WO2011088443A2 - Banques co-existantes pour le développement de phénotypes microbiens complexes - Google Patents

Banques co-existantes pour le développement de phénotypes microbiens complexes Download PDF

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WO2011088443A2
WO2011088443A2 PCT/US2011/021505 US2011021505W WO2011088443A2 WO 2011088443 A2 WO2011088443 A2 WO 2011088443A2 US 2011021505 W US2011021505 W US 2011021505W WO 2011088443 A2 WO2011088443 A2 WO 2011088443A2
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cells
library
vector
population
libraries
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WO2011088443A3 (fr
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Sergios Nicolaou
Stefan Gaida
Eleftherios T. Papoutsakis
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University Of Delaware
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • This invention relates generally to multi-library systems in microbial cells.
  • embodiments of the invention relate to the use of co-existing libraries to identify and develop complex phenotypes in microbial cells.
  • the resultant microbial strains can be used in industrial bioprocessing appl ications, such as the development of biofuels or bioremediation .
  • cellulosic biomass can be converted to biofuels and other useful chemicals.
  • biomass is collected a nd treated to release tangled lignin-cellulose fiber via am monia explosion, weak acid boiling, or steam treatment (Hahn-Hagerdal, Galbe et al. 2006).
  • the suspension is then digested by cellulolytic enzymes that hydrolyze the hemicellulosic a nd cellulosic biomass to five- and six- carbon sugars. These sugars can then be utilized for chemica l production .
  • the suga rs can be fermented by alcohol producing microorga nisms such as Escherichia coli (Ingram, Conway et al.
  • Clostridium acetobutylicum Zhao, Tomas et al. 2005
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Zymomonas mobilis Steppha nopoulos 2007
  • a common feature of these bioprocesses is the presence of molecules, as a resu lt of biomass pretreatment, that may be inhibitory to the fermentation process for the production of the desirable molecule(s).
  • Another common feature is the inhibitory nature of the desirable product (such as ethanol or butanol) or byproducts, which typically limit product titers, affect fermentation performance and operational options (e.g., continuous vs. batch), and profoundly impact process economics.
  • Development of host strains of microorganisms with superior tolerance to particular chemicals and stressful bioprocess conditions is an important and widely recognized goal, not only in the context of the production of biofuels and chemicals from biomass, but also for many bioremediation applications (Hosokawa, Park et al. 2002; Fernandes, Ferreira et al. 2003; Santos, Benndorf et al. 2004; Gupta, Singh et al. 2006).
  • Tolerance of microorganisms to chemicals is a complex, multigenic trait and is affected by several process parameters such as pH, temperature, osmotic pressure, and the presence of other small molecules.
  • host strains In order to develop industrial applications in the production of chemicals and biofuels, host strains must be developed that have the ability to produce the desirable chemicals at high concentrations.
  • bioremediation of toxic chemicals will require strains which must be capable of degrading the desirable chemical(s) at high concentrations, i.e., they must also be tolerant to these chemicals. It is likely that the mecha nism of tolerance varies widely among classes or even the same class of chemicals such as alcohols or halogenated hydrocarbons. Thus, it is critical to be able to develop strains which are tolerant to the desirable chemical(s) .
  • Tolerance to a toxic chemical is one of many possible complex phenotypes of practica l biotechnological interest.
  • examples of other such phenotypes include cells with novel capabilities in terms of substrate utilization so that they can directly utilize pretreated cellulose or hemicellulose to produce any desirable chemical or biofuel in the context of a biorefinery.
  • Another example is the ability of an organism to tolerate extensive anoxia or hyperoxia as may be encountered in bioprocessing applications.
  • An additional example is the development of prokaryotes (i.e., bacteria) which possess desirable eukaryotic traits (such as a complex yeast-cell wall or the ability to extensively glycosylate mammalian proteins) without loss of their core bacterial capabilities.
  • the robustness and prolonged productivity of the biocatalyst (the cells) under realistic bioprocessing conditions is an important issue.
  • the ability of cells to withstand "stressful" bioprocessing conditions without loss of productivity is a most significant goal .
  • Such conditions include: toxic substrates, accumulation of toxic products & byproducts, high or low pH, or high salt concentrations as encountered in most applications for the production of chemicals and biofuels as well as in bioremediation applications.
  • the difficulty is that the desirable phenotypic trait is determined by several genes or a complex regulatory circuit.
  • the ability to generate specific cellular phenotypes, which are determined by complex interactions among genes and other genetic and epigenetic elements is an important goal in modern biology a nd biotechnology.
  • An embod iment of the present invention provides a population of cells comprising at least two co-existing libraries.
  • Each cell in the population comprises a vector from each library, with each vector containing a gene insert.
  • the vectors have origins of replication that are compatible but different from each other.
  • Host cells that comprise multiple co-existing and interacting libraries preferably exhibit desired phenotypes, such as enhanced tolerance to a toxic chemical, a novel catabolic capabil ity, or a novel anabolic capabil ity, particularly phenotypes that can be used in industrial bioprocessing applications.
  • the present invention provides methods for constructing a population of cells having at least two co-existing libraries, comprising transforming a popu lation of cells with vectors from each library.
  • Each vector contains a gene insert and the vectors have origins of replication that a re compatible but different from each other. Additional methods com prise identifying and developing cells that exhibit a desired phenotype as a result of the interaction of the co-existing libraries.
  • Figure 1 Compleme ntation of dual knockouts with plasmid a nd fosmid libraries.
  • the approximate ch romosomal positions of knocked out genes in an E. coli chromosome are shown for each knockout combination in Examples 1 and 2.
  • the complementation of the dual knockouts in strain E. coli K- 12 MG1655, MapDMapE and AdapAAIysA are shown, which were complemented with two plasm id libraries.
  • the complementation of the dual knockouts in the strain E. coli Epi300Tl R , AdapBAIysA and AdapDAIysA are shown, which were complemented with a fosmid library and a plasmid library.
  • FIG. 2 Ethanol tolerant phenotype ch aracterization . Identified clones of a dual library enrichment were sub-cloned and cultivated under 48g/L ethanol in Example 2. A total of 4 biologica l replicates each with three technical replicates were performed . One typical growth profile is shown on the left side. The error bars indicate the range of the three technical replicates. Long time survivability was determ ined by counting CFU's of cells plated after 48 and 96 hours ethanol exposure. The results are shown in the right chart, whereby the error bars indicate the standard deviation of all replicates. Figure 3: The de novo lysine biosynthesis pathway of E. coll.
  • the target genes that were knocked out in pairs in Exam ples 1 and 2 are dapA, dapB, dapD, dapE, and lysA .
