Genetic transformation method for Huiarenaceae microflora and application of genetic transformation method

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a genetic transformation method aiming at Huiarenaceae microflora and application of the genetic transformation method. Specifically, a genetic transformation method aiming at Huiarenaceae microbe is established for the first time, exogenous plasmids are subjected to in-vitro methylation modification by using methylase in a cell-free system of the strain, and the activity of endonuclease is inhibited in combination with a combination mode of ATP consumption and EDTA chelation; the problem of exogenous plasmid degradation caused by a complex and unknown strain limitation modification system is effectively solved, and the immune barrier is successfully broken through. Meanwhile, by optimizing competent cell preparation conditions and electrotransformation parameters, the transformation efficiency reaches an operable level, and the requirements of molecular operation are met. The invention provides a key technical support for molecular modification of Huiaromatic Zhongaceae microflora, is helpful for excavating the metabolic potential of the Huiaromatic Zhongaceae microflora, improves the stress resistance, and promotes the industrial application of the Huiaromatic Zhongaceae microflora in the field of environmental governance such as combined pollution wastewater treatment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a genetic transformation method for Huifang Zhongke microorganism and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Huifang Zhongke Microbial ( Casimicrobium huifangae This strain is the type species of the genus *Casimicrobiaceae* within the phylum *Betaproteobacteria*, and is one of the 28 core functional groups in the activated sludge microbiome of urban wastewater treatment plants. This strain exhibits unique metabolic plasticity, combining metabolic diversity in mineralizing various carbohydrates and degrading various aromatic compounds with tolerance to multiple heavy metals and antibiotics. It can efficiently remove carbon, nitrogen, and phosphorus pollutants from wastewater, making it a strain with strong ability to treat complex polluted wastewater.

[0004] However, the genetic transformation and molecular operating system of this strain has not yet been established, hindering its molecular modification and improvement of stress resistance. Whole-genome methylation analysis revealed that this strain possesses nine restriction and modification systems (RM systems; including two type I systems, six type II systems, and one type III system), involving 18 related enzymes (including nine methyltransferases and nine restriction endonucleases). Their functions and recognition sequences are currently unclear, resulting in the inability to transform multiple broad-host-range plasmids into this strain.

[0005] In existing technologies, the following solutions are mainly used to address the problem of microbial restriction modification systems hindering plasmid introduction: First, commercially available methyltransferases are used to modify exogenous plasmids; second, relevant methyltransferases are expressed in *E. coli*, and in vivo methylation is performed after constructing a shuttle vector, or the methyltransferases are purified for in vitro methylation; third, the host's restriction modification system is removed through gene knockout or mutation. However, these methods all have limitations: they are more suitable for hosts with a small number of restriction modification systems, known methyltransferase functions, or easy expression in *E. coli*. For strains like *E. coli* (such as *E. coli*), which have multiple sets of unknown recognition sequence restriction modification systems and lack a mature genetic operating system, the above methods are insufficient to obtain fully methylated plasmids and cannot effectively knock out restriction modification systems, thus failing to meet the needs of genetic transformation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a genetic transformation method for *Huifang Zhongke Microbe* and its application. Specifically, this invention establishes for the first time a genetic transformation method for *Huifang Zhongke Microbe*. By utilizing the methyltransferases in the strain's own cell-free system to perform in vitro methylation modification of exogenous plasmids, and combining this with a combination of ATP consumption and EDTA chelation to inhibit endonuclease activity, the degradation problem of exogenous plasmids caused by the complexity and unknown nature of the restriction modification system of this strain is effectively solved. Based on the above research results, this invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: A first aspect of the present invention provides a genetic transformation method for *Huifang Zhongke Microbe*, the method comprising: S1. Prepare a cell-free system of Huifang Zhongke microorganisms and inhibit its endonuclease activity; S2. Using the treated cell-free system as the methyltransferase source, the exogenous plasmid was modified by in vitro methylation. S3. Preparation of Huifang Zhongke Microbial Competent Cells; S4. Introduce the methylated exogenous plasmid into competent cells.

