KR20200045357A - Microorganism including genetic modification that increases activity of haloacetate dehalogenase H-1 and method for reducing concentration of fluorinated compounds in sample - Google Patents

Abstract

Provided are a recombinant microorganism comprising genetic modification for increasing activity of haloacetate dehalogenase H-1, a composition used for reducing the concentration of fluorinated methane among samples comprising the recombinant microorganism, and a method for reducing the concentration of the fluorinated methane among the samples.

Description

Recombinant microorganism containing a genetic modification that increases the activity of haloacetate dehalogenase H-1, and a method for reducing the concentration of a fluorinated compound in a sample using the same {Microorganism including genetic modification that increases activity of haloacetate dehalogenase H -1 and method for reducing concentration of fluorinated compounds in sample}

Recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1, a composition for use in reducing the concentration of a fluorinated compound in a sample containing the recombinant microorganism, and fluorination in the sample It relates to a method for reducing the concentration of a compound.

Greenhouse gas emissions, which accelerate global warming, are one of the most serious environmental problems, and regulations on greenhouse gas emissions are being strengthened to regulate and prevent them. Among them, fluorine gas (F-gas) such as perfluorocarbons (PFCs), hydrofluorocarbons (HFCs), and sulfur hexafluoride (SF ₆ ) has low absolute emissions but a long half-life. It is reported that the global warming coefficient is so high that it has a more serious effect. In the semiconductor and electronic industries, which are the main sources of F-gas, the amount of F-gas generated tends to increase in excess of the GHG emission quota, and accordingly, the cost burden for disassembling greenhouse gas and purchasing emission rights is increasing every year.

However, the existing F-gas decomposition uses a thermal decomposition or catalytic thermal oxidation process, but there are limitations such as limited removal rate, secondary harmful substance emission, and high cost. In order to solve this, the introduction of a biological F-gas decomposition process using a microbial biocatalyst will overcome the limitations of the existing chemical decomposition process and more economical and eco-friendly treatment of F-gas will be possible.

Haloacetate dehalogenase H-1 catalyzes the chemical reaction of haloacetate + H ₂ O ⇔ glycolate + halide. Thus, the two substrates of this enzyme are haloacetate and H ₂ O, and the two products are glycorelite and halide. This enzyme may belong to a family of hydrolases that act on the halide bond in the carbon-halide compound.

However, even according to the above-described prior art, microorganisms, including genetic modification that increases the activity of haloacetate dehalogenase H-1 acting on the fluorinated compound, the concentration of the fluorinated compound in the sample using the same There is a need for compositions and methods to reduce.

One aspect provides a recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1.

Another aspect provides a composition for use in reducing the concentration of a fluorine-containing compound in a sample comprising recombinant microorganisms comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1.

Another aspect is a step of reducing the concentration of a fluorine-containing compound in a sample by contacting a recombinant microorganism containing a genetic modification that increases the activity of haloacetate dehalogenase H-1 with a sample containing a fluorine-containing compound. Provided is a method of reducing the concentration of a fluorine-containing compound in a sample.

The term "increase in activity", or "increased activity" as used herein refers to a detectable increase in the activity of a cell, protein, or enzyme. "Increase in activity", or "increased activity" is a cell, protein, or enzyme that does not have a given genetic modification (eg, native or "wild-type") "Shows the activity of a modified (eg, genetically engineered) cell, protein, or enzyme that is higher than the level of a comparable cell, protein, or enzyme of the same type, such as a cell, protein, or enzyme. "Cell activity" refers to the activity of a specific protein or enzyme in a cell. For example, the activity of the modified or engineered cell, protein, or enzyme is at least about 5% greater than the activity of the same type of unengineered cell, protein, or enzyme, eg, wild-type cell, protein, or enzyme. , About 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more. The activity of a specific protein or enzyme in a cell is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% of the activity of the same protein or enzyme in a parent cell, e.g., an unmanaged cell Or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more. Cells with increased activity of proteins or enzymes can be identified using any method known in the art.

Increased activity of enzymes or polypeptides can be obtained by increased expression or increased specific activity. The increase in expression may be due to introduction of a polynucleotide encoding an enzyme or polypeptide into a cell, an increase in the number of copies, or a variation in the regulatory region of the polynucleotide. The microorganism into which the gene is introduced may implicitly or not contain the gene. The gene may be operably linked to a regulatory sequence that enables its expression, such as a promoter, enhancer, poly adenylation site, or combinations thereof. Polynucleotides introduced from the outside or whose copy number is increased may be endogenous or exogenous. The endogenous gene refers to a gene existing on a genetic material contained in a microorganism. The exogenous gene refers to a gene that is introduced into the cell from the outside, and the introduced gene may be homologous or heterologous to the host cell to be introduced. “Heterologous” may mean foreign, not native.

