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WO2017010805A1 - Système basé sur un circuit génétique de prototypage de micro-organismes pour la recherche de nouvelles ressources de micro-organismes - Google Patents

Système basé sur un circuit génétique de prototypage de micro-organismes pour la recherche de nouvelles ressources de micro-organismes Download PDF

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WO2017010805A1
WO2017010805A1 PCT/KR2016/007613 KR2016007613W WO2017010805A1 WO 2017010805 A1 WO2017010805 A1 WO 2017010805A1 KR 2016007613 W KR2016007613 W KR 2016007613W WO 2017010805 A1 WO2017010805 A1 WO 2017010805A1
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Prior art keywords
phenol
gene
promoter
activity
sensor cell
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Korean (ko)
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이승구
나유진
김하성
성원재
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Priority claimed from KR1020160088411A external-priority patent/KR101937180B1/ko
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention is a substrate containing a "phenolic compound” that can be used to detect intracellular enzyme activity, a gene encoding a transcriptional regulator that is expressed by detecting a phenolic compound and a lower reporter gene operated by the transcriptional regulator Gene circuit comprising, sensor cell comprising the gene circuit, and new microbial strains having the activity of the target enzyme using the sensor cell co-cultured with the sensor cell to directly detect and search for the activity It is about how to.
  • sequence-based search technology is performed to identify DNA sequences, perform PCR reactions, and obtain amplified genes. screening).
  • genomic information is rapidly increasing, its utility is increasing day by day, and there is an advantage that only the desired gene can be specifically selected, but it can be used only when the exact information of the target gene is known.
  • the applicable subject is limited to a part of the genetic source.
  • activity-based screening techniques for selecting genes based on gene function, ie, enzyme activity
  • the method of separating microorganisms directly from environmental samples such as soil, river, factory wastewater, seawater and forest has been mainly used. Less than 1%, only a very small amount.
  • a strategy has been actively attempted to construct genetic resources in the form of metagenome libraries by separating DNA directly from environmental samples without culturing microorganisms. Accordingly, there is also a great interest in the development of screening techniques to directly detect industrially useful enzyme activity in the metagenome library.
  • High-speed assay techniques for enzymatic activity include (1) automated multi-analysis techniques using well-plates, (2) observing color or halo in solid media, and (3) deficient microorganisms.
  • the selective separation method used is mainly used. These methods have the advantage of clearly selecting the genes of the desired function because they are based on the actual activity of the enzyme, but there is a limitation in the versatility of the technology because it requires one detection technology to match the individual enzyme activity. In addition, the effect is further reduced when there is a problem such as low transcription, translation, or expression of foreign genes in the host cell or protein folding, secretion.
  • the present inventors have developed a gene circuit composed of a transcriptional regulator in which its expression is induced by phenol and a lower reporter gene actuated by the transcriptional regulator and a sensor cell comprising the same.
  • a transcriptional regulator in which its expression is induced by phenol and a lower reporter gene actuated by the transcriptional regulator and a sensor cell comprising the same.
  • the present invention provides a substrate comprising a "phenolic compound” that can be used to detect intracellular enzymatic activity, a gene encoding a transcriptional regulator that is expressed by detecting a phenolic compound, and a lower reporter gene operated by the transcriptional regulator.
  • the present invention is (1) the gene expression control region, the first promoter whose activity is regulated by the gene expression control region, and the expression is regulated by the activation of the first promoter, fluorescent protein
  • a first gene construct comprising; a reporter gene comprising a gene encoding a gene and a gene encoding an enzyme involved in nutritional requirements (auxotrophic);
  • a gene of a transcriptional regulator that regulates expression by the second promoter and the second promoter and binds to the gene expression control site only in the presence of a phenolic compound to activate the second promoter. It provides a genetic circuit for detecting the presence of a phenolic compound, including; a second gene construct.
  • the present invention also provides a sensor cell comprising the gene circuit.
  • the present invention provides a method for detecting and searching for a novel microbial strain retaining the activity of the target enzyme using a substrate comprising the "phenolic compound" that can be used for detecting the sensor cell and intracellular enzyme activity. do.
  • the present invention is a.
  • a reporter gene comprising a gene encoding a; a first gene construct comprising;
  • the present invention relates to a technology (MP-GESS: Microbe prototyping based genetic enzyme screening system) to easily identify and isolate a novel microbial resource retaining the target enzyme activity in nature using a genetic circuit.
  • MP-GESS Microbe prototyping based genetic enzyme screening system
  • phenolic compound is a substrate that can be used to detect the enzyme activity in the cell, phenol, 2-chlorophenol, 2-iodine phenol, 2-fluorophenol, o-cresol, 2-ethylphenol, m-cresol, 2-nitrophenol, catechol, 2-methoxyphenol, 2-aminophenol, 2,3-dichlorophenol, 3-chlorophenol, 2,3-dimethylphenol, 3-nitrophenol, 4-chloro Phenol, p-cresol, 2,5-dichlorophenol, 2,5-dimethylphenol and the like. Substrates containing such "phenolic compounds” are also referred to as "phenol-tag substrates.”
  • the reporter gene and the first promoter for regulating the expression of the reporter gene are operably linked.
  • the site where the transcriptional regulator binds to induce the expression of the downstream reporter gene activates the first promoter of the reporter gene such that the transcriptional regulator binds to express the downstream reporter gene. It is done.
