Sensors For The Detection Of Intracellular Metabolites

Patent metadata

Field	Value
Publication number	US-2016289776-A1
Application number	US-201615147177-A
Country	US
Kind code	A1
Filing date	May 5, 2016
Priority date	May 3, 2010
Publication date	Oct 6, 2016
Grant date	—

Abstract

The present invention relates to a cell which is genetically modified with respect to its wild type and which comprises a gene sequence coding for an autofluorescent protein, wherein the expression of the autofluorescent protein depends on the intracellular concentration of a particular metabolite.
The present invention also relates to a method for the identification of a cell having an increased intracellular concentration of a particular metabolite, a method for the production of a cell which is genetically modified with respect to its wild type with optimized production of a particular metabolite, a cell obtained by this method, a method for the production of metabolites and a method for the preparation of a mixture.

First claim

What is claimed is:

1 . A method for the identification of a cell having an increased intracellular concentration of a particular metabolite in a cell suspension, comprising the method steps:
i) provision of a cell suspension comprising cells which are genetically modified with respect to their wild type and which comprise a gene sequence coding for an autofluorescent protein, wherein the expression of the autofluorescent protein depends on the intracellular concentration of a particular metabolite; ii) genetic modification of the cells to obtain a cell suspension in which the cells differ with respect to the intracellular concentration of a particular metabolite; iii) identification of individual cells in the cell suspension having an increased intracellular concentration of this particular metabolite by detection of the intracellular fluorescence activity.

2 . The method according to claim 1 , wherein the genetic modification in method step ii) is carried out by non-targeted mutagenesis.

3 . The method according to claim 1 , further comprising the method step:
iv) separating off of the identified cells from the cell suspension.

4 . The method according to claim 3 , wherein the separating off is carried out by means of flow cytometry.

5 . The method according to claim 1 , wherein control of the expression of the gene sequence coding for the autofluorescent protein is effected as a function of the intracellular concentration of the particular metabolite at the transcription level.

6 . The method according to claim 1 , wherein the gene sequence coding for the autofluorescent protein is under the control of a heterologous promoter which, in the wild type of the cell, controls the expression of a gene of which the expression in the wild-type cell depends on the intracellular concentration of a particular metabolite.

7 . The method according to claim 6 , wherein control of the expression of the gene sequence coding for the autofluorescent protein is effected as a function of the intracellular concentration of the particular metabolite at the translation level.

8 . The method according to claim 5 , wherein the gene sequence coding for the autofluorescent protein is bonded functionally to a DNA sequence which, at the mRNA level, assumes the function of a riboswitch which regulates the expression of the gene sequence coding for the autofluorescent protein at the transcription level or the translation level.

9 . The method according to claim 1 , wherein the cell is a cell of the genus Corynebacterium or Escherichia.

10 . The method according to claim 1 , wherein the metabolite is chosen from the group consisting of amino acids, nucleotides, fatty acids and carbohydrates.

11 . The method according to claim 10 , wherein the metabolite is an amino acid.

12 . The method according to claim 11 , wherein the amino acid is L-lysine.

13 . The method according to claim 5 , wherein the promoter is the lysE promoter and the gene is the lysE gene.

14 . The method according to claim 1 , wherein the autofluorescent protein is green fluorescent protein (GFP) or a variant of this protein.

15 . A method for the production of a cell which is genetically modified with respect to its wild type with optimized production of a particular metabolite, comprising the method steps:
I) provision of a cell suspension comprising cells which are genetically modified with respect to their wild type and which comprise a gene sequence coding for an autofluorescent protein, wherein the expression of the autofluorescent protein depends on the intracellular concentration of a particular metabolite; II) genetic modification of the cells to obtain a cell suspension in which the cells differ with respect to their intracellular concentration of a particular metabolite; III) identification of individual cells in the cell suspension having an increased intracellular concentration of the particular metabolite by detection of the intracellular fluorescence activity; IV) separating off of the identified cells from the cell suspension; V) identification of those genetically modified genes G 1 to G n or those mutations M 1 to M m in the cells identified and separated off which are responsible for the increased intracellular concentration of the particular metabolite; VI) production of a cell which is genetically modified with respect to its wild type with optimized production of the particular metabolite, of which the genome comprises at least one of the genes G 1 to G n and/or at least one of the mutations M 1 to M m .

16 . The method according to claim 15 , wherein the genetic modification in method step II) is carried out by non-targeted mutagenesis.

17 . The method according to claim 15 , wherein control of the expression of the gene sequence coding for the autofluorescent protein is effected as a function of the intracellular concentration of the particular metabolite at the transcription level.

18 . The method according to claim 15 , wherein the gene sequence coding for the autofluorescent protein is under the control of a heterologous promoter which, in the wild type of the cell, controls the expression of a gene of which the expression in the wild-type cell depends on the intracellular concentration of a particular metabolite.

19 . The method according to claim 18 , wherein control of the expression of the gene sequence coding for the autofluorescent protein is effected as a function of the intracellular concentration of the particular metabolite at the translation level.

20 . The method according to claim 17 , wherein the gene sequence coding for the autofluorescent protein is bonded functionally to a DNA sequence which, at the mRNA level, assumes the function of a riboswitch which regulates the expression of the gene sequence coding for the autofluorescent protein at the transcription level or the translation level.

21 . The method according to claim 15 , wherein the cell is a cell of the genus Corynebacterium or Escherichia.

22 . The method according to claim 15 , wherein the metabolite is chosen from the group consisting of amino acids, nucleotides, fatty acids and carbohydrates.

23 . The method according to claim 22 , wherein the metabolite is an amino acid.

24 . The method according to claim 23 , wherein the amino acid is L-lysine.

25 . The method according to claim 17 , wherein the promoter is the lysE promoter and the gene is the lysE gene.

26 . The method according to claim 15 , wherein the autofluorescent protein is green fluorescent protein (GFP) or a variant of this protein.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Eggeling Lothar
• Bott Michael
• Binder Stephan
• Frunzke Julia
• Mustafi Nurije

Classifications

• A61P3/02

  Nutrients, e.g. vitamins, minerals · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• A23L33/175

  Amino acids · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title