  • the double knockouts AdapAAlysA and AdapDAdapE were generated in E. coli K-12 MG1655.
  • the double knockouts dapB NysA and [AdapD NysA were constructed in Epi300 T1 R .
  • Embod iments of the present invention provide novel methods for identifying and developing complex phenotypes in microbial cells by generating m ultiple co-existing and interacting libra ries in a single organism .
  • Methods according to the present invention enable the development of complex multigenic phenotypes via the interaction of dista l genes, and can allow for the interactions of multiple genomes, thus generating complex multi-transgenic phenotypes that can be beneficial in industrial and research applications. For example, construction of two or more co-existing and interacting E.
  • coli genomic libraries of various sizes of inserts (e.g . , between 3 kb and 8 kb) on vectors (e.g ., plasm ids) with com patible origins of replication in a host cell (e.g. , E. coli) can be used to identify and develop microbial phenotypes that require multigenic interactions, especially interactions between distantly located genomic loci .
  • appl icants used methods according to the invention to identify genes that can enhance the ethanol tolerance of E. coli by screening a combination of a fosmid library and a plasmid library that co-exist in a single cell. Sequencing identified the beneficial genes present in the enriched inserts, and the enhanced phenotype provided by these genes was demonstrated by better growth in ethanol as well as by higher survivability after 48 a nd 96 hours of ethanol exposure in comparison to the parent strain .
  • These embodiments of the present invention can be extended to other vector combinations, and can have more than two vectors present in a cell at a time.
  • the vectors can have different sizes, and can conta in d ifferent genomic inserts.
  • gene deletions can be made prior to the introduction of the libraries to enhance phenotypes and increase biochemical and biological capabilities further.
  • examples are included that extend uses of the present invention to multiple libra ries containing gene inserts from multiple species in one cell. Also, examples provided herein disclose uses of the present invention in relation to other phenotypes, particularly acid stress tolerance and oxidative stress tolerance.
  • a key aspect of the present invention provides a population of cells com prising at least two co-existing genomic, sub-genomic, or metagenomic libraries.
  • a "library” refers to a collection of vectors, each of which ca rries a gene insert (i.e., a fragment of DNA) from a genome, sub-genome, or metagenome of one or more source organisms, such that the collection of gene inserts present in the collection of vectors represents the genome, sub-genome, or metagenome of the one or more source orga nisms .
  • the libra ries are selected from the group consisting of genomic libraries, subgenomic libraries (e.g ., selected subgenomic libraries, enriched subgenomic libraries, or synthetically constructed subgenomic libra ries), metagenomic libraries (e.g., natural metagenomic libra ries or enriched metagenomic libraries), and com binations thereof.
  • the co-existing libraries can be constructed from the same genome or from different genomes.
  • the libraries can be constructed from orga nisms selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells, animal cells, and com binations thereof.
  • each cell in the population of cells preferably comprises at least two co-existing vectors from each of the at least two libraries (i.e. , one vector from each library co-existing in each cell). Each vector conta ins a gene insert.
  • Each gene insert in each vector preferably has a size between about 2 kb to about 52 kb (e.g ., a plasmid may contain an insert of about 3 kb to about 8 kb, a fosmid may contain an insert of about 35 to about 45 kb, or a cosmid may contain an insert of about 37 kb to about 52 kb). It is important that the multiple vectors present in each host cell are compatible with each other. As used herein, vectors that are "com patible" with each other in a single cell can co-exist and be co- expressed in the single cell.
  • each vector has an origin of replication that is compatible (i.e., replication can be initiated at both origins of replication within the cell) but different from each other (e.g ., a first vector has a first origin of replication and a second vector has a second orig in of replication, wherein the first origin of replication and the second origin of replication are different from each other) .
  • Each vector also preferably has a selection marker (e.g. , an antibiotic resistance) that is different from each other, so that the presence of all vectors in a cell ca n be confirmed by screening for each vector's selection ma rker.
  • At least one host cell in the population of cells preferably exhibits a desired phenotype as a result of the interaction(s) of the multiple libraries.
  • the vectors are selected from the group consisting of plasmids, fosmids, cosmids, other types of vectors known in the art, and combinations thereof.
  • two plasmid libraries each having inserts of about 3 kb to about 8 kb can be used .
  • a la rge fosmid library having inserts of about 35 kb to about 45 kb together with a plasmid library having inserts of about 3 kb to about 5 kb can be used .
  • a fosmid is a vector based on the F- factor replication origin (Kim, Shizuya et al .
  • a cosmid is a type of hybrid plasmid that contains cos sequences, which are DNA sequences originally from the Lambda phage. Cosmids are capable of containing about 37 to about 52 kb DNA.
  • the population of cells i .e., the host cells, are selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells, and a nimal cells, or, alternatively, from the group of genera consisting of Bacillus, Lactobacillus,
  • a population of cells comprises two co-existing genomic, sub-genomic, or metagenomic libraries (i.e., a first library and a second library) and each cell in the population of cells comprises a vector from each of the two libraries (i.e., a first vector from the first library contains a first gene insert and a second vector from the second library contains a second gene insert for a total of two gene inserts per cell).
  • the first gene insert and second gene insert each originates from a genome or sub-genome, which can be identical or different.
  • the first vector has a first origin of replication and the second vector has a second origin of replication, wherein the first origin of replication and the second origin of replication are compatible (i.e., replication can be initiated at both origins of replication within the cell) but different from each other.
  • the first vector and the second vector are compatible with each other, i.e., they can co-exist and be co-expressed in the same cell.
  • the first vector has a first selection marker and the second vector has a second selection marker, wherein the first selection marker and the second selection marker are different from each other.
  • At least one host cell in the population of cells preferably exhibits a desired phenotype as a result of the interaction of the at least two libraries, e.g., the interaction of expression of the gene insert in the first vector with expression of the gene insert in the second vector.
  • a "desired phenotype” may include, for example, a phenotype that is beneficial in industrial and research applications, such as the ability to withstand stressful bioprocessing conditions without loss of productivity, e.g., enhanced tolerance to a toxic chemical compared to the wild-type or parent strain of the host cell, a novel catabolic capability, a novel anabolic capabi lity, enhanced tolerance to accumulation of toxic products and byproducts compared to the wild-type or parent strain of the host cell, or enhanced tolerance to high or low pH or high salt concentrations compared to the wild-type or parent strain of the host cell.
