US 20050282260A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0282260 A1 Hicks et al. (43) Pub. Date: Dec. 22, 2005

(54) POLYPEPTIDES AND BIOSYNTHETIC (60) Provisional application No. 60/374,831, filed on Apr. PATHWAYS FOR THE PRODUCTION OF 23, 2002. MONATIN AND ITS PRECURSORS (75) Inventors: Paula M. Hicks, Eden Prairie, MN Publication Classification (US); Sara C. McFarlan, St. Paul, MN (US) (51) Int. Cl." ...... C12P 13/22; C12N 1/18 Correspondence Address: (52) U.S. Cl...... 435/121; 435/254.2 CARGILL, INCORPORATED LAW/24 15407 MCGINTY ROAD WEST (57) ABSTRACT WAYZATA, MN 55391 (US) Methods and compositions that can be used to make monatin (73) Assignee: Cargill, Incorporated from glucose, , indole-3-lactic acid, indole-3- Appl. No.: 11/114,922 pyruvate, and 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glu (21) taric acid, are provided. Methods are also disclosed for producing the indole-3-pyruvate and 2-hydroxy 2-(indol-3- (22) Filed: Apr. 26, 2005 ylmethyl)-4-ketoglutaric acid intermediates. Compositions Related U.S. Application Data provided include nucleic acid molecules, polypeptides, chemical Structures, and cells. Methods include in vitro and (63) Continuation-in-part of application No. 10/422,366, in Vivo processes, and the in Vitro methods include chemical filed on Apr. 23, 2003. reactions.

-- 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 Patent Application Publication Dec. 22, 2005 Sheet 1 of 9 US 2005/0282260 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

F.G. 1 Patent Application Publication Dec. 22, 2005 Sheet 2 of 9 US 2005/0282260 A1

U.S. Patent No. 4,371,614 Tryptophan EC 2.6.1.27 tryptophan aminotransferase EC 2.6.1.5 tyrosine (aromatic) aminotransferase EC 1.4.1.19 tryptophan dehydrogenase EC 1.4.1.2-4 EC 1.4.1.20 phenylalanine 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-amino acid 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 EC 2.6.1.21 D-alanine 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-tryptophan aminotransferase (no EC number) Monati Patent Application Publication Dec. 22, 2005 Sheet 3 of 9 US 2005/0282260 A1 F.G. 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 1.1.1.111 (3-imidazol-5-yl) lactate dehydrogenase EC 1.1.3.- lactate oxidase Chemical oxidation

Indole-3-pyruvate

FIG. 4

COOR O COOR, S COOR O 1sor, ?y - A ( ) OHO - . N N Enolate chemistry R R

R Boc, Cbz, etc. R2 and R3 = Alkyl, Aryl, etc. Patent Application Publication Dec. 22, 2005 Sheet 4 of 9 US 2005/0282260 A1

- 03-Apr-2002 OO FG. 5A 13:52:54

O 4 8 2 16 2O 24 Time (min)

F.G. 6

100 293 O Z a. 2 a 50

a

150 2OO 250 300 350 4OO Patent Application Publication Dec. 22, 2005 Sheet 5 of 9 US 2005/0282260 A1 FIG. 7A

08-Apr-2002 O O A 15:11:01 5 O O O 4 8 2 6 2O 24 FIG. 7B Time (min) 9 OO 2 B 168 of O O. 158 O) 9 50 130 g 74 229 R 21 l?12 \230 o 8 44 94. 293 C) O all.l. ill.i.l. tro- mind...... 25.2S. A1 lOO 150 200 250 3OO

FIG. 8

+TOF MS: 124 MCA scans from Sample 7 (293jd with Tryptophan) of car0709a.wif. Max. 9.3e4 counts. a=3-569708085.37750280e-004, to 2-1.246268795.88351.580 e+002 R;

2931144 90e4

7,084 Cargil Sample Sample 293 with Tryptophan as Internal Std 6.0 e4

5.0e4

4.0 e4

3.0e 4

2.0e 4 2.94.1210

1.0e 4

0.0 284 286 288 290 292 294 296 298 rhiz, amu Patent Application Publication Dec. 22, 2005 Sheet 6 of 9 US 2005/0282260 A1

VB342 10-Jul-2002 13:11:33 O710202a Scan ES 1CO FG. 9A 1.

%

O arrarauarry-sworwarrereverarmam-mm-rrrrrrrr OR10020 a 1. Scants 1OO F.G. 9B 124eB

%

O O7100203a SCan ES+ 1GO 1. FIG 9C %

- - - Tire 2.00 4.00 6.00 800 0.00 12.OO 4.00 1600 1800 2O.CO 22.00 24.00

F.G. 10

O 3 14OE+05 8 120E-05 a 1.00E+05 s 6.OOE+04 t 4.OOE+04 5, 200E+04 . s: 9. 0.00E+00 - - - Y1 Ris Ohr 70hr S -2.OOE--O4 (-) (+) () (+) (-) al -4.OOE--04 Time Patent Application Publication Dec. 22, 2005 Sheet 7 of 9 US 2005/0282260 A1 FIG. 11 tryptophan tryptophan oxidase O7 O catalase amino acid oxidase 'HO g indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) aspartate aspartate aminotranSferafe Se Ny Oxaloacetate monatin v Oxaloacetate \ decarboxylase 3. pyruvate + CO2 Patent Application Publication Dec. 22, 2005 Sheet 8 of 9 US 2005/0282260 A1 FIG. 12 tryptophan tryptophan oxidase Or O catalase amino acid Oxidase HO indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) lysine lysine &-aminotransferase | N) alysine monatin \spontaneous, - H20

1-Piperideine 6-carboxylate Patent Application Publication Dec. 22, 2005 Sheet 9 of 9 US 2005/0282260 A1 FIG. 13 tryptophan tryptophan oxidase ( O catalase or amino acid Oxidase | HO indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) CO 2 dehydrogenase NADH A s (reductive amination) A ...r" N) NAD 9/ mOnat1n / Formate dehydrogenase ammonium formate US 2005/0282260 A1 Dec. 22, 2005

POLYPEPTIDES AND BIOSYNTHETIC PATHWAYS reported to eliminate toxins and promote the production of FOR THE PRODUCTION OF MONATIN AND ITS hormones beneficial to women's health. PRECURSORS 0008 Tryptophan Derivatives CROSS-REFERENCE TO RELATED 0009 Chlorinated D-tryptophan has been identified as a APPLICATION nonnutritive Sweetener, and there is increasing interest in pursuing other derivatives as well. Monatin is a natural 0001. This application is a continuation-in-part of appli Sweetener that is similar in composition to the amino acid cation Ser. No. 10/422,366, filed Apr. 23, 2003, which tryptophan. It can be extracted from the bark of the roots of claims the benefit of U.S. Provisional Patent Application No. the South African shrub, Sclerochiton ilicifolius, and has 60/374,831, filed Apr. 23, 2002. In addition, this application promise in the food and beverage industry as a high is related to application Ser. No. 10/979,821, filed Nov. 3, intensity Sweetener. Some examples of patents on monatin 2004, which is also a continuation-in-part of application Ser. include: U.S. Pat. No. 5,994,559 Synthesis of monatin-A No. 10/422,366. The aforementioned applications are incor high intensity natural Sweetener, U.S. Pat. No. 4,975,298 porated by reference in their entireties. 3-(1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl)-indole com pounds, U.S. Pat. No. 5,128,164 Composition for human FIELD consumption containing 3-(1-amino-1,3-dicarboxy-3-hy 0002 This disclosure provides polypeptides and biosyn droxy-but-4-yl)-indole compounds; and U.S. Pat. No. 5,128, thetic pathways that are useful in the production of indole 482 Process for the production of 3-1(1-amino-1,3-dicar 3-pyruvate, 2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric boxy-3-hydroxy-but-4-yl)indole. acid (MP) and/or monatin. 0010 Some of the precursors of monatin described here can also be useful as Synthetic SweetenerS or as intermedi BACKGROUND ates in the Synthesis of monatin derivatives.

0003) Indole pyruvate. SUMMARY 0004 Indole-3-pyruvate is a strong antioxidant that is believed to counter act oxidative StreSS in tissues with high 0011. The disclosure provides several biosynthetic routes concentrations (Politi et al. “Recent advances in for making monatin from glucose, tryptophan, indole-3- Tryptophan Research', edited by G. A. Filippini et al. lactic acid, and/or through monatin precursorS Such as Plenum Press, New York, 1996, pp. 291-8). Indole pyruvate indole-3-pyruvate and 2-hydroxy 2-(indole-3-ylmethyl)-4- also is an intermediate in a pathway to produce indole-acetic ketoglutaric acid. Polypeptides and nucleic acid Sequences acid (IAA), the primary plant growth hormone auxin (dif that can be used to make monatin, indole-3-pyruvate, and fusible growth promoting factor). IAA is active in Submi 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glutaric acid are crogram amounts in a range of physiological processes disclosed. In an effort to be concise, where ever intermedi including apical dominance, tropisms, Shoot elongation, ates/products are identified in the Specification and claims induction of cambial cell division, and root initiation. Syn (e.g. monatin or monatin precursor) as being formed, the thetic auxins are used in horticulture to induce rooting and term “and/or salts thereof should be understood to be to promote the Set and development of fruit. At high con included where applicable. In other words, for example, the centrations the Synthetic auxins are effective herbicides phrase “indole-3-pyruvate is converted to monatin precur against broad-leafed plants. Natural auxins produced by Sor' should be understood to read “indole-3-pyruvic acid is fermentation may be considered more environmentally converted to monatin precursor and and/or Salts thereof.” A friendly than chemically produced herbicides. Growth regu perSon of ordinary skill, in fact, would appreciate that under lators had world sales in 1999 of 0.4 billion pounds (1.4 reaction conditions shown the Salts of the intermediateS/ billion U.S. dollars). products are in fact present or also present. 0012 Monatin can be produced by reacting a reaction 0005 Some examples of patents on indole acetic acid and mixture that includes one or more Suitable Substrates and derivatives thereof include: U.S. Pat. No. 5,843,782 Micro one or more Selected polypeptides. Suitable Substrates may propagation of rose plants, auxin used in culture medium include, but are not limited to, glucose, tryptophan, indole and U.S. Pat. No. 5,952,231 Micropropagation of rose 3-lactic acid, monatin precursors (Such as indole-3-pyruvate plants. and 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glutaric acid), 0006. In addition to plant related utilities, indole acetic and mixtures thereof. Suitable Substrates that are present in acid is useful in pharmaceutical applications. For example, the reaction mixture for producing monatin, may be added U.S. Pat. No. 5,173,497 “Method of preparing alpha-oxopy to the reaction mixture and/or may be produced in Situ in the rrolo2,3-Bindole acetic acids and derivatives” proposes the reaction mixture. The Selected polypeptides may be added to use of these compounds in the treatment of memory impair the reaction mixture and/or may be produced by microor ment Such as that associated with Alzheimer's disease and ganisms present in the reaction mixture (e.g., by fermenting senile dementia. The mechanism proposed in U.S. Pat. No. the reaction mixture with a microorganism that expresses the 5,173,497 is that these compounds inhibit the polypeptide Selected polypeptide). acetylcholinesterase and increase acetylcholine levels in the 0013 Monatin can be produced through indole-3-pyru brain. vate, 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glutaric acid 0007 Indole-3-carbinol is produced from indole-3-acetic (monatin precursor, MP, the alpha-keto form of monatin), acid by peroxidase-catalyzed oxidation, and can easily be indole-3-lactic acid, tryptophan, and/or glucose (FIG. 1). converted into diindolylmethane. Both compounds are Methods of producing or making monatin or its intermedi US 2005/0282260 A1 Dec. 22, 2005

ates shown in FIGS. 1-3 and 11-13 that involve converting 2.6.1.21), Synthase/lyase (EC 4.1.3.-), Such as 4-hydroxy-4- a Substrate to a first , and then converting the first methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy product to a Second product, and So on, until the desired end 2-oxoglutarate aldolase (EC 4.1.3.16), Synthase/lyase product is created, are disclosed. (4.1.2.-), D-tryptophan aminotransferase (Kohiba and Mito, 0014 FIGS. 1-3 and 11-13 show potential intermediate Proceedings of the 8" International Symposium on Vitamin products and end products in boxes. For example, a con B and Carbonyl , Osaka, Japan 1990), branched version from one product to another, Such as glucose to chain aminotransferase (BCAT, EC 2.6.1.42), phenylalanine tryptophan, tryptophan to indole-3-pyruvate, indole-3-pyru dehydrogenase (EC 1.4.1.20), glutamate dehydrogenase (EC vate to MP, MP to monatin, or indole-3-lactic acid (indole 1.4.1.2, 1.4.1.3, 1.4.1.4) and leucine (branched-chain) dehy lactate) to indole-3-pyruvate, can be performed by using drogenase (EC 1.4.1.9). these methods. These conversions can be facilitated chemi 0019. In another example, cells include one or more, for cally or biologically. The term “convert” refers to the use of example two or more, or three or more, of the following either chemical means or polypeptides in a reaction which activities: indolelactate dehydrogenase (EC 1.1.1.110), R-4- changes a first intermediate to a Second intermediate. The hydroxyphenylactate dehydrogenase (EC 1.1.1.222), 3-(4)- term “chemical conversion” refers to reactions that are not hydroxyphenylpyruvate reductase (EC 1.1.1.237), lactate actively facilitated by polypeptides. The term “biological dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol conversion” refers to reactions that are actively facilitated by 5-yl) lactate dehydrogenase (EC 1.1.1.111), lactate oxidase polypeptides (e.g., ). Conversions can take place in (EC 1.1.3.-), Synthase/lyase (4.1.3.-) Such as 4-hydroxy-4- vitro or in Vivo (e.g., by fermenting a nutrient broth with an methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy Suitable microorganism). When biological conversions are 2-oxoglutarate aldolase (EC 4.1.3.16), Synthase/lyase used the polypeptides and/or cells can be immobilized on (4.1.2.-), tryptophan dehydrogenase (EC 1.4.1.19), tryp Supports Such as by chemical attachment on polymer Sup tophan-phenylpyruvate transaminase (EC 2.6.1.28), tryp ports. The conversion can be accomplished using any reactor tophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) known to one of ordinary skill in the art, for example in a aminotransferase (EC 2.6.1.5), multiple Substrate ami batch or a continuous reactor. notransferase (EC 2.6.1.-), aspartate aminotransferase (EC 0.015 Methods are also provided that include contacting 2.6.1.1), branched-chain aminotransferase (BCAT, EC a first polypeptide with a Substrate and making a first 2.6.1.42), phenylalanine dehydrogenase (EC 1.4.1.20), product, and then contacting the first product created with a glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, 1.4.1.4 ), Second polypeptide and creating a Second product, and then leucine (branched-chain) dehydrogenase (EC 1.4.1.9), contacting the Second product created with a third polypep D-amino acid dehydrogenase (EC 1.4.99.1), D-tryptophan tide and creating a third product, for example monatin. The aminotransferase, and/or D-alanine aminotransferase (EC polypeptides used and the products produced are shown in 2.6.1.21). FIGS. 1-3 and 11-13. 0020. In addition, the disclosed cells can include one or more of the following activities, for example two or more or 0016 Polypeptides, and their coding sequences, that can three or more of the following activities: tryptophan ami be used to perform the conversions shown in FIGS. 1-3 and notransferase (EC 2.6.1.27), tyrosine (aromatic) aminotrans 11-13 are disclosed. In Some examples, polypeptides having ferase (EC 2.6.1.5), multiple substrate aminotransferase (EC one or more point mutations that allow the Substrate Speci 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), tryptophan ficity and/or activity of the polypeptides to be modified, are dehydrogenase (EC 1.4.1.19), tryptophan-phenylpyruvate used to make monatin. transaminase (EC 2.6.1.28), L-amino acid oxidase (EC 0.017) Isolated and recombinant cells that produce mona 1.4.3.2), tryptophan oxidase (no EC number), D-amino acid tin are disclosed. These cells can be any cell, Such as a plant, dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC animal, bacterial, yeast, algal, archaeal, or fungal cell. These 1.4.3.3), D-alanine aminotransferase (EC 2.6.1.21), indole cells may be used to Synthesize monatin by fermenting a lactate dehydrogenase (EC 1.1.1.110), R-4-hydroxyphenyl nutrient medium that includes the cell. The nutrient medium lactate dehydrogenase (EC 1.1.1.222), 3-(4)-hydroxyphe may include any Suitable molecule for Synthesizing monatin, nylpyruvate reductase (EC 1.1.1.237), lactate including but not limited to, glucose, tryptophan, indole-3- dehydrogenase (EC 1.1.1.27, 1.1.1.28, 1.1.2.3), (3-imidazol lactic acid, and/or monatin precursorS Such as indole-3- 5-yl) lactate dehydrogenase (EC 1.1.1.111), lactate oxidase pyruvate and 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glu (EC 1.1.3.-), Synthase/lyase (4.1.3.-) Such as 4-hydroxy-4- taric acid. methyl-2-oxoglutarate aldolase (EC 4.1.3.17) or 4-hydroxy 0.018. In a particular example, the disclosed cells include 2-oxoglutarate aldolase (EC 4.1.3.16), Synthase/lyase one or more of the following activities, for example two or (4.1.2.-), branched-chain aminotransferase (BCAT, EC more or three or more of the following activities: tryptophan 2.6.1.42), glutamate dehydrogenase (EC 1.4.1.2, 1.4.1.3, aminotransferase (EC 2.6.1.27), tyrosine (aromatic) ami 1.4.1.4), phenylalanine dehydrogenase (EC 1.4.1.20), leu notransferase (EC 2.6.1.5), multiple Substrate aminotrans cine (branched-chain) dehydrogenase (EC 1.4.1.9) and/or ferase (EC 2.6.1.-), aspartate aminotransferase (EC 2.6.1.1), D-tryptophan aminotransferase. tryptophan dehydrogenase (EC 1.4.1.19), tryptophan-phe 0021 Monatin can be produced by a method that includes nylpyruvate transaminase (EC 2.6.1.28), L-amino acid oxi contacting tryptophan and/or indole-3-lactic acid with a first dase (EC 1.4.3.2), tryptophan oxidase (no EC number, polypeptide, wherein the first polypeptide converts tryp Hadar et al., J. Bacteriol 125:1096-1104, 1976 and Furuya tophan and/or indole-3-lactic acid to indole-3-pyruvate et al., Biosci Biotechnol Biochem 64: 1486-93, 2000), (either the D or the L form of tryptophan or indole-3-lactic D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid acid can be used as the Substrate that is converted to oxidase (EC 1.4.3.3), D-alanine aminotransferase (EC indole-3-pyruvate; one of skill in the art will appreciate that US 2005/0282260 A1 Dec. 22, 2005 the polypeptides chosen for this step ideally exhibit the 0033 FIG. 8 is a chromatogram showing the high reso appropriate specificity), contacting the resulting indole-3- lution mass measurement of monatin produced enzymati pyruvate with a Second polypeptide, wherein the Second cally. polypeptide converts the indole-3-pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4-ketoglutaric acid (MP), and contact 0034 FIGS. 9A-9C are chromatograms showing the ing the MP with a third polypeptide, wherein the third chiral Separation of (A) R-tryptophan, (B) S-tryptophan, and polypeptide converts MP to monatin. Exemplary polypep (C) monatin produced enzymatically. tides that can be used for these conversions are shown in 0035 FIG. 10 is a bar graph showing the relative amount FIGS. 2 and 3. of monatin produced in bacterial cells following IPTG 0022. Another aspect of the invention provides compo induction. The (-) indicates a lack of Substrate addition (no Sitions Such as MP, cells that contain at least two polypep tryptophan or pyruvate was added). tides, or Sometimes at least three or at least four polypep 0036 FIGS. 11-12 are schematic diagrams showing tides, that are encoded on at least one exogenous nucleic pathways used to increase the yield of monatin produced acid Sequence. from tryptophan or indole-3-pyruvate. 0023 These and other aspects of the disclosure are appar 0037 FIG. 13 is a schematic diagram showing a pathway ent from the following detailed description and illustrative which can be used to increase the yield of monatin produced examples. from tryptophan or indole-3-pyruvate. BRIEF DESCRIPTION OF THE FIGURES SEQUENCE LISTING 0024 FIG. 1 shows biosynthetic pathways used to pro 0038. The nucleic and amino acid sequences listed in the duce monatin and/or indole-3-pyruvate. One pathway pro accompanying Sequence listing are shown using Standard duces indole-3-pyruvate via tryptophan, while another pro letter abbreviations for nucleotide bases, and three-letter duces indole-3-pyruvate via indole-3-lactic acid. Monatin is code for amino acids. Only one Strand of each nucleic acid Subsequently produced via a 2-hydroxy 2-(indol-3ylm Sequence is shown, but the complementary Strand is under ethyl)-4-ketoglutaric acid (MP) intermediate. stood to be included by any reference to the displayed 0.025 Compounds shown in boxes are substrates and Strand. products produced in the biosynthetic pathways. 0039 SEQ ID NOS: 1 and 2 show the nucleic acid and 0.026 Compositions adjacent to the arrows are cofactors, amino acid Sequences of an aminotransferase from or reactants that can be used during the conversion of a Sinorhizobium meliloti, respectively (tatA gene, called a Substrate to a product. The or reactant used will tyrosine or aromatic aminotransferase in literature). depend upon the polypeptide used for the particular Step of the biosynthetic pathway. The cofactor PLP (pyridoxal 0040 SEQ ID NOS: 3 and 4 show the nucleic acid and 5'-phosphate) can catalyze reactions independent of a amino acid Sequences of a tyrosine aminotransferase from polypeptide, and therefore, merely providing PLP can allow Rhodobacter Sphaeroides (2.4.1), respectively (by homol for the progression from Substrate to product. ogy with tatA (SEQ ID NOS: 1 and 2) predicted to be an "aspartate aminotransferase” by genomics Software). 0.027 FIG. 2 is a more detailed diagram of the biosyn thetic pathway that utilizes the MP intermediate. The Sub 0041) SEQ ID NOS: 5 and 6 show the nucleic acid and Strates for each Step in the pathways are shown in boxes. The amino acid Sequences of an aminotransferase from Rhodo polypeptides allowing for the conversion between Substrates bacter Sphaeroides (35053), respectively (novel, cloned are listed adjacent to the arrows between the Substrates. Each based on 2.4.1 sequence SEQ ID NOS 3 and 4). polypeptide is described by its common name and an 0042 SEQ ID NOS: 7 and 8 show the nucleic acid and enzymatic class (EC) number. amino acid Sequences of a broad Substrate aminotransferase 0028 FIG. 3 shows a more detailed diagram of the (bsat) from Leishmania major, respectively. biosynthetic pathway of the conversion of indole-3-lactic acid to indole-3-pyruvate. The Substrates are shown in 0043 SEQ ID NOS:9 and 10 show the nucleic acid and boxes, and the polypeptides allowing for the conversion amino acid Sequences of an aromatic aminotransferase between the Substrates are listed adjacent to the arrow (araT) from Bacillus subtilis, respectively. between the substrates. Each polypeptide is described by its 0044 SEQ ID NOS: 11 and 12 show novel nucleic acid common name and an enzymatic class (EC) number. and amino acid Sequences of an aromatic aminotransferase 0029 FIG. 4 shows one possible reaction for making MP (araT) from Lactobacillus amylovorus, respectively (by via chemical means. homology identified as an aromatic aminotransferase). 0045 SEQID NOS: 13 and 14 show the nucleic acid and 0030 FIGS.5A and 5B are chromatograms showing the amino acid Sequences of a multiple Substrate aminotrans LC/MS identification of monatin produced enzymatically. ferase (msa) from R. Sphaeroides (35053), respectively 0.031 FIG. 6 is an electrospray mass spectrum of enzy (identified as a multiple Substrate aminotransferase by matically Synthesized monatin. homology to Accession No. AAAE01000093.1, bp 14743 0032 FIGS. 7A and 7B show chromatograms of the 16155 and Accession No. ZP00005082.1). LC/MS/MS daughter ion analyses of monatin produced in 0046) SEQ ID NOS: 15-16 show primers used to clone an enzymatic mixture. the B. Subtilis D-alanine aminotransferase (dat) sequence. US 2005/0282260 A1 Dec. 22, 2005

0047 SEQ ID NOS: 17-18 show primers used to clone 0067 SEQ ID NOS: 79-80 show primers used to clone the S. meliloti tatA sequence. the E. coli tyrosine aminotransferase (tyrb). 0048 SEQ ID NOS: 19-20 show primers used to clone 0068 SEQ ID NOS: 81-82 show primers used to clone the B. Subtilis araTaminotransferase Sequence. the khg gene of Z. mobilis (ATCC 29191). 0049 SEQ ID NOS: 21-22 show primers used to clone 0069 SEQ ID NOS 83 and 84 show the nucleic acid and the Rhodobacter sphaeroides (2.4.1 and 35053) multiple amino acid sequences of the Z. mobiliskhg gene (Accession Substrate aminotransferase Sequences. No: AE008692.1 GI: 56542470) and gene product (Acces 0050 SEQ ID NOS: 23-24 show primers used to clone sion No.: AAV89621.1 GI: 56543467), respectively. the Leishmania major bsat Sequence. 0070 SEQ ID NOS: 85-86 show primers used to clone the E. coli yfdZ gene sequence deposited in NCBI as GI: 0051 SEQ ID NOS: 25-26 show primers used to clone 48994873 bases 2496317-2495079, coding for protein GI: the Lactobacillus amylovorus araT Sequence. 1788722 (protein ID AAC75438.1). 0.052 SEQ ID NOS: 27-28 show primers used to clone 0071 SEQID NOS: 87 and 88 show the nucleic acid and the R. Sphaeroides tatA sequences (both 2.4.1 and 35053). amino acid Sequences of an aldolase from Rhizobium legu 0053 SEQ ID NOS: 29-30 show primers used to clone minosarum biovar viciae rhiz23g02-plk 1009 341 the E. coli aspC sequence (gene sequence Genbank Acces (Sanger Institute), respectively. sion No.: AE000195.1, protein sequence Genbank Acces sion No.: AAC74014.1). DETAILED DESCRIPTION OF SEVERAL 0054 SEQID NOS: 31 and 32 show the nucleic acid and EMBODIMENTS amino acid sequences of aromatic aminotransferase (tyrB) from E. coli, respectively. Abbreviations and Terms 0055 SEQ ID NOS: 33-34 show primers used to clone 0072 The following explanations of terms and methods the E. coli tyrB Sequence. are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the 0056 SEQ ID NOS: 35-40 show primers used to clone present disclosure. AS used herein, “including” means “com polypeptides with 4-hydroxy-2-oxoglutarate aldolase prising.” In addition, the singular forms “a” or “an” or “the (KHG) (EC 4.1.3.16) activity. include plural references unless the context clearly dictates 0057 SEQ ID NOS: 41 and 42 show the nucleic acid otherwise. For example, reference to “comprising a protein' Sequences of tryptophanase (tna) from E. coli and tyrosine includes one or a plurality of Such proteins, and reference to phenol-lyase (tpl) from Citrobacter freundii, coding for “comprising the cell” includes reference to one or more cells proteins P00913 (GI: 401195) and P31013 (GI: 401201), and equivalents thereof known to those skilled in the art, and respectively. So forth. The term “about encompasses the range of experi mental error that occurs in any measurement. Unless other 0.058 SEQ ID NOS: 43–46 show primers used to clone wise Stated, all measurement numbers are presumed to have tryptophanase polypeptides and f-tyrosinase (tyrosine phe the word “about in front of them even if the word “about nol-lyase) polypeptides. is not expressly used. 0059 SEQ ID NOS: 47-54 show primers used to mutate tryptophanase polypeptides and B-tyrosinase polypeptides. 0073. Unless explained otherwise, all technical and sci entific terms used herein have the same meaning as com 0060 SEQ ID NOS: 55-64 show primers used to clone monly understood to one of ordinary skill in the art to which polypeptides with 4-hydroxy-4-methyl-2-oxoglutarate aldo this disclosure belongs. Although methods and materials lase (EC 4.1.3.17) activity. Similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, Suitable 0061 SEQID NOS: 65 and 66 show the nucleic acid and methods and materials are described below. The materials, amino acid Sequences of 4-hydroxy-4-methyl-2-oxoglut methods, and examples are illustrative only and not intended arate aldolase (proA) from C. testosteroni, respectively. to be limiting. Other features and advantages of the disclo 0062 SEQID NOS: 67-68 show primers used to clone C. Sure are apparent from the following detailed description testosterOni 4-hydroxy-4-methyl-2-oxoglutarate aldolase and the claims. (proA) in an operon with E. coli aspC in pET30 Xa/LIC. 0.074 cDNA (complementary DNA): A piece of DNA 0063 SEQID NOS: 69-72 show primers used to clone E. lacking internal, non-coding segments (introns) and regula coli aspC and C. testosteroni proA in pESC-his. tory Sequences which determine transcription. cDNA can be 0064 SEQ ID NOS: 73-74 show sequences added to the Synthesized in the laboratory by reverse transcription from 5' end of primers used to clone the genes disclosed herein. messenger RNA extracted from cells. 0075 Conservative substitution: a substitution of one 0065 SEQID NOS: 75 and 76 show the nucleic acid and amino acid for another amino acid in a polypeptide, which amino acid Sequences of the HEX gene and gene product substitution has little to no impact on the activity of the (NCBI accession number 1AHF A GI: 1127190) (HEX polypeptide. The Substitution is considered conservative AspC aminotransferase amino acid sequence), respectively. independent of whether the exchanged amino acids appear 0.066 SEQ ID NOS: 77-78 show primers used to clone Structurally or functionally similar. For example, ideally, a the E. coli aspartate aminotransferase (aspC) or the mutated tryptophan aminotransferase polypeptide including one or aspC aminotransferase (HEX) gene Sequence. more conservative Substitutions retains tryptophan ami US 2005/0282260 A1 Dec. 22, 2005 notransferase activity. A polypeptide can be produced to function of the . For example, functional equivalents contain one or more conservative Substitutions by manipu can be provided by Sequence alterations in an enzyme lating the nucleotide Sequence that encodes that-polypeptide Sequence, wherein the peptide with one or more Sequence using, for example, Standard procedures Such as Site-directed alterations retains a function of the unaltered peptide, Such mutagenesis or PCR or other methods known to those in the that it retains its enzymatic activity. In a particular example, art. a tryptophan aminotransferase functional equivalent retains 0.076 Non-limiting examples of amino acids which may the ability to convert tryptophan to indole-3-pyruvate. be Substituted for an original amino acid in a protein and 0082) Examples of sequence alterations include, but are which may be regarded as conservative Substitutions if there not limited to, conservative Substitutions, deletions, muta is little to no impact on the activity of the polypeptide tions, frameshifts, and insertions. In one example, a given include: Ala Substituted with ser or thr; arg substituted with polypeptide binds an antibody, and a functional equivalent is gln, his, or lys, asn Substituted with glu, gln, lys, his, asp; asp a polypeptide that binds the Same antibody. Thus a func Substituted with asn, glu, or gln, cys Substituted with Ser or tional equivalent includes peptides that have the same bind ala; gln Substituted with asn, glu, lys, his, asp, or arg, glu ing Specificity as a polypeptide, and that can be used as a Substituted with asn, gln lys, or asp; gly Substituted with pro; reagent in place of the polypeptide. In one example a his Substituted with asn, lys, gln, arg, tyr, ile Substituted with functional equivalent includes a polypeptide wherein the leu, met, Val, phe; leu. Substituted with ille, met, Val, phe, lyS binding Sequence is discontinuous, wherein the antibody Substituted with asn, glu, gln, his, arg; met Substituted with binds a linear epitope. Thus, if the peptide Sequence is ille, leu, Val, phe, phe Substituted with trip, tyr, met, ile, or leu, MPELANDLGL (amino acids 1-10 of SEQ ID NO: 12) a Ser Substituted with thr, ala; thr Substituted with Ser or ala; functional equivalent includes discontinuous epitopes, that trp Substituted with phe, tyr; tyr Substituted with his, phe, or can appear as follows (**=any number of intervening amino trp; and Val Substituted with met, ile, leu. COOH. In this example, the polypeptide is functionally 0077. Further information about conservative substitu tions can be found in, among other locations, Ben-Bassat et equivalent to amino acids 1-10 of SEQ ID NO: 12 if the al., (J. Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene three dimensional Structure of the polypeptide is Such that it 77:237-51, 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, can bind a monoclonal antibody that binds amino acids 1-10 1994), Hochuliet al., (Bio/Technology 6:1321-5, 1988), WO of SEO ID NO: 12. 00/67796 (Curd et al.) and in standard textbooks of genetics 0.083 Hybridization: The term “hybridization” as used and molecular biology. herein refers to a method of testing for complementarity in the nucleotide Sequence of two nucleic acid molecules, 0078 Derived: For purposes of the specification and based on the ability of complementary single-stranded DNA claims, a Substance is "derived from organism or Source if and/or RNA to form a duplex molecule. Nucleic acid hybrid any one or more of the following are true: 1) the Substance ization techniques can be used to obtain an isolated nucleic is present in the organism/Source; 2) the Substance is acid within the Scope of the disclosure. Briefly, any nucleic removed from the native host; or, 3) the substance is acid having Some homology to a Sequence Set forth in SEQ removed from the native host and is evolved, for example, ID NO: 11 can be used as a probe to identify a similar by mutagenesis. nucleic acid by hybridization under conditions of moderate 0079 Enzymatically Producing: The phrase “enzymati to high Stringency. Once identified, the nucleic acid then can cally producing,” or Similar phrases, Such as “produced be purified, Sequenced, and analyzed to determine whether enzymatically” or “enzymatically synthesized,” refers to the it is within the Scope of the present disclosure. production of product (Such as monatin) using at least one polypeptide, either in vitro (e.g., in test tube or reactor using 0084) Hybridization can be done by Southern or Northern one or more polypeptides) or in Vivo (e.g., in a whole cell or analysis to identify a DNA or RNA sequence, respectively, fermentation reaction). While “enzymatically producing” a that hybridizes to a probe. The probe can be labeled with a product does not exclude the use of chemical reagents or biotin, digoxygenin, a polypeptide, or a radioisotope Such as reactions, it includes the use of at least one polypeptide that *P. The DNA or RNA to be analyzed can be electrophoreti facilitates at least one reaction in the production of that cally Separated on an agarose or polyacrylamide gel, trans product. ferred to nitrocellulose, nylon, or other Suitable membrane, and hybridized with the probe using Standard techniques 0080 Exogenous: The term “exogenous” as used herein well known in the art Such as those described in Sections with reference to nucleic acid and a particular cell refers to 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, any nucleic acid that does not originate from that particular second edition, Cold Spring Harbor Laboratory, Plainview, cell as found in nature. Thus, non-naturally-occurring N.Y. Typically, a probe is at least about 20 nucleotides in nucleic acid is considered to be exogenous to a cell once length. For example, a probe corresponding to a contiguous introduced into the cell. Nucleic acid that is naturally 20 nucleotide sequence set forth in SEQ ID NO: 11 can be occurring also can be exogenous to a particular cell. For used to identify an identical or similar nucleic acid. In example, an entire chromosome isolated from a cell of addition, probes longer or shorter than 20 nucleotides can be perSon X is an exogenous nucleic acid with respect to a cell used. of person Y once that chromosome is introduced into Y's 0085. The disclosure also provides isolated nucleic acid cell. Sequences that are at least about 12 bases in length (e.g., at 0.081 Functionally Equivalent: Having an equivalent least about 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, function. In the context of an enzyme, functionally equiva 100, 250, 500, 750, 1000, 1500, 2000, 3000, 4000, or 5000 lent molecules include different molecules that retain the bases in length) and hybridize, under hybridization condi US 2005/0282260 A1 Dec. 22, 2005

tions, to the Sense or antisense Strand of a nucleic acid or genomic libraries, or gel Slices containing a genomic having the sequence set forth in SEQ ID NO: 11. The DNA restriction digest is not to be considered an isolated hybridization conditions can be moderately or highly Strin nucleic acid. gent hybridization conditions. 0091) Nucleic acid: The term “nucleic acid” as used 0.086 For the purpose of this disclosure, moderately herein encompasses both RNA and DNA including, without Stringent hybridization conditions mean the hybridization is limitation, cDNA, genomic DNA, and Synthetic (e.g., performed at about 42 C. in a hybridization solution con chemically synthesized) DNA. The nucleic acid can be taining 25 mM KPO (pH 7.4), 5xSSC, 5x Denhart's solu double-Stranded or Single-Stranded. Where Single-Stranded, tion, 50 lug/mL denatured, Sonicated Salmon Sperm DNA, the nucleic acid can be the Sense Strand or the antisense 50% formamide, 10% Dextran Sulfate, and 1-15 ng/mL Strand. In addition, nucleic acid can be circular or linear. probe (about 5x10 cpm/ug), while the washes are per formed at about 50 C. with a wash solution containing 0092 Operably linked: A first nucleic acid sequence is 2xSSC and 0.1% sodium dodecyl sulfate. “operably linked with a Second nucleic acid Sequence whenever the first nucleic acid Sequence is placed in a 0.087 Highly stringent hybridization conditions mean the functional relationship with the Second nucleic acid hybridization is performed at about 42 C. in a hybridization Sequence. For instance, a promoter is operably linked to a solution containing 25 mM KPO (pH 7.4), 5xSSC, 5x coding Sequence if the promoter affects the transcription of Denhart's Solution, 50 tug/mL denatured, Sonicated Salmon the coding Sequence. Generally, operably linked DNA sperm DNA, 50% formamide, 10% Dextran sulfate, and Sequences are contiguous and, where necessary to join two 1-15 ng/mL probe (about 5x10 cpm/ug), while the washes polypeptide-coding regions, in the same reading frame. are performed at about 65° C. with a wash solution con taining 0.2xSSC and 0.1% sodium dodecyl sulfate. 0093. Peptide Modifications: The present disclosure 0088 Isolated: The term “isolated” as used herein refers includes enzymes, as well as Synthetic embodiments thereof. to any Substance removed from its native host, the Substance In addition, analogues (non-peptide organic molecules), need not be purified. For example “isolated nucleic acid” derivatives (chemically functionalized peptide molecules refers to a naturally-occurring nucleic acid that is not obtained Starting with the disclosed peptide Sequences) and immediately contiguous with both of the Sequences with variants (homologs) having the desired enzymatic activity which it is immediately contiguous (one on the 5' end and can be utilized in the methods described herein. The peptides one on the 3' end) in the naturally-occurring genome of the disclosed herein include a sequence of amino acids, that can organism from which it is derived. For example, an isolated be either L- and/or D-amino acids, naturally occurring and nucleic acid can be, without limitation, a recombinant DNA otherwise. molecule of any length, provided one of the nucleic acid 0094 Peptides can be modified by a variety of chemical Sequences normally found immediately flanking that recom techniques to produce derivatives having essentially the binant DNA molecule in a naturally-occurring genome is Same activity as the unmodified peptides, and optionally removed or absent. Thus, an isolated nucleic acid includes, having other desirable properties. For example, carboxylic without limitation, a recombinant DNA that exists as a acid groups of the protein, whether carboxyl-terminal or side Separate molecule (e.g., a cDNA or a genomic DNA frag chain, may be provided in the form of a Salt of a pharma ment produced by PCR or restriction endonuclease treat ceutically-acceptable cation or esterified to form a C1-C16 ment) independent of other Sequences as well as recombi ester, or converted to an amide of formula NR1R2 wherein nant DNA that is incorporated into a vector, an R1 and R2 are each independently H or C1-C16 alkyl, or autonomously replicating plasmid, a virus (e.g., a retrovirus, combined to form a heterocyclic ring, Such as a 5- or adenovirus, or herpes virus), or into the genomic DNA of a 6-membered ring. Amino groups of the peptide, whether prokaryote or eukaryote. In addition, an isolated nucleic acid amino-terminal or Side chain, may be in the form of a can include a recombinant DNA molecule that is part of a pharmaceutically-acceptable acid addition Salt, Such as the hybrid or fusion nucleic acid Sequence. HCl, HBr, acetic, benzoic, toluene Sulfonic, maleic, tartaric 0089. The term “isolated” as used herein with reference and other organic Salts, or may be modified to C1-C16 alkyl to nucleic acid also includes any non-naturally-occurring or dialkyl amino or further converted to an amide. nucleic acid Since non-naturally-occurring nucleic acid 0095 Hydroxyl groups of the peptide side chains may be Sequences are not found in nature and do not have imme converted to C1-C16 alkoxy or to a C1-C 16 ester using diately contiguous Sequences in a naturally-ocurring well-recognized techniques. Phenyl and phenolic rings of genome. For example, non-naturally-occurring nucleic acid the peptide side chains may be Substituted with one or more Such as an engineered nucleic acid is considered to be halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, isolated nucleic acid. Engineered nucleic acid can be made C1-C16 alkoxy, carboxylic acids and esters thereof, or using common molecular cloning or chemical nucleic acid amides of Such carboxylic acids. Methylene groups of the Synthesis techniques. Isolated non-naturally-occurring peptide Side chains can be extended to homologous C2-C4 nucleic acid can be independent of other Sequences, or alkylenes. Thiols can be protected with any one of a number incorporated into a vector, an autonomously replicating of well-recognized protecting groups, Such as acetamide plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes groups. Those skilled in the art will also recognize methods virus), or the genomic DNA of a prokaryote or eukaryote. In for introducing cyclic Structures into the peptides of this addition, a non-naturally-occurring nucleic acid can include disclosure to Select and provide conformational constraints a nucleic acid molecule that is part of a hybrid or fusion to the Structure that result in enhanced Stability. For example, nucleic acid Sequence. a C- or N-terminal cysteine can be added to the peptide, So 0090. A nucleic acid existing among hundreds to millions that when oxidized the peptide will contain a disulfide bond, of other nucleic acid molecules within, for example, cDNA generating a cyclic peptide. Other peptide cyclizing methods US 2005/0282260 A1 Dec. 22, 2005

