Gene from acremonium chrysogenum encoding a protein with cephalosporin C acetylhydrolase activity and methods of use of such gene

• US 6,815,189 B1
• Filed: 03/01/2002
• Issued: 11/09/2004
• Est. Priority Date: 04/09/1999
• Status: Expired due to Fees

• Current Assignee: Antibioticos, S.A.U
• Original Assignee: Antibiotics SAU
• Inventors: Campoy Garcia, Sonia, Velasco Alvarez, Javier, Casqueiro Blanco, Francisco Javier, Gutierrez Martin, Santiago, Fierro Fierro, Francisco, Diez Garcia, Bruno, Martin Martin, Juan Francisco, Barredo Fuente, Jose Luis
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Swope, Rebecca E.
• Application Number:
  US09/958,413
• Time in Patent Office: 984 Days
• Field of Search: 435/196, 435/252.3, 435/252.33, 435/320.1, 435/47, 435/51, 536/23.2
• US Class Current: 435/196
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/58 derived from fungi
• CPC Class Codes: C12P 35/00 Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
• CPC Class Codes: C12P 35/02 by desacylation of the substituent in the 7 position
• CPC Class Codes: C12P 35/06 Cephalosporin C; Derivatives thereof

Abstract

An isolated DNA sequence that encodes a peptide with CPC-acetylhydrolase enzymatic activity is disclosed. Also, a method of expressing CPC-acetylhydrolase activity that includes the steps of (a) providing a microorganism that is susceptible to transformation with the isolated DNA sequence and that, upon transformation, expresses the CPC-acetylhydrolase activity encoded by the sequence, and (b) transforming the microorganism with the DNA sequence to cause expression of the CPC-acetylhydrolase activity in the microorganism is disclosed. Also, a method for producing a microorganism with increased capacity to aid in the production of cephalosporin, which method includes the steps of (a) providing a microorganism that has CPC-AH activity by virtue of expression of the DNA sequence or of a fragment thereof coding for the CPC-acetylhydrolase enzyme activity; and (b) inactivating the activity by disrupting expression of the DNA sequence is disclosed.

Claims

1. An isolated DNA sequence comprising SEQ ID NO:

4 or a fragment of SEQ ID NO;

4 encoding a peptide with cephalosporin C(CPC)-acetylhydrolase enzymatic activity.

2. The DNA sequence according to claim 1, wherein the sequence comprises SEQ ID NO:

4.

3. The DNA sequence according to claim 1, wherein the sequence encodes the polypeptide of SEQ ID NO:

5.

4. The DNA sequence according to claim 1, wherein the DNA sequence encodes the polypeptide of SEQ ID NO:

6.

5. A vector comprising the DNA sequence of claim 1.

6. The vector of claim 5, wherein the vector is a plasmid.

7. A vector comprising the DNA sequence of claim 2.

8. The vector of claim 7, wherein the vector is a plasmid.

9. A microorganism transformed with the vector of claim 5.

10. The transformed microorganism of claim 9, wherein the microorganism is E coli.

11. A microorganism transformed with the vector of claim 7.

12. The microorganism of claim 11, wherein the microorganism is E. coli.

13. A method of expressing CPC-acetylhydrolase activity comprising:

(a) providing a microorganism that is susceptible to transformation with the isolated DNA sequence of claim 1 and that, upon transformation, expresses the CPC-acetylhydrolase activity encoded by said DNA sequence; and

(b) transforming the microorganism with the isolated DNA sequence to cause expression of the CPC-acetylhydrolase activity in the microorganism.

14. The method according to claim 13, wherein the method further comprises using the expressed CPC-acetylhydrolase activity to prepare a deacetylated derivative of cephalosporin C or 7-aminocephalosporanic acid.

15. The method according to claim 14, wherein the derivative is deacetylcephalosporin C or deacetyl 7-aminocephalosporanic acid.

16. A method of expressing CPC-acetylhydrolase activity comprising:

(a) providing a microorganism that is susceptible to transformation with the isolated DNA sequence of claim 3 and that, upon transformation, expresses the CPC-acetylhydrolase activity encoded by said DNA sequence; and

(b) transforming the microorganism with the DNA sequence to cause expression of the CPC-acetylhydrolase activity in the microorganism.

17. The method according to claim 16, wherein the method further comprises using the expressed CPC-acetylhydrolase activity to prepare a deacetylated derivative of cephalosporin C or 7-aminocephalosporanic acid.

18. The method according to claim 17, wherein the derivative is deacetylcephalosporin C or deacetyl 7-aminocephalosporanic acid.

19. A method of expressing CPC-acetylhydrolase activity comprising:

(a) providing a microorganism that is susceptible to transformation with the isolated DNA sequence of claim 4 and that, upon transformation, expresses the CPC-acetylhydrolase activity encoded by said DNA sequence; and

(b) transforming the microorganism with the DNA sequence to cause expression of the CPC-acetylhydrolase activity in the microorganism.

20. The method according to claim 19, wherein the method further comprises using the expressed CPC-acetylhydrolase activity to prepare a deacetylated derivative of cephalosporin C or 7-aminocephalosporanic acid.

21. The method according to claim 20, wherein the derivative is deacetylcephalosporin C or deacetyl 7-aminocephalosporanic acid.