[go: up one dir, main page]

WO2024252009A1 - Modulation de mécanismes régulateurs globaux et de groupes de gènes biosynthétiques pour la production simplifiée de produits naturels - Google Patents

Modulation de mécanismes régulateurs globaux et de groupes de gènes biosynthétiques pour la production simplifiée de produits naturels Download PDF

Info

Publication number
WO2024252009A1
WO2024252009A1 PCT/EP2024/065835 EP2024065835W WO2024252009A1 WO 2024252009 A1 WO2024252009 A1 WO 2024252009A1 EP 2024065835 W EP2024065835 W EP 2024065835W WO 2024252009 A1 WO2024252009 A1 WO 2024252009A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
bgc
bacterium
fluorescent
constitutively expressed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/065835
Other languages
English (en)
Inventor
Helge Bode
Paushali CHAUDHURY
Edna BODE
Timo OBERMEIER
Alexander RILL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften
Publication of WO2024252009A1 publication Critical patent/WO2024252009A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the inventive method can be performed in high-throughput format allowing to speed-up the procedure.
  • Numerous medicines are directly originated or are inspired from bacterial natural products and their secondary metabolites. These secondary metabolites are known to be produced by bacteria in response to environmental stress or interaction with host, providing competitive advantages. Proteins essential for the production of the bioactive compound are usually encoded by large cryptic gene clusters that remain silent under normal laboratory conditions, which hinders the discovery of new secondary metabolites.
  • the corresponding biosynthetic gene cluster BGC can be identified biometric and act as an indicator or marker of bacterial capacity for the production of secondary metabolites.
  • cryptic BGCs are essentially present 5 - 10 times more than expressed BGCs, so that all bacterial genomes contain many more BGCs than there are secondary metabolites known from that particular strain. Understanding the modality of expression of these BGCs will not only allow discovery of new beneficial compounds but also revealing pathogenic mechanisms. The recognition of the global regulators controlling silent biosynthetic gene clusters can help to achieve these goals. In contrast to pathway-specific regulators which control the transcription of a small number of genes, global regulators control hundreds of genes.
  • GAR-P04397WO09 Application (final)2.docx Activation/inactivation of global regulators is related to significant changes in the production of secondary metabolites and to the induction of corresponding biosynthetic gene clusters.
  • global regulatory mechanisms dramatically affect the production of almost all secondary metabolites (SM, also called natural products (NPs)), in bacteria such as those of the genera Photorhabdus and Xenorhabdus.
  • NPs natural products
  • BGCs are silent under the conditions used in laboratories for growing bacterial or fungi strain, or the secondary metabolites are only produced in very minute amounts.
  • patent application US 2016348097 discloses compositions and methods for activating a silent gene or gene cluster with a bacteriophage and/or Streptomyces Antibiotic Regulatory Protein (SARP) transcription factor.
  • SARP Streptomyces Antibiotic Regulatory Protein
  • VRS-bAHL Gram-negative bacterial acyl-homoserine lactone quorum-sensing
  • RGMS reported-guided mutant selection
  • a robust and efficient method to find global regulators in bacteria such as bacteria and fungi, and for producing secondary metabolites at high levels is missing. This is particular relevant for the bacteria Photorhabdus and Xenorhabdus. It is the objective of the present invention to provide a method for screening for global regulator genes that can be manipulated to activate biosynthetic gene clusters (BGC) for producing target secondary metabolites at high level and / or inhibit background production of other not relevant metabolites.
  • BGC biosynthetic gene clusters
  • the present invention further comprises a method to elicit production of a secondary metabolite from a BGC based on deleting a global regulator and / or activating a BGC or based on activating a BGC and / or deleting a global regulator.
  • the present invention provides a recombination single plasmid for CRISPR/Cas based gene editing that allows efficient gene deletion and replacement.
  • the objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application. Brief description of the invention
  • the present invention relates to a method for finding global regulators in order to induce or increase production of target secondary metabolites.
  • the method comprises providing a bacterium indicator strain producing two fluorescent reporter signals under the same control of two different biosynthetic gene clusters (BGC), preferably highly expressed BGC; performing random mutagenesis in said
  • the invention also relates to a method to activate the production of target secondary metabolites, and to a CRISPR/Cas based single plasmid for gene editing to delete or inactivate a global regulator, to activate one or more BGC, to substitute a BGC with a fluorescent reporter, or for refactoring. Finally, the method can be performed in high-throughput format allowing to speed-up the procedure.
  • the present invention provides a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first BGC and of the second BGC in said
  • the method for screening for a global regulator (GR) gene in a bacterium comprises: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene;
  • BGC biosynthetic gene cluster
  • the method for screening for a global regulator (GR) gene in a bacterium comprises: a) replacing a gene sequence of a first highly expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second highly expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d);
  • the inventice method for screening for a global regulator (GR) gene in a bacterium comprises: a) replacing a gene sequence of a first biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first BGC and of the second BGC in said
  • the method further comprises after step h), the following step i): i) screening for production of secondary metabolites by HPLC / mass spectrometry analysis.
  • the method further comprises after step h), the following step i): i) screening for production of secondary metabolites regulated by said GR gene by HPLC / mass spectrometry analysis.
  • performing random mutagenesis of step d) comprises chemical random mutagenesis, or UV-mediated random mutagenesis, or error-prone PCR or transposon mutagenesis.
  • said BGC of step a) and/or b) encodes an enzyme responsible for production of secondary metabolites, wherein the enzyme is selected from the group comprising non- ribosomal peptide synthetase, terpene synthase/cyclase, polyketide synthase, ribosomally produced peptide (RiPP).
  • the secondary metabolite is an antibiotic, or an anti-cancer drug, or an immune suppressive drug, and is preferably selected from the group comprising Puromycin, Madumycin II, Xenoamicin, rupshomycin, rupshomycin derivatives, safracin, safracin derivatives.
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, Paenibacillus and Streptomyces.
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, Paenibacillus. In a preferred embodiment of the above inventive method, the bacterium is not Streptomyces. In a preferred embodiment of the above inventive method, the steps a) and b) are performed introducing in said bacterium a Cas gene and a first and second BGC
  • steps a) to e) are performed culturing and handling the bacteria in microtiter plates.
  • steps a) to e) are performed using an automated liquid handling robotics, and wherein said robotics enables high-throughput manipulation of liquid added to or removed from cultures comprising the bacteria.
  • the invention also relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or wherein activating a BGC of steps i
  • the invention also relates to a method to elicit production of a secondary metabolite by a bacterium, wherein the method comprises the following steps: i) deleting a global regulator (GR) gene in said bacterium, and ii) optionally activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium; or wherein the method comprises the following steps: i') essentially activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium, and
  • GR global regulator
  • BGC biosynthetic gene cluster
  • the GR gene is cyaA gene.
  • the BGC is xenoamicin.
  • deleting a GR gene of steps i) or ii’ comprises introducing in said bacterium at least one group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right.
  • the Cas is selected from the group comprising Cas9, Cas12 and Cas13.
  • step ii) or step i') comprises activating at least 2 BGCs in said bacterium.
  • said BGC is selected from the group comprising puromycin, madumycin II, xenoamicin, rupshomycin, rupshomycin derivatives, safracin, safracin derivatives.
  • said secondary metabolite is selected from the group comprising puromycin, madumycin II, xenoamicin, rupshomycin, rupshomycin derivatives, safracin, safracin derivatives.
  • the method further comprises culturing and handling the bacteria in microtiter plates.
  • the step i) - iii) are performed using an automated liquid handling robotics, and wherein said robotics enables high-throughput manipulation of liquid added to or removed from cultures comprising the bacteria.
  • the present invention provides a recombination single-plasmid to activate or silence a BGC in a bacterium by CRISPR/Cas-mediated homology-directed repair, the plasmid comprising: an origin of transfer sequence (oriT), a cas gene under control of a first inducible promoter, genes for a suitable recombinase system under control of a second inducible promoter, at least one positive selectable marker gene, at least one negative selectable marker gene, a crRNA system selected from the group consisting of: I) a crRNA system to activate a BGC comprising: at least one crRNA framework, at least one target spacer specific for a genome sequence positioned upstream to a BGC gene, homology region left complementary to a genome sequence positioned upstream to said B
  • the Cas is selected from the group comprising Cas9, Cas12, Cas13, dCas9.
  • Description of the invention The inventors have here developed a method to finding global regulators to increase secondary metabolite production.
  • the method is based on replacement of at least two constitutively expressed biosynthetic gene clusters by two different fluorescent reporters, followed by random mutagenesis and analysis for loss of fluorescence for both reporters ( Figure 1). BGC expression can be analysed by using fluorescence microscopy or FACS.
  • the disclosed method comprises random mutagenesis, e.g. transposon mutagenesis, screening for loss of fluorescence and mass spectrometry analysis to detect all secondary metabolites.
  • the disclosed method can also be performed in high-throughput in 96-well format or higher ( Figure 16A-C), allowing replacement of up to 96 BGCs or more in parallel.
  • the disclosed method allows direct bioactivity testing from crude extracts or simplified isolation of the secondary metabolites.
  • the inventors have also developed a single and easy to assemble vector to apply CRISPR/Cas ( Figures 1B, 3A-B, 6A-C) to generate bacterial indicator strains that express a fluorescent protein, for example mNeonGreen, in replacement for a biosynthetic gene cluster (Figure 17A-B) under the same control of said BGC.
  • a fluorescent protein for example mNeonGreen
  • the developed CRISPR/Cas single-plasmid can also be used to delete or inactivate a global regulator or part of it ( Figures 1D, 3B) , or to activate BGC expression by promoter exchange ( Figures1C, 1D, 3B, 6A-C, 7, 8, 9). Genome editing can be checked by PCR on isolated colonies ( Figure 5). Therefore, the present invention also provides a method to elicit production of a secondary metabolite from a BGC based on deleting a global regulator and / or activating a BGC or based on activating a BGC and / or deleting a global regulator, by using the described CRISPR/Cas single-plasmid ( Figure 3A-B).
  • Refactoring of mxn BGC led to a substantial increase in the production titer of madumycin to approximately 250 mg/l ( Figure 8 B, C and D, Figure 9).
  • Refactoring of xsc BGC (Example 6, Figure 10) allowed reaching safracin B production titers above 150 mg/l.
  • the safracin B amount of the Xenorhadus sp. TS4 strain harboring a single-plasmid for xsc BGC activation was 14 times higher than that of E. coli strain LZ84 harboring the xsc Cluster encoded on three expression plasmids ( Figure 11 B).
  • the inventive method for refactoring allowed discovering a novel BGC named rpmA-O, producing the compounds 19 - 26, wherein 26 is the primary product named rupshomycin.
  • Compounds 19 - 26 ( Figures 12, 13, 14) are not described in the prior art. Therefore, the inventive method has the advantage over the prior art to speed-up access to SM normally produced in very minute amounts, and /or from silent BCG, which is particularly relevant for SMs acting as antibiotics, anti-cancer or immune suppressive drugs.
  • the inventive method allows production of several secondary metabolites by a single bacterial multi-producer strain wherein at least 2 BGC are activated by promoter exchange and/or refactoring (Example 13).
