance other flavors such as lemon, or improve the temporal flavor profile. Data also indicate that monatin is quantitatively synergistic with cyclamates (which are used in Europe), but no significant quantitative synergy was noted with aspartame, saccharin, acesulfame-K, sucralose, or carbohydrate sweeteners.

Because monatin is not a carbohydrate, monatin can be used to lower the carbohydrate content in beverage compositions. In one embodiment, an amount of a beverage composition comprising monatin contains less calories and carbohydrates than the same amount of a beverage composition containing sugar (e.g., sucrose and/or high fructose corn syrup) in place ofthe monatin. In other embodiments, beverage compositions comprising monatin (e.g., comprising monatin and one or more carbohydrates) provide a mouthfeel, flavor and sweetness over time that is comparable to that provided by similar beverage compositions containing only carbohydrates as the sweetener.

Monatin is stable in a dry form, and has a desirable taste profile alone or when mixed with carbohydrates. It does not appear to irreversibly break down, but rather forms lactones and/or lactams at low pHs (in aqueous buffers) and reaches an equilibrium. It can racemize at the 4 position slowly over time in solution, but typically this occurs at high pHs. In general, the stability of monatin is comparable to or better than aspartame and the taste profile of monatin is comparable to or better than other quality sweeteners, such aspartame, alitame, and sucralose. Monatin does not have the undesirable aftertaste associated with some other high intensity sweeteners such as saccharin and stevioside.

In some embodiments, beverage compositions comprising monatin also include one or more ofthe following: buffers, bulking agents, thickeners, fats, flavorings, coloring agents (also called colorants or colors), sweeteners and flow agents. Beverage compositions can be formulated to have a particular sweetness profile, e.g., by tailoring the amount of monatin or other sweeteners present in the beverage or by tailoring the amount or type of other additives, including flavoring agents or acids, present in the composition. In other embodiments, all ingredients used in beverage compositions are food grade and generally recognized as safe.

In some embodiments, beverage compositions comprising monatin further comprise food grade antioxidants. Examples of such antioxidants include vitamin C (e.g., ascorbic acid, magnesium ascorbyl phosphate), erythorbate (isoascorbic acid), carotenoids such as lutein, lycopene and beta-carotene, tocopherols (e.g., α-tocopherol (natural vitamin E), γ- tocopherol, δ- tocopherol), hydroxycinnamates (e.g., neochlorogenic acid and chlorogenic acid), glutathione, phenolics (e.g., cocoa phenols, red wine phenols, phenolics in prunes), butylated hyroxyanisole (BHA), butylated hydroxytolulene (BHT), tertiary butylhydroquinone (TBHQ), propyl gallate, nisin, green tea extract and rosemary extract. In other embodiments, beverage compositions comprising monatin further comprise certain preservatives, such as sodium benzoate and/or potassium sorbate.

In other embodiments, beverage compositions comprising monatin further comprise one or more ingredients that prevent non-enzymatic browning reactions (e.g., browning due to Maillard reactions). Such ingredients may include, but are not limited to, sulfites and sulfϊting agents (e.g., sulfur dioxide, sodium sulfite, sodium or potassium bisulfite, metabisulfites, sulfhydryl-containing amino acids), calcium chloride and other inorganic halides, antioxidants, and compounds that affect the water activity (e.g., glycerol, sorbitol and trehalose).

In some embodiments, monatin-containing beverage concentrates such as dry beverage mixes can be readily dispersed to prepare chocolate beverages, fruit beverages, malted beverages, or lemonade. In other embodiments, a beverage concentrate is a beverage syrup that can be used to prepare carbonated soft drinks. A carbonated beverage can be prepared, for example, by diluting a beverage syrup containing water, monatin, and flavorings, with carbonated water. In some embodiments, the beverage syrup also contains other sweeteners and/or additives. Beverage syrups can be prepared, for example, by mixing all ofthe ingredients and heating to solubilize. Beverage syrups may include, for example, at least 80% water (e.g., at least 85%, 90%, or 95% water).

In certain embodiments, monatin is present in an amount that ranges from about 0.0003 to about 1 % ofthe beverage composition (i.e., about 3 to about 10,000 ppm) (e.g., about 0.0005 to about 0.2 %), including any particular value within that range (e.g., 0.0003 %, 0.005 %, 0.06 % or 0.2 % ofthe beverage composition). For example, a beverage composition may comprise 0.0005 to 0.005 % (e.g., 0.001 to 0.0045 %) ofthe R,R monatin, or 0.005 to 0.2 % (e.g., 0.01 to 0.175 %) of S,S monatin. One of skill in the art will recognize that combinations of sweeteners can be used to provide the desired taste and caloric count of a beverage composition. Thus, the amount of sweetener in a beverage composition depends upon the choice of sweeteners and desired sweetness intensity. Sweeteners are commercially available, e.g., through Cargill Inc. (Wayzata, MN) and McNeil Specialty (Fort Washington, PA). In one embodiment, a beverage composition includes a blend of monatin and a sweetener (e.g., sucrose or high fructose corn syrup). For example, a beverage composition can include monatin and a bulk sweetener.

Bulk sweeteners may be chosen from, for example, sugar sweeteners, sugarless sweeteners, lower glycemic carbohydrates, and a combination thereof. Sugar sweeteners can include, for example, a com sweetener, sucrose, dextrose (e.g., Cerelose dextrose), maltose, dextrin, maltodextrin, invert sugar, fructose, high fructose corn syrup, levulose, galactose, corn syrup solids, galactose, trehalose, isomaltulose, fructo-oligosaccharides (such as kestose or nystose), higher molecular weight fructo-oligosaccharides or a combination thereof. High fructose corn syrup (HFCS) and other corn derived sweeteners, for example, are combinations of dextrose (glucose) and fructose. In addition, sugar sweeteners include fruit sugars, maple syrup, and honey, or combinations thereof. In one embodiment, 0.0003 to 0.15 % monatin (e.g., 0.0006 to 0.004 % of R,R monatin) and 2 to 10 % (e.g., 3 to 10% or 4 to 6%) of sucrose or high fructose corn syrup can be used in a beverage composition.

In another embodiment, a beverage composition includes a sugarless sweetener and/or a lower glycemic carbohydrate (i.e., one with a lower glycemic index than glucose). Sugarless sweeteners or lower glycemic carbohydrates include, but are not limited to, D-tagatose, sorbitol (including amorphous and crystalline sorbitol), mannitol, xylitol, lactitol, erythritol, maltitol, hydrogenated starch hydrolysates, isomalt, D-psicose, 1,5 anhydro D-fructose or a combination thereof.

In certain embodiments, beverage compositions comprising monatin also comprise high intensity sweeteners. In some embodiments, high intensity sweeteners are at least 20 times sweeter than sucrose (i.e., 20 X sucrose). Such high intensity sweeteners include, but are not limited to, sucralose, aspartame, saccharin and its salts, salts of acesulfame (e.g., acesulfame K), alitame, thaumatin, dihydrochalcones (e.g., neohesperidin dihydrochalcone), neotame, cyclamic acid and its salts (i.e., cyclamates), stevioside (extracted from leaves of Stevia rebaudiana), mogroside (extracted from Lo Han Guo fruit), glycyrrhizin, phyllodulcin (extracted from leaves of Hydrangea macrophylla, about 400 to 600 X sucrose), monellin, mabinlin, brazzein, circulin, pentadin, either alone or in combination.

Sweetness enhancers, which only are sweet in the presence of other compounds such as acids, also can be used in a beverage composition. Non-limiting examples of sweetness enhancers (also known as sweetness potentiators) include curculin, miraculin, cynarin, chlorogenic acid, caffeic acid, strogins, arabinogalactan, maltol and dihyroxybenzoic acids. In certain embodiments, beverage compositions comprising monatin also include flavor enhancers or stabilizers, such as Sucramask™ or trehalose.

Food grade natural or artificial colorants may optionally be included in the beverage compositions. These colorants may be selected from those generally known and available in the art, including synthetic colors (e.g., azo dyes, triphenylmethanes, xanthenes, quinines, and indigoids), caramel color, titanium dioxide, red #3, red #40, blue #1, and yellow #5. Natural coloring agents such as beet juice (beet red), carmine, curcumin, lutein, carrot juice, berry juices, spice extractives (turmeric, annatto and/or paprika), and carotenoids, for example, may also be used. The type and amount of colorant selected will depend on the end product and consumer preference.

In some embodiments, beverage compositions also include one or more natural or synthetic flavorings. Suitable flavorings include citrus and non-citrus fruit flavors; spices; herbs; botanicals; chocolate, cocoa, or chocolate liquor; coffee; flavorings obtained from vanilla beans; nut extracts; liqueurs and liqueur extracts; fruit brandy distillates; aromatic chemicals, imitation flavors; and concentrates, extracts, or essences of any ofthe same. Citrus flavors include, for example, lemon, lime, orange, tangerine, grapefruit, citron or kumquat. Many flavorings are available commercially from, e.g., Rhodia USA (Cranbury, NJ); IFF (South Brunswick, NJ); Wild Flavors, Inc. (Erlanger, KY); Silesia Flavors, Inc. (Hoffman Estates, IL), Chr. Hansen (Milkwaukee, WI), and Firmenisch (Princeton, NJ).

For example, a beverage syrup for preparing a carbonated soft drink can include a natural cola flavor (e.g., from Kola nut extract) that can be used to impart a cola flavor to the beverage. In some embodiments, flavorings can be formed into an emulsion, which is then dispersed into the beverage syrup. Emulsion droplets usually have a specific gravity less than that ofthe water and therefore can form a separate phase. Weighting agents, emulsifiers, and emulsion stabilizers can be used to stabilize the flavor emulsion droplets. Examples of such emulsifiers and emulsion stabilizer include gums, pectins, cellulose, polysorbates, sorbitan esters and propylene glycol alginates. In some embodiments, cola flavor emulsions represent 0.8 to 1.5 % of a beverage syrup. In other embodiments, additional flavorings that can be used to enhance the cola flavor include citrus flavors, such as lemon, lime, orange, tangerine, grapefruit, citron or kumquat, and spice flavors such as clove and vanilla. In other embodiments, citrus flavors (e.g., natural lemon or lime flavor) represent about 0.03 to 0.06 % of a beverage syrup and spice flavors (e.g., vanilla) represent 0.5 to 1.5% of a beverage syrup.

The pH of a beverage syrup can be controlled by the addition of acids (e.g., inorganic or organic acids). Typically, the pH ofthe beverage syrup ranges from 2.5 to about 5 (e.g., 2.5 to about 4.0). A particularly useful inorganic acid includes phosphoric acid, which can be present in its undissociated form, or as an alkali metal salt (e.g., potassium or sodium hydrogen phosphate, or potassium or sodium dihydrogen phosphate salts). Non-limiting examples of organic acids that can be used include citric acid, malic acid, fiimaric acid, adipic acid, gluconic acid, glucuronolactone, hydroxycitric acid, tartaric acid, ascorbic acid, acetic acid or mixtures thereof. These acids can be present in their undissociated form or as their respective salts.

In some embodiments, the beverage syrup further comprise caffeine (e.g., from the natural cola flavor). Caffeine also can be added separately.

In one embodiment, a carbonated beverage may be prepared by diluting a beverage syrup with carbonated water such that the resulting beverage contains 15 to 25 % ofthe syrup and 75 to 85% water. Alternatively, non-carbonated water can be used to dilute the syrup to prepare the beverage then carbon dioxide can be introduced into the beverage to achieve carbonation. In another embodiment, the carbonated beverage typically is placed into a container such as a bottle or can and then sealed. Any conventional carbonation methodology can be used to make the carbonated beverages of this invention. In some embodiments, the beverage compositions can be dried beverage mixes. It is noted that "dry" material may contain residual levels of liquid. For instance, a beverage mix can be a malted beverage mix, chocolate-flavored beverage mix, or a powdered fruit drink mix such as Kool-Aid® or Crystal Light®. In one embodiment, dried beverage mixes can be prepared by wet-mixing liquid ingredients in solution and vacuum drying the ingredients to provide a dry cake, followed by pulverizing the dry cake to a base powder. Ingredients such as oil, emulsifiers, and water can be used to blend in further dry ingredients, such as adding a cocoa powder to the base powder.

In another embodiment, a base beverage powder that does not typically have a sweetener, such as a lemonade packet, which is typically combined with sucrose by the consumer, can be blended with a high intensity sweetener such as monatin. The blending can be facilitated, for example, by using a diluent or bulking agent such as maltodextrin, hydrolyzed starch, dextrose, polydextrin, and inulin.

In other embodiments, malted beverage mixes include dry beverage ingredients, such as, for example, a powdered protein source such as milk powder, skim milk powder, egg protein powder, vegetable or grain protein isolates such as soy protein isolates, malt powders, hydrolysed cereal powders, starch powders, other carbohydrate powders, vitamins, minerals, cocoa powders, and powdered flavoring agents, or any combination of such ingredients. Liquid malted beverage ingredients can include, for example, one or more of fats and oils, liquid malt extracts, liquid sweeteners such as honey and glucose syrup, and liquid protein sources such as vegetable protein concentrates, or any combination thereof. Suitable fats include, without limitation, partially or fully hydrogenated vegetable oils such as cotton seed oil, soybean oil, corn oil, sunflower oil, palm oil, canola oil, palm kernel oil, peanut oil, rice oil, safflower oil, coconut oil, rape seed oil, and their mid- and high-oleic counterparts; or any combination thereof. Animal fats such as butter fat also can be used. The amount of each malted beverage ingredient can vary depending on the desired formulation. In some embodiments, monatin can be combined with a bulk sweetener as discussed above.

In some embodiments, fruit beverage premixes include citric acid (e.g., 60 to 70 %), flavorings (e.g., 2 to 4 %), colorants (e.g., 0.001 to 1 %), monatin, calcium phosphate (e.g., 0 to 25 %), a clouding agent (e.g., 0 to 5%), and ascorbic acid (e.g., 0 to 2%). For example, a fruit beverage mix may include 64.9% citric acid, 20.5% calcium phosphate, 3.9% of a clouding agent, 0.78 ascorbic acid, 2.7% flavors, 0.1% colors, and monatin. In some embodiments, monatin can be combined with a bulk sweetener as discussed above. In another embodiment, to prepare a fruit beverage, the premix can be reconstituted with water such that the resulting beverage contains about 0.5 to 1.5 % (e.g., 0.75%) ofthe mix.

In one embodiment, a dry chocolate drink composition can include skimmed milk powder (e.g., about 20 to 30%), whey powder (e.g., 35 to 45%), coffee whitener (e.g., 10 to 15%), fat reduced cocoa powder (e.g., 15 to 20%), potassium bicarbonate (e.g., 0.1 to 10%), guar gum (e.g., 0.06 to 2%), carrageenan (e.g., 0.05 to 5%), flavors (e.g., chocolate and/or vanilla), and monatin. For example, a dry chocolate drink composition can include 26% skimmed milk powder, 40% whey powder, 12% coffee whitener, 18% fat reduced cocoa powder, 1% potassium bicarbonate, 0.6% guar gum, 0.5% carrageenan, chocolate flavor, vanilla flavor, and monatin. In another embodiment, to prepare a chocolate beverage, the premix can be reconstituted with water or milk such that the resulting beverage contains about 0.5 to 1.5 % (e.g., 0.8%) ofthe mix.

In some embodiments, mixtures of dry ingredients useful in preparing a beverage composition, mixtures of wet ingredients useful for the same, or liquid mixtures (dispersions) of dry and wet ingredients, are provided as compositions. Such compositions may be provided as an article of manufacture and can be packaged in appropriate containers (e.g., bags, buckets, cartons) for easy transport to points of sale and preparation and for easy pouring and/or mixing. The article of manufacture may contain optional objects, such as utensils; containers for mixing; or other optional ingredients. The articles of manufacture can include instructions for preparing beverage compositions.