include the formation of thioethers and carboxyl- and example, a DNA polymerase polypeptide. Primer pairs can amino-terminal amides and esters. be used for amplification of a nucleic acid Sequence, for example, by the polymerase chain reaction (PCR) or other 0.096 Peptidomimetic and organominetic embodiments nucleic-acid amplification methods known in the art. are also within the Scope of the present disclosure, whereby the three-dimensional arrangement of the chemical constitu 0100 Methods for preparing and using probes and prim ents of Such peptido- and organomimetics mimic the three erS are described, for example, in references Such as Sam dimensional arrangement of the peptide backbone and com brook et al. (ed.), Molecular Cloning: A Laboratory Manual, ponent amino acid Side chains, resulting in Such peptido- and 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, organomimetics of the proteins of this disclosure having Cold Spring Harbor, N.Y., 1989; Ausubeletal. (ed.), Current detectable enzyme activity. For computer modeling appli Protocols in Molecular Biology, Greene Publishing and cations, a pharmacophore is an idealized, three-dimensional Wiley-Interscience, New York (with periodic updates), definition of the Structural requirements for biological activ 1987; and Innis et al. (eds.), PCR Protocols: A Guide to ity. Peptido- and organomimetics can be designed to fit each Methods and Applications, Academic PreSS: San Diego, pharmacophore with current computer modeling Software 1990. PCR primer pairs can be derived from a known (using computer assisted drug design or CADD). See Sequence, for example, by using computer programs Walters, “Computer-Assisted Modeling of Drugs”, in intended for that purpose such as Primer (Version 0.5, (C) Klegerman & Groves (eds.), Pharmaceutical Biotechnology, 1991, Whitehead Institute for Biomedical Research, Cam 1993, Interpharm Press: Buffalo Grove, Ill., pp. 165-74 and bridge, Mass.). One of skill in the art will appreciate that the Ch. 102 in Munson (ed.), Principles of Pharmacology, 1995, Specificity of a particular probe or primer increases with the Chapman & Hall, for descriptions of techniques used in length, but that a probe or primer can range in size from a CADD. Also included within the scope of the disclosure are full-length Sequence to Sequences as short as five consecu mimetics prepared using Such techniques. In one example, a tive nucleotides. Thus, for example, a primer of 20 consecu mimetic mimics the enzyme activity generated by an tive nucleotides can anneal to a target with a higher speci enzyme or a variant, fragment, or fusion thereof. ficity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater Specificity, probes and 0097. ProA Aldolase: Although “ProA” and/or “ProA primers can be Selected that comprise, for example, 10, 20, aldolase' historically have been used to identify only the 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 4-hydroxy-4-methyl-2-oxoglutarate aldolase derived from 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, Comamonas testOSteroni, herein the term "ProA' and/or 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, “ProA aldolase' are used to mean any polypeptide with 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 4-hydroxy-4-methyl-2-oxoglutarate aldolase activity unless 1900, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, otherwise stated. Suitable examples of ProA or ProA aldo 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, lase include C. testosteroni ProA (SEQ ID NO: 66) and 2850, 2900, 3000, 3050, 3100, 3150, 3200, 3250, 3300, Sinorhizobium meliloti ProA (NCBI Accession No.: 3350, 3400, 3450, 3500, 3550, 3600-3650, 3700, 3750, CAC46344), or enzymes that display homology to C. test 3800, 3850, 3900, 4000, 4050, 4100, 4150, 4200, 4250, Osteroni ProA (SEQ ID NO: 66) and/or Sinorhizobium 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, meliloti ProA (NCBI Accession No.: CAC46344). For 4750, 4800, 4850, 4900, 5000, 5050, 5100, 5150, 5200, example, Suitable enzymes may have at least about 40%, 5250, 5300, 5350, 5400, 5450, or more consecutive nucle 50%, 60%, 70%, 80%, 90%, 95%, and/or 99% sequence otides. identity with C. testosteroni ProA (SEQ ID NO: 66) and/or Sinorhizobium meliloti ProA (NCBI Accession No.: 0101 Promoter: An array of nucleic acid control CAC46344). Sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid Sequences near the 0.098 Probes and primers: Nucleic acid probes and prim Start Site of transcription, Such as, in the case of a polymerase erS can be prepared readily based on the amino acid II type promoter, a TATA element. A promoter can include Sequences and nucleic acid Sequences provided herein. A distal enhancer or repressor elements which can be located "probe' includes an isolated nucleic acid containing a as much as Several thousand base pairs from the Start Site of detectable label or reporter molecule. Exemplary labels transcription. include, but are not limited to, radioactive isotopes, ligands, chemiluminescent agents, and polypeptides. Methods for 0102) Purified: The term “purified” as used herein does labeling and guidance in the choice of labels appropriate for not require absolute purity, but rather is intended as a various purposes are discussed in, for example, Sambrook et relative term. Thus, for example, a purified polypeptide or al. (ed.), Molecular Cloning: A Laboratory Manual 2nd ed., nucleic acid preparation can be one in which the Subject vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring polypeptide or nucleic acid is at a higher concentration than Harbor, N.Y., 1989, and Ausubel et al. (ed.) Current Proto the polypeptide or nucleic acid would be in its natural cols in Molecular Biology, Greene Publishing and Wiley environment within an organism or at a higher concentration Interscience, New York (with periodic updates), 1987. than in the environment from which it was removed. 0099. “Primers' are typically nucleic acid molecules hav 0.103 Recombinant: A “recombinant nucleic acid is one ing ten or more nucleotides (e.g., nucleic acid molecules having (1) a sequence that is not naturally occurring in the having between about 10 nucleotides and about 100 nucle organism in which it is expressed or (2) a sequence made by otides). A primer can be annealed to a complementary target an artificial combination of two otherwise-separated, shorter nucleic acid Strand by nucleic acid hybridization to form a Sequences. This artificial combination is often accomplished hybrid between the primer and the target nucleic acid Strand, by chemical Synthesis or, more commonly, by the artificial and then extended along the target nucleic acid Strandby, for manipulation of isolated Segments of nucleic acids, e.g., by US 2005/0282260 A1 Dec. 22, 2005 genetic engineering techniques. “Recombinant' is also used particular example, a homologous Sequence is aligned to a to describe nucleic acid molecules that have been artificially native Sequence, and the number of matches is determined manipulated, but contain the same regulatory Sequences and by counting the number of positions where an identical coding regions that are found in the organism from which nucleotide or amino acid residue is presented in both the nucleic acid was isolated. Sequences. The percent Sequence identity is determined by dividing the number of matches either by the length of the 0104 Sequence identity: The similarity between amino Sequence set forth in the identified sequence (e.g., SEQ ID acid Sequences is expressed in terms of the Similarity NO: 11), or by an articulated length (e.g., 100 consecutive between the Sequences, otherwise referred to as Sequence nucleotides or amino acid residues from a Sequence Set forth identity. Sequence identity is frequently measured in terms in an identified Sequence), followed by multiplying the of percentage identity (or Similarity or homology); the resulting value by 100. For example, a nucleic acid Sequence higher the percentage, the more Similar the two Sequences that has 1166 matches when aligned with the Sequence Set are. Homologs or variants of a peptide, such as SEQ ID NO: forth in SEQ ID NO: 11 is 75.0 percent identical to the 12, possess a relatively high degree of Sequence identity sequence set forth in SEQID NO: 11 (i.e., 1166-1554* 100= when aligned using Standard methods. 75.0). It is noted that the percent sequence identity value is 0105 Methods of alignment of sequences for comparison rounded to the nearest tenth. For example, 75.11, 75.12, are well known in the art. Various programs and alignment 75.13, and 75.14 is rounded down to 75.1, while 75.15, algorithms are described in: Smith and Waterman, Adv. Appl. 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It is Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. also noted that the length value will always be an integer. In 48:443-53, 1970; Pearson and Lipman, Proc. Natl. Acad. another example, a target Sequence containing a 20-nucle Sci. U.S.A. 85:2444-8, 1988; Higgins and Sharp, Gene otide region that aligns with 20 consecutive nucleotides 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, from an identified Sequence as follows contains a region that 1989; Corpet et al., Nucleic Acids Research 16:10881-90, shares 75 percent Sequence identity to that identified 1988; and Altschul et al., Nature Genet. 6:119-29, 1994. sequence (i.e., 15+20*100=75). 0106) The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschulet al., J. Mol. Biol. 215:403-10, 1990) 1 20 is available from Several Sources, including the National Target Sequence: AGGTCGTGTACTGTCAGTCA Center for Biotechnology Information (NCBI, Bethesda, | | | | | | | | | | | | | | | Md.) and on the Internet, for use in connection with the Identified Sequence: ACGTGGTGAACTGCCAGTGA Sequence analysis programs blastp, blastin, blastX, thlastin and thlastX. 0109 Specific binding agent: An agent that is capable of Specifically binding to any of the polypeptide described 0107 Variants of a peptide are typically characterized by herein. Examples include, but are not limited to, polyclonal possession of at least 50% sequence identity counted over antibodies, monoclonal antibodies (including humanized the full length alignment with the amino acid Sequence using monoclonal antibodies), and fragments of monoclonal anti the NCBI Blast 2.0, gapped blastp set to default parameters. bodies Such as Fab, F(ab'), and Fv fragments as well as any For comparisons of amino acid Sequences of greater than other agent capable of Specifically binding to an epitope of about 30 amino acids, the Blast 2 Sequences function is Such polypeptides. employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per 0110 Antibodies to the polypeptides provided herein (or residue gap cost of 1). When aligning short peptides (fewer fragments, variants, or fusions thereof) can be used to purify than around 30 amino acids), the alignment is performed or identify Such polypeptides. The amino acid and nucleic using the Blast 2 sequences function, employing the PAM30 acid Sequences provided herein allow for the production of matrix set to default parameters (open gap 9, extension gap Specific antibody-based binding agents that recognize the 1 penalties). Proteins with even greater Similarity to the polypeptides described herein. reference Sequences will show increasing percentage iden tities when assessed by this method, such as at least 80%, at 0111 Monoclonal or polyclonal antibodies can be pro least 90%, at least 95%, at least 98%, or even at least 99% duced to the polypeptides, portions of the polypeptides, or Sequence identity. When less than the entire Sequence is variants thereof. Optimally, antibodies raised against one or being compared for Sequence identity, homologs and Vari more epitopes on a polypeptide antigen will Specifically ants will typically possess at least 80% sequence identity detect that polypeptide. That is, antibodies raised against one over Short windows of 10-20 amino acids, and may possess particular polypeptide would recognize and bind that par sequence identities of at least 85%, at least 90%, at least ticular polypeptide, and would not Substantially recognize or 95%, or 98% depending on their similarity to the reference bind to other polypeptides. The determination that an anti Sequence. Methods for determining Sequence identity over body Specifically binds to a particular polypeptide is made Such short windows are described at the website that is by any one of a number of Standard immunoassay methods, maintained by the National Center for Biotechnology Infor for instance, Western blotting (See, e.g., Sambrook et al. mation in Bethesda, Md. One of skill in the art will appre (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., ciate that these Sequence identity ranges are provided for vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring guidance only, it is entirely possible that Strongly significant Harbor, N.Y., 1989). homologs could be obtained that fall outside of the ranges 0112 To determine that a given antibody preparation provided. (Such as a preparation produced in a mouse against a 0108 Similar methods can be used to determine the polypeptide having the amino acid Sequence Set forth in percent Sequence identity of a nucleic acid Sequence. In a SEQ ID NO: 12) specifically detects the appropriate US 2005/0282260 A1 Dec. 22, 2005 polypeptide (e.g., a polypeptide having the amino acid 0118 Stereoinverting aminotransferase: A “stereoinvert sequence set forth in SEQ ID NO: 12) by Western blotting, ing aminotransferase' is a polypeptide capable of preferen total cellular protein can be extracted from cells and Sepa tially or Selectively producing a chiral amino acid product rated by SDS-polyacrylamide gel electrophoresis. (Such as monatin) while using an opposite chirality Substrate 0113. The separated total cellular protein can then be as the amino donor. For example, a Stereoinverting ami transferred to a membrane (e.g., nitrocellulose), and the notransferase may be a D-phenylglycine aminotransferase antibody preparation incubated with the membrane. After (also called D-4-hydroxyphenylglycine aminotransferase) Washing the membrane to remove non-specifically bound that preferentially or Selectively uses L-glutamate as a antibodies, the presence of Specifically bound antibodies can Substrate to produce R,R monatin. Non-limiting examples of be detected using an appropriate Secondary antibody (e.g., Stereoinverting aminotransferases include D-methionine an anti-mouse antibody) conjugated to a polypeptide Such as aminotransferase (EC 2.6.1.41) and enzymes having D-phe alkaline phosphatase Since application of 5-bromo-4-chloro nylglycine aminotransferase activity or D-4-hydroxyphe 3-indolyl phosphate/nitro blue tetrazolium results in the nylglycine aminotransferase activity. production of a densely blue-colored compoundby immuno 0119 Transformed: A “transformed” cell is a cell into localized alkaline phosphatase. which a nucleic acid molecule has been introduced by, for 0114 Substantially pure polypeptides suitable for use as example, molecular biology techniques. Transformation an immunogen can be obtained from transfected cells, encompasses all techniques by which a nucleic acid mol transformed cells, or wild-type cells. Polypeptide concen ecule can be introduced into Such a cell including, without trations in the final preparation can be adjusted, for example, limitation, transfection with a viral vector, conjugation, by concentration on an Amicon filter device, to the level of transformation with a plasmid vector, and introduction of a few micrograms per milliliter. In addition, polypeptides naked DNA by electroporation, lipofection, and particle gun ranging in size from full-length polypeptides to polypeptides acceleration. having as few as nine amino acid residues can be utilized as 0120 Variants, fragments or fusion proteins: The dis immunogens. Such polypeptides can be produced in cell closed proteins, include variants, fragments, and fusions culture, can be chemically Synthesized using Standard meth thereof. DNA sequences (for example, SEQ ID NO: 11) ods, or can be obtained by cleaving large polypeptides into which encode for a protein (for example, SEQ ID NO: 12), Smaller polypeptides that can be purified. Polypeptides fusion protein, or a fragment or variant of a protein, can be having as few as nine amino acid residues in length can be engineered to allow the protein to be expressed in eukaryotic immunogenic when presented to an immune System in the cells, bacteria, insects, and/or plants. To obtain expression, context of a Major Histocompatibility Complex (MHC) the DNA sequence can be altered and operably linked to molecule Such as an MHC class I or MHC class II molecule. other regulatory Sequences. The final product, which con Accordingly, polypeptides having at least 9, 10, 11, 12, 13, tains the regulatory Sequences and the protein, is referred to 14, 15, 20, 25, 30,35, 40, 45, 50,55, 60, 70, 80,90, 100, 150, as a vector. This vector can be introduced into eukaryotic, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, bacteria, insect, and/or plant cells. Once inside the cell the 800, 900, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, vector allows the protein to be produced. or more consecutive amino acid residues of any amino acid Sequence disclosed herein can be used as immunogens for 0121 A fusion protein including a protein, Such as a producing antibodies. tryptophan aminotransferase (or variant, polymorphism, mutant, or fragment thereof), for example SEQ ID NO: 12, 0115 Monoclonal antibodies to any of the polypeptides linked to other amino acid Sequences that do not inhibit the disclosed herein can be prepared from murine hybridomas desired activity of the protein, for example the ability to according to the classic method of Kohler & Milstein convert tryptophan to indole-3-pyruvate. In one example, (Nature 256:495-7, 1975) or a derivative method thereof. the other amino acid Sequences are no more than about 10, 0.116) Polyclonal antiserum containing antibodies to the 12, 15, 20, 25, 30, or 50 amino acids in length. heterogeneous epitopes of any polypeptide disclosed herein 0.122 One of ordinary skill in the art will appreciate that can be prepared by immunizing Suitable animals with the a DNA sequence can be altered in numerous ways without polypeptide (or fragment thereof), which can be unmodified affecting the biological activity of the encoded protein. For or modified to enhance immunogenicity. An effective immu example, PCR can be used to produce variations in the DNA nization protocol for rabbits can be found in Vaitukaitis et al. Sequence which encodes an protein. Such variants can be (J. Clin. Endocrinol. Metab. 33:988-91,1971). variants optimized for codon preference in a host cell used 0117 Antibody fragments can be used in place of whole to express the protein, or other Sequence changes that antibodies and can be readily expressed in prokaryotic host facilitate expression. cells. Methods of making and using immunologically effec 0123 Vector: A nucleic acid molecule as introduced into tive portions of monoclonal antibodies, also referred to as a cell, thereby producing a transformed cell. A vector may “antibody fragments, are well known and include those include nucleic acid Sequences that permit it to replicate in described in Better & Horowitz (Methods Enzymol. the cell, Such as an origin of replication. A vector may also 178:476-96, 1989), Glockshuber et al. (Biochemistry include one or more Selectable marker genes and other 29:1362-7, 1990), U.S. Pat. No. 5,648,237 (“Expression of genetic elements known in the art. Functional Antibody Fragments”), U.S. Pat. No. 4,946,778 ("Single Polypeptide Chain Binding Molecules”), U.S. Pat. Overview of Biosynthetic Pathways No. 5,455,030 (“Immunotherapy Using Single Chain Polypeptide Binding Molecules”), and references cited 0124. As shown in FIGS. 1-3 and 11-13, many biosyn therein. thetic pathways can be used to produce monatin or its US 2005/0282260 A1 Dec. 22, 2005 intermediates such as indole-3-pyruvate or MP. For the converts L-tryptophan and phenylpyruvate to indole-3-pyru conversion of each Substrate (glucose, tryptophan, indole vate and L-phenylalanine; L-amino acid oxidase (also 3-lactic acid, indole-3-pyruvate, and MP) to each product termed Ophio-amino-acid oxidase and L-amino-acid:oxygen (tryptophan, indole-3-pyruvate, MP and monatin) Several (deaminating)) which converts an L-amino different polypeptides can be used. Moreover, these reac acid and H2O and O2 to a 2-oxo acid and NH and H2O, tions can be carried out in Vivo, in Vitro, or through a D-amino acid oxidase (also termed Ophio-amino-acid oxi combination of in Vivo reactions and in Vitro reactions, Such dase and D-amino-acid:oxygen oxidoreductase (deaminat as in vitro reactions that include non-enzymatic chemical ing)) which converts a D-amino acid and H2O and O2 to a reactions. Therefore, FIGS. 1-3 and 11-13 are exemplary, 2-oxo acid and NH and H2O, and tryptophan oxidase and show multiple different pathways that can be used to which converts L-tryptophan and H2O and O. to indole-3- obtain desired products. pyruvate and NH and HO. These classes also contain tyrosine (aromatic) aminotransferase, aspartate aminotrans 0.125 Glucose to Tryptophan ferase, D-amino acid (or D-alanine) aminotransferase, and 0.126 Many organisms can synthesize tryptophan from broad (multiple Substrate) aminotransferase which have glucose. The construct(s) containing the gene(s) necessary multiple aminotransferase activities, Some of which can to produce monatin, MP, and/or indole-3-pyruvate from convert tryptophan and a 2-oxo acid to indole-3-pyruvate glucose and/or tryptophan can be cloned into Such organ and an amino acid. isms. It is shown herein that tryptophan can be converted 0131 Eleven members of the aminotransferase class that into monatin. have Such activity are described below in Example 1, 0127. In other examples, an organism is engineered using including a novel aminotransferase shown in SEQ ID NOS: known polypeptides to produce tryptophan, or overproduce 11 and 12. Therefore, this disclosure provides isolated tryptophan. For example, U.S. Pat. No. 4,371,614 (herein nucleic acid and protein Sequences having at least 80%, at incorporated by reference) describes an E. coli Strain trans least 85%, at least 90%, at least 95%, at least 98%, or even formed with a plasmid containing a wild type tryptophan at least 99% sequence identity to SEQ ID NOS: 11 and 12. operOn. Also encompassed by this disclosure are fragments and fusions of SEQ ID NOS: 11 and 12 that retain aminotrans 0128 Maximum titers of tryptophan disclosed in U.S. ferase activity or encode a protein having aminotransferase Pat. No. 4,371,614 are about 230 ppm. Similarly, WO activity. Exemplary fragments include, but are not limited to 8701130 (herein incorporated by reference) describes an E. at least 10, 12, 15, 20, 25, 50, 100, 200, 500, or 1000 coli Strain that has been genetically engineered to produce contiguous nucleotides of SEQ ID NO: 11 or at least 6, 10, tryptophan and discusses increasing fermentative production 15, 20, 25, 50, 75, 100, 200, 300 or 350 contiguous amino of L-tryptophan. Those skilled in the art will recognize that acids of SEQ ID NO: 12. The disclosed sequences (and organisms capable of producing tryptophan from glucose are variants, fragments, and fusions thereof) can be part of a also capable of utilizing other carbon and energy Sources vector. The vector can be used to transform host cells, that can be converted to glucose or fructose-6-phosphate, thereby producing recombinant cells which can produce with Similar results. Exemplary carbon and energy Sources indole-3-pyruvate from tryptophan, and in Some examples include, but are not limited to, Sucrose, fructose, Starch, can further produce MP and/or monatin. cellulose, or glycerol. 0132) L-amino acid oxidases (1.4.3.2) are known, and 0129. Tryptophan to Indole-3-pyruvate Sequences can be isolated from Several different Sources, 0130 Several polypeptides can be used to convert tryp such as Vipera lebetine (sp P81375), Ophiophagus hannah tophan to indole-3-pyruvate. Exemplary polypeptides (sp P81383), Agkistrodon rhodostoma (spP81382), Crotalus include members of the enzyme classes (EC) 2.6.1.27, atrox (sp P56742), Burkholderia cepacia, Arabidopsis 1.4.1.19, 1.4.99.1, 2.6.1.28, 1.4.3.2, 1.4.3.3, 2.6.1.5, 2.6.1.-, thaliana, Caulobacter creSentus, Chlamydomonas rein 2.6.1.1 and 2.6.1.21. These classes include polypeptides hardtii, MuS musculus, Pseudomonas Syringae, and Rhodo termed tryptophan aminotransferase (also termed L-pheny coccuS Str. In addition, tryptophan oxidases are described in lalanine-2-oxoglutarate aminotransferase, tryptophan tran the literature and can be isolated, for example, from Copri Saminase, 5-hydroxytryptophan-ketoglutaric transaminase, nus Sp. SF-1, Chinese cabbage with club root disease, hydroxytryptophan aminotransferase, L-tryptophan ami Arabidopsis thaliana, and mammalian liver. One member of notransferase, L-tryptophan transaminase, and L-tryp the L-amino acid oxidase class that can convert tryptophan tophan:2-oxoglutarate aminotransferase) which converts to indole-3-pyruvate is discussed below in Example 5, as L-tryptophan and 2-oxoglutarate to indole-3-pyruvate and well as alternative Sources for molecular cloning. Many L-glutamate, D-tryptophan aminotransferase which con D-amino acid oxidase genes are available in databases for verts D-tryptophan and a 2-oxo acid to indole-3-pyruvate molecular cloning. and an amino acid; tryptophan dehydrogenase (also termed 0.133 Tryptophan dehydrogenases are known, and can be NAD(P)-L-tryptophan dehydrogenase, L-tryptophan dehy isolated, for example, from Spinach, Pisum Sativum, PrOSO drogenase, L-Trp-dehydrogenase, TDH and L-tryptophan pis juliflora, pea, mesquite, wheat, maize, tomato, tobacco, :NAD(P) oxidoreductase (deaminating)) which converts Chronobacterium violaceum, and Lactobacilli. Many L-tryptophan and NAD(P) to indole-3-pyruvate and NH3 D-amino acid dehydrogenase gene Sequences are known. and NAD(P)H; D-amino acid dehydrogenase, which con verts D-amino acids and FAD to indole-3-pyruvate and NH 0134 U.S. Pat. No. 5,728,555 discloses phenylalanine and FADH2, tryptophan-phenylpyruvate transaminase (also deaminases (EC 3.5.1.-), which can also convert tryptophan termed L-tryptophan-O-ketoisocaproate aminotransferase to indole-3-pyruvate and ammonium in the presence of and L-tryptophan:phenylpyruvate aminotransferase) which water. These broad Specificity enzymes can be isolated from US 2005/0282260 A1 Dec. 22, 2005 11

ProteuS microorganisms, Such as Proteus myxofaciens, Pro late-lyase also termed 4-hydroxy-2-oxoglutarate aldolase, teus mirabilis, Proteus vulgaris and Proteus morganii, and 2-oxo-4-hydroxyglutarate aldolase or KHG aldolase) con corresponding genes have been cloned and Sequenced. See verts glyoxylic acid and pyruvate to 4-hydroxy-2-ketoglu e.g., Proteus vulgaris deaminase (protein accession number: taric acid, and the polypeptide 4-hydroxy-4-methyl-2-oxo BAA90864.1 GI: 7007412; gene accession number: glutarate aldolase (EC 4.1.3.17, also termed 4-hydroxy-4- AB030003.1 GI: 700741. 1); Proteus mirabilis deaminase methyl-2-oxoglutarate pyruvate-lyase or ProA aldolase), (protein accession number: AAA86752.1 GI: 1015426; gene condenses two keto-acids Such as two pyruvates to 4-hy accession number: U35383.1 GI: 1015425). Providencia droxy-4-methyl-2-oxoglutarate. Reactions utilizing these and Morganella also contain L-deaminases that can convert are described herein. tryptophan to indole-3-pyruvate. See H. Drechsel, A. Thieken, R. Reissbrodt, G. Jung, and G. Winkelmann. J. 0.142 FIGS. 1-2 and 11-13 show schematic diagrams of these reactions in which a 3-carbon (C3) molecule is com Bacteriol., 175: 2727-2733 (1993). bined with indole-3-pyruvate. Many members of EC 4.1.2.- 0135). As shown in FIGS. 11-13, if an amino acid oxi and 4.1.3.-, particularly PLP-utilizing polypeptides, can dase, Such as tryptophan oxidase, is used to convert tryp utilize C3 molecules that are amino acids Such as Serine, tophan to indole-3-pyruvate, catalase can be added to reduce cysteine, and alanine, or derivatives thereof. Aldol conden or even eliminate the presence of hydrogen peroxide. sations catalyzed by representatives of EC 4.1.2.- and 4.1.3.- Indole-3-lactate to Indole-3-pyruvate require the three carbon molecule (i.e., C3 carbon Source) of 0.136) this pathway to be pyruvate or a derivative of pyruvate. 0.137 The reaction that converts indole-3-lactate to However, other compounds can Serve as a C3 carbon Source indole-3-pyruvate can be catalyzed by a variety of polypep and be converted to pyruvate. Alanine can be transaminated tides, Such as members of the 1.1.1.110, 1.1.1.27, 1.1.1.28, by many PLP-utilizing , including many of 1.1.2.3, 1.1.1.222, 1.1.1.237, 1.1.3.-, or 1.1.1.111 classes of those mentioned above, to yield pyruvate. Pyruvate and polypeptides. The 1.1.1.110 class of polypeptides includes ammonia can be obtained by beta-elimination reactions indolelactate dehydrogenases (also termed indolelactic acid: (Such as those catalyzed by tryptophanase or f3-tyrosinase) NAD" oxidoreductase). The 1.1.1.27, 1.1.1.28, and 1.1.2.3 of L-serine, L-cysteine, and derivatives of Serine and cys classes include lactate dehydrogenases (also termed lactic teine with Sufficient leaving groups, Such as O-methyl-L- acid dehydrogenases, lactate: NAD" oxidoreductase). The Serine, O-benzyl-L-serine, S-methylcysteine, S-benzylcyS 1.1.1.222 class contains (R)-4-hydroxyphenylactate dehy teine, S-alkyl-L-cysteine, O-acyl-L-serine, and 3-chloro-L- drogenase (also termed D-aromatic lactate dehydrogenase, alanine. Aspartate can serve as a Source of pyruvate in R-aromatic lactate dehydrogenase, and R-3-(4-hydroxyphe PLP-mediated beta-lyase reactions Such as those catalyzed nyl)lactate:NAD(P)' 2-oxidoreductase) and the 1.1.1.237 by tryptophanase (EC 4.1.99.1) and/or B-tyrosinase (EC class contains 3-(4-hydroxyphenylpyruvate)reductase (also 4.1.99.2, also termed tyrosine-phenol lyase). The rate of termed hydroxyphenylpyruvate reductase and 4-hydrox beta-lyase reactions can be increased by performing site yphenylactate:NAD" oxidoreductase). The 1.1.3.-class con directed mutagensis on the (4.1.99.1-2) polypeptides as tains lactate oxidases, and the 1.1.1.111 class contains described by Mouratou et al. (J. Biol. Chem 274: 1320-5, (3-imidazol-5-yl)lactate dehydrogenases (also termed (S)-3- 1999) and in Example 18. These modifications allow the (imidazol-5-yl)lactate:NAD(P) oxidoreductase). It is likely polypeptides to accept dicarboxylic amino acid Substrates. that Several of the polypeptides in these classes allow for the Lactate can also serve as a Source of pyruvate, and is production of indole-3-pyruvate from indole-3-lactic acid. oxidized to pyruvate by the addition of lactate dehydroge Examples of this conversion are provided in Example 4. nase and an oxidized cofactor or lactate oxidase and oxygen. 0138 Chemical reactions can also be used to convert Examples of these reactions are described below. For indole-3-lactic acid to indole-3-pyruvate. Such chemical example, as shown in FIG. 2 and FIGS. 11-13, ProA reactions include an oxidation Step that can be accomplished aldolase can be contacted with indole-3-pyruvate when using Several methods, for example: air oxidation using a B2 pyruvate is used as the C3 molecule. catalyst (China Chemical Reporter, v 13, n. 28, p 18 (1), 0143. The MP can also be generated using chemical 2002), dilute permanganate and perchlorate, or hydrogen reactions, Such as the aldol condensations provided in peroxide in the presence of metal catalysts. Example 8. 0139 Indole-3-pyruvate to 2-hydroxy 2-(indol-3ylm ethyl)-4-ketoglutaric acid (MP) 0144) MP to Monatin 0140) Several known polypeptides can be used to convert 0145 Conversion of MP to monatin can be catalyzed by indole-3-pyruvate to MP. Exemplary polypeptide classes one or more of tryptophan aminotransferases (2.6.1.27), include 4.1.3.-, 4.1.3.16, 4.1.3.17, and 4.1.2.-. These classes tryptophan dehydrogenases (1.4.1.19), D-amino acid dehy include carbon-carbon Synthases/lyases, Such as aldolases drogenases (1.4.99.1), glutamate dehydrogenases (1.4.1.2- that catalyze the condensation of two carboxylic acid Sub 4), phenylalanine dehydrogenase (EC 1.4.1.20), leucine Strates. Peptide class EC 4.1.3.- are Synthases/lyases that (branched-chain) dehydrogenase (EC 1.4.1.9), tryptophan form carbon-carbon bonds utilizing oxo-acid Substrates phenylpyruvate transaminases (2.6.1.28), or more generally (such as indole-3-pyruvate) as the electrophile, while EC members of the aminotransferase family (2.6.1.-), Such as 4.1.2.- are Synthases/lyases that form carbon-carbon bonds aspartate aminotransferase (EC 2.6.1.1), tyrosine (aromatic) utilizing aldehyde Substrates (Such as benzaldehyde) as the aminotransferase (2.6.1.5), D-tryptophan aminotransferase, electrophile. D-alanine aminotransferase(2.6.1.21) (FIG. 2) and branched-chain aminotransferase (BCAT, EC 2.6.1.42). 0141 For example, the polypeptide described in EP Eleven members of the aminotransferase class are described 1045-029 (EC 4.1.3.16, 4-hydroxy-2-oxoglutarate glyoxy below (Example 1), including a novel member of the class US 2005/0282260 A1 Dec. 22, 2005

shown in SEQ ID NOS: 11 and 12, and reactions demon 0154) The biosynthetic pathway for vitamin B (the pre Strating the activity of aminotransferase and dehydrogenase cursor of PLP) has been thoroughly studied in E. coli and enzymes are provided in Example 15. Some of the proteins have been crystallized (Laber et al., 0146 This reaction can also be performed using chemical FEBS Letters, 449:45-8, 1999). Two of the genes (epd or reactions. Amination of the keto acid (MP) is performed by gap B and SerC) are required in other metabolic pathways, reductive amination using ammonia and Sodium cyanoboro while three genes (pdxA, pdxB, and pdx.J) are unique to hydride. biosynthesis. One of the Starting mate rials in the E. coli pathway is 1-deoxy-D-xylulose-5-phos 0147 FIGS. 11-13 show additional polypeptides that can phate (DXP). Synthesis of this precursor from common 2 be used to convert MP to monatin, as well as providing and 3 carbon central metabolites is catalyzed by the increased yields of monatin from indole-3-pyruvate or tryp polypeptide 1-deoxy-D-xylulose-5-phosphate Synthase tophan. For example, if aspartate is used as the amino donor, (DSX). The other precursor is a threonine derivative formed aspartate aminotransferase can be used to convert the aspar from the 4-carbon Sugar, D-erythrose 4-phosphate. The tate to oxaloacetate (FIG. 11). The oxaloacetate is converted genes required for the conversion to phospho-4-hydroxyl-L to pyruvate and carbon dioxide by a decarboxylase, Such as threonine (HTP) are epd, pdxB, and serC. The last reaction oxaloacetate decarboxylase (FIG. 11) and 2-oxoglutarate for the formation of PLP is a complex intramolecular decarboxylase (EC 4.1.1.71). In addition, if lysine is used as condensation and ring-closure reaction between DXP and the amino donor, lysine epsilon aminotransferase (EC HTP, catalyzed by the gene products of pdxA and pdx.J. 2.6.1.36) can be used to convert the lysine to allysine (FIG. 12). The alysine is spontaneously converted to 1-piperi 0155 If PLP becomes a limiting nutrient during the deine 6-carboxylate (FIG. 12). An analogous process to that fermentation to produce monatin, increased expression of shown in FIG. 12 uses ornithine 8-aminotransferase (EC one or more of the pathway genes in a production host cell 2.6.1.13) in place of lysine epsilon aminotransferase, and can be used to increase the yield of monatin. A host organism ornithine Serves as the amino donor. If a polypeptide capable can contain multiple copies of its native pathway genes or of catalyzing reductive amination reactions (e.g., glutamate copies of non-native pathway genes can be incorporated into dehydrogenase) is used to convert MP to monatin, a the organism's genome. Additionally, multiple copies of the polypeptide that can recycle NAD(P)H and/or produce a Salvage pathway genes can be cloned into the host organism. volatile product (FIG. 13) can be used, such as formate dehydrogenase. 0156. One salvage pathway that is conserved in all organ isms recycles the various derivatives of Vitamin B to the Additional Considerations in the Design of the active PLP form. The polypeptides involved in this pathway Biosynthetic Pathways are pdxK kinase, pdxH oxidase, and pdxY kinase. Over expression of one or more of these genes can increase PLP 0148 Depending on which polypeptides are used to availability. generate indole-3-pyruvate, MP and/or monatin, cofactors, O157 Vitamin B levels can be elevated by elimination or Substrates, and/or additional polypeptides can be provided to repression of the metabolic regulation of the native biosyn the production cell to enhance product formation. thetic pathway genes in the host organism. PLP represses 0149 Removal of Hydrogen Peroxide polypeptides involved in the biosynthesis of the precursor 0150 Hydrogen peroxide (HO) is a product that, if threonine derivative in the bacterium Flavobacterium sp. generated, can be toxic to production cells and can damage strain 238-7. This bacterial strain, freed of metabolic control, the polypeptides or intermediates produced. The L-amino overproduces pyridoxal derivatives and can excrete up to 20 acid oxidase described above generates H2O as a product. mg/L of PLP. Genetic manipulation of the host organism Therefore, if L-amino acid oxidase is used, the resulting producing monatin in a similar fashion will allow the H.O. can be removed or its levels decreased to decrease increased production PLP without over-expression of the potential injury to the cell or product. biosynthetic pathway genes. 0151 Catalases can be used to reduce the level of HO 0158 Ammonium Utilization in the cell (FIGS. 11-13). The production cell can express a gene or cDNA sequence that encodes a catalase (EC 1.11. 0159. Tryptophanase reactions can be driven toward the 1.6), which catalyzes the decomposition of hydrogen per Synthetic direction (production of tryptophan from indole) oxide into water and oxygen gas. For example, a catalase can by making ammonia more available or by removal of water. be expressed from a vector transfected into the production Reductive amination reactions, Such as those catalyzed by cell. Examples of catalases that can be used to include, but glutamate dehydrogenase, can also be driven forward by an are not limited to: trQ9EV50 (Staphylococcus xylosus), excess of ammonium. trQ9KBE8 (Bacillus halodurans), trQ9URJ7 (Candida 0160 Ammonia can be made available as an ammonium albicans), trP77948 (Streptomyces coelicolor), trC9RBJ5 carbonate or ammonium phosphate Salt in a carbonate or (Xanthomonas campestris) (SwissProt Accession Nos.). phosphate buffered System. Ammonia can also be provided Biocatalytic reactors utilizing L-amino acid oxidase, as ammonium pyruvate or ammonium formate. Alterna D-amino acid oxidase, or tryptophan oxidase can also con tively, ammonia can be Supplied if the reaction is coupled tain a catalase polypeptide. with a reaction that generates ammonia, Such as glutamate 0152 Modulation of PLP (pyridoxal-5'-phosphate) Avail dehydrogenase, tryptophan dehydrogenase or branched ability chain dehydrogenase. Ammonia can be generated by addi tion of the natural substrates of EC 4.1.99.- (tyrosine or 0153. As shown in FIG. 1, PLP can be utilized in one or tryptophan), which will be hydrolyzed to phenol or indole, more of the biosynthetic steps described herein. The con pyruvate and NH. This also allows for an increased yield of centration of PLP can be supplemented so that PLP does not Synthetic product Over the normal equilibrium amount by become a limitation on the Overall efficiency of the reaction. allowing the enzyme to hydrolyze its preferred Substrate. US 2005/0282260 A1 Dec. 22, 2005

