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(12) United States Patent (10) Patent No.: US 9,296,993 B2 Chen Et Al

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(12) United States Patent (10) Patent No.: US 9,296,993 B2 Chen Et Al US009296993B2 (12) United States Patent (10) Patent No.: US 9,296,993 B2 Chen et al. (45) Date of Patent: Mar. 29, 2016 (54) ENGINEERED MINE REDUCTASES AND (56) References Cited METHODS FOR THE REDUCTIVE AMINATION OF KETONE AND AMINE U.S. PATENT DOCUMENTS COMPOUNDS 7,202,070 B2 4/2007 Rozzell, Jr. 7,423, 195 B2 9, 2008 Sticklen et al. (71) Applicant: Codexis, Inc., Redwood City, CA (US) 7,452,704 B2 11/2008 Esaki et al. 2005, 0124040 A1 6/2005 Esaki et al. (72) Inventors: Haibin Chen, Beijing (CN); Steven J. 2006/0205045 A1 9, 2006 Esaki et al. Collier, Concord, MA (US); Jovana 2007/OOO9995 A1 1/2007 Bogosian et al. Nazor, Santa Clara, CA (US); Joly Sukumaran, Singapore (SG); Derek OTHER PUBLICATIONS Smith, Singapore (SG); Jeffrey C. Moore, Westfield, NJ (US); Gregory Abrahamson, M.J., et al., “Development of an Amine Dehydrogenase Hughes, Scotch Plains, NJ (US); Jacob for Synthesis of Chiral Amines.” Angew. Chem. Intl. Ed., 51:3969 Janey, New York, NY (US); Gjalt W. 3972 2012. Huisman, Redwood City, CA (US); Asano, Y, et al., “A New NAD+-Dependent Opine Dehydrogenase Scott J. Novick, Palo Alto, CA (US); from Arthrobacter sp. Strain IC. J. Bacterol. 171 (8):4466-4471 Nicholas J. Agard, San Francisco, CA 1989. Baker, P.J., et al., “A role for quaternary structure in the substrate (US); Oscar Alvizo, Fremont, CA (US); specificity of leucine dehydrogenase.” Structure, 3(7):693-705 Gregory A. Cope, Menlo Park, CA 1995. (US); Wan Lin Yeo, Singapore (SG); Bevan, M., et al., “Structure and transcription of the nopaline Stefanie Ng Minor, Redwood City, CA synthase gene region of T-DNA. Nucleic Acids Research, (US) 11(2):369-385 1983. Britton, K.L., et al., “Crystallization of Arthrobacter sp. strain 1C (73) Assignee: Codexis, Inc., Redwood City, CA (US) N-(1-D-carboxyethyl)-L- norvaline dehydrogenase and its complex with NAD+.” Acta Cryst., D54:124-126 1998). (*) Notice: Subject to any disclaimer, the term of this Britton, K.L., et al., “Crystal structure and active site location of patent is extended or adjusted under 35 N-(1-D-carboxylethyl)-L-norvaline dehydrogenase.” Nature Struc U.S.C. 154(b) by 0 days. ture Biology, 5(7):593-601 1998). Brunhuber, N.M.W., et al., “Rhodococcus L-Phenylalanine (21) Appl. No.: 14/887,943 Dehydrogenase: Kinetics, Mechanism, and Structural Basis for Cata lytic Specifity,” Biochemistry, 39.9174-9187 (2000). (22) Filed: Oct. 20, 2015 Dairi, T., et al., "Cloning. Nucleotide Sequencing, and Expression of an Opine Dehydrogenase Gene from Arthrobacter sp. Strain 1C..” (65) Prior Publication Data Applied and Environmental Microbiology, 61 (8):3169-3171 (1995). Donkersloot, J.A., et al., "Cloning. Expression, Sequence Analysis, US 2016/004O140 A1 Feb. 11, 2016 and Site-directed Mutagenesis of the Tn5306-encoded Related U.S. Application Data N-(Carboxyethyl)ornithine Synthase from Lactococcus lactis K1.” J. Bio. Chem., 270(20): 12226-12234 (1995). (62) Division of application No. 13/890,944, filed on May Endo, N., et al., “Purification, characterization, and cDNA cloning of 9, 2013, now Pat. No. 9,193,957. opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.” Comparative Biochemistry and Physiology, Part B, (60) Provisional application No. 61/646,100, filed on May 147:293-307 2007. 