Microbial mutant strain and method for biodegrading cyclic ether using the same

Abstract

To provide a 1,4-dioxane-degrading microbial mutant strain having enhanced 1,4-dioxane degradation capability, and a method for biodegrading cyclic ether using the same.SOLUTION: The present invention provides a Pseudonocardia dioxanivorans CB1190-derived mutant strain with an increased gene expression level of THF monooxygenase α-subunit and / or GntR domain protein compared to the wild-type strain.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present disclosure relates to a microbial mutant strain and a method for biodegradably treating cyclic ethers using the same. More specifically, the present disclosure relates to a method for biodegradably treating cyclic ethers using a 1,4-dioxane-degrading microbial mutant strain having improved 1,4-dioxane-degrading ability, and the like.

Background Art

[0002] 1,4-Dioxane is a cyclic ether represented by the following formula (1). 1,4-Dioxane was added to the WHO environmental standards in 2002 as having a potential carcinogenic effect (Group 2B: Suspected carcinogenic to humans). 1,4-Dioxane is contained in the wastewater discharged from industrial plants such as the chemical product industry such as the production of photosensitive resins, the petrochemical industry such as the production of ethylene oxide and ethylene glycol, and the chemical fiber industry such as the production of polyester.

[0003]

Chem.

[0004] 1,4-Dioxane is considered to be difficult to decompose and remove when released into the environment. Therefore, the development of a method for simply and inexpensively decomposing 1,4-dioxane contained in wastewater is desired.

[0005] Patent Document 1 discloses a biodegradation treatment method for biodegradatively treating 1,4-dioxane contained in contaminated water and the like using N23 strain, which is a 1,4-dioxane-decomposing bacterium. Non-Patent Document 1 describes the 1,4-dioxane-decomposing bacterium Pseudonocardia dioxanivorans Strain CB1190, and reports the metabolic pathway of 1,4-dioxane and related gene groups. Pseudonocardia dioxanivorans Strain CB1190 is available from the Japan Collection of Microorganisms of the RIKEN BioResource Center (JCM 13855).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] The main object of the present disclosure is to provide a 1,4-dioxane-decomposing microbial mutant having improved 1,4-dioxane-decomposing ability and a biodegradation treatment method for cyclic ethers using the same.

Means for Solving the Problem

[0009] To solve the above problems, the present disclosure provides the following [1]-

[10] . [1] A mutant strain derived from Pseudonocardia dioxanivorans CB1190 in which the gene expression level of THF monooxygenase α-subunit and / or GntR domain protein is increased compared to the wild strain. [2] A mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to [1], which has a base substitution, insertion, or deletion at at least one position selected from the following positions in the gene sequences of the circular chromosome and circular plasmids 1-3 of Pseudonocardia dioxanivorans CB1190. Positions 115984, 350134, 350152, 350167, 350326, 350356, 350473, 350475, 598967, 642212, 1190528, 2082787, 2603923, 2634727, 2925050, 2932573, 2956189, 3035551, 3234928, 3507315, 5545605, 5640340, 5640341, 5640397, 5658991, 6194590 of the gene sequence of the circular chromosome shown in SEQ ID NO: 1; Positions 100129, 186574 of the gene sequence of circular plasmid 1 shown in SEQ ID NO: 2; Positions 6688, 6717, 6729 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO: 3; Position 31513 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO: 4. [3] A mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to [1] or [2], which is Pseudonocardia dioxanivorans MCI4804 strain (Accession No.: NITE P-03944).

[0010] [4] A method for biodegradably treating a cyclic ether contained in a treatment target, comprising: a step of contacting the cyclic ether with a mutant strain derived from Pseudonocardia dioxanivorans CB1190 of any one of [1] to [3]. [5] The method according to [4], wherein the cyclic ether is one or more selected from the group consisting of 1,4-dioxane, 1,3-dioxolane, and 2-chloromethyl-1,3-dioxolane. [6] The method according to [4] or [5], wherein the treatment target is wastewater or activated sludge.

[0011] [7] Pseudonocardia dioxanivorans MCI4804 strain (Accession No.: NITE P-03944).

