Method for improving methanol assimilation and acetic acid and butyric acid synthesis by using gene MESH1

Abstract

The invention discloses a method for improving methanol assimilation and acetic acid and butyric acid synthesis by using a gene MESH1. A fruit fly source (p) ppGpp gene MESH1 is reconstructed in bacillus methylophilus, and the content of (p) ppGpp in the bacillus methylophilus is interfered by mutating a strong promoter (pth1) in the bacillus methylophilus. Different promoter intensities result in different expression effects, wherein the expression effect of the promoter Pth13 is the best. According to the invention, when the expression of MESH1 is regulated by using the promoter Pth1, the methanol availability of the recombinant B.methylotrophicum is improved, compared with the methanol assimilation amount of an original strain, the methanol assimilation amount is improved by 1.4%, the yield of acetic acid is improved by 22.23%, and the yield of butyric acid is improved by 14.54%. The recombinant strain is subjected to promoter fermentation optimization, it is found that when the promoter Pth13 is used for regulating expression of MESH1, the methanol utilization performance of the recombinant B.methylotrophicum reaches the optimal value, the specific growth rate is increased by 1.38 times compared with that of an original strain, the methanol assimilation amount is increased by 25.2%, meanwhile, the acetic acid yield and the butyric acid yield are increased by 32.7% and 34.38% respectively compared with those of a control group, and good application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving methanol assimilation and acetic acid and butyric acid synthesis by utilizing gene MESH1. Background Art

[0002] Methanol (CH3OH), also known as wood alcohol, is a colorless, flammable liquid with a slight alcohol smell. It is the simplest alcohol compound. Methanol has a wide range of applications in industry and is often used as a solvent, fuel, and raw material for chemical synthesis. Methanol has a boiling point of 64.7°C, a melting point of -97.6°C, and a density of 0.791 g / cm³ (at 20°C). Methanol is miscible with most organic solvents such as water, alcohols, and ethers, and has good solubility. At the same time, methanol is one of the simplest energy storage molecules and can be used to produce a wide variety of products. Since methanol can be produced from biomass, many countries can produce and utilize biomethanol. Compared with fossil fuels, the combustion of biomethanol can reduce nitrogen oxide emissions by up to 80%, reduce carbon dioxide emissions by up to 95%, and eliminate sulfur oxide emissions. Green methanol can be sustainably produced from biomass (biomethanol) or synthesized from greenhouse gases such as carbon dioxide or methane. Compared with traditional biotechnology, methanol-based bioprocesses are independent of plant-derived sugars and can alleviate tight agricultural resources.

[0003] Butyribacterium methylotrphicum, classified as Clostridium in bacteriology, is an obligate anaerobic Gram-positive bacillus. Butyribacterium methylotrphicum is also a type of anaerobic natural methylotrophic bacteria, which can simultaneously use methanol, CO, CO2 and other carbon compounds for fermentation to produce organic acid substances. It can use methanol as the only carbon source to synthesize organic acids such as acetic acid and butyric acid. Therefore, Butyribacterium methylotrphicum is different from aerobic methylotrophic bacteria. The methanol metabolic pathway consumes less reducing power, which is mainly used for the accumulation of metabolites. At the same time, the strain has the advantages of high tolerance to methanol and fast methanol metabolism rate. Its main fermentation products are carboxylic acids, butyric acid and corresponding alcohols, etc., which has certain research value in the biomanufacturing industry.

[0004] However, current research mainly focuses on the fermentation conditions and culture medium screening of Methylobacterium butyricum, and does not involve related genetic engineering modifications, which limits the development and application of strains. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for improving methanol assimilation and the synthesis of acetate and butyrate by using the gene MESH1. It is the first time to construct and propose an artificially constructed (p)ppGpp pathway in methylbutyric acid bacteria, which improves the efficiency of methanol assimilation and product butyrate synthesis, lays a foundation for the future production of compounds by methylbutyric acid bacteria, and has far-reaching significance.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is: A method for improving methanol assimilation and acetic acid and butyric acid synthesis by using gene MESH1 comprises the following steps: Step 1, constructing the recombinant plasmid pXY3-thl-MESH1, optimizing the codon preference, synthesizing the MESH1 gene sequence by Qingke Biotechnology Company, and selecting primers to connect the MESH1 fragment to the vector pXY3 using the XHolI / XbaI restriction site; Step 2, constructing the recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1; Step 3, the recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1 is methylated and then transformed into Butyricum methylophilum to obtain recombinant Butyricum methylophilum BM / pXY3-thl1-MESH1 or BM / pXY3-thl3-MESH1.

