Library of a Collection of Cells

Abstract

The present invention relates to combinatorial gene expression libraries and methods for making these. Such libraries are useful in discovery of novel and/or enhanced metabolic pathways leading to the production of novel compounds for e.g., drug discovery and/or to the prosecution of known compounds in novel quantities or in novel compartments of the cells. The expression libraries in particular are composed of host cells capable of co-ordinated and controllable expression of large numbers of heterologous genes in the host cells.

1 - 111 . (canceled) 
     
     
         112 . A library comprising a collection of individual cells, the cells being denoted
 cell 1 , cell 2 , cell;, wherein i≧2,
 each cell comprising an expressible nucleotide sequence, wherein said expressible nucleotide sequence is comprised within 
   
       a) a concatemer of individual oligonucleotide cassettes, said concatemer comprising a nucleotide sequence of the following formula:
   [rs 2 -SP-PR-X-TR-SP-rs 1 ] n    
 wherein 
 rs 1  and rs 2  together denote a restriction site, 
 SP denotes a spacer of at least two bases, 
 X denotes an expressible nucleotide sequence, 
 PR denotes a promoter, capable of regulating the expression of X in the cell, 
 TR denotes a terminator, and 
 n≧2, or 
 
       b) a random combination of heterologous oligonucleotides having the general formula:
   [PR-X] 
 
       wherein X denotes an expressible nucleotide sequence, and PR denotes an independently controllable promoter being operably associated with X; 
       c) an expression cassette comprising a nucleotide sequence of the following formula:
   [rs 2 -SP-PR-X-TR-SP-rs 1 ] 
 wherein 
 rs 1  and rs 2  together denote a restriction site, 
 SP denotes a spacer of at least two bases, 
 PR denotes a promoter, capable of functioning in the cell, 
 X denotes an expressible nucleotide sequence, TR denotes a terminator, and 
 wherein said cell comprises at least two expression cassettes, wherein at least one of the expression cassettes comprises an expressible nucleotide sequence heterologous to the cell 
 
       and wherein at least one concatemer or independently controllable promoter or expression cassette of cell 1  is different from a concatemer or independently controllable promoter or expression cassette of cell 2 . 
     
     
         113 . The library according to  claim 112 , said library comprising a collection of sublibraries, wherein a sublibrary is a collection of individual cells having at least one phenotype in common, wherein the at least one phenotype is selected from the group consisting of the ability to grow on unusual substrates, the ability to grow on sublethal concentration of toxins, the ability to grow at a high temperature, the ability to grow at a low temperature, the ability to grow at elevated osmolality, the ability to grow at low osmolality, the ability to grow at high salinity, the ability to grow at low salinity, the ability to grow at elevated metal concentrations, the ability to grow at high CO 2  concentrations, the ability to grow at low CO 2  concentrations, the ability to grow at high O 2  concentrations, the ability to grow at low O 2  concentrations, the ability to provide special spectral properties, the ability to provide a special colour, the ability to have a deviating GST activity and the ability to have a deviating P450 activity. 
     
     
         114 . The library according to  claim 112 , wherein said cells are yeast cells, said yeast being selected from the group consisting of budding yeast,  Kluyveromyces marxianus, K. lactis, Candida utilis, Phaffia rhodozyma, Saccharomyces boulardii, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Candida paraffinica, Schwanniomyces castellii, Pichia stipitis, Candida shehatae, Rhodotorula glutinis, Lipomyces lipofer, Cryptococcos curvatus, Candida  spp. (e.g.  C. palmioleophila ),  Yarrowia lipolytica, Candida guilliermondii, Candida, Rhodotorula  spp.,  Saccharomycopsis  spp.,  Aureobasidium pullulans, Candida brumptii, Candida hydrocarbofumarica, Torulopsis, Candida tropicalis, Saccharomyces cerevisiae, Rhodotorula rubra, Candida flaveri, Eremothecium ashbyii, Pichia  spp.,  Kluyveromyces, Hansenula, Kloeckera, Pichia, Pachysolen  spp., or  Torulopsis bombicola  and mutants thereof. 
     
