Method for improving 1-deoxynojirimycin synthesis capability of bacillus amyloliquefaciens, ybaS gene and application

Abstract

The invention belongs to the technical field of industrial biology, and discloses a method for improving the 1-deoxynojirimycin synthesis capacity of bacillus amyloliquefaciens, a ybaS gene and application, and the method comprises the step of introducing the ybaS gene, an expression cassette containing the ybaS gene or a carrier containing the ybaS gene into the bacillus amyloliquefaciens. The core gene, namely the ybaS gene (the nucleotide sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No.2) participating in DNJ biosynthesis is identified and disclosed for the first time, the coded protein has unique functions, the key blank in a DNJ anabolism path is filled up, a brand new view is provided for analyzing a molecular mechanism for synthesizing DNJ by microorganisms, and the application of the ybaS gene in DNJ biosynthesis is developed. And a solid molecular foundation is laid for subsequent genetic engineering modification.

Description

Technical Field

[0001] The present invention belongs to the field of industrial biotechnology, and in particular, relates to a method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin, a ybaS gene and an application. The method covers a ybaS gene capable of promoting 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 a molecular formula of C6H 13 NO4, molecular weight is 163.2. 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 molecular structure of DNJ is similar to that of glucose, and it can form a more stable complex with the active center of α-glucosidase, reduce the binding of the enzyme with other sugars, and show strong α-glucosidase inhibitory activity. This inhibitory effect leads to carbohydrate absorption disorders and abnormal synthesis of glycoprotein oligosaccharide chains, which makes DNJ have biological activities such as inhibiting blood sugar increase, anti-tumor, and anti-virus.

[0003] At present, a variety of microorganisms have been reported to be able to produce DNJ, including Streptomyces lilacinus, Monascus purpurogenus, Bacillus subtilis, Escherichia coli and Bacillus amyloliquefaciens. However, these microorganisms generally have problems with low yield and long fermentation time in the production of DNJ. These problems limit the commercial production of DNJ, so it is particularly important to breed microbial strains with high DNJ production. Solving these problems can not only increase the yield of DNJ and reduce production costs, but also meet the demand for efficient and safe drugs in the field of medicine and health care products. Therefore, finding or engineering more efficient microbial strains to increase DNJ production has important economic and scientific value for solving the challenges currently facing DNJ production.

[0004] In Bacillus amyloliquefaciens, although part of the DNJ biosynthesis pathway has been revealed and the functions of the three key genes, gabT1, yktC1, and gutB1, have been clearly identified, the current engineering strain modification work is limited to these three genes, which limits the further improvement of DNJ production. Therefore, in-depth gene identification research is urgently needed to discover more potential modification targets and broaden the possibility of high-yield engineering modification of DNJ. Around these key genes, such as the ybaS gene, its role in DNJ biosynthesis has not yet been clarified, 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. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin, a ybaS gene and application.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] A ybaS gene for improving the biosynthesis efficiency of 1-deoxynojirimycin, wherein the nucleotide sequence of the ybaS gene is 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, wherein the amino acid sequence of the protein is shown in SEQ ID No.2.

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

[0010] A vector for improving the biosynthesis efficiency of 1-deoxynojirimycin comprising the ybaS gene as described above, wherein the vector is selected from a plasmid, a viral vector or an artificial chromosome.

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

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

[0013] A recombinant strain of Bacillus amyloliquefaciens comprising the ybaS gene described above and having improved ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin.

[0014] The method for constructing the recombinant strain as described above comprises the following steps:

[0015] Firstly, the overexpression plasmid pLH2387 containing the ybaS gene was introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electroporation, and the preliminary transformants were screened using LB medium containing 5 µg / mL chloramphenicol. The transformants were passaged at 42 ℃ for 3 times every 12 hours to induce single exchange between the plasmid and the genome, and the integration was confirmed by PCR. Subsequently, the double exchange recombination was promoted by further high-temperature passage, and the double exchange strains were screened by applying LB medium containing 25 µg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure was confirmed by PCR. Finally, the sensitive strains were screened by chloramphenicol sensitivity test, the residual plasmids were excluded, and the accuracy of gene editing was verified by sequencing to obtain the genetically stable ybaS overexpression recombinant strain.

