US 2005O112260A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0112260 A1 Abraham et al. (43) Pub. Date: May 26, 2005

(54) MONATIN TABLETOPSWEETENER (21) Appl. No.: 10/903,582 COMPOSITIONS AND METHODS OF MAKING SAME (22) Filed: Aug. 2, 2004 (75) Inventors: Timothy W. Abraham, Minnetonka, Related U.S. Application Data MN (US); Douglas C. Cameron, Plymouth, MN (US); Melanie J. (60) Provisional application No. 60/492,014, filed on Aug. Goulson, Dayton, MN (US); Paula M. 1, 2003. Hicks, Eden Prairie, MN (US); Michael O O G. Lindley, Crowthorne (GB); Sara C. Publication Classification McFarlan, St.Paul, MN (US); James 7

R. Millis, Plymouth, MN (US); John s ------A:

Rosazza, Iowa City, IA (US); Lishan ( 2) O O ------/5 Zhao, Carlsbad, CA (US); David P. Weiner, Del Mar, CA (US) (57) ABSTRACT Correspondence Address: The present invention relates to novel Sweetener composi CARGILL, INCORPORATED tions comprising monatin and methods for making Such LAW/24 compositions. The present invention also relates to Sweet 15407 MCGINTY ROAD WEST ener compositions comprising Specific monatin Stereoiso WAYZATA, MN 55391 (US) mers, Specific blends of monatin Stereoisomers, and/or monatin produced via a biosynthetic pathway in Vivo (e.g., (73) Assignee: Cargill, Inc. inside cells) or in vitro.

-- re Indole-3-lactic acid

PLP and amino acceptor or NAD(P) or HO and O, and FAD NAD(P) or HO and O, and FMN Indole-3-pyruvate

- C3 molecule PLP

2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid (MP)

PLP and amino donor or NAD(P)H +NH3 Patent Application Publication May 26, 2005 Sheet 1 of 14 US 2005/0112260 A1

-- st Tryptophan Indole-3-lactic acid

PLP and amino acceptor or NAD(P) or HO and O, and FAD NAD(P) or HO and O, and FMN Indole-3-pyruvate

+ C3 molecule PLP

2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid (MP)

PLP and amino donor or NAD(P)H +NH3

FIG. 1 Patent Application Publication May 26, 2005 Sheet 2 of 14 US 2005/0112260 A1

U.S. Patent No. 4,371,614 Tryptophan EC 2.6.1.27 tryptophan aminotransferase EC 2.6.1.5 (aromatic) aminotransferase EC 1.4.1.19 tryptophan dehydrogenase EC 1.4.1.2-4 EC 1.4.1.20 dehydrogenase EC 2.6.1.28 tryptophan-phenylpyruvate EC 2.6.1.- multiple aminotransferase EC 2.6.1.1 aspartate aminotransferase EC 1.4.3.2 L- oxidase tryptophan oxidase (no EC number) D-tryptophan aminotransferase (no EC number) EC 1.4.99.1 D-amino acid dehydrogenase EC 1.4.3.3 D-amino acid oxidase 2.6.1.21 D- aminotransferase Indole-3-pyruvate

EC 4.1.3.-, 4.1.2.-, synthase EC 4.1.3.16 4-Hydroxy-2-oxoglutarate glyoxylate-lyase EC 4.1.3.17 4-Hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase Chemical aldol condensation

2-hydroxy 2-(indol-3-ylmethyl)-4-ketoglutaric acid EC 2.6.1.27 tryptophan aminotransferase EC 2.6.1.5 tyrosine (aromatic) aminotransferase EC 1.4.1.19 tryptophan dehydrogenase EC 2.6.1.28 tryptophan-phenylpyruvate transaminase EC 2.6.1.- multiple substrate aminotransferase EC 2.6.1.1 aspartate aminotransferase EC 1.4.1.2-4 glutamate dehydrogenase EC 1.4.1.20 phenylalanine dehydrogenase EC 1.4.99.1 D-amino acid dehydrogenase EC 2.6.1.21 D-alanine aminotransferase D-typtophan aminotransferase (no EC number) Monati FIG. 2 Patent Application Publication May 26, 2005 Sheet 3 of 14 US 2005/0112260 A1

FIG 3 Indole-3-lactic acid

EC 1.1.1.110 indolelactate dehydrogenase EC 1.1.1.222 R-4-hydroxyphenylactate dehydrogenase EC 1.1.1.2373-(4)-hydroxyphenylpyruvate reductase EC 1.1.1.27, 1.1.1.28, 1.1.2.3 lactate dehydrogenase EC 1.1.1.11 l (3-imidazol-5-yl) lactate dehydrogenase EC 1.1.3.- lactate oxidase Chemical oxidation

Indole-3-pyruvate

FIG. 4

COOR 2 O COOR2 S COO O -N R3 COOR \ - R - y bh O N N Enolate chemistry R R R =Boc, Cbz, etc. R2 and R Alkyl, Aryl, etc. Patent Application Publication May 26, 2005 Sheet 4 of 14 US 2005/0112260 A1

FIG. 5A Agio?

lOO n/z = 293 5O O O 4. 8 2 6 2O 24 Time (min)

F.G. 6

OO 293

5 O

50 2OO 250 3OO 350 4OO Patent Application Publication May 26, 2005 Sheet 5 of 14 US 2005/0112260 A1

FIG. 7A

O8-Apr-2002 O O A 15:11:01 5 O O O 4 8 2 6 2O 24 FG. 7B Time (min)s 92 OO 2 B 168 es O OV) 58 9 50 30 g 174 229 21.212 230

aY4c) O awsw val "Itlar - d. III,na..... all. IV L.-...-L. . . 275,23.. . OO 50 200 250 300 n/z FG. 8

+T OF MS: 124 MCA scans from Sample 7 (293.jd with Tryptophan) of caroto9a.wif... Max 9.3e4 counts. a=3.569708085.37750280e-004, tos-1,246268795.88351580e 4002 r.

293,144 9.0 e4

8-0 ea

7,094 Cargill Sample Sample 293 with Tryptophan as Internal Std 6.0 e4

5.084

4.Oe4

3.0e4

2,084 i 294.1210

1.04

O. ------a------a ------all- ---A------Patent Application Publication May 26, 2005 Sheet 6 of 14 US 2005/0112260 A1

VB342 10-Jul-2002 13:11:33 O70202a t scans. 1CO FIG. 9A ka 2128

%

oria, 1: Scan ES+ 100 FG. 9B 1.

%

ori E" 1: Scan ES+ 10C o 15 FG.9C %

O 2OO ET BOO OOO 12.00 14.00 16.00 t 22.OO 240 Time

F.G. 10

O i 14OE--05 O 1.2OE+05 a 1.00E+05 8.00E--04 6.00E+04 5 4.00E+04 w5 2.OOE-04 2 O.OOE+00 ------S4 4hr 24hr 24hr 48hr 48hr 7Ohr 70hr -2.00E+04 (-) (+) (-) (+) (-) (+) a -4.00E+04 Time Patent Application Publication May 26, 2005 Sheet 7 of 14 US 2005/0112260 A1 FIG. 11 tryptophan tryptophan oxidase O 2 O catalase amino acid oxidase HO 6 2N/2 indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) aspartate aspartate aminotransferase Oxaloacetate monatin v Oxaloacetate \, decarboxylase 3. pyruvate + CO Patent Application Publication May 26, 2005 Sheet 8 of 14 US 2005/0112260 A1 F.G. 12 tryptophan tryptophan oxidase or O 5 catalase amino acid Oxidase HO indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) lysine &-aminotransferase alysine mOnatin \spontaneous, - H20

1-Piperideine 6-carboxylate Patent Application Publication May 26, 2005 Sheet 9 of 14 US 2005/0112260 A1 FIG. 13 tryptophan O tryptophan oxidase g catalase Or amino acid oxidase HO, indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) CO 2 dehydrogenase NADH (reductive amination) . g NH, NAD ) / monatin / Formate dehydrogenase ammonium formate Patent Application Publication May 26, 2005 Sheet 10 of 14 US 2005/0112260 A1 F.G. 14:

14 13 12 11 1O s i? 67 on 5 4. 3 2 1 O O 10 20 30 40 50 60 ppm R,R monatin Patent Application Publication May 26, 2005 Sheet 11 of 14 US 2005/0112260 A1

FIG. 15

12

10

6

Sensory Procedure: Sweetness matching using 20 trained assessors making judgements hin duplicate 2 Test and reference sohutions prepared in citric/citrate buffer at pH32

O 20 40 60 80 100 CONCENTRATION (ppm) Patent Application Publication May 26, 2005 Sheet 12 of 14 US 2005/0112260 A1 F.G. 16

12

O

2B e saccharin / DuBois et a, ACS Sy S Symposium Series 450, > pp. 261-277, 1991 N (stimuli presented in 4 de-lonized water) (stimuli presented in pH 3.2 buffer) 2

O O 250 500 2O 40 60 8O CONCENTRATION (ppm) Patent Application Publication May 26, 2005 Sheet 13 of 14 US 2005/0112260 A1

F.G. 17

WB342 04-Oct 2002 O8:53:24 RM of 1 Cane ES TC 10 S.9ses

Standard

1. MRM of ChanneS. 1. TC 2S4S/2R4R 4.36es

Standard

ne 2. 40 6.0 B.O 10.00 1200 14. 16. BOO 20.OO 22O 24.OO Patent Application Publication May 26, 2005 Sheet 14 of 14 US 2005/0112260 A1

F.G. 18

VB342 O7-Oct-2002 11:37:16 1:MRM of 1 Channel ES+ 100 2R4R 93.

% Standard

O

1:MRM of Channel ES+ 1G of 2S4S % Standard

2.OO 4. 6.00 B.O 10.00 2.0 14.OO 16.00 18too 20.00 22.0 24.00 e US 2005/0112260 A1 May 26, 2005

MONATIN TABLETOP SWEETENER 0006 Monatin is a naturally-occurring, high intensity COMPOSITIONS AND METHODS OF MAKING Sweetener. Monatin has four stereoisomeric forms: 2R, 4R SAME (the “RR stereoisomer” or “RR monatin”), 2S, 4S (the “S.S stereoisomer” or “S.S monatin”), 2R, 4S (the “R,S stereoi CROSS-REFERENCE TO RELATED Somer” or “R,S monatin”), and 2S, 4R (the “S.R stereoiso APPLICATIONS mer” or “S.R monatin”). As used herein, unless stated 0001) This application claims the benefit of U.S. Provi otherwise, “monatin” refers to all four stereoisomers of sional Patent Application 60/492,014 filed Aug. 1, 2003, the monatin, as well as any blends of any combination of entire disclosure of which is incorporated herein by refer monatin Stereoisomers (e.g., a blend of the R,R and S.S, CCC. Stereoisomers of monatin). 0007 Monatin has an excellent Sweetness quality. Mona FIELD OF INVENTION tin has a flavor profile that is as clean or cleaner that other 0002 The present invention relates to novel Sweetener known high intensity Sweeteners. The dose response curve compositions comprising monatin and methods for making of monatin is more linear, and therefore more Similar to Such compositions. The present invention also relates to Sucrose than other high intensity Sweeteners, Such as Sac Sweetener compositions comprising Specific monatin Stere charin. Monatin’s excellent SweetneSS profile makes mona oisomers, Specific blends of monatin Stereoisomers, and/or tin desirable for use in tabletop Sweeteners, foods, beverages monatin produced via a biosynthetic pathway in Vivo (e.g., and other products. inside cells) or in vitro. 0008 Different stereoisomers of monatin, including the R,R and S.S. Stereoisomers, have potential in the Sweetener BACKGROUND industry, either as Separate ingredients or in blends. Monatin, and blends of stereoisomers of monatin with other Sweet 0003. The use of non-caloric high intensity Sweeteners is eners, are thought to have Superior taste characteristics increasing due to health concerns raised over childhood and/or physical qualities, as compared to other high intensity obesity, type II diabetes, and related illnesses. Thus, a Sweeteners. For example, monatin is more Stable than demand exists for SweetenerS having a Sweetness signifi Equal(R) or NutraSweet(R) (aspartame, also known as cantly higher than that in conventional Sweeteners, Such as “APM'), has a cleaner taste than Sweet NLow(R) (saccha granulated Sugar (Sucrose). Many high intensity Sweeteners rin), and is more Sweet than Splenda(E) (Sucralose). Likewise, contain unpleasant off-flavors and/or unexpected and less monatin Sweeteners do not have the bitter aftertaste associ than-desirable Sweetness profiles. In attempts to overcome ated with Saccharin, or the metallic, acidic, astringent or these problems, the industry continues to conduct significant throatburning aftertastes of Some other high potency Sweet research into bitterness inhibitors, off-flavor masking tech eners. In addition, monatin Sweeteners do not exhibit the nologies, and Sweetener blends to achieve a SweetneSS licorice aftertaste associated with certain natural Sweeteners, profile Similar to Sucrose. Such as Stevioside and glycyrrhizin. Furthermore, unlike 0004 Monatin (2-hydroxy-2-(indol-3-ylmethyl)-4-ami aspartame Sweeteners, monatin Sweeteners do not require a noglutaric acid) is a naturally-occurring, high intensity phenylalanine warning for patients with phenylketonuria. Sweetener isolated from the plant Sclerochiton ilicifolius, Because of its intense Sweetness, the R,R Stereoisomer in found in the Transvaal Region of South Africa. Monatin particular should be economically competitive compared to contains no carbohydrate or Sugar, and nearly no calories, other high intensity Sweeteners. unlike Sucrose or other nutritive Sweeteners at equal Sweet 0009. In one aspect, the present invention provides CSS. Sweetener compositions containing R,R monatin and/or S.S monatin. For example, Such compositions may contain a SUMMARY Sweetness comparable to that of granulated Sugar (Sucrose), 0005 The present invention relates to Sweetener compo and therefore can be used "spoon-for-spoon” or "cup-for Sitions comprising monatin (2-hydroxy-2-(indol-3-ylm cup' in place of Sugar. “A Sweetness comparable” means ethyl)-4-aminoglutaric acid-also known as 4-amino-2-hy that an experienced Sensory evaluator, on average, will droxy-2-(1H-indol-3-ylmethyl)-pentanedioic acid, or determine that the SweetneSS presented in a first composition alternatively, based on an alternate numbering System, 4-hy is within a range of 80% to 120% of the Sweetness presented droxy-4-(3-indolylmethyl)), a compound hav a Second composition. The phrase “a SweetneSS comparable' ing the formula: relates to a determination ascertained by five or more experienced Sensory evaluators in a SweetneSS matching test, as discussed below, where the test is conducted with com positions (e.g., Solutions) having a range of 2-10%. Sucrose equivalents. Thus, for instance, 100 mg/mL of a composition comprising monatin provides “a Sweetness comparable' to 100 mg/mL of Sucrose if the monatin composition has a Sweetness falling with the range of Sweetness presented in 80-120 mg/mL of Sucrose. 0010. One assesses 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 US 2005/0112260 A1 May 26, 2005

temperature of 22 C.1. C. Test solutions, coded with 3 is known in the art that a "teaspoon' of Sucrose contains digit random number codes, are presented individually to approximately 4 grams of Sucrose. Alternatively, a Volume panelists, in random order. Sucrose reference Standards, of a ready-to-use formulation can provide a Sweetness ranging from 4.0-10.0% (w/v) Sucrose, increasing in steps of comparable to the same Volume of granulated Sugar. Alter 0.5% (w/v) sucrose are also provided. Panelists are asked to natively, a Single Serving packet of a monatin composition estimate Sweetness by comparing the Sweetness of the test (e.g., 1 gram) can provide a Sweetness comparable to about Solution to the Sucrose Standards. This is carried out by 0.9 to about 9.0 grams of granulated Sugar (Sucrose). The taking 3 Sips of the test Solution, followed by a sip of water, term “about encompasses the range of experimental error followed by 3 sips of Sucrose standard followed by a sip of that occurs in any measurement. Unless otherwise Stated, all water, etc. Panelists estimate the Sweetness to one decimal measurement numbers are presumed to have the word place, e.g., 6.8, 8.5. A five minute rest period is imposed “about” in front of them even if the word “about' is not between evaluating the test Solutions. Panelists are also expressly used. asked to rinse well and eat a cracker to reduce any potential 0014. In another aspect, the present invention provides a carry over effects. Using information obtained from the homogeneous tabletop Sweetener composition comprising panelists, the Sweetness intensity or potency is calculated by monatin. A "homogeneous' composition refers to a uniform dividing Sucrose equivalent value (SEV) (e.g., % Sucrose) composition. For example, a “homogeneous” tabletop by the % monatin at a particular point in a dose response Sweetener composition (e.g., a liquid) will contain the same CWC. concentration of monatin throughout the composition-any 0011. In some embodiments, the compositions comprise Sample obtained from that composition will have that con monatin that consists essentially of S.S or R,R monatin. In centration. other embodiments, the compositions contain predomi nantly S.S or R,R monatin. “Predominantly” means that of 0015. In another aspect of the present invention, a the monatin Sterioisomers present in the composition, the method of making a Sweetener composition is provided. The monatin contains greater than 90% of a particular Stereoi method includes biosynthetically producing monatin either Somer. In Some embodiments, the compositions are Substan in vivo or in vitro. A “biosynthetic pathway' comprises at tially free of S.S or R,R monatin. “Substantially free” means least one biological conversion Step. In Some embodiments, that of the monatin Sterioisomers present in the composition, the biosynthetic pathway is a multi-step proceSS and at least the composition contains less than 2% of a particular Stere one Step is a biological conversion Step. In other embodi oisomer. Additionally or alternatively, when used to describe ments, the biosynthetic pathway is a multi-step process monatin produced in a biosynthetic pathway, “Substantially involving both biological and chemical conversion Steps. In free” encompasses the amount of a stereoisomer (e.g., S.S Some embodiments, the monatin produced is a Stereoiso monatin) produced as a by- in a biosynthetic path merically-enriched monatin mixture. way involving chiral-specific (e.g., D-amino acid 0016. In another aspect of the present invention, several dehydrogenases or D-amino acid aminotransferases) and/or biosynthetic pathways exist for making monatin from Sub chiral-specific Substrates (e.g., one having a carbon in the Strates chosen from glucose, tryptophan, indole-3-lactic R-stereoconfiguration) to produce a different specific Ste acid, as well as indole-3-pyruvate and 2-hydroxy 2-(indole reostereoisomer (e.g., R,R monatin) 3-ylmethyl)-4-ketoglutaric acid (also known as “the mona tin precursor,”“MP” or the alpha-keto form of monatin). 0012. In another aspect of the present invention, a Sweet Examples of biosynthetic pathways for producing or making ener composition is provided, which includes a Stereoiso monatin or its intermediates are disclosed in FIGS. 1-3 and merically-enriched monatin mixture produced in a biosyn 11-13, which show potential intermediate products and end thetic pathway. "Stereoisomerically-enriched monatin products in boxes. For example, a conversion from one mixture” means that the mixture contains more than one product to another, Such as glucose to tryptophan, tryp monatin stereoisomer and at least 60% of the monatin tophan to indole-3-pyruvate, indole-3-pyruvate to MP, MP Steroisomers in the mixture is a particular Stereoisomer, Such to monatin, or indole-3-lactic acid (indole-lactate) to indole as R.R., S.S, S.R or R.S. In other embodiments, the mixture 3-pyruvate, occurs in these pathways. contains greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer. In 0017. These conversions within the biosynthetic path another embodiment, a Sweetener composition comprises an ways can be facilitated via chemical and/or biological con stereoisomerically-enriched R,R or S.S monatin. “Stereoi versions. The term “convert” refers to the use of either Somerically-enriched R,R monatin means that the monatin chemical means or at least one polypeptide in a reaction to comprises at least 60% R,R monatin. “Stereoisomerically change a first intermediate into a Second intermediate. enriched” S.S monatin means that the monatin comprises at Conversions can take place in Vivo or in vitro. The term least 60% S.S monatin. In other embodiments, “stereoiso “chemical conversion” refers to a reaction that is not merically-enriched' monatin comprises greater than 65%, actively facilitated by a polypeptide. The term “biological 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R,R or S.S conversion” refers to a reaction that is actively facilitated by monatin. one or more polypeptides. When biological conversions are 0013 In another aspect, the present invention also pro used, the polypeptides and/or cells can be immobilized on vides Sweetener formulations, for example, in pre-portioned Supports Such as by chemical attachment on polymer Sup packets or ready-to-use formulations, which include mona ports. The conversion can be accomplished using any reactor tin in the RR stereoisomer and/or S.S stereoisomer forms. known to one of ordinary skill in the art, for example in a For example, a single Serving packet formulation (usually batch or a continuous reactor. about 1 gram) can provide a Sweetness comparable to that 0018. Examples of polypeptides, and their coding contained in two teaspoons of granulated Sugar (Sucrose). It Sequences, that can be used to perform biological conver US 2005/0112260 A1 May 26, 2005 sions are shown in FIGS. 1-3 and 11-13. Polypeptides 1.1.1.237), lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28, having one or more point mutations that allow the Substrate 1.1.2.3), (3-imidazol-5-yl) lactate dehydrogenase (EC Specificity and/or activity of the polypeptides to be modified, 1.1.1.111), lactate oxidase (EC 1.1.3.-), Synthase/lyase (EC can be used to make monatin. Isolated and recombinant cells 4.1.3.-), Such as 4-hydroxy-2-oxoglutarate aldolase (EC expressing Such polypeptides can be used to produce mona 4.1.3.16) or 4-hydroxy-4-methyl-2-oxoglutarate aldolase tin. These cells can be any cell, Such as a plant, animal, (EC 4.1.3.17), and/or synthase/lyase (4.1.2.-). bacterial, yeast, algal, archaeal, or fungal cell. 0022. As further example, the cells can include one or 0.019 For example, monatin-producing cells can include more of the following aldolase activities: KHG aldolase, one or more (Such as two or more, or three or more) of the ProA aldolase, KDPG aldolase and/or related polypeptides following activities: tryptophan aminotransferase (EC (KDPH), transcarboxybenzalpyruvate hydratase-aldolase, 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase, trans-O- tryptophan dehydrogenase (EC 1.4.1.19), glutamate dehy hydroxybenzylidenepyruvate hydratase-aldolase, 3-hydrox drogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine yaspartate aldolase, benzoin aldolase, dihydroneopterin dehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate aldolase, L-threo-3-phenylserine benzaldehyde-lyase (phe transaminase (EC 2.6.1.28), multiple Substrate aminotrans nylserine aldolase), 4-hydroxy-2-oxovalerate aldolase, 1,2- ferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), dihydroxybenzylpyruvate aldolase, and/or 2-hydroxybenza L-amino acid oxidase (EC 1.4.3.2), tryptophan oxidase (no lpyruvate aldolase. EC number, Hadar et al., J. Bacteriol 125:1096-1104, 1976 0023 Monatin can be produced by methods that include and Furuya et al., BioSci Biotechnol Biochem 64: 1486-93, contacting tryptophan and/or indole-3-lactic acid with a first 2000), D-tryptophan aminotransferase (Kohiba and Mito, polypeptide, wherein the first polypeptide converts tryp Proceedings of the 8" International Symposium on Vitamin tophan and/or indole-3-lactic acid to indole-3-pyruvate B and Carbonyl , Osaka, Japan 1990), D-amino (either the D or the L form of tryptophan or indole-3-lactic acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase acid can be used as the Substrate that is converted to (EC 1.4.3.3), D-alanine aminotransferase (EC 2.6.1.21), indole-3-pyruvate; one of skill in the art will appreciate that Synthase/lyase (EC 4.1.3.-), Such as 4-hydroxy-2-oxoglut the polypeptides chosen for this step ideally exhibit the arate aldolase (EC 4.1.3.16) or 4-hydroxy-4-methyl-2-oxo appropriate specificity), contacting the resulting indole-3- glutarate aldolase (EC 4.1.3.17), and/or Synthase/lyase pyruvate with a Second polypeptide, wherein the Second (4.1.2.-). polypeptide converts the indole-3-pyruvate to 2-hydroxy 0020. In another example, cells can include one or more 2-(indol-3-ylmethyl)-4-ketoglutaric acid (MP), and contact (Such as two or more, or three or more) of the following ing the MP with a third polypeptide, wherein the third activities: indolelactate dehydrogenase (EC 1.1.1.110), R-4- polypeptide converts MP to monatin. Exemplary polypep hydroxyphenylactate dehydrogenase (EC 1.1.1.222), 3-(4)- tides that can be used for these conversions are shown in hydroxyphenylpyruvate reductase (EC 1.1.1.237), lactate FIGS. 2 and 3. 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 0024 Producing monatin in a biosynthetic pathway via (EC 1.1.3.-), Synthase/lyase (4.1.3.-) Such as 4-hydroxy-2- one or more biological conversions provides certain advan oxoglutarate aldolase (EC 4.1.3.16) or 4-hydroxy-4-methyl tages. For example, by using Specific polypeptides and/or 2-oxoglutarate aldolase (EC 4.1.3.17), Synthase/lyase certain Substrates in the biosynthetic pathway, one can (4.1.2.-), tryptophan aminotransferase (EC 2.6.1.27), produce a mixture enriched in a specific Stereoisomer, and/or tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryp produce a monatin mixture that is Substantially free of one tophan dehydrogenase (EC 1.4.1.19), glutamate dehydroge or more Stereoisomers. nase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4), phenylalanine dehydro 0025. A monatin composition may include impurities as genase (EC 1.4.1.20), tryptophan-phenylpyruvate a consequence of the method used for monatin Synthesis. transaminase (EC 2.6.1.28), multiple Substrate aminotrans Monatin compositions produced by purely Synthetic means ferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), (i.e., not involving at least one biological conversion) will D-tryptophan aminotransferase, D-amino acid dehydroge contain different impurities than monatin compositions pro nase (EC 1.4.99.1), and/or D-alanine aminotransferase (EC duced via a biosynthetic pathway. For example, based on 2.6.1.21). raw materials used, monatin compositions produced by purely Synthetic means may include petrochemical, toxic 0021. In addition, the cells can include one or more (such as two or more, or three or more) of the following activities: and/or other hazardous contaminants inappropriate for tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aro human consumption. Examples of Such contaminants are matic) aminotransferase (EC 2.6.1.5), tryptophan dehydro hazardous chemicals, Such as LDA, hydrogen-Pa/C, diaz genase (EC 1.4.1.19), glutamate dehydrogenase (EC 1.4.1.2, omethane, KCN, Grignard's reagent and Na/Hg. On the 1.4.1.3, 1.4.1.4), phenylalanine dehydrogenase (EC other hand, it is expected that a monatin composition pro 1.4.1.20), tryptophan-phenylpyruvate transaminase (EC duced via a biosynthetic pathway may contain edible or 2.6.1.28), multiple substrate aminotransferase (EC 2.6.1.-), potable impurities, but will not contain petrochemical, toxic aspartate aminotransferase (EC 2.6.1.1), L-amino acid oxi and/or other hazardous material. dase (EC 1.4.3.2), tryptophan oxidase, D-tryptophan ami 0026. It is expected that a method for producing monatin notransferase, D-amino acid dehydrogenase (EC 1.4.99.1), in a biosynthetic pathway via one or more biological con D-amino acid oxidase (EC 1.4.3.3), D-alanine aminotrans versions produces fewer toxic or hazardous contaminants ferase (EC 2.6.1.21), indolelactate dehydrogenase (EC and/or can provide a higher percentage of a particular 1.1.1.110), R-4-hydroxyphenylactate dehydrogenase (EC Stereoisomer, as compared to purely Synthetic means. For 1.1.1.222), 3-(4)-hydroxyphenylpyruvate reductase (EC example, it is expected that when making monatin using US 2005/0112260 A1 May 26, 2005

