CN109837289B

CN109837289B - Method for adjusting self-aggregation capability and mycoderm characteristics of bacillus natto

Info

Publication number: CN109837289B
Application number: CN201910233452.7A
Authority: CN
Inventors: 刘艳; 胡润; 薛正莲; 杨超英; 王洲; 钱森和; 胡刘秀; 李松; 杨自名; 冯静静; 高越
Original assignee: Anhui Polytechnic University
Current assignee: Anhui Polytechnic University
Priority date: 2019-03-26
Filing date: 2019-03-26
Publication date: 2021-12-28
Anticipated expiration: 2039-03-26
Also published as: CN109837289A

Abstract

The invention discloses a method for regulating the self-agglutination ability and biofilm characteristics of Bacillus natto. The method can change the self-agglutination ability and biofilm characteristics of Bacillus natto by changing the expression level and protein expression level of the non-specific DNA-binding protein _HBsu encoding gene, and further change the ability of Bacillus natto to synthesize vitamin K2. _The invention provides a new idea for the selection and breeding of excellent strains of vitamin K2, and has great practical significance and broad application prospects in the fields of food and biopharmaceuticals.

Description

Method for adjusting self-aggregation capability and mycoderm characteristics of bacillus natto

Technical Field

The invention belongs to the technical field of industrial microbial strain modification, and particularly relates to a method for regulating and controlling the self-agglutination capability and the mycoderm characteristic of bacillus natto.

Background

As an important class of fat-soluble vitamins, vitamin K₂Has important application in the fields of food and medicine. The health people are supplemented with appropriate amount of the health foodVitamin K₂The food can effectively reduce the incidence of fracture. In addition, more and more data are showing vitamin K₂Deficiency is a significant cause of bleeding disease and death in infants worldwide. In addition, in recent years, the research reports published by domestic and foreign scholars in top-level international periodicals such as Science and Nature show that similar to coenzyme Q, vitamin K₂As a membrane-bound electron carrier, the compound has the function of repairing damaged cell mitochondria, which plays an important role in protecting human beings from Parkinson's disease. Due to the application of the three aspects, the international market aims at the functional food vitamin K₂Has been increasing in recent years.

Researches show that a large amount of biofilms are generated by the self-agglutination of the static fermentation of the natto bacillus under the shake flask scale, and the formation of the biofilms obviously promotes the vitamin K₂And (4) synthesizing. Therefore, the self-agglutination capability and the mycoderm characteristic of the bacillus natto are regulated and controlled, and the vitamin K is promoted₂The anabolism of (2) has very important significance. However, the research on the self-agglutination capability and the mycoderm characteristics of the bacillus natto in China is still rarely reported at present.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a method for regulating and controlling the self-agglutination capability and the mycoderm characteristic of the bacillus natto, the self-agglutination capability and the mycoderm forming capability of the bacillus natto are obviously enhanced by regulating and controlling the expression level and the protein amount of an HBsu coding gene, and the vitamin K is finally improved₂The level of synthesis of (c).

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for regulating the self-aggregation capability and the mycoderm characteristic of Bacillus natto comprises the following steps:

a. culturing the bacillus natto in an LB liquid culture medium;

b. extracting the whole genome of the bacillus natto;

c. designing a primer pair by taking a non-specific DNA binding protein HBsu coding gene as a target gene for overexpression, and carrying out PCR amplification on a target gene fragment of the whole genome of the bacillus natto;

d. connecting the target gene fragment with a vector to construct an enhanced vector for enhancing the activity of HBsu;

e. the enhanced vector is used for transforming the bacillus natto, transformants are screened by utilizing the resistance genes on the vector, and the screened transformants are transferred to a slant culture medium;

f. selecting a bacterial colony from a slant culture medium and culturing a mutant strain on a seed culture medium;

g. the self-agglutination capability and the mycoderm characteristic of the mutant strain are regulated and controlled by regulating the temperature of the mutant strain when IPTG is added for culture.

The method for regulating and controlling the self-cohesive capacity of the mutant strain in the step g comprises the following steps: firstly inoculating the mutant strain into a fermentation culture medium for culture, then adding IPTG (isopropyl-beta-D-thiogalactoside) for culture at 15-30 ℃ until OD600 is 3.0, then sequentially centrifuging, washing and resuspending, controlling OD600 to be 2.5, finally standing the resuspension solution for 24 hours, measuring the bacterial liquid OD600 at 1cm below each culture solution after self-agglutination, and calculating the self-agglutination rate, wherein the calculation formula of the self-agglutination rate is as follows:

AAg (%) (from OD600 of supernatant after agglutination-OD 600 before agglutination)/OD 600 before self agglutination × 100%.

