WO2017186854A1 - Plateforme de sélection d'inhibiteur de protéase in vivo à haut débit - Google Patents

Plateforme de sélection d'inhibiteur de protéase in vivo à haut débit Download PDF

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WO2017186854A1
WO2017186854A1 PCT/EP2017/060069 EP2017060069W WO2017186854A1 WO 2017186854 A1 WO2017186854 A1 WO 2017186854A1 EP 2017060069 W EP2017060069 W EP 2017060069W WO 2017186854 A1 WO2017186854 A1 WO 2017186854A1
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protease
amino acid
polypeptide
acid sequence
seq
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Eric VAN DER HELM
Morten Sommer
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Danmarks Tekniske Universitet
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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    • 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
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • TITLE High throughput in vivo protease inhibitor selection platform
  • the invention relates to a recombinant microbial cell comprising a selection platform for screening for a protease inhibitor, wherein the platform comprises transgenes encoding a protease having selective peptide bond cleavage activity at a recognition site amino acid sequence; and transgenes encoding polypeptides conferring resistance to microbial growth inhibitors; wherein the polypeptides comprise the recognition site amino acid sequence cleavable by the protease.
  • Protease inhibitors are detected by their ability to inhibit protease specific cleavage and inactivation of the polypeptides whose activity is required for conferring resistance to the microbial growth inhibitors.
  • the invention further relates to recombinant microbial host cell libraries of metagenomic DNA that further comprise the selection platform; and the use of a recombinant microbial cell comprising the selection platform for screening for a protease inhibitor.
  • Metagenomics has proven to be a powerful tool in the search for novel bioactive compounds; since it sidesteps the problem that much of the microbial diversity found in nature cannot be reproduced and exploited using standard cultivation conditions in the laboratory.
  • novel proteases (Gupta R, et al., 2002) and small molecule antibiotics (Gillespie DE et al., 2002) has been driven by large metagenomic screens. These studies show that libraries constructed from DNA derived from microbial communities collected from the environment contain interesting biological pathways. Metagenomic library screening may be based on a functional screen, with the detection of expressed target genes in the cloning host.
  • Metagenomic libraries are also a source of genes expressing two or more enzymes performing several steps in a pathway; since the respective genes may be clustered within a cloned DNA fragment. This opens up to the possibility of detecting bioactive compounds produced by the coordinated action of enzymes in a biosynthetic pathway, expressed by clustered genes in a metagenomic library.
  • current techniques fail to screen libraries in a generic high-throughput fashion (Daniel R., 2005). Therefore there is a need for an in vivo platform for rapid detection of bioactive compounds that are synthesized by novel pathways residing in environment-derived metagenomic libraries, which is both sensitive and reliable.
  • the invention provides a recombinant microbial cell for screening for a protease inhibitor comprising :
  • transgene encoding a first polypeptide conferring resistance to a first microbial growth inhibitor
  • transgene encoding a second polypeptide conferring resistance to a second microbial growth inhibitor
  • first polypeptide and said second polypeptide comprise the recognition site amino acid sequence cleavable by said protease; and wherein cleavage of said sequence in said first polypeptide and said second
  • polypeptide inactivates resistance to said first microbial growth inhibitor and said second microbial growth inhibitor.
  • each cell of the library comprises:
  • transgene encoding a first polypeptide conferring resistance to a first microbial growth inhibitor
  • transgene encoding a second polypeptide conferring resistance to a second microbial growth inhibitor; wherein said first polypeptide and said second polypeptide comprise the recognition site amino acid sequence cleavable by said protease; and wherein cleavage of said sequence in said first polypeptide and in said second polypeptide inactivates resistance to said first microbial growth inhibitor and said second microbial growth inhibitor.
  • the invention provides a method for in vivo screening a compound or a compound library for an inhibitor of a target protease comprising the steps of:
  • each compartment with a growth medium supplemented with a first growth inhibitor and a second growth inhibitor; wherein the first polypeptide confers resistance to the first growth inhibitor and the second polypeptide confers resistance to the second growth inhibitor;
  • step (e) selection of cells growing on the selective medium in step (e).
  • the invention provides a method for in vivo screening for a protease inhibitor comprising :
  • step (b) optionally introducing the cells obtained of step (a) into a growth medium to produce a culture;
  • step (c) incubating the cells of step a) or the cell culture produced in step (b) on a growth medium supplemented with a first growth inhibitor and a second growth inhibitor; wherein the first polypeptide confers resistance to the first growth inhibitor and the second polypeptide confers resistance to the second growth inhibitor, and
  • the invention provides for the use of the genetically modified micro-organism according to the first or second embodiment for screening for protease inhibitors.
  • the protease according to the first, second, third, fourth or fifth embodiment may be a viral protease, selected from the group consisting of rhinovirus protease, human immunodeficiency virus protease, Rous Sarcoma Virus protease; Human Mammary Tumor Virus protease, Equine Infectious Anemia Virus protease, Epstein-Barr protease, Herpes Simplex Virus protease, Human Cytomegalovirus protease, Human Papilloma Virus protease, and Small pox virus protease.
  • rhinovirus protease selected from the group consisting of rhinovirus protease, human immunodeficiency virus protease, Rous Sarcoma Virus protease; Human Mammary Tumor Virus protease, Equine Infectious Anemia Virus protease, Epstein-Barr protease, Herpes Simplex Virus protease, Human Cytomegalovirus prote
  • Figure 1 Image of agarose size-separation gel of colony PCR products of the amplified region of the HRV 3Cpro gene on plasmid pEH500, showing in lane 1 : non-mutated amplified region of the HRV 3Cpro gene; lane 2 : amplified region of the mutated HRV 3Cpro gene comprising an IS1 (accession ID: J01730) DNA insert; lane 3 : amplified region of the mutated HRV 3Cpro gene comprising an IS5 (accession ID: J01730) DNA insert.
  • FIG. 2 Cartoon showing the mutation of the HRV 3Cpro gene on the selection platform plasmid pEH500.
  • IS1A is integrated into the ribosome binding site, thereby blocking translation.
  • IS5 is integrated in the J23101 promoter thereby blocking transcription.
  • Amplification of the constructed HRV 3Cpro gene on the selection platform plasmid pEH500 with the primers EH 146 and EH74 generates a PCT product of 673bp; while amplification of the mutant HRV 3Cpro gene comprising the ISIA or IS5 insert generates a product of 1449bp and 1872bp respectively.
  • FIG. 3 Diagram showing the B-factor of the kanamycin resistance protein (PDBID: 1ND4) as a function of the sequence position.
  • PDBID kanamycin resistance protein
  • FIG. 4 A diagram showing the growth of cells of four transformed E. coli strains, measured as numbers of CFUs, following cultivation on agar plates supplemented with spectinomycin and increasing concentrations of kanamycin.