It is expected that monatin contained in beverages, as compared to other sweeteners in beverages, will have a longer shelf-life, greater heat and acid stability, as well as better taste characteristics and marketing advantages. The invention will be further described in the following examples, which does not limit the scope ofthe invention described. EXAMPLES

EXAMPLE 1

Cloning and Expression of Tryptophan Aminotransferases This example describes methods that were used to clone tryptophan aminotransferases, which can be used to convert tryptophan to indole-3 -pyruvate.

Experimental Overview

Eleven genes encoding aminotransferases were cloned into E. coli. These genes were Bacillus subtilis D-alanine aminotransferase (dat, Genbank Accession No. Yl 4082.1 bp

28622-29470 and Genbank Accession No. NP_388848.1, nucleic acid sequence and amino acid sequence, respectively), Sinorhizobium meliloti (also termed Rhizobium melilotϊ) tyrosine aminotransferase (tatA, SEQ ID NOS: 1 and 2, nucleic acid sequence and amino acid sequence, respectively), Rhodobacter sphaeroides strain 2.4.1 tyrosine aminotransferase (tatA asserted by homology, SEQ ID NOS: 3 and 4, nucleic acid sequence and amino acid sequence, respectively), R. sphaeroides 35053 tyrosine aminotransferase (asserted by homology, SEQ ID NOS: 5 and 6, nucleic acid sequence and amino acid sequence, respectively), Leishmania major broad substrate aminotransferase (bsat, asserted by homology to peptide fragments from L. mexicana, SEQ ID NOS: 7 and 8, nucleic acid sequence and amino acid sequence, respectively), Bacillus subtilis aromatic aminotransferase (araT, asserted by homology, SEQ ID NOS: 9 and 10, nucleic acid sequence and amino acid sequence, respectively), Lactobacillus amylovorus aromatic aminotransferase (araT asserted by homology, SEQ ID NOS: 11 and 12, nucleic acid sequence and amino acid sequence, respectively), R. sphaeroides 35053 multiple substrate aminotransferase (asserted by homology, SEQ ID NOS: 13 and 14, nucleic acid sequence and amino acid sequence, respectively), Rhodobacter sphaeroides strain 2.4.1 multiple substrate aminotransferase (msa asserted by homology, Genbank Accession No. A AAE01000093.1, bp 14743-16155 and Genbank Accession No. ZP00005082.1, nucleic acid sequence and amino acid sequence, respectively), Escherichia coli aspartate aminotransferase (aspC, Genbank Accession No. AE000195.1 bp 2755-1565 and Genbank Accession No. AAC74014.1, nucleic acid sequence and amino acid sequence, respectively), and E. coli tyrosine aminotransferase (tyrB, SEQ ID NOS: 31 and 32, nucleic acid sequence and amino acid sequence, respectively). The genes were cloned, expressed, and tested for activity in conversion of tryptophan to indole-3 -pyruvate, along with commercially available enzymes. All eleven clones had activity.

Identification of Bacterial Strains that Can Contain Polypeptides with the Desired Activity No genes in the NCBI (National Center for Biotechnology Information) database were designated as tryptophan aminotransferases. However, organisms having this enzymatic activity have been identified. L-tryptophan aminotransferase (TAT) activity has been measured in cell extracts or from purified protein from the following sources: Rhizobacterial isolate from Festuca octoflora, pea mitochondria and cytosol, sunflower crown gall cells, Rhizobium leguminosarum biovar trifoli, Erwinia herbicola pv gypsophilae, Pseudomonas syringae pv. savastanoi, Agrobacterium tumefaciens, Azospirillum lipferum 8c brasilense, Enterobacter cloacae, Enterobacter agglomerans, Bradyrhizobium elkanii, Candida maltosa, Azotobacter vinelandii, rat brain, rat liver, Sinorhizobium meliloti, Pseudomonas fluorescens CHA0, Lactococcus lactis, Lactobacillus casei, Lactobacillus helveticus, wheat seedlings, barley, Phaseolus aureus (mung bean), Saccharomyces uvarum (carlsbergensis), Leishmania sp., maize, tomato shoots, pea plants, tobacco, pig, Clostridium sporogenes, and Streptomyces griseus. EXAMPLE 2

Conversion of Indole- -lactate to Indole-3 -pyruvate

As shown in FIGS. 1 and 3, indole-3 -lactic acid can be used to produce indole-3-pyruvate. Conversion between lactic acid and pyruvate is a reversible reaction, as is conversion between indole-3 -pyruvate and indole-3 -lactate. The oxidation of indole-lactate was typically followed due to the high amount of background at 340 nm from indole-3 -pyruvate.

The standard assay mixture contained 100 mM potassium phosphate, pH 8.0, 0.3 mM NAD⁺, 7 units of lactate dehydrogenase (LDH) (Sigma-L2395, St. Louis, MO), and 2 mM substrate in 0.1 mL. The assay was performed in duplicate in a UV-transparent microtiter plate, using a Molecular Devices SpectraMax Plus platereader. Polypeptide and buffer were mixed and pipetted into wells containing the indole-3 -lactic acid and NAD⁺ and the absorbance at 340 nm of each well was read at intervals of 9 seconds after brief mixing. The reaction was held at 25°C for 5 minutes. The increase in absorbance at 340 nm follows the production of NADH from NAD . Separate negative controls were performed without NAD⁺ and without substrate. D-LDH from Leuconostoc mesenteroides (Sigma catalog number L2395) appeared to exhibit more activity with the indole-derivative substrates than did L-LDH from Bacillus stearothermophilus (Sigma catalog number L5275).

Similar methods were utilized with D-lactic acid and NAD+ or NADH and pyruvate, the natural substrates of D-LDH polypeptides. The V_max for the reduction of pyruvate was 100- 1000 fold higher than the V_maχ for the oxidation of lactate. The V_maχ for the oxidation reaction of indole-3 -lactic with D-LDH was approximately one-fifth of that with lactic acid. The presence of indole-3 -pyruvate was also measured by following the change in absorbance at 327 (the enol-borate derivative) using 50 mM sodium borate buffer containing 0.5 mM EDTA and 0.5 mM sodium arsenate. Small, but repeatable, absorbance changes were observed, as compared to the negative controls for both L and D-LDH polypeptides.

Additionally, broad specificity lactate dehydrogenases (enzymes with activity associated with EC 1.1.1.27, EC 1.1.1.28, and/or EC 1.1.2.3) can be cloned and used to make indole-3- pyruvate from indole-3 -lactic acid. Sources of broad specificity dehydrogenases include E. coli, Neisseria gonorrhoeae, and Lactobacillus plantarum.

Alternatively, indole-3-pyruvate can be produced by contacting indole-3 -lactate with cellular extracts from Clostridium sporogenes which contain an indolelactate dehydrogenase (EC 1.1.1.110); or Trypanosoma cruzi epimastigotes cellular extracts which contain p- hydroxyphenylactate dehydrogenase (EC 1.1.1.222) known to have activity on indole-3 - pyruvate; or Pseudomonas acidovorans or E. coli cellular extracts, which contain an imidazol-5-yl lactate dehydrogenase (EC 1.1.1.111); or Coleus blumei, which contains a hydroxyphenylpyruvate reductase (EC 1.1.1.237); or Candida maltosa which contains a D- aromatic lactate dehydrogenase (EC 1.1.1.222). References describing such activities include, Nowicki et al. (FEMS Microbiol Lett 71 :119-24, 1992), Jean and DeMoss (Canadian J. Microbiol. 14 1968, Coote and Hassall (Biochem. J i l l: 237-9, 1969), Cortese et al. (CR. Seances Soc. Biol. Fil. 162 390-5, 1968), Petersen and Alfermann (Z. Naturforsch. C: Biosci.

43 501-4, 1988), and Bhatnagar et al. (J. Gen Microbiol 135:353-60, 1989). In addition, a lactate oxidase such as the one from Pseudomonas sp. (Gu et al J. Mol. Catalysis B:

Enzymatic: 18:299-305, 2002), can be utilized for oxidation of indole-3 -lactic to indole-3- pyruvate. EXAMPLE 3

Conversion of L-tryptophan to Indole-3-pyruvate utilizing L-amino acid oxidase This example describes methods used to convert tryptophan to indole-3 -pyruvate via an oxidase (EC 1.4.3.2), as an alternative to using a tryptophan aminotransferase as described in Example 1. L-amino acid oxidase was purified from Crotalus durissus (Sigma, St. Louis, MO, catalog number A-2805). The accession numbers of L-amino acid oxidases for molecular cloning include: CAD21325.1, AAL14831, NP_490275, BAB78253, A38314, CAB71136, JE0266, T08202, S48644, CAC00499, P56742, P81383, 093364, P81382, P81375, S62692, P23623, AAD45200, AAC32267, CAA88452, AP003600, and Z48565.

Reactions were performed in microcentrifuge tubes in a total volume of 1 mL, incubated for 10 minutes while shaking at 37°C. The reaction mix contained 5 mM L-tryptophan, 100 mM sodium phosphate buffer pH 6.6, 0.5 mM sodium arsenate, 0.5 mM EDTA, 25 mM sodium tetraborate, 0.016 mg catalase (83 U, Sigma C-3515), 0.008 mg FAD (Sigma), and 0.005- 0.125 Units of L-amino acid oxidase. Negative controls contained all components except tryptophan, and blanks contained all components except the oxidase. Catalase was used to remove the hydrogen peroxide formed during the oxidative deamination. The sodium tetraborate and arsenate were used to stabilize the enol-borate form of indole-3 -pyravate, which shows a maximum absorbance at 327 nm. Indole-3 -pyruvate standards were prepared at concentrations of 0.1 - 1 mM in the reaction mix.

The purchased L-amino acid oxidase had a specific activity of 540 μg indole-3 -pyruvate formed per minute per mg protein. This is the same order of magnitude as the specific activity of tryptophan aminotransferase enzymes. EXAMPLE 4

Converting Indole-3 -pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid with an Aldolase

This example describes methods that can be used to convert indole-3 -pyruvate to MP using an aldolase (lyase) (FIG. 2). Aldol condensations are reactions that form carbon-carbon bonds between the β-carbon of an aldehyde or ketone and the carbonyl carbon of another aldehyde or ketone. A carbanion is formed on the carbon adjacent to the carbonyl group of one substrate, and serves as a nucleophile attacking the carbonyl carbon ofthe second substrate (the electrophilic carbon). Most commonly, the electrophilic substrate is an aldehyde, so most aldolases fall into the EC 4.1.2.- category. Quite often, the nucleophilic substrate is pyruvate. It is less common for aldolases to catalyze the condensation between two keto-acids or two aldehydes.

However, aldolases that catalyze the condensation of two carboxylic acids have been identified. For example, EP 1045-029 describes the production of L-4-hydroxy-2- ketoglutaric acid from glyoxylic acid and pyruvate using a Pseudomonas culture (EC 4.1.3.16). In addition, 4-hydroxy-4-methyl-2-oxoglutarate aldolase (4-hydroxy-4-methyl-2- oxoglutarate pyravate lyase, EC 4.1.3.17) can catalyze the condensation of two keto acids. Therefore, similar aldolase polypeptides were used to catalyze the condensation of indole-3 - pyruvate with pyravate.

Cloning

4-Hydroxy-4-methyl-2-oxoglutarate pyruvate lyases (ProA aldolase, EC 4.1.3.17) and 4- hydroxy -2-oxoglutarate glyoxylate-lyase (KHG aldolase, EC 4.1.3.16) catalyze reactions very similar to the aldolase reaction of FIG. 2. Primers were designed with compatible overhangs for the pET30 Xa/LIC vector (Novagen, Madison, WI).

Activity Results with proA gene products Both the C. testosteroni proA and S. meliloti SMc00502 gene constructs had high levels of expression when induced with IPTG. The recombinant proteins were highly soluble, as determined by SDS-PAGE analysis of total protein and cellular extract samples. The C. testosteroni gene product was purified to > 95% purity. Because the yield ofthe S. meliloti gene product was very low after affinity purification using a His-Bind cartridge, cellular extract was used for the enzymatic assays.

Both recombinant aldolases catalyzed the formation of MP from indole-3 -pyruvate and pymvate. The presence of both divalent magnesium and potassium phosphate were required for enzymatic activity. No product was apparent when indole-3 -pyruvate, pyruvate, or potassium phosphate was absent. A small amount ofthe product was also formed in the absence of enzyme (typically one order of magnitude less than when enzyme was present). The product peak eluted from the reverse phase C18 column slightly later than the indole-3 - pymvate standard, the mass spectrum of this peak showed a collisionally-induced parent ion ([M + H]+) of 292.1 , the parent ion expected for the product MP. The major daughter fragments present in the mass spectrum included those with m/z =158 (lH-indole-3- carbaldehyde carbonium ion), 168 (3-buta-l,3-dienyl-lH-indole carbonium ion), 274 (292 - Η₂O), 256 (292 - 2 H₂O), 238 (292 - 3 H2O), 228 (292 - CH4O3), and 204 (loss of pyruvate). The product also exhibited a UV spectrum characteristic of other indole-containing compounds such as tryptophan, with the λ_max of 279-280 and a small shoulder at approximately 290 nm.

The amount of MP produced by the C. testosteroni aldolase increased with an increase in reaction temperature from room temperature to 37°C, amount of substrate, and amount of magnesium. The synthetic activity ofthe enzyme decreased with increasing pH, the maximum product observed was at pH 7. Based on tryptophan standards, the amount of MP produced under a standard assay using 20 μg of purified protein was approximately 10-40 μg per one mL reaction.

Due to the high degree of homology ofthe S. meliloti and C. testosteroni ProA aldolase coding sequences with the other genes described above, it is expected that all ofthe recombinant gene products can catalyze this reaction. Moreover, it is expected that aldolases that have threonine (T) at positions 59 and 87, arginine (R) at 119, aspartate (D) at 120, and histidine (H) at 31 and 71, (based on the numbering system of C. testosteroni) will have similar activity.

Activity Results with khg gene products

Both the B. subtilis and E. coli khg gene constructs had high levels of expression of protein when induced with IPTG, while the S. meliloti khg had a lower level of expression. The recombinant proteins were highly soluble, as judged by SDS-PAGE analysis of total proteins and cellular extracts. The B. subtilis and E. coli khg gene products were purified to > 95% purity; the yield ofthe S. meliloti gene product was not as high after affinity purification using a His-Bind cartridge. There is no evidence that magnesium and phosphate are required for activity for this enzyme. However, the literature reports performing the assays in sodium phosphate buffer, and the enzyme reportedly is bifunctional and has activity on phosphorylated substrates such as 2- keto-3-deoxy-6-phosphogluconate (KDPG). The enzymatic assays were performed as described above, and in some instances the phosphate was omitted. The results indicate that the recombinant KHG aldolases produced MP, but were not as active as the ProA aldolases. In some cases the level of MP produced by KHG was almost identical to the amount produced by magnesium and phosphate alone. Phosphate did not appear to increase the KHG activities. The Bacillus enzyme had the highest activity, approximately 20-25% higher activity than the magnesium and phosphate alone, as determined by SRM (see Example 10). The Sinorhizobium enzyme had the least amount of activity, which can be associated with folding and solubility problems noted in the expression. All three enzymes have the active site glutamate (position 43 in B. subtilis numbering system) as well as the lysine required for Shiff base formation with pyravate (position 130); however, the B. subtilis enzyme contains a threonine in position 47, an active site residue, rather than arginine. The B. subtilis KHG is smaller and appears to be in a cluster distinct from the S. meliloti and E. coli enzymes, with other enzymes having the active site threonine. The differences in the active site may be the reason for the increased activity ofthe B. subtilis enzyme.

Improvement of Aldolase Activity

Catalytic antibodies can be as efficient as natural aldolases, accept a broad range of substrates, and can be used to catalyze the reaction shown in FIG. 2.