[0007] As an improvement, the construction method of the recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1 in step 2 is: take the recombinant plasmid pXY3-thl-MESH1, use primers th11-F / th11-R or th13-F / th13-R to perform circular PCR amplification to obtain a plasmid fragment, and then obtain the recombinant plasmid pXY3-thl1-MESH1 or recombinant plasmid pXY3-thl3-MESH1 by digestion and transformation.

[0008] A further improvement is that the method for methylation modification and transformation in step 3 is: transforming the recombinant plasmid pXY3-thl1-MESH1 or the recombinant plasmid pXY3-thl3-MESH1 into Escherichia coli Top10 containing pMCljs, culturing and extracting the methylated plasmid, and then transforming it into Butyricum methylophyllum by electroporation to obtain reconstructed Butyricum methylophyllum BM / pXY3-thl1-MESH1 or BM / pXY3-thl3-MESH1.

[0009] The recombinant butyric acid bacteria of methylotrophic acid constructed by the above method belongs to the genus Clostridium in bacteriological classification and is an obligate anaerobic Gram-positive bacillus. It can simultaneously utilize a variety of carbon-one raw materials for fermentation, has high tolerance to methanol, metabolizes methanol at a fast rate, and the main fermentation products are carboxylic acids, butyric acid and corresponding alcohols.

[0010] The above-mentioned recombinant Butyricum methylotrophicum is used in the production of acetic acid and butyric acid by methanol assimilation and fermentation.

[0011] As an improvement, a single colony of the reconstructed Butyric acid bacteria methylotrophic acid was picked on the plate and inoculated into 2 ml YTF medium containing erythromycin. After culturing for 12-16 hours, all the bacterial liquid in the centrifuge tube was transferred to an ampoule and grown to an OD600 of 1-1.2. The bacterial liquid was poured into a 50 ml centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the culture was resuspended with PB medium and inoculated into 50 ml PB medium at an inoculum size of OD600=0.1. 100 mM methanol and 20 mM sodium bicarbonate were added. At regular intervals, 2 ml of the bacterial liquid was aspirated, and the cells were resuspended after centrifugation for determining the cell density. The supernatant was transferred to a new centrifuge tube for storage and used for high performance liquid chromatography detection of methanol and butyric acid.

[0012] Beneficial effects:

[0013] Compared with the prior art, the present invention uses the gene MESH1 to improve methanol assimilation and acetate and butyrate synthesis. The (p)ppGpp-mediated rigorous reaction in Methylobutyric acid bacteria is an important mechanism for bacteria to cope with stress conditions. By optimizing the intracellular (p)ppGpp content, intracellular metabolism and transport-related proteins can be regulated. Through the synergistic effect of multiple factors, the regulatory function is exerted at multiple levels, thereby regulating the growth and reproduction rate of bacteria, improving the efficiency of methanol assimilation and acetate and butyrate synthesis, which conforms to the concept of green and sustainable development and provides a new idea for the resource utilization of CO2. The specific advantages are as follows: 1. This method breaks through the traditional industrial microbial fermentation substrate and uses biomass production of green methanol, which is economical and environmentally friendly; 2. This method proposed for the first time to use the (p)ppGpp rigorous reaction pathway in Butyricum methylotrophicum, achieving a significant increase in methanol assimilation efficiency and maximum biomass; 3. By interfering with the (p)ppGpp content of Butyricum methylotrophicum, the efficiency of methanol assimilation and butyrate synthesis was further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Comparison of the growth of recombinant strains BM / pXY3-MESH1 (pth1 / pth11 / pth12 / pth13) and BM / pXY3 in methanol; Figure 2 Comparison of methanol consumption between the recombinant strains BM / pXY3-MESH1 (pth1 / pth11 / pth12 / pth13) and BM / pXY3; Figure 3 Comparison of acetic acid production in methanol between the recombinant strains BM / pXY3-MESH1 (pth1 / pth11 / pth12 / pth13) and BM / pXY3; Figure 4 Comparison of butyrate production in methanol between the recombinant strains BM / pXY3-MESH1 (pth1 / pth11 / pth12 / pth13) and BM / pXY3. DETAILED DESCRIPTION

[0015] The present invention is further illustrated by the following specific implementation modes, which are not intended to limit the present invention. Those skilled in the art may make various modifications or improvements based on the basic concept of the present invention, but all of them are within the scope of the present invention as long as they do not deviate from the basic concept of the present invention.

