High throughput screening for novel enzymes

Abstract

Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring co-encapsulation, a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.

What is claimed is:  
     
         1 . A method of screening for an agent that modulates the activity of a target cell component, wherein the target cell component and a selectable marker are expressed by a recombinant cell, the method comprising co-encapsulating the agent in a micro-environment with the recombinant cell expressing the target cell component and detectable marker and detecting the effect of the agent on the activity of the cell component.  
     
     
         2 . The method of  claim 1 , wherein the agent is an enzyme or small molecule.  
     
     
         3 . The method of  claim 1 , wherein the agent is derived from a multispecific expression library.  
     
     
         4 . The method of  claim 2 , wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.  
     
     
         5 . The method of  claim 1 , wherein the agent inhibits the activity of the target cell component.  
     
     
         6 . The method of  claim 1 , wherein the agent enhances the activity of the target cell component.  
     
     
         7 . The method of  claim 1 , wherein the agent is expressed from a recombinant cell co-encapsulated with the recombinant cell expressing the target cell component and detectable marker.  
     
     
         8 . The method of  claim 7 , wherein the recombinant cell is a eukaryotic cell.  
     
     
         9 . The method of  claim 7 , wherein the recombinant cell is a prokaryotic cell.  
     
     
         10 . The method of  claim 1 , wherein the micro-environment is a liposome, gel microdrop, bead, agarose, cell, ghost red blood cell or ghost macrophage.  
     
     
         11 . The method of  claim 10 , wherein the liposomes are prepared from one or more phospholipids, glycolipids, steroids, alkyl phosphates or fatty acid esters.  
     
     
         12 . The method of  claim 11 , wherein the phospholipids are selected from the group consisting of lecithin, sphingomyelin and dipalmitoyl.  
     
     
         13 . The method of  claim 11 , wherein the steroids are selected from the group consisting of cholesterol, cholesterol and lanosterol.  
     
     
         14 . The method of  claim 1 , wherein the detectable marker is a fluorescent dye, a visible dye, a bioluminescent material, a chemiluminescent material, a radioactive material, or an enzymatic substrate.  
     
     
         15 . The method of  claim 14 , wherein the bioluminescent material is green fluorescent protein (GFP) or red fluorescent protein (RFP).  
     
     
         16 . The method of  claim 14 , wherein detection of the fluorescent dye or a visible dye is carried out by fluorometric or spectrophotometric measurement.  
     
     
         17 . The method of  claim 1 , wherein the protein is a transducing protein.  
     
     
         18 . The method of  claim 17 , wherein the transducing protein is a G-protein.  
     
     
         19 . The method of  claim 1 , wherein the recombinant cell is a eukaryotic cell.  
     
     
         20 . The method of  claim 1 , wherein the recombinant cell is a prokaryotic cell.  
     
     
         21 . A method for enriching for target DNA sequences containing at least a partial coding region for at least one specified activity in a DNA sample comprising: 
 a) co-encapsulating in a micro-environment a mixture of target DNA obtained from a mixture of organisms with a mixture of DNA probes comprising a detectable marker and at least a portion of a DNA sequence encoding at least one enzyme having a specified enzyme activity;    b) incubating the co-encapsulated mixture under such conditions and for such time as to allow hybridization of complementary sequences; and    c) screening for the specified activity.    
     
     
         22 . The method of  claim 21 , further comprising transforming host cells with recovered target DNA to produce an expression library of a plurality of clones.  
     
     
         23 . The method of  claim 21 , wherein the organisms are microorganisms.  
     
     
         24 . The method of  claim 23 , wherein the microorganisms are uncultured microorganisms.  
     
     
         25 . The method of  claim 21 , further comprising screening the expression library for the specified enzyme activity.  
     
     
         26 . The method of  claim 21 , wherein the target DNA obtained from the DNA population is selected by: 
 a) converting double stranded DNA into single stranded DNA;    b) recovering from the converted single stranded DNA, single stranded target DNA which hybridizes to probe DNA;    c) converting recovered single stranded target DNA to double stranded DNA; and    d) transforming a host cell with the double stranded DNA of c).    
     
     
         27 . The method of  claim 21 , wherein screening is by FACS analysis.  
     
     
         28 . The method of  claim 21 , wherein said target DNA is gene cluster DNA.  
     
     
         29 . The method of  claim 24 , wherein the uncultured microorganisms are derived from an environmental sample.  
     
     
         30 . The method of  claim 23 , wherein the uncultured microorganisms comprise a mixture of terrestrial microorganisms or marine microorganisms or airborne microorganisms, or a mixture of terrestrial microorganisms, marine microorganisms and airborne microorganisms.  
     
     
         31 . The method of  claim 22 , wherein the clones comprise a construct selected from the group consisting of phage, plasmids, phagemids, cosmids, fosmids, viral vectors, and artificial chromosomes.  
     