Preferably, the original bacillus natto and the mutant strain are respectively inoculated into a fermentation medium, and the size of the self-aggregation characteristic of each strain is detected. The culture conditions were 37 ℃ for 2 days, IPTG was added to induce expression, and the cells were cultured at 15, 20, 25 and 30 ℃ until OD600 became 3.0. The cells were collected by centrifugation at 6000 Xg for 10min and washed twice with phosphate buffer. Finally, the cells were resuspended in phosphate buffer and the OD600 was controlled at 2.5. Placing 5mL of the heavy suspension in a glass test tube, standing for 24 hours at 4 ℃, and measuring the concentration of the bacterial liquid at a position 1cm below each culture liquid surface, wherein three samples are arranged in parallel.

The method for regulating and controlling the characteristics of the bacterial membrane in the step g comprises the following steps: firstly, culturing the mutant strain in an LB culture medium at 37 ℃ until OD600 is 0.02, then transferring to a fermentation culture medium for culturing until OD600 is 2.0, adding IPTG (isopropyl-beta-thiogalactoside) for shaking culture at 15-30 ℃, then removing a culture solution, sequentially washing, dyeing, standing, washing again until no purple color exists, finally adding acetic acid for treatment, and measuring the absorbance of the obtained solution under the visible light of 597nm (taking the original bacteria as a blank control), and recording the absorbance as the rate of mycoderm formation.

Preferably, the original bacillus natto and the mutant strain are taken as research objects, the bacillus natto and the mutant strain are cultured in LB culture medium until OD600 is 0.02, the bacillus natto and the mutant strain are transferred to a 5mL test tube containing TSB culture medium, IPTG is added when the bacillus natto and the mutant strain are cultured until OD600 is 2.0, the IPTG is added for culture, after shaking culture at 15, 20, 25 and 30 ℃ for 24 hours, the culture in the test tube is slowly removed, the test tube is washed by distilled water for 2-3 times, 3mL crystal violet solution (w/v%) with the mass volume ratio of 0.1% is added for staining after air drying, the test tube is kept still for 20min at room temperature, the stain is poured out, then the test tube is washed by distilled water for 2-3 times, floating color is washed, the test tube is inversely dried for 20min, residual moisture is removed, finally 2mL of 33% acetic acid (v/v%) is added for ultrasonic decolorization for 10min, the volume fraction of 33% acetic acid is taken as reference, and the absorbance of the obtained solution is measured under the visible light of 597nm (taking the original strain as the blank rate, and the strain film forming rate is taken as the reference.

Inoculating original Bacillus natto and mutant strain into fermentation medium, culturing at 37 deg.C for 2 days, adding IPTG, culturing at 15, 20, 25, and 30 deg.C for 3-5 days, and measuring vitamin K in fermentation liquid₂And (4) yield. And detecting the expression level of hbs in each strain by reverse transcription PCR or fluorescent quantitative PCR. Determination of vitamin K in fermentation broth₂The content is measured by HPLC, and the fluorescent quantitative PCR detection is carried out by SYBR Green Real-Time qPCR kit.

The fermentation medium is TSB, and the components (w/v%) are as follows: 17g of tryptone, 3g of soybean papain digest, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate and 2.5g of glucose.

And c, adding restriction enzyme cutting sites and protective base sequences of restriction enzymes on two sides of the target gene segment in the step c.

The vector in the step d is an inducible expression vector.

The mode of transforming the bacillus natto by the vector in the step e can be electro-stimulation transformation, protoplast transformation mediated by polyethylene glycol, isopropanol transformation or dimethyl sulfoxide transformation.

And e, selecting ampicillin and tetracycline resistance in the resistance gene screening in the step e.

The HBsu gene is SEQ ID No: 1, or a nucleotide sequence represented by the formula (I).

The primer pair in the step c is SEQ ID No: 2 and SEQ ID No: 3.

The self-agglutination capability and the pellicle characteristics of the bacillus natto are changed by changing the expression level and the protein expression quantity of a non-specific DNA binding protein HBsu coding gene. The mode of change is that the self-agglutination capability and the bacterial membrane characteristic of the natto bacillus are regulated and controlled by the expression level of over-expression non-specific DNA binding protein.

