A method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin, the ybaS gene and applications

Abstract

The present invention belongs to the field of industrial biotechnology, and discloses a method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin, the ybaS gene and its application. The method includes introducing the ybaS gene, an expression cassette containing the ybaS gene or a vector containing the ybaS gene into Bacillus amyloliquefaciens. The present invention for the first time identifies and reveals the core gene involved in DNJ biosynthesis - the ybaS gene (the nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2), and the protein encoded by it has unique functions, filling the key gap in the DNJ synthetic metabolic pathway, providing a new perspective for analyzing the molecular mechanism of microbial synthesis of DNJ, and laying a solid molecular foundation for subsequent genetic engineering modification.

Description

Technical Field

[0001] The present invention belongs to the field of industrial biotechnology. In particular, it is a method, ybaS gene and application for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin. This method encompasses the ybaS gene that can promote DNJ biosynthesis, as well as related biological materials, strains, implementation methods and application approaches, aiming to enhance the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin (DNJ). Background Art

[0002] 1-Deoxynojirimycin (DNJ) is a natural polyhydroxypiperidine iminosugar alkaloid with the molecular formula C6H 13 NO4 and a molecular weight of 163.2. The DNJ molecule contains multiple hydroxyl groups and has good water solubility. Especially after reacting with hydrochloric acid to form hydrochloride under acidic conditions, its water solubility is further enhanced. The DNJ molecular structure is similar to glucose and can form a more stable complex with the active center of α-glucosidase, reducing the binding of the enzyme to other sugars and showing strong α-glucosidase inhibitory activity. This inhibitory effect leads to carbohydrate absorption disorders and abnormal synthesis of glycoprotein oligosaccharide chains, making DNJ have biological activities such as inhibiting blood sugar elevation, anti-tumor, and anti-viral effects.

[0003] Currently, a variety of microorganisms have been reported to be able to produce DNJ, including Streptomyces lavendulae, Monascus purpureus, Bacillus subtilis, Escherichia coli, and Bacillus amyloliquefaciens, etc. However, these microorganisms generally have problems of low yield and long fermentation time in DNJ production. These problems limit the commercial production of DNJ. Therefore, it is particularly important to select and breed high-yield DNJ microbial strains. Solving these problems can not only increase the yield of DNJ, reduce production costs, but also meet the needs of the pharmaceutical and health product fields for highly efficient and safe drugs. Therefore, finding or engineering more efficient microbial strains to increase DNJ production has important economic and scientific value for solving the current challenges in DNJ production.

[0004] In Bacillus amyloliquefaciens, although part of the DNJ synthesis pathway has been revealed and the functions of three key genes, gabT1, yktC1, and gutB1, have been clearly identified, the current engineering strain transformation work is limited to these three genes, restricting the further improvement of DNJ yield. Therefore, in-depth gene identification research is urgently needed to discover more potential transformation targets and broaden the possibilities of DNJ high-yield engineering transformation. Around these key genes, such as the ybaS gene, its role in DNJ biosynthesis is not clear, but it may also be involved in this biosynthesis process. In particular, the function and mechanism of action of the protein encoded by the ybaS gene need to be revealed urgently. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin, the ybaS gene, and its applications.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0007] A ybaS gene for improving the biosynthesis efficiency of 1-deoxynojirimycin, the nucleotide sequence of the ybaS gene is as shown in SEQ ID No.1.

[0008] A protein encoded by the ybaS gene as described above and having the function of improving the biosynthesis efficiency of 1-deoxynojirimycin, the amino acid sequence of the protein is as shown in SEQ ID No.2.

[0009] An expression cassette for improving the biosynthesis efficiency of 1-deoxynojirimycin and containing the ybaS gene as described above, the expression cassette includes a promoter, the ybaS gene, and a terminator.

[0010] A vector for improving the biosynthesis efficiency of 1-deoxynojirimycin and containing the ybaS gene as described above, the vector is selected from plasmids, viral vectors, or artificial chromosomes.

[0011] A method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin by using the ybaS gene as described above, the method includes introducing the ybaS gene, the expression cassette containing the ybaS gene, or the vector containing the ybaS gene into Bacillus amyloliquefaciens.

[0012] Further, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, the classification name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC No.29550, the preservation date is: January 9, 2024, and the preservation unit is: China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] A recombinant strain of Bacillus amyloliquefaciens for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin and containing the ybaS gene as described above.

[0014] The construction method of the recombinant strain as described above includes the following steps:

[0015] First, the overexpression plasmid pLH2387 containing the ybaS gene was introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electroporation, and preliminary transformants were obtained by screening with LB medium containing 5 μg / mL chloramphenicol; the transformants were passaged at 42 °C, transferred every 12 hours for a total of 3 times, inducing single crossover between the plasmid and the genome, and confirmed by PCR verification; subsequently, further high-temperature passage promoted double crossover recombination, and the double crossover strains were screened by spreading on LB medium containing 25 μg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure was confirmed by PCR detection; finally, sensitive strains were screened by chloramphenicol sensitivity test to exclude residual plasmids, and the accuracy of gene editing was verified by sequencing, obtaining a genetically stable ybaS overexpression recombinant strain.

[0016] Use of the recombinant strain as described above in the fermentation production of 1-deoxynojirimycin.

[0017] A method for fermenting and producing 1-deoxynojirimycin using the recombinant strain as described above, comprising the following steps:

[0018] The preserved recombinant strain was taken out from the ultra-low temperature freezer and streaked on LB solid medium respectively, cultured at 37 °C for 12 h to grow single strains; the single colonies were transferred to 5 mL of fresh LB liquid medium and cultured at 37 °C and 200 r / min for 12 h for seed culture; according to the inoculation amount of 4%, the seed liquid of the cultured bacteria was inoculated into the fermentation medium respectively and cultured at 37 °C and 100 r / min for 72 h for 1-deoxynojirimycin fermentation;

[0019] Among them, the fermentation medium used: (NH4)2SO4: 4 g / L, lactose: 25 g / L, K2HPO4: 14 g / L, KH2PO4: 6 g / L, MgSO4·7H2O: 0.2 g / L, CaCl2: 0.15 g / L, MnSO4·H2O: 1.9 mg / L, FeCl3·6H2O: 37.8 mg / L, ZnCl2: 7 mg / L, the solvent is water, pH 7.0, sterilized at 121 °C for 20 min.