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

[0017] The method for producing 1-deoxynojirimycin by fermentation using the recombinant strain as described above comprises the following steps:

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

[0019] Among them, the fermentation medium used was: (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 was water, pH 7.0, and sterilized at 121 ℃ for 20 min.

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

[0021] 1. The present invention identifies and reveals for the first time the core gene involved in the biosynthesis of DNJ, the ybaS gene (nucleotide sequence see SEQ ID No. 1, amino acid sequence see SEQ ID No. 2). The protein encoded by the ybaS gene has unique functions, filling the key gap in the DNJ synthesis 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.

[0022] 2. The present invention uses gene knockout and overexpression technology to clearly verify the decisive role of the ybaS gene in DNJ synthesis: the ybaS gene knockout strain completely loses the ability to produce DNJ, while the engineered strain with the ybaS gene complemented has a 60% increase in DNJ production compared to the wild type, which is significantly better than the existing transformation strategy. This experimental data confirms for the first time from a 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 of low yield and lack of genetic modification targets in the current DNJ production, the present invention proposes an innovative solution, develops an expression cassette (including promoter and terminator) based on the ybaS gene and a matching vector system (plasmid vector), and establishes a genetic engineering modification method for Bacillus amyloliquefaciens LH-2 (CGMCC No. 29550), achieving the directional enhancement of DNJ synthesis ability. In addition, this technical solution is also widely applicable and can be extended to other Bacillus strains, providing broad application prospects for the industrial production of DNJ.

[0024] 4. The present invention uses precise gene knockout and complementation technology to systematically identify the function of the ybaS gene in Bacillus amyloliquefaciens. The goal is to clarify the specific role of the ybaS gene in DNJ biosynthesis and provide 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 production and provide new ideas and methods for related research in the field of industrial biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 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 This is the map of the knockout plasmid pLH2223 in the present invention;

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

[0029] Figure 5The figure is a PCR verification result of electroporation of the knockout plasmid pLH2223 into the LH-2 strain in the present invention; wherein, M is a DNA Marker; N is a negative control; P is a positive control, using the pLH2223 plasmid vector as a template; the bands numbered 1, 2, and 3 represent the genomic PCR amplification products of different transformants, respectively; the fragments of these products are consistent in size, all of which are 980 bp, which fully proves that the knockout plasmid pLH2223 has been successfully and stably transferred into the LH-2 strain;

[0030] Figure 6 The figure is a PCR verification result of electroporation of the overexpression plasmid pLH2387 into the LH-2 strain in the present invention; wherein, M is a DNA Marker; N is a negative control; P is a positive control, using the pLH2387 plasmid vector as a template; and 1, 2, and 3 are PCR amplification products of the transformants obtained after electroporation of the overexpression plasmid pLH2387 into the LH-2 strain, and the fragment size is 490 bp, which verifies the successful transfer of the overexpression plasmid;

[0031] Figure 7 The figure is a PCR verification result of the knockout plasmid pLH2223 in the present invention undergoing a single exchange in the recombinant bacteria; wherein, M represents a DNA Marker; N is a negative control; 1, 2, and 3 are PCR amplification products of the transformants, and the fragment size is 1330 bp. This result confirms that the knockout plasmid pLH2223 successfully undergoes a single exchange in the recombinant bacteria;

[0032] Figure 8 The figure is a PCR verification result of the overexpression plasmid pLH2387 in the present invention undergoing a single exchange in the recombinant bacteria; wherein M represents a DNA Marker; N is a negative control; 1 and 2 are PCR amplification products of the transformants, and the fragment size is 2760 bp. This result confirms that the overexpression plasmid pLH2387 successfully undergoes a single exchange in the recombinant bacteria;

[0033] Fig. 9 : is a PCR verification result diagram of the double exchange of the knockout plasmid pLH2223 in the present invention in the recombinant bacteria; wherein, M represents DNA Marker; N is a negative control; WT represents the genome of LH-2 as a template, and its fragment size is 1660 bp, as a reference; 1 is the PCR amplification product of the knockout strain, and the fragment size is 1400 bp, which confirms that the knockout plasmid pLH2225 has successfully undergone double exchange in the recombinant bacteria;