  • the tested strains are: E. coli transformed with pEH650 ( expressing rhinovirus protease (HRV 3Cpro) and a cleavable mKan55 protein ( ⁇ ); E. coli transformed with pEH670 expressing rhinovirus protease (HRV 3Cpro) and a non-cleavable wild-type Kan protein (O), as a positive control; and E.
  • Figure 5 A diagram showing the growth of cells of three transformed E. coli strains, measured as numbers of CFUs, following cultivation on agar plates supplemented with 50 ⁇ g/mL spectamycin, ( ⁇ ) with and (-) without 25 ⁇ g/mL tetracycline; and with increasing concentrations of kanamycin.
  • the tested strains in A E. coli transformed with pEH671 expressing HIV protease (HIV); cleavable HIV-TetR protein and un-cleavable wildtype KanR protein; B: E.
  • FIG. 6 A diagram of the pEH plasmid series.
  • Figure 7 A diagram showing the cell density (measured as OD 6 3o) of replicate cultures of E. coli transformed with pEH700 expressing rhinovirus protease (HRV 3Cpro); and cleavable mKan55 protein, following for 14 hours cultivation in a selective LB growth medium comprising 22.5 ⁇ g/mL tetracycline and 90 ⁇ g/mL kanamycin; and in the presence of increasing concentration of rupintrivir.
  • the cell density (OD 6 3o) of 8 replicate cell cultures was measured after 14 hours of growth and the OD 6 3o was normalized against growth at the lowest and highest OD630 measurement, error bars in SEM .
  • FIG 8 Cartoon showing the steps for using a reporter E. coli strain comprising the selection platform of the invention to screen a soil microbial population; as detailed in Example 4.
  • a reporter E. coli strain comprising the plasmid pEH800 containing a florescent marker is cultured on solid agar for 18 hours and overlaid on top of cultured soil microbes to facilitate reporter strain-soil microbe contact, and then cultured for 1 - 2 days. Growth of the reporter E. coli strain is detected in close association with a soil microbial colony producing a protease inhibitor.
  • Figure 9 Image of a O. lxLB-MOPS agar plate comprising a sample of soil microbes after 2 days culture (left). Image of the O. lxLB-MOPS agar plate comprising the soil sample; overlaid with solid agar comprising a culture of the reporter E. coli strain comprising the plasmid pEH800.
  • gi number (genlnfo identifier) is a unique integer which identifies a particular sequence, independent of the database source, which is assigned by NCBI to all sequences processed into Entrez, including nucleotide sequences from DDBJ/EMBL/GenBank, protein sequences from SWISS-PROT, PIR and many others.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length.
  • the two sequences to be compared must be globally aligned to give a best possible fit, by means of the insertion of gaps, where the aligned sequences can end in gaps.
  • sequence identity can be calculated as ((Nref-Ndif) 100)/(Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences.
  • Sequence identity can be calculated using the BLAST program e.g. the BLASTP program (Pearson W.R and DJ.
  • the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions.
  • substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1 : Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3 : Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine.
  • B-factors are used for indicating the relative vibrational motion of different parts of a protein structure. Atoms with low B-factors belong to a part of the structure that is well ordered. Atoms with large B-factors generally belong to part of the structure that is very flexible.
  • CFU colony forming unit(s).
  • the proteolytic activity of a target protease is specific for, or preferentially selective for, the cleavage of a peptide bond in a peptide or polypeptide that lies at a cleavage site within the protease's cognate recognition site amino acid sequence; also when said recognition site amino acid sequence is inserted into an antibiotic resistance protein.
  • a growth inhibitor/retardant is a component of external origin that can inhibit or retard the growth of a micro-organism and/or prevents cell survival (such as a toxin or antibiotic), that is not produced by the microorganism itself, but instead is supplied to the micro-organism either by addition to or its presence in the growth medium or environment in which the micro-organism is cultured.
  • cell survival such as a toxin or antibiotic
  • Metagenomic DNA genetic DNA recovered directly from environmental samples.
  • Native gene endogenous gene in a microbial cell genome, homologous to host micro-organism.
  • a gene (nucleic acid molecule comprising a coding sequence) is operably linked to a promoter when its transcription is under the control of the promoter and where transcription results in a transcript whose subsequent translation yields the product encoded by the gene.
  • Recognition site amino acid sequence a sequence of amino acids within a polypeptide that is recognised by a protease; such that peptide bond cleavage catalysed by the protease is limited to a cleavage site within this sequence.
  • the length of the sequence may be between 4 and 10 amino acid residues; preferably 4 and 15 amino acid residues or any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues.
  • the present invention relates to an in vivo platform for rapid detection of bioactive compounds, such as those synthesized by novel pathways residing in environmentally-derived metagenomic libraries, which is both sensitive and reliable.
  • a recombinant microbial cell comprising a selection platform suitable for use in screening for protease inhibitors
  • the invention provides a recombinant micro-organism suitable for screening for a protease inhibitor.
  • the micro-organism is genetically modified to comprise the following transgenes that function as a selection platform for protease inhibitors:
  • a transgene encoding a second polypeptide conferring resistance to a second microbial growth inhibitor.
  • the protease, encoded by the two transgenes is characterized by a proteolytic activity that is specific for, or preferentially selective for, the cleavage of a peptide bond that lies within a recognition site amino acid sequence in a peptide or polypeptide.
  • the nucleotide sequence of the two transgenes encoding the protease may be identical or divergent, the amino acid sequence of the encoded protease is the same.
  • the first polypeptide and said second polypeptide each comprise the cognate recognition site amino acid sequence that is cleavable by the protease encoded by the two transgenes.
  • the protease contacts its cognate recognition site amino acid sequence in the first polypeptide or the second polypeptide, it will cleave a peptide bond within the cognate recognition site amino acid sequence.
  • the resistance to the first and the second microbial growth inhibitor that is conferred by the first polypeptide and said second polypeptide respectively is abolished when the recognition site amino acid sequence in the first polypeptide and the second polypeptide is cleaved.
  • the two transgenes encoding the protease may be operatively linked to a constitutive or an inducible promoter. Accordingly the viability of the recombinant micro-organism that comprises a selection platform according to the invention, when contacted with the first and/or second microbial growth inhibitor will be determined by expression of a functional protease.
  • the first and the second transgene encoding the first and the second polypeptide respectively may be operatively linked to a constitutive or an inducible promoter. Accordingly the viability of the recombinant micro- organism that comprises a selection platform according to the invention, when contacted with the first and/or second microbial growth inhibitor will be dependent on expression of the first and/or the second polypeptide conferring resistance to the respective first and the second microbial growth inhibitor.
  • a recombinant micro-organism comprising a selection platform and expressing the protease according to the invention is not viable and cannot be reproduced in a growth medium that comprises lethal concentrations of the first and/or the second microbial growth inhibitor. Equally, for the purposes of reproduction and viability maintenance, the recombinant micro-organism is viable in growth or storage conditions that comprise sub-lethal concentrations, or is devoid of the first and the second microbial growth inhibitor.