[0020] The advantages and positive effects obtained by the present invention are:

[0021] 1. The present invention for the first time identified and revealed the core gene ybaS (nucleotide sequence shown in SEQ ID No.1, amino acid sequence shown in SEQ ID No.2) involved in DNJ biosynthesis, and the protein encoded by it has unique functions, filling the key gap in the DNJ synthetic metabolic pathway, providing a new perspective for analyzing the molecular mechanism of microbial DNJ synthesis, and laying a solid molecular foundation for subsequent genetic engineering transformation.

[0022] 2. The present invention clearly verified the decisive role of the ybaS gene in DNJ synthesis through gene knockout and overexpression techniques: the ybaS gene knockout strain completely lost the ability to produce DNJ, while the engineered strain with the ybaS gene complemented had a 60% increase in DNJ production compared to the wild type, and this improvement effect was significantly better than the existing transformation strategies. This experimental data first confirmed from the functional level that the ybaS gene is an essential element for DNJ biosynthesis, providing a reliable gene target for the construction of industrial high-yield strains.

[0023] 3. Aiming at the core problems such as low yield and lack of genetic transformation targets in current DNJ production, the present invention proposed an innovative solution, developed an expression cassette (including a promoter and a terminator) based on the ybaS gene and a supporting vector system (plasmid vector), and established a genetic engineering transformation method for Bacillus amyloliquefaciens LH-2 (CGMCC No. 29550), achieving the directional enhancement of DNJ synthesis ability. In addition, this technical solution also has wide applicability and can be extended to other Bacillus strains, providing a broad application prospect for the industrial production of DNJ.

[0024] 4. The present invention used precise gene knockout and complementation techniques to systematically identify the function of the ybaS gene in Bacillus amyloliquefaciens. The goal was to clarify the specific role of the ybaS gene in DNJ biosynthesis, providing a scientific basis and theoretical support for the development of new and more efficient DNJ production strategies. This research result is expected to break through the bottleneck of DNJ yield improvement and provide new ideas and methods for related research in the field of industrial biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the high performance liquid chromatography (HPLC) spectrum of DNJ in the fermentation broth in the present invention (variable wavelength detector, wavelength is 265 nm);

[0026] Figure 2 is a schematic diagram of the relative positions of the DNJ synthesis gene cluster and the genes on both sides in the present invention;

[0027] Figure 3 is the map of the knockout plasmid pLH2223 in the present invention;

[0028] Figure 4 is the map of the overexpression plasmid pLH2387 in the present invention;

[0029] Figure 5This is the PCR verification result diagram of the knockout plasmid pLH2223 electrotransformed into strain LH-2 in the present invention. Among them, M is the DNA Marker; N is used as the negative control; P is the positive control, with the pLH2223 plasmid vector as the template; the bands numbered 1, 2, and 3 respectively represent the genomic PCR amplification products of different transformants; the sizes of these product fragments are the same, all 980 bp, fully proving that the knockout plasmid pLH2223 has been successfully and stably transferred into strain LH-2;

[0030] Figure 6 This is the PCR verification result diagram of the overexpression plasmid pLH2387 electrotransformed into strain LH-2 in the present invention. Among them, M is the DNA Marker; N is the negative control; P is the positive control, with the pLH2387 plasmid vector as the template; and 1, 2, and 3 are the PCR amplification products of the transformants obtained after the overexpression plasmid pLH2387 was electrotransformed into strain LH-2, and the fragment sizes are all 490 bp. This result verifies the successful transfer of the overexpression plasmid;

[0031] Figure 7 This is the PCR verification result diagram of the single crossover of the knockout plasmid pLH2223 in the recombinant bacteria in the present invention. Among them, M represents the DNA Marker; N is the negative control; 1, 2, and 3 are the PCR amplification products of the transformants, and the fragment sizes are all 1330 bp. This result confirms that the single crossover of the knockout plasmid pLH2223 has occurred successfully in the recombinant bacteria;

[0032] Figure 8 This is the PCR verification result diagram of the single crossover of the overexpression plasmid pLH2387 in the recombinant bacteria in the present invention. Among them, M represents the DNA Marker; N is the negative control; 1 and 2 are the PCR amplification products of the transformants, and the fragment sizes are all 2760 bp. This result confirms that the single crossover of the overexpression plasmid pLH2387 has occurred successfully in the recombinant bacteria;

[0033] Figure 9 This is the PCR verification result diagram of the double crossover of the knockout plasmid pLH2223 in the recombinant bacteria in the present invention. Among them, M represents the DNA Marker; N is the negative control; WT represents using the genome of LH-2 as the template, and its fragment size is 1660 bp as a reference; 1 is the PCR amplification product of the strain with successful knockout, and the fragment size is 1400 bp. This result confirms that the double crossover of the knockout plasmid pLH2225 has occurred successfully in the recombinant bacteria;

[0034] Figure 10This is the PCR verification result diagram of the double crossover of the overexpression plasmid pLH2387 in the recombinant bacteria in the present invention. Among them, M represents DNA Marker; N is the negative control; WT indicates using the genome of LH-2 as the template, and its fragment size is 1590 bp as a reference; 1 is the PCR amplification product of the strain with successful overexpression integration, and the fragment size is 2900 bp. This result confirms that the overexpression plasmid pLH2387 has successfully undergone double crossover in the recombinant bacteria.

[0035] Figure 11 This is the comparison diagram of the DNJ shake flask fermentation yields of the strains LH-2, S4277, and S4452 in the present invention.

[0036] A Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, the classification name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC No. 29550, the preservation date: January 9, 2024, the preservation unit: China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation manners

[0037] The following further illustrates the present invention in combination with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0038] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically annotated in this article, those of ordinary skill in the art can refer to various common reference books, scientific and technological literatures, or relevant instructions, manuals, etc. before the application date of the present invention for implementation.