11, 2012. Endo, N., et al., “cDNA cloning and primary structure comparison of tauropine dehydrogenase and beta-alanopine dehydrogenase from (51) Int. C. the limpet Cellanagrata.” Fish Sci., 75:1471-1479 2009. C7H 2L/00 (2006.01) CI2N 15/52 (2006.01) (Continued) CI2N 9/06 (2006.01) CI2PI3/00 (2006.01) CI2P 13/06 (2006.01) Primary Examiner — Delia Ramirez CI2P 1 7/10 (2006.01) (74) Attorney, Agent, or Firm — Codexis, Inc. CI2P 1 7/12 (2006.01) CI2P 1 7/16 (2006.01) CI2P 17/18 (2006.01) (57) ABSTRACT (52) U.S. C. The present disclosure provides engineered polypeptides CPC ............. CI2N 9/0028 (2013.01); CI2P 13/001 having imine reductase activity, polynucleotides encoding (2013.01); C12Y 105/01 (2013.01) the engineered imine reductases, host cells capable of (58) Field of Classification Search expressing the engineered imine reductases, and methods of CPC ..... C12N 9/0028; C12P 13/001; C12P 13/02: using these engineered polypeptides with a range of ketone C12P 13/04; C12P 13/06; C12P 17/10: and amine Substrate compounds to prepare secondary and C12P 17/12: C12P 17/165; C12P 17/185; tertiary amine product compounds. C12P 17/188; C12Y 105/01023; C12Y 105/01024; C12Y 105/01028 See application file for complete search history. 6 Claims, No Drawings US 9,296,993 B2 Page 2 (56) References Cited Plese, B., et al., "Cloning and expression of a tauropine dehydrogenase from the marine sponge Suberites domuncula.” Mar OTHER PUBLICATIONS Biol. 153:1219-1232 (2008). Plese, B., et al., "Strombine dehydrogenase in the demosponge Goto. M., et al., "Crystal Structures of Delta1-Piperideine-2- Suberites domuncula: Characterization and kinetic properties of the carboxylate/Delta1-Pyrroline-2-carboxylate Reductase Belonging to enzyme crucial for anaerobic metabolism.” Comparative Biochem a New Family of NAD(P)H-dependent Oxidoreductases,” J. Biol. istry and Physiology, Part B, 154: 102-107 (2009). Chem., 280(49):40875-4.0884 2005). Smits, S.H.J., et al., “A Structural Basis for Substrate Selectivity and Kan-No, N., et al., "The amino acid sequence of tauropine Stereoselectivity in Octopine Dehydrogenase from Pecten dehydrogenase from the polychaete Arabella iricolor.” Comparative maximus,” J. Mol. Biol. 381:200-211 2008. Biochemistry and Physiology, Part B, 140:475-485 (2005). Smits, S.H.J., et al., “Insights into the Mechanism of Ligand Binding Kan-No, N., et al., "Tauropine dehydrogenase from the marine to Octopine Dehydrogenase from Pecten maximus by NMR and Sponge Halichondria japonica is a homolog of ornithine Crystallography.” PLoS One, 5(8): 1-10 |2010). cyclodeaminase/mu-crystallin.” Comparative Biochemistry and UniProt F4A2G3 dated Jun. 28, 2011. Physiology, Part B, 141:331-339 (2005). UniProt Q44297 dated Nov. 1, 1996. Kato, Y, et al., "Stereoselective synthesis of opine-type secondary Xuan, J.-W., et al., "Overlapping Reading Frames at the LYS5 Locus amine carboxylic acids by a new enzyme opine dehydrogenase Use in the Yeast Yarrowia lipolytica.” Molecular and Cellular Biology, of recombinant enzymes.” J. Mol. Catalysis B: Enzymatic, 1:151-160 10(9):4795-4806 1990). 1996). Yip, K.S.P. et al., “The structure of Pyrococcus furiosus glutamate Kimura, T., et al., "Complementary DNA Cloning and Molecular dehydrogenase reveals a key role for ion-pair networks in maintain Evolution of Opine Dehydrogenases in Some Marine Invertebrates.” ing enzyme stability at extreme temperatures.” Structure, Mar. Biotechnol. 