WO2016166303A1 - Riboswitch-controlled screening and selection of desired biocatalysts - Google Patents

Riboswitch-controlled screening and selection of desired biocatalysts Download PDF

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WO2016166303A1
WO2016166303A1 PCT/EP2016/058375 EP2016058375W WO2016166303A1 WO 2016166303 A1 WO2016166303 A1 WO 2016166303A1 EP 2016058375 W EP2016058375 W EP 2016058375W WO 2016166303 A1 WO2016166303 A1 WO 2016166303A1
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protein
cell
aptamer
expression
product
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French (fr)
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Sjoerd Constantijn Arnoud CREUTZBURG
John Van Der Oost
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Wageningen Universiteit
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • 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
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the field of enzymology and methods and techniques for cell- based screening for novel enzyme variants capable of more efficient and/or rapid production of product from substrate.
  • Enzymes enable efficient chemical transformations by reducing the activation energy required for a chemical reaction to proceed at a given temperature and reactant concentration. Consequently, many enzymes are usefully employed in the production of industrially important chemicals.
  • Enzymes are biological, often green and/or sustainable alternatives for chemical catalysts and are generally orders of magnitude more selective than their synthetic counterparts. Enzymes are unique because of their catalytic power and their extraordinary specificity. The specificity and (enantio- and regio-) selectivity of certain enzymatic transformations make them appealing for the production of fine chemicals, food additives and pharmaceutical intermediates. Theoretically, proteins can catalyse any conversion that is thermo-dynamically feasible. Hence, any product (natural or unnatural) can be generated with biocatalysts that possess the appropriate catalytic features, including the desired substrate specificity.
  • Riboswitches are regulatory segments of messenger RNA (mRNA) molecules that bind small molecules. They consist solely of RNA, sense their ligand in a specific binding pocket and undergo a conformational switch in response to ligand binding which results in a change in production of the proteins encoded by the downstream protein-coding mRNA. Riboswitches therefore regulate mRNA translation in response to the concentrations of their specific effector molecules and control a range of basic metabolic pathways.
  • a riboswitch controls the expression of a gene encoding an enzyme that catalyzes the production of a certain metabolite; this compound also serves as a ligand that binds to the riboswitch, controlling its conformation that directly affects the expression of the gene: a classical feedback loop.
  • a well-known example of this is the gene btuB, which encodes a transporter of vitamin B12, is controlled by a riboswitch that senses the intracellular concentration of vitamin B12 (Mandal and Breaker 2004 Nat Rev Mol Cell Biol 5:451 -463).
  • Natural riboswitches are typically located in the 5'-untranslated region (UTR) of a mRNA, controlling the translation of the downstream coding sequence.
  • UTR 5'-untranslated region
  • several synthetic riboswitches have been developed which harness the ability of riboswitches to regulate gene expression in response to exogenously applied stimuli.
  • An objective of the present invention is the provision of a more rapid and extensive process for the cell-based screening and selection of enzymes and enzyme variants.
  • the inventors have for the first time configured an intronic, self-splicing riboswitch for enzyme-product specificity by introducing an appropriate aptamer, and then used this in a sensing-expression construct, whereby the presence of an enzyme product in the cell triggers self-splicing of the intron sequence to restore the reading frame of the reporter gene and as such to drive expression of the gene product.
  • the sensing construct expresses a protein which marks the cell or permits its growth or survival in or on an otherwise selective media. In this way, introduction or the presence of such product sensing-reporter constructs in cells can be harnessed to provide a multi- parallel rapid screening of cells or libraries for desirable enzyme variants.
  • the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the imRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein; (ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide; (iii) identifying whether the cell expresse
  • the present invention directly addresses the problem of a limited screening capacity for enzymes with desired properties. This is accomplished by coupling the synthesis of a specific enzyme product to the expression of a detectable reporter by operably linking the specific binding of the product to the or each aptamer with excision of at least one intron which in the absence of product binding blocks translation of a downstream reporter. In this way, by selecting an appropriate selectable marker, product-dependent selection may be coupled to high-throughput screening systems such as product-dependent fluorescence or product-dependent host cell survival. Irrespective of the enzymatic reaction, this will allow screening of large libraries of cells and the rapid enrichment of enzymes with desired properties.
  • the utility of the present invention resides in the broad applicability of the selection systems, methods, nucleic acid constructs, kits and cells of the present invention to any protein which can be expressed in commonly used host cells, for example prokaryotic cells, fungal cells, plant cells or animal cells.
  • the selection systems and methods of the invention are applicable to any protein of interest provided that a suitable aptamer can be generated which will bind to the product produced by the action of the enzyme on reactants.
  • Proteins of interest are typically polypeptide macromolecules comprising 20 or more contiguous amino acid residues and may include, but are not limited to enzymes, structural proteins, binding proteins and/or surface-active proteins.
  • Proteins of interest may include those of therapeutic value or industrial value.
  • the protein of interest will be an enzyme.
  • the desired first protein is an enzyme and the product is generated from a substrate as the result of an enzyme reaction.
  • the substrate for the enzyme may be available in the cell either constitutively or upon induction.
  • the substrate for the enzyme may be provided to the cells.
  • the first protein may be a protein which is native to the host cell.
  • the first protein may be a heterologous protein.
  • the first protein may be a native protein of the host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated. In these circumstances, the first protein will be considered as a heterologous protein in the context of the mutated host cell.
  • the present invention is of broad applicability and host cells of the present invention may be derived from any genetically tractable organism which can be cultured. Therefore, in particular, commonly used host cell may be selected for use in accordance with the present invention including prokaryotic or eukaryotic cells which are genetically accessible and which can be cultured.
  • the approaches defined herein for the selection of cells which express a protein of interest may be applied to those cells which are able to serve as a host for production of the protein of interest (POI). It may therefore be applied to commonly used host cells, for example prokaryotic cells, fungal cells, plant cells and animal cells commonly used for recombinant heterologous protein expression.
  • Appropriate host cells may be prokaryotic or eukaryotic.
  • host cells will be selected from a prokaryotic cell, a fungal cell, a plant cell, a protist cell or an animal cell.
  • Preferred host cells for use in accordance with the present invention are commonly derived from species which typically exhibit high growth rates, are easily cultured and/or transformed, display short generation times, species which have established genetic resources associated with them or species which have been selected, modified or synthesized for optimal expression of heterologous protein under specific conditions.
  • an appropriate host cell may be selected based on the desired specific conditions or cellular context in which the protein of interest is to be deployed.
  • the host cell will be a prokaryotic cell.
  • the host cell is a bacterial cell.
  • the host cell is an Escherichia coli (E. coli) cell.
  • the polynucleotide sequence encoding the nucleic acid sensing construct will preferably be provided in an expression construct, e.g. an expression vector.
  • the polynucleotide may be provided in an expression vector.
  • Suitable expression vectors will vary according to the recipient host cell and suitably may incorporate regulatory elements which allow expression in the host cell of interest and preferably which facilitate high-levels of expression. Such regulatory sequences may be capable of influencing transcription or translation of a gene or gene product, for example in terms of initiation, accuracy, rate, stability, downstream processing and mobility.
  • Such elements may include, for example, strong and/or constitutive promoters, 5' and 3' UTR's, transcriptional and/or translational enhancers, transcription factor or protein binding sequences, start sites and termination sequences, ribosome binding sites, recombination sites, polyadenylation sequences, sense or antisense sequences, sequences ensuring correct initiation of transcription and optionally poly- A signals ensuring termination of transcription and transcript stabilisation in the host cell.
  • the regulatory sequences may be plant-, animal-, bacteria-, fungal- or virus derived, and preferably may be derived from the same organism as the host cell. Clearly, appropriate regulatory elements will vary according to the host cell of interest. For example, regulatory elements which facilitate high-level expression in prokaryotic host cells such as in E.
  • coli may include the pLac, T7, P(Bla), P(Cat), P(Kat), trp or tac promoters.
  • Regulatory elements which facilitate high-level expression in eukaryotic host cells might include the AOX1 or GAL1 promoter in yeast or the CMV- or SV40-promoters, CMV-enhancer, SV40-enhancer, Herpes simplex virus VIP16 transcriptional activator or inclusion of a globin intron in animal cells.
  • constitutive high-level expression may be obtained using, for example, the Zea mays ubiquitin 1 promoter or 35S and 19S promoters of cauliflower mosaic virus.
  • Suitable regulatory elements may be constitutive, whereby they direct expression under most environmental conditions or developmental stages, developmental stage specific or inducible.
  • the promoter is inducible, to direct expression in response to environmental, chemical or developmental cues, such as temperature, light, chemicals, drought, and other stimuli.
  • promoters may be chosen which permit expression of the protein of interest at particular developmental stages or in response to extra- or intra-cellular conditions, signals or externally applied stimuli.
  • a range of promoters exist for use in E. coli which give high- level expression at particular stages of growth (e.g. osmY stationary phase promoter) or in response to particular stimuli (e.g. HtpG Heat Shock Promoter).
  • Suitable expression vectors may comprise additional sequences encoding selectable markers which allow for the selection of said vector in a suitable host cell and/or under particular conditions.
  • Expression constructs comprising the polynucleotide sequence encoding the nucleic acid sensing construct may be located in plasmids (expression vectors) which are used to transform the host cell.
  • expression constructs comprising the polynucleotide sequence encoding the first desired protein may be located in plasmids (expression vectors) which are used to transform the host cell.
  • Methods of transformation may include but are not limited to; heat shock, electroporation, particle bombardment, chemical induction, microinjection and viral transformation.
  • the host cell may already express the desired first protein or be capable of expressing the desired first protein and may be subsequently transformed with the nucleic acid sensing construct.
  • the host cell may first be transformed with the nucleic acid sensing construct and the method may further comprise transforming the host cell with a polynucleotide sequence encoding the desired first protein.
  • transformation of the host cell with nucleic acid constructs encoding the nucleic acid sensing construct and the first desired protein may take place substantially simultaneously.
  • the host cell is transformed substantially simultaneously with the sensing and expression constructs.
  • the nucleic acid sensing construct comprises a polynucleotide sequence.
  • Polynucleotide sequences encoding the second protein may be isolated nucleic acid molecules and may be a DNA molecule, a cDNA molecule, an RNA molecule or synthetically produced DNA or RNA or a chimeric nucleic acid molecule.
  • the polynucleotide is an RNA, it will be understood that normally uracil (U) is to be used in place of thymine (T).
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form, or sense or anti-sense, and encompasses analogues of naturally occurring nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Such polynucleotides may be derived from any organism, including the host organism, or may be synthesised de novo.
  • the provision of a polynucleotide may comprise synthesis of a polynucleotide. This may be for example by modification of a pre-existing sequence, e.g. by site-directed mutagenesis or possibly by de novo synthesis.
  • polynucleotide sequences encoding the protein of interest may be prepared by any suitable method known to those of ordinary skill in the art, including but not limited to, for example, direct chemical synthesis or cloning for introduction into a desired host cell.
  • the starting polynucleotide sequence may be provided and subsequently modified ex vivo or alternatively in vivo for example by site directed mutagenesis or gene editing techniques.
  • methods of the present invention are based on a selectable marker, the gene of which is interrupted by one or more riboswitches with adjustable ribonuclease (RNase) activity and/or adjustable RNA ligase activity.
  • RNase adjustable ribonuclease
  • the RNase activity of these "self-splicing introns” is controlled by the concentration of the product produced by the first protein from a substrate.
  • the product-dependent splicing relies on the presence of a polynucleotide fragment of the riboswitch (the "aptamer").
  • the or each aptamer interacts with the product in a sequence specific manner. Consequently it will be understood that variation of the aptamer sequence will result in binding of (and control by) other products.
  • Product-dependent expression of the second protein which may be a selectable marker, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division.
  • the polynucleotide sequence encoding the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the mRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein.
  • the second protein which either encodes the selectable marker or induces expression of an amplification cascade resulting in the eventual expression of the selectable marker (e.g. antibiotic resistance protein).
  • the selectable marker e.g. antibiotic resistance protein
  • the selection system of the present invention is unique in that it is broadly applicable in that it allows for obtaining enzyme variants from large libraries (either from a natural metagenome library, or from a laboratory evolution library) capable of the specific generation of a certain product.
  • the key to the selection system is the specificity of the aptamer of the nucleic acid sensing construct.
  • Aptamers of the present invention are polynucleotide sequences which have a high binding affinity for the product of the enzyme of interest under specific conditions.
  • the aptamers may be DNA, cDNA, RNA, preferably RNA.
  • Suitable aptamers of the present invention are preferably 20-30nt in length; optionally they are 20nt, 21 nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt or 30nt in length.
  • aptamers which have specific binding affinity for the product of interest may be generated by any means known in the art.
  • aptamers of the present invention will be generated in a high-throughput manner by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) of the aptamer fragment. In this way the specificity of each sensing construct can theoretically be adjusted for a desired product.
  • the second protein may be a native protein of a host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated.
  • the second protein will be considered as a heterologous protein in the context of the mutated host cell.
  • the second protein may be a non-native, heterologous protein which is capable of being detected as part of a high-throughput system, for example Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • such selectable markers may include polynucleotide sequences encoding proteins which confer antibiotic resistance.
  • host cells expressing the protein of interest may be selected from those which do not express the protein of interest.
  • the product of the protein of interest binds to the one or more aptamers of the nucleic acid sensing construct in cells expressing the POI, resulting in self-splicing of the one or more introns located in the mRNA encoding the antibiotic resistance protein and lifting the block on faithful translation of the antibiotic resistance protein, permitting growth on substrates containing the relevant antibiotic.
  • selectable markers may include polynucleotide sequences encoding proteins to which the cell is fatally sensitive.
  • the presence of the desired product may be coupled to the restoration of translation of the reporter protein. In this way host cells expressing the protein of interest may be selected from those which do not express the protein of interest.
  • selectable markers may include polynucleotide sequences encoding proteins which promote cell growth and/or division.
  • the presence of the desired product may be coupled to the restoration of translation of the reporter protein. In this way host cells expressing the protein of interest may be selected from those which do not express the protein of interest.
  • suitable expression vectors may comprise sequences which enable visualisation or quantification of the expressed second protein (e.g. Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), Cyan Fluorescent Protein (CFP), or Luciferase fusion tags) in the chosen host cell.
  • the reporter protein may be an enzyme which can be used to generate an optical signal.
  • the expression vector may incorporate a polynucleotide reporter encoding a luminescent protein, such as a luciferase (e.g. firefly luciferase).
  • the reporter gene may be a chromogenic enzyme which can be used to generate an optical signal, e.g.
  • Tags used for detection of reporter protein expression may also be antigen peptide tags.
  • a cleavable tag may also be provided for affinity purification, e.g. a polyhistidine tag. It is envisaged that other types of label may also be used to indicate expression of the reporter protein including, for example, organic dye molecules or radiolabels.
  • preferred expression vectors will include sequences encoding a fluorescent protein, for example GFP which will enable the screening and optionally separation (selection) of a cell which expresses the protein of interest for example by Fluorescence Activated Cell Sorting (FACS).
  • FACS Fluorescence Activated Cell Sorting
  • the presence of a product generated by a protein of interest may be determined by the detection of expression of the second protein (reporter protein).
  • reporter protein reporter protein
  • the reporter protein which is eventually detected may in fact be a second protein, alternatively it may be a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth protein depending on the nature of the cascade system used, providing that the presence of the protein of interest is operably linked to the expression of the reporter protein.
  • the expression of the reporter protein will directly result in selection, for example where the selectable marker is a protein coding for a protein conferring antibiotic resistance.
  • the specific aptamer will bind the product generated by the POI with high affinity.
  • the insertion of one or more self-splicing introns comprising said aptamer into a gene encoding a substance required for survival on a particular substrate permits selection of cells expressing the POI on that substrate, for example into the coding sequence of a gene encoding the enzyme chloramphenicol acetyl transferase, which confers resistance to the antibiotic chloramphenicol. Cells not expressing the POI will not survive exposure to chloramphenicol.
  • the reporter gene will encode a protein conferring resistance to commonly used antibiotics.
  • the reporter gene will encode a protein conferring resistance to aminoglycoside antibiotics, in particular Kanamycin or Gentamicin.
  • the reporter gene may encode a protein conferring resistance to chloramphenicol, optionally the enzyme chloramphenicol acetyl transferase.
  • Other antibiotics which may be useful can include beta-lactam antibiotics, for example penicillins, cephalosporins, carbapenems, and monobactams.
  • the reporter construct may encode a protein which relieves an auxotrophic deficit which occurs when the host cell is grown under conditions where the desired product is absent.
  • the reporter construct encodes thyA which when expressed in the cell is able to overcome an auxotrophic deficit of thymidine monophosphate biosynthesis (see Figure 3).
  • the present invention provides a method wherein the cell is auxotrophic and the second protein or the third protein provides at least partial relief of the auxotrophy.
  • the cell may be auxotrophic in certain media, and the second protein, or alternatively a third protein (for example, an enzyme) complements the auxotrophy when expressed.
  • a third protein for example, an enzyme
  • auxotrophy is caused by interruption of the pyrimidine synthesis pathway, preferably by a knock-out of thyA and the second protein or the third protein is ThyA.
  • interruption of the pyrimidine synthesis pathway may be achieved by a reduction in the expression of other genes required for pyrimidine synthesis (for example pyrE and/or pyrF) and the second or third protein (encoding, for example, pyrE or pyrF) may provide at least partial relief of the auxotrophy.
  • the present invention provides a method, wherein cell growth and/or division is sensitive to a compound, e.g. antibiotic, and the second protein or the third protein provides at least some resistance to the compound.
  • the host cell is fatally sensitive to a compound, for example an antibiotic
  • the second protein provides complete resistance to the compound, permitting growth of cells expressing the product of interest on media containing the compound, for example an antibiotic, at concentrations which would typically inhibit growth of cells not possessing the protein conferring resistance.
  • the expression levels of the reporter protein may be determined.
  • the expression levels of the reporter protein may be proportional either to the expression levels of the protein of interest or to the binding affinity of the protein of interest for the aptamer.
  • the method chosen for expression analysis of the reporter protein allows for quantitative assessment of the level of expression.
  • the reporter protein may be directly determined, e.g. with GFP or luciferase.
  • the reporter protein will be detectable by a high-throughput screening method, for example, relying on the detection of an optical signal.
  • using an optical signal which is directly proportionate to the quantity of the expression product from the polynucleotide is a convenient method of measuring expression and is amenable to high throughput processing.
  • the measurement of reporter protein expression comprises the detection of an optical signal, for example a fluorescent signal, a luminescent signal or colour signal.
  • an optical signal for example a fluorescent signal, a luminescent signal or colour signal.
  • the optical signal is provided by a GFP reporter protein.
  • the method further comprises transforming the cell with an expression construct which comprises a polynucleotide sequence encoding the desired first protein.
  • the host cell may be transformed with such an expression construct either before, after or substantially simultaneously with the one or more sensing constructs.
  • the cell is transformed substantially simultaneously with the expression construct and one or more sensing constructs.
  • host cells can be transformed in any order sequentially with the expression construct and the one or more sensing constructs.
  • All of the reporter constructs may be either plasmid borne or integrated into the genome. More particularly one system involves an expression construct encoding a first protein (POI) and a sensing construct, incorporating a riboswitch, which encodes the reporter protein.
  • POI first protein
  • Another system involves an expression construct encoding a first protein (POI) and a primary reporter construct, incorporating a riboswitch which encodes T7 polymerase and a secondary reporter construct encoding a reporter protein under the control of T7 polymerase.