[0016] The techniques not mentioned in the examples are all conventional techniques in the art. In addition, the Butyric acid bacteria used were purchased from the ATCC Culture Collection, and materials such as Trans1-T1, pXY3, and Top10 are all commercial products and can be purchased directly.

[0017] Example 1 Construction of pXY3-thl-MESH1 plasmid The plasmid pXY3-thl-MESH1 was optimized according to codon preference, and the MESH1 gene sequence was synthesized by Qingke Biotechnology Co., Ltd. The existing laboratory plasmid PXY3 (its construction method is shown in Publication No. CN114480239B) was used as a template to amplify the (p)ppGpp hydrolase MESH1 coding sequence from Drosophila by conventional PCR.

[0018] MESH1 gene sequence: ATGGCAACCTATCCGAGTGCAAAATTCATGGAATGTCTGCAGTATGCCGCCTTTAAACATCGTCAGCAGCGTCGCAAAGATCCGCAGGAAACCCCGTATGTTAATCATGTGATTAATGTTAGCACCATTCTGAGTGTGGAAGCATGTATTACCGATGAAGGTGTTCTGATGGCCGCACTGCTGCATGATGTGGTGGAAGATACCGATGCAAGTTTTGAAGATGTTGAAAAACTGTTTGGCCCGGATGTTTGCGGTCTGGTTCGTGAAGTTACCGATGATAAAAGTCTGGAAAAACAGGAACGTAAACGCCTGCAGATTGAAAATGCAGCCAAAAGTAGCTGCCGCGCCAAACTGATTAAGCTGGCAGATAAACTGGATAATCTGCGCGATCTGCAGGTTAATACCCCGACCGGTTGGACCCAGGAACGTCGTGATCAGTATTTTGTGTGGGCAAAAAAGGTGGTTGATAATCTGCGTGGCACCAATGCAAATCTGGAACTGAAACTGGATGAAATTTTTCGTCAGCGCGGTCTGCTGTAA (p)ppGpp hydrolase MESH1 amino acid sequence: MATYPSAKFMECLQYAAFKHRQQRRKDPQETPYVNHVINVSTILSVEACITDEGVLMAALLHDVVEDTDASFEDVEKLFGPDVCGLVREVTDDKSLEKQERKRLQIENAAKSSCRAKLIKLADKLDNLRDLQVNTPTGWTQERRDQYFVWAKKVVDNLRGTNANLELKLDEIFRQRGLL The upstream primer has an XHolI restriction site and the sequence is as follows: TAGAGGATCCCCGGGTCTAGAATGGCGAACGAAGCAATAGAACA The downstream primer has an XbaI restriction site and the sequence is as follows: GGGGCCAGTGAATTCTCGAGTTACAGCAGACCGCG The reaction conditions were: 95°C for 3 min, 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 5 min; the obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered; the sequence and the expression vector pXY3 were digested with XHolI and XbaI from Takara, and the enzyme digestion reaction system was: 10×buffer H 2μl, XbaI 0.5μl, XHolI 0.5μl, pXY3 vector 3μl, H2O 14μl.

[0019] The enzyme digestion system was reacted at 37°C for 2 hours. The digestion products were connected, and the reaction system was: 10×Ligasebuffer 1μl, T4 DNA Ligase (Takara) 1μl, gene fragment 7μl, vector 1μl. The connection was reacted at 25°C for 3 hours. The connection product was transformed into Escherichia coli Trans1-T1. PCR screening of positive strains Trans1-T1-pXY3-thl-MESH1 and DNA sequencing were performed to verify that the recombinant plasmid was constructed correctly.

[0020] The positive strain was inoculated into 5 ml LB / Amp liquid medium, which consisted of 10 g / L peptone, 5 g / L yeast powder, and 5 g / L sodium chloride, and cultured overnight at 37°C and 200 rpm with shaking. After 24 hours, the plasmid pXY3-thl-MESH1 was extracted according to the instructions of the Tiangen Plasmid Extraction Kit.

[0021] Example 2 Construction of recombinant strain BM / pXY3-thl-MESH1 The plasmid pMCljs was heat-shock transformed into E. coli Top10 to obtain the recombinant strain Top10 / pMCljs. The recombinant strain was prepared into competent cells Top10 / pMCljs. The specific operation method is shown in the construction method disclosed in patent CN 113106047 A.