Aldolases can also be improved by directed evolution, for example as previously described for a KDPG aldolase (highly homologous to KHG described above) evolved by DNA shuffling and error-prone PCR to remove the requirement for phosphate and to invert the enantioselectivity. The KDPG aldolase polypeptides are useful in biochemical reactions since they are highly specific for the donor substrate (herein, pymvate), but are relatively flexible with respect to the acceptor substrate (i.e. indole-3 -pymvate) (Koeller & Wong, Nature 409:232-9, 2001). KHG aldolase has activity for condensation of pyruvate with a number of carboxylic acids. Mammalian versions ofthe KHG aldolase are thought to have broader specificity than bacterial versions, including higher activity on 4-hydroxy 4-methyl 2-oxoglutarate and acceptance of both stereoisomers of 4-hydroxy-2-ketoglutarate. Bacterial sources appear to have a 10-fold preference for the R stereoisomer. There are nearly 100 KHG homologs available in genomic databases, and activity has been demonstrated in Pseudomonas, Paracoccus, Providencia, Sinorhizobium, Morganella, E. coli, and mammalian tissues. These enzymes can be used as a starting point for tailoring the enantiospecificity that is desired for monatin production.

Aldolases that utilize pyruvate and another substrate that is either a keto acid and/or has a bulky hydrophobic group like indole can be "evolved" to tailor the polypeptide's specificity, speed, and selectivity. In addition to KHG and ProA aldolases demonstrated herein, examples of these enzymes include, but are not limited to: KDPG aldolase and related polypeptides (KDPH); transcarboxybenzalpyruvate hydratase-aldolase from Nocardioides st; 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (2'-carboxybenzalpyruvate aldolase) which condenses pymvate and 2-carboxybenzaldehyde (an aromatic ring-containing substrate); trans-O-hydroxybenzylidenepyruvate hydratase-aldolase from Pseudomonas putida and Sphingomonas aromaticivorans, which also utilizes pyruvate and an aromatic-containing aldehyde as substrates; 3-hydroxyaspartate aldolase (erythro-3-hydroxy-L-aspartate glyoxylate lyase), which uses 2-oxo acids as the substrates and is thought to be in the organism Micrococcus denitrificans; benzoin aldolase (benzaldehyde lyase), which utilizes substrates containing benzyl groups; dihydroneopterin aldolase; L-threo-3-phenylserine benzaldehyde-lyase (phenylserine aldolase) which condenses glycine with benzaldehyde; 4- hydroxy-2-oxo valerate aldolase; 1,2-dihydroxybenzylpyruvate aldolase; and 2- hydroxybenzalpyruvate aldolase.

A polypeptide having the desired activity can be selected by screening clones of interest using the following methods. Tryptophan auxotrophs are transformed with vectors carrying the clones of interest on an expression cassette and are grown on a medium containing small amounts of monatin or MP. Since aminotransferases and aldolase reactions are reversible, the cells are able to produce tryptophan from a racemic mixture of monatin. Similarly, organisms (both recombinant and wildtype) can be screened by ability to utilize MP or monatin as a carbon and energy source. One source of target aldolases is expression libraries of various Pseudomonas and rhizobacterial strains. Pseudomonads have many unusual catabolic pathways for degradation of aromatic molecules and they also contain many aldolases; whereas the rhizobacteria contain aldolases, are known to grow in the plant rhizosphere, and have many ofthe genes described for construction of a biosynthetic pathway for monatin.

EXAMPLE 5 Chemical Synthesis ofthe Monatin Precursor

Example 4 described a method of using an aldolase to convert indole-3 -pymvate to MP. This example describes an alternative method of chemically synthesizing MP. MP can be formed using a typical aldol-type condensation (FIG. 4). Briefly, a typical aldol-type reaction involves the generation of a carbanion ofthe pymvate ester using a strong base, such as LDA (lithium diisopropylamide), lithium hexamethyldisilazane or butyl lithium. The carbanion that is generated reacts with the indole-pymvate to form the coupled product.

Protecting groups that can be used for protecting the indole nitrogen include, but are not limited to: t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz). Blocking groups for carboxylic acids include, but are not limited to, alkyl esters (for example, methyl, ethyl, benzyl esters). When such protecting groups are used, it is not possible to control the stereochemistry ofthe product that is formed. However, if R2 and/or R3 are chiral protecting groups (FIG. 4), such as (S)-2-butanol, menthol, or a chiral amine, this can favor the formation of one MP enantiomer over the other.

EXAMPLE 6 Conversion of Tryptophan or Indole-3 -Pyruvate to Monatin

An in vitro process utilizing two enzymes, an aminotransferase and an aldolase, produced monatin from tryptophan and pyruvate. In the first step alpha-ketoglutarate was the acceptor ofthe amino group from tryptophan in a transamination reaction generating indole-3 - pymvate and glutamate. An aldolase catalyzed the second reaction in which pymvate was reacted with indole-3-pyravate, in the presence of Mg²⁺ and phosphate, generating the alpha- keto derivative of monatin (MP), 2-hydroxy-2-(indol-3-ylmethyl)-4-ketoglutaric acid. Transfer ofthe amino group from the glutamate formed in the first reaction produced the desired product, monatin. Purification and characterization ofthe product established that the stereoisomer formed was S,S-monatin. Alternative substrates, enzymes, and conditions are described as well as improvements that were made to this process. Enzymes

The aldolase, 4-hydroxy-4-methyl-2-oxoglutarate pymvate lyase (ProA aldolase, proA gene) (EC 4.1.3.17) from Comamonas testosteroni was cloned, expressed and purified as described in Example 4. The 4-hydroxy-2-oxoglutarate glyoxylate lyases (KHG aldolases) (EC 4.1.3.16) from B. subtilis, E. coli, and S. meliloti were cloned, expressed and purified as described in Example 4.

The aminotransferases used in conjunction with the aldolases to produce monatin were L- aspartate aminotransferase encoded by the E. coli aspC gene, the tyrosine aminotransferase encoded by the E. coli tyrB gene, the S. meliloti TatA enzyme, the broad substrate aminotransferase encoded by the L. major bsat gene, or the glutamic-oxaloacetic transaminase from pig heart (Type Ila). The cloning, expression and purification ofthe non- mammalian proteins are described in Example 1. Glutamic-oxaloacetic transaminase from pig heart (type Ila) was obtained from Sigma (# G7005).

Method using ProA aldolase and L-aspartate aminotransferase

The reaction mixture contained 50 mM ammonium acetate, pH 8.0, 4 mM MgCl₂, 3 mM potassium phosphate, 0.05 mM pyridoxal phosphate, 100 mM ammonium pyruvate, 50 mM tryptophan, 10 mM alpha-ketoglutarate, 160 mg of recombinant C. testosteroni ProA aldolase (unpurified cell extract, ~30% aldolase), 233 mg of recombinant E. coli L-aspartate aminotransferase (unpurified cell extract, ~40% aminotransferase) in one liter. All components except the enzymes were mixed together and incubated at 30°C until the tryptophan dissolved. The enzymes were then added and the reaction solution was incubated at 30°C with gentle shaking (100 rpm) for 3.5 hours. At 0.5 and 1 hour after the addition of the enzymes aliquots of solid tryptophan (50 mmoles each) were added to the reaction. All of the added tryptophan did not dissolve, but the concentration was maintained at 50 mM or higher. After 3.5 hours, the solid tryptophan was filtered off. Analysis ofthe reaction mixture by LC/MS using a defined amount of tryptophan as a standard showed that the concentration of tryptophan in the solution was 60.5 mM and the concentration of monatin was 5.81 mM (1.05 g).

The following methods were used to purify the final product. Ninety percent ofthe clear solution was applied to a column of BioRad AG50W-X8 resin (225 mL; binding capacity of 1.7 meq/mL). The column was washed with water, collecting 300 mL fractions, until the absorbance at 280 nm was <5% ofthe first flow through fraction. The column was then eluted with 1 M ammonium acetate, pH 8.4, collecting 4 300-mL fractions. All 4 fractions contained monatin and were evaporated to 105 mL using a roto-evaporator with a tepid water bath. A precipitate formed as the volume reduced and was filtered off over the course ofthe evaporation process.

Analysis ofthe column fractions by LC/MS showed that 99%) ofthe tryptophan and monatin bound to the column. The precipitate that formed during the evaporation process contained >97% tryptophan and <2% of monatin. The ratio of tryptophan to product in the supernatant was approximately 2:1.

The supernatant (7 mL) was applied to a 100 mL Fast Flow DEAE Sepharose (Amersham Biosciences) column previously converted to the acetate form by washing with 0.5 L 1 M NaOH, 0.2 L water, 1.0 L of 1.0 M ammonium acetate, pH 8.4, and 0.5 L water. The supernatant was loaded at <2 mL/min and the column was washed with water at 3-4 mL/min until the absorbance at 280 nm was ~0. Monatin was eluted with 100 mM ammonium acetate, pH 8.4, collecting 4 100-mL fractions.

Analysis ofthe fractions showed that the ratio of tryptophan to monatin in the flow through fractions was 85: 15 and the ratio in the eluent fractions was 7:93. Assuming the extinction coefficient at 280 nm of monatin is the same as tryptophan, the eluent fractions contained 0.146 nrmole of product. Extrapolation to the total 1 L reaction would produce -2.4 mmoles (~710 mg) of monatin, for a recovery of 68%.

The eluent fractions from the DEAE Sepharose column were evaporated to <20 mL. An aliquot ofthe product was further purified by application to a C₈ preparative reversed-phase column using the same chromatographic conditions as those described in Example 10 for the analytical-scale monatin characterization. Waters Fractionlynx™ software was employed to trigger automated fraction collection of monatin based on detection ofthe m/z = 293 ion. The fraction from the C₈ column with the corresponding protonated molecular ion for monatin was collected, evaporated to dryness, and then dissolved in a small volume of water. This fraction was used for characterization ofthe product. The resulting product was characterized using the following methods. UV/Visible Spectroscopy. UV/visible spectroscopic measurements of monatin produced enzymatically were carried out using a Gary 100 Bio UV/visible spectrophotometer. The purified product, dissolved in water, showed an absorption maximum of 280 nm with a shoulder at 288 nm, characteristics typical of indole containing compounds.

LC/MS Analysis. Analyses of mixtures for monatin derived from the in vitro biochemical reactions were carried out as described in Example 10. A typical LC/MS analysis of monatin in an in vitro enzymatic synthetic mixture is illustrated in FIG. 5. The lower panel of FIG. 5 illustrates a selected ion chromatogram for the protonated molecular ion of monatin at m/z = 293. This identification of monatin in the mixture was corroborated by the mass spectrum illustrated in FIG. 6. Analysis ofthe purified product by LC/MS showed a single peak with a molecular ion of 293 and absorbance at 280 nm. The mass spectrum was identical to that shown in FIG. 6.

MS/MS Analysis. LC/MS/MS daughter ion experiments, as described in Example 10, were also performed on monatin. A daughter ion mass spectrum of monatin is illustrated in FIG. 7. Tentative structural assignments of all fragment ions labeled in FIG. 7 were made. These include fragment ions of m/z = 275 (293 - H₂O), 257 (293-(2 x H₂O)), 230 (275-COOH), 212 (257-COOH), 168 (3-buta-l,3-dienyl-lH-indole carbonium ion), 158 (lH-indole-3- carbaldehyde carbonium ion), 144 (3-ethyl-lH-indole carbonium ion), 130 (3 -methylene- 1H- indole carbonium ion), and 118 (indole carbonium ion). Many of these are the same as those obtained for MP (Example 4), as expected if derived from the indole portion ofthe molecule. Some are 1 mass unit higher than those seen for MP, due to the presence of an amino group instead of a ketone.

Accurate Mass Measurement of Monatin. FIG. 8 illustrates the mass spectrum obtained for purified monatin employing an Applied Biosystems-Perkin Elmer Q-Star hybrid quadrapole/time-of-flight mass spectrometer. The measured mass for protonated monatin using tryptophan as an internal mass calibration standard was 293.1144. The calculated mass of protonated monatin, based on the elemental composition C₁₄Η₁ N₂O₅ is 293.1137. This is a mass measurement error of less than 2 parts per million (ppm), providing conclusive evidence ofthe elemental composition of monatin produced enzymatically.

NMR Spectroscopy. The NMR experiments were performed on a Varian Inova 500 MHz instrument. The sample of monatin (~3 mg) was dissolved in 0.5 mL of D₂O. Initially, the solvent (D₂O) was used as the internal reference at 4.78 ppm. Since the peak for water was large, the 1H-NMR was run with suppression ofthe peak for water. Subsequently, due to the broadness ofthe water peak, the C-2 proton of monatin was used as the reference peak, and set at the published value of 7.192 ppm.

For ¹³C-NMR, an initial run of several hundred scans indicated that the sample was too dilute to obtain an adequate ^I3C spectrum in the allotted time. Therefore, a heteronuclear multiple quantum coherence (HMQC) experiment was perfoπned, which enabled the correlation ofthe hydrogens and the carbons to which they were attached, and also providing information on the chemical shifts of the carbons.

A summary ofthe 1H and HMQC data is shown in Tables 1 and 2. By comparison to published values, the NMR data indicated that the enzymatically produced monatin was either (S,S), (R,R), or a mixture of both.

Chiral LC MS Analysis. To establish that the monatin produced in vitro was one stereoisomer, and not a mixture ofthe (R,R) and (S,S) enantiomers, chiral LC/MS analyses were carried out using the instrumentation described in Example 10.

Chiral LC separations were made using an Chirobiotic T (Advanced Separations Technology) chiral chromatography column at room temperature. Separation and detection, based on published protocols from the vendor, were optimized for the R- (D) and S- (L) stereoisomers of tryptophan. The LC mobile phase consisted of A) water containing 0.05% (v/v) trifluoroacetic acid; B) Methanol containing 0.05% (v/v) trifluoroacetic acid. The elution was isocratic at 70% A and 30% B. The flow rate was 1.0 mL/min, and PDA absorbance was monitored from 200 nm to 400 nm. The instrumental parameters used for chiral LC/MS analysis of tryptophan and monatin are identical to those described in Example 10 for LC/MS analysis. Collection of mass spectra for the region m/z 150-400 was utilized. Selected ion chromatograms for protonated molecular ions ([M + H]⁺ = 205 for both R- and S-tryptophan and [M + H] = 293 for monatin) allowed direct identification of these analytes in the mixtures.

The chromatograms of R- and S-tryptophan and monatin, separated by chiral chromatography and monitored by MS, are shown in FIG. 9. The single peak in the chromatogram of monatin indicates that the compound is one stereoisomer, with a retention time almost identical to S- tryptophan.

TABLE 1 1 H NMR data

¹ Vleggaar et α/^". (J.C.S. Perkin Trans. 1:3095-8, 1992). ² Takeshi and Shusuke (JP2002060382, 2002-02-26).

¹ Vleggaar et al. (J.C.S. Perkin Trans. 1:3095-8, 1992). Polarimetry. The optical rotation was measured on a Rudolph Autopol III polarimeter. The monatin was prepared as a 14.6 mg/mL solution in water. The expected specific rotation (MD²⁰) for S,S monatin (salt form) is -49.6 for a 1 g/mL solution in water (Vleggaar et al). The observed [α]o²⁰ was -28.1 for the purified, enzymatically produced monatin indicating that it was the S, S stereoisomer.

Improvements

The reaction conditions, including reagent and enzyme concentrations, were optimized and yields of 5-10 mg/mL were produced using the following reagent mix: 50 mM ammonium acetate pH 8.3, 2 mM MgCl₂, 200 mM pyravate (sodium or ammonium salt), 5 mM alpha- ketoglutarate (sodium salt), 0.05 mM pyridoxal phosphate, deaerated water to achieve a final volume of 1 mL after the addition ofthe enzymes, 3 mM potassium phosphate, 50 μg/mL of recombinant ProA aldolase (cell extract; total protein concentration of 167 μg/mL), 1000 μg/mL of L-aspartate aminotransferase encoded by the E. coli aspC gene (cell extract; total protein concentration of 2500 μg/mL), and solid tryptophan to afford a concentration of > 60 mM (saturated; some undissolved throughout the reaction). The mixture was incubated at 30°C for 4 hours with gentle stirring or mixing.

Substitutions The concentration of alpha-ketoglutarate can be reduced to 1 mM and supplemented with 9 mM aspartate with an equivalent yield of monatin. Alternative amino acid acceptors can be utilized in the first step, such as oxaloacetate.