[0008] In step S1, the preparation method of the Huifang Zhongke microbial cell-free system includes: culturing Huifang Zhongke microbial bacteria and adding exogenous plasmids to stimulate and induce the complete expression of the restriction modification system of the strain; collecting cells from different culture time periods; mixing and centrifuging to harvest cells; and centrifuging to collect the supernatant after cell wall disruption, which is the cell-free system. The exogenous plasmid can be a wide host range exogenous plasmid. In one specific embodiment of the present invention, the wide host range exogenous plasmid can be pBBR1MCS-2.

[0009] Furthermore, the cell wall disruption buffer comprises: 15-25 mM Tris-HCl, 15-25 mM NaCl, 5-15 mM β-mercaptoethanol, and 0.5-5 mM PMSF, with an alkaline pH. Alternatively, the cell wall disruption buffer comprises: 20 mM Tris-HCl, 20 mM NaCl, 10 mM β-mercaptoethanol, and 1 mM PMSF, with a pH of 8.0.

[0010] Furthermore, cells were collected after different culture periods: 5 hours, 10 hours, 15 hours, and 20 hours of culture.

[0011] Furthermore, firefly luciferase (with ATPase activity) and EDTA were used to inhibit endonuclease cleavage activity in a cell-free system (protein concentration approximately 4-8 mg / ml). Specifically, firefly luciferase (1-2 μL) was added and incubated (at 30°C for 10-30 min) to hydrolyze ATP in the cell-free system; then EDTA (10-90 mM) was added and incubated (at 30°C for 10-30 min) to chelate magnesium ions and other metal ions. This combined method effectively inhibited endonuclease cleavage activity in the cell-free system.

[0012] Furthermore, in step S2, the total volume of the reaction mixture for in vitro methylation modification is 100 μL, comprising: 20 μL of cell-free system treated with inhibition, 0.1 mM SAM, 0.01% BSA, 2-5 μg of exogenous plasmid, 20 mM Tris-HCl, 20 mM NaCl, 2 mM DTT, 10 mM EDTA, pH 8.0; the reaction conditions are incubation at 25-35℃ (preferably 30℃) for 12-18 hours, with SAM added 3-5 times during the process.

[0013] Furthermore, the plasmid is extracted from the reaction mixture to obtain the methylated plasmid.

[0014] In step S3, the method for preparing the competent cells includes: culturing *Huifang Zhongke Microbe* for a period of time (e.g., 2-6 hours), adding 0.2-2 mM EDTA, and continuing to culture for 5-10 hours until OD... 600nm The concentration was set at 0.4-0.6. Cells were harvested by centrifugation, resuspended in 10-20% glycerol solution containing 1-5 mM EDTA and washed to obtain competent cells.

[0015] In this invention, the culture medium used for culturing Huifang Zhongke microbes can be 3×R2A medium.

[0016] In step S4, the import method can be electroporation. Specifically, the electroporation buffer is a 15% glycerol solution, supplemented with 2 mM EDTA and 5 μg Type One Restriction Inhibitor; the electroporation conditions are voltage 1.25 kV, resistance 200 Ω, and capacitance 25 μF.

[0017] Experiments have verified that the above method can successfully transform exogenous plasmids into Huifang Zhongke microbial cells, with a transformation efficiency reaching an operable level (>1.0×10⁻⁶). 5 CFU / μg DNA).

[0018] A second aspect of the invention provides the application of the genetic transformation method in any one or more of the following: (a) Molecular modification of Huifang Zhongke microorganisms; (b) Improved stress resistance of Huifang Zhongke microorganisms; (c) Huifang Zhongke Microbial Environmental Pollution Control.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows: The above-mentioned technical solution establishes for the first time a genetic transformation method targeting *Huifang* microorganism. By utilizing the methyltransferases in the strain's own cell-free system to modify exogenous plasmids in vitro, and combining ATP consumption with EDTA chelation to inhibit endonuclease activity, it effectively solves the problem of exogenous plasmid degradation caused by the complexity and unknown nature of the strain's restriction modification system, successfully overcoming its immune barrier. Simultaneously, by optimizing the competent cell preparation conditions and electroporation parameters, the transformation efficiency reaches an operable level, meeting the requirements of molecular manipulation.