  • a gene encoding a transcriptional regulator that recognizes the phenolic compound and induces expression of a downstream reporter protein and a second promoter that regulates the expression of the transcriptional regulator are operably linked. It features.
  • the "transcription regulator” is a protein that preferably regulates the expression of the degradation enzyme of the substrate including the phenolic compound, and operates a promoter that controls the expression of the phenolic compound degradation enzyme by detecting phenol. And a protein inducing expression of a reporter linked to such a promoter.
  • genes that show the degradation activity of aromatic organic compounds such as phenol, xylene, toluene, and benzene are mainly found in the genus Pseudomonas and Acinetobacter, and these genes are multifunctional with multigene expression. It is composed of operon and expressed by ⁇ 54 dependent transcriptional regulation.
  • Representative transcriptional regulators include DmpR, DmpR variant, XylR, MopR, PhhR, PhlR, TbuT, etc.
  • DmpR which is involved in phenol degradation metabolism in Pseudomonas putida
  • XylR which is involved in metabolism of toluene and xylene
  • DmpR or XylR to detect toluene, xylene or phenol contaminated with the natural environment has been studied in the concept of microbial biosensor.
  • This NtrC family expression regulator is a combination of a domain that recognizes activators such as phenol and xylene (A domain), a domain that has ATPase activity (C domain), and a domain that binds to DNA (D domain). It is composed. Therefore, in the absence of a phenol molecule, the A domain inhibits transcription. However, when the phenol molecule binds to inhibit the A domain, the transcriptional activation function of the C and D domains appears. Recently, studies on improving specificity through research using genetics and genetic engineering methods using specificity of A domain have been known.
  • the transcriptional regulator preferably used in the present invention may be dmpR, or a variant thereof, which is a regulatory protein of P. putida-derived phenol-degrading operon.
  • dmpR is the ⁇ 54 -dependent transcriptional activity regulatory portion of the dmp operon gene, which shows the degradation activity of aromatic organic compounds such as phenol, xylene, toluene and benzene.
  • the dmp operon derived from P. putida consists of 15 genes, among which dmpKLMNOP codes for the enzymes required for phenol hydroxylation, and dmpQBCDEFGHI codes for the metalytic pathway that degrades catechol intermediates.
  • dmpKLMNOP of operon expression is ⁇ 54-dependent transcription factor in dmpR is activated by binding to the operator of the dmp dmpK top, transcription factors for dmpR itself is known as ⁇ 70 dependent.
  • the variant of dmpR is, for example, E135K, where the 135th E of the dmpR amino acid sequence is K-mutated, in addition to E172K, D135N, D135N and E172K, F65L, L184I, F42Y, R109C, L113V, D116N, F122L, K6E And one or more of F42S, Q10R and K117M, Q10R, D116G and K117R, D116V may be a variant in which amino acid residues are mutated, but is not limited thereto, and the variant may have a high affinity for a phenolic compound, thus Various variants, including single or multiple mutations at the periphery, as well as variants of various dmpRs
  • the "gene expression control site” is a site that controls the entire gene circuit.
  • the transcriptional regulator inhibits transcription, but when the phenolic molecule binds to inhibit the A domain, transcriptional activation function by C and D domains appears to bind to the OpR (Operator for Reporter) site. Which is modulated by ⁇ 54 dependency.
  • promoter means a promoter that regulates the expression of a transcriptional regulator or a promoter that controls the expression of a reporter protein.
  • a promoter of dmpR or dmp operon of Pseudomonas or a promoter for general protein expression may be High expression promoters, including trc, T7, lac and ara promoters, may be used for high expression of foreign proteins.
  • Phce which is a high expression vector that does not require an inducer, may be used.
  • ⁇ 54 -dependent promoter (PECO) of E. coli may be used as a promoter for regulating the expression of the reporter protein.
  • PPU Pseudomonas putida
  • yeast PYST
  • the like depending on the host of MP-GESS.
  • the gene circuit may include not only the promoter, but preferably, a ribosome binding site (RBS) and / or a transcription terminator that facilitates expression of the reporter protein. That is, as a site for controlling the expression of the regulatory protein, in addition to the promoter may include RBS and / or transcription terminator.
  • RBS ribosome binding site
  • protein expression starts with AUG (methionine) or GUG (valine), which is an initiation codon in mRNA, and the distinction between AUG or GUG where the ribosome is located at a residue inside the protein and AUG and GUG as protein initiation codon is the purine base of DNA.
  • AUG or GUG where the ribosome is located at a residue inside the protein and AUG and GUG as protein initiation codon is the purine base of DNA.
  • RBS or Shine-Dalgarno (SD) sequence
  • SD Shine-Dalgarno
  • the gene circuit constructed in the present invention uses a transcriptional regulator of Pseudomonas, which is different from E. coli, which is a host of the gene circuit, in the case of ⁇ 54 dependent gene expression.
  • ⁇ 54 binding sites and ⁇ 54 factors themselves are very different from E. coli, they are used in combination with Pseudomonas RBS (RBSPPU), or E. coli RBS (RBS ⁇ ), or all strains to facilitate the expression of reporter proteins in host E. coli.
  • Possible RBS (RBSx) can be used.
  • the transcription terminator may preferably be rrnBT1T2 or tL3, and in addition to this, the transcription terminator may be used to construct the present invention using any transcription terminator commonly used in the art.
  • the reporter protein is composed of a fluorescent protein and an enzyme involved in trophic composition.