  • a phenotype that is beneficial in industrial and research applications such as the ability to withstand stressful bioprocessing conditions without loss of productivity, e.g., enhanced tolerance to a toxic chemical compared to the wild-type or parent strain of the host cell, a novel catabolic capability, a novel anabolic capabi lity, enhanced tolerance to accumulation of toxic products and byproducts compared to the wild-type or parent strain of the
  • phenotypes include novel capabilities in terms of substrate utilization (e.g., the ability to directly utilize pretreated cellulose and/or hemicellulose to produce any desirable chemical or biofuel in the context of a biorefinery), the ability to tolerate extensive anoxia or hyperoxia in bioprocessing applications, or desirable eukaryotic traits (e.g., a complex yeast-cell wall or the ability to extensively and correctly glycosylate mammalian proteins).
  • substrate utilization e.g., the ability to directly utilize pretreated cellulose and/or hemicellulose to produce any desirable chemical or biofuel in the context of a biorefinery
  • desirable eukaryotic traits e.g., a complex yeast-cell wall or the ability to extensively and correctly glycosylate mammalian proteins.
  • a "beneficial gene insert” is a gene insert with expression that results in a desired phenotype.
  • the population of cells is large enough so that the population includes at least the number of clones necessary for at least 99% coverage of all possible combinations of all inserts of the at least two libraries to be represented in the population (e.g., so that at least 99% of the possible binary combinations of the gene inserts of two libraries are represented in the population) .
  • the population of cells is large enough so that the population includes at least the number of clones necessary for at least 75% , 85%, 90%, 95%, or 99% coverage of all combinations of all gene inserts of the at least two libra ries are represented in the population (e.g .
  • N ln(l - P)/ln(l - f), where N is the number of clones, P is the coverage probability, and f is the fraction of the insert size relative to the entire genome (Clarke and Carbon 1976).
  • N the number of clones
  • P the coverage probability
  • f the fraction of the insert size relative to the entire genome (Clarke and Carbon 1976).
  • Table 1 illustrates the number of clones required to generate a n E. coli genom ic library for any desirable genome % coverage and various average library insert sizes in the plasm id or fosmid based on the formula shown in Clarke, L. and J. Carbon ( 1976), "Colony Bank Conta ining Synthetic Col El Hybrid Plasmids Representative of Entire Escherichia Coli Genome," CeM 9( 1 ) : 91 -99.
  • the population of cells can be screened for a desired phenotype that is the result of an interaction of the gene inserts from the two libraries.
  • the popu lation of cells can a lso be screened to identify interactions between the two inserts (which generally are genes/genetic loci that are d istantly located on the chromosome of an orga nism) necessary for the development of a trait or a complex phenotype.
  • the interaction of two inserts in two plasmid-based genomic libraries was tested by their ability to "fix" (i .e., complement) two genetic disruptions which were generated by design in the lysine biosynthesis pathway of E. coli.
  • the population of cells comprises three libraries (i.e., a third library co-existing with the first and second libraries), with each cell in the population comprising a third vector from the third library.
  • the population of cells is preferably large enough so that all possible trinary combinations of all gene inserts of the three libraries are represented in the population. In other embodiments, the population of cells comprises more than three libraries.
  • a key aspect of the invention provides a host cell comprising at least two vectors from at least two libraries, with each vector conta ining a gene insert and each vector having an origin of replication, wherein the origins of replication of each vector are compatible but different from each other.
  • each vector has a selection marker, wherein the selection markers are different from each other.
  • the host cell preferably exhibits a desired phenotype, such as those phenotypes described above.
  • a host cell comprises a first vector from a first library and a second vector from a second library, with each vector containing a gene insert.
  • the first vector has a first origin of replication and the second vector has a second origin of replication, wherein the first origin of replication and the second origin of replication are compatible different from each other.
  • the first vector has a first selection marker and the second vector has a second selection marker, wherein the first selection ma rker and the second selection marker are different from each other.
  • the first vector and the second vector are compatible with each other, i.e. , they can co-exist and be co-expressed in the same cell.
  • the vectors are plasmids, each gene insert is preferably about 3 kb to about 8 kb, the first and second libraries a re genomic Escherichia coli libraries, and the host cell is a stra in of Escherichia coli.
  • the first vector is a plasm id and the second vector is a fosmid
  • the plasmid has a gene insert of about 3 kb to about 8 kb
  • the fosmid has a genetic insert of about 35 kb to about 45 kb
  • a nd the host E. coli cell exhibits enhanced tolerance in ethanol compared to the parent strain of the host cell.
  • Additional aspects of the invention provide methods for constructing a population of cells having at least two co-existing genomic, subgenomic, or
  • metagenomic libraries e.g. , a first library and a second library
  • the coexisting libraries may be constructed from the same genome or from different genomes. Additional embodiments include the further step of screening the population of cells for at least one cell having beneficial gene inserts that interact to generate a desired phenotype.
  • the desired phenotype may be selected from the group consisting of enhanced tolerance to a toxic chem ical compared to a wild-type or parent strain of the cell, a novel catabolic capabi lity, a novel anabolic capabil ity, and combinations thereof.
  • the desired phenotype comprises enhanced tolerance to a chemical that is used in an infrastructure l application, such as a biofuel application, industrial biocatalysis, or bioremediation.
  • Embodiments of a method according to the present invention may further include the add itional step of knocking out functions in the population of cells and screening for gene inserts that restore the knocked-out functions.
  • Additional embodiments of the present invention include the step of generating and identifying host cells having enhanced phenotypes, e.g., enhanced tolerance to a chemical that is generally toxic to microbes (e.g., ethanol) relative to wild-type or parent strains, due to the interaction of multiple co-existing libraries present in the population of cells.
  • This can be accomplished by culturing a population of cells conta ining multiple co-existing libraries in a medium that contains a chemical that is generally toxic to microbes (e.g., ethanol). The cells can then be cultured in media having increasing amounts of the toxic chemical.
  • the surviving cells i.e., the cells that are tolerant to increasing amounts of the toxic chemical
  • the beneficial gene inserts in the vectors of the surviving cells can be identified.
  • a "specialized library" of vectors containing the beneficial gene inserts can be constructed.
  • the beneficial gene inserts can be cloned together into host cells by transforming the host cells with specialized libraries comprising multiple vectors that contain the beneficial gene inserts (e.g., CLONE1 and CLONE2 as described in Example 2).
  • the transformed host cells preferably exhibit enhanced tolerance to the chemical that is normally toxic to the wild-type or parent strain of the host cells, due to the interaction of beneficial gene inserts present in the co-existing libraries.
  • host cells comprising multiple co-existing libraries can be identified for abilities to withstand other stressful bioprocessing conditions, such as high or low pH, or high salt concentrations.
