IP Library › Granted Patent US 12,344,874
Granted Patent B2
US 12,344,874 · App. 17/361,832 · Granted Jul 1, 2025

Transaminase biocatalysts

Inventors: Christopher K. Savile (Santa Clara, CA); Emily Mundorff (Garden City, NY); Jeffrey C. Moore (Westfield, NJ); Paul N. Devine (Tinton Falls, NJ); Jacob M. Janey (New York, NY)
Assignee: Codexis, Inc.
C12P17/182C12N9/1096C12N15/52C12P13/001C12Y206/01018C12N15/62Y02P20/52
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Quick Facts
Patent No.
US 12,344,874
App. No.
17/361,832
Granted
Jul 1, 2025
Kind
B2
Abstract

The present disclosure relates to polypeptides having transaminase activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides.

Claims (9)

1. A recombinant transaminase polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the amino acid sequence of the recombinant transaminase polypeptide further comprises a residue difference as compared to SEQ ID NO: 2 at residue position X65 and at residue position X81.

2. The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7 (8H)-yl]-1-(2,4,5-trifluorophenyl) butan-2-one (“ketoamide substrate”) to (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7 (8H)-yl]-1-(2,4,5-trifluorophenyl) butan-2-amine (“product”) under a defined reaction condition in the presence of an amino group donor isopropylamine to levels of the product detectable by HPLC-UV at 210 nm, where the reaction condition comprises about 2 g/L ketoamide substrate, about 0.5 M isopropylamine, about 22° C., about pH 7.5, about 5% DMSO, about 100 μM pyridoxal phosphate, and about 20 mg/mL of transaminase polypeptide.

3. The recombinant transaminase polypeptide of claim 2 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to the product with an activity that is equal to or greater than the activity of the polypeptide of SEQ ID NO: 4 under the defined reaction condition.

4. The recombinant transaminase polypeptide of claim 2 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 90% enantiomeric excess.

5. The recombinant transaminase polypeptide of claim 2 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 99% enantiomeric excess.

6. The transaminase polypeptide of claim 1 , wherein the amino acid sequence corresponds to the sequence of SEQ ID NO: 68, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

7. The transaminase polypeptide of claim 1 , wherein the amino acid sequence includes the following features: X69 is C or T; X122 is M or I; X223 is P; and X284 is G.

8. The transaminase polypeptide of claim 1 , wherein the amino acid sequence includes the following features: X69 is C or T; X122 is M or I; X223 is P; and X284 is G.

9. The transaminase polypeptide of claim 1 , wherein the amino acid sequence additionally comprises a residue difference as compared to SEQ ID NO:2 at one or more of the following residue positions: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X94; X96; X102; X117; X120; X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211; X215; X217; X225; X230; X252; X269; X273; X282, X292; X297; X302; X306; X321; and X329.

