Polypeptides having peroxygenase activity

Patent metadata

Field	Value
Publication number	US-9611459-B2
Application number	US-201314431925-A
Country	US
Kind code	B2
Filing date	Oct 8, 2013
Priority date	Oct 12, 2012
Publication date	Apr 4, 2017
Grant date	Apr 4, 2017

Abstract

The present invention relates to isolated polypeptides having peroxygenase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. A polynucleotide encoding a peroxygenase was isolated from Myceliophthora fergusii.

First claim

The invention claimed is:

1. A method of producing a polypeptide having peroxygenase activity, comprising:
(a) cultivating a microbial recombinant host cell under conditions conducive for production of the polypeptide, wherein
the microbial recombinant host cell comprises a nucleic acid construct or expression vector comprising a polynucleotide encoding the polypeptide,
the amino acid sequence of the polypeptide has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2, and
the polynucleotide is operably linked to one or more heterologous control sequences that direct the expression of the polypeptide in the recombinant host cell; and

(b) recovering the polypeptide.

2. The method of claim 1 , wherein the amino acid sequence of the polypeptide has at least 96% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

3. The method of claim 1 , wherein the amino acid sequence of the polypeptide has at least 97% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

4. The method of claim 1 , wherein the amino acid sequence of the polypeptide has at least 98% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

5. The method of claim 1 , wherein the amino acid sequence of the polypeptide has at least 99% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

6. The method of claim 1 , wherein the polypeptide comprises the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

7. The method of claim 1 , wherein the polypeptide is encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of (i) the sequence of nucleotides 52-1107 of SEQ ID NO: 1, wherein the high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C., and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

8. The method of claim 1 , wherein the polypeptide is a variant of the sequence of amino acids 1 to 244 of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions, and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

9. The method of claim 1 , wherein the polypeptide is a fragment of the sequence of amino acids 1 to 244 of SEQ ID NO: 2, and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2.

10. The method of claim 1 , wherein the amino acid sequence of the polypeptide includes the motif:

(SEQ ID NO: 7)

E-H-D-[G,A]-S-[L,I]-S-R,

or

R-[G,A]-P-C-P-X-[M,L]-N-[S,T]-L,

wherein X is any amino acid (SEQ ID NO: 16).

11. The method of claim 1 , wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

12. The method of claim 2 , wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

13. The method of claim 3 , wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

14. The method of claim 4 , wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

15. The method of claim 5 , wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , and Trichoderma.

16. The method of claim 11 , wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.

17. The method of claim 12 , wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.

18. The method of claim 13 , wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.

19. The method of claim 14 , wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.

20. The method of claim 15 , wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.

Assignees

• Novozymes As

Inventors

• Landvik Sara
• Oestergaard Lars Henrik
• Kalum Lisbeth

Classifications

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12P13/008

  containing a N-O bond, e.g. nitro (-NO2), nitroso (-NO) · CPC title

• C12Y111/02004

  Fatty-acid peroxygenase (1.11.2.4) · CPC title

• C12N9/0065 Primary

  acting on hydrogen peroxide as acceptor (1.11) · CPC title