US 2005O221455A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0221455A1 McFarlan et al. (43) Pub. Date: Oct. 6, 2005 (54) PRODUCTION OF MONATIN AND Related U.S. Application Data MONATIN PRECURSORS (60) Provisional application No. 60/513,406, filed on Oct. (75) Inventors: Sara C. McFarlan, St. Paul, MN (US); 21, 2003. Paula M. Hicks, Eden Prairie, MN (US); Mary Jo Zidwick, Wayzata, MN Publication Classification (US); Fernando A. Sanchez-Riera, Eden Prairie, MN (US); Douglas C. 51)1) Int. Cl.Cl." .............................. C12P 17/107/10; C12N 1/21 Cameron, Plymouth, MN (US); Mervyn L. deSouza, Plymouth, MN (52) U.S. Cl. .................. 435/121; 435/252.3; 435/252.33 (US); John Rosazza, Iowa City, IA (US); Steven J. Gort, Brooklyn Center, MN (US); Timothy W. Abraham, Minnetonka, MN (US) (57) ABSTRACT Correspondence Address: CARGILL, INCORPORATED Methods and compositions that can be used to make monatin LAW/24 or Salt thereof from glucose, tryptophan, indole-3-lactic 15407 MCGINTY ROAD WEST acid, indole-3-pyruvate, and 2-hydroxy 2-(indol-3-ylm ethyl)-4-ketoglutaric acid, are provided. Methods are also WAYZATA, MN 55391 (US) disclosed for producing the indole-3-pyruvate and 2-hy (73) Assignee: CARGILL, INC. droxy 2-(indol-3-ylmethyl)-4-keto glutaric acid intermedi ates. Compositions provided include nucleic acid molecules, (21) Appl. No.: 10/969,245 polypeptides, chemical Structures, and cells. Methods include in vitro and in Vivo processes, and the in Vitro (22) Filed: Oct. 21, 2004 methods include chemical 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 Oct. 6, 2005 Sheet 1 of 13 US 2005/0221455A1 -- - 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 Oct. 6, 2005 Sheet 2 of 13 US 2005/0221455A1 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) Monatin Patent Application Publication Oct. 6, 2005 Sheet 3 of 13 US 2005/0221455A1 FIG 3 Indole-3-lactic acid EC 1.1.1.110 indolelactate dehydrogenase EC 1.1.1.222 R-4-hydroxyphenylactate dehydrogenase EC 1.1.1.2373-(4)-hydroxyphenylpyruvate reductase EC 1.1.1.27, 1.1.1.28, 1.1.2.3 lactate dehydrogenase EC 1.1.1.111 (3-imidazol-5-yl) lactate dehydrogenase EC 1.1.3.- lactate oxidase Chemical oxidation Indole-3-pyruvate FIG. 4 COOR 2 O COOR O -'s s -COOR; COOR N -a-a-a- \ OH O N N Enolate chemistry R R R=Boc, Cbz, etc. R2 and R3 = Alkyl, Aryl, etc. Patent Application Publication Oct. 6, 2005 Sheet 4 of 13 US 2005/0221455A1 FIG. 5A gigo? O 4. 8 12 6 2O 24 Time (min) F.G. 6 OO 293 5 O l5O 2OO 25O 3OO 350 4OO n/z Patent Application Publication Oct. 6, 2005 Sheet 5 of 13 US 2005/0221455A1 FIG 7A 08-Apr-2002 l OO 15:11:01 5 O O 4. 8 12 6 2O 24 Time (min) 9 OO 2 B 68 te O O. 58 9 50 13O g 74 229 r 2ll 22 \so 257 e. O-H-III. Til. II . -. E - OO . 50 2OO 250 3OO nz FG. 8 +TOF MS: 124 MCA scans from Sample 7 (293jd with Tryptophan) of carO709a.wif... Max. 9,3e4 counts, a=3569708085.37750280e-004, to= -1.246268795.8835 1580e--002 R; 2931144 90e 4 8.0e 4 We 4 Cargill Sample Sample 293 with Tryptophan as Internal Std S.Oe 4 She 4 4,0e 4 3.Oe4 2.Oe4 2.94121) 1.0e4 0. 234 28s 288 29 2S2 294 29s 298 miz, an Patent Application Publication Oct. 6, 2005 Sheet 6 of 13 US 2005/0221455A1 WB3A2 10-Jul-2002 13:11:33 22a 1. Scal is O FG. 9A Y 1228 oria, : Scan ES 1. FG. 9B 124e25 ori O23a 1: ScarES O of FG. 9C O Time 2OO 4D 6. BOO 1.O 12. 40 1. B.OO 2O, 22. 