0.161 Removal of Products and Byproducts the gene coding for EC 4.1.99.1 (Kawasaki et al., J. Ferm. 0162 The conversion of tryptophan to indole-3-pyruvate and Bioeng, 82:604-6, 1996). Genetic modifications can be via a tryptophan aminotransferase may adversely affect the made to increase the level of intermediates Such as D-eryth production rate of indole-3-pyruvate because the reaction rose-4-phosphate and chorismate. produces glutamate and requires the co-Substrate 2-oxoglu 0169 Tryptophan production is regulated in most organ tarate (C.-ketoglutarate). Glutamate may cause inhibition of isms. One mechanism is via feedback inhibition of certain the aminotransferase, and the reaction will consume large enzymes in the pathway; as tryptophan levels increase, the amounts of the co-Substrate. Moreover, high glutamate con production rate of tryptophan decreases. Thus, when using a centrations are detrimental to downstream Separation pro host cell engineered to produce monatin via a tryptophan CCSSCS. intermediate, an organism can be used that is not Sensitive to tryptophan concentrations. For example, a Strain of Catha 0163 The polypeptide glutamate dehydrogenase ranthus roseus that is resistant to growth inhibition by (GLDH) converts glutamate to 2-oxoglutarate, thereby recy various tryptophan analogs was Selected by repeated expo cling the co-Substrate in the reaction catalyzed by tryptophan Sure to high concentrations of 5-methyltryptophan (Schal aminotransferase. GLDH also generates reducing equiva lenberg and Berlin, Z Naturforsch 34:541-5, 1979). The lents (NADH or NADPH) that can be used to generate resulting tryptophan Synthase activity of the Strain was leSS energy for the cell (ATP) under aerobic conditions. The effected by product inhibition, likely due to mutations in the utilization of glutamate by GLDH also reduces byproduct gene. Similarly, a host cell used for monatin production can formation. Additionally, the reaction generates ammonia, be optimized. which can Serve as a nitrogen Source for the cell or as a Substrate in a reductive amination for the final Step shown in 0170 Tryptophan production can be optimized through FIG. 1. Therefore, a production cell that over-expresses a the use of directed evolution to evolve polypeptides that are GLDH polypeptide can be used to increase the yield and 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 medium, but with high levels of non-metabolizable tryp 0164. In the tryptophan to monatin pathway, the amino tophan analogs. U.S. Pat. Nos. 5,756,345; 4,742,007; and donor of Step three (e.g., glutamate or aspartate) can be 4,371,614 describe methods used to increase tryptophan converted back to the amino acceptor required for Step 1 productivity in a fermentation organism. The last Step of (e.g., 2-oxo-glutarate or oxaloacetate), if an aminotrans tryptophan biosynthesis is the addition of Serine to indole; ferase from the appropriate enzyme classes is used. Utili therefore the availability of serine can be increased to Zation of two separate transaminases for this pathway, in increase tryptophan production. which the Substrate of one transaminase does not competi tively inhibit the activity of the other transaminase, can 0171 The amount of monatin produced by a fermentation increase the efficiency of this pathway. organism can be increased by increasing the amount of pyruvate produced by the host organism. Certain yeasts, 0.165 Many of the reactions in the described pathways Such as TrichoSpOron cutaneum (Wang et al., Lett. Appl. are reversible and will, therefore, reach an equilibrium Microbiol. 35:338-42, 2002) and Torulopsis glabrata (Liet between substrates and products. The yield of the pathway al., Appl Microbiol. Biotechnol. 57:451-9, 2001) overpro can be increased by continuous removal of the products duce pyruvate and can be used to practice the methods from the polypeptides. For example, Secretion of monatin disclosed herein. In addition, genetic modifications can be into the fermentation broth using a permease or other made to organisms to promote pyruvic acid production, Such transport protein, or Selective crystallization of monatin as those in E. coli strain W1485lip2 (Kawasaki et al., J. from a biocatalytic reactor Stream with concomitant recycle Ferm. and Bioeng. 82:604-6, 1996). of Substrates will increase the reaction yield. 0172 Controlling Chirality 0166 The removal of byproducts by additional enzy matic reactions or by Substitution of amino donor groups is 0173 The taste profile of monatin can be altered by another way to increase the reaction yield. Several examples controlling the Stereochemistry (chirality) of the molecule. are discussed in Example 21 and shown in FIGS. 11-13. For example, different monatin isomers may be desired in Ideally a byproduct is produced that is unavailable to react different blends of concentrations for different food systems. in the reverse direction, either by phase change (evapora Chirality can be controlled via a combination of pH and tion) or by Spontaneous conversion to an unreactive end polypeptides. product, Such as carbon dioxide. 0167 Modulation of the Substrate Pools 0168 The indole pool can be modulated by increasing production of tryptophan precursors and/or altering cata bolic pathways involving indole-3-pyruvate and/or tryp tophan. For example, the production of indole-3-acetic acid from indole-3-pyruvate can be reduced or eliminated by functionally deleting the gene coding for EC 4.1.1.74 in the host cell. Production of indole from tryptophan can be reduced or eliminated by functionally deleting the gene coding for EC 4.1.99.1 in the host cell. Alternatively, an excess of indole can be utilized as a Substrate in an in Vitro 0174 Racemization 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 US 2005/0282260 A1 Dec. 22, 2005 reprotonation of the alpha carbon, which can occur by a shift branched-chain aminotransferase (BCAT) (EC 2.6.1.42) in pH or by reaction with the cofactor PLP. In a microor and/or a branched-chain dehydrogenase (EC 1.4.1.9). For ganism, the pH is unlikely to Shift enough to cause the example, the branched-chain aminotransferase and/or a racemization, but PLP is abundant. Methods to control the branched-chain dehydrogenase may be used to facilitate a chirality with polypeptides depend upon the biosynthetic reaction of 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric route utilized for monatin production. acid. Suitable branched-chain aminotransferases (BCAT) 0175 When monatin is formed using the pathway shown (EC 2.6.1.42) may include AT-102 or AT-104. Suitable in FIG. 2, the following can be considered. In a biocatalytic branched-chain dehydrogenases (EC 1.4.1.9) may include reaction, the chirality of carbon-2 is determined by the AADH-110. enzyme that converts indole-3-pyruvate to MP. Multiple 0180. In another embodiment, 2-hydroxy 2-(indol-3-yl enzymes (e.g. from EC 4.1.2.-, 4.1.3.-) can convert indole methyl)-4-ketoglutaric acid or a Salt thereof may be pro 3-pyruvate to MP, thus, one can choose the enzyme that duced by a method that includes using an aldolase Such as forms the desired isomer. Alternatively, the enantiospecific a KHG aldolase. Suitable KHG aldolases may include Z. ity of the enzyme that converts indole-3-pyruvate to MP can mobilis KHG aldolase (Accession No.: AAV89621.1 GI: be modified through the use of directed evolution or cata 56543467) and/or a polypeptide comprising an amino acid lytic antibodies can be engineered to catalyze the desired sequence that is at least about 90% identical to Z. mobilis reaction. Once MP is produced (either enzymatically or by KHG aldolase (Accession No.: AAV89621.1 GI: 56543467) chemical condensation), the amino group can be added and that has Z. mobilis KHG aldolase activity. In some Stereospecifically using a transaminase, Such as those embodiments, Suitable aldolases may include polpeptides described herein. Either the R or S configuration of carbon-4 comprising an amino acid Sequence that is at least about can be generated depending on whether a D- or L-aromatic 95% identical or at least about 99% identical to Z. mobilis acid aminotransferase is used. Most aminotransferases are KHG aldolase (Accession No.: AAV89621.1 GI: 56543467), Specific for the L-isomer, however D-tryptophan ami where the polypeptide has Z. mobilis KHG aldolase activity. notransferases exist in certain plants (Kohiba and Mito, The Selected aldolase may be used to facilitate a reaction Proceedings of the 8th International Symposium on Vitamin involving a substrate for synthesis of 2-hydroxy 2-(indol-3- B and Carbonyl Catalysis, Osaka, Japan 1990). Moreover, ylmethyl)-4-ketoglutaric acid, Such as indole-3-pyruvate. D-alanine aminotransferases (2.6.1.21), D-methionine-pyru vate aminotransferases (2.6.1.41) and both (R)-3-amino-2- 0181. In other embodiments, monatin or a salt thereof methylpropanoate aminotransferase (2.6.1.61) and (S)-3- may be produced by a method that includes using an amino-2-methylpropanoate aminotransferase (2.6.1.22) aldolase to facilitate a reaction involving a Substrate for have been identified. Certain aminotransferases may only synthesis of monatin, in which more than 60% of the accept the Substrate for this reaction with a particular con monatin produced in the reaction is an R,R Stereoisomer of figuration at the C2 carbon. Therefore, even if the conver monatin. Suitable aldolases for the reaction may include sion to MP is not stereospecific, the stereochemistry of the KHG aldolase (EC 4.1.3.16), and suitable substrates for the final product can be controlled through the appropriate reaction may include indole-3-pyruvate. Selection of a transaminase. Since the reactions are revers 0182 In other embodiments, 2-hydroxy 2-(indol-3-ylm ible, the unreacted MP (undesired isomer) can be recycled ethyl)-4-keto glutaric acid may be produced by a method back to its constituents and a racemic mixture of MP can be that includes reacting a Suitable reaction mixture, the mix reformed. ture including: (a) a Suitable Substrate Such as tryptophan; (b) a first polypeptide chosen from HEXAspC aminotrans 0176) Activation of Substrates ferase (NCBI Accession No. 1AHF A GI: 1127190), YfdZ 0177 Phosphorylated substrates, such as phospho (NCBI Accession No. AAC75438.1), and a combination enolpyruvate (PEP), can be used in the reactions disclosed thereof, and (c) a second polypeptide chosen from KHG herein. Phosphorylated Substrates can be more energetically aldolase (EC 4.1.3.16), ProA aldolase (EC 4.1.3.17), and a favorable and, therefore, can be used to increase the reaction combination thereof. Where the Substrate includes tryp rates and/or yields. In aldol condensations, the addition of a tophan, the tryptophan may include D-tryptophan, L-tryp phosphate group Stabilizes the enol tautomer of the nucleo tophan, and a mixture thereof. The tryptophan may be added philic Substrate, making it more reactive. In other reactions, to the reaction mixture and/or producted in Situ by reacting a phosphorylated Substrate often provides a better leaving Suitable Substrates (e.g., glucose and Serine). In Some group. Similarly, Substrates can be activated by conversion embodiments, the tryptophan includes D-tryptophan. The to CoA derivatives or pyrophosphate derivatives. KHG aldolase may include Z. mobilis KHG aldolase (Accession No.: AAV8621.1 GI: 56543467) and/or a Illustrative Embodiments polypeptide comprising an amino acid Sequence that is at least about 90% identical to Z. mobilis KHG aldolase 0178. In one embodiment, monatin or a salt thereof may (Accession No.: AAV89621.1 GI: 56543467) and having Z. be produced by a method that includes using an aminotrans mobilis KHG aldolase activity. In some embodiments, suit ferase, such as HEXAspC aminotransferase (NCBI Acces able aldolases may include polpeptides comprising an amino sion No: 1AHF A GI: 1127190). For example, HEXAspC acid Sequence that is at least about 95% identical or at least aminotransferase may be used to facilitate at least one about 99% identical to Z. mobilis KHG aldolase (Accession reaction in which the reaction involves a Substrate Selected No.: AAV89621.1 GI: 56543467), where the polypeptide from tryptophan, 2-hydroxy 2-(indol-3-ylmethyl)-4-keto has Z. mobilis KHG aldolase activity. In some embodiments, glutaric acid, and a combination thereof. the ProA aldolase may include a C. testosteroni ProA 0179. In another embodiment, monatin or a salt thereof aldolase, a S. meliloti ProA aldolase, and a combination may be produced by a method that includes using a thereof. The selected polypeptides may be added to the US 2005/0282260 A1 Dec. 22, 2005 reaction mixture and/or expressed by microorganisms provided to facilitate the conversion of 2-hydroxy 2-(indol present in the reaction mixture. For example, the reaction 3ylmethyl)-4-ketoglutaric acid to monatin. mixture may include a nutrient medium, whereby the 0186. In other embodiments, monatin or a salt thereof Selected polypeptides are produced by fermenting the nutri may be produced by a method that includes reacting a ent medium with a Suitable microorganism that expresses reaction mixture, the mixture including: (a) a Substrate the polypeptide. selected from tryptophan, 2-hydroxy 2-(indol-3-ylmethyl)- 0183 In other embodiments, monatin or a salt thereof 4-ketoglutaric acid, and a combination thereof, and (b) an may be produced by reacting a reaction mixture that E. coli AspC polypeptide (NCBI Accession No. includes: (a) a Suitable Substrate Such as 2-hydroxy 2-(indol AAC74014.1). The E. coli AspC polypeptide (NCBI Acces 3-ylmethyl)-4-ketoglutaric acid; and (b) a polypeptide cho sion No. AAC74014.1) may include at least one substitution sen from YfdZ (NCBI Accession No. AAC75438.1), HEX at an amino acid position chosen from positions 39, 41, 47, AspCaminotransferase (NCBI Accession No: 1AHF A GI: 69, 109, 297, and a combination thereof, in which number 1127190), a branched-chain aminotransferase (BCAT) (EC ing of the amino acid positions is based on a pig cytosolic 2.6.1.42), a branched-chain dehydrogenase (EC 1.4.1.9), aspartate aminotransferase numbering System. In Suitable and a combination thereof. 2-hydroxy 2-(indol-3-ylmethyl)- embodiments, the E. coli AspC polypeptide may have 4-keto glutaric acid may be added to the reaction mixture aspartate aminotransferase activity. The E. coli AspC and/or may be Synthesized in situ. For example, the reaction polypeptide may include at least one of the following mixture may further include (c) indole-3-pyruvate; and (d) a substitutions: a Val 39 to Leu Substitution; a Lys 41 to Tyr Second polypeptide that is capable of converting indole-3- Substitution; a Thr 47 to Ile Substitution; an ASn 69 to Leu pyruvate and a C3 carbon source to 2-hydroxy 2-(indol-3- Substitution; a Thr 109 to Ser Substitution; an ASn 297 to Ser ylmethyl)-4-ketoglutaric acid. The indole-3-pyruvate may Substitution; and combinations thereof. The Substrate may be added to the reaction mixture and/or may be Synthesized be added to the reaction mixture and/or produced in Situ in Situ from Suitable Substrates. The Selected polypeptides from suitable substrates. The E. coli AspC polypeptide may may be added to the reaction mixture and/or may be be added to the reaction mixture and/or expressed by a expressed by microorganisms present in the reaction mix microorganism present in the reaction mixture (e.g., by ture. For example, the reaction mixture may include a fermenting the reaction mixture, which may include a nutri nutrient medium that is fermented with a microorganism that ent medium, with a microorganism that expresses the E. coli expresses one or more of the Selected polypeptides. In Some AspC polypeptide). embodiments, the reaction mixture may include an unpuri 0187. In further embodiments, 2-hydroxy 2-(indol-3-yl fied cell extract, Such as a cell extract that includes YfdZ methyl)-4-ketoglutaric acid or a Salt thereof may be pro (NCBI Accession No. AAC75438.1), HEXAspC ami duced by reacting a reaction mixture, the mixture including: notransferase (NCBI Accession No: 1AHF A GI: 1127190), (a) a Substrate Such as indole-3-pyruvate, and (b) a Suitable or a combination thereof. aldolase. For example, the aldolase may be chosen from 0184. In some embodiments, monatin or a salt thereof Bradyrhizobium japonicum str. USDA 110 (NCBI Accession may be produced by reacting a reaction mixture that No: GI: 27378.953); Sphingomonas (Pseudomonas) pauci includes: (a) a Suitable Substrate Such as 2-hydroxy 2-(indol mobilis (NCBI Accession No: GI: 19918963); Yersinia pes 3-ylmethyl)-4-ketoglutaric acid; and (b) a polypeptide cho tis KIM (NCBI Accession No: GI: 21956715); Ralstonia Sen from AT-101, AT-102, AT-103, AT-104, AADH-102, metallidurans CH34 (NCBI Accession No: GI: 48767386); AADH-110, AADH-112, AADH-113, and a combination Yersinia pseudotuberculosis IP32953 (NCBI Accession No: thereof. In Some embodiments, the Selected polypeptide may GI: 51594436); Rhizobium leguminosarum biovar viciae be AT-103. The monatin or a salt thereof produced by the rhiz23g02-plk 1009 341 (SEQ ID NO: 88); Novosphin method may be predominantly the R,R stereoisomer of gobium aromaticivorans DSM 12444 (Sphingomonas aro monatin. For example, in Some embodiments at least about maticivorans F199) (NCBI Accession No: GI: 48849026); 65% of the monatin produced in the method may be an R.R Pseudomonas putida KT2440 (NCBI Accession No: GI: Stereoisomer of monatin. 24984081); Magnetospirillum magnetotacticum MS-1 0185. In other embodiments, monatin or a salt thereof (NCBI Accession No: GI: 46200890); Rhodopseudomonas may be produced from a Substrate, (Such as tryptophan palustris CGA009 (NCBI Accession No: GI: 39937756); and/or indole-3-pyruvate), by enzymatically producing a Xanthomonas campestris ATCC-33913 (NCBI Accession monatin composition, in which at least about 80% of the No: GI: 21115297); Xanthomonas axonopodis citri 306 monatin or Salt thereof present in the monatin composition (NCBI Accession No: GI: 21110581); Streptomyces aver is an R,R Stereoisomer of monatin. In Suitable embodiments mitilis MA-4680 (NCBI Accession No: GI: 298.28159); and of the method, at least about 84% of the monatin or salt a combination thereof. The Substrate (e.g., indole-3-pyru thereof present in the monatin composition is an R,R Stere vate) may be added to the reaction mixture and/or may be oisomer of monatin. Suitable Substrates may include tryp produced in Situ from Suitable Substrates. The aldolase may tophan, which may include D-tryptophan, L-tryptophan, and be added to the reaction mixture and/or may be expressed by a mixture thereof. In Some embodiments, the Selected Sub a microorganism present in the reaction mixture (e.g., by Strate is D-trytophan. The method may include providing a fermenting the reaction mixture, which may include a nutri ProA aldolase (EC 4.1.3.17). For example, a ProA aldolase ent medium, with a microorganism that expresses a Selected (EC 4.1.3.17) may be provided to facilitate the conversion of aldolase). indole-3-pyruvate to 2-hydroxy 2-(indol-3ylmethyl)-4-keto 0188 In further embodiments, 2-hydroxy 2-(indol-3-yl glutaric acid. In Some embodiments, the method may methyl)-4-ketoglutaric acid or a Salt thereof may be pro include providing a transaminase Such as AT-103 (D-tran duced by reacting a reaction mixture, the mixture including: Saminase). For example, AT-103 (D-transaminase) may be (a) a Substrate Such as indole-3-pyruvate, and (b) a Suitable US 2005/0282260 A1 Dec. 22, 2005

aldolase. For example, Suitable aldolases may comprise an Y14082.1 bp 28622-29470 and Genbank Accession No. amino acid Sequence that is at least about 49% identical to NP 388848.1, nucleic acid sequence and amino acid C. testosteroni ProA (SEQ ID NO: 66) or an amino acid Sequence, respectively), Sinorhizobium meliloti (also termed sequence that is at least about 56% identical to Sinorhizo Rhizobium meliloti) tyrosine aminotransferase (tat.A, SEQ bium meliloti ProA (NCBI Accession No.: CAC46344), ID NOS: 1 and 2, nucleic acid Sequence and amino acid where the aldolase has 4-hydroxy-4-methyl-2-oxoglutarate Sequence, respectively), Rhodobacter Sphaeroides Strain lyase activity. In Some Suitable embodiments, the aldolases 2.4.1 tyrosine aminotransferase (tatA asserted by homology, may comprise an amino acid Sequence that is at least about SEQ ID NOS: 3 and 4, nucleic acid sequence and amino acid 60%, 70%, 80%, 90%, 95%, and/or 99% identical to C. Sequence, respectively), R. Sphaeroides 35053 tyrosine ami testosteroni ProA (SEQ ID NO: 66) and/or an amino acid sequence that is at least about 60%, 70%, 80%, 90%, 95%, notransferase (asserted by homology, SEQID NOS: 5 and 6, and/or 99% identical to Sinorhizobium meliloti ProA (NCBI nucleic acid Sequence and amino acid Sequence, respec Accession No.: CAC46344), where the aldolase has 4-hy tively), Leishmania major broad Substrate aminotransferase droxy-4-methyl-2-oxoglutarate lyase activity. In Some (bsat, asserted by homology to peptide fragments from L. embodiments, the aldolase comprises an amino acid mexicana, SEQID NOS: 7 and 8, nucleic acid sequence and sequence that is at least about 60% identical to C. test amino acid sequence, respectively), Bacillus Subtilis aro Osteroni ProA (SEQ ID NO: 66). In other embodiments, the matic aminotransferase (araT, asserted by homology, SEQ aldolase comprises an amino acid Sequence that is at least ID NOS: 9 and 10, nucleic acid sequence and amino acid about 70% identical to C. testosteroni ProA (SEQ ID NO: Sequence, respectively), Lactobacillus amylovorus aromatic 66). The Substrate (e.g., indole-3-pyruvate) may be added to aminotransferase (araT asserted by homology, SEQ ID the reaction mixture and/or produced in Situ from Suitable NOS: 11 and 12, nucleic acid Sequence and amino acid Substrates in the reaction mixture. The aldolase may be sequence, respectively), R. Sphaeroides 35053 multiple sub added to the reaction mixture and/or may be expressed by a Strate aminotransferase (asserted by homology, SEQ ID microorganism present in the reaction mixture (e.g., by NOS: 13 and 14, nucleic acid Sequence and amino acid fermenting the reaction mixture, which may include a nutri Sequence, respectively), Rhodobacter Sphaeroides Strain ent medium, with a microorganism that expresses a Selected 2.4.1 multiple Substrate aminotransferase (msa asserted by aldolase). homology, Genbank Accession No. AAAE01000093.1, bp 0189 In further embodiments, monatin or a salt thereof 14743-16155 and Genbank Accession No. ZP00005082:1, may be produced by a method that includes reacting a nucleic acid Sequence and amino acid Sequence, respec reaction mixture, the mixture including: (a) a Substrate Such tively), Escherichia coli aspartate aminotransferase (aspC, as tryptophan; and a Suitable deaminase (EC 3.5.1.-). In Genbank Accession No. AE000195.1 bp 2755-1565 and Some embodiments, the deaminase is derived from a micro Genbank Accession No. AAC74014.1, nucleic acid organism chosen from: Proteus spp., Providencia spp., Mor Sequence and amino acid sequence, respectively), and E. coli ganella spp., or combinations thereof. Suitable deaminases tyrosine aminotransferase (tyrB, SEQ ID NOS: 31 and 32, may include deaminases derived from Proteus spp. includ nucleic acid Sequence and amino acid Sequence, respec ing, but not limited to, Proteus myxofaciens, Proteus mira tively). bilis, Proteus vulgaris, Proteus morganii, and combinations thereof. The tryptophan may be added to the reaction 0193 The genes were cloned, expressed, and tested for mixture and/or may be produced in Situ from Suitable activity in conversion of tryptophan to indole-3-pyruvate, Substrates. The deaminase may be added to the reaction along with commercially available enzymes. All eleven mixture and/or may be expressed by a microorganism clones had activity. present in the reaction mixture (e.g., by fermenting the reaction mixture, which may include a nutrient medium, 0194 Identification of Bacterial Strains that Can Contain with a microorganism that expresses a selected deaminase). Polypeptides with the Desired Activity 0.195 No genes in the NCBI (National Center for Bio EXAMPLES technology Information) database were designated as tryp tophan aminotransferases. However, organisms having this Example 1 enzymatic activity have been identified. L-tryptophan ami notransferase (TAT) activity has been measured in cell Cloning and Expression of Tryptophan extracts or from purified protein from the following Sources: Aminotransferases Rhizobacterial isolate from Festuca Octoflora, pea mito chondria and cytosol, Sunflower crown gall cells, Rhizobium 0190. This example describes methods that were used to leguminosarum biovar trifoli, Erwinia herbicola pv gypso clone tryptophan aminotransferases, which can be used to philae, Pseudomonas Syringae pv. Savastanoi, Agrobacte convert tryptophan to indole-3-pyruvate. The genes were rium tumefaciens, Azospirillum lipferum & brasilense, cloned into the p.T 30 Xa/LIC vector to generate fusion Enterobacter cloacae, Enterobacter agglomerans, proteins with cleavable N-terminal HIS-Tag/T7-Tags. The Bradyrhizobium elkani, Candida maltosa, Azotobacter resulting proteins were purified using immobilized metal vinelandii, rat brain, rat liver, Sinorhizobium meliloti, affinity chromatography. Pseudomonas fluorescens CHAO, Lactococcus lactis, Lac tobacillus casei, Lactobacillus helveticus, wheat Seedlings, 0191 Experimental Overview barley, Phaseolus aureus (mung bean), Saccharomyces 0.192 Eleven genes encoding aminotransferases were uvarum (carlsbergensis), Leishmania sp., maize, tomato cloned into E. coli. These genes were Bacillus subtilis shoots, pea plants, tobacco, pig, CloStridium Sporogenes, D-alanine aminotransferase (dat, Genbank Accession No. and Streptomyces griseu.S. US 2005/0282260 A1 Dec. 22, 2005

0196) Isolation of Genomic DNA for Cloning -continued 0197) S. meliloti (ATCC number 9930) was grown in TY Sinorhizobium meliloti tatA primers: media at 25 C., pH 7.2. Cells were grown to an optical N term: density at 600 nm (OD) of 1.85 and a 2% inoculum was (SEQ ID NOS : 17 and 18) used for genomic DNA preparations. The Qiagen genomic 5'-GGTATTGAGGGTCGCATGTTCGACGCCCTCGCCCG tip 20/G kit (Valencia, Calif.) was used for genomic DNA and isolation. C. term: 0198 Bacillus subtilis 6051 (ATCC) was grown at 30° C. 5'-AGAGGAGAGTTAGAGCCTCAGAGACTGGTGAACTTG.C. in Bereto Nutrient Broth (Difco; Detroit, Mich.). The Qiagen Bacillus subtilis araT primers: genomic tip 20/G protocol was used to isolate the genomic N term: DNA with the following changes: the concentrations of (SEQ ID NOS : 19 and 20) proteinase K and lysozyme were doubled and incubation 5'-GGTATTGAGGGTCGCATGGAACATTTGCTGAATCC times were increased 2-3 fold. and

0199 Leishmania major ATCC 50122 genomic DNA C. term: was supplied by IDI, Inc. (Quebec, Canada) in TE buffer pH 5'-AGAGGAGAGTTAGAGCCTTAAACGCCGTTGTTTATCG. 8.0, 17 ng/ul. Rhodobacter sphaeroides misa 0200 Rhodobacter sphaeroides 2.4.1 (provided by Pro (both 2.4. 1. and 35053) : fessor Sam Kaplan, University of Texas, Houston), R. N term: Sphaeroides 35053 (ATCC number), and L. amylovorus (SEQ ID NOS: 21 and 22) genomic DNA was prepared by Standard phenol eXtraction. 5'-GGTATTGAGGGTCGCATGCGCGAGCCTCTTGCCCT Cells were harvested in late log phase, resuspended in TEN and buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM C. term: NaCl), and lysed by the addition of 0.024 mL sodium lauryl 5'-AGAGGAGAGTTAGAGCCTCAGCCGGGGAAGCTCCGGG. Sarcosine per mL cell Suspension. After extracting at least three times with an equal Volume of phenol Saturated with Leishmania major bsat : N term: TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), the DNA (SEQ ID NOS: 23 and 24) Solution was extracted once with 9:1 chloroform:octanol and 5'-GGTATTGAGGGTCGCATGTCCACGCAGGCGGCCAT three times with chloroform. The DNA was precipitated by and the addition of 0.1 volume of 3 M sodium acetate, pH 6.8 and 2 volumes ethanol. The precipitate was collected by C. term: centrifugation and washed once with 70% ethanol. Finally 5'-AGAGGAGAGTTAGAGCCTCACTCACGATTCACATTG.C. the DNA was dissolved in 0.10 mL distilled water. Lactobacillus amylovorus araT: 0201 Escherichia coli genomic DNA was isolated from N term: Strain DH10B (Invitrogen) and prepared using the Qiagen (SEQ ID NOS: 25 and 26) Genomic-tipTM (500/G) kit. From 30 mL of this strain grown 5'-GGTATTGAGGGTCGCATGCCAGAATTAGCTAATGA in LB to an ODs of 1.87, 0.3 mg of purified DNA was and obtained. The purified DNA was dissolved in Qiagen elution C. term: buffer (EB) at a concentration of 0.37 ug?.ul. 5'-AGAGGAGAGTTAGAGCCTTATTCGTCCTCTTGTAAAA. 0202 Polymerase Chain Reaction Protocol Rhodobacter sphaeroides tatA (both 2.4.1 and 35053 strains): 0203 Primers were designed with compatible overhangs N term: for the pET 30 Xa/LIC vector (Novagen, Madison, Wis.). (SEQ ID NOS: 27 and 28) The pe.T vector has a 12 base Single Stranded overhang on 5'-GGTATTGAGGGTCGCATGCGCTCTACGACGGCTCC the 5' side of the Xa/LIC site and a 15-base single stranded and overhang on the 3' side of the Xa/LIC site. The plasmid is designed for ligation independent cloning, with N-terminal C. term: His and S-tags and an optional C-terminal His-tag. The Xa 5'-AGAGGAGAGTTAGAGCCTCAGCCGCGCAGCACCTTGG. protease recognition site (IEGR) sits directly in front of the Escherichia coli aspC: Start codon of the gene of interest, Such that the fusion N term: protein tags can be removed. (SEQ ID NOS: 29 and 30) 5'-GGTATTGAGGGTCGCATGTTTGAGAACATTACCGC-3' 0204. The following sequences were added to the 5' ends and of the organism Specific Sequences when designing primers: forward primer, 5' GGTATTGAGGGTCGC (SEQ ID NO: C. term: 73); reverse primer: 5' AGAGGAGAGTTAGAGCC (SEQ 5'-AGAGGAGAGTTAGAGCCTTACAGCACTGCCACAATCG-3". ID NO: 74). Escherichia coli tyrB: N term: (SEQ ID NOS : 33 and 34) Bacillus subtilis dat primers: 5'-GGTATTGAGGGTCGCGTGTTTCAAAAAGTTGACGC N term: and (SEQ ID NOS : 15 and 16) 5'-GGTATTGAGGGTCGCATGAAGGTTTTAGTCAATGG-3' C. term: and 5'-AGAGGAGAGTTAGAGCCTTACATCACCGCAGCAAACG-3".

C. term: 5'-AGAGGAGAGTTAGAGCCTTATGAAATGCTAGCAGCCT-3". 0205 The gene derived from S. meliloti (tatA) was amplified using the following PCR protocol. In a 50 till reaction 0.1-0.5 ug template, 1.5 uM of each primer, 0.4 mM US 2005/0282260 A1 Dec. 22, 2005 each dNTP, 3.5 U Expand High Fidelity Polymerase (Roche, competent cells (Novagen, Madison, Wis.), and incubated Indianapolis, Ind.), and 1x ExpandTM buffer with Mg were on ice for 5 minutes. After mixing, the cells were trans used. The thermocycler program used included a hot start at formed by heat shock for 30 seconds at 42°C. The cells were 96° C. for 5 minutes, followed by 29 repetitions of the placed on ice for 2 minutes, and allowed to recover in 250 following steps: 94° C. for 30 seconds, 55 C. for 2 minutes, till of room temperature SOC for 30 minutes at 37 C. with and 72 C. for 2.5 minutes. After the 29 repetitions the Shaking at 225 rpm. Cells were plated on LB plates con sample was maintained at 72 C. for 10 minutes and then taining kanamycin (25-50 ug/mL). stored at 4 C. This PCR protocol produced a product of 0212 Plasmid DNA was purified using the Qiagen spin 1199 bp. miniprep kit and Screened for the correct inserts by restric 0206. The sequences of the genes derived from R. tion digest with Xhol and Xbal. The sequences of plasmids Sphaeroides (msa and tatA), L. amylovorus araT, and Bacil that appeared to have the correct insert were verified by lus araT were amplified using the following PCR protocol. dideoxy chain termination DNA sequencing. In a 50 till reaction, 0.1-0.5 lug template, 1.5 uM of each primer, 0.4 mM each dNTP, 3.5 U Expand High Fidelity TM 0213 SEQID NOS: 1-14 and 31-32 show nucleotide and Polymerase, and 1x ExpandTM buffer with Mg were added. corresponding amino acid Sequences of the recombinant The thermocycler program used included a hot start at 96 aminotransferases, any changes from the Genbank C. for 5 minutes, followed by 29 repetitions of the following Sequences were either Silent or generated conservative Sub steps: 94° C. for 30 seconds, 40-60° C. for 1 minute, 45 stitutions in the protein sequence. SEQ ID NOS: 11 and 12 Seconds (gradient thermocycler) and 72 C. for 2 minutes, are novel Sequences. 15 Seconds. After the 29 repetitions the Sample was main 0214 Gene Expression and Assays tained at 72 C. for 10 minutes and then stored at 4 C. 0215 Plasmid DNA, verified by sequence analysis, was 0207 For each R. Sphaeroides msa gene, the 42 C. and subcloned into E. coli expression hosts BLR(DE3) or 48 C. annealing temperatures produced multiple products, BL21 (DE3) (Novagen, Madison, Wis.). The cultures were but a distinct band at approximately 1464 bp. For L. amy grown and the plasmids were isolated using Qiagen mini lovorus araT, the 42 C., 48 C., and 56° C. annealing prep kit, and analyzed by restriction digest to confirm temperatures yielded Single products with intense bands at identity. 1173 bp. For B. subtilisaraT, the 40°C., 45° C., 50° C., 55° C. annealing temperatures generated Single intense products 0216) Induction was initially performed with L. amylo vorus araT, B. Subtilis araT, and S. meliloti tatA in both (1173 bp), from both genomic DNA and colonies. For L. BLR(DE3) and BL21(DE3) cells. A time course study was major bsat, the 55 C. annealing temperature gave the performed with cultures grown in LB containing kanamycin cleanest product (1239 bp). For Rhodobacter tatA genes, the (30 mg/L) to an ODoo of 0.5-0.8 and induced with 1 mM 50-55 C. annealing temperatures gave clean products at the IPTG (isopropyl thiogalacatoside) and Sampled at 0, 1, 2, correct size (1260 bp). For both E. coli genes and the B. and 4 hours post induction. Cells from 2.0 mL were resus Subtilis dat gene, an annealing temperature of 55-60C. was pended in 0.10 mL 120 mM Tris-HCI, pH 6.8 containing used, and the annealing time was shortened to 45 Seconds. 10% sodium dodecyl sulfate, 10% 2-mercaptoethanol, and Clean products of the correct sizes were obtained (approxi 20% glycerol, heated at 95 C. for 10 min, and cooled, and mately 1.3 kb for the E. coli genes, 850 bp for the dat gene). diluted with 0.10 mL HO. Aliquots of these total cellular 0208 Cloning protein samples were analyzed by SDS-PAGE using a 4-15% gradient gel. There were no significant differences in 0209. The PCR products were gel purified from 0.8 or 1% the amount of protein expressed between the 2 hour and 4 TAE-agarose gels using the Qiagen gel extraction kit (Valen hour induction, nor between the BLR(DE3) and BL21 (DE3) cia, Calif.). The PCR products were quantified by compari cells. Son to Standards on an agarose gel, and then treated with T4 DNA polymerase following the manufacturer's recom 0217 Cell extracts were also prepared from the 4 hour mended protocols for Ligation Independent Cloning Samples by Suspending cell pellets from 2 mL of culture in (Novagen, Madison, Wis.). 0.25 mL Novagen BugEusterTM reagent containing 0.25 till benzonase nuclease, incubating at room temperature for 20 0210 Briefly, approximately 0.2 pmol of purified PCR minutes with gentle Shaking, and centrifuging at 16,000xg to product was treated with 1 UT4 DNA polymerase in the presence of dGTP for 30 minutes at 22 C. The polymerase remove cell debris. The Supernatants (cell extracts) were removes Successive bases from the 3' ends of the PCR loaded onto 4-15% gradient gels for analysis of the cellular product. When the polymerase encounters a guanine residue, Soluble proteins. the 5' to 3' polymerase activity of the enzyme counteracts the 0218. The three clones, (L. amylovorus araT (SEQ ID exonuclease activity to effectively prevent further excision. NOS: 11 and 12), B. SubtilisaraT (SEQ ID NOS:9 and 10), This creates Single Stranded overhangs that are compatible and S. meliloti tatA (SEQ ID NOS: 1 and 2) showed soluble with the pT Xa/LIC vector. The polymerase is inactivated protein that corresponded to the correct size (approximately by incubating at 75 C. for 20 minutes. 45 kDa). The B. Subtilis araT gene product was over expressed at the highest level and/or was more Soluble than 0211 The vector and treated insert were annealed as recommended by Novagen. Approximately 0.02 pmol of the other two gene products. treated insert and 0.01 pmol vector were incubated for 5 0219. In subsequent expression methods, plasmid DNA minutes at 22°C., 6.25 mM EDTA (final concentration) was from positive clones was subcloned into BL21 (DE3) due to added, and the incubation at 22 C. was repeated. The the better growth characteristics of this host. Induction was annealing reaction (1 ul) was added to NovaBlueTM singles repeated using 1 mM IPTG with cultures grown in LB US 2005/0282260 A1 Dec. 22, 2005