D-amino acid dehydrogenases, D-alanine (aspartate) ami 0031. In other embodiments, 1 gram of the monatin notransferases, D-aromatic aminotransferases or D-me tabletop Sweetener composition comprises about 200 mg or thionine aminotranferases, one can obtain at least 60%. R.R less S.S monatin or salt thereof, and is substantially free of monatin and less than 40% S.S, S.R and/or R,S monatin. It R,R, S,R or R,S monatin or salt thereof. In another embodi is also expected, for example, that when making monatin ments, 1 gram of the monatin tabletop Sweetener composi using the above-mentioned D-enzymes, as well as at least tion comprises about 5 mg or leSS R,R monatin or Salt one Substrate (e.g., the monatin precursor) having a carbon thereof, and is substantially free of S.S, S.R or R,S monatin in the R-stereoconfiguration, one can obtain at least 95% or salt thereof. RR monatin and less than 5% S.S, S.R and/or R,S monatin. In contrast, it is expected that when making monatin by 0032. In other embodiments, composition comprising monatin or Salt thereof further comprise at least one ingre purely synthetic means, one obtains about 25%-50% of the dient chosen from bulking agents, carriers, fibers, Sugar desired Stereoisomer. alcohols, oligosaccharides, Sugars, non-monatin high inten 0027. In one embodiment, a method for producing mona sity Sweeteners, nutritive Sweeteners, flavorings, flavor tin via a biosynthetic pathway, for example, involving one or enhancers, flavor Stablizers, acidulants, anti-caking agents, more biological conversion, produces no petrochemical, and free-flow agents. For example, the monatin tabletop toxic or hazardous contaminants. “Petrochemical, toxic or Sweetener compositions may further comprise agglomerated hazardous contaminants' means any material that is petro dextrose with maltodextrin. chemical, toxic, hazardous and/or otherwise inappropriate for human consumption, including those contaminants pro 0033. In one embodiment, a Sweetener composition com Vided as raw material or created when producing monatin prises monatin and erthritol. In other embodiments, a mona via purely Synthetic means. In another embodiment, a tin composition comprises erythritol. In one embodiment, a method for producing monatin via a biosynthetic pathway, monatin composition comprises up to 99.7% erythritol. In for example, involving one or more biological conversion, another embodiment, a monatin composition comprises tre produces only edible or potable material. “Edible or potable halose. In another embodiment, a monatin composition material” means one or more compounds or material that are comprises cyclamate. fit for eating or drinking by humans, or otherwise Safe for 0034. In other embodiments, a tabletop Sweetener com human consumption. Examples of edible or potable material position comprises monatin or Salt thereof, where the mona include monatin, tryptophan, pyruvate, glutamate, other tin or Salt thereof consists essentially of R,R monatin or Salt amino acids, as well as other compounds or material that are thereof. Alternatively, the tabletop Sweetener composition naturally present in the body. comprises monatin or Salt thereof, wherein the monatin or 0028. In certain embodiments, monatin Sweetener com Salt thereof consists essentially of S.S monatin or Salt positions include tabletop pre-portioned packets and cubes thereof. In other embodiments, a tabletop Sweetener com of Sweetener, as well as tabletop ready-to-use “spoon-for position comprises monatin or Salt thereof, where the the Spoon' Sweeteners having approximately the Same Sweet monatin or Salt thereof is a Stereoisomerically-enriched R,R ness as Sucrose (Sugar) by Volume, which can be used as a or S.S monatin or Salt thereof. For example, the monatin Substitute for tabletop granulated Sugar. Such tabletop tabletop Sweetener composition may comprises at least 95% Sweeteners may be used to Sweeten coffee and tea (both hot RR monatin or salt thereof. and iced), cereal, fruit and home baked goods (cookies, 0035) In other embodiments, a tabletop Sweetener com muffins, cakes, etc.) and desserts. position comprises monatin or Salt thereof, wherein the SweetneSS is provided by monatin or Salt thereof produced in 0029. In one embodiment, a tabletop Sweetener compo a biosynthetic pathway. Sition comprises monatin or Salt thereof, wherein the com position provides a Sweetness comparable to about 0.9 to 0036). In other embodiments, a ready-to-use Sweetener about 9.0 grams of granulated Sugar. In another embodi composition comprises monatin or Salt thereof, wherein a ment, a 1 gram portion of the monatin tabletop Sweetener Volume of the composition provides a Sweetness comparable composition provides a Sweetness comparable to two tea to a Same Volume of granulated Sugar. In another embodi Spoons of granulated Sugar. In another embodiment, a 1 ment, 1 teaspoon of the monatin ready-to-use Sweetener gram portion of the monatin tabletop Sweetener composition composition contains less calories and carbohydrates than provides provides a Sweetness comparable to about 0.9 to about 1 teaspoon of granulated Sugar. about 9.0 grams of granulated Sugar, and contains leSS calories and carbohydrates than about 1 gram of granulated 0037. In other embodiments, a ready-to-use Sweetener composition comprises monatin or Salt thereof, wherein 1 Sugar. gram of the composition comprises about 3 to about 25 mg 0.030. In other embodiments, a tabletop Sweetener com S.S monatin or salt thereof, and from about 0 to about 0.625 position comprising monatin or Salt thereof comprises from mg R,R monatin or Salt thereof. Alternatively, for example, about 0 to about 200 mg S.S monatin or salt thereof, and the monatin ready-to-use Sweetener consists essentially of from about 0 to about 5 mg R,R monatin or salt thereof. For S.S monatin or salt thereof or R,R monatin or salt thereof. example, 1 gram of the tabletop Sweetener composition may In other embodiments, 1 gram of a ready-to-use monatin comprise from about 3 to about 200 mg S.S monatin or salt Sweetener composition comprises from about 5 to about 25 thereof, and from about 0 to about 5 mg R,R monatin or salt mg S.S monatin or Salt thereof. Alternatively, for example, thereof. Alternatively, for example, 1 gram of the tabletop 1 gram of a ready-to-use monatin Sweetener composition Sweetener composition may comprise from about 0 to about comprises from about 0.4 to about 0.625 R,R monatin or salt 200 mg S.S monatin or salt thereof, and from about 3 to thereof, and is substantially free of S.S, S.R or R,S monatin about 5 mg R,R monatin or salt thereof. or Salt thereof. Alternatively, for example, 1 gram of a US 2005/0112260 A1 May 26, 2005

ready-to-use monatin Sweetener composition comprises monatin or Salt thereof with at least one other ingredient that from about 0.5 to about 1 mg R,R monatin or salt thereof, is not monatin or Salt thereof. Such ingredients may include and is substantially free of S.S, S,R or R,S monatin or salt bulking agents, carriers, fibers, Sugar alcohols, oligosaccha thereof. rides, Sugars, non-monatin high intensity Sweeteners, nutri 0.038. In other embodiments, a Sweetener composition tive Sweeteners, flavorings, flavor enhancers, flavor Stabi comprises a Stereoisomerically-enriched monatin mixture, lizers, acidulants, anti-caking, free-flow agents, and any wherein the monatin mixture is produced via a biosynthetic combination thereof. In one embodiment, at least one other pathway. In one embodiment, the biosynthetic pathway is a ingredient is chosen from maltodextrin, dextrose, erythritol multi-step pathway and at least one Step of the multi-step and fiber. pathway is a chemical conversion. In another embodiment, 0043. In other embodiments, in a method for making a the Stereoisomerically-enriched monatin mixture is pre Sweetener composition, a portion of the Sweetener compo dominantly R,R monatin or salt thereof. Sition weighing about 1 gram comprises from about 0 mg to 0039. In other embodiments, a homogeneous tabletop about 200 mg S.S monatin or salt thereof and from about 0 Sweetener composition comprises monatin or Salt thereof, mg to about 5 mg R,R monatin or Salt thereof, and the wherein a Sample of the composition comprises monatin or portion provides a Sweetness comparable to two teaspoons Salt thereof in an amount ranging from more than 2 mg to of granulated Sugar. about 200 mg, and wherein the monatin or salt thereof in the 0044) In other embodiments, in a method for making a Sample provides a SweetneSS comparable to about 0.9 to Sweetener composition, 1 gram of the Sweetener composi about 9.0 grams of granulated Sugar. For example, a Sample tion comprises from about 0 to about 25 mg S.S monatin or of the homogeneous tabletop Sweetener composition may salt thereof and from about 0 to about 0.625 mg R,R monatin provide a Sweetness comparable to two teaspoons of granu or Salt thereof, wherein a Volume of the composition has a lated Sugar. In another embodiment, a Sample of the homo Sweetness comparable to a Same Volume of granulated Sugar. geneous tabletop Sweetener composition weighs about 1 Alternatively, in a method for making a Sweetener compo gram or has a volume of about 0.35 mL. In another embodi Sition, 1 gram of the composition comprises from about 0 to ment, a Sample of the homogeneous tabletop Sweetener about 25 mg S.S monatin or salt thereof and from about 0 to composition comprises an amount of monatin or Salt thereof about 0.625 mg R,R monatin or salt thereof, wherein 1 gram ranging from more than 2 mg to about 5 mg monatin or Salt of the composition has a Sweetness comparable to about 0.9 thereof. In another embodiment, the homogeneous tabletop to about 9.0 grams granulated Sugar. Alternatively, in a Sweetener composition is Substantially free of S.S monatin method for making a Sweetener composition, the amount of or salt thereof, or substantially free of R,R monatin or salt S.S monatin or salt thereof ranges from about 5 to about 200 thereof. mg S.S monatin or Salt thereof per 1 gram of the composi 0040. In other embodiments, a tabletop Sweetener com tion. position comprises monatin or Salt thereof, wherein a Sample 0045. In other embodiments, a method for making a of the composition comprises monatin or Salt thereof in an Sweetener composition further comprises combining S.S amount ranging from more than 2 mg to about 105 mg monatin or Salt thereof with at least one other ingredient monatin or Salt thereof, and wherein the monatin or Salt chosen from bulking agents, carriers, fibers, Sugar alcohols, thereof in the Sample provides a Sweetness comparable to oligosaccharides, Sugars, non-monatin high intensity Sweet one teaspoon of granulated Sugar. In another embodiment, a eners, nutritive Sweeteners, flavorings, flavor enhancers, Sample of the tabletop Sweetener composition comprising flavor Stablizers, acidulants, anti-caking, free-flow agents, monatin or salt thereof has a volume of about 0.35 ml, or is and any combination thereof, wherein the monatin or Salt a cube of granulated material. thereof comprises about 3 to about 200 mg of monatin or salt 0041. In other embodiments, a tabletop Sweetener com thereof per 1 gram of the composition. In other embodi position comprises a monatin composition produced in a ments, a method for making a Sweetener composition further biosynthetic pathway, wherein the monatin composition comprises combining R,R monatin or Salt thereof with at does not contain petrochemical, toxic or hazardous contami least one other ingredient chosen from bulking agents, nants. For example, the monatin tabletop Sweetener com carriers, fibers, Sugar alcohols, oligosaccharides, Sugars, position comprising monatin or Salt thereof may be pro non-monatin high intensity Sweeteners, nutritive Sweeteners, duced in a biosynthetic pathway and isolated from a flavorings, flavor enhancers, flavor Stablizers, acidulants, recombinant cell, wherein the recombinant cell does not anti-caking, free-flow agents, and any combination thereof, contain petrochemical, toxic or hazardous contaminants. wherein the amount of R,R monatin or Salt thereof ranges from about 3 to about 5 mg of R,R monatin or salt thereof 0042. In other embodiments, a method for making a per 1 gram of the composition. In other embodiments, a Sweetener composition comprises monatin or Salt thereof, method for making a Sweetener composition comprises from wherein the method comprises producing monatin or Salt about 0.4 to about 5 mg R,R monatin or salt thereof per 1 thereof from at least one Substrate chosen from glucose, gram of the composition. tryptophan, indole-3-lactic acid, indole-3-pyruvate and the monatin precursor. In other embodiments, the method fur 0046. In other embodiments, a method for making a ther comprises combining the monatin or Salt thereof with Sweetener composition comprises combining monatin or Salt erythritol. In other embodiments, the method further com thereof with at least one bulking agent chosen from dextrose prises combining the monatin or Salt thereof with trehalose. and maltodextrin, and wherein the monatin or Salt thereof In other embodiments, the method further comprises com comprises about 5 mg R,R monatin or Salt thereof per 1 gram bining the monatin or Salt thereof with cyclamate. In other of the composition. In other embodiments, a method for embodiments, the method further comprises combining the making a Sweetener composition comprises combining US 2005/0112260 A1 May 26, 2005

monatin or salt thereof with maltodextrin, and wherein the 0054 FIG. 3 shows a more detailed diagram of the monatin or salt thereof comprises from about 0.5 to about 1 biosynthetic pathway of the conversion of indole-3-lactic mg R,R monatin or Salt thereof per 1 gram of the compo acid to indole-3-pyruvate. The Substrates are shown in Sition. boxes, and the polypeptides allowing for the conversion 0047. In other embodiments, in a method for making a between the Substrates are listed adjacent to the arrow Sweetener composition comprising a monatin composition, between the substrates. Each polypeptide is described by its the method comprises producing the monatin composition in common name and an EC number. a biosynthetic pathway, and wherein the monatin composi 0055 FIG. 4 shows one possible reaction for making MP tion does not contain petrochemical, toxic or hazardous via chemical means. contaminants. Alternatively, the method comprises produc ing the monatin composition from a Substrate in a multi-step 0056 FIGS.5A and 5B are chromatograms showing the pathway, wherein one or more Steps in the multi-step path LC/MS identification of monatin produced enzymatically. way is a biological conversion, and wherein the monatin 0057 FIG. 6 is an electrospray mass spectrum of enzy composition does not contain petrochemical, toxic or haz matically Synthesized monatin. ardous contaminants. 0.058 FIGS. 7A and 7B are chromatograms of the 0.048. In other embodiments, in a method for making a LC/MS/MS daughter ion analyses of monatin produced in Sweetener composition comprising a monatin composition, an enzymatic mixture. the method comprises producing the monatin composition in a biosynthetic pathway, and wherein the monatin composi 0059 FIG. 8 is a chromatogram showing the high tion consists of monatin or salt thereof and other edible or resolution mass measurement of monatin produced enzy potable material. Alternatively, the method comprises pro matically. ducing the monatin composition from a Substrate in a 0060 FIGS. 9A-9C are chromatograms showing the multi-step pathway, wherein one or more Steps in the multi chiral Separation of (A) R-tryptophan, (B) S-tryptophan, and Step pathway is a biological conversion, and wherein the (C) monatin produced enzymatically. monatin composition consists of monatin or Salt thereof and 0061 FIG. 10 is a bar graph showing the relative amount other edible or potable material. of monatin produced in bacterial cells following IPTG 0049. In other embodiments, in a method for making a induction. The (-) indicates a lack of Substrate addition (no Sweetener composition comprising a monatin composition, tryptophan or pyruvate was added). the method comprises: (a) producing monatin or Salt thereof in a biosynthetic pathway in a recombinant cell; (b) isolating 0062 FIGS. 11-12 are schematic diagrams showing the monatin composition from the recombinant cell, wherein pathways used to increase the yield of monatin produced the monatin composition consists of monatin or Salt thereof from tryptophan or indole-3-pyruvate. and other edible or potable material. 0063 FIG. 13 is a schematic diagram showing a pathway 0050. It will be apparant to one of ordinary skill in the art that can be used to increase the yield of monatin produced from the teachings herein that specific embodiments of the from tryptophan or indole-3-pyruvate. present invention may be directed to one or a combination 0064 FIG. 14 presents a dose response curve obtained of the above-indicated aspects, as well as other aspects. with an R,R, Stereoisomer of monatin. 0065 FIG. 15 presents a dose response curve obtained BRIEF DESCRIPTION OF THE FIGURES with an R.R/S.S stereoisomer mixture of monatin. 0051 FIG. 1 shows biosynthetic pathways used to pro duce monatin and/or indole-3-pyruvate. One pathway pro 0066 FIG. 16 compares the dose response curve duces indole-3-pyruvate via tryptophan, while another pro obtained with an R.R/SS Stereoisomer mixture of monatin duces indole-3-pyruvate via indole-3-lactic acid. Monatin is to a dose response curve obtained with Saccharin. Subsequently produced via a MP intermediate. 0067 FIG. 17 shows reversed phase chromatography of Standards of Synthetically produced monatin. 0.052 Compounds shown in boxes are substrates and products produced in the biosynthetic pathways. Composi 0068 FIG. 18 shows chiral chromatography of monatin tions adjacent to the arrows are cofactors, or reactants that Standards. can be used during the conversion of a Substrate to a product. The or reactant used will depend upon the polypep DETAILED DESCRIPTION tide used for the particular Step of the biosynthetic pathway. The cofactor PLP (pyridoxal 5'-phosphate) can catalyze 0069. Overview of Biosynthetic Pathways for Monatin reactions independent of a polypeptide, and therefore, Production merely providing PLP can allow for the progression from 0070 The following explanations of terms and methods Substrate to product. are provided to better describe the present disclosure and to 0.053 FIG. 2 is a more detailed diagram of the biosyn guide those of ordinary skill in the art in the practice of the thetic pathway that utilizes the MP intermediate. The Sub present disclosure. AS used herein, “including” means “com Strates for each Step in the pathways are shown in boxes. The prising.” In addition, the singular forms “a” or “an” or “the polypeptides allowing for the conversion between Substrates include plural references unless the context clearly dictates are listed adjacent to the arrows between the Substrates. Each otherwise. polypeptide is described by its common name and an 0071. As shown in FIGS. 1-3 and 11-13, many biosyn enzymatic class (EC) number. thetic pathways can be used to produce monatin or its US 2005/0112260 A1 May 26, 2005 intermediates such as indole-3-pyruvate or MP. For the and L-phenylalanine; L-amino acid oxidase (also termed conversion of each Substrate (e.g., glucose, tryptophan, ophio-amino-acid oxidase and L-amino-acid: oxi indole-3-lactic acid, indole-3-pyruvate, and MP) to each doreductase (deaminating)) which converts an L-amino acid product (e.g., tryptophan, indole-3-pyruvate, MP and mona and HO and O. to a 2-oxo acid and NH and HO; tin), several different polypeptides can be used. Moreover, D-amino acid oxidase (also termed Ophio-amino-acid oxi these reactions can be carried out in Vivo, in vitro, or through dase and D-amino-acid:oxygen (deaminat a combination of in Vivo reactions and in vitro reactions, ing)) which converts a D-amino acid and H2O and O2 to a Such as in vitro reactions that include non-enzymatic chemi 2-oxo acid and NH and H2O, and tryptophan oxidase cal reactions. Therefore, FIGS. 1-3 and 11-13 are exem which converts L-tryptophan and HO and O. to indole-3- plary, and Show multiple different pathways that can be used pyruvate and NH and H2O. These classes also contain to obtain desired products. tyrosine (aromatic) aminotransferase, aspartate aminotrans ferase, D-amino acid (or D-alanine) aminotransferase, and 0072 Glucose to Tryptophan broad (multiple Substrate) aminotransferase which have 0.073 Many organisms can synthesize tryptophan from multiple aminotransferase activities, Some of which can glucose. The construct(s) containing the gene(s) necessary convert tryptophan and a 2-oxo acid to indole-3-pyruvate to produce monatin, MP, and/or indole-3-pyruvate from and an amino acid. glucose and/or tryptophan can be cloned into Such organ 0078 Eleven members of the aminotransferase class that isms. It is shown herein that tryptophan can be converted have Such activity are described below in Example 1, into monatin. including a novel aminotransferase shown in SEQ ID NOS: 0.074. In other examples, an organism can be engineered 11 and 12. Therefore, this disclosure provides isolated using known polypeptides to produce tryptophan, or over nucleic acid and amino acid Sequences having at least 80%, produce tryptophan. For example, U.S. Pat. No. 4,371,614 at least 85%, at least 90%, at least 95%, at least 98%, or even describes an E. coli Strain transformed with a plasmid at least 99% Sequence identity to the Sequences Set forth in containing a wild type tryptophan operon. SEQ ID NOS: 11 and 12, respectively. Also encompassed by this disclosure are fragments and fusions of the Sequences 0075) Maximum titers of tryptophan disclosed in U.S. set forth in SEQ ID NOS: 11 and 12 that encode a polypep Pat. No. 4,371,614 are about 230 ppm. Similarly, WO tide having aminotransferase activity or retaining ami 8701130 describes an E. coli strain that has been genetically notransferase activity. Exemplary fragments include, but are engineered to produce tryptophan and discusses increasing not limited to, at least 10, 12, 15, 20, 25, 50, 100, 200, 500, fermentative production of L-tryptophan. Those skilled in or 1000 contiguous nucleotides of SEQID NO: 11 or at least the art will recognize that organisms capable of producing 6, 10, 15, 20, 25, 50, 75, 100, 200, 300 or 350 contiguous tryptophan from glucose are also capable of utilizing other amino acids of SEQ ID NO: 12. The disclosed sequences carbon and energy Sources that can be converted to glucose (and variants, fragments, and fusions thereof) can be part of or fructose-6-phosphate, with Similar results. Exemplary a vector. The Vector can be used to transform host cells, carbon and energy Sources include, but are not limited to, thereby producing recombinant cells which can produce Sucrose, fructose, Starch, cellulose, or glycerol. indole-3-pyruvate from tryptophan, and in Some examples 0076) Tryptophan to Indole-3-Pyruvate can further produce MP and/or monatin. 0.077 Several polypeptides can be used to convert tryp 0079 L-amino acid oxidases (1.4.3.2) are known, and tophan to indole-3-pyruvate. Exemplary polypeptides Sequences can be isolated from Several different Sources, include, without limitation, members of the classes such as Vipera lebetine (sp P81375), Ophiophagus hannah (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, (sp P81383), Agkistrodon rhodostoma (spP81382), Crotalus 2.6.1.5, 2.6.1.-, 2.6.1.1, and 2.6.1.21. These classes include, atrox (sp P56742), Burkholderia cepacia, Arabidopsis without limitation, polypeptides termed tryptophan ami thaliana, Caulobacter creSentus, Chlamydomonas rein notransferase (also termed L-phenylalanine-2-oxoglutarate hardtii, MuS musculus, Pseudomonas Syringae, and Rhodo aminotransferase, tryptophan transaminase, 5-hydroxytryp coccuS Str. In addition, tryptophan oxidases are described in tophan-ketoglutaric transaminase, hydroxytryptophan ami the literature and can be isolated, for example, from Copri notransferase, L-tryptophan aminotransferase, L-tryptophan nus Sp. SF-1, Chinese cabbage with club root disease, transaminase, and L-tryptophan:2-oxoglutarate aminotrans Arabidopsis thaliana, and mammalian liver. One member of ferase) which converts L-tryptophan and 2-oxoglutarate to the L-amino acid oxidase class that can convert tryptophan indole-3-pyruvate and L-glutamate, D-tryptophan ami to indole-3-pyruvate is discussed below in Example 3, as notransferase which converts D-tryptophan and a 2-oxoacid well as alternative Sources for molecular cloning. Many to indole-3-pyruvate and an amino acid; tryptophan dehy D-amino acid oxidase genes are available in databases for drogenase (also termed NAD(P)-L-tryptophan dehydroge molecular cloning. nase, L-tryptophan dehydrogenase, L-Trp-dehydrogenase, 0080 Tryptophan dehydrogenases are known, and can be TDH and L-tryptophan:NAD(P) oxidoreductase (deaminat isolated, for example, from Spinach, Pisum Sativum, PrOSO ing)) which converts L-tryptophan and NAD(P) to indole pis juliflora, pea, mesquite, wheat, maize, tomato, tobacco, 3-pyruvate and NH and NAD(P)H; D-amino acid dehydro Chronobacterium violaceum, and Lactobacilli. Many genase, which converts D-amino acids and FAD to indole D-amino acid dehydrogenase gene Sequences are known. 3-pyruvate and NH and FADH, tryptophan phenylpyruvate transaminase (also termed L-tryptophan-O- 0081. As shown in FIGS. 11-13, if an amino acid oxi ketoisocaproate aminotransferase and dase, Such as tryptophan oxidase, is used to convert tryp L-tryptophan:phenylpyruvate aminotransferase) which con tophan to indole-3-pyruvate, catalase can be added to reduce verts L-tryptophan and phenylpyruvate to indole-3-pyruvate or even eliminate the presence of hydrogen peroxide. US 2005/0112260 A1 May 26, 2005

0082) Indole-3-lactate to Indole-3-pyruvate sations catalyzed by representatives of EC 4.1.2.- and 4.1.3.- require the three carbon molecule of this pathway to be 0.083. The reaction that converts indole-3-lactate to pyruvate or a derivative of pyruvate. However, other com indole-3-pyruvate can be catalyzed by a variety of polypep pounds can Serve as a C3 carbon Source and be converted to tides, Such as members of the 1.1.1.110, 1.1.1.27, 1.1.1.28, pyruvate. Alanine can be transaminated by many PLP 1.1.2.3, 1.1.1.222, 1.1.1.237, 1.1.3.-, or 1.1.1.111 classes of utilizing , including many of those mentioned polypeptides. The 1.1.1.110 class of polypeptides includes above, to yield pyruvate. Pyruvate and ammonia can be indolelactate dehydrogenases (also termed indolelactic acid: obtained by beta-elimination reactions (Such as those cata NAD" oxidoreductase). The 1.1.1.27, 1.1.1.28, and 1.1.2.3 lyzed by tryptophanase or f3-tyrosinase) of L-, L-cys classes include lactate dehydrogenases (also termed lactic teine, and derivatives of Serine and with Sufficient acid dehydrogenases, lactate: NAD" oxidoreductase). The leaving groups, Such as O-methyl-L-serine, O-benzyl-L- 1.1.1.222 class contains (R)-4-hydroxyphenylactate dehy Serine, S-methylcysteine, S-benzylcysteine, S-alkyl-L-cys drogenase (also termed D-aromatic lactate dehydrogenase, teine, O-acyl-L-serine, and 3-chloro-L-alanine. Aspartate R-aromatic lactate dehydrogenase, and R-3-(4-hydroxyphe can Serve as a Source of pyruvate in PLP-mediated beta nyl)lactate:NAD(P)"2-oxidoreductase) and the 1.1.1.237 lyase reactions Such as those catalyzed by tryptophanase class contains 3-(4-hydroxyphenylpyruvate) reductase (also (EC 4.1.99.1) and/or B-tyrosinase (EC 4.1.99.2, also termed termed hydroxyphenylpyruvate reductase and 4-hydrox tyrosine-phenol lyase). The rate of beta-lyase reactions can yphenylactate: NAD" oxidoreductase). The 1.1.3.- class be increased by performing site-directed mutagenesis on the contains lactate oxidases, and the 1.1.1.111 class contains (4.1.99.1-2) polypeptides as described by Mouratou et al. (J. (3-imidazol-5-yl) lactate dehydrogenases (also termed (S)- Biol. Chem 274: 1320-5, 1999) and in Example 8. These 3-(imidazol-5-yl)lactate:NAD(P)" oxidoreductase). It is modifications allow the polypeptides to accept dicarboxylic likely that Several of the polypeptides in these classes allow amino acid Substrates. Lactate can also serve as a Source of for the production of indole-3-pyruvate from indole-3-lactic pyruvate, and is oxidized to pyruvate by the addition of acid. Examples of this conversion are provided in Example lactate dehydrogenase and an oxidized cofactor or lactate 2. oxidase and oxygen. Examples of these reactions are 0084 Chemical reactions can also be used to convert described below. For example, as shown in FIG. 2 and indole-3-lactic acid to indole-3-pyruvate. Such chemical FIGS. 11-13, ProA aldolase can be contacted with indole reactions include an oxidation Step that can be accomplished 3-pyruvate when pyruvate is used as the C3 molecule. using several methods, for example: air oxidation using a B2 0089. The MP can also be generated using chemical catalyst (China Chemical Reporter, Vol. 13, no. 28, pg. reactions, Such as the aldol condensations provided in 18(1), 2002), dilute permanganate and perchlorate, or hydro Example 5. gen peroxide in the presence of metal catalysts. 0085 Indole-3-pyruvate to 2-hydroxy 2-(indol-3ylm 0090 MP to Monatin ethyl)-4-keto Glutaric Acid (MP) 0091 Conversion of MP to monatin can be catalyzed by one or more of tryptophan aminotransferases (2.6.1.27), 0086) Several known polypeptides can be used to convert tryptophan dehydrogenases (1.4.1.19), D-amino acid dehy indole-3-pyruvate to MP. Exemplary polypeptide classes drogenases (1.4.99.1), glutamate dehydrogenases (1.4.1.2- include 4.1.3.-, 4.1.3.16, 4.1.3.17, and 4.1.2.-. These classes 4), phenylalanine dehydrogenase (EC 1.4.1.20), tryptophan include carbon-carbon Synthases/, Such as aldolases phenylpyruvate transaminases (2.6.1.28), or more generally that catalyze the condensation of two carboxylic acid Sub members of the aminotransferase family (2.6.1.-) Such as Strates. Polypeptide class EC 4.1.3.- are Synthases/lyases aspartate aminotransferase (EC 2.6.1.1), tyrosine (aromatic) that form carbon-carbon bonds utilizing oxo-acid Substrates aminotransferase (2.6.1.5), D-tryptophan aminotransferase, (such as indole-3-pyruvate) as the electrophile, while EC or D-alanine (2.6.1.21) aminotransferase (FIG. 2). Eleven 4.1.2.- are Synthases/lyases that form carbon-carbon bonds members of the aminotransferase class are described below utilizing aldehyde Substrates (Such as benzaldehyde) as the (Example 1), including a novel member of the class shown electrophile. in SEQID NOS: 11 and 12, and reactions demonstrating the 0.087 For example, the polypeptide described in EP activity of aminotransferase and dehydrogenase enzymes 1045-029 (EC 4.1.3.16, 4-hydroxy-2-oxoglutarate glyoxy are provided in Example 7. late-lyase also termed 4-hydroxy-2-oxoglutarate aldolase, 0092. This reaction can also be performed using chemical 2-oxo-4-hydroxyglutarate aldolase or KHG aldolase) con reactions. Amination of the keto acid (MP) is performed by verts glyoxylic acid and pyruvate to 4-hydroxy-2-ketoglu reductive amination using ammonia and Sodium cyanoboro taric acid, and the polypeptide 4-hydroxy-4-methyl-2-oxo hydride. glutarate aldolase (EC 4.1.3.17, also termed 4-hydroxy-4- methyl-2-oxoglutarate pyruvate-lyase or ProA aldolase), 0093 FIGS. 11-13 show additional polypeptides that can condenses two keto-acids Such as two pyruvates to 4-hy be used to convert MP to monatin, as well as providing droxy-4-methyl-2-oxoglutarate. Reactions utilizing these increased yields of monatin from indole-3-pyruvate or tryp tophan. For example, if aspartate is used as the amino donor, lyases are described herein. aspartate aminotransferase can be used to convert the aspar 0088 FIGS. 1-2 and 11-13 show schematic diagrams of tate to oxaloacetate (FIG. 11). The oxaloacetate is converted these reactions in which a 3-carbon (C3) molecule is com to pyruvate and carbon dioxide by a decarboxylase, Such as bined with indole-3-pyruvate. Many members of EC 4.1.2.- oxaloacetate decarboxylase (FIG. 11). In addition, if lysine and 4.1.3.-, particularly PLP-utilizing polypeptides, can is used as the amino donor, lysine epsilon aminotransferase utilize C3 molecules that are amino acids Such as Serine, can be used to convert the lysine to allysine (FIG. 12). The cysteine, and alanine, or derivatives thereof. Aldol conden alysine is spontaneously converted to 1-piperideine 6-car US 2005/0112260 A1 May 26, 2005