  • novel phenotypes can be generated, such as novel capabilities in terms of substrate utilization (e.g., the ability to directly utilize pretreated cellulose and/or hemicellulose to produce any desirable chemical or biofuel in the context of a biorefinery), the ability to tolerate extensive anoxia or hyperoxia in bioprocessing applications, or desirable eukaryotic traits (e.g., a complex yeast-cell wall or the ability to extensively and correctly glycosylate mammalian proteins) .
  • Alternative embodiments may include the step of creating gene deletions in at least one of the libraries by the use of a transposon library, i.e., a collection of coexisting libraries can be introduced in a cell after gene deletions have been created by genomic means such as by the use of a transposon library. These deletions might disrupt competing biochemical pathways and can enhance production of any desirable product or produce a more desirable phenotype. The addition of multiple libraries after the transposon deletion can then improve the cellular phenotype further.
  • a transposon library i.e., a collection of coexisting libraries can be introduced in a cell after gene deletions have been created by genomic means such as by the use of a transposon library.
  • more than two genomic, subgenomic, or metagenomic libraries can be screened for traits that require more than two interacting, distantly located genetic loci.
  • genomic libraries of smal l (3-5 kb) inserts this can be readily implemented even with smal l inserts using enriched or specialized subgenomic libraries that contain genetic loci of interest to the desirable trait.
  • fosmids or cosmids that can carry la rge (30-52 kb) inserts may be employed.
  • Such fosmid or cosmid libraries can be screened together with plasmid libraries of 5-8 kb inserts.
  • libraries from two different organisms may be employed to examine interactions among genetic loci of different genomes. Some host cell modifications may be needed if all the
  • heterologous or allogeneic genes are to be expressed. This ca n be addressed in several ways, such as by expressing the heterologous library inserts using an inducible or constitutive promoter of the host strain, or by modifying the transcriptional machinery of the host cell to recognize the promoters of the heterologous library.
  • metagenomic libraries may be employed, especially libraries that are enriched for a specific desirable phenotype. It has been estimated that there are 10 30 microbes in the environment (Turnbaugh and Gordon 2008) and 10 31 phage particles that ca n shuttle genomic information between species (Dinsda le, Edwa rds et al . 2008). This enormous genetic diversity remains to be explored . Microbia l com munities evolve to survive in their specific environment and ca n provide the genetic material to identify novel genes with desirable functions. However, the grand majority of bacteria and other microbes cannot be cultured and thus the communities cannot be reproduced in the laboratory (Schmeisser, Steele et al. 2007).
  • Metagenomics aims to study the dynamic relationships ava ilable in a specific comm unity and determine the interactions and processes that allow for survival in that particular environment. To do this, the entire gene pool available is either sequenced (via next-generation sequencing (Mardis 2008 ; Gilad, Pritchard et al. 2009)), or cloned into vectors (BACs or cosmids) and screened in suitable hosts for specific traits. Metagenomic libra ries can be very useful in creati ng tolerant or other phenotypes by identifying novel genes from organisms that th rive in challenging environments or harsh conditions.
  • Jin et al. utilized a library to identify genes with glyphosate resistance (Jin, Lu et al, 2007).
  • Glyphosate a strong herbicide, is an analogue of phosphoenolpyruvate and a competitive inhibitor of 5-enolpyruvylshikimate 3- phosphate (EPSP).
  • EPP 5-enolpyruvylshikimate 3- phosphate
  • They utilized DNA extracted from soil exposed to the herbicide for over 15 years to construct a metag enomic library which was screened in an E. coli aroA knockout (Jin, Lu et al. 2007) .
  • One novel gene identified in this study exhibited higher resista nce to glyphosate up to 150 m M .
  • Chauhan et al. constructed an E.
  • Efficient library construction preferably uses high quality DNA that contains multiple copies of all genes in the metagenome, large inserts in the cloning vectors, and high tra nsformation efficiency in the host to facilitate coverage of all genes (Schloss and bottlesman 2003; Gabor, Liebeton et al. 2007) . It is often necessa ry to enrich for genomes, and techniques such as GC or
  • bromodeoxyuridine enrichment isotope labeling or enrichment based on a metabolite have been used (Schloss and bottlesman 2003 ; Gabor, Liebeton et al . 2007;
  • the inserts For efficient metagenomic library screening, it is preferable to have the inserts also be expressed in the host. Thus, the host can recognize multiple transcription factors and be promiscuous in its expression of transgenic sequences. It is also possible to include promoters in the plasmid l ibraries to ensure tra nscription and m inimize the number of genes which are not getting expressed.
  • Example 1 The approach described in Example 1 is based on co-existing plasmid libraries with relatively small libra ry inserts. By using this approach , multiple and distant genetic disruptions in the lysine biosynthesis pathway of E, coli were complemented (i.e., repaired) using E. coli libraries.
  • Another multi-library approach described in Example 2, is based on the co-existence of a fosmid-based libra ry with large inserts together with a plasmid-based library of small inserts.
  • Embod iments of the present invention described in Example 2 provide an ethanol tolerant phenotype in E. coli. These approaches facilitate the development of ethanol- tolerant E. coli (relative to the wild-type or parent strain of the host cell). Following selection of cells harboring library inserts under high ethanol concentrations, sequencing of the selected library identified the genes, which were reconfirmed by expressing the genes or program genes on suitably designed vectors for
  • Embodiments of the present invention described in Example 3 provide an ethanol tolerant phenotype in E. coli using genes and/or genetic loci from an ethanol-tolerant, Gram-positive lactobacillus organism, thus facilitating the development of ethanol-tolerant E. coli (relative to the wild-type or parent strain of the host cell) based on lactobacillus libraries. Furthermore, according to embodiments of the present invention, the cellular programs associated with the selected genes or genetic loci were examined with the aim of developing a mechanistic understanding of the tolerant phenotype.
  • Escherichia coli str. K-12 substr. MG1655 was grown to an ⁇ of 1.0
  • genomic phase and genomic DNA (gDNA) was isolated using the Charges witch® gDNA Mini Bacteria Kit (Invitrogen) according to the manufacturer's instructions.
  • Plasmid DNA was isolated using Hurricane plasmid mini and maxi-prep kits (Gerard Biotech) based on the supplier's protocol.
  • Genomic DNA (25 pg) was diluted in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) with 10% (v/v) glycerol to obtain a final volume of 750 ⁇ _.
  • a Nebulizer kit (Invitrogen) set at 5 psi was used to shear the gDNA for 30 seconds.