  • the fluorescent protein preferably, GFP, GFP UV , or RFP can be used, but as long as the object of the present invention can be achieved, the fluorescent protein that can be used in the present invention is not limited to these examples.
  • the enzymes involved in the nutritional constituents lose the ability of microorganisms to synthesize certain amino acids, nucleotide groups, vitamins, etc., due to mutations, and thus cannot grow in the medium, and thus, the components that cannot be synthesized as nutrients necessary for growth.
  • the required auxotroph represents an enzyme that allows the synthesis of the deficient substance.
  • enzymes involved in nutrient composition include but not limited to DAAT (D-amino acid aminotransferase) enzyme, which allows the glutamic acid nutrient component microorganism to survive in an environment deficient in glutamic acid. It doesn't happen.
  • DAAT D-amino acid aminotransferase
  • the reporter may be a dual reporter (dual reporter) consisting of both fluorescent proteins and enzymes involved in nutrition, and multiple reporters consisting of two or more reporters.
  • dual reporter dual reporter
  • any known method may be used for the sequential transformation of metagenome libraries and phenol sensing redesign gene circuits into suitable microbial hosts.
  • electroporation may be preferably used.
  • the present invention also provides a sensor cell comprising the gene circuit.
  • the genetic circuit may be provided in the form of a vector or a microorganism containing the vector.
  • the cell is preferably, but is not limited to any bacteria, such as E. coli, fungi, such as yeast, plant cells, animal cells and the like.
  • the Escherichia coli may be a trophyotrophic host cell, and preferably, glutamic acid trophyotrophic host cell, but is not limited thereto.
  • the present invention also provides a method for detecting and searching for a novel microbial strain retaining the activity of the target enzyme using the sensor cell.
  • the method is a
  • Step (2) may be co-cultured with the natural microbial strain or the environmental sample comprising the same is expected to have the target enzyme and mixed with the phenol-tag substrate and the sensor cell.
  • step (2) comprises: forming a colony by culturing in an environment in which the phenol-tag substrate is treated in a natural microorganism or an environmental sample comprising the same, which is expected to possess the target enzyme; And co-culturing the formed colonies and the sensor cells together.
  • the sensor cells can be co-cultured by spraying the sensor cell culture onto colonies formed from natural microorganisms or environmental samples comprising the same.
  • the sensor cells can be used immediately after thawing without storage after storage at low temperature.
  • the colony sensor cells after forming a colony by forming a colony on a culture medium containing tyrosine as a substrate, E. coli or Citrobacter prepundi or TPL, the colony sensor cells are sprayed and cultured, the sheet Fluorescence expressed by sensor cells in the vicinity of Lobacter Freundy or TPL producing E. coli was confirmed (FIG. 6D).
  • the nutrient may be glutamic acid, but is not limited thereto.
  • the phenol-tag substrate is designed according to the activity of the target enzyme in step (1).
  • the phenol-tag substrate includes a binding site that can be degraded by the activity of the enzyme of interest and phenol bound through the binding site.
  • the phenol-tag substrate by specifically designing the phenol-tag substrate to be suitable for the activity of the target enzyme, that is, by designing the binding site or functional group or the like in accordance with the activity of the enzyme to be searched, the microorganism which simply produces a phenolic compound.
  • the senor comprising the gene circuit in an environment in which a specific nutrient is deficient by treating the phenol-tag substrate to a natural microbial strain or an environmental sample including the same, which is expected to have a target enzyme in step (2). Incubate with cells. Since the genetic circuit introduced into the sensor cell contains the DAAT gene, even if the sensor cell is placed in an environment that is a trophogenic strain and lacks nutritional substances, the DAAT gene is expressed in the introduced genetic circuit. If you can, you can survive.
  • step (3) the sensor cell in which the expression of the reporter protein is induced by the phenolic compound produced by the activity of the target enzyme is identified and selected. If the target enzyme is present in a natural microorganism strain or an environmental sample containing the same, a phenolic compound will be produced by the activity of the target enzyme when the phenol-tag substrate designed as described above is treated, and the phenolic compound thus produced. Since the compound will induce the expression of the reporter protein in the sensor cell in the vicinity of the natural microorganism strain or the environmental sample containing the same, it is possible to search for a novel strain having the target enzyme by confirming the expression of the reporter protein as described above. .
  • step (4) the microbial strain adjacent to the sensor cell from which the expression of the reporter protein selected as described above is induced is isolated.
  • the microbial strain isolated as described above has a target enzyme.
  • the isolated microbial strain can be verified once again whether there is actually the activity of the target enzyme, 16s rRNA analysis can be identified which bacteria to the isolated microbial strain.
  • the timing of substrate addition can be controlled to separate the activation stages of the cell growth-redesigned genetic circuit in order to optimize the enzymatic reaction.
  • Compounds capable of producing phenolic compounds by enzymatic reactions include esters (-, -OOC-) and ethers (ether, -OC-) modified with phenolic hydroxyl groups (hydroxy, -OH).
  • Glycoside (-O-Glc), phospho-ester (-O-PO3), ortho-, meta-, para- position alkyl (-CH 3 ), hydroxyl (-OH), car Compound (-COOH), amino (-NH 2 ), thiol (-SH), amide (amide, -NH-CO- or -CO-NH-), sulfide (-S-SH), halogen group (- Phenol derivatives or benzene ring compounds in which Cl, -Br, and -F) are introduced, but are not limited thereto.