The term "copy number increase" may be due to the introduction or amplification of the gene, and also includes a case in which a gene that does not exist in an unmanaged cell is genetically manipulated. The introduction of the gene can be accomplished by mediating a vehicle such as a vector. The introduction may be a transient introduction in which the gene is not integrated into the genome or may be inserted into the genome. The introduction may be achieved, for example, by introducing a vector into which the polynucleotide encoding the desired polypeptide is inserted into the cell, and then the vector is cloned in the cell or the polynucleotide is integrated into the genome.

The introduction of the gene can be performed by known methods such as transformation, transfection, and electroporation. The gene may be introduced through a vehicle or may be introduced by itself. As used herein, "carrier" includes nucleic acid molecules capable of delivering other nucleic acids to which they are linked. In view of the nucleic acid sequence that mediates the introduction of a particular gene, it is contemplated herein that the carrier can be used interchangeably with vectors, nucleic acid constructs, and cassettes. Vectors include, for example, plasmid or virus-derived vectors. Plasmids include circular double-stranded DNA rings to which additional DNA can be linked. Vectors can include, for example, plasmid expression vectors, viral expression vectors, such as replication-defective retroviruses, adenoviruses, and adeno-associated viruses, or combinations thereof.

The manipulation of genes used herein can be by molecular biological methods known in the art.

The term “parent cell” refers to an original cell, eg, a cell that has not been genetically engineered of the same type with respect to the engineered microorganism. For a particular genetic modification, the "parent cell" is a cell that does not have the specific genetic modification, but may be the same for other situations. Thus, the parent cell is the starting material (starting material) to produce a genetically engineered microorganism with increased activity of a given protein (e.g., a protein having a sequence identity of at least about 85% with haloacetate dehalogenase H-1). material). The same comparison applies to other genetic modifications.

As used herein, the term “gene” refers to a nucleic acid fragment that expresses a specific protein, and 5′-non coding sequence and / or 3′-non coding sequence. The sequence may or may not contain a regulatory sequence.

In the present specification, “sequence identity” of a nucleic acid or a polypeptide refers to the same degree of base or amino acid residues between sequences after aligning both sequences in a specific comparison region so as to be maximally matched. Sequence identity is a value measured by optimally aligning and comparing two sequences in a specific comparison region, and a portion of the sequence in the comparison region may be added or deleted compared to a reference sequence. Percent sequence identity is, for example, comparing two optimally aligned sequences across the comparison region, determining the number of positions at the same amino acid or nucleotide in both sequences to obtain the number of matched positions Can be calculated by the step of dividing the number of matched positions by the total number of positions in the comparison range (i.e. range size), and multiplying the result by 100 to obtain a percentage of sequence identity. The percentage of sequence identity can be determined using known sequence comparison programs, examples of such programs include BLASTN (NCBI), BLASTP (NCBI), CLC Main Workbench (CLC bio), MegAlign ^TM (DNASTAR Inc), etc. You can.

The term "genetic modification" as used herein includes artificially altering the composition or structure of a cell's genetic material.

In the present specification,% represents w / w% unless otherwise specified.

One aspect provides a recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1 (haloacetate dehalogenase). Haloacetate dehalogenase H-1 may have a sequence identity of 85% or more with the amino acid sequence of SEQ ID NO: 1.

Haloacetate dehalogenase H-1 catalyzes the chemical reaction of haloacetate + H ₂ O ⇔ glycolate + halide. Thus, the two substrates of this enzyme are haloacetate and H ₂ O, and the two products are glycolate and halide. This enzyme may belong to a family of hydrolases that act on the halide bond in the carbon-halide compound. However, the recombinant microorganism is not necessarily interpreted as being limited to this particular mechanism in reducing the concentration of CHnF4-n (n is an integer from 0 to 3) in the sample. The haloacetate dehalogenase H-1 may belong to EC 3.8.1.3. The haloacetate dehalogenase H-1 may be foreign or intrinsic. The haloacetate dehalogenase H-1 may be selected from the group consisting of haloacetate dehalogenase H-1 derived from the genus Pseudomonas, Bacillus, Pseudomonas, Azotobacter, Agrobacterium, and Escherichia. The haloacetate dehalogenase H-1 may be derived from a strain selected from the group consisting of Pseudomonas saitens, Bacillus cereus, Bacillus thuringiensis, and Bacillus megaterium.