[0012] [8] A composition for biodegradably treating a cyclic ether, comprising a mutant strain derived from Pseudonocardia dioxanivorans CB1190 of [1] to [3]. [9] The composition according to [8], wherein the cyclic ether is one or more selected from the group consisting of 1,4-dioxane, 1,3-dioxolane, and 2-chloromethyl-1,3-dioxolane.

[10] The composition according to [8] or [9], which is used for biodegradation of a cyclic ether contained in wastewater or activated sludge. [Advantages of the Invention]

[0013] The present disclosure provides a 1,4-dioxane-degrading microbial mutant strain having improved 1,4-dioxane-degrading ability and a method for biodegradably treating a cyclic ether using the same. [Brief Description of the Drawings]

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0015] Hereinafter, preferred embodiments for carrying out the present disclosure will be described. Note that the embodiments described below show an example of typical embodiments of the present invention, and the scope of the present invention is not construed narrowly thereby.

[0016] The method for biodegradably treating cyclic ethers according to the present disclosure includes a procedure of bringing the cyclic ethers contained in the treatment target into contact with a microorganism having 1,4-dioxane-degrading ability (hereinafter also referred to as "1,4-dioxane-degrading bacterium").

[0017] As the 1,4-dioxane-degrading bacterium, a microorganism belonging to the genus Pseudonocardia is used. Examples of microorganisms belonging to the genus Pseudonocardia include Pseudonocardia dioxanivorans, Pseudonocardia benzenivorans, and Pseudonocardia antarctica. Among these, as strains belonging to Pseudonocardia dioxanivorans, Pseudonocardia dioxanivorans CB1190, preferably a strain having high 1,4-dioxane degradation ability obtained by introducing gene mutations into Pseudonocardia dioxanivorans CB1190 and screening using 1,4-dioxane degradation ability as an index can be used. As a method for introducing gene mutations, known methods can be employed. For example, methods by genetic recombination, methods of contacting cells with mutagenic agents (such as alkylating agents like N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS)), and methods of irradiating cells with radiation (such as X-rays and γ-rays) or ultraviolet rays can be mentioned.

[0018] In one embodiment, the 1,4-dioxane-degrading bacterium that has acquired high 1,4-dioxane degradation ability due to gene mutations in Pseudonocardia dioxanivorans CB1190 may show increased gene expression levels of THF monooxygenase α-subunit and / or GntR domain protein compared to the wild strain (Pseudonocardia dioxanivorans CB1190). The increase in the expression level of THF monooxygenase α-subunit (thmA) can be about 1.5 times that of the wild strain. Also, the increase in the expression level of GntR domain protein (GntR) can be 2 times, preferably 3 times, more preferably 4 times, and particularly preferably about 4.5 times that of the wild strain.

[0019] In addition, the 1,4-dioxane-degrading bacterium that has acquired high 1,4-dioxane degradation ability due to gene mutation in Pseudonocardia dioxanovorans CB1190 may have a base substitution, insertion, or deletion at at least one position selected from the following positions in one embodiment. Positions 115984, 350134, 350152, 350167, 350326, 350356, 350473, 350475, 598967, 642212, 1190528, 2082787, 2603923, 2634727, 2925050, 2932573, 2956189, 3035551, 3234928, 3507315, 5545605, 5640340, 5640341, 5640397, 5658991, 6194590 of the gene sequence of the circular chromosome shown in SEQ ID NO: 1; Positions 100129 and 186574 of the gene sequence of circular plasmid 1 shown in SEQ ID NO: 2; Positions 6688, 6717, and 6729 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO: 3; Position 31513 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO: 4. Details of the substitution, insertion, or deletion at each position are shown in Table 8 below. Note that both SEQ ID NO: 3 and SEQ ID NO: 4 are partial sequences of circular plasmid 2, and circular plasmid 2 further includes partial sequences other than these sequences. The 1,4-dioxane-degrading bacterium may have two or more, particularly preferably all, of these substitutions, insertions, or deletions.