  • ester compound ester, -OOC-
  • ether compound ether, -OC-
  • glycoside compound glycoside, -O
  • esterase lipase
  • glycosidase glycosidase
  • phosphatase pita It can be used for the purpose of detecting phytase activity.
  • the phenol-tag substrate is a new methyl group (-CH 3 ), hydroxyl group (-OH), carboxyl (-COOH), amino group (-NH 2 ), hydrogen sulfide in one position of ortho-, meta-, para- It may be a material prepared by introducing (-SH).
  • the phenolic compound linked through the amide group is amidase, pep It can be used for the purpose of detecting peptidase activity, and by using a phenol compound linked through a sulfide group, it can be used for the purpose of detecting intracellular redox levels.
  • a phenol compound linked to a new carbon bond ie, Ph-C- (R)
  • Ph-C- (R) Ph-C- (R)
  • Benzene ring material may be used as the phenol-tag substrate, and in this case, oxidase activity such as monooxygenase and dioxygenase, which are involved in the oxidation of aromatic compounds, may be detected.
  • Phenolic compounds bound to halogen such as chlorine (Cl), bromine (Br) and fluorine (F) may also be used as the substrate.
  • halogen such as chlorine (Cl), bromine (Br) and fluorine (F)
  • enzymatic activity of dehalogenation or isomerization that changes the position of halogen by acting on the above halogenated phenolic compound can be detected according to the present invention.
  • the activity of transferases that transfer covalent bonds to other organic molecules or syntheses that generate new covalent bonds can also be detected by the present invention. Therefore, using the various phenol-tag substrates presented in the present invention, specific hydrolase, oxido-reductase, isomerase, lyase, transfer Enzymes can be detected.
  • the substrate used for detecting the enzyme activity in the cell is a phenol-tag substrate, and represents various synthetic substrates including phenol, o / p-nitrophenol, o / p-chlorophenol and the like.
  • the phenolic material is produced from the original phenol-tag substrate. For example, when E. coli ⁇ -galactosidase (lacZ) acts on phenyl- ⁇ -glucoside, a phenol component is produced according to the enzyme activity.
  • an enzyme gene when introduced into a sensor cell containing a gene circuit for detecting a phenolic compound, and a phenol-tag substrate is treated to a microbial strain retaining the activity of a target enzyme,
  • concentration of the phenolic compound changes depending on the function and activity of the intracellular enzyme gene. Therefore, it is possible to easily identify microbial strains possessing the desired activity of the desired enzyme through the degree of reaction to fluorescence and nutrient composition by the expression-inducing function of the phenolic compound.
  • the fluorescent protein used as a reporter in the present invention and enzymes involved in nutritional urine composition can be applied not only to a high sensitivity measurement method, but also expressed in the cells because they are limited to specific cells without passing through the cell membrane. Individual characteristics of the foreign genes will be exercised. Therefore, since individual single cells serve as independent reactors and analyzers, a large amount of millions to tens of millions as a means of detecting the activity of expression-induced reporters by detecting phenolic compounds produced by enzymatic reactions. Samples may also be measured using a fluorescence flow cytometer (FACS), microcolony fluorescence image analysis, fluorescence spectrum analysis, mass screening using nutritional selection media.
  • FACS fluorescence flow cytometer
  • the gene circuit of the present invention and the sensor cell including the same include a fluorescent protein as a reporter gene together with related enzymes in nutritional components for controlling the survival of the sensor cell, the novel sensor having the activity of the sensor cell and the desired target enzyme
  • a fluorescent protein as a reporter gene together with related enzymes in nutritional components for controlling the survival of the sensor cell
  • the novel sensor having the activity of the sensor cell and the desired target enzyme
  • FIG. 1 is a schematic diagram showing the main configuration of the microbial prototyping-based enzyme gene screening system (MP-GESS) of the present invention.
  • MP-GESS microbial prototyping-based enzyme gene screening system
  • FIG. 2 is a schematic diagram showing an operation of the MP-GESS construct of the present invention.
  • Figure 3 is a graph showing the growth and fluorescence by phenol of the trophyotrophic host comprising the MP-GESS construct of the present invention.
  • FIG. 4A shows a system in which a phenolic compound inducing dmpR activation operates a GFP-coding gene or an antibiotic resistance gene
  • FIG. 4A (B) shows that a target enzyme in a cell produces phenol from a substrate molecule, DmpR is activated by phenol and indicates the expression of downstream GFP coding genes
  • 4B is a diagram illustrating the introduction of a D-AAT coding gene at the N-terminus of EGFP and transformation of this system with WM335, a D-glutamate trophic host, and FIG. 4B (B). Screening system based on cell-cell communication. Phenolic molecules are used not only as enzyme activity markers but also as signal transmitters, and phenol activates the transcription factor (dmpR) to induce the expression of D-AAT and GFP-coding genes.
  • dmpR transcription factor
  • 5 is a graph showing the extent of proliferation of transformants when antibiotic resistance genes or trophyotrophic genes are introduced into the phenotypic reporter.
  • Figure 6a shows a chemical reaction that shows the production of phenol, pyruvate and ammonia by the degradation of L-tyrosine by tyrosine-phenol lyase (TPL) enzyme.
  • Figure 6b shows two methods for detecting microorganisms with target enzyme activity using the sensor cells of the present invention.
  • 6c is a result of verifying activity of sensor cells confirmed based on a one-step protocol.