  • a sample of the DNA was run on a 0.7% agarose gel to confirm the distribution of fragment sizes, and the remaining DNA was concentrated via speed-vacuum centrifugation or microcon centrifugal columns (Millipore).
  • a 0.7% Crystal Violet gel (28 ⁇ . of crystal violet in 35mLTBE) was used to gel purify the desired fragments (3-6 kb). This protocol can be modified to obtain different genomic fragments by changing the pressure and shearing time of gDNA in the nebulizer.
  • Plasmid selection, origin of replication (ori) and plasmid construction In order to stably incorporate two genetically different plasmids in one host cell, the origins of replication (ori) of these vectors are preferably different but compatible. In addition, the chosen plasmids should carry different selection markers (e.g., antibiotic resistance genes) in order to select for positive transformants after each transformation (here: by electroporation) of the cells with each library. To minimize the effort to construct compatible libraries that can co-exist in a cell, the Gateway technology of Invitrogen (Carlsbad, CA) was used. A library constructed in a Gateway plasmid (the entry vector) can be shuffled into a variety of destination vectors. These destination vectors have to fulfill the aforementioned criteria concerning ori and a selection marker. The shuffling of library inserts is facilitated through a LR
  • the cassette also has a selection marker, a lethal ccdB gene leading to non- surviving negative transformants .
  • a commercially available vector containing such a recombination cassette is pDESTTM14 (Invitrogen, Carlsbad, CA), which was used as a destination vector for the first plasmid library.
  • the pDESTTM14 vector contains the ColE ori and an ampicillin resista nce gene.
  • a new destination vector with a compatible origin of replication and a different selection marker was constructed.
  • a compatible ori namely pl5A
  • pACYC184 was digested with restriction endonucleases Bsal and BsrBI to remove the chloramphenicol resistance gene. The two fragments were separated on a crystal violet gel . After blunting the digestion ends through a Klenow treatment, a recombination cassette, namely RfB purchased from Invitrogen, was ligated in the purified fragment, which contained the pl5A ori as well as a tetracycline resista nce gene.
  • RfB restriction endonucleases
  • This new desti nation plasm id was desig nated pACYCdest.
  • the sheared genomic DNA (gDNA) was polished by creating blunt ends that were dephosphorylated and adenylated. All enzymes used were obta ined from New England Biolabs ( NEB, Ipswich, MA).
  • a PCR mixture with T4 polymerase (34p L concentrated gDNA, 5 ⁇ ⁇ _ lOx BSA, 5pL NEB buffer 2, l pL T4 polymerase, 4pL ddH 2 0) was kept at 12°C for 2 hours, heated to 75°C for 20 minutes, and then 0,5 p L of CIP was added at 37°C to dephosphorylate the blunt ends for one hour.
  • the mixture was PCR purified (QIAGEN).
  • a second reaction was performed with Taq polymerase (50pL DNA, 5.7 pL lOx ThermoPol buffer, 1 p L lOmM dNTPs and 1 pL Taq Pol) at 72°C for 30 minutes, and the end PCR product was purified (QIAGEN, Germantown, MD, USA) .
  • Taq polymerase 50pL DNA, 5.7 pL lOx ThermoPol buffer, 1 p L lOmM dNTPs and 1 pL Taq Pol
  • the polished gDNA was used in a TOPO reaction with the pCR®8/GW/TOPO®
  • TA Cloning® kit (Invitrogen, Carlsbad, CA, USA) to construct a n entry vector for the Gateway® System with spectinomycin resistance.
  • the plasmids were transformed in 1 021505
  • the libraries can be shuttled from one plasmid to a nother using recombination reaction .
  • the Al MgL libra ry was shuttled to the destination vector pDESTTM14 (Invitrogen ; it has an Ampicillin resistance gene) using the LR
  • the recombi ned plasmids were transformed in TOP10 cells (Invitrogen), which are sensitive to the ccdB gene (and cannot survive with unrecombi ned plasm ids) to obta in a total of about 34,000 clo nes.
  • the library in the pDESTTM14 vector was desig nated A2MgL.
  • the Al MgL library was also shuffled in the pACYCdest plasmid via an LR recombination reaction to create the A4MgL library, which includes about 22,500 clones.
  • A2Mg L and A4MgL both contain E. colt str. K-12 substr. MG1655 DNA, but have different origins of replication and antibiotic resista nce.
  • A2MgL has the ColE origin of replication and the ampicillin resistance gene (Amp R ).
  • A4MgL has the pl5A origin of replication and contains the tetracycline resista nce gene (Tet R ).
  • Tet R tetracycline resista nce gene
  • the genes for the generation of the auxotrophs were selected by examining the biosynthetic pathway for lysine, which is illustrated in Figure 3. Five candidate genes were chosen, and were used to construct auxotrophs with two knocked out genes. Genes dapA (EC:4.2.1.52), dapB (EC : 1.3.1.26), dapD (EC: 2.3.1.117), dapE
  • genes are located far enough on the chromosome so that they cannot be part of a single plasmid-library insert (about 3-8 kb) as well as fosmid -library insert (about 35kb). Disruption of any of the aforementioned four genes prevents lysine biosynthesis ; thus lysine must be added to the culture medium for the cells to survive. Furthermore, because of the side pathway for peptidoglycan biosynthesis, knocking out dapA, dapB, dapD and dapE, leads to an add itional auxotrophy, for meso-diaminopimelic acid (meso-DAP), which must be added to the culture medium for the auxotrophic strains to survive. The mutant cells were grown on minimal medium supplemented with lysine and meso-DAP (Acord and Masters 2004) (Belinda B.B.G 2009) .
  • the distant dapA and lysA genes as well as dapD and dapE were disrupted to create double gene KOs.
  • Two libraries were cloned into these double KO auxotrophs, and the hypothesis was that each library would provide one of the missing genes.
  • the two co-existing library (A2MgL and A4MgL) system will "repair" the pathway.
  • the two-library system was tested with both double-KO auxotrophs to evaluate the feasibility of the method, namely to show that the proposed strategy is not gene specific and that both of the two different double KOs can be complemented .
  • double gene knockout (KO) E. coif strains To disrupt a metabolic pathway, double gene KO strains were constructed using the well-known PCR-product inactivation method (Datsenko and Wanner 2000) to produce lysine and meso-DAP a uxotrophs. Briefly, the Red helper plasmid (pKD20) that enhances recombination rates and represses exonuclease activity was transformed in E. coli str. K-12 substr. MG1655 cells (Datsenko and Wanner 2000) .
