An engineered bacterium for efficiently utilizing methanol to produce pyrroloquinoline quinone and a construction method thereof

Abstract

The present application relates to the technical field of metabolic engineering bacteria, and provides an engineered bacterium for efficiently utilizing methanol to produce pyrroloquinoline quinone and a construction method thereof. MutS Then, the bacterium is inoculated into a culture medium containing methanol, and is directionally cultured by increasing the methanol concentration in a 2 g / L gradient MutS The engineered bacterium obtained by gene back complementation. The engineered bacterium strain has not only significantly improved the tolerance and utilization efficiency of methanol, but also realized the significant improvement of PQQ yield by enhancing the synthesis capacity of methanol dehydrogenase coenzyme PQQ. The obtained engineered bacterium strain effectively solves the problem of slow growth of the original strain under the condition of high-concentration methanol, and has important industrial application value and prospect.

Description

Technical Field

[0001] This invention relates to the field of metabolic engineering bacteria, and in particular to an engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone and its construction method. Background Technology

[0002] Pyrroloquinoline quinone (PQQ) is an important cofactor of redox enzymes, widely involved in electron transport processes in the microbial respiratory chain, and possesses various physiological activities such as free radical scavenging, neuroprotection, improvement of mitochondrial function, and alleviation of chronic inflammation. Due to its broad health effects, PQQ has obtained certification from the US FDA and the EU EFSA, showing broad application prospects as a dietary supplement in the pharmaceutical, food, and health product fields. Currently, the industrial production of PQQ mainly relies on chemical synthesis, but this method has limitations such as complex reaction steps, numerous byproducts, and significant environmental pollution. In contrast, microbial fermentation has advantages such as being green and safe, cost-controllable, and easy to separate and purify, making it more in line with the development trend of green biomanufacturing. Among the microorganisms capable of synthesizing PQQ, methyltrophic Gram-negative bacteria have attracted much attention due to their unique metabolic characteristics. These strains can grow using methanol as the sole carbon source, and their production process is highly compatible with the clean energy technology route advocated by my country's "dual-carbon" strategy. This study focuses on the methyltrophic bacterium MB200, which efficiently assimilates formaldehyde via the ribulose monophosphate (RuMP) pathway and relies on PQQ as a coenzyme for methanol dehydrogenase (MDH) to catalyze the initial reaction of methanol oxidation, demonstrating its potential as a chassis cell for PQQ biosynthesis. Although targeted domestication holds promise for obtaining high-yielding strains, the natural DNA mismatch repair (MMR) system within microorganisms results in a low spontaneous mutation frequency, significantly prolonging the targeted evolution cycle, increasing experimental costs and the risk of contamination, and posing a significant challenge to the screening of superior mutant strains. Therefore, obtaining an efficient method for targeted microbial construction and screening is crucial and has significant practical implications for promoting the industrialization of PQQ production. Summary of the Invention

[0003] The purpose of this invention is to provide an engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone and its construction method. Through the above technical solution, mutant strains that can adapt to high methanol environments and efficiently synthesize PQQ can be obtained.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, using methyltrophic bacteria MB200 as the original strain and knocking out its mismatch repair gene. MutSThen, they were inoculated into a medium containing methanol, and the methanol concentration was increased in increments of 1-3 g / L for directional culture. MutS Engineered bacteria obtained through gene replacement.

[0005] This invention also provides a method for constructing the engineered bacteria that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, comprising the following steps: (1) Obtaining genes by PCR amplification MutSsps The expression fragment was then ligated with the enzyme-digested vector plasmid to obtain the recombinant plasmid pK18mob- MutSps ; (2) After the recombinant plasmid described in step (1) is transferred into the competent cells of methyltrophic bacteria MB200, the methyltrophic bacteria knockdown strain is obtained after resistance verification. (3) The methyl-trophic bacteria knockdown strain described in step (2) is inoculated into a culture medium containing 2-6 g / L methanol, and the methanol concentration is increased in an incremental gradient of 1-3 g / L for directional culture to screen for the optimal tolerant strain. (4) The recombinant plasmid pCM80- MutS The optimal tolerant strain described in step (3) is then introduced to obtain the product.