Assignments (1)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
Continuity (11)
Continuation 16807977 · Mar 3, 2020
Continuation 16195227 · Nov 19, 2018
Continuation 15915437 · Mar 8, 2018
Division 15376837 · Dec 13, 2016
Continuation 15140809 · Apr 28, 2016
Division 14818885 · Aug 5, 2015
Division 14518143 · Oct 20, 2014
Division 13604323 · Sep 5, 2012
Continuation 12714397 · Feb 26, 2010
Provisional Application 61155902 · Feb 26, 2009
Related Publication 20210332399A1 · Oct 28, 2021
References Cited (115)
US 4950606A · Stirling et al. · 1990 [cited by applicant]
US 5169780A · Stirling et al. · 1992 [cited by applicant]
US 5300437A · Stirling et al. · 1994 [cited by applicant]
US 5346828A · Stirling et al. · 1994 [cited by applicant]
US 5360724A · Matcham et al. · 1994 [cited by applicant]
US 5866512A · Matcham et al. · 1999 [cited by applicant]
US 5965432A · Kobayashi et al. · 1999 [cited by applicant]
US 6107521A · Lin et al. · 2000 [cited by applicant]
US 6117679A · Stemmer · 2000 [cited by applicant]
US 6133018A · Wu et al. · 2000 [cited by applicant]
US 6221638B1 · Yamada et al. · 2001 [cited by applicant]
US 6344351B2 · Yamada et al. · 2002 [cited by applicant]
US 6346402B1 · Iwasaki et al. · 2002 [cited by applicant]
US 6376246B1 · Crameri et al. · 2002 [cited by applicant]
US 6586182B1 · Patten et al. · 2003 [cited by applicant]
US 7169952B2 · Yamada et al. · 2007 [cited by applicant]
US 7172885B2 · Pannuri et al. · 2007 [cited by applicant]
US 7326708B2 · Cypes et al. · 2008 [cited by applicant]
US 7468459B2 · Xio et al. · 2008 [cited by applicant]
US 7588923B2 · Fotheringham et al. · 2009 [cited by applicant]
US 8293507B2 · Savile et al. · 2012 [cited by applicant]
US 8889380B2 · Savile et al. · 2014 [cited by applicant]
US 9133445B2 · Savile et al. · 2015 [cited by applicant]
US 9353355B2 · Savile et al. · 2016 [cited by applicant]
US 9550982B2 · Savile et al. · 2017 [cited by applicant]
US 9944963B2 · Savile et al. · 2018 [cited by applicant]
US 10160985B2 · Savile et al. · 2018 [cited by applicant]
US 10619176B2 · Savile et al. · 2020 [cited by applicant]
US 20050153417A1 · Davis et al. · 2005 [cited by applicant]
US 20060195947A1 · Davis et al. · 2006 [cited by applicant]
US 20080220990A1 · Fox · 2008 [cited by applicant]
US 20080248539A1 · Giver et al. · 2008 [cited by applicant]
US 20090123983A1 · Niddam-Hildesheim · 2009 [cited by applicant]
US 20090192326A1 · Perlman et al. · 2009 [cited by applicant]
US 20090221595A1 · Perlman et al. · 2009 [cited by applicant]
US 20090246837A1 · Robins et al. · 2009 [cited by applicant]
US 20090247532A1 · Huang et al. · 2009 [cited by applicant]
US 20090312196A1 · Colbeck et al. · 2009 [cited by applicant]
EP 1075534B1 · 2005 [cited by applicant]
EP 0857790B1 · 2005 [cited by applicant]
EP 404146B1 · 2006 [cited by applicant]
EP 1818411A1 · 2007 [cited by applicant]
EP 1045025B1 · 2008 [cited by applicant]
EP 0987332B1 · 2009 [cited by applicant]
EP 1819825B1 · 2010 [cited by applicant]
JP 63273486A · 1988 [cited by applicant]
KR 2006007124A · 2006 [cited by applicant]
WO 9946398A1 · 1999 [cited by applicant]
WO 200066760A1 · 2000 [cited by applicant]
WO 2006126498A1 · 2006 [cited by applicant]
WO 2007093372A1 · 2007 [cited by applicant]
WO 2008127646A2 · 2008 [cited by applicant]
WO 2009045507A2 · 2009 [cited by applicant]
WO 2009084024A2 · 2009 [cited by applicant]
WO 2010081053A2 · 2010 [cited by applicant]