  • a recombinant micro-organism comprising a selection platform and expressing both the protease and the first and the second polypeptide according to the invention will only grow under selective conditions (i.e.
  • the source of the protease inhibitor that contacts the protease expressed by the recombinant micro-organism may be extracellular; for example provided in the growth medium .
  • the protease inhibitor may be intracellular and synthesized within the micro-organism .
  • the first and/or the second microbial growth inhibitor can be an antibiotic, capable of inhibiting the growth of the recombinant micro-organism comprising a selection platform .
  • the microbial growth inhibitor is an antibiotic
  • the respective first and/or second polypeptides according to the invention are antibiotic resistance proteins.
  • the antibiotic and a gene encoding its cognate antibiotic resistance protein may be selected from the group consisting of: tetracycline (gene : tetA), kanamycin or neomycin (gene : aph(3')-II (synonyms : aphA-2, nptll, Neo, KanR) or aph(3')-III (synonyms : nptlll, neo, KanR)) ; tobramycin (phosphorylate (aminoglycoside phosphoryltransferase [APH]), acetylate (aminoglycoside acetyltransferase [AAC]), or adenylate (aminoglycoside nucleotidyltransferase [ANT] ; also referred to as aminoglyco
  • amino acid sequences of the antibiotic resistance proteins (as exemplified above), encoded by transgenes present in the selection platform, are modified by the insertion of a sequence of consecutive amino acids (i.e. peptide) whose sequence is recognized as a cleavage site by the target protease.
  • the insertion site of this sequence of consecutive amino acids is chosen according to three criteria : 1) the mod ified antibiotic resistance protein comprising the inserted sequence retains its functional properties vis-a-vis its cognate antibiotic, such that a host microbial cell expressing the modified antibiotic resistance protein is resistant to the cognate antibiotic; 2) the inserted sequence in the expressed and functional antibiotic resistance protein is both accessible to and cleavable by the target protease; 3) the inserted sequence in the expressed and functional antibiotic resistance protein is located at a position whereby its cleavage by the target protease leads to inactivation of the antibiotic resistance properties conferred by the protein.
  • the recognition site amino acid sequence inserted in the modified antibiotic resistance protein is from 4 to 20 amino acid residues in length.
  • the first polypeptide is a modified antibiotic resistance protein that confers resistance to the antibiotic tetracycline.
  • the modified tetracycline resistance protein expressed by the microbial cell is embedded in the cell inner membrane, where it is capable of preventing intracellular accumulation of tetracycline.
  • the modified protein, encoded by a transgene comprises two membrane spanning domains of six a-helixes (Tamura N, et al., 2001), and a cytoplasmic loop (or hinge) that is located between the two domains; and into which a recognition site amino acid sequence is inserted.
  • the recognition site amino acid sequence in the modified tetracycline resistance protein is one that is recognised by the target protease that is co- expressed in the cell.
  • the cytoplasmic loop (the domain suitable for insertion of the recognition site sequence) spans the amino acid residues: Gin 182 and Thr 218 , whereby a recognition site amino acid sequence can be inserted between any two amino acid residues in this loop (or domain).
  • the second modified antibiotic resistance protein confers resistance to the antibiotic kanamycin.
  • a modified kanamycin resistance protein comprising a recognition site sequence, and meeting the three functional criteria set out above, is provided by the present invention (see Example 1.4). Accordingly, when the unmodified kanamycin resistance protein has SEQ ID No. :4, the regions that were found to be suitable for insertion of the recognition site sequence, are located between the amino acid residues: Gly 55 I Ala 56 and Ala 87 1 Gly 88 , respectively.
  • a recombinant micro-organism comprising a selection platform according to the invention is preferably derived from a prokaryotic host micro-organism, for example a host cell of a genus selected from the group consisting of: Escherichia spp., (e.g. E. coli); Bacillus spp., (e.g. B. subtilis); Pseudomonas spp., (e.g. P. fluorescens, P. putida); Corynebacterium spp., (e.g. C. glutamicum); Agrobacterium spp., (e.g. A. tumefaciens); Caulobacter spp., (e.g.
  • C. vibrioides C. vibrioides
  • Burkholderia spp. e.g. B. graminis
  • Rhizobium spp. e.g. R. leguminosarum
  • Ralstonia spp. e.g. R. metallidurans
  • the selected host cell has a genome that is deficient of genes encoding proteases capable of degrading the first or second polypeptide.
  • the selection host cell has a genome and plasmids that is deficient of native endogenous genes encoding polypeptides that are capable of conferring resistance to the same microbial growth inhibitor as either of the first or the second polypeptide encoded by the first and second transgene.
  • the host cell is a strain of E. coli, due to the fact that (i) it has unparalleled fast growth kinetics; (ii) it easily achieves a high cell density; there are many molecular tools and protocols at hand for the high-level production of bioproducts in E. coli; and heterologous proteins can easily be expressed in E. coli.
  • the recombinant micro-organism comprising the selection platform is used for selecting inhibitors of a protease.
  • the protease, encoded by the two transgenes of the selection platform is the target protease.
  • Target proteases, for which inhibitors can be selected by the present invention include proteases that play a critical role in the development of disease in an animal.
  • target proteases include: (Human) Rhinovirus protease, human immunodeficiency virus (HIV) protease, Rous Sarcoma Virus (RSV) protease; Human Mammary Tumor Virus (HMTV) protease, and Equine Infectious Anemia Virus (EIAV) protease, Epstein-Barr protease, Herpes Simplex Virus (HSV) protease, Human Cytomegalovirus (HCMV); Human Papilloma Virus (HPV) protease; Small pox virus protease; kallikrein (that plays a role in inflammation, blood pressure control, coagulation and pain) and furin (processes latent precursor proteins into their biologically active products e.g.
  • HCV human immunodeficiency virus
  • RSV Rous Sarcoma Virus
  • HMTV Human Mammary Tumor Virus
  • EIAV Equine Infectious Anemia Virus
  • HSV Herpes
  • the target protease may also be an industrially relevant protease such as: renin, subtilisin, papain, chymosin and trypsin.
  • the selection platform comprises four genes, where two transgenes encode a protease; a transgene encodes a first polypeptide conferring resistance to a first microbial growth inhibitor; and a transgene encodes a second polypeptide conferring resistance to a second microbial growth inhibitor.
  • Each of the four transgenes may be located on plasmids, and all four transgenes may be located on the same plasmid.
  • one, two, three or four of the transgenes may be integrated into the chromosome of the host recombinant cell genome.
  • the plasmid is a self-replicating plasmid with an origin of replication; and is preferably provided with a plasmid selection marker gene for plasmid maintenance in the host cell.