[0020] The 1,4-dioxane-degrading bacterium that has acquired high 1,4-dioxane degradation ability due to gene mutation in Pseudonocardia dioxanovorans CB1190 may be Pseudonocardia dioxanovorans MCI4804 (also referred to as "PB-24-26" in the present disclosure). Pseudonocardia dioxanovorans MCI4804 has been deposited domestically at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (NPMD) (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture), which is a depositary institution based on Articles 27-2 and 27-3 of the Implementing Regulations of the Patent Law and an international depositary authority based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. The accession number of the above strain is NITE P-03944, and the receipt date is July 13, 2023. Note that Pseudonocardia dioxanovorans CB1190 can be obtained from the Japan Collection of Microorganisms at the RIKEN BioResource Center (JCM 13855).

[0021] Pseudonocardia dioxanovorans MCI4804 exhibits a 1,4-dioxane degradation specific activity (degradation rate per unit weight of cultured cells [mg-DOX / g-dcw / hr]) that is 1.35 to 1.55 times higher than that of the parent strain, Pseudonocardia dioxanovorans CB1190. In addition, Pseudonocardia dioxanovorans MCI4804 also exhibits high degradation specific activity against 1,3-dioxolane and 2-chloromethyl-1,3-dioxolane. Therefore, Pseudonocardia dioxanovorans MCI4804 or a composition containing the same can be particularly preferably used for the biodegradation of one or more cyclic ethers selected from the group consisting of 1,4-dioxane, 1,3-dioxolane, and 2-chloromethyl-1,3-dioxolane.

[0022] The 1,4-dioxane-degrading bacterium may be a mutant strain having an even higher 1,4-dioxane-degrading ability, which is obtained by further introducing gene mutations and screening using Pseudonocardia dioxanovorans MCI4804 as the parent strain. Alternatively, the 1,4-dioxane-degrading bacterium may be a recombinant strain obtained by genetically modifying Pseudonocardia dioxanovorans MCI4804 to improve its 1,4-dioxane-degrading ability. Examples of such genetic modification include modifications that enhance the expression of genes involved in the 1,4-dioxane degradation reaction (e.g., THF monooxygenase α-subunit: thmA) and genes of their transcriptional regulators (e.g., GntR domain protein: GntR). The gene partial sequences of thmA of Pseudonocardia dioxanovorans CB1190 are shown in SEQ ID NOs: 5 and 6. Both SEQ ID NO: 5 and SEQ ID NO: 6 are registered in the database as partial sequences of the thmA gene, and on the database, the thmA gene is registered as these two sequences interrupted by a stop codon. The gene partial sequence of GntR of Pseudonocardia dioxanovorans CB1190 is shown in SEQ ID NO: 7.

[0023] The 1,4-dioxane-degrading bacterium can be cultured by a conventionally known method. The medium used for the culture is not particularly limited and any conventionally known medium used for culturing the 1,4-dioxane-degrading bacterium can be used. The composition for biodegradation treatment of cyclic ethers containing the 1,4-dioxane-degrading bacterium may contain the 1,4-dioxane-degrading bacterium and a solvent such as a medium or a cryopreservation solution.

[0024] The medium contains components necessary for the growth and proliferation of 1,4-dioxane-decomposing bacteria. Examples of medium components for growing and proliferating 1,4-dioxane-decomposing bacteria include, as a carbon source, monosaccharides such as glucose and fructose; disaccharides such as lactose, sucrose, and maltose; oligosaccharides; polysaccharides such as starch, etc., as a nitrogen source, organic nitrogen sources such as yeast extract; free amino acids such as arginine, glutamic acid, and glutamine; inorganic nitrogen sources such as ammonium chloride, water-soluble vitamins such as vitamin C and vitamin B12 as a vitamin source; fat-soluble vitamins such as vitamin A and vitamin E, and salts or hydrates thereof such as phosphate ions, magnesium ions, manganese ions, iron ions, cobalt ions, and zinc ions as a mineral source. Cultivation can be carried out by inoculating the bacteria into the above-mentioned medium that has been appropriately sterilized, and it may be carried out under conventionally known conditions. The inoculation amount of the bacteria into the medium may be, for example, 0.1 to 10 (v / v)%, preferably 0.2 to 5 (v / v)%, more preferably about 0.2 to 1 (v / v)%. The cultivation conditions are set to a pH of 5.0 to 9.0, preferably 6.0 to 8.0, a temperature of 25 to 40 °C, preferably 28 to 32 °C, and aerobic cultivation. The cultivation time may be appropriately set according to the amount of the culture solution and the target number of cells, etc.

