Protein activity screening of clones having DNA from uncultivated microorganisms

Abstract

Disclosed is a process of screening clones having DNA from an uncultivated microorganism for a specified protein, e.g. enzyme, activity by screening for a specified protein, e.g. enzyme, activity in a library of clones prepared by (i) recovering DNA from a DNA population derived from at least one uncultivated microorganism; and (ii) transforming a host with recovered DNA to produce a library of clones which is screened for the specified protein, e.g. enzyme, activity.

What is claimed is:  
     
         1 . A protein having a desired activity produced using a method comprising: 
 culturing a gene expression library comprising a pool of expression constructs, each expression construct comprising a vector containing one or more cDNA or genomic DNA fragments, wherein the cDNA or genomic DNA fragments in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the cDNA or genomic DNA fragments are operably-associated with one or more regulatory regions that drives expression of genes encoded by the cDNA or genomic DNA fragments in an appropriate host organism;    detecting the protein activity encoded by the cDNA or genomic DNA fragments;    recovering the constructs expressing the desired protein activity; and    expressing the protein from the recovered construct.    
     
     
         2 . The protein of  claim 1 , wherein the protein activity is an enzymatic activity.  
     
     
         3 . The protein of  claim 2 , wherein the enzymatic activity is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase activity.  
     
     
         4 . The protein of  claim 1 , wherein the donor organisms are eukaryotic organisms.  
     
     
         5 . The protein of  claim 1 , wherein the donor organisms are microorganisms.  
     
     
         6 . The protein of  claim 4 , wherein the microorganisms are derived from an environmental sample.  
     
     
         7 . The protein of  claim 4 , wherein the microorganisms are a mixed population of uncultured organisms.  
     
     
         8 . The protein of  claim 1 , wherein the DNA fragment comprises one or more operons, or portions thereof.  
     
     
         9 . The protein of  claim 7 , wherein the operon or portions thereof encodes a complete or partial metabolic pathway.  
     
     
         10 . The protein of  claim 1 , wherein the organisms are fungi.  
     
     
         11 . The protein of  claim 1 , wherein the organisms are algae.  
     
     
         12 . The protein of  claim 1 , wherein the organisms are protozoan.  
     
     
         13 . The protein of  claim 5 , wherein the organisms are extremophiles  
     
     
         14 . The protein of  claim 12 , wherein the the organisms are thermophiles, hyperthermophiles, psychrophiles, or psychrotrophs.  
     
     
         15 . The protein of  claim 1 , wherein the host organism is a bacterial cell.  
     
     
         16 . The protein of  claim 14 , wherein the bacterial cell is an  E. coli , Bacillus, Streptomyces, or  Salmonella typhimurium  cell.  
     
     
         17 . The protein of  claim 1 , wherein the host organism is a fungal cell.  
     
     
         18 . The protein of  claim 16 , wherein the fungal cell is a yeast cell.  
     
     
         19 . The protein of  claim 1 , wherein the host cell is a Drosophila S2 or a Spodoptera S9 cell.  
     
     
         20 . The protein of  claim 1 , wherein the host organism is an animal cell.  
     
     
         21 . The protein of  claim 19 , wherein the animal cell is a CHO, COS or Bowes melanoma cell.  
     
     
         22 . The protein of  claim 1 , wherein the host cell is a viral cell.  
     
     
         23 . The protein of  claim 21 , wherein the viral cell is an adenovirus.  
     
     
         24 . The protein of  claim 1 , wherein the host organism is a plant cell.  
     
     
         25 . The protein of  claim 1 , wherein the step of expressing in the method comprises: 
 (a) transforming an appropriate host organism with the recovered constructs; and    (b) recovering the protein expressed by the host organism.    
     
     
         26 . A nucleic acid sequence having a desired activity identified using a method comprising: 
 culturing a gene expression library comprising a pool of expression constructs, each expression construct comprising a vector containing one or more nucleic acid sequences, wherein the nucleic acid sequences in the pool of expression constructs are derived from a plurality of species of donor organisms, and wherein the nucleic acid sequences are operably-associated with one or more regulatory regions that drives expression of genes encoded by the nucleic acid sequences in an appropriate host organism;    detecting the activity encoded by the nucleic acid sequences;    recovering the constructs expressing the desired activity; and    isolating the nucleic acid sequences having the desired activity.    
     
     
         27 . The nucleic acid sequence of  claim 26 , wherein the desired activity is an enzymatic activity.  
     
     
         28 . The nucleic acid sequence of  claim 27 , wherein the enzymatic activity is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase activity.  
     
     
         29 . The nucleic acid sequence of  claim 26 , wherein the donor organisms are eukaryotic organisms.  
     
     
         30 . The nucleic acid sequence of  claim 26 , wherein the donor organisms are microorganisms.  
     
     
         31 . The nucleic acid sequence of  claim 30 , wherein the microorganisms are derived from an environmental sample.  
     
     
         32 . The nucleic acid sequence of  claim 30 , wherein the microorganisms are a mixed population of uncultured organisms.  
     
     
         33 . The nucleic acid sequence of  claim 26 , wherein the nucleic acid sequence comprises one or more operons, or portions thereof.  
     
     
         34 . The nucleic acid sequence of  claim 33 , wherein the operon or portions thereof encodes a complete or partial metabolic pathway.  
     
     
         35 . The nucleic acid sequence of  claim 26 , wherein the organisms are fungi.  
     
     
         36 . The nucleic acid sequence of  claim 26 , wherein the organisms are algae.  
     
     
         37 . The nucleic acid sequence of  claim 26 , wherein the organisms are protozoan.  
     
     
         38 . The nucleic acid sequence of  claim 30 , wherein the microorganisms are extremophiles  
     
     
         39 . The nucleic acid sequence of  claim 30 , wherein the microorganisms are thermophiles, hyperthermophiles, psychrophiles, or psychrotrophs.  
     
     
         40 . The nucleic acid sequence of  claim 26 , wherein the host organism is a bacterial cell.  
     
     
         41 . The nucleic acid sequence of  claim 41 , wherein the bacterial cell is an  E. coli , Bacillus, Streptomyces, or  Salmonella typhimurium  cell.  
     
     
         42 . The nucleic acid sequence of  claim 26 , wherein the host organism is a fungal cell.  
     
     
         43 . The nucleic acid sequence of  claim 42 , wherein the fungal cell is a yeast cell.  
     
     
         44 . The nucleic acid sequence of  claim 26 , wherein the host cell is a Drosophila S2 or a Spodoptera S9 cell.  
     
     
         45 . The nucleic acid sequence of  claim 26 , wherein the host organism is an animal cell.  
     
     
         46 . The nucleic acid sequence of  claim 45 , wherein the animal cell is a CHO, COS or Bowes melanoma cell.  
     
     
         47 . The nucleic acid sequence of  claim 26 , wherein the host cell is a viral cell.  
     
     
         48 . The nucleic acid sequence of  claim 47 , wherein the viral cell is an adenovirus.  
     
     
         49 . The nucleic acid sequence of  claim 26 , wherein the host organism is a plant cell.