[0006] Preferably, the primers for PCR amplification in step (1) include MutSps -F sequence and MutSps -R sequence, the MutSps The -F sequence is shown in SEQ ID NO:1. MutSps The -R sequence is shown in SEQ ID NO:2.

[0007] Preferably, the amount of inoculation in step (3) is 0.5-1.5% v / v.

[0008] Preferably, the culture in step (3) is accompanied by an oscillation process, the oscillation speed is 180-220 rpm, and the culture temperature is 28-32℃.

[0009] Preferably, the recombinant plasmid pCM80- in step (4) MutS The construction steps include: using the genomic DNA of methyltrophic bacteria MB200 as a template, PCR amplification is performed to obtain... MutS The gene fragment is then ligated with the vector plasmid pCM80 to obtain the final product.

[0010] Preferably, the primers for the PCR amplification include MutS -F sequence and MutS -R sequence, the MutS The -F sequence is shown in SEQ ID NO:5. MutSThe -R sequence is shown in SEQ ID NO:6.

[0011] The present invention also provides a method for high-yield pyrroloquinoline quinone, comprising the following steps: The engineered bacterial culture solution is inoculated into the culture medium at an inoculation rate of 0.8-1.2% v / v and fermented at 28-32℃ for 220-260 hours to obtain the final product.

[0012] Preferably, the fermentation process is accompanied by an oscillation process, and the oscillation speed is 150-250 rpm.

[0013] Preferably, the concentration of the engineered bacteria solution is OD. 600 =2.0-2.6.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects: The technical solution of this invention uses methyltrophic bacteria MB200 as the original strain and knocks out its mismatch repair gene. MutS Then, they were inoculated into a medium containing methanol, and the methanol concentration was increased in increments of 2 g / L for directional culture. MutS The engineered bacteria obtained through gene feedback not only significantly improved the tolerance to methanol (capable of withstanding methanol concentrations up to 48 g / L) and utilization efficiency, but also significantly increased PQQ yield by enhancing the synthesis capacity of methanol dehydrogenase coenzyme PQQ. The engineered strain obtained in this invention also effectively solved the problem of slow growth of the original strain under high methanol concentration conditions, and has significant industrial application value and prospects.

[0015] The embodiments of the present invention also show that the extracellular PQQ content of the constructed engineered bacterial strain is 96 mg / L, while the control strain wild-type MB200 synthesized 20 mg / L PQQ, and the PQQ yield was significantly improved.

[0016] Biological Preservation Instructions

[0017] This invention relates to methyltrophic bacteria MB200, classified as follows: Methylobacterium sp. was deposited on November 4, 2005, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 1526. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the methanol tolerance acclimatization of the engineered bacterial strain MB200-P3 described in this invention; Figure 2The chromatograms are the high-performance liquid chromatography (HPLC) results of strains MB200 and MB200-P3 after fermentation as described in Example 5. Figure 3 The extracellular PQQ fermentation yield of strains MB200 and MB200-P3 described in Example 5 is shown. Detailed Implementation

[0019] This invention provides an engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, using methyltrophic bacteria MB200 as the original strain and knocking out its mismatch repair gene. MutS Then, they were inoculated into a medium containing methanol, and the methanol concentration was increased in increments of 1-3 g / L for directional culture. MutS Engineered bacteria obtained through gene replacement.

[0020] In this invention, the incremental gradient is preferably 1-3 g / L, more preferably 1.5-2.5 g / L, and even more preferably 2 g / L.

[0021] This invention also provides a method for constructing the engineered bacteria that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, comprising the following steps: (1) Obtaining genes by PCR amplification MutSsps The expression fragment was then ligated with the enzyme-digested vector plasmid to obtain the recombinant plasmid pK18mob- MutSps ; (2) After the recombinant plasmid described in step (1) is transferred into the competent cells of methyltrophic bacteria MB200, the methyltrophic bacteria knockdown strain is obtained after resistance verification. (3) The methyl-trophic bacteria knockdown strain described in step (2) is inoculated into a medium containing methanol, and the methanol concentration is increased in an incremental gradient of 1-3 g / L for directional culture to screen for the optimal tolerant strain. (4) The recombinant plasmid pCM80- MutS The optimal tolerant strain described in step (3) is then introduced to obtain the product.