  • Suitable selection marker genes are genes encoding antibiotic resistance proteins to antibiotics such as spectomycin, while excluding an antibiotic resistance protein that is expressed by any of the transgenes in the selection platform for conferring resistance to a microbial growth inhibitor.
  • the expression of the one, two, three, or all four transgenes of the selection platform is driven by a constitute promoter.
  • the drug target is a rhinovirus protease, which is a key component in the life cycle of this virus.
  • the rhinovirus protease cleaves the polypeptide precursor of the virus into functional structural proteins, and is essential for its virulent properties.
  • the rhinovirus protease is a key drug target for therapeutic treatment of rhinovirus infections (Binford SL et al., 2005), since the inhibition of this protease suppresses rhinovirus virulence.
  • a microbial selection platform for the in vivo selection of inhibitors of a rhinovirus protease, is composed of a microbial cell comprising two copies of a transgene encoding and being capable of expressing the rhinovirus protease; and two transgenes encoding and being capable of expressing a first and a second polypeptide, each corresponding to a first and second modified antibiotic resistance protein respectively, where each resistance protein confers resistance to each its own specific and distinct antibiotic.
  • the modified antibiotic resistance proteins encoded by the two transgenes are modified by the insertion of a sequence of consecutive amino acids (i.e. peptide) whose sequence is recognised as a cleavage site by the rhinovirus protease.
  • the rhinoprotease is a human rhinoprotease, where the two transgenes each encode an amino acid sequence having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 6.
  • the expression of both of the two transgenes is driven by a constitute promoter, for example the constitute promoter: J23101 [SEQ ID No. : 7] available from
  • the first modified antibiotic resistance protein confers resistance to the antibiotic tetracycline.
  • the modified tetracycline resistance protein comprises a rhino protease recognition sequence inserted in a cytoplasmic loop (or hinge) that is located between the two domains comprising the two membrane spanning domains of six a-helixes (see Section Ii).
  • a recognition site amino acid sequence cleavable by a rhinoprotease is selected from among :
  • Rhino protease recognition sequence any one of T, V or A; and where the cleavage site is most commonly between the P 1 and P 1 residues.
  • the transgene encoding a first polypeptide encodes a modified tetracycline resistance protein conferring tetracycline resistance.
  • the modified tetracycline resistance protein consists of: a tetracycline resistance protein and a cleavable recognition site amino acid sequence, wherein the amino acid sequence of the tetracycline resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No. : 2; and wherein the cleavable recognition site amino acid sequence comprising or consisting of [T/V/A] LFQGPV (SEQ ID No.
  • the modified tetracycline resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No. : 73; with the proviso that amino acid residues 190 - 198 are SEQ ID No. : 9.
  • the second modified antibiotic resistance protein encodes a modified kanamycin resistance protein (mKan55) conferring resistance to the antibiotic kanamycin.
  • the modified kanamycin resistance protein consists of: a kanamycin resistance protein and a cleavable recognition site amino acid sequence; wherein the amino acid sequence of the kanamycin resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 4, and wherein the cleavable recognition site amino acid sequence comprising or consisting of
  • [T/V/A]LFQGPV (SEQ ID No. : 8), or more preferably EVLFQGPVY (SEQ ID No. : 9), is located at a position in the modified kanamycin resistance protein corresponding to: between Gly 55
  • the modified kanamycin resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No. : 25; with the proviso that amino acid residues 56 -64 are SEQ ID No. : 9.
  • both of the transgenes encoding the first and second modified antibiotic resistance gene are driven by a constitute promoter, for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.iqem.orq/Promoters/Cataloq/Anderson. III
  • a constitute promoter for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.iqem.orq/Promoters/Cataloq/Anderson. III
  • the drug target is a HIV-1 protease, which is a key component in the viral life cycle.
  • the HIV-1 protease cleaves newly synthesized polyproteins at the appropriate places to create the mature protein components of an infectious HIV virion.
  • the HIV-1 protease is expressed by two transgenes, where each transgene encodes an amino acid sequence having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 11.
  • each transgene encodes an amino acid sequence having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 11.
  • the expression of both of the two transgenes is driven by a constitute promoter, for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.iqem.orq/Promoters/Cataloq/Anderson.
  • the first modified antibiotic resistance protein confers resistance to the antibiotic tetracycline.
  • the modified tetracycline resistance protein comprises a HIV-1 protease recognition sequence inserted in a cytoplasmic loop (or hinge) that is located between the two domains comprising the two membrane spanning domains of six a-helixes (see Section Ii).
  • a recognition site amino acid sequence cleavable by a HIV-1 protease comprises or consists of S Q N Y P I V (commonly cleaved between Y
  • the transgene encoding a first polypeptide encodes a modified tetracycline resistance protein conferring tetracycline resistance.
  • the modified tetracycline resistance protein consists of: a tetracycline resistance protein and a cleavable recognition site amino acid sequence, wherein the amino acid sequence of the tetracycline resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No.
  • the modified tetracycline resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No. : 76; with the proviso that amino acid residues 191 - 197 are SEQ ID No. : 12.
  • the second modified antibiotic resistance protein encodes a modified kanamycin resistance protein (mKan55) conferring resistance to the antibiotic kanamycin.
  • the modified kanamycin resistance protein consists of: a kanamycin resistance protein and a cleavable recognition site amino acid sequence; wherein the amino acid sequence of the kanamycin resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No.
  • cleavable recognition site amino acid sequence comprising or consisting of S Q N Y P I V, is located at a position in the modified kanamycin resistance protein corresponding to: between Gly 55 I Ala 56 or between Ala 87
  • both of the transgenes encoding the first and second modified antibiotic resistance gene are driven by a constitute promoter, for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.iqem.org/Promoters/Cataloq/Anderson. IV
  • a constitute promoter for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.iqem.org/Promoters/Cataloq/Anderson. IV
  • the drug target is a HSV-1, HSV-2, or HCMV protease, which is a key component in the life cycle of members of the Herpesviridae family of viruses. Synthesis of each of these herpesvirus proteases originates from a gene encoding a precursor form, where the gene contains within it nested genes encoding the substrate assembly protein. There are two primary cleavage sites in the precursor form, often referred to as the R site, for release of the mature protease, and M site, for maturation of the substrate assembly protein. The naturally occurring consensus cleavage sequence for both of these sites is (V,L,I)-X-A
  • the herpesvirus protease is essential for DNA encapsidation and replication of the virus.
  • the herpesvirus protease is an HSV-1 protease expressed by two transgenes, where each transgene encodes an amino acid sequence having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 15 (encoded by the HSV-1 UL26 gene).
  • each transgene encodes an amino acid sequence having at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No.
  • the first modified antibiotic resistance protein confers resistance to the antibiotic tetracycline.
  • the modified tetracycline resistance protein comprises a HSV-1 protease recognition sequence inserted in a cytoplasmic loop (or hinge) that is located between the two domains comprising the two membrane spanning domains of six a-helixes (see Section Ii).
