Novel efficient clostridium pasteurianum genetic transformation method

Abstract

The invention discloses a novel efficient clostridium pasteurianum genetic transformation method which comprises the following steps: culturing and collecting clostridium pasteurianum thalli, centrifuging, washing, adding SMP buffer for resuspension, adding carrier plasmids or methylated plasmids, uniformly mixing, carrying out ultrasonic treatment on the obtained suspension under an ice bath condition, and carrying out freeze drying to obtain the clostridium pasteurianum genetic transformation. And transferring the suspension subjected to ultrasonic treatment into an electric revolving cup for electric shock treatment, quickly adding the suspension into a culture medium after electric shock, resuscitating, coating resuscitated bacterial liquid onto an RCM culture medium solid plate containing chloramphenicol resistance, culturing, and counting the growth condition of bacterial colonies. A novel transformation system is adopted for the clostridium pasteurianum, ultrasonic treatment is performed before genetic vector electric shock transformation, shuttle vector plasmids are subjected to methylation treatment, the obtained transformation efficiency is far higher than that of single ultrasonic treatment or single electric shock treatment, and a new way for transferring exogenous genes into the clostridium pasteurianum is developed; the method is simple, high in operability and good in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically, is a novel and efficient genetic transformation method of Clostridium pasteurianum. Background Art

[0002] Clostridium pasteurianum ( Clostridium pasteurianum , DSM 525) was first discovered by French scientist Louis Pasteur in the early 1870s. It was originally discovered and named because it can produce butyrate through metabolism, so butyrate fermentation is one of the main metabolic pathways of Clostridium pasteurianum. In addition, Clostridium pasteurianum can produce a large amount of hydrogen during the fermentation process. This feature makes Clostridium pasteurianum potential in biohydrogen production research and helps to develop new technologies in the field of bioenergy. Bioenergy is clean and efficient, which helps to alleviate the energy crisis and reduce greenhouse gas emissions. To further tap the biological production potential of Clostridium, especially in improving bioenergy, it is necessary to rely on strain improvement technology.

[0003] Since Clostridium pasteurianum has a thick cell wall, there are a large number of non-specific nucleases on the cell wall, which hinder the entry of exogenous genes. It has a type II RM system, which includes a separate methyltransferase and an endonuclease, which can recognize and digest exogenous DNA of a specific sequence, seriously affecting the stability of exogenous genes in Clostridium. Because of its strict anaerobic and easy spore formation characteristics, it is very difficult to perform genetic transformation operations on it. Therefore, it is urgent to find a reliable and efficient genetic transformation method to break through the bottleneck of gene editing operations in Clostridium pasteurianum and make an important contribution to the green biosynthesis of bioenergy.

[0004] The premise of achieving genetic modification of strains is to establish a means of efficiently introducing exogenous DNA into host cells. The commonly used DNA transfer method in experiments is mainly electroporation transformation. As an efficient gene transfer method, electroporation transformation provides a way for genetic manipulation of Clostridium pasteurianum. Since the first electroporation transformation in Clostridium pasteurianum in the 1990s, researchers at home and abroad have made important progress in optimizing electroporation transformation conditions and constructing efficient vectors. In 2010, Heap et al. constructed a series of shuttle expression vectors suitable for Clostridium pasteurianum, which significantly improved the efficiency of electroporation transformation. In 2012, Cartman et al. optimized the composition of the electroporation buffer and increased the efficiency of electroporation transformation by more than 10 times by adding glycerol and sucrose. In 2017, Bruder et al. used the restriction modification system to develop a new electroporation transformation strategy to further improve the transformation efficiency. However, low transformation efficiency is still a bottleneck problem in the transformation of Clostridium pasteurianum, which has greatly affected the further genetic manipulation of Clostridium pasteurianum. Summary of the invention