When recombinant L. major broad substrate aminotransferase was used in place ofthe E. coli L-aspartate aminotransferase, similar yields of monatin were achieved. However, a second unidentified product (3-10% ofthe major product) with a molecular mass of 292 was also detected by LC-MS analysis. Monatin concentrations of 0.1-0.5 mg/mL were produced when the E. coli tyrB encoded enzyme, the S. meliloti tatA encoded enzyme or the glutamic- oxaloacetic transaminase from pig heart (type Ila) was added as the aminotransferase. When starting the reaction from indole-3 -pymvate, a reductive amination can be done for the last step with glutamate dehydrogenase and NADH (as in Example 7). The KHG aldolases from B. subtilis, E. coli, and S. meliloti were also used with the E. coli L- aspartate aminotransferase to produce monatin enzymatically. The following reaction conditions were used: 50 mM NH₄-OAc pH 8.3, 2 mM MgCl_2; 200 mM pymvate, 5 mM glutamate, 0.05 mM pyridoxal phosphate, deaerated water to achieve a final volume of 0.5 mL after the addition ofthe enzymes, 3 mM potassium phosphate, 20 μg/mL of recombinant B. subtilis KHG aldolase (purified), ca. 400 μg/mL of E. coli L-aspartate aminotransferase (AspC) unpurified from cell extract, and 12 mM indole-3 -pyruvate. The reactions were incubated at 30°C for 30 minutes with shaking. The amount of monatin produced using the B. subtilis enzyme was 80 ng/mL, and increased with increasing amounts of aldolase. If indole-3 -pymvate and glutamate were replaced by saturating amounts of tryptophan and 5 mM alpha-ketoglutarate, the production of monatin was increased to 360 ng/mL. Reactions were repeated with 30 μg/mL of each ofthe three KHG enzymes in 50 mM Tris pH 8.3, with saturating amounts of tryptophan, and were allowed to proceed for an hour in order to increase detection. The Bacillus enzyme had the highest activity as in Example 4, producing approximately 4000 ng/mL monatin. The E. coli KHG produced 3000 ng/mL monatin, and the S. meliloti enzyme produced 2300 ng/mL.

EXAMPLE 7

Interconversion between MP and Monatin The amination of MP to form monatin can be catalyzed by aminotransferases such as those identified in Examples 1 and 6, or by dehydrogenases that require a reducing cofactor such as NADH or NADPH. These reactions are reversible and can be measured in either direction. The directionality, when using a dehydrogenase enzyme, can be largely controlled by the concentration of ammonium salts.

Dehydrogenase activity. The oxidative deamination of monatin was monitored by following the increase in absorbance at 340 nm as NAD(P)+ was converted to the more chromophoric NAD(P)H. Monatin was enzymatically produced and purified as described in Example 6.

A typical assay mixture contained 50 mM Tris-HCl, pH 8.0 to 8.9, 0.33 mM NAD⁺ or

NADP⁺, 2 to 22 units of glutamate dehydrogenase (Sigma), and 10-15 mM substrate in 0.2 mL. The assay was performed in duplicate in a UV-transparent microtiter plate, on a Molecular Devices SpectraMax Plus platereader. A mix ofthe enzyme, buffer, and NAD(P)⁺ were pipetted into wells containing the substrate and the increase in absorbance at 340 nm was monitored at 10 second intervals after brief mixing. The reaction was incubated at 25°C for 10 minutes. Negative controls were carried out without the addition of substrate, and glutamate was utilized as a positive control. The type III glutamate dehydrogenase from bovine liver (Sigma # G-7882) catalyzed the conversion ofthe monatin to the monatin precursor at a rate of conversion approximately one-hundredth the rate ofthe conversion of glutamate to alpha-ketoglutarate.

Transamination activity. Monatin aminotransferase assays were conducted with the aspartate aminotransferase (AspC) from E. coli, the tyrosine aminotransferase (TyrB) from E. coli, the broad substrate aminotransferase (BSAT) from L. major, and the two commercially available porcine glutamate-oxaloacetate aminotransferases described in Example 1. Both oxaloacetate and alpha-ketoglutarate were tested as the amino acceptor. The assay mixture contained (in 0.5 mL) 50 mM Tris-HCl, pH 8.0, 0.05 mM PLP, 5 mM amino acceptor, 5 mM monatin, and 25 μg of aminotransferase. The assays were incubated at 30°C for 30 minutes, and the reactions were stopped by addition of 0.5 mL isopropyl alcohol. The loss of monatin was monitored by LC/MS (Example 10). The highest amount of activity was noted with L. major BSAT with oxaloacetate as the amino acceptor, followed by the same enzyme with alpha-ketoglutarate as the amino acceptor. The relative activity with oxaloacetate was: BSAT > AspC > porcine type Ila > porcine type I =TyrB. The relative activity with alpha- ketoglutarate was: BSAT > AspC > porcine type I > porcine type Ila > TyrB.

EXAMPLE 8

Production of Monatin from Tryptophan and C3 Sources Other than Pyruvate As described above in Example 6, indole-3 -pyravate or tryptophan can be converted to monatin using pyruvate as the C3 molecule. However, in some circumstances, pyravate may not be a desirable raw material. For example, pyruvate may be more expensive than other C3 carbon sources, or may have adverse effects on fermentations if added to the medium. Alanine can be transaminated by many PLP-enzymes to produce pyravate. Tryptophanase-like enzymes perform beta-elimination reactions at faster rates than other PLP enzymes such as aminotransferases. Enzymes from this class (4.1.99.-) can produce ammonia and pyravate from amino acids such as L-serine, L-cysteine, and derivatives of serine and cysteine with good leaving groups such as O-methyl-L-serine, O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine, S-alkyl-L-cysteine, O-acyl-L-serine, 3-chloro-L-alanine.

Processes to produce monatin using EC 4.1.99.- polypeptides can be improved by mutating the β-tyrosinase (TPL) or tryptophanase according to the method of Mouratou et al (J. Biol. Chem 274:1320-5, 1999). Mouratou et al. describe the ability to covert the β-tyrosinase into a dicarboxylic amino acid β-lyase, which has not been reported to occur in nature. The change in specificity was accomplished by converting valine (V) 283 to arginine (R) and arginine (R) 100 to threonine (T). These amino acid changes allow for the lyase to accept a dicarboxylic amino acid for the hydrolytic deamination reaction (such as aspartate). Aspartate, therefore, can also be used as a source of pyruvate for subsequent aldol condensation reactions.

Additionally, cells or enzymatic reactors can be supplied with lactate and an enzyme that converts lactate to pymvate. Examples of enzymes capable of catalyzing this reaction include lactate dehydrogenase and lactate oxidase.

The reaction mixture consisted of 50 mM Tris-Cl pH 8.3, 2 mM MgCl₂, 200 mM C3 carbon source, 5 mM alpha-ketoglutarate, sodium salt, 0.05 mM pyridoxal phosphate, deaerated water to achieve a final volume of 0.5 mL after the addition ofthe enzymes, 3 mM potassium phosphate pH 7.5, 25 μg of cmde recombinant C. testosteroni ProA aldolase as prepared as in Example 4, 500 μg of crude L-aspartate aminotransferase (AspC) as prepared in Example 1, and solid tryptophan to afford a concentration of > 60 mM (saturated; some undissolved throughout the reaction). The reaction mix was incubated at 30°C for 30 minutes with mixing. Serine, alanine, and aspartate were supplied as 3 -carbon sources. Assays were performed with and without secondary PLP enzymes (purified) capable of performing beta- elimination and beta-lyase reactions (tryptophanase (TNA), double mutant tryptophanase, β- tyrosinase (TPL)). The results are shown in Table 3: TABLE 3

The monatin produced from alanine and serine as 3 -carbon sources was verified by

LC/MS/MS daughter scan analysis, and was identical to the characterized monatin produced in Example 6. Alanine was the best alternative tested, and was transaminated by the AspC enzyme. The amount of monatin produced was increased by addition ofthe tryptophanase, which is capable of transamination as a secondary activity. The amount of monatin produced with serine as a carbon source nearly doubled with the addition ofthe tryptophanase enzymes, even though only one-fifth ofthe amount of tryptophanase was added in comparison to the aminotransferase. AspC is capable of some amount of beta-elimination activity alone. The results with aspartate indicate that the tryptophanase activity on aspartate does not increase with the same site-directed mutations as previously suggested for β- tyrosinase. It is expected that the mutant β-tyrosinase will have higher activity for production of monatin.

EXAMPLE 9

Chemical Synthesis of Monatin

The addition of alanine to indole-3-pymvic acid produces monatin, and this reaction can be performed synthetically with a Grignard or organolithium reagent.

For example, to 3-chloro- or 3-bromo-alanine which has been appropriately blocked at the carboxyl and amino groups, is added magnesium under anhydrous conditions. Indole-3 - pymvate (appropriately blocked) is then added to form the coupled product followed by removal ofthe protecting groups to form monatin. Protecting groups that are particularly useful include THP (tetrahydropyranyl ether) which is easily attached and removed.

EXAMPLE 10 Detection of Tryptophan, Monatin, andMP

This example describes methods used to detect the presence of monatin, or its precursor 2- hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid.

LC/MS Analysis Analyses of mixtures for monatin, MP, and/or tryptophan derived from in vitro or in vivo biochemical reactions were performed using a Waters/Micromass liquid chromatography- tandem mass spectrometry (LC/MS/MS) instrument including a Waters 2690 liquid chromatograph with a Waters 996 Photo-Diode Array (PDA) absorbance monitor placed in series between the chromatograph and a Micromass Quattro Ultima triple quadrupole mass spectrometer. LC separations were made using a Supelco Discovery C₁₈ reversed-phase chromatography column, 2.1mm x 150 mm, or an Xterra MS C₈ reversed-phase chromatography column, 2.1mm x 250 mm, at room temperature. The LC mobile phase consisted of A) water containing 0.05% (v/v) trifluoroacetic acid and B) methanol containing 0.05% (v/v) trifluoroacetic acid.

The gradient elution was linear from 5% B to 35% B, 0-9 min, linear from 35% B to 90% B, 9-16 min, isocratic at 90% B, 16-20 min, linear from 90% B to 5% B, 20-22 min, with a 10 min re-equilibration period between runs. The flow rate was 0.25 mL/min, and PDA absorbance was monitored from 200 nm to 400 nm. All parameters ofthe ESI-MS were optimized and selected based on generation of protonated molecular ions ([M + H]⁺) ofthe analytes of interest, and production of characteristic fragment ions.

The following instrumental parameters were used for LC/MS analysis of monatin: Capillary: 3.5 kV; Cone: 40 V; Hex 1: 20 V; Aperture: 0 V; Hex 2: 0 V; Source temperature: 100°C; Desolvation temperature: 350°C; Desolvation gas: 500 L/h; Cone gas: 50 L/h; Low mass resolution (Ql): 15.0; High mass resolution (Ql): 15.0; Ion energy: 0.2; Entrance: 50V; Collision Energy: 2; Exit: 50V; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): 3.5; Multiplier: 650. Uncertainties for reported mass/charge ratios (m/z) and molecular masses are ± 0.01%. Initial detection ofthe alpha-keto acid form of monatin (MP) and monatin in the mixtures was accomplished by LC/MS monitoring with collection of mass spectra for the region m/z 150-400. Selected ion chromatograms for protonated molecular ions ([M + H]⁺ = 292 for MP, [M + H]⁺ = 293 for monatin) allowed direct identification of these analytes in the mixtures.

MS/MS Analysis

LC/MS/MS daughter ion experiments were performed on monatin as follows. A daughter ion analysis involves transmission ofthe parent ion (e.g., m/z = 293 for monatin) of interest from the first mass analyzer (Ql) into the collision cell ofthe mass spectrometer, where argon is introduced and chemically dissociates the parent into fragment (daughter) ions. These fragment ions are then detected with the second mass analyzer (Q2), and can be used to corroborate the structural assignment ofthe parent. Tryptophan was characterized and quantified in the same way via transfrnission and fragmentation of m/z ^~ 205.

The following instrumental parameters were used for LC/MS/MS analysis of monatin: Capillary: 3.5 kV; Cone: 40 V; Hex 1 : 20 V; Aperture: 0 V; Hex 2: 0 V; Source temperature: 100 °C; Desolvation temperature: 350 °C; Desolvation gas: 500 L/h; Cone gas: 50 L/h; Low mass resolution (Ql): 13.0; High mass resolution (Ql): 13.0; Ion energy: 0.2; Entrance: -5 V; Collision Energy: 14; Exit: IV; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): 3.5; Multiplier: 650.

High-Throughput Determination of Monatin and Tryptophan

High-throughput analyses (< 5 mm/sample) of mixtures for monatin and tryptophan derived from in vitro or in vivo reactions were carried out using instrumentation described above, and the same parameters as described for LC/MS/MS. LC separations were made using a 4.6 mm x 50 mm Advanced Separation Technologies Chirobiotic T column at room temperature. The LC mobile phase consisted of A) water containing 0.25% acetic acid; B) Methanol containing 0.25% acetic acid. The isocratic elution was at 50% B, 0-5 min. The flow rate was 0.6 mL/min. All parameters ofthe ESI-MS/MS system were optimized and selected based on optimal in-source generation ofthe protonated molecular ion of tryptophan and the internal standard ²H₅-tryptophan, as well as collision-induced production of amino acid-specific fragment ions for multiple reaction monitoring (MRM) experiments. The following instrumental parameters were used for LC/MS/MS analysis of monatin and tryptophan in the positive ion multiple reaction monitoring (mrm) mode: Capillary: 3.5 kV; Cone: 20 V; Hex 1: 15 V; Aperture: 1 V; Hex 2: 0 V; Source temperature: 100 °C; Desolvation temperature: 350 °C; Desolvation gas: 500 L/h; Cone gas: 40 L/h; Low mass resolution (Ql): 12.0; High mass resolution (Ql): 12.0; Ion energy: 0.2; Entrance: - 5 V; Collision Energy: 14; Exit: 1 V; Low mass resolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): 0.5; Multiplier: 650. MRM parameters: Interchannel delay: 0.03 s; InterScan delay: 0.03 s; Dwell: 0.05 s.

Accurate Mass Measurement of Monatin. High resolution MS analysis was carried out using an Applied Biosystems-Perkin Elmer Q- Star hybrid quadrupole/time-of-flight mass spectrometer. The measured mass for protonated monatin used tryptophan as an internal mass calibration standard. The calculated mass of protonated monatin, based on the elemental composition C₁₄H₁₇N₂O₅ is 293.1137. Monatin produced using the biocatalytic process described in Example A showed a measured mass of 293.1144. This is a mass measurement error of less than 2 parts per million (ppm), providing conclusive evidence ofthe elemental composition of monatin produced enzymatically.

EXAMPLE 11

Production of Monatin in Bacteria This example describes methods used to produce monatin in E. coli cells. One skilled in the art will understand that similar methods can be used to produce monatin in other bacterial cells. In addition, vectors containing other genes in the monatin synthesis pathway (FIG. 2) can be used.

Trp-1 + glucose medium, a minimal medium that has been used for increased production of tryptophan in E. coli cells (Zeman et al. Folia Microbiol. 35:200-4, 1990), was prepared as follows. To 700 mL nanopure water the following reagents were added: 2 g (NH₄)₂SO₄, 13.6 g KH₂PO₄, 0.2 g MgSO_4*7H₂0, 0.01 g CaCl_2*2H₂0, and 0.5 mg FeSO_4*7H₂0. The pH was adjusted to 7.0, the volume was increased to 850 mL, and the medium was autoclaved. A 50% glucose solution was prepared separately, and sterile-filtered. Forty mL was added to the base medium (850 mL) for a 1 L final volume. A 10 g/L L-tryptophan solution was prepared in 0.1 M sodium phosphate pH 7, and sterile- filtered. One-tenth volume was typically added to cultures as specified below. A 10% sodium pymvate solution was also prepared and sterile-filtered. A 10 mL aliquot was typically used per liter of culture. Stocks of ampicillin (100 mg/mL), kanamycin (25 mg/mL) and IPTG (840 mM) were prepared, sterile-filtered, and stored at -20°C before use. Tween 20 (polyoxyethylene 20-Sorbitan monolaurate) was utilized at a 0.2% (vol/vol) final concentration. Ampicillin was used at non-lethal concentrations, typically 1-10 μg/mL final concentration.