[0025] The above-mentioned cultivation (main cultivation) can also be carried out after pre-cultivation if necessary. The method of pre-cultivation is not particularly limited. For example, it is preferably carried out aerobically under the control of a pH of 6.0 to 8.0 and a temperature of 28 to 32 °C in a medium containing carbon sources such as glucose and fructose, nitrogen sources such as yeast extract, free amino acids such as arginine, glutamic acid, and glutamine, vitamins, and inorganic salts.

[0026] In the biodegradation treatment method of cyclic ethers according to the present disclosure, 1,4-dioxane can be decomposed by contacting a 1,4-dioxane-decomposing bacterium or a composition containing the same with a cyclic ether in an appropriate reaction solution. Specifically, the 1,4-dioxane-decomposing bacterium is added to the treatment target as a composition in the culture solution, or added to the treatment target as the separated cells obtained by centrifuging the culture solution, or the separated cells obtained by centrifuging the culture solution are dried and added to the treatment target as dried cells, and then stirring is performed as necessary to advance the decomposition reaction of the cyclic ether. Alternatively, the bacterium or the composition is immobilized or fixed on a carrier, and the carrier is immersed in the treatment target, or the carrier is placed at a position where it can contact the treatment target in the flow path of the treatment target to cause a decomposition reaction of 1,4-dioxane. As a method for immobilizing or fixing the bacterium or the composition, any known method can be used without particular limitation as long as it is a method for immobilizing or fixing the 1,4-dioxane-decomposing bacterium so that it does not flow out from the carrier. Specific immobilization or fixation methods include, for example, the attached biofilm method using a carrier on which the bacterium or the composition adheres to form a biofilm, the supported culture method of mixing the carrier and the medium to culture the bacterium, the carrier binding method of binding the bacterium or the composition to a water-insoluble carrier, the method of enclosing the bacterium or the composition in the pores of the carrier under reduced pressure, the method of forming a crosslink in the bacterial cell by a reagent having two or more functional groups for immobilization, the inclusive immobilization method of confining the bacterium or the composition inside a polymer gel or film, etc., and further, carrier binding methods classified into covalent binding methods, physical adsorption methods, ionic binding methods, and biochemical specific binding methods by binding means, etc. can be mentioned.

[0027] The treatment target is not particularly limited, but may be industrial wastewater, domestic wastewater, pit wastewater, contaminated soil and groundwater, etc. Industrial wastewater may be wastewater from business establishments such as the chemical product industry such as photosensitive resin production, the petrochemical industry such as ethylene oxide and ethylene glycol production, and the oilification recycling of different waste plastics, and the chemical fiber industry such as polyester production. The 1,4-dioxane-decomposing bacterium and the composition for 1,4-dioxane decomposition treatment containing the same according to the present disclosure have high 1,4-dioxane decomposition activity, and thus can decompose and remove cyclic ethers such as 1,4-dioxane contained in the treatment target with higher efficiency compared to the prior art.

Example

[0028] [Example 1: Acquisition of Mutant Strain with Improved 1,4-Dioxane Degrading Ability] (1) First-stage Selection Using Pseudonocardia dioxanovorans CB1190 as the parent strain, a mutant library was created by introducing random mutations, and mutants showing improved 1,4-dioxane degrading ability were screened. Random mutations were introduced by ultraviolet irradiation or exposure to alkylating mutagens (EMS and NTG). The strains with introduced random mutations were statically cultured on ISP4 agar medium (Becton Dickinson BD Difco) for about 12 days, and mutants were isolated from about 2000 colonies.