[0034] Fig.101 is a PCR verification result diagram of double exchange of the overexpression plasmid pLH2387 in the recombinant bacteria in the present invention; wherein, M represents DNA Marker; N is a negative control; WT represents the genome of LH-2 as a 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, which confirms that the overexpression plasmid pLH2387 has successfully undergone double exchange in the recombinant bacteria;

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

[0036] A Bacillus amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, its classification name is: Bacillus amyloliquefaciens, its preservation number is: CGMCC No.29550, its preservation date is: January 9, 2024, and its preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0038] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0039] A ybaS gene for improving the biosynthesis efficiency of 1-deoxynojirimycin, wherein the nucleotide sequence of the ybaS gene is 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, wherein the amino acid sequence of the protein is shown in SEQ ID No.2.

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

[0042] A vector for improving the biosynthesis efficiency of 1-deoxynojirimycin comprising the ybaS gene as described above, wherein the vector is selected from a plasmid, a viral vector or an artificial chromosome.

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

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

[0045] A recombinant strain of Bacillus amyloliquefaciens comprising the ybaS gene described above and having improved ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin.

[0046] The method for constructing the recombinant strain as described above comprises the following steps:

[0047] Firstly, the overexpression plasmid pLH2387 containing the ybaS gene was introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electroporation, and the preliminary transformants were screened using LB medium containing 5 µg / mL chloramphenicol. The transformants were passaged at 42 ℃ for 3 times every 12 hours to induce single exchange between the plasmid and the genome, and the integration was confirmed by PCR. Subsequently, the double exchange recombination was promoted by further high-temperature passage, and the double exchange strains were screened by applying LB medium containing 25 µg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure was confirmed by PCR. Finally, the sensitive strains were screened by chloramphenicol sensitivity test, the residual plasmids were excluded, and the accuracy of gene editing was verified by sequencing to obtain the genetically stable ybaS overexpression recombinant strain.

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

[0049] The method for producing 1-deoxynojirimycin by fermentation using the recombinant strain as described above comprises the following steps:

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

[0051] Among them, the fermentation medium used was: (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 was water, pH 7.0, and sterilized at 121 ℃ for 20 min.

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

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

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

[0055] Grind the collected cow dung samples, pass them through a 60-100 mesh sieve, take 1g of the sample and add it to 15-50mL LB liquid culture medium with glass beads, shake it thoroughly, place it in a water bath at 80-90℃ for 10-15 min, and then culture it on a shaker at 28℃ (120rpm) for 24 h. After the culture is completed, place it in a water bath at 80-90℃ for 10-15 min again, dilute it appropriately by the 10-fold dilution method, and take the diluted 10 5 , 10 6 and 10 7 Spread 0.1 mL of each of the three dilutions on LB solid medium, invert and culture at 28-37°C for 2-5 days, and observe every day. Select white colonies with irregular, bumpy, and uneven edges, and purify them through 3-5 rounds of streaking and subculture as candidate strains.

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

[0057] Among them, the sample processing and detection methods refer to the national standard detection method "Detection of 1-deoxynojirimycin in mulberry leaf extract" (GB / T40642-2021), in which the liquid chromatography detection wavelength is set to 265 nm.

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

[0059] The fermentation medium formula consists of: 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, and the pH is adjusted to 4.0~9.0.

[0060] Experimental results: A total of 600 bacterial strains were isolated in this experiment, and 50 strains producing DNJ were isolated, among which the highest DNJ content was 1.5 g / L. The HPLC spectrum is shown in Figure 1As shown, the strain was selected and numbered LH-2. The strain LH-2 was inoculated into LB liquid medium and cultured at 28-37 °C for 24 h. The bacteria were collected by centrifugation (8,000×g, 10 min). The genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen, DP302), and the 16S rRNA gene was amplified with universal primers 27F and 1492R (as shown in Table 1) (PCR conditions: 94 °C pre-denaturation for 5 min; 94 °C 30 s, 55 °C 30 s, 72 °C 90 s, 30 cycles; 72 °C final extension for 10 min). The amplified product was purified after verification by 1% agarose gel electrophoresis and sent to Jinweizhi Biotechnology Co., Ltd. for bidirectional sequencing. The obtained 16S rDNA sequence (SEQ ID No. 3) was submitted to the NCBI database for BLASTn homology comparison (parameter setting: E-value ≤ 1e-50, coverage ≥ 95%). As shown in Table 2 (only five strains with higher homology are listed in the table), the sequence had 100% similarity with the 16S rRNA gene of different Bacillus amyloliquefaciens strain H and other Bacillus amyloliquefaciens, so the strain was identified as Bacillus amyloliquefaciens.

