Overexpression of phytase genes in yeast systems

• US 8,993,300 B2
• Filed: 05/17/2013
• Issued: 03/31/2015
• Est. Priority Date: 06/25/1998
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Lei, Xingen
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US13/897,172
• Publication Number:
  US 20130244303A1
• Time in Patent Office: 683 Days
• Field of Search: 435/196
• US Class Current: 435/196
• CPC Class Codes: A23K 20/189 Enzymes
• CPC Class Codes: A23K 50/30 for swines
• CPC Class Codes: A23K 50/60 for weanlings
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12P 7/18 polyhydric

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.

Claims

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.