Transaminase biocatalysts

Patent metadata

Field	Value
Publication number	US-2019071702-A1
Application number	US-201816195227-A
Country	US
Kind code	A1
Filing date	Nov 19, 2018
Priority date	Feb 26, 2009
Publication date	Mar 7, 2019
Grant date	—

Abstract

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

First claim

What is claimed is:

1 . A recombinant transaminase polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2.

2 . The recombinant transaminase polypeptide of claim 1 , wherein the amino acid sequence of the recombinant transaminase polypeptide comprises a residue difference as compared to SEQ ID NO: 2 at one or more residue positions selected from: X8, X94, X136, and X209.

3 . The recombinant transaminase polypeptide of claim 2 , wherein the amino acid residue differences of the recombinant transaminase polypeptide are selected from the following: X8 is proline; X94 is leucine, isoleucine, or valine; X136 is leucine; and X209 is leucine.

4 . 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.

5 . The recombinant transaminase polypeptide of claim 1 , 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.

6 . The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 90% enantiomeric excess.

7 . The recombinant transaminase polypeptide of claim 1 , wherein said recombinant transaminase polypeptide is capable of converting the ketoamide substrate to product in at least 99% enantiomeric excess.

8 . A recombinant polynucleotide encoding the recombinant transaminase polypeptide of claim 1 .

9 . An expression vector comprising the recombinant polynucleotide of claim 8 .

10 . The expression vector of claim 9 , further comprising at least one control sequence.

11 . The expression vector of claim 10 , wherein said control sequence comprises a promoter.

12 . The expression vector of claim 10 , wherein said control sequence comprises a secretion signal.

13 . A host cell comprising the expression vector of claim 9 .

14 . The host cell of claim 13 , wherein said host cell is a bacterial cell.

15 . The host cell of claim 14 , wherein said bacterial cell is E.coli .

Assignees

• Codexis Inc

Inventors

• Savile Christopher K
• Mundorff Emily
• Moore Jeffrey C
• Devine Paul N
• Janey Jacob M

Classifications

• C12P13/001

  Amines; Imines · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P17/182 Primary

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

• C12Y206/01018

  Beta-alanine-pyruvate transaminase (2.6.1.18) · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title