[0061] The Bacillus amyloliquefaciens LH-2 is named LH-2, classified as Bacillus amyloliquefaciens, deposited at CGMCC No. 29550, deposited on January 9, 2024, and deposited at China National Center for the Administration of Microbiological Culture Collection, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0062] Table 1 Sequences of primers used

[0063]

[0064] Table 2 BLASTn homology comparison results of 16S rRNA sequences 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 resolved, and the study clearly revealed the core role of the gabT1, yktC1 and gutB1 gene clusters in DNJ biosynthesis. However, existing studies still lack systematic exploration of whether genes in the flanking regions of the gene clusters are involved in DNJ synthesis. Based on this, the present invention first discovered the ybaS gene (SEQ ID No. 1) flanking the gabT1-yktC1-gutB1 gene cluster 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), which encodes an NAD(P)H-dependent reductase (amino acid sequence shown in SEQ ID No. 2). Its specific role in DNJ synthesis has not yet been clarified. Therefore, the present invention aims to identify the function of the ybaS gene in DNJ biosynthesis by knocking out and complementing the ybaS gene in Bacillus amyloliquefaciens LH-2, and to provide a new strategy for increasing DNJ production. Figure 2 .

[0068] Example 3 Construction of ybaS gene editing vector

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

[0070] First, using the genome sequence of Bacillus amyloliquefaciens LH-2 (the genome sequencing has been completed by Suzhou GENEWIZ Biotechnology Co., Ltd. / GENEWIZ) as a template, 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, PhantaMax Super-Fidelity Polymerase (Nanjing Novigene, catalog number P505-d1) was used to amplify the upstream and downstream homologous arms of the ybaS gene (SEQ ID No. 4 / No. 5), and the PCR amplification system is shown in Table 3. The PCR amplification products of the upstream and downstream homology arms of the ybaS gene (SEQ ID No. 4 / No. 5) were recovered by a common DNA product purification kit (Tiangen, DP204) to obtain purified DNA fragments, which were then sequentially connected to the BamHI site of the pKSU plasmid using a ClonExpress Ultra One Step Cloning Kit V (Nanjing Novogene, Cat. No. C117-01) to successfully construct the ybaS gene knockout vector pLH2223 ( 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 ClonExpress Ultra One Step Cloning Kit V one-step cloning system

[0074]

[0075] Special note: Plasmid pKSU is the core tool vector for genetic manipulation of Bacillus amyloliquefaciens. It has become the preferred system for gene editing of this species due to its high transformation efficiency, multiple cloning site compatibility and stable replication characteristics. This plasmid was first reported by Zhang et al. in Applied Microbiology and Biotechnology in 2014. The markerless gene replacement technology developed by them was successfully applied to the genome streamlining and optimization of poly-γ-glutamic acid synthesis pathway (Zhang, W., Gao, WX, Feng, J., Zhang, C., He, YL, Cao, MF, 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 Biotechnol98: 8963–8973.). Currently, the genetic operating system based on pKSU has been included in the Bacillus Genetic Stock Center (BGSC).