Cameron, M., et al., “The highly stereospecific enzyme catalysed transamination of 4-fluorophenylglyoxylic acid to 4-(S)-fluorephenylglycine”, Journal of Molecular Catalysis, 14: 1-5, 2001. [cited by applicant]
Cho, B.-K., et al., “Asymmetric Synthesis of L-Homophenylalanine by Equilibrium-Shift Using Recombinant Aromatic L-Amino Acid Transaminase”, Biotechnology and Bioengineering, 83(2):226-234, 2003. [cited by applicant]
Cho, B.-K., et al., “Engineering Aromatic L-Amino Acid Transaminase for the Asymmetric Synthesis of Constrained Analogs of L-Phenylalanine”, Biotechnology and Bioengineering, 94(5):842-850, 2006. [cited by applicant]
Cho, B.-K., et al., “Enzymatic Resolution for the Preparation of Enantiomerically Enriched D-β-Heterocyclic Alanine Derivatives Using [cited by applicant]
Cho, B.-K., et al., “Redesigning the Substrate Specificity of ω-Aminotransferase for the Kinetic Resolution of Aliphatic Chiral Amines”, Biotechnology and Bioengineering, 99(2):275-284, 2008. [cited by applicant]
Cho, B.-K., et al., “Simultaneous Synthesis of Enantiomerically Pure (S)-Amino Acids and (R)-Amines Using Coupled Transaminase Reactions”, Biotechnology and Bioengineering, 81(7):783-789, 2003. [cited by applicant]
Christen, P., et al., “From Cofactor to Enzymes. The Molecular Evolution of Pyridoxal-5′-Phosphate-Dependent Enzymes”, The Chemical Record, 1:436-447, 2001. [cited by applicant]
Crump, S.P et al., “Biocatalytic Production of Amino Acids by Transamination”, In Biocatalytic Production of Amino Acids and Derivatives; Rozzell, J. D., Wagner, F., Eds.; Wiley: New York, 1992; pp. 43-58. [cited by applicant]
Eliot, A.C., et al., “The Dual-Specific Active Site of 7,8-Diaminopelargonic Acid Synthase and the Effect of the R391A Mutation”, Biochemistry, 41:12582-12589, 2002. [cited by applicant]
Fadnavis, N.W., et al., “Asymmetric synthesis of nonproteinogenic amino acids with L-amino acid transaminase: synthesis of (2S)-2-amino-4-oxo-4-phenylbutyric and (3E,2S)-2-amino-4-phenylbutenoic acids”, Tetrahedron: Asy… [cited by applicant]
Fernandez, F.J., et al., “Structural Studies of the Catalytic Reaction Pathway of a Hyperthermophilic Histidinol-phosphate Aminotransferase”, The Journal of Biological Chemistry, 279(20):21478-21488, 2004. [cited by applicant]
Goto, M., et al., “Crystal Structures of Branched-Chain Amino Acid Aminotransferase Complexed with Glutamate and Glutarate: True Reaction Intermediate and Double Substrate Recognition of the Enzyme”, Biochemistry, 42:37… [cited by applicant]
Hansen, K.B., et al., “First generation process for the preparation of the DPP-IV inhibitor Sitagliptin”, Organic Process Research & Development, 9:634-639, 2005. [cited by applicant]
Maruyama, K., et al., “Structures of [cited by applicant]
Hirotsu K., et al., “Dual Substrate Recognition of Aminotransferases”, The Chemical Record, 5:160-172, 2005. [cited by applicant]
Höhne, M., et al., “Efficient Asymmetric Synthesis of Chiral Amines by Combining Transaminase and Pyruvate Decarboxylase”, ChemBioChem, 9:363-365, 2008. [cited by applicant]
Hwang, B.-Y., et al., “High-throughput screening method for the identification of active and enantioselective ω- transaminases”, Enzyme and Microbial Technology, 34:429-436, 2004. [cited by applicant]
Hwang, B.-Y., et al., “Revisit of aminotransferase in the genomic era and its application to biocatalysis”, Journal of Molecular Catalysis B: Enzymatic, 37:47-55, 2005. [cited by applicant]
Hwang, J.-Y., et al., “Simultaneous Synthesis of 2-Phenylethanol and L-Homophenylalanine Using Aromatic Transaminase With Yeast Ehrlich Pathway”, Biotechnology and Bioengineering, 102(5):1323-1329, 2009. [cited by applicant]