Fresh plates of E. coli BL21(DE3)::C. testosteroni proA/pET 30 Xa/LIC (described in Example 4) were prepared on LB medium containing 50 μg/mL kanamycin. Overnight cultures (5 mL) were inoculated from a single colony and grown at 30°C in LB medium with kanamycin. Typically a 1 to 50 inoculum was used for induction in trp-1 + glucose medium. Fresh antibiotic was added to a final concentration of 50 mg/L. Shake flasks were grown at 37°C prior to induction.

Cells were sampled every hour until an OD₆₀₀ of 0.35-0.8 was obtained. Cells were then induced with 0.1 mM IPTG, and the temperature reduced to 34 °C. Samples (1 mL) were collected prior to induction (zero time point) and centrifuged at 5000 x g. The supernatant was frozen at -20°C for LC/MS analysis. Four hours post-induction, another 1 mL sample was collected, and centrifuged to separate the broth from the cell pellet. Tryptophan, sodium pyravate, ampicillin, and Tween were added as described above.

The cells were grown for 48 hours post-induction, and another 1 mL sample was taken and prepared as above. At 48 hours, another aliquot of tryptophan and pyravate were added. The entire culture volume was centrifuged after approximately 70 hours of growth (post- induction), for 20 minutes at 4°C and 3500 rpm. The supernatant was decanted and both the broth and the cells were frozen at -80°C. The broth fractions were filtered and analyzed by LC/MS. The heights and areas ofthe [M+H]⁺ = 293 peaks were monitored as described in Example 10. The background level ofthe medium was subtracted. The data was also normalized for cell growth by plotting the height ofthe [M+H]⁺ = 293 peak divided by the optical density ofthe culture at 600 nm. Higher levels of monatin were produced when pymvate, ampicillin, and Tween were added 4 hours post induction rather than at induction. Other additives such as PLP, additional phosphate, or additional MgCl₂ did not increase the production of monatin. Higher titers of monatin were obtained when tryptophan was utilized instead of indole-3 -pymvate, and when the tryptophan was added post-induction rather than at inoculation, or at induction. Prior to induction, and 4 hours post-induction (at time of substrate addition), there was typically no detectable level of monatin in the fermentation broth or cellular extracts. Negative controls were done utilizing cells with pET30a vector only, as well as cultures where tryptophan and pymvate were not added. A parent MS scan demonstrated that the compound with (m+l)/z = 293 was not derived from larger molecules, and daughter scans (performed as in Example 10) were similar to monatin made in vitro.

The effect of Tween was studied by utilizing 0, 0.2% (vol/vol), and 0.6% final concentrations of Tween-20. The highest amount of monatin produced by shake flasks was at 0.2% Tween. The ampicillin concentration was varied between 0 and 10 μg/mL. The amount of monatin in the cellular broth increased rapidly (2.5 X) between 0 and 1 μg/mL, and increased 1.3 X when the ampicillin concentration was increased from 1 to 10 μg/mL.

A time course experiment showing typical results is shown in FIG. 10. The amount of monatin secreted into the cell broth increased, even when the values are normalized for cell growth. By using the molar extinction coefficient of tryptophan, the amount of monatin in the broth was estimated to be less than 10 μg/mL. The same experiment was repeated with the cells containing vector without pro A insert. Many ofthe numbers were negative, indicating the peak height at m/z=293 was less in these cultures than in the medium alone (FIG. 10). The numbers were consistently lower when tryptophan and pyruvate were absent, demonstrating that monatin production is a result of an enzymatic reaction catalyzed by the aldolase enzyme.

The in vivo production of monatin in bacterial cells was repeated in 800 mL shake flask experiments and in fermentors. A 250 mL sample of monatin (in cell-free broth) was purified by anion exchange chromatography and preparative reverse-phase liquid chromatography. This sample was evaporated, and submitted for high resolution mass analysis (described in Example 6). The high resolution MS indicated that the metabolite being produced is monatin.

In vitro assays indicate that aminotransferase needs to be present at higher levels than aldolase (see Example 6), therefore the aspartate aminotransferase from E. coli was overexpressed in combination with the aldolase gene to increase the amount of monatin produced. Primers were designed to introduce C. testosteroni proA into an operon with α^pC/pET30 Xa/LIC, as follows: 5' primer: ACTCGGATCCGAAGGAGATATACATATGTACGAACTGGGACT (SEQ ID NO: 67) and 3* primer: CGGCTGTCGACCGTTAGTCAATATATTTCAGGC (SEQ ID NO: 68).

The 5' primer contains a BamHI site, the 3' primer contains a Sail site for cloning. PCR was performed as described in Example 4, and gel purified. The spC/pET30 Xa/LIC constract was digested with BamHI and Sail, as was the PCR product. The digests were purified using a Qiagen spin column. The pro A PCR product was ligated to the vector using the Roche Rapid DNA Ligation kit (Indianapolis, IN) according to manufacturer's instructions.

Chemical transformations were done using Novablues Singles (Novagen) as described in Example 1. Colonies were grown up in LB medium containing 50 mg/L kanamycin and plasmid DNA was purified using the Qiagen spin miniprep kit. Clones were screened by restriction digest analysis and sequence was confirmed by Seqwright (Houston, TX). Constructs were subcloned into BLR(DE3), BLR(DE3)pLysS, BL21 (DE3) and

BL21(DE3)pLysS (Novagen). he proA/nET30 Xa/LIC constract was also transformed into BL21(DE3)pLysS.

Initial comparisons of BLR(DE3) shake flask samples under the standard conditions described above demonstrated that the addition ofthe second gene (aspC) improved the amount of monatin produced by seven-fold. To hasten growth, BL21(DE3)-derived host strains were used. The pro A clones and the two gene operon clones were induced in Trp-1 medium as above, the pLysS hosts had chloramphenicol (34 mg/L) added to the medium as well. Shake flask experiments were performed with and without the addition of 0.2% Tween- 20 and 1 mg/L ampicillin. The amount of monatin in the broth was calculated using in vitro produced purified monatin as a standard. SRM analyses were performed as described in Example 10. Cells were sampled at zero, 4 hours, 24 hours, 48 hours, 72 hours, and 96 hours of growth. The results are shown in Table 4 for the maximum amounts produced in the culture broths. In most instances, the two gene constract gave higher values than the pro A construct alone. The pLysS strains, which should have leakier cell envelopes, had higher levels of monatin secreted, even though these strains typically grow at a slower rate. The additions of Tween and ampicillin were beneficial.

Table 4 Amount of Monatin Produced by E. coli Bacteria

EXAMPLE 12 Production of Monatin in Yeast This example describes methods used to produce monatin in eukaryotic cells. One skilled in the art will understand that similar methods can be used to produce monatin in any cell of interest. In addition, other genes can be used (e.g., those listed in FIG. 2) in addition to, or alternatively to those described in this example.

The pESC Yeast Epitope Tagging Vector System (Stratagene, La Jolla, CA) was used to clone and express the E. coli aspC and C. testosteroni proA genes into Saccharomyces cerevisiae. The pESC vectors contain both the GALl and the GAL10 promoters on opposite strands, with two distinct multiple cloning sites, allowing for expression of two genes at the same time. The pESC-His vector also contains the His3 gene for complementation of histidine auxotrophy in the host (YPH500). The GALl and GAL10 promoters are repressed by glucose and induced by galactose; a Kozak sequence is utilized for optimal expression in yeast. The pESC plasmids are shuttle vectors, allowing the initial constract to be made in E. coli (with the bla gene for selection); however, no bacterial ribosome binding sites are present in the multiple cloning sites.

The following primers were designed for cloning into pESC-His (restriction sites are underlined, Kozak sequence is in bold): aspC (BamHVSaH), GALl: 5'- CGCGGATCCATAATGGTTGAGAACATTACCG-3' (SEQ ID NO: 69) and 5'- ACGCGTCGACTTACAGCACTGCCACAATCG-3' (SEQ ID NO: 70). proA (EcoRVNotT), GAL10: 5'-CCGGAATTCATAATGGTCGAACTGGGAGTTGT-3' (SEQ ID NO: 71) and 5'-GAATGCGGCCGCTTAGTCAATATATTTCAGGCC-3' (SEQ ID NO: 72).

The second codon for both mature proteins was changed from an aromatic amino acid to valine due to the introduction ofthe Kozak sequence. The genes of interest were amplified using pET30 Xa/LIC miniprep DNA from the clones described in Examples 1 and Example 4 as template. PCR was performed using an Eppendorf Master cycler gradient thermocycler and the following protocol for a 50 μL reaction: 1.0 μL template, 1.0 μM of each primer, 0.4 mM each dNTP, 3.5 U Expand High Fidelity Polymerase (Roche, Indianapolis, IN), and IX Expand™ buffer with Mg. The thermocycler program used consisted of a hot start at 94°C for 5 minutes, followed by 29 repetitions ofthe following steps: 94°C for 30 seconds, 50°C for 1 minute 45 seconds, and 72°C for 2 minutes 15 seconds. After the 29 repetitions the sample was maintained at 72°C for 10 minutes and then stored at 4°C. The PCR products were purified by separation on a 1% TAE-agarose gel followed by recovery using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).

The pESC-His vector DNA (2.7 μg) was digested with BamHl/Sall and gel-purified as above. The aspC PCR product was digested with BamHVSall and purified with a QIAquick PCR Purification Column. Ligations were performed with the Roche Rapid DNA Ligation Kit following the manufacturer's protocols. Desalted ligations were electroporated into 40 μl Electromax DH10B competent cells (Invitrogen) in a 0.2 cm Biorad disposable cuvette using a Biorad Gene Pulser II with pulse controller plus, according to the manufacturer's instructions. After 1 hour of recovery in 1 mL of SOC medium, the transformants were plated on LB medium containing 100 μg/mL ampicillin. Plasmid DNA preparations for clones were done using QIAprep Spin Miniprep Kits. Plasmid DNA was screened by restriction digest, and sequenced (Seqwright) for verification using primers designed for the vector.

The aspC /pESC-His clone was digested with EcoRl and N , as was the proA PCR product. DΝA was purified as above, and ligated as above. The two gene constract was transformed into DH10B cells and screened by restriction digest and DΝA sequencing.

The construct was transformed into S. cerevisiae strain YPH500 using the S.c. EasyComp™ Transformation Kit (Invitrogen). Transformation reactions were plated on SC-His minimal medium (Invitrogen pYES2 manual) containing 2% glucose. Individual yeast colonies were screened for the presence of the proA and aspC genes by colony PCR using the PCR primers above. Pelleted cells (2 μl) were suspended in 20 μL of Y-Lysis Buffer (Zymo Research) containing 1 μl of zymolase and heated at 37°C for 10 minutes. Four μL of this suspension was then used in a 50 μL PCR reaction using the PCR reaction mixture and program described above.

Five mL cultures were grown overnight on SC-His + glucose at 30°C and 225 rpm. The cells were gradually adjusted to growth on raffinose in order to minimize the lag period prior to induction with galactose. After approximately 12 hours of growth, absorbance measurements at 600 nm were taken, and an appropriate volume of cells was spun down and resuspended to give an OD of 0.4 in the fresh SC-His medium. The following carbon sources were used sequentially: 1% raffinose + 1 % glucose, 0.5% glucose + 1.5% raffinose, 2% raffinose, and finally 1% raffinose + 2% galactose for induction.

After approximately 16 hours of growth in induction medium, the 50 mL cultures were divided into duplicate 25 mL cultures, and the following were added to only one ofthe duplicates: (final concentrations) 1 g/L L-tryptophan, 5 mM sodium phosphate pH 7.1, 1 g/L sodium pyruvate, 1 mM MgCl₂. Samples of broths and cell pellets from the non-induction medium, and from the 16 hour cultures prior to addition of substrates for the monatin pathway, were saved as negative controls. In addition, constructs containing only a functional aspC gene (and a truncated pro A gene) were utilized as another negative control. The cells were allowed to grow for a total of 69 hours post-induction. Occasionally the yeast cells were induced at a lower OD, and only grown for 4 hours prior to addition of tryptophan and pyruvate. However, these monatin substrates appear to inhibit growth and the addition at higher OD was more effective.

The cell pellets from the cultures were lysed with 5 mL of YeastBuster™ + 50 μl THP (Novagen) per gram (wet weight) of cells following manufacturer's protocols, with the addition of protease inhibitors and benzonase nuclease as described in previous examples. The culture broth and cell extracts were filtered and analyzed by SRM as described in Example 10. Using this method, no monatin was detected in the broth samples, indicating that the cells could not secrete monatin under these conditions. The proton motive force may be insufficient under these conditions or the general amino acid transporters may be saturated with tryptophan. Protein expression was not at a level that allowed for detection of changes using SDS-PAGE.

Monatin was detectable (approximately 60 ng/mL) transiently in cell extracts ofthe culture with two functional genes, when tryptophan and pyruvate were added to the medium.

Monatin was not detected in any ofthe negative control cell extracts. In vitro assays for monatin were performed in duplicate with 4.4 mg/mL of total protein (about double what is typically used for E. coli cell extracts) using the optimized assay described in Example 6. Other assays were performed with the addition of either 32 μg/mL C. testosteroni ProA aldolase or 400 μg/mL AspC aminotransferase, to determine which enzyme was limiting in the cell extract. Negative controls were performed with no addition of enzyme, or the addition of only AspC aminotransferase (the aldol condensation can occur to some extent without enzyme). Positive controls were performed with partially pure enzymes (30-40%), using 16 μg/mL aldolase and 400 μg/mL aminotransferase.

In vitro results were analyzed by SRM. The analysis of cell extracts showed that tryptophan was effectively transported into the cells when it was added to the medium post-induction, resulting in tryptophan levels two orders of magnitude higher than those in which no additional tryptophan was added. The results for in vitro monatin analysis are shown in Table 5 (numbers indicate ng/mL). Table 5 Monatin production with yeast cell extracts

Positive results were obtained with the full two-gene constract cell extracts with and without substrate added to the growth medium. These results, in comparison to the positive controls, indicate that the enzymes were expressed at levels of close to 1% ofthe total protein in yeast. The amount of monatin produced when the cell extract ofthe aspC constract (with truncated proA) was assayed with aldolase was significantly greater than when cell extracts were assayed alone, and indicates that the recombinant AspC aminotransferase comprises approximately 1-2% ofthe yeast total protein. The cell extracts of uninduced cultures had a small amount of activity when assayed with aldolase due to the presence of native aminotransferases in the cells. When assayed with AspC aminotransferase, the activity ofthe extracts from uninduced cells increased to the amount of monatin produced by the negative control with AspC (ca. 200 ng/mL). In contrast, the activity observed when assaying the two gene constract cell extract increases more when aminotransferase is supplemented than when aldolase is added. Since both genes should be expressed at the same level, this indicates that the amount of monatin produced is maximized when the level of aminotransferase is higher than that of aldolase, in agreement with results shown in Example 6.

The addition of pymvate and tryptophan not only inhibits cellular growth, but apparently inhibits protein expression as well. The addition ofthe pESC-Trp plasmid can be used to correct for tryptophan auxotrophy ofthe YPH500 host cells, to provide a means of supplying tryptophan with fewer effects on growth, expression, and secretion. EXAMPLE 13

Improvement of Enzymatic Processes using Coupled Reactions In theory, if no side reactions or degradation of substrates or intermediates occurs, the maximum amount of product formed from the enzymatic reaction illustrated in FIG. 1 is directly proportional to the equilibrium constants of each reaction, and the concentrations of tryptophan and pymvate. Tryptophan is not a highly soluble substrate, and concentrations of pymvate greater than 200 mM appear to have a negative effect on the yield (see Example 6).