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

[0077] Using pKSU as the starting vector, the ybaS gene overexpression vector pLH2387 integrated into the pksJ gene target site (SEQ ID No. 6) was designed and constructed. The specific process is as follows:

[0078] Using the genomic DNA of Bacillus amyloliquefaciens LH-2 (the extraction method is shown in Example 1) as a template, PhantaMax Super-Fidelity Polymerase (Nanjing Novozyme, catalog number P505-d1) was used to amplify the upstream and downstream homologous arms of the pksJ gene (SEQ ID No. 7 / No. 8) with pksJUP-F / pksJUP-R and pksJDN-F / pksJDN-R primers (see Table 1); the amyS promoter (SEQ ID No. 9) was amplified with primers amsy-F / asmy-R (see Table 1); the ybaS gene (SEQ ID No. 1) was amplified with primers ybas-F / ybas-R (see Table 1), and the DNA fragments were purified by a common DNA product purification kit (Tiangen, DP204), and then purified by ClonExpress Ultra One Step Cloning Kit. The V3 one-step cloning kit (Nanjing Novogene, Cat. No. C117-01) was used to sequentially connect the upstream and downstream homology arms of the pksJ gene, the amyS promoter, and the ybaS gene 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 strain LH-2 stored at -80℃, make three-zone lines on LB solid medium, and culture it in a 37°C incubator for 12-24 hours until a clear single colony appears. Pick a single clone and inoculate it into 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 place on ice for 30 min. Then centrifuge at 8000 r / min and 4 °C for 20 min to collect the bacterial cells.

[0083] 3) Add about 60 mL of elution buffer (the formula is: 91.0 g sorbitol, 91.0 g mannitol, 50 g glycerol, dilute to 0.5 L with water), mix well, centrifuge again for 20 min under the same conditions to collect the cells. Repeat this operation twice.

[0084] 4) Add 500 μL of the resuspension prepared for electroporation (the formula is: 0.91 g sorbitol, 0.91 g mannitol, 0.1 g glycerol, 1.4 g PEG6000, dilute to 10 mL with water), and gently resuspend the bacteria thoroughly.

[0085] 7) Aliquot the competent cells into sterile EP tubes at 100 μL each and store at -80 °C for later use.

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

[0087] 1) Thaw the competent cells naturally on ice, add appropriate amount of pLH2223 or pLH2387 plasmid (no more than 200 ng), and mix thoroughly.

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

[0089] 3) Perform electroporation at 2500 V, then add 1 mL of pre-cooled electroporation bacterial recovery liquid culture medium (0.38 mol / L mannitol dissolved in LBS culture medium) into the electroporation cup and mix by pipetting.

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

[0091] 5) Centrifuge at 8000 r / min for 2 min to concentrate the cells, take 100 μL of the bacterial solution and spread it on LB solid medium containing chloramphenicol (5 μg / mL) and culture it statically. 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 by electroporation in the previous step into LB liquid medium containing 5 μg / mL chloramphenicol and culture at 30°C overnight;

[0094] 2) PCR verification was performed on the overnight culture using primers p1 / p2 to ensure that the ybaS knockout plasmid pLH2223 and the overexpression plasmid pLH2387 were successfully electroporated 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 Novozyme Biotech Co., Ltd. The specific method is shown in Table 5. The results are shown in Table 5. Figure 5 and Figure 6 As shown, the results showed that pLH2223 and pLH2387 were electroporated into Bacillus amyloliquefaciens LH-2.

[0095] 3) The correct transformants of the two plasmids were transferred to fresh LB liquid medium containing 5 μg / mL chloramphenicol, and subcultured at 42°C, with transfer every 12 hours for a total of 3 times to promote single exchange of plasmids in the strain.

[0096] 4) After diluting the bacterial suspension of the two plasmids in step 3 appropriately, take 100 μL and spread it on LB solid medium containing 5 μg / mL chloramphenicol. Incubate at 30 °C for 48 h. Single colonies can be seen growing. Using primers p3 / p4, PCR detection of single colonies of knockout plasmid pLH2223 is performed. The results are as follows: Figure 7 As shown; primers p5 / p6 were used to perform PCR detection on a single colony of the overexpression plasmid pLH2387, and the results were as follows Figure 8 The results are shown in Figure 7 and Figure 8 As shown, the results showed that pLH2223 and pLH2387 completed single exchange in Bacillus amyloliquefaciens LH-2, respectively, and single exchange transformants of the two plasmids were screened and obtained.

[0097] 5) Transfer the obtained single exchange transformants of the two plasmids to LB liquid medium respectively, and subculture at 42°C, transferring once every 12 hours for a total of 3 times to promote double exchange of single exchange transformants.