[0029] Each mutant was precultured in 2 mL of ISP2 liquid medium (Becton Dickinson BD Difco) at 30 °C, 180 rpm for 2 days. The whole culture solution was inoculated into the activation medium dispensed 2 mL per well of a 24-well microtiter plate at 0.1 mL (5%) each, and cultured at 30 °C, 180 rpm for 4 days. 3 mL of 1,4-dioxane-containing decomposition test medium 1 was added to the wet bacterial cells obtained by centrifuging the culture solution to remove the supernatant, and reacted at 30 °C, 180 rpm for 2 hours. The residual concentration of 1,4-dioxane in the reaction solution was measured by gas chromatography analysis, and about 100 mutants with high 1,4-dioxane decomposition amounts were selected in descending order.

[0030] Activation Medium Composition Glucose 10 g Yeast extract (BD Difc) 3 g Sodium Glutamate 10 g KH2PO4 0.5 g 0.5 g of MgSO4·7H2O 0.01 g of FeSO4·7H2O 0.01 g of MnSO4·5H2O 0.01 g of ZnSO4·7H2O 0.01 g of CoCl2·6H2O 1.0 g of 1,4 - dioxan - 2 - one 1 L of distilled water

[0031] Composition of the decomposition test medium 1 0.1 g of 1,4 - dioxane 1.0 g of K2HPO4 1.0 g of (NH4)2SO4 0.2 g of MgSO4·7H2O 0.01 g of FeCl3 0.05 g of NaCl 0.05 g of CaCl2·6H2O 1 L of distilled water (pH 7.2)

[0032] Gas chromatography Apparatus: Agilent GC6890N Column: Restek FAMEWAX 30m×0.32mm I.D., film thickness 0.25μm Inlet temperature: 110°C, detector temperature (FID): 220°C Oven temperature: 35°C (0 min) → 5°C / min → 60°C (0 min) Carrier gas flow rate: He 2.0 ml / min Injection volume 1 μl, split ratio: 1:100

[0033] After culturing the selected mutant strain in liquid culture in the same manner as described above, the culture broth was centrifuged to remove the supernatant, and 2 mL of degradation test medium 2 (1,4-dioxane 100 mg / L, 1,3-dioxolane 500 mg / L, 2-chloromethyl-1,3-dioxolane 200 mg / L, 20 mM Na phosphate buffer pH 7.0) was added to the wet bacterial cells. The mixture was reacted at 30 °C and 180 rpm for 16 hours. The residual concentrations of 1,4-dioxane, 1,3-dioxolane, and 2-chloromethyl-1,3-dioxolane in the reaction solution were measured by HPLC analysis. Also, the total amount of bacterial cells in the reaction solution was recovered, and the dry cell weight (dcw) of the dried bacterial cells by air-drying to a constant weight was measured.

[0034] HPLC Apparatus: Shimadzu LC-20 HPLC system Column: COSMOSIL 5C 18 -PAQ 4.6 mm I.D.×250 mm, 30 °C Eluent: water 1.0 mL / min, Detector: RI (RID-10A) Injection volume: 10 μL

[0035] As an index for improving the 1,4-dioxane (DOX) degradation ability of the mutant strain, the degradation specific activity (degradation rate per culture cell weight [mg-DOX / g-dcw / hr]) was calculated, and PB-24-10 was obtained as the mutant strain showing the highest degradation specific activity.

[0036] (2) Second-stage selection The parent strain was changed from Pseudonocardia dioxanovorans CB1190 to PB-24-10, and the second-stage selection was carried out in the same procedure as in the first stage. PB-24-19 was obtained as the mutant strain showing high DOX degradation specific activity.

[0037] (3) Third-stage selection The parent strain was changed from PB-24-10 to PB-24-19, and the third-stage selection was carried out in the same procedure as in the second stage. As the mutant strain showing the highest DOX degradation ratio activity, PB-24-26 was obtained. This mutant strain was named Pseudonocardia dioxanivorans MCI4804 (Accession number: NITE P-03944).

[0038] Table 1 shows the degradation ratio activities of 1,4-dioxane (DOX), 1,3-dioxolane (DXL), and 2-chloromethyl-1,3-dioxolane (CMD) by mutant strains PB-24-10, PB-24-19, and PB-24-26 (MCI4804).

