Method for Producing Recombinant 11-De-O-Methyltomaymycin

• US 20150111298A1
• Filed: 05/13/2013
• Published: 04/23/2015
• Est. Priority Date: 05/14/2012
• Status: Active Grant

• Current Assignee: Sanofi
• Original Assignee: Sanofi
• Inventors: Broenstrup, Mark, Mller, Rolf, Lattemann, Claus, Werner, Stefan, Harmrols, Kirsten
• Granted Patent: US 10,550,397 B2
• Time in Patent Office: Days
• US Class Current: 435/471
• CPC Class Codes: A61K 31/445 Non condensed piperidines, e.g. piperocaine
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/76 for Actinomyces; for Streptomyces

Abstract

The present invention provides a tomaymycin biosynthetic gene cluster of Streptomyces species FH6421, and its use for producing 11-de-O-methyltomaymycin.

Claims

1. A nucleic acid molecule comprising at least one nucleic acid selected from the group consisting of:

(a) a nucleic acid comprising at least one of the Open Reading Frames (ORFs) 1 to 19 of SEQ ID NO;

2 that encodes proteins of SEQ ID NOs;

4 to 22 or a variant or fragment thereof, whereby the variant or fragment encodes a functionally active variant or fragment of a protein of SEQ ID NOs;

4 to 22,

(b) a nucleic acid encoding at least one of the proteins of SEQ ID NOs;

4 to 22 or a functionally active variant or fragment thereof,

(c) a nucleic acid encoding a protein that is at least 70%, 80%, 90%, 95% or 97% identical in amino acid sequence to a protein or fragment thereof encoded by the nucleic acid of (a) or (b),

(d) a nucleic acid that hybridizes under stringent conditions with a nucleic acid of (a) to (c),

(e) a nucleic acid that is complementary to a nucleic acid of (a) to (d).

2. The nucleic acid according to claim 1 comprising the tomaymycin biosynthetic gene cluster of SEQ ID NO:

2 or having a variant or fragment sequence of SEQ ID NO;

2 harboring a variant or fragment of at least one of ORFs 1 to 19, whereby the variant or fragment encodes a functionally active variant or fragment of a protein of SEQ ID NOs;

4 to 22.

3. An expression vector comprising a nucleic acid of claim 1.

4. A cell comprising the expression vector according to claim 3.

5. The cell of claim 4, wherein the cell is Streptomyces species FH6421.

6. A method for producing a cell that harbors a tomaymycin biosynthetic gene cluster or a functionally active variant or fragment thereof.

7. The method according to claim 6, wherein the cell harbors the tomaymycin biosynthetic gene cluster of SEQ ID NO:

1, SEQ ID NO;

2, or SEQ ID NO;

3, or a functionally active variant or fragment thereof.

8. The method according to claim 7, wherein the cell is a Streptomyces strain harboring the tomaymycin biosynthetic gene cluster of SEQ ID NO:

1, SEQ ID NO;

2, or SEQ ID NO;

3, or a functionally active variant or fragment thereof.

9. The method according to claim 8, wherein the Streptomyces strain is selected from the group consisting of Streptomyces achromogenes var. tomaymyceticus, Streptomyces species FH6421, and Streptomyces albus/pStW102tc.

10. The method according to claim 6, wherein the cell harbors at least one ORF of SEQ ID NO:

1, SEQ ID NO;

2, or SEQ ID NO;

3, or functionally active variant or fragment thereof, and is capable of producing 11-de-O-methyltomaymycin.