[0098] 6) Dilute the bacterial suspension of the two plasmids in step 5) appropriately, spread them on LB solid medium containing 25 μg / mL 5-fluorouracil, and culture them at 30 °C for 48 h. Single colonies can be seen growing. Perform PCR detection on the single colonies of the two plasmids to screen for double exchange strains. Using primers p7 / p8, perform PCR detection on the single colony of the knockout plasmid pLH2223. The results are as follows: Fig. 9 As shown; primers p9 / p10 were used to perform PCR detection on a single colony of the overexpression plasmid pLH2387, and the results were as follows Fig.10 The results are shown in Fig. 9 and Fig.10As shown, it was shown that double exchange transformants of two plasmids were obtained, thereby obtaining ybaS gene knockout and overexpression recombinant transformants.

[0099] 7) The ybaS gene knockout and overexpression recombinant transformants obtained in 6) were inoculated into LB liquid medium, cultured overnight at 30 °C, and diluted and spread on LB solid medium. After culture at 30 °C for 48 h, single colonies were visible. The single colonies were respectively inoculated on LB solid medium and LB solid medium containing 5 μg / mL chloramphenicol to determine whether the recombinant transformants were sensitive to chloramphenicol. The chloramphenicol-sensitive strains were selected and sent for sequencing verification. Finally, the ybaS gene knockout recombinant strain S4277 and the overexpression recombinant strain S4452 were 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 on LB solid medium, and incubate at 37 °C for 12 h to grow single strains. Transfer the single colonies of the three bacteria to 5 mL fresh LB liquid medium, incubate at 37 °C and 200 r / min for 12 h for seed culture. Inoculate the seed liquid of the three cultured bacteria into 30 mL fermentation medium (250 mL triangular flask) at an inoculation rate of 4% (v / v, volume ratio), incubate at 37 °C and 100 r / min for 72 h, and perform DNJ fermentation. Set up 3 shake flask experiments for each strain fermentation. The fermentation medium used was: (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 was water, pH 7.0, and sterilized at 121 ℃ for 20 min.

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

[0106] The fermentation broth was centrifuged at 12000 r / min for 5 min, and the supernatant was then aspirated for later use. Next, 35 µL of the fermentation supernatant or DNJ standard solution was taken and added to a 1.5 mL centrifuge tube. Then, 200 µL of 0.4 mol / L potassium borate buffer (pH 8.5) and 250 µL of 5 mmol / L derivatization reagent fluorenylmethoxycarbonyl chloride solution (FMOC-Cl, dissolved in 50% acetonitrile) were added in sequence. After thorough mixing, the mixture was placed in a constant temperature water bath at 25 °C for 20 minutes. After the reaction was completed, 25 µL of 1 mol / L glycine aqueous solution was added to terminate the reaction, and then 100 µL of 1% (V / V, volume concentration) acetic acid aqueous solution was added and the volume was adjusted to 800 µL with water. Finally, the solution was filtered using a microporous filter membrane with a pore size of 0.22 µm, and the filtrate was collected as a subsequent test sample.

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

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

[0109] After 72 hours of shake flask fermentation, the DNJ titer of the starting strain LH-2 was 1.5 g / L, the DNJ titer of the recombinant strain S4277 with the ybaS gene knocked out was almost 0, and the DNJ titer of the strain S4452 overexpressing ybaS was 2.4 g / L. Compared with the wild-type Bacillus amyloliquefaciens LH-2, the DNJ production of the overexpressed strain S4452 increased by 60% ( Fig.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 technology, the function of ybaS as an indispensable gene for DNJ synthesis was systematically verified, and after the gene was complemented, the yield of DNJ was significantly improved. 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, which greatly enhanced 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] GAAATTAAAAAGCTGGCGGATGAAGGACGCATCCGCTGGATTCCCCGGAGAATTGAAATGAAAGATCTCAAGCCCGCTTTTTCATTATTGCCGCGACAAATGACCGAGGCGTGAATCAGGAGATAGCCGCAAACGCTTCTGAAACGCAGCTGGTCAACTGTGTAAGCAAGGCTGAACAAGGCAGCGTATATATGCCGAAGATCATCCGCAAAGGGCGCATTCAAGTATCAGTATCAACAAGCGGGGCAAG CCCCGCACATACGAAAAGACTGGCTGAAAACATTGAGCCTTTGATGACTGATGATTTGGCTGAAGAAGTGGATCGATTGTTTGAGAAAAGAAGAAGACCATAAAAATACCTTGTCTGTCATCAGACAGGGTATTTTTTATGCTGTCCAGACTGTCCGCTGTGTAAAAAATAGGAATAAAGGGGGGTTGTTATTATTTTACTGATATGTAAAATATAATTTGTATAAGAAAATGAGAGGGAGAGGAAACATG