[0022] The recombinant plasmid pXY3-thl-MESH1 was transformed into the competent cell Top10 / pMCljs to obtain the recombinant bacteria Top10 / pMCljs / pXY3-thl-MESH1.

[0023] The recombinant strain Top10 / pMCljs / pXY3-thl-MESH1 was inoculated into 5 ml of LB medium containing a final concentration of 50 ug / mL ampicillin resistance and 100 ug / mL streptomycin hydrochloride. After culturing at 37°C for 12 hours, the plasmid was extracted to obtain the methylated plasmid pXY3-thl-MESH1. Finally, the methylated plasmid pXY3-thl-MESH1 was transformed into Butyricum methylophyllus to obtain the methylated recombinant strain BM / pXY3-thl-MESH1.

[0024] Example 3 Construction of recombinant plasmid pXY3-thl1-MESH1, recombinant plasmid pXY3-thl2-MESH1, and recombinant plasmid pXY3-thl3-MESH1 (1) Using the constructed plasmid pXY3-thl-MESH1 as a template, the plasmid fragment pXY3-thl1-MESH1 was amplified by directed circular PCR, and then the original plasmid template was removed by digestion and transformed into a plate for testing and verification of the transformant.

[0025] The sequence of the upstream primer th11-F is: CTTTTAACAAAATATATTGATAAAAATTATAATAGTGTGTATAATTAAGTTGTTAGAGAAAAC The sequence of the downstream primer th11-R is: ACACTATTATAATTTTTATCAATATATTTTGTTAAAAGCTGCAGCACATTAAGTATATACTAT The reaction conditions were: 95°C for 3 min, 95°C for 15 s, 55°C for 15 s, 72°C for 180 s, for a total of 30 cycles; 72°C for 5 min; the obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered; The digestion system was reacted at 37°C for 2 hours to remove the original plasmid template. The reaction system was: 10×Ligasebuffer 2μl, Dpnl 2μl, gene fragment 16μl. The ligation product was transformed into Escherichia coli Trans1-T1. PCR screening of positive strains Trans1-T1-pXY3-thl1-MESH1 and DNA sequencing were performed to verify that the recombinant plasmid was constructed correctly.

[0026] The positive strain was inoculated into 5 ml LB / Amp liquid medium, which consisted of 10 g / L peptone, 5 g / L yeast powder, and 5 g / L sodium chloride, and cultured overnight at 37°C and 200 rpm with shaking. After 24 hours, the plasmid pXY3-thl1-MESH1 was extracted according to the instructions of the Tiangen Plasmid Extraction Kit.

[0027] (2) The recombinant plasmid pXY3-thl2-MESH1 was constructed according to the construction method of the recombinant plasmid pXY3-thl1-MESH1 except for the difference in primers.

[0028] The sequence of the upstream primer th12-F is: TTTTTAACGAAAAATATTGATAAAAATAAAAATTGTCCGtataattaagttgttagagaaaacg The sequence of the downstream primer th12-R is: CGGACAATTTTTATTTTTATCAATATTTTTCGTTAAAAActgcagcacattaagtatatactat (3) The recombinant plasmid pXY3-thl3-MESH1 was constructed according to the construction method of the recombinant plasmid pXY3-thl1-MESH1 except for the difference in primers.

[0029] The sequence of the upstream primer th13-F is: TTTCCAGCGAGATGTATTGATAAAACTCTAACTAAATGCTATAATTATATTGTTAGAGAAAACGTATAAATTAG The sequence of the downstream primer th13-R is: AATATAATTATAGCATTTAGTTAGAGTTTTATCAATACATCTCGCTGGAAACTGCAGCACATTAAGTATATACTAT Example 4 Construction of recombinant strain BM / pXY3-thl1-MESH1, recombinant strain BM / pXY3-thl2-MESH1, and recombinant strain BM / pXY3-thl3-MESH1 The plasmid pMCljs was heat-shock transformed into E. coli Top10 to obtain the recombinant strain Top10 / pMCljs. The recombinant strain was prepared into competent cells Top10 / pMCljs. The specific operation method is shown in the construction method disclosed in patent CN 113106047 A.

[0030] The recombinant plasmids pXY3-thl1-MESH1, pXY3-thl2-MESH1, and pXY3-thl3-MESH1 were transformed into the competent state Top10 / pMCljs to obtain the methylated recombinant strains Top10 / pMCljs / pXY3-thl1-MESH1, Top10 / pMCljs / pXY3-thl2-MESH1, and Top10 / pMCljs / pXY3-thl3-MESH1.