boxylate (FIG. 12). If a polypeptide capable of catalyzing condensation and ring-closure reaction between DXP and reductive amination reactions (e.g., glutamate dehydroge HTP, catalyzed by the gene products of pdxA and pdx.J. nase) is used to convert MP to monatin, a polypeptide that 0102) If PLP becomes a limiting nutrient during the can recycle NAD(P)H and/or produce a volatile product fermentation to produce monatin, increased expression of (FIG. 13) can be used, such as formate dehydrogenase. one or more of the pathway genes in a production host cell 0094. Additional Considerations in the Design of the can be used to increase the yield of monatin. A host organism Biosynthetic Pathways can contain multiple copies of its native pathway genes or copies of non-native pathway genes can be incorporated into 0.095 Depending on which polypeptides are used to the organism's genome. Additionally, multiple copies of the generate indole-3-pyruvate, MP, and/or monatin, cofactors, Salvage pathway genes can be cloned into the host organism. Substrates, and/or additional polypeptides can be provided to the production cell to enhance product formation. In addi 0103) One salvage pathway that is conserved in all organ tion, genetic modification can be designed to enhance pro isms recycles the various derivatives of Vitamin B to the duction of products such as indole-3-pyruvate, MP, and/or active PLP form. The polypeptides involved in this pathway are pdxK kinase, pdxH oxidase, and pdxY kinase. Over monatin. Similarly, a host cell used for monatin production expression of one or more of these genes can increase PLP can be optimized. availability. 0096 Removal of Hydrogen Peroxide 0104 Vitamin B levels can be elevated by elimination or 0097 Hydrogen peroxide (HO) is a product that, if repression of the metabolic regulation of the native biosyn generated, can be damaging to production cells, polypep thetic pathway genes in the host organism. PLP represses tides or products (e.g., intermediates) produced. The polypeptides involved in the biosynthesis of the precursor L-amino acid oxidase described above generates H2O as a derivative in the bacterium Flavobacterium sp. product. Therefore, if L-amino acid oxidase is used, the strain 238-7. This bacterial strain, freed of metabolic control, resulting H2O can be removed or its levels decreased to overproduces pyridoxal derivatives and can excrete up to 20 reduce potential injury to the cell or product. mg/L of PLP. Genetic manipulation of the host organism producing monatin in a similar fashion will allow the 0.098 Catalases can be used to reduce the level of H.O. increased production PLP without over-expression of the in the cell (FIGS. 11-13). The production cell can express a biosynthetic pathway genes. gene or cDNA sequence that encodes a catalase (EC 1.11.1.6), which catalyzes the decomposition of hydrogen 0105 Ammonium Utilization peroxide into water and oxygen gas. For example, a catalase 0106 Tryptophanase reactions can be driven toward the can be expressed from a vector transfected into the produc Synthetic direction (production of tryptophan from indole) tion cell. Examples of catalases that can be used include, but by making ammonia more available or by removal of water. are not limited to: trQ9EV50 (Staphylococcus xylosus), Reductive amination reactions, Such as those catalyzed by trQ9 KBE8 (Bacillus halodurans), trQ9URJ7 (Candida glutamate dehydrogenase, can also be driven forward by an albicans), trP77948 (Streptomyces coelicolor), trC9RBJ5 excess of ammonium. (Xanthomonas campestris) (SwissProt Accession Nos.). 0107 Ammonia can be made available as an ammonium Biocatalytic reactors utilizing L-amino acid oxidase, carbonate or ammonium phosphate Salt in a carbonate or D-amino acid oxidase, or tryptophan oxidase can also con phosphate buffered System. Ammonia can also be provided tain a catalase polypeptide. as ammonium pyruvate or ammonium formate. Alterna 0099 Modulation of Pyridoxal-5'-phosphate (PLP) tively, ammonia can be Supplied if the reaction is coupled Availability with a reaction that generates ammonia, Such as glutamate dehydrogenase or tryptophan dehydrogenase. Ammonia can 0100. As shown in FIG. 1, PLP can be utilized in one or be generated by addition of the natural substrates of EC more of the biosynthetic steps described herein. The con 4.1.99.-(tyrosine or tryptophan), which will be hydrolyzed to centration of PLP can be supplemented so that PLP does not phenol or indole, pyruvate and NH. This also allows for an become a limitation on the Overall efficiency of the reaction. increased yield of Synthetic product over the normal equi librium amount by allowing the enzyme to hydrolyze its 0101 The biosynthetic pathway for vitamin B (the pre preferred Substrate. cursor of PLP) has been thoroughly studied in E. coli, and Some of the proteins have been crystallized (Laber et al., 0108) Removal of Products and Byproducts FEBS Letters, 449:45-8, 1999). Two of the genes (epd or gap B and SerC) are required in other metabolic pathways, 0109 The conversion of tryptophan to indole-3-pyruvate while three genes (pdxA, pdxB, and pdx.J) are unique to via a tryptophan aminotransferase can adversely affect the biosynthesis. One of the Starting mate production rate of indole-3-pyruvate because the reaction rials in the E. coli pathway is 1-deoxy-D-xylulose-5-phos produces glutamate and requires the co-Substrate 2-oxoglu phate (DXP). Synthesis of this precursor from common 2 tarate (a-ketoglutarate). Glutamate can cause inhibition of and 3 carbon central metabolites is catalyzed by the the aminotransferase, and the reaction can consume large polypeptide 1-deoxy-D-xylulose-5-phosphate Synthase amounts of the co-Substrate. Moreover, high glutamate con (DXS). The other precursor is a threonine derivative formed centrations can be detrimental to downstream Separation from the 4-carbon Sugar, D-erythrose 4-phosphate. The proceSSeS. genes required for the conversion to phospho-4-hydroxyl-L 0110. The polypeptide glutamate dehydrogenase threonine (HTP) are epd, pdxB, and serC. The last reaction (GLDH) converts glutamate to 2-oxoglutarate, thereby recy for the formation of PLP is a complex intramolecular cling the co-Substrate in the reaction catalyzed by tryptophan US 2005/0112260 A1 May 26, 2005 aminotransferase. GLDH also generates reducing equiva Sure to high concentrations of 5-methyltryptophan (Schal lents (NADH or NADPH) that can be used to generate lenberg and Berlin, Z Naturforsch 34:541-5, 1979). The energy for the cell (ATP) under aerobic conditions. The resulting tryptophan Synthase activity of the Strain was leSS utilization of glutamate by GLDH also reduces byproduct effected by product inhibition, likely due to mutations in the formation. Additionally, the reaction generates ammonia, gene. Similarly, a host cell used for monatin production can which can Serve as a nitrogen Source for the cell or as a be optimized. Substrate in a reductive amination for the final Step shown in FIG. 1. Therefore, a production cell that over-expresses a 0117 Tryptophan production can be optimized through GLDH polypeptide can be used to increase the yield and the use of directed evolution to evolve polypeptides that are leSS Sensitive to product inhibition. For example, Screening reduce the cost of media and/or separation processes. can be performed on plates containing no tryptophan in the 0111. In the tryptophan to monatin pathway, the amino medium, but with high levels of non-metabolizable tryp donor of Step three (e.g., glutamate or aspartate) can be tophan analogs. U.S. Pat. Nos. 5,756,345; 4,742,007; and converted back to the amino acceptor required for Step 1 4,371,614 describe methods used to increase tryptophan (e.g., 2-oxo-glutarate or oxaloacetate), if an aminotrans productivity in a fermentation organism. The last Step of ferase from the appropriate enzyme classes is used. Utili tryptophan biosynthesis is the addition of Serine to indole; Zation of two separate transaminases for this pathway, in therefore the availability of serine can be increased to which the Substrate of one transaminase does not competi increase tryptophan production. tively inhibit the activity of the other transaminase, can increase the efficiency of this pathway. 0118. The amount of monatin produced by a fermentation organism can be increased by increasing the amount of 0112 Many of the reactions in the described pathways pyruvate produced by the host organism. Certain yeasts, are reversible and can, therefore, reach an equilibrium Such as TrichoSporon cutaneum (Wang et al., Lett. Appl. between substrates and products. The yield of the pathway Microbiol. 35:338-42, 2002) and Torulopsis glabrata (Liet can be increased by continuous removal of the products al., Appl Microbiol. Biotechnol. 57:451-9, 2001) overpro from the polypeptides. For example, Secretion of monatin duce pyruvate and can be used to practice the methods into the fermentation broth using a permease or other disclosed herein. In addition, genetic modifications can be transport protein, or Selective crystallization of monatin made to organisms to promote production, Such from a biocatalytic reactor Stream with concomitant recycle as those in E. coli strain W1485lip2 (Kawasaki et al., J. of Substrates will increase the reaction yield. Ferm. and Bioeng. 82:604-6, 1996). 0113 Removal of byproducts via additional enzymatic 0119) Controlling Chirality reactions or via Substitution of amino donor groups is another way to increase the reaction yield. Several examples 0120) The taste profile of monatin can be altered by are discussed in Example 13 and shown in FIGS. 11-13. For controlling its Stereochemistry (chirality). For example, dif example, a byproduct can be produced that is unavailable to ferent monatin isomers may be desired in different blends of react in the reverse direction, either by phase change (evapo concentrations for different food systems. Chirality can be ration) or by spontaneous conversion to an unreactive end controlled via a combination of pH and polypeptides. product, Such as carbon dioxide.

0114) Modulation of the Substrate Pools C2 HO O 0115 The indole pool can be modulated by increasing N production of tryptophan precursors and/or altering cata bolic pathways involving indole-3-pyruvate and/or tryp OH tophan. For example, the production of indole-3-acetic acid NH2 from indole-3-pyruvate can be reduced or eliminated by functionally deleting the gene coding for EC 4.1.1.74 in the N N. host cell. Production of indole from tryptophan can be O OH 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 0121 at the C-4 position of monatin (see or in Vivo proceSS in combination with increased amounts of numbered molecule above) can occur by deprotonation and the gene coding for EC 4.1.99.1 (Kawasaki et al., J. Ferm. reprotonation of the alpha carbon, which can occur by a shift and Bioeng, 82:604-6, 1996). In addition, genetic modifi in pH or by reaction with the cofactor PLP bound to an cations can be made to increase the level of intermediates enzyme Such as a racemase or free in Solution. In a micro Such as D-erythrose-4-phosphate and chorismate. organism, the pH is unlikely to shift enough to cause the racemization, but PLP is abundant. Methods to control the 011.6 Tryptophan production is regulated in most organ chirality with polypeptides depend upon the biosynthetic isms. One mechanism is via feedback inhibition of certain route utilized for monatin production. enzymes in the pathway; as tryptophan levels increase, the production rate of tryptophan decreases. Thus, when using a 0122) When monatin is formed using the pathway shown host cell engineered to produce monatin via a tryptophan in FIG. 2, the following can be considered. In a biocatalytic intermediate, an organism can be used that is not Sensitive to reaction, the chirality of carbon-2 can be determined by an tryptophan concentrations. For example, a Strain of Catha enzyme that converts indole-3-pyruvate to MP. Multiple ranthus roseus that is resistant to growth inhibition by enzymes (e.g., from EC 4.1.2.-, 4.1.3.-) can convert indole various tryptophan analogs was Selected by repeated expo 3-pyruvate to MP, thus, the enzyme that forms the desired US 2005/0112260 A1 May 26, 2005

isomer can be chosen. Alternatively, the enantiospecificity of mize cost. Monatin may be used in combination with other the enzyme that converts indole-3-pyruvate to MP can be Sweeteners and/or other ingredients to generate a temporal modified through the use of directed evolution, or catalytic profile Similar to Sucrose, or for other benefits. antibodies can be engineered to catalyze the desired reac tion. Once MP is produced (either enzymatically or by 0128. For example, monatin may be blended with other chemical condensation), the amino group can be added nutritive and nonnutritive Sweeteners to achieve particular Stereospecifically using a transaminase, Such as those flavor profiles or calorie targets. Thus, Sweetener composi described herein. Either the R or S configuration of carbon-4 tions can include combinations of monatin with one or more can be generated depending on whether a D- or L-aromatic of the following Sweetener types: (1) Sugar alcohols (such as acid aminotransferase is used. Most aminotransferases are erythritol, Sorbitol, maltitol, mannitol, lactitol, Xylitol, iso Specific for the L-isomer; however, D-tryptophan ami malt, low glycemic syrups, etc.); (2) other high intensity notransferases exist in certain plants (Kohiba and Mito, Sweeteners (Such as aspartame, Sucralose, Saccharin, Proceedings of the 8th International Symposium on Vitamin aceSulfame-K, Stevioside, cyclamate, neotame, thaumatin, B and Carbonyl Catalysis, Osaka, Japan 1990). Moreover, alitame, dihydrochalcone, monellin, glycyrrihizin, mogro D-alanine aminotransferases (2.6.1.21), D--pyru Side, phyllodulcin, mabinlin, braZZein, circulin, pentadin, vate aminotransferases (2.6.1.41), and both (R)-3-amino-2- etc.) and (3) nutritive Sweeteners (such as Sucrose, tagatose, methylpropanoate aminotransferase (2.6.1.61) and (S)-3- invert Sugar, fructose, corn Syrup, high fructose corn Syrup amino-2-methylpropanoate aminotransferase (2.6.1.22) (HFCS), glucose/dextrose, trehalose, isomaltulose, etc.). have been identified. Certain aminotransferases may only Monatin may be used in Such blends as a taste modifier to accept the Substrate for this reaction with a particular con SuppreSS aftertaste, enhance other flavorS Such as lemon, or figuration at the C2 carbon. Therefore, even if the conver improve the temporal flavor profile. Data also indicate that sion to MP is not stereospecific, the stereochemistry of the monatin is quantitatively Synergistic with cyclamates (which final product can be controlled through the appropriate are used in Europe), but no significant quantitative Synergy Selection of a transaminase. Since the reactions are revers was noted with aspartame, Saccharin, aceSulfame-K, Sucral ible, the unreacted MP (undesired isomer) can be recycled ose, or carbohydrate Sweeteners. Because monatin is not a back to its constituents, and a racemic mixture of MP can be carbohydrate, monatin can be used to partially or fully reformed. replace carbohydrate-containing Sweeteners. 0.129 Monatin is stable in a dry form, and has a desirable 0123 Activating Substrates taste profile alone or when mixed with carbohydrates. It does 0.124 Phosphorylated substrates, such as phospho not appear to irreversibly break down, but rather forms enolpyruvate (PEP), can be used in the reactions disclosed lactones and/or lactams at low pHs (in aqueous buffers) and herein. Phosphorylated Substrates can be more energetically reaches an equilibrium. It can racemize at the 4 position favorable and, therefore, can be used to increase the reaction Slowly over time in Solution, but typically this occurs at high rates and/or yields. In aldol condensations, the addition of a pHS. In general, the Stability of monatin is comparable to or phosphate group Stabilizes the enol tautomer of the nucleo better than aspartame (Equal(F) and the taste profile of philic Substrate, making it more reactive. In other reactions, monatin is comparable to or better than other quality Sweet a phosphorylated Substrate can provide a better leaving eners, Such aspartame (Equal(F), alitame, and Sucralose group. Similarly, Substrates can be activated by conversion (Splenda(R). Monatin does not have the undesirable after to CoA derivatives or pyrophosphate derivatives. taste associated with Some other high intensity Sweeteners 0.125 Use of Monatin in a Sweetener Composition Such as Saccharin and Stevioside. 0.130 Formulations of monatin Sweeteners may be used 0.126 The S.S stereoisomer of monatin is approximately as tabletop Sugar Substitutes. Generally, when making table 50-200 times Sweeter than sucrose by weight. The R.R stereoisomer of monatin is approximately 2000-2400 times top Sugar Substitutes, one also employs bulking agents Sweeter than Sucrose by weight. AS discussed above, the and/or carries to dilute the monatin and allow it to be easily Sweetness of the monatin is calculated using experienced measured. Sensory evaluators in a Sweetness comparison procedure, 0131. In one embodiment, one may prepare convenient where a test Sweetener Solution is matched for SweetneSS Single-Serving packets formulated to provide a Sweetness intensity against one of a Series of reference Solutions. The comparable to that in 2 teaspoons (~8 grams) of granulated Solutions may be prepared, for example, using a buffer Sugar. Because S.S is 50-200 times Sweeter than Sucrose, comprising 0.16% (v/w) and 0.02% (V/w) sodium 40-160 mg of S.S monatin delivers a Sweetness comparable citrate at ~pH 3.0. The intensity SweetneSS is calculated as to that in 8 grams of granulated Sugar. Thus, in one embodi the slope in a dose response curve, where the % Sucrose is ment, allowing for +/-25% Sweetness optimization, Single divided by the % monatin. See e.g., FIG. 15 (R.R/S.S Serving packet 1 gram formulations of monatin may com monatin dose response curve); FIG. 14 (R,R monatin dose prise approximately 40-200 mg of S.S monatin. response curve). The above-mentioned monatin Sweetness, as compared to Sucrose Sweetness, is obtained by measuring 0.132. Likewise, because R.R is 2000-2400 times Sweeter than Sucrose, 3.3-4.0 mg of R,R monatin delivers a Sweet the slope at 8% sucrose equivalence values (“SEV”). neSS comparable to that in 8 grams of Sugar. Thus, in another 0127. Monatin is soluble in aqueous solutions in concen embodiment, allowing for +/-25% Sweetness optimization, trations that are appropriate for consumption. Various blends Single-Serving packet 1 gram formulations of monatin may of monatin StereoisomerS may be qualitatively better in comprise approximately 3.3-5.0 mg of R,R monatin. In certain matrices, or in blending with other Sweeteners. another embodiment, packet formulations may comprise Blends of monatin with other Sweeteners may be used to 40-200 mg of S.S monatin, 3.3-5.0 mg of R,R monatin or a maximize the Sweetness intensity and/or profile, and mini combination thereof in the same or lesser amounts per gram US 2005/0112260 A1 May 26, 2005 total weight, to provide a Sweetness comparable to that in 2 0142. In other embodiments, one can make monatin teaspoons of granulated Sugar. compositions in the form of cubes for use, for example, in restaurants. The cubes weigh approximately 8 grams and are 0.133 Tabletop Sweetener packets often contain a total of of equivalent size to a Standard cube of granulate Sugar, 1 gram and a mixture of high-intensity Sweetener and one or which is 2.2 cmx2.2 cmx1 cm. These cube formulations more bulking agents or carriers. A number of bulking agents contain monatin in amounts similar to those described above or combinations of agents may be used in preparing the for the ready-to-use formulations, So that the cubes contain formulations. For example, in Some embodiments, monatin a Sweetness comparable to that in the Standard Sugar cube. can be spray dried with maltodextrin and/or dextrose. Dex Alternatively, one may also make /2 size cubes (4 grams, 1.1 trose, for example, provides greater density and Still allows cmx2.2 cmx1 cm). These formulations may be prepared to for one packet (~1 g) of Sweetener to round down to 0 deliver a Sweetness comparable to that in a Standard Sugar Calories per Serving. cube (and therefore contain approximately two times the 0134. In an embodiment, a formulation for a monatin Sweetness of Sucrose by weight). containing packet (1 g total) includes: 0.143 Monatin tabletop formulations, such as those for 0135) dextrose (nutritive) 0-99.7 wt % packets or for the ready-to-use compositions, may also 0136 maltodextrin 0-99.7 wt % include other bulking agents or carriers. For instance, the 0137 3.3-5.0 mg R,R monatin, 40-200 mg S.S mona formulations may include one or more fibers, Such as inulin, tin or a combination thereof in the same or lesser arabinogalactan, hydrolyzed guar gum, polydextrose, micro amountS. crystalline cellulose, Solka floc, Starch, modified Starch, 0.138. In other embodiments, ready-to-use formulations resistant Starch, resistant maltodextrin, etc. Many fibers are of tabletop Sweeteners are designed to have a Sweetness and highly soluble in water, have low viscosity, and bland flavor. Volume comparable to that of granulated Sugar (Sucrose). Fibers are especially useful as a bulking agent because they Such formulations can be used "spoon-for-spoon' in place are less caloric than other bulking agents. of granulated Sugar, which is especially useful in baking 014.4 For example, inulin delivers only 1.3 Calories per recipes. Compared to packet (~1 g) formulations, the ready gram compared to 4 Calories per gram for dextrose. De to-use “spoon-for-spoon' formulations generally contain Sugared inulin, which contains <2% mono- and disaccha more dilutant materials, Such as bulking agents and/or rides delivers only 1.1 Calories per gram is a particularly carriers, which allow for common household measurements, good choice. Certain fibers, Such as inulin, have the potential Such as by teaspoon, tableSpoon or cup. These formulations to impart a more granular Sugar-like appearance to the typically have a leSS dense and less granular appearance than formulation than maltodextrin, which is commonly used in the packet versions. Thus, the ready-to-use “spoon-for cup-for-cup Sugar Substitutes. FiberS also provide the added Spoon' formulations may include different ingredients to benefits of fiber fortification, improved gut health, improved reduce the density and allow the formulation to be used as absorption, etc. Use of non-digestable carbohy an equal replacement for Sugar. drates, Such as fibers or resistant Starch derivatives, also 0139 For example, because dextrose is more dense, one results in leSS Sugar content for those concerned with gly may want to use maltodextrin alone when preparing the cemic modulation (e.g., diabetics). For example, one may ready-to-use “spoon-for-spoon' formulations. The light den use resistant Starch, resistant maltodextrin and inulin to sity of maltodextrin allows the Sweetener formulation to be obtain lower calorie, low glycemic and low insulinogenic used in equal Volumes to Sugar without accumulating the carriers or dilutants. same (high) level of calories. This approach is especially 0145 Likewise, monatin formulations may include one useful when the monatin Sweetener is used by the Spoonful or more Sugar alcohols, Such as erythritol, Sorbitol, Xylitol, or cupful, Such as in baking applications. maltitol, mannitol and lactitol. In one embodiment, one may 0140. Because S.S is 50-200 times Sweeter than Sucrose, use erythritol in place of other carriers or bulking agents. 5-20 mg of SS monatin delivers a Sweetness comparable to Erythritol provides certain advantages over dextrose or that in 1 gram of granulated Sugar. Thus, in one embodiment, maltodextrin. For example, erythritol provides little to no allowing for +/-25% Sweetness optimization, ready-to-use calories, as compared to dextrose and maltodextrin-eryth “Spoon-for-spoon' formulations may comprise about 5-25 ritol provides 0-0.2 Calories per gram, while dextrose pro mg of S.S monatin/gram total weight. Likewise, because vides 4 Calories per gram, and maltodextrin provides 2.8-3.2 R.R is 2000-2400 times Sweeter than sucrose, 0.4-0.5 mg of Calories per gram. See ALTERNATIVE SWEETENERS, R,R monatin delivers a SweetneSS comparable to that in 1 3. Ed., edited by Lyn O'Brien Nabors, Chapter 13 “Eryth gram of Sugar. Thus, in another embodiment, allowing for ritol” by M. E. Embuscado and S. K. Patil (Marcel Dekker, +/-25% Sweetness optimization, ready-to-use “spoon-for Inc, New York 2001). Also, when maltodextrin is sprayed spoon” formulations may comprise about 0.4–0.625 mg of dried, it looks like a powder rather than granular Sugar. R,R monatin. In another embodiment, the ready-to-use Erythritol, on the other hand, is monocrystalized and there formulations may comprise 5-25 mg of S.S monatin, 0.4- fore looks like Sugar. Thus, one can add monatin directly on 0.625 mg of R,R monatin or a combination thereof in the top of erythritol. Same or lesser amounts per gram total weight, to provide a 0146 In addition, the formulations may also include Sweetness comparable to that in granulated Sugar. oligosaccharides, Such as fructooligosaccharides, maltooli 0.141. In another embodiment, a formulation for a ready gosaccharide, isomaltooligosaccharides, galatooligSaccha to-use Sweetener contains approximately 0.2-13 mg monatin rides, Soybean oligosaccharides, and lactooligosaccharides. per every half gram Serving size, spray-dried onto approxi The formulations also may include Sugars, Such as Sucrose, mately 500 mg maltodextrin. invert Sugar, trehalose, isomerized Sugar, glucose, fructose, US 2005/0112260 A1 May 26, 2005 lactose, malt Sugar, D-xylose and isomerized lactose, etc. If D-alanine aminotransferase (dat, Genbank Accession No. tablets are made, lactose can be added to increase bulk and Y14082.1 bp 28622-29470 and Genbank Accession No. shape the tablets. NP 388848.1, nucleic acid sequence and amino acid 0147 The monatin and bulking agent combination may Sequence, respectively), Sinorhizobium meliloti (also termed be produced by any means, including dry-mixed, co-spray Rhizobium meliloti) tyrosine aminotransferase (tat.A, SEQ dried, co-freeze dried, agglomerated, blended, co-dried, ID NOS: 1 and 2, nucleic acid Sequence and amino acid extruded or other process. Sequence, respectively), Rhodobacter Sphaeroides Strain 2.4.1 tyrosine aminotransferase (tatA asserted by homology, 0148 One also may add flavorings and acidulants, such SEQ ID NOS: 3 and 4, nucleic acid sequence and amino acid as cream of tartar, to the formulation mixtures for improved Sequence, respectively), R. Sphaeroides 35053 tyrosine ami flavor, Stability, and/or baking performance. One may also notransferase (asserted by homology, SEQID NOS: 5 and 6, add flavor enhancers or stabilizers, Such as SucramaskTM. nucleic acid Sequence and amino acid Sequence, respec 014.9 The formulations may also include anti-caking or tively), Leishmania major broad Substrate aminotransferase free-flow agents, Such as Silicon dioxide (silica), calcium (bsat, asserted by homology to peptide fragments from L. Silicate, magnesium Silicate, calcium carbonate, Sodium mexicana, SEQID NOS: 7 and 8, nucleic acid sequence and aluminosilicate, calcium aluminosilicate, potassium alumi amino acid sequence, respectively), Bacillus Subtilis aro nosilicate, monocalcium phosphate, dicalcium phosphate, matic aminotransferase (araT, asserted by homology, SEQ tricalcium phosphate, talc, mannitol, etc. ID NOS: 9 and 10, nucleic acid sequence and amino acid Sequence, respectively), Lactobacillus amylovorus aromatic 0150. In some embodiments, monatin is used in baking. aminotransferase (araT asserted by homology, SEQ ID In certain embodiments, honey or molasses is included in NOS: 11 and 12, nucleic acid Sequence and amino acid baking recipes to promote browning and good texture. For sequence, respectively), R. Sphaeroides 35053 multiple sub example, certain recipes may call for the addition of a Strate aminotransferase (asserted by homology, SEQ ID tableSpoon of molasses to imitate the coloration due to NOS: 13 and 14, nucleic acid Sequence and amino acid normal browning of SugarS. Sequence, respectively), Rhodobacter Sphaeroides Strain 0151. In another embodiments, monatin is used to replace 2.4.1 multiple Substrate aminotransferase (msa asserted by many of the polyols that are currently used in candies and homology, Genbank Accession No. AAAE01000093.1, bp other no Sugar foods. These polyols have a laxation effect, 14743-16155 and Genbank Accession No. ZP00005082.1, and in the case of erythritol, also have a cooling effect. nucleic acid Sequence and amino acid Sequence, respec Erythritol has substantially less laxation effect than other tively), Escherichia coli aspartate aminotransferase (aspC, polyols. Other polyols have cooling effect as much as Genbank Accession No. AE000195.1 bp 2755-1565 and erythritol. Erythritol, as compared to traditional polyols, Genbank Accession No. AAC74014.1, nucleic acid Such as maltitol, contains fewer calories and a lower glyce Sequence and amino acid sequence, respectively), and E. coli mic index. Monatin will even further reduce the caloric tyrosine aminotransferase (tyrB, SEQ ID NOS: 31 and 32, value and the glycemic index, without Significantly increas nucleic acid Sequence and amino acid Sequence, respec ing the cariogenicity. However, when replacing polyols or tively). other traditional Sweeteners (Such as Sugars) with high intensity Sweeteners, bulking agents are required to maintain O157 The genes were cloned, expressed, and tested for both the volume and mouthfeel of the product. The most activity in conversion of tryptophan to indole-3-pyruvate, common bulking agents include inulin, maltodextrin, poly along with commercially available enzymes. All eleven dextrose, and Sorbitol. Inulin can also improve uptake of clones had activity. dietary calcium by the body. Resistant Starches and slowly 0158 Identification of Bacterial Strains That Can Con digestible Starches can also be utilized as bulking agents or tain Polypeptides with the Desired Activity diluents of high intensity Sweeteners. Monatin can be used in combination with a number of Sweeteners, carbohydrates, 0159) No genes in the NCBI (National Center for Bio and bulking agents. technology Information) database were designated as tryp 0152. It is expected that monatin tabletop blends, as tophan aminotransferases. However, organisms having this compared to other Sweeteners, will have a longer shelf-life, enzymatic activity have been identified. L-tryptophan ami greater heat and acid Stability, as well as better taste char notransferase (TAT) activity has been measured in cell acteristics and marketing advantages. extracts or from purified protein from the following Sources: Rhizobacterial isolate from Festuca Octoflora, pea mito EXAMPLES chondria and cytosol, Sunflower crown gall cells, Rhizobium leguminoSarum biovar trifoli, Erwinia herbicola pv gypso Example 1 philae, Pseudomonas Syringae pv. Savastanoi, Agrobacte rium tumefaciens, Azospirillum lipferum & brasilense, 0153. Cloning and Expression of Tryptophan Ami Enterobacter cloacae, Enterobacter agglomerans, notransferases Bradyrhizobium elkani, Candida maltosa, Azotobacter 0154) This example describes methods that were used to vinelandii, rat brain, rat liver, Sinorhizobium meliloti, clone tryptophan aminotransferases, which can be used to Pseudomonas fluorescens CHAO, Lactococcus lactis, Lac convert tryptophan to indole-3-pyruvate. tobacillus casei, Lactobacillus helveticus, wheat Seedlings, barley, Phaseolus aureus (mung bean), Saccharomyces 0155 Experimental Overview uvarum (carlsbergensis), Leishmania sp., maize, tomato 0156 Eleven genes encoding aminotransferases were shoots, pea plants, tobacco, pig, CloStridium Sporogenes, cloned into E. coli. These genes were Bacillus subtilis and Streptomyces griseu.S. US 2005/0112260 A1 May 26, 2005

Example 2 Microbiol. 14 1968, Coote and Hassall (Biochem. J. 111: 237-9, 1969), Cortese et al. (C.R. Seances Soc. Biol. Fil. 162 0160 Conversion of Indole-3-lactate to Indole-3-pyru 390-5, 1968), Petersen and Alfermann (Z. Naturforsch. C. Vate BioSci. 43 501-4, 1988), and Bhatnagar et al. (J. Gen 0161. As shown in FIGS. 1 and 3, indole-3-lactic acid Microbiol 135:353-60, 1989). In addition, a lactate oxidase can be used to produce indole-3-pyruvate. Conversion such as the one from Pseudomonas sp. (Gu et al. J. Mol. between lactic acid and pyruvate is a reversible reaction, as Catalysis B: Enzymatic: 18:299-305, 2002), can be utilized is conversion between indole-3-pyruvate and indole-3-lac for oxidation of indole-3-lactic to indole-3-pyruvate. tate. The oxidation of indole-lactate was typically followed due to the high amount of background at 340 nm from Example 3 indole-3-pyruvate. 0166 Conversion of L-tryptophan to Indole-3-pyruvate 0162 The standard assay mixture contained 100 mM Utilizing L-Amino Acid Oxidase potassium phosphate, pH 8.0, 0.3 mM NAD", 7 units of 0.167 This example describes methods used to convert lactate dehydrogenase (LDH) (Sigma-L2395, St. Louis, tryptophan to indole-3-pyruvate via an oxidase (EC 1.4.3.2), Mo.), and 2 mM Substrate in 0.1 mL. The assay was as an alternative to using a tryptophan aminotransferase as performed in duplicate in a UV-transparent microtiter plate, described in Example 1. L-amino acid oxidase was purified using a Molecular Devices SpectraMax Plus platereader. from CrOtalus duriSSuS (Sigma, St. Louis, Mo., catalog Polypeptide and buffer were mixed and pipetted into wells number A-2805). The accession numbers of L-amino acid containing the indole-3-lactic acid and NAD" and the absor oxidases for molecular cloning include: CAD21325.1, bance at 340 nm of each well was read at intervals of 9 AAL14831, NP 490275, BAB78253, A38314, CAB71136, seconds after brief mixing. The reaction was held at 25 C. JE0266, TO8202, S48644, CAC00499, P56742, P81383, for 5 minutes. The increase in absorbance at 340 nm follows 093364, P81382, P81375, S62692, P23623, AAD45200, the production of NADH from NAD". Separate negative AAC32267, CAA88452, APO03600, and Z4.8565. controls were performed without NAD" and without Sub strate. D-LDH from LeuconoStoc mesenteroides (Sigma 0168 Reactions were performed in microcentrifuge tubes catalog number L2395) appeared to exhibit more activity in a total volume of 1 mL, incubated for 10 minutes while with the indole-derivative Substrates than did L-LDH from shaking at 37 C. The reaction mix contained 5 mM L-tryp Bacillus Stearothermophilus (Sigma catalog number tophan, 100 mM sodium phosphate buffer pH 6.6, 0.5 mM Sodium arsenate, 0.5 mM EDTA, 25 mMSodium tetraborate, L5275). 0.016 mg catalase (83 U, Sigma C-3515), 0.008 mg FAD 0163 Similar methods were utilized with D-lactic acid (Sigma), and 0.005-0.125 Units of L-amino acid oxidase. and NAD+ or NADH and pyruvate, the natural Substrates of Negative controls contained all components except tryp D-LDH polypeptides. The V for the reduction of pyru tophan, and blanks contained all components except the vate was 100-1000 fold higher than the V for the oxida oxidase. Catalase was used to remove the hydrogen peroxide tion of lactate. The V for the oxidation reaction of formed during the oxidative deamination. The Sodium tet indole-3-lactic with D-LDH was approximately one-fifth of raborate and arsenate were used to Stabilize the enol-borate that with lactic acid. The presence of indole-3-pyruvate was form of indole-3-pyruvate, which shows a maximum absor also measured by following the change in absorbance at 327 bance at 327 nm. Indole-3-pyruvate Standards were prepared (the enol-borate derivative) using 50 mM sodium borate at concentrations of 0.1-1 mM in the reaction mix. buffer containing 0.5 mM EDTA and 0.5 mM sodium 0169. The purchased L-amino acid oxidase had a specific arsenate. Small, but repeatable, absorbance changes were activity of 540 ug indole-3-pyruvate formed per minute per observed, as compared to the negative controls for both L mg protein. This is the same order of magnitude as the and D-LDH polypeptides. Specific activity of tryptophan aminotransferase enzymes. 0164. Additionally, broad specificity lactate dehydroge nases (enzymes with activity associated with EC 1.1.1.27, Example 4 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 0170 Converting Indole-3-pyruvate to 2-hydroxy 2-(in of broad Specificity dehydrogenases include E. coli, Neis dol-3-ylmethyl)-4-ketoglutaric Acid with an Aldolase Seria gonorrhoeae, and Lactobacillus plantarum. 0171 This example describes methods that can be used to 0.165 Alternatively, indole-3-pyruvate can be produced convert indole-3-pyruvate to MP using an aldolase (lyase) by contacting indole-3-lactate with cellular extracts from (FIG. 2). Aldol condensations are reactions that form car CloStridium Sporogenes which contain an indolelactate bon-carbon bonds between the B-carbon of an aldehyde or dehydrogenase (EC 1.1.1.110); or Trypanosoma Cruzi epi ketone and the carbonyl carbon of another aldehyde or maStigOtes cellular extracts which contain p-hydroxypheny ketone. A carbanion is formed on the carbon adjacent to the lactate dehydrogenase (EC 1.1.1.222) known to have activ carbonyl group of one Substrate, and Serves as a nucleophile ity on indole-3-pyruvate, or Pseudomonas acidovOranS or E. attacking the carbonyl carbon of the Second Substrate (the coli cellular extracts, which contain an imidazol-5-yl lactate electrophilic carbon). Most commonly, the electrophilic dehydrogenase (EC 1.1.1.111); or Coleus blumei, which substrate is an aldehyde, so most aldolases fall into the EC contains a hydroxyphenylpyruvate reductase (EC 4.1.2.- category. Quite often, the nucleophilic Substrate is 1.1.1.237); or Candida maltosa which contains a D-aromatic pyruvate. It is leSS common for aldolases to catalyze the lactate dehydrogenase (EC 1.1.1.222). References describ condensation between two keto-acids or two aldehydes. ing such activities include, Nowicki et al. (FEMS Microbiol 0172 However, aldolases that catalyze the condensation Lett 71:119-24, 1992), Jean and DeMoss (Canadian J. of two carboxylic acids have been identified. For example, US 2005/0112260 A1 May 26, 2005