[0005] To overcome the deficiencies in the prior art, a novel and efficient genetic transformation method for Clostridium pasteurianum is provided. The genetic transformation method comprises the following steps: (1) Inoculate the Clostridium pasteurianum seed solution into 2×YTG medium and culture at 37°C for 2 h. Then add glycine to the medium and continue culturing until the bacterial solution OD 600 The value is 0.6; (2) Collect Clostridium pasteurianum cells by refrigerated centrifugation, wash with SMP buffer, and then refrigerated centrifuge and wash twice; (3) After washing, add SMP buffer to resuspend, then add vector plasmid and mix well; (4) The suspension obtained in step (3) was ultrasonicated for 20 s in an ice bath with an ultrasonic input power of 200 W and an ultrasonic frequency of 40 kHz; (5) Transfer the suspension after ultrasound into an electric rotating cup, add ethanol to mix well, and let it stand for 5 min; then perform electric shock with a voltage of 1.8 kV, a capacitance of 25 µF, a resistance of ∞ Ω, and a time of 10-18 ms. After electric shock, quickly add 2×YTG culture medium and let it stand in a 37°C incubator for recovery for 12-16 h; (6) Apply the revived bacterial solution to a solid plate containing RCM medium containing chloramphenicol resistance, incubate in a 37°C incubator for 36-48 hours, and count the growth of colonies. The chloramphenicol concentration used is preferably 15-25 ug / mL.

[0006] Furthermore, in the above-mentioned novel and efficient genetic transformation method of Clostridium pasteurianum, the preparation method of the Clostridium pasteurianum seed solution in step (1) is as follows: inoculating the frozen wild-type Clostridium pasteurianum into 2×YTG medium, culturing at 37°C for 12-14 h, and making the bacterial solution OD 600 The value is 0.8-1, and the added amount of glycine is preferably 12.5 g / L.

[0007] Furthermore, in the above-mentioned novel and efficient genetic transformation method of Clostridium pasteurianum, the refrigerated centrifugation conditions are preferably: centrifugation at 4°C and 8500 g for 10 min.

[0008] Furthermore, in the novel and efficient genetic transformation method for Clostridium pasteurianum, the plasmid is pMTL-IV, methylated pMTL-IV, pMTL 85151 or methylated pMTL 85151, preferably methylated pMTL-IV or methylated pMTL 85151.

[0009] The methylated pMTL-IV or methylated pMTL 85151 plasmid is a vector plasmid pMTL-IV or pMTL 85151 subjected to methylation treatment (CpG) in order to circumvent the restriction modification system inside Clostridium pasteurianum and prevent it from degrading the vector. The treatment method is: using a CpG methyltransferase kit (purchased from New England Biolabs, NEBuffer / SAM solution are both items in the kit), the plasmid is methylated, and the following ingredients are added according to the formula ratio: NEBuffer 2 (10×) 5 uL, SAM solution 5 uL, plasmid 1 ug, methyltransferase 1 uL, enzyme-free water is added to 50 uL, mixed evenly, incubated at 37°C for 1 hour, and incubated at 65°C for 20 minutes to stop the reaction, and the methylated plasmid can be obtained.

[0010] Furthermore, in the above-mentioned novel and efficient genetic transformation method of Clostridium pasteurianum, the composition of the 2×YTG medium in step (1) is: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose, and 0.2 g / L cysteine.

[0011] Furthermore, in the above-mentioned novel and efficient genetic transformation method of Clostridium pasteurianum, the SMP buffer components in step (2) and step (3) are: 92.4 g / L sucrose, 0.093 g / L magnesium chloride, and 0.78 g / L sodium dihydrogen phosphate.

[0012] Furthermore, in the above-mentioned novel and efficient genetic transformation method of Clostridium pasteurianum, the RCM medium described in step (6) is a reinforced Clostridium medium purchased from Haibo Biotechnology.

[0013] Furthermore, in the novel and efficient genetic transformation method of Clostridium pasteurianum, the amount of the plasmid in step (3) is as follows: 9.5 mL of 2×YTG medium is cultured to a bacterial solution OD of 600 The amount of bacteria collected when the value is 0.6 is resuspended in 580 μL SMP buffer and then added with 1-2 ug of plasmid. The optimal amount is 1 ug.