[0039] A ybaS gene for improving the biosynthesis efficiency of 1-deoxynojirimycin, and the nucleotide sequence of the ybaS gene is as shown in SEQ ID No. 1.

[0040] A protein encoded by the ybaS gene as described above and having the function of improving the biosynthesis efficiency of 1-deoxynojirimycin, and the amino acid sequence of the protein is as shown in SEQ ID No. 2.

[0041] An expression cassette for improving the biosynthesis efficiency of 1-deoxynojirimycin and containing the ybaS gene as described above, and the expression cassette contains a promoter, the ybaS gene, and a terminator.

[0042] A vector for improving the biosynthesis efficiency of 1-deoxynojirimycin and containing the ybaS gene as described above, and the vector is selected from plasmids, viral vectors, or artificial chromosomes.

[0043] A method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin by using the ybaS gene as described above, the method comprising introducing the ybaS gene, an expression cassette containing the ybaS gene, or a vector containing the ybaS gene into Bacillus amyloliquefaciens.

[0044] Preferably, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, the classification name is: Bacillus amyloliquefaciens, the preservation number is: CGMCC No. 29550, the preservation date is: January 9, 2024, and the preservation unit is: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] A recombinant strain of Bacillus amyloliquefaciens that contains the ybaS gene as described above and has improved ability to synthesize 1-deoxynojirimycin.

[0046] The construction method of the recombinant strain as described above includes the following steps:

[0047] First, the overexpression plasmid pLH2387 containing the ybaS gene is introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electrotransformation, and the primary transformants are screened using LB medium containing 5 μg / mL chloramphenicol; the transformants are passaged at 42 °C, transferred every 12 hours for a total of 3 times, to induce single crossover between the plasmid and the genome, and integration is confirmed by PCR verification; subsequently, further high-temperature passage promotes double crossover recombination, and the double crossover strains are screened by spreading on LB medium containing 25 μg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure is confirmed by PCR detection; finally, sensitive strains are screened by chloramphenicol sensitivity test to exclude residual plasmids, and the gene editing accuracy is verified by sequencing to obtain a genetically stable ybaS overexpression recombinant strain.

[0048] The application of the recombinant strain as described above in the fermentation production of 1-deoxynojirimycin.

[0049] A method for fermenting and producing 1-deoxynojirimycin by using the recombinant strain as described above, including the following steps:

[0050] The preserved recombinant strains were taken out from the ultra-low temperature refrigerator and streaked on LB solid medium respectively, and cultured on the medium at 37 °C for 12 h to grow single strains; the single colonies were transferred to 5 mL of fresh LB liquid medium and cultured at 37 °C and 200 r / min for 12 h for seed culture; according to the inoculation amount of 4%, the seed solutions of the cultured bacteria were inoculated into the fermentation medium respectively and cultured at 37 °C and 100 r / min for 72 h for 1-deoxynojirimycin fermentation;

[0051] Among them, the fermentation medium used: (NH4)2SO4: 4 g / L, lactose: 25 g / L, K2HPO4: 14 g / L, KH2PO4: 6 g / L, MgSO4·7H2O: 0.2 g / L, CaCl2: 0.15 g / L, MnSO4·H2O: 1.9 mg / L, FeCl3·6H2O: 37.8 mg / L, ZnCl2: 7 mg / L, the solvent is water, pH 7.0, sterilized at 121 °C for 20 min.

[0052] Specifically, the related preparation and detection are as follows:

[0053] Screening and identification of strain Bacillus amyloliquefaciens LH-2 in Example 1

[0054] It was isolated from cow dung in a cattle farm (address: Xiazhuang'er Village, Wangcun Township, Laishui County, Baoding City, Hebei Province, China).

[0055] The collected cow dung samples were ground and passed through a 60-100 mesh sieve. 1 g of the sample was added to 15-50 mL of LB liquid medium with glass beads, shaken well and then placed in a water bath at 80-90 °C for 10-15 min, and then shaken on a shaker at 28 °C (120 rpm) for 24 h. The cultured product after the end of the culture was placed in a water bath at 80-90 °C for 10-15 min again, appropriately diluted by the 10-fold dilution method, and 0.1 mL of the diluted solutions of three dilution factors of 10 5 、10 6 and 10 7 times were spread on LB solid medium and cultured upside down at 28-37 °C for 2-5 days, and observed every day. Colonies that were white, irregular, uneven, and had uneven edges were selected and used as candidate strains after 3-5 rounds of streak passage purification.

[0056] Use an inoculation loop to inoculate the candidate strain into a test tube containing LB liquid medium, and culture it at 100 - 200 rpm and 28 - 37 °C for 8 - 24 h for seed culture. Inoculate 100 - 1000 μL of the seed liquid into a 500 mL baffled shake flask (containing 50 - 200 mL of fermentation medium), and culture it at 28 - 37 °C and 100 - 200 rpm for 2 - 3 days to obtain the primary screening fermentation broth. Take 1 mL of the fermentation broth, centrifuge it at 10000 rpm for 10 min to remove the bacteria, and use the supernatant for DNJ detection.

[0057] Among them, the sample treatment and detection method refer to the national standard detection method "Detection of 1-Deoxynojirimycin in Mulberry Leaf Extracts" (GB / T 40642-2021), and the detection wavelength of liquid chromatography is set at 265 nm.

[0058] The composition of the LB medium is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, adjust the pH to 7.0, and 2% agar powder needs to be added to the solid medium.

[0059] The composition of the fermentation medium is: 0.1 - 100 g / L starch, 0.1 - 20 g / L soy peptone, 0.01 - 10.0 g / L (NH4)2SO4, 0.01 - 20.0 g / L K2HPO4, 0.01 - 20.0 g / L KH2PO4, 0.01 - 10 g / L MgSO4·7H2O or MgSO4, 0.01 - 5 g / L CaCl2, 0.01 - 100 mg / L MnSO4, 0.01 - 500 mg / L Fe2(SO4)3, 0.01 - 100 mg / L ZnCl2, adjust the pH to 4.0 - 9.0.