  • a recognition site amino acid sequence cleavable by a HSV-1 protease is (V,L,I)-X-A
  • the transgene encoding a first polypeptide encodes a modified tetracycline resistance protein conferring tetracycline resistance.
  • the modified tetracycline resistance protein consists of: a tetracycline resistance protein and a cleavable recognition site amino acid sequence, wherein the amino acid sequence of the tetracycline resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100% sequence identity to SEQ ID No. : 2; and wherein the cleavable recognition site amino acid sequence comprising or consisting of (V,L,I)-X-A
  • YLQA ⁇ SELF (SEQ ID No. : 16) is located at a position in the cytoplasmic loop in the modified tetracycline resistance protein corresponding to: Gin 182 and Thr 218 in the unmodified tetracycline resistance protein having SEQ ID No. : 2.
  • the second modified antibiotic resistance protein encodes a modified kanamycin resistance protein (mKan55) conferring resistance to the antibiotic kanamycin.
  • the modified kanamycin resistance protein consists of: a kanamycin resistance protein and a cleavable recognition site amino acid sequence; wherein the amino acid sequence of the kanamycin resistance protein has at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% sequence identity to SEQ ID No. : 4, and wherein the cleavable recognition site amino acid sequence comprising or consisting of (V,L,I)-X-A
  • both of the transgenes encoding the first and second modified antibiotic resistance gene are driven by a constitute promoter, for example the constitute promoter: J23101 [SEQ ID No. : 7] available from http://parts.igem.org/Promoters/Catalog/Anderson.
  • the invention provides a method for the in vivo screening of a compound or a compound library for an inhibitor of a target protease comprising the steps of: a. providing cells of the recombinant micro-organism comprising a selection platform of the invention (as described in sections I, II, III or IV);
  • each compartment with a growth medium supplemented with a first growth inhibitor cognate to said first resistance protein and a second growth inhibitor cognate to said second resistance protein; wherein the first resistance protein confers resistance to the first growth inhibitor and the second resistance protein confers resistance to the second growth inhibitor;
  • the compound library comprises at least 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375 or 400 compounds.
  • the first growth inhibitor and the second growth inhibitor are antibiotics, which may be selected from the group consisting of: tetracycline, kanamycin, neomycin, tobramycin, chloramphenicol, spectinomycin, gentamycin, ampicillin and carbenicillin.
  • the growth medium in step c) is either solid or liquid.
  • Potential inhibitors of the target protease are identified by the detection of cell growth on the selective medium in step (d) in said single-compartment container or in one or more compartments of said multi-compartment container.
  • a recombinant microbial cell library comprising a selection platform
  • the invention provides a recombinant microbial host cell library of non-host DNA fragments wherein each cell of the library comprises the transgenes of the selection platform as described above in sections I - IV.
  • the library is a metagenomics DNA library where the DNA fragments of metagenomic DNA are cloned into a self-replicating vector (e.g. plasmid, cosmid or fosmid).
  • a self-replicating vector e.g. plasmid, cosmid or fosmid.
  • the average size of the metagenomic DNA insert in a plasmid is about 1.5 kb; but can be up to about 40 kb for a fosmid which is sufficiently large to encompass a cluster of genes potentially expressing one, two or more steps in a metabolic pathway.
  • the recombinant microbial host cell library is conveniently constructed in cells of a recombinant micro-organism comprising the selection platform according to the invention.
  • the self-replicating vector into which the metagenomic DNA is cloned is provided with a resistance marker gene, which is different from the resistance markers that are expressed by transgenes in the selection platform for conferring resistance to the first and the second microbial growth inhibitor. VII Methods for screening a metagenomics DNA library for production of a protease inhibitor using the recombinant micro-organism of the invention
  • the invention provides a method for the in vivo screening for an inhibitor of a target protease comprising the steps of:
  • step (b) introducing the cells obtained in step (b) into a growth medium to produce a cell culture
  • the self-replicating library of non-host DNA fragments may be a metagenomics DNA library where the DNA fragments of metagenomic DNA are cloned into a self-replicating vector (e.g. plasmid, cosmid or fosmid).
  • the first growth inhibitor and the second growth inhibitor are antibiotics, which may be selected from the group consisting of: tetracycline, kanamycin, neomycin, tobramycin, chloramphenicol, spectinomycin, gentamycin, ampicillin and carbenicillin.
  • the cells of the cell culture produced in step (c), that are incubated on a growth medium in step (d) are plated on a growth medium that is solid, at a plating density that ensures the growth of single colonies.
  • a suitable plating density is about 0.5 to 1.5* 10 6 cells/cm 2 ; which generates an overgrown plate when the growth medium is devoid of a growth-inhibiting antibiotic.
  • Single colonies that grow on the selective medium in step (d) can be selected; and the non-host DNA fragment cloned in a self-replicating plasmid of the library (and present in cells of the selected colony) can be isolated, amplified and sequenced.
  • Cells of a recombinant microbial host cell library that give rise to cell colonies when cultured under selective conditions are expected to produce an inhibitor that inactivates the target protease.
  • Determination of the nucleotide sequence of the non-host DNA fragment cloned in a self-replicating plasmid of the library and present in the cell colonies, and annotation of the sequence using BLAST/pfam/antismash, provides a first step towards the identification of a potential inhibitor.
  • the inhibitor may be the direct protein product of the expressed gene(s) in the non-host DNA fragment; or the inhibitor may be the product of one or more enzyme expressed by the genes in the non-host DNA fragment.
  • the phenotype of the isolated plasmid may be confirmed by transformation into cells of the original selection strain comprising the selection platform.
  • identity of the potential inhibitor is determined using techniques such as mass-spectrometry and NMR to determine its chemical structure.
  • Integration and self-replicating vectors suitable for cloning and introducing a first, second, third or additional DNA molecules, DNA fragments and transgenes into a micro-organism for the constructing the recombinant micro- organism of the invention comprising a selection platform, are commercially available and known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning : A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989). Cells of a micro-organism are genetically engineered by the introduction into the cells of heterologous DNA (RNA).
  • RNA heterologous DNA
  • a first, second, third or additional transgene or DNA molecule(s) according to the invention can be introduced into a cell or cells on plasmids or optionally integrated into the host cell genome using methods and techniques that are standard in the art.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc.
  • Representative members of microbial communities are propagated under laboratory conditions, whereby cells of the microbial community to be screened are co-cultivated with cells comprising the selection platform of the invention.
  • Bioactive compounds produced by members of a soil microbial community, that allow survival and/or growth of the co-cultivated cells comprising the screening platform in the presence of growth inhibitors, are those that inhibit the proteases encoded by the selection platform. Survival and/or growth of cells comprising the screening platform, is detected by virtue of incorporating a constitutively expressed fluorescent marker gene into the screening platform of the cells of the invention (Example 4). This allows growth of the screening platform cells to be distinguished from growth of soil bacteria.
