Biocatalysts and methods for the synthesis of substituted lactams

Patent metadata

Field	Value
Publication number	US-9624478-B2
Application number	US-201514799180-A
Country	US
Kind code	B2
Filing date	Jul 14, 2015
Priority date	Sep 8, 2011
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

The present disclosure relates to transaminase polypeptides capable of aminating a dicarbonyl substrate, and polynucleotides, vectors, host cells, and methods of making and using the transaminase polypeptides.

First claim

What is claimed is:

1. An engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:4 and a substitution at positon X192 selected from X192A, X192G, X192H, X192K, X192N, X192Q, X192R, and X192S.

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:4 selected from: X42G; X54P; X54R; X69A; X69G; X69T; X122M; X124F; X124L; X124N; X124R; X126A; X126T; X136Y, X136F; X150S; X152C; X152G; X152I; X152L; X152S; X156A; X156S; X156T; X157L; and X165N.

3. The engineered polypeptide of claim 1 , in which the amino acid sequence further comprises a combination of residue differences selected from:
(a) X124W and X327L;
(b) X209M and X300G;
(c) X122F, X223V and X284A;
(d) XI92A, X215H and X311T;
(e) X62N, X124F, X126A and X136L;
(f) X124W, X126A, X136L, X192A and X284A; and
(g) X124W, X126A, X136L, X152R/X152L/X152I, and X192A.

4. The engineered polypeptide of claim 1 , in which the amino acid sequence further comprises at least one or more residue differences as compared to SEQ ID NO:4 selected from: X54A; X54L; X56D; X56E; X61G; X61W; X62A; X62L; X62N; X62S; X64C; X68I; X122F, X122W; X122Y; X124K; X124M; X124S; X124W; X126C; X126I; X136K; X136L; X136M; X139E; X140K; X143V; X150L; X155I; X155L; X159G; X160L; X176C; X199L; X199V; X209F; X209M; X209Q; X209V; X223S; X223T; X223V; X227I; X282L, X282V; X284A; X284P; X284S; X284T; and X284V.

5. An engineered polynucleotide encoding the polypeptide of claim 1 .

6. A vector comprising the polynucleotide of Claim 5 .

7. A host cell comprising the vector of claim 6 .

8. A method of converting compound 2 to compound 1,

comprising contacting compound 2 with the engineered polypeptide of claim 1 under suitable reaction conditions to produce compound 1.

9. A method of converting compound 2 to compound 1d and compound 1a

comprising contacting compound 2 with the engineered polypeptide of claim 1 under suitable reaction conditions to produce compound 1d and 1a, under suitable reaction conditions wherein said engineered polypeptide comprises at least 1.5 fold the activity of SEQ ID NO:4.

Assignees

• Codexis Inc

Inventors

• Cabirol Fabien Louis
• Chen Haibin
• Gohel Anupam
• Salim Paulina Elena
• Smith Derek J
• Janey Jacob
• Kosjek Birgit
• Tang Weng Lin
• Hsieh Helen
• Pham Son Q

Classifications

• C12P13/001

  Amines; Imines · CPC title

• C12N9/1096 Primary

  transferring nitrogenous groups (2.6) · CPC title

• C12P13/005

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12P17/12

  containing a six-membered hetero ring · CPC title