  • Such multi- producer strains have the advantage of being easier GAR-P04397WO09 Application (final)2.docx to handle compared to culture of several mono-producing strains or handling of many extracts of individual compounds to be combined.
  • the inventive method also allows direct bioactivity testing from crude extracts or simplified isolation of SMs that can also be achieved in high-throughput in 96-well format or higher. Deletion of crucial global regulators allows a much “cleaner” production of desired SM due to the lack of production of interfering SMs.
  • the inventive CRISPR/Cas single plasmid allows multiple rounds of gene cluster optimization, that is especially important for multiple transcriptional units, such as for mxn ( Figures 8, 9), xsc ( Figure 10) or rpm ( Figure 12, 13) BGCs.
  • the inventive CRISPR/Cas single plasmid also allows direct conjugation of the plasmid from E. coli to the recipient strain without any integration of the plasmid or its parts into the genome.
  • the developed inventive CRISPR/Cpf1 single plasmid allows transformation of Photorhabdus and Xenorhabdus, wherein Photorhabdus had poor transformation efficiency and Xenorhabdus was not transformable at all with the prior art methods (Example 3).
  • Global regulators and global regulatory mechanisms that affect natural product biosynthesis can be transcription factors (TF), chaperones, metabolic switches, signalling compounds binding to TFs.
  • Exemplary global regulator are Hfq, which is a RNA chaperone mediating interaction of mRNA and sRNA (Example 7) ; ArcZ, a sRNA partner; DNA methyltransferase Dam1; the cAMP synthase CyaA (Example 12).
  • Exemplary global regulators are transcription factors that bind to the regulatory element of DNA of a BGC and help to coordinate the responses of several genes to direct the production of biosynthesis of secondary metabolites.
  • Bacterial indicator strain refers to a strain wherein a first BGC of interest is replaced by a first fluorescent reporter and the second BGC of interest is replaced by a second fluorescent reporter. "Bacterial indicator strain” is used interchangeably with "bacterial reporter strain”.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the screening of fluorescent reporter strains is preferably performed by fluorescence activated cell sorting, fluorescence spectroscopy, stereo microscopy, or fluorescence imaging.
  • Table 11 reports some fluorescent proteins that can be inserted in a BGC according to the disclosed method. Therefore, the present invention also relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: GAR-P04397WO09 Application (final)2.docx a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit
  • An embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, GAR-P04397WO09 Application (final)2.docx g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • Random mutagenesis can be performed by a method selected from chemical random mutagenesis, UV mediated random mutagenesis, transposon mutagenesis, or error prone PCR.
  • Transposon mutagenesis allows isolating mutants easily with antibiotic resistance caused by transposon insertion successfully.
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • a still more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated
  • a further more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • a still more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations
  • Biosynthetic gene cluster A “biosynthetic gene cluster” (BGC) can be defined as a physically clustered group of two or more genes in a particular genome that together encode a biosynthetic pathway for the production of a specialized metabolite.
  • a "silent BCG” refers to silent or cryptic BGC under standard laboratory growth conditions, so the SM for which are responsible are not produced.
  • Selective activation of a BGC refers to the selective stimulation of the expression of a particular BGC in order to obtain production of the SM for which is responsible at high level.
  • BGC BGC that can be modulated with the present invention: - encoding enzymes responsible for production of secondary metabolytes, wherein the enzyme is selected from the group comprising non-ribosomal peptide synthetase, terpene synthase/cyclase, polyketide synthase, ribosomally produced peptide (RiPP).
  • Non-ribosomal peptides are not directly encoded in the genome like typical proteins or peptides but are produced by metabolic pathways encoded by BGCs.
  • NRPs are a large family of structurally diverse and pharmacologically GAR-P04397WO09 Application (final)2.docx useful natural products with broad biological activities.
  • Prominent examples are the antibiotic daptomycin or the immunosuppressant cyclosporine A2. They are assembled by multifunctional enzyme complexes called non-ribosomal peptide synthetases (NRPSs) that are organized in a modular fashion.
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent
  • An alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: GAR-P04397WO09 Application (final)2.docx a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • a preferred alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated
  • a further preferred alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations
  • a further more preferred alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • An aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • a further aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • Another aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • Secondary metabolites are natural products (NP) synthesized mainly by bacteria, fungi and plants. They are molecules of low molecular weight with GAR-P04397WO09 Application (final)2.docx diverse chemical structures and biological activities. Exemplary SMs are pigments, antibiotics, anti-cancer or immune suppressive drugs with applications in medicine, biotechnology and agriculture. Secondary metabolites play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs).
  • NRPSs nonribosomal peptide synthetases
  • Exemplary SMs are: Safracin compounds, comprising Safracin B that can be used for the semisynthesis of the two chemoterapeutics Ecteinascidin 743 and (–)- Jorumycin ( Figures 10, 11), rupshomycin and derivatives produced from rpm BGC ( Figure 12-14), Madumycin II ( Figure 8, 9), Xenoamicine ( Figure 22), Puromycin, Stilbenes ( Figure 2, 4), GameXPeptides (gxpS, Figure 7A, Table 10), glidobactin (glbA, Figure 7A), Xenocoumacins (xcnA, Figure 7B, Table 10), Rhabdopeptides (rxpA, Figure 7B), Indigoidine, (indC, Figure 6, Table 10).
  • Safracin compounds comprising Safracin B that can be used for the semisynthesis of the two chemoterapeutics Ecteinascidin 743 and (–)- Jorumycin ( Figures 10, 11), r
  • indA ⁇ indC E. chrysanthemi
  • V. indigofera igiA ⁇ igiE
  • Photorhabdus luminescens pluripotent proteasome inhibitors and are regarded as promising candidates for anticancer drug development.
  • GLNPs glidobactin-like natural products
  • cepafungin I glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development.
  • BGC biosynthetic gene cluster
  • the plu1881 has the same function as the homologue glbB, i.e. catalysis of the 4-hydroxylation reaction of L-lysine.
  • the biosynthesis of xenortides A-D consists of two NRPS coded by genes XndA and XndB.
  • the XndA consists of a condensation, adenylation, methylation, and thiolation domain, and has been implicated for the loading of N-methylleucine (xenortides A-B) or N-methylvaline (xenortides C-D).
  • the XndB consists of a condensation, adenylation, methylation, thiolation, and terminal condensation domains.
  • XndB has been implicated in elongation with N-methylphenylalanine, as well as the final condensation of the enzyme-bound peptide with either decarboxylated phenylalanine (phenylethylamine in xenortides A and C) or decarboxylated tryptophan (tryptamine in xenortides B and D), ending the biosynthesis.
  • Xenorhabdus and Photorhabdus GxpS, an NRPS with five modules, is responsible for the biosynthesis of GameXPeptides (Table 10), which are a class of cyclic pentapeptides composed of valine, leucine, and phenylalanine.
  • RXP are rhabdopeptide/xenortide-like peptides (Table 10).
  • Other natural products produced by XP silathride, xenoautoxin, phenylethylamide, tryptamide, rhabdopeptide, and PAX.
  • a preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • a more preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • a still more preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; GAR-P04397WO09 Application (final)2.docx e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • a further preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • a further more preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • a further still more preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • the present invention also relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR
  • the present invention further relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the present invention alternatively relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the present invention preferably relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, GAR-P04397WO09 Application (final)2.docx f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • An embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations generated in
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • a still more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • a further more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • a further still more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • Preferred bacteria for the present invention are Gram negative bacteria (Pseudomonas, Xenorhabdus, Photorhabdus, Serratia, Vibrio etc.) and other strains that produce natural products, such as myxobacteria, cyanobacteria, Pseudomonades, Bacillus, Paenibacillus or Streptomyces.
  • Gram negative bacteria Pseudomonas, Xenorhabdus, Photorhabdus, Serratia, Vibrio etc.
  • other strains that produce natural products such as myxobacteria, cyanobacteria, Pseudomonades, Bacillus, Paenibacillus or Streptomyces.
  • an aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations generated in
  • a further aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a further aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • a further aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, GAR-P04397WO09 Application (final)2.docx f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a particular aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a preferable aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • An alternative aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a further preferred aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a particular aspect of present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a still more particular aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • a further more particular aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • An alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a preferred embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a more preferred embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a still more preferred embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a further more preferred embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; GAR-P04397WO09 Application (final)2.docx e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; GAR-P04397WO09 Application (final)2.docx e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; GAR-P04397WO09 Application (final)2.docx e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a further more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • a further still more particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: GAR-P04397WO09 Application (final)2.docx a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutation
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • BGC
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention also relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • the present invention particularly relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, GAR-P04397WO09 Application (final)2.docx g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the bacterium is selected from the group comprising Xenorhabdus, Photorhabdus, Pseudomonas, Serratia, Vibrio, myxobacteria, cyanobacteria, Bacillus, and Paenibacillus.
  • CRISPR/Cas based gene editing CRISPR-Cas systems are native to bacteria and Archaea and provide adaptive immunity against viruses and plasmids.
  • the CRISPR-Cas endonuclease system is utilized in genomic engineering as follows: the gRNA complex (either a crRNA:tracrRNA complex or an sgRNA) binds to Cas9, inducing a conformational change that activates Cas9 and opens the DNA binding cleft, the protospacer domain of the crRNA (or sgRNA) aligns with the complementary target DNA and GAR-P04397WO09 Application (final)2.docx Cas9 binds the PAM sequence, initiating unwinding of the target DNA followed by annealing of the protospacer domain to the target, after which cleavage of the target DNA occurs.
  • the Cas9 contains two domains, homologous to endonucleases HNH and RuvC respectively, wherein the HNH domain cleaves the DNA strand complementary to the crRNA and the RuvC-like domain cleaves the non-complementary strand. This results in a double-stranded break in the genomic DNA. When repaired by non-homologous end joining (NHEJ) the break is typically repaired in an imprecise fashion, resulting in the DNA sequence being shifted by 1 or more bases, leading to disruption of the natural DNA sequence and, in many cases, leading to a frameshift mutation if the event occurs in a coding exon of a protein-encoding gene.