6:493-502 (2005). 3(11): 1147-1158 (1995). Mihara, H., et al., "N-Methyl-L-amino acid dehydrogenase from International Search Report for International Application No. PCT/ Pseudomonasputida: A novel member of an unusual NAD(P)-depen US2013/040377 dated Jul. 18, 2013. dent oxidoreductase superfamily.” FEBS Journal, 272:1117-1123 Branden, C., et al., “Introduction to Protein Structure”. Published by 2005). Garland Publishing, Inc., New York, New York, p. 247 (1991). Muller, A., et al., “Putative reaction mechanism of heterologously Seffernick, J.L., et al., “Melamine Deaminase and Atrazine expressed octopine dehydrogenase from the great scallop, Pecten Chlorohydrolase: 98 Percent Identical but Functionally Different.” J. maximus (L).” FEBS Journal, 274:6329-6339 2007). Bacteria, 183:2405-2410 |2001). Peterson, P.E., et al., “The structure of bovine glutamate Witkowski, A., et al., "Conversion of a beta-ketoacyl synthase to a dehydrogenase provides insights into the mechanism of allostery.” malonyl decarboxylase by replacement of the active-site cysteine Structure, 7:769-782 1999. with glutamine.” Biochemistry, 38(36): 11643-50 1999). US 9,296,993 B2 1. 2 ENGINEERED IMINE REDUCTASES AND from Cellana grata (BADH); and tauropine dehydrogenase METHODS FOR THE REDUCTIVE from Suberites domuncula (Taul DH). The crystal structure of AMINATION OF KETONE AND AMINE the opine dehydrogenase CENDH has been determined (see COMPOUNDS Britton et al., “Crystal structure and active site location of N-(1-D-carboxyethyl)-L-norvaline dehydrogenase.” Nat. 1. CROSS REFERENCE TO RELATED Struct. Biol. 5(7): 593-601 (1998)). Another enzyme, N-me APPLICATIONS thyl L-amino acid dehydrogenase from Pseudomonas putida (NMDH) is known to have activity similar to opine dehydro The present application is a Divisional of U.S. patent appli genases, reacting with O-keto acids and alkyl amines, but cation Ser. No. 13/890,944, filed May 9, 2013, now U.S. Pat. 10 appears to have little or no sequence homology to opine No. 9,193.957, which claims benefit under 35 U.S.C. S 119(e) dehydrogenases and amino acid dehydrogenases. NMDH has of US Pat. Applin. Ser. No. 61/646,100, filed May 11, 2012, been characterized as belonging to a new Superfamily of the contents of both of which are incorporated herein by NAD(P) dependent oxidoreductase (see e.g., U.S. Pat. No. reference. 7,452,704 B2: Esaki et al., FEBS Journal 2005, 272, 1117 15 1123). 2. TECHNICAL FIELD There is a need in the art for biocatalysts and processes for using them, under industrially applicable conditions, for the The disclosure relates to engineered polypeptides having synthesis of chiral secondary and tertiary amines. imine reductase activity useful for the conversion of various ketone and amine Substrates to secondary and tertiary amine 5. SUMMARY products. The present disclosure provides novel biocatalysts and 3. REFERENCE TO SEQUENCE LISTING, associated methods to use them for the synthesis of chiral TABLE OR COMPUTER PROGRAM secondary and tertiary amines by direct reductive amination 25 using an unactivated ketone and an unactivated amine as The official copy of the Sequence Listing is submitted substrates.
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