A bacillus pumilus strain with high 1-deoxynojirimycin yield and application thereof

Abstract

This invention discloses a high-yield 1-deoxynojirimycin-producing *Bacillus pumilus* strain and its applications, belonging to the field of microbiology. This invention, through screening, ARTP and NTG mutagenesis, obtained a *Bacillus pumilus* strain with significantly increased 1-deoxynojirimycin production. Bacillus pumilus DNJ-BP01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38218, was used to inoculate the seed culture of Bacillus pumilus DNJ-BP01 into a fermentation medium containing 5% beef extract supplemented with 2.5% mannitol as the carbon source and 3% potassium nitrate as the nitrogen source. After 76 hours of aerobic fermentation, the yield of 1-deoxynojirimycin reached 0.892 g / L. This invention provides a new fermentation strain for the efficient industrial production of 1-deoxynojirimycin and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of Bacillus pumilus that produces high levels of 1-deoxynojirimycin and its applications. Background Technology

[0002] 1-Deoxynojirimycin (1-DNJ), a natural nitrogen-containing heterocyclic compound, is a highly valuable α-glucosidase inhibitor with irreplaceable roles in pharmaceuticals and functional foods. Its core efficacy lies in effectively regulating blood sugar levels by inhibiting the breakdown and absorption of carbohydrates in the intestines, making it a key active ingredient in the treatment of type II diabetes. It also shows broad clinical application prospects in anti-obesity, antiviral (such as hepatitis B and C viruses), and anti-tumor applications, leading to continuously rising market demand. With the rising global incidence of chronic diseases and the upgrading of health consumption, the market size of 1-DNJ-related products is expanding at an average annual double-digit growth rate, creating an urgent need for low-cost, high-purity 1-DNJ raw material supply. However, the current industrial production of 1-DNJ faces significant technological bottlenecks. Traditional production methods mainly include two pathways: natural plant extraction and chemical synthesis. Natural extraction uses mulberry leaves and other raw materials, which suffers from cumbersome extraction steps, low product concentration, and high production costs. Furthermore, it is limited by natural conditions such as the origin of raw materials and climate, making it difficult to achieve large-scale and stable supply. While chemical synthesis can overcome dependence on raw materials, it involves complex reaction steps, low synthesis efficiency, and the presence of enantiomers in the product makes separation and purification difficult, resulting in product purity that fails to meet pharmaceutical-grade standards. It also causes environmental pollution problems, which is inconsistent with the concept of green production. Microbial fermentation has become the mainstream development direction for 1-DNJ production due to its advantages such as mild culture conditions, low production costs, and environmental friendliness.

[0003] Mutagenesis breeding, a classic method for improving microbial strains, offers advantages such as ease of operation, high mutation rate, and short cycle time, making it particularly suitable for the rapid optimization of industrial strains. Among these techniques, ambient pressure room temperature plasma (ARTP) mutagenesis generates a highly active plasma jet at 25-40°C through helium discharge, inducing changes in microbial cell wall permeability and multi-point DNA mutations. It features high mutation rate, good genetic stability, and no chemical residues. Nitrosoguanidine (NTG), a classic chemical mutagen, efficiently induces base substitution mutations in microbial genes, significantly altering the metabolic phenotype of the strain. Studies have shown that combined ARTP and NTG mutagenesis can exert a synergistic effect, achieving mutation accumulation through iterative mutagenesis. Compared to single mutagenesis techniques, it can more efficiently screen for high-yielding mutant strains and has been successfully applied to the improvement of various secondary metabolite-producing strains. For example, after ARTP-NTG combined mutagenesis, the yield of a certain strain increased by more than 70%, with good genetic stability. Based on this, this study uses ARTP and NTG combined mutagenesis technology to target and improve Bacillus pumilus, aiming to obtain a high-yield 1-DNJ superior strain and solve the pain points of existing production technology. Summary of the Invention The purpose of this invention is to provide a high-yield 1-deoxynojirimycin Bacillus strain and its application, in order to solve the problems existing in the prior art. This invention provides a new fermentation strain for the industrial and efficient production of 1-deoxynojirimycin, with a yield of 0.892 g / L, and has broad prospects for industrial application.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a short-spore bacillus that produces 1-deoxynojirimycin ( Bacillus pumilus DNJ-BP01, the Bacillus pumilus DNJ-BP01, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38218, deposited on April 13, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The present invention also provides a microbial agent comprising the above-mentioned Bacillus pumilus DNJ-BP01.