In the microorganism, the genetic modification may be to increase the expression of the gene encoding haloacetate dehalogenase H-1. The genetic modification may be to increase the number of copies of the haloacetate dehalogenase H-1 gene. The genetic modification may be to increase the number of copies of a gene encoding a polypeptide having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, respectively. The gene may have a sequence identity of 85% or more with the nucleotide sequence of SEQ ID NO: 2, respectively. The genetic modification may be that a gene encoding a haloacetate dehalogenase H-1 is introduced, for example, a vehicle such as a vector. The gene encoding the haloacetate dehalogenase H-1 may exist within or outside the chromosome. The gene encoding the introduced haloacetate dehalogenase H-1 may be plural, for example, 2 or more, 5 or more, 10 or more, 10 or more, 50 or more, 100 or more, or 1000 or more.

The microorganism may be of Escherichia genus, Bacillus genus, Xanthobacter genus, Pseudomonas genus, Azotobacter genus, or Agrobacterium genus. The microorganism may be Pseudomonas saitens, E. coli, or X. autotrophicus.

The recombinant microorganism can reduce the concentration of the fluorine-containing compound in the sample. The reduction may be achieved by introducing the hydroxyl group into carbon by the protein acting on the C-F or C-H bond of the fluorine-containing compound, or by accumulating the fluorine-containing compound in the cells of microorganisms. In addition, the reduction may include cutting the C-F bond of the fluorine-containing compound, converting the fluorine-containing compound to another substance, or accumulating the fluorine-containing compound in cells. The sample may be in a liquid or gaseous state. The sample may be factory wastewater or waste gas. Any of the samples may include any of the fluorine-containing compounds.

The fluorine-containing compound may be a compound represented by the following Chemical Formulas 1 to 3.

<Formula 1> <Formula 2>

C (R ₁ ) (R ₂ ) (R ₃ ) (R ₄ ) (R ₅ ) (R ₆ ) (R ₇ ) C- [C (R ₁₁ ) (R ₁₂ )] _n -C (R ₈ ) ( R ₉ ) (R ₁₀ )

<Formula 3>

S (R ₁₃ ) (R ₁₄ ) (R ₁₅ ) (R ₁₆ ) (R ₁₇ ) (R ₁₈ )

In the above equations,

n is an integer of 0 to 10, and when n is 2 to 10, R11 among the plurality of R11 may have each other or be different, and R12 of the plurality of R12 may have each other or different,

R ₁ , R ₂ , R ₃ and R ₄ are independently of each other F, Cl, Br, I, or H, and at least one of R ₁ , R ₂ , R ₃ and R ₄ is F,

R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are independently F, Cl, Br, I, or H, and R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , one or more of R ₁₁ and R ₁₂ is F,

R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ are each independently F, Cl, Br, I, or H, and R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ One or more of them are F.

The fluorine-containing compound may be a fluorinated methane compound represented by CHnF4-n (n is an integer from 0 to 3). The fluorine-containing compound may include, for example, one or more selected from the group consisting of CH ₃ F, CH ₂ F ₂ , CHF ₃ , CF ₄ and SF ₆ .

Another aspect is a composition for use in reducing the concentration of a fluorine-containing compound in a sample comprising a recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1 (haloacetate dehalogenase). Gives

In the above composition, the recombinant microorganism, sample, and fluorine-containing compound are as described above.

In the above composition, the term "reduction" is to reduce the concentration of the fluorine-containing compound in the sample, which includes completely removing it. The sample can be gas or liquid. The sample may be one that does not contain the microorganism. The composition may further include a substance that increases the solubility of the fluorine-containing compound in the medium or culture.

The composition may be to reduce the concentration of the fluorine-containing compound in the sample by contacting the sample. The contact may be made in a liquid or solid phase. The contacting can be made, for example, by contacting the sample with a culture of a microorganism cultured in a medium. The culture may be performed under conditions for propagating microorganisms. The contact may be performed in a closed container. The contacting may include incubating or incubating the recombinant microorganism with a sample containing a fluorine-containing compound. The contact includes culturing in a sealed container in a condition where the recombinant microorganisms proliferate.

Another aspect is that a recombinant microorganism containing a genetic modification that increases the activity of haloacetate dehalogenase H-1 (haloacetate dehalogenase) is contacted with a fluorine-containing compound-containing sample to increase the concentration of the fluorine-containing compound in the sample. It provides a method for reducing the concentration of a fluorine-containing compound in a sample comprising a step of reducing.