  • NHEJ non-homologous end joining
  • the break may also be repaired by homology directed recombination (HDR), which permits insertion of new genetic material based upon exogenous DNA introduced into the cell with the Cas9/gRNA complex, which is introduced into the cut site created by Cas9 cleavage
  • HDR homology directed recombination
  • This type V CRISPR-associated system contains Cpf1, which is a ⁇ 1300 amino acid protein—slightly smaller than Cas9 from S. pyogenes.
  • the PAM recognition sequence of Cpf1 is TTTN, in contrast to the NGG PAM recognition domain of S. pyogenes Cas9.
  • the Cpf1 system is also remarkably simple in that it does not utilize a separate tracrRNA, and only requires a single short crRNA of 40-45 base length that both specifies target DNA sequence and directs binding of the RNA to the Cpf1 nuclease. In contrast to Cas9 which produces blunt-ended cleavage products, Cpf1 facilitates double stranded breaks with 4-5 nucleotide overhangs. The advantage of this is that it may ensure proper orientation as well as providing microhomology during non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • the single strand guide RNA oligonucleotide consists of a constant region of 20 nt and a target region of 21-24 nt for an overall length of 41-44 nt.
  • GAR-P04397WO09 Application (final)2.docx A further suitable Cas for the present invention is Cas13.
  • the term "crRNA framework" (crRNA FW) refers to a nucleotide sequence comprising a constitutive or inducible promoter, a crRNA leader, direct repeat, a spacer dummy (comprising a BsaI or BsmbI or any other type II restriction sites for insertion of target spacers A and B), direct repeat, a terminator.
  • crRNA array refers to a group of nucleotide sequences comprising: - a crRNA framework; - "target” specific target spacers A and/or B inserted inside said crRNA framework; - optionally further elements such as promoter system as defined herein, translational enhancer, fluorescent reporter gene.
  • a crRNA array is target specific.
  • the target specific crRNA array is formed in the plasmid for gene editing from the elements of the homology arms left and right.
  • the "target” can be for example a gene of a BGC for BGC activation, or a gene of a BGC for silencing or replacement with a FR gene, or a GR for GR deletion.
  • crRNA system refers to a group of dsDNA fragments (dsDNA nucleotide sequences) comprising: at least one crRNA framework, at least one target spacer A or B, target specific homology region left, target specific homology region right, and optionally a fluorescent reporter gene, or a promoter system or enhancer sequence positioned between said homology region left and said homology region right.
  • a "crRNA leader” sequence can be an AT-rich sequence, but can also be part of the UTR of the promoter, so it is not necessary as an independent component.
  • a "direct repeat” refers to a 36 bp long direct repeat that is an essential part of the crRNA framework and must be encoded upstream of any target specific spacer to be recognized by the Cas protein, such as Cpf1. A repeat after the spacer is not necessary if the spacer has already been shortened to the mature length of 23 bp.
  • GAR-P04397WO09 Application (final)2.docx The term “target spacer” refers to a nucleic acid sequence having the function of "target specific crRNA”.
  • a “target spacer” is selected from the genome of the target organism and is located distally after a PAM sequence (in the best-case TTTV). Best editing results can be achieved selecting one spacer for each leading and lagging strand, i.e.
  • target spacer A and target spacer B It should be avoided to have three or more “T” in the sequence.
  • a GC content of 50% should be aimed at.
  • a "target spacer” has a nucleic acid sequence with a length between 23 and 31 bp depending on whether there is a direct repeat after the target spacer.
  • a "spacer dummy” refers to a polynucleotide sequence which should not have homology to the target host; and must be accessible for any kind of cloning (Gibson, Golden Gate, Gateway, Restriction cloning).
  • a "spacer dummy” can comprise a reporter gene (e.g. mCherry) or a toxin (e.g.
  • a "terminator” or “transcriptional terminator” refers to a target host adapted terminator or standard terminator from the iGEM library (http://parts.igem.org/Terminators/Catalog).
  • the term “homology arm left” (HA-L) refers to a synthetic dsDNA fragment comprising the following elements in this order: spacer with restriction site (e.g. BsaI site), target spacer A or B (TS-A or TS-B), direct repeat (DR), optionally a terminator, homology region left (HR-L), spacer with restriction site (e.g. BsaI site).
  • the target spacer A or B and DR form the "target specific crRNA array" in the assembled single plasmid, e.g. pAR20 in the Examples of this invention.
  • the term "homology arm right” (HA-R) refers to a synthetic dsDNA fragment comprising the following elements in this order: spacer with restriction site (e.g. BsaI site), homology region right (HR-R), a constitutive promoter (e.g. J23119), direct repeat (DR), target spacer A or B (TS-A or TS-B), spacer with restriction site (e.g. BsaI site).
  • the constitutive promoter, crRNA leader, DR, and target spacer form the "target specific crRNA array" in the assembled single plasmid, e.g. pAR20 in the Examples of this invention.
  • GAR-P04397WO09 Application (final)2.docx The term "homology region” left or right refers to a nucleic acid sequence which is contained in the single plasmid for CRISPR/Cas gene editing and is complementary to the target BGC or global regulatory gene. It is used for repair after CRISPR induced double strand break. A nucleic acid sequence for "homology region" left or right depends on the recombination genes and the used strains.
  • a nucleic acid sequence for "homology region” left or right can comprise at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp (basepair).
  • a nucleic acid sequence for "homology region” left or right can comprise at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1700, 1800, 1900, 2000 bp.
  • a nucleic acid sequence for "homology region" left or right preferably comprises between 100 and 500 bp, 100 and 600 bp, 100 and 700 bp, 100 and 800 bp, 100 and 900 bp, 100 and 1000 bp, 100 and 1100 bp, 100 and 1200 bp, 100 and 1300 bp, 100 and 1400 bp, 100 and 1500 bp, 100 and 1600 bp, 100 and 1700 bp, 100 and 1800 bp, 100 and 1900 bp, 100 and 2000 bp, 50 and 500 bp, 50 and 600 bp, 50 and 700 bp, 50 and 800 bp, 50 and 900 bp, 50 and 1000 bp, 50 and 1100 bp, 50 and 1200 bp, 50 and 1300 bp, 50 and 1400 bp, 50 and 1500 bp, 50 and 1600 bp, 50 and 1700 bp, 50 and 1800 bp, 50 and 1900 bp, 50 and 2000 bp
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, GAR-P04397WO09 Application (final)2.docx f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • BGC
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • BGC
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • BGC
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention further relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR
  • the present invention also relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the present invention is also directed to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention preferably relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a
  • the present invention more preferably relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention still more preferably relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; GAR-P04397WO09 Application (final)2.docx b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention further more preferably relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; GAR-P04397WO09 Application (final)2.docx d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention also preferentially relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations generated in
  • the present invention more preferentially relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • the present invention more preferentially relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • the present invention still more preferentially relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the BGC
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; GAR-P04397WO09 Application (final)2.docx e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a bacter
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulate
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • BGC bios
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • BGC bios
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • BGC
  • a further aspect of present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • a still more particular aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates
  • a particular aspect of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • An alternative embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a preferred embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • An embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); GAR-P04397WO09 Application (final)2.docx h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, GAR-P04397WO09 Application (final)2.docx f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; GAR-P04397WO09 Application (final)2.docx c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a a
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: GAR-P04397WO09 Application (final)2.docx a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in
  • a particular embodiment of the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity
  • the present invention relates to a method for screening for a global regulator (GR) gene in a bacterium, the method comprising: a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene; b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals; c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals; d) performing random mutagenesis in said bacterium indicator strain to generate random mutations; e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal, f) isolating said non-fluorescent mutant bacteria, g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d); h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of
  • helper plasmid is a plasmid for a bacterium that provides functions required for efficient recombination.
  • an helper plasmid contains the gene encoding the endonuclease Cas12a (Cpf1) under an inducible promoter, and a lambda red (gam, exo, bet) recombination system under an inducible promoter.
  • a "donor plasmid” is a plasmid carrying a crRNA sequence for a specific target gene under a constitutive promoter or a strong constitutive promoter or an inducible promoter, as well as homology regions left and right to the target gene serving as repair templates after double strand break.
  • Promoter refers to genetic elements controlling the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced.
  • a promoter can be specific for a species or genus, but often a promoter from a bacterium can function well in a distantly related bacterium.
  • a GAR-P04397WO09 Application (final)2.docx promoter from Bacillus subtilis or a phage that normally grows on B. subtilis can function well in E. coli.
  • "Constitutive promoter” refers to promoters that are always active. Suitable examples are rpsL (30S ribosomal subunit protein S12(V00355.1)), Amp (bla, BBa_I14018) promoter Suitable promoters to carry out the methods described herein are disclosed in Shimada T. et al., The Whole Set of Constitutive Promoters Recognized by RNA Polymerase RpoD Holoenzyme of Escherichia coli.
  • Further constitutive artificial promoter can be found in Anderson library: ‘Promoters/Catalog/Anderson’, http://parts.igem.org/Promoters/Catalog/Anderson. Promoters from primary metabolism or from ribosomal proteins can be used herein, as for example: glyceraldehyde-3-phosphate dehydrogenase promoter (GenBank: CP097883.1) or ribosomal protein S12 promoter (GenBank: CP097884.1). "Strong constitutive promoter” are for examples the strong constitutive from Bacillus subtilis phage SP01, and coliphage lambda PR, and PJ23119 from the Anderson library.
  • Inducible promoters refer to promoters that are only active under specific circumstances and which can be switched from an OFF to an ON state, or from an ON to an OFF state.
  • Inducible promoters suitable for expression of cpf1 and recombinases in the present invention are AraC - P BAD (Meyer AJ 2019, Nature Chemical Biology 15, 196 - 204), LacI – P tac ( Meyer AJ 2019) , the xylose-inducible expression system: xutR - Pxut, Genbank MT344942, and MN857504, and the L-rhamnose-inducible promoter from Burkholderia, P rhaBAD , controlled by AraC/XylS-family transcriptional regulators rhaS and rhaR (Hogan AM et al., 2021.
  • Inducible promoter systems have been described in the prior art (e.g. in Meyer AJ 2019), and comprise the following elements: a 3'-Terminator, a repressor / regulator gene (Table 6), a RBS specific for the regulator (Table 6), a strong constitutive promotor for expression of the regulator (Table 6), a terminator to separate regulator regulation from target gene regulation: (ex.: L3S2P21), a Insulator element (examples listed below), a strong RBS for target gene, such as BBa_B0064.