  • POI first protein
  • primary reporter construct incorporating a riboswitch which encodes T7 polymerase and a secondary reporter construct encoding a reporter protein under the control of T7 polymerase.
  • the present invention makes use of riboswitches; stretches of RNA that can adopt different conformational states, depending on the presence or absence of a binding molecule (metabolite, ligand) and provides an unprecedented, adjustable in vivo selection system based on RNA-based translational control by specific synthetic riboswitches.
  • riboswitches are not usually required to completely switch off expression of their genes and therefore the control of gene expression exerted by natural riboswitches is known to be incomplete.
  • the present invention provides synthetic riboswitches with improved stringency, which are appropriate for use in selecting cells expressing a protein of interest. Surprisingly, this is achieved by incorporating at least two, optionally more than two, i.e. multiple copies of the riboswitches in sequential arrangement.
  • the self-splicing riboswitches whose splicing is under the control of an aptamer, have the effect in use of the sensing constructs and system of the invention of reducing the background levels of growth of cells transformed with the constructs, compared to constructs where there is a riboswitch is located in the UTR.
  • This reduction in background compared to the UTR located riboswitch constructs is preferably at least a 5% reduction, when measured in terms of growth rate of cells; more preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more.
  • the background level of growth of cells is reduced, by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.
  • the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the mRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein; (ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide; (iii) identifying whether the cell expresse
  • the two self-splicing introns comprise the same aptamer.
  • the three self- splicing introns comprise the same aptamer.
  • the splicing activities of the introns may desirably be under the control of more than one aptamer, more than two aptamers, more than three aptamers.
  • the sequences of the aptamers may be the same.
  • the sequences of the aptamers may be different from one another.
  • the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct and a further construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which acts on a regulatory element, for example a promoter, of the further construct comprising a nucleotide sequence which encodes a third protein and which is under expression control of a promoter, wherein the promoter is under operable control of the second protein and which when expressed by the cell, directly or indirectly marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct is interrupted by at
  • suitable promoters will generate strong expression in response to stimulation by expression of the second protein. It will be appreciated that such amplification cascades may include further levels of amplification provided by additional promoters under expression control of one or more reporter proteins as desired.
  • the second protein is Phage T7 DNA dependent RNA polymerase and the promoter is PT7.
  • the PT7 promoter controls the expression of a third protein, which is a reporter protein, for example GFP.
  • the present invention provides a method wherein the second protein or the third protein generates a selectable phenotype; preferably a detectable marker protein, e.g. GFP.
  • the expression of the reporter will be proportional to the binding affinity for the aptamer. Even more preferably, such a detectable marker will enable cells expressing the protein of interest to be identified and isolated by a high-throughput system such as FACS.
  • the present invention provides a method of screening cells for expression of a desired first protein which generates a product from a substrate, comprising a first round of selecting cells according to a method of the invention, followed by subjecting cells selected from the first round to a second round of selecting cells according to a method of the invention.
  • the method of screening cells further comprises a third or subsequent rounds of selecting cells whereby cells selected in the previous round are subjected to the further round(s) according to a method of the invention.
  • the sequence of the or each aptamer may be varied to select for different parameters or binding affinities for the product or for binding affinity to a different product.
  • the aptamer is a first aptamer with a first binding affinity for the product.
  • the aptamer is a second aptamer with a second binding affinity for the product.
  • the aptamer is a third aptamer with a third binding affinity for the product.
  • the binding affinity of the first aptamer for the product is higher than that of the second aptamer.
  • the present invention provides a cell which is capable of expressing a desired first protein, the first protein being a protein which generates a product from a substrate, wherein the cell comprises a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self- splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein.
  • the nucleotide sequence of the second protein is interrupted by at least two self-splicing introns. More preferably, the two or more self-splicing introns contain the same aptamer. More preferably the cells of the invention further comprise a construct which comprises a nucleotide sequence encoding a third protein under expression control of a promoter, wherein the promoter is under operable control of the second protein. Desirably, the cell may express the desired first protein constitutively. Alternatively, it is envisaged that the cell may express the first protein in response to induction, for example in response to a chemical compound (e.g.
  • DMSO Dimethyl sulfoxide
  • Doxycycline e.g. Doxycycline
  • Muristerone A e.g. Doxycycline
  • Ponasterone A e.g. Doxycycline
  • environmental e.g. glucose elevation; hypoxia
  • developmental cues e.g. cell density; growth phase
  • Suitable inducers also include, but are not limited to; Rhamnose, Arabinose or Isopropyl ⁇ -D-l -thiogalactopyranoside (IPTG).
  • the first protein may be a protein which is native to the host cell or a heterologous protein.
  • the first protein may be a native protein of the host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated. In these circumstances, the first protein will be considered as a heterologous protein in the context of the mutated host cell.
  • the present invention also provides a system for screening or selecting cells for expression of a desired enzyme which produces a product from a substrate, comprising: (i) a library of cells; (ii) a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by a cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein when a cell comprises the sensing construct, the presence of the product in the cell switches on expression of the second protein; and (iii) a device for observing, measuring and/or isolating cells from the library.
  • the cells are genetically modified and form a library of cells expressing variants of the enzyme.
  • the host cell may be used as a vehicle for conducting the screening of heterologous enzyme variants.
  • the cells comprise an heterologous expression construct comprising a nucleic acid with a nucleotide sequence encoding the enzyme.
  • the present invention provides a system for screening or selecting cells for expression of a desired heterologous enzyme which produces a product from a substrate, comprising: (i) cells comprising a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein; and (ii) a library of polynucleotide constructs for expression of one or more desired heterologous enzymes and/or variants in the cells; and (iii) a device for observing, measuring and/or isolating cells.
  • the present invention also provides a nucleic acid construct for detecting the presence of a substance in a cell, comprising a nucleotide sequence encoding a protein, which when the construct is contained in the cell and the protein is expressed, the protein marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self-splicing intron, and the self-splicing intron comprises an aptamer which has binding affinity for the substance, whereby the substance when present in the cell switches on expression of the protein.
  • said nucleic acid construct has at least two self-splicing introns each comprising an aptamer having binding affinity for the substance. More preferably the or each self-splicing intron in the nucleic acid construct is the T4 td gene self-splicing intron. Even more preferably the or each aptamer of the nucleic acid construct binds theophylline.
  • the present invention provides a kit comprising a first container comprising a nucleic acid construct comprising a nucleotide sequence encoding a protein (the reporter), which when the construct is contained in a cell and the protein expressed, the protein directly or indirectly marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self- splicing intron.
  • the kit further comprises a set of instructions.
  • the kit further comprises a second container comprising at least two binding partners; wherein the at least two binding partners are capable of specifically binding sequences which flank the sequence of the at least one aptamer.
  • the binding partners are nucleic acid primers adapted to bind specifically to the nucleic acid sequences adjacent to the at least one aptamer sequence or aptamer.
  • the kit may include other reagents for performing amplification, e.g. PCR reagents.
  • the kit may include a nucleic acid joining enzyme such as a ligase.
  • the nucleic acid construct may be provided as part of a plasmid or a functional expression vector.
  • the kit may further comprise a third container comprising a cell as herein defined.
  • the aptamer may bind theophylline and the kit therefore further comprises a fourth container comprising theophylline.
  • the specific binding partners enable the amplification (for example by polymerase chain reaction) of desired aptamer sequences fused to sequences which are complementary to the sequences flanking the one or more aptamer sequences, such that the desired aptamers may be ligated into the nucleic acid sensing construct at the appropriate positions within the one or more self-splicing introns.
  • This ligation may be achieved for example by Gibson assembly or alternatively by restriction enzyme-based ligation protocols.
  • a variety of suitable PCR amplification-based technologies are well known in the art. PCR applications are routine in the art and the skilled person will be able to select appropriate polymerases, buffers, reporter moieties and reaction conditions.
  • Figure 1 shows an illustration of one of the mechanisms of a riboswitch located in the 5'-UTR of bacterial mRNA.
  • Figure 2 shows the structures of two introns. as shown in Thompson et al., 2002 BMC Biotechnol 2:21 . This drawing is from ⁇ 2002 Thompson et al; licensee BioMed Central ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL: http://www.biomedcentral.eom/1472-6750/2/21 .
  • Panel A shows the parental self- splicing intron of phage T4 (which does not need an inducer to splice).
  • Panel B shows an engineered variant, in which the P6a-loop (see panel A, box) has been replaced by a theophylline-binding aptamer; binding of theophylline induces a conformational change that triggers splicing activity (see panel A, arrows indicate cleavage in loops P1 and P10).
  • Figure 3 shows part of the pyrimidine synthesis pathway.
  • dTMP is a key link in the synthesis of DNA.
  • the pathway from thymine to dTMP is not supported by E. coli, while the pathway from DNA to dTMP does not support growth.
  • Bacteria can therefore be grown under non-selective conditions by adding thymidine (and indeed not thymine) to the medium, a compound which has a very low abundance in common medium components such as tryptone, peptone and yeast extract.
  • Figure 4 shows the relationship between growth rate of single intron thyA constructs (i.e. those with a single self-splicing intron) and promoter strength.
  • E. coli DH10B- AthyA carrying the reporter constructs were grown with different amounts of theophylline and monitored for 20h.
  • Figure 5 shows a comparison of the phage T4 td intron with the mutant td intron.
  • the mutant td intron differs only slightly from the phage T4 td intron in the 5' and 3' flanking sequence and retains its activity.
  • the flanking regions which are part of the riboswitch, but not of the intron (they are part of the exons) are WT and mutated in intron 1 and 2 respectively.
  • Figure 6 shows the growth rate of both single and double intron thyA constructs under control of the P ta ci promoter under varying theophylline concentrations.
  • Intron 1 (0) is the intron at (F171 - P175)
  • intron 2 (A) is the intron at (H51 - I55).
  • a construct featuring no intron in the thyA gene ( ⁇ ) is also shown.
  • Figure 7 shows vector maps for dTMP auxotrophy complementation.
  • Panel A shows the intron insertion like in the phage T4 td intron situation.
  • Panel B shows the introduction of the functional intron upstream of the wild type position, such that the introduction is silent in the sense that no amino acids were changed in the protein sequence into which the intron is inserted.
  • Panel C shows the tandem introns for improved control of expression.
  • Figure 8 shows the structure of a self-splicing intron (Cech et a/., 1994).
  • sequences have been inserted that affect the splicing activity.
  • Insertion of ligand- binding RNA fragments (aptamers) controls the splicing by a conformational change, triggered by the presence or absence of a specific ligand.
  • Figure 9 shows the structure of a small RNA fragment with an aptamer.
  • the aptamer sequence can be varied using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and screened for novel specificities.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • FIG. 10 shows the structures of self-splicing constructs.
  • Panel A shows a schematic of the generic riboswitch-based screening and selection system for obtaining specific enzymes.
  • the system incorporates a library of genes encoding a specific enzyme which, when expressed, generates a product (P) from a substrate (S).
  • the sensing construct shown features two product-dependent self-splicing introns.
  • the activity of the self-splicing introns is under control of at least one ligand- dependent aptamer (1 , 2). In this example, when bound by the product splicing of the mRNA transcript is activated.
  • the splicing restores the open reading frame (ORF) and thus permits translation of the mRNA encoding a second protein, in this case a functional reporter.
  • ORF open reading frame
  • P product, S
  • substrate substrate
  • Panel B shows a single self-splicing intron construct.
  • Panel C shows a double self-splicing intron construct.
  • Panel D compares the structures of pSC024, pSC026, pSC018 and pSC022.
  • Figure 11 shows a schematic of the "reporter cascade" described for GFPuv production, although any protein may be produced.
  • the T7 polymerase gene is carried on the bacterial genome.
  • the enzyme's functionality depends on maturation (RNA splicing that restores open reading frame), which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method.
  • Figure 12 shows the vector map for the reporter plasmid pSC028-GFPuv-term.
  • pSC028-GFPuv-term was constructed using pACYC184 as a base.
  • Figure 13 shows the sensitivity of the cascade, showing the theophylline dependency of the cascade in response to differing concentrations of Rhamnose. The cascade is controlled by one theophylline dependent intron at the first position depicted in Figure 1 1 . In particular expression of GFPuv using the single intron driven cascade over a wide range of rhamnose and theophylline concentrations is shown.
  • An in vivo biosensor is usually composed of a control element and a reporter gene.
  • the reporter gene can confer antibiotic resistance, fluorescence, auxotrophy complementation or luminescence.
  • the control element can act on several stages in the protein production process. Protein based control elements like Lacl typically intervene with the transcription of the gene, while inteins and post-translational modification deal with activation of the protein itself. In between, there is the control on translational level predominantly performed by riboswitches. The riboswitches are mostly located in the 5'-UTR sequestering and releasing the Shine-Dalgarno sequence to block or allow translation by the ribosome.
  • Figure 1 shows the operation of a generalised riboswitch, illustrating how structural changes in the RNA fragment induced by the binding of a signal metabolite may result in the reduced accessibility of the Shine-Dalgarno sequence and the blockage of translation.
  • 5' UTR 5' untranslated region
  • mRNA transcripts form secondary structures (e.g. stem-loop structures) via intra-molecular base paring.
  • the structure of an RNA fragment may change upon specific binding of a ligand, and may affect the accessibility of the ribosome binding site (Shine-Dalgarno sequence) and as such the translation efficiency (riboswitch on/off).
  • Variation of the sequence of the signal metabolite (ligand)-binding sub- fragment ('aptamer') may adjust the specificity of the riboswitch.
  • anti-RBS anti-Ribosome Binding Site
  • anti-terminator may be part of the aptamer domain. Altering the aptamer domain will change the anti-RBS or anti-terminator rendering the riboswitch inactive. Randomising the 5'-UTR and testing for riboswitch activity is one solution to that problem.
  • Another possibility is changing a ribozyme, either a synthetic or a natural one, into a riboswitch by attaching an aptamer domain. This creates an allosteric ribozyme also referred to as aptazyme.
  • aptazyme that was based on the hammerhead ribozyme was designed by Ogawa and Maeda (Ogawa and Maeda, 2007 Bioorg Med Chem Lett 17:3156-3160.) (This aptazyme is based on SD sequestering and the block is released by the endonuclease activity of the ribozyme upon induction. A different approach using the same mechanism can be applied in eukaryotes cutting of the poly-A tail upon induction. A type of synthetic riboswitch that has not been studied extensively is the group I aptazyme. This aptazyme is a modified version of a group I self-splicing intron.
  • the intron that was modified is derived from the phage T4 td gene encoding thymidylate synthase (see Figure 2).
  • This gene has a homologue in E. coli named thyA.
  • the P6a-loop (see panel A, box) has been replaced by a theophylline- binding aptamer.
  • Binding of theophylline induces a conformational change that triggers splicing activity (see panel A, arrows indicate cleavage in loops P1 and P10) (Thompson et al., 2002 BMC Biotechnol 2:21 ) (REF Thompson 2002).
  • This system has many properties that make it suitable as an in vivo biosensor. Contrary to riboswitches that block the Ribosome Binding Site (RBS), there is only one way leakage can occur; when blocking the RBS the block may be released by complete unfolding of part of the mRNA. When the intron is unfolded it does not splice out of the mRNA, still disallowing functional translation. The leakage that will occur in both instances is when the aptamer is not completely destabilised when no ligand is present and the switch is flipped in the absence of a trigger.
  • the gene the intron is naturally present in is an essential gene that can be complemented easily by adding thymidine to the medium or supplying the gene on a plasmid.
  • thyA is a gene encoding thymidylate synthase, a crucial part of the pyrimidine synthesis pathway. It catalyses the reaction from dU P to dTMP using THF as a cofactor (see Figure 3).
  • dTMP is a key link in the synthesis of DNA.
  • the pathway from thymine to dTMP is not supported by E. coli, while the pathway from DNA to dTMP does not support growth.
  • dTMP can also be derived from thymidine when this is added to the medium, but not thymine as E. coli DH10B lacks the enzyme to convert thymine into thymidine.
  • the ThyA deficient bacteria cannot grow on rich medium without addition of thymidine.
  • the relationship between the growth rate of single intron constructs (i.e. those with a single self-splicing intron) and promoter strength was determined.
  • the theophylline dependent phage T4 td intron was designed according to Thompson et al (2002) BMC Biotechnol. 2: 21 .
  • the intron flanking regions are identical on protein level between the thyA gene of E. coli and the td gene of phage T4 allowing for introduction of the intron into thyA with silent mutations only.
  • Reporter constructs carry a p15A origin of replication derived from pACYC184, a kanamycin resistance gene from pET24d and the 5'-UTR and CDS of E. coli thyA.
  • E. coli DH10B-AthyA carrying the reporter constructs were grown with different amounts of theophylline and monitored for 20h. The log phase growth rate was determined in biological triplicate for each construct and theophylline concentration (Figure 4).
  • the constructs with promoters Pt ac i ( ⁇ ) and P te t (O) show near maximum growth while not being induced and maximum growth with slight induction.
  • the Piacuvs (0) construct shows the highest theophylline dependency having no growth without induction and a dynamic range of 0 mM - 0.4 mM theophylline resulting in growth rate 0 h "1 - 0.69 h "1 .
  • the promoters P ara , Pbia, eat and Pi ac do not support log phase growth nor does the negative control (frame shifted thyA).
  • E. coli DH10B containing the frame-shifted thyA serves as positive control (closed square) ( Figure 4).
  • ThyA expression results in more growth. More growth does not necessarily mean that the ThyA expression is high enough to sustain the growth. No growth above OD 6 oo of 0.040 was observed for bacteria carrying thyA under control of the Para, bia, Peat and Pi ac promoters. These promoters do not support log phase growth, but can extend the period the bacteria can grow on the carry-over thymidine depending on the promoter strength and induction by theophylline.
  • the concentration of mature imRNA depends on the concentration of immature mRNA and the maturation rate.
  • the concentration of immature mRNA is mostly dependent on promoter strength, while the maturation rate is dependent on theophylline induction.
  • the maturation rate does not equal zero when the no inducer is present.
  • This leakage is shown by the constructs having a strong promoter in front of the coding sequence. Where there no leakage, promoter strength should have no effect when no inducer is present.
  • the weak promoter constructs do not generate enough mature mRNA even when the maturation rate is high.
  • the amount of ThyA is not enough to reach the minimal concentration of dTTP required in the cell.
  • E. coli DH10B-AthyA carrying the Pi ac uv5 construct was not observed in microtiter plates, it sometimes was observed in 5 ml_ cultures in a 50 imL Greiner tube. Evaporation is a serious issue in the microtiter plate only causing problems after several hours of growth. By that time all exponential growth was finished already and carry-over thymidine was consumed staggering the growth. The bacteria in the Greiner tube did not suffer from evaporation, so a very small subpopulation having slightly increased expression may become dominant overnight indicating that the background expression of ThyA is only just below the minimal requirement, sometimes exceeding it. While this background growth may not be interfering with competition experiments between induced and uninduced bacteria, it may lead to false negatives.
  • the top candidate position was identified as HLRSI (amino acids 51-55) with the intron in frame 2 and only one mutation in the intron flanking region changing a wobble base pair into a U-A base pair.
  • Two unique restriction sites could be mutated close to the insertion site: Psp1406l upstream and Pstl downstream.
  • a construct with a tandem intron at (H51 - I55) and (F171 - P175) and a construct with the (H51 - I55) intron only were made. In both cases, the thyA gene was under control of the Ptaci promoter.
  • the constructs were tested in E. coli DH10B-AthyA according to the same protocol as the single intron constructs ( Figure 6).