[0006] The present invention also provides the application of the above-mentioned Bacillus pumilus DNJ-BP01 or the above-mentioned bacterial agent in the production of 1-deoxynojirimycin.

[0007] The present invention also provides a method for producing 1-deoxynojirimycin, comprising the step of fermentation using the above-mentioned Bacillus pumilus DNJ-BP01 or the above-mentioned inoculum.

[0008] Furthermore, it includes the following steps: A seed culture of the Bacillus pumilus DNJ-BP01 was prepared, and the seed culture was inoculated into a fermentation medium for aerobic fermentation. The fermentation broth was collected and purified to obtain 1-deoxynojirimycin.

[0009] Furthermore, the inoculation amount of the seed liquid is 10%.

[0010] Furthermore, the carbon source of the fermentation medium is 5% beef extract and 2.5% mannitol.

[0011] Furthermore, the nitrogen source of the fermentation medium is 3% KNO3.

[0012] Furthermore, the fermentation medium comprises 5% beef extract, 2.5% mannitol, 3% KNO3 and 0.5% magnesium sulfate.

[0013] Furthermore, the aerobic fermentation time is 76 hours and the temperature is 37°C.

[0014] The present invention discloses the following technical effects: This invention, through screening, ARTP and NTG mutagenesis, yielded a *Bacillus pumilus* strain with significantly increased 1-deoxynojirimycin production. Bacillus pumilus DNJ-BP01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38218, was used to inoculate the seed culture of Bacillus pumilus DNJ-BP01 into a fermentation medium containing 5% beef extract supplemented with 2.5% mannitol as the carbon source and 3% KNO3 as the nitrogen source. After 76 hours of aerobic fermentation, the yield of 1-deoxynojirimycin reached 0.892 g / L. This invention provides a new fermentation strain for the efficient industrial production of 1-deoxynojirimycin and has broad application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The ARTP mutagenesis mortality curve for Bacillus pumilus 642; Figure 2 The NTG mutagenesis mortality curves for Bacillus pumilus 1-F9; Figure 3 Figure showing the effect of different carbon sources on the DNJ titer produced by fermentation of Bacillus pumilus DNJ-BP01; Figure 4 Figure showing the effect of different supplementary carbon sources and precursors on the DNJ titer produced by Bacillus pumilus DNJ-BP01 fermentation; Figure 5 The figure shows the effect of different nitrogen sources on the DNJ titer produced by fermentation of Bacillus pumilus DNJ-BP01. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1: Screening and Identification of 1-DNJ Producing Strains 1. Strain screening Single colonies were selected for fermentation and cultured at 30°C for 24 h. 30 μL of fermentation broth was mixed with 50 μL of 0.1M potassium phosphate (pH=7.0) buffer, followed by the addition of 20 μL of α-glucosidase (40 U / mL), and incubated at 37°C for 5 min. Then, 50 μL of p-nitrophenyl-α-glucopyranoside (12 mM) was added, and the reaction was carried out at 37°C for 30 min. The reaction was terminated by adding 50 μL of sodium carbonate (200 mM). The absorbance of each well was measured at 405 nm using a microplate reader. Lower absorbance indicated higher 1-DNJ yield.

[0023] The fermentation products of the strain were further derivatized using the FMOC-Cl derivatization method, and the presence of 1-DNJ in the fermentation products was detected by high-performance liquid chromatography (HPLC). The derivatization method is as follows: First, 100 μL of standard or fermentation broth that had been ethanol-precipitated and concentrated to 5 times its original volume was placed in a 1.5 mL centrifuge tube. Then, 175 μL of 0.4 mol / L, pH 8.5 potassium borate buffer solution and 250 μL of 5 mmol / L FMOC-Cl acetonitrile derivatization reagent were added sequentially, mixed thoroughly for 30 s, and reacted in a 25℃ constant temperature water bath for 25 min. Next, 100 μL of 0.1 mol / L glycine solution was added, and the reaction was carried out for 20 min to neutralize the excess FMOC-Cl. Then, 75 μL of 1% acetic acid aqueous solution and 300 μL of deionized water were added, and mixed thoroughly. Finally, the mixture was filtered through a 0.45 μm organic phase filter membrane to obtain the derivatized sample solution for later use.