[0020] The above-mentioned technical solution does not rely on commercially available methyltransferases or E. coli expression systems, nor does it require gene knockout of restriction modification systems. It is simple to operate, low in cost, and the methylation modification is highly targeted, ensuring the stability of exogenous plasmids in Huifang Zhongke microorganisms. This technical solution provides key technical support for the molecular modification of Huifang Zhongke microorganisms, helping to explore its metabolic potential, improve its stress resistance, and promote its industrial application in environmental governance fields such as the treatment of complex polluted wastewater. Therefore, it has good practical application value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This is a graph showing the degradation detection of unmethylated DNA fragments containing type I RM system-targeting motifs in a cell-free system of Huifang Zhongke microorganisms after ATP depletion treatment with luciferase (which has ATPase activity). Note: "+" indicates the addition of luciferase to the reaction incubation system; "Indicates that it was not added to the reaction incubation system. (a) The DNA fragment contains the type I RM system-targeting motif fragment 5'-GAGGCCAGTTTGCT-3'(GAGNNNNNNNTGCT); (b) The DNA fragment contains the type I RM system-targeting motif fragment 5'-GAGAGCTATTG-3'(GAGNNNNRTTG). Cell-free systems that have depleted ATP through luciferase treatment no longer degrade the target DNA fragment."

[0023] Figure 2 This describes the degradation detection of unmethylated plasmids in the EDTA-treated Huifang Zhongke microbial cell-free system. " " indicates the negative control group, reflecting the degradation of exogenous plasmids in a reaction system without EDTA; "+" indicates the positive control group, where the reaction system does not contain EDTA. In this positive control group, exogenous plasmids were first added to experimental groups 1-5 and the negative control, respectively. After the reaction, the exogenous plasmids were added to the positive control group, reflecting the absence of plasmid degradation. The EDTA concentrations for lanes 1-5 were 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM, respectively. EDTA can effectively inhibit endonuclease activity in a cell-free system.

[0024] Figure 3 This is a graph showing the degradation detection of methylated plasmids in the Huifang Zhongke microbial cell-free system. Note: "This indicates the negative control group, whose plasmids were not methylated (incubated overnight in a non-methylated system containing 10 mM EDTA, where endonuclease activity was inhibited, without the addition of methyl donor SAM). After recovering the methylated plasmids, they were incubated in a system without EDTA and with the addition of Mg." 2+ The plasmids were subjected to cleavage experiments in a crude cell extract containing endonuclease activity. "1" represents the experimental group, in which the plasmids were incubated overnight in a cell-free system with endonuclease activity inhibited by 10 mM EDTA. The plasmids were then methylated with 80 μM methyl donor SAM. After recovering the methylated plasmids, they were incubated in a Mg2+-free system without EDTA. 2+ The cleavage experiment was conducted in a cell-free system with endonuclease activity. "+" indicates the positive control group, and the procedure for the methylation reaction was the same as the negative control group. During the cleavage experiment, the exogenous plasmid was first added to both the negative control group and the experimental group. After the reaction was complete, the exogenous plasmid was added to the positive control group. The positive control group indicates that the plasmid was not degraded. Methylated plasmids are not degraded in the cell-free system.

[0025] Figure 4 The growth curve of Huifang Zhongke Microbe (a) and the effect of cells at different growth stages on electroconversion efficiency (b) are shown.

[0026] Figure 5 The effects of voltage (a) and resistance (b) on the electroconversion efficiency of Huifang Zhongke microorganisms are shown.