24 FIG 10 1.4OE-05 1.2OE-05 1.OOE-05 8.OOE-04 6.OOE-04 4.OOE+04 2.OOE-04 O.OOE+OO 4hr 24hr 24hr 48hr 48hr 70hr 70hr -2.OOE-04 (-) (+) () (+) () (+) -4.OOE-04 Time Patent Application Publication Oct. 6, 2005 Sheet 7 of 13 US 2005/0221455A1 FIG 11 tryptophan tryptophan oxidase Or (, O s catalase amino acid Oxidase HO indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) aspartate 2 aspartate aminOtranSferaSefe Ny Oxaloacetate monatin v Oxaloacetate \ decarboxylase 3. pyruvate + CO Patent Application Publication Oct. 6, 2005 Sheet 8 of 13 US 2005/0221455A1 F.G. 12 tryptophan tryptophan oxidase or O catalase amino acid oxidase I HO indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) lysine lysine &-aminotransferaseAf 7 Ny allysineh monat1n \spontaneous, - H20 1 -Piperideine 6-carboxylate Patent Application Publication Oct. 6, 2005 Sheet 9 of 13 US 2005/0221455A1 F.G. 13 tryptophan O tryptophan oxidase catalase or amino acid oxidase H.O. indole-3-pyruvate pyruvate ProA aldolase monatin precursor (MP) CO 2 dehydrogenase a NADH (reductive amination) > NAD (9/ monat1n / Formate dehydrogenase ammonium formate Patent Application Publication Oct. 6, 2005 Sheet 10 of 13 US 2005/0221455 A1 FIG. 14 Pyruvate production by E. coli strains 2YT media + lipoate 1 0.6 0.5 S bySY S.w bySY N. b wa in SYY SC bySY wa GS9) & &SS. Qss 2NN SS. QSS- A& $SS. M s SS2 M s sa 24 hr growth 48 hr growth Patent Application Publication Oct. 6, 2005 Sheet 11 of 13 US 2005/0221455A1 FIG. 15 g/L pyruvate divided by OD600 C d So So So So do O - a N C N CU O N OO 7692 7692 (DE3) O Va. 7692 lipA C 7692 lipA (DE3) D BW251 13 O in C BW25113 lipA- 8 C. S. BW25113 lipA (DE3) to 7692 e MC a 7692 (DE3) (D 7692 lipA- o sm 7692 lipA (DE3) 3. 8 BW25113 ass 2. 9 BW25113 lipA O s BW25113 lipA (DE3) S S O 7692 C O 7692 (DE3) () 7692 lipA d 7692 lipA (DE3) BW 25113 BW251 13 lipA BW251 13 lipA- (DE3) Patent Application Publication Oct. 6, 2005 Sheet 12 of 13 US 2005/0221455A1 FIG. 16 ATGTACGAACTGGGAGTTGTCTACCGCAATATCCAGCGCGCCGACCGCGC TGCTGCTGACGGCCTGGCCGCCCTGGGCTCCGCCACCGTGCACGAGGCCA TGGGCCGCGTCGGTCTGCTCAAGCCCTATATGCGCCCCATCTATGCCGGCA AGCAGGTCTCGGGCACCGCCGTCACGGTGCTGCTGCAGCCCGGCGACAAC TGGATGATGCATGTGGCTGCCGAGCAGATTCAGCCCGGCGACATCGTGGT CGCAGCCGTCACCGCAGAGTGCACCGACGGCTACTTCGGCGATCTGCTGG CCACCAGCTTCCAGGCGCGCGGCGCACGTGCGCTGATCATCGATGCCGGC GTGCGCGACGTGAAGACGCTGCAGGAGATGGACTTTCCGGTCTGGAGCAA GGCCATCTCTTCCAAGGGCACGATCAAGGCCACCCTGGGCTCGGTCA ACA TCCCCATCGTCTGCGCCGGCATGCTGGTCACGCCCGGTGACGTGATCGTGG CCGACGACGACGGCGTGGTCTGCGTGCCCGCCGCGCGTGCCGTGGAAGTG CTGGCCGCCGCCCAGAAGCGTGAAAGCTTCGAAGGCGAAAAGCGCGCCA AGCTGGCCTCGGGCATCCTCGGCCTGGATATGTACAAGATGCGCGAGCCC CTGGAAAAGGCCGGCCTGAAATATATTGACTAA Patent Application Publication Oct. 6, 2005 Sheet 13 of 13 US 2005/0221455 A1 - FIG. 17 MYELGVVYRNIQRADRAAADGLAALGSATVHEAMGRVGLLKPYMRPIYAG KQVSGTAVTVLLQPGDNWMMHVAAEQIQPGDIVVAAVTAECTDGYFGDLL ATSFQARGARALIIDAGVRDVKTLOEMDFPVWSKAISSKGTIKATLGSVNIPIV CAGMLVTPGDVIVADDDGVVCVPAARAVEVLAAAQKRESFEGEKRAKLASG ILGLDMYKMREPLEKAGLKYD US 2005/0221455 A1 Oct. 6, 2005 PRODUCTION OF MONATIN AND MONATIN pentanedioic acid, or alternatively, based on an alternate PRECURSORS numbering System, 4-hydroxy-4-(3-indolylmethyl)glutamic 0001. This application claims priority of U.S. provisional acid), a compound having the formula: patent application No. 60/513,406, filed Oct. 21, 2003, which is incorporated herein by reference for all purposes. TECHNICAL FIELD 0002 This disclosure provides methods and materials that are useful in the production of indole-3-pyruvate, 2-hy droxy 2-(indol-3ylmethyl)-4-ketoglutaric acid and/or mona tin. BACKGROUND 0.003 Indole-3-pyruvate is a strong antioxidant that is believed to counter act oxidative StreSS in tissues with high 0007 Monatin also has the following chemical names: oxygen concentrations (Politi et al. “Recent advances in 3-(1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl)-indole; Tryptophan Research', edited by G. A. Filippini et al. 4-(indole-3-ylmethyl)-4-hydroxy-glutamic acid; and 3-(1- Plenum Press, New York, 1996, pp. 291-8). Indole pyruvate amino-1,3-dicarboxy-3-hydroxybutane-4-yl)-indole. also is an intermediate in a pathway to produce indole-acetic acid (IAA), the primary plant growth hormone auxin (dif 0008 Monatin is a naturally-occurring, high intensity fusible growth promoting factor).