  • Intron 1 (0) is the intron at (F171 - P175)
  • intron 2 ( ⁇ ) is the intron at (H51 - I55).
  • Introns 1 and 2 in tandem (O) shows a theophylline dependency with a dynamic range of 0 mM - 0.4 mM theophylline resulting in a growth rate of 0 h "1 - 0.67 h "1 .
  • Less leakage is observed with Intron 2, but less maximum growth rate as well, indicating a lower splice rate.
  • No intron in the thyA gene ( ⁇ ) results in maximum growth regardless of the theophylline concentration.
  • the tandem intron completely erases the background growth even with the P tac i promoter. Aside from the absence of background growth on microtiter plate, no bacterial growth was observed lacking both thymidine and theophylline.
  • the difference in position means that the surrounding parts of the mRNA will have different secondary and tertiary structures as well as a different translation speed. This may affect the splicing rate of the intron.
  • the difference in sequence will affect the ThyA production in two ways.
  • the intron splice rate is directly dependent on the intron flanking region (Pichler and Schroeder, 2002, J Biol Chem 277:17987-17993) which is not the same for intron 1 and 2. Furthermore, by introducing the silent mutations for the restriction sites and the intron flanking region, the amino acid sequence may not be altered, but the codon usage is. The difference in codon usage may influence the ThyA expression either in a positive or negative way.
  • the phage T4 td intron is therefore a useful tool for selection of E. coli that have a small molecule inside their plasma membrane.
  • the ability of this system to completely select against bacteria that have no such small molecule present makes it relatively straightforward to select for the bacteria that do. Leakage and fully- induced expression can be carefully adjusted so bacteria without small molecule do not grow at all, whilst the bacteria with small molecule do. It was shown that the Piacuv5 promoter can balance the leakage and the induced expression so that the dynamic range is between 0 mM and 0.4 mM theophylline resulting in a growth rate between 0 h "1 and 0.69 h "1 on microtiter plate.
  • Tandem introns are significantly more effective in reducing background splicing, while maintaining the dynamic range in both inducer concentration and growth rate.
  • Thymidine and theophylline were purchased from Sigma-Aldrich (St. Louis, MO).
  • a plasmid containing the E. coli thyA gene interrupted by a modified phage T4 td intron between G173 and L174 was commissioned at GeneArt (pMA-ThyA-SI001 ) as well as an intron version containing a theophylline responsive aptamer (pMA-ThyA- Theo).
  • Plasmid pET24d was purchased from Novagen.
  • Plasmid pRham C-His was purchased from Lucigen. Enzymes were purchased from Thermo Scientific and used according to the manufacturer's instructions, unless stated otherwise.
  • E. coli DH 10B T1 R was purchased from Invitrogen (C6400-03) and used for plasmid propagation and standard molecular techniques, as well as a parent strain for the thyA deficient E. coli DH10B-AthyA strain. Transformation was performed with a ECM 63 electroporator (BTX) at 2500 V, 200 ⁇ and 25 ⁇ J F, 2 mm cuvettes, 20-40 ⁇ _ of electro-competent cells and recovery in LB.
  • BTX ECM 63 electroporator
  • Bacteria were generally grown at 37°C on LB medium (Miller) containing the appropriate antibiotics: kanamycin (50 mg/L), ampicillin (100 mg/L), chloramphenicol (35 mg/L) and tetracycline (15 mg/L).
  • kanamycin 50 mg/L
  • ampicillin 100 mg/L
  • chloramphenicol 35 mg/L
  • tetracycline 15 mg/L
  • the auxotrophic E. coli DH 10B- AthyA was complemented with thymidine (100 mg/L) when necessary. Construction of reporter plasmids
  • the reporter plasmids pSC018a-g - Theo were constructed using pACYC184 as a base.
  • the steps include exchange of the chloramphenicol acetyltransferase [cat) for the aminoglycoside 3'-phosphotransferase (kari) from pET24d (Novagen), exchanging the TeiA(C) for the thyA gene encoded on the pMA-ThyA-SI001 plasmid and exchanging the 6b hairpin for the theophylline responsive aptamer from pMA- ThyA-Theo).
  • Promoter variants were made by polymerase chain reaction (PCR) and ligating the PCR product into pSC018f-Theo ( Figure 7) between the Kpnl and Bcul sites.
  • pSC022f-Theo Figure 7) was constructed by cloning a second theophylline responsive intron into pSC018f-Theo between R53 and S54 using the Psp1406l and Pstl sites. The second intron was generated by PCR using pMA-ThyA-Theo as a template.
  • pSC024f and pSC026f-Theo were constructed by using pSC018f-Theo and pSC022f-Theo respectively as template for PCR.
  • DNA purification was performed with the DNA Clean & Concentrator-5 kit of Zymo Research (D4004) or the ZymocleanTM Gel DNA Recovery Kit (D4002). Plasmid was isolated with the Plasmid Miniprep kit of Thermo Scientific (#K0503). Ligation was performed at 22°C for 1 h, followed by 10 min heat inactivation. All plasmids were verified by PCR and/or restriction analysis and sequencing by GATC Biotech (Konstanz, Germany).
  • the thyA deficient strain DH10B-AthyA was made according to a standard protocol (Datsenko and Wanner (2000) PNAS 97: 6640-6645) with the exception of the PCR template and the competent cells protocol and the PCR template for the insertion cassette. Electro-competent cells were made by growing DH10B T1 R (Invitrogen) containing pKD46 at 30°C on 16 g/L peptone, 10 g/L yeast extract and appropriate antibiotic to an OD 6 oo of 0.4 and cooled down to 4°C, washed with ultrapure water once and 10% glycerol twice. Finally the bacteria were concentrated 250x in 10% glycerol.
  • DH10B T1 R containing pKD46 was transformed with a PCR product generated from pMA-RQ-Lox71 -kan-Lox66, kindly provided by Teunke van Rossum, containing a kanamycin resistance gene flanked by Lox71 and Lox66.
  • the Lox sites can be recombined by ere recombinase removing kanamycin resistance, but do not form a functional Lox site.
  • Transformed bacteria were recovered in LB medium containing thymidine (100 mg/L) for 2.5 h at 37°C and plated on LB agar plates containing kanamycin (50 mg/L) and thymidine (20 mg/L).
  • Colonies were verified for thyA deficiency by plating on LB agar plates containing kanamycin (50 mg/L). Plasmid curation was assessed by growing on LB agar plates containing ampicillin (100 mg/L) and thymidine (20 g/L).
  • Electro-competent cells were made from DH10B T1 R -AthyA-kan growing on medium containing kanamycin (50 mg/L) and thymidine (100 mg/L) at 37°C and transformed with pJW168 containing the ere recombinase. Auxotrophy, recombination of the Lox sites and plasmid curation were assessed by plating on LB agar medium, LB agar containing kanamycin (50 mg/L) and thymidine (20 mg/L) and plating on LB agar medium containing ampicillin (50 mg/L) and thymidine (20 mg/L). Electro-competent cells were made of the knock-out strain and transformed with the auxotrophy reporter constructs.
  • E. coli DH10B-AthyA containing a reporter construct of the pSC series were grown overnight at 37°C on LB medium containing kanamycin (50 mg/L) and thymidine (100 mg/L).
  • a 10 ⁇ 4 dilution was made and grown in with a variable amount of theophylline in a 96 well microtiter plate (Greiner) in a final volume of 200 ⁇ . Culture plates were incubated under continuous shaking for 20h at 37°C and the OD 6 oo was measured every 10 minutes in a Synergy MX plate reader.
  • a lower limit ODeoo was set to 0.040 AU to negate false positive growth.
  • Group I self-splicing introns are RNA molecules with catalytic activity: i.e. RNA ribozymes. These introns catalyze their own excision from precursors such as mRNA.
  • the well characterized T4 self-splicing intron has been demonstrated to adopt a specific 3D-structure that is required for catalytic activity ( Figure 8).
  • Figure 8 sequences have been inserted that affect the splicing activity.
  • Insertion of ligand-binding RNA fragments (aptamers) controls the splicing by a conformational change, triggered by the presence or absence of a specific ligand. This ligand could be the product of an enzymatic reaction.
  • the T4 self-splicing intron has been engineered into a functional catalytic riboswitch by inserting a theophylline-binding aptamer (Figure 5).
  • the recombinant riboswitch was still able to splice itself, but its cleavage activity was triggered by a conformational change upon binding of the aptamer ligand, i.e. theophylline (Thompson et al. 2002 BMC Biotechnol. 2: 21 ).
  • the molecular mechanism of the ligand-dependent riboswitch activity is the reversible disruption of the RNA structure ( Figure 5).
  • the intron/aptamer fusion was integrated in a reporter gene.
  • autotrophic markers can be used (essential genes for amino acids or nucleotides are deleted in microbial hosts; growth in the absence of these amino acids or nucleotides is only possible when the corresponding gene is complemented in a plasmid).
  • an E. coli thyA knockout strain the thyA gene encodes an essential enzyme in biosynthesis of thymidine (one of the four bases of DNA nucleotides).
  • the thymidine auxotrophy is complemented by a plasmid-borne thyA gene.
  • the present invention involves the coupling of the presence of a product (of a certain enzyme) to growth; growth is enabled through a product-specific riboswitch.
  • the specificity of riboswitch variants is selected for by a laboratory or 'directed' evolution approach (random variation of 25-50 nt aptamer sequence), using Systematic Evolution of Ligands by Exponential Enrichment, (SELEX) to screen riboswitch libraries for the desired specificity and functionality.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • theophylline controls self-splicing of the riboswitch, thereby restoring the reading frame of the gene encoding a second protein which may encode a selectable marker (e.g. ThyA), resulting in translation of a functional reporter, and as such in growth. In this way, the production of a specific ligand is coupled to growth. Variation of the sequence of the ligand-binding fragment of the riboswitch ('the aptamer') will result in a wide range of aptamers with different ligand specificities.
  • Each mRNA only possesses a single 5'UTR and this limits the potential positions for riboswitches to be inserted. It has been shown that the introduction of more than one riboswitch can confer improved stringency on the activity of the riboswitch, by removing background levels of non-induced splicing (and therefore expression), as there is only one 5'-UTR and multiple positions for an intron, instead of inserting the riboswitch in the 5' UTR, use of the coding sequence allows multiple riboswitches to be inserted into the coding sequence and therefore improved flexibility and stringency of the engineered switch.
  • a specific ligand e.g.
  • theophylline controls self-splicing of the riboswitch, thereby restoring the reading frame of the gene encoding a selection marker (e.g. ThyA), resulting in translation of a functional reporter, and as such in growth. In this way, production of a specific ligand is coupled to growth. Variation of the ligand-binding fragment of the riboswitch ('the aptamer') will result in a wide range of aptamers with different ligand specificity.
  • Auxotrophy complementation is based on interruption of an important step in the pyrimidine synthesis pathway ( Figure 3).
  • the thyA gene is knocked out in the host strain E. coli K12 substrain DH10B and complemented by a either a plasmid encoded thyA copy or thymidine supplement in the growth medium.
  • the plasmid encoded thyA gene is interrupted by a theophylline responsive self- splicing intron; single or in tandem.
  • the vector maps are depicted in Figure 7.
  • the pSC018-Theo ( Figure 7A) contains one intron in the coding sequence and does slow down the growth significantly when not induced, however during prolonged incubation (overnight) the non-induced bacteria grow to the a similar density as the induced bacteria. While induction does give a growth advantage, the selection is not black and white.
  • a single intron insertion on another position (Figure 7B) yields a similar picture as an intron inserted on the wild type position. Only two introns in tandem ( Figure 7C) provide enough control to have no growth at all while the bacteria are not induced and a dose dependent growth when they are.
  • Example 6 Riboswitch-based screening and selection system for obtaining specific enzymes
  • the aptamer sequence ( Figure 10A) of the self-splicing intron can be varied to bind different specific ligands. This can be achieved for example by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and screened for novel specificities to obtain aptamers which bind a product (e.g. enzyme) of interest.
  • This aptamer may then be incorporated into a nucleic acid sensing construct based on the self- splicing intron system described above, whereby product-dependent expression of the construct results in the expression of a detectable reporter. This may as described above relieve an auxotrophic deficit or on expression may mark the host cell, such that those cells where the desired product is present may be readily identified.
  • FIG. 1 1 A schematic of the generic riboswitch-based screening and selection system for obtaining specific enzymes is shown in Figure 1 1 .
  • the system incorporates a library of genes encoding a specific enzyme which, when expressed, generates a product (P) from a substrate (S).
  • the sensing construct shown features two product- dependent self-splicing introns.
  • the activity of the self-splicing introns is under control of at least one ligand-dependent aptamer (1 , 2).
  • the splicing restores the open reading frame (ORF) and thus permits translation of the mRNA encoding a second protein, in this case a functional reporter.
  • the second protein may usefully be a selectable marker, for instance resulting in a detectable change in colour, fluorescence or luminescence (such as mRFP, GFP, LacZ), auxotrophy complementation (thyA) or antibiotic resistance.
  • Linking the expression of the product to a selectable marker in this way allows the use of the system as a high-throughput screen.
  • product-specific aptamers could be used for specific, high-throughput selection system for the discovery of enzymes that generate this product.
  • a 'leakage-proof expression system for the ligand-induced production of toxic proteins can be developed based on translational control by the described self-splicing riboswitches. Generation of an enzyme-specific RNA aptamer
  • Allosteric SELEX is conducted to find an aptamer for an enzyme of interest, for example 2-nitrophenol (ONP).
  • This compound is generated by the well- studied hydrolysis of O-nitrophenyl- -D-galactopyranoside (ONPG), catalysed by ⁇ - galactosidase (LacZ).
  • ONP has limited toxicity and can penetrate the bacterial membranes, as can ONPG, making this a suitable proof of principle reaction.
  • SELEX is used to generate a functional aptamer to be grafted on the riboswitch platform.
  • the process comprises the steps of;
  • oligonucleotide sequences of a fixed length comprising randomly generated sequences of a fixed length. These sequences may include both naturally occurring and/or synthetic nucleic acids, which may expand the number of candidate aptamers in the library. These sequences can optionally be flanked by constant 5' and 3' end sequences, which may serve as primers for amplification. However, flanking sequences are may desirably be omitted due to their stabilizing effect on otherwise unstable candidates.
  • Candidate oligonucleotide sequences of the library are exposed to the target enzyme in a first round of selection. Candidate sequences which do not bind to the enzyme specifically are removed. This may suitably be achieved by affinity chromatography or other means of separation. Sequences which do bind the enzyme of interest specific manner are eluted and amplified by PCR.
  • Single-stranded amplification products are generated from the PCR amplification products, typically by using biotinylated reverse primers in the amplification step, followed by differential elution of the nucleic acid strands.
  • the single-stranded amplification products provide the candidates for the next round of selection.
  • Subsequent rounds of selection are conducted at varying degrees of stringency, depending on the required application and/or binding characteristics. Normally, the stringency of elution conditions in subsequent rounds of selection is increased in order to select for the tightest-binding sequences.
  • the same approach to aptamer generation may be repeated for other enzymes of interest. This may involve several rounds of optimisation in order to generate that specifically bind to a target enzyme or enzymes.
  • ThyA Being an enzyme, a single molecule of ThyA can convert a vast amount of dUMP to dTMP, thereby enhancing the signal.
  • reporters like GFPuv, this is not the case as the bacteria are only as fluorescent as there are GFPuv molecules.
  • Signal below the detection limit is a problem in case of GFPuv as is illustrated in Table 2.
  • the fluorescence of the bacteria themselves (pSC012) is in the same order of magnitude as the induction independent self-splicing intron (pSC034f-SI001 ). The induction by theophylline is not significantly observed in pSC034f-Theo.
  • RNA dependent RNA polymerase from phage T7.
  • the GFPuv expression is controlled by T7 polymerase and the T7 polymerase is in turn controlled by the theophylline responsive intron ( Figure 12).
  • the T7 polymerase gene is carried on the bacterial genome.
  • the enzyme's functionality depends on maturation, which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method.
  • T7 polymerase A few copies of the T7 polymerase will result in a myriad of GFPuv molecules, so a small change in T7 polymerase concentration will result in a large change in GFPuv concentration, which can be measured. Since the T7 polymerase is a very processive enzyme, it needs a tight control of expression. The polymerase is controlled on the transcription level (L-rhamnose dependent promoter) and translation level (theophylline dependent intron). Read-through GFPuv expression is reduced by an upstream terminator.
  • the reporter plasmid pSC028-GFPuv-term was constructed using pACYC184 as a base (vector map shown in Figure 13).
  • the cat gene conferring chloramphenicol resistance was exchanged with the kanamycin resistance gene from pET24d.
  • the tetA(C) gene was replaced causing an insertion site flanked by Acc65l and Bcul.
  • the pGFPuv Ndel- Xhol- was used as template for a polymerase chain reaction (PCR) adding an Ndel site to the 5'-end of the GFPuv gene and a Bcul site to the 3'- end.
  • the PCR fragment was ligated to pRham-CHis (Lucigen) digested with Ndel.
  • This ligation added the 5'-UTR to the GFPuv gene.
  • a PCR was performed on the ligation reaction adding an Acc65l site, a terminator and PT7 promoter in front of the 5'-UTR and a Bcul site to the 3'-end of the GFPuv gene.
  • the secondary PCR fragment was digested with Acc65l and Bcul and ligated into the Acc65l/Bcul insertion site.
  • the T7 terminator from pET24d was amplified by PCR adding a Bcul site to the 5'-end and an Xbal site on the 3'-end. The terminator was ligated into the Bcul site 3' of the GFPuv gene, causing the Bcul site to persist on the 5'-end of the terminator and to be removed on the 3'-end of the terminator.
  • the performance of the cascade was measured using GFPuv expression ( Figure 14). Specifically, the theophylline dependency of the cascade at 0.8 mg/L Rhamnose was measured.
  • the Ptacl serves as benchmark for a strong promoter dependent on E. coli RNA polymerase and is the pSC034f construct. Additionally, expression of GFPuv over a wide range of rhamnose and theophylline concentrations was determined.
  • the cascade is controlled by one theophylline dependent intron at the first position depicted in Figure 12. The system is virtually off when either rhamnose or theophylline is absent, although the absence of L- rhamnose is more important than the absence of theophylline.
  • the background observed when fully induced with rhamnose is about 7.5% of the maximum expression. Also there is a correlation between the background expression in the absence of one inducer and the expression level upon induction. There is a good dose-dependent relationship and the fully induced system yields a signal 5-6 times the signal observed from the strong Tacl promoter. A small amount of theophylline causes a measurable signal already, which can be distinguished by techniques like FACS to separate the bacteria producing the enzyme of interest from the rest.
  • the reporter plasmid pSC028-GFPuv-term ( Figure 13) was constructed using pACYC184 as a base.
  • the cat gene conferring chloramphenicol resistance was exchanged with the kanamycin resistance gene from pET24d.
  • the tetA(C) gene was replaced causing an insertion site flanked by Acc65l and Bcul.
  • the pGFPuv Ndel " Xhol " was used as template for a polymerase chain reaction (PCR) adding an Ndel site to the 5'-end of the GFPuv gene and a Bcul site to the 3'-end.
  • the PCR fragment was ligated to pRham-CHis (Lucigen) digested with Ndel.
  • This ligation adds the 5'-UTR to the GFPuv gene.
  • a PCR was performed on the ligation reaction adding an Acc65l site, a terminator and Pj7 promoter in front of the 5'-UTR and a Bcul site to the 3'-end of the GFPuv gene.
