Method for screening and quantifying various enzyme activities using a genetic enzyme screening system

A method of screening target enzyme activity 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 a phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a phenolic compound-degrading enzyme regulatory protein which recognizes a phenolic compound, (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 phenolic compound-degrading enzyme regulatory protein, a region to which the phenolic compound-degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene;
• (b) providing a clone or gene library containing a gene encoding an enzyme to be screened;
• (c) introducing the clone or gene library into microorganisms containing the artificial gene circuit for detecting the phenolic compound to prepare recombinant microorganisms;
• (d) treating the recombinant microorganisms with a phenol-release compound capable of liberating a phenolic compound by an enzymatic reaction;
• and
• (e) detecting the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction.

A method of quantifying target enzyme activity using an artificial genetic circuit, the method comprising the steps of:

• (a) providing an artificial genetic circuit for detecting a phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a phenolic compound-degrading enzyme regulatory protein which recognizes a phenolic compound, (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 phenolic compound-degrading enzyme regulatory protein, a region to which the phenolic compound-degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene;
• (b) providing a clone or gene library containing a gene encoding an enzyme to be screened;
• (c) introducing the clone or gene library and the artificial gene circuit for detecting the phenolic compound into host microorganisms to prepare recombinant microorganisms;
• (d) treating the recombinant microorganisms with a phenol-release compound capable of liberating a phenolic compound by an enzymatic reaction;
• and
• (e) quantifying the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction.

A method of quantifying target enzyme activity 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 a phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a phenolic compound-degrading enzyme regulatory protein which recognizes a phenolic compound, (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 phenolic compound-degrading enzyme regulatory protein, a region to which the phenolic compound-degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene;
• (b) providing a clone or gene library containing a gene encoding an enzyme to be screened;
• (c) introducing the clone or gene library into microorganisms containing the artificial gene circuit for detecting the phenolic compound to prepare recombinant microorganisms;
• (d) treating the recombinant microorganisms with a phenol-release compound capable of liberating a phenolic compound by an enzymatic reaction;
• and
• (e) quantifying the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction.

A method of screening a target enzyme capable of liberating a phenolic compound by the reaction of an enzyme from a metagenomic library, the method comprising the steps of:

• (a) providing a metagenomic library from a natural environment;
• (b) providing an artificial genetic circuit for detecting a phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a phenolic compound-degrading enzyme regulatory protein which recognizes a phenolic compound, (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 phenolic compound-degrading enzyme regulatory protein, a region to which the phenolic compound-degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene;
• (c) introducing the metagenomic library and the artificial genetic circuit into host microorganisms to construct a library of transformed microorganisms;
• (d) treating the library of transformed microorganisms with a phenol-release compound capable of liberating a phenolic compound by an enzymatic reaction;
• (e) measuring the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction, thereby performing high-throughput screening of microorganisms having activity of liberating the phenolic compound by the enzymatic reaction;
• and
• (f) collecting a gene of the enzyme, which is capable of liberating the phenolic compound by an enzymatic reaction, from the screened microorganisms, and then identifying the gene by sequencing.

A method of screening a target enzyme capable of liberating a phenolic compound by the reaction of an enzyme from a metagenomic library, the method comprising the steps of:

• (a) providing a metagenomic library from a natural environment;
• (b) providing microorganisms containing in their chromosomal DNA or cytoplasm an artificial genetic circuit for detecting a phenolic compound, the artificial genetic circuit comprising (i) a gene encoding a phenolic compound-degrading enzyme regulatory protein which recognizes a phenolic compound, (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 phenolic compound-degrading enzyme regulatory protein, a region to which the phenolic compound-degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter regulating the expression of the reporter gene;
• (c) introducing the metagenomic library into the microorganisms containing in their chromosomal DNA or cytoplasm the artificial genetic circuit to construct a library of transformed microorganisms;
• (d) treating the library of transformed microorganisms with a phenol-release compound capable of liberating a phenolic compound by an enzymatic reaction;
• (e) measuring the activity of the reporter protein whose expression was induced by sensing the phenolic compound liberated by the enzymatic reaction, thereby performing high-throughput screening of microorganisms having activity of liberating the phenolic compound by the enzymatic reaction;
• and
• (f) collecting a gene of the enzyme, which is capable of liberating the phenolic compound by the enzymatic reaction, from the screened microorganisms, and then identifying the gene by sequencing.