Neurospora expression system

• US 5,695,965 A
• Filed: 06/02/1992
• Issued: 12/09/1997
• Est. Priority Date: 03/13/1990
• Status: Expired due to Term

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Hawaii Biotechnology Group Incorporated
• Inventors: Koo, Kenneth, Stuart, W. Dorsey, Ivy, John M.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US07/896,455
• Time in Patent Office: 2,016 Days
• Field of Search: 435/69.1, 435/172.3, 435/252.7, 435/69.4, 435/69.52, 435/69.6, 435/254.4, 435/320.1, 435/219, 536/24.1
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)

Abstract

Neurospora crassa transformations are employed for production of heterologous proteins, where DNA constructs are prepared using a foreign gene under transcriptional and translational regulatory regions functional in Neurospora. A functional signal sequence is provided for secretion of the heterologous gene product. An auxotrophic Neurospora host is employed and transformed by the combination of the subject construct and a complementing gene. The resulting transformants provide for efficient secretion of the heterologous product.

Claims

1. A method of producing a mammalian peptide in a Neurospora fungus host, said method comprising:

transforming by DNA uptake Neurospora spheroplasts with (1) a DNA construct comprising as operably linked components a constitutive promoter functional in said fungus host, a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said promoter and a transcriptional termination regulatory region functional in said host, and (2) a second DNA sequence comprising a marker gene which is expressed in said host;

selecting for transformed host cells comprising said construct by means of said marker gene;

identifying stably transformed host cells; and

growing said stably transformed host cells whereby said first DNA sequence is expressed, and said cells produce said mammalian peptide.

2. A method of producing a mammalian peptide in a Neurospora crassa host, said method comprising:

transforming by DNA uptake N. crassa spheroplasts with (1) a DNA construct comprising as operably linked components a β

-tubulin promoter functional in said host, a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said β

-tubulin promoter and a transcriptional termination regulatory region functional in said host, and (2) a second DNA sequence comprising a marker gene which is expressed in said host;

selecting for transformed host cells comprising said construct by means of said marker gene;

identifying stably transformed host cells; and

growing said stably transformed host cells whereby said first DNA sequence is expressed, and said cells produce said mammalian peptide.

3. A DNA construct comprising a Neurospora β

-tubulin promoter and a DNA sequence encoding a mammalian peptide under the transcriptional control of said promoter.

4. A DNA construct according to claim 3, wherein said mammalian peptide is preprochymosin.

5. A stably transformed Neurospora cell containing a DNA construct comprising as operably linked components β

-tubulin promoter functional in said cell, a first DNA sequence encoding a mammalian peptide under the transcriptional control of said β

-tubulin promoter and a transcriptional termination regulatory region functional in said cell; and

a second DNA sequence comprising a marker gene expressed in said cell.

6. A Neurospora cell according to claim 5, wherein said mammalian peptide is preprochymosin.

7. A Neurospora cell according to claim 5, which produces a mature mammalian peptide as a result of transcription of said first DNA sequence and translation of the resulting RNA.

8. A Neurospora cell according to claim 7, wherein said mature mammalian peptide is chymosin.

9. A method of producing and secreting in a Neurospora crassa host a mammalian peptide said method comprising:

transforming by DNA uptake N. crassa spheroplasts with (1) a DNA construct comprising as operably linked components a β

-tubulin promoter, an open reading frame comprising a signal sequence for secretion joined to and in reading frame with a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said promoter and a transcriptional termination regulatory region functional in said host, and (2) a second DNA sequence comprising a marker gene which is expressed in said host;

selecting for transformed host cells comprising said construct by means of said marker gene;

identifying stably transformed host cells; and

growing said stably transformed host cells whereby said first DNA sequence is expressed, and said cells produce and secrete said peptide.