[0039] [Table 1]

[0040] [Example 2: Evaluation of 1,4-dioxane degradation ability of mutant strain PB-24-26 (MCI4804)] Pseudonocardia dioxanivorans CB1190 or mutant strain PB-24-26 (MCI4804) was inoculated into 20 mL of ISP2 liquid medium and cultured with shaking at 30 °C and 180 rpm for 5 days. 10 mL of the culture solution was inoculated into 100 mL of activation medium, and shaking culture was started at 30 °C and 180 rpm. The culture solution was collected 24 hours and 48 hours later. 10 mL of the collected culture solution was centrifuged to remove the supernatant and the cells were recovered, and the dry cell weight (dcw) of the cells was measured. In addition, 85 mL of the collected culture solution was centrifuged to remove the supernatant and the cells were recovered. The wet cells were washed with 20 mM Na phosphate buffer (pH 7.0) and then suspended in 20 mL of the same buffer. The cell suspension was transferred to a 200 mL flask, 1,4-dioxane was added (100 mg / L), and the shaking reaction was started at 30 °C and 180 rpm. The residual concentration of 1,4-dioxane in the reaction solution was measured by HPLC analysis 6 hours later. Table 2 shows the results of calculating the degradation ratio activity of 1,4-dioxane.

[0041] [Table 2]

[0042] [Example 3: Expression analysis of the 1,4-dioxane-degrading gene of mutant strain PB-24-26 (MCI4804)] Pseudonocardia dioxanovorans CB1190 or mutant strain PB-24-26 (MCI4804) was cultured in 100 mL of activation medium, and the culture broth was collected at the start of culture (0 h), 6 h, 24 h, and 48 h after the start of culture. 5 mL of the collected culture broth was centrifuged to remove the supernatant and the cells were recovered, and total RNA extraction, cDNA synthesis, and gene expression quantification by real-time PCR were performed using a commercially available kit. Referring to Non-Patent Document 1, the expression of the gene involved in the 1,4-dioxane degradation reaction (THF monooxygenase α-subunit: thmA), its transcriptional regulator gene (GntR domain protein: GntR), and the housekeeping gene (RNA polymerase sigma70 factor: rpoD) was quantified. The primer sequences for each gene are shown in Table 3.

[0043]

Table 3

[0044] The expression levels of thmA and GntR were compared by relative quantification using the ΔΔCt method (delta-delta Ct method). The difference between the ΔCt values [Ct(thmA) - Ct(rpoD)], [(Ct(GntR) - Ct(rpoD)] of the 0 h sample of CB1190 and the ΔCt values of each sample was taken as the ΔΔCt value, and 2 ( -ΔΔ Ct) was calculated as the relative ratio. The results are shown in Tables 4, 5 and Figures 1, 2. When the relative ratios were compared between samples at the same culture time, PB-24-26 (MCI4804) showed higher expression levels of thmA and GntR than CB1190.

[0045]

Table 4

[0046]

Table 5

[0047] [Example 4: Continuous Degradation Test of 1,4 - Dioxane by Mutant Strain PB - 24 - 26 (MCI4804)] A model wastewater containing 1,4 - dioxane (DOX), 1,3 - dioxolane (DXL), and 2 - chloromethyl - 1,3 - dioxolane (CMD) was prepared by makeup, and a continuous biodegradation test was carried out using the cultured cells of PB - 24 - 26 (MCI4804).

[0048] PB - 24 - 26 (MCI4804) was inoculated into 100 mL of activated medium and cultured with shaking at 30 °C and 180 rpm for 3 days, and then the whole amount (5%) was inoculated into 2000 mL of activated medium. Culturing was carried out at 30 °C, 200 rpm, aeration rate of 5 L / min, and pH 7.0 (controlled by adding 5N sulfuric acid and 1N NaOH solution) for 96 hours. After centrifugation to recover the cells, a small amount of a part of them was measured for air - dried constant weight, and as a result, the dry cell weight (dcw) was 11 g.