  • the secondary PCR fragment was digested with Acc65l and Bcul and ligated into the Acc65l/Bcul insertion site.
  • the T7 terminator from pET24d was amplified by PCR adding a Bcul site to the 5'-end and an Xbal site on the 3'-end.
  • the terminator was ligated into the Bcul site 3' of the GFPuv gene, causing the Bcul site to persist on the 5'-end of the terminator and to be removed on the 3'-end of the terminator.
  • the plasmid pRham-CHis (Lucigen) was used as base for constructing the T7 polymerase variants.
  • the CDS is flanked by an Ndel site and 6xHis tag on the 5'- end and a Bglll site on the 3'-end.
  • the intron positions are between G201 and L202 flanked by Pscl and Hindlll, between G449 and L450 flanked by Bsu15l and Xagl and between G671 and L672 flanked by Eco88l and Pstl. All have CAAGGGT as 5' intron flank instead of wild type CTTGGGT.
  • the 3' intron flanks are CTAC, CTAC and CTAA respectively.
  • E. coli DH10B-T7His-Theo4 was grown overnight at 37°C in LB medium containing kanamycin (50 mg/L).
  • a 96 well 2 mL culture plate (Greiner) was filled with a concentrate of theophylline and L-rhamnose.
  • LB medium containing kanamycin and overnight grown bacteria were added so that the final concentration of kanamycin was 50 mg/L, the bacteria had a final dilution of 10 "3 and the theophylline and L- rhamnose were diluted to 1x in 500 [it total volume.
  • Culture plates were incubated at 37°C overnight under continuous shaking. The bacteria were centrifuged for 10 minutes at 4700 rpm in a Sorval Legend centrifuge.
  • the supernatant was cleared and the cell pellet was resuspended in 500 [it 50 mM Tris-HCI pH 7.5. After resuspension, the plates were incubated at 37°C for 1 hour to allow maturation of the GFPuv. 100 ⁇ of suspension was pipetted into a 96 well black plate with clear bottom (Perkin Elmer) and measured with a Synergy MX plate reader. The cell density was measured by scattering at 600 nm and the fluorescence was measured at an excitation wavelength of 385 nm with a width of 20 nm and an emission wavelength of 508 nm with 20 nm width with a gain of 50.
  • E. coli DH10B-T7His-Theo4 diluted from an overnight culture were grown overnight in a 2 mL culture plate containing a variable amount of L-rhamnose and theophylline. The medium was cleared and the bacteria were resuspended in 50 mM Tris-HCI pH 7.5. The fluorescence was measured at an excitation wavelength of 385 nm and an emission wavelength of 508 nm. The cell density was measured by scattering at 600 nm. GFPuv fluorescence showed a strong dependency on both L-rhamnose and theophylline (Figure 15).
  • the fluorescence observed without any induction at all does not significantly differ from the fluorescence observed for the GFPuv reporter plasmid alone. This implies that the double control on the T7 polymerase - transcription control by L-rhamnose and translation control by theophylline - succeeds to a large extent in keeping the T7 polymerase inactive.
  • the transcription of T7 polymerase itself dictates the dynamic range in fluorescence caused by theophylline. This dynamic range can be adjusted according to the requirements of the application. At any L-rhamnose concentration, the fold-change caused by theophylline is around 15 times.
  • the cascade being a multi-component system with two types of control makes it very hard to model the dependency on both L- rhamnose and theophylline and to have reproducible results.
  • the transcription of T7His polymerase may be fixed with a constitutive promoter, while the functional translation will be ligand dependent. Since the output dynamic range can be adjusted relatively easily, the intron controlled T7His polymerase can be employed as a generic tool. The intron lowers the maximum translation quite severely, so not all reporter genes will show enough signal when put under control of a ligand dependent intron directly.
  • Enzymes like ThyA or LacZ can handle the lower translation efficiency, but all genes that need an at least decent expression to function, like GFPuv, can now be put under control of one enzyme.
  • An additional advantage is the exchangeability of the reporter plasmids. Expression from these reporter plasmids can be easily adjusted by mutating the T7 promoter.

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Abstract

An intronic, self-splicing riboswitch is configured for enzyme-product specificity by using an appropriate aptamer. The riboswitch is used in a sensing-expression construct in a transformed cell, whereby the presence of an enzyme product in the cell triggers self- splicing of the intron sequence to restore the reading frame of the reporter gene. This then drives expression of the gene product. The sensing construct expresses a protein which marks the cell or permits its growth or survival in or on an otherwise selective media. In this way, introduction or the presence of such product sensing-reporter constructs in cells can be harnessed to provide a multi-parallel rapid screening of cells or libraries for desirable enzyme variants. A "reporter cascade" is set up for GFPuv production. The T7 polymerase gene is carried on the bacterial genome. The enzyme's functionality depends on maturation (RNA splicing that restores open reading frame), which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method.

Description

RIBOSWITCH-CONTROLLED SCREENING AND SELECTION OF DESIRED
BIOCATALYSTS
Field of the Invention
The invention relates to the field of enzymology and methods and techniques for cell- based screening for novel enzyme variants capable of more efficient and/or rapid production of product from substrate.
Background to the Invention
Enzymes enable efficient chemical transformations by reducing the activation energy required for a chemical reaction to proceed at a given temperature and reactant concentration. Consequently, many enzymes are usefully employed in the production of industrially important chemicals.
Enzymes are biological, often green and/or sustainable alternatives for chemical catalysts and are generally orders of magnitude more selective than their synthetic counterparts. Enzymes are unique because of their catalytic power and their extraordinary specificity. The specificity and (enantio- and regio-) selectivity of certain enzymatic transformations make them appealing for the production of fine chemicals, food additives and pharmaceutical intermediates. Theoretically, proteins can catalyse any conversion that is thermo-dynamically feasible. Hence, any product (natural or unnatural) can be generated with biocatalysts that possess the appropriate catalytic features, including the desired substrate specificity.
Although enzymes are faster and more environmentally friendly than traditional chemical catalysts, finding the right enzyme for generating the desired product with a high specificity and a high yield, however, represents a major challenge. Industrial processes frequently take place under extreme conditions (e.g. high temperature, non-neutral pH). Consequently, even when appropriate enzymes are available, they generally require optimisation to function optimally in an industrial setting. Unfortunately, only a limited set of enzymes is available for bioconversion applications. Methods aimed at exploiting natural evolution (screening and selection of metagenome libraries) or using laboratory evolution methods (screening and selection of libraries of enzyme variants) have shown some promise in extending the pool of available enzymes with optimal features. However, the screening of large mutant libraries for variants with the desired functionality remains a major limitation of these approaches; screening millions of mutant enzymes can be complicated, expensive and time-consuming. Often a high-throughput screening assay is simply not available, especially when the product of interest can only be demonstrated by sophisticated analytical tools (Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR)). Therefore, the development of an efficient screening and selection method to isolate appropriate enzyme variants with the desired properties from metagenome and laboratory evolution libraries populated by millions of variants is a major bottleneck in the exploitation of biocatalysis potential.
In recent years the range of available screening and selection strategies has expanded significantly. Of particular interest are in vivo reporter systems in which the activity of a reporter is controlled by the activity of the enzyme of interest. A number of dedicated selection systems have been established by engineering synthetic signal-transduction systems. In some of these systems the enzyme of interest directly modifies the transcriptional regulator resulting in expression of a reporter or alternatively, directly modifies the reporter, altering its activity. In others the product of an intracellular enzyme is detected by a sensor-domain of a transcriptional regulator, that upon binding of the product induces transcription of a reporter or selection system (reviewed in van Rossum et al. 2013 FEBS J. 280:2979- 2996). However, only a limited number of specific transcriptional regulators are available and the specificity of these transcriptional regulators is not adjustable, which places limits on the application of such a selection system.
An alternative approach has focused on the use of riboswitches; messenger RNA (mRNA) molecules comprising regulatory elements that regulate their own activity in response to concentrations of their products. Riboswitches are regulatory segments of messenger RNA (mRNA) molecules that bind small molecules. They consist solely of RNA, sense their ligand in a specific binding pocket and undergo a conformational switch in response to ligand binding which results in a change in production of the proteins encoded by the downstream protein-coding mRNA. Riboswitches therefore regulate mRNA translation in response to the concentrations of their specific effector molecules and control a range of basic metabolic pathways. Usually, a riboswitch controls the expression of a gene encoding an enzyme that catalyzes the production of a certain metabolite; this compound also serves as a ligand that binds to the riboswitch, controlling its conformation that directly affects the expression of the gene: a classical feedback loop. A well-known example of this is the gene btuB, which encodes a transporter of vitamin B12, is controlled by a riboswitch that senses the intracellular concentration of vitamin B12 (Mandal and Breaker 2004 Nat Rev Mol Cell Biol 5:451 -463).
Natural riboswitches are typically located in the 5'-untranslated region (UTR) of a mRNA, controlling the translation of the downstream coding sequence. Inspired by these naturally occurring cases, several synthetic riboswitches have been developed which harness the ability of riboswitches to regulate gene expression in response to exogenously applied stimuli. Again, they reside in the 5'-UTR of a reporter gene, they are induced by ligand-dependent structural rearrangements and they block translation of the reporter transcript either by masking the Shine-Dalgarno sequence or by nucleolytic cleavage by the riboswitch/ribozyme (Suess et al., 2004 Nucleic Acids Res 32:1610-1614; Ogawa and Maeda, 2007 Bioorg Med Chem Lett 17:3156- 3160; Topp and Gallivan 2008 RNA 14:2498-2503; Mandal and Breaker 2004 Nat Rev Mol Cell Biol 5:451-463). A review of current knowledge of engineered riboswitches and their application in gene expression is provided by Groher & Suess 2014, Biochimica et Biophysica Acta 1839:964 - 973. Figure 1 of Groher & Suess illustrates and briefly describes common mechanisms of engineered riboswitches in bacteria. Figure 2 illustrates and briefly describes common mechanism in eukaryotes.
Building on the results of previous studies showing that regions of secondary structure in mRNA 5'-UTRs could cause substantial reductions in expression in prokaryotic and eukaryotic cells (Paraskeva et al. 1998 PNAS 95:951-956; Stripecke et al. 1994 Mol. Cell. Biol. 14:5898-5909; De Smit and Van Duin 1990 PNAS 87:7668-7672) ligand-inducible gene expression was accomplished in yeast by introduction of a small-molecule binding RNA into the 5'-UTR of a gene (Werstuck and Green, 1998 Science 282:296-298). This concept was extended to a variety of organisms by generating synthetic riboswitches which were responsive to theophylline (Desai and Gallivan 2004 J. Am. Chem. Soc. 126:13247-13254; Suess et al. 2004 Nucleic Acids Res. 32:1610-1614; Thompson et al. 2002 BMC Biotechnol. 2: 21 ), tetracyclines (Suess et al. 2003 Nucleic Acids Res. 31 :1853- 1858) or dyes (Werstuck and Green, 1998 Science 282:296-298).
So far these switches have been developed with a view to remotely improving the control of heterologous gene expression in host cells and therefore the focus of this research has largely been centered on the development of high-throughput screens or selections to isolate synthetic riboswitches that respond to a variety of exogenously applied ligands (Thompson et al. 2002 BMC Biotechnol. 2002 2: 21 ; Ogawa and Maeda, 2007 Bioorg. Med. Chem. Lett. 17: 3156-3160).
However, the development of riboswitches that sense and respond to the presence of an endogenously expressed product generated by an enzyme of interest under specific conditions would represent a major breakthrough in the generation of smarter selection systems, allowing the screening of large libraries of cells for enzymes with desired characteristics. An objective of the present invention is the provision of a more rapid and extensive process for the cell-based screening and selection of enzymes and enzyme variants.
The inventors have for the first time configured an intronic, self-splicing riboswitch for enzyme-product specificity by introducing an appropriate aptamer, and then used this in a sensing-expression construct, whereby the presence of an enzyme product in the cell triggers self-splicing of the intron sequence to restore the reading frame of the reporter gene and as such to drive expression of the gene product. The sensing construct expresses a protein which marks the cell or permits its growth or survival in or on an otherwise selective media. In this way, introduction or the presence of such product sensing-reporter constructs in cells can be harnessed to provide a multi- parallel rapid screening of cells or libraries for desirable enzyme variants. Summary of the Invention
Accordingly, the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the imRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein; (ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide; (iii) identifying whether the cell expresses the second protein.
The present invention directly addresses the problem of a limited screening capacity for enzymes with desired properties. This is accomplished by coupling the synthesis of a specific enzyme product to the expression of a detectable reporter by operably linking the specific binding of the product to the or each aptamer with excision of at least one intron which in the absence of product binding blocks translation of a downstream reporter. In this way, by selecting an appropriate selectable marker, product-dependent selection may be coupled to high-throughput screening systems such as product-dependent fluorescence or product-dependent host cell survival. Irrespective of the enzymatic reaction, this will allow screening of large libraries of cells and the rapid enrichment of enzymes with desired properties.
Protein of Interest (First Protein)
The utility of the present invention resides in the broad applicability of the selection systems, methods, nucleic acid constructs, kits and cells of the present invention to any protein which can be expressed in commonly used host cells, for example prokaryotic cells, fungal cells, plant cells or animal cells. The selection systems and methods of the invention are applicable to any protein of interest provided that a suitable aptamer can be generated which will bind to the product produced by the action of the enzyme on reactants. Proteins of interest are typically polypeptide macromolecules comprising 20 or more contiguous amino acid residues and may include, but are not limited to enzymes, structural proteins, binding proteins and/or surface-active proteins. The methods of the present invention will be useful in the selection and production of a large number of different proteins in the agricultural, chemical, industrial and pharmaceutical fields. Proteins of interest may include those of therapeutic value or industrial value. Preferably, the protein of interest will be an enzyme. More preferably, the desired first protein is an enzyme and the product is generated from a substrate as the result of an enzyme reaction. Where the first protein is an enzyme which generates a product from a substrate, it is envisaged that the substrate for the enzyme may be available in the cell either constitutively or upon induction. Alternatively, where the first protein is an enzyme which generates a product from a substrate, the substrate for the enzyme may be provided to the cells.
The first protein may be a protein which is native to the host cell. Alternatively, the first protein may be a heterologous protein. The first protein may be a native protein of the host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated. In these circumstances, the first protein will be considered as a heterologous protein in the context of the mutated host cell.
Host Cells
Advantageously, the present invention is of broad applicability and host cells of the present invention may be derived from any genetically tractable organism which can be cultured. Therefore, in particular, commonly used host cell may be selected for use in accordance with the present invention including prokaryotic or eukaryotic cells which are genetically accessible and which can be cultured. The approaches defined herein for the selection of cells which express a protein of interest may be applied to those cells which are able to serve as a host for production of the protein of interest (POI). It may therefore be applied to commonly used host cells, for example prokaryotic cells, fungal cells, plant cells and animal cells commonly used for recombinant heterologous protein expression. Appropriate host cells may be prokaryotic or eukaryotic. Preferably, host cells will be selected from a prokaryotic cell, a fungal cell, a plant cell, a protist cell or an animal cell. Preferred host cells for use in accordance with the present invention are commonly derived from species which typically exhibit high growth rates, are easily cultured and/or transformed, display short generation times, species which have established genetic resources associated with them or species which have been selected, modified or synthesized for optimal expression of heterologous protein under specific conditions. In preferred embodiments of the invention where the protein of interest is eventually to be used in specific industrial, agricultural, chemical or therapeutic contexts, an appropriate host cell may be selected based on the desired specific conditions or cellular context in which the protein of interest is to be deployed. Preferably the host cell will be a prokaryotic cell. In preferred embodiments the host cell is a bacterial cell. Preferably the host cell is an Escherichia coli (E. coli) cell.
Expression Vectors
In order that expression of the nucleic acid sensing construct can be carried out in the chosen host cell, the polynucleotide sequence encoding the nucleic acid sensing construct will preferably be provided in an expression construct, e.g. an expression vector. In some embodiments, the polynucleotide may be provided in an expression vector. Suitable expression vectors will vary according to the recipient host cell and suitably may incorporate regulatory elements which allow expression in the host cell of interest and preferably which facilitate high-levels of expression. Such regulatory sequences may be capable of influencing transcription or translation of a gene or gene product, for example in terms of initiation, accuracy, rate, stability, downstream processing and mobility.
Such elements may include, for example, strong and/or constitutive promoters, 5' and 3' UTR's, transcriptional and/or translational enhancers, transcription factor or protein binding sequences, start sites and termination sequences, ribosome binding sites, recombination sites, polyadenylation sequences, sense or antisense sequences, sequences ensuring correct initiation of transcription and optionally poly- A signals ensuring termination of transcription and transcript stabilisation in the host cell. The regulatory sequences may be plant-, animal-, bacteria-, fungal- or virus derived, and preferably may be derived from the same organism as the host cell. Clearly, appropriate regulatory elements will vary according to the host cell of interest. For example, regulatory elements which facilitate high-level expression in prokaryotic host cells such as in E. coli may include the pLac, T7, P(Bla), P(Cat), P(Kat), trp or tac promoters. Regulatory elements which facilitate high-level expression in eukaryotic host cells might include the AOX1 or GAL1 promoter in yeast or the CMV- or SV40-promoters, CMV-enhancer, SV40-enhancer, Herpes simplex virus VIP16 transcriptional activator or inclusion of a globin intron in animal cells. In plants, constitutive high-level expression may be obtained using, for example, the Zea mays ubiquitin 1 promoter or 35S and 19S promoters of cauliflower mosaic virus.
Suitable regulatory elements may be constitutive, whereby they direct expression under most environmental conditions or developmental stages, developmental stage specific or inducible. Preferably, the promoter is inducible, to direct expression in response to environmental, chemical or developmental cues, such as temperature, light, chemicals, drought, and other stimuli. Suitably, promoters may be chosen which permit expression of the protein of interest at particular developmental stages or in response to extra- or intra-cellular conditions, signals or externally applied stimuli. For example, a range of promoters exist for use in E. coli which give high- level expression at particular stages of growth (e.g. osmY stationary phase promoter) or in response to particular stimuli (e.g. HtpG Heat Shock Promoter).
Suitable expression vectors may comprise additional sequences encoding selectable markers which allow for the selection of said vector in a suitable host cell and/or under particular conditions.
Transformation of the host cell with a heterologous gene sequence
Expression constructs comprising the polynucleotide sequence encoding the nucleic acid sensing construct may be located in plasmids (expression vectors) which are used to transform the host cell. Similarly, expression constructs comprising the polynucleotide sequence encoding the first desired protein may be located in plasmids (expression vectors) which are used to transform the host cell. Methods of transformation may include but are not limited to; heat shock, electroporation, particle bombardment, chemical induction, microinjection and viral transformation.
The host cell may already express the desired first protein or be capable of expressing the desired first protein and may be subsequently transformed with the nucleic acid sensing construct.
Alternatively, the host cell may first be transformed with the nucleic acid sensing construct and the method may further comprise transforming the host cell with a polynucleotide sequence encoding the desired first protein. Equally, transformation of the host cell with nucleic acid constructs encoding the nucleic acid sensing construct and the first desired protein may take place substantially simultaneously. Preferably the host cell is transformed substantially simultaneously with the sensing and expression constructs.