  • Standard PCR to produce DNA molecules was carried using PfuX7 polymerase (Norholm MHH 2010).
  • PCR reactions products were either purified using gel extraction or PCR cleanup using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co. KG).
  • PCR amplified DNA molecules were inserted into plasmids by USER cloning (Bitinaite J et al., 2009). USER cloning was carried out according to the protocol described by Bitinaite et al. (2009) in a 10 ⁇ _ reaction containing 1 ⁇ _ USERTM Enzyme (New England Biolabs Inc.), USER primers were designed using AMUSER (Genee HJ et al., 2015).
  • Plasmid DNA was purified using the QIAprep® Spin Miniprep Kit. Synthetic DNA oligios for PCR were ordered from Integrated DNA Technologies Inc. E. coli
  • Example 1 Construction and use of a selection platform for rhinovirus protease inhibitors
  • the drug target, rhinovirus protease cleaves the rhinovirus pre-polypeptide into its component functional proteins which is an essential step in the infection process.
  • a microbial selection platform for the in vivo selection of inhibitors of the rhinovirus protease, is composed of a microbial cell comprising a transgene capable of expressing the rhinovirus protease; and a transgene encoding a modified tetracyline resistance protein.
  • the modified tetracyline resistance protein, encoded by a transgene comprises a rhino protease recognition site inserted into a cytoplasmic loop located between the two membrane spanning domains of the resistance protein.
  • a transgene (HRV 3Cpro) encoding a human rhinovirus 3C protease was synthesized using a codon usage compatible with expression in Escherichia coli by GeneArtTM (Thermo Fisher Scientific Inc.) and its sequence was verified.
  • the mTetR gene with its native promoter from plasmid pBR322 and the HRV 3Cpro gene comprising the promoter 323101 [SEQ ID No. : 7] were cloned together with an pl5A origin of replication, and a spectinomycin resistance gene (specR) for plasmid selection purposes, to create plasmid pEH500 (Table 2) and Figure 6.
  • the sequence of the rhinoprotease corresponds to Uniprot ID P04936 (Human rhinovirus 2) position 1508-1689 (protease 3C); modified with an engineered start codon, coding for a methionine leading the sequence.
  • Two commonly used synonyms are HRV 3C protease and HRV 3C proteinase.
  • the plasmid pEH500 was transformed into electrocompetent host cells of the E. coli ToplO strain by electroporation.
  • E. coli ToplO cells containing the selection plasmid comprising a spectomycin resistance (specR) gene (pEH500 in table 2; figure 6) were grown overnight at 37°C with shaking in 10 ml_ Luria Broth (LB) medium supplemented with 50 ⁇ g/mL spectinomycin. The next day 0.5 ml_ of the overnight culture was transferred to a shaking flask with 250 ml_ fresh LB medium supplemented with 50 ⁇ g/mL spectinomycin. The culture was allowed to grow in a shake flask for ⁇ 5 hours to a cell density of OD600 ⁇ 0.6. The shake flask was incubated on ice for 15 minutes, with intermittent gentle shaking to ensure homogenous cooling.
  • spectrumR spectomycin resistance
  • Plasmid pZE21 and the plasmids of each DNA plasmid library comprised an antibiotic resistance (kanR) gene for selection of transformants.
  • each of the above combinations of cells and DNA was electroporated using a BioRad MicroPulserTM with the EC2 method (2.5 kV). Directly after electroporation, the cells were rapidly suspended in 450 ⁇ _ pre-warmed SOC media and incubated for 1 hour at 37°C at 100 rpm. 9 ml_ of LB medium supplemented with 50 ⁇ g/mL spectinomycin and 50 ⁇ g/mL kanamycin was added to the 500 ⁇ _ of recovered cells and cultured overnight at 37°C for phenotypic expression.
  • 100 ⁇ _ of each transformed culture were plated out on agar plates supplemented with 50 ⁇ g/mL tetracycline and 50 ⁇ g/mL spectinomycin, or plates only comprising 50 ⁇ g/mL spectinomycin that were non-selective for the tet R selection platform.
  • the nucleotide sequence of amplified fragment of the enlarged HRV 3Cpro gene was determined using primers EH 146 and EH74 and annotated using BlastP. Insertion sequences were further investigated using ISFinder (Siguier P et al., 2006).
  • the nucleotide sequence of the circa 1449 base pair amplified fragment corresponded to a mutated HRV 3Cpro operon containing an inserted sequence corresponding to IS1 (accession ID: J01730) DNA.
  • the nucleotide sequence of the circa 1872 base pair amplified fragment corresponded to a mutated HRV 3Cpro operon containing an inserted sequence corresponding to IS5 (accession ID: J01735) DNA. Since both inserted sequences correspond to E. coli genome sequences, their identification in the enlarged HRV 3Cpro gene is likely to be due to their transfer from the E. coli genome to the pEH500 selection plasmid.
  • the frequency of insertion of the IS element is around 10 6 (Machida Y et al., 1982), the frequency of insertion of two IS in one plasmid is predicted to be > 10 12 , which is well above the selective range (10 9 ) required for this platform. If the insertion frequency creating false positives is reduced to > 10 12 (by inserting 2 copies of the transgene encoding the target protease), then screening a metagenomic library of cells comprising the selection platform on plasmid pEH700 to select for putative protease inhibitors, at a plating density of 10 9 cells/plate, would only give rise a single false positive in 1 : 1000 plates.
  • kanamycin resistance gene was selected as a candidate antibiotic resistance gene for potential inclusion in the selection platform .
  • Use of the kanamycin resistance gene in the selection platform requires that the expressed kanamycin resistance (KanR) protein comprises a rhinovirus protease cleavage site.
  • KanR kanamycin resistance
  • a suitable site for insertion of a rhinovirus protease cleavage site into a KanR protein was investigated by analysis of the crystal structure of the KanR protein, aminoglycoside-3'-phosphotransferase-IIa using Pymol (PDBID : 1N D4) (Nurizzo D et al ., 2003) .
  • PDBID aminoglycoside-3'-phosphotransferase-IIa using Pymol
  • Primers EH76-EH93 were used to insert the rhino protease cleavage site sequence into nine loops of the kanamycin resistance gene cloned in the pZE21 plasmid (Lutz et al. , 1997) to yield a potentially cleavable kanamycin resistance protein (Table 4) yielding plasmids pEH450-pEH458, as follows.
  • Primers EH76-EH93 were designed to contain the nucleotide sequence coding for the rhinoprotease cleavage site in the 5' end of the primer.
  • the insertion of the rhino protease cleavage site in the kanamycin resistance gene on the pZE21 plasmid was performed with one PCR reaction, next the plasmid template was digested with Dpnl and the reaction was incubated with USER enzyme as described above yielding a nicked circular plasmid with the rhino protease cleavage site inserted in the kanR gene.