The method changes the vitamin K synthesized by the bacillus natto by adjusting the self-aggregation capability and the mycoderm characteristics of the bacillus natto₂The ability of the cell to perform.

The invention has the beneficial effects that: according to the invention, the self-agglutination capability and the bacterial membrane characteristic of the bacillus natto are regulated and controlled through the expression level of over-expression non-specific DNA binding protein, so that a mutant strain capable of enriching and generating a biological membrane is obtained; the mutant strain obtained by the invention has greatly improved self-agglutination rate, prolonged logarithmic growth phase of the strain and improved biofilm formation capability. After the biofilm forms in the later stage of fermentation, vitamin K₂The yield is also obviously improved. The method regulates and controls the expression level of nonspecific DNA binding protein (HBsu) by temperature induction, and finally influences the synthesis of vitamin K by Bacillus natto₂The ability of the cell to perform. The invention is vitamin K₂The breeding of the excellent strain provides a new idea, and has great practical significance and wide application prospect in the fields of food and biological pharmacy.

Drawings

The description includes the following figures, the contents shown are respectively:

FIG. 1 shows that different expression levels of hbs gene were measured by fluorescence quantitative PCR for original strain and mutant strains obtained at different induction temperatures;

FIG. 2 shows that different expression levels of HBsu were measured by Western blot for original bacteria and mutants obtained at different induction temperatures.

Detailed Description

The following examples are included to provide further detailed description of the embodiments of the invention and to provide those skilled in the art with a more complete, concise and complete understanding of the principles and spirit of the invention, and to facilitate its practice.

Example 1

Taking bacillus natto BS-13 as an original strain, and sequentially operating according to the following steps:

1. culturing the bacillus natto in 50mL LB culture medium and culturing for 48h at 37 ℃ by a shaking table.

2. Collecting culture solution, centrifuging, collecting thallus precipitate, extracting genome DNA with kit, and storing at-20 deg.C.

3. The nonspecific DNA binding protein HBsu coding gene hbs is used as a target gene, and primer pairs are designed as follows:

Primer-F 5'-GCGTCGACATGAACAAAACAGAACTTATCAATGCG 3'(SEQ ID No：2)

Primer-R 5'-GAATTCCGTTATTTTCCGGCAACTGCGTCTTTAAG 3'(SEQ ID No：3)

PCR amplification was performed using Taq DNA polymerase using genomic DNA as a template, and the desired fragment was approximately 279 bp.

4. The vector pHY-P43 was used to transform Bacillus subtilis WB600 competent cells, spread on ampicillin and tetracycline resistant plates, and incubated overnight (12-16h) in a 37 ℃ incubator. Picking single colony from the transformation plate, extracting plasmid with plasmid extraction kit after liquid culture, and storing at-20 ℃.

5. The vector and the target fragment are cut by SalI and EcoRI respectively and then are connected into an enhanced vector pHY-P43 under the action of T4DNA ligase.

6. The obtained enhanced vector enters the bacillus natto BS-13 by an electrotransformation method, transformants are screened by ampicillin and tetracycline resistance, and the screened transformants are transferred to a slant culture medium for storage.

7. Colonies were picked from the slant medium and cultured in 50mL of LB seed medium in a 37 ℃ incubator for 48 hours.

8. Inoculating the bacillus natto mutant strain liquid into 500mL conical flasks containing 50mL fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at 37 ℃ at 220 r/min. The fermentation medium was TSB, with the composition (w/v%): 17g of tryptone, 3g of soybean papain digest, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate and 2.5g of glucose. The cells were collected by centrifugation at 6000 Xg for 10min after adding IPTG at 15 ℃ and then cultured until OD600 became 3.0, and the cells were washed twice with a phosphate buffer. Finally, the cells were resuspended in phosphate buffer, and the OD600 of the cells before self-agglutination was controlled to 2.5. Placing 5mL of the heavy suspension in a glass test tube, standing at 4 ℃ for 24 hours, and determining that the bacterial liquid OD600 at 1cm under the culture liquid after self-agglutination is 5.5-6.5, and the self-agglutination rate of the mutant strain is 160-.