     
         115 . The library according to  claim 112 , wherein substantially all rs 1 -rs 2  sequences are recognised by the same restriction enzyme. 
     
     
         116 . The library according to  claim 112 , wherein at least one cassette in one cell comprises an intron between the promoter and the expressible nucleotide sequence. 
     
     
         117 . The library according to  claim 112 , wherein at least two expressible nucleotide sequences come from different expression states, wherein the different expression states represent at least two different tissues. 
     
     
         118 . The library according to  claim 112 , wherein at least one concatemer and/or random combination of heterologous oligonucleotides and/or expression cassette is integrated into the host genome. 
     
     
         119 . The library according to  claim 112 , wherein at least one concatemer and/or random combination of heterologous oligonucleotides and/or expression cassette is integrated into an artificial chromosome in the host cell. 
     
     
         120 . The library according to  claim 112 , wherein the random combinations are made from a two dimensional array of promoters and heterologous expressible nucleotide sequences. 
     
     
         121 . The library according to  claim 112 , comprising an externally controllable promoter. 
     
     
         122 . A method of producing a library comprising a collection of individual cells, comprising the steps:
 i) providing a population of nucleotide cassettes having the general formula
   [rs 2 -SP-PR-X-TR-SP-rs 1 ], 
   wherein   rs 1  and rs 2  together denote a restriction site,   SP denotes a spacer of at least two bases,   X denotes an expressible nucleotide sequence,   PR denotes a promoter, capable of regulating the expression of X in the cell,   TR denotes a terminator, and   ii) assembling random sub-sets of the cassettes into concatemers comprising at least two cassettes,   iii) ligating the concatemers into vectors,   iv) introducing vectors into host cells,   v) mixing at least two cells so that at least one concatemer of a first cell comprises a random sub-set of cassettes being different from a random sub-set of cassettes of a concatemer of a second cell.   
     
     
         123 . The method according to  claim 122 , whereby the vectors comprise an artificial chromosome. 
     
     
         124 . A method of producing a library comprising a collection of individual cells, comprising the steps:
 inserting at least two expressible nucleotides into the cloning site of at least two primary vectors comprising a cassette, the cassette comprising a nucleotide sequence of the general formula in 5′→3′ direction:
   [RS1-RS2-SP-PR-CS-TR-SP-RS2-RS1′).
 
   wherein   RS1 and RS1′ denote restriction sites,   RS2 denote a restriction site different from RS1 and RS1′,   SP denotes a spacer sequence of at least two nucleotides,   PR denotes a promoter, CS denotes a cloning site, and TR denotes a terminator;   i) excising the cassettes using at least a restriction enzyme specific for RS1, RS1′ RS2 and RS2′ obtaining expression cassettes having the general formula
   [rs 2 -SP-PR-X -TR-SP-rs 1 ], 
   wherein   rs 1  and rs 2  together denote a restriction site, and wherein X denotes an expressible nucleotide sequence,   ii) inserting the expression cassettes into a vector,   iii) transferring the expression cassettes into at least two host cells, and   iv) mixing at least two host cells having different cassettes.   
     
     
         125 . A method of producing a library comprising a collection of individual cells, comprising the steps:
 i) providing at least one expressible nucleotide sequence,   ii) ligating at least one expressible nucleotide sequence to a controllable promoter capable of functioning in a host cell obtaining a first expression construct,   iii) ligating at least one expressible nucleotide sequence to another independently controllable promoter capable of functioning in a host cell, obtaining a second expression construct,   iv) inserting constructs of step ii) and iii) into at least two host cells,   v) mixing at least two cells having a different combination of independently controllable promoter and expressible nucleotide sequences.   
     
     
         126 . An expression library obtainable by the method of  claim 122 . 
     
     
         127 . An expression library obtainable by the method of  claim 123 . 
     
     
         128 . An expression library obtainable by the method of  claim 124 . 
     
     
         129 . An expression library obtainable by the method of  claim 125 .