EP 1045-029 describes the production of L-4-hydroxy-2- (T) at positions 59 and 87, (R) at 119, aspartate (D) ketoglutaric acid from glyoxylic acid and pyruvate using a at 120, and (H) at 31 and 71, (based on the Pseudomonas culture (EC 4.1.3.16). In addition, 4-hydroxy numbering system of C. testosteroni) will have similar 4-methyl-2-oxoglutarate aldolase (4-hydroxy-4-methyl-2- activity. oxoglutarate pyruvate lyase, EC 4.1.3.17) can catalyze the condensation of two keto acids. Therefore, Similar aldolase 0181 Activity Results. With KHG Gene Products polypeptides were used to catalyze the condensation of 0182 Both the B. Subtilis and E. coli khg gene constructs indole-3-pyruvate with pyruvate. had high levels of expression of protein when induced with 0173 Cloning IPTG, while the S. meliloti khg had a lower level of expression. The recombinant proteins were highly Soluble, 0.174 4-Hydroxy-4-methyl-2-oxoglutarate pyruvate as judged by SDS-PAGE analysis of total proteins and lyases (ProA aldolase, EC 4.1.3.17) and 4-hydroxy-2-oxo cellular extracts. The B. Subtilis and E. coli khg gene glutarate glyoxylate-lyase (KHG aldolase, EC 4.1.3.16) products were purified to >95% purity; the yield of the S. catalyze reactions very Similar to the aldolase reaction of meliloti gene product was not as high after affinity purifi FIG. 2. Primers were designed with compatible overhangs cation using a His-Bind cartridge. for the pET30 Xa/LIC vector (Novagen, Madison, Wis.). 0183 There is no evidence that magnesium and phos 0175 Activity Results. With proA Gene Products phate are required for activity for this enzyme. However, the 0176 Both the C. testosteroni proA and S. meliloti literature reports performing the assays in Sodium phosphate SMcO0502 gene constructs had high levels of expression buffer, and the enzyme reportedly is bifunctional and has when induced with IPTG. The recombinant proteins were activity on phosphorylated Substrates Such as 2-keto-3- highly soluble, as determined by SDS-PAGE analysis of deoxy-6-phosphogluconate (KDPG). The enzymatic assays total protein and cellular extract Samples. The C. testosterOni were performed as described above, and in Some instances gene product was purified to >95% purity. Because the yield the phosphate was omitted. The results indicate that the of the S. meliloti gene product was very low after affinity recombinant KHG aldolases produced MP, but were not as purification using a His-Bind cartridge, cellular extract was active as the ProA aldolases. In Some cases the level of MP produced by KHG was almost identical to the amount used for the enzymatic assayS. produced by magnesium and phosphate alone. Phosphate 0177 Both recombinant aldolases catalyzed the forma did not appear to increase the KHG activities. The Bacillus tion of MP from indole-3-pyruvate and pyruvate. The pres enzyme had the highest activity, approximately 20-25% ence of both divalent magnesium and potassium phosphate higher activity than the magnesium and phosphate alone, as were required for enzymatic activity. No product was appar determined by SRM (see Example 10). The Sinorhizobium ent when indole-3-pyruvate, pyruvate, or potassium phos enzyme had the least amount of activity, which can be phate was absent. A Small amount of the product was also associated with folding and solubility problems noted in the formed in the absence of enzyme (typically one order of expression. All three enzymes have the glutamate magnitude less than when enzyme was present). (position 43 in B. Subtilis numbering system) as well as the 0.178 The product peak eluted from the reverse phase C lysine required for Shiff base formation with pyruvate 18 column slightly later than the indole-3-pyruvate Standard, (position 130); however, the B. Subtilis enzyme contains a the mass spectrum of this peak showed a collisionally threonine in position 47, an active site residue, rather than induced parent ion (IM+H+) of 292.1, the parent ion arginine. The B. Subtilis KHG is smaller and appears to be expected for the product MP. The major daughter fragments in a cluster distinct from the S. meliloti and E. coli enzymes, present in the mass spectrum included those with m/z=158 with other enzymes having the active site threonine. The (1H-indole-3-carbaldehyde carbonium ion), 168 (3-buta-1, differences in the active site may be the reason for the 3-dienyl-1H-indole carbonium ion), 274 (292-H2O), 256 increased activity of the B. Subtilis enzyme. (292-2HO), 238 (292-3HO), 228 (292-CH403), and 204 0.184 Improvement of Aldolase Activity (loss of pyruvate). The product also exhibited a UV spec trum characteristic of other indole-containing compounds 0185. Catalytic antibodies can be as efficient as natural Such as tryptophan, with the was of 279-280 and a Small aldolases, accept a broad range of Substrates, and can be shoulder at approximately 290 nm. used to catalyze the reaction shown in FIG. 2. 0179 The amount of MP produced by the C. testosteroni 0186 Aldolases can also be improved by directed evo aldolase increased with an increase in reaction temperature lution, for example as previously described for a KDPG from room temperature to 37 C., amount of substrate, and aldolase (highly homologous to KHG described above) amount of magnesium. The Synthetic activity of the enzyme evolved by DNA shuffling and error-prone PCR to remove decreased with increasing pH, the maximum product the requirement for phosphate and to invert the enantiose observed was at pH 7. Based on tryptophan standards, the lectivity. The KDPG aldolase polypeptides are useful in amount of MP produced under a Standard assay using 20 lig biochemical reactions since they are highly Specific for the of purified protein was approximately 10-40 ug per one mL donor substrate (herein, pyruvate), but are relatively flexible reaction. with respect to the acceptor Substrate (i.e. indole-3-pyruvate) (Koeller & Wong, Nature 409:232-9, 2001). KHG aldolase 0180. Due to the high degree of homology of the S. has activity for condensation of pyruvate with a number of meliloti and C. testosterOni ProA aldolase coding Sequences carboxylic acids. Mammalian versions of the KHG aldolase with the other genes described above, it is expected that all are thought to have broader Specificity than bacterial Ver of the recombinant gene products can catalyze this reaction. Sions, including higher activity on 4-hydroxy 4-methyl Moreover, it is expected that aldolases that have threonine 2-oxoglutarate and acceptance of both Stereoisomers of US 2005/0112260 A1 May 26, 2005

4-hydroxy-2-ketoglutarate. Bacterial Sources appear to have 0191 Protecting groups that can be used for protecting a 10-fold preference for the R isomer. There are nearly 100 the indole nitrogen include, but are not limited to: t-buty KHG homologs available in genomic databases, and activity loxycarbonyl (Boc), and benzyloxycarbonyl (Cbz). Block has been demonstrated in Pseudomonas, ParacOccus, Provi ing groups for carboxylic acids include, but are not limited dencia, Sinorhizobium, Morganella, E. coli, and mammalian to, alkyl esters (for example, methyl, ethyl, benzyl esters). tissues. These enzymes can be used as a starting point for When Such protecting groups are used, it is not possible to tailoring the enantiospecificity that is desired for monatin control the stereochemistry of the product that is formed. production. However, if R2 and/or R3 are chiral protecting groups (FIG. 0187 Aldolases that utilize pyruvate and another sub 4), Such as (S)-2-butanol, menthol, or a chiral amine, this can Strate that is either a keto acid and/or has a bulky hydro favor the formation of one MP enantiomer over the other. phobic group like indole can be “evolved” to tailor the polypeptide's Specificity, Speed, and Selectivity. In addition Example 6 to KHG and ProA aldolases demonstrated herein, examples 0.192 Conversion of Tryptophan or Indole-3-Pyruvate to of these enzymes include, but are not limited to: KDPG Monatin aldolase and related polypeptides (KDPH); transcarboxy benzalpyruvate hydratase-aldolase from Nocardioides st; 0193 An in vitro process utilizing two enzymes, an 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (2-car aminotransferase and an aldolase, produced monatin from boxybenzalpyruvate aldolase) which condenses pyruvate tryptophan and pyruvate. In the first Step alpha-ketoglutarate and 2-carboxybenzaldehyde (an aromatic ring-containing was the acceptor of the amino group from tryptophan in a Substrate); trans-O-hydroxybenzylidenepyruvate hydratase transamination reaction generating indole-3-pyruvate and aldolase from Pseudomonas putida and Sphingomonas aro glutamate. An aldolase catalyzed the Second reaction in maticivorans, which also utilizes pyruvate and an aromatic which pyruvate was reacted with indole-3-pyruvate, in the containing aldehyde as Substrates, 3-hydroxyaspartate presence of Mg" and phosphate, generating the alpha-keto aldolase (erythro-3-hydroxy-L-aspartate glyoxylate lyase), derivative of monatin (MP), 2-hydroxy-2-(indol-3-ylm which uses 2-oxoacids as the Substrates and is thought to be ethyl)-4-ketoglutaric acid. Transfer of the amino group from in the organism MicrococcuS denitrificans; benzoin aldolase the glutamate formed in the first reaction produced the (benzaldehyde lyase), which utilizes Substrates containing desired product, monatin. Purification and characterization benzyl groups, dihydroneopterin aldolase, L-threo-3-phe of the product established that the isomer formed was nylserine benzaldehyde-lyase (phenylserine aldolase) which S.S.-monatin. Alternative Substrates, enzymes, and condi condenses with benzaldehyde, 4-hydroxy-2-oxoval tions are described as well as improvements that were made erate aldolase; 1,2-dihydroxybenzylpyruvate aldolase, and to this process. 2-hydroxybenzalpyruvate aldolase. 0194 Enzymes 0188 A polypeptide having the desired activity can be 0.195 The aldolase, 4-hydroxy-4-methyl-2-oxoglutarate Selected by Screening clones of interest using the following pyruvate lyase (ProA aldolase, proA gene) (EC 4.1.3.17) methods. Tryptophan auxotrophs are transformed with vec from Comamonas teStoSteroni was cloned, expressed and tors carrying the clones of interest on an expression cassette purified as described in Example 4. The 4-hydroxy-2-oxo and are grown on a medium containing Small amounts of glutarate glyoxylate lyases (KHG aldolases) (EC 4.1.3.16) monatin or MP. Since aminotransferases and aldolase reac from B. Subtilis, E. coli, and S. meliloti were cloned, tions are reversible, the cells are able to produce tryptophan expressed and purified as described in Example 4. from a racemic mixture of monatin. Similarly, organisms (both recombinant and wildtype) can be screened by ability 0196. The aminotransferases used in conjunction with the to utilize MP or monatin as a carbon and energy Source. One aldolases to produce monatin were L-aspartate aminotrans Source of target aldolases is expression libraries of various ferase encoded by the E. coli aspC gene, the tyrosine Pseudomonas and rhizobacterial Strains. Pseudomonads aminotransferase encoded by the E. coli tyrB gene, the S. have many unusual catabolic pathways for degradation of meliloti Tata enzyme, the broad Substrate aminotransferase aromatic molecules and they also contain many aldolases, encoded by the L. major bsat gene, or the glutamic-Oxalo whereas the rhizobacteria contain aldolases, are known to acetic transaminase from pig heart (Type Ia). The cloning, grow in the plant rhizosphere, and have many of the genes expression and purification of the non-mammalian proteins described for construction of a biosynthetic pathway for are described in Example 1. Glutamic-Oxaloacetic transami monatin. nase from pig heart (type Ia) was obtained from Sigma (if G7005). Example 5 0.197 Method Using ProA Aldolase and L-Aspartate 0189 Chemical Synthesis of the Monatin Precursor Aminotransferase 0190. Example 4 described a method of using an aldolase 0198 The reaction mixture contained 50 mM ammonium to convert indole-3-pyruvate to MP. This example describes acetate, pH 8.0, 4 mM MgCl, 3 mM potassium phosphate, an alternative method of chemically synthesizing MP MP 0.05 mM pyridoxal phosphate, 100 mM ammonium pyru can be formed using a typical aldol-type condensation (FIG. vate, 50 mM tryptophan, 10 mM alpha-ketoglutarate, 160 4). Briefly, a typical aldol-type reaction involves the gen mg of recombinant C. testosteroniProA aldolase (unpurified eration of a carbanion of the pyruvate ester using a Strong cell extract, ~30% aldolase), 233 mg of recombinant E. coli base, Such as LDA (lithium diisopropylamide), lithium hex L-aspartate aminotransferase (unpurified cell extract, ~40% amethyldisilaZane or butyl lithium. The carbanion that is aminotransferase) in one liter. All components except the generated reacts with the indole-pyruvate to form the enzymes were mixed together and incubated at 30° C. until coupled product. the tryptophan dissolved. The enzymes were then added and US 2005/0112260 A1 May 26, 2005 the reaction solution was incubated at 30° C. with gentle were carried out using a Cary 100 Bio UV/visible spectro shaking (100 rpm) for 3.5 hours. At 0.5 and 1 hour after the photometer. The purified product, dissolved in water, addition of the enzymes aliquots of solid tryptophan (50 showed an absorption maximum of 280 nm with a shoulder mmoles each) were added to the reaction. All of the added at 288 nm, characteristics typical of indole containing com tryptophan did not dissolve, but the concentration was pounds. maintained at 50 mM or higher. After 3.5 hours, the solid tryptophan was filtered off. Analysis of the reaction mixture 0205 LC/MS Analysis. Analyses of mixtures for monatin by LC/MS using a defined amount of tryptophan as a derived from the in vitro biochemical reactions were carried Standard showed that the concentration of tryptophan in the out as described in Example 10. A typical LC/MS analysis Solution was 60.5 mM and the concentration of monatin was of monatin in an in vitro enzymatic Synthetic mixture is 5.81 mM (1.05 g). illustrated in FIG. 5. The lower panel of FIG. 5 illustrates 0199 The following methods were used to purify the a Selected ion chromatogram for the protonated molecular final product. Ninety percent of the clear Solution was ion of monatin at m/z=293. This identification of monatin in applied to a column of BioRad AG50W-X8 resin (225 mL.; the mixture was corroborated by the mass Spectrum illus binding capacity of 1.7 meg/mL). The column was washed trated in FIG. 6. Analysis of the purified product by LC/MS with water, collecting 300 mL fractions, until the absorbance showed a Single peak with a molecular ion of 293 and at 280 nm was <5% of the first flow through fraction. The absorbance at 280 nm. The mass Spectrum was identical to column was then eluted with 1 Mammonium acetate, pH that shown in FIG. 6. 8.4, collecting 4300-mL fractions. All 4 fractions contained monatin and were evaporated to 105 mL using a roto 0206 MS/MS Analysis. LC/MS/MS daughter ion experi evaporator with a tepid water bath. A precipitate formed as ments, as described in Example 10, were also performed on the volume reduced and was filtered off over the course of monatin. A daughter ion maSS spectrum of monatin is the evaporation process. illustrated in FIG. 7. Tentative structural assignments of all 0200 Analysis of the column fractions by LC/MS fragment ions labeled in FIG. 7 were made. These include showed that 99% of the tryptophan and monatin bound to the fragment ions of m/z=275 (293-HO), 257 (293-(2xH2O)), column. The precipitate that formed during the evaporation 230 (275-COOH), 212 (257-COOH), 168 (3-buta-1,3-di process contained >97% tryptophan and <2% of monatin. enyl-1H-indole carbonium ion), 158 (1H-indole-3-carbalde The ratio of tryptophan to product in the Supernatant was hyde carbonium ion), 144 (3-ethyl-1H-indole carbonium approximately 2:1. The Supernatant (7 ml) was applied to a ion), 130 (3-methylene-1H-indole carbonium ion), and 118 100 mL Fast Flow DEAE Sepharose (Amersham Bio (indole carbonium ion). Many of these are the same as those Sciences) column previously converted to the acetate form obtained for MP (Example 4), as expected if derived from by washing with 0.5 L 1 M NaOH, 0.2 L water, 1.0 L of 1.0 the indole portion of the molecule. Some are 1 mass unit Mammonium acetate, pH 8.4, and 0.5 L water. The Super higher than those seen for MP, due to the presence of an natant was loaded at <2 mL/min and the column was washed amino group instead of a ketone. with water at 3-4 mL/min until the absorbance at 280 nm. 0207. Accurate Mass Measurement of Monatin. FIG. 8 was -0. Monatin was eluted with 100 mM ammonium illustrates the mass spectrum obtained for purified monatin acetate, pH 8.4, collecting 4 100-mL fractions. employing an Applied Biosystems-Perkin Elmer Q-Star 0201 Analysis of the fractions showed that the ratio of hybrid quadrupole/time-of-flight mass spectrometer. The tryptophan to monatin in the flow through fractions was measured mass for protonated monatin using tryptophan as 85:15 and the ratio in the eluent fractions was 7:93. ASSum an internal mass calibration standard was 293.1144. The ing the extinction coefficient at 280 nm of monatin is the calculated mass of protonated monatin, based on the Same as tryptophan, the eluent fractions contained 0.146 elemental composition CHNOs is 293.1137. This is a mmole of product. Extrapolation to the total 1 L reaction mass measurement error of less than 2 parts per million would produce ~2.4 mmoles (~710 mg) of monatin, for a (ppm), providing conclusive evidence of the elemental com recovery of 68%. position of monatin produced enzymatically. 0202) The eluent fractions from the DEAE Sepharose 0208 NMR Spectroscopy. The NMR experiments were column were evaporated to <20 mL. An aliquot of the performed on a Varian Inova 500 MHz instrument. The product was further purified by application to a Cs prepara sample of monatin (-3 mg) was dissolved in 0.5 ml of D.O. tive reversed-phase column using the Same chromatographic Initially, the solvent (DO) was used as the internal reference conditions as those described in Example 10 for the ana at 4.78 ppm. Since the peak for water was large, the lytical-Scale monatin characterization. Waters Fractionl "H-NMR was run with suppression of the peak for water. ynx"M Software was employed to trigger automated fraction Subsequently, due to the broadness of the water peak, the collection of monatin based on detection of the m/z=293 ion. C-2 proton of monatin was used as the reference peak, and The fraction from the Cs column with the corresponding set at the published value of 7.192 ppm. protonated molecular ion for monatin was collected, evapo rated to dryness, and then dissolved in a Small Volume of 0209 For C-NMR, an initial run of several hundred water. This fraction was used for characterization of the Scans indicated that the Sample was too dilute to obtain an product. adequate C spectrum in the allotted time. Therefore, a heteronuclear multiple quantum coherence (HMOC) experi 0203 The Resulting Product was Characterized Using ment was performed, which enabled the correlation of the the Following Methods. hydrogens and the carbons to which they were attached, and 0204 UV/Visible Spectroscopy. UV/visible spectro also providing information on the chemical shifts of the Scopic measurements of monatin produced enzymatically carbons. US 2005/0112260 A1 May 26, 2005 18

0210. A summary of the H and HMOC data is shown in 0214) Tables 2 and 3. By comparison to published values, the NMR data indicated that the enzymatically produced mona TABLE 3 tin was either (S,S), (R,R), or a mixture of both. 'C NMR data (from HMQC spectrum 0211 Chiral LC/MS Analysis. To establish that the mona tin produced in vitro was one isomer, and not a mixture of Cargill Vleggaar et al.' the (R,R) and (S,S) enantiomers, chiral LC/MS analyses Atom Öc Öc were carried out using the instrumentation described in 2 126.1 126.03 Example 10. 3 : 110.31 4 120.4 120.46 0212 Chiral LC separations were made using an Chiro 5 12O2 120.25 6 122.8 122.74 biotic T (Advanced Separations Technology) chiral chroma 7 112.8 112.79 tography column at room temperature. Separation and detec 8 : 137.06 tion, based on published protocols from the Vendor, were 9 : 129.23 optimized for the R-(D) and S- (L) isomers of tryptophan. 10a 36.4 36.53 12 39.5 39.31 The LC mobile phase consisted of A) water containing 13 54.9 54.89 0.05% (v/v) trifluoroacetic acid; B) Methanol containing 14 : 175.30 0.05% (v/v) trifluoroacetic acid. The elution was isocratic at 15 : 18118 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 "Vleggaar et al. (J.C.S. Perkin Trans. 1: 3095-8, 1992). instrumental parameters used for chiral LC/MS analysis of tryptophan and monatin are identical to those described in 0215 Polarimetry. The optical rotation was measured on Example 10 for LC/MS analysis. Collection of mass spectra a Rudolph Autopol III polarimeter. The monatin was pre for the region m/z 150-400 was utilized. Selected ion pared as a 14.6 mg/mL Solution in water. The expected chromatograms for protonated molecular ions (IM+H"=205 specific rotation (Ia)”) for S.S monatin (salt form) is -49.6 for both R- and S-tryptophan and M+H=293 for monatin) for a 1 g/mL Solution in water (Vleggaar et al). The observed allowed direct identification of these analytes in the mix al" was -28.1 for the purified, enzymatically produced tureS. monatin indicating that it was the S, S isomer. 0213 The chromatograms of R- and S-tryptophan and 0216) Improvements monatin, Separated by chiral chromatography and monitored by MS, are shown in FIG. 9. The single peak in the 0217. The reaction conditions, including reagent and chromatogram of monatin indicates that the compound is enzyme concentrations, were optimized and yields of 5-10 one isomer, with a retention time almost identical to S-tryp mg/mL were produced using the following reagent mix: 50 tophan. mM ammonium acetate pH 8.3, 2 mM MgCl2, 200 mM

TABLE 2 H NMR data

HO

Cargill Vleggaar et al.' Takeshi et al.’ Atom 8H J(HH) Hz, 8, J(HH) Hz, 8, 2 7.192 (1H, s) 7.192 (s) 7.18 (s) 4 7.671 (d) 7.99 7.686 (d) 7.9 7.67 (d) 8.0 5 7.104 (dd) 7.99 7.102 (dd) 8.0, 8.0 7.11 (dd) 7.5, 7.5 6 7.178 (dd) * 7.176 (dd) 8.0, 8.0 7.17 (dd) 7.5, 7.5 7 7.439 (d) 7.99 7.439 (d) 8.1 7.43 (d) 8.0 10a 3.242 (d) 14.5 3.243 (d) 14.3 3.24 (d) 14.5 10b 3.033 (d) 14.5 3.051 (d) 14.3 3.05 (d) 14.5 12 2.626 (dd) 15.5, 1.5 2.651 (dd) 15.3, 1.7 2.62 (dd) 15.5, 1.8 2.015 (dd) 15.0, 12.0 2.006 (dd) 15.3, 11.7 2.01 (dd) 15.5, 12.0 13 3.571 (dd) 10.75*, 1.5 3.168 (dd) 11.6, 1.8 3.57 (dd) 12.0, 1.8 "Vleggaar et al. (J.C.S. Perkin Trans. 1:3095-8, 1992). Takeshi and Shusuke (JP2002060382, 2002-02-26). US 2005/0112260 A1 May 26, 2005 pyruvate (Sodium or ammonium salt), 5 mM alpha-ketoglu Examples 1 and 6, or by dehydrogenases that require a tarate (Sodium salt), 0.05 mM pyridoxal phosphate, deaer reducing cofactor such as NADH or NADPH. These reac ated water to achieve a final volume of 1 mL after the tions are reversible and can be measured in either direction. addition of the enzymes, 3 mM potassium phosphate, 50 The directionality, when using a dehydrogenase enzyme, can Aug/mL of recombinant ProA aldolase (cell extract; total be largely controlled by the concentration of ammonium protein concentration of 167 ug/mL), 1000 ug/mL of L-as Salts. partate aminotransferase encoded by the E. coli aspC gene (cell extract; total protein concentration of 2500 ug/mL), and 0224 Dehydrogenase activity. The oxidative deamina solid tryptophan to afford a concentration of >60 mM tion of monatin was monitored by following the increase in (Saturated; Some undissolved throughout the reaction). The absorbance at 340 nm as NAD(P)+ was converted to the mixture was incubated at 30° C. for 4 hours with gentle more chromophoric NAD(P)H. Monatin was enzymatically Stirring or mixing. produced and purified as described in Example 6. 0225. A typical assay mixture contained 50 mM Tris 0218. Substitutions HCl, pH 8.0 to 8.9, 0.33 mM NAD" or NADP", 2 to 22 units 0219. The concentration of alpha-ketoglutarate can be of glutamate dehydrogenase (Sigma), and 10-15 mM Sub reduced to 1 mM and supplemented with 9 mM aspartate Strate in 0.2 mL. The assay was performed in duplicate in a with an equivalent yield of monatin. Alternative amino acid UV-transparent microtiter plate, on a Molecular Devices acceptors can be utilized in the first Step, Such as Oxaloac SpectraMax Plus platereader. A mix of the enzyme, buffer, etate. and NAD(P)' were pipetted into wells containing the Sub Strate and the increase in absorbance at 340 nm was moni 0220. When recombinant L. major broad substrate ami tored at 10 second intervals after brief mixing. The reaction notransferase was used in place of the E. coli L-aspartate was incubated at 25 C. for 10 minutes. Negative controls aminotransferase, Similar yields of monatin were achieved. were carried out without the addition of Substrate, and However, a second unidentified product (3-10% of the major glutamate was utilized as a positive control. The type III product) with a molecular mass of 292 was also detected by glutamate dehydrogenase from bovine liver (Sigma if LC-MS analysis. Monatin concentrations of 0.1-0.5 mg/mL G-7882) catalyzed the conversion of the monatin to the were produced when the E. coli tyrB encoded enzyme, the monatin precursor at a rate of conversion approximately S. melilotitat A encoded enzyme or the glutamic-Oxaloacetic one-hundredth the rate of the conversion of glutamate to transaminase from pig heart (type IIa) was added as the alpha-ketoglutarate. aminotransferase. When starting the reaction from indole 3-pyruvate, a reductive amination can be done for the last 0226 Transamination activity. Monatin aminotransferase Step with glutamate dehydrogenase and NADH (as in assays were conducted with the aspartate aminotransferase Example 7). (AspC) from E. coli, the tyrosine aminotransferase (TyrB) from E. coli, the broad substrate aminotransferase (BSAT) 0221) The KHG aldolases from B. subtilis, E. coli, and S. from L. major, and the two commercially available porcine meliloti were also used with the E. coli L-aspartate ami glutamate-Oxaloacetate aminotransferases described in notransferase to produce monatin enzymatically. The fol Example 1. Both Oxaloacetate and alpha-ketoglutarate were lowing reaction conditions were used: 50 mM NH-OAc tested as the amino acceptor. The assay mixture contained pH 8.3, 2 mM MgCl2, 200 mM pyruvate, 5 mM glutamate, (in 0.5 mL) 50 mM Tris-HCl, pH 8.0, 0.05 mM PLP, 5 mM 0.05 mM pyridoxal phosphate, deaerated water to achieve a amino acceptor, 5 mM monatin, and 25 ug of aminotrans final volume of 0.5 mL after the addition of the enzymes, 3 ferase. The assays were incubated at 30° C. for 30 minutes, mM potassium phosphate, 20 tug/mL of recombinant B. and the reactions were stopped by addition of 0.5 mL Subtilis KHG aldolase (purified), ca. 400 tug/mL of E. coli isopropyl alcohol. The loSS of monatin was monitored by L-aspartate aminotransferase (AspC) unpurified from cell LC/MS (Example 10). The highest amount of activity was extract, and 12 mM indole-3-pyruvate. The reactions were noted with L. major BSAT with oxaloacetate as the amino incubated at 30° C. for 30 minutes with shaking. The amount acceptor, followed by the same enzyme with alpha-ketoglu of monatin produced using the B. Subtilis enzyme was 80 tarate as the amino acceptor. The relative activity with ng/mL, and increased with increasing amounts of aldolase. Oxaloacetate was: BSATYASpC>porcine type IIadporcine If indole-3-pyruvate and glutamate were replaced by Satu type I=TyrB. The relative activity with alpha-ketoglutarate rating amounts of tryptophan and 5 mM alpha-ketoglutarate, was: BSATsAspC>porcine type I>porcine type IIad TyrB. the production of monatin was increased to 360 ng/mL. Reactions were repeated with 30 tug/mL of each of the three EXAMPLE 8 KHG enzymes in 50 mM Tris pH 8.3, with Saturating 0227 Production of Monatin from Tryptophan and C3 amounts of tryptophan, and were allowed to proceed for an Sources Other Than Pyruvate hour in order to increase detection. The Bacillus enzyme had the highest activity as in Example 4, producing approxi 0228. As described above in Example 6, indole-3-pyru mately 4000 ng/mL monatin. The E. coli KHG produced Vate or tryptophan can be converted to monatin using 3000 ng/mL monatin, and the S. meliloti enzyme produced pyruvate as the C3 molecule. However, in Some circum 2300 ng/mL. stances, pyruvate may not be a desirable raw material. For example, pyruvate may be more expensive than other C3 EXAMPLE 7 carbon Sources, or may have adverse effects on fermenta tions if added to the medium. Alanine can be transaminated 0222 Interconversion Between MP and Monatin by many PLP-enzymes to produce pyruvate. 0223) The amination of MP to form monatin can be 0229. Tryptophanase-like enzymes perform beta-elimi catalyzed by aminotransferases Such as those identified in nation reactions at faster rates than other PLP enzymes Such US 2005/0112260 A1 May 26, 2005 2O as aminotransferases. Enzymes from this class (4.1.99.-) can amount of monatin produced was increased by addition of produce ammonia and pyruvate from amino acids Such as the tryptophanase, which is capable of transamination as a L-serine, L-cysteine, and derivatives of Serine and cysteine Secondary activity. The amount of monatin produced with with good leaving groupS. Such as O-methyl-L-serine, Serine as a carbon Source nearly doubled with the addition of O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine, the tryptophanase enzymes, even though only one-fifth of S-alkyl-L-cysteine, O-acyl-L-serine, 3-chloro-L-alanine. the amount of tryptophanase was added in comparison to the aminotransferase. AspC is capable of Some amount of beta 0230 Processes to produce monatin using EC 4.1.99.- elimination activity alone. The results with aspartate indi polypeptides can be improved by mutating the P-tyrosinase cate that the tryptophanase activity on aspartate does not (TPL) or tryptophanase according to the method of Moura increase with the Same site-directed mutations as previously tou et al. (J. Biol. Chem 274:1320-5, 1999). Mouratou et al. Suggested for B-tyrosinase. It is expected that the mutant describe the ability to covert the B-tyrosinase into a dicar B-tyrosinase will have higher activity for production of boxylic amino acid B-lyase, which has not been reported to monatin. occur in nature. The change in Specificity was accomplished by converting (V) 283 to arginine (R) and arginine EXAMPLE 9 (R) 100 to threonine (T). These amino acid changes allow for the lyase to accept a dicarboxylic amino acid for the 0234 Chemical Synthesis of Monatin hydrolytic deamination reaction (Such as aspartate). Aspar 0235. The addition of alanine to indole-3-pyruvic acid tate, therefore, can also be used as a Source of pyruvate for produces monatin, and this reaction can be performed Syn Subsequent aldol condensation reactions. thetically with a Grignard or organolithium reagent. 0231. Additionally, cells or enzymatic reactors can be 0236. For example, to 3-chloro- or 3-bromo-alanine Supplied with lactate and an enzyme that converts lactate to which has been appropriately blocked at the carboxyl and pyruvate. Examples of enzymes capable of catalyzing this amino groups, is added magnesium under anhydrous con reaction include lactate dehydrogenase and lactate oxidase. ditions. Indole-3-pyruvate (appropriately blocked) is then 0232) The reaction mixture consisted of 50 mM Tris-Cl added to form the coupled product followed by removal of pH 8.3, 2 mM MgCl, 200 mM C3 carbon source, 5 mM the protecting groups to form monatin. Protecting groups alpha-ketoglutarate, Sodium Salt, 0.05 mM pyridoxal phos that are particularly useful include THP (tetrahydropyranyl phate, deaerated water to achieve a final volume of 0.5 mL ether) which is easily attached and removed. after the addition of the enzymes, 3 mM potassium phos phate pH 7.5, 25 lug of crude recombinant C. testosteroni EXAMPLE 10 ProA aldolase as prepared as in Example 4, 500 lug of crude 0237) Detection of Tryptophan, Monatin, and MP L-aspartate aminotransferase (AspC) as prepared in Example 1, and Solid tryptophan to afford a concentration of 0238. This example describes methods used to detect the >60 mM (saturated; some undissolved throughout the reac presence of monatin, or its precursor 2-hydroxy 2-(indol-3- tion). The reaction mix was incubated at 30° C. for 30 ylmethyl)-4-ketoglutaric acid. minutes with mixing. Serine, alanine, and aspartate were 0239). LC/MS Analysis Supplied as 3-carbon Sources. ASSays were performed with and without secondary PLP enzymes (purified) capable of 0240 Analyses of mixtures for monatin, MP, and/or performing beta-elimination and beta-lyase reactions (tryp tryptophan derived from in vitro or in vivo biochemical reactions were performed using a WaterS/Micromass liquid tophanase (TNA), double mutant tryptophanase, f-tyrosi chromatography-tandem mass spectrometry (LC/MS/MS) nase (TPL)). The results are shown in Table 4: instrument including a Waters 2690 liquid chromatograph with a Waters 996 Photo-Diode Array (PDA) absorbance TABLE 4 monitor placed in Series between the chromatograph and a Production of monatin utilizing alternative C3-carbon sources Micromass Quattro Ultima triple quadrupole mass Spec trometer. LC Separations were made using a Supelco Dis Additional PLP Relative covery Cs reversed-phase chromatography column, 2.1 C3-carbon source Enzyme Activity mmx 150 mm, or an Xterra MSCs reversed-phase chroma Ole None O% tography column, 2.1 mmx250 mm, at room temperature. pyruvate None 100% serine None 3% The LC mobile phase consisted of A) water containing serine 11 tug wildtype TNA (1 U) 5.1% 0.05% (v/v) trifluoroacetic acid and B) methanol containing serine 80 ug double mutant TNA 4.6% 0.05% (v/v) trifluoroacetic acid. alanine None 32% alanine 11 tug wildtype TNA 41.7% 0241 The gradient elution was linear from 5% B to 35% alanine 80 ug mutant TNA 43.9% B, 0-9 min, linear from 35% B to 90% B, 9-16 min, isocratic aspartate 110 ug wildtype TNA (10 U) 7.7% at 90% B, 16-20 min, linear from 90% B to 5% B, 20-22 aspartate 5 U wildtype TPL (crude) 5.1% min, with a 10 min re-equilibration period between runs. The aspartate 80 ug mutant TNA 3.3% flow rate was 0.25 mL/min, and PDA absorbance was monitored from 200 nm to 400 nm. All parameters of the 0233. The monatin produced from alanine and serine as ESI-MS were optimized and selected based on generation of 3-carbon sources was verified by LC/MS/MS daughter scan protonated molecular ions (IM+H") of the analytes of analysis, and was identical to the characterized monatin interest, and production of characteristic fragment ions. produced in Example 6. Alanine was the best alternative 0242. The following instrumental parameters were used tested, and was transaminated by the AspC enzyme. The for LC/MS analysis of monatin: Capillary: 3.5 kV, Cone: 40 US 2005/0112260 A1 May 26, 2005

