Library of a collection of cells

• US 20040241672A1
• Filed: 01/06/2004
• Published: 12/02/2004
• Est. Priority Date: 01/25/2001
• Status: Active Grant

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Nielsen, Soren S V, Sorensen, Alexandra M P Santana, Goldsmith, Neil
• Granted Patent: US 7,838,287 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention relates to combinatorial gene expression libraries and methods for making these. The library comprises a collection of individual cells, the cells being denoted cell1,cell2 . . . , cell, wherein i>2, each cell comprising at least one concatemer of individual oligonucleotide cassettes, each concatemer comprising a nucleotide sequence of the following formula: (rs2-SP-PR-X-TR-SP-rs1) n wherein rs1 and rs2 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, and wherein at least one concatemer of cell1 is different from a concatemer cell2. 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 production 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 gene in the host cells co-ordinated and controllable expression of large numbers of heterologous gene i host cells.

Claims

1-111. -111. (Cancelled).

112. A library comprising a collection of individual cells, the cells being denoted

cell₁, cell₂, . . . , cell_i, 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;

[rs₂-SP-PR-X-TR-SP-rs₁]_n

wherein

rs₁ and rs₂ 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 cassettes comprising a nucleotide sequence of the following formula;

[rs₂-SP-PR-X-TR-SP-rs₁]

wherein

rs₁ and rs₂ 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 cell₁ is different from a concatemer or independently controllable promoter or expression casette of cell₂.

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 CO₂ concentrations, the ability to grow at low CO₂ concentrations, the ability to grow at high O₂ concentrations, the ability to grow at low O₂ 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 rs₁-rs₂ 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 [rs₂-SP-PR-X-TR-SP-rs₁], wherein rs₁ and rs₂ 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.

126. An expression library obtainable by the method of claim 122.

127. An expression library obtainable by the method of claim 123.

124. A method of producing a library comprising a collection of individual cells, comprising the steps:

i) 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.

ii) excising the cassettes using at least a restriction enzyme specific for RS1, RS1′

RS2 and RS2′

obtaining expression cassettes having the general formula [rs₂-SP-PR-X-TR-SP-rs₁], wherein rs₁ and rs₂ together denote a restriction site, and wherein X denotes an expressible nucleotide sequence,

iii) inserting the expression cassettes into a vector,

iv) transferring the expression cassettes into at least two host cells, and

v) mixing at least two host cells having different cassettes.

128. An expression library obtainable by the method of claim 124.

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.

129. An expression library obtainable by the method of claim 125.