Engineered transaminase polypeptides for industrial biocatalysis

Patent metadata

Field	Value
Publication number	US-12371678-B2
Application number	US-202117379563-A
Country	US
Kind code	B2
Filing date	Jul 19, 2021
Priority date	Feb 28, 2013
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

Abstract

The present disclosure provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The disclosure also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.

First claim

What is claimed is:

1. An engineered polypeptide having transaminase activity comprising an amino acid sequence having at least 80% sequence identity to reference sequence of SEQ ID NO: 2 and a combination of amino acid residue differences X42A/G, X54P, and X152S/T as compared to SEQ ID NO:2 and further comprises one or more residue differences as compared to SEQ ID NO:2 selected from: X33L, X36C, X41C/R, X44Q, X51K, X76S, X122Q, X148Q, X150A, X155K, X156R, X160P, X215G, X218M, and X328I.

2. The engineered polypeptide of claim 1 , in which the amino acid sequence further comprises one or more residue differences as compared to SEQ ID NO:2, selected from: X5K, X41F/M/N, X48D/E/G/K/T, X49T, X55L, X108V, X117G, X122F, X126A, X150F, X155A/I/L/T/V, X156Q, X164P, X165N, X182T, X215H/L, X241R, X267V, X270T, X273H, and X325M.

3. The engineered polypeptide of claim 1 , further comprising
(a) an amino acid residue difference as compared to SEQ ID NO:2 selected from G36C, I41C, I41F, I41K, I41M, I41N, I41R, P48D, P48E, P48G, P48K, P48T, A51K, M122F, M122Q, Y148Q, Q155A, Q155I, Q155K, Q155T, Q155V, C215H, C215L, Y273H, L325M, and A241R; or
(b) a combination of residue differences selected from: A5K, S49T, Q155T, and W156Q; P33L, I41C, S150F, Q155K, F160P, and C215G; P33L, I41K, S150F, Q155I, F160P, and C215L; P33L, P48G, S150F, Q155T, and C215H; P33L, A109S, S150F, Q155K, W156Q, and C215H; P33L, E117G, S150F, Q155I, W156Q, and C215G; P33L, S150F, Q155I, W156Q, and C215G; P33L, S150F, Q155K, W156Q, and C215H; P33L, S150F, Q155L, W156Q, and C215H; P33L, S150F, Q155L, W156Q, C215H, and A241R; G36C, P48G, S150F, Q155I, and C215H; G36C, P48K, S150F, Q155T, and C215H; G36C, S150F, Q155I, C215H, and A241R; G36C, S150F, Q155K, C215H, and A241R; G36C, S150F, Q155T, and A241R; G36C, S150F, Q155V, and C215H; I41C, S49T, S150F, Q155I, F160P, C215G, and I267V; I41C, S49T, S150F, Q155K, W156Q, C215G and I267V; I41C, 1108V, S150F, and Q155K; I41C, I108V, S150F, Q155K, W156Q, C215G, and I267V; I41C, I108V, S150F, Q155T, W156Q, and C215G; I41C, E117G, S150F, Q155K, and F160P; 141C, E117G, S150F, Q155K, and C215L; I41C, E117G, S150F, Q155L, and C215L; I41C, S150F, Q155I, and C215G; I41C, S150F, Q155I, and C215L; I41C, S150F, Q155K, W156Q, C215G, and I267V; I41C, S150F, Q155K, and C215L; I41C, S150F, Q155K, and C215G; I41C, S150F, Q155L, F160P, C215G, and I267V; I41C, S150F, Q155T, W156Q, F160P, and C215L; I41C, S150F, Q155T, W156Q, and C215L; I41F, M122Q, S150F, Q155V, W156Q, and C215G; I41F, S150F, Q155L, W156Q, V171I, and C215G; I41F, S150F, Q155L, W156Q, V171I, C215G, and A241R; I41F, S150F, Q155I, W156Q, and C215G; I41K, P48E, S150F, Q155K, and W156Q; I41K, P48E, S150F, Q155L, and C215L; I41K, I108V, E117G, S150F, Q155K, and C215L; I41K, I108V, S150F, Q155T, and C215G; 141K, E117G, S150F, Q155L, and C215G; 141K, E117G, S150F, Q155K, C215L, and I267V; I41K, S150F, Q155K, W156Q, and C215G; I41K, S150F, Q155K, F160P, C215G, and I267V; I41K, S150F, Q155K, and C215L; I41K, S150F, and Q155T; I41K, S150F, Q155T, and F160P; I41K, S150F, Q155T, and C215G; I41K, S150F, Q155T, C215G, and I267V; I41K, S150F, Q155K, W156Q, and C215G; 141N, S150F, Q155I, and F160P; I41N, E117G, S150F, Q155T; and W156Q; I41N, S49T, S150F, Q155L, F160P, D165N, and C215L; A44Q, I108V, S150F, Q155T, and I267V; A44Q, I108V, S150F, and Q155T; A44Q, I108V, S150F, Q155T, and I267V; A44Q, S150A, and Q155T; A44Q, S150F, and Q155T; P48G, S150F, Q155L, W156Q, and C215H; P48G, S150F, and Q155T; S49T, I108V, E117G, S150F, Q155L, F160P, and C215L; S49T, 1108V, E117G, S150F, Q155K, W156Q, and C215G; S49T, I108V, E117G, S150F, Q155T, W156Q, C215G, and I267V; S49T, Q155T, and W156Q; I55L, T126A, Q155T, L218M, and A270T; F60V, S150F, Q155L, W156Q, and C215G; T68A, S150F, Q155L, W156Q, and C215G; T69S, S150F, Q155L, W156Q, and C215G; N76S, T126A, Q155T, S182T, L218M, A270T, and V328I; I108V, S150F, Q155K, and C215H; I108V, S150F, and Q155T; I108V, S150F, Q155T, and I267V; I108V, S150F, Q155V, W156Q, and F160P; E117G, and Q155T; E117G, Q155T, and W156Q; M122Q, S150F, Q155I, W156Q, C215G, and A241R; M122Q, S150F, Q155L,W156Q, V171I, C215G, and A241R; M122Q, S150F, Q155V, W156Q, V171I, C215G, and A241R; T126M, S150F, Q155L, W156Q, and C215G; P135I, F136Y, S150F, Q155L, W156Q, W192F, and C215G; F136I, S150F, Q155L, W156Q, and C215G; F136I, S150F, Q155L, W156Q, C215G, and G224I; F136I, S150F, Q155L, W156Y, C215G, S282V, and G284I; F136I, S150F, Q155L, W156Y, C215G, and G284P; F136Y, S150F, Q155L, W156Q, C215G, S282V, and G284P; S150A, Q155T, and I267V; S150F, Q155I, W156Q, F160P, C215L, and I267V; S150F, Q155I, W156Q, V171I, C215G, and A241R; S150F, Q155I, W156Q, and C215L; S150F, Q155I, F160P, and C215G; S150F, Q155I, and C215H; S150F, Q155K, and W156Q; S150F, Q155K, W156Q, and 1267V; S150F, Q155L, W156Q, G193M, and C215G; S150F, Q155L, W156Q, and C215G; S150F, Q155L, W156Q, C215G, S282V, and G284I; S150F, Q155L, W156Q, C215G, and T283S; S150F, Q155L, W156Q, C215G, and G284I; S150F, Q155L, W156Y, and C215G; S150F, Q155L, and C215H; S150F, and Q155T; S150F, Q155T, C215G, and I267V; S150F, Q155T, and I267V; S150F, Q155T, W156Q, F160P, C215L, and I267V; S150F, Q155T, W156Q, C215G, and I267V; S150F, Q155T, and W156R; S150F, Q155T, F160P, and C215G; S150F, Q155T, F160P, and C215L; S150F, Q155T, C215G, and I267V; S150F, Q155T, and I267V; Q155I, and W156S; Q155K, and W156S; Q155L, and W156S; Q155T; Q155T, and F160P; Q155T, and R164P; W156Q; Q155T, and W156S; Q155T, and R164P; Q155T, S182T, L218M, and A270T; Q155T, and C215G; Q155T, and C215L; and Q155V, and W156S.