Nucleic Acid Sensing Construct
The nucleic acid sensing construct comprises a polynucleotide sequence. Polynucleotide sequences encoding the second protein may be isolated nucleic acid molecules and may be a DNA molecule, a cDNA molecule, an RNA molecule or synthetically produced DNA or RNA or a chimeric nucleic acid molecule. In embodiments where the polynucleotide is an RNA, it will be understood that normally uracil (U) is to be used in place of thymine (T). Throughout, the term "polynucleotide" as used herein refers to a deoxyribonucleotide or ribonucleotide polymer in single- or double-stranded form, or sense or anti-sense, and encompasses analogues of naturally occurring nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Such polynucleotides may be derived from any organism, including the host organism, or may be synthesised de novo. The provision of a polynucleotide may comprise synthesis of a polynucleotide. This may be for example by modification of a pre-existing sequence, e.g. by site-directed mutagenesis or possibly by de novo synthesis.
In all embodiments of the invention, polynucleotide sequences encoding the protein of interest may be prepared by any suitable method known to those of ordinary skill in the art, including but not limited to, for example, direct chemical synthesis or cloning for introduction into a desired host cell. Alternatively, the starting polynucleotide sequence may be provided and subsequently modified ex vivo or alternatively in vivo for example by site directed mutagenesis or gene editing techniques.
Self-splicing Introns
Advantageously, methods of the present invention are based on a selectable marker, the gene of which is interrupted by one or more riboswitches with adjustable ribonuclease (RNase) activity and/or adjustable RNA ligase activity. The RNase activity of these "self-splicing introns" is controlled by the concentration of the product produced by the first protein from a substrate. The product-dependent splicing relies on the presence of a polynucleotide fragment of the riboswitch (the "aptamer"). The or each aptamer interacts with the product in a sequence specific manner. Consequently it will be understood that variation of the aptamer sequence will result in binding of (and control by) other products.
Product-dependent expression of the second protein, which may be a selectable marker, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division. The polynucleotide sequence encoding the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the mRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein.
Therefore, in the presence of the product, splicing of the or each intron will be induced, restoring the reading frame of the selectable marker transcript, resulting in faithful translation and therefore expression of the second protein, which either encodes the selectable marker or induces expression of an amplification cascade resulting in the eventual expression of the selectable marker (e.g. antibiotic resistance protein). In any aspect of the invention, in the absence of expression of the first protein substantially no second protein is expressed and there is substantially no marking or growth and/or division of the host cell. Similarly, where there is first protein expression, there may be no product and substantially no second protein is expressed, in this situation there is substantially no marking or growth and/or division of the host cell.
Aptamers
The selection system of the present invention is unique in that it is broadly applicable in that it allows for obtaining enzyme variants from large libraries (either from a natural metagenome library, or from a laboratory evolution library) capable of the specific generation of a certain product. The key to the selection system is the specificity of the aptamer of the nucleic acid sensing construct. Aptamers of the present invention are polynucleotide sequences which have a high binding affinity for the product of the enzyme of interest under specific conditions. The aptamers may be DNA, cDNA, RNA, preferably RNA. Suitable aptamers of the present invention are preferably 20-30nt in length; optionally they are 20nt, 21 nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt or 30nt in length. Although a new aptamer has to be developed for each new product, aptamers which have specific binding affinity for the product of interest may be generated by any means known in the art. Preferably, aptamers of the present invention will be generated in a high-throughput manner by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) of the aptamer fragment. In this way the specificity of each sensing construct can theoretically be adjusted for a desired product.
Second Protein (Reporter Protein)
In the context of methods of the invention, it will be appreciated that the second protein may be a native protein of a host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated. In these circumstances, the second protein will be considered as a heterologous protein in the context of the mutated host cell. However, the second protein may be a non-native, heterologous protein which is capable of being detected as part of a high-throughput system, for example Green Fluorescent Protein (GFP).
In embodiments where the functional expression of a particular protein of interest (POI) in a host cell is coupled to cell survival and/or growth, such selectable markers may include polynucleotide sequences encoding proteins which confer antibiotic resistance. In this way host cells expressing the protein of interest may be selected from those which do not express the protein of interest. In a simplified example of such an embodiment, the product of the protein of interest binds to the one or more aptamers of the nucleic acid sensing construct in cells expressing the POI, resulting in self-splicing of the one or more introns located in the mRNA encoding the antibiotic resistance protein and lifting the block on faithful translation of the antibiotic resistance protein, permitting growth on substrates containing the relevant antibiotic. In other embodiments where the expression of a particular POI in a host cell is coupled to relief of an auxotrophic deficit, it will be appreciated that such selectable markers may include polynucleotide sequences encoding proteins to which the cell is fatally sensitive. In these embodiments of the invention, the presence of the desired product may be coupled to the restoration of translation of the reporter protein. In this way host cells expressing the protein of interest may be selected from those which do not express the protein of interest.
In other embodiments where the expression of a particular POI in a host cell is coupled to promotion of cell growth and/or division, it will be appreciated that such selectable markers may include polynucleotide sequences encoding proteins which promote cell growth and/or division. In these embodiments of the invention, the presence of the desired product may be coupled to the restoration of translation of the reporter protein. In this way host cells expressing the protein of interest may be selected from those which do not express the protein of interest.
In alternative embodiments, suitable expression vectors may comprise sequences which enable visualisation or quantification of the expressed second protein (e.g. Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), Cyan Fluorescent Protein (CFP), or Luciferase fusion tags) in the chosen host cell. The reporter protein may be an enzyme which can be used to generate an optical signal. Alternatively, the expression vector may incorporate a polynucleotide reporter encoding a luminescent protein, such as a luciferase (e.g. firefly luciferase). Alternatively, the reporter gene may be a chromogenic enzyme which can be used to generate an optical signal, e.g. a chromogenic enzyme (such as beta-galactosidase (LacZ) or beta-glucuronidase (Gus)). Tags used for detection of reporter protein expression may also be antigen peptide tags. A cleavable tag may also be provided for affinity purification, e.g. a polyhistidine tag. It is envisaged that other types of label may also be used to indicate expression of the reporter protein including, for example, organic dye molecules or radiolabels. In particular, preferred expression vectors will include sequences encoding a fluorescent protein, for example GFP which will enable the screening and optionally separation (selection) of a cell which expresses the protein of interest for example by Fluorescence Activated Cell Sorting (FACS).
Heterologous protein expression analysis
Subsequently, in preferred embodiments of the present invention the presence of a product generated by a protein of interest may be determined by the detection of expression of the second protein (reporter protein). It will be appreciated that in embodiments where a signaling or expression cascade is used to amplify the expression signal of the reporter protein (i.e. where, dependent on the binding of the product of a first protein to the one or more aptamers of the polynucleotide sequence, the expression of a second protein is operably linked to the expression of one or more further reporter proteins any of which may be detectable). Therefore it will be appreciated that the reporter protein which is eventually detected may in fact be a second protein, alternatively it may be a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth protein depending on the nature of the cascade system used, providing that the presence of the protein of interest is operably linked to the expression of the reporter protein.
In certain embodiments, the expression of the reporter protein will directly result in selection, for example where the selectable marker is a protein coding for a protein conferring antibiotic resistance. Preferably, the specific aptamer will bind the product generated by the POI with high affinity. The insertion of one or more self-splicing introns comprising said aptamer into a gene encoding a substance required for survival on a particular substrate permits selection of cells expressing the POI on that substrate, for example into the coding sequence of a gene encoding the enzyme chloramphenicol acetyl transferase, which confers resistance to the antibiotic chloramphenicol. Cells not expressing the POI will not survive exposure to chloramphenicol. However, it will be appreciated that a number of selectable markers exist which have application in the present invention. Preferably the reporter gene will encode a protein conferring resistance to commonly used antibiotics. Preferably, the reporter gene will encode a protein conferring resistance to aminoglycoside antibiotics, in particular Kanamycin or Gentamicin. Alternatively, the reporter gene may encode a protein conferring resistance to chloramphenicol, optionally the enzyme chloramphenicol acetyl transferase. Other antibiotics which may be useful can include beta-lactam antibiotics, for example penicillins, cephalosporins, carbapenems, and monobactams.
Alternatively, the reporter construct may encode a protein which relieves an auxotrophic deficit which occurs when the host cell is grown under conditions where the desired product is absent. Preferably, the reporter construct encodes thyA which when expressed in the cell is able to overcome an auxotrophic deficit of thymidine monophosphate biosynthesis (see Figure 3).
Advantageously, the present invention provides a method wherein the cell is auxotrophic and the second protein or the third protein provides at least partial relief of the auxotrophy. Alternatively, the cell may be auxotrophic in certain media, and the second protein, or alternatively a third protein (for example, an enzyme) complements the auxotrophy when expressed. Many suitable auxotrophic cells are known in the art. Preferably the auxotrophy is caused by interruption of the pyrimidine synthesis pathway, preferably by a knock-out of thyA and the second protein or the third protein is ThyA. Alternatively, interruption of the pyrimidine synthesis pathway may be achieved by a reduction in the expression of other genes required for pyrimidine synthesis (for example pyrE and/or pyrF) and the second or third protein (encoding, for example, pyrE or pyrF) may provide at least partial relief of the auxotrophy. Alternatively, the present invention provides a method, wherein cell growth and/or division is sensitive to a compound, e.g. antibiotic, and the second protein or the third protein provides at least some resistance to the compound. Preferably, the host cell is fatally sensitive to a compound, for example an antibiotic, and the second protein provides complete resistance to the compound, permitting growth of cells expressing the product of interest on media containing the compound, for example an antibiotic, at concentrations which would typically inhibit growth of cells not possessing the protein conferring resistance. In other embodiments where the expression of the reporter protein does not directly determine cell survival or growth on certain substrates, the expression levels of the reporter protein may be determined. In some instances the expression levels of the reporter protein may be proportional either to the expression levels of the protein of interest or to the binding affinity of the protein of interest for the aptamer. Preferably, the method chosen for expression analysis of the reporter protein allows for quantitative assessment of the level of expression. In some instances, physical expression of the reporter protein may be directly determined, e.g. with GFP or luciferase. In preferred embodiments of the invention, the reporter protein will be detectable by a high-throughput screening method, for example, relying on the detection of an optical signal. Preferably, using an optical signal which is directly proportionate to the quantity of the expression product from the polynucleotide is a convenient method of measuring expression and is amenable to high throughput processing.
Accordingly, in a preferred embodiment of the invention, the measurement of reporter protein expression comprises the detection of an optical signal, for example a fluorescent signal, a luminescent signal or colour signal. In a particularly preferred embodiment the optical signal is provided by a GFP reporter protein.
Optionally, the method further comprises transforming the cell with an expression construct which comprises a polynucleotide sequence encoding the desired first protein. It will be understood that the host cell may be transformed with such an expression construct either before, after or substantially simultaneously with the one or more sensing constructs. Preferably the cell is transformed substantially simultaneously with the expression construct and one or more sensing constructs. In alternative embodiments host cells can be transformed in any order sequentially with the expression construct and the one or more sensing constructs. All of the reporter constructs may be either plasmid borne or integrated into the genome. More particularly one system involves an expression construct encoding a first protein (POI) and a sensing construct, incorporating a riboswitch, which encodes the reporter protein. Another system involves an expression construct encoding a first protein (POI) and a primary reporter construct, incorporating a riboswitch which encodes T7 polymerase and a secondary reporter construct encoding a reporter protein under the control of T7 polymerase.
Although synthetic riboswitches are known, many of them are 'leaky' and so are not appropriate for use in selection systems.
Advantageously, the present invention makes use of riboswitches; stretches of RNA that can adopt different conformational states, depending on the presence or absence of a binding molecule (metabolite, ligand) and provides an unprecedented, adjustable in vivo selection system based on RNA-based translational control by specific synthetic riboswitches. In their natural function, riboswitches are not usually required to completely switch off expression of their genes and therefore the control of gene expression exerted by natural riboswitches is known to be incomplete. However, background levels of cell survival, growth and/or marking of the cells (where visual detection of a reporter is used) due to incomplete riboswitch control of expression (for example in the absence of the binding product), negatively influence the efficiency of selection systems which depend on selective expression or cell growth. For the purpose of developing an accurate screening or selection system, a tightly controlled on/off switch is desirable. Indeed, the more stringent the control by the riboswitch the better. Advantageously, the present invention provides synthetic riboswitches with improved stringency, which are appropriate for use in selecting cells expressing a protein of interest. Surprisingly, this is achieved by incorporating at least two, optionally more than two, i.e. multiple copies of the riboswitches in sequential arrangement. The self-splicing riboswitches whose splicing is under the control of an aptamer, have the effect in use of the sensing constructs and system of the invention of reducing the background levels of growth of cells transformed with the constructs, compared to constructs where there is a riboswitch is located in the UTR. This reduction in background compared to the UTR located riboswitch constructs is preferably at least a 5% reduction, when measured in terms of growth rate of cells; more preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more. When two self-splicing introns are present as the riboswitches under aptamer control, then when compared to equivalent constructs and systems where there is just one riboswitch under the aptamer control, then the background level of growth of cells is reduced, by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%.
Accordingly the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the mRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein; (ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide; (iii) identifying whether the cell expresses the second protein; wherein the sensing construct comprises at least two said self-splicing introns whose splicing activity is under the control of an aptamer. Preferably, the two self-splicing introns comprise the same aptamer. In certain situations it may be desirable to further enhance control of expression by introduction of at least three self-splicing introns whose splicing activity is under the control of an aptamer. Preferably, the three self- splicing introns comprise the same aptamer. Alternatively, it will be appreciated that in embodiments with more than one self-splicing intron, the splicing activities of the introns may desirably be under the control of more than one aptamer, more than two aptamers, more than three aptamers. In this situation, the sequences of the aptamers may be the same. Alternatively, the sequences of the aptamers may be different from one another.
Signal Amplification Cascade
In another aspect of the present invention it may be necessary or desirable to amplify the signal generated by binding of the product, to facilitate detection of product binding to the aptamer. This may be achieved by the use of a signal amplification cascade. Accordingly, the present invention provides a method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising: (i) transforming an host cell with a nucleic acid sensing construct and a further construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which acts on a regulatory element, for example a promoter, of the further construct comprising a nucleotide sequence which encodes a third protein and which is under expression control of a promoter, wherein the promoter is under operable control of the second protein and which when expressed by the cell, directly or indirectly marks the cell and/or permits or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct is interrupted by at least one intron which is a self- splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the mRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein; (ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide; (iii) identifying whether the cell expresses the second protein; wherein the method further comprises transforming the cell with a further construct which comprises a nucleotide sequence which encodes a third protein and which is under expression control of a promoter, wherein the promoter is under operable control of the second protein. Ideally, suitable promoters will generate strong expression in response to stimulation by expression of the second protein. It will be appreciated that such amplification cascades may include further levels of amplification provided by additional promoters under expression control of one or more reporter proteins as desired. In preferred embodiments, the second protein is Phage T7 DNA dependent RNA polymerase and the promoter is PT7. Preferably the PT7 promoter controls the expression of a third protein, which is a reporter protein, for example GFP. Accordingly, the present invention provides a method wherein the second protein or the third protein generates a selectable phenotype; preferably a detectable marker protein, e.g. GFP. Preferably, the expression of the reporter will be proportional to the binding affinity for the aptamer. Even more preferably, such a detectable marker will enable cells expressing the protein of interest to be identified and isolated by a high-throughput system such as FACS.
It will be understood by a person of ordinary skill, that cells selected from a first round of selection may be subjected to one or more further rounds of selection in order to further refine or purify the cells selected by the first round of selection or alternatively to select for different parameters than were selected for in the first round of selection. Consequently, in a further aspect, the present invention provides a method of screening cells for expression of a desired first protein which generates a product from a substrate, comprising a first round of selecting cells according to a method of the invention, followed by subjecting cells selected from the first round to a second round of selecting cells according to a method of the invention. Preferably, the method of screening cells further comprises a third or subsequent rounds of selecting cells whereby cells selected in the previous round are subjected to the further round(s) according to a method of the invention.
In any subsequent round of selecting cells the sequence of the or each aptamer may be varied to select for different parameters or binding affinities for the product or for binding affinity to a different product. Preferably in at least the first round, the aptamer is a first aptamer with a first binding affinity for the product. More preferably, in at least the second, third or subsequent round(s) the aptamer is a second aptamer with a second binding affinity for the product. Even more preferably in at least a third or subsequent round(s) the aptamer is a third aptamer with a third binding affinity for the product. Still more preferably, the binding affinity of the first aptamer for the product is higher than that of the second aptamer.
In another aspect the present invention provides a cell which is capable of expressing a desired first protein, the first protein being a protein which generates a product from a substrate, wherein the cell comprises a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self- splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein. Preferably the nucleotide sequence of the second protein is interrupted by at least two self-splicing introns. More preferably, the two or more self-splicing introns contain the same aptamer. More preferably the cells of the invention further comprise a construct which comprises a nucleotide sequence encoding a third protein under expression control of a promoter, wherein the promoter is under operable control of the second protein. Desirably, the cell may express the desired first protein constitutively. Alternatively, it is envisaged that the cell may express the first protein in response to induction, for example in response to a chemical compound (e.g. Dimethyl sulfoxide (DMSO), Doxycycline, Muristerone A; Ponasterone A), or to environmental (heat shock, high or low pH) physiological (e.g. glucose elevation; hypoxia) or developmental cues (e.g. cell density; growth phase). Suitable inducers also include, but are not limited to; Rhamnose, Arabinose or Isopropyl β-D-l -thiogalactopyranoside (IPTG). As indicated elsewhere, the first protein may be a protein which is native to the host cell or a heterologous protein. Alternatively, the first protein may be a native protein of the host cell in which expression of the native protein has been silenced, for example, the polynucleotide sequence encoding that protein has been disrupted, deleted or mutated. In these circumstances, the first protein will be considered as a heterologous protein in the context of the mutated host cell.
In a further aspect, the present invention also provides a system for screening or selecting cells for expression of a desired enzyme which produces a product from a substrate, comprising: (i) a library of cells; (ii) a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by a cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein when a cell comprises the sensing construct, the presence of the product in the cell switches on expression of the second protein; and (iii) a device for observing, measuring and/or isolating cells from the library. In preferred embodiments the cells are genetically modified and form a library of cells expressing variants of the enzyme. Rather than using sensing constructs to detect the presence of endogenously produced enzyme variants native to the host cell, the host cell may be used as a vehicle for conducting the screening of heterologous enzyme variants. Accordingly, in preferred embodiments the cells comprise an heterologous expression construct comprising a nucleic acid with a nucleotide sequence encoding the enzyme.
In a further aspect the present invention provides a system for screening or selecting cells for expression of a desired heterologous enzyme which produces a product from a substrate, comprising: (i) cells comprising a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein; and (ii) a library of polynucleotide constructs for expression of one or more desired heterologous enzymes and/or variants in the cells; and (iii) a device for observing, measuring and/or isolating cells. In a further aspect the present invention also provides a nucleic acid construct for detecting the presence of a substance in a cell, comprising a nucleotide sequence encoding a protein, which when the construct is contained in the cell and the protein is expressed, the protein marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self-splicing intron, and the self-splicing intron comprises an aptamer which has binding affinity for the substance, whereby the substance when present in the cell switches on expression of the protein. Preferably said nucleic acid construct has at least two self-splicing introns each comprising an aptamer having binding affinity for the substance. More preferably the or each self-splicing intron in the nucleic acid construct is the T4 td gene self-splicing intron. Even more preferably the or each aptamer of the nucleic acid construct binds theophylline.
In another aspect the present invention provides a kit comprising a first container comprising a nucleic acid construct comprising a nucleotide sequence encoding a protein (the reporter), which when the construct is contained in a cell and the protein expressed, the protein directly or indirectly marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self- splicing intron. Optionally the kit further comprises a set of instructions.
