Engineered proline hydroxylase polypeptides and methods

Patent metadata

Field	Value
Publication number	US-10995349-B2
Application number	US-202016913761-A
Country	US
Kind code	B2
Filing date	Jun 26, 2020
Priority date	May 8, 2012
Publication date	May 4, 2021
Grant date	May 4, 2021

Abstract

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

First claim

What is claimed is:

1. An engineered polypeptide having proline hydroxylase activity, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 and, wherein the engineered polypeptide has a residue difference at position X3 of SEQ ID NO:2.

2. The engineered polypeptide of claim 1 , wherein the residue difference at position X3 is X3S.

3. The engineered polypeptide of claim 1 , wherein the amino acid sequence comprises one or more combination of features selected from the group consisting of:
(a) X103L and X166Q;
(b) X52P and X255Y;
(c) X4E/L/S and X115A;
(d) X25R and X58A;
(e) X29A and X166T/Q/L;
(f) X115H/D/G and X121F;
(h) X103L, X131Y/F, and X166T/Q/L;
(i) X26T, X103L and X166T/Q/L;
(j) X25R, X66Q, X92V and X115E; and
(k) X25R, X66Q, X92V, X103L, X115E, and X166Q.

4. The engineered polypeptide of claim 1 , further comprising one or more residue differences as compared to the sequence of SEQ ID NO: 2 at residue positions selected from: X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205.

5. The engineered polypeptide of claim 4 , wherein the residue differences at residue positions X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205 are selected from X17V, X24R, X24S, X26R, X26W, X62Q, X88R, X98F, X98T, X114N, X140L, X151A, X151H, X186G, X188G, and X205V.

6. The engineered polypeptide of claim 1 , wherein the polypeptide converts substrate compound (2), (2S)-piperidine-2-carboxylic acid,

to product compound (1), (2S,5S)-5-hydroxypiperidine-2-carboxylic acid,

under suitable reaction conditions.

7. The engineered polypeptide of claim 6 , the polypeptide converts substrate compound (2) to product compound (1) with at least 2 fold the activity of SEQ ID NO:2, and wherein the amino acid sequence comprises one or more residue differences selected from the group consisting of: X4Q; X4L; X5I; X5L; X24S; X25R; X30P; X66Q; X86S; X92V; X103L; X103Q; X113E; X115E; X150S; X166Q; X151S; X225L; and X270E.

8. The engineered polypeptide of claim 1 , wherein the polypeptide converts substrate compound (2)

to product compound (1)

in excess of product compound (1a), (2S,3R)-3-hydroxypiperidine-2-carboxylic acid,

wherein the amino acid sequence comprises one or more residue differences selected from the group consisting of: X103L; X115E; X131Y and X166Q.

9. The engineered polypeptide of claim 1 , wherein the polypeptide forms product compound (1),

in diastereomeric excess of product compound (1R), (2S,5R)-5-hydroxypiperidine-2-carboxylic acid,

Assignees

• Codexis Inc

Inventors

• Chen Haibin
• Bong Yong Koy
• Cabirol Fabien L
• Gohel Prafulchandra Anupam
• Li Tao
• Moore Jeffrey C
• Quintanar-Audelo Martina
• Hong Yang
• Collier Steven J
• Smith Derek

Classifications

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• Y02P20/52

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

• C12Y114/11002

  Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase · CPC title