In the above method, the sample containing the recombinant microorganism and the fluorine-containing compound is as described above.

In the above method, the contact may be made in a liquid or solid phase. The contacting can be made, for example, by contacting the sample with a culture of a microorganism cultured in a medium. The culture may be performed under the condition that the microorganisms proliferate. The contact may be performed in a closed container. The contact may be made when the growth stage of the microorganism is an exponential phase or a stationary phase. The culture may be performed under aerobic or anaerobic conditions. The contact may be performed in a condition where the recombinant microorganism is viable in a closed container. The viable condition may be a condition that allows the recombinant microorganism to proliferate or rest sate.

In the above method, the sample may be in a liquid or gaseous state. The sample may be factory wastewater or waste. The sample includes not only passive contact with the culture of the microorganism, but also active contact. The sample may be, for example, sparging in the culture medium of the microorganism. That is, the sample may be blown through a medium or a culture medium. The sparging may be blown from the bottom of the medium or culture medium to the top. The sparging may be injecting while dropping the sample.

In the above method, the contacting can be carried out batchwise or continuously. The contacting, for example, contacting the contacted sample obtained in the reducing step with a recombinant microorganism comprising a genetic modification that increases the activity of fresh haloacetate dehalogenase H-1 (haloacetate dehalogenase). It may include a step. The contact with the fresh microorganism may be made two or more times, for example, 2, 3, 5, or 10 times or more. The contact can be continued or repeated until the desired reduced concentration of the fluorine-containing compound in the sample is achieved.

Another aspect provides a method of producing a microorganism comprising introducing a genetic modification that increases the activity of haloacetate dehalogenase (H-1) into the microorganism. The method may be a method for producing a microorganism, comprising introducing a gene encoding a haloacetate dehalogenase H-1 into the microorganism. The step of introducing a gene encoding the haloacetate dehalogenase H-1 may be to introduce a vehicle containing the gene into the microorganism. In the above method, the genetic modification may include amplifying the gene, manipulating the regulatory sequence of the gene, or manipulating the sequence of the gene itself. The manipulation may be nucleotide insertion, substitution, conversion or addition.

Another aspect provides an isolated promoter polynucleotide comprising the nucleotide sequence of SEQ ID NO: 8.

Another aspect provides a recombinant vector comprising an isolated promoter polynucleotide comprising the nucleotide sequence of SEQ ID NO: 8. The vector can be a plasmid, or a virus-derived vector.

Another aspect provides a recombinant host cell comprising a recombinant vector comprising an isolated promoter polynucleotide comprising the nucleotide sequence of SEQ ID NO: 8. The host cell may be of the genus Peudomonas, Escherichia, Bacillus, or Xanthobacter. The host cell may be heterologous to Peudomonas saitens. The vector may be a polynucleotide encoding a protein operably linked to the promoter polynucleotide.

Another aspect is culturing the host cell to produce a protein; And isolating the protein from the culture. The host cell may include a vector, and the vector may include a polynucleotide encoding a protein operably linked to the promoter polynucleotide.

Recombinant microorganisms according to one aspect may be used to remove fluorine-containing compounds from a sample.

Compositions according to other aspects can be used to reduce the concentration of a fluorine-containing compound in a sample.

According to another aspect, a method of reducing the concentration of a fluorine-containing compound in a sample can effectively reduce the concentration of a fluorine-containing compound in a sample.

1 is a schematic diagram of a glass dimloss screw tube reflux condenser.
2 shows a vector map of the pBBR122 vector.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Pseudomonas saitens  origin Haloacetate Dehalogenase  H-1 ( deh  H-1) Recombinant microorganism expressing the gene and CF in the sample ₄  remove

One. Pseudomonas from saitens Deh  Confirmation of inducible expression of H-1 gene

The cultured: (KCTC Korea Collection for Type Culture ) with Pseudomonas saitens entrusted to the accession number of KCTC 13107BP in conditions of CF ₄ present and CF ₄ present in September 2016 a dated international deposit under the Budapest Treaty agency Korea Bio Resource Center It was confirmed whether the expression is induced. This strain is also referred to as Pseudomonas saitens SF1 strain.