[0014] The present invention has the advantages that: 1. The present invention adopts a novel "ultrasound + electric shock" transformation system for Clostridium pasteurianum. Ultrasound treatment is first performed before the electric shock transformation of the genetic carrier. Experimental results show that the transformation efficiency obtained by the novel genetic transformation system is much higher than the transformation efficiency of ultrasonic treatment or electric shock treatment alone, and even has the effect of "1+1 is much greater than 2", which opens up a new way to transfer exogenous genes into Clostridium pasteurianum; 2. Methylation treatment (CpG) of the shuttle vector plasmid suitable for Clostridium pasteurianum can significantly improve the transformation efficiency of Clostridium pasteurianum; 3. The genetic transformation method of the present invention is simple, highly operable and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Attached Figure 1 The growth of bacterial colonies on the plates under the three different transformation system conditions in Example 1, with the plates from left to right being electric shock, ultrasound, and ultrasound + electric shock; Attached Figure 2 is the number of colonies counted under the conditions of three different transformation systems in Example 1; Attached Figure 3 The growth of bacterial colonies on the plates under the three different vector plasmid concentrations in Example 2, with the plates from left to right using 0.5 ug, 1 ug, and 2 ug of plasmid pMTL-IV; Attached Figure 4 is the number of colonies and transformants counted under the conditions of three different vector plasmid concentrations in Example 2; Attached Figure 5 The growth of colonies on the plates under the four different vector plasmid conditions in Example 3, from left to right, the plates are the ones using plasmid pMTL-IV, methylated pMTL-IV, pMTL 85151, and methylated pMTL 85151; Attached Figure 6 is the number of colonies counted under the conditions of using plasmid pMTL-IV and methylated pMTL-IV in Example 3; Attached Figure 7 The growth of bacterial colonies on the plates under the conditions of three different chloramphenicol concentrations in Example 4, the plates from left to right are chloramphenicol concentrations of 15 ug / mL, 25 ug / mL, and 35 ug / mL; Attached Figure 8 is the number of transformants counted under the three different chloramphenicol concentration conditions in Example 4; The meaning of CFU / ug DNA in the figure is: the number of colonies produced by each unit ug of plasmid DNA. DETAILED DESCRIPTION

[0016] The specific embodiments provided by the present invention are described in detail below in conjunction with the accompanying drawings. Unless otherwise specified, the reagents and consumables used in the examples are all easily available on the market, and if no specific experimental methods or conditions are specified, they are carried out according to the conventional methods or conditions described in the literature in the field or according to the product instructions.

[0017] The 2×YTG medium used in the following examples contains: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose, 0.2 g / L cysteine. The amount of glycine added is 12.5 g / L. The SMPbuffer contains: 92.4 g / L sucrose, 0.093 g / L magnesium chloride, and 0.78 g / L sodium dihydrogen phosphate. The RCM medium is a reinforced Clostridium medium purchased from Haibo Biotechnology. The ultrasonic operation is performed using an ultrasonic cleaner (Kunshan Shumei). Example 1

[0018] This example provides a novel and efficient genetic transformation method for Clostridium pasteurianum. The growth of colonies under three transformation system conditions, namely, "electric shock", "ultrasound", and "ultrasound + electric shock", is compared. Specifically, the following steps are included: (1) Take out the wild-type Clostridium pasteurianum from the -80℃ refrigerator and inoculate it into 2×YTG medium. Incubate it at 37℃ overnight for 14h until the OD value of the bacterial solution reaches 600 The value is 1.0.

[0019] (2) Transfer 500 μL of seed solution to 12 test tubes containing 9 mL of 2×YTG medium. Incubate at 37°C for 2 h. Add glycine to the medium and incubate for another 2 h until the OD value of the bacterial solution reaches 0. 600 The value is 0.6.

[0020] (3) Pre-cool the centrifuge and centrifuge the 12 tubes of cultured bacterial suspension at 4°C and 8500 g for 10 min.

[0021] (4) Wash each tube of Clostridium cells collected with 5 mL SMP buffer, centrifuge using the method in step ③, and wash twice.

[0022] (5) After washing, add 580 μL SMP buffer to each tube for resuspending, add 1 ug plasmid pMTL 85151, and mix well.

[0023] (6) Ultrasonicate the 8 tubes of suspension in an ultrasonic cleaner in an ice bath for 20 s with the following parameters: ultrasonic input power 200 W, ultrasonic frequency 40 kHz.

[0024] (7) Transfer the 4 tubes of sonicated suspension and the 4 tubes of non-sonicated suspension into an electric rotating cup, add 30 μL of 96% ethanol, mix well, and let stand for 5 min.