  • 6D is a result of verifying activity of sensor cells confirmed based on a two-step protocol.
  • Figure 7a shows a chemical reaction showing the production of p-nitrophenol and cellobiose by the degradation of pNPG2 by fibrinolytic enzymes
  • Figure 7b shows the process of searching for microorganisms having fibrinolytic activity in soil samples .
  • FIG. 8 is a photograph showing fluorescence images of four colonies isolated from soil from metagenome samples.
  • 9 is a graph showing fibrinolytic activity on various substrates of the seven candidate microorganisms selected.
  • Figure 11 shows the results of sequence similarity analysis with other strains by comparison of the whole strain of the ORF sequence.
  • FIG. 12 is a comparison of the functions of Pseudomonas fluorescence Pf0-1 strains found to be the most similar strains by sequence comparison analysis with new strains. It is a graph showing the top ten functions of the function (left) only in the strain (left) and the function (right) only in the Pseudomonas fluorescein Pf0-1 strain.
  • 13A and 13B are photographs showing images of Pseudomonas genus # 46-2 strains using a scanning electron microscope and a transmission electron microscope, respectively.
  • FIG. 14A shows the reaction of phenylphosphate to phenol and phosphate by phosphatase
  • FIG. 14B shows seven candidate microorganisms selected as sensor cells
  • FIG. 14C shows sensor cells as negative control.
  • FIG. 14D is a result of confirming the presence of sensor cells by PCR of 10 selected colonies of FIGS. 14B and 14C.
  • FIG. 15A shows green fluorescence and bright field images of 36 colonies selected using microscopic analysis with GFP filters after incubating soil samples and sensor cells using the two step protocol of Example 8, FIG. Specific enzyme activity of the candidate colonies in dogs was measured using their crude extracts.
  • Example 1 microbial prototyping-based enzyme gene screening system ( MP -GESS)
  • D-amino acid aminotransferase was added to the GESS gene construct using dmpR or dmpR variants, which are transcription regulators of Pseudomonas putida- derived phenol-degrading operons.
  • a dual reporter-type GESS construct was constructed using additional amino-acid aminotransferase (DAAT) gene. The DAAT gene is expressed in the presence of phenol.
  • DAAT additional amino-acid aminotransferase
  • glutamic acid was used auxotrophic E. coli (Escherichia coli, WM335) as a host cell of the MP-GESS construct.
  • FIG. 1 A schematic diagram of the MP-GESS configuration is shown in FIG. 1, and Table 1 shows the components of the MP-GESS and the characteristics of each configuration.
  • Transcription regulator dmpR or dmpR variants Pseudomonas Derived / Phenolic Compounds Reporter Protein DAAT, EGFP Host cell survival, fluorescence expression including MP-GESS Control part Electronic regulator coupling site Transcription Factor Binding / Reporter Expression Activation Promoter 1 Promoter for expression of transcriptional regulator (P X ) Transcription Factor Expression Promoter 2 Reporter Protein Expression Promoter (P R ) Reporter Protein Expression Ribosomal binding site RBS Promote reporter protein expression Transcription Terminator rrnBT1T2, tL3 (t) Warrior Termination Explode tag ssrA DAAT half-life adjustment
  • the operating aspect of the MP-GESS construct is shown in FIG. Specifically, the MP-GESS construct was introduced into glutamic acid trophic coliform to make sensor cells. Cultured sensor cells are cultured in a medium without glutamic acid, and if killed, cultivated with a phenol-producing strain and a phenol-degradable substrate is added, the phenol produced by the phenol-producing strain is used to control the MP-GESS. Since the DAAT gene of the truck expresses and the sensor cell survives, and the EGFP expresses and fluoresces, it is possible to search for a phenol-producing strain depending on the survival of the sensor cell.
  • the MP-GESS construct is the top of the N-terminus of pGESSv4 (ACS Synthetic Biology (2014) 3 (3): 163-167) or pGESS (E135K) (ACS Synthetic Biology (2014) 3 (3): 163-167) It can be produced by inserting the DAAT gene into.
  • the fragment of 6,185bp was amplified by PCR using pGESSv4 as a template and the following SEQ ID NO: 1 and SEQ ID NO: 2.
  • the DAAT gene was obtained by the following procedure. DNA of Bacillus subtilis 168 (Microorganism Resource Center, Korean Collection for Type Cultures) was used as a template, and after amplifying the PCR product of about 800 bp using the sequences of SEQ ID NO: 3 and SEQ ID NO: 4, The final 852 bp length PCR product was obtained by performing PCR once more using the 800 bp PCR product as a template and the sequences of SEQ ID NO: 5 and SEQ ID NO: 6 having homology with the vector pGESSv4.
  • the sequence of SEQ ID NO: 6 contains an ssrA tag (underlined) that controls the life of the DAAT.
  • the ssrA tag is a kind of destruction tag that can control the half-life of the protein when inserted into the C-terminus of the protein, and can control the half-life of the fluorescent protein in Escherichia coli or Pseudomonas.
  • SEQ ID NO: 7 AANDENYALAA amino acid sequence (ssrA tag; amino acid sequence corresponding to the underline of the sequence of SEQ ID NO: 6) by controlling the half-life of DAAT to reduce the non-specific response of MP-GESS.
  • the amplified and secured pGESSv4 vector and DAAT gene were cloned by Gibson assembly method (Gibson Assembly Master Mix, NEB, UK) to complete the production of MP-GESS construct.