1 is a schematic diagram of a glass dimloss screw tube reflux condenser. As shown in FIG. 1, 50 ml of LB medium in a glass Dimroth screw tube reflux cooler 10 (reactor length 350 mm, exterior diameter 35 mm, internal volume 200 mL) autoclaved and vertically disposed, and After 220 ppm of CF ₄ gas was injected, the LB medium was circulated. The LB medium was supplied to the inlet 12 at the top of the cooler 10 and flowed through the inner wall of the cooler 10 and discharged to the outlet 14 at the bottom of the cooler 10. The discharged LB medium was re-supplied to the inlet 12 along the circulation line 18. Although not shown in Figure 1, the inner screw tube of the cooler 10 was connected to a constant temperature zone of 30 ℃ to maintain the temperature. Circulation was driven by pump 16. The circulation rate of LB medium was maintained at 4 mL / min. After the specified time, that is, after 24 hours, the CF ₄ gas content in the cooler was confirmed by GC-MS. There was no change in the CF ₄ gas content.

Subsequently, the P.saitens SF1 and the control microorganisms were inoculated by concentrating the microorganisms having an initial concentration of OD ₆₀₀ 1.0 in the LB medium in the cooler twice to a total concentration of OD ₆₀₀ 2.

The circulation rate of the cell-containing culture was 4 mL / min, and the temperature inside the cooler was maintained at 30 ° C. After inoculating the strain, 220 ppm of CF ₄ gas was injected, and then the LB medium was circulated and after 5 hours, the amount of deh H-1 mRNA expressed from P.saitens SF1 was measured. The control group was the same as the experimental group, except that CF ₄ gas was not injected.

 The amount of mRNA was measured by quantitative RT-PCR method using 16s rRNA as an internal standard. Total RNA was extracted using an RNAeasy mini kit (Qiagen, USA), and contacted with RNase-free DNase I (Bio-rad, USA) to remove DNA left in the RNA. The first strand cDNA was synthesized using a random hexamer and reverse transcriptase (Applied Biosystems, USA), and then amplified using CFX96 Real Time System (Applied Biosystems, USA).

As a result, when CF ₄ was injected, the relative expression of deh H-1 mRNA increased by about 36 fold over the mRNA expression in the untreated control. This is a value obtained by normalizing 16s rRNA to an internal standard to obtain a ΔCt value and calculating 2 ^ ΔΔCt based on the difference (ΔΔCt) between the Ct values of the two strains. This increase in expression indicates that the expression of the deh H-1 gene is driven by CF ₄ .

2. Pseudomonas from saitens Deh  H-1 gene expression Increased  CF among microorganisms and samples using the same ₄ Removal of

(One) deh  Construction of microorganisms containing the H-1 gene

After culturing P.saitens SF1 in LB medium at 30 ° C. at 230 rpm overnight, the genomic DNA was separated by a method using a total DNA extraction kit (Invitrogen Biotechnology), and this genomic DNA was templated. And SEQ ID NOs: 3 and 4; Alternatively, PCR was performed using the nucleotide sequences of SEQ ID NOs: 5 and 6 as a primer set to amplify the deh H-1 gene. In this case, when the primer sets of SEQ ID NOs: 3 and 4 were used, the deh H-1 gene open reading frame (ORF) containing no natural promoter was amplified, and when the primer sets of SEQ ID NOs: 5 and 4 were used, The deh H-1 gene containing the natural promoter was amplified. The deh H-1 gene has the nucleotide sequence of SEQ ID NO: 2, and the deh H-1 protein has the amino acid sequence of SEQ ID NO: 1.

The amplified gene was linked to the pBBR122 vector (MoBiTec GmbH, Germany) fragments amplified by PCR using SEQ ID NOs: 6 and 7, respectively, through an InFusion Cloning Kit (Clontech Laboratories, Inc.). The pBBR122 vector was developed by Dr. Camille of the Pasteur Institute in France and is commercially available. The pBBR122 vector has a broad bacterial host range.

The basic vector pBBR122 is constructed to be resistant to chloramphenicol and kanamycin antibiotics, and by introducing the 4318 gene into the chloramphenicol locus, expression of pBBR122_Pcat :: 4318 induced by the chloramphenicol promoter (Pcat) was obtained. . The chloramphenicol promoter (Pcat) and chloramphenicol genes were replaced with 4318 natural promoter and 4318 gene to obtain pBBR122_P4318 :: 4318.

That is, pBBR122_Pcat :: 4318 is a pBBR122 vector into which the deh H-1 gene linked to the Pcat promoter is introduced, and pBBR122_P4318 :: 4318 represents a pBBR122 vector into which the deh H-1 gene linked to the natural promoter of the deh H-1 gene is introduced. . The native promoter of the deh H-1 gene has the nucleotide sequence of SEQ ID NO: 8. 2 shows a vector map of the pBBR122 vector.

