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, tryptophan, 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 glutamate dehydrogenase EC 1.4.1.20 phenylalanine dehydrogenase EC 2.6.1.28 tryptophan-phenylpyruvate transaminase - EC 2.6.1-multiple substrate aminotransferase EC 2.6.1.1 aspartate aminotransferase EC 1.4.3.2 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.-lyase, 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 oxygen 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.