[0031] The recombinant strain was inoculated into 5 ml of LB medium containing a final concentration of 50 ug / mL ampicillin resistance and 100 ug / mL streptomycin hydrochloride. After culturing at 37°C for 12 hours, the plasmid was extracted to obtain the methylated plasmid. Finally, the methylated plasmid was transformed into Butyricum methylophyllus to obtain the methylated recombinant strains BM / pXY3-thl1-MESH1, BM / pXY3-thl2-MESH1, and BM / pXY3-thl3-MESH1.

[0032] Example 5 Methanol-catalyzed synthesis of products by recombinant strains Single colonies of the recombinant strain BM / pXY3-thl-MESH1, recombinant strain BM / pXY3-thl1-MESH1, recombinant strain BM / pXY3-thl2-MESH1, and recombinant strain BM / pXY3-thl3-MESH1 were picked up on the plate, inoculated into 2 ml YTF medium containing erythromycin with a final concentration of 30 ug / mL. After culturing for 12-16 hours, all the bacterial liquid in the centrifuge tube was transferred to an ampoule, grown to OD600 of 1-1.2, poured into a 50 ml centrifuge tube, centrifuged at 4000 rpm for 10 min, discarded the supernatant, resuspended with PB medium, and inoculated into 50 ml PB medium at an inoculum size of OD600 = 0.1, and then 100 mM methanol + 20 mM sodium bicarbonate were added. The culture was repeated every 24 hours. h, aspirate 2 ml of bacterial solution, centrifuge, resuspend in 2 ml of ultrapure water, detect its OD600, and transfer the supernatant to a new centrifuge tube for storage.

[0033] Cell growth determination: After a certain fermentation period, take 2 ml of bacterial solution and centrifuge to remove the supernatant, then resuspend it with ultrapure water and measure its OD600 to determine its cell concentration.

[0034] Determination of methanol content: Agilent high performance liquid chromatography, the chromatographic column is Biorad HPX-87H, the mobile phase is 0.8 g / L H2SO4 solution, the column temperature is 60℃, the injection volume is 20 μL, the flow rate is 0.5mL / min, and the detector is a differential detector.

[0035] For the determination of acetic acid and butyric acid content, the detector is an ultraviolet detector.

[0036] The specific calculation formula is: Methanol consumption calculation formula: (theoretical methanol - actual methanol consumption) / theoretical methanol The formula for calculating the increase is: (the amount of experimental product - the amount of control group product) / the amount of control group product.

[0037] For example, in the logarithmic growth phase, the methanol residue of the control strain BM / pXY3 is 1.456 g / L, so the methanol consumption is: (3.24-1.456) / 3.24=55% The methanol residue of the recombinant strain BM / pXY3-thl-MESH1 is 1.5 g / L, so the methanol consumption is: (3.24-1.5) / 3.24=53.7% Similarly, the methanol consumption of the recombinant strain BM / pXY3-thl1-MESH1 was 73.7%, the methanol consumption of the recombinant strain BM / pXY3-thl2-MESH1 was 42.5%, and the methanol consumption of the recombinant strain BM / pXY3-thl3-MESH1 was 80.2%.

[0038] The catalytic synthesis of different recombinant strains is shown in Figure 3-4 shown.

[0039] The acetic acid production of Pth1 is 1.039 g / L, and the control pxy3 is 0.85 g / L, so the calculation method is (1.039-0.85) / 0.85=22.23% The regulation of promoter Pth1 increased the production of acetate and butyrate in the recombinant strain by 22.23% and 14.54%, respectively.

[0040] The promoter Pth11 was used to regulate the acetic acid production of the recombinant strain by 18.35% and the butyric acid production by 29.91%.

[0041] When the promoter Pth12 was used to regulate the expression of MESH1, the methanol utilization performance of the recombinant B. methylotrophicum was reduced, with the acetic acid production reduced by 42.4% and the butyric acid production reduced by 10.16% compared with the original strain.