This vector is introduced into a fluorine-containing hydrocarbon, that is, a CF ₄ biodegradable microorganism, Pseudomonas saitens (SF1), through electroporation, and cultured in an LB plate medium containing 50 µg / mL of kanamicin to enhance kanamicin resistance. Visible strains were selected. Recombinant Pseudomonas thus prepared The saitens (SF1) strains were named SF1_pBBR122, SF1_pBBR122_Pcat :: 4318 and SF1_pBBR122_P4318 :: 4318, respectively. SF1_pBBR122 is a control strain into which an empty PBBR122 vector is introduced.

(2) CF using recombinant microorganisms ₄  remove

The three strains obtained in section (1) were subjected to primary evaluation in a 125 mL flask. In the 125 mL flask, the strain was inoculated with 25 mL (OD ₆₀₀ 5.0) in the form of LB medium, and 220 ppm CF ₄ was injected, followed by shaking culture at 30 ° C and 220 rpm. After about 3 days, the reduced CF ₄ amount was confirmed by GC-MS (Gas Chromatography Mass-Spectrum) to calculate the removal rate. The removal rate was calculated from Equation 1 below and the results are shown in Table 1.

The secondary evaluation was incubated for 3 days by inoculating the strain in a glass dimrose screw reflux cooler as described in FIG. 1, as described in Section 1. Injection of CF ₄ and confirm the CF ₄ gas content inside the rear cooler about 3 days to GC-MS (Gas Chromatography Mass- Spectrum) and an CF ₄ removal rate calculated from equation (1) and the results are shown in Table 2. Hereinafter, the results of Tables 1 and 2 are also referred to as a flask experiment and a cyclic process experiment result.

<Equation 1>

CF ₄ removal rate = [(Initial CF ₄ content-CF ₄ content after reaction) / Initial CF ₄ content] × 100

Strain CF4 removal rate (%) 0h 66h SF1_pBBR122 0 3.13 SF1_pBBR122_Pcat :: 4318 0 10.81 SF1_pBBR122_P4318 :: 4318 0 12.59

Strain CF4 removal rate (%) 0h 66h SF1_pBBR122 0 20.24 SF1_pBBR122_Pcat :: 4318 0 21.85 SF1_pBBR122_P4318 :: 4318 0 22.59

As shown in Tables 1 and 2, when performing the flask experiment and the gas phase circulation process experiment, the experimental group significantly reduced the concentration of CF ₄ compared to the control group. In addition, among the recombinant microorganisms into which the deh H-1 gene was introduced, the removal efficiency was better when it was connected to the deh H-1 self promoter than to the Pcat promoter. This seems to be because the expression of deh H-1 gene is induced by the deh H-1 promoter in the presence of CF ₄ .