[0049] Using a small - scale MBR (Membrane Bioreactor: Membrane - Separation Activated Sludge Process) device, wastewater treatment operation was carried out under the following conditions. The configuration of the small - scale MBR device is shown in Figure 3. Liquid volume in the treatment tank: 2.8 L, aeration rate: 1.5 L / min, 30 °C (temperature - controlled circulating water) Membrane module: Hollow - fiber membrane made of PVDF (SADF membrane), membrane area: 0.021 m 2 Withdrawal flow rate: 1.9 mL / min (HRT: 24 hr), Raw water supply: Intermittent pump operation linked to the water - level gauge (set value 20 mm), Raw water composition (model wastewater) DOX: 14 mg / L, DXL: 40 mg / L, CMD: 16 mg / L PB - 24 - 26 cultured cells; 11 g - dcw (4.1 g - dcw / L treatment tank)

[0050] The time-course changes in the concentrations of 1,4-dioxane (DOX), 1,3-dioxolane (DXL), and 2-chloromethyl-1,3-dioxolane (CMD) in the treatment water were measured by HPLC analysis. The results are shown in Figs. 4 - 6. DOX and DXL were not detected until the operation time reached 78 hours, and complete decomposition continued (see Figs. 4 and 5). CMD was not detected until the operation time reached 102 hours, and complete decomposition continued (see Fig. 6).

[0051] [Example 5: Genome Analysis of Mutant Strain PB-24-26 (MCI4804)] Genomic DNA was extracted and purified from the cultured cells of PB-24-26 (MCI4804) using a commercially available kit, and long-read sequencing was performed using GridION from Oxford Nanopore Technologies.

[0052] [Procedure for Preparing a Sequence Library for Long Reads] From the genomic DNA obtained from the cultured cells, DNA fragments of 15 kbp or less were removed using BluePippin (Sage Science), and DNA fragments of 15 - 50 kbp were recovered. A sequencing library was prepared from the recovered DNA fragments using the Ligation Sequencing kit (SQK-LSK110) from Oxford Nanopore Technologies. [Long-Read Sequencing] Approximately 400 ng of the prepared library was applied to a FLO-MIN106 (R9.4.1) RevD flow cell for GridION from Oxford Nanopore Technologies, and sequencing was performed using a GridION sequencer. Basecalling of the sequencing data by GridION was performed in High-Accuracy mode.

[0053] [Procedure for Preparing a Sequence Library for Short Reads] The genomic DNA obtained from the cultured strain was fragmented using a DNA Shearing System M220 (Covaris Inc.). A sequencing library for NovaSeq was prepared from the fragmented DNA using an Illumina TruSeq DNA PCR-Free Library Prep kit. The prepared NovaSeq library was subjected to agarose electrophoresis, and DNA fragments of approximately 500 bp were recovered from the gel using a Zymoclean Large Fragment DNA Recovery Kit (Zymo Research). [Short-read sequencing] The recovered DNA fragments were sequenced using an Illumina NovaSeq 6000 sequencer with a read length of 150 bp in paired-end mode.

[0054] [Assembly method] The long-read data obtained from the GridION sequencer were assembled using assembly software (Canu version 2.2), and the sequences were corrected using Nanopolish version 0.13.3 to construct a genomic sequence. Furthermore, the accuracy of the genomic sequence was improved using Pilon version 1.23 with the short-read data obtained from the NovaSeq 6000 sequencer. As a result, a complete genomic sequence of PB-24-26 consisting of a total of four sequences, a circular chromosome of 7,102,409 bp, a circular plasmid (plasmid 1) of 192,350 bp, a circular plasmid (plasmid 2) of 142,361 bp, and a circular plasmid (plasmid 3) of 15,063 bp, was obtained.

[0055] [Comparison of the complete genomic sequences of CB1190 and PB-24-26] For the two sequences of the obtained genomic sequence of PB-24-26 and the already published genomic sequence of CB1190, automatic gene annotation was performed using DFAST, and gene homology analysis was performed using OrthoFinder 2 to create a list of genes possessed by the two genomes.

[0056] When comparing the lengths of the genomic sequences of CB1190 and PB-24-26, it was found that the length of the chromosome of PB24-26 was 5,838 bp longer as shown in Table 6. The main cause of this difference in length was that, as shown in Table 7, the number of genomic copies of transposases belonging to the IS (Insertion Sequence) family of IS110, IS256, IS630, and IS982 and other repeat sequences had increased in PB24-26.