     
         32 . The method of  claim 21 , wherein the target DNA comprises one or more operons, or portions thereof, of the DNA population.  
     
     
         33 . The method of  claim 32 , wherein the operon or portions thereof encodes a complete or partial metabolic pathway.  
     
     
         34 . The method of  claim 24 , wherein the uncultured microorganisms comprise extremophiles.  
     
     
         35 . The method of  claim 34 , wherein the extremophiles are selected from the group consisting of thermophiles, hyperthermophiles, psychrophiles, barophiles, and psychrotrophs.  
     
     
         36 . The method of  claim 26 , wherein the host cell is selected from the group consisting of a bacterium, fungus, plant cell, insect cell and animal cell.  
     
     
         37 . The method of  claim 21 , wherein the target DNA encodes a protein.  
     
     
         38 . The method of  claim 37 , wherein the protein is an enzyme.  
     
     
         39 . The method of  claim 38  wherein the enzyme is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.  
     
     
         40 . The method of  claim 21 , wherein the micro-environment is a liposome, gel microdrop, bead, agarose, cell, ghost red blood cell or ghost macrophage.  
     
     
         41 . The method of  claim 40 , wherein the liposomes are prepared from one or more phospholipids, glycolipids, steroids, alkyl phosphates or fatty acid esters.  
     
     
         42 . The method of  claim 41 , wherein the phospholipids are selected from the group consisting of lecithin, sphingomyelin and dipalmitoyl.  
     
     
         43 . The method of  claim 41 , wherein the steroids are selected from the group consisting of cholesterol, cholestanol and lanosterol.  
     
     
         44 . The method of  claim 21 , wherein the detectable marker is a fluorescent dye, a visible dye, a bioluminescent material, a chemiluminescent material, a radioactive material, or an enzymatic substrate.  
     
     
         45 . The method of  claim 44 , wherein the bioluminescent material is green fluorescent protein (GFP) or red fluorescent protein (RFP).  
     
     
         46 . The method of  claim 45 , wherein detection of the fluorescent dye or a visible dye is carried out by fluorometric or spectrophotometric measurement.  
     
     
         47 . A method of screening for an agent that modulates the interaction of a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, comprising co-encapsulating the agent with the first test protein and second test protein in a suitable microenvironment and determining the ability of the agent to modulate the interaction of the first test protein linked to a DNA binding moiety with the second test protein covalently linked to a transcriptional activation moiety, wherein the agent enhances or inhibits the expression of a detectable protein, and wherein the enhancement or inhibition is detected by FACS analysis.  
     
     
         48 . The method of  claim 47 , wherein the agent is an enzyme or small molecule.  
     
     
         49 . The method of  claim 48 , wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.  
     
     
         50 . The method of  claim 47 , wherein the agent inhibits the activity of the first protein or the second protein.  
     
     
         51 . The method of  claim 47 , wherein the agent enhances the activity of the first protein or the second protein.  
     
     
         52 . The method of  claim 47 , wherein the agent is expressed from a recombinant cell co-encapsulated with the recombinant cell expressing the target protein and detectable marker.  
     
     
         53 . The method of  claim 52 , wherein the recombinant cell is a eukaryotic cell.  
     
     
         54 . The method of  claim 52 , wherein the recombinant cell is a prokaryotic cell.  
     
     
         55 . The method of  claim 47 , wherein the micro-environment is a liposome, gel microdrop, bead, agarose, cell, ghost red blood cell or ghost macrophage.  
     
     
         56 . The method of  claim 55 , wherein the liposomes are prepared from one or more phospholipids, glycolipids, steroids, alkyl phosphates or fatty acid esters.  
     
     
         57 . The method of  claim 56 , wherein the phospholipids are selected from the group consisting of lecithin, sphingomyelin and dipalmitoyl.  
     
     
         58 . The method of  claim 56 , wherein the steroids are selected from the group consisting of cholesterol, chlorestanol and lanosterol.  
     
     
         59 . The method of  claim 47 , wherein the detectable marker is a fluorescent dye, a visible dye, a bioluminescent material, a chemiluminescent material, a radioactive material, or an enzymatic substrate.  
     
     
         60 . The method of  claim 59 , wherein the bioluminescent material is green fluorescent protein GFP) or red fluorescent protein (RFP).  
     
     
         61 . The method of  claim 59 , wherein detection of the fluorescent dye or a visible dye is carried out by fluorometric or spectrophotometric measurement.  
     
     
         62 . A method of identifying a molecule that modulates the activity of a target cell component comprising co-encapsulating the molecule and a cell expressing a target cell component of interest and a detectable marker, in a microenvironment under conditions that allow the molecule and the target cell component to interact, and detecting an effect of the molecule on the activity of the cell component, thereby identifying a molecule that modulates the activity of the target cell component.