                         SEQUENCE LISTING <110> Samsung Electronics Co., Ltd.   <120> Microorganism including genetic modification that increases        activity of haloacetate dehalogenase H-1 and method for reducing        concentration of fluorinated compounds in sample <130> PN123779KR <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 272 <212> PRT <213> Pseudomonas saitens <400> 1 Met Ser Ala Ser Glu Thr Phe Thr His Thr Gln Leu Pro Leu Thr Ile 1 5 10 15 Asp Gly Val Gln Leu Asn Ile Ala Ala Ile His Arg Arg Gly Glu Leu             20 25 30 Ala Pro Ile Val Phe Leu His Gly Phe Gly Ser Thr Lys Glu Asp Tyr         35 40 45 Ala Gly Ile Ala Leu Gln Ala Glu Phe Asp Gly His Pro Phe Val Ala     50 55 60 Phe Asp Ala Pro Gly Cys Gly Glu Thr Leu Cys Ser Asp Leu Ser Arg 65 70 75 80 Val Ser Ile Pro Leu Leu Val Gln Val Thr Leu Gln Val Leu Glu His                 85 90 95 Phe Asp Ile Glu Arg Phe His Leu Val Gly His Ser Met Gly Gly Leu             100 105 110 Ala Ala Leu Leu Leu Ala His Gln Phe Pro Gly Arg Val Leu Ser Phe         115 120 125 Thr Asp Ile Glu Gly Asn Ile Ala Pro Glu Asp Cys Phe Leu Ser Arg     130 135 140 Gln Ile Asp Asp Tyr Pro Ser Asp Asp Pro Glu Val Phe Phe Ala Ala 145 150 155 160 Phe Ile Lys Arg Val Arg His Ala Pro Ala Tyr Ala Ser Ala Leu Tyr                 165 170 175 Ser Ala Ser Leu Arg His Lys Val Arg Ala Glu Ala Val Arg Gly Ile             180 185 190 Phe Ser Thr Ile Val Asp Leu Ser Asp Asn Ala Asp Leu Met Gly Lys         195 200 205 Phe Leu Gly Leu Lys Cys Pro Arg Met Phe Met Tyr Gly Glu Gln Asn     210 215 220 Ser Ser Leu Ser Tyr Leu Ala His Ile Gln Ala Gln Gly Val Arg Leu 225 230 235 240 Ala Pro Ile Pro Val Cys Gly His Phe Pro Met Tyr Ser Asn Pro Ile                 245 250 255 Ala Met Trp Gln Gln Ile Ala Asp Phe His Arg Ala Ser Gln Ala Gly             260 265 270 <210> 2 <211> 819 <212> DNA <213> Pseudomonas saitens <400> 2 gtgtctgcat ccgaaacatt cactcatacc caacttccgc taacgattga cggtgtgcaa 60 ctgaacatcg ccgccattca caggcgtggc gagttggcgc cgatcgtttt tctgcatggt 120 tttggttcga ccaaagagga ctatgccggg atcgcgctgc aggcggaatt cgacggtcac 180 ccctttgttg ccttcgacgc acccggctgc ggtgaaaccc tctgcagtga cctgtccagg 240 gtgtccatcc cgctgttggt gcaggtgaca ctgcaagtgc tggagcattt cgatatcgaa 300 cgcttccatc tggtcggtca ctctatgggg gggttggcgg ctttgctgct ggctcatcag 360 tttccggggc gggtactgag cttcaccgac atagagggca atattgcccc ggaagactgc 420 tttctcagtc gtcagatcga cgattatccg agcgatgatc ccgaggtgtt tttcgcggct 480 ttcatcaagc gtgtcaggca tgccccggcg tatgccagtg cattgtattc ggcgagcctg 540 agacacaagg tgcgtgccga ggcggtgcga ggcatttttt cgacgatagt tgatctgtcc 600 gataacgccg acctgatggg taagtttctt ggcttgaagt gcccgcgaat gtttatgtac 660 ggcgagcaga actcatcact ttcctacctt gcgcatatcc aggctcaggg cgtacgcctt 720 gcgccaatcc ctgtgtgcgg gcatttcccc atgtattcca acccgatcgc gatgtggcaa 780 cagatcgctg attttcaccg cgccagccag gccggttaa 819 <210> 3 <211> 45 <212> DNA <213> Artificial <220> <223> primer <400> 3 gagctaagga agctaaaact agtgtgtctg catccgaaac attca 45 <210> 4 <211> 45 <212> DNA <213> Artificial <220> <223> primer <400> 4 aactgcctta aaaaaatcta gattaaccgg cctggctggc gcggt 45 <210> 5 <211> 45 <212> DNA <213> Artificial <220> <223> primer <400> 5 gagctaagga agctaaaact agttcctcgg gtgagtaaaa aacag 45 <210> 6 <211> 28 <212> DNA <213> Artificial <220> <223> Primer <400> 6 tctagatttt tttaaggcag ttattggt 28 <210> 7 <211> 29 <212> DNA <213> Artificial <220> <223> primer <400> 7 actagtttta gcttccttag ctcctgaaa 29 <210> 8 <211> 306 <212> DNA <213> Pseudomonas saitens <400> 8 tcctcgggtg agtaaaaaac aggcctgagg gcctgatgct ggccaggcgg gcatatctgc 60 tcgactggcc ggcggcaaac aatccaatcc gatgccggtt ccggcatcgg atttttttat 120 gcggggtgcg tgcgccctgg catgcagcat aaatagtgtg ggcagaggaa attcgagtga 180 taggttttgc gtatcaaaac cttccacgat caggatttta caggctgctc gaatcccttg 240 agattgcgta ccgagctgcg gcaactctcg tgcggcagtc attttatcgt tcaaggggct 300 attaac 306

Claims (20)

A recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1 having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1. The microorganism according to claim 1, wherein the genetic modification is a genetic modification that increases the expression of the gene encoding the haloacetate dehalogenase H-1. The microorganism according to claim 1, wherein the genetic modification is a genetic modification that increases the number of copies of the gene encoding the haloacetate dehalogenase H-1. The method according to claim 1, wherein the haloacetate dehalogenase H-1 is selected from the group consisting of haloacetate dehalogenase H-1 derived from the genus Bacillus, Pseudomonas, Azotobacter, Agrobacterium, and Escherichia. . The microorganism according to claim 1, wherein the haloacetate dehalogenase H-1 is derived from a strain selected from the group consisting of Peudomonas saitens, Bacillus cereus, Bacillus megaterium, and Bacillus thuringiensis. The microorganism according to claim 1, wherein the haloacetate dehalogenase H-1 belongs to EC 3.8.1.3. The microorganism according to claim 3, wherein the gene has 85% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1, Peudomonas genus, Escherichia genus, Bacillus genus, or Xanthobacter (Xanthobacter) belong to the genus. A composition for use in reducing the concentration of a fluorine-containing compound in a sample, comprising a recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1, wherein the fluorine-containing compound Is a compound represented by the following formulas 1 to 3:
<Formula 1><Formula2>
C (R ₁ ) (R ₂ ) (R ₃ ) (R ₄ ) (R ₅ ) (R ₆ ) (R ₇ ) C- [C (R ₁₁ ) (R ₁₂ )] _n -C (R ₈ ) ( R ₉ ) (R ₁₀ )
<Formula 3>
S (R ₁₃ ) (R ₁₄ ) (R ₁₅ ) (R ₁₆ ) (R ₁₇ ) (R ₁₈ )
In the above equations,
n is an integer of 0 to 10, and when n is 2 to 10, R11 among the plurality of R11 may have each other or be different, and R12 of the plurality of R12 may have each other or different,
R ₁ , R ₂ , R ₃ and R ₄ are independently of each other F, Cl, Br, I, or H, and at least one of R ₁ , R ₂ , R ₃ and R ₄ is F,
R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are independently F, Cl, Br, I, or H, and R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , one or more of R ₁₁ and R ₁₂ is F,
R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ are each independently F, Cl, Br, I, or H, and R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ One or more of them are F. The composition according to claim 9, wherein the genetic modification is a genetic modification of the expression of the gene encoding the haloacetate dehalogenase H-1. The composition of claim 9, wherein the haloacetate dehalogenase H-1 belongs to EC 3.8.1.3. 10. The composition of claim 9, wherein the reduction comprises the protein cleaving the C-F bond of the fluorine-containing compound, converting the fluorine-containing compound to another substance, or accumulating the fluorine-containing compound in cells. The composition of claim 9, wherein the sample is in a liquid or gaseous state. The composition according to claim 9, wherein the microorganism belongs to the genus Peudomonas, genus Escherichia, Bacillus or Xanthobacter. Contacting a recombinant microorganism comprising a genetic modification that increases the activity of haloacetate dehalogenase H-1 with a fluorine-containing compound-containing sample to reduce the concentration of the fluorine-containing compound in the sample, As a method for reducing the concentration of the fluorine-containing compound in the sample,
The fluorine-containing compound is a method represented by the following formulas 1 to 3:
<Formula 1><Formula2>
C (R ₁ ) (R ₂ ) (R ₃ ) (R ₄ ) (R ₅ ) (R ₆ ) (R ₇ ) C- [C (R ₁₁ ) (R ₁₂ )] _n -C (R ₈ ) ( R ₉ ) (R ₁₀ )
<Formula 3>
S (R ₁₃ ) (R ₁₄ ) (R ₁₅ ) (R ₁₆ ) (R ₁₇ ) (R ₁₈ )
In the above equations,
n is an integer of 0 to 10, and when n is 2 to 10, R11 among the plurality of R11 may have each other or be different, and R12 of the plurality of R12 may have each other or different,
R ₁ , R ₂ , R ₃ and R ₄ are independently of each other F, Cl, Br, I, or H, and at least one of R ₁ , R ₂ , R ₃ and R ₄ is F,
R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are independently F, Cl, Br, I, or H, and R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , one or more of R ₁₁ and R ₁₂ is F,
R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ are each independently F, Cl, Br, I, or H, and R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ One or more of them are F. 16. The method of claim 15, wherein the genetic modification is a genetic modification that increases the expression of the gene encoding the haloacetate dehalogenase H-1. 16. The method of claim 15, wherein the haloacetate dehalogenase H-1 belongs to EC 3.8.1.3. 16. The method of claim 15, wherein said contacting is performed in a closed container. 16. The method of claim 15, wherein the contacting comprises culturing or incubating the recombinant microorganism with a sample containing the fluorine-containing compound. 16. The method of claim 15, wherein said contacting comprises culturing in conditions in which the recombinant microorganism grows in a closed container.

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