Overexpression of phytase genes in yeast systems

Patent metadata

Field	Value
Publication number	US-8993300-B2
Application number	US-201313897172-A
Country	US
Kind code	B2
Filing date	May 17, 2013
Priority date	Jun 25, 1998
Publication date	Mar 31, 2015
Grant date	Mar 31, 2015

Abstract

The present invention relates to a method of producing a heterologous protein or polypeptide having phytase activity in a yeast system. The invention also provides proteins having phytase activity which have increased thermostability. Yeast strains which produce a heterologous phytase and the vectors used to produce the phytase are also provided.

First claim

What is claimed:

1. A method of producing a phytase in fungal cells, the method comprising:
providing a polynucleotide encoding an Escherichia coli phytase;
expressing the polynucleotide in the fungal cells; and
isolating the expressed Escherichia coli phytase wherein the Escherichia coli phytase catalyzes the release of phosphate from phytate.

2. The method of claim 1 wherein the phytase is an Escherichia coli AppA phytase.

3. The method of claim 1 wherein the fungal cells are from a filamentous fungus.

4. The method of claim 3 wherein the filamentous fungus is an Aspergillus species or a Neurospora species.

5. The method of claim 1 wherein the polynucleotide is incorporated into a nucleic acid vector.

6. The method of claim 1 wherein the fungal cells are cultured in a growth medium.

7. The method of claim 6 wherein the Escherichia coli phytase is secreted from the fungal cells into the growth medium.

8. The method of claim 7 wherein the Escherichia coli phytase is purified from the growth medium.

9. The method of claim 8 wherein the Escherichia coli phytase is purified from the growth medium at a concentration greater than 300 U/ml.

10. The method of claim 1 wherein the Escherichia coli phytase has an optimum activity at a temperature range of 57 degrees C. to 65 degrees C.

11. The method of claim 1 wherein the Escherichia coli phytase retains at least 40% of its activity after heating the phytase for 15 minutes at 80 degrees C.

12. The method of claim 1 wherein the Escherichia coli phytase retains at least 60% of its activity after heating the phytase for 15 minutes at 60 degrees C.

13. A method of converting phytate to inorganic phosphate, the method comprising expressing an Escherichia coli phytase in fungal cells;
isolating the expressed Escherichia coli phytase from the fungal cells; and
contacting the isolated Escherichia coli phytase with phytate wherein the phytase catalyzes the release of phosphate from phytate.

14. The method of claim 13 wherein the phytase is an Escherichia coli AppA phytase.

15. The method of claim 13 wherein the fungal cells are from a filamentous fungus.

16. The method of claim 15 wherein the filamentous fungus is an Aspergillus species or a Neurospora species.

17. The method of claim 13 wherein the fungal cells are cultured in a growth medium.

18. The method of claim 17 wherein the Escherichia coli phytase is secreted from the fungal cells into the growth medium.

19. The method of claim 18 wherein the Escherichia coli phytase is purified from the growth medium.

20. The method of claim 19 wherein the Escherichia coli phytase is purified from the growth medium at a concentration greater than 300 U/ml.

21. The method of claim 13 wherein the Escherichia coli phytase has an optimum activity at a temperature range of 57 degrees C. to 65 degrees C.

22. The method of claim 13 wherein the Escherichia coli phytase retains at least 40% of its activity after heating the phytase for 15 minutes at 80 degrees C.

23. The method of claim 13 wherein the Escherichia coli phytase retains at least 60% of its activity after heating the phytase for 15 minutes at 60 degrees C.

Assignees

• Cornell Res Foundation Inc

Inventors

• Lei Xingen

Classifications

• A23K50/60

  for weanlings · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• C12P7/18

  polyhydric · CPC title

• A23K20/189

  Enzymes · CPC title

• A23K50/30

  for swines · CPC title