[0022] In this invention, the primers for PCR amplification in step (1) include MutSps -F sequence and MutSps -R sequence, the MutSps The -F sequence is shown in SEQ ID NO:1, specifically 5'-TATAAGCTTACGCTCGCCATCGACGCG-3'; MutSps The -R sequence is shown in SEQ ID NO:2, specifically 5'-TATCTGCAGGGCGATGAAGGCTTCGCC-3'.

[0023] In this invention, it is preferred to prepare a logarithmic seed culture by shaking culture of the methyl-trophic bacteria knockdown strain described in step (2), wherein the shaking culture temperature is 28-32℃, more preferably 29-31℃, and even more preferably 30℃; and the shaking culture speed is 180-220 rpm, more preferably 190-210 rpm, and even more preferably 200 rpm.

[0024] In this invention, the logarithmic seed culture is preferably inoculated into MM medium and cultured with shaking. The inoculation amount is preferably 0.5-1.5% v / v, more preferably 0.8-1.2% v / v, and even more preferably 1% v / v. The shaking culture time is preferably 36-48 h, more preferably 38-42 h, and even more preferably 40 h. The shaking culture temperature is preferably 28-32℃, more preferably 29-31℃, and even more preferably 30℃. The shaking culture speed is preferably 180-220 rpm, more preferably 190-210 rpm, and even more preferably 200 rpm.

[0025] In this invention, the MM culture medium comprises: (NH4)2HPO4 3.0 g / L, K2HPO4 2.0 g / L, NaCl 1.0 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.010 g / L, MnSO4·6H2O 0.005 g / L, vitamin B1 10 μg / L, nicotinic acid 20 μg / L, calcium pantothenate 200 μg / L, vitamin B2 20 μg / L, biotin 10 μg / L, and para-aminobenzoic acid 10 μg / L.

[0026] In this invention, the strain cultured to the logarithmic phase in the aforementioned MM medium is further inoculated into MM medium containing methanol for further culture. The inoculation amount is preferably 0.5-1.5% v / v, more preferably 0.8-1.2% v / v, and even more preferably 1% v / v. The methanol concentration is preferably 2-6 g / L, more preferably 3-5 g / L, and even more preferably 4 g / L. The culture process is accompanied by oscillation, with the oscillation speed preferably 180-220 rpm, more preferably 190-210 rpm, and even more preferably 200 rpm. The culture temperature is preferably 28-32℃, more preferably 29-31℃, and even more preferably 30℃.

[0027] In this invention, it is preferred to carry out directional culture by increasing the methanol concentration in an incremental gradient of 1-3 g / L, and the concentration increment is further preferably 1.5-2.5 g / L, and even more preferably 2 g / L.

[0028] In this invention, the recombinant plasmid pCM80- MutS The construction steps include: using the genomic DNA of methyltrophic bacteria MB200 as a template, PCR amplification is performed to obtain... MutS The gene fragment is then ligated with the vector plasmid pCM80 to obtain the final product.

[0029] In this invention, the primers for PCR amplification include MutS -F sequence and MutS -R sequence, the MutS The -F sequence is shown in SEQ ID NO:5, specifically 5'-CATAAGCTTCAGGCGGCGGATACG-3'; MutS The -R sequence is shown in SEQ ID NO:6, specifically 5'-CGCCTGCAGATCAGCGACGCTCTATA-3'.

[0030] The present invention also provides a method for high-yield pyrroloquinoline quinone, comprising the following steps: The engineered bacterial culture solution is inoculated into the culture medium at an inoculation rate of 0.8-1.2% v / v and fermented at 28-32℃ for 220-260 hours to obtain the final product.