[0025] (8) Then, perform electric shock. The electric shock procedure is as follows: voltage 1.8 kV, capacitance 25 µF, resistance ∞ Ω, and time 12 ms. After electric shock, quickly add 5 mL of 2×YTG medium and let it rest in a 37°C incubator for 16 h to recover.

[0026] (9) Spread the revived bacterial suspension onto a solid plate containing 25 ug / mL chloramphenicol-resistant RCM medium and culture in a 37°C incubator for 40 h.

[0027] (10) Observe and count the growth of bacterial colonies on the plates under three different transformation systems (electric shock, ultrasound, and ultrasound + electric shock).

[0028] Figure 1 This is the growth of colonies on the plate under three different transformation system conditions. From left to right, the plates are electric shock, ultrasound, and ultrasound + electric shock. Figure 2 The data are the colony counts under three different transformation systems, and the data are the average values ​​of four parallel groups under three conditions. The results show that the transformation efficiency of the "ultrasound + electric shock" transformation system is much higher than that of ultrasonic treatment or electric shock treatment alone, and even has a significant effect of "1+1 is much greater than 2". Example 2

[0029] This embodiment provides a novel and efficient genetic transformation method for Clostridium pasteurianum, and compares the growth of colonies under different vector plasmid concentrations. The method specifically includes the following steps: (1) Take out the wild-type Clostridium pasteurianum from the -80℃ refrigerator and inoculate it into 2×YTG medium. Incubate it at 37℃ overnight for 14h until the OD value of the bacterial solution reaches 600 The value is 1.0.

[0030] (2) Transfer 500 μL of seed solution to 12 test tubes containing 9 mL of 2×YTG medium. Incubate at 37°C for 2 h. Add glycine to the medium and incubate for another 2 h until the OD value of the bacterial solution reaches 0. 600 The value is 0.6.

[0031] (3) Pre-cool the centrifuge and centrifuge the cultured bacterial solution at 4°C and 8500 g for 10 min.

[0032] (4) Wash each tube of Clostridium cells collected with 5 mL SMP buffer, centrifuge using the method in step ③, and wash twice.

[0033] (5) After washing, add 580 μL SMP buffer to each tube for resuspending. Divide the 12 tubes into three groups, with 4 tubes in each group. Add 0.5 ug, 1 ug, and 2 ug of plasmid pMTL-IV to the three groups respectively and mix well.

[0034] (6) The suspension was ultrasonically treated in an ultrasonic cleaner for 20 s in an ice bath with the following parameters: ultrasonic input power 200 W, ultrasonic frequency 40 kHz.

[0035] (7) After transferring the sonicated suspension into an electric rotating cup, add 30 μL of 96% ethanol, mix well, and let stand for 5 min.

[0036] (8) Then, perform electric shock. The electric shock procedure is as follows: voltage 1.8 kV, capacitance 25 µF, resistance ∞ Ω, time 10 ms. After electric shock, quickly add 5 mL of 2×YTG medium and let it stand in a 37°C incubator for 14 h to recover.

[0037] (9) Take half of the revived bacterial solution from each tube and spread it on a solid plate containing 25 ug / mL chloramphenicol-resistant RCM medium and culture it in a 37°C incubator for 36 h.

[0038] (10) Observe and count the colony growth on the plate under three different vector plasmid concentrations.

[0039] Figure 3 This is the growth of colonies on the plate under three different vector plasmid concentrations. From left to right, the plates use 0.5 ug, 1 ug, and 2 ug of plasmid pMTL-IV. Figure 4 The colony counts and transformant counts were calculated under three different vector plasmid concentrations. The data are the average values ​​of four parallel groups under three conditions. As can be seen from the figure, when 1 ug and 2 ug of plasmid were added, the colony counts were better than those of 0.5 ug of plasmid, and the best result was when 1 ug of plasmid was added. Example 3

[0040] This embodiment provides a novel and efficient genetic transformation method for Clostridium pasteurianum, and compares the growth of colonies under different vector plasmid conditions, which specifically includes the following steps: (1) Take out the wild-type Clostridium pasteurianum from the -80℃ refrigerator and inoculate it into 2×YTG medium. Incubate it at 37℃ overnight for 14h until the OD value of the bacterial solution reaches 600 The value is 1.0.