In another embodiment the kit further comprises a second container comprising at least two binding partners; wherein the at least two binding partners are capable of specifically binding sequences which flank the sequence of the at least one aptamer. In preferred embodiments, the binding partners are nucleic acid primers adapted to bind specifically to the nucleic acid sequences adjacent to the at least one aptamer sequence or aptamer. Additionally, the kit may include other reagents for performing amplification, e.g. PCR reagents. Also, the kit may include a nucleic acid joining enzyme such as a ligase.
The nucleic acid construct may be provided as part of a plasmid or a functional expression vector.
The kit may further comprise a third container comprising a cell as herein defined.
The aptamer may bind theophylline and the kit therefore further comprises a fourth container comprising theophylline.
Suitably the specific binding partners enable the amplification (for example by polymerase chain reaction) of desired aptamer sequences fused to sequences which are complementary to the sequences flanking the one or more aptamer sequences, such that the desired aptamers may be ligated into the nucleic acid sensing construct at the appropriate positions within the one or more self-splicing introns. This ligation may be achieved for example by Gibson assembly or alternatively by restriction enzyme-based ligation protocols. A variety of suitable PCR amplification-based technologies are well known in the art. PCR applications are routine in the art and the skilled person will be able to select appropriate polymerases, buffers, reporter moieties and reaction conditions.
Detailed Description
The invention will now be described in detail with reference to the examples and to the drawings in which:
Figure 1 shows an illustration of one of the mechanisms of a riboswitch located in the 5'-UTR of bacterial mRNA.
Figure 2 shows the structures of two introns. as shown in Thompson et al., 2002 BMC Biotechnol 2:21 . This drawing is from © 2002 Thompson et al; licensee BioMed Central ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL: http://www.biomedcentral.eom/1472-6750/2/21 . Panel A shows the parental self- splicing intron of phage T4 (which does not need an inducer to splice). Panel B shows an engineered variant, in which the P6a-loop (see panel A, box) has been replaced by a theophylline-binding aptamer; binding of theophylline induces a conformational change that triggers splicing activity (see panel A, arrows indicate cleavage in loops P1 and P10).
Figure 3 shows part of the pyrimidine synthesis pathway. dTMP is a key link in the synthesis of DNA. The pathway from thymine to dTMP is not supported by E. coli, while the pathway from DNA to dTMP does not support growth. Bacteria can therefore be grown under non-selective conditions by adding thymidine (and indeed not thymine) to the medium, a compound which has a very low abundance in common medium components such as tryptone, peptone and yeast extract.
Figure 4 shows the relationship between growth rate of single intron thyA constructs (i.e. those with a single self-splicing intron) and promoter strength. E. coli DH10B- AthyA carrying the reporter constructs were grown with different amounts of theophylline and monitored for 20h.
Figure 5 shows a comparison of the phage T4 td intron with the mutant td intron. The mutant td intron differs only slightly from the phage T4 td intron in the 5' and 3' flanking sequence and retains its activity. The flanking regions which are part of the riboswitch, but not of the intron (they are part of the exons) are WT and mutated in intron 1 and 2 respectively. Figure 6 shows the growth rate of both single and double intron thyA constructs under control of the Ptaci promoter under varying theophylline concentrations. Intron 1 (0) is the intron at (F171 - P175), intron 2 (A) is the intron at (H51 - I55). A construct featuring no intron in the thyA gene (□) is also shown. Figure 7 shows vector maps for dTMP auxotrophy complementation. Panel A shows the intron insertion like in the phage T4 td intron situation. Panel B shows the introduction of the functional intron upstream of the wild type position, such that the introduction is silent in the sense that no amino acids were changed in the protein sequence into which the intron is inserted. Panel C shows the tandem introns for improved control of expression.
Figure 8 shows the structure of a self-splicing intron (Cech et a/., 1994). In loop 6a, sequences have been inserted that affect the splicing activity. Insertion of ligand- binding RNA fragments (aptamers) controls the splicing by a conformational change, triggered by the presence or absence of a specific ligand.
Figure 9 shows the structure of a small RNA fragment with an aptamer. The aptamer sequence can be varied using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and screened for novel specificities.
Figure 10 shows the structures of self-splicing constructs. Panel A shows a schematic of the generic riboswitch-based screening and selection system for obtaining specific enzymes. The system incorporates a library of genes encoding a specific enzyme which, when expressed, generates a product (P) from a substrate (S). The sensing construct shown features two product-dependent self-splicing introns. The activity of the self-splicing introns is under control of at least one ligand- dependent aptamer (1 , 2). In this example, when bound by the product splicing of the mRNA transcript is activated. The splicing restores the open reading frame (ORF) and thus permits translation of the mRNA encoding a second protein, in this case a functional reporter. Abbreviations; P; product, S; substrate. Panel B shows a single self-splicing intron construct. Panel C shows a double self-splicing intron construct. Panel D compares the structures of pSC024, pSC026, pSC018 and pSC022. Figure 11 shows a schematic of the "reporter cascade" described for GFPuv production, although any protein may be produced. The T7 polymerase gene is carried on the bacterial genome. The enzyme's functionality depends on maturation (RNA splicing that restores open reading frame), which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method.
Figure 12 shows the vector map for the reporter plasmid pSC028-GFPuv-term. pSC028-GFPuv-term was constructed using pACYC184 as a base. Figure 13 shows the sensitivity of the cascade, showing the theophylline dependency of the cascade in response to differing concentrations of Rhamnose. The cascade is controlled by one theophylline dependent intron at the first position depicted in Figure 1 1 . In particular expression of GFPuv using the single intron driven cascade over a wide range of rhamnose and theophylline concentrations is shown.
Example 1 - Evaluation of T4 Intron-controlled ThyA auxotrophy
An in vivo biosensor is usually composed of a control element and a reporter gene. The reporter gene can confer antibiotic resistance, fluorescence, auxotrophy complementation or luminescence. The control element can act on several stages in the protein production process. Protein based control elements like Lacl typically intervene with the transcription of the gene, while inteins and post-translational modification deal with activation of the protein itself. In between, there is the control on translational level predominantly performed by riboswitches. The riboswitches are mostly located in the 5'-UTR sequestering and releasing the Shine-Dalgarno sequence to block or allow translation by the ribosome. For example, Figure 1 shows the operation of a generalised riboswitch, illustrating how structural changes in the RNA fragment induced by the binding of a signal metabolite may result in the reduced accessibility of the Shine-Dalgarno sequence and the blockage of translation. Several cases have been described where the 5' untranslated region (5' UTR) of mRNA transcripts form secondary structures (e.g. stem-loop structures) via intra-molecular base paring. The structure of an RNA fragment may change upon specific binding of a ligand, and may affect the accessibility of the ribosome binding site (Shine-Dalgarno sequence) and as such the translation efficiency (riboswitch on/off). Variation of the sequence of the signal metabolite (ligand)-binding sub- fragment ('aptamer') may adjust the specificity of the riboswitch.
It may be difficult to alter the ligand of these riboswitches, since the anti-Ribosome Binding Site (anti-RBS) or anti-terminator may be part of the aptamer domain. Altering the aptamer domain will change the anti-RBS or anti-terminator rendering the riboswitch inactive. Randomising the 5'-UTR and testing for riboswitch activity is one solution to that problem. Another possibility is changing a ribozyme, either a synthetic or a natural one, into a riboswitch by attaching an aptamer domain. This creates an allosteric ribozyme also referred to as aptazyme. An aptazyme that was based on the hammerhead ribozyme was designed by Ogawa and Maeda (Ogawa and Maeda, 2007 Bioorg Med Chem Lett 17:3156-3160.) (This aptazyme is based on SD sequestering and the block is released by the endonuclease activity of the ribozyme upon induction. A different approach using the same mechanism can be applied in eukaryotes cutting of the poly-A tail upon induction. A type of synthetic riboswitch that has not been studied extensively is the group I aptazyme. This aptazyme is a modified version of a group I self-splicing intron. The intron that was modified is derived from the phage T4 td gene encoding thymidylate synthase (see Figure 2). This gene has a homologue in E. coli named thyA. In the engineered variant, derived from the parental self-splicing intron of phage T4 (both shown in Figure 2), the P6a-loop (see panel A, box) has been replaced by a theophylline- binding aptamer. Binding of theophylline induces a conformational change that triggers splicing activity (see panel A, arrows indicate cleavage in loops P1 and P10) (Thompson et al., 2002 BMC Biotechnol 2:21 ) (REF Thompson 2002).
This system has many properties that make it suitable as an in vivo biosensor. Contrary to riboswitches that block the Ribosome Binding Site (RBS), there is only one way leakage can occur; when blocking the RBS the block may be released by complete unfolding of part of the mRNA. When the intron is unfolded it does not splice out of the mRNA, still disallowing functional translation. The leakage that will occur in both instances is when the aptamer is not completely destabilised when no ligand is present and the switch is flipped in the absence of a trigger. The gene the intron is naturally present in is an essential gene that can be complemented easily by adding thymidine to the medium or supplying the gene on a plasmid. By default, no large amount of thymidine is present in most widely used media like LB, so all experiments can be performed on rich media. The selection allows for a great number of variants to be tested without expensive equipment or labour intensive experiments. A property the td intron based riboswitch shares with other riboswitches is the transferability to other organisms, as riboswitches are unaffected by post-translational modification. Group I introns have the extra advantage that no species-specific elements like SD sequence or poly-A tail are involved. This experiment focuses on the exact conditions the theophylline responsive T4 td intron needs to function as selection tool in E. coli.
Auxotrophy complementation with thyA under control of a theophylline dependent intron
thyA is a gene encoding thymidylate synthase, a crucial part of the pyrimidine synthesis pathway. It catalyses the reaction from dU P to dTMP using THF as a cofactor (see Figure 3). As a precursor of dTTP, which is required for DNA synthesis dTMP is a key link in the synthesis of DNA. The pathway from thymine to dTMP is not supported by E. coli, while the pathway from DNA to dTMP does not support growth. dTMP can also be derived from thymidine when this is added to the medium, but not thymine as E. coli DH10B lacks the enzyme to convert thymine into thymidine. The ThyA deficient bacteria cannot grow on rich medium without addition of thymidine.
The relationship between the growth rate of single intron constructs (i.e. those with a single self-splicing intron) and promoter strength was determined. The theophylline dependent phage T4 td intron was designed according to Thompson et al (2002) BMC Biotechnol. 2: 21 . The intron flanking regions are identical on protein level between the thyA gene of E. coli and the td gene of phage T4 allowing for introduction of the intron into thyA with silent mutations only. Reporter constructs carry a p15A origin of replication derived from pACYC184, a kanamycin resistance gene from pET24d and the 5'-UTR and CDS of E. coli thyA. A terminator and promoters of different strength were placed upstream of the 5'-UTR. All promoters are listed in Table 1 . E. coli DH10B-AthyA carrying the reporter constructs were grown with different amounts of theophylline and monitored for 20h. The log phase growth rate was determined in biological triplicate for each construct and theophylline concentration (Figure 4).
The constructs with promoters Ptaci (Δ) and Ptet (O) show near maximum growth while not being induced and maximum growth with slight induction. The Piacuvs (0) construct shows the highest theophylline dependency having no growth without induction and a dynamic range of 0 mM - 0.4 mM theophylline resulting in growth rate 0 h"1 - 0.69 h"1. The promoters Para, Pbia, eat and Piac do not support log phase growth nor does the negative control (frame shifted thyA). E. coli DH10B containing the frame-shifted thyA serves as positive control (closed square) (Figure 4). Code Name Sequence
- 35box -10box
Consensus seq TfGA iNNNNNNNNNNNNNNNNN - 7 i ΓΑΛΓ
SC019a P_a ra CTGMCGCTTTTTATCGCAACTC - - TCTACT
SC019b P_bla TTCMAM T ATG TAT CCGCTCATGA -6A CMM T
SC019C P_cat /ITOM ITAAGATCACTACCGGGCG77¾F7"7T
SC019d P_lac TFWaCTTTATGCTTCCGGCTCGWreFF
SC019e P_lacUV5 niAC ICTTTATGCTTCCGGCTCGr l Γ Wr
SC019f P_tacl TFGMOtATTAATCATCGGCTCG - - TMTMAT
SC019g P_tet 7T(MC_AGCTTATCATCGATAAGC - TTTAAT
Table 1. Promoter sequences used in the expression of reporter constructs in E. coli
In all cases a higher ThyA expression results in more growth. More growth does not necessarily mean that the ThyA expression is high enough to sustain the growth. No growth above OD6oo of 0.040 was observed for bacteria carrying thyA under control of the Para, bia, Peat and Piac promoters. These promoters do not support log phase growth, but can extend the period the bacteria can grow on the carry-over thymidine depending on the promoter strength and induction by theophylline.
Theophylline dependent log phase growth was observed for Piacuvs, Ptaci and Ptet. These promoters more closely resemble the consensus sequence of the -35 and -10 regions. To observe better growth when the auxotrophy complementation is under control of a stronger promoter is to be expected. The promoters Ptaci and Ptet are strong enough to support log phase growth without induction by theophylline, while the Piacuvs promoter does not. The positive control, E. coli DH10B with the frame- shifted thyA gene, appears to be slightly theophylline dependent. This effect is marginal and is most likely caused by the position the samples have on the microtiter plate rather than the theophylline concentration (Figure 4).
Auxotrophy complementation indirectly depends on the concentration of mature mRNA. The concentration of mature imRNA depends on the concentration of immature mRNA and the maturation rate. The concentration of immature mRNA is mostly dependent on promoter strength, while the maturation rate is dependent on theophylline induction. The maturation rate does not equal zero when the no inducer is present. This leakage is shown by the constructs having a strong promoter in front of the coding sequence. Where there no leakage, promoter strength should have no effect when no inducer is present. The weak promoter constructs do not generate enough mature mRNA even when the maturation rate is high. The amount of ThyA is not enough to reach the minimal concentration of dTTP required in the cell. A concentration below the minimal requirement will result in thymine-less death. It appears there is a fine line between never enough ThyA and always enough ThyA. The balance is matched rather well with the Piacuv promoter. No growth is observed when no inducer is present and maximum growth is observed at full induction. (Figure 4).
Although the growth of E. coli DH10B-AthyA carrying the Piacuv5 construct was not observed in microtiter plates, it sometimes was observed in 5 ml_ cultures in a 50 imL Greiner tube. Evaporation is a serious issue in the microtiter plate only causing problems after several hours of growth. By that time all exponential growth was finished already and carry-over thymidine was consumed staggering the growth. The bacteria in the Greiner tube did not suffer from evaporation, so a very small subpopulation having slightly increased expression may become dominant overnight indicating that the background expression of ThyA is only just below the minimal requirement, sometimes exceeding it. While this background growth may not be interfering with competition experiments between induced and uninduced bacteria, it may lead to false negatives.
Example 2 - Introduction of a second intron into the thyA coding sequence
The strong promoters Ptaci and Ptet showed leakage exceeding the minimal requirement of ThyA (Figure 4). A second intron was introduced into the coding region of thyA. No other part of the thyA gene matches the native flanking regions of the phage T4 id intron, so another strategy was applied. The absence of an easily identified insertion site presents a challenge as the intron ribozyme is composed not only of the intron region itself, but of the 3' flank of exon 1 and the 5' flank of exon 2 as well. The intron flanks are therefore part of both the ribozyme and the coding region (Figure 5).
Previously, Pichler et al. (Pichler and Schroeder, 2002, J Biol Chem 277:17987- 17993) showed that the flanking sequence does not need to match the native flanking sequence perfectly for a functional phage T4 td intron. Next to some tolerance in the intron flanking regions, the coding sequence can be composed of different codons. An algorithm was written to analyse the thyA coding sequence for possible locations for the intron to be inserted. The position had to match several requirements: 1 ) Mutations in the coding sequence were to be translationally silent for both the intron flanking regions and the restriction sites to clone the second intron into the thyA gene; 2) The flanking regions of the second intron had to match the flanking regions of the native intron as closely as possible; 3) No mutations in the flanking regions were allowed other than described by Pichler et al. (Pichler and Schroeder, 2002, J Biol Chem 277:17987-17993); and 4) Possibility of silent introduction a restriction site next to either flank was preferential.
The top candidate position was identified as HLRSI (amino acids 51-55) with the intron in frame 2 and only one mutation in the intron flanking region changing a wobble base pair into a U-A base pair. Two unique restriction sites could be mutated close to the insertion site: Psp1406l upstream and Pstl downstream. A construct with a tandem intron at (H51 - I55) and (F171 - P175) and a construct with the (H51 - I55) intron only were made. In both cases, the thyA gene was under control of the Ptaci promoter. The constructs were tested in E. coli DH10B-AthyA according to the same protocol as the single intron constructs (Figure 6).
The growth rate of both single and double intron constructs under control of the Ptaci promoter was measured (Figure 6). Intron 1 (0) is the intron at (F171 - P175), intron 2 (Δ) is the intron at (H51 - I55). Introns 1 and 2 in tandem (O) shows a theophylline dependency with a dynamic range of 0 mM - 0.4 mM theophylline resulting in a growth rate of 0 h"1 - 0.67 h"1. Less leakage is observed with Intron 2, but less maximum growth rate as well, indicating a lower splice rate. No intron in the thyA gene (□) results in maximum growth regardless of the theophylline concentration. A reduction of background was discovered with the second intron (Figure 6). The tandem intron completely erases the background growth even with the Ptaci promoter. Aside from the absence of background growth on microtiter plate, no bacterial growth was observed lacking both thymidine and theophylline. The second intron at (H51 - I55) alone shows a slightly lower ThyA expression compared to the first intron at (F171 - P175). This result may be caused by the difference in position, the difference in sequence or both. The difference in position means that the surrounding parts of the mRNA will have different secondary and tertiary structures as well as a different translation speed. This may affect the splicing rate of the intron. The difference in sequence will affect the ThyA production in two ways. Firstly, the intron splice rate is directly dependent on the intron flanking region (Pichler and Schroeder, 2002, J Biol Chem 277:17987-17993) which is not the same for intron 1 and 2. Furthermore, by introducing the silent mutations for the restriction sites and the intron flanking region, the amino acid sequence may not be altered, but the codon usage is. The difference in codon usage may influence the ThyA expression either in a positive or negative way.
The phage T4 td intron is therefore a useful tool for selection of E. coli that have a small molecule inside their plasma membrane. The ability of this system to completely select against bacteria that have no such small molecule present makes it relatively straightforward to select for the bacteria that do. Leakage and fully- induced expression can be carefully adjusted so bacteria without small molecule do not grow at all, whilst the bacteria with small molecule do. It was shown that the Piacuv5 promoter can balance the leakage and the induced expression so that the dynamic range is between 0 mM and 0.4 mM theophylline resulting in a growth rate between 0 h"1 and 0.69 h"1 on microtiter plate. However, this particular promoter is not expected to support Log phase growth and is not so preferred. A direct route to manage balance between leakage and full expression is the introduction of a second intron at an upstream position. Tandem introns are significantly more effective in reducing background splicing, while maintaining the dynamic range in both inducer concentration and growth rate. Materials and methods
Chemicals and plasmids
Thymidine and theophylline and were purchased from Sigma-Aldrich (St. Louis, MO). A plasmid containing the E. coli thyA gene interrupted by a modified phage T4 td intron between G173 and L174 was commissioned at GeneArt (pMA-ThyA-SI001 ) as well as an intron version containing a theophylline responsive aptamer (pMA-ThyA- Theo). Plasmid pET24d was purchased from Novagen. Plasmid pRham C-His was purchased from Lucigen. Enzymes were purchased from Thermo Scientific and used according to the manufacturer's instructions, unless stated otherwise.
