Methods and means for metabolic engineering and improved product formation by micro-organisms

Patent metadata

Field	Value
Publication number	US-9834773-B2
Application number	US-22405007-A
Country	US
Kind code	B2
Filing date	Feb 14, 2007
Priority date	Feb 14, 2006
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

Described are methods and means for metabolic engineering and improved product formation by a filamentous micro-organism or a low G+C gram-positive bacterium. Disclosed is that DasR and DasR binding sites play an important and universal role in the control of gene expression in micro-organisms. Based on this finding, provided are multiple useful applications, such as a method for regulating the expression of a gene of interest, a method for controlling metabolism, a method for decreasing undesired expression and many more. Moreover, provided are means that can be used to establish said methods: for example a micro-organism in which the DasR binding site in operable linkage with a particular gene has been modified to obtain increased or decreased expression of a protein (being a desired or undesired protein) encoded by said gene.

First claim

What is claimed is:

1. A method for regulating expression of a gene of interest in a micro-organism comprising a DasR protein, wherein the gene of interest is in operable linkage to a DasR-binding site, the method comprising:
providing the micro-organism with a compound selected from the group consisting of glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, N-acetylglucosamine, and multimers thereof, such that the expression of the gene of interest is regulated;
screening for the production of a secondary metabolite; and
wherein the DasR-binding site is selected from the group consisting of:
NN(T/A)GG(T/A)(C/G)T(A/G)N(A/T)C(C/A)(A/C)N(SEQ ID NO:1);
(A/G)N(T/A)(G/T)(G/A)T(C/A)TA(G/T)A(C/T)(C/A)(A/T)N(T/C) (SEQ ID NO: 4);
A(T/A)(T/C)(G/A)(G/A)TATATA(C/T)(C/T)(A/G)(A/T)T (SEQ ID NO: 5);
A(T/C)(T/C)(G/T)(G/A)T(A/C)TA(T/G)A(C/T)(C/A)(A/G)(A/G)T (SEQ ID NO: 6); and
(A/T)(T/A)T(G/A)(G/T)(A/C)TA(T/G)N(C/A)(C/T)A(A/T)(T/A) (SEQ ID NO: 7).

2. The method according to claim 1 wherein the compound is selected from the group consisting of glucosamine-6-phosphate, N-acetylglucosamine, and multimers thereof.

3. The method according to claim 1 , further comprising providing the micro-organism with a polynucleotide encoding a DasR protein.

4. The method according to claim 1 , wherein the microorganism is a Streptomyces , a Nocardia , a Thermobifido , an Amycolatopsis , a Planobispora , a Streptoverticillium , a Rhodococcus , or a Corynebacterium.

5. The method according to claim 1 , wherein the micro-organism is a low G+C gram-positive bacterium.

6. The method according to claim 1 , further comprising introducing into the micro-organism the DasR-binding site in operable linkage with the gene of interest.

7. The method according to claim 1 , wherein the micro-organism is a Streptomyces , a Nocardia , a Thermobifido , a Amycolatopsis , a Planobispora , a Streptoverticillium , a Rhodococcus , a Corynebacterium , or a low G+C gram-positive bacterium.

8. The method according to claim 1 , further comprising purifying the secondary metabolite.

Assignees

• Rigali Sebastien
• Titgemeyer Mannfred Friedrich Bruno
• Van Wezel Gilles Philippus
• Univ Leiden
• Univ Of Liège
• Friedrich-Alexander-Universität Erlangen-Nürnberg

Inventors

• Rigali Sebastien
• Titgemeyer Mannfred Friedrich Bruno
• Van Wezel Gilles Philippus

Classifications

• C07K14/36

  from Actinomyces; from Streptomyces (G) · CPC title

• C12N1/38

  Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence) · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title