V; Hex 1: 20 V; Aperture: 0 V; Hex 2: 0 V; Source (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2): temperature: 100° C.; Desolvation temperature: 350° C.; 0.5; Multiplier: 650. MRM parameters: Interchannel delay: Desolvation gas: 500 L/h; Cone gas: 50 L/h; Low mass 0.03 s; Interscan delay: 0.03 s; Dwell: 0.05 s. resolution (Q1): 15.0; High mass resolution (Q1): 15.0; Ion energy: 0.2; Entrance: 50V; Collision Energy: 2; Exit: 50V; 0248. Accurate Mass Measurement of Monatin. Low mass resolution (Q2): 15; High mass resolution (Q2): 0249 High resolution MS analysis was carried out using 15; Ion energy (Q2): 3.5; Multiplier: 650. Uncertainties for an Applied Biosystems-Perkin Elmer Q-Star hybrid quadru reported mass/charge ratios (m/z) and molecular masses are pole/time-of-flight mass spectrometer. The measured mass +0.01%. Initial detection of the alpha-keto acid form of for protonated monatin used tryptophan as an internal mass monatin (MP) and monatin in the mixtures was accom calibration Standard. The calculated mass of protonated plished by LC/MS monitoring with collection of mass monatin, based on the elemental composition CH7NO. spectra for the region m/z 150-400. Selected ion chromato is 293.1137. Monatin produced using the biocatalytic pro grams for protonated molecular ions (IM+H"=292 for MP, ceSS described in Example A showed a measured mass of IM+H=293 for monatin) allowed direct identification of 293.1144. This is a mass measurement error of less than 2 these analytes in the mixtures. parts per million (ppm), providing conclusive evidence of the elemental composition of monatin produced enzymati 0243 MS/MS Analysis cally. 0244 LC/MS/MS daughter ion experiments were per formed on monatin as follows. A daughter ion analysis EXAMPLE 11 involves transmission of the parent ion (e.g., m/z=293 for monatin) of interest from the first mass analyzer (Q1) into 0250) Production of Monatin in Bacteria the collision cell of the mass spectrometer, where argon is 0251 This example describes methods used to produce introduced and chemically dissociates the parent into frag monatin in E. coli cells. One skilled in the art will under ment (daughter) ions. These fragment ions are then detected Stand that Similar methods can be used to produce monatin with the Second mass analyzer (Q2), and can be used to in other bacterial cells. In addition, vectors containing other corroborate the Structural assignment of the parent. Tryp genes in the monatin Synthesis pathway (FIG. 2) can be tophan was characterized and quantified in the Same way via used. transfmission and fragmentation of m/z=205. 0252) Trp-1+glucose medium, a minimal medium that 0245 The following instrumental parameters were used has been used for increased production of tryptophan in E. for LC/MS/MS analysis of monatin: Capillary: 3.5 kV; coli cells (Zeman et al. Folia Microbiol. 35:200-4, 1990), Cone: 40 V; Hex 1: 20 V; Aperture: 0 V; Hex 2: 0 V; Source was prepared as follows. To 700 mL nanopure water the temperature: 100° C.; Desolvation temperature: 350° C.; following reagents were added: 2 g (NH4)2SO4, 13.6 g. Desolvation gas: 500 L/h; Cone gas: 50 L/h; Low mass KHPO, 0.2 g MgSO.7HO, 0.01 g CaCl2.H2O, and 0.5 resolution (Q1): 13.0; High mass resolution (Q1): 13.0; Ion mg FeSO.7H2O. The pH was adjusted to 7.0, the volume energy: 0.2; Entrance: -5 V, Collision Energy: 14; Exit: 1V; was increased to 850 mL, and the medium was autoclaved. Low mass resolution (Q2): 15; High mass resolution (Q2): A 50% glucose Solution was prepared Separately, and Sterile 15; Ion energy (Q2): 3.5; Multiplier: 650. filtered. Forty mL was added to the base medium (850 mL) 0246 High-Throughput Determination of Monatin and for a 1 L final volume. Tryptophan 0253 A10 g/L L-tryptophan solution was prepared in 0.1 0247 High-throughput analyses (<5 min/sample) of mix M sodium phosphate pH 7, and sterile-filtered. One-tenth tures for monatin and tryptophan derived from in vitro or in Volume was typically added to cultures as Specified below. Vivo reactions were carried out using instrumentation A 10%. Sodium pyruvate Solution was also prepared and described above, and the same parameters as described for Sterile-filtered. A 10 mL aliquot was typically used per liter LC/MS/MS. LC separations were made using a 4.6 mmx50 of culture. Stocks of amplicillin (100 mg/mL), kanamycin mm Advanced Separation Technologies Chirobiotic T col (25 mg/mL) and IPTG (840 mM) were prepared, sterile umn at room temperature. The LC mobile phase consisted of filtered, and stored at -20° C. before use. Tween 20 (poly A) water containing 0.25% acetic acid; B) Methanol con oxyethylene 20-Sorbitan monolaurate) was utilized at a taining 0.25% acetic acid. The isocratic elution was at 50% 0.2% (vol/vol) final concentration. Ampicillin was used at B, 0-5 min. The flow rate was 0.6 mL/min. All parameters non-lethal concentrations, typically 1-10 ug/mL final con of the ESI-MS/MS system were optimized and selected centration. based on optimal in-Source generation of the protonated molecular ion of tryptophan and the internal standard 2H 0254 Fresh plates of E. coli BL21 (DE3):C. testosteroni tryptophan, as well as collision-induced production of amino proA?pET 30 Xa/LIC (described in Example 4) were pre acid-specific fragment ions for multiple reaction monitoring pared on LB medium containing 50 lug/mL kanamycin. (MRM) experiments (Table 1). The following instrumental Overnight cultures (5 mL) were inoculated from a single parameters were used for LC/MS/MS analysis of monatin colony and grown at 30° C. in LB medium with kanamycin. and tryptophan in the positive ion multiple reaction moni Typically a 1 to 50 inoculum was used for induction in toring (mrm) mode: Capillary: 3.5 kV, Cone: 20 V, Hex 1: trp-1+glucose medium. Fresh antibiotic was added to a final 15 V, Aperture: 1 V; Hex 2: 0 V; Source temperature: 100° concentration of 50 mg/L. Shake flasks were grown at 37 C.; Desolvation temperature: 350° C.; Desolvation gas: 500 C. prior to induction. L/h; Cone gas: 40 L/h; Low mass resolution (Q1): 12.0; 0255 Cells were sampled every hour until an ODoo of High mass resolution (Q1): 12.0, Ion energy: 0.2; Entrance: 0.35–0.8 was obtained. Cells were then induced with 0.1 mM -5 V, Collision Energy: 14; Exit: 1 V; Low mass resolution IPTG, and the temperature reduced to 34° C. Samples (1 ml) US 2005/0112260 A1 May 26, 2005 22 were collected prior to induction (Zero time point) and parative reverse-phase liquid chromatography. This Sample centrifuged at 5000xg. The Supernatant was frozen at -20° was evaporated, and Submitted for high resolution mass C. for LC/MS analysis. Four hours post-induction, another analysis (described in Example 6). The high resolution MS 1 mL Sample was collected, and centrifuged to Separate the indicated that the metabolite being produced is monatin. broth from the cell pellet. Tryptophan, Sodium pyruvate, 0261. In vitro assays indicate that aminotransferase needs ampicillin, and Tween were added as described above. to be present at higher levels than aldolase (see Example 6), 0256 The cells were grown for 48 hours post-induction, therefore the aspartate aminotransferase from E. coli was and another 1 mL Sample was taken and prepared as above. overexpressed in combination with the aldolase gene to At 48 hours, another aliquot of tryptophan and pyruvate increase the amount of monatin produced. Primers were were added. The entire culture Volume was centrifuged after designed to introduce C. teStoSterOni proA into an operon approximately 70 hours of growth (post-induction), for 20 with aspC/pET30 Xa/LIC, as follows: 5' primer: ACTCG minutes at 4 C. and 3500 rpm. The Supernatant was GATCCGAAGGAGATATACATATGTAC decanted and both the broth and the cells were frozen at -80 GAACTGGGACT (SEQ ID NO: 67) and 3' primer: C. The broth fractions were filtered and analyzed by LC/MS. CGGCTGTCGACCGTTAGTCAATATATTTCAGGC The heights and areas of the M+H=293 peaks were (SEQ ID NO: 68). The 5' primer contains a BamHI site, the monitored as described in Example 10. The background 3' primer contains a SalI site for cloning. PCR was per level of the medium was Subtracted. The data was also formed as described in Example 4, and gel purified. The normalized for cell growth by plotting the height of the aspC/pET30 Xa/LIC construct was digested with BamHI M+H=293 peak divided by the optical density of the and SalI, as was the PCR product. The digests were purified culture at 600 nm. using a Qiagen Spin column. The proA PCR product was 0257 Higher levels of monatin were produced when ligated to the vector using the Roche Rapid DNA Ligation pyruvate, amplicillin, and Tween were added 4 hours post kit (Indianapolis, Ind.) according to manufacturer's instruc induction rather than at induction. Other additives Such as tions. Chemical transformations were done using Novablues PLP, additional phosphate, or additional MgCl, did not Singles (Novagen) as described in Example 1. Colonies increase the production of monatin. Higher titers of monatin were grown up in LB medium containing 50 mg/L kana were obtained when tryptophan was utilized instead of mycin and plasmid DNA was purified using the Qiagen Spin indole-3-pyruvate, and when the tryptophan was added miniprep kit. Clones were Screened by restriction digest post-induction rather than at inoculation, or at induction. analysis and Sequence was confirmed by Seqwright (Hous Prior to induction, and 4 hours post-induction (at time of ton, Tex.). Constructs were subcloned into BLR(DE3), Substrate addition), there was typically no detectable level of BLR(DE3)pLysS, BL21 (DE3) and BL21 (DE3)pLysS monatin in the fermentation broth or cellular extracts. Nega (Novagen). TheproA?pET30 Xa/LIC construct was also tive controls were done utilizing cells with peT30a vector transformed into BL21 (DE3)pLysS. only, as well as cultures where tryptophan and pyruvate were 0262 Initial comparisons of BLR(DE3) shake flask not added. A parent MS Scan demonstrated that the com Samples under the Standard conditions described above pound with (m+1)/Z=293 was not derived from larger mol demonstrated that the addition of the Second gene (aspC) ecules, and daughter Scans (performed as in Example 10) improved the amount of monatin produced by seven-fold. To were Similar to monatin made in vitro. hasten growth, BL21 (DE3)-derived host strains were used. 0258. The effect of Tween was studied by utilizing 0, The proA clones and the two gene operon clones were 0.2% (vol/vol), and 0.6% final concentrations of Tween-20. induced in Trp-1 medium as above, the pySS hosts had The highest amount of monatin produced by Shake flaskS chloramphenicol (34 mg/L) added to the medium as well. was at 0.2% Tween. The amplicillin concentration was varied Shake flask experiments were performed with and without between 0 and 10 ug/mL. The amount of monatin in the the addition of 0.2% Tween-20 and 1 mg/L amplicillin. The cellular broth increased rapidly (2.5x) between 0 and 1 amount of monatin in the broth was calculated using in Vitro tug/mL, and increased 1.3xwhen the amplicillin concentra produced purified monatin as a Standard. SRM analyses tion was increased from 1 to 10 ug/mL. were performed as described in Example 10. Cells were Sampled at Zero, 4 hours, 24 hours, 48 hours, 72 hours, and 0259 A time course experiment showing typical results is 96 hours of growth. shown in FIG. 10. The amount of monatin Secreted into the cell broth increased, even when the values are normalized 0263. The results are shown in Table 5 for the maximum for cell growth. By using the molar extinction coefficient of amounts produced in the culture broths. In most instances, tryptophan, the amount of monatin in the broth was esti the two gene construct gave higher values than the proA mated to be less than 10 ug/mL. The same experiment was construct alone. The pySS Strains, which should have repeated with the cells containing vector without proA leakier cell envelopes, had higher levels of monatin Secreted, insert. Many of the numbers were negative, indicating the even though these Strains typically grow at a slower rate. The peak height at m/Z=293 was less in these cultures than in the additions of Tween and amplicillin were beneficial. medium alone (FIG. 10). The numbers were consistently lower when tryptophan and pyruvate were absent, demon TABLE 5 Strating that monatin production is a result of an enzymatic reaction catalyzed by the aldolase enzyme. Amount of Monatin Produced by E. coli Bacteria 0260 The in vivo production of monatin in bacterial cells Construct Host Tween + Amp tug/mL monatin time was repeated in 800 mL shake flask experiments and in proA BL21 (DE3) O41 72 hr fermentors. A 250 mL sample of monatin (in cell-free broth) proA BL21 (DE3) -- 1.58 48 hr was purified by anion eXchange chromatography and pre US 2005/0112260 A1 May 26, 2005 23

mM each dNTP, 3.5 U Expand High Fidelity Polymerase TABLE 5-continued (Roche, Indianapolis, Ind.), and IX Expand TM buffer with Mg. The thermocycler program used consisted of a hot start Amount of Monatin Produced by E. coli Bacteria at 94 C. for 5 minutes, followed by 29 repetitions of the Construct Host Tween + Amp tug/mL monatin time following steps: 94 C. for 30 seconds, 50° C. for 1 minute proA BL21 (DE3)pLyss 1.04 48 hr 45 seconds, and 72 C. for 2 minutes 15 seconds. After the proA BL21 (DE3)pLyss -- 1.60 48 hr 29 repetitions the sample was maintained at 72 C. for 10 aspC-proA BL21 (DE3) O.O9 48 hr minutes and then stored at 4 C. The PCR products were aspC-proA BL21 (DE3) -- O.58 48 hr purified by Separation on a 1%. TAE-agarose gel followed by aspC-proA BL21 (DE3)pLysS 1.39 48 hr aspC-proA BL21 (DE3)pLysS -- 6.68 48 hr recovery using a QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.). 0269. The pESC-His vector DNA (2.7 ug) was digested EXAMPLE 12 with BamHI/SalI and gel-purified as above. The aspC PCR product was digested with BamHI/SalI and purified with a 0264) Production of Monatin in Yeast QIAquick PCR Purification Column. Ligations were per 0265. This example describes methods used to produce formed with the Roche Rapid DNA Ligation Kit following monatin in eukaryotic cells. One skilled in the art will the manufacturer's protocols. DeSalted ligations were elec understand that Similar methods can be used to produce troporated into 40 ul Electromax DH10B competent cells monatin in any cell of interest. In addition, other genes can (Invitrogen) in a 0.2 cm Biorad disposable cuvette using a be used (e.g., those listed in FIG. 2) in addition to, or Biorad Gene Pulser II with pulse controller plus, according alternatively to those described in this example. to the manufacturer's instructions. After 1 hour of recovery 0266 The pESC Yeast Epitope Tagging Vector System in 1 mL of SOC medium, the transformants were plated on (Stratagene, La Jolla, Calif.) was used to clone and express LB medium containing 100 tug/mLampicillin. Plasmid DNA the E. coli aspC and C. teStoSterOni proA genes into Sac preparations for clones were done using OIAprep Spin charomyces cerevisiae. The pSC vectors contain both the Miniprep Kits. Plasmid DNA was screened by restriction GAL1 and the GAL10 promoters on opposite strands, with digest, and Sequenced (Seqwright) for verification using two distinct multiple cloning Sites, allowing for expression primerS designed for the vector. of two genes at the same time. The pSC-His vector also contains the His3 gene for complementation of histidine 0270. The aspC/pESC-His clone was digested with auxotrophy in the host (YPH500). The GAL1 and GAL10 EcoRI and Not, as was the proA PCR product. DNA was promoters are repressed by glucose and induced by galac purified as above, and ligated as above. The two gene tose, a Kozak Sequence is utilized for optimal expression in construct was transformed into DH 10B cells and Screened yeast. The pSC plasmids are shuttle Vectors, allowing the by restriction digest and DNA sequencing. initial construct to be made in E. coli (with the bla gene for 0271 The construct was transformed into S. cerevisiae Selection); however, no bacterial ribosome binding sites are strain YPH500 using the S.c. EasyCompTM Transformation present in the multiple cloning sites. Kit (Invitrogen). Transformation reactions were plated on 0267 The following primers were designed for cloning SC-His minimal medium (Invitrogen pYES2 manual) con into pESC-His (restriction sites are underlined, Kozak taining 2% glucose. Individual yeast colonies were Screened Sequence is in bold): for the presence of the proA and aspC genes by colony PCR using the PCR primers above. Pelleted cells (2 ul) were suspended in 20 lull of Y-Lysis Buffer (Zymo Research) aspC (BamHI/Sall), GAL1 : containing 1 ul of Zymolase and heated at 37 C. for 10 (SEQ ID NO: 69) minutes. Four ul of this Suspension was then used in a 50 5'-CGCGGATCCATAATGGTTGAGAACATTACCG-3' till PCR reaction using the PCR reaction mixture and and program described above. (SEQ ID NO : 70) 5'-ACGCGTCGACTTACAGCACTGCCACAATCG-3'. 0272 Five mL cultures were grown overnight on SC-His--glucose at 30° C. and 225 rpm. The cells were proA (EcoRVNotI), GAL10: gradually adjusted to growth on raffinose in order to mini (SEQ ID NO : 71.) 5'-CCGGAATTCATAATGGTCGAACTGGGAGTTGT-3' mize the lag period prior to induction with galactose. After and approximately 12 hours of growth, absorbance measure ments at 600 nm were taken, and an appropriate Volume of (SEQ ID NO: 72) cells was spun down and resuspended to give an OD of 0.4 5'-GAATGCGGCCGCTTAGTCAATATATTTCAGGCC-3'. in the fresh SC-His medium. The following carbon sources were used sequentially: 1% raffinose--1% glucose, 0.5% 0268. The second codon for both mature proteins was glucose--1.5% raffinose, 2% raffinose, and finally 1% raffi changed from an aromatic amino acid to Valine due to the nose-2% galactose for induction. introduction of the Kozak Sequence. The genes of interest were amplified using peT30 Xa/LIC miniprep DNA from 0273. After approximately 16 hours of growth in induc the clones described in Examples 1 and Example 4 as tion medium, the 50 mL cultures were divided into duplicate template. PCR was performed using an Eppendorf Master 25 mL cultures, and the following were added to only one of cycler gradient thermocycler and the following protocol for the duplicates: (final concentrations) 1 g/L L-tryptophan, 5 a 50 till reaction: 1.0 till template, 1.0 uM of each primer, 0.4 mM Sodium phosphate pH 7.1, 1 g/L Sodium pyruvate, 1 US 2005/0112260 A1 May 26, 2005 24 mM MgCl2. Samples of broths and cell pellets from the 0277 Positive results were obtained with the full two non-induction medium, and from the 16 hour cultures prior gene construct cell extracts with and without Substrate added to addition of Substrates for the monatin pathway, were to the growth medium. These results, in comparison to the Saved as negative controls. In addition, constructs containing positive controls, indicate that the enzymes were expressed only a functional aspC gene (and a truncated proA gene) at levels of close to 1% of the total protein in yeast. The were utilized as another negative control. The cells were amount of monatin produced when the cell extract of the allowed to grow for a total of 69 hours post-induction. aspC construct (with truncated proA) was assayed with Occasionally the yeast cells were induced at a lower OD, aldolase was significantly greater than when cell extracts and only grown for 4 hours prior to addition of tryptophan were assayed alone, and indicates that the recombinant and pyruvate. However, these monatin Substrates appear to AspC aminotransferase comprises approximately 1-2% of inhibit growth and the addition at higher OD was more the yeast total protein. The cell extracts of uninduced effective. cultures had a Small amount of activity when assayed with 0274 The cell pellets from the cultures were lysed with aldolase due to the presence of native aminotransferases in 5 mL of YeastBusterTM+50 ul THP (Novagen) per gram (wet the cells. When assayed with AspC aminotransferase, the weight) of cells following manufacturer's protocols, with activity of the extracts from uninduced cells increased to the the addition of protease inhibitors and benzonase nuclease as amount of monatin produced by the negative control with described in previous examples. The culture broth and cell AspC (ca. 200 ng/ml). In contrast, the activity observed extracts were filtered and analyzed by SRM as described in when assaying the two gene construct cell extract increases Example 10. Using this method, no monatin was detected in more when aminotransferase is Supplemented than when the broth Samples, indicating that the cells could not Secrete aldolase is added. Since both genes should be expressed at monatin under these conditions. The proton motive force the same level, this indicates that the amount of monatin may be insufficient under these conditions or the general produced is maximized when the level of aminotransferase amino acid transporters may be Saturated with tryptophan. is higher than that of aldolase, in agreement with results Protein expression was not at a level that allowed for shown in Example 6. detection of changes using SDS-PAGE. 0278. The addition of pyruvate and tryptophan not only 0275 Monatin was detectable (approximately 60 ng/mL) inhibits cellular growth, but apparently inhibits protein transiently in cell extracts of the culture with two functional expression as well. The addition of the pSC-Trp plasmid genes, when tryptophan and pyruvate were added to the can be used to correct for tryptophan auxotrophy of the medium. Monatin was not detected in any of the negative YPH500 host cells, to provide a means of Supplying tryp control cell extracts. In vitro assays for monatin were tophan with fewer effects on growth, expression, and Secre performed in duplicate with 4.4 mg/mL of total protein tion. (about double what is typically used for E. coli cell extracts) using the optimized assay described in Example 6. Other EXAMPLE 13 assays were performed with the addition of either 32 tug/mL 0279 Improvement of Enzymatic Processes using C. testosteroni ProA aldolase or 400 lug/mL AspC ami Coupled Reactions notransferase, to determine which enzyme was limiting in the cell extract. Negative controls were performed with no 0280. In theory, if no side reactions or degradation of addition of enzyme, or the addition of only ASpC ami Substrates or intermediates occurs, the maximum amount of notransferase (the aldol condensation can occur to Some product formed from the enzymatic reaction illustrated in extent without enzyme). Positive controls were performed FIG. 1 is directly proportional to the equilibrium constants with partially pure enzymes (30-40%), using 16 ug/mL of each reaction, and the concentrations of tryptophan and aldolase and 400 tug/mL aminotransferase. pyruvate. Tryptophan is not a highly Soluble Substrate, and 0276. In vitro results were analyzed by SRM. The analy concentrations of pyruvate greater than 200 mM appear to sis of cell extracts showed that tryptophan was effectively have a negative effect on the yield (see Example 6). transported into the cells when it was added to the medium 0281 Ideally, the concentration of monatin is maximized post-induction, resulting in tryptophan levels two orders of with respect to Substrates, in order to decrease the cost of magnitude higher than those in which no additional tryp Separation. Physical Separations can be performed Such that tophan was added. The results for in vitro monatin analysis the monatin is removed from the reaction mixture, prevent are shown in Table 6 (numbers indicate ng/mL). ing the reverse reactions from occuring. The raw materials