containing kanamycin at 50 mg/L, inducing when the ODoo the effective linear range of the assay. In these cases a > reached approximately 0.8. Cells were harvested after 4 precedes the Specific activity number. hours of growth at 37 C., centrifuged at 3000 rpm for 10 minutes (4° C.), washed with TEGGP buffer (50 mM TABLE 1. Tris-HCl(pH 7.0), 0.5 mM EDTA, 100 mg/L glutathione, 5% glycerol, with Roche complete protease inhibitor cock Specific Activities of Clones in Cell Extracts (CE) and Purified (P) tail), and flash frozen in-80 C. ethanol. and Commercial Enzyme Specific 0220 Samples were resuspended in 5 mL/g wet cell Activity weight of BugEusterTM (Novagen) reagent containing 5 (ug I3P/mg uL/mL protease inhibitor cocktail set #3 (Calbiochem-Nova Enzyme protein/min) Note biochem Corp., San Diego, Calif.) and 1 u/mL benzonase L. major bsat CE >49.3 nuclease. Samples were incubated at room temperature for L. major bsat P >428O 20 minutes on an orbital shaker. Insoluble cell debris was S. meliloti tatACE >28.6 removed by centrifugation at 16,000xg for 20 minutes at 4 S. meliloti tatAP >931 C. R. Sphaeroides 2.4.1 tatACE >41.2 R. Sphaeroides 2.4.1 tatAP 1086 R. sphaeroides 35053 tatACE >623 0221) Cell extracts were analyzed by SDS-PAGE, and R. sphaeroides 35053 tatAP >486 assayed for tryptophan aminotransferase activity by follow L. amylovorus araT CE 1.26 ing production of indole-pyruvic acid using the following L. amylovorus araTP O little protein after protocol. One mL reactions were carried out in 50 mM His-Bind cartridge B. SubtilisaraT CE O undetectable sodium tetraborate (pH 8.5), 0.5 mM EDTA, 0.5 mM B. SubtilisaraTP 1.5-4.5 Sodium arsenate, 50 uM pyridoxal phosphate, 5 mM C.-ke R. Sphaeroides 2.4.1 msa CE 2.05 very little soluble protein toglutarate, and 5 mM L-tryptophan. The reactions were R. Sphaeroides 2.4.1 msa P O no protein after His-Bind initiated by the addition of cell free extracts or purified cartridge R. sphaeroides 35053 msa CE 3.97 very little soluble protein enzyme and were incubated 30 minutes at 30° C. 20% TCA R. sphaeroides 35053 msa P O no protein after His-Bind (200 uD) was added to stop the reaction, and the precipitated cartridge protein was removed by centrifugation. The absorbance at E. coli aspC (P) 8OO 327 nm was measured and compared to a Standard curve of E. coli tyrB (P) 1. not very soluble freshly prepared indole-3-pyruvate in the assay buffer. Con B. Subtilis D-aminotransf(P) 2.7 using D-tryptophan as substrate trol reactions without the Substrate tryptophan or using broad range transaminase 22 Sigma cat #T 7684 cell-free extracts from clones transformed with pT30a Porcine type II-A 1.5 Sigma G7005 alone were also performed. Porcine type I 1. Sigma G2751 0222. Due to background from the native E. coli ami notransferases in cell extracts, the recombinant fusion pro 0225. An alignment comparing all of the recombinant teins each containing an aminotransferase protein fused to proteins cloned illustrates that there are not many highly the pET30 amino terminal HIS-Tag/S-Tag were purified conserved areas between the araT, tatA, bSat, and msa using immobilized metal affinity chromatography with His Sequences. An alignment of highest activity recombinant Bind cartridges following manufacturer's protocols proteins: Rhodobacter tatA gene product homologs, L. (Novagen, Madison, Wis.). The HIS-Tag sequence of the major broad Substrate aminotransferase, and the Sinorhizo fusion proteins binds to the divalent Ni" cations immobi bium meliloti tyrosine aminotransferase showed Several con lized on IDA-based His-Bind resin. The eluent fractions Served regions, however they are only approximately were desalted on PD-10 (Amersham Biosciences, Piscat 30-43% identical at the protein level. The availability of the away, N.J.) columns and eluted in 50 mM Tris, pH 7.0. broad range, D-specific (D-alanine) aminotransferase can be Purified proteins were analyzed by SDS-PAGE and assayed useful in the production of other Stereoisomers of monatin for aminotransferase activity. (see Examples 9-15). 0223 Results from the 37° C. induction with 1 mM IPTG (4 hours) demonstrate that L. major bsat, S. meliloti tatA, E. Example 2 coli aspC, and both R. Sphaeroides tatA clones have signifi cant levels of tryptophan aminotransferase activity. The araT 0226. This example describes methods that were used to protein from B. Subtilis was over-expressed and soluble, but Subclone and analyze a mutated version of the E. coli aspC showed little enzymatic activity. The L. amylovorus araT gene, HEX, cloned in the vector pET30 Xa/LIC. The prod gene product appeared to be Soluble in the cell extract, but uct of this gene carries 6 mutations to its and was purification using a His-Bind cartridge resulted in only Small rationally designed to have increased aminotransferase amounts of protein with the correct molecular weight. The activity with aromatic amino acids, based on homology to msa gene products were insoluble and further expression the TyrB aromatic (tyrosine) aminotransferase from E. coli. experiments were done at 24 C. to minimize inclusion body Two of these positions (Thr 109 and ASn 297; pig cytosolic formation. Several concentrations of IPTG between 10 uM aspartate aminotransferase (AAT) numbering System) are and 1 mM were used to maximize the amount of Soluble invariant in all known aspartate aminotransferase enzymes, protein. but were modified to mimic the E. coli TyrB sequence (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, 0224 Table 1 lists the specific activities measured in Thr 47, and ASn 69) line the active site pocket of E. coli micrograms of indole-3-pyruvate (13P) formed per milli AspC and are replaced by amino acids with more hydro gram protein per minute. In Some cases, very Small amounts phobic side chains found in TyrB (Leu39, Tyr 41, Ile 47, and of recombinant protein showed high levels of activity above Leu 69). US 2005/0282260 A1 Dec. 22, 2005 2O

0227 Cloning inhibitor cocktail set III and 0.33 ul/10 mL r-Lysozyme 0228. The HEX gene cloned in pUC19 was provided by following the Novagen recommended protocol. After incu Professor J F Kirsch (Department of Molecular and Cell bation at 25 C. for 15 min with gentle shaking, the cell Biology, University of California, Berkeley, Berkeley, Calif. debris was pelleted by centrifugation at 21,000xg for 20 min 94720-3206) and used as the template for the cloning of the at 4 C. The Supernatant (cell free extract) was analyzed by gene into p.T30 Xa/LIC. See James J. Onuffer and Jack F. SDS-PAGE on 4-15% gradient gels (Bio-Rad) to detect Kirsch, Redesign of the Substrate specificity of Escherichia soluble protein levels of the recombinant fusion protein. The coli aspartate aminotransferase to that of Escherichia coli level of expression was high (approximately 30-40% of the tyrosine aminotransferase by homology modeling and Site soluble protein) and similar to that observed for the aspC directed mutagenesis, Protein Science, 4: 1750-1757 (1995). aminotransferase protein. 0233. The HIS-HEX protein was purified from cell 0229. The primers designed for cloning the E. coli aspC extract (prepared as described above) using immobilized gene into the pET30 Xa/LIC vector (Novagen, Madison, metal affinity chromatography with His-Bind 900 cartridges Wis.) described in Example 1 (SEQ ID NOS: 29 and 30) following the manufacturer's protocol (Novagen, Madison, were used to subclone the HEX gene into the same vector. Wis.). (The HIS-Tag sequence of the HIS6-HEX protein The following PCR protocol was used for gene amplifica binds to the divalent Ni" cations immobilized on IDA tion: In a 50 till reaction, 50 ng DNA template, 1.0 uM of each primer, 0.2 mM each dNTP, 1 Upful Jltra HF Poly based His-Bind resin). The eluent fractions were desalted on merase (Stratagene), 2.1 U Expand High Fidelity TM Poly PD-10 (Amersham Biosciences, Piscataway, N.J.) columns merase (Roche Molecular Biochemicals, Indianapolis, Ind. and eluted in 50 mM Tris-HCl, pH 7.0. The purified protein ), and 1x ExpandTM buffer with Mg were added. The was analyzed by SDS-PAGE for purity and the amount of thermocycler program utilized a hot start of 94 C. for 5 protein in the purification fractions was determined using the minutes; followed by 10 cycles of a denaturing step at 94 Pierce BCA assay with bovine serum albumin as the stan C. (30 Sec), an annealing step at 50 C. (1 min), and an dard. extension step at 72° C. (1 min 30 sec); 15 cycles of a 0234. The purified HIS-HEX protein and unpurified cell denaturing step at 94° C. (30 sec), an annealing step at 55 extracts were analyzed for tryptophan aminotransferase C. (1 min), and an extension step at 72° C. (1 min 30 sec) activity using the following protocol: The reaction mixture that increased 5 Sec per cycle; 10 cycles of a denaturing Step contained, in 1.0 mL, 50 mM sodium tetraborate, pH 8.5, 5 at 94° C. (30 sec), an annealing step at 55° C. (1 min), and mM alpha-ketoglutarate, 0.05 mM pyridoxal phosphate, 0.5 an extension step at 72° C. (2 min 45 sec); and finally a mM sodium arsenate, 0.5 mM EDTA and enzyme. All finishing step at 72° C. (7 min). The amplified DNA was components except the enzyme were mixed together, the purified from a 1% agarose gel using a Qiagen QIAquick enzyme was added to start the reaction and the reaction Gel Extraction Kit (Valencia, Calif.). The PCR product was Solution was incubated at 30° C. for 30 min. The reaction quantified by measuring the absorbance at 260 nm, treated was stopped by the addition of 0.2 mL of 20% trichloro with T4 DNA polymerase, and annealed with the vector acetic acid, the mixture was centrifuged at 21,000 rpm, and following the manufacturer's recommended protocols for the clear Supernatant was carefully removed. The absor Ligation Independent Cloning (Novagen, Madison, Wis.) bance of the Supernatant was measured at 327 nm (absor and previously described in Example 1. bance maximum for indole-3-pyruvate). All reactions were run in duplicate. For comparison the HIS-tyrb and HIS 0230 Transformation of the annealing reaction into elec aspC aminotransferases described in Example 1 were also trocompetent DH 10B was performed under standard condi analyzed using the same protocol. The results are shown in tions using a 0.1 cm cuvette and a Bio-Rad Gene Pulser II Table 2. Absorbance at 327 is proportional to indole-3- System as described in the Bio-Rad electroporation manual. pyruvate concentration, and can be used to determine rela Clones containing the HEX gene were identified by restric tive activities of various enzymes for a particular experi tion analysis and confirmed by DNA sequencing. SEQ ID ment. NOS 75 and 76 show nucleotide and the corresponding amino acid Sequences of the HEX gene and gene product (HEXAspCaminotransferase, also called HEX protein). See TABLE 2 also NCBI accession number 1AHF A GI: 1127190 (amino pig aminotransferase acid Sequence). aminotransferase gene in reaction Absorbance at 327 mm 0231 Gene Expression and Assays HIS-HEX, purified 9.1 1.171 HIS-HEX, cell extract 22.8 1683 HIS-aspC purified 9.1 O834 0232 Plasmid DNA (verified by sequence analysis) was HIS-aspC, cell extract 22.8 1.42 subcloned into expression host BL21 (DE3) (Novagen). The HIS-tyrB purified 9.1 O.OO8 cultures were grown in LB medium with 50 mg/L kanamy HIS-tyrB, cell extract 22.8 O346 cin and the plasmids were isolated using a Qiagen Spin plasmid miniprep kit and Subsequently analyzed by restric tion digest to confirm identity. Induction experiments were 0235. The results listed in Table 2 show that the HEX carried out with the BL21 (DE3) construct grown in LB AspC aminotransferase has approximately 40% higher medium containing 50 mg/L kanamycin at 37 C. Protein activity for tryptophan than the AspC protein when purified expression was induced using 0.2 mM IPTG after the ODoo enzymes are used in the assay. The increase in activity is reached approximately 0.6. The cells were grown for 4 hours Significantly leSS when cell extracts are the Source of the at 30° C. and harvested by centrifugation. The cells were enzymes. This may be due to interfering activity from native then lysed using Bugbuster" reagent (Novagen) containing proteins of the host E. coli strain. The tyrB gene product is 1 u/mL benzonase nuclease, 5 u/mL. Calbiochem protease leSS Stable than the ASpC protein and its lack of Stability may US 2005/0282260 A1 Dec. 22, 2005

be reflected in the low level of activity observed for this Quick Ligation kit (NEB). The ligated DNA was trans enzyme. Furthermore, the His fusion tag may interfere with formed into chemically competent TOP10F" cells (Invitro the correct conformation of the protein and adversely affect gen; Carlsbad, Calif.). Clones with an insert were identified its activity, See data below. The increased activity of the by running purified plasmid DNA on a 1% agarose gel. HEX mutant of ASpC on tryptophan may explain the Clones with an insert were confirmed by DNA sequencing. improvement in amounts of monatin formed when using this enzyme as described below in Examples 9-10 (Tables 6 and 0240 Gene Expression and Assays 7). The HEX mutant of AspC is the optimal enzyme for S.S 0241 Plasmid DNA (verified by sequence analysis) was monatin production in reactions with C. testosteroni ProA subcloned into expression host BL21 (DE3) (Novagen). The aldolase. cultures were grown in LB medium with 50 mg/L kanamy cin. Induction experiments were carried out with the Example 3 BL21 (DE3) construct grown in LB medium containing 50 mg/L kanamycin at 37 C. Protein expression was induced 0236. This example describes methods that were used to using 0.1 mM IPTG after the ODoo reached approximately Subclone, express and analyze aspC, tyrB, and HEX ami 0.5. The cells were grown for 4 hours at 30° C. and harvested notransferase genes and gene products that were constructed by centrifugation. The cells were then lysed using Bug without amino terminal HIS tags. The aminotransferase buster" reagent (Novagen) containing 1 u/mL benzonase activity was measured by following the formation of the nuclease, 5 u/mL Calbiochem protease inhibitor cocktail co-product of the reaction, glutamate, by HPLC as described set III and 0.33 ill /10 mL r-Lysozyme following the in Example 18. Novagen recommended protocol. After incubation at 25 C. 0237) Cloning for 15 min with gentle shaking, the cell debris was pelleted by centrifugation at 21,000xg for 20 min at 4 C. The 0238. The following primers were designed for cloning Supernatant (cell free extract) was analyzed by SDS-PAGE the E. coli aspC, tyrB and HEX genes into the peT30a on 4-15% gradient gels (Bio-Rad) to detect soluble protein vector (Novagen, Madison, Wis.) (the same primers were levels of the recombinant fusion protein. The level of used for aspC and HEX): expression was high (approximately 25-40% of the total soluble protein) for all 3 proteins and similar to that aspC/HEX primers: observed for the corresponding pET30 HIS-tagged con N ter: StructS. (SEQ ID NO: 77) 0242. The untagged AspC and TyrB proteins were 5'-GCGGAACATATGTTTGAGAACATTACCGCC-3"; assayed for tryptophan aminotransferase activity using the C. term: following protocol. The formation of the co-product of the (SEQ ID NO: 78) reaction, glutamate, was measured using the HPLC fluores 5'-ATAACCGGATCCTTACAGCACTGCCACAATCG-3"; cence detection method described in Example 18 rather than tyrB primers: measuring the formation of the indole-3-pyruvate as N term: described above in Example 1. In this reaction tryptophan (SEQ ID NO: 79) reacts Stoichiometrically with alpha-ketoglutarate to gener 5'-GCGGCGCATATGGTGTTTCAAAAAGTTGACGC-3'; ate indole-3-pyruvate and glutamate. Glutamate is more C. term: Stable in aqueous Solution than indole-3-pyruvate and thus (SEQ ID NO: 80) its measurement may afford a more accurate measure of 5'-CCAATAGGATCCTTACATCACCGCAGCAAACG-3'. enzyme activity. The reaction mixture contained, in 1.0 mL, 50 mM Tris-HCl, pH 8.0, 5 mM alpha-ketoglutarate, 0.05 0239). An ATG start codon was added 5' to the coding mM pyridoxal phosphate, 5 mM tryptophan and enzyme. All sequence of tyrB for compatibility with the restriction components except the enzyme were mixed together, the enzyme and for higher expression levels. The following enzyme was added to start the reaction and the reaction PCR protocol was used for gene amplification: In a 100 till Solution was incubated at 30° C. for 60 min. The reaction reaction, 50 ng DNA template, 1.0 uM of each primer, 0.2 was stopped by the addition of 0.15 mL of 20% trichloro mM each dNTP, 1 U Pfu Turbo Polymerase (Stratagene; acetic acid, the mixture was centrifuged at 21,000 rpm, and LaJolla, Calif.), and 1x Cloned Pful buffer were added. The the clear Supernatant was carefully removed. The results thermocycler program utilized a hot start of 94 C. for 5 (corrected for background levels of glutamate and for the minutes; followed by 25 cycles of a denaturing step at 94 dilution from the addition of trichloroacetic acid to precipi C. (30 Sec), an annealing step at 55 C. (1 min), and an tate the proteins) are shown in Table 3. extension Step at 72 C. (2 min) and finally a finishing Step at 72° C. (7 min). The amplified DNA was purified using a TABLE 3 Qiagen QIAquick(R) PCR Purification Kit (Valencia, Calif.). The purified DNA and purified plasmid DNA (pET30 puri Aminotransferase gene tug aminotransferase glutamatel; ug/mL fied using a Qiagen QIAprep(R) Spin Miniprep Kit) was tyrB 50 310 digested with the Nde and BamHI according to the manu aspC 50 328 facturer's directions (NEB; Beverly, Mass.). Digestion of the pT30a vector with NdeI removes the amino terminal HIS-tag region. The digested DNA was purified from a 1% 0243 This result is surprising as other researchers have agarose gel using a Qiagen QIAquickCE) Gel Extraction Kit observed that the TyrB aminotransferase exhibits higher (Valencia, Calif.). The purified DNA product was quantified activity with tryptophan as the Substrate than AspC (Hayashi by measuring the absorbance at 260 nm, and ligated using a et al (1993) Biochemistry 32:12229-1239). The unexpect US 2005/0282260 A1 Dec. 22, 2005 22 edly low level of activity may due to protein instability. make indole-3-pyruvate from indole-3-lactic acid. Sources However, the level of activity found with the untagged TyrB of broad Specificity dehydrogenases include E. coli, Neis protein is considerably higher than that seen with the tagged Seria gonorrhoeae, and Lactobacillus plantarum. version above (see Table 1). Because tryptophan reacts Stoichiometrically with alpha-ketoglutarate to generate 0248 Alternatively, indole-3-pyruvate can be produced indole-3-pyruvate and glutamate in the aminotransferase by contacting indole-3-lactate with cellular extracts from reaction, the concentration of indole-3-pyruvate formed (as CloStridium Sporogenes which contain an indolelactate shown in Table 1) and the concentration of glutamate formed dehydrogenase (EC 1.1.1.110); or Trypanosoma Cruzi epi (as shown in Table 3) reflect the same activity. However, maStigOtes cellular extracts which contain p-hydroxypheny glutamate is more Stable in aqueous Solution than indole-3- lactate dehydrogenase (EC 1.1.1.222) known to have activ pyruvate and, thus, its measurement may afford a more ity on indole-3-pyruvate, or Pseudomonas acidovOranS or E. accurate measure of enzyme activity. The relative activity coli cellular extracts, which contain an imidazol-5-yl lactate (or ratio) of TyrB to AspC using either detection method dehydrogenase (EC 1.1.1.111); or Coleus blumei, which should yield the same number, and it is clear that the tagged contains a hydroxyphenylpyruvate reductase (EC version of TyrB has less activity on tryptophan in compari 1.1.1.237); or Candida maltosa which contains a D-aromatic Son to tagged AspC, while the untagged TyrB has nearly the lactate dehydrogenase (EC 1.1.1.222). References describ Same activity as the untagged AspC. ing such activities include, Nowicki et al. (FEMS Microbiol Lett 71:119-24, 1992), Jean and DeMoss (Canadian J. Example 4 Microbiol. 14 1968, Coote and Hassall (Biochem. J. 111: 237-9, 1969), Cortese et al. (C.R. Seances Soc. Biol. Fil. 162 Conversion of Indole-3-lactate to Indole-3-pyruvate 390-5, 1968), Petersen and Alfermann (Z. Naturforsch. C. 0244. As shown in FIGS. 1 and 3, indole-3-lactic acid BioSci. 43 501-4, 1988), and Bhatnagar et al. (J. Gen can be used to produce indole-3-pyruvate. Conversion Microbiol 135:353-60, 1989). In addition, a lactate oxidase between lactic acid and pyruvate is a reversible reaction, as such as the one from Pseudomonas sp. (Gu et al. J. Mol. is conversion between indole-3-pyruvate and indole-3-lac Catalysis B: Enzymatic: 18:299-305, 2002), can be utilized tate. The oxidation of indole-lactate was typically followed for oxidation of indole-3-lactic to indole-3-pyruvate. due to the high amount of background at 340 nm from indole-3-pyruvate. Example 5 0245. The standard assay mixture contained 100 mM potassium phosphate, pH 8.0, 0.3 mM NAD", 7 units of Conversion of L-tryptophan to Indole-3-pyruvate lactate dehydrogenase (LDH) (Sigma-L2395, St. Louis, Utilizing L-amino acid oxidase Mo.), and 2 mM Substrate in 0.1 mL. The assay was performed in duplicate in a UV-transparent microtiter plate, 0249. This example describes methods used to convert using a Molecular Devices SpectraMax Plus platereader. tryptophan to indole-3-pyruvate via an oxidase (EC 1.4.3.2), Polypeptide and buffer were mixed and pipetted into wells as an alternative to using a tryptophan aminotransferase as containing the indole-3-lactic acid and NAD" and the absor described in Example 1. L-amino acid oxidase was purified bance at 340 nm of each well was read at intervals of 9 from CrOtalus duriSSuS (Sigma, St. Louis, Mo., catalog seconds after brief mixing. The reaction was held at 25 C. number A-2805). The accession numbers of L-amino acid for 5 minutes. The increase in absorbance at 340 nm follows oxidases for molecular cloning include: CAD21325.1, the production of NADH from NAD". Separate negative AAL14831, NP 490275, BAB78253, A38314, CAB71136, controls were performed without NAD" and without Sub JE0266, TO8202, S48644, CAC00499, P56742, P81383, strate. D-LDH from LeuconoStoc mesenteroides (Sigma O93364, P81382, P81375, S62692, P23623, AAD45200, catalog number L2395) appeared to exhibit more activity AAC32267, CAA88452, APO03600, and Z4.8565. with the indole-derivative Substrates than did L-LDH from 0250 Reactions were performed in microcentrifuge tubes Bacillus Stearothermophilus (Sigma catalog number in a total volume of 1 mL, incubated for 10 minutes while L5275). shaking at 37 C. The reaction mix contained 5 mM L-tryp 0246 Similar methods were utilized with D-lactic acid tophan, 100 mM sodium phosphate buffer pH 6.6, 0.5 mM and NAD+ or NADH and pyruvate, the natural Substrates of Sodium arsenate, 0.5 mM EDTA, 25 mMSodium tetraborate, D-LDH polypeptides. The V for the reduction of pyru 0.016 mg catalase (83 U, Sigma C-3515), 0.008 mg FAD vate was 100-1000 fold higher than the V for the oxida (Sigma), and 0.005-0.125 Units of L-amino acid oxidase. tion of lactate. The V for the oxidation reaction of Negative controls contained all components except tryp indole-3-lactic with D-LDH was approximately one-fifth of tophan, and blanks contained all components except the that with lactic acid. The presence of indole-3-pyruvate was oxidase. Catalase was used to remove the hydrogen peroxide also measured by following the change in absorbance at 327 formed during the oxidative deamination. The Sodium tet (the enol-borate derivative) using 50 mM sodium borate raborate and arsenate were used to Stabilize the enol-borate buffer containing 0.5 mM EDTA and 0.5 mM sodium form of indole-3-pyruvate, which shows a maximum absor arsenate. Small, but repeatable, absorbance changes were bance at 327 nm. Indole-3-pyruvate Standards were prepared observed, as compared to the negative controls for both L at concentrations of 0.1-1 mM in the reaction mix. and D-LDH polypeptides. 0251 The purchased L-amino acid oxidase had a specific 0247. Additionally, broad specificity lactate dehydroge activity of 540 ug indole-3-pyruvate formed per minute per nases (enzymes with activity associated with EC 1.1.1.27, mg protein. This is the same order of magnitude as the EC 1.1.1.28, and/or EC 1.1.2.3) can be cloned and used to Specific activity of tryptophan aminotransferase enzymes. US 2005/0282260 A1 Dec. 22, 2005 23

Example 6 0257) Cloning Conversion of D-tryptophan to Indole-3-pyruvate 0258 4-Hydroxy-4-methyl-2-oxoglutarate pyruvate Utilizing D-amino acid oxidase lyases (e.g., ProA aldolase, EC 4.1.3.17) and 4-hydroxy-2- oxoglutarate glyoxylate-lyases (e.g., KHG aldolase, EC 0252) This example describes methods used to convert 4.1.3.16) catalyze reactions very similar to the aldolase D-tryptophan to indole-3-pyruvate via an oxidase (EC reaction of FIG. 2. Primers were designed with compatible 1.4.3.3), as an alternative to using L-tryptophan as the overhangs for the pET30 Xa/LIC vector (Novagen, Madi Starting Substrate for the reaction. D-amino acid oxidase was son, Wis.). The design of these primers is described above in purchased from BioCatalytics (Pasadena, Calif., catalog Example 1. AOD-101). 0253) Reactions were performed in microcentrifuge tubes 0259. The following primers were designed for pET30 in a total volume of 1 mL and were incubated for 20 minutes Xa/LIC cloning: while shaking at 30° C. The reaction mix contained 5 mM 0260) 1. Pseudomonas Straminea (Pseudomonas Ochra L-tryptophan, 50 mM sodium tetraborate buffer pH 8, 0.5 ceae NGJI) proA gene (Genbank Accession No.: 12964663 mM sodium arsenate, 0.5 mM EDTA, 25.5 till tech grade Version: 12964663) and Comamonas testosteroni proA gene catalase (1000 U, BioCatalytics CAT-101), 0.008 mg FAD (SEQ ID NOS: 65-66, nucleic acid sequence and amino acid (Sigma), and approximately 10 mgs of D-amino acid oxi Sequence, respectively) dase crude preparation. The D-amino acid oxidase prepara tion contained a large amount of insoluble material. Nega tive controls contained all components except oxidase. forward Samples were run in duplicate. (SEQ ID NOS: 55 and 56) 5'-GGTATTGAGGGTCGCATGTACGAACTGGGAGTTGT-3' 0254 The samples were spun to remove debris and the and Supernatant was diluted 10-fold prior to absorbance mea surements at 327 nm. The diluted samples had absorbances ewese of between 0.789-0.926 while the negative controls (undi 5'-AGAGGAGAGTTAGAGCCTTAGTCAATATATTTCAGGC-3'. luted) had OD's of 0.418 and 0.416. As expected, broad Specificity D-oxidases can be used to efficiently convert 0261) 2. Sinorhizobium meliloti 1021 SMcO0502 gene D-tryptophan to indole-3-pyruvate. (homologous to proA, Genbank Accession Nos.: 15074579 and CAC46344, nucleic acid Sequence and amino acid Example 7 Sequence, respectively) Converting Indole-3-pyruvate to 2-hydroxy forward 2-(indol-3-ylmethyl)-4-ketoglutaric acid with an (SEQ ID NOS : 61 and 62) Aldolase 5'-GGTATTGAGGGTCGCATGAGCGTGGTTCACCGGAA-3' and 0255. This example describes methods that can be used to convert indole-3-pyruvate to the 2-hydroxy 2-(indol-3-ylm ewese ethyl)-4-ketoglutaric acid monatin precursor (MP) using an 5'-AGAGGAGAGTTAGAGCCTCAATCGATATATTTCAGTC-3'. aldolase (lyase) (FIG. 2). Aldol condensations are reactions that form carbon-carbon bonds between the B-carbon of an 0262. 3. Sphingomonas sp. LB126 flaz gene (Genbank aldehyde or ketone and the carbonyl carbon of another Accession No.: 7573247 Version: 7573247, codes for a aldehyde or ketone. A carbanion is formed on the carbon putative acyl ) adjacent to the carbonyl group of one Substrate, and Serves as a nucleophile attacking the carbonyl carbon of the Second substrate (the electrophilic carbon). Most commonly, the forward (SEQ ID NOS: 57 and 58) electrophilic Substrate is an aldehyde, So most aldolases fall 5'-GGTATTGAGGGTCGCATGTCCGGCATCGTTGTCCA-3' into the EC 4.1.2.-category. Quite often, the nucleophilic and Substrate is pyruvate. It is less common for aldolases to catalyze the condensation between two keto-acids or two ewese aldehydes. 5'-AGAGGAGAGTTAGAGCCTCAGACATATTTCAGTCCCA-3". 0256 However, aldolases that catalyze the condensation of two carboxylic acids have been identified. For example, 0263 4. Arthrobacter keyseri pcmE gene (Genbank EP 1045-029 describes the production of L-4-hydroxy-2- Accession No.: AF331043 Version: AF331043.1, codes for ketoglutaric acid from glyoxylic acid and pyruvate using a an oxalocitramalate aldolase) Pseudomonas culture (EC 4.1.3.16). In addition, 4-hydroxy 4-methyl-2-oxoglutarate aldolase (4-hydroxy-4-methyl-2- forward oxoglutarate pyruvate lyase, EC 4.1.3.17) can catalyze the (SEQ ID NOS: 59 and 60) condensation of two keto acids. Therefore, Similar aldolase 5'-GGTATTGAGGGTCGCATGCGACTGAACAACCTCGG-3' polypeptides were used to catalyze the condensation of and indole-3-pyruvate with pyruvate. The activity or enan ewese tiospecificity of these enzymes can be modified for produc 5'-AGAGGAGAGTTAGAGCCTCAGTTCTCCACGTATTCCA-3". tion of a Specific Stereoisomer of monatin. US 2005/0282260 A1 Dec. 22, 2005 24

0264) 5. Yersinia pestis strain C092 YPO0082 gene (Gen according to the protocol described by Eaton (J. Bacteriol. bank Accession No.: 15978115 Version: 15978115, codes 183:3689-3703, 2001). Sinorhizobium meliloti 1021 (ATCC for a possible transferase) 51124) was grown at 26° C. in ATCC TY medium and hydroxybenzoate medium. Yersinia pestis strain CO92 (ATCC) is grown at 26° C. in ATCC medium 739 Horse forward blood agar. Bacillus Subtilis 6051 (ATCC) was grown at 30° (SEQ ID NOS: 63 and 64) 5'-GGTATTGAGGGTCGCATGAGCCTGGTTAATATGAA-3' C. in Bereto Nutrient Broth (Difco; Detroit, Mich.). E. coli and genomic DNA was isolated from strain DH10B (Invitrogen) as described in Example 1. ewese 5'-AGAGGAGAGTTAGAGCCTTATGACTTTAACGCGTTGA-3". 0270. The PCR, cloning, and screening protocols described in Example 1 were used to clone the C. test 0265 6. Bacillus subtilis khg gene (Genbank Accession OSteroni and the S. meliloti proA Sequences, as well as the E. Nos. Z99115.1 GI: 2634.478, 126711-127301 and coli, B. Subtilis, and S. meliloti khg Sequences. The same CAB14127.1, nucleic acid Sequence and amino acid methods can be used to clone the other Sequences described Sequence, respectively) above. For the C. teStoSteroni proA gene, the annealing and extension conditions for PCR were 40-60 C. for 1 minute, 45 seconds (gradient thermocycler) and 72 C. for 2 min forward utes, 15 Seconds. (SEQ ID NOS: 35 and 36) 5'-GGTATTGAGGGTCGCATGGAGTCCAAAGTCGTTGA-3' 0271 Positive clones were sequenced using dideoxy and chain termination sequencing (Seqwright, Houston, Tex.) ewese with S-tag and T7 terminator primers (Novagen), and inter 5'-AGAGGAGAGTTAGAGCCTTACACTTGGAAAACAGCCT-3". nal primers from Integrated DNA Technologies, Inc. (Cor alville, Iowa). 0266 7. E. coli khg gene (Genbank Accession Nos. AE000279.1 1331-1972 and AAC74920. 1, nucleic acid 0272 Expression and Activity Assays and amino acid sequence, respectively) 0273 Plasmid DNA (verified by sequence analysis) was subcloned into expression host BL21 (DE3) (Novagen). The forward cultures were grown in LB medium with 50 mg/L kanamy (SEQ ID NOS : 37 and 38) cin, the plasmids isolated using a Qiagen Spin plasmid 5'-GGTATTGAGGGTCGCATGAAAAACTGGAAAACAAG-3' miniprep kit and Subsequently analyzed by restriction digest and to confirm identity. Induction experiments were done with

ewese the BL21 (DE3) constructs grown in LB medium containing 5'-AGAGGAGAGTTAGAGCCTTACAGCTTAGCGCCTTCTA-3". 50 mg/L kanamycin at 37 C. Protein expression was induced using 0.1 mM IPTG after the ODoo reached approximately 0.6. The cells were grown for 4 hours at 30 0267 8. S. meliloti khg gene (Genbank Accession Nos. C. and harvested by centrifugation. The cells were then lysed AL591792.1 GI: 15075850, 65353-64673 and CAC47463.1, using Bugbuster" reagent (Novagen) and the His-tag nucleic acid and amino acid sequence, respectively) recombinant proteins were purified using His-Bind car tridges as described above (Example 1). Purified proteins forward were desalted on PD-10 disposable columns and eluted in 50 (SEQ ID NOS: 39 and 40) mM Tris-HCl buffer, pH 7.3 with 2 mM MgCl. 5'-GGTATTGAGGGTCGCATGCGAGGGGCATTATTCAA-3' and 0274) The proteins were analyzed by SDS-PAGE on 4-15% gradient gels to detect soluble protein levels at the ewese 5'-AGAGGAGAGTTAGAGCCTCAGCCCTTGAGCGCGAAG-3'. predicted MW of the recombinant fusion protein. 0275. The proteins were assayed for activity using 0268 Genomic DNA from the organisms described in indole-3-pyruvate and Sodium pyruvate as Substrates. The 1-2 and 6-8, above, was purified using the Qiagen Genomic assay mixture contained 100 mM Tris-HCl (pH 7-pH 8.9), tipTM (Valencia, Calif.) protocol. Using similar techniques 0-8 mM MgCl, 3 mM potassium phosphate (pH 8), and 6 the genomic DNA from organisms described in 3-5 can be mM of each substrate in 1 mL. The reaction was started by purified. adding varying amounts of polypeptide (for example from 0269 Pseudomonas Straminea (ATCC 33636) was 10 to 100 lug), and was incubated at 25° C.-37° C. for 30 grown at 30° C. in Nutrient Broth and hydroxybenzoate minutes, filtered, and then frozen at -80 C. medium. Comamonas testosteroni (ATCC 49249) was 0276 Activity Results with ProA Gene Products grown at 26 C. in Nutrient Broth and hydroxybenzoate medium. Sphingomonas sp. LB 126 (Flemish Institute for 0277 Both the C. testosteroni proA and S. meliloti Technological Research, VITO, B-2400 Mol, Belgium) is SMcO0502 gene constructs had high levels of expression grown according to the method described by Wattiau et al. when induced with IPTG. The recombinant proteins were (Research in Microbiol. 152:861-72, 2001). Arthrobacter highly soluble, as determined by SDS-PAGE analysis of keyseri (Gulf Ecology Division, National Health and Envi total protein and cellular extract Samples. The C. teStoSteroni ronmental Effects Research Laboratory, U.S. Environmental gene product was purified to >95% purity. Because the yield Protection Agency, Gulf Breeze, Fla. 32561, USA) is grown of the S. meliloti gene product was very low after affinity US 2005/0282260 A1 Dec. 22, 2005 25 purification using a His-Bind cartridge, cellular extract was 0290 percent identity to C. testosteroni ProA: 65 used for the enzymatic assayS. 0291 percent identity to S. meliloti ProA: 64 0278 Both recombinant aldolases catalyzed the forma tion of MP from indole-3-pyruvate and pyruvate. The pres 0292 Aldolase Source: Yersinia pestis KIM ence of both divalent magnesium and potassium phosphate 0293 gene: AE013606.1 GI: 21956705 were required for enzymatic activity. No product was appar ent when indole-3-pyruvate, pyruvate, or potassium phos 0294 protein: AAM83650.1 GI: 21956715 phate was absent. A Small amount of the product was also 0295 percent identity to C. testosteroni ProA: 56 formed in the absence of enzyme (typically one order of magnitude less than when enzyme was present). 0296 percent identity to S. meliloti ProA: 57 0297 Aldolase Source: Ralstonia metallidurans CH34 0279. Using the LC/MS method described in Example 18, the product peak eluted from the reverse phase C18 0298 gene: NZ AAAI02000016.1 GI: 48767334 column slightly later than the indole-3-pyruvate Standard, 0299 protein: ZP 00271743.1 GI: 48767386 the mass spectrum of this peak showed a collisionally induced parent ion (IM+H+) of 292. 1, the parent ion 0300 percent identity to C. testosteroni ProA: 60 expected for the product MP. The major daughter fragments 0301 percent identity to S. meliloti ProA: 57 present in the mass spectrum included those with m/z=158 (1H-indole-3-carbaldehyde carbonium ion), 168 (3-buta-1, 0302 Aldolase Source: Yersinia pseudotuberculosis IP 3-dienyl-1H-indole carbonium ion), 274 (292-H2O), 256 32953 (292-2 HO), 238 (292-3 H2O), 228 (292-CH4O3), and 204 (loss of pyruvate). The product also exhibited a UV spec 0303) gene: NC 006155.1 GI: 51594359 trum characteristic of other indole-containing compounds 0304 protein: YP 068627.1 GI: 51594436 Such as tryptophan, with the was of 279-280 and a Small percent identity to C. testosteroni ProA: 56 shoulder at approximately 290 nm. 0305 0306 percent identity to S. meliloti ProA: 57 0280. The amount of MP produced by the C. testosteroni aldolase increased with an increase in reaction temperature 0307 Aldolase Source: Rhizobium leguminosarum bio from room temperature to 37 C., amount of substrate, and var viciae rhiz23g02-plk 1009 341 amount of magnesium. The Synthetic activity of the enzyme 0308 (Sanger Institute) decreased with increasing pH, the maximum product observed was at pH 7. Based on tryptophan standards, the amount of MP produced under a Standard assay using 20 lig gene : of purified protein was approximately 10-40 ug per one mL ATGGGCATCGTCGTACAGAACATACCACGGGCGG (SEQ ID NO : 87) reaction. AAGCTGATGTGATCGACAGGCTCGCCAAATCAGG 0281. Due to the high degree of homology of the S. meliloti and C. testosterOni ProA aldolase coding Sequences CGTCGCGACGGTCCACGAAGCCCAGGGGCGCAAA. with the other genes described above, it is expected that all GGCATGCTCGCCAGCCATATGAGACCAATCTATT of the recombinant gene products can catalyze this reaction. Moreover, it is expected that aldolases that have threonine CAGGTGCGCAGATCGCCGGCTCCGCCATTACGAT (T) at positions 59 and 87, arginine (R) at 119, aspartate (D) at 120, and histidine (H) at 31 and 71, (based on the CTCCGCACCGCCCGGTGATAACTGGATGATCCAT numbering system of C. testosteroni) will have similar activity. Additional homologs have been Sequenced and GTGGCGATCGAGCAGATCCAGGCCGGCGACATCC deposited in NCBI. Their genes could be cloned and the TGGTGCTTTCGCCGACCTCGCCCTGTGACAACGG corresponding gene products are expected to have similar activity. Identification numbers, as well as percent identity to TTATTTCGGCGACCGCTTGCCACCCGGCGCGG the C. testosteroni ProA protein and percent identity to the S. meliloti ProA protein, are provided below as examples of GCGCGAGGTTGCCGCGGCCTTGTCATCGACGCCG genes and proteins that are expected to have similar aldolase GTGTCCGCGATATCAGGGATCTGACCCAGATGCA activity: GTTCCCCGTGTGGTCCAAGGCCGTGTCCGCGCAG 0282 Aldolase Source: Bradyrhizobium japonicum str. USDA 110 (protein blr3842) GGGACCGTCAAGGAAACGCTCGGTTCGGTCAACG 0283 gene: NC 004.463.1:42.60815.4261498 TTCCGATCGTCGCGCTGGCGCCTTCACGAAGC 0284) protein: GI: 27378953 (NP 770482.1) CGGCGACATCATCGTCGCCGACGACGACGGGGTG 0285 percent identity to C. testosteroni ProA: 63 TGCGTGGTGAAGCTCAACGCGGCCGAGGAGGTTC 0286 percent identity to S. meliloti ProA: 63 TGACTGCTGCCGAGAACCGTGTGGCGAACGAGGA 0287 Aldolase Source: Sphingomonas (Pseudomonas) GGCCAAGCGGCAACGCCTCGCCGCCGGCGAACTC paucimobilis GGGCTCGATATCTATGACATGCGGTCGAAGCTCC 0288 gene: GI: 19918959 (AB073227.1:3738.4424) GGGAAAAGGGGCTTAAATATGTATGA 0289 protein: GI: 19918963 (BAB88738.1) US 2005/0282260 A1 Dec. 22, 2005 26