[0060] Experimental results: A total of 600 colonies were isolated in this experiment, and 50 strains producing DNJ were isolated. Among them, the highest content of DNJ was 1.5 g / L, and its HPLC chromatogram is as Figure 1As shown, select the single strain with the number LH-2. Inoculate the strain LH-2 into LB liquid medium and culture it with shaking at 28-37 °C for 24 h. Then collect the bacteria by centrifugation (8,000×g, 10 min). Extract genomic DNA using a bacterial genomic DNA extraction kit (Tiangen, DP302), and amplify the 16S rRNA gene with universal primers 27F and 1492R (as shown in Table 1) (PCR conditions: pre-denaturation at 94 °C for 5 min; 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, 30 cycles; final extension at 72 °C for 10 min). After verifying the amplified product by 1% agarose gel electrophoresis, purify it and send it to Genewiz Biotechnology Co., Ltd. to complete bidirectional sequencing. The obtained 16S rDNA sequence (SEQ ID No.3) was submitted to the NCBI database for BLASTn homology comparison (parameter settings: E-value ≤ 1e-50, coverage ≥ 95%). As shown in Table 2 (only 5 strains with higher homology are listed in the table), the similarity of this sequence to the 16S rRNA genes of different Bacillus amyloliquefaciens strains such as Bacillus amyloliquefaciens strain H reached 100%. Therefore, the strain was identified as Bacillus amyloliquefaciens.

[0061] The Bacillus amyloliquefaciens LH-2, its name is LH-2, the taxonomic name is: Bacillus amyloliquefaciens, the deposit number is: CGMCC No. 29550, the deposit date: January 9, 2024, the deposit unit: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0062] Table 1 Sequences of primers used

[0063]

[0064] Table 2 BLASTn homology comparison results of the 16S rRNA sequence of strain LH-2

[0065]

[0066] Example 2 Discovery of the ybaS gene

[0067] In Bacillus amyloliquefaciens, the metabolic pathway of DNJ has been partially analyzed, and the research has clearly revealed the core role of the gabT1, yktC1, and gutB1 gene clusters in the biosynthesis of DNJ. However, existing research still lacks a systematic exploration of whether the genes in the flanking regions of the gene clusters are involved in DNJ synthesis. Based on this, through whole-genome sequencing analysis of the DNJ-producing Bacillus amyloliquefaciens strain LH-2 (CGMCC No. 29550) (completed by Suzhou Genewiz Biotechnology Co., Ltd. / GENEWIZ), the ybaS gene (SEQ ID No. 1) located on the flank of the gabT1-yktC1-gutB1 gene cluster was first discovered, and it encodes a NAD(P)H-dependent reductase (the amino acid sequence is shown in SEQ ID No. 2). Its specific role in DNJ synthesis has not been clarified. Therefore, in the present invention, by knocking out and complementing the ybaS gene in Bacillus amyloliquefaciens LH-2, it aims to identify the function of this gene in DNJ biosynthesis and provide a new strategy for increasing DNJ production. A schematic diagram of the relative positions of the gene cluster and the genes on both sides can be seen in Figure 2 。

[0068] Example 3 Construction of the ybaS gene editing vector

[0069] (1) Construction of the ybaS gene knockout vector pLH2223

[0070] First, using the genomic sequence of Bacillus amyloliquefaciens LH-2 (the genomic sequencing has been completed by Suzhou Genewiz Biotechnology Co., Ltd. / GENEWIZ.) as a template, the ybasUP-F / ybasUP-R and ybasDN-F / ybasDN-R primers were designed (see Table 1). Using the genomic DNA of Bacillus amyloliquefaciens LH-2 (the extraction method is shown in Example 1) as a template, the upstream and downstream homologous arms of the ybaS gene (SEQ ID No. 4 / No. 5) were amplified using PhantaMax Super-Fidelity Polymerase (Nanjing Novoprotein, product number P505-d1), and the PCR amplification system is shown in Table 3. The PCR amplification products of the upstream and downstream homologous arms of the ybaS gene (SEQ ID No. 4 / No. 5) obtained were recovered using a common DNA product purification kit (Tiangen, DP204) to obtain purified DNA fragments, and then were sequentially ligated to the BamHI site of the pKSU plasmid through the ClonExpress Ultra One Step Cloning Kit V (Nanjing Novoprotein, product number C117-01), and the ybaS gene knockout vector pLH2223 was successfully constructed ( Figure 3). The one-step cloning system of ClonExpress Ultra One Step Cloning Kit V is shown in Table 4.

[0071] Table 3 PCR amplification system of Phanta Max Super-Fidelity Polymerase

[0072]

[0073] Table 4 One-step cloning system of ClonExpress Ultra One Step Cloning Kit V

[0074]

[0075] Special note: Plasmid pKSU, as the core tool vector for genetic manipulation of Bacillus amyloliquefaciens, has become the preferred system for gene editing in this species due to its high transformation efficiency, multi-cloning site compatibility, and stable replication characteristics. This plasmid was first reported by Zhang et al. in 2014 in "Applied Microbiology and Biotechnology", and the markerless gene replacement technology they developed was successfully applied to the genome reduction and optimization of the poly-γ-glutamic acid synthesis pathway (Zhang, W., Gao, W.X., Feng, J., Zhang, C., He, Y.L., Cao, M.F., et al. (2014) A markerless gene replacement method for Bacillus amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production. Appl Microbiol Biotechnol 98: 8963–8973.). Currently, the genetic operation system based on pKSU has been included in the International Bacillus Genetic Stock Center (BGSC).