  • the vanillic Acid promoter system (SEQ ID NO 131, 132) used in the examples of the present invention comprises the following elements: PvanCC modified (Promoter), BBa_B0064 (RBS), RiboJ (Insulator), RiboJ51 (Insulator).
  • the expression of a gene cluster can be increased by additionally introducing "enhancer sequences" such as a T7g10 transcriptional enhancer, or a "translation enhancement by a Dictyostelium gene sequence", TED sequence.
  • the transcriptional enhancer T7g10 (also abbreviated as g10T7 UTR), (SEQ ID NO:135, comprising BBa_K1758100, sequence available online) comprises a 5' untranslated region (5'-UTR) and a strong ribosomal binding site from bacteriophage T7 gene 10 (g10-L).
  • the TED sequence derived by a Dictyostelium has to be placed upstream of the Shine–Dalgarno sequence located between the promoter and the initiation codon of a target gene in order to achieve an enhancement in gene expression.
  • Suitable TED sequences are mlcR sequences, such as mlcR10, mlcR20, mlcR25, mlcR30, mlcR40, mlcR45, mlcR60, and mlcR74, previously described by Kondo and Yumura 2019, Applied Microbiology and Biotechnology 103:3501–3510. Further enhancement sequences and structures has been described in Xiao et al., ACS Synth. Biol., 9, 1051 ⁇ 1058 The wording "refactoring" refers to the manipulation of a BGC cluster comprising: replacing natural promoters with constitutive or readily inducible promoters andheterologous expression in strains optimized for heterologous expression.
  • 2019 a Tc tetR P tet PLaclR tet1 TetR is a Meyer repressor AJ from E. coli 2019 Ara AraC AM , PBAD PLaclR ara1, AraC is Meyer (L- araE e1 activator AJ arabinose) /repressor 2019 from E. coli.
  • Cho BetI AM P BetI P J23100 bet2 BetI is a TetR Meyer (choline family AJ chloride) repressor 2019 from E. coli.
  • Nar TtgR AM PTtg PJ23100 ttg2 TtgR is a Meyer (naringenin) TetR family AJ repressor 2019 from P. putida DHBA PcaU AM P3B5B PJ23100 pca2 PcaU is a Meyer (3,4- IcIR family AJ dihydroxybe repressor 2019 nzoic acid) from Acineto- bacter sp.
  • Sal NahR AM PSalTTC PJ23100 nah2 NahR is a Meyer (sodium LysR family AJ salicylate) activator from 2019 P.
  • cln1 CinR is a Meyer (3- LuxR family AJ Hydroxytetr activator from 2019 adecanoyl- Rhizobium homoserine leguminosaru lactone)
  • Suitable Insulators are RiboJ, RiboJ10, RiboJ51, RiboJ53, BydvJ, ElvJ. (Meyer AJ, 2019), LtsvJ, SccJ, SarJ, PlmJ, and VtmoJ (Lou et al., Nat Biotechnol 2012, 30 (11), 1137-42).
  • Terminators are B0053, ECK120017009, ECK120033736, ECK120033737, IOT, L3S2P21, L3S3P21, L3S2P56, L3S3P41, L3S1P52, L3S2P41. (Meyer AJ, 2019).
  • the system of the Xylose inducible promotor PxylA comprises a modified 5-UTR containing g10-L RBS (RBS from gene 10 of the T7 phage 5), a strong ribosomal binding site from bacteriophage T7. This sequence greatly enhances translation of a following gene.
  • Suitable recombinase systems Recombineering system based on three Rac bacteriophage RecET-like operons: RecETheBDU8, RecEThTJI49 and RecETh1h2eYI23, optionally combined with exonuclease inhibitors Plu ⁇ or Red ⁇ . RecETheBDU8 from Burkholderia sp.
  • BDU8 harbours a four-gene operon predicted to encode: the YqaJ viral recombinase family protein RecEBDU8 (protein ID: KVE53656.1; locus tag: WS71_06320); the recombinase RecTBDU8 (protein ID: KVE53655.1; locus tag: WS71_06315); and two hypothetical proteins hBDU8 (protein ID: KVE53654.1; locus tag: WS71_06310) and eBDU8 (protein ID: KVE53653.1; locus tag: WS71_06305).
  • TJI49 is predicted to encode the following three proteins: the hypothetical protein RecETJI49 (protein ID: EGD06616.1; locus tag: B1M_00520),; the phage-related DNA recombination protein RecTTJI49 (protein ID: EGD06615.1; locus tag: B1M_00515); and the hypothetical protein hTJI49 (protein ID: EGD06614.1; locus tag: B1M_00510).
  • the third operon, RecETh1h2eYI23 from Burkholderia cordobensis YI23 contains genes encoding the putative 5′-3′ specific dsDNA exonuclease RecEYI23 (protein ID: AET91062.1; locus tag: BYI23_B004550); the putative recombinase protein RecTYI23 (protein ID: AET91060.1; locus tag: BYI23_B004530),; three hypothetical proteins h1YI23 (protein ID: AET91061.1; locus tag: BYI23_B004540), h2YI23 (protein ID: AET91063.1; locus tag: BYI23_B004560) and eYI23 (protein ID: AET91059.1; locus tag: BYI23_B004520).
  • a number of selectable marker genes are known in the art and several antibiotic resistance markers satisfy these criteria, including those conferring resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin, ampicillin, tetracycline, neomycin, ZeocinTM, and the like.
  • a preferred negative selectable marker gene is SacB.
  • SacB Bacillus subtilis levansucrase
  • SacB encoded levansucrase converts sucrose to levans, which is harmful to the bacteria, and allows plasmid selection on sucrose.
  • the sacB gene including its native promoter can be amplified with AR432/433 from pEP17-KM.
  • a further preferred negative selectable marker gene is mutated PheS, which encodes ⁇ -subunit of phenylalanyl tRNA synthase. Counterselection is with 0.1% p-chlorophenylalanine), GenBank EU329004.1.
  • Single plasmid for gene editing A "single-plasmid" refers to a plasmid comprising elements of helper plasmid, donor plasmid, optionally repair template, and positive and negative selectable markers.
  • a single plasmid for replacement of a BGC with a fluorescent reporter comprises the following sequences: - first crRNA array comprising a crRNA FW (J23119, DR, DR, Term) with a BGC specific TS-A inserted in the spacer dummy of the crRNA FW.
  • - BGC specific HR-L complementary to a genome sequence positioned upstream said BGC gene
  • - Fluorescent Reporter gene for example a gene encoding for a protein of Table 11
  • BGC specific HR-R complementary to a genome sequence positioned downstream said BGC gene
  • second crRNA array comprising a crRNA FW (J23119, DR, DR, Term) with a BGC specific TS-B inserted in the spacer dummy of the crRNA FW.
  • a single plasmid for activation of a BGC by promoter exchange comprises the following sequences: - first crRNA array comprising a crRNA FW (J23119, DR, DR, Term) with a BGC specific TS-A inserted in the spacer dummy of the crRNA FW.
  • - BGC specific HR-L complementary to a genome sequence positioned upstream to said BGC gene;
  • - Promoter system e.g.: repressor with constitutive promoter and Terminator all in reverse direction; an Inducible Promoter;
  • - BGC specific HR-R complementary to a genome sequence positioned inside said BGC gene;
  • second crRNA array comprising a crRNA FW (J23119, DR, DR, Term) with a BGC specific TS-B inserted in the spacer dummy of the crRNA FW.
  • a single plasmid for inactivation or deletion of a GR comprises the following sequences: - first crRNA array comprising a crRNA FW (J23119, DR, DR, Term) with a GR specific TS-A inserted in the spacer dummy of the crRNA FW.
  • a preferred embodiment of the present invention is directed to a recombination single-plasmid to activate or silence a BGC in a bacterium by CRISPR/Cas- mediated homology-directed repair, the plasmid comprising: an origin of transfer sequence (oriT), a cas gene under control of a first inducible promoter, GAR-P04397WO09 Application (final)2.docx genes for a suitable recombinase system under control of a second inducible promoter, at least one positive selectable marker gene, at least one negative selectable marker gene, a crRNA system selected from the group consisting of: I) a crRNA system to activate a BGC comprising: at least one crRNA framework, at least one target spacer specific for a genome sequence positioned upstream to a BGC gene, homology region left complementary to a genome sequence positioned upstream to said BGC gene, homology region right complementary to a genome sequence positioned inside said BGC gene, and a promoter system positioned between said homology
  • a more preferred embodiment of the present invention is directed to a recombination single-plasmid to activate or silence a BGC in a bacterium by CRISPR/Cas- mediated homology-directed repair, the plasmid comprising: an origin of transfer sequence (oriT), a cas gene under control of a first inducible promoter, genes for a suitable recombinase system under control of a second inducible promoter, at least one positive selectable marker gene, at least one negative selectable marker gene, a crRNA system selected from the group consisting of: I) a crRNA system to activate a BGC comprising: at least one crRNA framework, GAR-P04397WO09 Application (final)2.docx at least one target spacer specific for a genome sequence positioned upstream to a BGC gene, homology region left complementary to a genome sequence positioned upstream to said BGC gene, homology region right complementary to a genome sequence positioned inside said BGC gene, and a promoter system positioned between said homo
  • An embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii') comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right
  • the method to elicit production of a secondary metabolite by a bacterium comprises the following steps: i) deleting a global regulator (GR) gene in said bacterium, and ii) optionally activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium; or wherein the method comprises the following steps: i') essentially activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii') comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific
  • a preferred embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or GAR-P04397WO09 Application (final)2.docx wherein
  • a more preferred embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or wherein activating a B
  • a further more preferred embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating at least 2 BGCs in said bacterium; or wherein the method comprises the following steps: i') essentially activating at least 2 BGCs in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; GAR-P04397WO09 Application (final)2.docx wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair
  • a more preferred alternative embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or wherein activating a
  • An embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating at least 2 BGCs in said bacterium; or wherein the method comprises the following steps: i') essentially activating at least 2 BGCs in said bacterium, and GAR-P04397WO09 Application (final)2.docx ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR
  • a particularly more preferred embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or wherein activating a
  • a preferable embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating at least 2 BGCs in said bacterium; or wherein the method comprises the following steps: i') essentially activating at least 2 BGCs in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific
  • a further more preferable embodiment of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or GAR-P0
  • a more preferred aspect of the present invention relates to a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting a GR gene in said bacterium, and ii) optionally activating at least 2 BGCs in said bacterium; or wherein the method comprises the following steps: i') essentially activating at least 2 BGCs in said bacterium, and ii') optionally deleting a GR gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a GR gene of steps i) or ii’) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right; and/or wherein activ
  • a particularly preferred embodiment of the present invention is a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting CyaA gene in said bacterium, and GAR-P04397WO09 Application (final)2.docx ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a CyaA gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a CyaA gene of steps i) or i') comprises introducing in said bacterium at least one CyaA specific siRNA, or a group of sequences comprising a Cas gene, at least one CyaA specific crRNA array, a pair of CyaA specific
  • a particularly more preferred embodiment of the present invention is a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting CyaA gene in said bacterium, and ii) optionally activating a BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating a BGC in said bacterium, and ii') optionally deleting a CyaA gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a CyaA gene of steps i) or i') comprises introducing in said bacterium at least one CyaA specific siRNA, or a group of sequences comprising a Cas gene, at least one CyaA specific crRNA array, a pair of CyaA specific homology regions left and right; ; and/or wherein activ
  • a further more preferred embodiment of the present invention is a method to elicit production of a secondary metabolite from a BGC in a bacterium, wherein the method comprises the following steps: i) deleting CyaA gene in said bacterium, and ii) optionally activating xenoamicin BGC in said bacterium; or wherein the method comprises the following steps: i') essentially activating xenoamicin BGC in said bacterium, and ii') optionally deleting a CyaA gene in said bacterium; and the method further comprises: iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene; wherein deleting a CyaA gene of steps i) or i') comprises introducing in said bacterium at least one CyaA specific siRNA, or a group of comprising a Cas gene, at least one CyaA specific crRNA array,
  • Rupshomycin derivatives Another aspect of the present invention is directed to rupshomycin and its derivatives discovered and obtained by the inventive methods described herein. Said compounds (see Figures 12 – 14, and 23 – 25) are the produc of a novel BGC named rpmA-O and exhibit interesting biological activities. Therefore, thee compounds are particularly useful as aantibiotic, or anti-drug, or immune suppressive drug.