[0057]

Table 6

[0058]

Table 7

[0059] The two genomic sequences were genome-aligned with MUMmer3, and SNVs between strains were detected with the show-snps option. The SNVs found were verified using 7,473,688 pairs of short-read data from the NovaSeq 6000 sequencer of PB-24-26. The short-read data was mapped to the genomic sequence of CB1190 using the read mapping tool BWA-MEM, and the results were visually confirmed for each SNV with the visualization tool IGV to determine validity.

[0060] As shown in Table 8, it was found that there were differences of 24 SNPs (single nucleotide polymorphisms) and 4 InDels (insertions and deletions) in the chromosome, 1 SNP and 1 InDel in plasmid 1, and 4 SNPs in plasmid 2. For plasmid 3, the sequences were completely identical between the two strains. Among these 29 SNPs and 5 InDels, there were also many non-synonymous substitutions within genes that changed the amino acids, and it is thought that these differences in the genomic sequences have led to differences in the dioxane degradation ability of the two strains.

[0061]

Table 8

Sequence Listing Free Text

[0062] Accession No. 1: Nucleotide sequence of the circular chromosome of Pseudonocardia dioxanivorans CB1190 Accession No. 2: Nucleotide sequence of the circular plasmid 1 of Pseudonocardia dioxanivorans CB1190 Accession No. 3: Partial nucleotide sequence of the circular plasmid 2 of Pseudonocardia dioxanivorans CB1190 Accession No. 4: Partial nucleotide sequence of the circular plasmid 2 of Pseudonocardia dioxanivorans CB1190 Accession No. 5: Gene partial sequence of the THF monooxygenase α-subunit of Pseudonocardia dioxanivorans CB1190 Accession No. 6: Gene partial sequence of the THF monooxygenase α-subunit of Pseudonocardia dioxanivorans CB1190 Accession No. 7: Gene sequence of the GntR domain protein of Pseudonocardia dioxanivorans CB1190 Accession No. 8: Nucleotide sequence of the thmA forward primer Accession No. 9: Nucleotide sequence of the thmA reverse primer Accession No. 10: Nucleotide sequence of the GntR forward primer Accession No. 11: Nucleotide sequence of the GntR reverse primer Accession No. 12: Nucleotide sequence of the rpoD forward primer Accession No. 13: Nucleotide sequence of the rpoD reverse primer

Claims

1. A mutant strain derived from Pseudonocardia dioxanivorans CB1190, in which the gene expression level of THF monooxygenase α-subunit and / or GntR domain protein is increased compared to the wild strain.

2. The mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to claim 1, having a base substitution, insertion, or deletion at at least one position selected from the following positions in the gene sequences of the circular chromosome and circular plasmids 1-3 of Pseudonocardia dioxanivorans CB1190. Positions 115984, 350134, 350152, 350167, 350326, 350356, 350473, 350475, 598967, 642212, 1190528, 2082787, 2603923, 2634727, 2925050, 2932573, 2956189, 3035551, 3234928, 3507315, 5545605, 5640340, 5640341, 5640397, 5658991, 6194590 of the gene sequence of the circular chromosome shown in SEQ ID NO: 1; Positions 100129, 186574 of the gene sequence of circular plasmid 1 shown in SEQ ID NO: 2; Positions 6688, 6717, 6729 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO: 3; Position 31513 of the gene partial sequence of circular plasmid 2 shown in SEQ ID NO:

4.

3. The mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to claim 2, which is Pseudonocardia dioxanivorans MCI4804 strain (Accession No.: NITE P-03944).

4. A method for biodegradable treatment of a cyclic ether contained in a treatment target, comprising the step of contacting the cyclic ether with the mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to any one of claims 1-3.

5. The method according to claim 4, wherein the cyclic ether is one or more selected from the group consisting of 1,4-dioxane, 1,3-dioxolane, and 2-chloromethyl-1,3-dioxolane.

6. The method according to claim 5, wherein the treatment target is wastewater or activated sludge.

7. A composition for biodegradative treatment of cyclic ethers, comprising a mutant strain derived from Pseudonocardia dioxanivorans CB1190 according to any one of claims 1 to 3.

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