0336 Aldolase Source: Xanthomonas axonopodis citri -continued 306 protein: MGIV VQNIPRAEADVIDRLAKSGVATVHEAQGRK (SEQ ID NO: 88) 0337 gene: AE012066.1 GI: 21110580 GMLASHMRPIYSGAQIAGSAITISAPPGDNWMIH 0338 protein: AAM38990.1 GI: 21110581

WAIEQIQAGDILVLSPTSPCDNGYFGDLLATSAR 0339 percent identity to C. testosteroni ProA: 61

ARGCRGLVIDAGVRDIRDLTQMQFPVWSKAVSAQ 0340 percent identity to S. meliloti ProA: 62 0341 Aldolase Source: Streptomyces avermitilis GTWKETLGSWNWPIWCAGAFIEAGDIIWADDDGW MA-468O CVWKLNAAEEWLTAAENRWANEEAKRQRLAAGEL 0342 gene: NC 003155.3 GI: 57833846 GLDIYDMRSKLREKGLKYWW 0343 protein: NP 822793.1 GI: 29828159 0309 percent identity to C. testosteroni ProA: 58 0344 percent identity to C. testosteroni ProA: 49 0310 percent identity to S. meliloti ProA: 61 0345 percent identity to S. meliloti ProA: 56 0346 Activity Results with khg Gene Products 0311 Aldolase Source: Novosphingobium aromati civorans DSM 12444 (Sphingomonas aromaticivorans F199 0347 Both the B. Subtilis and E. coli khg gene constructs Contains Same Gene) had high levels of expression of protein when induced with IPTG, while the S. meliloti khg had a lower level of 0312 gene: NZ AAAV02000003.1 GI: 48848843 expression. The recombinant proteins were highly Soluble, as judged by SDS-PAGE analysis of total proteins and 0313 protein: ZP 00303270.1 GI: 48849026 cellular extracts. The B. Subtilis and E. coli khg gene products were purified to >95% purity; the yield of the S. 0314 percent identity to C. testosteroni ProA: 68 meliloti gene product was not as high after affinity purifi 0315 percent identity to S. meliloti ProA: 63 cation using a His-Bind cartridge. 0348 There is no evidence that magnesium and phos 0316 Aldolase Source: Pseudomonas putida KT2440 phate are required for activity for this enzyme. However, the 0317 gene: AE016783.1 GI: 26557027 literature reports performing the assays in Sodium phosphate buffer, and the enzyme reportedly is bifunctional and has 0318 protein: AAN68126.1 GI: 2498.4081 activity on phosphorylated Substrates Such as 2-keto-3- 0319 percent identity to C. testosteroni ProA: 57 deoxy-6-phosphogluconate (KDPG). The enzymatic assays were performed as described above, and in Some instances 0320 percent identity to S. meliloti ProA: 60 the phosphate was omitted. The results indicate that the recombinant KHG aldolases produced MP, but were not as 0321) Aldolase Source: Magnetospirillum magnetotacti active as the ProA aldolases. In Some cases the level of MP cum MS-1 produced by KHG was almost identical to the amount produced by magnesium and phosphate alone. Phosphate 0322 gene: NZ AAAP01003877.1 GI: 23016465 did not appear to increase the KHG activities. The Bacillus 0323 protein: ZP 00056301.2 GI: 46200890 enzyme had the highest activity, approximately 20-25% higher activity than the magnesium and phosphate alone, as 0324 percent identity to C. testosteroni ProA: 73 determined by LC/MS/MS (see Example 18). The Sinorhizobium enzyme had the least amount of activity, 0325 percent identity to S. meliloti ProA: 59 which can be associated with folding and solubility prob 0326 Aldolase Source: Rhodopseudomonas palustris lems noted in the expression. All three enzymes have the CGAO09 active site glutamate (position 43 in B. Subtilis numbering system) as well as the lysine required for Shiff base forma 0327 gene: NC 005296.1 GI: 39933080 tion with pyruvate (position 130); however, the B. Subtilis enzyme contains a threonine in position 47, an active site 0328) protein: NP 950032.1 GI: 39937756 residue, rather than arginine. The B. Subtilis KHG is smaller and appears to be in a cluster distinct from the S. meliloti and 0329 percent identity to C. testosteroni ProA: 74 E. coli enzymes, with other enzymes having the active site 0330 percent identity to S. meliloti ProA: 58 threonine. The differences in the active site may be the reason for the increased activity of the B. Subtilis enzyme. 0331 Aldolase Source: Xanthomonas campestris ATCC 33.913 0349) Improvement of Aldolase Activity 0350 Catalytic antibodies can be as efficient as natural 0332) gene: AE012524.1 GI: 21115292 aldolases, accept a broad range of Substrates, and can be 0333) protein: AAM43251.1 GI: 21115297 used to catalyze the reaction shown in FIG. 2. 0334) percent identity to C. testosteroni ProA: 63 0351 Aldolases can also be improved by directed evo lution, for example as previously described for a KDPG 0335) percent identity to S. meliloti ProA: 64 aldolase (highly homologous to KHG described above) US 2005/0282260 A1 Dec. 22, 2005 27 evolved by DNA shuffling and error-prone PCR to remove monatin or MP. Since aminotransferases and aldolase reac the requirement for phosphate and to invert the enantiose tions are reversible, the cells are able to produce tryptophan lectivity. The KDPG aldolase polypeptides are useful in from a racemic mixture of monatin. Similarly, organisms biochemical reactions Since they are highly Specific for the (both recombinant and wildtype) can be screened by ability donor substrate (herein, pyruvate), but are relatively flexible to utilize MP or monatin as a carbon and energy Source. One with respect to the acceptor Substrate (i.e. indole-3-pyruvate) Source of target aldolases is expression libraries of various (Koeller & Wong, Nature 409:232-9, 2001). KHG aldolase Pseudomonas and rhizobacterial Strains. Pseudomonads has activity for condensation of pyruvate with a number of have many unusual catabolic pathways for degradation of carboxylic acids and aldehydes. Mammalian versions of the KHG aldolase are thought to have broader enantiospecificity aromatic molecules and they also contain many aldolases, than many bacterial versions, including higher activity on whereas the rhizobacteria contain aldolases, are known to 4-hydroxy 4-methyl 2-oxoglutarate and acceptance of both grow in the plant rhizosphere, and have many of the genes Stereoisomers of 4-hydroxy-2-ketoglutarate. Most bacterial described for construction of a biosynthetic pathway for Sources appear to have a 10-fold preference for a particular monatin. configuration of the fusion product. An exception is the enzyme from the bacterium Zymomonas mobilis which Example 8 shows less Substrate Selectivity (accepting a broad range of Substrates) as well as relaxed Substrate Sterochemical Chemical Synthesis of the Monatin Precursor requirements, Similar to the enzymes isolated from mam malian sources such as rat liver (ref: Shelton et al (1996) J 0355 Example 7 described a method of using an aldolase Am Chem Soc, 118(9):2117-2125. There are nearly 100 to convert indole-3-pyruvate to the 2-hydroxy 2-(indol-3- KHG homologs available in genomic databases, and activity ylmethyl)-4-ketoglutaric acid monatin precursor (MP). This has been demonstrated in Pseudomonas, ParacOccus, Provi example describes an alternative method of chemically dencia, Sinorhizobium, Morganella, E. coli, and mammalian synthesizing MP. tissues. These enzymes can be used as a starting point for tailoring the enantiospecificity that is desired for monatin 0356) MP can be formed using a typical aldol-type con production. densation (FIG. 4). Briefly, a typical aldol-type reaction involves the generation of a carbanion of the pyruvate ester 0352 Aldolases that utilize pyruvate and another sub using a strong base, Such as LDA (lithium diisopropyla strate that is either a keto acid and/or has a bulky hydro mide), lithium hexamethyldisilazane or butyl lithium. The phobic group like indole can be “evolved” to tailor the carbanion that is generated reacts with the indole-pyruvate polypeptide's Specificity, Speed, and Selectivity. In addition to form the coupled product. to KHG and ProA aldolases demonstrated herein, examples of these enzymes include, but are not limited to: KDPG 0357 Protecting groups that can be used for protecting aldolase and related polypeptides (KDPH); transcarboxy the indole nitrogen include, but are not limited to: t-buty benzalpyruvate hydratase-aldolase from Nocardioides st; loxycarbonyl (Boc), and benzyloxycarbonyl (Cbz). Block 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (2-car ing groups for carboxylic acids include, but are not limited boxybenzalpyruvate aldolase) which condenses pyruvate to, alkyl esters (for example, methyl, ethyl, benzyl esters). and 2-carboxybenzaldehyde (an aromatic ring-containing When Such protecting groups are used, it is not possible to Substrate); trans-O-hydroxybenzylidenepyruvate hydratase control the stereochemistry of the product that is formed. aldolase from Pseudomonas putida and Sphingomonas aro However, if R2 and/or R3 are chiral protecting groups (FIG. maticivorans, which also utilizes pyruvate and an aromatic 4), Such as (S)-2-butanol, menthol, or a chiral amine, this can containing aldehyde as Substrates, 3-hydroxyaspartate favor the formation of one MP enantiomer over the other. aldolase(erythro-3-hydroxy-L-aspartate glyoxylate lyase), which uses 2-oxoacids as the Substrates and is thought to be Example 9 in the organism MicrococcuS denitrificans, benzoin aldola Se(benzaldehyde lyase), which utilizes Substrates containing benzyl groups, dihydroneopterin aldolase, L-threo-3-phe Conversion of Tryptophan or Indole-3-Pyruvate to nylserine benzaldehyde-lyase(phenylserine aldolase) which Monatin condenses glycine with benzaldehyde, 4-hydroxy-2-oxoval 0358 An in vitro process utilizing two enzymes, an erate aldolase; 1,2-dihydroxybenzylpyruvate aldolase, and aminotransferase and an aldolase, produced monatin from 2-hydroxybenzalpyruvate aldolase. tryptophan and pyruvate. In the first Step alpha-ketoglutarate was the acceptor of the amino group from tryptophan in a 0353 Using assays similar to those described above, and transamination reaction generating indole-3-pyruvate and the detection methods described in Example 18, isocitrate glutamate. An aldolase catalyzed the Second reaction in lyase, N-acetylneuraminic acid Synthase, citrate lyase, tryp which pyruvate was reacted with indole-3-pyruvate, in the tophanase and certain mutants, beta-tyrosinase and certain presence of Mg" and phosphate, generating the alpha-keto mutants, PLP, catalytic aldolase antibodies, tryptophan Syn derivative of monatin (MP), 2-hydroxy-2-(indol-3-ylm thase(s) did not appear to detectably convert indole-3- ethyl)-4-ketoglutaric acid. Transfer of the amino group from pyruvate to MP under the conditions tested. the glutamate formed in the first reaction produced the 0354) A polypeptide having the desired activity can be desired product, monatin. Purification and characterization Selected by Screening clones of interest using the following of the product established that the isomer formed was methods. Tryptophan auxotrophs are transformed with vec S.S.-monatin. Alternative Substrates, enzymes, and condi tors carrying the clones of interest on an expression cassette tions are described as well as improvements that were made and are grown on a medium containing Small amounts of to this process. US 2005/0282260 A1 Dec. 22, 2005 28

0359 Enzymes umn previously converted to the acetate form by Washing 0360 The aldolase, 4-hydroxy-4-methyl-2-oxoglutarate with 0.5 L 1 M NaOH, 0.2 L water, 1.0 L of 1.0 M pyruvate lyase (ProA aldolase, proA gene) (EC 4.1.3.17) ammonium acetate, pH 8.4, and 0.5 L water. The Supernatant from Comamonas teStoSteroni was cloned, expressed and was loaded at <2 mL/min and the column was washed with purified as described in Example 7. The 4-hydroxy-2-oxo water at 3-4 mL/min until the absorbance at 280 nm was ~0. glutarate glyoxylate lyases (KHG aldolases) (EC 4.1.3.16) Monatin was eluted with 100 mM ammonium acetate, pH from B. Subtilis, E. coli, and S. meliloti were cloned, 8.4, collecting 4 100-mL fractions. expressed and purified as described in Example 7. 0367 Analysis of the fractions showed that the ratio of 0361 The aminotransferases used in conjunction with the tryptophan to monatin in the flow through fractions was aldolases to produce monatin were L-aspartate aminotrans 85:15 and the ratio in the eluent fractions was 7:93. ASSum ferase encoded by the E. coli aspC gene, the tyrosine ing the extinction coefficient at 280 nm of monatin is the aminotransferase encoded by the E. coli tyrB gene, the S. Same as tryptophan, the eluent fractions contained 0.146 meliloti Tata enzyme, the broad Substrate aminotransferase mmole of product. Extrapolation to the total 1 L reaction encoded by the L. major bsat gene, or the glutamic-Oxalo would produce ~2.4 mmoles (~710 mg) of monatin, for a acetic transaminase from pig heart (Type IIa). The cloning, recovery of 68%. expression and purification of the non-mammalian proteins 0368. The eluent fractions from the DEAE Sepharose are described in Example 1. Glutamic-Oxaloacetic transami column were evaporated to <20 mL. An aliquot of the nase from pig heart (type IIa) was obtained from Sigma (if product was further purified by application to a Cs prepara G7005). tive reversed-phase column using the Same chromatographic 0362 Method Using ProA aldolase and L-aspartate ami conditions as those described in Example 18 for the ana notransferase lytical-Scale monatin characterization. Waters Fractionl 0363 The reaction mixture contained 50 mM ammonium ynx"M Software was employed to trigger automated fraction acetate, pH 8.0, 4 mM MgCl, 3 mM potassium phosphate, collection of monatin based on detection of the m/z=293 ion. 0.05 mM pyridoxal phosphate, 100 mM ammonium pyru The fraction from the Cs column with the corresponding vate, 50 mM tryptophan, 10 mM alpha-ketoglutarate, 160 protonated molecular ion for monatin was collected, evapo mg of recombinant C. testosteroniProAaldolase (unpurified rated to dryness, and then dissolved in a Small Volume of cell extract, ~30% aldolase), 233 mg of recombinant E. coli water. This fraction was used for characterization of the L-aspartate aminotransferase (unpurified cell extract, ~40% product. aminotransferase) in one liter. All components except the 0369 The resulting product was characterized using the enzymes were mixed together and incubated at 30° C. until following methods. the tryptophan dissolved. The enzymes were then added and the reaction solution was incubated at 30° C. with gentle 0370 UV/Visible Spectroscopy. UV/visible spectro shaking (100 rpm) for 3.5 hours. At 0.5 and 1 hour after the Scopic measurements of monatin produced enzymatically addition of the enzymes aliquots of solid tryptophan (50 were carried out using a Cary 100 Bio UV/visible spectro mmoles each) were added to the reaction. All of the added photometer. The purified product, dissolved in water, tryptophan did not dissolve, but the concentration was showed an absorption maximum of 280 nm with a shoulder maintained at 50 mM or higher. After 3.5 hours, the solid at 288 nm, characteristics typical of indole containing com tryptophan was filtered off. Analysis of the reaction mixture pounds. by LC/MS using a defined amount of tryptophan as a 0371 LC/MS Analysis. Analyses of mixtures for monatin Standard showed that the concentration of tryptophan in the derived from the in vitro biochemical reactions were carried Solution was 60.5 mM and the concentration of monatin was out as described in Example 18. A typical LC/MS analysis 5.81 mM (1.05 g). of monatin in an in vitro enzymatic Synthetic mixture is 0364. The following methods were used to purify the illustrated in FIG. 5. The lower panel of FIG. 5 illustrates final product. Ninety percent of the clear Solution was a Selected ion chromatogram for the protonated molecular applied to a column of BioRad AG50W-X8 resin (225 mL.; ion of monatin at m/z=293. This identification of monatin in binding capacity of 1.7 meg/mL). The column was washed the mixture was corroborated by the mass Spectrum illus with water, collecting 300 mL fractions, until the absorbance trated in FIG. 6. Analysis of the purified product by LC/MS at 280 nm was <5% of the first flow through fraction. The showed a Single peak with a molecular ion of 293 and column was then eluted with 1 Mammonium acetate, pH absorbance at 280 nm. The mass Spectrum was identical to 8.4, collecting 4300-mL fractions. All 4 fractions contained that shown in FIG. 6. monatin and were evaporated to 105 mL using a roto evaporator with a tepid water bath. A precipitate formed as 0372 MS/MS Analysis. LC/MS/MS daughter ion experi the volume reduced and was filtered off over the course of ments, as described in Example 18, were also performed on the evaporation process. monatin. A daughter ion maSS spectrum of monatin is illustrated in FIG. 7. Tentative structural assignments of all 0365 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-(2xHO)), 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-1-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. ion), 130 (3-methylene-1H-indole carbonium ion), and 118 0366 The Supernatant (7 ml) was applied to a 100 mL (indole carbonium ion). Many of these are the same as those Fast Flow DEAE Sepharose (Amersham Biosciences) col obtained for MP (Example 7), as expected if derived from US 2005/0282260 A1 Dec. 22, 2005 29 the indole portion of the molecule. Some are 1 mass unit NMR data indicated that the enzymatically produced mona higher than those seen for MP, due to the presence of an tin was either (S,S), (R,R), or a mixture of both. amino group instead of a ketone. 0377 Chiral LC/MS Analysis. To establish that the 0373). Accurate Mass Measurement of Monatin. FIG. 8 illustrates the mass spectrum obtained for purified monatin monatin produced in Vitro was one isomer, and not a mixture employing an Applied Biosystems-Perkin Elmer Q-Star of the (R,R) and (S,S) enantiomers, chiral LC/MS analyses hybrid quadrupole/time-of-flight mass spectrometer. The were carried out using the instrumentation described in measured mass for protonated monatin using tryptophan as Example 18. an internal mass calibration standard was 293.1144. The calculated mass of protonated monatin, based on the 0378 Chiral LC separations were made using an Chiro elemental composition CHNOs is 293.1137. This is a biotic T (Advanced Separations Technology) chiral chroma mass measurement error of less than 2 parts per million tography column at room temperature. Separation and detec (ppm), providing conclusive evidence of the elemental com tion, based on published protocols from the Vendor, were position of monatin produced enzymatically. optimized for the R-(D) and S-(L) isomers of tryptophan. 0374 NMR Spectroscopy. The NMR experiments were The LC mobile phase consisted of A) water containing performed on a Varian Inova 500 MHz instrument. The 0.05% (v/v) trifluoroacetic acid; B) Methanol containing sample of monatin (-3 mg) was dissolved in 0.5 ml of D.O. 0.05% (v/v) trifluoroacetic acid. The elution was isocratic at Initially, the solvent (DO) was used as the internal reference 70% A and 30% B. The flow rate was 1.0 mL/min, and PDA at 4.78 ppm. Since the peak for water was large, the absorbance was monitored from 200 nm to 400 nm. The "H-NMR was run with suppression of the peak for water. instrumental parameters used for chiral LC/MS analysis of Subsequently, due to the broadness of the water peak, the tryptophan and monatin are identical to those described in C-2 proton of monatin was used as the reference peak, and Example 18 for LC/MS analysis. Collection of mass spectra set at the published value of 7.192 ppm. for the region m/z 150-400 was utilized. Selected ion chromatograms for protonated molecular ions (IM+H"=205 0375 For C-NMR, an initial run of several hundred Scans indicated that the Sample was too dilute to obtain an for both R- and S-tryptophan and M+H=293 for monatin) adequate 'C spectrum in the allotted time. Therefore, a allowed direct identification of these analytes in the mix heteronuclear multiple quantum coherence (HMOC) experi tureS. ment was performed, which enabled the correlation of the 0379 The chromatograms of R- and S-tryptophan and hydrogens and the carbons to which they were attached, and monatin, Separated by chiral chromatography and monitored also providing information on the chemical shifts of the by MS, are shown in FIG. 9. The single peak in the carbons. chromatogram of monatin indicates that the compound is 0376) A summary of the H and HMOC data is shown in one isomer, with a retention time almost identical to S-tryp Tables 4 and 5. By comparison to published values, the tophan.

TABLE 4

H NMR data

NH 1 HO O

Cargill Veggaar et al.' Takeshi et al.’ Atom 8H J(HH) Hz ÖH. J(HH) Hz, 8, J(HH) Hz 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 2005/0282260 A1 Dec. 22, 2005 30

0380 3-pyruvate, a reductive amination can be done for the last Step with glutamate dehydrogenase and NADH (as in TABLE 5 Example 15). 'C NMR data from HMQC spectrum 0387. The KHG aldolases from B. subtilis, E. coli, and S. meliloti were also used with the E. coli L-aspartate ami Cargill Vleggaar et al.' notransferase to produce monatin enzymatically. The fol Atom Öc Öc lowing reaction conditions were used: 50 mM NH-OAc 2 126.1 126.03 pH 8.3, 2 mM MgCl, 200 mM pyruvate, 5 mM glutamate, 3 : 110.31 0.05 mM pyridoxal phosphate, deaerated water to achieve a 4 120.4 120.46 5 12O2 120.25 final volume of 0.5 mL after the addition of the enzymes, 3 6 122.8 122.74 mM potassium phosphate, 20 lug/mL of recombinant B. 7 112.8 112.79 Subtilis KHG aldolase (purified), ca. 400 ug/mL of E. coli 8 : 137.06 L-aspartate aminotransferase (AspC) unpurified from cell 9 : 129.23 10a 36.4 36.53 extract, and 12 mM indole-3-pyruvate. The reactions were 12 39.5 39.31 incubated at 30° C. for 30 minutes with shaking. The amount 13 54.9 54.89 of monatin produced using the B. Subtilis enzyme was 80 14 : 175.30 ng/mL, and increased with increasing amounts of aldolase. 15 : 18118 If indole-3-pyruvate and glutamate were replaced by Satu "Vleggaar et al. (J. C. S. Perkin Trans. 1: 3095-8, 1992). rating amounts of tryptophan and 5 mM alpha-ketoglutarate, the production of monatin was increased to 360 ng/mL. Reactions were repeated with 30 tug/mL of each of the three 0381 Polarimetry. The optical rotation was measured on KHG enzymes in 50 mM Tris pH 8.3, with Saturating a Rudolph Autopol III polarimeter. The monatin was pre amounts of tryptophan, and were allowed to proceed for an pared as a 14.6 mg/mL Solution in water. The expected hour in order to increase detection. The Bacillus enzyme had specific rotation (IC)) for S.S monatin (salt form) is the highest activity as in Example 7, producing approxi -49.6 for a 1 g/mL solution in water (Vleggaar et al). The mately 4000 ng/mL monatin. The E. coli KHG produced observed ol' was -28.1 for the purified, enzymatically 3000 ng/mL monatin, and the S. meliloti enzyme produced produced monatin indicating that it was the S.S isomer. 2300 ng/mL. 0382 Improvements Example 10 0383. The reaction conditions, including reagent and enzyme concentrations, were optimized and yields of 5-10 Conversion of Tryptophan to Monatin Using ProA mg/mL were produced using the following reagent mix: 50 Aldolase and AspC, TyrB, or HEXAspC mM ammonium acetate pH 8.3, 2 mM MgCl2, 200 mM Aminotransferases With and Without Amino pyruvate (Sodium or ammonium salt), 5 mM alpha-ketoglu Terminal HIS Tags tarate (Sodium salt), 0.05 mM pyridoxal phosphate, deaer ated water to achieve a final volume of 1 mL after the 0388. The in vitro process utilizing two enzymes to addition of the enzymes, 3 mM potassium phosphate, 50 produce monatin from tryptophan, described in Example 9, Aug/mL of recombinant ProA aldolase (cell extract; total was further examined using three aminotransferases con protein concentration of 167 ug/mL), 1000 ug/mL of L-as Structed with and without amino terminal HIS tags. partate aminotransferase encoded by the E. coli aspC gene 0389. Enzymes (cell extract; total protein concentration of 2500 ug/mL), and solid tryptophan to afford a concentration of >60 mM 0390 The proA aldolase gene from Comamonas test (Saturated; Some undissolved throughout the reaction). The OSteroni was cloned and expressed as described in Example mixture was incubated at 30° C. for 4 hours with gentle 7. The gene product of this gene construct carries the amino Stirring or mixing. terminal HIS tag of the peT30 system. 0384 Substitutions 0391 The aspC and tyrB aminotransferase genes con structed with the pET30 amino terminal HIS tag system 0385) The concentration of alpha-ketoglutarate can be were cloned and expressed as described in Example 1. The reduced to 1 mM and supplemented with 9 mM aspartate HEX gene constructed with the pET30amino terminal HIS with an equivalent yield of monatin. Alternative amino acid tag System was cloned and expressed as described in acceptors can be utilized in the first Step, Such as Oxaloac Example 2. Untagged versions of the three aminotransferase etate. genes were cloned and expressed as described in Example 3. 0386 When recombinant L. major broad substrate ami 0392 Gene Expression and Assay of Monatin Production notransferase was used in place of the E. coli L-aspartate aminotransferase, Similar yields of monatin were achieved. 0393). Using Cell Extracts Containing Aminotransferase However, a second unidentified product (3-10% of the major Protein product) with a molecular mass of 292 was also detected by 0394 Cell extracts containing the proA, aspC, tyrB, and LC-MS analysis. Monatin concentrations of 0.1-0.5 mg/mL HEX gene products were prepared from cultures grown in were produced when the E. coli tyrB encoded enzyme, the LB medium with 50 mg/L kanamycin. The proteins were S. melilotitat A encoded enzyme or the glutamic-Oxaloacetic induced by the addition of 0.2 mM IPTG after the ODoo transaminase from pig heart (type IIa) was added as the reached approximately 0.5. The cells were grown for 4 hours aminotransferase. When Starting the reaction from indole at 30° C. after induction and harvested by centrifugation. US 2005/0282260 A1 Dec. 22, 2005

The washed cells were lysed using BugbusterTM reagent (Novagen) containing 1 u/mL benzonase nuclease, 5 TABLE 6-continued lil /mL. Calbiochem protease inhibitor cocktail set III and 0.33 u/10 mL r-Lysozyme following the Novagen recom Aminotransferase gene Aminotransferase: tug/mL Monatin: g/L mended protocol. After incubation at 25 C. for 15 min with HIS-tyrB 3O O.OO1 gentle Shaking, the cell debris was pelleted by centrifugation HIS-HEX 3OO 1581 at 21,000xg for 20 min at 4 C. and the Supernatant was HIS-HEX 3O O.487 carefully removed (cell extract). ND* 0395. The protein concentration was estimated using the below level of detection Pierce BCA Protein Assay Kit in a 96-well plate format. The total assay volume per well was 200 ul. Two hundred till of 0397 When non-optimal media, inducer concentrations, working reagent was added to 10 till protein Solution in each or induction times were utilized the level of expression of well. Bovine serum albumin (Pierce catalog #23209) was AspC or ProA decreased (e.g. to approximately 10-20% of utilized for the standard curve determination (0 to 1.0 the total Soluble protein). In these cases additional amounts mg/mL). The absorbance of the assay Samples and Standards of cell extracts were used with equivalent results. were measured at 562 nm. 0398. Using Purified Aminotransferase Proteins 0396 The formation of monatin from tryptophan was examined using unpurified enzymes. The level of expression 0399. The aminotransferases were purified from cell for the two proteins was high in the following example (30 extracts of the tagged peT30 constructs (prepared as to 40% of the total soluble protein). However, other cell described above) using immobilized metal affinity chroma extracts could also be used in which the level of expression tography with His-Bind cartridges following manufacturer's is lower, for example from 5 to 30% of the total soluble protocols (Novagen, Madison, Wis.). The HIS-Tag protein, or higher, for example greater than 40% of the total sequence of the fusion proteins binds to the divalent Ni’" Soluble protein. The reaction mixture contained, in one mL, cations immobilized on IDA-based His-Bind resin. The 100 mM sodium acetate, pH 8.0, 4 mM MgCl, 3 mM eluent fractions were desalted on PD-10 (Amersham Bio potassium phosphate, 0.05 mM pyridoxal phosphate, 200 sciences, Piscataway, N.J.) columns and eluted in 50 mM mM sodium pyruvate, 50 mM tryptophan, 10 mM alpha Tris-HCl, pH 7.0. The purified proteins were analyzed by ketoglutarate, 112 lug of recombinant C. testOSterOni ProA SDS-PAGE for purity and the amount of protein in the aldolase (unpurified cell extract containing ~30% aldolase fractions was determined using the Pierce BCA assay with (calculated as percent of the total Soluble protein)) and either bovine Serum albumin as the Standard. 1000 ug or 10 ug of recombinant aminotransferase (unpu rified cell extract containing ~40% aminotransferase (cal 0400. The formation of monatin from tryptophan was culated as percent of the total Soluble protein)). The tryp examined using purified AspC, HEXAspC, or TyrB ami tophan was added as a Solid. All components except the notransferase and unpurified ProA aldolase. The reaction enzymes were mixed together and incubated at 30° C. until mixture contained, in 0.5 mL, 50 mM Tris-HCl, pH 8.0, 4 the tryptophan dissolved. The enzymes were then added and mM MgCl, 3 mM potassium phosphate, 0.05 mM pyri the reaction solution was incubated at 30° C. with gentle doxal phosphate, 200 mM sodium pyruvate, -50 mM tryp Shaking for 1 h. The reaction mixtures were analyzed for tophan, 5 mM alpha-ketoglutarate, 165 lug of recombinant C. monatin formation by LC/MS/MS MRM as described in testOSteroni ProAaldolase (unpurified cell extract containing Example 18. Table 6 lists the activity of the enzymes as the ~30% aldolase (calculated as percent of total soluble pro concentration of product formed in the 1 h incubation. These tein)) and either 64 ug or 10 ug of purified recombinant E. results show that there is no significant difference between coli aminotransferase. The tryptophan was added as a Solid. the amount of monatin formed from tryptophan when the All components except the enzymes were mixed together AspC or HEXAspC aminotransferases are fused with the and incubated at 30° C. until the tryptophan dissolved. The pET30 amino terminal HIS tag or when they have been enzymes were then added and the reaction Solution was expressed as the native proteins. In contrast, the tagged TyrB incubated at 30° C. with gentle shaking for 2 h. The reaction protein produces leSS product as compared to the untagged mixtures were diluted 10-fold and analyzed for monatin TyrB protein. The untagged TyrB produces approximately formation by LC-MS/MS MRM as described in Example 25-75% the level of product observed in the AspC or 18. Table 7 lists the activity of the enzymes as the concen HEXASpC aminotransferase reaction mixtures, depending tration of product formed in the 2 h incubation. on the total amount of aminotransferase enzyme added. TABLE 7 TABLE 6 Aminotransferase Aminotransferase: gene pig?ml Monatin: g/L Aminotransferase gene Aminotransferase; lug/mL Monatin g/L HIS-aspC 64 O.38O aspC 3OO 1.085 HIS-aspC 1O O.OO15 aspC 3O O.186 HIS-tyrB 1O O.OO19 tyrB 3OO O.751 HIS-HEX 64 0.457 tyrB 3O O.O87 HIS-HEX 1O O.O879 HEX 3OO 1.514 O.OO32 HEX 3O O.411 HIS-aspC 3OO 1.101 HIS-aspC 3O O.213 HIS-tyrB 3OO O.OO2 04.01 The results of Table 7 show that with limiting quantities of purified aminotransferases and a large excess of US 2005/0282260 A1 Dec. 22, 2005 32 the aldolase (unpurified) the HEXAspC aminotransferase 0407. The following PCR protocol was used for gene reaction produces Several-fold more monatin than the AspC amplification: In a 50 till reaction, 200 ng DNA template, 1.6 aminotransferase reaction (barely measurable vs 0.0879 uM of each primer, 0.4 mM each dNTP, 0.5 Upful Jltra HF g/L). With larger aminotransferase concentrations the Polymerase (Stratagene), 2.8 U Expand High Fidelity"M increase is about 20%. Similar, though, leSS Striking differ Polymerase (Roche Molecular Biochemicals, Indianapolis, ences were observed with the unpurified enzymes (see Table Ind.), and 1x ExpandTM buffer with MgCl, were added. The 6). thermocycler program utilized a hot start of 94 C. for 3 minutes; followed by 8 cycles of a denaturing step at 94 C. 0402. The proA aldolase gene from Comamonas test (30 sec), an annealing step at 53 C. (30 sec), and an OSterOni and the HEX aspartate aminotransferase gene with extension step at 72° C. (1 min 30 sec); 20 cycles of a out the HIS tag system were also cloned into a peT23a denaturing step at 94° C. (30 sec), an annealing step at 59 (Novagen) derivative at the NdeI and XhoI sites of the C. (30 sec), and an extension step at 72° C. (1 min 30 sec); multiple cloning Sequence. Cell extracts of these constructs and finally a finishing step at 72° C. (7 min). The amplified were prepared from cultures induced with either IPTG or DNA was purified from a 1% agarose gel using a Qiagen lactose and were used as the Source of enzymes for the QIAquick(R) Gel Extraction Kit (Valencia, Calif.). The puri enzymatic production of monatin. The reactions were car fied DNA and purified plasmid DNAs (purified using a ried out in 50 mL using one of three sulfonate buffers Qiagen QIAprep(R) Spin Miniprep Kit) were digested with (MOPS, 3-(N-morpholio)propanesulfonic acid; HEPES, the Nde and XhoI according to the manufacturer's direc 4-(2-hydroxyethyl)poperazine-1-sulfonic acid; TAPS, 2-hy tions (NEB; Beverly, Mass.). Digestion of the pET30a droxy-1,1-bis(hydroxymethyl)ethyl)amino)-1-propane vector with Nde removes the amino terminal HIS-tag sulfonic acid) at a pH range of 7.5 to 8.9. The reactions region. The digested DNA was purified from a 1% agarose contained 50 mM buffer, 2 mM MgCl2, 200 mM pyruvate, gel using a Qiagen QIAquickCE) Gel Extraction Kit (Valencia, 5 mM glutamate, 0.05 mM pyridoxal phosphate, deaerated Calif.). The purified DNA product was quantified by mea water to achieve a final volume of 50 mL after the addition Suring the absorbance at 260 nm, and ligated using a Rapid of the enzymes, 3 mM potassium phosphate, 194 ug/mL of DNA Ligation Kit (Roche). Transformation of the ligation ProA aldolase cell extract (containing ~50 ug/mL aldolase) reactions into electrocompetent DH 10B was performed and either 281 tug/mL or 2810 ug/mL of HEXAspC ami under Standard conditions using a 0.2 cm cuvette and a notransferase cell extract (containing ~50 ug/mL or ~500 Bio-Rad Gene Pulser II system as described in the Bio-Rad Aug/mL aminotransferase). Solid tryptophan (0.5 g) was electroporation manual. Clones containing the khg gene added to the reaction mixtures just before the addition of the were identified by restriction analysis and confirmed by enzymes and at time intervals after the reactions were DNA sequencing. SEQ ID NOS 83 and 84 show nucleotide initiated. The reactions were Stirred at room temperature and and the corresponding amino acid Sequences of the khg gene aliquots were withdrawn for analysis by LC-MS as and gene product. described above. Under these conditions, the pH optimum for monatin production was between 8.2 and 8.5. The 0408 Gene Expression and Assays enzymes continued to produce monatin for Several days after 04.09 Plasmid DNA (verified by sequence analysis) was the reactions were initiated and produced up to 6 g/L of transformed into expression host BL21 (DE3) according to product. manufacturer's protocols (Novagen). The cultures were grown in LB medium with 50 mg/L kanamycin. Induction Example 11 experiments were carried out with the BL21 (DE3) construct 0403. This example describes methods that were used to grown in LB medium containing 50 mg/L kanamycin at 37 clone and analyze the KHG aldolase from Zymomonas C. Protein expression was induced using 0.2 mM IPTG after mobilis (ATCC 29191). This enzyme was used in combina the ODoo reached approximately 0.6. The cells were grown tion with an aminotransferase to produce monatin from for 4 hours at 30° C. and harvested by centrifugation. The tryptophan. cells were then lysed using Bugbuster" reagent (Novagen) containing 1 u/mL benzonase nuclease, and 5 ul/mL 0404 Cloning Calbiochem protease inhibitor cocktail set III following the 04.05) The khg gene from Z. mobilis (ATCC 29191) was Novagen recommended protocol. The Supernatant (cell free cloned in a Similar fashion to that described in Example 2. extract) was analyzed by SDS-PAGE on 4-15% gradient gels The genomic DNA was isolated using the method of Mekal (Bio-Rad) to detect soluble protein levels of the recombinant anoS J J, Duplication and amplification of toxin genes in fusion protein. The Z. mobolis aldolase was expressed efficiently in E. coli and accounted for approximately 30% Vibrio cholerae, Cell 35:253-263 (1983). of the Soluble protein as judged on a SDS polyacrylamide 0406. The following primers were designed for cloning gel. the Z. mobilis khg gene into pET30a and peT28 vectors 0410 The formation of monatin from tryptophan was (Novagen, Madison, Wis.) examined using unpurified enzymes. The reaction mixtures contained, in one mL, 100 mM sodium acetate, pH 8.0, 4 N term: mM MgCl, 3 mM potassium phosphate, 0.05 mM pyri (SEQ ID NO: 81) doxal phosphate, 100 mM sodium pyruvate, 50 mM tryp 5'-GGCCGGCATATGCGTGATATCGATTCCGTAAT-3"; tophan, 10 mM alpha-ketoglutarate, 239 ug of recombinant C. term: HEXASpC aminotransferase (unpurified cell extract con (SEQ ID NO: 82) taining ~30-40% aldolase (calculated as percent of the total 5'-GGAATTCTCGAGTTAGGCAACAGCAGCGCG-3'. soluble protein)) and either recombinant Z. mobilis khg aldolase (10 or 100 ug of unpurified cell extract containing US 2005/0282260 A1 Dec. 22, 2005 33