  • GAR-P04397WO09 Application (final)2.docx Particularly preferred is when the compound of the above shown group is a (2E)- thiazolidine isomer. Particularly preferred is when the compound of the above shown group is a (2Z)- thiazolidine isomer.
  • Protein Locus Size (aa) Proposed function MxnA XNC1_RS07585 81 acyl carrier protein MxnB XNC1_RS07580 408 beta-ketoacyl-ACP synthase MxnC XNC1_RS07575 411 HMG-CoA synthase-like protein MxnD XNC1_RS20555 262 enoyl-CoA hydratase MxnE XNC1_RS07565 250 enoyl-CoA hydratase MxnF XNC1_RS07560 1980 PKS MxnG XNC1_RS07555 2407 Mixed trans-AT type I PKS/NRPS MxnH XNC1_RS07550 2302 NRPS MxnI fabD 286 Acyltransferase MxnJ XNC1_RS07540 400 major facilitator superfamily transporter MxnK XNC1_RS07535 229 thioeste
  • Protein NCBI Reference Sequence Size (aa) Proposed function XscA Xets_RS15840 1055 non-ribosomal peptide synthetase XscB Xets_RS15835 1073 non-ribosomal peptide synthetase XscC Xets_RS15830 1444 non-ribosomal peptide synthetase XscD Xets_RS15825 352 putative hydroxylase GAR-P04397WO09 Application (final)2.docx XscE Xets_RS15820 66 MbtH family NRPS accessory protein XscF Xets_RS15815 355 O-methyltransferase XscG Xets_RS15810 343 O-methyltransferase XscH Xets_RS15805 180 dihydrofolate reduct
  • TTTA nem Deletion NO 194 TS-B atophila TTTA NO 195 TS-A TTTG dprA promoter exchange NO 196 TS-B TTTC NO 197 TS-A TTTA cyaA_XDUO Deletion NO 198 TS-B TTTG NO 199 TS-A TTTC xabA promoter exchange NO 200 TS-B TTTA Table 7c Sequence list of synthetic dsDNA fragments used for CRISPR/Cas12 mediated genome editing SEQ ID Name Type of Editing /Description crRNA PJ23119, crRNA leader, direct repeat, spacer- NO 119 framework dummy (BsaI sites), direct repeat, terminator Adapter with BsaI site, [selected target spacer (23-31 bp)], direct repeat, TERMINATOR, placeholder (lowercase), [homologues region HA-L (up to 500 bp)], GS-linker sequence I, Adapter NO
  • Non-fluorescent mutants are identified, isolated and then analysed by whole-genome sequencing to identify global regulators. Non-fluorescent mutants are also screened for the production of Natural Products by HPLC/MS analysis.
  • MS mass spectroscopy
  • Figure 3 shows A) schematic representation of pAR20 plasmid that can be used for genome editing with CRISPR/Cpf1 approach digested with BsaI.
  • the approach starts with conjugation of the target specific pAR20 into the strain to be edited (with or without previous global regulator (GR) deletion, day 1); selection of conjugates and induction of CRISPR/Cpf1 components followed by selection of correct edited strains (day 3); plasmid curing with sucrose (day 5); screening for new or known compounds (day 6); entering a new editing cycle (day 7).
  • Figure 4 shows overview of MS spectra of deletion mutants.
  • FIG. 1 BPCs of X. nematophila wild type and deletion mutants.
  • Figure 5 shows gel electrophoresis images of deletion and promoter exchange mutants after colony PCR.
  • Figure 6 shows single plasmid CRISPR/Cpf1 genome editing method for promoter exchange upstream of indC.
  • C Schematic representation of the genome editing approach mediated by pAR20; the sequence between the homology arms are replaced by the promoter sequence; detailed genotype of pAR20 can be found in Table 1.
  • Figure 8 shows strain engineering towards madumycin production in X. nematophila.
  • C condensation domain
  • CY cyclization domain
  • E epimerization domain
  • MT methylation domain
  • PCP peptidylcarrier protein domain
  • ACP small black circle
  • KS ⁇ -ketoacyl carrier protein synthase domain
  • KR ketoreductase domain
  • DH dehydratase domain
  • OX oxidation domain
  • TE thioesterase domain.
  • Figure 9 shows structure elucidation and proposed biosynthesis pathway of the formation of madumycin II (16) in X. nematophila.
  • C MS/MS fragmentation comparison of produced compound 16 and its chemical standards.
  • Figure 10 shows A) the xsc BGC responsible for the production of safracin A (SAC-A, 2) and safracin B (SAC-B, 1) in Xenorhabdus sp. TS4.
  • B) shows strains I – III and corresponding EICs for SAC-A and SAC-B production; I: wild type stain with silent xsc BGC; II: Bidirectional promoter exchange between xscA and xscJ, production of SAC-A and B; III: Additional promoter exchange upstream of xscK while deleting xscH; producing SAC-B exclusively.
  • FIG. 12 shows stepwise activation and characterization of the rupshomycin biosynthetic gene cluster (rpm).
  • rpm rupshomycin biosynthetic gene cluster
  • I native and silent BGC rpm under laboratory conditions.
  • II PvanCC promoter exchange upstream of rpmA, no production observed after induction.
  • III PvanCC promoter exchange upstream of rpmD, no production observed after induction.
  • IV promoter exchange upstream of rpmA with exchange of the intergenic region between rpmC and rpmD by RiboJ51 and T7g10 UTR, production of 19 observed after induction.
  • V Ptac promoter exchange upstream of rpmA with exchange of the intergenic region between rpmC and rpmD by RiboJ51 and T7g10 UTR; feeding of several benzoic acid derivatives (all 1mM) leading to the production of compounds 20 – 24 after induction.
  • VI strain V with deletion of rpmG, conjugated with expression vector expressing rpmA and rpmJ; detection of compound 25 after induction.
  • VII strain V with deletion of rpmMNO and feeding of 3,4- AHBA; no production observed, culture dies after induction.
  • VIII strain VII conjugated with expression vector expressing rpmM and rpmN.
  • IX strain VII conjugated with expression vector expressing rpmO.
  • rpmG Hydroxylation in 5-position is catalyzed by rpmG (shaded); Removal of 3 amino group is catalyzed by RpmB and RpmM in the presence of Trp; C: condensation domain, CY: cyclization domain, PCP: peptidylcarrier protein domain, ACP (small black circle): acyl carrier protein domain, KS: ⁇ -ketoacyl carrier protein synthase domain, KR: ketoreductase domain, DH: dehydratase domain, TE: thioesterase domain.
  • FIG. 15A shows schematic representation of the high-throughput genome editing approach using microtiter plates. Overnight cultures grown in a deepwell plate are diluted 1:25 in 100 ⁇ l and transferred to a 96 well microtiter plate. Cultures are incubated in a microplate reader at 30 °C while growth is recorded.
  • Figure 17 A shows fluorescence imaging of pigmented Photorhabdus luminescens TTO1 indicator strains.
  • Strains were constructed using the CRISPR-Cas12 approach described in this patent. In these strains, BGCs stlCDE, ppyS, AQ MT were replaced by an mNeonGreen gene. Strains were spotted onto LB agar plates and imaged at different days using an automated Leica Thunder Imager stereo microscope. Selected GAR-P04397WO09 Application (final)2.docx indicator strains develop fluorescence over time and are thus suited for screening. Intensities were manually adjusted for good contrast for each strain, while the intensity range was kept identical over the time course.
  • B) shows imaging of non-pigmented Xenorhabdus nematophila indicator strains.
  • Strains were constructed using the CRISPR-Cas12 approach described in this patent. In these strains, BGCs rxp, ppyS, Odilorhabdin (odlC) were replaced by a mNeonGreen gene. Strains were spotted onto LB agar plates and imaged at different days using an automated Leica Thunder Imager stereo microscope. Selected indicator strains develop fluorescence over time and are thus suited for screening. Intensities were manually adjusted for good contrast for each strain, while the intensity range was kept identical over the time course.
  • Figure 18 shows the method to obtain multi-producer strains.
  • FIG. 19 shows suitable crRNA frameworks: A) A crRNA framework consisting of 5 components: 1. host specific promoter, constitutive or inducible, or derived from a housekeeping gene; 2. A leader sequence; 3. cas12 specific direct repeat; 4.
  • two distinct crRNA arrays can be created (to target leading and lagging strand). R repair templates for the double-strand break is located in between.
  • FIG. 23 shows chemical structures of further rupshomycin derivatives 31 – 44 prepared according to the methods described herein.
  • Figure 24 A) shows extracted-ion chromatograms (EIC) of rupshomycin derivatives 26, 31, and epoxy-rupshomycin derivatives 45 – 48; B) chemical structures of epoxy-rupshomycin derivatives 45 – 48; C) mass spectra of compounds 26, 31, 46 and 47.
  • EIC extracted-ion chromatograms
  • Figure 25 upper part shows extracted-ion chromatograms (EIC) of rupshomycin derivatives 49 – 52; lower part shows chemical structures of rupshomycin derivatives 49 – 52.
  • EIC extracted-ion chromatograms
  • Promoter exchange mutants were induced by adding L-arabinose (0.2%, v/v) to the cultures. All plasmids and strains used in this study are listed in Table 2. Production of selected metabolites took place in XPP3 medium or BactoTM CD Supreme Fermentation Production Medium (SFPM) for 72 h at 28 °C.
  • Transposon Mutants Construction Transposon mutagenesis was performed by conjugating Xenorhabdusbendingtiae and Photorhabdus laumondii with a pool of donor E.
  • coli APA752 (harboring the pKMW3 (mariner transposon vector library with ⁇ 3 million unique 20mer DNA barcodes flanked by common PCR priming sites) in WM3064 (conjugation donor strain)).
  • APA752 was a gift of Prof. Adam Deutschbauer (Berkeley, California, USA).
  • the donor strain APA752 and the recipient strains X.gglingtiae and P. laumondii were grown to mid-log-phase (OD 0.6 to 0.8) then mixed at a donor/recipient ratio of 1:3 for 24 h at 30°C on LB agar plates.
  • the conjugation reaction mixtures were scraped into LB and the cells plated on LB agar plates supplemented with 100 ⁇ g/ml Kanamycin, and incubated the plates at 30°C. After 2 days of growth, non-fluorescent colonies were selected and those were grown in 10ml LB with 100 ⁇ g/ml Kanamycin at 30°C to a final OD of 1.5. Final volume of 20% glycerol was added, 2ml -80°C freezer stocks were made, and simultaneously cell pellets were collected for genomic DNA extraction for Whole genome sequencing. Genomic DNA extraction For sequencing of transposon-insertion mutants, genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions.
  • Flow cytometry measurements were performed on a BD Fortessa Flow Cytometer (BD Bioscience) to select reporter strains and optimize growth condition, e.g. time point for maximal expression of reporter genes and media with highest expression level. For this, cells were grown for 1-3 days in low-salt LB medium. Other media, such as minimal media (M9 and derivatives), insect cell culture media (Sf900 II) or GAR-P04397WO09 Application (final)2.docx defined production media (XPPM or XPP3) can also be used, depending on the expression profile of the reporter genes. The samples were washed once with PBS (pelleting at 5000 rcf for one minute) and diluted 1:300 with PBS before measuring.
  • PBS pelleting at 5000 rcf for one minute
  • Reporter strains carrying for example mNeonGreen and mScarlet were measured using the 488 nm and 561 nm laser lines (100 mW) and the bandpass filter 510/20 and 586/15 respectively. Gating was set according to forward scatter, sideward scatter and fluorescence signal.30,000 events were acquired. The data were recorded using the BD FACS DIVA software (BD Bioscience). This kind of experiment is suitable for high throughput approaches (see M4). FACS for collecting of possible candidates with deletion of global regulators. Fluorescence activated cell sorting measurements were performed on a BD FACSAria TM Fusion Flow Cytometer (BD Biosciences). Reporter strains carrying mNeonGreen and mScarlet were measured after transposon mutagenesis.
  • Table 1 Methods for screening of fluorescent reporter strains
  • Method Sensitivity Throughput Time effort M1 Fluorescence imaging of colony High Low High smear
  • M2 Stereo microscopy of colonies on Medium Medium Medium solid agar
  • M3 Fluorescence spectroscopy using Medium - Medium - Medium a microplate reader High High
  • M4 FACS High High Low M1) Fluorescence imaging of colony smear
  • This screening method is based on manual fluorescence microscopy imaging using an upright or inverted fluorescence microscope with a high magnification objective and a sensitive detector (preferably cooled EMCCD or sCMOS camera).
  • Fluorescence signal of bacterial cells is recorded using appropriate filter sets (here: Zeiss GFP filter cube for mNeonGreen or Zeiss Texas Red filter cube for mScarlet-I). This approach has a low throughput, but equipment is available in many laboratories.
  • M2 Stereo microscopy of colonies on solid agar
  • colonies on solid agar can be imaged using a stereo microscope with a white light and fluorescence module.
  • Systems with automated stages increase the throughput as they facilitate screening of entire plates.
  • a Leica ThunderImager M205FC with an LED illumination system can be used. Non-fluorescent colonies are identified in overlays of fluorescence and white light images and can be subsequently picked and cultured.
  • Microtiter plates (96-well or 384-well format) are inoculated with single colonies from selection agar plates. Plates are incubated at optimized conditions for 1 – 3 days and subsequently measured using a microplate reader with absorption and fluorescence modules. For this, a Tecan Spark system with a fluorescence module can be used. Measuring the OD600 for each well reports on the biomass, while the fluorescence signal in two spectral windows is recorded to screen for loss of reporter protein fluorescence.