[0076] (2) Construction of the overexpression vector pLH2387 of the ybaS gene

[0077] Using pKSU as the starting vector, the specific process of designing and constructing the overexpression vector pLH2387 of the ybaS gene integrated into the pksJ gene target (SEQ ID No.6) is as follows:

[0078] Using the genomic DNA of Bacillus amyloliquefaciens LH-2 (the extraction method is shown in Example 1) as a template, and using PhantaMax Super-Fidelity Polymerase (Nanjing Novoprotein, product number P505-d1), the upstream and downstream homologous arms of the pksJ gene (SEQ ID No.7 / No.8) were amplified with the primers pksJUP-F / pksJUP-R and pksJDN-F / pksJDN-R (see Table 1); the amyS promoter (SEQ ID No.9) was amplified with the primers amsy-F / asmy-R (see Table 1); the ybaS gene (SEQ ID No.1) was amplified with the primers ybas-F / ybas-R (see Table 1), and the purified DNA fragments were recovered by a common DNA product purification kit (Tiangen, DP204). Then, through the ClonExpress Ultra One Step Cloning Kit V3 one-step cloning kit (Nanjing Novoprotein, product number C117-01), the upstream and downstream homologous arms of the pksJ gene, the amyS promoter, and the ybaS gene were successively ligated to the BamHI site of the pKSU plasmid to obtain the ybaS gene overexpression vector pLH2387 ( Figure 4 ).

[0079] Example 4 Construction of ybaS gene knockout and overexpression recombinant strains

[0080] (1) Preparation of competent cells of Bacillus amyloliquefaciens LH-2

[0081] 1) Take out the preserved strain LH-2 from -80 °C, streak it on the LB solid medium in three zones, and place it in a 37 °C incubator for 12 - 24 h until clear single colonies appear. Pick a monoclonal colony and inoculate it into the LBS medium (containing 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, 91.0 g / L sorbitol, and the solvent is water.) and culture it overnight.

[0082] 2) Transfer the bacterial solution to a sterile centrifuge tube and ice-bath for 30 min. Then centrifuge at 8000 r / min at 4 °C for 20 min to collect the bacterial cells.

[0083] 3) Add approximately 60 mL of eluent (its formula is: containing 91.0 g of sorbitol, 91.0 g of mannitol, 50 g of glycerol, and made up to 0.5 L with water). After mixing, centrifuge again for 20 min under the same conditions and collect the thalli. Repeat this operation twice.

[0084] 4) Add 500 μL of the resuspension solution for preparing electrocompetent cells (its formula is: containing 0.91 g of sorbitol, 0.91 g of mannitol, 0.1 g of glycerol, 1.4 g of PEG6000, and made up to 10 mL with water), and gently resuspend the thalli thoroughly.

[0085] 7) Aliquot the obtained electrocompetent cells into sterile EP tubes at 100 μL per portion and store them at -80 °C for later use.

[0086] (2) Electrotransformation of the ybaS gene editing vector into Bacillus amyloliquefaciens

[0087] 1) Take the electrocompetent cells and let them thaw naturally on ice. Add appropriate amounts of pLH2223 or pLH2387 plasmids (not exceeding 200 ng) respectively and mix well.

[0088] 2) Transfer the above system to a pre-chilled sterile electroporation cuvette (2 mm specification) and place it on ice for 2 - 3 min.

[0089] 3) Perform electroporation at a voltage of 2500 V. Then add 1 mL of pre-chilled electrotransformation bacterial resuscitation liquid medium (0.38 mol / L mannitol dissolved in LBS medium) to the electroporation cuvette and pipette to mix well.

[0090] 4) Transfer the above bacterial solution to an EP tube and culture it at 30 °C and 180 r / min for 3 h.

[0091] 5) Centrifuge at 8000 r / min for 2 min to concentrate the thalli. Take 100 μL of the bacterial solution and spread it on an LB solid medium containing chloramphenicol (5 μg / mL), and let it stand for culture. Culture it at 30 °C for 48 h, and transformants can be seen growing.

[0092] (3) Screening and verification of ybaS gene knockout strains and overexpression recombinant strains

[0093] 1) Inoculate the transformants obtained from the previous step of electroporation into an LB liquid medium containing 5 μg / mL chloramphenicol and culture them overnight at 30 °C;

[0094] 2) The overnight cultured bacterial solution was verified by PCR using the p1 / p2 primer pair to ensure whether the ybaS knockout plasmid pLH2223 and the overexpression plasmid pLH2387 were successfully electrotransformed into the competent cells of Bacillus amyloliquefaciens LH-2. The enzyme preparation used for PCR amplification verification was 2×Rapid Taq Master Mix from Nanjing Novoprotein Scientific Inc., and the specific method is shown in Table 5. The results are as Figure 5 and Figure 6 shown. The results indicated that pLH2223 and pLH2387 were respectively electrotransformed into Bacillus amyloliquefaciens LH-2.

[0095] 3) The correct transformants of the two plasmids obtained were respectively transferred into fresh LB liquid medium containing 5 μg / mL chloramphenicol and subcultured at 42 °C, transferred every 12 h for a total of 3 times. This promoted single crossover of the plasmids in the strain.

[0096] 4) After appropriately diluting the bacterial solutions of the two plasmids in step 3), 100 μL was taken and spread on LB solid medium containing 5 μg / mL chloramphenicol and cultured at 30 °C for 48 h, and single colonies could be seen growing. Using the primers p3 / p4, PCR detection was performed on the single colonies of the knockout plasmid pLH2223, and the results are as Figure 7 shown; using the primers p5 / p6, PCR detection was performed on the single colonies of the overexpression plasmid pLH2387, and the results are as Figure 8 shown. The results are as Figure 7 and Figure 8 shown. The results indicated that pLH2223 and pLH2387 respectively completed single crossover in Bacillus amyloliquefaciens LH-2, and single-crossover transformants of the two plasmids were screened and obtained.

[0097] 5) The single-crossover transformants of the two plasmids obtained were respectively transferred into LB liquid medium and subcultured at 42 °C, transferred every 12 h for a total of 3 times. This promoted double crossover of the single-crossover transformants.