~30% aldolase (calculated as percent of the total soluble pH for the phosphate buffered Samples was approximately 7. protein)) or ProA aldolase (11.2 or 112 tug of unpurified cell None of the aldolases appeared to have Significant activity at extract containing 30% aldolase (calculated as percent of the pH 5, the Sample containing ProA aldolase was slightly total Soluble protein)). The tryptophan was added as a Solid. above the negative control but probably not above experi All components except the enzymes were mixed together mental error. In potassium phosphate, the ProA aldolase and incubated at 30° C. until the tryptophan dissolved. The produced 73.4 ppm monatin with a ratio of R.R.S.R of 1.7:1. enzymes were then added and the reaction Solution was The KHG aldolases produced 0.03-0.6 ppm monatin, with incubated at 30° C. with gentle shaking for 1 h or 22 h. The approximate ratios of 2:1-4:1 for R.R.S.R production. reaction mixtures were diluted 10-fold and analyzed for monatin formation by LC/MS/MS MRM as described in Example 12 Example 18. Table 8 lists the activity of the enzymes as the concentration of product formed in the incubations. Production of S.S Monatin Using Another ASpartate Aminotransferase from E. coli TABLE 8 0413 A putative PLP-dependent aminotransferase hav fig fig Incubation Monatin: ing homology to both aspartate and aromatic aminotrans Aldolase gene aldolase aminotransferase time (h) pig?mL ferases was cloned from E. coli MG1655 and the recombi Z. mobilis khg 1O 239 O.73O nant protein was tested for activity in producing SS Z. mobilis khg 1O 239 24 18.150 monatin. Primers were designed based on the yfdZ gene Z. mobilis khg 1OO 239 O.800 Z. mobilis khg 1OO 239 24 18.630 sequence deposited in NCBI as GI: 48994873 bases Z. mobilis khg O 239 ind 2496317-2495079, coding for protein GI: 1788722 (protein Z. mobilis khg O 239 24 <0.2 ID AAC75438.1). Z. mobilis khg O O O.98O Z. mobilis khg O O 24 16.990 C. testosteroni proA 11.2 239 214 5' primer: C. testosteroni proA 11.2 239 24 1464 TGA, CCC. TCT AGA TAA GAA. GGA. GAT (SEQ ID NO: 85) C. testosteroni proA 112 239 746 C. testosteroni proA 112 239 24 1160 ATA CAT ATG GCT GAC ACT CGC CCT C. testosteroni proA O 239 <0.2 C. testosteroni proA O 239 24 1790 GAA. C. C. testosteroni proA O O O.990 C. testosteroni proA O O 24 17.470 3' primer: TTC TCA AGC TTT TAT TCC GCG TTT (SEQ ID NO: 86) 0411) The results listed in Table 8 show that the Z. mobilis TCG TGA ATA GT TTG KHG aldolase is catalyzing the formation of monatin, though at low levels compared to the reactions in which the 0414 Genomic DNA from MG 1655 was prepared using ProA aldolase is present. AS can be seen by comparison of standard techniques and 100 ng was used in a 100 till PCR the results in rows 2 and 4, when a higher concentration of reaction which also contained 1x rTth buffer, 1 mM mag the KHG aldolase was added to the reaction, more monatin nesium acetate, 0.3 mM each dNTP, 0.75 uM of each primer, was formed. Two peaks with the monatin MS/MS signature 0.5 uL Pfu Polymerase (Stratagene), and 4 Units rTth were observed in the LC/MS/MS chromatograms from the Polymerase (Applied Biosystems). Eight rounds of PCR reactions in which the Z. mobilis KHG aldolase was present, utilized a 56° C. annealing temperature followed by 22 Suggesting that more than one isomer of monatin was rounds of PCR using a 60° C. annealing temperature. The formed by this enzyme. The results shown in Table 8 reflect extension step was done at 68 C. for two minutes and 15 the sum of these two peaks. The 24 hour samples from the Seconds. The PCR product was gel purified using a Qiagen Z. mobilis assay, when analyzed using chiral techniques, QIAquick Gel Extraction Kit (Valencia, Calif.) and eluted in showed that the majority of the Sample was S.S.-monatin. 50 uL EB buffer. The purified PCR product and pTRC99a There was, however a small amount of R.S.-monatin indi (Pharmacia Biotech) vector were digested overnight at 37 cating that the Z. mobilis aldolase is capable of making the C. with Xba and HindIII in 1x NEB buffer 2 plus BSA. The R-monatin precursor (R-MP) stereoisomer. digestion product was purified using a Qiagen QIAquick(E) PCR Purification Kit and eluted in 32 till 0.5xEB. Ligations 0412. The Z. mobilis KHG was further characterized by were performed using a Quick Ligation Kit (NEB) and a 6:1 comparison to the E. coli homolog described in Example 7, insert:Vector ratio. The ligation reaction was purified using as well as with the Pro Aaldolase. The following were added a PCR Purification Kit and electroporated into DH.10B per 1 mL of reaction mixture: approximately 60 lug aldolase competent cells. Two till were plated on LB plates contain (supplied in cellular extracts), 4 mM MgCl, 50 mMD-tryp ing 100 tug/mL ampicillin. Colonies were screened by PCR tophan, 0.5 mg BioCatalytics D-aminotransferase (AT-103), using primers derived from the pTRC99a vector. Miniprep 100 mM sodium pyruvate, 100 mM potassium phosphate DNA was sequenced to confirm correct insertion into the buffer pH 7.5 or 100 mM sodium acetate buffer pH 8, 0.05 mM PLP, 3 mM potassium phosphate (only to the acetate VectOr. reactions), and 10 mM O-ketoglutarate. Experiments were 0415 Cells were grown in LB medium containing ampi run in duplicate, with negative controls in which no aldolase cillin to an OD of 0.4 and induced with 1 mM IPTG for 3 was added. Samples were incubated overnight (20 hours) at hours at 37 C. Cellular extracts were produced using 30° C. with gentle shaking and filtered prior to LC/MS/MS BugEusterTM (Novagen) according to manufacturer's pro analysis and FDAA derivitization. The actual pH of the tocols, and protein concentration was determined using a Sodium acetate Samples was approximately 5, while the final Pierce BCA assay with 0-2 mg/mL BSA standards. Expres US 2005/0282260 A1 Dec. 22, 2005 34 sion was verified by SDS-PAGE analysis of soluble protein extract of the recombinant ProA enzyme. The reactions were and it was estimated that the putative aminotransferase incubated overnight at 30° C. with gentle shaking (100 rpm). (YfdZ) comprised approximately 10% of the soluble pro The samples were filtered and submitted for reverse phase tein. LC/MS/MS analysis as described in Example 18. The results are presented below: 0416) The yfdz gene product was assayed in both 100 mM sodium acetate and 50 mM potassium phosphate buffers (pH 7.5) for production of monatin and compared with the HEXAspC protein prepared as described in Example 2. Fifty Enzyme Aug/mL monatin produced tug of unpurified aminotransferase was assayed in each AT-101 173.05 buffer with 20 mM L-tryptophan, 4 mM MgCl2, 3 mM AT-102 122.05 potassium phosphate (for acetate buffer only), 0.05 mM PLP, AT-103 369.7 10 mM O-ketoglutarate, 100 mM sodium pyruvate, and 50 AT-104 133.05 AT-105 15.2 pug ProA aldolase (described in Example 7) provided as a AT-106 78.35 cellular extract. The pH of the acetate samples had to be negative 73.25 adjusted from 6 to 7.5 prior to addition of enzymes. The 1 mL samples (run in duplicate) were incubated with gentle shaking at 30° C. for 1 hour. Negative controls were also 0418 AT-101, AT-102, AT-103, and AT-104 aminotrans done in each buffer and did not contain any aminotransferase ferases clearly produced more monatin than the negative other than what is present in the cellular extracts of the ProA control. AT-105 produced leSS monatin than the negative aldolase. Samples were filtered and analyzed by LC/MS/MS control, presumably because lysine was utilized as the (MRM) as described in Example 18. After subtraction of amino donor, which is not Suitable for native E. coli ami background amounts of monatin produced (between 410 notransferases in the cellular extract. Similarly, one would 469 ng/mL monatin), the HEXAspC aminotransferase was expect the background to be lower for the reactions with found to produce 1285 ng/mL monatin in phosphate buffer, AT-103 which were provided D-glutamate. The results were while the YfdZ protein produced 426 ng/mL monatin. In further analyzed to determine ratios of S.R/R,S versus Sodium acetate buffer, the HEXAspC aminotransferase pro R.R/S,S monatin, on the basis of the peak areas of the two duced 815 ng/mL monatin while the putative aminotrans Stereoisomer pools that resolve during the chromatographic ferase produced 446 ng/mL. The results confirm that the Separation. Of the total monatin produced, the negative yfdz gene product is in fact an aminotransferase that behaves control contained approximately 99.7% R.R/SS monatin. Similarly to the aspartate aminotransferase described in AT-102 showed the next highest specificity (89% RR/SS Example 1, with activity towards both tryptophan and mona peak) followed by AT-101 and AT-104 (~80%). The reac tin. tions utilizing the broad Specificity D-aminotransferase, AT-103, produced 69% R,R/SS monatin in comparison to Example 13 the mixed isomers. This enzyme is homologous to the Bacillus Subtilis DAT enzyme described in Example 1, Comparison of Monatin Production from which is known to have a broad specificity for D-amino indole-3-pyruvate Using a Commercially Available acids and was shown in Example 1 to accept D-tryptophan Transaminase Library as a Substrate. Chiral analysis was performed using the methodology described in Example 18, which verified that 0417. A transaminase library was purchased from Bio the D-aminotransferase was making R,R monatin as Catalytics (Pasadena, Calif.) and the enzymes were tested expected. Further experimentation with S.S monatin or R.R for production of monatin in coupled reactions using the monatin as a substrate verified that the BioCatalytics ProA aldolase from C. testosteroni. The library consisted of: enzyme is highly Selective for the D-configuration, as AT-101, a broad range L-aminotransferase, AT-102, a expected (results described in Example 15). branched chain L-transaminase (i.e., a branched-chain ami notransferase (BCAT, EC 2.6.1.42)); AT-103, a broad range 0419. To decrease the amount of S.S monatin or R,S D-transaminase, AT-104, a branched chain L-transaminase monatin produced as byproducts in coupled reactions with (i.e., a branched-chain aminotransferase (BCAT, EC AT-103 (the broad range D-transaminase) and the ProA 2.6.1.42)); AT-105, lysine-6-aminotransferase; and AT-106, aldolase, the aldolase was purified using His-Bind car a broad range L-transaminase. In reactions with AT-103, tridges, as described in Example 1. The purified enzyme D-glutamate was used as the amino acid donor. For reactions should not contain wildtype aminotransferase activities that with AT-105, L-lysine was used as the amino donor. All can be present in cellular extracts. The His-Bind eluent was other reactions utilized L-glutamate as the co-Substrate. desalted to remove imidazole using PD-10 columns (G25 Enzymes and additional components/Substrates were added Sephadex, Amersham-Pharmacia) and was eluted in 50 mM directly to the reaction buffer provided in the kit, which Tris-Cl, pH 7. Experiments were carried out in duplicate in contained 100 mM potassium phosphate buffer pH 7.5, 100 a volume of 1 mL and contained 100 mM Tris-Cl buffer, pH mM amino donor, and 0.1 mM PLP. To one mL of reaction 7.8, 50 ug ProA aldolase, 4 mg indole-3-pyruvate, 1 or 2 mg buffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, D-aminotransferase, 200 mM sodium pyruvate, 2 mM approximately 50 lug ProA provided in a cellular extract, 1 MgCl, 3 mM potassium phosphate, 0.1 mM PLP, and 14.7 AiL 2 M MgCl2, and 2 mg of aminotransferase enzyme to be mg of D-glutamate. The tubes were incubated at 30° C. with tested. All reactions were performed in duplicate, and a gentle Shaking. Two hour time points were taken and frozen negative control reaction was done with no additional ami immediately at -20° C. The pH was adjusted at two hours notransferase added. Background production of monatin is from 5 to between 7-8 using NaOH, and the assays were due to native E. coli aminotransferases present in the cellular incubated overnight. Samples were filtered and analyzed for US 2005/0282260 A1 Dec. 22, 2005 35 monatin as described in Example 18. The two hour Samples did not have detectable amounts of monatin, probably due to TABLE 10 the low pH. The overnight Samples contained approximately 190 ng/mL monatin when 1 mg of D-aminotransferase was Comparison of D-aminotransferase enzymes used, and approximately 84% was R,R monatin and 16% Enzyme time (hr) Monatin (ppb) % RR Monatin was S.R monatin. When 2 mg of D-aminotransferase were B. Sub DATHIS 1. 512 not determined used, 540 ng/mL monatin was produced, approximately 71 B. Sub DAT untagged 1. 1056 not determined BioCatalytics AT-103 1. 2353 not determined % was R,R monatin. B. Sub DATHIS 2 894 --80-90% B. Sub DAT untagged 2 1913 --80% 0420 Similar experiments were conducted using Bio BioCatalytics AT-103 2 6887 92.5% catalytics Aminotransferase buffer, which contained 100 B. Sub DATHIS 16 3O14 31 mM potassium phosphate pH 7.5, 0.1 mM PLP, and 100 mM B. Sub DAT untagged 16 5612 33 glutamate. Solid indole-pyruvate and D-aminotransferase BioCatalytics AT-103 16 16131 66 were added as above. ProA aldolase (50 ug), MgCl, and 50 mM pyruvate were added from Stock Solutions. The assays 0423. The removal of the HIS-6 tag appears to have were treated as above, although no pH adjustment was improved the activity of the B. Subtilis D-aminotransferase; required in this case. A negative control was done with just however, the BioCatalytics D-aminotransferase homolog the BioCatalytics supplied enzyme and buffer, which did not clearly had the highest activity. It also showed more Sub produce any monatin. The experimental results are shown in strate specificity for the R-MP. Increased incubation times Table 9. appear to reduce the enantiomeric excess of R,R monatin that is produced. TABLE 9 0424. Other homologous D-amino acid aminotrans ferases from Bacillus species have been characterized. The Production of Monatin from Indole-3-Pyruvate in Phosphate Buffer enzyme from Bacillus sp. YM-1 was found to have similar Substrate Specificity to the B. Subtilis enzyme, and therefore Total it is expected that this enzyme would work for production of Mg D-aminotransferase time (hrs) monatin (ng/mL) % R, R monatin. Additionally, the B. YM-1 and B. Subtilis have been shown to be more specific enzymes, and do not have O 2 O infa as broad of Substrate Specificity as homologs from B. 1. 2 678O not determined Sphaericus and other Species. Therefore, it is expected that 2 2 1317O 55% all of the Bacillus homologs will be active in the enzymatic O 16 O infa pathway to monatin. See K. Yonaha, H. Misono, T. Yama 1. 16 15OOO not determined moto, and K. Soda, JBC, 250: 6983-6989 (1975); K. 2 16 2893O 51% Tanizawa, Y. Masu, S. Asano, H. Tanaka, and K. Soda, JBC, 264. 2445-2449 (1989). AT-103, one homologous enzyme tested (above), was shown to catalyze the production of 0421. The production of monatin in phosphate buffer is monatin. Many genes are known that code for D-amino acid clearly higher than that in Tris buffered systems. aminotransferases in Bacillus species, including B. anthra cis, B. cereus, B. halodurans, B. licheniformis, B. Sphaeri 0422 To compare activities of the cloned B. Subtilis DAT cus, B. Stearothermophilus, B. Subtilis, B. thuringiensis, B. from Example 1 with the BioCatalytics enzyme additional YM-1/YM-2, Geobacillus sp., thermophilic sp., and assays were done. The B. Subtilis dat gene was also Sub Oceanobacillus sp. cloned to remove the His-6 tag, as described for the AspC and HEXASpC aminotransferases in Example 1. Untagged Example 14 and tagged enzyme were produced in BL21 (DE3), as described in Example 1. Cellular extracts were made and Production of Monatin Using Commercially total protein assays were done to estimate protein concen Available Dehydrogenase Enzymes tration as described previously. Duplicate one mL reactions 0425 Production of monatin from indole-3-pyruvate and were done which contained: 500 tug D-aminotransferase, 50 pyruvate, using BioCatalytics amino acid dehydrogenase lug ProA aldolase, 100 mM potassium phosphate pH 7.5, 3 enzymes coupled with the C. testosteroni Pro Aaldolase, was mM MgCl, 4 mg indole-3-pyruvate, 200 mM sodium assayed under the following conditions: 6-7 mg/ml dehy pyruvate, 7.35 mg (50 mM) D-glutamate, and 0.1 mM PLP. drogenase enzyme, 5 mg NADH or NADPH, 50 tugaldolase Samples were incubated at 30° C. for 1 hr, 2 hr., and (unpurified, see Example 7), 3 mM potassium phosphate overnight, and were filtered for LC/MS/MS analysis. The buffer, 2 mM MgCl, 4 mg indole-3-pyruvate, and 20 mg pyruvate were added to one mL of AADH reaction buffer Samples contained only the S.R and R,R Stereoisomers of (100 mM bicarbonate, pH 9.5, 200 mM NHCl). Negative monatin, as determined by the FDAA derivitization protocol controls contained no amino acid dehydrogenase enzyme. described in Example 18. The results are summarized in Samples were incubated at 30° C. at 100 rpm overnight. Table 10 below. The % RR was determined by peak areas Experiments were performed in duplicate. The dehydroge that were Separated by reverse phase chromatography, and nases tested were AADH-110, AADH-111, AADH-112, and the composition of the overnight Samples was further veri AADH-113. AADH-110 and 111 have been defined as broad fied by the FDAA derivitization technique. Specificity enzymes, while 112 and 113 are glutamate dehy US 2005/0282260 A1 Dec. 22, 2005 36 drogenases. AADH-110 produced the most monatin (as the same enzyme with alpha-ketoglutarate as the amino measured by LC/MS/MS) in comparison to the negative acceptor. The relative activity with Oxaloacetate was: HIS controls, approximately 0.36 tug/mL. The NADH-utilizing BSAT-HIS-AspC>porcine type IIadporcine type I=HIS glutamate dehydrogenase, AADH-112, showed higher activ TyrB. The relative activity with alpha-ketoglutarate was: ity than the NADPH utilizing glutamate dehydrogenase HIS-BSAT>HIS-AspC>porcine type I>porcine type (AADH-113). AADH-111 did not appear to produce more IadHIS-TyrB. monatin than the negative control under the conditions assayed. 0430. Using assays similar to those described above, and the detection methods described in Example 18, two enzymes, S. meliloti Tata and R. Sphaeroides Tata, did not Example 15 appear to detectably convert monatin to MP under the conditions tested. This lack of detectable activity, however, Interconversion Between MP and Monatin may be due to the fact that MP is sometimes difficult to 0426. The amination of MP to form monatin can be detect because it is unstable in an aqueous Solution. In catalyzed by aminotransferases, or by dehydrogenases that reactions using approximately 50 lug each of purified ProA require a reducing cofactor such as NADH or NADPH. See aldolase and purified S. meliloti aminotransferase (under Examples 1 and 9, 10, 12-14. These reactions are reversible conditions similar to Example 9), tryptophan and pyruvate and can be measured in either direction. The directionality, were converted to 6 ppm monatin in two hours, and 40 ppm when using a dehydrogenase enzyme, can be largely con monatin in overnight reactions. The R. Sphaeroides tryp tophan aminotransferase produced approximately 10-fold trolled by the concentration of ammonium Salts. more monatin, indicating a higher activity in converting MP 0427 Dehydrogenase activity. The oxidative deamina to monatin. tion of monatin was monitored by following the increase in absorbance at 340 nm as NAD(P)+ was converted to the 0431. The aminotransferase activity and substrate speci more chromophoric NAD(P)H. Monatin was enzymatically ficity of the untagged TyrB and ASpC proteins were also produced and purified as described in Example 9. measured by following the formation of the co-product glutamate using the protocol described in Example 3 in 0428. A typical assay mixture contained 50 mM Tris which S.S.-monatin (5 mM) was added as the substrate HCl, pH 8.0 to 8.9, 0.33 mM NAD" or NADP', 2 to 22 units instead of tryptophan. In this aminotransferase reaction of glutamate dehydrogenase (Sigma), and 10-15 mM Sub S.S.-monatin reacts Stoichiometrically with the amino accep Strate in 0.2 mL. The assay was performed in duplicate in a tor alpha-ketoglutarate to form MP and glutamate. Thus, if UV-transparent microtiter plate, on a Molecular Devices 1 tumole of glutamate is formed, 1 umole of MP should also SpectraMax Plus platereader. A mix of the enzyme, buffer, be formed. The results are listed in Table 11. and NAD(P)' were pipetted into wells containing the Sub Strate and the increase in absorbance at 340 nm was moni TABLE 11 tored at 10 second intervals after brief mixing. The reaction was incubated at 25 C. for 10 minutes. Negative controls Aminotransferase gene pig protein glutamatel; tig/mL were carried out without the addition of Substrate, and tyrB 50 211.8 glutamate was utilized as a positive control. The type III aspC 50 543 glutamate dehydrogenase from bovine liver (Sigma if G-7882) catalyzed the conversion of the monatin to MP at a rate of conversion approximately one-hundredth the rate of 0432 Comparison of these results with those described in the conversion of glutamate to alpha-ketoglutarate. Attempts Example 3 indicate that the AspC has a 6-fold higher to produce monatin from indole-3-pyruvate utilizing substrate preference for tryptophan over S.S.-monatin (328.2 glutamate dehydrogenaseS resulted in production of detect Aug/mL/54.3 ug/mL). TyrB, on the other hand, only shows able amounts of tryptophan, indicating that glutamate dehy 1.5-fold preference for tryptophan (310.1 lug/mL/211.8 drogenases, or mutants thereof, could potentially be utilized Aug/mL) and shows Substantially more activity than the AspC for deamination of tryptophan rather than using an oxidase protein when monatin is the Substrate. or an aminotransferase for this Step in the pathway. 0433 Conversion of Monatin to MPUsing Commercially 0429 Transamination activity. Monatin aminotransferase Available Dehydrogenases assays were conducted with the aspartate aminotransferase (HIS-AspC) from E. coli, the tyrosine aminotransferase 0434. Several amino acid dehydrogenases from BioCata (HIS-TyrB) from E. coli, the broad substrate aminotrans lytics (AADH-101-110) were assayed for conversion of ferase (HIS-BSAT) from L. major, and the two commer monatin to MP. Assays were performed in 100 mM sodium cially available porcine glutamate-Oxaloacetate aminotrans bicarbonate buffer, pH 10.0, 10-20 mM NAD", 20-200 mM ferases described in Example 1. Both oxaloacetate and monatin prepared as described in Example 9, and 20 mg/ml alpha-ketoglutarate were tested as the amino acceptor. The enzyme. Reactions were incubated at 30-45 C. for 2 hours, assay mixture contained (in 0.5 mL) 50 mM Tris-HCl, pH and then 30 C. for 16 hours. The reaction progress was 8.0, 0.05 mM PLP, 5 mM amino acceptor, 5 mM monatin, monitored by following absorbance changes at 340 nm due and 25 ug of aminotransferase. The assays were incubated at to production of NADH. Negative controls were done in 30° C. for 30 minutes, and the reactions were stopped by which enzyme was absent, or AADH-101 was present but addition of 0.5 mL isopropyl alcohol. The loss of monatin either NAD" or monatin were omitted. Positive controls was monitored by LC/MS or LC/MS/MS (Example 18). The were done using AADH-101 with valine as the substrate. highest amount of activity was noted with L. major HIS Although low (~1-4%) in comparison to the positive con BSAT with oxaloacetate as the amino acceptor, followed by trols, AADH-102 (an aromatic L-amino acid dehydroge US 2005/0282260 A1 Dec. 22, 2005 37 nase) and AADH-110 (a broad specificity branched chain Example 16 L-amino acid dehydrogenase; EC 1.4.1.9) appeared to have Some activity on S.S.-monatin and could potentially be Production of Monatin from Tryptophan and C3 evolved to improve activity in a biocatalytic process. The Sources Other than Pyruvate reductive amination reaction, which is what is needed pro 0438. As described above in Example 9, indole-3-pyru duction of monatin from MP, is typically a much faster Vate or tryptophan can be converted to monatin using reaction than the oxidative deamination reaction that was pyruvate as the C3 molecule. However, in Some circum measured here. MP was not detectable by LC/MS; however, stances, pyruvate may not be a desirable raw material. For it has been found to be unstable and difficult to assay. example, pyruvate may be more expensive than other C3 carbon Sources, or may have adverse effects on fermenta 0435 Conversion of Monatin and C-KG to MP and tions if added to the medium. Alanine can be transaminated Glutamate Using Commercially Available Aminotrans by many PLP-enzymes to produce pyruvate. ferases 0439 Tryptophanase-like enzymes perform beta-elimi 0436 AT-101, AT-102, AT-103, and AT-104 were pur nation reactions at faster rates than other PLP enzymes Such as aminotransferases. Enzymes from this class (4.1.99.-) can chased from BioCatalytics (Pasadena, Calif.). AT-101 is a produce ammonia and pyruvate from amino acids Such as broad range L-transaminase, AT-102 is a branched-chain L-serine, L-cysteine, and derivatives of Serine and cysteine aminotransferase (EC 2.6.1.42), AT-103 is a broad range with good leaving groupS. Such as O-methyl-L-serine, D-aminotransferase, and AT-104 is a branched-chain ami O-benzyl-L-serine, S-methylcysteine, S-benzylcysteine, notransferase (EC 2.6.1.42). These enzymes all were active S-alkyl-L-cysteine, O-acyl-L-serine, 3-chloro-L-alanine. in production of monatin from indole-pyruvate and pyruvate 0440 Processes to produce monatin using EC 4.1.99.- when coupled with aldolase enzyme (see Example 13). The polypeptides can be improved by mutating the B-tyrosinase enzymes were tested for activity on S.S and R,R monatin (TPL) or tryptophanase according to the method of Moura that was produced chemically. Reactions were performed in tou et al. (J. Biol. Chem 274:1320-5, 1999). Mouratou et al. a total Volume of 0.5 mL, and run in duplicate. The assay describe the ability to covert the B-tyrosinase into a dicar contained 50 mM Tris pH 7.8, 0.08 mM PLP, 10 mM boxylic amino acid B-lyase, which has not been reported to O-ketoglutarate (C.-KG), 5 mM monatin, and 1 mg/ml ami occur in nature. The change in Specificity was accomplished notransferase enzyme. Negative controls did not contain by converting valine (V) 283 to arginine (R) and arginine aminotransferase enzyme. The Samples were incubated for 2 (R) 100 to threonine (T). These amino acid changes allow hrs at 30° C. at 100 rpm shaking. Samples were filtered and for the lyase to accept a dicarboxylic amino acid for the LC/MS/MS analysis was run to ascertain glutamate levels hydrolytic deamination reaction (Such as aspartate). Aspar (as described in Example 18). Glutamate levels should tate, therefore, can also be used as a Source of pyruvate for correlate stoichiometrically with MP production. The nega Subsequent aldol condensation reactions. tive controls contain Some background levels of glutamate 0441. Additionally, cells or enzymatic reactors can be from the cell extracts of the aldolase, as well as glutamate Supplied with lactate and an enzyme that converts lactate to production from native E. coli aminotranferases. When R.R pyruvate. Examples of enzymes capable of catalyzing this was used as the reaction Substrate, the only significant reaction include lactate dehydrogenase and lactate oxidase. glutamate produced in comparison to the negative control 0442) Isolation of Genomic DNA was by the D-transaminase (AT-103), approximately 1.1 0443) Tryptophanase polypeptides have previously been lug/ml was detected. AT-101 and AT-104 produced slightly reported in, for example, Mouratou et al. (JBC 274: 1320-5, more glutamate than the negative controls. The D-transami 1999). To isolate genes that encode tryptophanase polypep nase enzyme showed no detectable activity on S.S.-monatin. tides, genomic DNA from E. coli DH10B was used as a The aminotransferases are enantioselective for the chiral template for PCR as described in Example 1. carbon that contains the amino moiety, as expected. All of the L-aminotransferases showed activity on S.S.-monatin. 0444 The gene for tyrosine-phenol lyase was isolated AT-101 produced 75 ug/ml glutamate, AT-102 produced 102 from C. freundii (ATCC catalog number 8090, Designation tug/ml glutamate, and AT-104 produced 64 ug/ml glutamate. ATCC 13316; NCTC 9750) and grown on Nutrient agar (Difco 0001) and nutrient broth (Difco 0003) at 37° C. to an 0437 Similar reactions using 25 mM R.R-monatin, 100 OD of 2.0. The genomic DNA was purified using a Qiagen mM potassium phosphate, pH 7.5, 0.08 mM PLP, 50 mM Genomic-tipTM 100/G kit. C.-ketoglutarate, and 4 mg/mL AT-103 enzyme produced 0445 PCR Amplification of Coding Sequences 0.145, 0.268, 0.391, and 0.593 mM glutamate (determined by the LC-post column fluorescence method described in 0446 Primers were designed with compatible overhangs Example 18) at 1, 2, 3, and 19 hrs incubation at 30° C. for the pET 30 Xa/LIC vector (Novagen, Madison, Wis.) as Phosphate appears to have increased the D-aminotransferase described above in Example 1. activity when compared to Tris-Cl buffer. In parallel experi 0447 E. colitma (SEQ ID NO: 41). N-terminal primer for ments using 1 mg/mL ASpC and S.S.-monatin as a Substrate, pET30 Xa/LIC cloning: 5'-GGTATTGAGGGT CGC ATG 0.11-0.18 mM glutamate was formed during the incubation GAA AAC TTT AAA CAT CT-3' (SEQ ID NO: 43). time. Increasing the AspC concentration to 2 mg/ml C-terminal primer for pT30 Xa/LIC cloning: 5'-AGAGGA increased glutamate concentrations to 1.2 mM in a 4 hour GAGTTAGAG. CCTTAAACTTCT TTA AGTTTT G-3 reaction. (SEQ ID NO: 44). US 2005/0282260 A1 Dec. 22, 2005 38

0448 C. freundii tpl (SEQID NO: 42). N-terminal primer 04.58 Mutagenesis of Tryptophanase: Example 1.6A for pET30 Xa/LIC cloning: 5'-GGTATTGAGGGT CGC 0459. The mutagenesis protocol provided below intro ATGAATTATCCGGCAGAACC-3' (SEQ ID NO: 45). duced two point mutations in the amino acid Sequence. The C-terminal primer for pET 30 Xa/LIC cloning: 5'-AGA first point mutation changed arginine (R) at position 103 to GGAGAGTTAGAGCCTTAGATGTAATCAAAGCGTG threonine (T) and the Second point mutation changed valine 3' (SEQ ID NO: 46). (V) at position 299 to arginine (R) (numbering system for E. 0449) The Eppendorf MastercyclerTM Gradient 5331 coli mature protein). Mutagenesis experiments were per Thermal Cycler was used for all PCR reactions. In 50L was formed by ATG Laboratories (Eden Prairie, Minn.). Muta added 0.5 lug template (genomic DNA), 1.0 uM of each tions were introduced Sequentially by PCR of gene frag primer, 0.4 mM each dNTP, 3.5 U Expand High Fidelity ments and reassembly of the fragments was accomplished Polymerase (Roche), 1x Expand buffer with Mg, and 5% by PCR as well. Primers for converting arginine (R) 103 to DMSO (final concentration). The thermocycler PCR pro threonine (T): gram used was as follows: 96° C. hot start (5 minutes), 94' C.-30 seconds, 40-60 C.-1 minute 45 seconds, 72 C.-2 (SEQ ID NO: 47) minutes 15 seconds; 30 repetitions. The final polymerization 5'-CCAGGGCACCGGCGCAGAGCAAATCTATATT-3' Step was for 7 minutes, and the Samples were then Stored at and 4° C. (SEQ ID NO : 48) 0450 Cloning 5'-TGCGCCGGTGCCCTGGTGAGTCGGAATGGT-3'. 0451 Cloning and positive clone identification proce 0460 Primers for converting valine (V)299 to arginine dures detailed above in Example 1 were used to identify the (R): appropriate clones.

0452 Gene Expression and Activity Assays (SEQ ID NO: 49) 5'-TCCTGCACGCGGCAAAGGGTTCTGCACTCGGT-3' 0453 Plasmid DNA (verified by sequence analysis) was and subcloned into the expression host BL21(DE3) (Novagen). The cultures were grown in LB medium with 30 mg/L (SEQ ID NO : 50) kanamycin, the plasmids were isolated using a Qiagen 5'-CTTTGCCGCGTGCAGGAAGGCTTCCCGACA-3'. miniprep kit, and analyzed by restriction digest to confirm identity. 0461) Mutants were screened by restriction digest with Xba I/HindIII and SphI, and verified by sequencing. 0454) Induction experiments were done with the BL21(DE3) expression host, the constructs were grown in 0462 Mutagenesis of Tyrosine Phenol Lyase (B-tyrosi LB medium containing 50 mg/L kanamycin at 37 C. nase): Example 16B Protein expression was induced using 0.1 mM IPTG after 0463 Two point mutations were made to the tyrosine the ODoo of the culture reached approximately 0.6. The phenol lyase amino acid Sequence. These mutations con cells were grown for 4 hours at 30° C. and harvested by verted arginine (R) at position 100 to threonine (T) and centrifugation. The cells were then lysed in 5 mL/g wet cell valine (V) at position 283 to arginine (R) (in C. freundii weight BugBuster" (Novagen) reagent containing 5 ul/mL mature protein Sequence). protease inhibitor cocktail set #III (Calbiochem) and 1 AiL/mL benzonase nuclease (Novagen), and the His-tagged 0464) Primers for the R100T conversion were: recombinant proteins were purified using the His-Bind car tridges as described above in Example 1. Purified proteins were desalted on a PD-10 (G25 Sephadex, Amersham Bio (SEQ ID NO: 51) sciences) column and eluted in 100 mM Tris-Cl buffer, pH 5'-AGGGGACCGGCGCAGAAAACCTGTTATCG-3' 8.0. The proteins were analyzed by SDS-PAGE on 4-15% and gradient gels to check for Soluble protein levels at the (SEQ ID NO : 52) predicted MW of the recombinant fusion protein. 5'-AGGGGACCGGCGCAGAAAACCTGTTATCG-3'. 0455 Mutagenesis 0465 Primers for the V283R conversion were: 0456 Some members of polypeptide class 4.1.99.-(tryp tophanase and B-tyrosinase) will perform the beta-lyase (SEQ ID NO: 53) reaction with aspartate or similar amino acids without any 5'-GTTAGTCCGCGTCTACGAAGGGATGCCAT-3' modification. However, Some members of the class may and need to be mutagenized to allow for the use of the Substrates and/or the creation of the product. Moreover, in Some cases (SEQ ID NO: 54) polypeptides that can perform the conversion can be further 5'-GTAGACGCGGACTAACTCTTTGGCAGAAG-3'. optimized by mutagenesis. 0466. The methods described above were used, and the 0457 Site directed mutagenesis was performed based on clones were screened by Kpn/SacI digestion, and BstXI 3D structure analysis of PLP-binding polypeptides. Two digestion. The Sequences were verified by dideoxy chain examples for changing the Substrate Specificity of the termination Sequencing. Recombinant protein was produced polypeptides are shown below. as described above for the wildtype enzymes. US 2005/0282260 A1 Dec. 22, 2005 39