  • pKD20 Red helper plasmid
  • PCR products (one for each gene to be disrupted) were generated by PCR using the pKD4 plasmid as a template (Datsenko and Wa nner 2000) . These products have flanking homology regions to the genes targeted, as well as a kanamycin antibiotic resistance marker.
  • the first gene was knocked out by a homologous recombination (mediated through the gene products originating from the pKD20 plasmid) of the PCR product (introduced by electroporation) with the target gene.
  • Transformants were grown on rich media (LB) supplemented with the appropriate antibiotic and lysine or meso-DAP at 37°C, which leads to loss of the heat sensitive pKD20 plasm id.
  • the introduced kanamycin resista nce gene located in the genome at the position of the knocked out ta rget gene) was then removed through the action of the enzymes originating from the
  • AdapAAIysA and MG165S AdapDAdapE strategy for tra nsforming the cells with the two libraries.
  • the double knockout (AdapAAIysA) requires supplementation of both lysine and meso-DAP for cell growth, so these were supplied in the media for the following experiments in concentrations of 10 ⁇ g/ml and 50 pg/ml, respectively.
  • the double knockout AdapDAdapE only requires the supplementation with meso-DAP.
  • the cells from these double KO strains were made electrocompetent using standard laboratory techniques (Sambrook, Fritsch et al, 1989) and the first library (A2MgL) was transformed into the cells by
  • electroporation in a 0.2 cm cuvette with settings of 2.5 kV, 200 Sz para llel resistance, and a 25 mF capacitor.
  • cells were first outgrown in liquid rich (LB) media supplemented with meso-DAP and lysine (for strain MG1655AdapAAIysA) or meso-DAP alone (for strain MG1655AdapDAdapE) but without antibiotics at 37°C for one hour in order to allow the cells to recover from the electroporation stress.
  • LB liquid rich
  • the library containing cells were transferred in 250m l LB
  • the total number of colonies (clones) obta ined was approximately 32,800 for strain MG1655AdapAA IysA, and approximately 370,000 for strain MG1655Adap DAdapE. These numbers of colonies shows that there was more than 5-fold and 60-fold, respectively, of coverage by the library insert in these clones (as stated above, the coverage is estimated using the formula N - ln(l - P)/ln( l - f), where N is the number of colonies, P is the coverage probability, and f ⁇ s the fraction of the insert size relative to the entire genome (Clarke and Carbon 1976). A 99% coverage with an average library size of 3.5 kb requires 6100 clones. The colony numbers above far exceed this number.
  • A2MgL library were first exam ined for com plementation of one of the KOs, by using either meso-DAP or lysine aiming to confirm that sing le inserts containing the knocked out genes are present.
  • the MG1655Adap AAlysA cells containing A2Mg L were grown in minimal M9 media supplemented with either meso-DAP or lysine. For both sing le supplemented cultures growth was observed confirming the presence of dapA and lysA on different fragments of the library.
  • the first strategy involved outgrowth of the cells containing the first library (A2MgL) only in the presence of meso-DAP.
  • outgrowth of the cells conta ining the first library (A2MgL) was carried out in the presence of both meso-DAP and lysine. It wa s hypothesized that the first strategy mig ht expedite the selection process by enabling partial enrichment of cells containing an insert that incl udes the lysA gene.
  • strain MG1655AdapAAIysA containing the first library (A2MgL) were again made electrocompetent, and the second library (A4Mg l_) was transformed into the cells as described for the A2MgL library.
  • the total number of clones varied between the two complementation strateg ies as well as between biological replicates depending largely on the competency of the prepared cells as well as storing the cells at -85°C or not prior to the 2 nd electroporation .
  • MG1655AdapAAIysA containing A2Mg L and outgrown in the presence of both suppl ements (lysi ne and meso-DAP), in order to increase the number of transformed cells, the number of electroporations was increased, and the electrocompetent cells were not stored at - 85°C prior to the final electroporation . Under these conditions a total of 83 m illion transformants was obta ined.
  • the cells of strain MG1655AdapAAIysA conta ining both libraries (A2Mg L and A4MgL) were outgrown to an optical density of about 2 at 600 nm before screening.
  • the cells of strai n MG1655AdapDAdapE were grown in liquid LB media supplemented with meso-DAP when transforming into the cells both with A2 gL and the A4MgL library.
  • the transformation of cells of strain MG1655Ada pDAdapE containing A2 g L with the second library A4Mg L resulted in approximately 6.8 million transforma nts.
  • Aga in the cells of stra in MG1655AdapDAdapE containing both libraries (A2MgL as well as A4Mg L) were outgrown to an optical density of 2.2 at 600 nm before screening .
  • the two knockout strains containing members of both A2MgL and A4Mg L libra ries were grown to an absorbance of about 2 at 600 n m (exponential phase), washed twice with M9 minimal media to remove any residual substrates (meso-DAP or lysine), and then plated on M9 misl media agar plates containing ampicillin a nd tetracycline.
  • chemical competent cells either TOP10 cells from Invitrogen or NEB 10-beta cells from New England Biolabs
  • the library inserts were subjected to Sanger sequencing with primers annealing outside the LR recombination sites on the plasmids.
  • the resulting sequence data were analyzed using Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information (NCBI)) in order to determine the exact genomic location of the sequences of the isolated library inserts.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • the lysA gene was found on three different inserts among the few isolates tested. The sizes of these inserts are 4.6kb, 3.5kb and 3.1kb. The 3.5kb insert is identical to the 3.5kb insert found by the first complementation strategy. Again, the dapA was found in the A4Mg l_ library with an insert size of 4.3kb, which was identical to the insert found in the first complementation strategy.
  • the complete dapD gene was found in inserts of the A4MgL library: two different inserts with a size of 3.8kb a nd 4.1kb were identified.
  • the complete dapE gene was found in inserts of the A2MgL library. Again, two disti nct library inserts, one with a size of 3.1 kb and a 3.3-kb insert, were isolated. Therefore complementation of the
  • MG1655dapDAdap E cells could be restored by several combinations of library inserts from the two libraries, despite the fact the number of colonies containing both libraries - was smaller than what would be necessary for 99% genome coverage for all possible binary inserts.
  • strains were true lysine and meso- diaminopimelate auxotrophs. Both strains were infected with a packaged fosmid library using standa rd techniques with a fosmid library that contained Escherichia coli K-12
  • the fosmid library was constructed by cloning E. coli K- 12 MG1655 DNA in the pCCl FosTM vector according to the manufacturer's instruction (Epicentre
  • the Epi300 T1 R double knockout strains were grown to early exponential phase (OD 600 ⁇ 1), washed twice in M9 minimal media to remove residual nutrients (lysine as well as meso-DAP) and finally plated on M9 minimal media plates for selection of complemented clones with a non- auxotrophic phenotype.
