Sensor for NADP (H) and development of alcohol dehydrogenases

Patent metadata

Field	Value
Publication number	US-10385349-B2
Application number	US-201314424559-A
Country	US
Kind code	B2
Filing date	Aug 16, 2013
Priority date	Aug 28, 2012
Publication date	Aug 20, 2019
Grant date	Aug 20, 2019

Abstract

The present invention relates to an NADP(H) nanosensor comprising
i) a nucleic acid sequence to which a regulator is capable of binding, wherein the oxidation state of the regulator depends on the NADP(H) availability; ii) a promoter sequence following the nucleic acid sequence i), to which an RNA polymerase is capable of binding, wherein the affinity of the RNA polymerase for the promoter sequence is influenced by the oxidation state of the regulator; iii) a nucleic acid sequence which is under the control of the promoter sequence ii) and which codes for an autofluorescent protein.

The present invention also relates to a cell, a method for isolating genes which code for NADP(H)-dependent enzymes, and the use of an NADP(H) nanosensor.

First claim

The invention claimed is:

1. A cell comprising an NADPH(H) nanosensor, wherein the NADP(H) nanosensor comprises:
i) a nucleic acid to which a regulator is capable of binding, wherein the oxidation state of the regulator depends on the cell's intra-cellular NADP(H) availability;
ii) a promoter following the nucleic acid i), to which an RNA polymerase is capable of binding, wherein the affinity of the RNA polymerase for the promoter is influenced by the oxidation state of the regulator;
iii) a nucleic acid which is under the control of the promoter ii) and which codes for an autofluorescent protein, wherein the autofluorescent protein is selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), enhanced blue fluorescent protein (EBFP), enhanced cyan fluorescent protein (ECFP), DsRed, HcRed, AsRed, Am Cyan, ZsGreen, AcGFP and Zs Yellow, or a photoreceptor protein which contains a LOY domain,
wherein components i) and ii) together have a nucleic acid sequence according to SEQ. ID. No. 01,
wherein the NADP(H) nanosensor is present in the cell in the episomal form,
wherein the cell, in addition to the NADP(H)-nanosensor, comprises a gene which codes for an NADP(H)-dependent enzyme that has been introduced into the cell, and
wherein the cell is Escherichia coli ( E. coli ).

2. The cell according to claim 1 , wherein the NADP(H) nanosensor comprises:
(α1) the E. coli gene for SoxR (soxR);
(α2) components i) and ii) downstream of (α1);
(α3) at least the first 5 nucleotides of the soxS gene from E. coli following (α2);
(α4) a nucleic acid which codes for an autofluorescent protein, following (α2) or (α3) and which is under the control of the soxS promoter, as component iii).

3. The cell according to claim 1 , wherein the NADP(H) nanosensor comprises:
(β1) the E. coli gene for SoxR (soxR);
(β2) components i) and ii) downstream of (β1);
(β3) the soxS gene from E. coli following (β2) and under the control of the soxS promoter or a part of this gene;
(β3′) a further nucleic acid following (β3) which at the mRNA level corresponds to a ribosome binding site;
(β4) a nucleic acid, which codes for an autofluorescent protein, following (β3′) and which is under the control of the soxS promoter, as component iii).

4. The cell according to claim 2 or 3 , wherein component (α1) or (β1) is selected from the group consisting of:
a) a nucleic acid sequence according to SEQ. ID. No. 02, and
b) a nucleic acid sequence coding for a polypeptide with an amino acid sequence according to SEQ. ID. No. 03.

5. The cell according to claim 1 , wherein the nucleic acid (iii) which codes for an auto fluorescent protein is the gene coding for enhanced yellow fluorescent protein (EYFP).

6. The cell according to claim 1 , wherein the NADP(H) dependent enzyme is selected from the group consisting of alcohol dehydrogenases, aldehyde dehydrogenases, lactate dehydrogenases, enoate reductases, epoxide reductases, diaminopimelate dehydrogenases, amino acid dehydrogenases, aldehyde oxidoreductases, alkane reductases, amine reductases, epoxide dehydrogenases, carboxylic acid dehydrogenases, hydroxy acid ketoreductases and hydroxy acid dehalogenases.

Assignees

• Forschungszentrum Juelich Gmbh

Inventors

• Siedler Solvej
• Schendzielorz Georg
• Binder Stephan
• Eggeling Lothar
• Bringer-Meyer Stephanie
• Bott Michael

Classifications

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title

• C12Q1/68 Primary

  involving nucleic acids · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• G06N5/045

  Explanation of inference; Explainable artificial intelligence [XAI]; Interpretable artificial intelligence · CPC title