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.

1 - 15 . (canceled)  
     
     
         16 . A method for identifying bioactivities or biomolecules using high throughput screening of nucleic acid comprising: 
 a) generating a normalized environmental gene library containing a plurality of clones in a host, wherein the nucleic acid for generating the library is naturally occurring and obtained from a mixed population of uncultured organisms;    b) encapsulating a bioactive substrate and at least one clone from a) in a gel microdroplet, wherein a bioactivity or biomolecule produced by the clone is detectable by a change in fluorescence of the substrate prior to contacting with the at least one clone as compared to after the contacting; and    c) screening the microdroplet with an assay or an analyzer that detects the presence therein of the change in fluorescence of the substrate, wherein the change indicates the identity of the bioactivity or biomolecule.    
     
     
         17 . The method of  claim 16 , further comprising transferring a plurality of clones to a myceliate bacteria or a myceliate fungi.  
     
     
         18 . The method of  claim 16 , wherein in the host is selected from the group consisting of bacteria, yeast, fungi, myceliate bacteria, and myceliate fungi.  
     
     
         19 . The method of  claim 16 , wherein the bioactivity is provided by an enzyme that is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidazes, lignin peroxidases, diarylpropane peroxidazes, epozide hydrolazes, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.  
     
     
         20 . The method of  claim 16 , wherein the gene library is an expression library.  
     
     
         21 . The method of  claim 20 , wherein the expression library contains DNA obtained from extremophiles.  
     
     
         22 . The method of  claim 21 , wherein the extremophiles are thermophiles.  
     
     
         23 . The method of  claim 21 , wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.  
     
     
         24 . The method of  claim 16 , wherein the bioactive substrate comprises C12FDG.  
     
     
         25 . The method of  claim 16 , wherein the bioactive substrate comprises a lipophilic tail.  
     
     
         26 . The method of  claim 16 , wherein the clones are heated before step b).  
     
     
         27 . The method of  claim 26 , wherein the heating is at about 70° C.  
     
     
         28 . The method of  claim 27 , wherein the heating occurs for about 30 minutes.  
     
     
         29 . The method of  claim 16 , wherein the analyzer comprises a fluorescent analyzer.  
     
     
         30 . The method of  claim 29 , wherein the fluorescent analyzer is a FACS apparatus.  
     
     
         31 . The method of  claim 16 , wherein the library is biopanned before step b).  
     
     
         32 . The method of  claim 31 , wherein the myceliate bacteria is a  Streptomyces  sp.  
     
     
         33 . The method of  claim 31 , wherein the  Streptomyces  sp. is  Streptomyces venezuelae.    
     
     
         34 . The method of  claim 16 , further comprising co-encapsulating an indicator cell in step c).  
     
     
         35 . The method of  claim 16 , wherein the analyzer is a chromogenic analyzer.  
     
     
         36 . The method of  claim 16 , wherein the assay is an immunoassay.