[0027] Figure 6 This study investigated the effects of different electroporation buffers on the electroconversion efficiency of *Microbes huifangensis*. The test buffers included 15% glycerol, EB (270 mM sucrose, 15% glycerol, pH 7.0), PEB (1 mM Na2HPO4, 270 mM sucrose, 15% glycerol, pH 7.0), and SMP (1 mM Na2HPO4, 1 mM MgCl2, 270 mM sucrose, 15% glycerol, pH 7.0).

[0028] Figure 7 The images show the growth of Huifang Zhongke Microbe transformants containing pBBR1MCS-2 on kanamycin plates (a) and selected single-colony PCR verification (b). PCR verification was performed using universal primers M13F / M13R for pBBR1MCS-2. Detailed Implementation

[0029] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. In the following embodiments, the molecular biology techniques and methods used are all conventional methods known in the art. For those not specifically described, please refer to *Molecular Cloning: A Laboratory Manual* (Sambrook and Russell, 2001). Those skilled in the art can use other conventional techniques, methods, and reagents in the art based on the embodiments provided by the invention, and are not limited to the specific embodiments of the present invention. The examples given in this invention are merely preferred embodiments of the invention. It should be noted that the following description is only for explaining the invention and is not intended to limit the invention in any way. Any simple modifications, equivalent changes, and modifications made to the embodiments based on the technical essence of the invention are within the scope of the technical solution of the present invention. Unless otherwise specified, the materials, reagents, plasmids, strains, kits, etc., used in the embodiments are obtained commercially.

[0032] Example 1: Preparation of Huifang Zhongke Microbial Cell-Free System (1) Cell culture and harvesting (a) Take Huifang Zhongke microbial glycerol tubes, stored at 80℃, were inoculated at a rate of 2% into 5 ml of antibiotic-free 3×R2A liquid medium and cultured at 30℃ with shaking at 200 rpm until OD reached. 600nm After the value is around 0.6, the activated strain is passaged once more.

[0033] (b) Inoculate the activated culture into 50 mL of 3×R2A liquid medium at an inoculation rate of 2%, and culture at 30°C with constant temperature shaking at 200 rpm for 10 h. Then, inoculate the entire seed culture into 0.5 L of 3×R2A liquid medium.

[0034] (c) Four hours after inoculation, add 5-10 μg of the broad host range exogenous plasmid pBBR1MCS-2 to the culture to stimulate the complete expression of the restriction modification system of the induced strain. Collect 50 ml of cells after 5, 10, 15 and 20 hours of culture, centrifuge at 9000 rpm for 15 min at 4℃, discard the supernatant and retain the cell pellet.

[0035] (2) Centrifugation to break the cell wall The harvested cells were resuspended in 10 ml of cell disruption buffer (20 mM Tris-HCl, 20 mM NaCl, 10 mM β-mercaptoethanol, 1 mM PMSF, pH 8.0), then combined and centrifuged again. The collected cells were resuspended in 6 mL of cell disruption buffer and disrupted using a high-pressure homogenizer (operating pressure 1500 bar) until the lysate was clear. The cells were then centrifuged at 9000 rpm for 30 min at 4°C. The supernatant was collected after centrifugation and placed on ice for later use. This liquid is the cell-free system (protein concentration approximately 4-8 mg / ml).

[0036] (3) Inhibition of endonuclease cleavage activity Add 1-2 μL of firefly luciferase (Beyotime Biotechnology Co., Ltd., catalog number: S0026) to the cell-free system obtained above, mix well, and incubate at 30°C to hydrolyze ATP in the cell-free system; after 30 min, add 50 mM EDTA and mix well, then incubate at 30°C with slow shaking (50 rpm) for 20 min to chelate magnesium ions and other metal ions. This combination method can prevent the cleavage activity of endonuclease in the cell-free system. Place on ice for later use.

[0037] Example 2: In vitro methylation modification of exogenous plasmids using the Huifang Zhongke microbial cell-free system (1) Culture pBBR1MCS-2-carrying cells in LB medium containing 50 μg / L kanamycin. E. coli JM110 cells were cultured at 37°C with shaking at 200 rpm for 16 hours, followed by centrifugation at 4°C and 9000 rpm for 20 min to harvest the cells. Plasmids were then extracted using a plasmid extraction kit (Tiangen Biotech Co., Ltd.) according to the instructions for subsequent experiments.