0467. The reaction mixture consisted of 50 mM Tris-Cl ditions. Indole-3-pyruvate (appropriately blocked) is then pH 8.3, 2 mM MgCl2, 200 mM C3 carbon source, 5 mM added to form the coupled product followed by removal of alpha-ketoglutarate, Sodium Salt, 0.05 mM pyridoxal phos the protecting groups to form monatin. Protecting groups phate, deaerated water to achieve a final volume of 0.5 mL that are particularly useful include THP (tetrahydropyranyl after the addition of the enzymes, 3 mM potassium phos ether) which is easily attached and removed. phate pH 7.5, 25 lug of crude recombinant C. testosteroni ProA aldolase as prepared in Example 7,500 lug of crude Example 18 L-aspartate aminotransferase (AspC) as prepared in Example 1, and Solid tryptophan to afford a concentration of Detection of Monatin, MP, Tryptophan, and >60 mM (saturated; some undissolved throughout the reac tion). The reaction mix was incubated at 30° C. for 30 minutes with mixing. Serine, alanine, and aspartate were 0471. This example describes methods used to detect the Supplied as 3-carbon Sources. ASSays were performed with presence of monatin, or its precursor 2-hydroxy 2-(indol-3- and without secondary PLP enzymes (purified) capable of ylmethyl)-4-keto glutaric acid, as well tryptophan and performing beta-elimination and beta-lyase reactions (tryp glutamic acid. It also describes a method for the Separation tophanase (TNA), double mutant tryptophanase, f-tyrosi and detection of the four Stereoisomers of monatin. nase (TPL)). The results of the LC/MS analyses of the 0472 LC/MS Analysis of Monatin, MP, and Tryptophan reaction mixtures are shown in Table 12: 0473 Analyses of mixtures for monatin, MP, and/or tryptophan derived from in vitro or in vivo biochemical TABLE 12 reactions were performed using a WaterS/Micromass liquid Production of Monatin utilizing alternative C3-carbon sources chromatography-tandem mass spectrometry (LC/MS/MS) instrument including a Waters 2795 liquid chromatograph Additional PLP with a Waters 996 Photo-Diode Array (PDA) absorbance C3-carbon source Enzyme Relative Activity monitor placed in Series between the chromatograph and a Ole None O% Micromass Quattro Ultima triple quadrupole mass Spec pyruvate None 100% serine None 3% trometer. LC Separations were made using an Xterra MSCs serine 11 tug wildtype TNA (1 U) 5.1% reversed-phase chromatography column, 2.1 mmx250 mm, serine 80 ug double mutant TNA 4.6% or a Supelco Discovery Cs reversed phase chromatography alanine None 32% column, 2.1 mmx 150 mm at room temperture or at 40 C. alanine 11 tug wildtype TNA 41.7% alanine 80 ug mutant TNA 43.9% The LC mobile phase consisted of A) water containing aspartate 110 ug wildtype TNA (10 U) 7.7% 0.05% (v/v) trifluoroacetic acid and B) methanol containing aspartate 5 U wildtype TPL (crude) 5.1% 0.05% (v/v) trifluoroacetic acid. aspartate 80 ug mutant TNA 3.3% 0474 The gradient elution was linear from 5% B to 35% B, 0-4 min, linear from 35% B to 60% B, 4-6.5 min, linear 0468. The monatin produced from alanine and serine as from 60% B to 90% B, 6.5-7 min, isocratic at 90% B 7-11 3-carbon sources was verified by LC/MS/MS daughter scan min, linear from 90% B to 95% B, 11-12 min, linear from analysis, and was identical to the characterized monatin 95% B to 5% B, 12-13 min, with a 5 min re-equilibration produced in Example 9. Alanine was the best alternative period between runs. The flow rate was 0.25 mL/min, and tested, and was transaminated by the AspC enzyme. The PDA absorbance was monitored from 200 nm to 400 nm. All amount of monatin produced was increased by addition of parameters of the ESI-MS were optimized and selected the tryptophanase, which has a transamination Secondary based on generation of protonated molecular ions (IM+H") activity. The amount of monatin produced with Serine as a of the analytes of interest, and production of characteristic carbon source nearly doubled with the addition of the fragment ions. tryptophanase enzymes, even though only one-fifth of the 0475. The following instrumental parameters were used amount of tryptophanase was added in comparison to the for LC/MS analysis of monatin: Capillary: 3.5 kV, Cone: 40 aminotransferase. AspC is capable of Some amount of beta V; Hex 1:20 V; Aperture: 0 V; Hex 2:0 V; Source tempera elimination activity alone. The results with aspartate indi ture: 100 C.; Desolvation temperature: 350° C.; Desolva cate that the tryptophanase activity on aspartate does not tion gas: 500 L/h; Cone gas: 50 L/h; Low mass resolution increase with the Same site-directed mutations as previously (Q1): 15.0; High mass resolution (Q1): 15.0; Ion energy: Suggested for B-tyrosinase. It is expected that the mutant 0.2; Entrance: 50V; Collision Energy: 2; Exit: 50V; Low B-tyrosinase will have higher activity for production of mass resolution (Q2): 15; High mass resolution (Q2): 15; monatin. Ion energy (Q2): 3.5; Multiplier: 650. Uncertainties for reported mass/charge ratios (m/z) and molecular masses are Example 17 +0.01%. Initial detection of the alpha-keto acid form of monatin (MP) and monatin in the mixtures was accom Chemical Synthesis of Monatin plished by LC/MS monitoring with collection of mass 0469 The addition of alanine to indole-3-pyruvic acid spectra for the region m/z 150-400. Selected ion chromato produces monatin, and this reaction can be performed Syn grams for protonated molecular ions (IM+H"=292 for MP, thetically with a Grignard or organolithium reagent. M+H"=293 for monatin, M+H=205 for tryptophan) allowed direct identification of these analytes in the mix 0470 For example, to 3-chloro- or 3-bromo-alanine tures. Subsequent methods for monatin and MP detection which has been appropriately blocked at the carboxyl and used multiple reaction monitoring (MRM) LC/MS/MS amino groups, is added magnesium under anhydrous con methodology (see below). US 2005/0282260 A1 Dec. 22, 2005 40

0476 LC/MS/MS Analysis for Monatin for protonated monatin used tryptophan as an internal mass calibration Standard. The calculated mass of protonated 0477 LC/MS/MS daughter ion experiments were per monatin, based on the elemental composition CH7NOs formed on monatin as follows. A daughter ion analysis is 293.1137. Monatin produced using the biocatalytic pro involves transmission of the parent ion (e.g., m/z=293 for ceSS described in Example 9 showed a measured mass of monatin) of interest from the first mass analyzer (Q1) into 293.1144. This is a mass measurement error of less than 2 the collision cell of the mass spectrometer, where argon is parts per million (ppm), providing conclusive evidence of introduced and chemically dissociates the parent into frag the elemental composition of monatin produced enzymati ment (daughter) ions. These fragment ions are then detected cally. with the Second mass analyzer (Q2), and can be used to corroborate the Structural assignment of the parent. Tryp 0485 Chiral LC/MS/MS (MRM) Measurement of Mona tophan was characterized and quantified in the Same way via tin transmission and fragmentation of m/z=205. 0486) Determination of the stereoisomer distribution of 0478. The following instrumental parameters were used monatin in in Vitro and in Vivo reactions was accomplished for LC/MS/MS analysis of monatin: Capillary: 3.5 kV; by derivitization with 1-fluoro-2-4-dinitrophenyl-5-L-ala Cone: 40 V; Hex 1:20 V; Aperture: 0 V; Hex 2:0 V; Source nine amide (FDAA), followed by reversed-phase LC/MS/ temperature: 100° C.; Desolvation temperature: 350° C.; MS MRM measurement. Desolvation gas: 500 L/h; Cone gas: 50 L/h; Low mass resolution (Q1): 13.0; High mass resolution (Q1): 13.0; Ion 0487. Derivitization of Monatin with FDAA energy: 0.2; Entrance: -5 V, Collision Energy: 14; Exit: 1V; 0488 To 50 till of sample or standard was added 200 till Low mass resolution (Q2): 15; High mass resolution (Q2): of a 1% solution of FDAA in acetone. Forty lull of 1.0 M 15; Ion energy (Q2): 3.5; Multiplier: 650. Sodium bicarbonate was added, and the mixture incubated 0479 LC/MS/MS Multiple Reaction Monitoring for 1 h at 40 C. with occasional mixing. The sample was removed and cooled, and neutralized with 20 u, of 2.0 M 0480. To increase the sensitivity and selectivity of mona HCl (more HCl may be required to effect neutralization of tin detection, an LC/MS/MS method employing MRM mea a buffered biological mixture). After degassing is complete, Surements has been developed. LC Separations were per samples were ready for analysis by LC/MS/MS. formed as described in previous Sections. Instrumental parameters for ESI-MS/MS were set up as described in the 0489 LC/MS/MS Multiple Reaction Monitoring for the previous Section, except that low and high mass resolution Determination of the Stereoisomer Distribution of Monatin Settings for Q1 and Q2 are set to 12.0 to maximize Sensi in In Vitro and In Vivo Reactions. tivity. Five monatin-Specific parent-to daughter transitions are used to Specifically detect monatin in in Vitro and in vivo 0490 Analyses were performed using the LC/MS/MS reactions. The transitions are 293.1 to 158.3, 293.1 to 168.2, instrumentation described in previous Sections. LC Separa 293.1 to 2112, 293.1 to 230.2, and 293.1 to 257.2. tions capable of Separating all four Stereoisomers of monatin (specifically FDAA-monatin) were performed on a Phenom 0481 High-Throughput Determination of Monatin, Tryp enex Luna (5 um) C18 reversed phase chromatography tophan, and Glutamic Acid (Glutamate) column at 40°C. The LC mobile phase consisted of A) water 0482 High-throughput analyses (<5 min/sample) of mix containing 0.05% (mass/volume) ammonium acetate and B) tures for monatin, tryptophan, and/or glutamic acid derived acetonitrile. The gradient elution was linear from 2% B to from in vitro or in Vivo reactions were carried out using 34% B, 0-33 min, linear from 34% B to 90% B, 33-34 min, instrumentation described above, and the same MS param isocratic at 90% B34-44 min, and linear from 90% B to 2% eters as described for LC/MS/MS Multiple Reaction Moni B, 44-46 min, with a 16 min re-equilibration period between toring. LC Separations were made using a 4.6 mmx50 mm runs. The flow rate was 0.25 mL/min, and PDA absorbance Advanced Separation Technologies Chirobiotic T column at was monitored from 200 nm to 400 nm. All parameters of room temperature. The LC mobile phase consisted of A) the ESI-MS were optimized and selected based on genera water containing 0.25% acetic acid; B) Methanol containing tion of protonated molecular ions (IM+H") of FDAA 0.25% acetic acid. The isocratic elution was at 50% B, 0-5 monatin, and production of characteristic fragment ions. min. The flow rate was 0.6 mL/min. All parameters of the 0491. The following instrumental parameters were used ESI-MS/MS system were optimized and selected based on for LC/MS analysis of monatin in the negative ion ESI/MS optimal in-Source generation of the protonated molecular mode: Capillary: 2.0 kV; Cone: 25 V; Hex 1:10 V; Aperture: ions of tryptophan and monatin and the internal Standard 0 V; Hex 2:0 V; Source temperature: 100° C.; Desolvation “Hs-tryptophan or Ha-glutamic acid, as well as collision temperature: 350° C.; Desolvation gas: 500 L/h; Cone gas: induced production of analyte-specific fragment ions for 50 L/h; Low mass resolution (Q1): 12.0; High mass reso multiple reaction monitoring (MRM) experiments (204.7 to lution (Q1): 12.0; Ion energy: 0.2; Entrance: -5V, Collision 146.4 for tryptophan, 209.7 to 151.4 for Hs-tryptophan, Energy: 20, Exit: 1V; Low mass resolution (Q2): 12; High 147.6 to 102.4 for glutamic acid, 150.6 to 105.4 for H mass resolution (Q2): 12; Ion energy (Q2): 3.0; Multiplier: glutamic acid, monatin-specific transitions listed in the 650. Three FDAA-monatin-specific parent-to daughter tran previous Section). Sitions are used to Specifically detect FDAA-monatin in in vitro and in vivo reactions. The transitions are 543.6 to 0483 Accurate Mass Measurement of Monatin. 268.2, 543.6 to 499.2, and 543.6 to 525.2. Identification of 0484 High resolution MS analysis was carried out using FDAA-monatin StereoisomerS is based on chromatographic an Applied Biosystems-Perkin Elmer Q-Star hybrid quadru retention time as compared to purified monatin Stereoiso pole/time-of-flight mass spectrometer. The measured mass mers, and mass spectral data. US 2005/0282260 A1 Dec. 22, 2005

0492 Liquid Chromatography-Post Column Fluores 0498 Cells were sampled every hour until an ODoo of cence Detection of Amino Acids Including Glutamate 0.35–0.8 was obtained. Cells were then induced with 0.1 mM 0493 Liquid chromatography with post-column fluores IPTG, and the temperature reduced to 34° C. Samples (1 ml) cence detection for the determination of glutamic acid in in were collected prior to induction (Zero time point) and vitro and in vivo reactions was performed on a Waters 2690 centrifuged at 5000xg. The Supernatant was frozen at -20 LC system or equivalent combined with a Waters 474 C. for LC/MS analysis. Four hours post-induction, another Scanning fluorescence detector, and a Waters post-column 1 mL Sample was collected, and centrifuged to Separate the reaction module. LC Separations were performed on, an broth from the cell pellet. Tryptophan, Sodium pyruvate, Interaction-Sodium loaded ion exchange column at 60° C. ampicillin, and Tween were added as described above. Mobile phase A was Pickering Na 328 buffer (Pickering Laboratories, Inc.; Mountain View, Calif.). Mobile phase B 0499. The cells were grown for 48 hours post-induction, was Pickering Na 740 buffer. The gradient elution was from and another 1 mL Sample was taken and prepared as above. 0% B to 100% B, 0-20 min, isocratic at 100% B, 20-30 min, At 48 hours, another aliquot of tryptophan and pyruvate and linear from 100% B to 0% B, 30-31 min, with a 20 min were added. The entire culture Volume was centrifuged after re-equilibration period between runs. The flow rate for the approximately 70 hours of growth (post-induction), for 20 mobile phase was 0.5 mL/min. The flow rate for the OPA minutes at 4 C. and 3500 rpm. The Supernatant was post-column derivitization solution was 0.5 mL/min. The decanted and both the broth and the cells were frozen at -80 fluorescence detector settings were EX338 nm and Em 425 C. The broth fractions were filtered and analyzed by LC/MS. nm. Norleucine was employed as an internal Standard for the The heights and areas of the M+H=293 peaks were analysis. Identification of amino acids was based on chro monitored as described in Example 18. The background matographic retention time data for purified Standards. level of the medium was Subtracted. The data was also normalized for cell growth by plotting the height of the Example 19 M+H=293 peak divided by the optical density of the Production of Monatin in Bacteria culture at 600 mn. 0494. This example describes methods used to produce 0500 Higher levels of monatin were produced when monatin in E. coli cells. One skilled in the art will under pyruvate, amplicillin, and Tween were added 4 hours post Stand that Similar methods can be used to produce monatin induction rather than at induction. Other additives Such as in other bacterial cells. In addition, vectors containing other PLP, additional phosphate, or additional MgCl, did not genes in the monatin Synthesis pathway (FIG. 2) can be increase the production of monatin. Higher titers of monatin used. were obtained when tryptophan was utilized instead of 0495 Trp-1+glucose medium, a minimal medium that indole-3-pyruvate, and when the tryptophan was added has been used for increased production of tryptophan in E. post-induction rather than at inoculation, or at induction. coli cells (Zeman et al. Folia Microbiol. 35:200-4, 1990), Prior to induction, and 4 hours post-induction (at time of was prepared as follows. To 700 mL nanopure water the Substrate addition), there was typically no detectable level of following reagents were added: 2 g (NH4)2SO4, 13.6 g. monatin in the fermentation broth or cellular extracts. Nega KHPO, 0.2 g MgSO.7HO, 0.01 g CaCl2.H2O, and 0.5 tive controls were done utilizing cells with peT30a vector mg FeSO.7H2O. The pH was adjusted to 7.0, the volume only, as well as cultures where tryptophan and pyruvate were was increased to 850 mL, and the medium was autoclaved. not added. A parent MS Scan demonstrated that the com A 50% glucose Solution was prepared Separately, and Sterile pound with (m+1)/Z=293 was not derived from larger mol filtered. Forty mL was added to the base medium (850 mL) ecules, and daughter Scans (performed as in Example 18) for a 1 L final volume. were Similar to monatin made in vitro. 0496 A10 g/L L-tryptophan solution was prepared in 0.1 0501) The effect of Tween was studied by utilizing 0, M sodium phosphate pH 7, and sterile-filtered. One-tenth 0.2% (vol/vol), and 0.6% final concentrations of Tween-20. Volume was typically added to cultures as Specified below. The highest amount of monatin produced by Shake flaskS A 10%. Sodium pyruvate Solution was also prepared and was at 0.2% Tween. The amplicillin concentration was varied Sterile-filtered. A 10 mL aliquot was typically used per liter between 0 and 10 ug/mL. The amount of monatin in the of culture. Stocks of amplicillin (100 mg/mL), kanamycin cellular broth increased rapidly (2.5x) between 0 and 1 (25 mg/mL) and IPTG (840 mM) were prepared, sterile tug/mL, and increased 1.3.x when the amplicillin concentra filtered, and stored at -20° C. before use. Tween 20 (poly tion was increased from 1 to 10 ug/mL. oxyethylene 20-Sorbitan monolaurate) was utilized at a 0502. A time course experiment showing typical results is 0.2% (vol/vol) final concentration. Ampicillin was used at shown in FIG. 10. The amount of monatin Secreted into the non-lethal concentrations, typically 1-10 ug/mL final con cell broth increased, even when the values are normalized centration. for cell growth. By using the molar extinction coefficient of 0497 Fresh plates of E. coli BL21 (DE3):C. testosteroni tryptophan, the amount of monatin in the broth was esti pro Alpe.T 30 Xa/LIC (described in Example 7) were pre mated to be less than 10 ug/mL. The same experiment was pared on LB medium containing 50 lug/mL kanamycin. repeated with the cells containing vector without proA Overnight cultures (5 mL) were inoculated from a single insert. Many of the numbers were negative, indicating the colony and grown at 30° C. in LB medium with kanamycin. peak height at m/Z=293 was leSS in these cultures than in the Typically a 1 to 50 inoculum was used for induction in medium alone (FIG. 10). The numbers were consistently trp-1+glucose medium. Fresh antibiotic was added to a final lower when tryptophan and pyruvate were absent, demon concentration of 50 mg/L. Shake flasks were grown at 37 Strating that monatin production is a result of an enzymatic C. prior to induction. reaction catalyzed by the aldolase enzyme. US 2005/0282260 A1 Dec. 22, 2005 42

0503) The in vivo production of monatin in bacterial cells was repeated in 800 mL shake flask experiments and in TABLE 13 fermentors. A 250 mL sample of monatin (in cell-free broth) was purified by anion eXchange chromatography and pre Amount of Monatin Produced by E. coli Bacteria parative reverse-phase liquid chromatography. This Sample Construct Host Tween + Amp tug/mL Monatin time was evaporated, and Submitted for high resolution mass proA BL21 (DE3) O41 72 hr analysis (described in Example 9). The high resolution MS proA BL21 (DE3) -- 1.58 48 hr indicated that the metabolite being produced is monatin. proA BL21 (DE3)pLyss 1.04 48 hr proA BL21 (DE3)pLyss -- 1.60 48 hr 0504. In vitro assays indicate that aminotransferase needs aspC-proA BL21 (DE3) O.09 48 hr to be present at higher levels than aldolase (see Example 9), aspC-proA BL21 (DE3) -- O.58 48 hr therefore the aspartate aminotransferase from E. coli was aspC-proA BL21 (DE3)pLysS 1.39 48 hr overexpressed in combination with the aldolase gene to aspC-proA BL21 (DE3)pLysS -- 6.68 48 hr increase the amount of monatin produced. Primers were designed to introduce C. testosterOni proA into an operon with aspC/pET30 Xa/LIC, as follows: 5' primer: ACTCG Example 20 GATCCGAAGGAGATATACATATGTAC GAACTGGGACT (SEQ ID NO: 67) and 3' primer: Production of Monatin in Yeast CGGCTGTCGACCGTTAGTCAATATATTTCAGGC (SEQ ID NO: 68). The 5' primer contains a BamHI site, the 0507. This example describes methods used to produce 3' primer contains a SalI site for cloning. PCR was per monatin in eukaryotic cells. One skilled in the art will formed as described in Example 7, and gel purified. The understand that Similar methods can be used to produce aspC/pET30 Xa/LIC construct was digested with BamHI monatin in any cell of interest. In addition, other genes can and SalI, as was the PCR product. The digests were purified be used (e.g., those listed in FIG. 2) in addition to, or using a Qiagen Spin column. The proA PCR product was alternatively to those described in this example. ligated to the vector using the Roche Rapid DNA Ligation 0508. The pESC Yeast Epitope Tagging Vector System kit (Indianapolis, Ind.) according to manufacturer's instruc (Stratagene, La Jolla, Calif.) was used to clone and express tions. Chemical transformations were done using Novablues the E. coli aspC and C. teStoSteroni proA genes into Sac Singles (Novagen) as described in Example 1. Colonies charomyces cerevisiae. The pSC vectors contain both the were grown up in LB medium containing 50 mg/L kana GAL1 and the GAL10 promoters on opposite strands, with mycin and plasmid DNA was purified using the Qiagen Spin two distinct multiple cloning Sites, allowing for expression miniprep kit. Clones were Screened by restriction digest of two genes at the same time. The pSC-His vector also analysis and sequence was confirmed by Seqwright (Hous contains the His3 gene for complementation of histidine ton, Tex.). Constructs were subcloned into BLR(DE3), auxotrophy in the host (YPH500). The GAL1 and GAL10 BLR(DE3)pLysS, BL21 (DE3) and BL21 (DE3)pLysS promoters are repressed by glucose and induced by galac (Novagen). The proA?pET30 Xa/LIC construct was also tose, a Kozak Sequence is utilized for optimal expression in transformed into BL21(DE3)pLysS. yeast. The pSC plasmids are shuttle vectors, allowing the 0505) Initial comparisons of BLR(DE3) shake flask initial construct to be made in E. coli (with the bla gene for Samples under the Standard conditions described above Selection); however, no bacterial ribosome binding sites are demonstrated that the addition of the Second gene (aspC) present in the multiple cloning sites. improved the amount of monatin produced by seven-fold. To 0509. The following primers were designed for cloning hasten growth, BL21 (DE3)-derived host strains were used. The proA clones and the two gene operon clones were into pESC-His (restriction sites are underlined, Kozak induced in Trp-1 medium as above, the pySS hosts had sequence is in bold): aspC (BamHI/Sall), GAL1: chloramphenicol (34 mg/L) added to the medium as well. Shake flask experiments were performed with and without (SEQ ID NO: 69) the addition of 0.2% Tween-20 and 1 mg/L amplicillin. The 5'-CGCGGATCCATAATGGTTGAGAACATTACCG-3' amount of monatin in the broth was calculated using in vitro and produced purified monatin as a Standard. SRM analyses (SEQ ID NO : 70) were performed as described in Example 18. Cells were 5'-ACGCGTCGACTTACAGCACTGCCACAATCG-3'. Sampled at Zero, 4 hours, 24 hours, 48 hours, 72 hours, and 96 hours of growth. 0510) proA (EcoRI/NotI), GAL10: 0506) The results are shown in Table 13 for the maximum amounts produced in the culture broths. In most instances, (SEQ ID NO : 71.) the two gene construct gave higher values than the ProA 5'-CCGGAATTCATAATGGTCGAACTGGGAGTTGT-3' construct alone. The pySS Strains, which should have and leakier cell envelopes, had higher levels of monatin Secreted, (SEQ ID NO: 72) even though these Strains typically grow at a slower rate. The 5'-GAATGCGGCCGCTTAGTCAATATATTTCAGGCC-3'. additions of Tween and ampicillin were beneficial. US 2005/0282260 A1 Dec. 22, 2005 43

0511. The second codon for both mature proteins was SC-His medium. The following carbon Sources were used changed from an aromatic amino acid to Valine due to the sequentially: 1% raffinose--1% glucose, 0.5% glucose--1.5% introduction of the Kozak Sequence. The genes of interest raffinose, 2% raffinose, and finally 1% raffinose--2% galac were amplified using peT30 Xa/LIC miniprep DNA from tose for induction. the clones described in Examples 1 and Example 7 as template. PCR was performed using an Eppendorf Master 0516. After approximately 16 hours of growth in induc cycler gradient thermocycler and the following protocol for tion medium, the 50 mL cultures were divided into duplicate a 50 till reaction: 1.0 till template, 1.0 uM of each primer, 0.4 25 mL cultures, and the following were added to only one of mM each dNTP, 3.5 U Expand High Fidelity Polymerase the duplicates: (final concentrations) 1 g/L L-tryptophan, 5 (Roche, Indianapolis, Ind.), and 1x Expand TM buffer with mM Sodium phosphate pH 7.1, 1 g/L Sodium pyruvate, 1 Mg. The thermocycler program used consisted of a hot start mM MgCl2. Samples of broths and cell pellets from the at 94 C. for 5 minutes, followed by 29 repetitions of the non-induction medium, and from the 16 hour cultures prior following steps: 94 C. for 30 seconds, 50° C. for 1 minute to addition of Substrates for the monatin pathway, were 45 seconds, and 72 C. for 2 minutes 15 seconds. After the Saved as negative controls. In addition, constructs containing 29 repetitions the sample was maintained at 72 C. for 10 only a functional aspC gene (and a truncated proA gene) minutes and then stored at 4 C. The PCR products were were utilized as another negative control. The cells were purified by Separation on a 1%. TAE-agarose gel followed by allowed to grow for a total of 69 hours post-induction. recovery using a QIAquick Gel Extraction Kit (Qiagen, Occasionally the yeast cells were induced at a lower OD, Valencia, Calif.). and only grown for 4 hours prior to addition of tryptophan 0512. The pESC-His vector DNA (2.7 ug) was digested and pyruvate. However, these monatin Substrates appear to with BamHI/Sall and gel-purified as above. The aspC PCR inhibit growth and the addition at higher OD was more product was digested with BamHI/SalI and purified with a effective. QIAquick PCR Purification Column. Ligations were per formed with the Roche Rapid DNA Ligation Kit following 0517. The cell pellets from the cultures were lysed with the manufacturer's protocols. DeSalted ligations were elec 5 mL of YeastBusterTM+50 ul THP (Novagen) per gram (wet troporated into 40 ul Electromax DH10B competent cells weight) of cells following manufacturer's protocols, with (Invitrogen) in a 0.2 cm Biorad disposable cuvette using a the addition of protease inhibitors and benzonase nuclease as Biorad Gene Pulser II with pulse controller plus, according described in previous examples. The culture broth and cell to the manufacturers instructions. After 1 hour of recovery extracts were filtered and analyzed by SRM as described in in 1 mL of SOC medium, the transformants were plated on Example 18. Using this method, no monatin was detected in LB medium containing 100 tug/mLampicillin. Plasmid DNA the broth Samples, indicating that the cells could not Secrete preparations for clones were done using OIAprep Spin monatin under these conditions. The proton motive force Miniprep Kits. Plasmid DNA was screened by restriction may be insufficient under these conditions or the general digest, and Sequenced (Seqwright) for verification using amino acid transporters may be Saturated with tryptophan. primerS designed for the Vector. Protein expression was not at a level that allowed for detection of changes using SDS-PAGE. 0513. The aspC/pESC-His clone was digested with EcoRI and Not, as was the proA PCR product. DNA was 0518 Monatin was detectable (approximately 60 ng/mL) purified as above, and ligated as above. The two gene transiently in cell extracts of the culture with two functional construct was transformed into DH10B cells and Screened genes, when tryptophan and pyruvate were added to the by restriction digest and DNA sequencing. medium. Monatin was not detected in any of the negative control cell extracts. In vitro assays for monatin were 0514. The construct was transformed into S. cerevisiae performed in duplicate with 4.4 mg/mL of total protein strain YPH500 using the S.c. EasyCompTM Transformation (about double what is typically used for E. coli cell extracts) Kit (Invitrogen). Transformation reactions were plated on using the optimized assay described in Example 9. Other SC-His minimal medium (Invitrogen pYES2 manual) con assays were performed with the addition of either 32 tug/mL taining 2% glucose. Individual yeast colonies were Screened C. testosteroni ProA aldolase or 400 lug/mL AspC ami for the presence of the proA and aspC genes by colony PCR notransferase, to determine which enzyme was limiting in using the PCR primers above. Pelleted cells (2 ul) were the cell extract. Negative controls were performed with no suspended in 20uL of Y-Lysis Buffer (Zymo Research) addition of enzyme, or the addition of only ASpC ami containing 1 ul of Zymolase and heated at 37 C. for 10 notransferase (the aldol condensation can occur to Some minutes. Four ul of this Suspension was then used in a 50 extent without enzyme). Positive controls were performed till PCR reaction using the PCR reaction mixture and with partially pure enzymes (30-40%), using 16 ug/mL program described above. aldolase and 400 tug/mL aminotransferase. 0515. Five mL cultures were grown overnight on SC-His 0519 In vitro results were analyzed by SRM. The analy + glucose at 30° C. and 225 rpm. The cells were gradually sis of cell extracts showed that tryptophan was effectively adjusted to growth on raffinose in order to minimize the lag transported into the cells when it was added to the medium period prior to induction with galactose. After approxi post-induction, resulting in tryptophan levels two orders of mately 12 hours of growth, absorbance measurements at 600 magnitude higher than those in which no additional tryp nm were taken, and an appropriate Volume of cells was spun tophan was added. The results for in vitro monatin analysis down and resuspended to give an OD of 0.4 in the fresh are shown in Table 14 (numbers indicate ng/mL). US 2005/0282260 A1 Dec. 22, 2005

TABLE 1.4 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

0520 Positive results were obtained with the full two and catalysts can then be regenerated. Due to the Similarity gene construct cell extracts with and without Substrate added of monatin in size, charge, and hydrophobicity to Several of to the growth medium. These results, in comparison to the the reagents and intermediates, physical Separations will be positive controls, indicate that the enzymes were expressed difficult unless there is a high amount of affinity for monatin at levels of close to 1% of the total protein in yeast. The (Such as an affinity chromatography technique). However, amount of monatin produced when the cell extract of the the monatin reactions can be coupled to other reactions Such aspC construct (with truncated ProA) was assayed with that the equilibrium of the system is shifted toward monatin aldolase was significantly greater than when cell extracts production. The following are examples of processes for were assayed alone, and indicates that the recombinant improving the yield of monatin obtained from tryptophan or AspC aminotransferase comprises approximately 1-2% of indole-3-pyruvate. the yeast total protein. The cell extracts of uninduced cultures had a Small amount of activity when assayed with 0524 Coupled Reactions Using Oxaloacetate Decar aldolase due to the presence of native aminotransferases in boxylase (EC 4.1.1.3) the cells. When assayed with AspC aminotransferase, the 0525 FIG. 11 is an illustration of the pathway to produce activity of the extracts from uninduced cells increased to the monatin in which Oxaloacetate decarboxylase is added to amount of monatin produced by the negative control with remove the co-product oxaloacetate formed during the con AspC (ca. 200 ng/ml). In contrast, the activity observed version of MP to monatin. Tryptophan oxidase and catalase when assaying the two gene construct cell extract increases are utilized to drive the reaction in the direction of indole more when aminotransferase is Supplemented than when 3-pyruvate production. Catalase is used in exceSS Such that aldolase is added. Since both genes should be expressed at hydrogen peroxide is not available to react in the reverse the same level, this indicates that the amount of monatin direction or to damage the enzymes or intermediates. Oxy produced is maximized when the level of aminotransferase gen is regenerated during the catalase reaction. Alterna is higher than that of aldolase, in agreement with results tively, indole-3-pyruvate can be used as the initial Substrate. shown in Example 9. 0526 In this pathway, aspartate is used as the amino 0521. The addition of pyruvate and tryptophan not only donor for the amination of MP in a reaction catalyzed by inhibits cellular growth, but apparently inhibits protein aspartate aminotransferase. Ideally, an aminotransferase that expression as well. The addition of the pSC-Trp plasmid has a low Specificity for the tryptophan/indole-3-pyruvate can be used to correct for tryptophan auxotrophy of the reaction in comparison to the MP/monatin reaction is used YPH500 host cells, to provide a means of Supplying tryp So that the aspartate does not act as an amino donor to tophan with fewer effects on growth, expression, and Secre reaminate the indole-3-pyruvate. Oxaloacetate decarboxy tion. lase (from Pseudomonas sp.) can be added to convert the oxaloacetate to pyruvate and carbon dioxide. Since CO is Example 21 volatile it is not available for reaction with the enzymes, thus decreasing or even preventing the reverse reactions. The Improvement of Enzymatic Processes. Using pyruvate produced in this Step can also Serve as a Substrate Coupled Reactions in the aldol condensation reaction to form MP. Other decar boxylase enzymes can be used; homologs are known to exist 0522. In theory, if no side reactions or degradation of in Actinobacillus actinomycetemcomitans, Aquifex aeolicus, Substrates or intermediates occurs, the maximum amount of Archaeoglobusfulgidus, Azotobacter vinelandii, Bacteroi product formed from the enzymatic reaction illustrated in desfragilis, several Bordetella species, Campylobacterie FIG. 1 is directly proportional to the equilibrium constants juni, Chlorobium tepidum, Chloroflexus aurantiacus, of each reaction and the concentrations of tryptophan and Enterococcusfaecalis, Fusobacterium nucleatum, Klebsiella pyruvate. Tryptophan is not a highly Soluble Substrate and pneumoniae, Legionella pneumophila, MagnetococcuS concentrations of pyruvate greater than 200 mM appear to MC-1, Mannheimia haemolytica, Methylobacillus flagella have a negative effect on the yield (see Example 9). tus KT, Pasteurella multocida Pm70, Petrotoga miotherma, 0523 Ideally, the concentration of monatin is maximized Porphyromonas gingivalis, Several Pseudomonas Species, with respect to Substrates in order to decrease the cost of Several PyrococcuS Species, RhodoCOccus, Several Salmo Separation. Physical Separations can be performed Such that nella Species, Several StreptococcuS Species, Thermochro the monatin is removed from the reaction mixture, prevent matium tepidum, Thermotoga maritima, Treponema palli ing the reverse reactions from occurring. The raw materials dum, and Several Vibrio Species. US 2005/0282260 A1 Dec. 22, 2005 45

0527 Tryptophan aminotransferase assays were per 0.05 mM PLP, 3 mM potassium phosphate, 3 mM MgCl, 25 formed with the HIS-tagged aspartate aminotransferase tug/mL aminotransferase, 50 ug/mL. C. teStoSteroni ProA (ASpC) from E. coli, the HISs-tagged tyrosine aminotrans aldolase, 4 Units/mL of decarboxylase, 5-200 m U/mL ferase (TyrB) from E. coli, the HIS-tagged broad substrate L-amino acid oxidase (Sigma # A-2805), 168 U/mL catalase aminotransferase (BSAT) from L. major, and the two com (Sigma # C-3515), and 0.008 mg FAD. Reactions were mercially available porcine glutamate-Oxaloacetate ami carried out for 30 minutes at 30° C. Improvement was notransferases as described in Example 1. Both oxaloacetate observed with the addition of decarboxylase. The greatest and alpha-ketoglutarate were tested as amino acceptors. The amount of monatin was produced when 50 m U/mL of ratio of activity using monatin (Example 15) versus activity oxidase was used. Improvements were similar to those using tryptophan was compared to determine which enzyme observed when indole-3-pyruvate was used as the Substrate. had the highest Specificity for the monatin aminotransferase In addition, the amount of monatin produced increased when reaction. These results indicated that the enzyme with the 1) the tryptophan level was low (i.e., tryptophan concentra highest Specificity for the monatin reaction verses the tryp tions below the K of the aminotransferase enzyme and tophan reaction is the Porcine type II-A glutamate-Oxaloac therefore less competitive with MP in the active site of the etate aminotransferase, GOAT (Sigma # G7005). This speci aminotransferase), and 2) the ratio of oxidase to aldolase and ficity was independent of which amino acceptor was aminotransferase was maintained at a level Such that indole utilized. Therefore, this enzyme was used in the coupled 3-pyruvate could not accumulate. reactions with Oxaloacetate decarboxylase. 0531. Whether starting with either indole-3-pyruvate or 0528. A typical reaction starting from indole-3-pyruvate tryptophan, the amount of monatin produced in assays with contained (final concentrations) 50 mM Tris-Cl pH 7.3, 6 incubation times of 1-2 hours increased when 2-4 times the mM indole-3-pyruvate, 6 mM sodium pyruvate, 6 mM amounts of all the enzymes were used while maintaining the aspartate, 0.05 mM PLP 3 mM potassium phosphate, 3 mM Same enzyme ratio. Using either Substrate, concentrations of MgCl2, 25 ug/mL aminotransferase, 50 lug/mL. C. test approximately 1 mg/mL of monatin were achieved. The Osteroni ProA aldolase, and 3 Units/mL of decarboxylase amount of tryptophan produced if Starting from indole (Sigma # O4878). The reaction mixtures were incubated for pyruvate was typically less than 20% of the amount of 1 hour at 26 C. In Some cases, the decarboxylase was product, showing the benefit of utilizing coupled reactions. omitted or the aspartate was Substituted with alpha-ketoglu With further optimization and control of the concentrations tarate (as negative controls). The aminotransferase enzymes of intermediates and Side reactions, the productivity and described above were also tested in place of the GOAT to yield can be improved further. confirm earlier Specificity experiments. Samples were fil tered and analyzed by LC/MS as described in Example 18. 0532. In place of Oxaloacetate, alpha-ketoglutarate can be The results demonstrate that the GOAT enzyme produced utilized as the amino acceptor, with the enzyme 2-oxoglu the highest amount of monatin per mg of protein, with the tarate decarboxylase (EC 4.1.1.71). In this case, Succinate least amount of tryptophan produced as a byproduct. In Semialdehyde and carbon dioxide are produced. This reac addition, there was a 2-3 fold increase in product formation tion Scheme is expected to also prevent the reverse reactions when the decarboxylase enzyme was added. The E. coli from occurring, but does not have the benefit of providing AspC enzyme also produced greater amounts of monatin in pyruvate as a byproduct. Numerous gene Sequences are comparison to the other aminotransferases. published that encode this enzyme. 0529) Monatin production was increased by: 1) periodi 0533 Coupled Reactions. Using Lysine Epsilon Ami cally adding 2 mM additions of indole-pyruvate, pyruvate, notransferase (EC 2.6.1.36) and aspartate (every half hour to hour), 2) performing the 0534) Lysine epsilon aminotransferase (L-Lysine 6-tran reactions in an anaerobic environment or with degassed Saminase) is found in Several organisms, including Rhodo buffers, 3) allowing the reactions to proceed for several coccus, Mycobacterium, Streptomyces, Nocardia, Flavobac hours, and 4) using freshly prepared decarboxylase that has terium, Candida utilis, and Streptomyces. It is utilized by not been freeze-thawed multiple times. The decarboxylase organisms as the first Step in the production of Some beta was inhibited by concentrations of pyruvate greater than 12 lactam antibiotics (Rius and Demain, J. Microbiol. Biotech., mM. At concentrations of indole-3-pyruvate higher than 4 7:95-100, 1997). This enzyme converts lysine to L-2-ami mM, Side reactions with indole-3-pyruvate were hastened. noadipate 6-semialdehyde (allysine), by a PLP-mediated The amount of indole-3-pyruvate used in the reaction could transamination of the C-6 of lysine when alpha-ketoglutarate be increased if the amount of aldolase was also increased. is the amino acceptor. Allysine is unstable and Spontane High levels of phosphate (50 mM) and aspartate (50 mM) ously undergoes an intramolecular dehydration to form were inhibitory to the decarboxylase enzyme reaction. The 1-piperideine 6-carboxylate, a cyclic molecule. This effec amount of decarboxylase enzyme added could be reduced to tively inhibits any reverse reaction from occurring. The 0.5 U/mL with no decrease in monatin production in a one reaction scheme is depicted in FIG. 12. Alternatively, hour reaction. The amount of monatin produced increased lysine-pyruvate 6-transaminase (EC 2.6.1.71), can also be when the temperature was increased from 26 C. to 30° C. used. and from 30° C. to 37 C. However, at 37° C. the side 0535 A typical reaction contained in 1 mL: 50 mM reactions of indole-3-pyruvate were also hastened. The Tris-HCl pH 7.3, 20 mMindole-3-pyruvate, 0.05 mM PLP, amount of monatin produced increased with increasing pH 6 mM potassium phosphate pH 8, 2-50 mM sodium pyru from 7 to 7.3 and was relatively stable from pH 7.3-8.3. vate, 1.5 mM MgCl, 50 mM lysine, 100 lug aminotrans 0530 A typical reaction starting with tryptophan ferase (lysine epsilon aminotransferase LAT-101, BioCata included (final concentrations) 50 mM Tris-Cl pH 7.3, 20 lytics Pasadena, Calif.), and 200 ug C. testosteroni ProA mM tryptophan, 6 mM aspartate, 6 mM Sodium pyruvate, aldolase. The amount of monatin produced increased with US 2005/0282260 A1 Dec. 22, 2005 46 increasing concentrations of pyruvate. The maximum pane serves as the amino acid donor in the conversion of MP amount using these reaction conditions (at 50 mM pyruvate) to monatin in a reaction catalyzed by an omega-amino acid was 10-fold less than what was observed with coupled aminotransferase (EC 2.6.1.18) such as those described by reactions with oxaloacetate decarboxylase (approximately in U.S. Pat. Nos. 5,360,724 and 5,300,437, one of the 0.1 mg/mL). resulting products would be acetone, a more volatile product 0536. In the LC/MS analysis of the reaction mixtures, a than the Substrate, aminopropane. The temperature could be peak with M+H=293 eluted at the expected time for raised periodically for Short periods to Selectively volatilize monatin and the mass spectrum contained Several of the the acetone, thereby alleviating equilibrium. Acetone has a Same fragments observed with other enzymatic processes. A boiling point of 47 C., a temperature not likely to degrade Second peak with the correct mass to charge ratio (293) the intermediates if used for short periods of time. Most eluted slightly earlier than what is typically observed for the aminotransferases that can utilize alpha-ketoglutarate as an S.S.-monatin produced in Example 9, Suggestiong the pres amino acceptor also have activity on MP. Similarly, if a ence of another isomer of monatin. Very little tryptophan glyoxylate/aromatic acid aminotransferase (EC 2.6.1.60) is was produced by this enzyme. However, this enzyme may be used with glycine as the amino donor, glyoxylate is pro able to use pyruvate as a Substrate (producing alanine as a duced. This product is unstable and has a lower boiling point byproduct). Also, the enzyme is known to be unstable. than that of glycine. Improvements can be made by performing directed evolu tion experiments to increase Stability, reduce the activity Example 22 with pyruvate, and increase the activity with MP. These reactions can also be coupled to L-amino acid oxidase/ Recombinant Expression catalase as described above. 0542. With publicly available enzyme cDNA and amino 0537. An analogous process to that shown in FIG. 12 acid Sequences, and the enzymes and Sequences disclosed utilizes ornithine 8-aminotransferase (EC 2.6.1.13) in place herein, Such as SEQ ID NOS: 11 and 12, as well as variants, of lysine epsilon aminotransferase, and ornithine Serves as polymorphisms, mutants, fragments and fusions thereof, the the amino donor in this case. The alpha-keto product, expression and purification of any protein, Such as an L-glutamic acid Semialdehyde, Spontaneously cyclizes to enzyme, by Standard laboratory techniques is enabled. One form A-pyrroline-5-carboxylate. Alternative reaction skilled in the art will understand that enzymes and fragments Schemes using these types of enzymes are described else thereof can be produced recombinantly in any cell or organ where for production of non-proteinogenic amino acid ism of interest, and purified prior to use, for example prior preparation (T. Li, A. B. Kootstra, I. G. Fotheringham, to production of SEQ ID NO: 12 and derivatives thereof. Organic Process Research & Development, 6: 533-538 0543 Methods for producing recombinant proteins are (2002)). These schemes require the addition of another well known in the art. Therefore, the scope of this disclosure aminotransferase with higher activity for the amino acid of includes recombinant expression of any protein or fragment interest (Such as HEXASpC for monatin) and glutamate. thereof, such as an enzyme. For example, see U.S. Pat. No. 0538) Other Coupled Reactions 5,342,764 to Johnson et al.; U.S. Pat. No. 5,846,819 to 0539 Another coupling reaction that can improve mona Pausch et al.; U.S. Pat. No. 5,876,969 to Fleer et al. and tin yield from tryptophan or indole-pyruvate is shown in Sambrook et al. (Molecular Cloning: A Laboratory Manual, FIG. 13. Formate dehydrogenase (EC 1.2.1.2 or 1.2.1.43) is Cold Spring Harbor, N.Y., 1989, Ch. 17). a common enzyme. Some formate dehydrogenases require 0544 Briefly, partial, full-length, or variant cDNA NADH while others can utilize NADPH. Glutamate dehy Sequences, which encode for a protein or peptide, can be drogenase was shown to catalyze the interconversion ligated into an expression vector, Such as a bacterial or between MP and monatin in previous examples, using eukaryotic expression vector. Proteins and/or peptides can ammonium based buffers. The presence of ammonium for be produced by placing a promoter upstream of the cDNA mate and formate dehydrogenase is an efficient System for Sequence. Examples of promoters include, but are not lim regeneration of cofactors, and the production of carbon ited to lac, trp, tac, trc, major operator and promoter regions dioxide is an efficient way to decrease the rate of the reverse of phage lambda, the control region of fall coat protein, the reactions (Bommarius et al., Biocatalysis 10:37, 1994 and early and late promoters of SV40, promoters derived from Galkin et al. Appl. Environ. Microbiol. 63:4651-6, 1997). In polyoma, adenovirus, retrovirus, baculovirus and Simian addition, large amounts of ammonium formate can be dis Virus, the promoter for 3-phosphoglycerate kinase, the pro solved in the reaction buffer. The yield of monatin produced moters of yeast acid phosphatase, the promoter of the yeast by glutamate dehydrogenase reactions (or similar reductive alpha-mating factors and combinations thereof. aminations) could be improved by the addition of formate dehydrogenase and ammonium formate. 0545 Vectors suitable for the production of intact native proteins include pKC30 (Shimatake and Rosenberg, 1981, 0540 Suitable enzymes to catalyze similar reductive Nature 292:128), pKK177-3 (Amann and Brosius, 1985, aminations were demonstrated in Examples 14 and 15. Gene 40:183) and pET-3 (Studier and Moffatt, 1986, J. Mol. These included broad specificity branched chain dehydro Biol. 189:113). A DNA sequence can be transferred to other genases (EC 1.4.1.9) as well as an aromatic (phenylalanine) cloning vehicles, Such as other plasmids, bacteriophages, dehydrogenase (EC 1.4.1.20). It is expected that broad cosmids, animal viruses and yeast artificial chromosomes Specificity D-amino acid dehydrogenases (1.4.99.1) can also (YACs) (Burke et al., 1987, Science 236:806-12). These be utilized to catalyze this reaction. vectors can be introduced into a variety of hosts including 0541. Other processes can be used to drive the equilib Somatic cells, and Simple or complex organisms, Such as rium toward monatin production. For instance, if aminopro bacteria, fingi (Timberlake and Marshall, 1989, Science US 2005/0282260 A1 Dec. 22, 2005 47