TABLE 6 Monatin production with yeast cell extracts aspC two-gene construct + aldolase + AspC construct + aldolase + AspC repressed (glucose medium) O 888.3 173.5 O 465.2 829 24 hr induced O 2832.8 642.4 O 1375.6 9146.6 69 hr induced O 4937.3 340.3 71.9 1652.8 23693.5 69 hr + subs. O 556.9 659.1 21.9 755.6 16688.2 + control (purified enzymes) 21853 21853 - control (no enzymes) O 254.3 O 254.3 US 2005/0112260 A1 May 26, 2005 25 and catalysts can then be regenerated. Due to the Similarity which amino acceptor was utilized. Therefore, this enzyme of monatin in size, charge, and hydrophobicity to Several of was used in the coupled reactions with oxaloacetate decar the reagents and intermediates, physical Separations will be boxylase. difficult unless there is a high amount of affinity for monatin (Such as an affinity chromatography technique). However, 0286 A typical reaction starting from indole-3-pyruvate the monatin reactions can be coupled to other reactions Such included (final concentrations) 50 mM Tris-Cl pH 7.3, 6 mM that the equilibrium of the system is shifted toward monatin indole-3-pyruvate, 6 mM Sodium pyruvate, 6 mM aspartate, production. The following are examples of processes for 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl, 25 improving the yield of monatin obtained from tryptophan or tug/mL aminotransferase, 50 ug/mL. C. teStoSteroni ProA indole-3-pyruvate. aldolase, and 3 Units/mL of decarboxylase (Sigma 04878). The reactions were allowed to proceed for 1 hour at 26 C. 0282 Coupled Reactions Using Oxaloacetate Decar In Some cases, the decarboxylase was omitted or the aspar boxylase (EC 4.1.1.3) tate was Substituted with alpha-ketoglutarate (as negative controls). The aminotransferase enzymes described above 0283 FIG. 11 is an illustration of the reaction. Tryp were also tested in place of the GOAT to confirm earlier tophan oxidase and catalase are utilized to drive the reaction Specificity experiments. Samples were filtered and analyzed in the direction of indole-3-pyruvate production. Catalase is by LC/MS as described in Example 10. The results demon used in exceSS Such that hydrogen peroxide is not available strate that the GOAT enzyme produced the highest amount to react in the reverse direction or to damage the enzymes or of monatin per mg of protein, with the least amount of intermediates. Oxygen is regenerated during the catalase tryptophan produced as a byproduct. In addition, there was reaction. Alternatively, indole-3-pyruvate can be used as the a 2-3 fold benefit from having the decarboxylase enzyme Substrate. added. The E. coli ASpC enzyme also produced large 0284 Aspartate is used as the amino donor for the amounts of monatin in comparison to the other aminotrans amination of MP, and an aspartate aminotransferase is ferases. utilized. Ideally, an aminotransferase that has a low speci ficity for the tryptophan/indole-3-pyruvate reaction in com 0287 Monatin production was increased by: 1) periodi parison to the MP to monatin reaction is used so that the cally adding 2 mM additions of indole-pyruvate, pyruvate, aspartate is not utilized to reaminate the indole-3-pyruvate. and aspartate (every half hour to hour), 2) performing the Oxaloacetate decarboxylase (from Pseudomonas sp.) can be reactions in an anaerobic environment or with degassed added to convert the Oxaloacetate to pyruvate and carbon buffers, 3) allowing the reactions to proceed overnight, and dioxide. Since CO is volatile, it is not available for reaction 4) using freshly prepared decarboxylase that has not been with the enzymes, decreasing or even preventing the reverse freeze-thawed multiple times. The decarboxylase was inhib reactions. The pyruvate produced in this step can also be ited by concentrations of pyruvate greater than 12 mM. At utilized in the aldol condensation reaction. Other decarboxy concentrations of indole-3-pyruvate higher than 4 mM, Side lase enzymes can be used, and homologs are known to exist reactions with indole-3-pyruvate were hastened. The amount in Actinobacillus actinomycetemcomitans, Aquifex aeolicus, of indole-3-pyruvate used in the reaction could be increased Archaeoglobus fulgidus, Azotobacter vinelandii, Bacteroi if the amount of aldolase was also increased. High levels of des fragilis, Several Bordetella Species, Campylobacter phosphate (50 mM) and aspartate (50 mM) were found to be jejuni, Chlorobium tepidum, Chloroflexus aurantiacus, inhibitory to the decarboxylase enzyme. The amount of Enterococcus faecalis, Fusobacterium nucleatum, Kleb decarboxylase enzyme added could be reduced to 0.5 U/mL Siella pneumoniae, Legionella pneumophila, MagnetOCOc with no decrease in monatin production in a one hour cus MC-1, Mannheimia haemolytica, Methylobacillus reaction. The amount of monatin produced increased when flagellatus KT, Pasteurella multocida Pm70, Petrotoga mio the temperature was increased from 26 C. to 30° C. and therma, Porphyromonas gingivalis, Several Pseudomonas from 30° C. to 37 C.; however, at 37° C. the side reactions Species, Several PyrococcuS Species, RhodoCOccus, Several of indole-3-pyruvate were also hastened. The amount of Salmonella Species, Several StreptococcuS Species, Thermo monatin produced increased with increasing pH from 7 to chromatium tepidum, Thermotoga maritima, Treponema 7.3, and was relatively stable from pH 7.3-8.3. pallidum, and Several Vibrio Species. 0288 A typical reaction starting with tryptophan 0285) Tryptophan aminotransferase assays were per included (final concentrations) 50 mM Tris-Cl pH 7.3, 20 formed with the aspartate aminotransferase (AspC) from E. mM tryptophan, 6 mM aspartate, 6 mM Sodium pyruvate, coli, the tyrosine aminotransferase (TyrB) from E. coli, the 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl, 25 broad substrate aminotransferase (BSAT) from L. major, and tug/mL aminotransferase, 50 ug/mL. C. teStoSteroni ProA the two commercially available porcine glutamate-Oxaloac aldolase, 4 Units/mL of decarboxylase, 5-200 m U/mL etate aminotransferases as described in Example 1. Both L-amino acid oxidase (Sigma A-2805), 168 U/mL catalase Oxaloacetate and alpha-ketoglutarate were tested as the (Sigma C-3515), and 0.008 mg FAD. Reactions were carried amino acceptor. The ratio of activity using monatin out for 30 minutes at 30°C. Improvement was observed with (Example 7) versus activity using tryptophan was compared, the addition of decarboxylase. The greatest amount of mona to determine which enzyme had the highest Specificity for tin was produced when 50 m U/mL of oxidase was used. the monatin aminotransferase reaction. These results indi Improvements were similar to those observed when indole cated that the enzyme with the highest Specificity for the 3-pyruvate was used as the Substrate. In addition, the amount monatin reaction verses the tryptophan reaction is the Por of monatin produced increased when 1) the tryptophan level cine type II-A glutamate-Oxaloacetate aminotransferase, was low (i.e., below the K of the aminotransferase enzyme GOAT (Sigma G7005). This specificity was independent of and therefore unable to compete with MP in the active site), US 2005/0112260 A1 May 26, 2005 26 and 2) the ratio of oxidase to aldolase and aminotransferase FIG. 13. Formate dehydrogenase (EC 1.2.1.2 or 1.2.1.43) is was maintained at a level Such that indole-3-pyruvate could a common enzyme. Some formate dehydrogenases require not accumulate. NADH while others can utilize NADPH. Glutamate dehy drogenase catalyzed the interconversion between the mona 0289 Whether starting with either indole-3-pyruvate or tin precursor and monatin in previous examples, using tryptophan, the amount of monatin produced in assays with ammonium based buffers. The presence of ammonium for incubation times of 1-2 hours increased when 2-4 times the mate and formate dehydrogenase is an efficient System for amounts of all the enzymes were used while maintaining the regeneration of cofactors, and the production of carbon Same enzyme ratio. Using either Substrate, concentrations of dioxide is an efficient way to decrease the rate of the reverse approximately 1 mg/mL of monatin were achieved. The reactions (Bommarius et al., Biocatalysis 10:37, 1994 and amount of tryptophan produced if Starting from indole Galkin et al. Appl. Environ. Microbiol. 63:4651-6, 1997). In pyruvate was typically less than 20% of the amount of addition, large amounts of ammonium formate can be dis product, which shows the benefit of utilizing coupled reac solved in the reaction buffer. The yield of monatin produced tions. With further optimization and control of the concen by glutamate dehydrogenase reactions (or similar reductive trations of intermediates and Side reactions, the productivity aminations) can be improved by the addition of formate and yield can be improved greatly. dehydrogenase and ammonium formate. 0290 Coupled reactions using lysine epsilon aminotrans ferase (EC 2.6.1.36) Lysine epsilon aminotransferase 0295) Other processes can be used to drive the equilib (L-Lysine 6-transaminase) is found in several organisms, rium toward monatin production. For instance, if aminopro including RhodoCOccus, Mycobacterium, Streptomyces, pane is utilized as the amino acid donor in the conversion of Nocardia, Flavobacterium, Candida utilis, and Streptomy MP to monatin with an omega-amino acid aminotransferase ceS. It is utilized by organisms as the first Step in the (EC 2.6.1.18) such as those described by in U.S. Pat. Nos. production of Some beta-lactam antibiotics (Rius and 5,360,724 and 5,300,437, one of the resulting products Demain, J. Microbiol. Biotech., 7:95-100, 1997). This would be acetone, a more Volatile product than the Substrate, enzyme converts lysine to L-2-aminoadipate 6-semialde aminopropane. The temperature can be raised periodically hyde (allysine), by a PLP-mediated transamination of the for short periods to flash off the acetone, thereby alleviating C-6 of lysine, utilizing alpha-ketoglutarate as the amino equilibrium. Acetone has a boiling point of 47 C., a acceptor. Allysine is unstable and Spontaneously undergoes temperature not likely to degrade the intermediates if used for short periods of time. Most aminotransferases that have an intramolecular dehydration to form 1-piperideine 6-car activity on alpha-ketoglutarate also have activity on the boxylate, a cyclic molecule. This effectively inhibits any monatin precursor. Similarly, if a glyoxylate/aromatic acid reverse reaction from occuring. The reaction Scheme is aminotransferase (EC 2.6.1.60) is used with glycine as the depicted in FIG. 12. An alternative enzyme, lysine-pyruvate amino donor, glyoxylate is produced which is relatively 6-transaminase (EC 2.6.1.71), can also be used. unstable and has a highly reduced boiling point in compari 0291. A typical reaction contained in 1 mL: 50 mM Son to glycine. Tris-HCl pH 7.3, 20 mMindole-3-pyruvate, 0.05 mM PLP, 6 mM potassium phosphate pH 8, 2-50 mM sodium pyru EXAMPLE 1.4 vate, 1.5 mM MgCl, 50 mM lysine, 100 lug aminotrans ferase (lysine epsilon aminotransferase LAT-101, BioCata Packet Formulations Comprising Monatin lytics Pasadena, Calif.), and 200 ug C. testosteroni ProA 0296 Packet formulations comprising monatin are pre aldolase. The amount of monatin produced increased with pared to deliver the Sweetness of about 2 teaspoons of Sugar increasing concentrations of pyruvate. The maximum (approximately 8 grams). amount using these reaction conditions (at 50 mM pyruvate) was 10-fold less than what was observed with coupled 0297 A. Packet Formulation with S.S Monatin (Per reactions using oxaloacetate decarboxylase (approximately Gram): 0.1 mg/mL). 0298) 787 mg Dextrose 0292 A peak with M+H"=293 eluted at the expected time for monatin and the mass Spectrum contained Several of 0299) 200 mg S.S monatin the same fragments observed with other enzymatic pro 0300 10 mg Cream of Tartar cesses. A Second peak with the correct mass to charge ratio (293) eluted slightly earlier than what is typically observed 0301) 3 mg Calcium Silicate for the S.S monatin produced in Example 6, and may 0302) B. Packet Formulation with R.R and SS Monatin indicate the presence of another isomer of monatin. Very little tryptophan was produced by this enzyme. However, (Per Gram) there is likely Some activity on pyruvate (producing alanine 0303 700 mg Dextrose as a byproduct). Also, the enzyme is known to be unstable. Improvements can be made by performing directed evolu 0304 292 mg Maltodextrin tion experiments to increase Stability, reduce the activity 0305) 4 mg R,R monatin with pyruvate, and increase the activity with MP. These reactions can also be coupled to L-amino acid oxidase/ 0306 4 mg S, S monatin catalase as described above. 0307 C. Packet Formulation with R.R and SS Monatin 0293). Other Coupled Reactions (Per Gram) 0294. Another coupling reaction that can improve mona 0308 992 mg agglomerated dextrose with maltodex tin yield from tryptophan or indole-pyruvate is shown in trin US 2005/0112260 A1 May 26, 2005 27

0309 4 mg R,R monatin 0310 4 mg SS monatin -continued 0311 * For example, UnideX(R) Agglomerated Dextrose Ingredients % w/w 02540 (2034) from CPC International, Inc. Baking Powder 1.24 Salt O.31 0312 D. Packet Formulation with R.R Monatin (Per R.R Monatin (to desired taste) O.O1O-O.O40 Gram): 0313 500 mg Dextrose 0330 For every 100 g of cake, 10-40 mg of R,R monatin 0314. 495 mg Maltodextrin are used (provided by, for example, 3-10 packets of tabletop monatin Sweetener). 0315) 5 mg R,R monatin 0316 E. Half-Sized Cube with R.R Monatin (Per Gram): EXAMPLE 16 0317) 999 mg Maltodextrin Ready-To-Use Formulations Comprising Monatin 0331 “Spoon for spoon” ready-to-use formulations are 0318) 1 mg R,R monatin prepared to be used in place of granulated Sugar. Because EXAMPLE 1.5 monatin formulations may have the same Volume of Sugar, one may add these formulations Straight to beverages or Recipes for Packet Formulations Comprising food, or use them directly in baking as a direct and equal Monatin Substitute for granulated Sugar. 03.19. The following are examples of how monatin packet 0332 A. Ready-to-use formulation with SS monatin (per formulations, Such as those disclosed above, may be used as gram): Sugar Substitutes in baking and other recipes. The monatin 0333 188 mg Hydrolyzed Starch containing products produced using the following recipes are proposed to be acceptable and of good quality compared 0334 800 mg Maltodextrin to the same products prepared with Sugar. 0335) 10 mg S.S monatin 0320 Lemonade: 0336 2 mg Sodium Gluconate 0321) Mix 2 tablespoons of lemon juice and 3 packets (3 0337 B. Ready-to-use formulation with R,R monatin g) of a monatin packet formulation with /cup of water in a (per gram): tall glass until dissolved. Add ice. The monatin-Sweetened lemonade will be nearly equivalent in Sweetness and equally 0338 999.5 mg Maltodextrin preferred to the lemonade Sweetened with 6 teaspoons (24 g) 0339) 0.5 mg R,R monatin Sucrose and will have significantly fewer calories (about 0 0340 C. Ready-to-use formulation with S.S/R,R blend Calories versus 96 Calories). (per gram): 0322) Whipped Topping 0341 972.5 mg Maltodextrin 0323 /; cup ice water 0342 20 mg Cream of Tartar 0324 4 teaspoon lemon juice 0343 4 mg Calcium Silicate 0325 % teaspoon vanilla 0344 3.1 mg S.S monatin 0326 /3 cup nonfat dry milk powder 0345 0.4 mg R,R monatin (g 3 packets monatin Sugar Substitute (per Example EXAMPLE 1.7 Recipes for Ready-To-Use Formulations 0328 Combine water, lemon juice and vanilla. Stir in Comprising Monatin nonfat dry milk powder. Beat 10 minutes or until stiff. Add 0346) The following are examples of how monatin monatin Sugar Substitute packets and beat 2 minutes. “Spoon for Spoon” ready-to-use formulations, Such as those 0329 Sucrose-free sponge cake data sheet disclosed above, may be used as Sugar Substitutes in baking and other recipes. The monatin-containing products pro duced using the following recipes are proposed to be accept able and of good quality compared to the same products Ingredients % w/w prepared with Sugar. cake flour 2042 water 1862 0347 French Silk Pie whole egg 18.2 0348 Crust: polydextrose 17.23 High Ratio Shortening 1344 0349) 1 cup crushed graham crackers Sorbitol Powder 8.62 Skimmed Milk Powder 1.66 0350 1 cup chopped pecans 0351 1 stick butter, melted US 2005/0112260 A1 May 26, 2005 28

0352 Mix crust ingredients and pat into 9" pie plate. 0381 1154-ounce can crushed pineapple Bake at 300 for 30 minutes. 0382 1 6-ounce can frozen orange juice concentrate, 0353 Filling: thawed 0354 2 squares unsweetened chocolate 0383) 3 cups skim milk 0355] 2 sticks butter 0384 % cup evaporated skim milk 0356) 1%cups monatin ready-to-use formulation (per 0385) / cup monatin ready-to-use formulation (per Example 16B) Example B) 0357 2 teaspoons vanilla 0386 % teaspoon vanilla 0358) 4 pasteurized eggs 0387 Stir together skim milk, undrained pineapple, 0359 Melt the unsweetened chocolate and cool to room evaporated Skim milk, orange juice concentrate, monatin temperature. Cream butter and monatin Sugar Substitute. Sugar Substitute and Vanilla in a large bowl until the monatin Add Vanilla and melted chocolate to the creamed mixture. Sugar Substitute dissolves. Freeze in 4- or 5-quart ice cream Add eggs one at a time beating each egg 4 minutes. Pour freezer according to manufacturer's directions. Makes 15 (1. over cool crust and refrigerate overnight. cup) Servings. 0360 Blueberry Muffins: 0388) Lemon Mousse 0361 Note: Sugar can be important in baked goods for 0389) 1 12-ounce can evaporated milk texture, browning, as well as Sweetening. In this formula, a Small amount of honey is used to compensate for the lack of 0390 2 Tablespoons grated lemon rind Sugar in the formula and promote browning and good 0391) Juice from 2 lemons teXture. 0392) 1 cup monatin ready-to-use formulation (per 0362 2 cups all-purpose flour Example A) 0363) 2 tsp baking powder 0393 12 graham crackers, crushed 0364) % tsp salt 0394 Pour milk on jelly roll pan and place in freezer until 0365) /2 cup light margarine, Softened a sliver of ice forms, about 2 hours. Transfer milk to chilled bowl and whip. Add lemon rind and juice slowly while 0366) 1 cup monatin ready-to-use formulation mixing. Add monatin Sugar Substitute and continue whip 0367) /4 cup honey ping until Soft peaks form. Transfer to Serving dish(es) and sprinkle with graham cracker crumbs. May be served chilled 0368) 2 whole large eggs or frozen. 0369) 1 tsp Vanilla EXAMPLE 1.8 0370 /2 cup skim milk 0371) 1 cup blueberries, fresh or frozen Dessert and Confections Products Comprising Monatin 0372 Preheat oven to 350 F. Line 10 muffin cups with paper liners. Sift together flour, baking powder, Salt, Set 0395 Ready-to-use monatin compositions may also be aside. Beat together margarine, monatin Sweetener and used commercially in the production of consumer packaged honey with an electric mixer until light and fluffy. Add eggs goods. one at a time beating well after each addition. Stir in Vanilla. Alternately stir in flour mixture and milk, beginning and 0396 Instant chocolate pudding data sheet ending with flour mixture. Fold in berries. Spoon batter into paper lined muffin cups, and bake until golden brown and a toothpick inserted in middle comes out clean, about 25-30 Ingredients % w/w minutes. Cool in pan 10 minutes on wire rack. Remove muffins from pan. Cool completely on wire rack. Makes 10 Cold Swelling Starch (Ultratex 4) S.OO muffins. Skimmed Milk Powder 14.OO Cocoa Powder 2.5 0373) Strawberry Banana Smoothie: Maltodextrin 3.5 Chocolate flavoring 0.4 0374 1 cup orange juice Xanthan Gum O.2O Salt O.15 0375] 1 cup fat-free plain yogurt Lecithin Powder 0.25 Disodium Phosphate 0.17 0376) 1 frozen banana R.R Monatin (to desired taste) 0.005-0.010 0377 1 cup frozen strawberries Water to 1OO 0378 6 packets monatin formulation or 4 cup monatin ready-to-use formulation 0397 For every 100 g of pudding, this corresponds to 0379 Place all items in a blender and blend until Smooth. 5-10 mg of R,R monatin, (provided by, for example, 2-3 Makes 2 servings. This smoothie has about 33% fewer packets of tabletop monatin Sweetener). Calories than the same formula prepared with /4 cup Sugar 0398 Sugar-free chocolate coating 0380 Pineapple Orange Sherbet 0399. Ingredients US 2005/0112260 A1 May 26, 2005 29

0400. 4.0-10% milk solids TABLE 7 0401 15.0-20% chocolate flavoring or chocolate liquors Coffee formulation Concentration (%; 0402) 0-10% cocoa butter Ingredient Supplier wfw) g/700 ml 0403. 0-2% cocoa Classic Roast Folger (R) 5.41 37.87 Coffee 0404 5-10% mannitol Water 94.59 662.13 04.05 0.3-0.5% lecithin 0422 Sweeteners were added to coffee at the following concentrations: 0406 12-25% polydextrose (such as polydextrose K) 0423 Aspartame 0.025% (w/v) 0407 0.10-0.5% S.S monatin OR 0.007-0.015% R.R 0424) Sucralose 0.0082% (w/v) monatin or blends thereof 0425) R,R monatin 0.0020, 0.0025, 0.0030% (w/v) plus 1 g maltodextrin 0408) 20-60% vegetable oil 0426 R,R monatin/erythritol 0.0020, 0.0025, 0.0030% 04.09 For every 100 mg of coating, approximately 2-4 (w/v) plus 1 g erythritol packets of tabletop Sweetener are used. 0427 (ii) Iced Tea 0428. An ice tea formulation was developed to evaluate 0410 Sugar-free chocolate Sweetener performance (Table 8). 0411 Ingredients TABLE 8 0412 2-3 tablespoons cocoa Iced Tea formulation 0413 2 tablespoons butter (no substitutes) Ingredient Supplier Concentration (w/v) Citric acid O.2OO 0414 2 tablespoons liquid heavy whipping cream Sodium citrate O.O2O Tea extract Assam Plantextrakt O.150 0415 4 teaspoons pure vanilla extract 28SOO2 Natural black tea flavour Rudolph Wild O.OSO extract 311083O4O1OOOO 0416) 1 tablespoon peanut butter (crunchy or creamy) Sodium benzoate (20% w/w) 0.075 Sweetener As required 0417 6 packets tabletop monatin Water To volume

EXAMPLE 1.9 0429 Sweeteners were added to tea at the following concentrations: Evaluation of Monatin in Table-Top Sweetener Applications 0418 Monatin table-top formulations, including R.R Aspartame 0.0450% (w/v) monatin or R,R monatin/erythritol combinations, were Sucralose 0.0170% (w/v) RR monatin 0.0030, 0.0035, 0.0040% (w/v) plus assessed relative to other known Sweeteners (aspartame and 1 g maltodextrin Sucralose) in coffee and iced tea. The key sensory param R,R, monatinferythritol 0.0030, 0.0035, 0.0040% (w/v) plus eters assessed included Sweetness quality, aftertaste, bitter 1 g erythritol taste and its aftertaste. Qualitative evaluation has been carried out. 0430 Sensory Evaluation 0419 Product Formulations 0431. The evaluation of these coffee and tea drinks was carried out by a Small (n=6) panel of experienced sensory 0420 (i) Coffee evaluators who evaluated the coffee products on one tasting 0421 Standard coffee was used in which to evaluate occasion and the tea products on a Subsequent occasion. The Sweetener performance (Table 7). results of these evaluations are Summarized in Table 9.

TABLE 9 Sensory evaluation of coffee and tea (200 ml serving size)

Product Sweetenerf concentration Comments Coffee Aspartame/250 ppm Balanced sweetness profile. Very low level of bitterness, probably due to inhibition by US 2005/0112260 A1 May 26, 2005 30

TABLE 9-continued Sensory evaluation of coffee and tea (200 ml serving size Product Sweetenerf concentration Comments APM. Flat, even coffee flavor delivery. Typical APM aftertaste that is perceived at the back of the tongue. Sucralose/82 ppm Slow sweetness onset allows stronger coffee notes to be perceived. Bitter coffee notes quite clearly apparent in the aftertaste, although balanced somewhat by the lingering sweet character of sucralose. Monatin (25 ppm) + Maltodextrin Balanced sweetness profile. Clear coffee (1 g) (0.5%) flavor in the aftertaste. Stronger coffee flavor overall than with either of the other Sweeteners, although this may be (at least in part) due to the limited bitterness inhibiting capacity of monatin. Monatin (25 ppm) + Erythritol More coffee flavor in monatin sample. (1 g) (0.5%) Sweetness is less delayed with monatinferythritol combination than with monatin?maltodextrin. Erythritol smoothes out the coffee flavor and makes the Sweetness onset a little faster. Iced Tea Aspartame/450 ppm Good temporal characteristics although the typical aspartame flavor is clearly apparent. Balanced, though quite subtle tea flavour. No evidence of flavor enhancement. Sucralose/170 ppm Delay in Sweetness onset means first impressions are of acidity. Product flavor and Overall impression somewhat out of balance because of sweetness profile not matching acidity or flavor profiles. Monatin (40 ppm) + Maltodextrin Sweetness and flavor profiles very (1 g) (0.5%) balanced. The lemon flavor notes are clearly enhanced over those of the other Sweeteners. Monatin (40 ppm) + Erythritol Sweetness and flavor profiles balanced. (1 g) (0.5%) Lemon flavor notes even more enhanced than monatin?maltodextrin alone. The astringency in the aftertaste is greatly reducedfeliminated.

0432 Discussion duplicate. Test and reference Solutions were prepared in 0433 Monatin delivers unexpected performance benefits, citric/citrate buffer at pH 3.2. See FIG. 16. The more linear including clear Sensory benefits, in table-top Sweetener response of R,R/S.S monatin, as compared to Saccharin, is formulations. When used in a table-top formulation added to consistent with the delivery of a more Sugar-like taste coffee, a clear increase in the level of coffee flavor is character. The plateau above 10% SEV indicates absence/ perceived. This benefit is further enhanced through addition low levels of “mixture-Suppressing off-tastes and after of low concentrations of erythritol, which are able to balance tastes. The shape of monatin's dose-response curve is simi and round the flavor and to Speed up Sweetness onset times. lar to those of aspartame, Sucralose and alitame, all of which In iced-tea, and particularly acidified acid tea (i.e., with are “quality Sweeteners. lemon), monatin enhances the lemon flavor notes. Again, 0437. With R.R/SS monatin as a sole Sweetener in the erythritol blending with monatin confers additional flavor model System (pH 3.2), the following characteristics were benefits. observed: (1) slight delay in Sweet taste onset; (2) Sweet taste 0434 Monatin delivers improved sensory properties decay was quite rapid; (3) slight "aspartame-like' aftertaste, (e.g., less aftertaste, less off-taste), and/or improved Solu Slightly Sweet aftertaste, no bitterneSS in the aftertaste; and bility and Stability characteristics. (4) residual cooling Sensation in unflavored Systems. 0435 Monatin Sweetened coffee contains close to 0 Calo EXAMPLE 21 ries, as compared to 32 Calories in coffee Sweetened with 2 teaspoons (~8 g) of Sucrose. Stability of Monatin at pH 3 with Increasing Temperatures EXAMPLE 2.0 0438 Asample of synthetic monatin was subjected to pH 3 at temperatures of 25° C., 50° C. and 100° C. At room Sweetness Dose Response Curve of Monatin and temperature and pH 3, a 14% loss in monatin was observed Saccharin over a period of 48 hours. This loss was attributed to lactone 0436 Sweetness of monatin and saccharine was assessed formation. At 50 C. and pH 3, a 23% loss in monatin was using 20 trained Sensory evaluators, making judgements in observed over a period of 48 hours. This loss was attributed US 2005/0112260 A1 May 26, 2005

to lactone formation and the buildup of an unknown com 0449 Tables 10 and 11 present results of the stability pound after about 15.5 minutes. At 100° C. and pH 3, nearly Studies in the phosphate citrate buffers. At each pH and after all monatin was lost after 24 hours. The major detectable 100 days storage at 40 C. in the dark, the percentage component was an unknown at 15.5 minutes. retention of monatin Sweetness was greater than that retained with aspartame. At pH 4.0, the loSS of Sweetness of EXAMPLE 22 the monatin Solution appeared almost to have Stabilized Since there was very little change in measured Sweetness Sensory Stability of Monatin and Aspartame at pH intensity between Days 17 and 100, whereas the aspartame 2.5, 3.0, 4.0 at 40° C. Solution continued to lose Sweetness. 0439. The sensory stability of monatin solutions prepared at pH 2.5, 3.0 and 4.0 and stored at 40°C. was monitored for TABLE 10 100 days. Loss of Sweetness from these solutions was Sensory Stability of Monatin: Sweetness after 100 Days Storage at 40 C. compared with the losses of Sweetness from aspartame Retention of Retention of Solutions prepared and Stored under identical conditions. SEV Monatin SEV Aspartame 0440 The sensory stability of monatin (8% SEV, -55 Time Monatin Sweetness Aspartame Sweetness ppm, Synthetic blend containing approximately 96% of the pH (days) (% sucrose) (%) (% sucrose) (%) 2R,4R/2S, 4S enantiometric pair and 4% of the 2R,4S/2S,4R A. enantiometric pair) in phosphate/citrate buffers having a pH 2.5 O 7.35 7.34 of 2.5, 3.0, and 4.0 was examined after storage at 40 C. The 1. 6.86 93.3 6.90 94.O 2 6.7O 91.2 6.8O 92.6 Stability of monatin was compared to that of aspartame (400 3 6.50 88.4 6.60 89.9 ppm) in the same buffers. Three Sucrose reference Solutions 4 6.26 85.2 6.29 85.7 were prepared in the same phosphate/citrate buffers as the 7 6.08 82.7 6.O1 81.9 monatin and aspartame Solutions. All prepared Solutions 8 5.98 81.4 5.98 81.5 9 5.89 80.1 5.97 813 were Stored in the dark. 11 5.78 78.6 5.86 79.8 50 4.61 62.7 4.19 57.1 0441 Buffer compositions: 1OO 2.10 28.6 O.80 10.9 B. 0442 pH2.5 Phosphoric acid (75% solution) 3.0 O 7.08 7.15 0.127% (w/v) 1. 7.05 99.6 6.90 96.5 2 6.60 93.2 6.87 96.1 0443 Tri-sodium citrate monohydrate 0.005% 3 6.47 91.4 6.60 92.3 (w/v) 4 6.49 91.6 6.43 89.9 7 6.04 85.3 6.17 86.3 0444 pH3.0 Phosphoric acid (75% solution) 8 5.93 83.8 5.93 82.9 0.092% (w/v) 9 5.88 83.1 5.94 83.1 11 5.88 83.1 5.83 81.5 0445 Tri-sodium citrate monohydrate 0.031% 50 5.12 72.3 4.71 65.9 (w/v) 1OO 4.10 57.9 2.20 30.8 C. 0446 pH4.0 Phosphoric acid (75% solution) 4.0 O 7.40 7.10 0.071% (w/v) 3 7.08 95.7 6.75 95.1 0447 Tri-sodium citrate monohydrate 0.047% 8 6.42 86.8 6.23 87.8 11 6.36 85.9 6.02 84.8 (w/v) 17 6.10 82.4 5.75 81.0 24 6.25 84.5 5.85 82.4 0448. The Sweetness of each Sweetener relative to 50 6.14 82.9 5.29 74.5 Sucrose was assessed in duplicate by a Small panel (n=8) of 1OO 5.8O 78.4 4.10 57.7 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 0450 were presented individually to panelists, in random order. Sucrose reference standards, ranging from 4.0-10.0% (w/v) TABLE 11 Sucrose, increasing in Steps of 0.5% (w/v) Sucrose were also Stability: Amount of Sweetness remaining after 100 days storage at provided. Panelists were asked to estimate Sweetness by Stated pH at 40 C. comparing the Sweetness of the test Solution to the Sucrose Standards. This was carried out by taking 3 Sips of the test pH Sweetener Sweetness Retained (%) solution, followed by a sip of water, followed by 3 sips of 2.5 Aspartame 11 Sucrose Standard followed by a Sip of water, etc. Panelists 2.5 Monatin 29 3.0 Aspartame 31 were encouraged to estimate the Sweetness to one decimal 3.0 Monatin 58 place, e.g., 6.8, 8.5. A five minute rest period was imposed 4.0 Aspartame 58 between evaluating the test Solutions. Panelists were also 4.0 Monatin 78 asked to rinse well and eat a cracker to reduce any potential carry over effects. US 2005/0112260 A1 May 26, 2005 32