[0098] 6) After appropriately diluting the bacterial solutions of the two plasmids in step 5), they were spread on LB solid medium containing 25 μg / mL 5-fluorouracil and cultured at 30 °C for 48 h, and single colonies could be seen growing. PCR detection was performed on the respective single colonies of the two plasmids to screen for double-crossover strains. Using the primers p7 / p8, PCR detection was performed on the single colonies of the knockout plasmid pLH2223, and the results are as Figure 9 shown; using the primers p9 / p10, PCR detection was performed on the single colonies of the overexpression plasmid pLH2387, and the results are as Figure 10 shown. The results are as Figure 9 and Figure 10As shown, it indicates that double crossover transformants of two plasmids are obtained, thus obtaining ybaS gene knockout and overexpression recombinant transformants.

[0099] 7) Inoculate the ybaS gene knockout and overexpression recombinant transformants obtained in 6) into LB liquid medium, culture overnight at 30 °C, and dilute and spread on LB solid medium, culture at 30 °C for 48 h, and single colonies can be seen growing. Spot inoculate the single colonies onto LB solid medium and LB solid medium containing 5 μg / mL chloramphenicol respectively to determine whether the recombinant transformants are sensitive to chloramphenicol, select chloramphenicol-sensitive strains, and send them for sequencing verification. Finally, the ybaS gene knockout recombinant strain S4277 and the overexpression recombinant strain S4452 are obtained.

[0100] Table 5 PCR verification system

[0101]

[0102] Example 5 DNJ shake flask fermentation of strains LH-2, S4277 and S4452

[0103] (1) Shake flask fermentation experiment

[0104] Take out the preserved strains LH-2, S4277 and S4452 from the ultra-low temperature refrigerator, streak inoculate them on LB solid medium respectively, and culture at 37 °C for 12 h to grow single strains. Transfer the single colonies of the three strains to 5 mL of fresh LB liquid medium respectively, culture at 37 °C and 200 r / min for 12 h for seed culture. Inoculate the seed solutions of the three cultured strains into 30 mL of fermentation medium (in 250 mL Erlenmeyer flasks) according to the inoculation amount of 4% (v / v, volume ratio), and culture at 37 °C and 100 r / min for 72 h for DNJ fermentation. Set 3 replicates of shake flask experiments for the fermentation of each strain. The fermentation medium used is: (NH4)2SO4: 4 g / L, lactose: 25 g / L, K2HPO4: 14 g / L, KH2PO4: 6 g / L, MgSO4·7H2O: 0.2 g / L, CaCl2: 0.15 g / L, MnSO4·H2O: 1.9 mg / L, FeCl3·6H2O: 37.8 mg / L, ZnCl2: 7 mg / L, the solvent is water, pH 7.0, sterilize at 121 °C for 20 min.

[0105] (2) Fermentation broth treatment and determination method

[0106] Centrifuge the fermentation broth at 12,000 r / min for 5 min, and then aspirate the supernatant for standby. Next, take 35 µL of the fermentation supernatant or DNJ standard solution and add it to a 1.5 mL centrifuge tube. Then, sequentially add 200 µL of 0.4 mol / L potassium borate buffer (pH 8.5) and 250 µL of 5 mmol / L derivatization reagent 9-fluorenylmethyl chloroformate solution (abbreviated as FMOC-Cl, which has been dissolved in acetonitrile with a volume concentration of 50%). After thorough mixing, place the mixture in a constant temperature water bath at 25 °C for 20 minutes. After the reaction, add 25 µL of 1 mol / L glycine aqueous solution to terminate the reaction, then add 100 µL of 1% (V / V, volume concentration) acetic acid aqueous solution, and make up the volume to 800 µL with water. Finally, filter the solution using a microporous membrane with a pore size of 0.22 µm, and collect the filtrate as the subsequent detection sample.

[0107] Determine the detection sample by high performance liquid chromatography. The chromatographic column is ZORBAX SB-C18 (specification: 5 µm, 4.6×150 mm), the detector is an ultraviolet detector, and the detection wavelength is 254 nm. The mobile phase is acetonitrile - 0.1% acetic acid (1:1, V / V), the flow rate is 1.0 mL / min, and the injection volume is 20 µL.

[0108] (3)Analysis of DNJ shake flask fermentation results

[0109] After 72 hours of shake flask fermentation, among them, the DNJ titer of the starting strain LH-2 is 1.5 g / L, the DNJ titer of the recombinant strain S4277 with the ybaS gene knocked out is almost 0, and the DNJ titer of the strain S4452 overexpressing ybaS is 2.4 g / L. Compared with the wild-type Bacillus amyloliquefaciens LH-2, the DNJ production of the overexpressing strain S4452 increased by 60% ( Figure 11 ), verifying the key role of the ybaS gene in the synthesis of DNJ by Bacillus amyloliquefaciens.

[0110] In summary, the present invention not only successfully identified and revealed the key gene ybaS for the biosynthesis of 1-deoxynojirimycin (DNJ) in Bacillus amyloliquefaciens, filling an important gap in the synthetic metabolic pathway; moreover, by using gene knockout and overexpression techniques, the function of ybaS as an essential gene for DNJ synthesis was systematically verified, and after the gene was complemented, the production of DNJ was significantly increased. In addition, the present invention also innovatively developed an expression cassette and vector system with ybaS as the core, and established a new strategy for genetic engineering transformation of Bacillus amyloliquefaciens, greatly enhancing the synthesis efficiency of DNJ. This technical solution has wide applicability and strong application potential, opening up a broad road for the industrial production of DNJ, and has important strategic significance and value.