  • the parent strai n Epi300 T1 R requires leucine ( 100 g/ml) and th iamine (10 ⁇ g m l) to grow in minimal media so these supplements were added to the plates.
  • Colonies obta ined in this manner were able to produce their own lysine and meso-diaminopimelate via the restored native pathway, which demonstrates the usefulness of embodiments of the present invention .
  • This demonstrates aga in that genes present in the two different libraries can effectively interact with each other in order to prod uce a desired phenotype.
  • Figure 1 illustrates complementation of dual knockouts with plasmid and fosmid libra ries. The approximate ch romosomal position of the knocked out genes in the E. coli chromosome are shown for each knockout combination.
  • the complementation of the dual knockouts in strain E. coli K-12 MG1655, MapDMapE and dapA lysA are shown, which were complemented with two plasmid libraries, namely A4MgL (contains a pl5A origin of replication) and A2MgL (contains a ColE origin of replication).
  • A4MgL contains a pl5A origin of replication
  • A2MgL contains a ColE origin of replication
  • coli Epi300Tl R dapBC lysA and MapDNysA, are shown, which were complemented with a fosmid library (F-plasmid origin of replication) and a plasmid library (A2Mgl_, ColE origin of replication).
  • the fosmid and plasid libraries were able to interact to restore the disrupted pathway and provide the essential genes for growth .
  • the dual fosmid and plasmid library designated B2FosMg, contains a fosmid and a compatible plasmid containing E. coli MG1655 genomic DNA.
  • Ethanol was chosen as the stressor chemical due to its importance as a biofuel and the ability of E. coli to ferment ethanol from a va riety of sugar sources (Ingram , Gomez et al . 1998) .
  • ethanol tolerance is a complex phenotype arising from the interplay of multiple genes and response systems (Stephanopoulos 2007; Nicolaou, Gaida et al. 2010) .
  • the B2FosMg strain containing a fosm id and a plasmid library was enriched in progressively higher ethanol concentrations up to
  • genes were enriched during etha nol screening of multiple genomic libraries.
  • Epi300 T1 R cells containing two genomic libraries (fosmid and plasm id library) were screened for ethanol tolerance in increasing ethanol concentrations up to 5.25%.
  • Plasmids a nd fosm ids of 10 su rviving clones were isolated .
  • Genomic regions included in the library inserts were identified by sequencing and the genes present were identified using the KEGG data base. Incomplete genes in the enriched fragments are listed in parentheses.
  • a subset of the genes (underlined in Table 4, namely, sfsB, murA, yrbA, yrbB, and yrbC) identified in the enriched fosmid inserts of the ethanol tolerance screening were cloned into a pl5A ori based plasm id, namely CLONE 2.
  • This plasmid is com patible with the plasmids of the plasmid library of B2FosMgL and was used in further ethanol tolerance assays.
  • the set of genes above provide information as to the methods of ethanol tolerance being developed in the cell, and can be used to improve the phenotype .
  • the predicted DNA-binding tra nscriptional regulator yrbA belongs to the bolA
  • murA codes for UDP-N- acetylglucosamine enolpyruvyl transferase, which catalyzes the comm itted step for peptidoglycan biosynthesis (Brown, Vivas et al. 1995) .
  • the stressed cells might be altering their cell shape and also counteracting the fluidizing effects of the ethanol by synthesizing more peptidoglycan to create a more resistant cell wall .
  • the plasmids enriched contain the arabinose genes, which are not known to enhance ethanol tolerance.
  • E the strain used for this demonstration, E.
  • coli Epi300 T1 R has a ⁇ ara, /eu)7697 genotype and is missing the arabinose genes.
  • the presence of these genes in the genomic libraries complement the missing genes and enhance the phenotype of the cells, by restoring them to a more wild type status .
  • the genes identified might not have a direct effect on tolerance, but give rise to more robust cells.
  • embod iments of the present invention allow for the selection of genes that will improve the overa ll cellular phenotype.
  • CLONE 1 contains a genomic fragment with the araD, araA, araB, araC, & yabl genes that was isolated during a single library enrichment. This clone was used because it contains a more complete operon, allowing for better expression of the relevant genes, compared to the plasmid found during the dual library enrichment.
  • CLONE 2 contains sfsB, murA, yrbA, yrbB and yrbC, which is a smal l subset of the genes identified in the enriched fosmids.
  • gus Arabidopsis thaliana ⁇ -glucuronisase
  • pENTRTM Gus Invitrogen
  • the gus was incorporated to yield control plasmids of compatible size, whereby the expression and its potential influence on the phenotype was eliminated by exclusion of necessary signa ls for transcription (promoteriess gus).
  • Control plasmids were necessary to create strains, which can be exposed to similar environments, e.g . antibiotic stress.
  • Figure 2 i llustrates ethanol tolerant phenotype characte rization .
  • Identified clones of the dual library enrichment were sub-cloned agai n and cultivated under 48g/L etha nol .
  • a total of 4 biologica l replicates each with three technical replicates were performed .
  • One typical growth profile is shown on the left side.
  • the error bars indicate the range of the three technical replicates.
  • Long time survivability was determined by counting CFU's of cells plated after 48 and 96 hours ethanol exposure. The results are shown in the rig ht chart, whereby the error bars indicate the standard derivation of all replicates.
  • Epi300 T1 R allowed for higher (better) growth in ethanol, as evidenced by the higher optica l density achieved by the strain harboring CLON E1 and CLON E2. Also, higher survivability was observed after 48 a nd 96 hours, as illustrated by the higher number of colony forming units (CFU ) formed. CFU are created when viable cells are present, and since the strain with CLONE1 and CLONE2 produces more colonies, it ca n be concluded that it has higher survivability in ethanol due to more viable cells at the time point of plating .
  • CFU colony forming units
  • the libraries with the beneficial genes will be enriched and can easily be identified.
  • interactions can be readily identified something which can only be achieved by using co-existi ng genomic libraries as described in embodiments of this invention.
  • the genes identified in this example restore the genetic background of the parent E. coli Epi300 T1 R stra in to a more wild-type status by com plementing the A(ara, leu) genes that have been deleted in this cloning strain .
  • the method used here provides the missing genes a nd allows for improvement in the phenotype of the strain used, namely E. coll Epi300 T1 R .
  • the phenotype under investigation is improved in the genomic background .
  • the genes identified might not confer a better phenotype to other strai ns, but for the specific strain used under this genomic background, performance was improved.
  • the screening can be repeated to be specific to that strain for maximum benefits.