[0041] (2) Transfer 500 μL of seed solution to 12 test tubes containing 9 mL of 2×YTG medium. Incubate at 37°C for 2 h. Add glycine to the medium and incubate for another 2 h until the OD value of the bacterial solution reaches 0. 600 The value is 0.6.

[0042] (3) Pre-cool the centrifuge and centrifuge the cultured bacterial solution at 4°C and 8500 g for 10 min.

[0043] (4) Wash each tube of Clostridium cells collected with 5 mL SMP buffer, centrifuge using the method in step ③, and wash twice.

[0044] (5) After washing, add 580 μL SMP buffer to each tube for resuspending. Divide the 12 tubes into four groups, with 3 tubes in each group. Add 1 ug of plasmid pMTL-IV, methylated plasmid pMTL-IV, plasmid pMTL 85151, and methylated plasmid pMTL85151 to each group, respectively, and mix well.

[0045] (6) The suspension was ultrasonically treated in an ultrasonic cleaner for 20 s in an ice bath with the following parameters: ultrasonic input power 200 W, ultrasonic frequency 40 kHz.

[0046] (7) After transferring the sonicated suspension into an electric rotating cup, add 30 μL of 96% ethanol, mix well, and let stand for 5 min.

[0047] (8) Then, perform electric shock. The electric shock procedure is as follows: voltage 1.8 kV, capacitance 25 µF, resistance ∞ Ω, time 18 ms. After electric shock, quickly add 5 mL of 2×YTG medium and let it stand in a 37°C incubator for 12 h to recover.

[0048] (9) Take half of the revived bacterial solution from each tube and spread it on a solid plate containing 25 ug / mL chloramphenicol-resistant RCM medium and culture it in a 37°C incubator for 48 h.

[0049] (10) Observe and count the colony growth on the plate under four different vector plasmid conditions.

[0050] Figure 5 This is the growth of colonies on the plate under four different vector plasmid conditions. From left to right, the plates use plasmid pMTL-IV, methylated pMTL-IV, pMTL 85151, and methylated pMTL 85151. Figure 6 The colony counts were calculated using plasmid pMTL-IV and methylated pMTL-IV. The data are the average values ​​of three parallel groups. In this example, two shuttle vectors suitable for Clostridium pasteurianum were selected, and methylated (CpG) was performed on them respectively, and then transformed into Clostridium pasteurianum respectively. As can be seen from the figure, the methylated vectors obtained more colonies on the plate, which can significantly improve the transformation efficiency of Clostridium pasteurianum. Example 4

[0051] This embodiment provides a novel and efficient genetic transformation method for Clostridium pasteurianum, and compares the growth of colonies under different chloramphenicol concentrations, which specifically includes the following steps: (1) Take out the wild-type Clostridium pasteurianum from the -80℃ refrigerator and inoculate it into 2×YTG medium. Incubate it at 37℃ overnight for 14h until the OD value of the bacterial solution reaches 600 The value is 1.0.

[0052] (2) Transfer 500 μL of seed solution to two test tubes containing 9 mL of 2×YTG medium. Incubate at 37°C for 2 h. Add glycine to the medium and incubate for another 2 h until the OD value of the bacterial solution reaches 0. 600 The value is 0.6.

[0053] (3) Pre-cool the centrifuge and centrifuge the cultured bacterial solution at 4°C and 8500 g for 10 min.

[0054] (4) Wash each tube of Clostridium cells collected with 5 mL SMP buffer, centrifuge using the method in step ③, and wash twice.

[0055] (5) After washing, add 580 μL SMP buffer to each tube for resuspending. Add 1 ug of plasmid pMTL-IV to each tube and mix well.

[0056] (6) The suspension was ultrasonically treated for 20 s in an ultrasonic cleaner under ice bath conditions with the following parameters: ultrasonic input power 200 W, ultrasonic frequency 40 kHz.

[0057] (7) After transferring the sonicated suspension into an electric rotating cup, add 30 μL of 96% ethanol, mix well, and let stand for 5 min.

[0058] (8) Then, the cells were electroporated with the following procedure: voltage 1.8 kV, capacitance 25 µF, resistance ∞ Ω, time 16 ms. After electroporation, 5 mL of 2×YTG medium was quickly added and the cells were allowed to recover in a 37°C incubator for 15 h.