[0042] When the promoter Pth13 was used to regulate the expression of MESH1, the methanol utilization performance of the recombinant B. methylotrophicum reached the optimal value, the specific growth rate increased by 1.38 times compared with the original strain, the methanol consumption increased by 80.2%, and the acetic acid production and butyric acid production increased by 32.7% and 34.38% respectively compared with the control group. The growth phenotypes of the recombinant strain BM / pXY3-thl-MESH1, the recombinant strain BM / pXY3-thl1-MESH1, the recombinant strain BM / pXY3-thl2-MESH1, and the recombinant strain BM / pXY3-thl3-MESH1 are as follows Figure 1 As shown. The growth of the recombinant strain BM / pXY3-thl2-MESH1 was roughly the same as that of the control group, and the cell growth concentrations of the recombinant strains BM / pXY3-thl-MESH1, BM / pXY3-thl1-MESH1, and BM / pXY3-thl3-MESH1 were all higher than those of the control. This indicates that different promoter regulation affects the growth and metabolism of cells, among which the growth of the recombinant strain BM / pXY3-thl3-MESH1 is the most obvious.

[0043] In summary, the present invention improves the ability of Methylbutyric acid bacteria to assimilate methanol and synthesize acetic acid and butyric acid by artificially constructing MESH1 to interfere with the (p)ppGpp pathway by comparing promoters of different strengths. Although the promoter strength is: pth11>pth13>pth12, the present invention proves that pth13 has a better effect, which further proves that it is not the stronger the strength, the better the effect, but that different promoters regulate ppGpp to achieve a moderate effect. It lays a foundation for the future use of Methylbutyric acid bacteria to produce compounds, which has far-reaching significance.

Claims

1. A method for improving methanol assimilation and acetic acid and butyric acid synthesis using gene MESH1, characterized in that: The following steps are involved: Step 1: construct the recombinant plasmid pXY3-thl-MESH1, optimize it according to the codon preference, synthesize the MESH1 gene sequence by Qingke Biotechnology Company, select primers to connect the MESH1 fragment to the vector pXY3 using the XHolI / Xba I restriction site; Step 2: construct recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1; Step 3, the recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1 is methylated and then transformed into Butyricum methylophilum to obtain recombinant Butyricum methylophilum BM / pXY3-thl1-MESH1 or BM / pXY3-thl3-MESH1.

2. The method of claim 1, wherein the method comprises: The construction method of the recombinant plasmid pXY3-thl1-MESH1 or pXY3-thl3-MESH1 in step 2 is: take the recombinant plasmid pXY3-thl-MESH1, use primers th11-F / th11-R or th13-F / th13-R to perform circular PCR amplification to obtain a plasmid fragment, and then obtain the recombinant plasmid pXY3-thl1-MESH1 or recombinant plasmid pXY3-thl3-MESH1 by digestion and transformation.

3. The method of claim 1, wherein the method comprises: The method for methylation modification and transformation in step 3 is: transforming the recombinant plasmid pXY3-thl1-MESH1 or the recombinant plasmid pXY3-thl3-MESH1 into Escherichia coli Top10 containing pMCljs, culturing and extracting the methylated plasmid, and then transforming it into Butyricum methylotrophicum by electroporation to obtain reconstructed Butyricum methylotrophicum BM / pXY3-thl-MESH1, BM / pXY3-thl1-MESH1 or BM / pXY3-thl3-MESH1.

4. The recombinant Butyricum methylophilus constructed according to any one of claims 1 to 3, characterized in that: The recombinant butyric acid bacteria of methylotrophic bacteria belongs to the genus Clostridium in bacteriological classification and is an obligate anaerobic Gram-positive bacillus. It can simultaneously utilize a variety of carbon-one raw materials for fermentation, has high tolerance to methanol, metabolizes methanol at a fast rate, and the main fermentation products are carboxylic acid, butyric acid and corresponding alcohols.

5. Use of the recombinant Butyricum methylotrophicum described in claim 4 in the production of acetic acid and butyric acid by methanol assimilation and fermentation.

6. The use according to claim 5, characterized in that: The specific steps of the application are as follows: a single colony of the reconstructed methyl-butyric acid bacteria was picked on a plate and inoculated into a 2 ml YTF medium containing erythromycin. After culturing for 12-16 hours, all the bacterial liquid in the centrifuge tube was transferred to an ampoule, grown to an OD600 of 1-1.2, poured into a 50 ml centrifuge tube, centrifuged at 4000 rpm for 10 minutes, discarded the supernatant, resuspended with PB medium and inoculated into 50 ml PB medium at an inoculum size of OD600=0.1, and then 100 mM methanol and 20 mM sodium bicarbonate were added. At regular intervals, 2 ml of bacterial liquid was drawn, and the bacterial liquid was resuspended after centrifugation for determining the cell density. The supernatant was transferred to a new centrifuge tube for storage and used for high performance liquid chromatography detection of methanol and butyric acid.

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