[0024] The detection method is as follows: Select C 18 Analytical column (4.6 × 250 mm, 5 μm); injection volume 20 μL; isocratic elution; mobile phase ratio: 50% mobile phase A (acetonitrile), 50% mobile phase B (deionized water containing 0.1% acetic acid); flow rate 1 mL / min; UV detector, wavelength 254 nm. Peak times of the 1-DNJ standard derivatives were compared.

[0025] 2. Screening Results A strain producing 1-DNJ was obtained through screening and named strain 642.

[0026] Example 2: ARTP mutagenesis of strain 642 Strain 642 was inoculated into LB medium and cultured overnight at 37°C. 500 μL of the seed culture was then transferred to an Erlenmeyer flask containing 50 mL of fresh LB medium. The culture was centrifuged at 4000 rpm and 4°C for 15 min to collect the pellet. The cells were then washed twice with water. Finally, the cells were resuspended in physiological saline. The resuspended cells were then diluted with physiological saline to dilute the OD. 600The absorbance was adjusted to 0.4-0.6. The diluted sample was exposed to a plasma beam for 0, 30, 35, 40, and 45 seconds. After treatment, the sample was spread onto solid LB plates and incubated overnight at 37°C. Strains with low absorbance were initially screened using a microplate reader for large-scale culture. These strains were first inoculated into 50 mL of LB medium and cultured at 37°C and 220 rpm for 12 h. Then, they were inoculated into 1-DNJ fermentation medium and fermented at 30°C and 150 rpm for 72 h. The 1-DNJ content was then determined by HPLC.

[0027] like Figure 1 As shown, the results showed that the lethality rate reached 99% after ARTP mutagenesis for 35 s. Among the strains obtained after mutagenesis for 35 s, a strain with a higher 1-DNJ yield than the original strain (strain 642) was screened out. Its 1-DNJ yield was 260 mg / L (the 1-DNJ yield of strain 642 was 210 mg / L), and it was named strain 1-F9.

[0028] Example 3: NTG mutagenesis of strain 1-F9 Strain 1-F9 was inoculated into LB medium and cultured overnight at 37°C. 500 μL of the seed culture was then transferred to an Erlenmeyer flask containing 50 mL of fresh LB medium. The culture was centrifuged at 5000 rpm and 4°C for 1.5 min to collect the pellet. The cells were resuspended in sterile LB medium, and sterile diluted OD was added. 600 The concentration was increased to 0.2. Subsequently, the cells were diluted 1000-fold with sterile LB medium, and 500 μL of the cell suspension was treated with 50 µL of NTG (0.4 g / L) at 37°C in the dark for 40-70 min. The culture medium was centrifuged at 4000 rpm for 1.5 min to collect the precipitate, resuspended on sterile LB medium, and spread onto solid LB plates. The plates were incubated overnight at 37°C. Strains with low absorbance were initially screened using a microplate reader for large-scale culture. They were first inoculated into 50 mL of LB medium and cultured at 37°C and 220 rpm for 12 h, and then inoculated into 1-DNJ fermentation medium and fermented at 30°C and 150 rpm for 72 h. The 1-DNJ content was then determined by HPLC.

[0029] like Figure 2 As shown, the results showed that the lethality rate reached 99% after 60 min of NTG mutagenesis. Among the strains obtained after 60 min of mutagenesis and plate preparation, a strain 1-DNJ with a higher yield than strain 1-F9 was screened, with a yield of 290 mg / L (the yield of strain 642 was 210 mg / L, and the yield of strain 1-F9 was 260 mg / L), and it was named strain DNJ-BP01.