244:1313-7), invertebrates, plants (Gasser and Fraley, 1989, 52:466) strontium phosphate (Brash et al., 1987, Mol. Cell Science 244:1293), and mammals (Pursel et al., 1989, Sci Biol. 7:2013), electroporation (Neumann et al., 1982, EMBO ence 244:1281-8), which are rendered transgenic by the J. 1:841), lipofection (Felgner et al., 1987, Proc. Natl. Acad. introduction of the heterologous cDNA. Sci USA 84:7413), DEAE dextran (McCuthan et al., 1968, J. 0546 For expression in mammalian cells, a cDNA Natl. Cancer Inst. 41:351), microinjection (Mueller et al., Sequence can be ligated to heterologous promoters, Such as 1978, Cell 15:579), protoplast fusion (Schafner, 1980, Proc. the simian virus SV40, promoter in the pSV2 vector (Mul Natl. Acad. Sci. USA 77:2163-7), or pellet guns (Klein et al., ligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072 1987, Nature 327:70). Alternatively, the cDNA can be 6), and introduced into cells, Such as monkey COS-1 cells introduced by infection with Virus Vectors, for example (Gluzman, 1981, Cell 23:175-82), to achieve transient or long-term expression. The Stable integration of the chimeric retroviruses (Bernstein et al., 1985, Gen. Engrg. 7:235) such gene construct can be maintained in mammalian cells by as adenoviruses (Ahmad et al., 1986, J. Virol. 57:267) or biochemical Selection, Such as neomycin (Southern and Herpes (Spaete et al., 1982, Cell 30:295). Berg, 1982, J. Mol. Appl. Genet. 1:327-41) and mycophoe 0548. In view of the many possible embodiments to nolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. which the principles of our disclosure may be applied, it USA 78:2072-6). should be recognized that the illustrated embodiments are 0547 The transfer of DNA into eukaryotic, such as only particular examples of the disclosure and should not be human or other mammalian cells, is a conventional tech taken as a limitation on the Scope of the disclosure. Rather, nique. The vectors are introduced into the recipient cells as the Scope of the disclosure is in accord with the following pure DNA (transfection) by, for example, precipitation with claims. We therefore claim as our invention all that comes phosphate (Graham and vander Eb, 1973, Virology within the Scope and Spirit of these claims.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 91 <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

atgttct coa togctoccgct titcc.gaag.cg gaggittatgc ggcticaggac cqag cacggc 1080 US 2005/0282260 A1 Dec. 22, 2005 48

-continued 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 3O 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 5 O 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 Lieu Arg Lieu Ala Ala Asp Lieu. 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 2005/0282260 A1 Dec. 22, 2005 50

-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 5 O 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 Gly Ala Wal 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 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

<210 SEQ ID NO 5

US 2005/0282260 A1 Dec. 22, 2005 52

-continued

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 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

<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 2005/0282260 A1 Dec. 22, 2005 54

-continued

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 Leu 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 2005/0282260 A1 Dec. 22, 2005 55

-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 3O 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 5 O 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 Leu Glu Ala Val Thr Asn Gly Phe Asp Asp Ala Lieu. Ile Met Arg Glu US 2005/0282260 A1 Dec. 22, 2005 56

-continued

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 totact gatgt aaaagcttca 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 <213> ORGANISM: Lactobacillus amylovorus US 2005/0282260 A1 Dec. 22, 2005 57

-continued

<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 3O 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 5 O 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 Lys Val Lieu Glin Glu Asp Glu

US 2005/0282260 A1 Dec. 22, 2005 59

-continued

5 O 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 Leu Arg Ile Asp Ala Leu Glu Ala Ala Leu 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 450 455 460 US 2005/0282260 A1 Dec. 22, 2005 60

-continued

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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic US 2005/0282260 A1 Dec. 22, 2005 61

-continued primer

<400 SEQUENCE: 20 agaggagagt tagagc citta aacgc.cgttg tittatcg 37

<210> SEQ ID NO 21 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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 US 2005/0282260 A1 Dec. 22, 2005 62

-continued

&220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 26 agaggagagt tagagc citta titcgtc.citct totaaaa 37

EQ ID NO 27 ENGTH 35 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

EQUENCE: 27 gg tatt gagg gtc.gcatgcg citctacgacg gcticc 35

EQ ID NO 28 ENGTH 37 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<400 SEQUENCE: 28 agaggagagt tagagcctica gcc.gc.gcago accittgg 37

<210 SEQ ID NO 29 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 29 gg tatt gagg gtc.gcatgtt to agaacatt accgc 35

<210 SEQ ID NO 30 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 30 agaggagagt tagagc citta cago actocc acaatcg 37

<210> SEQ ID NO 31 &2 11s LENGTH 1194 &212> TYPE DNA <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 31 gtgtttcaaa aagttgacgc citacgctggc gaccc.gatto ttacgctitat ggagc gttitt 60 aaagaag acc citc.gcagoga caaagtgaat ttalagtatcg gtctgtact a caacgaagac 120 ggaattatto cacaactgca agcc.gtgg.cg gaggcggaag cqc.gc.ctgaa toc goagcct 18O catggc gott cqctittattt accgatggaa gqgctta act gctato.gc.ca toccattgcg 240

US 2005/0282260 A1 Dec. 22, 2005 64

-continued 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 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 35 US 2005/0282260 A1 Dec. 22, 2005 65

-continued 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 36 agaggagagt tagagc citta cacttggaaa acagoct 37

<210 SEQ ID NO 37 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 38 agaggagagt tagagc citta cagottagcg cct tota 37

EQ ID NO 39 ENGTH 35 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

EQUENCE: 39 gg tatt gagg gtc.gcatgcg agggg catta ttcaa 35

EQ ID NO 40 ENGTH 36 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<400 SEQUENCE: 40 agaggagagt tagagcctica gcc cittgagc gcgaag 36

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

<400 SEQUENCE: 41 atggaaaact ttaaac atct coctogalaccg titcc.gcattc gtgttattga gcc agtaaaa 60

US 2005/0282260 A1 Dec. 22, 2005 67

-continued aacgcc tact ttatcaaaga gcaa.gagcag g gotttgaga acaagag cat cqc.cgagatc 720 gtgcatgaga t gttcagota C go cqacggit totac catga gtggtaaaaa agacitgtctg 78O gtgaac atcg gcggct tcct gtgcatgaac gatgacgaaa tottctottctgccaaagag 840 ttagtcgtgg totacgaagg gatgc catct tacgg.cggcc tiggcc.ggacg tdatatggaa 9 OO gc gatggcga ttggcctg.cg tdaagccatg cagtacgaat atattgagca cog.cgtgaag 96.O caggttc.gct acctggg.cga taagctgaaa gocgctdgcg taccgattgttgaac cqgta 1020 ggcggtoacg cqg tatto ct c gatgcgc.gt cqcttctg.cg agcatctgac goaagatgag 1080 titcc.cggcac aaagtctggc tigc.ca.gcatc tatgtggaaa ccggcgtgcg cagtatggag 1140 cgcggaatta totctg.cggg cc.gtaataac gtgaccggtgaacaccacag accgaaactg 1200 gaaaccgtgc gtctgacitat tocacgtogt gtttatacct acgcacatat ggatgttgtg 1260 gctgacggta ttattaaact ttaccago ac aaagaagata titcgcgggct gaagtttatt 1320 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: Description of Artificial Sequence: Synthetic prlmer

<400 SEQUENCE: 43 gg tatt gagg gtc.gcatgga aaactittaaa catct 35

EQ ID NO 44 ENGTH 37 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

EQUENCE: 44 agaggagagt tagagc citta aacttctitta agttittg 37

EQ ID NO 45 ENGTH 35 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 46 agaggagagt tagagc citta gatgtaatca aag.cgtg 37 US 2005/0282260 A1 Dec. 22, 2005 68

-continued

<210> SEQ ID NO 47 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 47 ccaggg cacc gg.cgcagagc aaatctatat it 31

EQ ID NO 48 ENGTH 30 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<400 SEQUENCE: 48 tgcgc.cggtg CCCtggtgag toggaatggit 30

EQ ID NO 49 ENGTH 32 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 51 aggggaccgg cqcagaaaac citgttatcg 29

<210> SEQ ID NO 52 &2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 52 US 2005/0282260 A1 Dec. 22, 2005 69

-continued 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 54 gtag acgcgg actaactcitt togcagaag 29

EQ ID NO 55 ENGTH 35 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic rimer

EQUENCE: 55 gg tatt gagg gtc.gcatgta C gaactggga gttgt 35

EQ ID NO 56 ENGTH 37 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer US 2005/0282260 A1 Dec. 22, 2005 70

-continued

<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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 61 gg tatt gagg gtc.gcatgag cqtggttcac cqgaa 35

<210> SEQ ID NO 62 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 62 agaggagagt tagagcctica atcgatatat titcagtc 37

<210 SEQ ID NO 63 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 63 gg tatt gagg gtc.gcatgag cotggittaat atgaa 35

<210> SEQ ID NO 64 &2 11s LENGTH 37 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE US 2005/0282260 A1 Dec. 22, 2005 71

-continued <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 64 agaggagagt tagagc citta toactittaac gogttga 37

<210 SEQ ID NO 65 &2 11s LENGTH 684 &212> TYPE DNA <213> ORGANISM: Comamonas testosteroni

<400 SEQUENCE: 65 atgtacgaac toggagttgt citaccgcaat atccagogcg cc gaccgc.gc tigctgctgac 60 ggcctggc.cg ccctgggcto C gocaccgtg cac gaggcca toggcc.gc.gt c ggtotgctic 120 aag.ccctata toc gocccat citatgcc.ggc aag caggtot cqggcaccgc cqtcacggtg 18O citgctgcago Coggcgacaa citggatgatg catgtggctd cogag cagat totagoccggc 240 gacatcgtgg togcago.cgt caccgcagag tdcaccgacg gct actt.cgg cqatctgctg 3OO gccaccagot to caggcgcg cqgcgcacgt gcgct gatca to gatgc.cgg cqtgcgcgac 360 gtgaagacgc tigcaggagat ggactittc.cg gtctggagca aggccatcto titcca agggc 420 acgatcaagg ccaccctggg citcggtoaac atc.cc catcg totgcgc.cgg catgctggto 480 acgc.ccggtg acgtgatcgt gg.ccgacgac gacgg.cgtgg totgcgtgcc cqc.cgc.gc.gt 540 gcc.gtggaag togctggcc.gc C gCCC agaag C gtgaaagct tcgaaggcga aaag.cgc.gc.c 600 aagctggcct c gggcatcct c ggcc toggat atgtacaaga tigcgc gagcc cct ggaaaag 660 gcc.ggcct ga aatatattga citaa 684

<210 SEQ ID NO 66 &2 11s LENGTH 227 &212> TYPE PRT <213> ORGANISM: Comamonas testosteroni

<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 3O 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 5 O 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 US 2005/0282260 A1 Dec. 22, 2005 72

-continued

Thr Pro Gly Asp Val Ile Val Ala Asp Asp Asp Gly Val Val Cys Wal 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 67 actcggat.cc gaaggagata tacatatgta C galactogga ct 42

EQ ID NO 68 ENGTH 33 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic rimer

EQUENCE: 68 cggctgtcga cc.gttagtica atatatttica ggc 33

EQ ID NO 69 ENGTH: 31 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer US 2005/0282260 A1 Dec. 22, 2005 73

-continued

<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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic 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: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 74 agaggagagt tagagcc. 17

<210 SEQ ID NO 75 &2 11s LENGTH 1191. &212> TYPE DNA <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 75 atgtttgaga acattaccgc cqctoctocc gaccc.gatto tdggcctggc cqatctgttt 60 cgtgcc gatgaacgtoccgg caaaattaac citcgggattg gtc.totatta C gatgaga.cg 120 ggcaaaatcc cqgtactgac cagogtgaaa aaggctdaac agitatctgct c gaaaatgaa 18O accaccaaac tttacctogg cattgacggc atc.cctdaat ttggtogctg. cactcaggaa 240 citgctgtttg gtaaaggtag cqc cotgatc aatgacaaac gtgctcqcac ggcacagact 3OO cc.ggggggct citggcgcact acgc.gtggct gcc gattitcc togcaaaaaa taccago gtt 360 aag.cgtgttgt gggtgagcaa cccaagctgg cc galaccata agagcgt.ctt taactctgca 420 ggtotggaag titcgtgaata cqctt attat gatgcggaaa atcacactct to actitc gat 480 gcactgatta acagoctogaa toaa.gctdag gotgg.cgacg tagtgctgtt coatggctoc 540 tgccatalacc caa.ccggitat cqacccitacg citggaacaat ggcaaac act gg cacaactic 600 to cqttgaga aaggctggitt accgctgttt gactitc.gctt accagggittt toccc.gtggit 660 citggaagaag atgctgaagg actg.cgc.gct titcgcggcta to cataaaga gctgattgtt 720 US 2005/0282260 A1 Dec. 22, 2005 74

-continued gccagttcct actictaaaaa citttggcc to tacaacgagc gtgttgg.cgc titgtactctg 78O gttgctg.ccg acagtgaaac cqttgatcgc gcattcagoc aaatgaaagc gg.cgattic.gc 840 gctaac tact citt.ccccacc agcacacggc gottctgttg ttgccaccat cotgagcaac 9 OO gatgcgttac gtgcgatttg ggaacaagag citgactogata toc go cagog tattoag.cgt. 96.O atgcgtcagt tottcgtcaa tacgctgcag gaaaaaggcg caaac cqcga cittcagottt 1020 atcatcaaac agaacgg cat gttcticcittc agtgg cctoga caaaagaaca agtgctg.cgt. 1080 citgcgc gaag agtttggcgt. atatgcggitt gottctgg to go.gtaaatgt gg.ccgggatg 1140 acaccagata acatggctico gotgtgcgaa gogattgttgg cagtgct gta a 1.191

<210 SEQ ID NO 76 &2 11s LENGTH 396 &212> TYPE PRT <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 76 Met Phe Glu Asn. Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Lieu 1 5 10 15 Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Lieu Gly 2O 25 3O Ile Gly Leu Tyr Tyr Asp Glu Thr Gly Lys Ile Pro Val Leu Thir Ser 35 40 45 Wall Lys Lys Ala Glu Glin Tyr Lieu Lieu Glu Asn. Glu Thir Thr Lys Lieu 5 O 55 60 Tyr Lieu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Glin Glu 65 70 75 8O Leu Lleu Phe Gly Lys Gly Ser Ala Lieu. Ile Asn Asp Lys Arg Ala Arg 85 90 95 Thr Ala Glin Thr Pro Gly Gly Ser Gly Ala Lieu Arg Val Ala Ala Asp 100 105 110 Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro 115 120 125 Ser Trp Pro Asn His Lys Ser Val Phe Asin Ser Ala Gly Leu Glu Val 130 135 1 4 0 Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Lieu. Asp Phe Asp 145 15 O 155 160 Ala Lieu. Ile Asn. Ser Lieu. Asn. Glu Ala Glin Ala Gly Asp Val Val Lieu 1.65 170 175 Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu 18O 185 19 O Glin Trp Glin Thr Lieu Ala Glin Leu Ser Val Glu Lys Gly Trp Lieu Pro 195 200 2O5 Leu Phe Asp Phe Ala Tyr Glin Gly Phe Ala Arg Gly Lieu Glu Glu Asp 210 215 220 Ala Glu Gly Lieu Arg Ala Phe Ala Ala Met His Lys Glu Lieu. Ile Val 225 230 235 240 Ala Ser Ser Tyr Ser Lys Asn. Phe Gly Lieu. Tyr Asn. Glu Arg Val Gly 245 250 255 Ala Cys Thr Lieu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe 260 265 27 O Ser Glin Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Ser Pro Pro Ala US 2005/0282260 A1 Dec. 22, 2005 75

-continued

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

SEQ ID NO 77 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 77 gc ggaacata totttgagaa cattaccgcc 30

SEQ ID NO 78 LENGTH 32 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 78 ataa.ccggat cottacagoa citgccacaat cq 32

SEQ ID NO 79 LENGTH 32 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 79 gc gg.cgcata toggtgtttca aaaagttgac go 32

SEQ ID NO 80 LENGTH 32 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 80 ccaataggat cottacatca cog cagoaaa cq 32 US 2005/0282260 A1 Dec. 22, 2005 76

-continued <210> SEQ ID NO 81 &2 11s LENGTH 32 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 81 ggcc.gg cata togc gtgatat cq attcc.gta at 32

<210> SEQ ID NO 82 &2 11s LENGTH 30 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<400 SEQUENCE: 82 ggaattcticg agittaggcaa cagoagcgc.g 30

<210 SEQ ID NO 83 &2 11s LENGTH 627 &212> TYPE DNA <213> ORGANISM: Zymomonas mobilis <400 SEQUENCE: 83 atgcgtgata to gatticcgt aatgc gtttg gcaccggitta toccggtoct c gtcattgaa 60 gatattgctg atgcaaaacc tatcgcagaa gotttggttg citggtggtot galacgttctt 120 gaagtaacgc titcgcacccc ttgttgctott gaa.gc.catca agatcat gala agaagttcc.g 18O ggtgcc gttg ttggtgcc.gg tacggttctgaacgcaaaaa togcto gacca agctdaggaa 240 gctggttgcg aatttittcgt tagcc.cgggit citgaccgctd accitcggcaa goatgctgtt 3OO gcc.ca.gaaag cagotttgct tcc aggtgtt gctaatgctd citgatgtgat gcttggtott 360 gaccttgg to ttgatcgctt caa attctitc ccggctdaaa atato ggtgg tttacct gcc. 420 citgaagttcca togcttctgt titt cogtcag gttcgtttct gcc.cg accgg cqgitatcacc 480 cc.gacgtoag citcctaaata tottgaaaac cc.gto: cattc tittgcgt.cgg togtagctogg 540 gttgttcc.gg citggcaaacc agatgtc.gca aaaatcacgg cactc.gctaa agaagcttct 600 gctttcaa.gc gcgctgctgt toccitaa 627

<210> SEQ ID NO 84 &2 11s LENGTH 208 &212> TYPE PRT <213> ORGANISM: Zymomonas mobilis <400 SEQUENCE: 84 Met Arg Asp Ile Asp Ser Val Met Arg Leu Ala Pro Val Met Pro Val 1 5 10 15 Leu Val Ile Glu Asp Ile Ala Asp Ala Lys Pro Ile Ala Glu Ala Lieu 2O 25 3O Val Ala Gly Gly Leu Asn Val Leu Glu Val Thr Leu Arg Thr Pro Cys 35 40 45 Ala Leu Glu Ala Ile Lys Ile Met Lys Glu Val Pro Gly Ala Val Val 5 O 55 60 Gly Ala Gly Thr Val Lieu. Asn Ala Lys Met Lieu. Asp Glin Ala Glin Glu US 2005/0282260 A1 Dec. 22, 2005 77

-continued

65 70 75 8O Ala Gly Cys Glu Phe Phe Val Ser Pro Gly Leu Thr Ala Asp Leu Gly 85 90 95 Lys His Ala Val Ala Glin Lys Ala Ala Lieu Lleu Pro Gly Val Ala Asn 100 105 110 Ala Ala Asp Wal Met Lieu Gly Lieu. Asp Leu Gly Lieu. Asp Arg Phe Lys 115 120 125 Phe Phe Pro Ala Glu Asn. Ile Gly Gly Lieu Pro Ala Lieu Lys Ser Met 130 135 1 4 0 Ala Ser Val Phe Arg Glin Val Arg Phe Cys Pro Thr Gly Gly Ile Thr 145 15 O 155 160 Pro Thr Ser Ala Pro Lys Tyr Leu Glu Asn Pro Ser Ile Leu Cys Val 1.65 170 175 Gly Gly Ser Trp Val Val Pro Ala Gly Lys Pro Asp Wall Ala Lys Ile 18O 185 19 O Thr Ala Lieu Ala Lys Glu Ala Ser Ala Phe Lys Arg Ala Ala Val Ala 195 200 2O5

EQ ID NO 85 ENGTH 52 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

EQUENCE: 85 tgaccotcta gataagaagg agatatacat atggctgaca citc.gc.cctga ac 52

EQ ID NO 86 ENGTH 39 YPE DNA RGANISM: Artificial Sequence EATURE THER INFORMATION: Description of Artificial Sequence: Synthetic Lille

<400 SEQUENCE: 86 ttctdaagct tittatto.cgc gtttitcgtga atatgtttg 39

<210 SEQ ID NO 87 &2 11s LENGTH 672 &212> TYPE DNA <213> ORGANISM: Rhizobium leguminosarum <400 SEQUENCE: 87 atgggcatcg togtacagaa cataccacgg gcggaagctd atgttgatcga caggotcgc.c 60 aaatcaggcg togcgacggit coacgaagcc cagggg.cgca aaggcatcct c gccago cat 120 atgagaccaa totattoagg togc goagatc gcc.ggcticcg ccattacgat citcc.gcaccg 18O cc.cggtgata actggatgat coatgtgg.cg atcgagcaga toc aggc.cgg cqa catcctg 240 gtgcttitcgc cqacct cqcc citgttgacaac ggittattitcg gcg acct gct toccaccitcg 3OO gc gciggg.cgc gaggttgcc.g. c ggccittgtc atcgacgc.cg gtgtc.cgcga tat cagg gat 360 citgacccaga tigcagttc.cc cqtgtggtcc aaggcc.gtgt cog cqcaggg gaccgtoaag 420 gaaacgctcg gttcgg to aa cqttc.cgatc gtctg.cgctd gcgc.citt cat cqaag.ccggc 480 US 2005/0282260 A1 Dec. 22, 2005 78

-continued gaCatcatcg togc.cgacga C gacggggtg togcgtggtga agctcaacgc gg.ccgaggag 540 gttctgacitg citgcc.gagaa cc.gtgtgg.cg aac gaggagg ccaag.cggca acgc.citc.gc.c 600 gcc.ggc gaac togggctcga tat citatgac atgcggtoga agcto cqgga aaaggggctt 660 aaatatgtat ga 672

<210 SEQ ID NO 88 <211& LENGTH 224 &212> TYPE PRT <213> ORGANISM: Rhizobium leguminosarum <400 SEQUENCE: 88 Met Gly Ile Val Val Glin Asn. Ile Pro Arg Ala Glu Ala Asp Wal Ile 1 5 10 15 Asp Arg Lieu Ala Lys Ser Gly Val Ala Thr Val His Glu Ala Glin Gly 2O 25 3O Arg Lys Gly Met Leu Ala Ser His Met Arg Pro Ile Tyr Ser Gly Ala 35 40 45 Glin Ile Ala Gly Ser Ala Ile Thir Ile Ser Ala Pro Pro Gly Asp Asn 5 O 55 60 Trp Met Ile His Val Ala Ile Glu Glin Ile Glin Ala Gly Asp Ile Leu 65 70 75 8O Val Leu Ser Pro Thr Ser Pro Cys Asp Asin Gly Tyr Phe Gly Asp Leu 85 90 95 Leu Ala Thir Ser Ala Arg Ala Arg Gly Cys Arg Gly Lieu Val Ile Asp 100 105 110 Ala Gly Val Arg Asp Ile Arg Asp Lieu. Thr Glin Met Glin Phe Pro Val 115 120 125 Trp Ser Lys Ala Val Ser Ala Glin Gly Thr Val Lys Glu Thr Leu Gly 130 135 1 4 0 Ser Val Asn Val Pro Ile Val Cys Ala Gly Ala Phe Ile Glu Ala Gly 145 15 O 155 160 Asp Ile Ile Val Ala Asp Asp Asp Gly Val Cys Val Val Lys Lieu. Asn 1.65 170 175 Ala Ala Glu Glu Val Lieu. Thr Ala Ala Glu Asn Arg Val Ala Asn. Glu 18O 185 19 O Glu Ala Lys Arg Glin Arg Lieu Ala Ala Gly Glu Lieu Gly Lieu. Asp Ile 195 200 2O5 Tyr Asp Met Arg Ser Lys Lieu Arg Glu Lys Gly Lieu Lys Tyr Val Trp 210 215 220

<210 SEQ ID NO 89 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<400 SEQUENCE: 89 aggtogtgta citgtcagtica 20

<210 SEQ ID NO 90 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence US 2005/0282260 A1 Dec. 22, 2005 79

-continued

&220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<400 SEQUENCE: 90 acgtggtgaa citgc.ca.gtga 20

<210 SEQ ID NO 91 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic 6xHis tag <400 SEQUENCE: 91 His His His His His His 1 5

We claim: (c) a second polypeptide chosen from KHG aldolase (EC 1. A method for producing monatin or Salt thereof com 4.1.3.16), ProA aldolase (EC 4.1.3.17), and a combi prising using a HEXASpC aminotransferase (NCBI Acces nation thereof. sion No: 1AHF A GI: 1127190) to facilitate at least one 8. The method of claim 7, wherein the tryptophan com reaction chosen from a reaction involving tryptophan, a prises D-tryptophan. reaction involving 2-hydroxy 2-(indol-3-ylmethyl)-4-keto 9. The method of claim 7, wherein the KHG aldolase is Z. mobilis KHG aldolase (Accession No.: AAV8621.1 GI: glutaric acid, and a combination thereof. 56543467) or a polypeptide comprising an amino acid 2. A method for producing monatin or Salt thereof com sequence that is at least about 90% identical to Z. mobilis prising using a branched-chain aminotransferase (BCAT) KHG aldolase (Accession No.: AAV89621.1 GI: 56543467) (EC 2.6.1.42) or a branched-chain dehydrogenase (EC and having Z. mobilis KHG aldolase activity. 1.4.19) to facilitate a reaction of 2-hydroxy 2-(indol-3- 10. The method of claim 9, wherein the ProA aldolase is ylmethyl)-4-ketoglutaric acid. chosen from a C. testosteroni ProA aldolase, a S. meliloti 3. The method of claim 2, wherein the branched-chain ProA aldolase, and a combination thereof. aminotransferase (BCAT) (EC 2.6.1.42) is AT-102 or 11. A method for producing monatin comprising reacting AT-104. a reaction mixture, the mixture comprising: 4. The method of claim 2, wherein the branched-chain (a) 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid; dehydrogenase (EC 1.4.1.9) is AADH-110. and 5. A method for producing 2-hydroxy 2-(indol-3-ylm ethyl)-4-keto glutaric acid comprising using a Z. mobilis (b) a polypeptide chosen from YfdZ (NCBI Accession KHG aldolase to facilitate a reaction of indole-3-pyruvate, No. AAC75438.1), HEXAspC aminotransferase (NCBI Accession No: 1AHF A GI: 1127190), a wherein the Z. mobilis KHG aldolase is Z. mobilis KHG branched-chain aminotransferase (BCAT) (EC aldolase (Accession No.: AAV89621.1 GI: 56543467) 2.6.1.42), a branched-chain dehydrogenase (EC or a polypeptide comprising an amino acid Sequence 1.4.1.9), and a combination thereof. that is at least about 90% identical to Z. mobilis KHG 12. The method of claim 11, wherein the reaction mixture aldolase (Accession No.: AAV89621.1 GI: 56543467) further comprises: and has Z. mobilis KHG aldolase activity. (c) indole-3-pyruvate; and 6. A method for producing monatin or Salt thereof com prising using a KHG aldolase (EC 4.1.3.16) to facilitate a (d) a second polypeptide that is capable of converting reaction of indole-3-pyruvate, wherein more than 60% of the indole-3-pyruvate and a C3 carbon source to 2-hydroxy monatin produced in the reaction is an R,R Stereoisomer of 2-(indol-3-ylmethyl)-4-ketoglutaric acid. monatin. 13. The method of claim 11, the mixture comprising an 7. A method for producing 2-hydroxy 2-(indol-3-ylm unpurified cell extract that includes YfdZ (NCBI Accession ethyl)-4-keto glutaric acid comprising reacting a reaction No. AAC75438.1), HEXAspC aminotransferase (NCBI mixture, the mixture comprising: Accession No: 1AHF A GI: 1127190), or a combination thereof (a) tryptophan; 14. A method for producing monatin or Salt thereof comprising reacting a reaction mixture, the mixture com (b) a first polypeptide chosen from HEXAspC ami prising: notransferase (NCBI Accession No: 1AHF A GI: 1127190), YfdZ (NCBI Accession No. AAC75438.1), (a) 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid; and a combination thereof, and and US 2005/0282260 A1 Dec. 22, 2005

(b) a polypeptide chosen from AT-101, AT-102, AT-103, 30. A method for producing 2-hydroxy 2-(indol-3-ylm AT-104, AADH-102, AADH-110, AADH-112, AADH ethyl)-4-keto glutaric acid comprising reacting a reaction 113, and a combination thereof. mixture, the mixture comprising: 15. The method of claim 14, wherein the chosen polypep (a) indole-3-pyruvate; and tide is AT-103. 16. The method of claim 15, wherein more than 65% of (b) an aldolase, chosen from Bradyrhizobium japonicum the monatin produced in the method is an R,R Stereoisomer str. USDA 110 (NCBI Accession No: GI: 27378953); of monatin. Sphingomonas (Pseudomonas) paucimobilis (NCBI 17. A method for producing monatin or salt thereof from Accession No: GI: 19918963); Yersinia pestis KIM tryptophan or indole-3-pyruvate comprising enzymatically (NCBI Accession No: GI: 21956715); Ralstonia met producing a monatin composition, wherein at least about allidurans CH34 (NCBI Accession No: GI: 48767386); 80% of the monatin or salt thereof present in the monatin Yersinia pseudotuberculosis IP 32953 (NCBI Acces composition is an R,R Stereoisomer of monatin. sion No: GI: 51594436); Rhizobium leguminosarum 18. The method of claim 17, wherein the tryptophan biovar viciae rhiz23g02-plk 1009 341 (SEQID NO: comprises D-tryptophan. 88); Novosphingobium aromaticivorans DSM 12444 19. The method of claim 17, wherein at least about 84% (Sphingomonas aromaticivorans F199) (NCBI Acces of the monatin or Salt thereof present in the monatin com sion No: GI: 48849026); Pseudomonasputida KT2440 position is an R,R Stereoisomer of monatin. (NCBI Accession No: GI: 24984081); Magnetospiril 20. The method of claim 19 wherein the method further lum magnetotacticum MS-1 (NCBI Accession No: GI: comprises providing a ProA aldolase (EC 4.1.3.17) to facili 46200890); Rhodopseudomonas palustris CGAO09 tate the conversion of indole-3-pyruvate to 2-hydroxy 2-(in (NCBI Accession No: GI: 39937756); Xanthomonas dol-3ylmethyl)-4-ketoglutaric acid. campestris ATCC-33913 (NCBI Accession No: GI: 21. The method of claim 19 wherein the method further 21115297); Xanthomonas axonopodis citri 306 (NCBI comprises providing AT-103 (D-transaminase) to facilitate Accession No: GI: 21110581); Streptomyces avermiti the conversion of 2-hydroxy 2-(indol-3ylmethyl)-4-keto lis MA-4680 (NCBI Accession No: GI: 298.28159); and glutaric acid to monatin. a combination thereof. 22. A method for producing monatin or Salt thereof 31. A method for producing 2-hydroxy 2-(indol-3-ylm comprising reacting a reaction mixture, the mixture com ethyl)-4-keto glutaric acid comprising reacting a reaction prising: mixture that includes: (a) tryptophan, 2-hydroxy 2-(indol-3-ylmethyl)-4-keto (a) indole-3-pyruvate; and glutaric acid, or a combination thereof, and (b) an aldolase comprising an amino acid Sequence that is at least about 49% identical to C. testosteroni ProA (b) an E. coli AspC polypeptide (NCBI Accession No. (SEQ ID NO: 66) or an amino acid sequence that is at AAC74014.1) having at least one substitution at an least about 56% identical to Sinorhizobium meliloti amino acid position chosen from positions 39, 41, 47, ProA (NCBI Accession No.: CAC46344), wherein the 69, 109, 297, and a combination thereof, wherein aldolase has 4-hydroxy-4-methyl-2-oxoglutarate lyase numbering of the amino acid positions is based on a pig activity. cytosolic aspartate aminotransferase numbering Sys 32. The method of claim 31, wherein the amino acid tem. sequence is at least about 60% identical to C. testosteroni 23. The method of claim 22, wherein the E. coli AspC ProA (SEQ ID NO: 66). polypeptide has aspartate aminotransferase activity. 33. The method of claim 31, wherein the amino acid 24. The method according to claim 22, wherein the AspC sequence is at least about 70% identical to C. testosteroni polypeptide comprises a Val 39 to Leu substitution. ProA (SEQ ID NO: 66). 25. The method according to claim 22, wherein the AspC 34. A method of producing monatin or Salt thereof com polypeptide comprises a LyS 41 to Tyr Substitution. prising reacting a reaction mixture that includes tryptophan 26. The method according to claim 22, wherein the AspC and a deaminase (EC 3.5.1.-) that is derived from a micro polypeptide comprises a Thr 47 to Ile Substitution. organism chosen from: Proteus spp., Providencia spp., Mor 27. The method according to claim 22, wherein the AspC ganella spp., or combinations thereof. polypeptide comprises an ASn 69 to Leu Substitution. 35. The method of claim 34, wherein the Proteus spp. 28. The method according to claim 22, wherein the AspC includes Proteus myxofaciens, Proteus mirabilis, Proteus polypeptide comprises a Thr 109 to Ser Substitution. vulgaris, and Proteus morganii. 29. The method according to claim 22, wherein the AspC polypeptide comprises an ASn 297 to Ser Substitution. k k k k k