[0031] In this invention, the fermentation temperature is preferably 28-32℃, more preferably 29-31℃, and even more preferably 30℃; the fermentation time is preferably 220-260h, more preferably 230-250h, and even more preferably 240h.

[0032] In this invention, the fermentation culture is accompanied by an oscillation process, and the oscillation speed is preferably 150-250 rpm, more preferably 180-220 rpm, and even more preferably 200 rpm.

[0033] In this invention, the concentration of the engineered bacterial solution is preferably OD. 600 =2.0-2.6, further preferred OD 600 =2.3-2.55, with OD being a further preferred value. 600 =2.5.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Construction of recombinant plasmid pk18mob- MutSps

[0036] (a) Extraction of the genome of methyltrophic bacteria MB200

[0037] Methyltrophic bacteria MB200 (CGMCC No. 1526) were cultured to the logarithmic phase. The bacterial cells were collected into 1.5 mL centrifuge tubes and centrifuged at 13000 rpm / min for 30 s. The supernatant was discarded. In this experiment, total DNA of the MB200 strain was extracted using a bacterial genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0038] (II) Constructing pk18mob- MutSps Knockout plasmid

[0039] Methyltrophic bacteria ( Methylorubrum rhodesianum Using the MB200 genome (GENBANK number PRJNA894144) as a template, SEQ ID NO:1 shows MutSps -F and SEQ ID NO:2 are shown MutSps -R represents the upstream and downstream primers (Table 1), PCR amplification MutSsps The target band was recovered using a DNA purification kit (purchased from Shanghai Bioengineering Co., Ltd.) to obtain the gel recovery product. PCR amplification conditions: 98℃ for 2 min, 98℃ for 15 s, 56℃ for 2 min, 72℃ for 4 min, 30 cycles; 72℃ for 2 min. The total volume of the PCR system described in this invention is 50 μl: the MB200 genome template is 1 μl. MutSps -F、 MutSps 1 μl each of -R, 22 μl of double-distilled water, and 25 μl of thermostable DNA polymerase (Novizan). pK18mob plasmid and MutSsps The fragments were double-digested with HindIII and PstI (purchased from Sangon Biotech (Shanghai) Co., Ltd.) for 4 h, recovered by gel, and then the two fragments were mixed and ligated using T4 ligase (purchased from Xiamen Lulong Biotechnology Development Co., Ltd.) at 16℃ for 4 h. The ligated product was then transformed into E. coli DH5α competent cells and ligated using pK18- as shown in SEQ ID NO:3. MutSp -F sequence and pK18- as shown in SEQ ID NO:4 MutSp -R sequence primers were used to verify and obtain positive clones. The knockout plasmid pK18mob- was extracted. MutSps The sequencing results were completely consistent with the designed plasmid DNA sequence.

[0040] Table 1 Primer sequence information

[0041] (III) Constructing pCM80- MutS plasmid recombinant plasmid

[0042] Methyltrophic bacteria (Methylorubrum rhodesianum Using the MB200 genome (PRJNA894144) as a template, as shown in SEQ ID NO:5 MutS -F and as shown in SEQ ID NO:6 MutS -R represents the upstream and downstream primers (Table 1), PCR amplification MutS The target band was recovered using a DNA purification kit to obtain the gel extraction product. PCR amplification conditions: 98℃ for 2 min, 98℃ for 15 s, 56℃ for 2 min, 72℃ for 4 min, 30 cycles; 72℃ for 2 min. pCM80 plasmid and MutS The fragments were digested with HindIII and PstI for 4 hours, recovered by gel electrophoresis, and then the two fragments were mixed and ligated using T4 ligase at 16°C for 4 hours. The ligated product was then transformed into *E. coli* DH5α competent cells and ligated using pCM80- (nucleotide sequence as shown in SEQ ID NO:7). Muts -F sequence and pCM80 as shown in SEQ ID NO:8- Muts -R sequence primers were used to verify and obtain positive clones. Recombinant pCM80- was extracted. MutS The plasmid sequencing results were completely consistent with the designed plasmid DNA sequence.