Iwasaki, A., et al., “Microbial synthesis of (R)- and (S)-3,4-dimethoxyamphetamines through stereoselective transamination”, Biotechnology Letters, 24: 1845-1846, 2003. [cited by applicant]
Iwasaki, A., et al., “Microbial synthesis of chiral amines by (R)-specific transamination with [cited by applicant]
Käck, H., et al., “Crystal Structure of Diaminopelargonic Acid Synthase: Evolutionary Relationships between Pyridoxal-5′-phosphate-dependent Enzymes”, J. Mol. Biol., 291:857-876, 1999. [cited by applicant]
Koszelewski, D., et al., “Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ω-Transaminases”, Adv. Synth. Catal., 350:2761-2766, 2008. [cited by applicant]
Koszelewski, D., et al., “Deracemisation of α-Chiral Primary Amines by a One-Pot, Two-Step Cascade Reaction Catalysed by ω-Transaminases”, Eur. J. Org. Chem., pp. 2289-2292, 2009. [cited by applicant]
Koszelewski, D., et al., “Formal Asymmetric Biocatalytic Reductive Amination”, Agnew. Chem. Int. Ed., 47:9337-9340, 2008. [cited by applicant]
Koszelewski, D., et al., “Synthesis of Optically Active Amines Employing Recombinant ω-Transaminases in [cited by applicant]
Liu, W., et al., “Crystal Structures of Unbound and Aminooxyacetate-Bound [cited by applicant]
Liu, W., et al., “Kinetic and Crystallographic Analysis of Active Site Mutants of [cited by applicant]
Matsui, I., et al., “The Molecular Structure of Hyperthermostable Aromatic Aminotransferase with Novel Substrate Specificity from Pyrococcus horikoshii”, The Journal of Biological Chemistry, 275(7): 4871-4879, 2000. [cited by applicant]
Mihara, H., et al., “N-Methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily”, FEBS Journal, 272:1117-1123, 2005. [cited by applicant]
Nobe, Y., et al., “The Novel Substrate Recognition Mechanism Utilized by Aspartate Aminotransferase of the Extreme Thermophile Thermus thermophilus HB8”, The Journal of Biological Chemistry, 273(45):29554-29564, 1998. [cited by applicant]
Noland, B.W., et al., “Structural Studies of [cited by applicant]
Okada, K., et al., “Structures of [cited by applicant]
Okada, K., et al., “Three-Dimensional Structure of [cited by applicant]
Okamoto, A., et al., “Crystal Structures of Paracoccus denitrificans Aromatic Amino Acid Aminotransferase: A Substrate Recognition Site Constructed by Rearrangement of Hydrogen Bond Network”, J. Mol. Biol., 280:443-461,… [cited by applicant]
Okamoto, A., et al., “The Active Site of Paracoccus denitrificans Aromatic Amino Acid Aminotransferase Has Contrary Properties: Flexibility and Rigidity”, Biochemistry, 38:1176-1184, 1999. [cited by applicant]
Oue, S., et al., “Paracoccus denitrificans Aromatic Amino Acid Aminotransferase: A Model Enzyme for the Study of Dual Substrate Recognition Mechanism”, J. Biochem, 121:161-171, 1997. [cited by applicant]
Peisach, D., et al., “Crystallographic Study of Steps along the Reaction Pathway of D-Amino Acid Aminotransferase”, Biochemistry, 37:4958-4967, 1998. [cited by applicant]
Sandmark, J., et al., “Conserved and Nonconserved Residues in the Substrate Binding Site of 7,8-Diaminopelargonic Acid Synthase from [cited by applicant]
Sandmark, J., et al., “Structural Basis for the Inhibition of the Biosynthesis of Biotin by the Antibiotic Amiclenomycin”, The Journal of Biological Chemistry, 277(45):43352-43358, 2002. [cited by applicant]
