Method for genetically manipulating peptide synthetases

• US 5,652,116 A
• Filed: 07/06/1994
• Issued: 07/29/1997
• Est. Priority Date: 07/13/1993
• Status: Expired due to Fees

• Current Assignee: Eniricerche S.P.A.
• Original Assignee: Eniricerche S.P.A.
• Inventors: De Ferra, Francesca, Grandi, Guido, Galli, Giuliano, Cosmina, Paola
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US08/268,542
• Time in Patent Office: 1,119 Days
• Field of Search: 435/183, 435/69.1, 435/172.3
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Disclosed is a novel method for the in vivo and in vitro synthesis of modified peptides, using either engineered microorganisms capable of expressing peptide synthetases with modified substrate specificity, or peptide synthetases extracted from said microorganisms.

Claims

1. A method for biosynthesizing a novel peptide which comprises:

(a) constructing a gene encoding a peptide synthetase with an amino acid specificity different than any known peptide synthetase;

(b) inserting said gene into a vector;

(c) transforming a microorganism with said vector;

(d) isolating transformants which express said peptide synthetase; and

(e) cultivating said transformants to produce said novel peptide.

2. The method of claim 1, wherein in step (a), said gene is obtained by at least one step of:

deleting one or more activation domain coding regions,

substituting one or more activation domain coding regions from the same or different peptide synthetase, or

adding one or more activation domain coding regions from the same or different peptide synthetase.

3. The method of claim 1, wherein said novel peptide has antibiotic, antifungal or immunosuppressive properties.

4. The method of claim 1, wherein said novel peptide comprises the same amino acid residues as surfactin but a different primary sequence.

5. The method of claim 1, wherein said vector is an expression plasmid.

6. The method of claim 1, wherein said vector is an integration plasmid.

7. The method of claim 1, wherein said microorganism is a bacterium, fungus or yeast.