[0130] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 contents disclosed in the embodiments.

Claims

1. A ybaS gene for improving the biosynthesis efficiency of 1-deoxynojirimycin, characterized in that: The nucleotide sequence of the ybaS gene is shown in SEQ ID No.

1.

2. A protein encoded by the ybaS gene according to claim 1 having the function of improving the biosynthesis efficiency of 1-deoxynojirimycin, characterized in that: The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. An expression cassette for improving the biosynthesis efficiency of 1-deoxynojirimycin comprising the ybaS gene according to claim 1, characterized in that: The expression cassette comprises a promoter, a ybaS gene and a terminator.

4. A vector for improving the biosynthesis efficiency of 1-deoxynojirimycin comprising the ybaS gene according to claim 1, characterized in that: The vector is selected from a plasmid, a viral vector or an artificial chromosome.

5. A method for improving the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin using the ybaS gene as claimed in claim 1, characterized in that: The method comprises introducing the ybaS gene, an expression cassette comprising the ybaS gene or a vector comprising the ybaS gene into Bacillus amyloliquefaciens.

6. The method according to claim 5, characterized in that: The amyloliquefaciens is Bacillus amyloliquefaciens LH-2, its name is: LH-2, its classification name is: Bacillus amyloliquefaciens, its preservation number is: CGMCC No.29550, its preservation date is: January 9, 2024, and its preservation unit is: General Microbiology Center of China National Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

7. A recombinant strain of Bacillus amyloliquefaciens comprising the ybaS gene according to claim 1, which improves the ability of Bacillus amyloliquefaciens to synthesize 1-deoxynojirimycin.

8. The method for constructing a recombinant strain according to claim 7, characterized in that: The steps include: Firstly, the overexpression plasmid pLH2387 containing the ybaS gene was introduced into the competent cells of Bacillus amyloliquefaciens LH-2 by electroporation, and the preliminary transformants were screened using LB medium containing 5 µg / mL chloramphenicol. The transformants were passaged at 42 ℃ for 3 times every 12 hours to induce single exchange between the plasmid and the genome, and the integration was confirmed by PCR. Subsequently, the double exchange recombination was promoted by further high-temperature passage, and the double exchange strains were screened by applying LB medium containing 25 µg / mL 5-fluorouracil, and the stable integration of the ybaS gene overexpression structure was confirmed by PCR. Finally, the sensitive strains were screened by chloramphenicol sensitivity test, the residual plasmids were excluded, and the accuracy of gene editing was verified by sequencing to obtain the genetically stable ybaS overexpression recombinant strain.

9. Use of the recombinant strain according to claim 7 in the fermentation production of 1-deoxynojirimycin.

10. A method for producing 1-deoxynojirimycin by fermentation using the recombinant strain according to claim 7, characterized in that: The steps include: The recombinant strains preserved in the ultra-low temperature refrigerator were taken out, streaked and inoculated on LB solid medium, and cultured at 37 °C for 12 h to grow single strains; the single colony was transferred to 5 mL of fresh LB liquid medium, and cultured at 37 °C and 200 r / min for 12 h for seed culture; the seed liquid of the cultured bacteria was inoculated into the fermentation medium at an inoculation rate of 4%, and cultured at 37 °C and 100 r / min for 72 h to ferment 1-deoxynojirimycin; Among them, the fermentation medium used was: (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 was water, pH 7.0, and sterilized at 121 ℃ for 20 min.

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