  • Table 4 Construction of a selection plasmid comprising a cleavable kanamycin resistance protein
  • EH80 SEQ ID No:46 AGGGCCCUGGAATAAAACCTCGCCCGTCGTGGCCA
  • EH83 SEQ ID No:49 AGGGCCCUGTGTACGGAAGGGACTGGCTGCTATTG
  • EH101 SEQ ID No :64 ACCCCAGAGUCCCGCTCAGAAGAACTCGTC
  • EH103 SEQ ID No :66 AGAACCAUGGAAAAAATCCTTAGCTTTCGCTA
  • EH104 SEQ ID No :67 AC 1 C 1 bbbbU 1 CbACAbb 1 CbCAbACb 1 1 1 1 bCA
  • Primers EH97 and EH98 were used to amplify each of the nine modified KanR genes (cloned in pEH450-pEH458) encoding a KanR protein comprising a rhinovirus protease cleavable recognition site amino acid sequence, and each amplified gene was then inserted into pEH500 amplified using primers EH99 and EH 100 to yield plasmids pEH650 - EH658, respectively.
  • a wild-type KanR gene was amplified using primers EH97 and EH98 and inserted into pEH500 amplified using EH99 and EH 100 yielding plasmid pEH670 (Table 3).
  • the pEH650-pEH658 plasmids comprising each of the nine modified KanR genes and plasmid pEH670 comprising the wild-type KanR gene were then transformed into electro-competent host cells of the E. coli ToplO strain by electroporation.
  • the mKanR-0 gene encoding the modified kan R protein comprises a rhinovirus protease cleavage sequence inserted between residue glycine 55 and residue alanine 56 of the native KanR protein.
  • the mKan55 gene was amplified from pEH450 using primers EH 101 and EH 102 and inserted in a strain expressing the HIV protease instead of the rhino protease (pEH lOO) that was amplified with EH 103 and EH 104 finally yielding pEH672.
  • Plasmid pEH lOO containing the DNA sequence encoding the template HIV protease sequence from the pPolo plasmid obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH : pPolo from Dr. Bruce Korant. Catalog number 238).
  • the wild type kanamycin resistance gene was amplified from pZE21 using primers EH 101 and EH 102 and inserted into pEH lOO amplified with EH 103 and EH 104 yielding pEH671.
  • E. coli ToplO cells transformed with each of plasmids pEH650 - EH658 and pEH670 were grown overnight at 37°C with 250 RPM shaking in LB medium supplemented with 50 ⁇ g/mL spectinomycin. The next day, the cells were 10- fold serially diluted in LB medium and 5 ⁇ of the culture was spotted on LB- agar plates supplemented with 50 ⁇ g/mL spectinomycin and with either 0, 10, 50 or 100 ⁇ g/mL kanamycin concentration.
  • the false positive rate needs to be 10 "8 or below in order to avoid a high proportion of those cells giving rise to colonies are in fact false positives.
  • a robust screen may be characterized as one where: a 5 ⁇ _ aliquot derived from an overnight culture of cells comprising the selection platform plasmid alone (e.g.
  • pEH700 will not grow on media selective for the resistance genes expressed on the selection platform plasmid.
  • the screen is not sufficiently robust if growth is detected, since the obtained cells will be due to the growth of false positive (mutant) cells.
  • HRV 3Cpro rhinoprotease transgene
  • Primers EH 105 and EH 106 were used to amplify the HRV 3Cpro DNA molecule, which was inserted into the pEH650 (pEH650 is constructed from pEH500 by amplification with EH99 and EH 100 and used in a USER ligation with the mkanR55 transgene amplified with primers EH 107 and EH 108 plasmid
  • * gbEH2 gene is the reverse complement of SEQ ID No. : 5; ** mKanR55 gene in pEH700 is the reverse complement of SEQ ID No: 24; *** In pEH671 mKanR55 was substituted by a wild-type KanR gene (SEQ ID No. : 3).
  • dual antibiotic selection platform plasmids comprising the HIV prot target gene were constructed as positive (pEH671) and negative (pEH672) controls, where the selection platform plasmid comprised a gene encoding a modified TetR protein comprising a HIV protease cleavage site sequence (mTetR-HIV) and either a gene encoding the cleavable mKanR protein or a gene encoding the wild type non-cleavable KanR protein.
  • mTetR-HIV HIV protease cleavage site sequence
  • E. coli cells expressing the plasmid comprising the dual selection platform and two copies of the target protease gene was assayed by spotting an overnight culture of the cells on LB medium agar plates supplemented with 50 g/mL spectinomycin, with or without 25 ⁇ g/mL tetracycline, and combined with increasing concentrations of kanamycin. Growth, measured as CFUs, was determined in order to define the antibiotic concentration required for optimal selective pressure.
  • E. coli cells expressing the plasmid comprising the HIV protease, and the dual selection platform (pEH672) showed 100% growth at 200 ⁇ g/mL kanamycin and 25 ⁇ g/mL tetracycline (Figure 5B). Since growth was unaffected by the addition of kanamycin, this demonstrates that the HIV protease was unable to cleave and inactivate the mKan R protein containing the rhinoprotease cleavage recognition site sequence, thereby confirming the protease specificity of the assay.
  • this double protease, dual selection platform of the invention is able to screen a library of 10 7 cells without giving rise to any false positives.
  • Example 2 Use of the selection platform of the invention to detect a rhinovirus protease inhibitor
  • Rupintrivir (Axon Medchem cat nr: 1571, The Netherlands) was dissolved in DMSO and added to the culture media at increasing concentrations.
  • the cultures comprising 3 replicates grown at 273 ⁇ rupintrivir and 8 replicates for each of the lower rupintrivir concentrations, were grown in an Bio-Tek ELx808 spectrophotometer with continues shaking interrupted every 5 minutes to measure the optical density (OD 6 3o) -
  • the optical density after 14 hours of growth was normalized against the OD 6 3o of the 3 replicates grown at the highest rupintrivir concentration (273 ⁇ ) and and normalized with a minimum value of the OD 6 3o when grown at 0.03 ⁇ rupintrivir. Growth of the E.
  • coli ToplO strain transformed with plasmid pEH700, on selective medium comprising two antibiotics, was strongly dependent the addition of rupintrivir, a known rhinovirus protease inhibitor.
  • a dose response curve, shown in Figure 7 has an EC50 of 6.6 ⁇ [5.3;8.3] 95% CI.
  • Example 3 Use of the selection platform of the invention to screen a metagenomic library for a rhinovirus protease inhibitor
  • Cells of E. coli ToplO strain comprising plasmid pEH700, were transformed with each metagenomic library and amplified as described in Example 1.2.