[0030] Example 4: Identification and Preservation of Strain DNJ-BP01 1. Strain identification After culturing strain DNJ-BP01 for 2 days, the bacterial cells were collected by centrifugation, ground into powder using liquid nitrogen, and genomic DNA was extracted from the strain using a modified SDS method. Using the genomic DNA as a template, PCR amplification was performed using universal primers 727F (5'-CAGAGTTTGATCCTGGCT-3', SEQ ID NO.1) and 1540R (5'-AGGAGGTGATCCAGCCGCA-3', SEQ ID NO.2).

[0031] PCR amplification reaction program: 94℃ pre-denaturation for 5 min; 30 cycles of 94℃ for 1 min, 60℃ for 1 min, 72℃ for 1 min; final extension at 72℃ for 10 min.

[0032] After the PCR products were separated by 1% agarose gel electrophoresis, the target band was purified using an agarose gel DNA recovery kit. The target nucleic acid fragment was sent to Sangon Biotech Co., Ltd. for sequencing. The obtained gene sequence was compared with the existing 16S rDNA sequence in the NCBI database using BLAST. Phylogenetic analysis was performed using MEGA 4.1 software, and a phylogenetic tree was constructed using the Neighbor-Joining method.

[0033] The sequence was submitted to the GenBank database, obtaining the GenBank accession number KU3244601. Homology and phylogenetic analysis were performed using NCBI BLAST with known related sequences in GenBank. Bacillus pumilus The highest similarity (100%) was found with WN-5-1-1 (KU570367.1). Based on morphological characteristics, strain DNJ-BP01 was identified as *Bacillus pumilus*. Bacillus pumilus ), named Bacillus pumilus ( Bacillus pumilus )DNJ-BP01.

[0034] 2. Strain preservation Bacillus pumilus ( Bacillus pumilus DNJ-BP01 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38218, on April 13, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0035] Example 5: Optimization of carbon source composition for DNJ production by Bacillus pumilus DNJ-BP01 strain Using 3-6% beef extract, glycerol, and glucose as carbon sources, the DNJ-BP01 strain was inoculated into LB medium and cultured overnight at 37°C. 500 μL of seed culture was then inoculated into an Erlenmeyer flask containing 50 mL of fresh fermentation medium and cultured at 30°C for 72 h. The culture was centrifuged at 8000 rpm and 4°C for 15 min to collect the supernatant. 5 μL of the fermentation supernatant was mixed with 75 μL of 0.1M potassium borate (pH=8.5) buffer, and 20 μL of α-glucosidase (40 U / mL) was added. The mixture was incubated at 37°C for 5 min; then 50 μL of p-nitrophenyl-α-glucopyranoside (12 mM) was added, and the reaction was continued at 37°C for 30 min; finally, 50 μL of sodium carbonate (200 mM) was added to terminate the reaction. The absorbance of each well was measured at 405 nm using a microplate reader to determine the inhibition rate. This process was used to screen for the optimal carbon source.

[0036] like Figure 3 As shown, the results revealed that the inhibition rate was highest when 5% beef extract was used as the carbon source.

[0037] Example 6 Optimization of precursor components for DNJ production by Bacillus pumilus DNJ-BP01 strain Based on the addition of 5% beef extract, mannitol and sorbitol were used as supplementary carbon sources and precursors, respectively, at 1-3%. The DNJ-BP01 strain was inoculated into LB medium and cultured overnight at 37°C. 500 μL of seed culture was inoculated into an Erlenmeyer flask containing 50 mL of fresh fermentation medium and cultured at 30°C for 72 h. The culture was centrifuged at 8000 rpm and 4°C for 15 min to collect the supernatant. 5 μL of the fermentation supernatant was mixed with 75 μL of 0.1M potassium borate (pH=8.5) buffer, and 20 μL of α-glucosidase (40 U / mL) was added. The mixture was incubated at 37°C for 5 min; then 50 μL of p-nitrophenyl-α-glucopyranoside (12 mM) was added, and the reaction was carried out at 37°C for 30 min; finally, 50 μL of sodium carbonate (200 mM) was added to terminate the reaction. The inhibition rate was determined by measuring the absorbance of each well at 405 nm using a microplate reader. This allows us to select the most suitable carbon source type.