  • FACS FACS allows for sorting of non-fluorescent bacteria from liquid cultures at high throughput. After transposon mutagenesis, bacteria are grown in liquid medium including appropriate selection markers for 1-3 days. As FACS can separate individual cells with specific properties, culturing on solid agar is not required. Non- fluorescent cells can be sorted either directly into well plates or into reaction tubes followed by streaking onto solid agar to isolate individual mutant clones. Sorting into well plates can be combined with M3 to verify the loss of fluorescence. In the examples described herein was used a BD FACSAria TM Fusion Flow Cytometer.
  • E. coli DH10B were used as cloning for plasmid construction
  • E. coli ST18 was used as donor strain for conjugation to transfer plasmids into Photorhabdus laumondii subsp. laumondii TTO1 and Xenorhabdus nematophila ATCC 19061.
  • E. coli was cultured in LB medium at 37 °C, and Photorhabdus/Xenorhabdus was cultured in LB medium at 30 °C.
  • Plasmid Construction for BGC/ fluorescence reporter replacement based on pAR20 series The empty pAR20 was constructed in silico and synthesized as whole plasmid by Double Helix Technologies (DOULIXTM). To overcome multiple assembly rounds, the repair template containing homologues regions left and right (HR-L, HR-R) was fused together with target specific crRNA sequence on a synthetic dsDNA fragment ( Figure 1B). The position of the left/ upstream homologues region was selected appropriately to conserve the native regulation of the gene and generate a fusion protein from partial origin gene and the fluorescent reporter. For this purpose, 350 bp of the UTR and and 150 bp of the coding gene, including the start codon, were selected.
  • the right homologues region was selected depending on the desired deletion size.
  • the sequence does not necessarily have to be deleted as long as the PAM sequences of the selected spacers are mutated and another double strand break is prevented.
  • the selection criteria were target spacer with 31 bp length (23 bp minimum length) and TTTY as PAM sequence. At least one spacer sequence is selected per editing procedure. Selecting two spacer sequences, targeting each leading and lagging strand, the best results are achieved.
  • Target spacer, homology repair templates and fluorescent reporter gene were ordered as synthetic dsDNA gene fragments, from Twist Bioscience and Integrated DNA GAR-P04397WO09 Application (final)2.docx Technologies, respectively ( Figure 1B).
  • the NEB Golden Gate assembly mix containing pAR20 and dsDNA inserts was prepared according to the manufacturer's instructions resulting in a final editing plasmid ( Figure 1B). After the reaction, 1-2 ⁇ l of the mix was transformed into chemically or electro competent E. coli cells. Correct assembly was checked by colony PCR using primer pair AR533/534. Regulatory seuqences and homology repair templates are listed in Table 7c. Selected target spacer can be found in Table 7b.
  • TS sequence was selected as follows: A region 50 - 1000 bp upstream of the cluster of interest were screened and annotated using the “CRISPR Sites” prediction ( Figure 1C). Two sequences were selected, oriented in opposite directions to target the leading and lagging strand respectively. The average GC content was between 30-60%, sequences with more than two Ts in a row were excluded. The left homologous region were then identified upstream of the most left selected target spacers with a minimal distance of 50 bp to the PAM sequence.
  • the length was 500 (+/-100) bp and the average GC content was between 30 and 60% (Figure 1C).
  • the right homologues region is located downstream of the most right target spacer, but it must initiate with the start codon of the gene to be activated ( Figure 1C).
  • target spacer sequences located in the later homologous regions were also selected.
  • artificial silent mutations were introduced into the PAM sequence to prevent the CRISPR complex from 'attacking' the repair template.
  • Target spacer homology repair templates and regulatory sequence (inducible promoter and regulator) ordered as synthetic dsDNA gene fragments, were obtained from Twist Bioscience and Integrated DNA Technologies, respectively ( Figure 1C).
  • the assembly is carried out with the NEB Golden Gate assembly mix containing pAR20 and dsDNA inserts was prepared according to the manufacturer's instructions resulting in a final editing plasmid ( Figure 1C). After the reaction, 1-2 ⁇ l of the mix was transformed into chemically or electro competent E. coli cells. Correct assembly was checked by colony PCR using primer pair AR533/534. Regulatory seuqences and homology repair templates are listed in Table 7c. Selected target spacer can be found in Table 7b.
  • TS sequence was selected as follows: within the coding sequence of the BGC/gene of interest were screened and annotated using the “CRISPR Sites” prediction ( Figure 2C). Two sequences were selected, oriented in opposite directions to target the leading and lagging strand respectively ( Figure 2C). The average GC content was between 30-60%, sequences with more than two “T”s in a row were excluded.
  • the left homologous region were then identified upstream of the most left selected target spacers with a minimal distance of 50 bp to the PAM sequence, the region can be located within the coding sequence for a partial deletion or before the start codon for a complete deletion.
  • the length was 500bp (+/-50bp) and the average GC content was between 30 and 60% (complete deletion, Figure 2C).
  • the right homologues region is located downstream of the most right target spacer, the region can be located within the coding sequence for a partial deletion or behind the stopp codon for a complete deletion (complete deletion, Figure 2C). In cases where the described distances could not be maintained, e.g.
  • Target spacer sequences located in the later homologous regions were also selected.
  • artificial silent mutations were introduced into the PAM sequence to prevent the CRISPR complex from 'attacking' the repair template.
  • Target spacer, homology repair ordered as synthetic dsDNA gene fragments were obtained from Twist Bioscience and Integrated DNA Technologies, respectively ( Figure 2C).
  • the assembly is carried out with the NEB Golden Gate assembly mix containing pAR20 and dsDNA inserts was prepared according to the manufacturer's instructions resulting in a final editing plasmid. After the reaction, 1-2 ⁇ l of the mix was transformed into chemically or electro competent E. coli cells. Correct assembly was checked by colony PCR using primer pair AR533/534.
  • Electro transformation and biparental conjugation pAR20 plasmids could be introduced into the target bacterial strains either by electro transformation (BTXTM ECMTM 630 Exponential Decay Wave Electroporator) or by conjugation. Electro competent Photorhabdus and Xenorhabdus cells were prepared following standard protocols. For conjugation of the pAR20 derivatives overnight cultures of E. coli ST18, having a hemA deletion mutation, and Photorhabdus/Xenorhabdus were used, respectively.
  • the optical density of the two cultures was measured and set to a GAR-P04397WO09 Application (final)2.docx ratio of 4:1 Recipient/Donor in a total volume of 1 ml.
  • the cell mixture was centrifuged at 5000 x g and washed twice with 1 ml LB. Finally, the cell pellet was resuspended in 100 ⁇ l LB and spotted onto a LB agar plate containing 50 ⁇ g/ml 5- aminolevulinic acid, ALA. The next day, the cell mass was scraped off and resuspended in 1 ml LB.
  • a dilution of 1:50 was plated on a selection plate with 25 ⁇ g/ml kanamycin (Km) and without ALA in order to select only for Photorhabdus/Xenorhabdus mutants and counterselecting for E. coli, due to the hemA deletion causing a defect in tetrapyrrole biosynthesis.
  • Cpf1 (Cas12a) assisted gene editing BGC activation, BGC/FR replacement, BGC deletion
  • pAR20 series and plasmid curing For general gene editing, a strain carrying a gene-specific pAR20 vector was inoculated as a 5 ml overnight culture in a 50 mlenmeyer flask.
  • this culture was diluted to a final OD of 0.5 in 10 ml LB containing 25 ⁇ g/ml kanamycin. The culture was then incubated at 30 °C until an OD of 0.8 - 1.0 was reached. After addition of AHT, the culture was further incubated at 25 °C for 1h. Subsequently, L-arabinose was added and incubated for another 3 h at the same temperature. Finally, 50 ⁇ l of the cell suspension was plated on a selection plate with kanamycin and arabinose. Successful editing approaches were confirmed by colony PCR.
  • Example 1 Transposon mutagenesis and mutant screening based on loss- of-fluorescence Many bacterial strains potentially producing interesting secondary metabolites form non-pigmented colonies, rendering visual identification by color unsuitable. Replacing sequences of BGCs, such as NPRS, in such non-pigmented strains by fluorescent reporters, i.e. fluorescent proteins, allows for identification of global regulator mutants using fluorescence microscopy, spectroscopy or fluorescence- assisted cell sorting (FACS) .
  • FACS fluorescence- assisted cell sorting
  • mutants can be constructed using classical methods, e.g. suicide plasmids, or the CRISPR/Cas- GAR-P04397WO09 Application (final)2.docx based method presented here to increase the throughput.
  • suicide plasmids or the CRISPR/Cas- GAR-P04397WO09 Application (final)2.docx based method presented here to increase the throughput.
  • final 2.docx based method presented here to increase the throughput.
  • indicator strains in which highly expressed NRPS encoding genes are replaced by fluorescent reporters, so that the fluorescent reporter is under control of the same regulatory elements as the original NPRS encoding gene.
  • Suitable indicator strains can also be created by replacing another gene being part of the BGC instead of NPRS.
  • BGCs responsible for Rhabdopeptide production was chosen as model BGC due to their high expression level. Growth conditions were optimized in 96-well format using a microplate reader Tecan Spark, to ensure that reporters were expressed at detectable levels.
  • Figure 17A shows Photorhabdus luminescens TTO1 indicator strains where BGCs stlCDE (Isopropylstilbene), ppyS (pyrones), and AQMT4 (anthraquinone) were replaced by a mNeonGreen gene.
  • Figure 17B shows Xenorhabdus nematophila indicator strains, where BGCs rxpA-C (XNC1_2228 – 2230) (Rhabdopeptides (9-12)), ppyS, and odlC (Odilorhabdins) were replaced by a mNeonGreen gene. Afterwards, the constructed indicator strains were subjected to transposon mutagenesis (Method section). Following transposon mutagenesis, cultures were plated out on LB agar plates including appropriate selection markers, incubated at suitable conditions (see Methods) and colonies were screened for the absence of fluorescence signal as described in the Method section above.
  • Absence of fluorescence signal of one reporter might be the result of transposon integration into the fluorescent reporter itself or the promoter region of the respective NRPS. Absence of both reporter signals, however, might indicate transposon integration into the genomic locus of a global regulator.
  • Candidate mutant bacteria colonies having lost fluorescence emission were verified by sequencing and HPLC-MS in order to identify global regulator gene: If no production of secondary metabolites is found in the mutants by HPLC/MS analysis, suggesting that the transposon might have hit the global regulators involved in the global regulatory pathways, WGS was performed in those mutants GAR-P04397WO09 Application (final)2.docx in order to identify the transposon insertion sites and thus the global regulator gene affecting the global regulation of secondary metabolite biosynthesis.
  • Example 2 Optimization of the two-plasmid-based CRISPR/Cpf1 method for Photorhabdus The present method represents an improvement of the CRISPR/Cpf1 editing method described by Ao X et al. 2018 (Front. Microbiol. 9:2307).
  • a helper and a donor plasmid were used to perform genome editing supported by the lambda recombination system.
  • the helper plasmid p46Cpf1-OP2 encodes an E.
  • the donor plasmid is based on a high copy pUC vector carrying the crRNA sequence for a specific target gene, as well as homology regions to the target serving as repair templates after double strand break.
  • Cas12a processes the transcript from the donor plasmid to generate mature crRNAs. Guided by the crRNA, Cas12a finds the genomic target and induces a double-strand break.
  • StlA is a phenylalanine ammonia lyase involved in the biosynthesis of isopropylstilbene (IPS; 1) produced by all Photorhabdus strains ( Figure 2B) and its deletion results in overproduction of the orange anthraquinone pigment.
  • IPS isopropylstilbene
  • luminescens comprises a stilbene epoxidase gene (plu2236), which is adjacent to stlA (plu2234), a phenylalanine ammonia lyase that converts phenylalanine to cinnamic acid, initiating phenylpropanoid/stilbene biosynthesis.
  • a donor plasmid (pAR18, Figure 2C) was designed based on the pTargetF (Addgene # 62226) vector for constitutive expression of crRNA (specific for a target DNA), with gentamycin resistance as well as the sacB gene as counter-selection marker enabling rapid loss of the donor plasmid allowing for several rounds of GAR-P04397WO09 Application (final)2.docx genome editing.