9. Culturing Bacillus natto BS-13 and mutant strain LB culture medium until OD600 is 0.02, transferring to 5mL test tube containing TSB culture medium, culturing until OD600 is 2.0, adding IPTG (isopropyl thiogalactoside) for culturing, after shaking culturing for 24 hours at 15 ℃, slowly removing the culture in the test tube, washing with distilled water for 2-3 times, drying in the air, adding 3mL of crystal violet solution (w/v%) with the mass volume ratio of 0.1% for dyeing, standing at room temperature for 20min, washing with distilled water for 2-3 times after pouring off the dyeing agent, washing off the floating color, inverting and drying in the air for 20min, removing residual moisture, finally adding 2mL of 33% acetic acid (v/v%), performing ultrasonic decoloration for 10min by taking the volume fraction of 33% acetic acid as a reference, the absorbance of the obtained solution is measured under the visible light of 597nm, and the mutant strain bacterial film forming rate (taking Bacillus natto BS-13 as a blank control) OD597 is 1.3-1.8.

10. Inoculating Bacillus natto BS-13 and mutant strain into fermentation medium, culturing at 37 deg.C for 2 days, adding IPTG, culturing at 15 deg.C for 3-5 days, and culturing to obtain mutant strain vitamin K₂The yield is 37.8-59.4mg/L, which is improved by 2.1-3.3 times compared with BS-13.

Example 2

Primer-F 5'-GCGTCGACATGAACAAAACAGAACTTATCAATGCG 3'(SEQ ID No：2)

Primer-R 5'-GAATTCCGTTATTTTCCGGCAACTGCGTCTTTAAG 3'(SEQ ID No：3)

8. Inoculating the bacillus natto mutant strain liquid into 500mL conical flasks containing 50mL fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at 37 ℃ at 220 r/min. The fermentation medium was TSB, with the composition (w/v%): 17g of tryptone, 3g of soybean papain digest, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate and 2.5g of glucose. The cells were collected by centrifugation at 6000 Xg for 10min after adding IPTG at 20 ℃ and then cultured until OD600 became 3.0, and the cells were washed twice with a phosphate buffer. Finally, the cells were resuspended in phosphate buffer, and the OD600 of the cells before self-agglutination was controlled to 2.5. Placing 5mL of the heavy suspension in a glass test tube, standing at 4 ℃ for 24 hours, and determining that the bacterial liquid OD600 at 1cm under the culture liquid after self-agglutination is 5.2-7.0, and the self-agglutination rate of the mutant strain is 110-180%.

9. Culturing Bacillus natto BS-13 and mutant strain LB culture medium until OD600 is 0.02, transferring to 5mL test tube containing TSB culture medium, culturing until OD600 is 2.0, adding IPTG (isopropyl thiogalactoside) for culturing, after shaking culturing for 24 hours at 20 ℃, slowly removing the culture in the test tube, washing with distilled water for 2-3 times, drying in the air, adding 3mL of crystal violet solution (w/v%) with the mass volume ratio of 0.1% for dyeing, standing at room temperature for 20min, washing with distilled water for 2-3 times after pouring off the dyeing agent, washing off the floating color, inverting and drying in the air for 20min, removing residual moisture, finally adding 2mL of 33% acetic acid (v/v%), performing ultrasonic decoloration for 10min by taking the volume fraction of 33% acetic acid as a reference, the absorbance of the obtained solution is measured under the visible light of 597nm, and the mutant strain bacterial film forming rate (taking Bacillus natto BS-13 as a blank control) OD597 is 1.2-2.6.

10. Inoculating Bacillus natto BS-13 and mutant strain into fermentation medium, culturing at 37 deg.C for 2 days, adding IPTG, culturing at 20 deg.C for 3-5 days, and culturing to obtain mutant strain vitamin K₂The yield is 50.4-77.4mg/L, which is improved by 2.8-4.3 times compared with BS-13.

Example 3

Primer-F 5'-GCGTCGACATGAACAAAACAGAACTTATCAATGCG 3'(SEQ ID No：2)

Primer-R 5'-GAATTCCGTTATTTTCCGGCAACTGCGTCTTTAAG 3'(SEQ ID No：3)

8. Inoculating the bacillus natto mutant strain liquid into 500mL conical flasks containing 50mL fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at 37 ℃ at 220 r/min. The fermentation medium was TSB, with the composition (w/v%): 17g of tryptone, 3g of soybean papain digest, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate and 2.5g of glucose. The cells were collected by centrifugation at 6000 Xg for 10min after adding IPTG at 25 ℃ and then cultured until OD600 became 3.0, and the cells were washed twice with a phosphate buffer. Finally, the cells were resuspended in phosphate buffer, and the OD600 of the cells before self-agglutination was controlled to 2.5. Placing 5mL of the heavy suspension in a glass test tube, standing at 4 ℃ for 24 hours, and determining that the bacterial liquid OD600 at 1cm under the culture liquid after self-agglutination is 5.2-6.3, and the self-agglutination rate of the mutant strain is 110-150%.

