P450-BM3 variants with improved activity

Patent metadata

Field	Value
Publication number	US-10450550-B2
Application number	US-201816152709-A
Country	US
Kind code	B2
Filing date	Oct 5, 2018
Priority date	Jul 7, 2015
Publication date	Oct 22, 2019
Grant date	Oct 22, 2019

Abstract

The present invention provides improved P450-BM3 variants with improved activity. In some embodiments, the P450-BM3 variants exhibit improved activity over a wide range of substrates.

First claim

What is claimed is:

1. A recombinant cytochrome P450-BM3 variant having at least 90% sequence identity to a polypeptide sequence comprising the sequence set forth in SEQ ID NO:2.

2. The recombinant cytochrome P450-BM3 variant of claim 1 , wherein said variant oxidizes at least three organic substrates.

3. The recombinant cytochrome P450-BM3 variant of claim 2 , wherein said organic substrate is selected from loratadine, imatinib, geftinib, and diclofenac.

4. A recombinant polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant having at least 90% sequence identity to a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 1.

5. A recombinant polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant of claim 1 .

6. The recombinant polynucleotide sequence of claim 4 , wherein said sequence comprises SEQ ID NO: 1.

7. An expression vector comprising the polynucleotide sequence of claim 4 .

8. The vector of claim 7 , wherein said polynucleotide sequence is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.

9. The vector of claim 8 , wherein said host cell is a prokaryotic or eukaryotic cell.

10. The vector of claim 9 , wherein said host cell is a prokaryotic cell.

11. The vector of claim 10 , wherein said host cell is E. coli.

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

13. A method for producing at least one recombinant cytochrome P450-BM3 variant comprising culturing a host cell under conditions such that at least one recombinant cytochrome P450-BM3 variant of claim 1 is produced by said host cell.

14. The method of claim 13 , further comprising the step of recovering said at least one recombinant cytochrome P450-BM3 variant.

Assignees

• Codexis Inc

Inventors

• Osborne Robert
• Mitchell Vesna
• Htwe Khin Yu Naing
• Zhang Xiyun
• Milczek Erika M
• Moore Jeffrey C

Classifications

• C12Y114/14001

  Unspecific monooxygenase (1.14.14.1) · CPC title

• C12Y106/02004

  NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/0071 Primary

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