  • the MP-GESS plasmid was transformed into E. coli WM335 strain.
  • Escherichia coli WM335 strain is a glutamic acid trophic strain and does not grow unless additional glutamic acid is added to the growth medium (Biosci Biotechnol Biochem. 1998 Jan; 62 (1): 193-5).
  • a single colony of Escherichia coli WM335 strain containing MP-GESS construct was added to LB medium and 0.1 mg / ml of D-glutamic acid, followed by shaking culture at 37 ° C. for 24 hours to be used as a seed.
  • M9 liquid medium ((1l Na 2 HPO 4 ⁇ 7H 2 O 6.78g, KH 2 PO 4 3g, NaCl 0.5g, NH 4 Cl 1g, 2mM MgSO 4 , 0.1 mM CaCl 2 , 0.4% (w / v) Glucose, 0.01% (w / v) thiamine is added to the suspension) to determine whether the reaction to phenol.
  • the glutamic acid trophic host cell into which the MP-GESS construct was introduced did not grow when glutamic acid was absent, but it was confirmed that fluorescence was expressed while growing only when the DAAT gene was expressed by phenol (FIG. 3). From the above results, it can be seen that glutamic acid trophic constituent host cells into which the MP-GESS construct of the present invention is introduced, that is, sensor cells, are viable by phenol.
  • Cm- (chloramphenicol) and Tc- (tetracycline) resistance genes were amplified by PCR from pACYC184 (New England Biolabs, Ipswitch, MA, USA), and the Km- (kanamycin) resistance gene was pET27b (EMD Millipore, Darmstadt, Germany). Amplified by PCR.
  • PGESSv4 a DmpR-based GESS plasmid (ACS Synthetic Biology (2014) 3 (3): 163-167), was used as a template, and SEQ ID NO: 10 (Vector-F1: aaggagatatacatatggtgagcaagggcg) and SEQ ID NO: 11 (Vector-R1: atgtatatctccttctccaggttggcggat) PCR was performed using the primers to amplify 6,185 bp fragments.
  • PGESS-Cm, pGESS-Tc, and pGESS-Km were introduced into the N-terminus of EGFP in the amplified antibiotic resistance gene using the same cloning method as in pGESS-DAAT of Example ⁇ 1-1>. Construct was constructed. Next, DH5 ⁇ Escherichia coli cells containing each plasmid were incubated overnight at 37 ° C., and the preculture was diluted 10 6 fold, with different combinations of phenol (1-1000 ⁇ M) and antibiotics (0, 10, 20). , And 30 ⁇ g / mL), and spread spread to LB-plate selection medium. pGESS-Tc was investigated for the detection of tyrosine phenolases.
  • DH5 ⁇ Escherichia coli cells comprising pGESS-Tc transformed with pHCEIIB-TPL were placed on LB solid medium containing 1 mM tyrosine, 10 ⁇ M PLP, 30 ⁇ g / mL tetracycline and incubated at 30 ° C. for 48 hours.
  • the cells with pGESS-Cm and pGESS-Tc increased the survival in proportion to the phenol concentration of the medium, showing very little viability in the absence of phenol (Fig. 5 (A), (B)) .
  • Cells with pGESS-Cm showed similar profiles as cells with pGESS-Tc, whereas cells transformed with pGESS-Km showed the lowest viability among the three tested strains (FIG. 5).
  • pGESS-Tc higher fluorescence intensity of colonies was observed at increased phenol concentrations.
  • Cells with pGESS-DAAT increased the number of colonies with increasing phenol concentration.
  • antibiotic resistance genes can be used to screen for microorganisms with specific enzymatic activity, but by adding antibiotic resistance genes as an additional reporter with green fluorescent protein, any metagenomic gene with antibiotic resistance activity can be expressed in sensor cell colonies. It can lead to formation, which can lead to false positive rates.
  • E. coli host-based metagenome screening it is not possible to grow natural microorganisms on plates containing antibiotics. Therefore, in order to screen microorganisms having a specific enzymatic activity in the metagenome microorganisms, it is more efficient to introduce a trophic constitutive gene as in Example ⁇ 1-2> rather than to use an antibiotic resistance gene. Additional experiments were performed using MP-GESS with genes encoding AAT and fluorescent reporters.
  • TPL tyrosin-phenol lyase
  • the MP-GESS vector is as shown in Example ⁇ 1-2> Introduced glutamic trophyotrophic Escherichia coli cells, ie, sensor cells, were cultured and cultured with 0.75% (w / v) agar solution, and placed in a solid medium in which TPL producing cells were grown. After culturing for 12 hours, survival and fluorescence expression of sensor cells into which MP-GESS constructs were introduced by fluorescence microscopy were observed (one-step protocol of FIG. 6B).
  • the treatment time of the sensor cells was the same as in Example ⁇ 2-1> except that the colony was formed after 12 hours of incubation with TPL-producing Citrobacter preoundi cells or TPL-producing E. coli (Fig. 6b two-step protocol).
  • Example 3> One-step protocol based microbial discovery: microbial discovery with fibrinolytic activity in soil samples Example ⁇ 1- 2> of Coculture with sensor cells and microorganisms to be searched)
  • Example 3 Whether or not the seven candidate microorganisms selected in Example 3 has fibrinolytic activity was confirmed using various substrates. After culturing the selected candidate microorganisms at 30 °C, and treated with celLyticB, lysozyme, DNase, respectively, the lysate was used as coenzyme solution. After reacting pNPG2 with pNPG3 (para-nitrophenyl cellotrioside) as a substrate for 2 hours at 45 ° C, the degraded pNP (para-nitrophenol) was measured at 405 nm at an optical density (Victor).