  • the donor plasmid comprises homology regions left and right (HR-L and HR-R) to the target serving as repair templates after double strand break. Two crRNA sequences were used for each deletion to enable targeting of the leading and lagging strand, respectively.
  • a synthetic framework consisting of the constitutive promoter J23119, a transcriptional terminator and the direct repeats of the crRNA sequence.
  • the inventors modified a standard CRISPR array replacing the spacer sequence between the direct repeats by two BsaI restriction sites containing spacer sequence, (also named spacer dummy, SD) ( Figure 2C, 2D).
  • the stlA specific target spacers were determined by "Annotate & Predict" function of Geneious Prime, "TTTN” was set as PAM site and a target spacer length of 23-31 bp.
  • the homology repair arms (HA-L/HA- R; ⁇ 500 bp) were each coupled with one of the crRNAs and synthesized as a dsDNA fragment (Figure 2C). Since the original p46Cpf1-OP2 of Ao et al., 2018 was not transformable into Photorhabdus, the inventors first exchanged the origin of replication from pSC101 to p15A resulting in helper plasmid pAR16, assuming it would increase transformation efficiency. The stlA-specific donor pAR18, assembled by the Golden Gate reaction, was transformed, together with helper pAR16 into Photorhabdus laumondii TTO1 by electroporation (Figure 2D).
  • OD 0.1, 0.2 and 0.5 after inoculation from an overnight culture were selected. Since Photorhabdus has a much slower doubling time compared to E. coli, the required OD600 of 0.8-1.0 could not be achieved within a single working day when the starting OD600 was below 0.5. Therefore, 0.5 was chosen as the starting OD600 of the liquid culture for the following experiments.
  • the inducers for cpf1 (Ara) and lambda red (AHT) were added. As the system was previously tested (Ao et al., 2018) only the overall method instead of the individual components of the system, was checked.
  • plasmid pAR20 combining the components from the donor plasmid pAR18 and helper plasmid p46Cpf1-OP2 was constructed.
  • a major advantage of the method is the possibility to conjugate the plasmid directly from E. coli to the recipient strain without any integration of the plasmid or its parts into the genome.
  • the BsaI digested pAR20 ( Figure 3) can be used for CRISPR/Cpf1 mediated genome editing such as deletions, promoter exchange and replacement of sequences with fluorescent reporters.
  • CRISPR/Cpf1 mediated genome editing such as deletions, promoter exchange and replacement of sequences with fluorescent reporters.
  • the inventors wanted to evaluate the potential of the method for medium and large deletions and whether the editing process can be speed up.
  • the selected and successfully deleted genes/genome segments include (Table 3, Figure 4): gxpS (plu3263, 15.4 kb deletion) in TT01 and xcnA (XNC1_1711, 7.5 kb deletion) in Xenorhabdus nematophila, rxpABC (XNC1_2228 – 2230, 15.7 kb deletion) in Xenorhabdus nematophila Efficiency was determined by dividing the number of positive edited colonies by the total number of colonies tested. The corresponding agarose gel electrophoresis pictures are shown in Figure 5. Target spacers used for each approach are listed in Table 7b. Large gene deletions up to 15 kb in P. laumondii TTO1 and X.
  • Example 4 Induction of secondary metabolites by insertion of regulatory sequences in target BGC.
  • the use of small regulatory sequences like inducible promoters is particularly important in natural product research and for biosynthesis elucidation.
  • various inducible promoters described in Table 1 were tested, in particular those featuring an insulator (riboJ) and a strong ribosomal binding site (RBS) to switch on and off different genes and gene clusters.
  • indC which encodes a NRPS involved in the biosynthesis of the blue pigment indigoidine. This gene is silent under laboratory conditions but can be activated by the introduction of a foreign promoter.
  • the homology regions “selected” are positioned 500 bp upstream of the indC coding sequence (HR-L) and 500 bp downstream of the start codon (HR-R), while the distance between both can be variable (Figure 6A).
  • a sequence of the VA inducible promoter system (SEQ ID NO 131) was assembled between the homology arms during golden gate assembly ( Figure 6B-C).
  • the sequence between the homology regions was replaced by the promoter sequence comprising the repressor vanC in reverse direction with the related elements: PJ23100, RBS, insulator and a terminator, and the inducible PvanCC with an insulator and RBS element "in frame" with indC.
  • the colonies were tested by PCR and showed 100% editing (Figure 5). After induction of the promoter with vanillic acid, the typical blue pigment was observed (Figure 6D).
  • VA inducible promoter system (SEQ ID NO 131) assembled with HA-L and HA-R mxnB_L/R: SEQ ID NO 159-160 on pAR20 backbone.
  • the inventors revealed the previously identified mass of 526.2 m/z [M+H] + as the sodium adduct of the mass of 504.2 m/z [M+H] + .
  • the according editing plasmid was GAR-P04397WO09 Application (final)2.docx based on pAR20 with mxnK specific HA-L and HA-R (SEQ ID NOs 161-162). Production of madumycin II was performed in XPP3 medium with 2% (v/v) XAD-16 for in situ extraction.
  • Example 6 Activation and refactoring of the silent BGC xsc (safracin) In addition to "simple" and multiple promoter exchanges, complex BGC activations are also possible with the method developed herein.
  • TS4 is such an example due to its bidirectional architecture ( Figure 10A and 10B I). Safracins have been previously described and characterized in Pseudomonas fluorescens. Like the mxn BGC above, this xsc (Xenorhabdus safracin cluster) BGC is not active under laboratory conditions ( Figure 10B I). For activation, a strong inducible PvanCC promoter was introduced upstream of xscA and a strong constitutive promoter (proD) in the opposite direction upstream of xscI ( Figure 10B II) in Xenorhabdus sp. TS4.
  • SAC-B was deleted with simultaneous promoter exchange before xscK (Fig 10 B III).
  • xscK ⁇ xscH specific HA-L and HA-R SEQ ID NOs 169-170
  • SEQ ID NO 136 SEQ ID NO 136 as promoter sequence.
  • High SAC-B production is very valuable as SAC-B can be used as starting material for semisynthetic approaches to produce potent antitumor drugs (ET-743 and (-)-jorumycin, Figure 11A).
  • coli strain LZ84 harboring the xsc Cluster encoded on three expression plasmids (pCola_PBad_ xscA-H, pACYC_PBad_xscIJ, pCDF_PBad_xscK, Table 2).
  • Triplicates were made from both strains and cultivated in XPP3 production medium according to the previously described “Metabolite production, extraction and HPLC–MS/MS with absorbing Amberlite GAR-P04397WO09 Application (final)2.docx XAD-16 resin” method.
  • TS4 strains were cultivated at 28 °C and LZ84 strains at 22 °C.
  • Example 7 Activation and refactoring of the silent novel rpm BGC (rupshomycin)
  • rpm BGC trans-AT NRPS/PKS hybrid cluster
  • rpmA-rpmO trans-AT NRPS/PKS hybrid cluster
  • an inducible promoter (Pvan modified, SEQ ID NO 131, SEQ ID NO 171- 172) was inserted upstream of rpmA ( Figure 12 II), not resulting in production of a new compound. Thereafter, a promoter exchange with Pvan modified (SEQ ID NO 132, NO 173-174) was performed before rpmD ( Figure 12 III), also without success. As it was presumed the presence of another regulatory sequence between rpmC and rpmD, this sequence was replaced by ribozyme RiboJ51 and the T7g10 translational enhancer in the PvanCC_rpmA strain, generating mutant strain "IV" ( Figure 12 IV).
  • rpmMN and rpmO were cloned, individually into pAR30bad expression vectors (Primer sequenced can be found in Table 7a), followed by their conjugation into the ⁇ rpmM-O mutant, and analysis of the growth of the resulting strains "VIII” and "IX” in presence of 3,4-AHBA and/or IPTG, to induce rpm BGC ( Figure 12 VIII and IX, Figure 14 C and D). The influence of the vector was excluded by conjugating an empty vector into the strains as a control.
  • fraction F1 (10.8 mg) was further purified with analytical GAR-P04397WO09 Application (final)2.docx HPLC (3 mL/min, A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid, gradient: 25% of B for 2 min then 25% to 55% of B in 12 min) leading to the isolation of fraction F1F1 (0.6 mg) containing an E/Z-isomer mixture of 33 in ratio of 1:1.
  • 32 white amorphous solid
  • Example 9 High-throughput conjugation
  • the robot Rotor + system from Singer was used. Overnight cultures of donor strains were grown 10 ml LB containing 50 ⁇ g/ml Km and 50 ⁇ g/ml ALA acid in 96 well deep well plates. The culture was pelleted the next day and washed twice with ddH2O.
  • the pellet was resuspended in 100 ⁇ l LB and the suspension was spotted robotically in 7x7 patches to a LB agar plate containing 50 ⁇ g/ml ALA ( Figure 15B).
  • Figure 15B The same procedure was used for the recipient spotted on top of donor strains. After overnight incubation at 30 °C, patches were scraped through the Singer ROTOR+ benchtop robot and transferred to microtiter plates containing 100 ⁇ l LB with 25 ⁇ g/ml Km. After further incubation overnight, the suspension was spotted through the ROTOR+ onto LB agar containing 25 ⁇ g/ml Km. Colonies were visible after 1 - 2 days depending on the strain.
  • Example 11 Screening in high-throughput format of mutated bacteria This Example describes selection in high-throughput format ( Figure 15A), i.e.
  • the strains carrying gene-specific CRISPR/Cpf1 -pAR20 were inoculated in a 1 ml overnight culture in 96 deep well plates. The next day, the cultures were diluted 1:25 in 100 ⁇ l fresh LB containing 25 ⁇ g/ml Km in 96-well microtiter plates (e.g. Greiner Flat bottom plates). Growth was monitored by incubation in a Tecan microtiter plate reader (Tecan Spark).
  • the absorbance at 595 nm was measured every 15 minutes while the plate was incubated at 220 rpm at 30 °C between measurements. Once the absorbance doubled relative to the initial value, Cpf1 expression inducer AHT was added, the incubation temperature lowered to 25 °C and the plate was then incubated at 220 rpm. After another hour of incubation, L-arabinose was added and plates were incubated for another 3 hours. After the incubation period, two dilutions (1:4 and 1:20) of the original cultures were prepared in microtiter plates.
  • the activatable BGCs can be combined following the inventive methods for promoter exchange and refactoring (see Examples 5 - 7) in a single strain, leading to strains producing exclusively and only metabolites with desired bioactivities, like antibiotic, antifungal, nematicidal (or any other) activity ( Figure 18).
  • These bacterial strains can be used to generate extracts or compound mixtures with superior bioactivity than the individual metabolites alone and can be applied in medicine, animal health and agriculture.
  • Such a multi-producer strain (or an extract derived thereof) has the advantage of being easier to handle compared to culture of several mono-producing strains or handling of many extracts of individual compounds to be combined.
  • Example 14 Activation of the silent BGC dpr (deoxy-puromycin) in Xenorhabdus nematophila
  • a promoter exchange was performed in a global regulator cyaA and or hfq deficient mutant of Xenrohabdus nematophila upstream of the silent BGC dpr ( Figure 20A).
  • Deletion of the GRs were done with the pAR20 system, with the: ⁇ hfq specific HA-L and R having SEQ ID NOs 181-182 or ⁇ cyaA specific HA-L and R having SEQ ID NO 183.184.
  • the activation of the dprA BGC by insertion of the Pvan promoter (SEQ ID NO 131) upstream of dprA was done together with the dprA specific HA-L and R having SEQ ID NO 185 - 186, also based on the pAR20 editing system.
  • M1 was not produced.
  • cultivation was carried out in 13 C and 15 N medium (ISOGRO®-13C Powder - Growth Medium; ISOGRO®-15N Powder -Growth Medium).
  • the MS/MS spectra are shown in Figure 20C.
  • mass shifts could be assigned to several mass signals.
  • the altered masses result from the incorporation of heavy 13 C/ 15 N isotopes into the respective molecule, whereby the magnitude of the shift correlates with the number of isotopes incorporated.
  • the molecular formula of M1 was determined as C21H27N7O4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé de recherche de régulateurs globaux afin d'induire ou d'augmenter la production de métabolites secondaires cibles. Le procédé comprend la fourniture d'une souche d'indicateur de bactérie produisant deux signaux rapporteurs fluorescents sous la même régulation de deux regroupements de gènes biosynthétiques (BGC) différents, de préférence un BGC hautement exprimé, la réalisation d'une mutagenèse aléatoire dans ladite souche d'indicateur de bactérie, et la sélection de bactéries mutantes ne produisant pas lesdits signaux rapporteurs. L'invention concerne également un procédé permettant d'activer la production de métabolites secondaires cibles, et un plasmide unique à base de CRISPR/Cas permettant de supprimer ou d'inactiver un régulateur global et/ou d'activer un BGC. Enfin, le procédé selon l'invention peut être mis en œuvre dans un format haut débit permettant d'accélérer la procédure.
PCT/EP2024/065835 2023-06-08 2024-06-07 Modulation de mécanismes régulateurs globaux et de groupes de gènes biosynthétiques pour la production simplifiée de produits naturels Pending WO2024252009A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23178208 2023-06-08
EP23178208.7 2023-06-08