Bacterial strains and media
E. coli DH 10B T1 R was purchased from Invitrogen (C6400-03) and used for plasmid propagation and standard molecular techniques, as well as a parent strain for the thyA deficient E. coli DH10B-AthyA strain. Transformation was performed with a ECM 63 electroporator (BTX) at 2500 V, 200 Ω and 25 \J F, 2 mm cuvettes, 20-40 μΙ_ of electro-competent cells and recovery in LB. Bacteria were generally grown at 37°C on LB medium (Miller) containing the appropriate antibiotics: kanamycin (50 mg/L), ampicillin (100 mg/L), chloramphenicol (35 mg/L) and tetracycline (15 mg/L). In addition, the auxotrophic E. coli DH 10B- AthyA was complemented with thymidine (100 mg/L) when necessary. Construction of reporter plasmids
The reporter plasmids pSC018a-g - Theo were constructed using pACYC184 as a base. The steps include exchange of the chloramphenicol acetyltransferase [cat) for the aminoglycoside 3'-phosphotransferase (kari) from pET24d (Novagen), exchanging the TeiA(C) for the thyA gene encoded on the pMA-ThyA-SI001 plasmid and exchanging the 6b hairpin for the theophylline responsive aptamer from pMA- ThyA-Theo).
Promoter variants were made by polymerase chain reaction (PCR) and ligating the PCR product into pSC018f-Theo (Figure 7) between the Kpnl and Bcul sites. pSC022f-Theo (Figure 7) was constructed by cloning a second theophylline responsive intron into pSC018f-Theo between R53 and S54 using the Psp1406l and Pstl sites. The second intron was generated by PCR using pMA-ThyA-Theo as a template. pSC024f and pSC026f-Theo (Figure 7) were constructed by using pSC018f-Theo and pSC022f-Theo respectively as template for PCR. Ligation of the PCR products into pSC018f between the Acc65l and Mlul site removed the intron between G173 and L174, leaving no intron in pSC024f and one intron between R53 and S54 in pSC026f-Theo. A frame-shift construct in the thyA gene of pSC024f was constructed by digestion with Mlul, Klenow fragment 5' fill-in and re-ligation of the plasmid.
DNA purification was performed with the DNA Clean & Concentrator-5 kit of Zymo Research (D4004) or the Zymoclean™ Gel DNA Recovery Kit (D4002). Plasmid was isolated with the Plasmid Miniprep kit of Thermo Scientific (#K0503). Ligation was performed at 22°C for 1 h, followed by 10 min heat inactivation. All plasmids were verified by PCR and/or restriction analysis and sequencing by GATC Biotech (Konstanz, Germany).
Construction of the thymidine synthase deficient strain
The thyA deficient strain DH10B-AthyA was made according to a standard protocol (Datsenko and Wanner (2000) PNAS 97: 6640-6645) with the exception of the PCR template and the competent cells protocol and the PCR template for the insertion cassette. Electro-competent cells were made by growing DH10B T1 R (Invitrogen) containing pKD46 at 30°C on 16 g/L peptone, 10 g/L yeast extract and appropriate antibiotic to an OD6oo of 0.4 and cooled down to 4°C, washed with ultrapure water once and 10% glycerol twice. Finally the bacteria were concentrated 250x in 10% glycerol. DH10B T1 R containing pKD46 was transformed with a PCR product generated from pMA-RQ-Lox71 -kan-Lox66, kindly provided by Teunke van Rossum, containing a kanamycin resistance gene flanked by Lox71 and Lox66. The Lox sites can be recombined by ere recombinase removing kanamycin resistance, but do not form a functional Lox site. Transformed bacteria were recovered in LB medium containing thymidine (100 mg/L) for 2.5 h at 37°C and plated on LB agar plates containing kanamycin (50 mg/L) and thymidine (20 mg/L). Colonies were verified for thyA deficiency by plating on LB agar plates containing kanamycin (50 mg/L). Plasmid curation was assessed by growing on LB agar plates containing ampicillin (100 mg/L) and thymidine (20 g/L).
Electro-competent cells were made from DH10B T1 R -AthyA-kan growing on medium containing kanamycin (50 mg/L) and thymidine (100 mg/L) at 37°C and transformed with pJW168 containing the ere recombinase. Auxotrophy, recombination of the Lox sites and plasmid curation were assessed by plating on LB agar medium, LB agar containing kanamycin (50 mg/L) and thymidine (20 mg/L) and plating on LB agar medium containing ampicillin (50 mg/L) and thymidine (20 mg/L). Electro-competent cells were made of the knock-out strain and transformed with the auxotrophy reporter constructs.
E. coli DH10B-AthyA growth assays
E. coli DH10B-AthyA containing a reporter construct of the pSC series were grown overnight at 37°C on LB medium containing kanamycin (50 mg/L) and thymidine (100 mg/L). A 10~4 dilution was made and grown in with a variable amount of theophylline in a 96 well microtiter plate (Greiner) in a final volume of 200 μί. Culture plates were incubated under continuous shaking for 20h at 37°C and the OD6oo was measured every 10 minutes in a Synergy MX plate reader. As carry-over thymidine allows the knock-out strain to grow without ThyA, a lower limit ODeoo was set to 0.040 AU to negate false positive growth. Growth rate (μ) was calculated from at least 1 h of log phase growth exceeding an OD60o of 0.040 according to ln(C) = ln(C0)■ μ■ t
Example 3 - Development of a synthetic riboswitch-ribozvme hybrid
Group I self-splicing introns are RNA molecules with catalytic activity: i.e. RNA ribozymes. These introns catalyze their own excision from precursors such as mRNA. The well characterized T4 self-splicing intron has been demonstrated to adopt a specific 3D-structure that is required for catalytic activity (Figure 8). In loop 6a, sequences have been inserted that affect the splicing activity. Insertion of ligand-binding RNA fragments (aptamers) controls the splicing by a conformational change, triggered by the presence or absence of a specific ligand. This ligand could be the product of an enzymatic reaction.
The T4 self-splicing intron has been engineered into a functional catalytic riboswitch by inserting a theophylline-binding aptamer (Figure 5). The recombinant riboswitch was still able to splice itself, but its cleavage activity was triggered by a conformational change upon binding of the aptamer ligand, i.e. theophylline (Thompson et al. 2002 BMC Biotechnol. 2: 21 ). The molecular mechanism of the ligand-dependent riboswitch activity is the reversible disruption of the RNA structure (Figure 5). Replacing the stem-loop by an aptamer destabilizes its formation, but the stem-loop can be restored by a ligand binding to the aptamer. However, the formation of the stem-loop can occur without binding of the ligand. The stem-loop without the bound ligand is less stable than the ligand bound stem-loop, but can cause background provided the screening or selection method is sensitive enough.
To test for riboswitch functionality, the intron/aptamer fusion was integrated in a reporter gene. Next to the aforementioned antibiotic resistance marker, also autotrophic markers can be used (essential genes for amino acids or nucleotides are deleted in microbial hosts; growth in the absence of these amino acids or nucleotides is only possible when the corresponding gene is complemented in a plasmid). For the riboswitch test, an E. coli thyA knockout strain; the thyA gene encodes an essential enzyme in biosynthesis of thymidine (one of the four bases of DNA nucleotides). The thymidine auxotrophy is complemented by a plasmid-borne thyA gene. When using a plasmid with ThyA that was interrupted by the hybrid riboswitch, it was demonstrated that the thyA knockout strain of E. coli could survive on minimal medium containing theophylline (without thymidine), but not on minimal medium lacking both theophylline and thymidine (Thompson et al. 2002 BMC Biotechnol. 2: 21 ). Hence, the presence of the riboswitch ligand theophylline, allows for growth. Example 4 - Tandem riboswitch provides improved stringency
In the aforementioned experiments (Thompson et al. 2002 BMC Biotechnol. 2: 21 ), there was a background level cell growth was observed in the absence of the theophylline ligand (Figure 9). This background was subtracted from the growth observed in the presence of the ligand. Theophylline-induced growth was approximately 40% of the parental intron, so maximally 40% of the wild type growth. In an experiment based on the latter publication, the induction of the thyA gene by theophylline turned out to be far from stringent. Non-induced splicing of the intron was thought to be the cause of growth on minimal medium lacking both theophylline and thymidine. As a solution to the problem of non-induced splicing, a suitable second insertion site was identified, for the insertion of a second self-splicing intron.
A comparison of a single intron construct with a construct of the present invention featuring a tandem self-splicing intron was made (Figure 9). Growth (OD600) was plotted against time (minutes) in the presence (1 mM) and absence (0 mM) of theophylline for the construct designed by Thompson et al. (Thompson et al. 2002 BMC Biotechnol. 2: 21 ) utilising one theophylline-dependent intron in thyA, in a thyA- deficient E. coll DH10B strain (Figure 9A). On this time scale the theophylline- induced bacteria outcompeted the non-induced bacteria with ease, but the non- induced bacteria continued growth until the OD600 reached a value comparable to the induced bacteria overnight (not shown). Growth was measured in the same way for a construct containing an E. coli thyA gene interrupted by two theophylline- dependent introns, which was transformed into a thyA-deficient E. coli DH10B strain. The overnight growth was plotted against the theophylline concentration in the medium (Figure 9B). The growth shows a non-linear relationship with theophylline concentration. The line drawn through the first five points represents a Michaelis- Menten-like kinetics profile with an apparent kd value of 0.1 -0.2 mM theophylline (Figure 9B). Surprisingly, the absence of growth in the absence of theophylline indicates a selection method with no detectable background (Figure 9B) in contrast with the prior art construct (Figure 9A) where a low level of cell growth continues in the absence of theophylline). Above 1 mM, theophylline became slightly toxic. The introduction of a "tandem-riboswitch" resulted in a 70% recovery of growth compared to wild type with theophylline induction, while no growth at all was observed without induction by theophylline, i.e. black and white selection. The present invention involves the coupling of the presence of a product (of a certain enzyme) to growth; growth is enabled through a product-specific riboswitch. The specificity of riboswitch variants is selected for by a laboratory or 'directed' evolution approach (random variation of 25-50 nt aptamer sequence), using Systematic Evolution of Ligands by Exponential Enrichment, (SELEX) to screen riboswitch libraries for the desired specificity and functionality. Instead of inserting the riboswitch in the 5' UTR, one or more riboswitches are inserted into the coding sequence encoding the second protein. The presence of a specific ligand (e.g. theophylline) controls self-splicing of the riboswitch, thereby restoring the reading frame of the gene encoding a second protein which may encode a selectable marker (e.g. ThyA), resulting in translation of a functional reporter, and as such in growth. In this way, the production of a specific ligand is coupled to growth. Variation of the sequence of the ligand-binding fragment of the riboswitch ('the aptamer') will result in a wide range of aptamers with different ligand specificities. Several applications have been described for the use of the theophylline-dependent group I intron (Thompson et al. 2002 BMC Biotechnol. 2: 21 ): (i) Gene therapy induced by several different drugs, (ii) monitoring of drug uptake and efficacy, and (iii) monitoring of gene expression in vivo. Instead, we aim to detect the production of non-native metabolites. The presence of non-native metabolite indicates the presence of a gene and the corresponding protein/enzyme that converts the provided substrate, which is either derived from E. coli metabolism or supplied externally. Detection and selection of every new enzyme product requires a specific aptamer domain. However, the system should be easily adaptable to fit the interaction between the aptamer and another ligand by a high-throughput SELEX procedure. The aforementioned use of multiple introns is an essential, unprecedented optimization of the system.
Each mRNA only possesses a single 5'UTR and this limits the potential positions for riboswitches to be inserted. It has been shown that the introduction of more than one riboswitch can confer improved stringency on the activity of the riboswitch, by removing background levels of non-induced splicing (and therefore expression), as there is only one 5'-UTR and multiple positions for an intron, instead of inserting the riboswitch in the 5' UTR, use of the coding sequence allows multiple riboswitches to be inserted into the coding sequence and therefore improved flexibility and stringency of the engineered switch. The presence of a specific ligand (e.g. theophylline) controls self-splicing of the riboswitch, thereby restoring the reading frame of the gene encoding a selection marker (e.g. ThyA), resulting in translation of a functional reporter, and as such in growth. In this way, production of a specific ligand is coupled to growth. Variation of the ligand-binding fragment of the riboswitch ('the aptamer') will result in a wide range of aptamers with different ligand specificity.
Example 5 - Auxotrophy complementation
Auxotrophy complementation is based on interruption of an important step in the pyrimidine synthesis pathway (Figure 3). The thyA gene is knocked out in the host strain E. coli K12 substrain DH10B and complemented by a either a plasmid encoded thyA copy or thymidine supplement in the growth medium.
The plasmid encoded thyA gene is interrupted by a theophylline responsive self- splicing intron; single or in tandem. The vector maps are depicted in Figure 7. The pSC018-Theo (Figure 7A) contains one intron in the coding sequence and does slow down the growth significantly when not induced, however during prolonged incubation (overnight) the non-induced bacteria grow to the a similar density as the induced bacteria. While induction does give a growth advantage, the selection is not black and white. A single intron insertion on another position (Figure 7B) yields a similar picture as an intron inserted on the wild type position. Only two introns in tandem (Figure 7C) provide enough control to have no growth at all while the bacteria are not induced and a dose dependent growth when they are. Example 6 - Riboswitch-based screening and selection system for obtaining specific enzymes
The aptamer sequence (Figure 10A) of the self-splicing intron can be varied to bind different specific ligands. This can be achieved for example by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and screened for novel specificities to obtain aptamers which bind a product (e.g. enzyme) of interest. This aptamer may then be incorporated into a nucleic acid sensing construct based on the self- splicing intron system described above, whereby product-dependent expression of the construct results in the expression of a detectable reporter. This may as described above relieve an auxotrophic deficit or on expression may mark the host cell, such that those cells where the desired product is present may be readily identified.
A schematic of the generic riboswitch-based screening and selection system for obtaining specific enzymes is shown in Figure 1 1 . The system incorporates a library of genes encoding a specific enzyme which, when expressed, generates a product (P) from a substrate (S). The sensing construct shown features two product- dependent self-splicing introns. The activity of the self-splicing introns is under control of at least one ligand-dependent aptamer (1 , 2). In this example, when bound by the product splicing of the mRNA transcript is activated. The splicing restores the open reading frame (ORF) and thus permits translation of the mRNA encoding a second protein, in this case a functional reporter. As indicated in the figure, the second protein may usefully be a selectable marker, for instance resulting in a detectable change in colour, fluorescence or luminescence (such as mRFP, GFP, LacZ), auxotrophy complementation (thyA) or antibiotic resistance. Linking the expression of the product to a selectable marker in this way allows the use of the system as a high-throughput screen. In principle, product-specific aptamers could be used for specific, high-throughput selection system for the discovery of enzymes that generate this product. Moreover, a 'leakage-proof expression system for the ligand-induced production of toxic proteins can be developed based on translational control by the described self-splicing riboswitches. Generation of an enzyme-specific RNA aptamer
The generation of a biosensor for the screening/selection system has a solid proof of principle. Allosteric SELEX is conducted to find an aptamer for an enzyme of interest, for example 2-nitrophenol (ONP). This compound is generated by the well- studied hydrolysis of O-nitrophenyl- -D-galactopyranoside (ONPG), catalysed by β- galactosidase (LacZ). ONP has limited toxicity and can penetrate the bacterial membranes, as can ONPG, making this a suitable proof of principle reaction. SELEX is used to generate a functional aptamer to be grafted on the riboswitch platform.
The process comprises the steps of;
Synthesis of a large oligonucleotide library comprising randomly generated sequences of a fixed length. These sequences may include both naturally occurring and/or synthetic nucleic acids, which may expand the number of candidate aptamers in the library. These sequences can optionally be flanked by constant 5' and 3' end sequences, which may serve as primers for amplification. However, flanking sequences are may desirably be omitted due to their stabilizing effect on otherwise unstable candidates. Candidate oligonucleotide sequences of the library are exposed to the target enzyme in a first round of selection. Candidate sequences which do not bind to the enzyme specifically are removed. This may suitably be achieved by affinity chromatography or other means of separation. Sequences which do bind the enzyme of interest specific manner are eluted and amplified by PCR.
Single-stranded amplification products are generated from the PCR amplification products, typically by using biotinylated reverse primers in the amplification step, followed by differential elution of the nucleic acid strands. The single-stranded amplification products provide the candidates for the next round of selection.
Subsequent rounds of selection are conducted at varying degrees of stringency, depending on the required application and/or binding characteristics. Normally, the stringency of elution conditions in subsequent rounds of selection is increased in order to select for the tightest-binding sequences. The same approach to aptamer generation may be repeated for other enzymes of interest. This may involve several rounds of optimisation in order to generate that specifically bind to a target enzyme or enzymes.
Example 7 - Intron controlled expression cascade
Being an enzyme, a single molecule of ThyA can convert a vast amount of dUMP to dTMP, thereby enhancing the signal. For other reporters, like GFPuv, this is not the case as the bacteria are only as fluorescent as there are GFPuv molecules. Signal below the detection limit is a problem in case of GFPuv as is illustrated in Table 2. The fluorescence of the bacteria themselves (pSC012) is in the same order of magnitude as the induction independent self-splicing intron (pSC034f-SI001 ). The induction by theophylline is not significantly observed in pSC034f-Theo. Possibly there is a difference in expression between the induction independent self-splicing intron and the non-induced and induced theophylline dependent intron, but it cannot be concluded from these data. When no intron is present in the GFPuv (pSC034f) the fluorescence is multiple orders of magnitude higher than the background fluorescence.
Figure imgf000043_0001
Table 2 - Direct interruption of GFPuv
To enhance the signal from GFPuv, a cascade was made using DNA dependent RNA polymerase from phage T7. The GFPuv expression is controlled by T7 polymerase and the T7 polymerase is in turn controlled by the theophylline responsive intron (Figure 12). The T7 polymerase gene is carried on the bacterial genome. The enzyme's functionality depends on maturation, which is controlled by theophylline in case of the heterologous expression system, or a compound derived from an enzymatic reaction when the system is utilised as a screening method. A few copies of the T7 polymerase will result in a myriad of GFPuv molecules, so a small change in T7 polymerase concentration will result in a large change in GFPuv concentration, which can be measured. Since the T7 polymerase is a very processive enzyme, it needs a tight control of expression. The polymerase is controlled on the transcription level (L-rhamnose dependent promoter) and translation level (theophylline dependent intron). Read-through GFPuv expression is reduced by an upstream terminator.
The reporter plasmid pSC028-GFPuv-term was constructed using pACYC184 as a base (vector map shown in Figure 13). The cat gene conferring chloramphenicol resistance was exchanged with the kanamycin resistance gene from pET24d. The tetA(C) gene was replaced causing an insertion site flanked by Acc65l and Bcul. The pGFPuv Ndel- Xhol- was used as template for a polymerase chain reaction (PCR) adding an Ndel site to the 5'-end of the GFPuv gene and a Bcul site to the 3'- end. The PCR fragment was ligated to pRham-CHis (Lucigen) digested with Ndel. This ligation added the 5'-UTR to the GFPuv gene. A PCR was performed on the ligation reaction adding an Acc65l site, a terminator and PT7 promoter in front of the 5'-UTR and a Bcul site to the 3'-end of the GFPuv gene. The secondary PCR fragment was digested with Acc65l and Bcul and ligated into the Acc65l/Bcul insertion site. Finally, the T7 terminator from pET24d was amplified by PCR adding a Bcul site to the 5'-end and an Xbal site on the 3'-end. The terminator was ligated into the Bcul site 3' of the GFPuv gene, causing the Bcul site to persist on the 5'-end of the terminator and to be removed on the 3'-end of the terminator.