0451. The respective buffers were effective at maintain 1.10. Triangle tests are performed with two monatin/eryth ing pH, as Seen in Table 12: ritol Samples and one monatin/maltodextrin Sample. Tri angle tests are known in the art. It is expected that ready TABLE 12 to-use cubes prepared with monatin/erythritol will have a color and crystalline quality that closely resembles Sucrose. Sweetener Nominal pH Actual pH (after 50 days) EXAMPLE 24 Monatin 2.5 2.39 3.0 3.13 Chromatography of Stereoisomers of Monatin 4.0 4.28 Aspartame 2.5 2.49 0457 Sample Preparation-Approximately 50-75 ug of 3.0 3.13 lyophilized material was placed in a microcentrifuge tube. 4.0 4.19 To this 1.0 mL of HPLC grade methanol was added. The Solution was Vortexed for 30 minutes, centrifuged and an aliquot of the Supernatant was removed for analysis. 0452) If a pseudo-first order breakdown reaction is 0458 Reversed Phase HPLC-Chromatography of two assumed, a plot of log percentage retention versus time distinct diastereomer peaks (R,R/S.S and R.S/S,R) was (log,% RTN v. t) allows estimation of the half-life (t/2) and accomplished using a 2.1x250 mm XterraTM MS C-5 um rate constant (k) of Sweetness loSS under any given set of (Waters Corporation) HPLC column. Detection was carried conditions. In So doing, the kinetics of monatin and aspar out using an Ultima" triple quadrupole mass spectrometer tame Sweetness loSS may be Summarized as follows in Table from Micromass. Mobile phase was delivered by the fol 13. lowing gradient: TABLE 13

Rate constant Sweetener PH Half-life (t/2; days) (k; day") Time (min Monatin 2.5 65 days 0.011 day' O 9 16 2O 21 3.0 115 days 0.006 day' 4.0 230 days 0.003 day' O.05% TEA A% 95 65 1O 1O 95 Aspartame 2.5 55 days 0.013 day' Methanol, 0.05% TFA B 9% 5 35 90 90 5 3.0 75 days 0.009 day Flow mL/min 0.25 O.25 0.25 O.25 0.25 4.0 140 days 0.005 day 0459 Chiral HPLC-Chromatography of two distinct 0453 At each pH and after 100 days storage at 40°C., the monatin Stereoisomers (R,R and S,S) was accomplished percentage retention of monatin Sweetness is greater than using a 250x4.6 mm Chirobiotic T(Advanced Separations that retained from aspartame. At pH 4.0, the loSS of Sweet Technologies, Inc.) HPLC column. Detection was carried neSS of the monatin Solution appears almost to have Stabi out using an Ultima triple quadrupole mass spectrometer lized Since there has been very little change in measured from Micromass. Mobile phase consisted of Methanol with Sweetness intensity between Days 17 and 100, whereas the 0.2% Acetic acid and 0.05% ammonium hydroxide. aspartame Solution continues to lose Sweetness. 0460 Mass Spectrometry (MS/MS)- The presence of 04.54 Estimates of the half-life of monatin and aspartame monatin was detected by a Selected Reaction Monitoring indicate that Sweetness derived from monatin is lost at a (SRM) experiment. The protonated molecular ion of mona slower rate than that from aspartame. Half-life estimates for tin (IM+H") has a m/z=293.3. Fragmentation of this monatin Sweetness at pH 2.5, 3.0 and 4.0 were 65 days, 115 molecular ion produces a significant ion at m/Z=257.3 aris days and 230 days, respectively. ASpartame half-life esti ing from multiple dehydrations of the molecular ion. This mates were 55 days, 75 days and 140 days under the same transition has been shown to be very Specific to monatin and conditions. was chosen as the transition (293.3 to 257.3) for monitoring during the SRM experiment. This method of detection was 0455 Thus, under acidic conditions and storage at 40 C., employed for both reversed phase and chiral Separations of monatin delivers a more Stable Sweetness than does aspar monatin. tame. 0461 Results. The standard samples of R.S/S.R and S.S/R,R were evaluated under Reversed Phase HPLC. The EXAMPLE 23 Samples were prepared by derivatization and enzymatic resolution. Chromatograms for Standard Solutions are dis Production of Erythritol/Monatin Granules played in FIG. 17. Following the reversed phase analysis, 0456. A solution of 2000 g erythritol, 16 g R,R monatin chiral chromatography was performed to evaluate specific and 5 L water is prepared in a small tank at 40 C. 58 kg of isomers present in the Samples. Chiral chromatography of erythritol is put into the basket of a fluid bed. The air Standard S.S and R,R, monatin Solutions are displayed in temperature of the fluid bed is set at 65 C. The monatin/ FIG. 18. erythritol Solution is sprayed on the fluid bed at 25 kg/hr for EXAMPLE 25 17 minutes. Additional heating time of 20 minutes is required to dry the powder. Product is sieved over a 1250 Stability of Monatin at High Temperature (80° C.) mm sieve. See also EP 0 325 790 B1 (Mitsubishi, Nikken and Neutral pH 1993). The calculated relative Sweetness of the monatin/ 0462. A 100 milliliter solution of 75 ppm monatin at pH erythritol product (as compared to Sucrose) is approximately 7 was used as a Stock Solution. The Synthetic monatin Sample US 2005/0112260 A1 May 26, 2005 33 contained approximately 96% of the 2R,4R/2S,4S enantio pearance of monatin over time is highly dependent on pH meric pair and 4% of the 2R,4S/2S,4R enantiomeric pair. Since the primary byproducts are cyclization and possibly Samples were incubated at 80° C. and pH 7 for the duration very Small levels of racemization. During the experiment at of the experiment and Samples were withdrawn at 0, 1, 2, 3, 80 C. and pH 7, no change in concentration of racemic 4 hours and 1, 2, 4, 7, 14, 21 and 35 days. All experimental RR/SS monatin or lactones thereof was detected within the conditions were run in duplicate. limits of precision afforded by using LC-MS for quantita 0463 Separation and Quantification Using LC-MS using tion. Based on this data, it is expected that at high tempera Reverse Phase Chromatography Aresponse curve was estab tures and neutral pH, monatin Stability is acceptable for lished for both detected diastereomer peaks of the synthetic baking. monatin. A range of 5-150 ppm was bracketed with the Synthetic monatin Standard dissolved in DI water. Separation 0465. In view of the many possible embodiments to of the two diastereomer peaks was accomplished using a which the principles of this disclosure may be applied, it 3.9x1 50 mm Novapak C18 (Waters Corporation) HPLC should be recognized that the illustrated embodiments are column. Ultraviolet-Visible (UV) and Mass Spectrometer only particular examples of the disclosure and should not be (MS) detectors were used in Series for detection and quan taken as a limitation on the Scope of the disclosure. For titation. Monatin and its lactone peak each have a UV at example, from the teachings of the disclosure herein, it 279 nm that aided in precise detection. Quantification was would be apparant to a perSon of ordinary skill that a done by acquiring Selected Ion Monitoring (SIM) scan of monatin Sweetener compostion can be made in liquid con 293.3 m/z and 275.3 m/z in positive-ion electrospray mode. centrate form, rather than in a Solid form, wherein a chosen 0464 Results-At a neutral pH (common for baking and Volume of the liquid concentrate, e.g., 1 mL or 0.35 mL, desserts), the degree of degradation of monatin was deter would have a Sweetness comparable to a chosen amount, mined to be insignificant even after 7-35 days. The disap Such as two teaspoons, of granulated Sugar.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS 74 <210> SEQ ID NO 1 &2 11s LENGTH 1170 &212> TYPE DNA <213> ORGANISM: Sinorhizobium meliloti

<400 SEQUENCE: 1 atgttcgacg cc citc.gc.ccg ccaag.ccgac gatc.ccttgc titttcc toat cqgcct gttc 60 aggaaggatg agcgc.ccc.gg aaagg to gat citcggcgtag gagt citatcg cqac gag acc 120 ggacgcacgc cqatctt.ccg gg.ccgtoaag gogg.cggaaa agcggcttct cqaaacacag 18O gacagdaagg cctatat cqg ccc.cgaaggg gaccitcgtot ttctogatcg gotctgggaa 240 Ctcgt.cggcg gC gacac gat cqagcggagc catgttgcgg gC gtcCagaC gCCC gg.cggC 3OO to cqgcgc.gc to cqtttggc gg.cgg acctic atc.gc.ccgca toggcggcc g aggcatctgg 360 citcgggctgc cq agctggcc galaccacgcg cc gatcttca aggcggcc.gg gcto gatato 420 gccaccitacg acttctt.cga cattcc.gtog cagtcggtoa tott.cgataa totggtgagc 480 gcqctggaag gogcc.gcatc cqgcgatgc g g togctgctgc atgcaa.gct g ccacaa.ccc.g 540 accgg.cggcg to Ctgagcga agcacaatgg atggagatcg cc.gc.gctggt gg.ccgagcgc 600 ggcctgctgc cqctogtoga totc.gccitat caggggttcg go cqcggcct cqaccaggat 660 gtogcgggcc to cqg catct to toggcgt g g toccggaag cqctcgtogc ggtttcct gc 720 togaagttcct tcgggctitta to go gag.cgc goggg.cgcga tott.cgc.gcg gaccagotcg 78O

act gcct cqg cqgacagggit gcgctcaaac citc.gcgggcc to go acgcac cago tattoc 840

atgcc.gc.cgg at cacgg.cgc agcc.gtogt g c gacgatcc ttgacg acco ggaacticagg 9 OO

cgcgactgga cqgaggagct cqagacgatg cqgctCagga tacgggcct CC gg.cggtcg 96.O

cittgcc.gagg gacitcc.gcac cogctgg cag agccitcggcg cagtc.gc.cga to aggagggc 1020 US 2005/0112260 A1 May 26, 2005 34

-continued atgttctoca togctg.ccgct titc.cgaag.cg gaggittatgc ggcto aggac cqagcacggc 1080 atctatatgc cqgcatcc.gg cc.gcatcaac atc.gc.cgggc tigaagacggc ggaagcc.gc.c 1140 gagattgc.cg gcaagttcac cagtc.tctga 1170

<210> SEQ ID NO 2 &2 11s LENGTH 389 &212> TYPE PRT <213> ORGANISM: Sinorhizobium meliloti

<400 SEQUENCE: 2 Met Phe Asp Ala Leu Ala Arg Glin Ala Asp Asp Pro Leu Lleu Phe Lieu 1 5 10 15 Ile Gly Lieu Phe Arg Lys Asp Glu Arg Pro Gly Lys Val Asp Leu Gly 2O 25 30 Val Gly Val Tyr Arg Asp Glu Thr Gly Arg Thr Pro Ile Phe Arg Ala 35 40 45 Wall Lys Ala Ala Glu Lys Arg Lieu Lieu Glu Thr Glin Asp Ser Lys Ala 50 55 60 Tyr Ile Gly Pro Glu Gly Asp Leu Val Phe Lieu. Asp Arg Lieu Trp Glu 65 70 75 8O Leu Val Gly Gly Asp Thr Ile Glu Arg Ser His Val Ala Gly Val Glin 85 90 95 Thr Pro Gly Gly Ser Gly Ala Leu Arg Leu Ala Ala Asp Leu. Ile Ala 100 105 110 Arg Met Gly Gly Arg Gly Ile Trp Lieu Gly Lieu Pro Ser Trp Pro Asn 115 120 125 His Ala Pro Ile Phe Lys Ala Ala Gly Lieu. Asp Ile Ala Thr Tyr Asp 130 135 1 4 0 Phe Phe Asp Ile Pro Ser Glin Ser Val Ile Phe Asp Asn Leu Val Ser 145 15 O 155 160 Ala Leu Glu Gly Ala Ala Ser Gly Asp Ala Val Lieu Lleu. His Ala Ser 1.65 170 175 Cys His Asn Pro Thr Gly Gly Val Leu Ser Glu Ala Gln Trp Met Glu 18O 185 19 O Ile Ala Ala Lieu Val Ala Glu Arg Gly Lieu Lleu Pro Leu Val Asp Lieu 195 200 2O5 Ala Tyr Glin Gly Phe Gly Arg Gly Lieu. Asp Glin Asp Wall Ala Gly Lieu 210 215 220 Arg His Lieu Lieu Gly Val Val Pro Glu Ala Lieu Val Ala Val Ser Cys 225 230 235 240 Ser Lys Ser Phe Gly Lieu. Tyr Arg Glu Arg Ala Gly Ala Ile Phe Ala 245 250 255 Arg Thr Ser Ser Thr Ala Ser Ala Asp Arg Val Arg Ser Asn Lieu Ala 260 265 27 O Gly Leu Ala Arg Thr Ser Tyr Ser Met Pro Pro Asp His Gly Ala Ala 275 280 285 Val Val Arg Thr Ile Lieu. Asp Asp Pro Glu Lieu Arg Arg Asp Trp Thr 29 O 295 3OO Glu Glu Lieu Glu Thr Met Arg Lieu Arg Met Thr Gly Lieu Arg Arg Ser 305 310 315 320 Leu Ala Glu Gly Lieu Arg Thr Arg Trp Glin Ser Leu Gly Ala Val Ala 325 330 335

US 2005/0112260 A1 May 26, 2005 36

-continued

Glin Pro Ala Asp Lys Ile Leu Glin Lieu. Ile Glin Met Phe Arg Glu Asp 35 40 45 Ala Arg Ala Asp Lys Ile Asp Leu Gly Val Gly Val Tyr Lys Asp Pro 50 55 60 Thr Gly Lieu. Thr Pro Wal Met Arg Ala Wall Lys Ala Ala Glu Lys Arg 65 70 75 8O Leu Trp Glu Val Glu Thir Thr Lys Thr Tyr Thr Gly Leu Ala Asp Glu 85 90 95 Pro Ala Tyr Asn Ala Ala Met Ala Lys Lieu. Ile Leu Ala G Ala Wall 100 105 1 : Pro Ala Asp Arg Val Ala Ser Val Ala Thr Pro Gly Gly Thr Gly Ala 115 120 125 Val Arg Glin Ala Leu Glu Lieu. Ile Arg Met Ala Ser Pro Glu Ala Thr 130 135 1 4 0 Val Trp Ile Ser Asn Pro Thir Trp Pro Asn His Leu Ser Ile Val Lys 145 15 O 155 160 Tyr Leu Gly Ile Pro Met Arg Glu Tyr Arg Tyr Phe Asp Ala Glu Thr 1.65 170 175 Gly Ala Val Asp Ala Glu Gly Met Met Glu Asp Leu Ala Glin Val Lys 18O 185 19 O Ala Gly Asp Val Val Lieu Lleu. His Gly Cys Cys His Asn Pro Thr Gly 195 200 2O5 Ala Asn Pro Asn Pro Val Glin Trp Lieu Ala Ile Cys Glu Ser Lieu Ala 210 215 220 Arg Thr Gly Ala Val Pro Leu. Ile Asp Leu Ala Tyr Glin Gly Phe Gly 225 230 235 240 Asp Gly Lieu Glu Met Asp Ala Ala Ala Thr Arg Lieu Lleu Ala Thr Arg 245 250 255 Leu Pro Glu Val Lieu. Ile Ala Ala Ser Cys Ser Lys Asn. Phe Gly Ile 260 265 27 O Tyr Arg Glu Arg Thr Gly Ile Lieu. Ile Ala Ile Gly Glu Ala Ala Gly 275 280 285 Arg Gly Thr Val Glin Ala Asn Lieu. Asn. Phe Lieu. Asn Arg Glin Asn Tyr 29 O 295 3OO Ser Phe Pro Pro Asp His Gly Ala Arg Leu Val Thr Met Ile Leu Glu 305 310 315 320 Asp Glu Thir Lieu Ser Ala Asp Trp Lys Ala Glu Lieu Glu Glu Val Arg 325 330 335 Lieu. Asn Met Lieu. Thir Lieu Arg Arg Glin Leu Ala Asp Ala Leu Glin Ala 340 345 35 O Glu Thr Gly Ser Asn Arg Phe Gly Phe Val Ala Glu His Arg Gly Met 355 360 365 Phe Ser Arg Leu Gly Ile Thr Pro Ala Glu Val Glu Arg Leu Arg Thr 370 375 38O Glu His Gly Val Tyr Met Val Gly Asp Ser Arg Lieu. Asn. Ile Ala Gly 385 390 395 400 Lieu. Asn Arg Thr Thr Val Pro Val Lieu Ala Arg Ala Val Ala Lys Wal 405 410 415 Leu Arg Gly

US 2005/0112260 A1 May 26, 2005 38

-continued

100 105 110 Pro Ala Asp Arg Val Ala Ser Val Ala Thr Pro Gly Gly Thr Gly Ala 115 120 125 Val Arg Glin Ala Leu Glu Lieu. Ile Arg Met Ala Ser Pro Glu Ala Thr 130 135 1 4 0 Val Trp Ile Ser Asn Pro Thir Trp Pro Asn His Leu Ser Ile Val Lys 145 15 O 155 160 Tyr Leu Gly Ile Pro Met Arg Glu Tyr Arg Tyr Phe Asp Ala Glu Thr 1.65 170 175 Gly Ala Val Asp Ala Glu Gly Lieu Met Glu Asp Leu Ala Glin Val Lys 18O 185 19 O Ala Gly Asp Val Val Lieu Lleu. His Gly Cys Cys His Asn Pro Thr Gly 195 200 2O5 Ala Asn Pro Asn Pro Val Glin Trp Lieu Ala Val Cys Glu Ser Lieu Ala 210 215 220 Arg Thr Gly Ala Val Pro Leu. Ile Asp Leu Ala Tyr Glin Gly Phe Gly 225 230 235 240 Asp Gly Lieu Glu Met Asp Ala Ala Ala Thr Arg Lieu Lleu Ala Thr Arg 245 250 255 Leu Pro Glu Val Lieu. Ile Ala Ala Ser Cys Ser Lys Asn. Phe Gly Ile 260 265 27 O Tyr Arg Glu Arg Thr Gly Ile Leu. Ile Ala Ile Gly Glu Ala Ala Gly 275 280 285 Arg Gly Thr Val Glin Ala Asn Lieu. Asn. Phe Lieu. Asn Arg Glin Asn Tyr 29 O 295 3OO Ser Phe Pro Pro Asp His Gly Ala Arg Leu Val Thr Met Ile Leu Glu 305 310 315 320 Asp Glu Thir Lieu Ser Ala Asp Trp Lys Ala Glu Lieu Glu Glu Val Arg 325 330 335 Lieu. Asn Met Lieu. Thir Lieu Arg Arg Glin Leu Ala Asp Ala Leu Glin Ala 340 345 35 O Glu Thr Gly Ser Asn Arg Phe Gly Phe Val Ala Glu His Arg Gly Met 355 360 365 Phe Ser Arg Leu Gly Ile Thr Pro Ala Glu Val Glu Arg Leu Arg Thr 370 375 38O Glu His Gly Val Tyr Met Val Gly Asp Ser Arg Lieu. Asn. Ile Ala Gly 385 390 395 400 Lieu. Asn Arg Thr Thr Val Pro Val Lieu Ala Arg Ala Val Ala Lys Wal 405 410 415 Leu Arg Gly

<210 SEQ ID NO 7 &2 11s LENGTH 1239 &212> TYPE DNA <213> ORGANISM: Leishmania major <400 SEQUENCE: 7 atgtc.catgc aggcggcc at gaccacgg.cg gag.cgctggc agaagattca ggcacaagct 60 cc.cgatgtca tottcgatct c gcaaaacgc gcc.gcc.gctd coaaggg.ccc caaggccaac 120 citcg to attg gtgcct accq C gacgagcag ggcc.gtocct atcc.gctac g c gtggtoc.gc 18O aaggctgagc agcttctott go acatgaat citcgacitacg agtacct acc catctogggc 240

US 2005/0112260 A1 May 26, 2005 40

-continued Leu Ala Ala Pro Asp Gly Ser Val Phe Ile Lieu. His Glin Cys Ala His 18O 185 19 O Asn Pro Thr Gly Val Asp Pro Ser Glin Glu Gln Trp Asn Glu Ile Ala 195 200 2O5 Ser Leu Met Leu Ala Lys His His Glin Val Phe Phe Asp Ser Ala Tyr 210 215 220 Glin Gly Tyr Ala Ser Gly Ser Lieu. Asp Thr Asp Ala Tyr Ala Ala Arg 225 230 235 240 Leu Phe Ala Arg Arg Gly Ile Glu Val Lieu Lieu Ala Glin Ser Phe Ser 245 250 255 Lys Asn Met Gly Lieu. Tyr Ser Glu Arg Ala Gly Thr Lieu Ser Lieu Lieu 260 265 27 O Leu Lys Asp Llys Thr Lys Arg Ala Asp Wall Lys Ser Wal Met Asp Ser 275 280 285 Lieu. Ile Arg Glu Glu Tyr Thr Cys Pro Pro Ala His Gly Ala Arg Lieu 29 O 295 3OO Ala His Lieu. Ile Leu Ser Asn. Asn. Glu Lieu Arg Lys Glu Trp Glu Ala 305 310 315 320 Glu Leu Ser Ala Met Ala Glu Arg Ile Arg Thr Met Arg Arg Thr Val 325 330 335 Tyr Asp Glu Lieu Lleu Arg Lieu Glin Thr Pro Gly Ser Trp Glu His Val 340 345 35 O Ile Asin Glin Ile Gly Met Phe Ser Phe Leu Gly Lieu Ser Lys Ala Glin 355 360 365 Cys Glu Tyr Cys Glin Asn His Asn Ile Phe Ile Thr Val Ser Gly Arg 370 375 38O Ala Asn Met Ala Gly Lieu. Thr His Glu Thir Ala Lieu Met Leu Ala Glin 385 390 395 400 Thir Ile Asn Asp Ala Val Arg Asn. Wall Asn Arg Glu 405 410

<210 SEQ ID NO 9 <211& LENGTH: 1182 &212> TYPE DNA <213> ORGANISM: Bacillus subtilis

<400 SEQUENCE: 9 atggaacatt togctgaatcc gaaagcaaga gagat.cgaaa titt caggaat acgcaaattic 60 to gaatcttg tagcc.caa.ca cqaagacg to atttcactta caatcggcca gcc to attitt 120 ttcacaccgc atcatgtgaa agctdcc.gca aaaaaag.cca ttgatgaaaa cqtgacgtoa 18O tatacticcga atgcc.ggcta cct ggagctg agacaagctg togcagottta tatgaagaaa 240 aaag.cggatt toaactata togctgaatct galaattatca totacaac agg cqcaa.gc.cala 3OO gccattgatg citgcattcc.g. gacgattitta totcc.cggtg atgaagttcat tatgc.caggg 360 ccitatttatc cqggctata accitattatc aatttgtgc g g g gccaa.gcc tdt cattgtt 420 gatactacgt cacacggctt taagcttacc gcc.cggctga ttgaagatgc tict gacaccc 480 aacaccaagt gtgtcgtgct tccittatcc.g. tcaaaccota ccggcgtgac tittatctgaa 540 gaagaact ga aaag catcgc agctotcitta aaagg cagaa atgtc.ttcgt attgttctgat 600 gaaatataca gtgaattaac atatgacaga cc.gcattact coatcgcaac citatttgcgg 660 gatcaaacga ttgtcattaa C gg gttgtca aaatcacaca gcato accgg ttggaga att 720 US 2005/0112260 A1 May 26, 2005 41

-continued ggatttitt at ttgcaccgaa agacattgca aag cacattt taalaggttca totaatacaat 78O gtgtcgtgcg cctoratcc at ttctoaaaaa gocgc.gcttg aagctgtcac aaacggctitt 840 gacgatgcat tdattatgag agaacaatac aaaaaacg.to togactatot titatgaccgt. 9 OO cittgtttcca tagg acttga cqtagittaaa cc.gtocggtg cgttittatat cittcc cittct 96.O attaaatcat ttggaatgac titcatttgat tittagtatgg citcttittgga agacgctggc 1020 gtgg cacticg togc.cgggcag citc gttctica acatatggtg aaggatatgt aaggctgtct 1080 tittgcatgct caatggacac gotgagagaa gqcctag acc gtttagaatt atttgtatta 1140 aaaaaacgtg aag caatgca gacgataaac aacgg.cgttt aa 1182

<210> SEQ ID NO 10 &2 11s LENGTH 393 &212> TYPE PRT <213> ORGANISM: Bacillus subtilis

<400 SEQUENCE: 10 Met Glu His Lieu Lleu. Asn Pro Lys Ala Arg Glu Ile Glu Ile Ser Gly 1 5 10 15 Ile Arg Llys Phe Ser Asn Lieu Val Ala Gln His Glu Asp Val Ile Ser 2O 25 30 Leu Thir Ile Gly Gln Pro Asp Phe Phe Thr Pro His His Val Lys Ala 35 40 45 Ala Ala Lys Lys Ala Ile Asp Glu Asn Val Thr Ser Tyr Thr Pro Asn 50 55 60 Ala Gly Tyr Lieu Glu Lieu Arg Glin Ala Val Glin Leu Tyr Met Lys Lys 65 70 75 8O Lys Ala Asp Phe Asn Tyr Asp Ala Glu Ser Glu Ile Ile Ile Thir Thr 85 90 95 Gly Ala Ser Glin Ala Ile Asp Ala Ala Phe Arg Thr Ile Leu Ser Pro 100 105 110 Gly Asp Glu Val Ile Met Pro Gly Pro Ile Tyr Pro Gly Tyr Glu Pro 115 120 125 Ile Ile Asn Leu Cys Gly Ala Lys Pro Val Ile Val Asp Thr Thr Ser 130 135 1 4 0 His Gly Phe Lys Lieu. Thr Ala Arg Lieu. Ile Glu Asp Ala Lieu. Thr Pro 145 15 O 155 160 Asn Thr Lys Cys Val Val Leu Pro Tyr Pro Ser Asn Pro Thr Gly Val 1.65 170 175 Thr Lieu Ser Glu Glu Glu Lieu Lys Ser Ile Ala Ala Lieu Lleu Lys Gly 18O 185 19 O Arg Asn Val Phe Val Leu Ser Asp Glu Ile Tyr Ser Glu Leu Thr Tyr 195 200 2O5 Asp Arg Pro His Tyr Ser Ile Ala Thr Tyr Leu Arg Asp Gln Thr Ile 210 215 220 Val Ile Asin Gly Leu Ser Lys Ser His Ser Met Thr Gly Trp Arg Ile 225 230 235 240 Gly Phe Lieu Phe Ala Pro Lys Asp Ile Ala Lys His Ile Leu Lys Wal 245 250 255 His Glin Tyr Asn. Wal Ser Cys Ala Ser Ser Ile Ser Glin Lys Ala Ala 260 265 27 O US 2005/0112260 A1 May 26, 2005 42

-continued Leu Glu Ala Val Thr Asn Gly Phe Asp Asp Ala Lieu. Ile Met Arg Glu 275 280 285 Glin Tyr Lys Lys Arg Lieu. Asp Tyr Val Tyr Asp Arg Lieu Val Ser Met 29 O 295 3OO Gly Leu Asp Val Val Lys Pro Ser Gly Ala Phe Tyr Ile Phe Pro Ser 305 310 315 320 Ile Lys Ser Phe Gly Met Thr Ser Phe Asp Phe Ser Met Ala Leu Leu 325 330 335 Glu Asp Ala Gly Val Ala Leu Val Pro Gly Ser Ser Phe Ser Thr Tyr 340 345 35 O Gly Glu Gly Tyr Val Arg Leu Ser Phe Ala Cys Ser Met Asp Thr Leu 355 360 365 Arg Glu Gly Lieu. Asp Arg Lieu Glu Lieu Phe Val Lieu Lys Lys Arg Glu 370 375 38O Ala Met Gln Thr Ile Asn Asn Gly Val 385 390

<210> SEQ ID NO 11 &2 11s LENGTH 1176 &212> TYPE DNA <213> ORGANISM: Lactobacillus amylovorus <400 SEQUENCE: 11 atgccagaat tagctaatga tittaggatta agcaaaaaga toactgatgt aaaag Cttca 60 ggaattagaa totttgataa caaagtttca gctattocto goattatcaa attgactittg 120 ggtgaaccag atatgaatac toctogagcat gttaa.gcaag cqgct attaa gaatattgca 18O gataatgatt cacactatoc tocacaaaag ggaaagcttgaattaagaaa agctato agt 240 aaatatttga aaaagattac toggaattgaa tatgatccag aaacagaaat cqtagtaa.ca 3OO gttggtgcaa citgaag caat taacgctacc ttgtttgcta ttactaatcc gggtgacaag 360 gttgcaattic citacgc.cagt cittittctota tattgg.cccg tdgctacact tactgatgcc 420 gattatgttt tatgaatac toc agaagat ggttittaagt talacaccitaa gaagttagaa 480 gaaactatoa aagaaaatcc aacaattaaa goagtaattt toga attatcc aacta acco a 540 actggtgttgaatatagoga agatgaaatt aaagctttgg cita aggtaat taaagataat 600 catctgtacg taattaccga tigaaatttac agtactittga cittacggtgt aaaac actitt 660 tdaattgcca gcttaattcc agaaagagca atttatatot citggitttatc taaatcacat 720 gc gatgacitg gttatcgttt aggctatott gcc.gg acct g caaaaattat ggcagaaatt 78O ggtaaagttc atggccittat ggtgacgact acgacggatt catcacaagc tigcc.gcaatt 840 gaag cactitg alacacgg act to atgaccct gagaaatata gggaagttta toaaaag.cgt. 9 OO cgtgactato ttittaaagga attagcc.gag atagagatgc aag cagttaa goc agaaggt 96.O gcattittata totttgctaa aattic cagct aagtatggca aagacgatat galaatttgcc 1020 ttggatttag cittittaaaga aaaagtgggt atc acto cag gtagtgcatt togtoctogt 1080 ggtgaagg to atattagatt atctitatgca toaagtgatgaaaacttgca toaggcaatg 1140 aagc gaatga agaaagttitt acaagaggac gaataa 1176

<210> SEQ ID NO 12 &2 11s LENGTH 391 &212> TYPE PRT US 2005/0112260 A1 May 26, 2005 43

-continued <213> ORGANISM: Lactobacillus amylovorus <400 SEQUENCE: 12 Met Pro Glu Lieu Ala Asn Asp Leu Gly Lieu Ser Lys Lys Ile Thr Asp 1 5 10 15 Wall Lys Ala Ser Gly Ile Arg Ile Phe Asp Asn Lys Wal Ser Ala Ile 2O 25 30 Pro Gly Ile Ile Lys Leu Thir Leu Gly Glu Pro Asp Met Asn Thr Pro 35 40 45 Glu His Wall Lys Glin Ala Ala Ile Lys Asn. Ile Ala Asp Asn Asp Ser 50 55 60 His Tyr Ala Pro Glin Lys Gly Lys Lieu Glu Lieu Arg Lys Ala Ile Ser 65 70 75 8O Lys Tyr Lieu Lys Lys Ile Thr Gly Ile Glu Tyr Asp Pro Glu Thr Glu 85 90 95 Ile Val Val Thr Val Gly Ala Thr Glu Ala Ile Asn Ala Thr Leu Phe 100 105 110 Ala Ile Thr Asn Pro Gly Asp Llys Val Ala Ile Pro Thr Pro Val Phe 115 120 125 Ser Leu Tyr Trp Pro Val Ala Thr Lieu Ala Asp Ala Asp Tyr Val Lieu 130 135 1 4 0 Met Asn. Thir Ala Glu Asp Gly Phe Lys Lieu. Thr Pro Lys Lys Lieu Glu 145 15 O 155 160 Glu Thir Ile Lys Glu Asn Pro Thir Ile Lys Ala Val Ile Lieu. Asn Tyr 1.65 170 175 Pro Thr Asn Pro Thr Gly Val Glu Tyr Ser Glu Asp Glu Ile Lys Ala 18O 185 19 O Leu Ala Lys Val Ile Lys Asp Asn His Leu Tyr Val Ile Thr Asp Glu 195 200 2O5 Ile Tyr Ser Thr Leu Thir Tyr Gly Val Lys His Phe Ser Ile Ala Ser 210 215 220 Lieu. Ile Pro Glu Arg Ala Ile Tyr Ile Ser Gly Lieu Ser Lys Ser His 225 230 235 240 Ala Met Thr Gly Tyr Arg Leu Gly Tyr Val Ala Gly Pro Ala Lys Ile 245 250 255 Met Ala Glu Ile Gly Lys Val His Gly Leu Met Val Thr Thr Thr Thr 260 265 27 O Asp Ser Ser Glin Ala Ala Ala Ile Glu Ala Leu Glu His Gly Lieu. Asp 275 280 285 Asp Pro Glu Lys Tyr Arg Glu Val Tyr Glu Lys Arg Arg Asp Tyr Val 29 O 295 3OO Leu Lys Glu Lieu Ala Glu Ile Glu Met Glin Ala Wall Lys Pro Glu Gly 305 310 315 320 Ala Phe Tyr Ile Phe Ala Lys Ile Pro Ala Lys Tyr Gly Lys Asp Asp 325 330 335 Met Lys Phe Ala Lieu. Asp Leu Ala Phe Lys Glu Lys Val Gly Ile Thr 340 345 35 O Pro Gly Ser Ala Phe Gly Pro Gly Gly Glu Gly His Ile Arg Lieu Ser 355 360 365 Tyr Ala Ser Ser Asp Glu Asn Lieu. His Glu Ala Met Lys Arg Met Lys 370 375 38O