[0038] (2) Using the cell-free system with inhibited endonuclease cleavage activity prepared in Example 1 as the enzyme source of methyltransferase, the exogenous plasmid pBBR1MCS-2 was methylated in vitro. The total volume of the reaction mixture was 100 μL, which contained 20 μL of the cell-free system prepared by the above method, 20 μL of 0.5 mM SAM, 10 μL of 0.1% BSA, 10 μL of pBBR1MCS-2 (5 μg), 20 μL of buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM DTT, 50 mM EDTA, pH 8.0), and 20 μL of ddH2O. The reaction mixture was incubated in a water bath at 30°C for 12-18 hours, with SAM added 3-5 times in between to completely methylate the exogenous plasmid.

[0039] (3) Extract plasmids from the above reaction solution, first extract once with an equal volume of chloroform, take the supernatant and add an equal volume of isopropanol. After precipitation at 20℃ for 2-3 hours, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash the precipitate with 75% ethanol, centrifuge at 12000 rpm for 10 min, discard the supernatant, dry, and dissolve the precipitate in 15 µL ddH2O. This plasmid is the methylated plasmid and can be used for subsequent electroconversion experiments.

[0040] (4) The methylated plasmid extracted above was cut and detected using the Huifang Zhongke microbial cell-free system (preparation method is described in Example 1) without restriction of endonuclease. The reaction mixture totaled 100 µL and contained 20 µL of 5× cutting buffer (100 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 10 mM DTT, pH 8.0), 10 µL of 0.1% BSA, 10 µL of pBBR1MCS-2 (0.5-1 µg) modified by in vitro methylation, 20 µL of Huifang Zhongke microbial cell-free system without restriction of endonuclease, and 40 µL of ddH2O. After reacting at 30℃ for 45 min, the plasmid was extracted by chloroform extraction, isopropanol precipitation, washing with 75% ethanol, drying and dissolving in ddH2O. The integrity of the plasmid was then detected by agarose gel electrophoresis.

[0041] Example 3: Preparation of competent cells from Huifang Zhongke microorganisms and electroconversion of exogenous plasmids (1) Preparation of competent cells (a) will Huifang Zhongke Microorganisms were inoculated into antibiotic-free 3×R2A medium (2% inoculum) at 80℃ and cultured with shaking at 30℃ (200 rpm). OD 600nm When the concentration reaches 0.4-0.6, passage the cells again to reactivate them and restore their metabolic activity.

[0042] (b) Take an equal amount of activated bacterial solution and inoculate it into 50 mL of fresh 3×R2A liquid medium for the preparation of competent cells.

[0043] (c) Incubate at 30℃ with shaking. After 4 hours of inoculation, add 0.4 mM EDTA to the culture and continue incubation for about 2-4 hours. When OD 600nm When the concentration reaches 0.4, the culture is placed in an ice bath for 10 min, and then the bacterial solution is transferred to a sterile 2 mL centrifuge tube.

[0044] (d) After centrifugation at low temperature (4℃, 9000 rpm, 6 min), discard the supernatant, aspirate the culture medium from the bottom of the centrifuge tube, place it on ice, add 2 mL of pre-cooled 15% glycerol solution containing 2 mM EDTA to resuspend, and let it stand on ice for 10 min.

[0045] (e) Repeat the previous step twice, washing the cells a total of three times. For the last wash, resuspend the cells (100 μL) in the remaining solution at the bottom.

[0046] (f) Aliquot the resuspended cells into pre-cooled 1.5 mL EP tubes, 25 μL per tube, which are the competent cells.