Ideally, the concentration of monatin is maximized with respect to substrates, in order to decrease the cost of separation. Physical separations can be performed such that the monatin is removed from the reaction mixture, preventing the reverse reactions from occurring. The raw materials and catalysts can then be regenerated. Due to the similarity of monatin in size, charge, and hydrophobicity to several ofthe reagents and intermediates, physical separations will be difficult unless there is a high amount of affinity for monatin (such as an affinity chromatography technique). However, the monatin reactions can be coupled to other reactions such that the equilibrium ofthe system is shifted toward monatin production. The following are examples of processes for improving the yield of monatin obtained from tryptophan or indole-3 -pymvate.

Coupled reactions using oxaloacetate decarboxylase (EC 4.1.1.3) FIG. 11 is an illustration ofthe reaction. Tryptophan oxidase and catalase are utilized to drive the reaction in the direction of indole-3 -pyruvate production. Catalase is used in excess such that hydrogen peroxide is not available to react in the reverse direction or to damage the enzymes or intermediates. Oxygen is regenerated during the catalase reaction. Alternatively, indole-3 -pymvate can be used as the substrate.

Aspartate is used as the amino donor for the amination of MP, and an aspartate aminotransferase is utilized. Ideally, an aminotransferase that has a low specificity for the tryptophan/indole-3 -pyruvate reaction in comparison to the MP to monatin reaction is used so that the aspartate is not utilized to reaminate the indole-3 -pymvate. Oxaloacetate decarboxylase (from Pseudomonas sp.) can be added to convert the oxaloacetate to pymvate and carbon dioxide. Since CO₂ is volatile, it is not available for reaction with the enzymes, decreasing or even preventing the reverse reactions. The pymvate produced in this step can also be utilized in the aldol condensation reaction. Other decarboxylase enzymes can be used, and homologs are known to exist in Actinobacillus actinomycetemcomitans, Aquifex aeolicus, Archaeoglobus fulgidus, Azotobacter vinelandii, Bacteroides fragilis, several Bordetella species, Campylobacter jejuni, Chlorobium tepidum, Chloroflexus aurantiacus, Enter ococcus faecalis, Fusobacterium nucleatum, Klebsiella pneumoniae, Legionella pneumophila, Magnetococcus MC-1, Mannheimia haemolytica, Methylobacillus flagellatus KT, Pasteurella multocida Pm70, Petrotoga miotherma, Porphyromonas gingivalis, several Pseudomonas species, several Pyrococcus species, Rhodococcus, several Salmonella species, several Streptococcus species, Thermochromatium tepidum, Thermotoga maritima, Treponema pallidum, and several Vibrio species.

Tryptophan aminotransferase assays were performed with the aspartate aminotransferase (AspC) from E. coli, the tyrosine aminotransferase (TyrB) from E. coli, the broad substrate aminotransferase (BSAT) from L. major, and the two commercially available porcine glutamate-oxaloacetate aminotransferases as described in Example 1. Both oxaloacetate and alpha-ketoglutarate were tested as the amino acceptor. The ratio of activity using monatin (Example 7) versus activity using tryptophan was compared, to determine which enzyme had the highest specificity for the monatin aminotransferase reaction. These results indicated that the enzyme with the highest specificity for the monatin reaction verses the tryptophan reaction is the Porcine type II-A glutamate-oxaloacetate aminotransferase, GOAT (Sigma G7005). This specificity was independent of which amino acceptor was utilized. Therefore, this enzyme was used in the coupled reactions with oxaloacetate decarboxylase.

A typical reaction starting from indole-3 -pymvate included (final concentrations) 50 mM Tris-Cl pH 7.3, 6 mM indole-3 -pymvate, 6 mM sodium pyravate, 6 mM aspartate, 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl₂, 25 μg/mL aminotransferase, 50 μg/mL C. testosteroni ProA aldolase, and 3 Units/mL of decarboxylase (Sigma 04878). The reactions were allowed to proceed for 1 hour at 26°C. In some cases, the decarboxylase was omitted or the aspartate was substituted with alpha-ketoglutarate (as negative controls). The aminotransferase enzymes described above were also tested in place ofthe GOAT to confirm earlier specificity experiments. Samples were filtered and analyzed by LC/MS as described in Example 10. The results demonstrate that the GOAT enzyme produced the highest amount of monatin per mg of protein, with the least amount of tryptophan produced as a byproduct. In addition, there was a 2-3 fold benefit from having the decarboxylase enzyme added. The E. coli AspC enzyme also produced large amounts of monatin in comparison to the other aminotransferases .

Monatin production was increased by: 1) periodically adding 2 mM additions of indole- pyravate, pyravate, and aspartate (every half hour to hour), 2) performing the reactions in an anaerobic environment or with degassed buffers, 3) allowing the reactions to proceed overnight, and 4) using freshly prepared decarboxylase that has not been freeze-thawed multiple times. The decarboxylase was inhibited by concentrations of pyruvate greater than 12 mM. At concentrations of indole-3 -pyravate higher than 4 mM, side reactions with indole-3 -pyruvate were hastened. The amount of indole-3 -pymvate used in the reaction could be increased if the amount of aldolase was also increased. High levels of phosphate (50 mM) and aspartate (50 mM) were found to be inhibitory to the decarboxylase enzyme. The amount of decarboxylase enzyme added could be reduced to 0.5 U/mL with no decrease in monatin production in a one hour reaction. The amount of monatin produced increased when the temperature was increased from 26°C to 30°C and from 30°C to 37°C; however, at 37°C the side reactions of indole-3 -pyravate were also hastened. The amount of monatin produced increased with increasing pH from 7 to 7.3, and was relatively stable from pH 7.3- 8.3.

A typical reaction starting with tryptophan included (final concentrations) 50 mM Tris-Cl pH 7.3, 20 mM tryptophan, 6 mM aspartate, 6 mM sodium pyravate, 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl₂, 25 μg/mL aminotransferase, 50 μg/mL C. testosteroni ProA aldolase, 4 Units/mL of decarboxylase, 5-200 mU/mL L-amino acid oxidase (Sigma A- 2805), 168 U/mL catalase (Sigma C-3515), and 0.008 mg FAD. Reactions were carried out for 30 minutes at 30°C. Improvement was observed with the addition of decarboxylase. The greatest amount of monatin was produced when 50 mU/mL of oxidase was used. Improvements were similar to those observed when indole-3 -pyruvate was used as the substrate. In addition, the amount of monatin produced increased when 1) the tryptophan level was low (i.e., below the K_m ofthe aminotransferase enzyme and therefore unable to compete with MP in the active site), and 2) the ratio of oxidase to aldolase and aminotransferase was maintained at a level such that indole-3 -pyravate could not accumulate. Whether starting with either indole-3 -pymvate or tryptophan, the amount of monatin produced in assays with incubation times of 1-2 hours increased when 2-4 times the amounts of all the enzymes were used while maintaining the same enzyme ratio. Using either substrate, concentrations of approximately 1 mg/mL of monatin were achieved. The amount of tryptophan produced if starting from indole-pyruvate was typically less than 20% of the amount of product, which shows the benefit of utilizing coupled reactions. With further optimization and control ofthe concentrations of intermediates and side reactions, the productivity and yield can be improved greatly.

Coupled reactions using lysine epsilon aminotransferase (EC 2.6.1.36)

Lysine epsilon aminotransferase (L-Lysine 6-transaminase) is found in several organisms, including Rhodococcus, Mycobacterium, Streptomyces, Nocardia, Flavobacterium, Candida utilis, and Streptomyces. It is utilized by organisms as the first step in the production of some beta-lactam antibiotics (Rius and Demain, J Microbiol. Biotech., 7:95-100, 1997). This enzyme converts lysine to L-2-aminoadipate 6-semialdehyde (allysine), by a PLP-mediated transamination ofthe C-6 of lysine, utilizing alpha-ketoglutarate as the amino acceptor. Allysine is unstable and spontaneously undergoes an intramolecular dehydration to form 1- piperideine 6-carboxylate, a cyclic molecule. This effectively inhibits any reverse reaction from occurring. The reaction scheme is depicted in FIG. 12. An alternative enzyme, lysine- pymvate 6-transaminase (EC 2.6.1.71), can also be used.

A typical reaction contained in 1 mL: 50 mM Tris-HCl pH 7.3, 20 mM indole-3 -pyravate, 0.05 mM PLP, 6 mM potassium phosphate pH 8, 2-50 mM sodium pyravate, 1.5 mM MgCl₂, 50 mM lysine, 100 μg aminotransferase (lysine epsilon aminotransferase LAT-101, BioCatalytics Pasadena, CA), and 200 μg C. testosteroni ProA aldolase. The amount of monatin produced increased with increasing concentrations of pyravate. The maximum amount using these reaction conditions (at 50 mM pyravate) was 10-fold less than what was observed with coupled reactions using oxaloacetate decarboxylase (approximately 0.1 mg/mL).

A peak with [M+H]⁺ = 293 eluted at the expected time for monatin and the mass spectrum contained several ofthe same fragments observed with other enzymatic processes. A second peak with the correct mass to charge ratio (293) eluted slightly earlier than what is typically observed for the S,S monatin produced in Example 6, and may indicate the presence of another stereoisomer of monatin. Very little tryptophan was produced by this enzyme. However, there is likely some activity on pyravate (producing alanine as a byproduct). Also, the enzyme is known to be unstable. Improvements can be made by performing directed evolution experiments to increase stability, reduce the activity with pymvate, and increase the activity with MP. These reactions can also be coupled to L-amino acid oxidase/catalase as described above.

Other coupled reactions Another coupling reaction that can improve monatin yield from tryptophan or indole- pyruvate is shown in FIG. 13. Formate dehydrogenase (EC 1.2.1.2 or 1.2.1.43) is a common enzyme. Some formate dehydrogenases require NADH while others can utilize NADPH. Glutamate dehydrogenase catalyzed the interconversion between the monatin precursor and monatin in previous examples, using ammonium based buffers. The presence of ammonium formate and formate dehydrogenase is an efficient system for regeneration of cofactors, and the production of carbon dioxide is an efficient way to decrease the rate ofthe reverse reactions (Bommarius et al, Biocatalysis 10:37, 1994 and Galkin et al. Appl. Environ. Microbiol 63:4651-6, 1997). In addition, large amounts of ammonium formate can be dissolved in the reaction buffer. The yield of monatin produced by glutamate dehydrogenase reactions (or similar reductive aminations) can be improved by the addition of formate dehydrogenase and ammonium formate.

Other processes can be used to drive the equilibrium toward monatin production. For instance, if aminopropane is utilized as the amino acid donor in the conversion of MP to monatin with an omega-amino acid aminotransferase (EC 2.6.1.18) such as those described by in US patents 5,360,724 and 5,300,437, one ofthe resulting products would be acetone, a more volatile product than the substrate, aminopropane. The temperature can be raised periodically for short periods to flash off the acetone, thereby alleviating equilibrium. Acetone has a boiling point of 47°C, a temperature not likely to degrade the intermediates if used for short periods of time. Most aminotransferases that have activity on alpha- ketoglutarate also have activity on the monatin precursor. Similarly, if a glyoxylate/aromatic acid aminotransferase (EC 2.6.1.60) is used with glycine as the amino donor, glyoxylate is produced which is relatively unstable and has a highly reduced boiling point in comparison to gly cine.

EXAMPLE 14: Dose Response Curve

Solutions of monatin (mixture of approximately 96% ofthe 2R,4R/2S, 4S enantiometric pair and 4%o ofthe 2R,4S/2S,4R enantiometric pair — also called "racemic mix" of monatin") at

15, 30, 45, 60, 75 and 90 ppm were prepared in a pH 3.2 model soft drink system that contained 0.14% (w/v) citric acid and 0.04% (w/v) sodium citrate. The sweetness of monatin relative to sucrose was determined using the sweetness estimation methodology described below. All assessments were carried out in duplicate by a panel (n = 6-8) of trained panelists experienced in this sweetness determination procedure. All samples were served at a temperature of 20°C ± 1°C.

Monatin solutions were coded and presented individually to panelists, in random order.

Sucrose reference standards, ranging from 2.0 - 11.0% (w/v) sucrose, increasing in steps of 0.5%> (w/v) sucrose also were provided. Panelists were asked to estimate sweetness by comparing the sweetness ofthe test solution to the sucrose standards. This was carried out by taking 3 sips ofthe test solution, followed by a sip of water, followed by 3 sips of sucrose standard followed by a sip of water, etc. Panelists were encouraged to estimate the sweetness to one decimal place, e.g., 6.8, 8.5. A five minute rest period was imposed between evaluating the test solutions. Panelists also were asked to rinse well and eat a cracker to reduce any potential carry over effects. The sucrose equivalence values (SEVs) and standard deviations are summarized in Table 6.

The blends were all judged to exhibit rapid onset to sweetness and sweetness build to maximum intensity. The decay of sweetness also was rapid. Most ofthe mixtures were judged less fruity than sucrose, except the monatin/glucose blend. A slight lingering sweetness aftertaste was noted, very slight bitter/metallic notes. No licorice or cooling aftertaste was noted. TABLE 6 Monatin Dose Response Data

EXAMPLE 15: Blending of Monatin with Carbohydrate Sweeteners

Blends of monatin (as described in Example 14) with sucrose, HFCS (55% fructose), and glucose syrup (63 dextrose equivalents, DE) equisweet to 10.0% (w/v) sucrose were prepared. For each carbohydrate sweetener, the monatimsweetener ratio was adjusted so that monatin delivered 25, 50, and 75% ofthe total sweetness. Sweetness parity to 10.0 % (w/v) sucrose was determined using the sweetness estimation method described in Example 14. As in Example 14, all assessments were carried out in the pH 3.2 model soft drink system, using 6-8 panelists, each tasting in duplicate. Results are presented as Tables 7-9. Monatin compared similarly to sucralose, with a slight delay in onset of sweetness.

TABLE 7 Equisweet Blends of Monatin and Sucrose

TABLE 9 E uisweet Blends of Monatin and Glucose S ru

The quality of equisweet monatin/carbohydrate (50:50) blends then was assessed relative to sucrose by a small panel of trained assessors. This evaluation was carried out "double blind." The sucrose-sweetened system was identified as the control and all other products randomly coded. Panelists were asked to assess the randomly coded sample relative to the control for the following attributes: Sweetness Profile: Onset, build and decay; Flavor Profile: Acidity, bitterness and other characteristics; Mouthfeel; and Aftertaste. Panelists also were asked to assign a score (1; poor - 5; good) for the quality ofthe sweetener system. A summary ofthe comments made and scores given is presented as Table 10.

TABLE 10 Taste Profiling of Monatin/Carbohydrate Blends

EXAMPLE 16: Time Intensity Profile of Monatin in a Soft Drink System

Solutions of 80 ppm monatin (racemic mix of monatin described in Example 14), 10.0% (w/v) sucrose and 200 ppm sucralose were prepared in the pH 3.2 model soft drink system described in Example 14. The time intensity profile of these solutions then was assessed using the following procedure. Six panelists were included in the study. These panelists were screened for their general sensory acuity and selected for their sensitivity to sweetness intensity and sweetness quality differences. All were experienced in methods of sweetener assessment and had received special training in time intensity evaluations. Training sessions were carried out initially to familiarize the panel with the method of evaluation and scoring the samples over time using a computerized data entry system.

Samples of each solution (13 mL) were coded and presented individually to panelists, in random order. For each panelist, immediately after swallowing, the computer recorded timed intensity readings on the scale of 0-100 each second, up to 60 seconds. Each solution was evaluated in duplicate. The results ofthe time intensity evaluation are summarized as Table 11. TABLE 11 Time Intensity Study Results

These results indicate that the temporal taste attributes of monatin are comparable to sucrose, which is indicative of a high quality sweetener. Additionally, monatin compares favorably to sucralose, a commonly used high intensity sweetener.

EXAMPLE 17: Preparation of Cola and Lemon/lime Beverages Containing Monatin Cola and lemon/lime beverages were prepared using the following formulations and sweetened with sucrose, HFCS (55% fructose), aspartame, sucralose, monatin (racemic mix described in Example 14), monatin/sucrose, or monatin/HFCS. One part of syrup was added to 5.5 parts carbonated water and evaluated.