  • Embodiments of the present invention can use genomic DNA from a variety of species, including but not limited to Bacillus, Lactobacillus, Deinococcus, Clostridium, Actinomyces, Streptomyces, Pseudomonas, Escherichia, Rhodococcus, Nocardioides, Saccha romyces , Klyveromyces, Pichla, and Sterigmatomyces, and com binations thereof.
  • genomic DNA of Lactobacillus plantarum was utilized.
  • Lactobacil li include some of the most ethanol-tolerant known organisms (Gold, Meagher et al. 1992; Couto, Pina et a l. 1997; G-Alegria, Lopez et al . 2004) despite the perception that yeast are overall more tolerant to ethanol .
  • the molecular basis of their tolerance rema ins unknown .
  • genes from L. plantarum that confer higher tolerance were identified .
  • genomic libraries of L. plantarum DNA were constructed using the techniques mentioned in Example 1. Briefly, L. plantarum genomic DNA was isolated using standard molecular biology techniques.
  • the DNA was sheared using a Nebulizer kit, gel extracted to obtained the desirable DNA size, polished to create blunt ends that were adenylated a nd phosphorylated, and fina lly cloned into the pCR®8/GW/TOPO® vector obtai ned from Invitrogen.
  • This initial library was designated A1PI, and was then shuttled via LR reactions to other vectors, namely pDESTTM14 (Invitrogen) and pACYCdest (constructed by us) to generate the A2PI and A3PI libraries, respectively. The details on how these reactions were performed are included in the section
  • a L. plantarum fosmid library was also constructed in the pCCl FosTM vector according to the manufactu rer's instruction (Epicentre Biotechnologies, Madison, WI), and was designated BFosLp.
  • the libra ries constructed were screened to investigate the expression of L.
  • the L. plantarum libraries were screened based on a tested protocol. Briefly, the screening was performed by inoculating 200 ⁇ of frozen-stock aliquot of the library in 10 mL of LB media with appropriate antibiotic(s) and allowing for aerobic culture at 37°C for 12 hours. A 1 mL aliquot was then transferred into 9 mL of fresh LB (with antibiotic(s)) conta ining a small amount of ethanol . After 12 hours of aerobic g rowth, another transfer was made into fresh media with higher ethanol content. This process of serial transfers into media with higher ethanol concentrations was repeated until the cultures cou ld not survive. Frozen stocks were made after each transfer and the inserts included in the clones were sequenced and identified.
  • the libra ries described in embodiments of this invention are modular, allowing for the generation of, for example, dual plasmid (i.e,. plasmid -plasmid libraries) or plasmid-fosmid combination .
  • They can contain DNA from various species, thus allowing for genomic fragments from ma ny orga nisms to be tested simultaneously.
  • the L. plantarum libraries can be used with the previously mentioned E. coli libraries to create strains harboring libraries conta ining DNA from two different strains.
  • the A2PI library can be coupled with the A4MgL library, since they have different origins of replication and antibiotic markers, making them com patible.
  • the L. plantarum fosmid can be cloned in tandem with an E. coli plasmid library.
  • combi nations of libraries allow for more complex phenotypes to develop and be screened for with selection protocols.
  • Another embodiment of the present invention was tested by screening the mu lti-library system for survival in low pH environments.
  • the dual plasmid E. coli libraries (A2MgL and A4MgL) as well as the fosmid and plasmid libraries (B2FosMg) introduced in E. coli K-12 MG1655 were screened for a phenotype of increased acid resista nce compa red to the wild-type strain E. coli K-12 MG1655.
  • the screening was performed by sequential passes through an acidified (pH 2) minimal media (M9 media) supplemented with 1.5mM glutamate with subsequent outgrowth in rich media (LB med ia supplemented with 0.4% glucose, adjusted to pH 5) as described in literature (Castanie-Cornet, Penfound et al. 1999). Stationary growth phase libraries were exposed to the acidified minimal media for 4 hours in order to enrich for surviving clones. After the exposure to this harsh environment the culture was recovered by outgrowth in rich media for 20 hours before being stressed again. Survival of the library was monitored throughout the screening by p lating dilution series before and after the pass through the acid ic minimal media and counting CFU's .
  • Sequencing revealed the enrichment of several genes, whereby two genes, namely did and hdfR, were observed for multiple isolated clones.
  • these two aforementioned genes were found consistently a nd repeatedly in both plasmid libraries, A2MgL and A4MgL, whereby did was also enriched in the screening of the plasmid a nd fosmid library system . This demonstrates that the screening method yields a consistent selection of inserts with the same functional characteristics.
  • the hdfR is a known regulator of the glutamate depended acid resistance system in E. coli (Krin, Danch in et al. 2010) , but the function of did with rega rd to acid resistance is unknown thus far.
  • This example shows the potential and application of embod iments of the present invention to identify genes responsible for an enhanced phenotype, whether they are known or unknown. Once genes are identified, they can be used to enhance the phenotype according to methods described herein.
  • ROS reactive oxygen species
  • HSP33 heat shock protein
  • the library culture was stressed for 4 hours and a 10% inoculum of surviving cells was tra nsferred into fresh media . This allowed the cells that survived the oxidative stress to replicate and overta ke the fresh media. The process was repeated with increasing concentration of sodium hypoch lorite and after 15 rounds of enrichment clones were isolated by plating culture on agar plates. The clones were tested for enhanced oxidative stress tolerance.

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Abstract

Une population de cellules comprend au moins deux banques génomiques, sous-génomiques, ou métagénomiques co-existantes et interagissantes. Chaque cellule de ladite population comprend un vecteur provenant de chaque banque, chaque vecteur contenant un insert de type gène. Les vecteurs ont des origines de réplication qui sont compatibles mais différentes les unes des autres. Les méthodes ci-décrites permettent d'identifier et de développer des cellules qui présentent un phénotype recherché, tel qu'une tolérance améliorée à un agent chimique toxique, une nouvelle fonction catabolique, ou une nouvelle fonction anabolique, en particulier, des phénotypes qui peuvent être utilisés dans des applications de biotraitement industrielles.
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GALL ET AL.: 'Parallel mapping of genotypes to phenotypes contributing to overall biological fitness' METAB ENG. vol. 10, no. 6, 23 August 2008, pages 382 - 393 *
LIU ET AL.: 'Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways' MOL GENET GENOMICS vol. 282, no. 3, 11 June 2009, pages 233 - 244 *
LIU ET AL.: 'Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae' APPL MICROBIOL BIOTECHNOL. vol. 81, no. 4, 23 September 2008, pages 743 - 753 *
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