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.

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.