Lemon/Lime Syrup Formulation: Ingredient % wt/vol citric acid 2.400 sodium citrate 0.500 sodium benzoate 0.106 Flavor 0.450 (Lemon/Lime Flavor 730301-H ex. Givaudan Roure) Sweeteners see below Water to 100.000

Cola Syrup Formulation: Ingredient % wt/vol Phosphoric Acid 0.650 (75% solution) citric acid 0.066 sodium citrate 0.300 sodium benzoate 0.106 Cola Flavor A 1.100 (A01161 ex. Givaudan Roure) Cola Flavor B 1.100 (B01162 ex. Givaudan Roure) Sweeteners see below Water to 100.000

Sweetener concentration in lemon/lime or cola carbonate: sucrose 10% HFCS (55% Fmctose) 10% (solids) Aspartame 500 ppm Sucralose 200 ppm Monatin 67 ppm (in lemon/lime); 80 ppm (in cola) Monatin/sucrose 30.8 ppm/5.0% Monatin/HFCS 30.8 ppm/5.0% (solids) Assessments were carried out 'double blind' by a panel of trained tasters. The sucrose- sweetened product was identified as the control and all other products randomly coded. Panelists were asked to assess the randomly coded sample relative to the control for the following attributes:

Flavor Profile: Acidity Bitterness Other Characteristics

Sweetness Profile: Onset Build Intensity Decay

Mouthfeel Aftertaste

Panelists also were asked assign a score (1; poor — 5; good) for the quality ofthe sweetener system. A summary ofthe comments generated together with the average score awarded is presented in Tables 12 and 13 for lemon/lime carbonates and colas, respectively. In the lemon/lime flavor, monatin was comparable in flavor to aspartame. Blends of monatin/carbohydrate rated higher. In the cola, monatin was similar to aspartame.

TABLE 12 Taste Profiling Lemon/Lime Carbonates

TABLE 12 continued: Taste Profiling Lemon/Lime Carbonates

TABLE 13

Taste Profiling of Colas

TABLE 13 continued: Taste Profiling of Colas

Discussion

The monatin used in this example elicited a clean, sweet taste profile, essentially free from bitterness, cooling and licorice flavors often observed in natural high intensity sweeteners. The blend of monatin stereoisomers used in this example produced a smooth, regular dose response curve with a relative sweetness intensity 1250x sweeter than sucrose at 10.0% (w/v) SEV.

The results ofthe time intensity study showed that the monatin exhibited a time/sweetness intensity profile broadly similar to that of sucrose and sucralose. In comparison with sucrose, monatin took slightly longer to achieve maximum intensity and exhibited a slower rate of decay, with a higher perceived sweetness at the end of the evaluation (60s). However, the differences observed were not statistically significant.

When blended with carbohydrate sweeteners, the monatin delivered a sweetness intensity 1500 — 2000x sucrose. The resulting blends produced a very good quality sweetness and flavor profile. Little delay in sweetness onset was observed with only a low level of lingering sweetness detectable. Blends of monatin and carbohydrate sweeteners can be used, for example, to prepare mid-calorie beverages.

The evaluated monatin performed well both as a sole sweetener and when blended with carbohydrate sweeteners. In lemon/lime carbonates the product solely sweetened with monatin had a very similar taste profile to both the aspartame and sucralose sweetened drinks. The monatin/sucrose drink was particularly good and was actually judged more acceptable than the sucrose control product. It is expected that monatin will enhance the lemon/lime flavor in blends with other carbohydrate sweeteners. In the cola system, blending monatin with HFCS produced a drink as acceptable as the HFCS control.

Example 18: Sensory stability of monatin in water

The sensory stability of monatin (racemic mix described in Example 14) in water (8% SEV) was studied after storage at room temperature for 0 to 6 hours. The SEV was monitored (as described above in Example 14) at either 0-1 hours or 5-6 hours after preparing a monatin solution. There was no detectable loss of monatin SEV after 6 hours in room temperature; these data were corroborated by analytical studies using LC/MS (e.g., no lactonization was observed).

EXAMPLE 19: Preparation of a Malted Beverage Premix

A malted beverage premix is prepared using the ingredients listed in Table 14. TABLE 14

EXAMPLE 20: Preparation of a Chocolate Flavored Beverage Premix

A chocolate flavored beverage premix is prepared using the ingredients listed in Table 15. Non-dairy creamers can include vegetable oil, thickening agents, lecithin, protein, vitamins, minerals, emulsifiers (such as lecithin, DATEM and mono- and diglycerides) and bulking agents (e.g., com symp solids, low-calorie bulking agents). TABLE 15

EXAMPLE 21: Preparation of an Orange Flavored Beverage Premix

An orange flavored beverage premix is prepared using the ingredients listed in Table 16. TABLE 16

An orange beverage can be made by mixing approximately 1 oz. ofthe dry mix in 8 oz. water, then stirring or shaking until fully hydrated. Thus, the final ready-to-drink beverage has from about 66 to about 440 ppm S,S monatin, from about 6 to about 13 ppm R,R, or a mixture thereof.

EXAMPLE 22: Preparation of Lemonade Using a Monatin Sweetener

One may prepare convenient single-serving packets of sweetener comprising monatin, where the sweetener is formulated to provide a sweetness comparable to that in 2 teaspoons (~8 grams) of granulated sugar. Because S,S is 50-200 times sweeter than sucrose, 40-160 mg of S,S monatin delivers a sweetness comparable to that in 8 grams of granulated sugar. Thus, for example, allowing for +/— 25% sweetness optimization, single-serving packet 1 gram formulations of monatin may comprise approximately 40-200 mg of S,S monatin.

Likewise, because R,R is 2000-2400 times sweeter than sucrose, 3.3-4.0 mg of R,R monatin delivers a sweetness comparable to that in 8 grams of sugar. Thus, in another embodiment, allowing for +/- 25% sweetness optimization, single-serving packet 1 gram formulations of monatin may comprise approximately 3.3-5.0 mg of R,R monatin. In another embodiment, packet formulations may comprise 40-200 mg of S,S monatin, 3.3-5.0 mg of R,R monatin or a combination thereof in the same or lesser amounts per gram total weight, to provide a sweetness comparable to that in 2 teaspoons of granulated sugar.

To make lemonade, mix 2 tablespoons of lemon juice and 3 packets (3 g) of a monatin packet formulation with ³λ cup of water in a tall glass until dissolved. Add ice. The monatin-sweetened lemonade will be nearly equivalent in sweetness and equally preferred to the lemonade sweetened with 6 teaspoons (24 g) sucrose and will have significantly fewer calories (about 0 Calories versus 96 Calories).

EXAMPLE 23: Evaluation of R,R Monatin-Containing Sweeteners In Coffee and Iced Tea.

Monatin sweetener formulations, comprising R,R monatin or R,R monatin/erythritol combinations, were assessed relative to other known sweeteners (aspartame and sucralose) in coffee and iced tea. The key sensory parameters assessed included sweetness quality, aftertaste, bitter taste and its aftertaste. Qualitative evaluation was carried out.

Product Formulations (i) Coffee Standard coffee was used in which to evaluate sweetener performance (Table 17).

Table 17. Coffee formulation

Sweeteners were added to coffee at the following concentrations: Aspartame 0.025% (w/v) Sucralose 0.0082% (w/v) R,R monatin 0.0020, 0.0025, 0.0030% (w/v) plus 1 g maltodextrin R,R monatin/erythritol 0.0020, 0.0025, 0.0030% (w/v) plus 1 g erythritol

(ii) Iced Tea An ice tea formulation was developed to evaluate sweetener performance (Table 18).

Table 18. Iced Tea formulation

Sweeteners were added to tea at the following concentrations: Aspartame 0.0450% (w/v) Sucralose 0.0170% (w/v) R,R monatin 0.0030, 0.0035, 0.0040% (w/v) plus 1 g maltodextrin R,R, monatin/erythritol 0.0030, 0.0035, 0.0040% (w/v) plus 1 g erythritol Sensory Evaluation

The evaluation of these coffee and tea drinks was carried out by a panel (n = 6) of experienced sensory evaluators who evaluated the coffee products on one tasting occasion and the tea products on a subsequent occasion. The results of these evaluations are summarized in Table 19. Table 19. Sensory evaluation of coffee and tea (200 mL serving size)

Discussion

Monatin delivered unexpected performance benefits, including clear sensory benefits, in sweetener formulations. When monatin was added to coffee, a clear increase in the level of coffee flavor was perceived. This benefit was further enhanced through addition of low concentrations of erythritol, which were able to balance and round the flavor and to speed up sweetness onset times. In iced-tea, and particularly acidified acid tea, monatin enhanced the lemon flavor notes. Again, erythritol blending with monatin conferred additional flavor benefits.

Monatin delivers improved sensory properties (e.g., less aftertaste, less off-taste, no flavor masking) in commonly consumed beverages such as tea and coffee. Monatin sweetened coffee contains close to 0 Calories, as compared to 32 Calories in coffee sweetened with 2 teaspoons (~8 g) of sucrose.

It is expected that in beverage compositions, monatin exhibits enhancement of all ci rus flavors, as well as provides a more favorable time/intensity profile for sweetness, as compared to aspartame or sucralose. It is further expected that in beverage compositions, a blend of monatin and erythritol further enhances citrus flavors and provides more favorable sweetness profiles, as compared to aspartame or sucralose. It is expected that blends of monatin and erythritol will exhibit these benefits in any beverage composition, such as soft drinks, carbonated beverages, syrups, dry beverage mixes, and slush beverages maintained at lower temperatures.

EXAMPLE 24: Evaluation of R, R Monatin in Beverages Beverages (cola, lemon-lime and orange) were formulated and sweetened with aspartame, sucralose or R,R monatin. Qualitative evaluation was carried out.

Product Formulations

Soft drink formulations developed and evaluated are presented in Table 20. The term "throw" refers to dilution in water. For example, a throw of "1+4" means 1 part concentrate formulation to 4 parts water. Thus, if a concentrate formulation includes 0.021 % wt/vol (i.e., 210 ppm) of R,R monatin, for example, a throw of 1+4 makes a diluted beverage containing 42 ppm (210 ppm/5) R,R monatin. Table 20. Soft drink formulations (concentrates)

Final ready-to-drink beverages (after throw) contained sweetener concentrations as follows:

Lemon/lime Aspartame 500 ppm Sucralose 200 ppm R,R Monatin 42 ppm Orangeade Aspartame 550 ppm Sucralose 220 ppm R,R Monatin 45 ppm Cola Aspartame 550 ppm Sucralose 220 ppm R,R Monatin 45 ppm Sensory Evaluation of Beverages Evaluation of these soft drinks was carried out by a panel (n = 6) of assessors who evaluated each set of drinks on separate tasting occasions. Results ofthe evaluations are summarized in Table 21. Table 21. Sensory evaluation of soft drinks

Discussion

In lemon/lime, orangeade and cola beverages, monatin delivered a sweet taste similar in quality to aspartame and slightly better than that of sucralose, both of which are high quality sweeteners. In the lemon/lime beverage, less aftertaste was noted in the monatin formulation than in the aspartame formulation. Moreover, the potency of R,R monatin is greater than that of aspartame and sucralose.

EXAMPLE 25: Sweetness dose response curve of monatin and saccharin

Sweetness of monatin and saccharin was assessed using 20 trained sensory evaluators, making judgements in duplicate. Test and reference solutions were prepared in citric/citrate buffer at pH 3.2. See FIG. 16. The more linear response of R,R/S,S monatin, as compared to saccharin, is consistent with the delivery of a more sugarlike taste character. The plateau above 10% SEV indicates absence/low levels of "mixture-suppressing" off-tastes and aftertastes. The shape of monatin's dose- response curve is similar to those of aspartame, sucralose and alitame, all of which are "quality" sweeteners.

With R,R/S,S monatin as a sole sweetener in the model system (pH 3.2), the following characteristics were observed: (1) slight delay in sweet taste onset; (2) sweet taste decay was quite rapid; (3) slight "aspartame-like" aftertaste, slightly sweet aftertaste, no bitterness in the aftertaste; and (4) residual cooling sensation in un- flavored systems.

EXAMPLE 26: Stability of monatin at pH 3 with increasing temperatures

A sample of synthetic monatin was subjected to pH 3 at temperatures of 25°C, 50°C and 100°C. At room temperature and pH 3, a 14% loss in monatin was observed over a period of 48 hours. This loss was attributed to lactone formation. At 50°C and pH 3, a 23%o loss in monatin was observed over a period of 48 hours. This loss was attributed to lactone formation and the buildup of an unknown compound after about 15.5 minutes. At 100°C and pH 3, nearly all monatin was lost after 24 hours. The major detectable component was an unknown at 15.5 minutes. EAAMfLE 27: Sensory stability of monatin and aspartame at pH 2.5, 3.0, 4.0 at 40°C

The sensory stability of monatin solutions prepared at pH 2.5, 3.0 and 4.0 and stored at 40°C was monitored for 100 days. Loss of sweetness from these solutions was compared with the losses of sweetness from aspartame solutions prepared and stored under identical conditions.

The sensory stability of monatin (8% SEV, -55 ppm, synthetic blend containing approximately 96% ofthe 2R,4R/2S, 4S enantiometric pair and 4% ofthe

2R,4S/2S,4R enantiometric pair) in phosphate/citrate buffers having a pH of 2.5, 3.0, and 4.0 was examined after storage at 40°C. The stability of monatin was compared to that of aspartame (400 ppm) in the same buffers. Three sucrose reference solutions were prepared in the same phosphate/citrate buffers as the monatin and aspartame solutions. All prepared solutions were stored in the dark.

Buffer compositions: pH 2.5 Phosphoric acid (75% solution) 0.121% (w/v) Tri-sodium citrate monohydrate 0.005% (w/v) pH 3.0 Phosphoric acid (75% solution) 0.092% (w/v) Tri-sodium citrate monohydrate 0.031% (w/v) pH 4.0 Phosphoric acid (75% solution) 0.071% (w/v) Tri-sodium citrate monohydrate 0.047% (w/v)

The sweetness of each sweetener relative to sucrose was assessed in duplicate by a panel (n = 8) of trained sensory evaluators experienced in the sweetness estimation procedure. All samples (in the same buffers) were served in duplicate at a temperature of 22°C ± 1°C. Monatin (test) solutions, coded with 3 digit random number codes were presented individually to panelists, in random order. Sucrose reference standards, ranging from 4.0 - 10.0% (w/v) sucrose, increasing in steps of 0.5%) (w/v) sucrose were also provided. Panelists were asked to estimate sweetness by comparing the sweetness of the test solution to the sucrose standards. This was carried out by taking 3 sips of the test solution, followed by a sip of water, followed by 3 sips of sucrose standard followed by a sip of water, etc. Panelists were encouraged to estimate the sweetness to one decimal place, e.g., 6.8, 8.5. A five minute rest period was imposed between evaluating the test solutions. Panelists were also asked to rinse well and eat a cracker to reduce any potential carry over effects.

Tables 22 and 23 present results ofthe stability studies in the phosphate citrate buffers. At each pH and after 100 days' storage at 40°C in the dark, the percentage retention of monatin sweetness was greater than that retained with aspartame. At pH 4.0, the loss of sweetness ofthe monatin solution appeared almost to have stabilized since there was very little change in measured sweetness intensity between Days 17 and 100, whereas the aspartame solution continued to lose sweetness.

TABLE 22 Sensory Stability of Monatin: Sweetness after 100 Days Storage at 40°C

A.

B.

C.

TABLE 23

Stability: Amount of sweetness remaining after 100 days storage at stated pH at 40^c

The respective buffers were effective at maintaining pH, as seen in Table 24:

TABLE 24

If a pseudo-first order breakdown reaction is assumed, a plot of log_n percentage retention versus time (log_n%RTN v. t) allows estimation of the half-life (tV₂) and rate constant (k) of sweetness loss under any given set of conditions. In so doing, the kinetics of monatin and aspartame sweetness loss may be summarized as follows in Table 25.