10. A stably transformed Neurospora cell containing a DNA construct comprising as operable components a Neurospora β

-tubulin promoter and a first DNA sequence encoding a mammalian peptide under the transcriptional control of said promoter and a transcriptional termination regulatory region functional in said cell; and

a second DNA sequence comprising a marker gene which is expressed in said cell.

11. A stably transformed Neurospora crassa cell containing a DNA construct comprising as operable components a β

-tubulin promoter and a first DNA sequence encoding a mammalian peptide under the transcriptional control of said promoter and a transcriptional termination regulatory region functional in said cell; and

a second DNA sequence comprising a marker gene which is expressed in said cell.

12. An isolated DNA construct comprising a Neurospora β

-tubulin promoter and wherein said DNA construct is substantially free of a β

-tubulin open reading frame which expresses β

-tubulin.

13. A method of obtaining a mammalian peptide, said method comprising:

isolating said mammalian peptide from a culture broth of stably transformed Neurospora fungus cells which produce said mammalian peptide, wherein said Neurospora fungus cells are derived from spheroplasts transformed by DNA uptake with a DNA construct comprising as operably linked components, a promoter functional in said Neurospora fungus cells, a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said promoter and a transcriptional termination regulatory region functional in said Neurospora fungus cells, a second DNA sequence comprising a marker gene which is expressed in said Neurospora fungus cells and a third DNA sequence encoding a secretion signal peptide joined to and in reading frame with said first DNA sequence.

15. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes chymosin.

16. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes blood protein Factor VIII.

17. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes tissue plasminogen activator.

18. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes serum albumin.

19. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes a growth factor.

20. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes an interleukin.

21. The method according to claim 13 or claim 14, wherein said first DNA sequence encodes a neurotrophin.

22. The method according to claim 13 or claim 14, wherein said promoter is a constitutive promoter.

24. The method according to claim 13 or claim 14, wherein said promoter is derived from a grg-1 promoter.

25. The method according to any one of claims 13 or 14, wherein said spheroplasts are derived from an auxotrophic mutant of a Neurospora fungus, and said a marker gene provides complementation of said auxotrophic mutant to prototropy.

26. The method according to claim 25, wherein said auxotrophic mutant is unable to synthesize an essential amino acid.

14. A method of obtaining a mammalian peptide, said method comprising:

isolating said mammalian peptide from a culture broth of stably transformed Neurospora crassa cells which produce said mammalian peptide, wherein said Neurospora crassa cells are derived from spheroplasts transformed by DNA uptake with a DNA construct comprising as operably linked components, a promoter functional in said Neurospora crassa cells, a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said promoter and a transcriptional termination regulatory region functional in said Neurospora crassa cells, a second DNA sequence comprising a marker gene which is expressed in said Neurospora crassa cells and a third DNA sequence encoding a secretion signal peptide joined to and in reading frame with said first DNA sequence.

23. The method according to claim 14, wherein said constitutive promoter is the β

-tubulin promoter.

27. A method of obtaining a mammalian peptide, said method comprising:

isolating said mammalian peptide from a culture broth of stably transformed Neurospora fungus cells which produce said mammalian peptide, wherein said Neurospora fungus cells are derived from spheroplasts transformed by DNA uptake with a DNA construct comprising as operably linked components, a Neurospora β

-tubulin promoter, a first DNA sequence encoding a mammalian peptide under the transcriptional regulation of said promoter and a transcriptional termination regulatory region functional in said Neurospora fungus cells, a second DNA sequence comprising a marker gene which is expressed in said Neurospora fungus cells and a third DNA sequence encoding a secretion signal peptide joined to and in reading frame with said first DNA sequence.

28. The method according to claim 27, wherein said mammalian peptide is preprochymosin.

29. The method according to claim 27, wherein said secretion signal peptide is derived from a secretion signal peptide of chymosin.

30. Stably transformed Neurospora fungus cells which produce a mammalian peptide.

31. Stably transformed Neurospora fungus cells which secrete a mammalian peptide.