[0047] (2) Electroconversion of exogenous plasmids (a) Pre-cool the 1 mm electroporation cuvette on ice. Mix 25 μL of freshly prepared competent cells with 1 μL of the methylated plasmid pBBR1MCS-2 obtained above in electroporation buffer (15% glycerol; 2 mMEDTA; 5 μg Type One Restriction Inhibitor, LGC Biosearch Technologies, catalog number: 75928-006), then transfer to the 1 mm electroporation cuvette and place on ice for 10 min before electroporation.

[0048] (b) Set the BioRad electroporation instrument parameters to 1.25 kV voltage, 200 Ω resistance, and 25 μF capacitance for electric shock.

[0049] (c) Immediately after the electroshock, the cells were recovered with 1 mL of 3×R2A liquid medium, transferred to 1.5 mL EP tubes, and then cultured on a 30°C constant temperature shaker for about 4 h to revive the cells.

[0050] (d) The bacterial culture was serially diluted, and 100 μL of each solution was spread onto selection medium containing 50 μg / mL kanamycin resistance. The cultures were incubated at 30°C for 3-4 days. The number of transformants was counted, and the electroconversion efficiency was calculated. The formula for calculating the electroconversion efficiency is:

[0051] (e) Using this optimized method, plasmid pBBR1MCS-2 was successfully transformed into Huifang Zhongke Microbial cells, and the transformation efficiency reached an operable level (>1.0×10⁻⁶). 5 CFU / μg DNA).

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetic transformation method for *Huifang Zhongke Microbe*, the method comprising: S1. Prepare a cell-free system of Huifang Zhongke microorganisms and inhibit its endonuclease activity; S2. Using the treated cell-free system as the methyltransferase source, the exogenous plasmid was modified by in vitro methylation. S3. Preparation of Huifang Zhongke Microbial Competent Cells; S4. Introduce the methylated exogenous plasmid into competent cells.

2. The genetic transformation method as described in claim 1, characterized in that, In step S1, the preparation method of the Huifang Zhongke microbial cell-free system includes: culturing Huifang Zhongke microbial bacteria and adding exogenous plasmids to stimulate and induce the complete expression of the restriction modification system of the strain; collecting cells from different culture time periods; mixing and centrifuging to harvest cells; and centrifuging to collect the supernatant after cell wall disruption, which is the cell-free system.

3. The genetic transformation method as described in claim 2, characterized in that, The exogenous plasmid is a wide host range exogenous plasmid, and more specifically, the wide host range exogenous plasmid is pBBR1MCS-2.

4. The genetic transformation method as described in claim 2, characterized in that, The components of the cell wall disruption buffer are: 15-25 mM Tris-HCl, 15-25 mM NaCl, 5-15 mM β-mercaptoethanol, 0.5-5 mM PMSF, and the pH is alkaline.

5. The genetic transformation method as described in claim 1, characterized in that, Firefly luciferase and EDTA were used to inhibit endonuclease cleavage activity in a cell-free system.

6. The genetic transformation method as described in claim 1, characterized in that, In step S2, the reaction conditions are a 25-35℃ incubator for 12-18 hours, with SAM added 3-5 times during the process; Furthermore, the plasmid is extracted from the reaction mixture to obtain the methylated plasmid.

7. The genetic transformation method as described in claim 1, characterized in that, In step S3, the method for preparing the competent cells includes: culturing *Huifang Zhongke Microbe* for a period of time, adding 0.2-2 mM EDTA, and continuing to culture for 5-10 hours until OD... 600nm The concentration was set at 0.4-0.

6. Cells were harvested by centrifugation, resuspended in 10-20% glycerol solution containing 1-5 mM EDTA and washed to obtain competent cells.

8. The genetic transformation method as described in claim 1, characterized in that, In step S4, the import method is electrical conversion.

9. The genetic transformation method as described in claim 1, characterized in that, The culture medium used for culturing Huifang Zhongke microbes was 3×R2A medium.

10. The use of the genetic transformation method according to any one of claims 1-9 in any one or more of the following: (a) Molecular modification of Huifang Zhongke microorganisms; (b) Improved stress resistance of Huifang Zhongke microorganisms; (c) Huifang Zhongke Microbial Environmental Pollution Control.

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