US 2005/0112260 A1 May 26, 2005 45

-continued Ala Leu Gly Val His Arg Asn. Thr Val Asp Ala Ala Tyr Glin Glu Lieu 50 55 60 Leu Thr Glin Gly Trp Leu Glin Ala Glu Pro Ala Arg Gly Thr Phe Val 65 70 75 8O Ala Glin Asp Leu Pro Glin Gly Met Lieu Val His Arg Pro Ala Pro Ala 85 90 95 Pro Val Glu Pro Val Ala Met Arg Ala Gly Leu Ala Phe Ser Asp Gly 100 105 110 Ala Pro Asp Pro Glu Lieu Val Pro Asp Lys Ala Lieu Ala Arg Ala Phe 115 120 125 Arg Arg Ala Lieu Lleu Ser Pro Ala Phe Arg Ala Gly Ala Asp Tyr Gly 130 135 1 4 0 Asp Ala Arg Gly Thr Ser Ser Lieu Arg Glu Ala Lieu Ala Ala Tyr Lieu 145 15 O 155 160 Ala Ser Asp Arg Gly Val Val Ala Asp Pro Ala Arg Lieu Lieu. Ile Ala 1.65 170 175 Arg Gly Ser Glin Met Ala Leu Phe Lieu Val Ala Arg Ala Ala Lieu Ala 18O 185 19 O Pro Gly Glu Ala Ile Ala Val Glu Glu Pro Gly Tyr Pro Leu Ala Trp 195 200 2O5 Glu Ala Phe Arg Ala Ala Gly Ala Glu Val Arg Gly Val Pro Val Asp 210 215 220 Gly Gly Gly Lieu Arg Ile Asp Ala Leu Glu Ala Ala Lieu Ala Arg Asp 225 230 235 240 Pro Arg Ile Arg Ala Val Tyr Val Thr Pro His His Glin Tyr Pro Thr 245 250 255 Thr Val Thr Met Gly Ala Ala Arg Arg Lieu Glin Leu Lleu Glu Lieu Ala 260 265 27 O Glu Arg His Arg Lieu Ala Lieu. Ile Glu Asp Asp Tyr Asp His Glu Tyr 275 280 285 Arg Phe Glu Gly Arg Pro Val Lieu Pro Leu Ala Ala Arg Ala Pro Glu 29 O 295 3OO Gly Lieu Pro Lieu. Ile Tyr Val Gly Ser Lieu Ser Lys Lieu Lleu Ser Pro 305 310 315 320 Gly Ile Arg Lieu Gly Tyr Ala Lieu Ala Pro Glu Arg Lieu Lieu. Thir Arg 325 330 335 Met Ala Ala Ala Arg Ala Ala Ile Asp Arg Glin Gly Asp Ala Pro Leu 340 345 35 O Glu Ala Ala Lieu Ala Glu Lieu. Ile Arg Asp Gly Asp Lieu Gly Arg His 355 360 365 Ala Arg Lys Ala Arg Arg Val Tyr Arg Ala Arg Arg Asp Leu Lieu Ala 370 375 38O Glu Arg Lieu. Thir Ala Glin Leu Ala Gly Arg Ala Ala Phe Asp Leu Pro 385 390 395 400 Ala Gly Gly Lieu Ala Lieu Trp Lieu Arg Cys Ala Gly Val Ser Ala Glu 405 410 415 Thir Trp Ala Glu Ala Ala Gly Glin Ala Gly Lieu Ala Lieu Lleu Pro Gly 420 425 43 O Thr Arg Phe Ala Leu Glu Ser Pro Ala Pro Glin Ala Phe Arg Lieu Gly 435 4 40 4 45 Tyr Ala Ala Lieu. Asp Glu Gly Glin Ile Ala Arg Ala Val Glu Ile Leu US 2005/0112260 A1 May 26, 2005 46

-continued

450 455 460 Ala Arg Ser Phe Pro Gly 465 470

<210 SEQ ID NO 15 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 15 gg tatt gagg gtc.gcatgaa gottt tagtc. aatgg 35

<210> SEQ ID NO 16 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 16 agaggagagt tagagc citta toaaatgcta gcagoct 37

<210 SEQ ID NO 17 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 17 gg tatt gagg gtc.gcatgtt cqacgc.cc to go.ccg 35

<210> SEQ ID NO 18 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 18 agaggagagt tagagcctica gag actdgtgaacttgc 37

<210 SEQ ID NO 19 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 19 gg tatt gagg gtc.gcatgga acatttgctgaatcc 35

<210> SEQ ID NO 20 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 20 agaggagagt tagagc citta aacgc.cgttg tittatcg 37 US 2005/0112260 A1 May 26, 2005 47

-continued

<210> SEQ ID NO 21 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 21 gg tatt gagg gtc.gcatgcg cqagc citctt gcc ct 35

<210> SEQ ID NO 22 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 22 agaggagagt tagagcctica gcc.ggggaag Citc.cggg 37

<210> SEQ ID NO 23 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 23 gg tatt gagg gtc.gcatgtc. cacgcaggcg gcc at 35

<210> SEQ ID NO 24 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 24 agaggagagt tagagcctica citcacgatto acattgc 37

<210> SEQ ID NO 25 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 25 gg tatt gagg gtc.gcatgcc agaattagct aatga 35

<210> SEQ ID NO 26 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 26 agaggagagt tagagc citta titcgtc.citct totaaaa 37

<210 SEQ ID NO 27 &2 11s LENGTH 35

US 2005/0112260 A1 May 26, 2005

-continued gtgagcaatt cqttct cqaa aattittcticc citttacggcg agc.gc.gtcgg cqgactittct 78O gttatgtgtg aagatgcc ga agcc.gctggc cqc gtactgg ggcaattgaa agcaa.cagtt 840 cgcc.gcaact acticcagocc gcc gaattitt gotgcgcagg togtogctgc agtgctgaat 9 OO gacgaggcat togaaag.ccag citggctgg.cg gaagtagaag agatgcgtac togcattctg 96.O gcaatgcg to aggaattggit galagg tatta agcacagaga tigccagaacg caattitc gat 1020 tatctgctta atcagogcgg catgttcagt tataccggitt taagtgcc.gc. tcaggttgac 1080 cgacitacgtg aagaatttgg totctatotc atc.gc.ca.gcg gtc.gcatctg tdtc.gc.cggg 1140 ttaaatacgg caaatgtaca acgtgtggca aaggc gtttg ctg.cggtgat gtaa 1194

<210> SEQ ID NO 32 &2 11s LENGTH 397 &212> TYPE PRT <213> ORGANISM: E. coli

<400 SEQUENCE: 32 Val Phe Glin Lys Val Asp Ala Tyr Ala Gly Asp Pro Ile Lieu. Thir Lieu 1 5 10 15 Met Glu Arg Phe Lys Glu Asp Pro Arg Ser Asp Llys Val Asn Lieu Ser 2O 25 30 Ile Gly Lieu. Tyr Tyr Asn. Glu Asp Gly Ile Ile Pro Glin Leu Glin Ala 35 40 45 Val Ala Glu Ala Glu Ala Arg Lieu. Asn Ala Glin Pro His Gly Ala Ser 50 55 60 Leu Tyr Lieu Pro Met Glu Gly Lieu. Asn. Cys Tyr Arg His Ala Ile Ala 65 70 75 8O Pro Leu Lieu Phe Gly Ala Asp His Pro Val Lieu Lys Glin Glin Arg Val 85 90 95 Ala Thir Ile Glin Thr Lieu Gly Gly Ser Gly Ala Lieu Lys Val Gly Ala 100 105 110 Asp Phe Leu Lys Arg Tyr Phe Pro Glu Ser Gly Val Trp Val Ser Asp 115 120 125 Pro Thr Trp Glu Asn Arg Val Ala Ile Phe Ala Gly Ala Gly Phe Glu 130 135 1 4 0 Val Ser Thr Tyr Pro Trp Tyr Asp Glu Ala Thr Asn Gly Val Arg Phe 145 15 O 155 160 Asn Asp Leu Lieu Ala Thr Lieu Lys Thr Lieu Pro Ala Arg Ser Ile Val 1.65 170 175 Leu Lieu. His Pro Cys Cys His Asn Pro Thr Gly Ala Asp Lieu. Thir Asn 18O 185 19 O Asp Gln Trp Asp Ala Val Ile Glu Ile Leu Lys Ala Arg Glu Lieu. Ile 195 200 2O5 Pro Phe Leu Asp Ile Ala Tyr Glin Gly Phe Gly Ala Gly Met Glu Glu 210 215 220 Asp Ala Tyr Ala Ile Arg Ala Ile Ala Ser Ala Gly Lieu Pro Ala Lieu 225 230 235 240 Val Ser Asn Ser Phe Ser Lys Ile Phe Ser Leu Tyr Gly Glu Arg Val 245 250 255 Gly Gly Lieu Ser Val Met Cys Glu Asp Ala Glu Ala Ala Gly Arg Val 260 265 27 O US 2005/0112260 A1 May 26, 2005 50

-continued Leu Gly Glin Leu Lys Ala Thr Val Arg Arg Asn Tyr Ser Ser Pro Pro 275 280 285 Asn Phe Gly Ala Glin Val Val Ala Ala Val Lieu. Asn Asp Glu Ala Lieu 29 O 295 3OO Lys Ala Ser Trp Leu Ala Glu Val Glu Glu Met Arg Thr Arg Ile Leu 305 310 315 320 Ala Met Arg Glin Glu Leu Val Lys Val Leu Ser Thr Glu Met Pro Glu 325 330 335 Arg Asin Phe Asp Tyr Leu Leu Asn Glin Arg Gly Met Phe Ser Tyr Thr 340 345 35 O Gly Lieu Ser Ala Ala Glin Val Asp Arg Lieu Arg Glu Glu Phe Gly Val 355 360 365 Tyr Lieu. Ile Ala Ser Gly Arg Met Cys Wall Ala Gly Lieu. Asn. Thir Ala 370 375 38O Asn Val Glin Arg Val Ala Lys Ala Phe Ala Ala Wal Met 385 390 395

<210 SEQ ID NO 33 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400> SEQUENCE: 33 gg tatt gagg gtc.gc.gtgtt toaaaaagtt gacgc 35

<210> SEQ ID NO 34 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 34 agaggagagt tagagc citta catcaccgca gcaaacg. 37

<210 SEQ ID NO 35 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 35 gg tatt gagg gtc.gcatgga gtccaaagttc gttga 35

<210 SEQ ID NO 36 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 36 agaggagagt tagagc citta cacttggaaa acagoct 37

<210 SEQ ID NO 37 &2 11s LENGTH 35 &212> TYPE DNA US 2005/0112260 A1 May 26, 2005 51

-continued <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 37 gg tatt gagg gtc.gcatgaa aaactggaaa acaag 35

<210 SEQ ID NO 38 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 38 agaggagagt tagagc citta cagottagcg cct tota 37

<210 SEQ ID NO 39 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 39 gg tatt gagg gtc.gcatgcg agggg catta ttcaa 35

<210> SEQ ID NO 40 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 40 agaggagagt tagagcctica gcc cittgagc gcgaag 36

<210> SEQ ID NO 41 <211& LENGTH 1416 &212> TYPE DNA <213> ORGANISM E. coli

<400 SEQUENCE: 41 atggaaaact ttaaa.catct ccctdaac cq titcc.gcattc gtgttattga gcc agtaaaa 60 cgtaccacco gc gottatcg tgaagaggca attattaaat cc.gg tatgaa ccc.gttcctg 120 citggatagog aagatgttitt tatcg attta citg accgaca gCggCaccgg ggcggtgacg 18O cagagcatc aggctg.cgat gatgcgcggC gacgaagcct acagcgg Cag togtagctac 240 tatgcgittag cc.gagt cagt gaaaaatato tittggittatc aatacaccat tdcgacticac

Cagg gcc.gtg gcgcagagca aatctatatt cc.ggtactga ttaaaaaacg. Cgagcaggaa 360 alaaggcctgg atc.gcagdaa aatggtgg.cg ttcticta act atttctittga taccacgcag 420 ggccatagoc agatcaacgg citgtaccgtg cgtaacgtot atatoaaaga agc ctitc gat 480 acggg.cgtgc gttacgacitt taaaggcaac tittgaccittg agggattaga acgcgg tatt 540 gaagaagttg gtocgaataa cgtgc.cgitat atcgttgcaa ccatcaccag taactctgca 600 ggtggtcago cggitttcact ggcaaactta aaag.cgatgt acago atc.gc gaagaaatac 660 gatatto.cgg tggtaatgga tittgctgaaa acgccitatitt catcaag cag 720

US 2005/0112260 A1 May 26, 2005 53

-continued tacgagcc ga agcagttgcg tittctttact gcacgctittg attacatcta a 1371.

<210> SEQ ID NO 43 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 43 gg tatt gagg gtc.gcatgga aaactittaaa catct 35

<210> SEQ ID NO 44 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 44 agaggagagt tagagc citta aacttctitta agttittg 37

<210> SEQ ID NO 45 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 45 gg tatt gagg gtc.gcatgaa ttatc.cggca galacc 35

<210> SEQ ID NO 46 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 46 agaggagagt tagagc citta gatgtaatca aag.cgtg 37

<210> SEQ ID NO 47 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 47 ccaggg cacc gg.cgcagagc aaatctatat it 31

<210> SEQ ID NO 48 &2 11s LENGTH 30 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 48 tgcgc.cggtg CCCtggtgag toggaatggit 30

<210 SEQ ID NO 49 US 2005/0112260 A1 May 26, 2005 54

-continued

&2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 49 to citgcacgc ggcaaagggit totgcacticg gt 32

<210 SEQ ID NO 50 &2 11s LENGTH 30 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 50 citttgcc.gcg togcaggaagg citt.ccc.gaca 30

<210 SEQ ID NO 51 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 51 aggggaccgg cqc agaaaac Ctgttatcg 29

<210> SEQ ID NO 52 &2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 52 totgc.gc.cgg toccctgg to agtcggaaca at 32

<210 SEQ ID NO 53 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 53 gttagt cogc gtctacgaag g gatgccat 29

<210> SEQ ID NO 54 &2 11s LENGTH 29 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 54 gtag acgcgg actaactcitt togcagaag 29

<210 SEQ ID NO 55 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE US 2005/0112260 A1 May 26, 2005 55

-continued <223> OTHER INFORMATION: primer <400 SEQUENCE: 55 gg tatt gagg gtc.gcatgta C gaactggga gttgt 35

<210 SEQ ID NO 56 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 56 agaggagagt tagagc citta gtcaatatat titcaggc 37

<210 SEQ ID NO 57 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 57 gg tatt gagg gtc.gcatgtc. c.gg catcgtt gtc.ca 35

<210 SEQ ID NO 58 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 58 agaggagagt tagagcctica gacatattitc agtcc.ca 37

<210 SEQ ID NO 59 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 59 gg tatt gagg gtc.gcatgcg actgaacaac citcgg 35

<210 SEQ ID NO 60 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 60 agaggagagt tagagcctica gttcticcacg tatto.ca 37

<210> SEQ ID NO 61 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 61

US 2005/0112260 A1 May 26, 2005 57

-continued <400 SEQUENCE: 66 Met Tyr Glu Lieu Gly Val Val Tyr Arg Asn. Ile Glin Arg Ala Asp Arg 1 5 10 15 Ala Ala Ala Asp Gly Lieu Ala Ala Lieu Gly Ser Ala Thr Val His Glu 2O 25 30 Ala Met Gly Arg Val Gly Leu Leu Lys Pro Tyr Met Arg Pro Ile Tyr 35 40 45 Ala Gly Lys Glin Val Ser Gly. Thr Ala Val Thr Val Leu Leu Glin Pro 50 55 60 Gly Asp Asn Trp Met Met His Val Ala Ala Glu Glin Ile Glin Pro Gly 65 70 75 8O Asp Ile Val Val Ala Ala Val Thr Ala Glu Cys Thr Asp Gly Tyr Phe 85 90 95 Gly Asp Lieu Lieu Ala Thr Ser Phe Glin Ala Arg Gly Ala Arg Ala Lieu 100 105 110 Ile Ile Asp Ala Gly Val Arg Asp Wall Lys Thr Lieu Glin Glu Met Asp 115 120 125 Phe Pro Val Trp Ser Lys Ala Ile Ser Ser Lys Gly Thr Ile Lys Ala 130 135 1 4 0 Thr Leu Gly Ser Val Asn Ile Pro Ile Val Cys Ala Gly Met Leu Val 145 15 O 155 160 Thr Pro Gly Asp Val Ile Val Ala Asp Asp Asp Gly Val Val Cys Val 1.65 170 175 Pro Ala Ala Arg Ala Val Glu Val Lieu Ala Ala Ala Glin Lys Arg Glu 18O 185 19 O Ser Phe Glu Gly Glu Lys Arg Ala Lys Lieu Ala Ser Gly Ile Leu Gly 195 200 2O5 Leu Asp Met Tyr Lys Met Arg Glu Pro Leu Glu Lys Ala Gly Lieu Lys 210 215 220 Tyr Ile Asp 225

<210 SEQ ID NO 67 <211& LENGTH 42 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 67 actcggat.cc gaaggagata tacatatgta C galactogga ct 42

<210 SEQ ID NO 68 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer <400 SEQUENCE: 68 cggctgtcga cc.gttagtica atatatttica ggc 33

<210 SEQ ID NO 69 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial sequence US 2005/0112260 A1 May 26, 2005 58

-continued

&220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 69 cgcggatcca taatggttga galacattacc g 31

<210 SEQ ID NO 70 &2 11s LENGTH 30 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 70 acgc.gtcgac ttacago act gcc acaatcg 30

<210 SEQ ID NO 71 &2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 71 cc.ggaattica taatggtoga actgggagtt gt 32

<210 SEQ ID NO 72 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 72 gaatgcggcc gottagtcaa tatatttcag goc 33

<210 SEQ ID NO 73 &2 11s LENGTH 15 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 73 ggtattgagg gtc.gc. 15

<210> SEQ ID NO 74 &2 11s LENGTH 17 &212> TYPE DNA <213> ORGANISM: Artificial sequence &220s FEATURE <223> OTHER INFORMATION: primer

<400 SEQUENCE: 74 agaggagagt tagagcc. 17 US 2005/0112260 A1 May 26, 2005 59

1. A tabletop Sweetener composition comprising monatin 20. The tabletop Sweetener composition of claim 2, or Salt thereof, wherein the composition provides a Sweet wherein the Sweetness is provided by monatin or salt thereof neSS comparable to about 0.9 to about 9.0 grams of granu produced in a biosynthetic pathway. lated Sugar. 21. A ready-to-use Sweetener composition comprising 2. The tabletop Sweetener composition of claim 1, monatin or Salt thereof, wherein a Volume of the composi wherein a 1 gram portion of the composition provides a tion provides a Sweetness comparable to a Same Volume of Sweetness comparable to two teaspoons of granulated Sugar. granulated Sugar. 3. The tabletop Sweetener composition of claim 1, 22. The ready-to-use Sweetener composition of claim 21, wherein 1 gram of the composition contains less calories and wherein 1 teaspoon of the composition contains less calories carbohydrates than about 1 gram of granulated Sugar. and carbohydrates than about 1 teaspoon of granulated 4. The tabletop Sweetener composition of claim 1, Sugar. wherein 1 gram of the composition comprises from about 0 23. The ready-to-use Sweetener composition of claim 21, to about 200 mg S.S monatin or salt thereof, and from about wherein 1 gram of the composition comprises about 3 to 0 to about 5 mg R,R monatin or salt thereof. about 25 mg S.S monatin or salt thereof, and from about 0 5. The tabletop Sweetener composition of claim 1, to about 0.625 mg R,R monatin or salt thereof wherein 1 gram of the composition comprises from about 3 24. The ready-to-use Sweetener composition of claim 21, to about 200 mg S.S monatin or salt thereof, and from about wherein the monatin or Salt thereof consists essentially of 0 to about 5 mg R,R monatin or salt thereof. S.S monatin or salt thereof or R,R monatin or salt thereof. 6. The tabletop Sweetener composition of claim 1, 25. The ready-to-use Sweetener composition of claim 21, wherein 1 gram of the composition comprises from about 0 wherein 1 gram of the composition comprises from about 5 to about 200 mg S.S monatin or salt thereof, and from about to about 25 mg S.S monatin or salt thereof. 3 to about 5 mg R,R monatin or salt thereof. 26. The ready-to-use Sweetener composition of claim 21, 7. The tabletop Sweetener composition of claim 1, wherein 1 gram of the composition comprises from about wherein 1 gram of the composition comprises about 200 mg 0.4 to about 0.625 R,R monatin or salt thereof, and wherein or leSS S.S monatin or Salt thereof, and wherein the monatin the monatin or salt thereof is substantially free of S.S, S.R or R,S monatin or salt thereof. or salt thereof is substantially free of R,R, S,R or R,S 27. The ready-to-use Sweetener composition of claim 21, monatin or Salt thereof. wherein 1 gram of the composition comprises from about 8. The tabletop Sweetener composition of claim 1, 0.5 to about 1 mg R,R monatin or salt thereof, and wherein wherein 1 gram of the composition comprises about 5 mg or the monatin or salt thereof is substantially free of S.S, S.R leSS R,R monatin or Salt thereof, and wherein the monatin or or R,S monatin or salt thereof. salt thereof is substantially free of S.S, S,R or R,S monatin 28. The ready-to-use Sweetener composition of claim 21, or salt thereof. further comprising erythritol. 9. The tabletop Sweetener composition of claim 1, further comprising at least one ingredient chosen from bulking 29. The ready-to-use Sweetener composition of claim 28, agents, carriers, fibers, Sugar alcohols, oligosaccharides, wherein the composition comprises up to 99.7% erythritol. Sugars, non-monatin high intensity Sweeteners, nutritive 30. The ready-to-use Sweetener composition of claim 21, Sweeteners, flavorings, flavor enhancers, flavor Stablizers, further comprising trehalose. acidulants, anti-caking agents, and free-flow agents. 31. The ready-to-use Sweetener composition of claim 21, 10. The tabletop Sweetener composition of claim 1, fur further comprising cyclamate. ther comprising erythritol. 32. A Sweetener composition comprising a Stereoisomeri cally-enriched monatin mixture, wherein the monatin mix 11. The tabletop Sweetener composition of claim 10, ture is produced via a biosynthetic pathway. wherein the composition comprises up to 99.7% erythritol. 33. The Sweetener composition of claim 32, wherein the 12. The tabletop Sweetener composition of claim 1, fur biosynthetic pathway is a multi-step pathway and at least ther comprising trehalose. one Step of the multi-step pathway is a chemical conversion. 13. The tabletop Sweetener composition of claim 1, fur 34. The Sweetener composition of claim 32, wherein the ther comprising cyclamate. mixture is predominantly R,R monatin or Salt thereof. 14. The tabletop Sweetener composition of claim 1, fur 35. A homogeneous tabletop Sweetener composition com ther comprising agglomerated dextrose with maltodextrin. prising monatin or Salt thereof, wherein a Sample of the 15. The tabletop Sweetener composition of claim 1, composition comprises monatin or Salt thereof in an amount wherein the monatin or Salt thereof consists essentially of ranging from more than 2 mg to about 200 mg, and wherein RR monatin or salt thereof. the monatin or Salt thereof in the Sample provides a Sweet 16. The tabletop Sweetener composition of claim 1, neSS comparable to about 0.9 to about 9.0 grams of granu wherein the monatin or Salt thereof consists essentially of lated Sugar. S.S monatin or salt thereof. 36. The composition of claim 35, wherein the monatin or 17. The tabletop Sweetener composition of claim 1, Salt thereof in the Sample provides a Sweetness comparable wherein the monatin or Salt thereof is a Stereoisomerically to two teaspoons of granulated Sugar. enriched R,R monatin or salt thereof. 37. The composition of claim 35, wherein the sample 18. The tabletop Sweetener composition of claim 1, weighs about 1 gram. wherein the monatin or Salt thereof is a Stereoisomerically 38. The composition of claim 35, wherein the sample enriched S.S monatin or salt thereof. comprises an amount of monatin or Salt thereof ranging from 19. The tabletop Sweetener composition of claim 17, more than 2 mg to about 5 mg monatin or Salt thereof. wherein the monatin or salt thereof comprises at least 95% 39. The composition of claim 38, wherein the composi RR monatin or salt thereof. tion is substantially free of S.S monatin or salt thereof. US 2005/0112260 A1 May 26, 2005 60

40. The tabletop Sweetener of claim 35, wherein the 0.625 mg R,R monatin or salt thereof, and wherein a volume composition is Substantially free of R,R monatin or Salt of the composition has a SweetneSS comparable to a same thereof. Volume of granulated Sugar. 41. A tabletop Sweetener composition comprising monatin 55. The method of claim 50, wherein 1 gram of the or Salt thereof, wherein a Sample of the composition com composition comprises from about 0 to about 25 mg S.S prises monatin or Salt thereof in an amount ranging from monatin or salt thereof and from about 0 to about 0.625 mg more than 2 mg to about 105 mg monatin or Salt thereof, and R,R monatin or Salt thereof, and wherein 1 gram of the wherein the monatin or Salt thereof in the Sample provides composition has a SweetneSS comparable to about 0.9 to a Sweetness comparable to one teaspoon of granulated Sugar. about 9.0 grams granulated Sugar. 42. The tabletop Sweetener of claim 35 or 41, wherein the 56. The method of claim 53, wherein the amount of S.S sample has a volume of about 0.35 ml. monatin or salt thereof ranges from about 5 to about 200 mg 43. The tabletop Sweetener of claim 35 or 41, wherein the S.S monatin or Salt thereof per 1 gram of the composition. Sample is a cube of granulated material. 57. The method of claim 56, wherein the method further 44. A tabletop Sweetener composition comprising a mona comprises combining S.S monatin or Salt thereof with at tin composition produced in a biosynthetic pathway, and least one other ingredient chosen from bulking agents, wherein the monatin composition does not contain petro carriers, fibers, Sugar alcohols, oligosaccharides, Sugars, chemical, toxic or hazardous contaminants. non-monatin high intensity Sweeteners, nutritive Sweeteners, 45. Atabletop Sweetener composition comprising monatin flavorings, flavor enhancers, flavor Stablizers, acidulants, or salt thereof, wherein the monatin or salt thereof is anti-caking, free-flow agents, and any combination thereof, produced in a biosynthetic pathway and isolated from a wherein the monatin or Salt thereof comprises about 3 to recombinant cell, and wherein the recombinant cell does not about 200 mg of monatin or salt thereof per 1 gram of the contain petrochemical, toxic or hazardous contaminants. composition. 46. A method for making a Sweetener composition com 58. The method of claim 53, wherein the method further prising monatin or Salt thereof, wherein the method com comprises combining R,R monatin or Salt thereof with at prises producing monatin or Salt thereof from at least one least one other ingredient chosen from bulking agents, Substrate chosen from glucose, tryptophan, indole-3-lactic carriers, fibers, Sugar alcohols, oligosaccharides, Sugars, acid, indole-3-pyruvate and the monatin precursor. non-monatin high intensity Sweeteners, nutritive Sweeteners, 47. The method of claim 46, wherein the method further flavorings, flavor enhancers, flavor Stablizers, acidulants, comprises combining the monatin or Salt thereof with eryth anti-caking, free-flow agents, and any combination thereof, ritol. wherein the amount of R,R monatin or Salt thereof ranges 48. The method of claim 46, wherein the method further from about 3 to about 5 mg of R,R monatin or salt thereof comprises combining the monatin or Salt thereof with tre per 1 gram of the composition. halose. 59. The method of claim 50, wherein the monatin or salt 49. The method of claim 46, wherein the method further thereof comprises from about 0.4 to about 5 mg R,R monatin comprises combining the monatin or Salt thereof with cycla or Salt thereof per 1 gram of the composition. mate. 60. The method of claim 46, wherein the method further 50. The method of claim 46, wherein the method further comprises combining the monatin or Salt thereof with at comprises combining the monatin or Salt thereof with at least one bulking agent chosen from dextrose and malto least one other ingredient that is not monatin or Salt thereof. dextrin, and wherein the monatin or Salt thereof comprises 51. The method of claim 50, wherein the at least one other about 5 mg R,R monatin or Salt thereof per 1 gram of the ingredient is chosen from bulking agents, carriers, fibers, composition. Sugar alcohols, oligosaccharides, Sugars, non-monatin high 61. The method of claim 46, wherein the method further intensity Sweeteners, nutritive Sweeteners, flavorings, flavor comprises combining the monatin or Salt thereof with mal enhancers, flavor Stabilizers, acidulants, anti-caking, free todextrin, and wherein the monatin or Salt thereof comprises flow agents, and any combination thereof. from about 0.5 to about 1 mg R,R monatin or salt thereof per 52. The method of claim 50, wherein the at least one other 1 gram of the composition. ingredient is chosen from maltodextrin, dextrose, erythritol 62. A method for making a Sweetener composition com and fiber. prising a monatin composition, wherein the method com 53. The method of claim 50, wherein a portion of the prises: (a) producing monatin or Salt thereof in a biosyn Sweetener composition weighing about 1 gram comprises thetic pathway in a recombinant cell; (b) isolating the from about 0 mg to about 200 mg SS monatin or salt thereof monatin composition from the recombinant cell, wherein the and from about 0 mg to about 5 mg R,R monatin or salt monatin composition consists of monatin or Salt thereof and thereof, and wherein the portion provides a Sweetness com other edible or potable material. parable to two teaspoons of granulated Sugar. 63. A monatin composition comprising monatin and 54. The method of claim 50, wherein 1 gram of the erythritol. Sweetener composition comprises from about 0 to about 25 mg S.S monatin or salt thereof and from about 0 to about