4. The engineered polypeptide of claim 1 , wherein said engineered polypeptide is capable of converting a substrate compound (2)

to a product compound (1)

under suitable reaction conditions.

5. The engineered polypeptide of claim 4 , which is capable of converting compound (2) to compound (1) with at least 1.2 fold the activity of SEQ ID NO:2 under suitable reaction conditions.

6. The engineered polypeptide of claim 5 , in which the suitable reaction conditions comprise compound (2) loading of at least 50 g/L, 1 mM PLP, 50% DMSO (v/v), 1.5 M isopropylamine, pH 11, and 55° C.

7. The engineered polypeptide of claim 1 , in which the amino acid sequence comprises SEQ ID NO: 130.

8. The engineered polypeptide of claim 1 , in which the polypeptide is immobilized on a solid support.

9. The engineered polypeptide of claim 8 , in which the solid support is a bead or resin comprising polymethacrylate with epoxide functional groups, polymethacrylate with amino epoxide functional groups, styrene/DVB copolymer or polymethacrylate with octadecyl functional groups.

10. A process for preparing a compound of structural Formula (I):

having the indicated stereochemical configuration at the stereogenic center marked with an *; in an enantiomeric excess of at least 70% over the opposite enantiomer,
wherein
Z is OR 2 or NR 2 R 3 ;
R1 is C1-8 alkyl, aryl, heteroaryl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, or a 5- to 6-membered heterocyclic ring system optionally containing an additional heteroatom selected from O, S, and N, the heterocyclic ring being unsubstituted or substituted with one to three substituents independently selected from oxo, hydroxy, halogen, C1-4 alkoxy, and C1-4 alkyl, wherein alkyl and alkoxy are unsubstituted or substituted with one to five fluorines;
R2 and R3 are each independently hydrogen, C1-8 alkyl, aryl, or aryl-C1-2 alky

Assignees

• Codexis Inc

Inventors

• Quintanar-Audelo Martina
• Eberhard Ellen
• Nazor Jovana
• Smith Derek
• Wang Cuixia

Classifications

• C07D487/04

  Ortho-condensed systems · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12P17/182

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title