[0043] Example 2: Construction of recombinant strain MB200-A1

[0044] Competent cells were prepared from the methyltrophic bacterium MB200 strain and electroporated into pk18mob- MutSps The plasmid was cultured on MM basal medium plates containing 50 mg / mL kanamycin at 30°C for 240 h. Single colonies were obtained and verified by PCR using primers shown in SEQ ID NO:3 and SEQ ID NO:4. The correct methyltrophic bacterium strain Δ MB200 was confirmed. Muts (Methyltrophic bacteria knockdown strain), named recombinant strain MB200-A1.

[0045] The MM culture medium consists of: (NH4)2HPO4 3.0 g / L, K2HPO4 2.0 g / L, NaCl 1.0 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.010 g / L, MnSO4·6H2O 0.005 g / L, vitamin B1 10 μg / L, nicotinic acid 20 μg / L, calcium pantothenate 200 μg / L, vitamin B2 20 μg / L, biotin 10 μg / L, and para-aminobenzoic acid 10 μg / L.

[0046] The kanamycin solution was prepared as follows: 0.5 g of kanamycin sulfate was weighed, dissolved in double-distilled water, and the solution was brought to a final volume of 10 mL. The solution was then filtered through a sterile 0.22 μm inorganic phase filter membrane for sterilization and dispensed into 1.5 mL sterile EP tubes. The entire procedure was performed aseptically in a laminar flow hood and the solution was stored at -20°C. The kanamycin concentration was 50 mg / mL, and the working concentration was 25 μg / mL.

[0047] Example 3: Targeted domestication to obtain highly methanol-tolerant mutant strains

[0048] Single colonies of the recombinant strain MB200-A1 were streaked and inoculated into shake tubes to prepare a seed culture in the logarithmic growth phase. This seed culture was then transferred at a 1% inoculum to 100 mL of MM liquid medium and cultured at 30°C and 200 rpm for 40 h with shaking. Once it entered the logarithmic growth phase, it was further transferred at a 1% inoculum to MM medium with a methanol concentration increased by 4 g / L and cultured for another 40 h under the same conditions (30°C, 200 rpm).

[0049] When the methanol concentration in the culture medium increased to a level that significantly inhibited the growth of the bacterial strain, the methanol concentration gradient was adjusted to 2 g / L to alleviate the stress of high methanol concentration on the bacterial cells. The corresponding bacterial strain was preserved under each methanol concentration condition; if the strain could not grow at a certain concentration, the induction culture was restarted from the previously preserved lower concentration bacterial solution. After multiple rounds of passage and directed induction, a mutant strain tolerant to high methanol concentrations (the optimal tolerant strain) was finally screened. Using the wild-type strain MB200 as a control, directed induction experiments were conducted simultaneously under the same conditions. Finally, a mutant engineered strain that could grow normally in 48 g / L methanol medium was obtained and named MB200-X3.

[0050] Example 4: Construction of Complementary Strains

[0051] Competent cells were prepared from the methyltrophic bacterium MB200-X3 strain and electroporated into pCM80- MutS The plasmid was cultured at 30°C for 240 h on MM basal medium plates containing 25 mg / mL tetracycline. Single colonies were obtained and verified by PCR using primers as shown in SEQ ID NO:7 and SEQ ID NO:8. The correct single colony strain was named recombinant engineered strain MB200-P3.

[0052] Example 5: Mutant strain PQQ yield during shake-flask fermentation

[0053] The recombinant engineered strain MB200-P3 and wild-type strain MB200 constructed in Example 4 were cultured at 30℃ and 200rpm for 36h to prepare seed liquid. The prepared seed liquid was inoculated into 30mL Erlenmeyer flasks of MM liquid medium at an inoculation rate of 1% (v / v) and cultured at 30℃ and 200rpm for 240h to prepare fermentation broth.

[0054] Take 1 mL of the above fermentation broth, centrifuge at 14000 rpm for 2 min, filter through a filter membrane, and take 200 μL into a liquid chromatography injection bottle for high performance liquid chromatography detection (chromatographic conditions: mobile phase 65% methanol, 35% water, Shimadzu C18 column (4.6×250 mm, 5 μm), flow rate 0.6 mL / min, column temperature 25℃, injection volume 10 μL). The fermentation yield of the engineered strain PQQ is calculated by converting it with the area of ​​PQQ standard (purchased from Chenguang Biotechnology Co., Ltd.).