[0038] like Figure 4 As shown, the results showed that when 2.5% mannitol was added as a supplementary carbon source and precursor, the inhibition rate reached 64.42%.

[0039] Example 7 Optimization of nitrogen source composition for DNJ production by Bacillus pumilus DNJ-BP01 strain Yeast extract (2-6%), tryptone, and KNO3 were used as single nitrogen sources in the fermentation medium. *Bacillus pumilus* DNJ-BP01 was inoculated into LB medium and cultured overnight at 37°C. 500 μL of seed culture was then inoculated into an Erlenmeyer flask containing 50 mL of fresh fermentation medium and cultured at 30°C for 72 h. The culture was centrifuged at 8000 rpm and 4°C for 15 min to collect the supernatant. 5 μL of the fermentation supernatant was mixed with 75 μL of 0.1 M potassium borate (pH=8.5) buffer, and 20 μL of α-glucosidase (40 U / mL) was added. The mixture was incubated at 37°C for 5 min; then 50 μL of p-nitrophenyl-α-glucopyranoside (12 mM) was added, and the reaction was carried out at 37°C for 30 min; finally, 50 μL of sodium carbonate (200 mM) was added to terminate the reaction. The inhibition rate was determined by measuring the absorbance of each well at 405 nm using a microplate reader. This allows us to select the most suitable nitrogen source type.

[0040] like Figure 5 As shown, the results showed that when 4% yeast extract was added as a nitrogen source, the inhibition rate reached 54.24%. This may be due to the rich amino acids and growth factors provided by yeast extract. Although KNO3 was slightly less effective than yeast extract, it was cheaper, so 3% KNO3 was chosen.

[0041] Example 8 Production of 1-DNJ by Bacillus pumilus DNJ-BP01 strain in a fermenter A 10 L tank fermentation experiment was conducted on *Bacillus pumilus* strain DNJ-BP01 using an inorganic salt culture medium supplemented with 50 g / L beef extract, 25 g / L mannitol as the carbon source, and 30 g / L potassium nitrate as the nitrogen source. The fermentation process is as follows: (1) Seed culture preparation: Inoculate a single colony from a plate into a 500 mL shake flask containing 200 mL of culture medium and incubate at 37°C and 220 rpm for 12 h; (2) Tank fermentation: The seed culture was inoculated into a fermenter containing 7 L of culture medium at an inoculation rate of 10%. The fermentation temperature was 37℃, the stirring speed was 200 rpm, and the aeration rate was 2 vvm. The DNJ titer in the supernatant reached its highest value of 892 mg / L after 76 h of fermentation.

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A short-spore bacillus producing 1-deoxynojirimycin ( Bacillus pumilus DNJ-BP01, characterized in that, The Bacillus pumilus DNJ-BP01 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38218, on April 13, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial agent, characterized in that, It contains the short-lived Bacillus DNJ-BP01 as described in claim 1.

3. The use of Bacillus pumilus DNJ-BP01 as described in claim 1 or the bacterial agent as described in claim 2 in the production of 1-deoxynojirimycin.

4. A method for producing 1-deoxynojirimycin, characterized in that, The method includes the step of fermentation using Bacillus pumilus DNJ-BP01 as described in claim 1 or the inoculum as described in claim 2.

5. The method as described in claim 4, characterized in that, Includes the following steps: A seed culture of the Bacillus pumilus DNJ-BP01 was prepared, and the seed culture was inoculated into a fermentation medium for aerobic fermentation. The fermentation broth was collected and purified to obtain 1-deoxynojirimycin.

6. The method as described in claim 5, characterized in that, The inoculation amount of the seed solution is 10%.

7. The method as described in claim 5, characterized in that, The carbon source of the fermentation medium is 5% beef extract and 2.5% mannitol.

8. The method as described in claim 5, characterized in that, The nitrogen source for the fermentation medium is 3% KNO3.

9. The method as described in claim 5, characterized in that, The fermentation medium comprises 5% beef extract, 2.5% mannitol, 3% KNO3 and 0.5% magnesium sulfate.

10. The method as described in claim 5, characterized in that, The aerobic fermentation time was 76 hours and the temperature was 37°C.

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