Publications (1)

Publication Number Publication Date
WO2024252009A1 true WO2024252009A1 (fr) 2024-12-12

Family

ID=86764727

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/065835 Pending WO2024252009A1 (fr) 2023-06-08 2024-06-07 Modulation de mécanismes régulateurs globaux et de groupes de gènes biosynthétiques pour la production simplifiée de produits naturels

Country Status (1)

Country Link
WO (1) WO2024252009A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025132327A1 (fr) * 2023-12-20 2025-06-26 Johannes Gutenberg-Universität Mainz Utilisation de composés d'extinction de quorum (qq) pour éviter la formation de biofilms, d'encrassement biologique ou de biocorrosion sur des surfaces

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2510069A1 (fr) 2002-12-20 2004-07-08 Pharma Mar, S.A. Famille de gene impliquee dans la biosynthese de la safracine et ses utilisations en genie genetique
CN103074395A (zh) 2011-10-26 2013-05-01 上海医药工业研究院 一种用于生产Safracin B的发酵培养基
WO2019192281A1 (fr) 2018-04-03 2019-10-10 浙江大学 Procédé d'extraction d'un promoteur fort du métabolisme secondaire basé sur un transcriptome de streptomyces, et son application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2510069A1 (fr) 2002-12-20 2004-07-08 Pharma Mar, S.A. Famille de gene impliquee dans la biosynthese de la safracine et ses utilisations en genie genetique
CN103074395A (zh) 2011-10-26 2013-05-01 上海医药工业研究院 一种用于生产Safracin B的发酵培养基
WO2019192281A1 (fr) 2018-04-03 2019-10-10 浙江大学 Procédé d'extraction d'un promoteur fort du métabolisme secondaire basé sur un transcriptome de streptomyces, et son application

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. EU329004.1
"Genbank", Database accession no. MT344942
AO X ET AL., FRONT. MICROBIOL, vol. 9, 2018, pages 2307
HOGAN AM ET AL., APPL ENVIRON MICROBIOL, vol. 87, 2021
JIA YIN ET AL., NUCLEIC ACIDS RESEARCH, vol. 43, no. 6, 2015, pages e36
JINGJING ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 292, no. 48, 2017, pages 19708 - 20
KONDOYUMURA, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 103, 2019, pages 3501 - 3510
LI SICONG ET AL: "Exploration of Hygromycin B Biosynthesis Utilizing CRISPR-Cas9-Associated Base Editing", ACS CHEMICAL BIOLOGY, vol. 15, no. 6, 10 April 2020 (2020-04-10), pages 1417 - 1423, XP093191868, ISSN: 1554-8929, DOI: 10.1021/acschembio.0c00071 *
LIU ZHENYU ET AL: "Recent Advances in Silent Gene Cluster Activation in Streptomyces", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, vol. 9, 18 February 2021 (2021-02-18), XP055856266, DOI: 10.3389/fbioe.2021.632230 *
LOPATNIUK MARIA ET AL: "Effect of "ribosome engineering" on the transcription level and production ofS. albusindigenous secondary metabolites", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 103, no. 17, 19 July 2019 (2019-07-19), pages 7097 - 7110, XP036859180, ISSN: 0175-7598, [retrieved on 20190719], DOI: 10.1007/S00253-019-10005-Y *
LOU ET AL., NAT BIOTECHNOL, vol. 30, no. 11, 2012, pages 1137 - 42
MEYER AJ, NATURE CHEMICAL BIOLOGY, vol. 15, 2019, pages 196 - 204
NAT CHEM BIOL, vol. 15, no. 2, 2019, pages 196 - 204
NAT. CHEM., vol. 14, 2022, pages 701 - 712
PARK JIMIN ET AL., ACS SYNTHETIC BIOLOGY, vol. 10, no. 8, 2021, pages 1859 - 73
RUIJUAN LI ET AL., MICROB. BIOTECHNOL., vol. 14, no. 4, 2021, pages 1809 - 1826
SHI YUE ET AL: "Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527", BIOTECHNOLOGY AND BIOENGINEERING, vol. 120, no. 5, 18 February 2023 (2023-02-18), Hoboken, USA, pages 1411 - 1422, XP093191811, ISSN: 0006-3592, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1002/bit.28347> DOI: 10.1002/bit.28347 *
SHIMADA T ET AL.: "The Whole Set of Constitutive Promoters Recognized by RNA Polymerase RpoD Holoenzyme of Escherichia coli", PLOS ONE, vol. 9, no. 3, 2014, pages e90447, XP055755657
XIANG ET AL., COMMUNICATIONS BIOLOGY, vol. 5, no. 1, 2022, pages 901
XIAO ET AL., ACS SYNTH. BIOL., vol. 9, pages 1051 - 1058
XU JINGJING ET AL: "Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes", vol. 292, no. 48, 1 December 2017 (2017-12-01), US, pages 19708 - 19720, XP093096386, ISSN: 0021-9258, Retrieved from the Internet <URL:http://dx.doi.org/10.1074/jbc.M117.809145> DOI: 10.1074/jbc.M117.809145 *
YIN ET AL., ISCIENCE, vol. 14, 26 April 2019 (2019-04-26), pages 1 - 14
YIN ET AL., PHOTORHABDUS LUMINESCENS LAMBDA RED-LIKE OPERON, 2019

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025132327A1 (fr) * 2023-12-20 2025-06-26 Johannes Gutenberg-Universität Mainz Utilisation de composés d'extinction de quorum (qq) pour éviter la formation de biofilms, d'encrassement biologique ou de biocorrosion sur des surfaces

Similar Documents

Publication Publication Date Title
Liu et al. Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria
Thomason et al. Multicopy plasmid modification with phage λ Red recombineering
US10961513B2 (en) Methods and compositions for recombinase-based genetic diversification
JP2001520055A (ja) 微生物ゲノム、それに関する組成物、および方法
Bian et al. Rational and efficient site‐directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo
Wang et al. Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria
Welker et al. Cyanobacteria as a source of natural products
WO2024252009A1 (fr) Modulation de mécanismes régulateurs globaux et de groupes de gènes biosynthétiques pour la production simplifiée de produits naturels
Bode et al. easyPACId, a simple method for Induced Production, isolation, identification, and testing of Natural products from Proteobacteria
Rill et al. Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling
US20080206803A1 (en) Metabolic genes and related methods and compositions
Zheng et al. Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva
Reichmuth et al. Dibenzothiophene biodesulfurization pathway improvement using diagnostic GFP fusions
WO2023065725A1 (fr) Procédé d&#39;édition multiplex du génome bactérien fondé sur la technique de recombinaison de l&#39;adn double brin et son application
KR102359972B1 (ko) 화합물의 제조를 위한 조성물 및 방법
CN119120564A (zh) 一种黑暗诱导的增强型发光表达载体及其构建方法和应用
Weihmann et al. The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida
US20200102557A1 (en) Modulation of gene expression
Huo et al. Identifying amino acid overproducers using rare-codon-rich markers
Victoria et al. Engineering the highly productive cyanobacterium Synechococcus sp. PCC 11901
US20090298117A1 (en) Methods for heterologous expression of secondary metabolites
Karbalaei-Heidari et al. Genomically integrated orthogonal translation in Escherichia coli, a new synthetic auxotrophic chassis with altered genetic code, genetic firewall, and enhanced protein expression
Nalley et al. Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans
WO2003064623A2 (fr) Methodes et vecteurs permettant de faciliter la recombinaison a un site specifique
KR20230100688A (ko) CRISPRi 시스템 기반 표적 유전자 스크리닝 방법 및 이의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24732249

Country of ref document: EP

Kind code of ref document: A1