[0111] The sequences used in the present invention are as follows:

[0112] SEQ ID No.1 ybaS fragment:

[0113] ATGATGTGGCTTCAGAAGATGAACCAAGTGTTGGGGAGAATCATGCCGCTTTTGACTCCGTCAAGTGTGGCGGCGGGTGTGCTGTTATCTCAGCATATCAGCTGGATGGCCGGCGCTGTGCCGTGGATTTTTGCTTTTATTACATTTACGGGCAGTCTGAGTGCCAATTTTCAATCGCTGAAGCGAAGTATTGAGCAGCCGCTCCCGATGATCATGGCATTGTTTGTTCTGCATATTTTCATGCCTGTTTTTGCATGGGGAAGCGGTCATCTGATATTCAGCGGTGACCCTTTAACTGTGACGGGGCTGATAATGGGTGTGGTGATTCCGACCGGAATTACGAGTTTGATTTGGGCCGCGATGTATAAGGGGAATGTCGGATTGACGCTGTCTATAATCCTGGTTGATACCGTACTGTCTCCTTTTATCGTGCCGCTGAGCCTTTCTGTCCTGGCCGGGGCAAATGTCCAGATGGATGTGTGGGGAATGATGAAAGGGCTTTTGGAAATGGTGGTGCTTCCGTCTATCGCAGGGATGATGGTCAACCAGTTTTCCCCGCCGGTCAGGACTCAGGCGATAAGCCGGACGCTGTCTCCTTTTTCAAAACTCTGTCTGATGATAGTCATTGCCATTAACAGTTCGGAAATCGCGCCGTATGTTACGCACTTGGATATCAAATTTGCGGGCATTGCGGCCGTGGTATTTTTTATTGCCATGACGGGTTATGCTGTGGCTTGGCTGATCGGAAGGCTGTTGAAGCGGAGCCAGGATGAAATTGTCTCTCTGATTTATACGGGAGGCATGCGCAATATCAGTGCTGGGGCCGTTCTCGCCGTTTCGTTTTTTCCGTCGCAAGTGGCGGTTCCGGTCGTCATCGGGATGCTTTTTCAGCAAATCCTTGCCGCTTTATTCGGCTATTTGCTGAGGCGCTTTGAACTTAAGCCGGTCGTTATGAAGTATGAAAAAAACCGCTCAGCCACGTGA

[0114] Amino acid sequence of ybaS of SEQ ID No.2:

[0115] MMWLQKMNQVLGRIMPLLTPSSVAAGVLLSQHISWMAGAVPWIFAFITFTGSLSANFQSLKRSIEQPLPMIMALFVLHIFMPVFAWGSGHLIFSGDPLTVTGLIMGVVIPTGITSLIWAAMYKGNVGLTLSIILVDTVLSPFIVPLSLSVLAGANVQMDVWGMMKGLLEMVVLPSIAGMMVNQFSPPVRTQAISRTLSPFSKLCLMIVIAINSSEIAPYVTHLDIKFAGIAAVVFFIAMTGYAVAWLIGRLLKRSQDEIVSLIYTGGMRNISAGAVLAVSFFPSQVAVPVVIGMLFQQILAALFGYLLRRFELKPVVMKYEKNRSAT

[0116] 16S rDNA sequence of SEQ ID No.3:

[0117]

[0118] Nucleotide sequence upstream of the ybaS gene of SEQ ID No.4:

[0119] TGGCCGTGATGGTTATGGTGTCCATCGGCACTTTTGATTGGTCATCGATTAAAGGCTTGCGGAAAGCGCCTCTTACGGATTCAATCGTTATGGTTATTACCGTCTTGACGGTCGTCCTGACAAATGATTTATCGAAGGGTGTTTTCGTGGGTGTGTTATTAAGCGCAATCTTTTTCACGGCTAAAATTTCTAAGCTGAAGATTGTATCACATTCCGAACAGAAAGATGTCAGAACATATAAGGTAACGGGACAGATCTTTTTTGCTTCTGTATCTGAGCTGATGAATGCCATCGATTATGATGAAGGTGTGAAACGGATTGTCATTGATTTGACAGGTGCCCACGTATGGGATGATTCAGGAGCGGCGGCTCTTGAGAAAATTGTCGCCAAATGTAAAGAATACGGAATTGAGGCTGAATTAAAGGGATTAAATCAGAAAAGCCGAGATTTAATGAAGCAAATGGCATAGAATTTGTGAAGTCCGGATGAATATATCCGGGCTTTTTTTCGGTTAAATTTGCCGGAGTGCATTGTTATAATGAATGAGACTTGAAAACCGTATATAGGAGACTTGCATATGATGTGGCTTCAGAAGATGAACCAAGTGTTGGGGAGAATCATGCCGCTTTTGACTCCG

[0120] Nucleotide sequence downstream of the ybaS gene of SEQ ID No.5:

[0121] AGGCGCTTTGAACTTAAGCCGGTCGTTATGAAGTATGAAAAAAACCGCTCAGCCACGTGAGCGGTTTTTTCCGTACAGCCAGCTTAGAGCTGAAATGAATTGTCTTTTGAACGCTTCTTGATGGTGCACGGCGCCCTCTTCAATCGTGAAACAGAGCTGAGAGTGAGAGAAGCCTTTTTGCCTCATGCTTTCATGCACGTCTCTATTTTGTCTGACCATAGTGTTTTGGATGTTGTTTTTATGCTCAGCTTCACGGCTGCCGACAGACATATAGATGCGCTGCCGTGTCTGCCGCAAAGGTGCGGAGCGGACATAGTCTGCCGATTTTTCATACCAGAAGGACCCGGAGATACAGCCGATTCTGCCGAATATCTCAGGCTTCTGTAACATCGCGTACATTGAAAACAATCCGCCTAATGACGCGCCGATCAGTCCTCTTGTTTCCGGGTCCGGAGACATGTGATGCATATCTTCAACCGTTGGGATGATGGTATTTGTCAGGTCTTCAAGAAAGAGGCTCCCTTTGCCGCCGAAATCATGAAACGCAGGGCTCAGGGAAGGCGCAGGCCATGGTGTGTACTCATCGAGCCTGTTATCCGGTTTGATGCCGGCGAAAATCAATTCAGGAAGTGTTCCGT

[0122] SEQ ID No.6 pksJ locus:

[0123]

[0124] SEQ ID No.7 Upstream homologous arm of pksJ:

[0125] CAGAAGACATTCAAAACCGGAAATGGGACTTATCCTCCATGCGTTACATGCTGAACGGAGGGGAGGCCACGGTTGCGAAAGTCGGGCGCAGAATTATGGAGCTTTTAGAGCCGCATGGTCTGCCGGCTAATGCCATCCGTCCCGCATGGGGAATGTCCGAAACATCTTCAGGCGTTATTTTCTCAGATGAGTTTACACTGGAAAACACGAGTGATGATGATCGTTTCGTGGAGATCGGTCTCCCGATTCCGGGGTTTAATATGAGAATTACGGATGACCGCAATCAGGTGGTGGAAGAAGGAGAAATCGGACGTTTCCAAGTGTCGGGTCTGACTGTAACAAGCGGTTATTATGAGCGTCCGGACTTAAACGAGAGTGTCTTTACAGAAGACGGCTGGTTTGAAACGGGAGATCTCGGCTTTCTGCGTGAAGGCCGCCTGACGATAACGGGCCGAACGAAAGATGCCATCATTATCAACGGTGTGAATTATTACAGCCAC

[0126] SEQ ID No.8 Downstream homologous arm of pksJ:

[0127] TCATTACATAATAACAAGCAAGAAGACGGCCGTTCAGAATCGGCTTCTGCGGAGAAAGAGATTGAACGGGATTTTATCCGTTTTCTGAAAGAGGAACTTTCTATCGCGGATGAACTCGTTGATCCGCATACACCGCTTCAAAGTCTTGGCGTCAATTCCATCAAGATGATGAAACTCGCCAGATCGATTGAAAAAACGTATCATATCCGGCTGACGGCGCGAGAGCTGCACAAGAACCCGACCATTGGCGCTTTAGCGGCTTATACCGCTGAAAAAGCGGGTAGCACCTCCGCTGATCATCATCCCGCAAAAGCCGAGCTGCCGGCAGAACGTGAGAAACAGAAAACGGCGCCAGCGCTTTCAGAAGTGCAAAAAGGGCTGTGGACATTGCAGAAAATGTCGCCTGAAACGACTGCATACCATGTCCCGCTCTGTTTCCGATTCACATCAGGAATCAACAAAGAGAAAATGAAGCAGGCGTTTCAGCTCGTATTAACACA

[0128] SEQ ID No.9 amyS promoter:

[0129] GAAATTAAAAAGCTGGCGGATGAAGGACGCATCCGCTGGATTCCCCGGAGAATTGAAATGAAAGATCTCAAGCCCGCTTTTTTCATTATTGCCGCGACAAATGACCGAGGCGTGAATCAGGAGATAGCCGCAAACGCTTCTGAAACGCAGCTGGTCAACTGTGTAAGCAAGGCTGAACAAGGCAGCGTATATATGCCGAAGATCATCCGCAAAGGGCGCATTCAAGTATCAGTATCAACAAGCGGGGCAAGCCCCGCACATACGAAAAGACTGGCTGAAAACATTGAGCCTTTGATGACTGATGATTTGGCTGAAGAAGTGGATCGATTGTTTGAGAAAAGAAGAAGACCATAAAAATACCTTGTCTGTCATCAGACAGGGTATTTTTTATGCTGTCCAGACTGTCCGCTGTGTAAAAAATAGGAATAAAGGGGGGTTGTTATTATTTTACTGATATGTAAAATATAATTTGTATAAGAAAATGAGAGGGAGAGGAAACATG

[0130] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin by using the ybaS gene, which is characterized in that: The method includes introducing ybaS the ybaS gene, an expression cassette containing the ybaS gene, or a vector containing the ybaS gene into Bacillus amyloliquefaciens Bacillus amyloliquefaciens ; The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, whose name is: LH-2, and the classification name is: Bacillus amyloliquefaciens , with the preservation number: CGMCC No. 29550, preservation date: January 9, 2024, preservation unit: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The said ybaS The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A recombinant strain of Bacillus amyloliquefaciens containing the ybaS gene and having an improved ability to synthesize 1-deoxynojirimycin The said ybaS The nucleotide sequence of the gene is shown in SEQ ID No. 1; The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, whose name is: LH-2, and the classification name is: Bacillus amyloliquefaciens , with the preservation number: CGMCC No. 29550, preservation date: January 9, 2024, preservation unit: China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

3. The method for constructing the recombinant strain according to claim 2, wherein: comprising the following steps: First, the overexpression plasmid pLH2387 containing ybaS gene was introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electroporation, and the primary transformants were screened using LB medium containing 5 µg / mL chloramphenicol; the transformants were passaged at 42 °C, transferred every 12 hours for a total of 3 times, inducing single crossover between the plasmid and the genome, and confirmed by PCR verification; subsequently, further high-temperature passage promoted double crossover recombination, and the double crossover strains were screened by spreading on LB medium containing 25 µg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure was confirmed by PCR detection; finally, sensitive strains were screened by chloramphenicol sensitivity test to exclude residual plasmids, and the gene editing accuracy was verified by sequencing to obtain genetically stable ybaS overexpression recombinant strains.

4. Use of the recombinant strain according to claim 2 in the fermentative production of 1-deoxynojirimycin 5. A method for fermenting and producing 1-deoxynojirimycin using the recombinant strain as described in claim 2, characterized in that: comprising the following steps: Take out the recombinant strain from the ultra-low temperature refrigerator, streak inoculate it on the LB solid medium respectively, culture it at 37 °C for 12 h to grow single strains; transfer the single colonies to 5 mL of fresh LB liquid medium, culture it at 37 °C and 200 r / min for 12 h for seed culture; inoculate the cultured seed liquid of the bacteria into the fermentation medium at an inoculation amount of 4 %, and culture it at 37 °C and 100 r / min for 72 h for the fermentation of 1-deoxynojirimycin; Among them, the fermentation medium used: (NH4)2SO4: 4 g / L, lactose: 25 g / L, K2HPO4: 14 g / L, KH2PO4: 6 g / L, MgSO4·7H2O: 0.2 g / L, CaCl2: 0.15 g / L, MnSO4·H2O: 1.9 mg / L, FeCl3·6H2O: 37.8 mg / L, ZnCl2: 7 mg / L, the solvent is water, pH 7.0, sterilize at 121 °C for 20 min

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