[0055] High performance liquid chromatography detection results as follows Figure 2 As shown, after analysis and conversion, the extracellular PQQ content of strain MB200-P3 was 96 mg / L, while the extracellular PQQ content of the control strain wild-type MB200 was 20 mg / L (e.g., Figure 3 ).

[0056] In summary, the technical solution of this invention uses methyltrophic bacteria MB200 as the original strain and knocks out its mismatch repair gene. MutS Then, they were inoculated into a medium containing methanol, and the methanol concentration was increased in increments of 2 g / L for directional culture. MutS The engineered bacteria obtained through gene feedback. The engineered bacterial strain described in this invention not only significantly improves methanol tolerance and utilization efficiency, but also significantly increases PQQ yield by enhancing the synthesis capacity of methanol dehydrogenase coenzyme PQQ.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, characterized in that, Using methyltrophic bacteria MB200 as the original strain, its mismatch repair gene MutS was knocked out. The bacteria were then inoculated into a medium containing methanol, and the methanol concentration was increased in an incremental gradient of 1-3 g / L for directional culture. Finally, the MutS gene was reintroduced to obtain the engineered bacteria.

2. The method for constructing an engineered bacterium that efficiently utilizes methanol to produce high yields of pyrroloquinoline quinone, as described in claim 1, is characterized in that... Includes the following steps: (1) The MutSsps gene expression fragment was obtained by PCR amplification and then ligated with the enzyme-digested vector plasmid to obtain the recombinant plasmid pK18mob-MutSps; (2) After the recombinant plasmid described in step (1) is transferred into the competent cells of methyltrophic bacteria MB200, the methyltrophic bacteria knockdown strain is obtained after resistance verification. (3) The methyl-trophic bacteria knockdown strain described in step (2) is inoculated into a culture medium containing 2-6 g / L methanol, and the methanol concentration is increased in an incremental gradient of 1-3 g / L for directional culture to screen for the optimal tolerant strain. (4) The recombinant plasmid pCM80-MutS is introduced into the optimal tolerant strain described in step (3) to obtain the desired strain.

3. The construction method according to claim 2, characterized in that, The primers for PCR amplification in step (1) include the MutSps-F ​​sequence and the MutSps-R sequence, wherein the MutSps-F ​​sequence is shown in SEQ ID NO:1 and the MutSps-R sequence is shown in SEQ ID NO:

2.

4. The construction method according to claim 2, characterized in that, The amount of inoculation in step (3) is 0.5-1.5% v / v.

5. The construction method according to claim 2, characterized in that, The culture in step (3) is accompanied by an oscillation process, the oscillation speed is 200 rpm, and the culture temperature is 28-32℃.

6. The construction method according to claim 2, characterized in that, The construction steps of the recombinant plasmid pCM80-MutS in step (4) include: using the genomic DNA of methyltrophic bacteria MB200 as a template, the MutS gene fragment is amplified by PCR and then ligated with the vector plasmid pCM80 to obtain the MutS gene fragment.

7. The construction method according to claim 5, characterized in that, The primers for the PCR amplification include the MutS-F sequence and the MutS-R sequence, wherein the MutS-F sequence is shown in SEQ ID NO:5 and the MutS-R sequence is shown in SEQ ID NO:

6.

8. A method for high-yield pyrroloquinoline quinone, characterized in that, Includes the following steps: The engineered bacterial culture of claim 1 is inoculated into the culture medium at an inoculation rate of 0.8-1.2% v / v and fermented at 28-32℃ for 220-260 h to obtain the culture.

9. The method according to claim 7, characterized in that, The fermentation process is accompanied by an oscillation process at a speed of 200 rpm.

10. The method according to claim 7, characterized in that, The concentration of the engineering bacteria bacterial liquid is OD 600 =2.0-2.6.

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