  • pNPG2 para-nitrophenyl cellotrioside
  • the negative control group was prepared by adding a substrate to the buffer without coenzyme solution, using no substrate (None) and nothing (NC) in each candidate microbial sample, and 0.02% c-tech as a positive control group.
  • PC no substrate
  • NC nothing
  • filter paper (1 ⁇ 3 cm), 1% (w / v) Avicel and 2% (w / v) CMC were also used as substrates.
  • Each reaction was carried out at 45 °C, after the enzyme reaction the sample was measured for activity by the DNS reducing sugar quantification method.
  • pNPG2, Avicel, and filter paper are substrates of exo fibrinase
  • pNPG3 are substrates of endo type fibrinase.
  • the # 25-1 and # 46-2 strains reacted with Avicel, and the activities against pNPG2 and pNPG3 were confirmed in samples except for the # 34-1 and # 46-1 strains.
  • the strain # 46-2 had a high activity on the filter paper, and because of this property may be useful for crystalline fiber breakdown (Fig. 9). From the above results, it was confirmed that the strain of the fibrinase activity can be searched by the E. coli WM335 strain containing MP-GESS sensor cell of the present invention.
  • Example 5 Example Identification of species of candidate microorganisms selected in 3
  • 16s rRNA analysis was performed to identify the microbial species of colonies selected in Example 4.
  • 16s rRNA sequencing was performed on 6 microbial resources except # 34-1 which were not cultured.
  • Table 2 shows the similarity results between each candidate microorganism and previously known microorganisms.
  • # 25-1 showed more than 97% similarity to Bacillus cereus ATCC14579 (T), and # 34-2 and # 46-1 showed more than 99% similarity to Pseudomonas thringiensis ATCC10792 It was analyzed.
  • # 46-2 which has a relatively high activity on the filter paper, it was analyzed to be 93% similar to Pseudomonas koreensis Ps 9-14 (T), confirming that strain # 46-2 was a novel microorganism. It was deposited in the microbial resource center with the accession number KCTC32541. Microorganisms # 25-2 and # 43 were found to be more than 98% similar to Pseudomonas koreensis Ps 9-14.
  • the sensor cell of the present invention MP-GESS-containing E. coli WM335 can be detected strains with fibrinolytic activity, the detected strains are similar to Bacillus cereus, Pseudomonas trigiensis, Pseudomonas corriensis, It was also found that new microorganisms with degrading activity could be explored.
  • Example 6 Example Of new microorganisms selected in 3 NGS Analysis, whole genome analysis and functional difference analysis with existing microorganisms
  • RAST is a tool that takes a continuation as an input value, estimates the ORF, and classifies / analyzes the function of the estimated ORF by a method called 'subsystem technique'. As of May 2015, it has 1,151 validated subsystems, which are divided into three levels with 27 categories. The function of the estimated ORF may belong to one or more subsystems. For strain # 46-2, subsystem classifications were defined in 2,941 ORFs, 52% of a total of 5,692 ORFs, with the remaining 48% not being subsystem classifications. Was not ORF. This ratio and the distribution of 2,941 subsystems are shown in FIG. The most common categories were amino acids and their derivatives, followed by carbohydrates, cofactors, vitamins, prosthetic groups and pigments.
  • strain # 46-2 shows the Genomic Coverage value obtained by dividing the number of identified genes by the total number of genes of each compared strain. It was confirmed that about 84% of the genes of P. fluorescence Pf0-1 were similar to strain # 46. Thus, the selected strain # 46-2 was found to be a novel microorganism with cellulase enzyme activity but different from the existing Pseudomonas species.
  • the novel strain # 46-2 which was selected as the E. coli WM335 strain containing MP-GESS, the sensor cell of the present invention, was found to be the most similar genus in P. koreensis sp. It was observed by electron microscopy to confirm whether or not.
  • # 46-2 strain was identified as the most similar genus (genus) in 16srRNA Analysis P. koreensis sp .
  • the phenotype with more flagella compared to that shown (Fig. 13). This is similar to that of P. koreensis Ps9-14 (T) with multiple flagella.
  • the microbial resources were rod-shaped and bulging, which is believed to produce polymer material due to the slime colony form.
  • Example 8 Microbial Screening Based on Two-Step Protocol: Screening of Microbes with Phosphatase Activity in Soil Samples (Example 1 after culturing microorganisms to detect enzyme activity, forming colonies Spraying sensor cells of -2)
  • phosphatase Since phosphatase has an activity of decomposing phenylphosphate into phenol and phosphate (FIG. 14A), microorganisms having phosphatase activity were searched using sensor cells.
  • the cultured sensor cells were centrifuged at 3000 rpm for 15 minutes and then washed by addition of 5 mL of dLB5. After one more wash step, the sensor cell had an OD 600 of about 1 before spraying. Thereafter, the sensor cells were sprayed onto a solid plate in which soil microorganisms formed colonies by using an atomizer. After 12 hours of incubation at 37 ° C., the sample was AZ100M fluorescence multi-zoom microscope (Nikon) equipped with a GFP filter. It was observed using. As expected from the TPL results, cyclic colonies that emit green fluorescence were formed around the edges of candidate colonies with phosphatase activity (FIG. 14B).
