Method of screening and quantifying various enzymatic activities using artificial genetic circuits

Patent metadata

Field	Value
Publication number	US-9783859-B2
Application number	US-201013376783-A
Country	US
Kind code	B2
Filing date	Jun 8, 2010
Priority date	Jun 8, 2009
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

A method of detecting and quantifying various enzymatic activities using a constructed artificial genetic circuit GESS (genetic enzyme screening system) for sensing phenolic compounds and a method of screening a trace of activities of target enzymes from a metagenome using the artificial genetic circuit, thereby securing target enzyme genes. When the method for screening and quantifying target enzymatic activity is used, useful genes can be screened from various genetic communities, including environmental or metagenomic libraries, at a single cell level in high throughput (million/day). Further, the sensitivity of the genetic circuit to phenol derivatives and the expression thereof can be controlled, and thus the genetic circuit can rapidly sense and quantify various enzymatic activities. Thus, the method can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.

First claim

What is claimed is:

1. A method of detecting, screening, or quantifying the activity of one or more biosynthesis enzymes performing enzymatic reactions using compounds having a phenol group bound thereto as substrates, using an artificial genetic circuit, the method comprising the steps of:
(a) providing an artificial genetic circuit for detecting said phenolic compound or microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting said phenolic compound, the artificial genetic circuit comprising:
(i) a gene encoding a transcriptional regulator to which said phenolic compound binds,
(ii) at least one reporter gene selected from the group consisting of fluorescence protein-encoding genes and antibiotic resistance genes, and
(iii) a gene expression regulatory region consisting of a promoter regulating the expression of said transcriptional regulator,
a promoter regulating the expression of the reporter gene, and
a region, located between the promoter regulating the expression of the reporter gene and reporter gene, to which said transcriptional regulator binds to induce the expression of a downstream reporter gene,
wherein the phenolic compound binds to (i) transcriptional regulator to induce binding of the transcriptional regulator to the (iii) gene expression regulatory region and activate the promoter of the reporter gene such that the reporter gene located downstream of the gene expression regulatory region is expressed;
(b) providing a clone or gene library containing one or more of a gene encoding a biosynthesis enzyme for said phenolic compound;
(c) introducing the clone or gene library and the artificial gene circuit for detecting said phenolic compound into host microorganisms to prepare recombinant microorganisms or introducing the clone or gene library into the microorganisms containing the artificial gene circuit for detecting said phenolic compound to prepare recombinant microorganisms;
(d) treating the recombinant microorganisms with a compound capable of liberating said phenolic compound by an enzymatic reaction of the biosynthesis enzyme for production of phenolic compound that is to be detected, screened, or quantified; and
(e) detecting or quantifying the activity of the reporter protein whose expression was induced by sensing said phenolic compound liberated by the enzymatic reaction of the biosynthesis enzyme for production of phenolic compound that is to be detected, screened, or quantified.

2. A method of screening target enzyme activity of one or more biosynthesis enzymes performing enzymatic reactions using compounds having a phenol group bound thereto as substrates, using an artificial genetic circuit, the method comprising the steps of:
(a) providing microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting the phenolic compound, the artificial genetic circuit comprising
(i) a gene encoding a transcriptional regulator to which the phenolic compound binds,
(ii) at least one reporter gene selected from the group consisting of fluorescence protein-encoding genes and antibiotic resistance genes, and
(iii) a gene expression regulatory region consisting of a promoter regulating the expression of the transcriptional regulator, a promoter regulating the expression of the reporter gene, and a region, located between the promoter regulating the expression of the reporter gene and reporter gene, to which said transcriptional regulator binds to induce the expression of a downstream reporter gene,
wherein the phenolic compound binds to (i) transcriptional regulator to induce binding of the transcriptional regulator to the (iii) gene expression regulatory region and activate the promoter of the reporter gene such that the reporter gene located downstream of the gene expression regulatory region is expressed;
(b) providing a clone or gene library containing one or more of a gene encoding a biosynthesis enzyme for the production of phenolic compound;
(c) introducing the clone or gene library into microorganisms containing the artificial gene circuit for detecting the phenolic compound to prepare recombinant microorganisms or introducing the clone or gene library into the microorganisms containing the artificial gene circuit for detecting said phenolic compound to prepare recombinant microorganisms;
(d) treating the recombinant microorganisms with a compound capable of liberating the phenolic compound by an enzymatic reaction of the biosynthesis enzyme for the production of phenolic compound that is to be detected, screened, or quantified; and
(e) detecting the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction of the biosynthesis enzyme for the production of phenolic compound that is to be detected, screened, or quantified.

3. A method of quantifying target enzyme activity of one or more biosynthesis enzymes performing enzymatic reactions using compounds having a phenol group bound thereto as substrates using an artificial genetic circuit, the method comprising the steps of:
(a) providing an artificial genetic circuit for detecting the phenolic compound or microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting the phenolic compound, the artificial genetic circuit comprising
(i) a gene encoding a transcriptional regulator to which the phenolic compound binds,
(ii) at least one reporter gene selected from the group consisting of fluorescence protein-encoding genes and antibiotic resistance genes, and
(iii) a gene expression regulatory region consisting of a promoter regulating the expression of the transcriptional regulator, a promoter regulating the expression of the reporter gene, and a region, located between the promoter regulating the expression of the reporter gene and reporter gene, to which said transcriptional regulator binds to induce the expression of a downstream reporter gene,
wherein the phenolic compound binds to (i) transcriptional regulator to induce binding of the transcriptional regulator to the (iii) gene expression regulatory region and activate the promoter of the reporter gene such that the reporter gene located downstream of the gene expression regulatory region is expressed;
(b) providing a clone or gene library containing one or more of a gene encoding a biosynthesis enzyme for the production of phenolic compound;
(c) introducing the clone or gene library and the artificial gene circuit for detecting the phenolic compound into host microorganisms to prepare recombinant microorganisms or introducing the clone or gene library into the microorganisms containing the artificial gene circuit for detecting the phenolic compound to prepare recombinant microorganisms;
(d) treating the recombinant microorganisms with a compound capable of liberating the phenolic compound by an enzymatic reaction of the biosynthesis enzyme for the production of phenolic compound that is to be detected, screened, or quantified; and
(e) quantifying the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction of the biosynthesis enzyme for the production of phenolic compound that is to be detected, screened, or quantified.

4. A method of quantifying target enzyme activity of one or more biosynthesis enzymes performing enzymatic reactions using compounds having a phenol group bound thereto as substrates, using an artificial genetic circuit, the method comprising the steps of:
(a) providing microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting the phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a transcriptional regulator to which

Assignees

• Lee Seung Goo
• Rha Eugene
• Choi Su Lim
• Song Jae Jun
• Choi Jong Hyun
• Kim Hee Sik
• Korea Res Inst Bioscience & Biotechnology

Inventors

• Lee Seung Goo
• Rha Eugene
• Choi Su Lim
• Song Jae Jun
• Choi Jong Hyun
• Kim Hee Sik

Classifications

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title