The performance of the cascade was measured using GFPuv expression (Figure 14). Specifically, the theophylline dependency of the cascade at 0.8 mg/L Rhamnose was measured. The Ptacl serves as benchmark for a strong promoter dependent on E. coli RNA polymerase and is the pSC034f construct. Additionally, expression of GFPuv over a wide range of rhamnose and theophylline concentrations was determined. The cascade is controlled by one theophylline dependent intron at the first position depicted in Figure 12. The system is virtually off when either rhamnose or theophylline is absent, although the absence of L- rhamnose is more important than the absence of theophylline. The background observed when fully induced with rhamnose is about 7.5% of the maximum expression. Also there is a correlation between the background expression in the absence of one inducer and the expression level upon induction. There is a good dose-dependent relationship and the fully induced system yields a signal 5-6 times the signal observed from the strong Tacl promoter. A small amount of theophylline causes a measurable signal already, which can be distinguished by techniques like FACS to separate the bacteria producing the enzyme of interest from the rest.
It is unlikely that an enzyme of interest will provide a concentration as high as 1 mM for every small molecule that is screened. As the enzyme of interest functions inside the cell, the cell membranes acting as a barrier will help the production of GFPuv rather than diminishing it. Usually aptamers have a dissociation constant in the low μΜ range, so for maximum signal the intracellular theophylline concentration does not have to be 1 mM, but much less.
Example 8 - Amplification of the signal using a T7 GFPuv Cascade Construction of reporter plasmids
The reporter plasmid pSC028-GFPuv-term (Figure 13) was constructed using pACYC184 as a base. The cat gene conferring chloramphenicol resistance was exchanged with the kanamycin resistance gene from pET24d. The tetA(C) gene was replaced causing an insertion site flanked by Acc65l and Bcul. The pGFPuv Ndel" Xhol" was used as template for a polymerase chain reaction (PCR) adding an Ndel site to the 5'-end of the GFPuv gene and a Bcul site to the 3'-end. The PCR fragment was ligated to pRham-CHis (Lucigen) digested with Ndel. This ligation adds the 5'-UTR to the GFPuv gene. A PCR was performed on the ligation reaction adding an Acc65l site, a terminator and Pj7 promoter in front of the 5'-UTR and a Bcul site to the 3'-end of the GFPuv gene. The secondary PCR fragment was digested with Acc65l and Bcul and ligated into the Acc65l/Bcul insertion site. Finally, the T7 terminator from pET24d was amplified by PCR adding a Bcul site to the 5'-end and an Xbal site on the 3'-end. The terminator was ligated into the Bcul site 3' of the GFPuv gene, causing the Bcul site to persist on the 5'-end of the terminator and to be removed on the 3'-end of the terminator. The plasmid pRham-CHis (Lucigen) was used as base for constructing the T7 polymerase variants. The CDS is flanked by an Ndel site and 6xHis tag on the 5'- end and a Bglll site on the 3'-end. The intron positions are between G201 and L202 flanked by Pscl and Hindlll, between G449 and L450 flanked by Bsu15l and Xagl and between G671 and L672 flanked by Eco88l and Pstl. All have CAAGGGT as 5' intron flank instead of wild type CTTGGGT. The 3' intron flanks are CTAC, CTAC and CTAA respectively.
GFPuv fluorescence
E. coli DH10B-T7His-Theo4 was grown overnight at 37°C in LB medium containing kanamycin (50 mg/L). A 96 well 2 mL culture plate (Greiner) was filled with a concentrate of theophylline and L-rhamnose. LB medium containing kanamycin and overnight grown bacteria were added so that the final concentration of kanamycin was 50 mg/L, the bacteria had a final dilution of 10"3 and the theophylline and L- rhamnose were diluted to 1x in 500 [it total volume. Culture plates were incubated at 37°C overnight under continuous shaking. The bacteria were centrifuged for 10 minutes at 4700 rpm in a Sorval Legend centrifuge. The supernatant was cleared and the cell pellet was resuspended in 500 [it 50 mM Tris-HCI pH 7.5. After resuspension, the plates were incubated at 37°C for 1 hour to allow maturation of the GFPuv. 100 μί of suspension was pipetted into a 96 well black plate with clear bottom (Perkin Elmer) and measured with a Synergy MX plate reader. The cell density was measured by scattering at 600 nm and the fluorescence was measured at an excitation wavelength of 385 nm with a width of 20 nm and an emission wavelength of 508 nm with 20 nm width with a gain of 50. The background fluorescence and background scattering were subtracted and the fluorescence was divided by the scattering at 600 nm. The background fluorescence of bacteria without either GFPuv or T7His polymerase was negligible, but the fluorescence caused by other components than GFPuv in the bacteria was still subtracted. Results
The theophylline dependency of the cascade in response to differing concentrations of Rhamnose was measured. E. coli DH10B-T7His-Theo4 diluted from an overnight culture were grown overnight in a 2 mL culture plate containing a variable amount of L-rhamnose and theophylline. The medium was cleared and the bacteria were resuspended in 50 mM Tris-HCI pH 7.5. The fluorescence was measured at an excitation wavelength of 385 nm and an emission wavelength of 508 nm. The cell density was measured by scattering at 600 nm. GFPuv fluorescence showed a strong dependency on both L-rhamnose and theophylline (Figure 15). The fluorescence observed without any induction at all does not significantly differ from the fluorescence observed for the GFPuv reporter plasmid alone. This implies that the double control on the T7 polymerase - transcription control by L-rhamnose and translation control by theophylline - succeeds to a large extent in keeping the T7 polymerase inactive. The transcription of T7 polymerase itself dictates the dynamic range in fluorescence caused by theophylline. This dynamic range can be adjusted according to the requirements of the application. At any L-rhamnose concentration, the fold-change caused by theophylline is around 15 times. The cascade being a multi-component system with two types of control makes it very hard to model the dependency on both L- rhamnose and theophylline and to have reproducible results. For a final application as a biosensor, the transcription of T7His polymerase may be fixed with a constitutive promoter, while the functional translation will be ligand dependent. Since the output dynamic range can be adjusted relatively easily, the intron controlled T7His polymerase can be employed as a generic tool. The intron lowers the maximum translation quite severely, so not all reporter genes will show enough signal when put under control of a ligand dependent intron directly. Enzymes like ThyA or LacZ can handle the lower translation efficiency, but all genes that need an at least decent expression to function, like GFPuv, can now be put under control of one enzyme. An additional advantage is the exchangeability of the reporter plasmids. Expression from these reporter plasmids can be easily adjusted by mutating the T7 promoter.

Claims

A method of selecting a cell in culture for expression of a desired first protein which generates a product from a substrate, comprising:
(i) transforming an host cell with a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, directly or indirectly marks the cell and/or permits and/or promotes cell growth and/or division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on splicing of the imRNA transcript, thereby restoring the open reading frame that results in functional expression of the second protein;
(ii) culturing the cell for a period of time such that cells expressing the desired first protein grow and divide;
(iii) identifying whether the cell expresses the second protein.
A method as claimed in claim 1 , wherein the sensing construct comprises at least two said self-splicing introns whose splicing activity is under the control of an aptamer.
A method as claimed in claim 1 or claim 2, further comprising transforming the cell with an expression construct which comprises a polynucleotide sequence encoding the desired first protein.
A method as claimed in claim 3, wherein the cell is transformed substantially simultaneously with the sensing and expression constructs.
A method as claimed in any preceding claim, wherein the desired first protein is an enzyme and the product is generated from a substrate as the result of an enzyme reaction.
6. A method as claimed in any preceding claim, wherein the cell is prokaryotic or eukaryotic.
7. A method as claimed in any of claims 2 to 6, wherein the two self-splicing introns each comprise an aptamer and wherein the aptamer is the same.
8. A method as claimed in any preceding claim, comprising transforming the cell with a further construct which comprises a nucleotide sequence which encodes a third protein and which is under expression control of a promoter, wherein the promoter is under operable control of the second protein.
9. A method as claimed in claim 8, wherein the second protein is Phage T7 DNA dependent RNA polymerase and the promoter is PT7.
10. A method as claimed in any preceding claim, wherein the second protein or the third protein generates a selectable phenotype; preferably a detectable marker protein, e.g. GFP.
1 1 .A method as claimed in claim 10, wherein the cells are identified and isolated by FACS.
12. A method as claimed in any of claims 1 to 10, wherein the cell is auxotrophic and the second protein or the third protein provides at least partial relief of the auxotrophy.
13. A method as claimed in claim 12, wherein the auxotrophy is caused by
interruption of the pyrimidine synthesis pathway, preferably knock out of thyA and the second protein or the third protein is ThyA.
14. A method as claimed in any of claims 1 to 10, wherein cell growth and/or division is sensitive to a compound, e.g. antibiotic, and the second protein or the third protein provides at least some resistance to the compound.
15. A method as claimed in any preceding claim, wherein the first protein is an enzyme and a substrate for the enzyme is provided to the cells.
16. A method as claimed in any preceding claim, wherein in the absence of expression of first protein substantially no second protein is expressed and there is substantially no marking or growth and/or division of the host cell.
17. A method as claimed in any preceding claim, wherein the or each aptamer binds theophylline.
18. A method of screening cells for expression of a desired first protein which generates a product from a substrate, comprising a first round of selecting cells according to a method of any of claims 1 to 17, followed by subjecting cells selected from the first round to a second round of selecting cells according to a method of any of claims 1 to 17.
19. A method of screening cells as claimed in claim 18, comprising third or
subsequent rounds of selecting cells whereby cells selected in the previous round are subjected to the further round(s) according to a method of any of claims 1 to 17.
20. A method of screening cells as claimed in claim 18 or claim 19, wherein in at least the first round, the aptamer is a first aptamer with a first binding affinity for the product.
21 .A method of screening cells as claimed in any of claims 18 to 20, wherein in at least the second, third or subsequent round(s) the aptamer is a second aptamer with a second binding affinity for the product.
22. A method of screening cells as claimed in any of claims 19 to 21 , wherein in at least a third or subsequent round(s) the aptamer is a third aptamer with a third binding affinity for the product.
23. A method of screening cells as claimed in any of claims 18 to 22, wherein the binding affinity of the first aptamer for the product is higher than that of the second aptamer.
24. A cell which is capable of expressing a desired first protein, the first protein being a protein which generates a product from a substrate, wherein the cell comprises a nucleic acid sensing construct, the sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, directly or indirectly, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self- splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein.
25. A cell as claimed in claim 24, wherein the nucleotide sequence of the second protein is interrupted by at least two self-splicing introns.
26. A cell as claimed in claim 25, wherein the two or more self-splicing introns contain the same aptamer.
27. A cell as claimed in any of claims 25 to 26, further comprising a construct which comprises a nucleotide sequence encoding a third protein under expression control of a promoter, wherein the promoter is under operable control of the second protein.
28. A system for screening or selecting cells for expression of a desired enzyme which produces a product from a substrate, comprising:
(i) a library of cells;
(ii) a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by a cell, marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein when a cell comprises the sensing construct, the presence of the product in the cell switches on expression of the second protein; and
(iii) a device for observing, measuring and/or isolating cells from the library.
29. A system as claimed in claim 28, wherein cells are genetically modified and form a library of cells expressing variants of the enzyme.
30. A system as claimed in claim 29, wherein cells comprise an heterologous
expression construct comprising a nucleic acid with a nucleotide sequence encoding the enzyme.
31 .A system for screening or selecting cells for expression of a desired
heterologous enzyme which produces a product from a substrate, comprising:
(i) cells comprising a sensing construct comprising a polynucleotide sequence which encodes a second protein which, when expressed by the cell, directly or indirectly marks the cell and/or permits or promotes cell division, the polynucleotide sequence of the second protein of the sensing construct being interrupted by at least one intron which is a self-splicing intron and whose splicing activity is under the control of an aptamer, the aptamer having binding affinity for the product; wherein the presence of the product in the cell switches on expression of the second protein; and
(ii) a library of polynucleotide constructs for expression of one or more desired heterologous enzymes and/or variants in the cells; and
(iii) a device for observing, measuring and/or isolating cells.
32. A nucleic acid construct for detecting the presence of a substance in a cell, comprising a nucleotide sequence encoding a protein, which when the construct is contained in the cell and the protein is expressed, the protein marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self-splicing intron, and the self- splicing intron comprises an aptamer which has binding affinity for the substance, whereby the substance when present in the cell switches on expression of the protein.
33. A nucleic acid construct as claimed in claim 32, having at least two self- splicing introns each comprising an aptamer having binding affinity for the substance.
34. A nucleic acid construct as claimed in claim 32 or claim 33, wherein the or each self-splicing intron is the T4 fd gene self-splicing intron.
35. A kit comprising a first container comprising a nucleic acid construct
comprising a nucleotide sequence encoding a protein, which when the construct is contained in a cell and the protein expressed, the protein marks the cell and/or selects, permits or promotes cell division; wherein the protein coding sequence has at least one self-splicing intron; optionally further comprising a set of instructions.
36. A kit as claimed in claim 35, further comprising a second container comprising at least two binding partners; wherein the at least two binding partners are capable of specifically binding sequences which flank the sequence of the at least one aptamer.
37. A kit as claimed in claim 35 or claim 36, wherein the nucleic acid construct is provided as part of an expression vector.
38. A kit as claimed in any of claims 35 to 37, wherein the kit further comprises a third container comprising a cell of any of claims 24 to 27.
39. A kit as claimed in any of claims 35 to 37, wherein the aptamer binds
theophylline and the kit further comprises a fourth container comprising theophylline.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018025085A3 (en) * 2016-08-03 2018-04-12 Meiragtx Uk Ii Limited High throughput cell-based screening for aptamers
US10494646B2 (en) 2015-02-02 2019-12-03 Meiragtx Uk Ii Limited Regulation of gene expression by aptamer-mediated modulation of alternative splicing
WO2022074113A1 (en) * 2020-10-08 2022-04-14 Wageningen Universiteit Universal riboswitch for inducible gene expression

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064956A2 (en) * 2000-03-01 2001-09-07 Amgen Inc. The identification and use of effectors and allosteric molecules for the alteration of gene expression
WO2004033653A2 (en) * 2002-10-10 2004-04-22 Oxford Biomedica (Uk) Limited Gene regulation with aptamer and modulator complexes for gene therapy
WO2007073163A1 (en) * 2005-12-21 2007-06-28 Eidgenössische Technische Hochschule Zürich Selection of biocatalysts for chemical synthesis
WO2008116220A2 (en) * 2007-03-22 2008-09-25 Yale University Methods and compositions related to riboswitches that control alternative splicing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001064956A2 (en) * 2000-03-01 2001-09-07 Amgen Inc. The identification and use of effectors and allosteric molecules for the alteration of gene expression
WO2004033653A2 (en) * 2002-10-10 2004-04-22 Oxford Biomedica (Uk) Limited Gene regulation with aptamer and modulator complexes for gene therapy
WO2007073163A1 (en) * 2005-12-21 2007-06-28 Eidgenössische Technische Hochschule Zürich Selection of biocatalysts for chemical synthesis
WO2008116220A2 (en) * 2007-03-22 2008-09-25 Yale University Methods and compositions related to riboswitches that control alternative splicing

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
CHEAH M T ET AL: "Control of alternative RNA splicing and gene expression by eukaryotic riboswitches", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 447, no. 7143, 24 May 2007 (2007-05-24), pages 497 - 500,1, XP002584633, ISSN: 0028-0836, [retrieved on 20070429], DOI: 10.1038/NATURE05769 *
CHRISTIAN BERENS ET AL: "RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression", BIOTECHNOLOGY JOURNAL, vol. 10, no. 2, 1 February 2015 (2015-02-01), DE, pages 246 - 257, XP055279945, ISSN: 1860-6768, DOI: 10.1002/biot.201300498 *
DESAI S K ET AL: "Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, US, vol. 126, 1 January 2004 (2004-01-01), pages 13,247 - 13,254, XP002982412, ISSN: 0002-7863, DOI: 10.1021/JA048634J *
DONG-SUK KIM ET AL: "An antificial riboswitch for controlling pre-mRNA splicing", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 11, no. 11, 1 November 2005 (2005-11-01), pages 1667 - 1677, XP008126231, ISSN: 1355-8382, DOI: 10.1261/RNA.2162205 *
GROHER FLORIAN ET AL: "Synthetic riboswitches - A tool comes of age", BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, vol. 1839, no. 10, Sp. Iss. SI, October 2014 (2014-10-01), pages 964 - 973, XP002759901 *
KIM DONG-SUK ET AL: "Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing", BMC MOLECULAR BIOLOGY, BIOMED CENTRAL LTD, GB, vol. 9, no. 1, 12 February 2008 (2008-02-12), pages 23, XP021033460, ISSN: 1471-2199 *
OGAWA ET AL: "Aptazyme-based riboswitches as label-free and detector-free sensors for cofactors", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, AMSTERDAM, NL, vol. 17, no. 11, 10 May 2007 (2007-05-10), pages 3156 - 3160, XP022068412, ISSN: 0960-894X, DOI: 10.1016/J.BMCL.2007.03.033 *
STEPHAN VAN SINT FIET ET AL: "Selection of biocatalysts for chemical synthesis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 103, no. 6, 30 January 2006 (2006-01-30), pages 1693 - 1698, XP002420997, ISSN: 0027-8424, DOI: 10.1073/PNAS.0504733102 *
THOMPSON K M ET AL: "GROUP I APTAZYMES AS GENETIC REGULATORY SWITCHES", BMC BIOTECHNOLOGY, BIOMED CENTRAL LTD. LONDON, GB, vol. 2, 4 December 2002 (2002-12-04), pages 1 - 12, XP008062371, ISSN: 1472-6750, DOI: 10.1186/1472-6750-2-21 *
VAN ROSSUM TEUNKE ET AL: "Reporter-based screening and selection of enzymes.", THE FEBS JOURNAL JUL 2013, vol. 280, no. 13, July 2013 (2013-07-01), pages 2979 - 2996, XP002759900, ISSN: 1742-4658 *
WEIGAND JULIA E ET AL: "Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD, GB, vol. 35, no. 12, 1 June 2007 (2007-06-01), pages 4179 - 4185, XP002615441, ISSN: 0305-1048, [retrieved on 20070612], DOI: 10.1093/NAR/GKM425 *
WELZ R ET AL: "Ligand binding and gene control characteristics of tandem riboswitches in Bacillus anthracis", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 13, no. 4, 16 February 2007 (2007-02-16), pages 573 - 582, XP002615439, ISSN: 1355-8382, [retrieved on 20070216], DOI: 10.1261/RNA.407707 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10494646B2 (en) 2015-02-02 2019-12-03 Meiragtx Uk Ii Limited Regulation of gene expression by aptamer-mediated modulation of alternative splicing
US11248239B2 (en) 2015-02-02 2022-02-15 Meiragtx Uk Ii Limited Regulation of gene expression by aptamer-mediated modulation of alternative splicing
WO2018025085A3 (en) * 2016-08-03 2018-04-12 Meiragtx Uk Ii Limited High throughput cell-based screening for aptamers
US11371077B2 (en) 2016-08-03 2022-06-28 Meiragtx Uk Ii Limited High throughput cell-based screening for aptamers
WO2022074113A1 (en) * 2020-10-08 2022-04-14 Wageningen Universiteit Universal riboswitch for inducible gene expression

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