  • any soil microorganisms C8, C9 and C10 emit autofluorescence rather than contain sensor cells since the genes for the vectors contained in the sensor cells have not been amplified.
  • the two-step protocol is characterized by the size of colonies formed by sensor cells and metagenome microorganisms, and between the sensor cells and metagenome microorganisms in colony formation. The difference between can be clearly indicated, and thus, more effective guidelines can be suggested in selecting candidate microorganisms having target enzyme activity.
  • the seven selected colonies were further examined by 16s rRNA analysis. Seven colonies were floated on sterile toothpicks and streaked in dLB medium without substrate, phenol or D-glutamic acid. At this stage, the sensor cells naturally die off and single colonies were isolated for further 16s rRNA analysis. Table 3 shows the results of 16s rRNA analysis of each colony.
  • Aeromonas As a result, four types of genera, Aeromonas, Pseudomonas, Shewanella, and Escherichia, were identified from seven selected microorganisms. In particular, the genus Shewanella and Aeromonas have been identified in aquatic environments.
  • phosphatase Since phosphatase has an activity of decomposing phenylphosphate into phenol and phosphate, the cells selected by the sensor cells were recovered to confirm whether the selected cells have phosphatase activity.
  • the selected cells were recovered and suspended in 50 mM HEPES buffer (pH 7.5), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) was added as a protease inhibitor. Suspended cells were disrupted by sonication on ice (Fisher Scientific, Pittsburgh, Pa.). Cell debris was removed by centrifugation at 15,000 ⁇ g for 20 minutes at 4 ° C., and the supernatant was filtered through a 0.45- ⁇ m filter. Protein concentrations were quantified according to the methods described in Analytical Biochemistry 72, 248-254, Bradford.
  • PMSF phenylmethylsulfonyl fluoride
  • the catalytic activity of the crude extract was confirmed based on the amount of pNP released from 0.2 mL HEPES buffer (pH 7.5) containing 1 mM pNP-phosphate in a round bottomed 1.5-ml tube.
  • the crude extract enzyme reaction was carried out at 25 ° C. for 10 minutes and terminated by addition of 1 M Na 2 CO 3 .
  • the reaction solution was clarified by centrifugation at 16,300 ⁇ g for 15 minutes and the absorbance change was measured at 420 nm using Victor V Multilabel Plate Reader (PerkinElmer Life Sciences, Waltham, Massachusetts, USA).
  • One unit of enzyme was defined as the activity required to produce 1 nmol of pNP as product per minute under specific assay conditions.
  • FIG. 15A shows the phosphatase activity of crude extracts of 36 colonies selected.
  • 15B shows the phosphatase activity (U / mg) of the selected colonies with 16s rRNA analysis of the selected colonies.
  • Five major strains Shigella sonnei , Shigella flexneri , Rheinheimera tangshanensis , Rheinheimera soli , Escherichia fergusonii ) has been shown to exhibit a clear phosphatase activity in the range of 10-30 U / mg.
  • Shigella flexneri is one of the well-known sources of nonspecific acidic phosphatase of bacteria, which is used for the production of various phosphorylation products.
  • Rheinheimera soli is known to have weak enzymatic activity against acid phosphatase
  • Escherichia fergusonii has also been reported to have acidic phosphatase activity.
  • Shigella sonnei and Rheinheimera Since no information on phosphatase activity of tangshanensis has been published, Shigella sonnei and Rheinheimera Further studies of tangshanensis may reveal new phosphatase.

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Abstract

La présente invention concerne : un circuit génétique comprenant un gène codant un facteur régulateur de transcription exprimé par détection de phénol, et un gène rapporteur aval activé par le facteur régulateur de transcription; une cellule de capteur comprenant le circuit génétique; et un procédé pour détecter et rechercher, directement à partir d'échantillons naturels, de nouvelles souches de micro-organismes ou ressources génétiques ayant une activité enzymatique cible, en utilisant la cellule de capteur et un substrat marqueur du phénol. Le circuit génétique et la cellule de capteur le comprenant, de la présente invention, comprennent, comme gènes rapporteurs, une protéine fluorescente associée à une enzyme liée à l'auxotrophie pour contrôler la survie de la cellule de capteur, ce qui permet, par un procédé simple de co-culture de la cellule de capteur et d'une souche inconnue de microorganismes ayant une activité enzymatique cible souhaitée et par mesure de la fluorescence de celle-ci, une identification facile de nouvelles souches de micro-organismes ou ressources génétiques ayant l'activité enzymatique cible correspondante. De plus, par l'introduction du système à double rapporteur, une confirmation visible et plus claire, quant à savoir si l'enzyme cible est activée, est validée. De plus, lors de la recherche de ressources utiles directement dans la nature, on peut développer au maximum la possibilité d'expression génique d'enzyme cible, ainsi que la simplicité d'expérimentation étant donné qu'il n'y a pas de processus de séparation d'ADN et de construction de bibliothèque et que l'utilisation d'antibiotiques est limitée, et de minimiser les problèmes liés aux OGM/OVM provoqués par des micro-organismes de recombinaison en utilisant directement des micro-organismes naturels ayant une haute activité.
PCT/KR2016/007613 2015-07-13 2016-07-13 Système basé sur un circuit génétique de prototypage de micro-organismes pour la recherche de nouvelles ressources de micro-organismes Ceased WO2017010805A1 (fr)

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