Engineered peptide synthetases and their use for the non-ribosomal production of peptides

• US 5,795,738 A
• Filed: 06/26/1996
• Issued: 08/18/1998
• Est. Priority Date: 08/09/1995
• Status: Expired due to Fees

• Current Assignee: Eniricerche S.P.A.
• Original Assignee: Eniricerche S.P.A.
• Inventors: Rodriguez, Francesco, Grandi, Guido, de Ferra, Francesca
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Stole, Einar
• Application Number:
  US08/670,901
• Time in Patent Office: 783 Days
• Field of Search: 435/69.1, 435/69.7, 435/252.3, 435/320.1, 536/23.2
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/93 Ligases (6)

Abstract

The present invention relates to the construction of functional engineered peptide synthetases capable of displaying a correct activity and their use for the non-ribosomal production of modified peptides.

Claims

1. A method for the non-ribosomal production of a peptide of predefined amino acid sequence and length comprising the steps of:

(a) providing a first DNA sequence containing the coding region of at least one amino acid activating domain of a peptide synthetase;

(b) providing a second DNA sequence containing the coding region of the carboxy-terminus of the last amino acid binding domain of a peptide synthetase downstream of the amino acid sequence of SEQ ID No. 1;

(c) fusing the 3'"'"'-end of the first DNA sequence to the 5'"'"'-end of the second DNA sequence to form a third DNA sequence;

(d) inserting the third DNA sequence into a vector selected from a group consisting of an expression or an integration vector to form a recombinant vector;

(e) transforming a microorganism with a recombinant vector of step (d);

(f) culturing the transformed microorganism to produce the novel peptide synthetase; and

(g) producing the peptide having a predefined sequence and length using the novel peptide synthetase.

2. The method of claim 1, wherein the vector of step (d) is an integration vector and the novel peptide synthetase is produced by homologous recombination with a first peptide synthetase gene in the microorganism to form a second DNA peptide synthetase gene, wherein the second peptide synthetase encodes a different number of amino acid activating domains than the first peptide synthetase.

3. The method of claim 1, wherein the vector of step (d) is an expression vector and the third DNA sequence encodes a full-length peptide synthetase in which the number and the order of the DDAs is established by the order and number of the amino acids in the peptide to be obtained.

4. The method of claim 1, wherein the step (c) the second DNA sequence is fused to the 3'"'"'-end of the first DNA sequence downstream from the region encoding the amino acid sequence of SEQ ID No. 1.

5. The method of claim 4, wherein at least 30 nucleotides are present between the 5'"'"'-end of the second DNA sequence and the coding region of the amino acid sequence of SEQ ID No. 1 of the last DDA of the first DNA sequence.

6. The method of claim 1, wherein the last amino acid activating domain of the first DNA sequence comprises a region catalyzing the epimerization of the amino acid.

7. The method of claim 1, wherein the last amino acid activating domain of the first DNA sequence comprises a region catalyzing the methylation of the amino acid.

8. The method of claim 1, wherein the microorganism is selected from the group consisting of a bacterium, a fungus, or a yeast.

9. The method of claim 1, wherein the peptide of predefined sequence and length is produced while the new peptide synthetase is immobilized on a solid insoluble support.

10. The method of claim 1, wherein the peptides of predefined sequence and length have antibiotic, antifungal, immunosuppressive or surfactant properties.

11. A peptide synthetase produced by a method comprising the steps of:

(a) providing a first DNA sequence containing the coding region of at least one amino acid activating domain of a peptide synthetase;

(b) providing a second DNA sequence containing the coding region of the carboxy-terminus of the last amino acid binding domain of a peptide synthetase downstream of the amino acid sequence of SEQ ID No. 1;

(c) fusing the 3'"'"'-end of the first DNA sequence to the 5'"'"'-end of the second DNA sequence to form a third DNA sequence;

(d) inserting the third DNA sequence into a vector selected from a group consisting of an expression or an integration vector to form a recombinant vector;

(e) transforming a microorganism with a recombinant vector of step (d);

(f) expressing the novel active peptide synthetase by culturing the transformed microorganism of step (e).

12. A peptide synthetase comprising the first four amino acid activation domains of the surfactin synthetase, wherein the fourth amino acid activation domain is fused in the region downstream from the motif encoding the amino acid sequence SEQ ID No. 1 with the carboxy-terminus of the last amino acid binding domain of the surfactin synthetase, wherein the carboxy-terminus region contains at least 260 amino acid residues.

13. The peptide synthetase of claim 12, wherein said peptide synthetase catalyses the production and secretion of an analog of the surfactin having an altered number of amino acids.