[0059] (9) Divide the two tubes of bacterial suspension into three equal parts after recovery, and spread each part onto RCM medium solid plates with chloramphenicol concentrations of 15 ug / mL, 25 ug / mL, and 35 ug / mL, respectively, and culture in a 37°C incubator for 44 h.

[0060] (10) Observe and count the colony growth on the plate under three different chloramphenicol concentrations.

[0061] Figure 7 This is the growth of colonies on the plate under three different chloramphenicol concentrations. From left to right, the plate has chloramphenicol concentrations of 15 ug / mL, 25 ug / mL, and 35 ug / mL. Figure 8The data are the number of transformants counted under three different chloramphenicol concentrations, and the data are the average of two parallel groups. As can be seen from the figure, more transformants were obtained when the chloramphenicol concentration was 15 ug / mL and 25 ug / mL, and the optimal chloramphenicol concentration was 25 ug / mL.

[0062] In summary, the genetic transformation system of Clostridium pasteurianum provided by the present invention is different from the traditional means. The present invention uses a transformation method combining ultrasound + electric shock, and optimizes the condition parameters including culture conditions, plasmid dosage, plasmid type and methylation treatment. The experimental results show that the transformation efficiency of the experimental method of the present invention can reach more than 600 CFU / ugDNA, which is 2 orders of magnitude higher than the traditional electric shock method.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A novel and efficient genetic transformation method for Clostridium pasteurianum, characterized in that: The following steps are involved: (1) Inoculate the Clostridium pasteurianum seed solution into 2×YTG medium and culture at 37°C for 2 h. Then add glycine to the medium and continue culturing until the bacterial solution OD 600 The value is 0.6; (2) Collect Clostridium pasteurianum cells by refrigerated centrifugation, wash with SMP buffer, and then refrigerated centrifuge and wash twice; (3) After washing, add SMP buffer to resuspend, then add vector plasmid and mix well; (4) The suspension obtained in step (3) was ultrasonicated for 20 s in an ice bath with an ultrasonic input power of 200 W and an ultrasonic frequency of 40 kHz; (5) Transfer the suspension after ultrasound into an electric rotating cup, add ethanol to mix well, and let it stand for 5 min; then perform electric shock with a voltage of 1.8 kV, a capacitance of 25 µF, a resistance of ∞ Ω, and a time of 10-18 ms. After electric shock, quickly add 2×YTG culture medium and let it stand in a 37°C incubator for recovery for 12-16 h; (6) Spread the revived bacterial liquid onto a solid plate containing RCM medium containing chloramphenicol resistance, culture in a 37°C incubator for 36-48 h, and count the colony growth.

2. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The preparation method of the Clostridium pasteurianum seed solution in step (1) is as follows: inoculate the frozen wild-type Clostridium pasteurianum into 2×YTG medium, and culture at 37°C for 12-14 h until the OD of the bacterial solution reaches 600 The value is 0.8-1.

3. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1 or 2, characterized in that: The 2×YTG culture medium contains: 16 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, 5 g / L glucose, and 0.2 g / L cysteine.

4. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The amount of glycine added in step (1) is 12.5 g / L.

5. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The refrigerated centrifugation conditions in step (2) are: 4°C, 8500 g centrifugation for 10 min.

6. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The SMP buffer described in step (2) and step (3) comprises: 92.4 g / L sucrose, 0.093 g / L magnesium chloride, and 0.78 g / L sodium dihydrogen phosphate.

7. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The plasmid described in step (3) is pMTL-IV, methylated pMTL-IV, pMTL 85151 or methylated pMTL 85151.

8. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 7, characterized in that: The plasmid is methylated pMTL-IV or methylated pMTL 85151.

9. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, 7 or 8, characterized in that: The amount of plasmid used in step (3) is: 9.5 mL of 2×YTG medium is cultured until the bacterial solution OD is 600 The amount of bacteria collected when the value is 0.6 was resuspended in 580 μL SMP buffer and 1-2 ug of plasmid was added.

10. The novel and efficient genetic transformation method of Clostridium pasteurianum according to claim 1, characterized in that: The concentration of chloramphenicol used in step (6) is 15-25 ug / mL.

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