  • Cells of E. coli ToplO strain - pEH700, transformed with each metagenomic library were then plated out on agar plates comprising LB medium supplemented with 50 ⁇ g/mL spectinomycin, 200 ⁇ g/mL kanamycin and 25 ⁇ / ⁇ _ tetracycline and then cultured for at least 24 hours at 37°C.
  • DNA was isolated from each detected colonies, and the DNA fragment present in the respective plasmid pZE31 was sequenced and annotated using deFUME (van der Helm E et al., 2015). Selected fragments were sub-cloned and retransformed in strains containing pEH700 to verify their activity.
  • a fosmid library was constructed using the CopyControlTM Fosmid Library Production Kit (Epicentre). Briefly DNA was isolated from soil collected at 10 cm depth at coordinates N : 55.870655, E12.497390 and DNA fragments between 30-40kb were cloned into the CopyControlTM pCClFOSTMvector according to the manufactures instructions. This library was introduced in the EPI300TM-T1R E. coli strain (as described by Wild J et al., 2002) containing the pEH700 plasmid.
  • Example 4 Use of the selection platform of the invention to screen a soil microbial population for a rhinovirus protease inhibitor
  • An alternative approach to screening and detecting bioactive compounds, produced by the coordinated action of enzymes in microbial biosynthetic pathways found in nature, is instead to perform the screen on the microbial communities themselves that express these diverse pathways.
  • This approach allows the detection of bioactive compounds that are produced in situ by biosynthetic pathways encoded by larger gene clusters; thereby encompassing DNA fragment sizes beyond the capacity of standard vector- based functional metagenomics.
  • Bioactive compounds produced by members of a soil microbial community, that allow survival and/or growth of the co-cultivated cells comprising the screening platform in the presence of growth inhibitors, are those that inhibit the proteases encoded by the selection platform. Survival and/or growth of cells comprising the screening platform, is detected by virtue of incorporating a constitutively expressed fluorescent marker gene into the screening platform of the cells of the invention. This allows growth of the screening platform cells to be distinguished from growth of soil bacteria.
  • a reporter gene comprising a constitutive promoter BBa_J23107 [SEQ ID No. : 7] operably linked to a coding sequence (SEQ ID No: 79) encoding the mCherry red monomer protein (SEQ ID No: 80), was incorporated into the plasmid pEH700 to create pEH800.
  • the plasmid was transformed into the cells E. coli ToplO host to create the pEH800 E. coli reporter strain.
  • the microbial community was derived from a soil sample by mixing 3g of soil obtained from 1cm below the soil surface with 30 ml_ sterile 0.85% NaCI and 15 mL 50% glycerol; and collecting the aqueous phase.
  • aqueous phase of the soil sample was plated on a solid phase comprising O. lxLB - MOPS medium which was prepared by dissolving 10.45g MOPS, 7.5g agar, and lg LB in 500 mL dH 2 0, after which the solution was adjusted to pH 7.0 with NaOH; and then incubated at 37°C for 2 days.
  • Cultivation of the E. coli reporter strain was initiated by cultivation of 10 7 CFUs from an overnight culture of the strain on solid agar plates.
  • the solid agar plates were prepared as follows: Agar medium comprising 2xYT (Yeast extract-try ptone medium) supplemented with 50 ⁇ g/mL spectinomycin were cast in a Petri dish lined with a plastic liner (50 mL per dish) and allowed to solidify. After culture for 18 hours, a ⁇ dH 2 0 comprising 90 ⁇ g/mL kanamycin and 22.5 ⁇ g/mL tetracycline was applied to the surface of each culture plate, which was then allowed to dry. The cells of the E.
  • Block TM, Grafstrom RH Novel Bacteriological Assay for Detection of Potential Antiviral Agents. Antimicrob Agents Chemother 1990, 34.
  • Machida Y, Machida C, Ohtsubo H, Ohtsubo E Factors determining frequency of plasmid cointegration mediated by insertion sequence IS1. Proc Natl Acad Sci U S A 1982, 79: 277-281.
  • Norholm MHH A mutant Pfu DNA polymerase designed for advanced uracil- excision DNA engineering. 2010.
  • Wild J, Hradecna Z, and Szybalski W Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones, Genome Research 2002, 12: 1434- 1444.

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Abstract

La présente invention concerne une cellule microbienne recombinante comprenant une plateforme de sélection pour le criblage d'un inhibiteur de protéase, la plateforme comprenant des transgènes codant pour une protéase ayant une activité sélective de clivage de liaison peptidique au niveau d'une séquence d'acides aminés de site de reconnaissance; et des transgènes codant pour des polypeptides conférant une résistance à des inhibiteurs de croissance microbienne; les polypeptides comprenant la séquence d'acides aminés de site de reconnaissance clivable par la protéase. Les inhibiteurs de protéase sont détectés par leur capacité à inhiber le clivage spécifique par la protéase et l'inactivation des polypeptides dont l'activité est nécessaire pour conférer une résistance aux inhibiteurs de croissance microbienne. L'invention concerne en outre des banques de cellules hôtes microbiennes recombinées d'ADN métagénomique qui comprennent en outre la plateforme de sélection; et l'utilisation d'une cellule microbienne recombinante comprenant la plateforme de sélection pour le criblage d'un inhibiteur de protéase.
PCT/EP2017/060069 2016-04-27 2017-04-27 Plateforme de sélection d'inhibiteur de protéase in vivo à haut débit WO2017186854A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019185750A1 (fr) * 2018-03-27 2019-10-03 Danmarks Tekniske Universitet Plateforme à haut débit pour sélectionner des régulateurs de l'activité associée à crispr-cas

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003099846A2 (fr) * 2002-05-22 2003-12-04 Keck Graduate Institute Systeme et procede de criblage d'inhibiteurs de proteases et de proteases
US6699702B1 (en) * 1999-01-08 2004-03-02 Bristol-Myers Squibb Co. Prokaryotic system designed to monitor protease activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6699702B1 (en) * 1999-01-08 2004-03-02 Bristol-Myers Squibb Co. Prokaryotic system designed to monitor protease activity
WO2003099846A2 (fr) * 2002-05-22 2003-12-04 Keck Graduate Institute Systeme et procede de criblage d'inhibiteurs de proteases et de proteases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OK-KYU SONG ET AL: "DEVELOPMENT OF AN IN VIVO ASSAY SYSTEM SUITABLE FOR SCREENING INHIBITORS OF HEPATITIS C VIRAL PROTEASE", MOLECULAR AND CELLS, KOREAN SOCIETY FOR MOLECULAR SOCIETY, KR, vol. 6, no. 2, 1 January 1996 (1996-01-01), pages 183 - 189, XP002923443 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019185750A1 (fr) * 2018-03-27 2019-10-03 Danmarks Tekniske Universitet Plateforme à haut débit pour sélectionner des régulateurs de l'activité associée à crispr-cas

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