TABLE 25

At each pH and after 100 days storage at 40°C, the percentage retention of monatin sweetness is greater than that retained from aspartame. At pH 4.0, the loss of sweetness ofthe monatin solution appears almost to have stabilized since there has been very little change in measured sweetness intensity between Days 17 and 100, whereas the aspartame solution continues to lose sweetness.

Estimates ofthe half-life of monatin and aspartame indicate that sweetness derived from monatin is lost at a slower rate than that from aspartame. Half-life estimates for monatin sweetness at pH 2.5, 3.0 and 4.0 were 65 days, 115 days and 230 days, respectively. Aspartame half-life estimates were 55 days, 75 days and 140 days under the same conditions.

Thus, under acidic conditions and storage at 40°C, monatin delivers a more stable sweetness than does aspartame. Monatin has a better stability than aspartame in colas and other beverages having a lower pH, as well as at higher temperatures. Because monatin exhibits better stability than aspartame, and reaches an equilibrium and does not irreversibly break down at pH 3, it is expected that monatin provides a long-term stable sweetness at a low pH in beverages, such as cola beverages.

It was further found (data not shown) that when exposed to ultra violet (UV) light, monatin in phosphoric/citrate buffer at pH 3.0 (at ambient temperature) is similarly stable or slightly more stable than aspartame. UV instability can be accelerated by certain flavor systems. UV-absorbing packaging material, colorants and/or antioxidants can protect against UV light-induced flavor interactions in monatin- containing beverages.

EXAMPLE 28: Chromatography of stereoisomers of monatin

Sample Preparation — Approximately 50-75 μg of lyophilized material was placed in a microcentrifuge tube. To this 1.0 mL of HPLC grade methanol was added. The solution was vortexed for 30 minutes, centrifuged and an aliquot ofthe supernatant was removed for analysis. Reversed Phase HPLC— Chromatography of two distinct diastereomer peaks (R,R/S,S and R,S/S,R) was accomplished using a 2.1 x 250mm Xterra™ MS C₈ 5μm (Waters Corporation) HPLC column. Detection was carried out using an Ultima^τ triple quadrupole mass spectrometer from Micromass. Mobile phase was delivered by the following gradient:

Time (min) 0 9 16 20 21 0.05%TFA A% 95 65 10 10 95 Methanol, 0.05% TFA B% 5 35 90 90 5 Flow mL/min 0.25 0.25 0.25 0.25 0.25

Chiral HPLC— Chromatography of two distinct monatin stereoisomers (R,R and S,S) was accomplished using a 250 x 4.6 mm Chirobiotic T(Advanced Separations

Technologies, Inc.) HPLC column. Detection was carried out using an Ultima triple quadrupole mass spectrometer from Micromass. Mobile phase consisted of Methanol with 0.2%) Acetic acid and 0.05% ammonium hydroxide.

Mass Spectrometry (MS/MS) — The presence of monatin was detected by a Selected Reaction Monitoring (SRM) experiment. The protonated molecular ion of monatin ([M+H]⁺) has a m/z=293.3. Fragmentation of this molecular ion produces a significant ion at m/z=251.3 arising from multiple dehydrations ofthe molecular ion. This transition has been shown to be very specific to monatin and was chosen as the transition (293.3 to 257.3) for monitoring during the SRM experiment. This method of detection was employed for both reversed phase and chiral separations of monatin.

Results — The standard samples of R,S/S,R and S,S/R,R were evaluated under Reversed Phase HPLC. The samples were prepared by derivatization and enzymatic resolution. Chromatograms for standard solutions are displayed in FIG. 17. Following the reversed phase analysis, chiral chromatography was performed to evaluate specific stereoisomers present in the samples. Chiral chromatography of standard S,S and R,R, monatin solutions are displayed in FIG. 18. EXAMPLE 29: Stability of monatin at high temperature (80 °C) and neutral pH

A 100 milliliter solution of 75 ppm monatin at pH 7 was used as a stock solution. The synthetic monatin sample contained approximately 96% ofthe 2R,4R/2S,4S enantiomeric pair and 4% ofthe 2R,4S/2S,4R enantiomeric pair. Samples were incubated at 80°C and pH 7 for the duration ofthe experiment and samples were withdrawn at 0, 1, 2, 3, 4 hours and 1, 2, 4, 7, 14, 21 and 35 days. All experimental conditions were run in duplicate.

Separation and Quantification Using LC-MS using Reverse Phase Chromatography — A response curve was established for both detected diastereomer peaks ofthe synthetic monatin. A range of 5-150 ppm was bracketed with the synthetic monatin standard dissolved in DI water. Separation ofthe two diastereomer peaks was accomplished using a 3.9 x 150mm Novapak C18 (Waters Corporation) HPLC column. Ultraviolet- Visible (UV) and Mass Spectrometer (MS) detectors were used in series for detection and quantitation. Monatin and its lactone peak each have a Vmax at 279 nm that aided in precise detection. Quantification was done by acquiring Selected Ion Monitoring (SIM) scan of 293.3 m/z and 275.3 m/z in positive- ion electrospray mode.

Results - At a neutral pH, the degree of degradation of monatin was determined to be insignificant even after 7-35 days. The disappearance of monatin over time is highly dependent on pH since the primary byproducts are cyclization and possibly very small levels of racemization. During the experiment at 80°C and pH 7, no change in concentration of racemic RR/SS monatin or lactones thereof was detected within the limits of precision afforded by using LC-MS for quantitation.

Due to the thermal stability of monatin at neutral pH, it is expected that monatin has a suitable stability for beverages at a neutral pH (such as dairy or powdered beverage compositions). It is also expected that monatin has longer shelf life in these beverage compositions, as compared to other high intensity sweeteners (e.g., aspartame). In addition, it is expected that monatin will be more stable during processing conditions, such as heat filling. EXAMPLE 30: Other Soft Drink Formulations (Concentrates)

Formulation A:

Throw 1+4. The diluted ready-to-drink beverage contains 1980 ppm S,S monatin.

Formulation B:

Throw 1+4. The diluted ready-to-drink beverage contains 80 ppm of monatin racemic mix. Formulation C:

Throw 1+4. The diluted ready-to-drink beverage contains 550 ppm S,S monatin and 32 ppm R,R monatin. In view ofthe many possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are only particular examples ofthe disclosure and should not be taken as a limitation on the scope ofthe disclosure.

Claims

Claims:

1. A beverage composition comprising monatin or salt thereof.

2. The beverage composition of claim 1, wherein an amount ofthe composition contains less calories and carbohydrates than the same amount ofthe beverage composition containing sucrose or high fructose corn syrup in place ofthe monatin or salt thereof at comparable sweetness.

3. The beverage composition of claim 1 , wherein the composition further comprises a citrus flavor, and wherein the monatin or salt thereof is present in an amount that enhances the flavor provided by the citrus flavor.

4. The beverage composition of claim 1, wherein the composition further comprises a citrus flavor and a carbohydrate, and wherein the monatin or salt thereof and the carbohydrate are present in an amount that enhances the flavor provided by the citrus flavor.

5. The beverage composition of claim 4, wherein the carbohydrate is chosen from erythritol, maltodextrin, sucrose and a combination thereof.

6. A carbonated beverage, comprising a symp composition in an amount ranging from about 15% to about 25%> by weight ofthe carbonated beverage, wherein the syrup composition comprises monatin or salt thereof.

7. The beverage composition of claim 1, wherein the composition comprises from about 3 to about 10000 ppm monatin or salt thereof.

8. The beverage composition of claim 1, wherein the beverage composition is a syrup or dry beverage mix, and wherein the composition comprises from about 10 to about 10000 ppm monatin or salt thereof.

. The beverage composition of claim 8, wherein the beverage composition is a syrup, and wherein the syrup is a concentrate adapted for dilution in a drink in a range of about 1 part syrup: 3 parts drink to about 1 part syrup: 5.5 drink.

10. The beverage composition of claim 9, wherein the syrup comprises from about 600 to about 10000 ppm S,S monatin or salt thereof.

11. The beverage composition of claim 9, wherein the symp comprises from about 18 to about 300 ppm R,R monatin or salt thereof.

12. The beverage composition of claim 1, wherein the beverage composition is a syrup comprising from about 0 to about 10000 ppm S,S monatin or salt thereof, and from 0 to about 300 ppm R,R monatin or salt thereof.

13. The beverage composition of claim 1, wherein the beverage composition is a dry beverage mix comprising from about 10 to about 10000 ppm monatin or salt thereof.

14. The beverage composition of claim 13, wherein the dry beverage mix comprises from about 600 to about 10000 ppm S,S monatin or salt thereof.

15. The beverage composition of claim 13, wherein the dry beverage mix comprises from about 10 to about 450 ppm R,R monatin or salt thereof.

16. The beverage composition of claim 1, wherein the beverage composition is a dry beverage mix comprising from about 0 to about 10000 ppm S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

17. The beverage composition of claim 7, wherein the composition is substantially free of R,R monatin or salt thereof.

18. The beverage composition of claim 7, wherein the composition is substantially free of S,S monatin or salt thereof.

19. The beverage composition of claim 1, wherein the composition comprises from about 3 to about 450 ppm R,R monatin or salt thereof.

20. The beverage composition of claim 19, wherein the composition comprises from about 6 to about 225 ppm R,R monatin or salt thereof.

21. The beverage composition of claim 1, wherein the composition comprises from about 3 to about 10000 ppm S,S monatin or salt thereof.

22. The beverage composition of claim 21, wherein the composition comprises from about 60 to about 4600 ppm of S,S monatin or salt thereof.

23. The beverage composition of claim 1, wherein the composition comprises from about 0 to about 10000 ppm of S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

24. The beverage composition of claim 1, where the composition is a ready-to-drink composition comprising from about 3 to about 2000 ppm monatin or salt thereof.

25. The ready-to-drink composition of claim 24, where the ready-to-drink composition comprises from about 5 to about 50 ppm R,R monatin or salt thereof.

26. The ready-to-drink composition of claim 24 , where the ready-to-drink composition comprises from about 60 to about 2000 ppm S,S monatin or salt thereof.

27. The beverage composition of claim 1, wherein the composition comprises about 450 or less ppm R,R monatin or salt thereof, and wherein the monatin or salt thereof is substantially free of S,S, S,R or R,S monatin or sal^ thereof.

28. The beverage composition of claim 1, wherein the composition comprises about 10000 or less ppm S,S monatin or salt thereof, and wherein the monatin or salt thereof is substantially free of R,R, S,R or R,S monatin or salt thereof.

29. The beverage composition of claim 1, wherein the monatin or salt thereof consists essentially of R,R monatin or salt thereof.

30. The beverage composition of claim 1, wherein the monatin or salt thereof consists essentially of S,S monatin or salt thereof.

31. The beverage composition of claim 1, wherein the monatin or salt thereof is a stereoisomerically-enriched R,R monatin or salt thereof.

32. The beverage composition of claim 1, wherein the monatin or salt thereof is a stereoisomerically-enriched S,S monatin or salt thereof.

33. The beverage composition of claim 1, wherein the monatin or salt thereof comprises at least 95% R,R monatin or salt thereof.

34. The beverage composition of claim 1, wherein the monatin or salt thereof comprises at least 95%) S,S monatin or salt thereof.

35. The beverage composition of claim 1, wherein the monatin or salt thereof is produced in a biosynthetic pathway.

36. The beverage composition of claim 1, wherein the beverage composition further comprises erythritol, trehalose, a cyclamate, D-tagatose or combination thereof.

37. The beverage composition of claim 1, wherein the composition is non-cariogenic.

38. A beverage composition comprising a stereoisomerically-enriched monatin mixture, wherein the monatin mixture is produced via a biosynthetic pathway.

39. The beverage composition of claim 38, wherein the biosynthetic pathway is a multi-step pathway and at least one step ofthe multi-step pathway is a chemical conversion.

40. The beverage composition of claim 38, wherein the mixture is predominantly R,R monatin or salt thereof.

41. The beverage composition of claim 38, wherein the mixture is predominantly S,S monatin or salt thereof.

42. A beverage composition comprising a monatin composition produced in a biosynthetic pathway, wherein the monatin composition does not contain petrochemical, toxic or hazardous contaminants.

43. A beverage composition comprising monatin or salt thereof, wherein the monatin or salt thereof is produced in a biosynthetic pathway and isolated from a recombinant cell, and wherein the recombinant cell does not contain petrochemical, toxic or hazardous contaminants.

44. The beverage composition of claim 1, wherein the monatin or salt thereof is a blend of R,R and S,S, monatin or salt thereof.

45. The beverage composition of claim 1, wherein the composition further comprises a bulk sweetener, a high-intensity sweetener, a lower glycemic carbohydrate, a flavoring, an antioxidant, caffeine, a sweetness enhancer or a combination thereof.

46. The beverage composition of claim 45, wherein the flavoring is chosen from a cola flavor, a citrus flavor and a combination thereof, wherein the bulk sweetener is chosen from com sweeteners, sucrose, dextrose, invert sugar, maltose, dextrin, maltodextrin, fructose, levulose, high fructose corn syrup, com symp solids, levulose, galactose, trehalose, isomaltulose, fructo-oligosaccharides and a combination thereof, wherein the high-intensity sweetener is chosen from sucralose, aspartame, saccharin, acesulfame K, alitame, thaumatin, dihydrochalcones, neotame, cyclamates, stevioside, mogroside, glycyrrhizin, phyllodulcin, monellin, mabinlin, brazzein, circulin, pentadin and a combination thereof, wherein the lower glycemic carbohydrate is chosen from D-tagatose, sorbitol, mannitol, xylitol, lactitol, erythritol, maltitol, hydrogenated starch hydrolysates, isomalt, D-psicose, 1,5 anhydro D-fructose and a combination thereof, and wherein the sweetness enhancer is chosen from curculin, miraculin, cynarin, chlorogenic acid, caffeic acid, strogins, arabinogalactan, maltol, dihyroxybenzoic acids and a combination thereof.

47. The beverage composition of claim 1, wherein the beverage composition comprises a blend of monatin or salt thereof and a non-monatin sweetener.

48. The beverage composition of claim 47, wherein the non-monatin sweetener is chosen from sucrose and high fructose corn syrup.

49. A method for making a beverage composition comprising monatin or salt thereof, wherein the method comprises producing monatin or salt thereof from at least one substrate chosen from glucose, tryptophan, indole-3-lactic acid, indole-3 -pyruvate and the monatin precursor.

50. A method for making a beverage composition comprising monatin or salt thereof, wherein the method comprises producing monatin or salt thereof through a biosynthetic pathway.

51. A method for making a beverage composition comprising monatin or salt thereof, wherein the method comprises producing monatin or salt thereof using at least one biological conversion.

52. A method for making a beverage composition comprising monatin or salt thereof, wherein the method comprises producing monatin or salt thereof using only biological conversions.

53. The method of claim 49, wherein the method further comprises combining the monatin or salt thereof with at least one other ingredient that is not monatin or salt thereof.

54. The method of claim 49, wherein the method further comprises combining the monatin or salt thereof with erythritol, trehalose, a cyclamate, D-tagatose, maltodextrin or combination thereof.

55. The method of claim 53, wherein the at least one other ingredient is chosen from bulking agents, bulk sweeteners, liquid sweeteners, lower glycemic carbohydrates, high intensity sweeteners, thickeners, fats, oils, emulsifiers, antioxidants, sweetness enhancers, colorants, flavorings, caffeine, acids, powders, flow agents, buffers, protein sources, flavor enhancers, flavor stabilizers and a combination thereof.

56. The method of claim 53, wherein the beverage composition comprises from about 0 to about 10000 ppm of S,S monatin or salt thereof, and from about 0 to about 450 ppm R,R monatin or salt thereof.

57. A method for making a beverage composition comprising a monatin composition, wherein the method comprises: (a) producing monatin or salt thereof in a biosynthetic pathway in a recombinant cell; (b) isolating the monatin composition from the recombinant cell, wherein the monatin composition consists of monatin or salt thereof and other edible or potable material.

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