Savile, C.K., et al., “Biocatalytic asymmetric synthesis of chiral amines from ketones applied to Sitagliptin manufacture”, Science, 329: 205-309, including supporting materials, 2010. [cited by applicant]
Shin J.-S., et al., “Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17”, Appl. Microbiol. Biotechnol., 61:463-471, 2003. [cited by applicant]
Shin, J.-S., et al., “Asymmetric Synthesis of Chiral Amines With ω-Transaminase”, Biotechnology and Bioengineering, 65(2):206-211, 1999. [cited by applicant]
Shin, J.-S., et al., “Exploring the Active Site of Amine: Pyruvate Aminotransferase on the Basis of the Substrate Structure-Reactivity Relationship: How the Enzyme Controls Substrate Specificity and Stereoselectivity”, … [cited by applicant]
Sivaraman, J., et al., “Crystal Structure of Histidinol Phosphate Aminotransferase (HisC) from [cited by applicant]
Sugio, S., et al., “Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 Å resolution: implication of the structural role of Leu201 in transamination”, Protein Engineering, 11(8):613-619, 1998. [cited by applicant]
Taylor, P.P., et al., “Novel biosynthetic approaches to the production of unnatural amino acids using transaminases”, Tibtech, 16:412-418, 1998. [cited by applicant]
Truppo, M.D., et al., “Efficient kinetic resolution of racemic amines using a transaminase in combination with an amino acid oxidase”, Chemical Communications, pp. 2127-2129, 2009. [cited by applicant]
Truppo, M.D., et al., “Efficient production of chiral amines at concentrations of 50g/L using transaminases”, Adv. Synth, Catal., 351:1-5, 2009. [cited by applicant]
Truppo, M.D., et al., “Rapid determination of both the activity and enantioselectivity of ketoreductases”, Angew. Chem. Int. Ed. Engl., 47(14):2639-41, 2008. [cited by applicant]
Truppo, M.D., et al., “Rapid screening and scale-up of transaminase catalysed reactions,” Organic & Biomolecular Chemistry, 7:395-398, 2009. [cited by applicant]
Truppo, M.D., “Rapid Screening and Process Development of Biocatalytic Reactions”, Thesis submitted Univ. of Manchester, Faculty of Engineering and Physical Sciences, 1-296, 2009. [cited by applicant]
Ura, H., et al., “Substrate Recognition Mechanism of Thermophilic Dual-Substrate Enzyme”, J. Biochem., 130:89-98, 2001. [cited by applicant]
Ura, H., et al., “Temperature Dependence of the Enzyme-Substrate Recognition Mechanism”, J. Biochem., 129:173-178, 2001. [cited by applicant]
Van Ophem, P.W., et al., “Effects of the E177K Mutation in D-Amino Acid Transaminase. Studies on an Essential Coenzyme Anchoring Group That Contributes to Stereochemical Fidelity”, Biochemistry, 38:1323-1331, 1999. [cited by applicant]
Yonaha, K., et al., “Properties of the Bound Coenzyme and Subunit Structure of ω-Amino Acid:Pyruvate Aminotransferase”, The Journal of Biological Chemistry, 258(4):2660-2665, 1983. [cited by applicant]
Yun, H., et al., “Asymmetric synthesis of (S)-α-methylbenzylamine by recombinant [cited by applicant]
Yun, H., et al., “Kinetic Resolution of (R,S)-sec-Butylamine Using Omega-Transaminase From Vibrio fluvialis JS17 Under Reduced Pressure”, Biotechnology and Bioengineering, 87(6):772-778, 2004. [cited by applicant]
Yun, H., et al., “Synthesis of Enantiomerically Pure trans-(1R,2R)- and cis-(1S,2R)-1-Amino-2-Indanol by Lipase and ω-Transaminase”, Biotechnology and Bioengineering, 93(2):391-395, 2006. [cited by applicant]
Yun, H., et al., “Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which is Resistant to Product Inhibition by Aliphatic Ketones”, Applied and Environmental Microbiology, 71(… [cited by applicant]