9. Culturing Bacillus natto BS-13 and mutant strain LB culture medium until OD600 is 0.02, transferring to 5mL test tube containing TSB culture medium, culturing until OD600 is 2.0, adding IPTG (isopropyl thiogalactoside) for culturing, after shaking culturing for 24 hours at 25 ℃, slowly removing the culture in the test tube, washing with distilled water for 2-3 times, drying in the air, adding 3mL of crystal violet solution (w/v%) with the mass volume ratio of 0.1% for dyeing, standing at room temperature for 20min, washing with distilled water for 2-3 times after pouring off the dyeing agent, washing off the floating color, inverting and drying in the air for 20min, removing residual moisture, finally adding 2mL of 33% acetic acid (v/v%), performing ultrasonic decoloration for 10min by taking the volume fraction of 33% acetic acid as a reference, the absorbance of the obtained solution is measured under the visible light of 597nm, and the mutant strain bacterial film forming rate (taking Bacillus natto BS-13 as a blank control) OD597 is 1.4-2.1.

10. Inoculating Bacillus natto BS-13 and mutant strain into fermentation medium, culturing at 37 deg.C for 2 days, adding IPTG, culturing at 25 deg.C for 3-5 days, and culturing to obtain mutant strain vitamin K₂The yield is 32.4-43.2mg/L, which is improved by 1.8-2.4 times compared with BS-13.

Example 4

1. culturing the bacillus natto in 50mL LB culture medium at 37 ℃ for 48h by a shaking table;

Primer-F 5'-GCGTCGACATGAACAAAACAGAACTTATCAATGCG 3'(SEQ ID No：2)

Primer-R 5'-GAATTCCGTTATTTTCCGGCAACTGCGTCTTTAAG 3'(SEQ ID No：3)

8. Inoculating the bacillus natto mutant strain liquid into 500mL conical flasks containing 50mL fermentation medium according to the inoculation amount of 5%, and culturing for 2 days at 37 ℃ at 220 r/min. The fermentation medium was TSB, with the composition (w/v%): 17g of tryptone, 3g of soybean papain digest, 5g of sodium chloride, 2.5g of potassium dihydrogen phosphate and 2.5g of glucose. The cells were collected by centrifugation at 6000 Xg for 10min after adding IPTG at 30 ℃ and then cultured until OD600 became 3.0, and the cells were washed twice with a phosphate buffer. Finally, the cells were resuspended in phosphate buffer, and the OD600 of the cells before self-agglutination was controlled to 2.5. Placing 5mL of the heavy suspension in a glass test tube, standing at 4 ℃ for 24 hours, and determining that the bacterial liquid OD600 at 1cm under the culture liquid after self-agglutination is 5.1-5.8, and the self-agglutination rate of the mutant strain is 130-.

9. Culturing Bacillus natto BS-13 and mutant strain LB culture medium until OD600 is 0.02, transferring to 5mL test tube containing TSB culture medium, culturing until OD600 is 2.0, adding IPTG (isopropyl thiogalactoside) for culturing, after shaking culturing for 24 hours at 30 ℃, slowly removing the culture in the test tube, washing with distilled water for 2-3 times, drying in the air, adding 3mL of crystal violet solution (w/v%) with the mass volume ratio of 0.1% for dyeing, standing at room temperature for 20min, washing with distilled water for 2-3 times after pouring off the dyeing agent, washing off the floating color, inverting and drying in the air for 20min, removing residual moisture, finally adding 2mL of 33% acetic acid (v/v%), performing ultrasonic decoloration for 10min by taking the volume fraction of 33% acetic acid as a reference, the absorbance of the obtained solution is measured under the visible light of 597nm, and the mutant strain bacterial film forming rate (taking Bacillus natto BS-13 as a blank control) OD597 is 1.1-1.6.

10. Inoculating Bacillus natto BS-13 and mutant strain into fermentation medium, culturing at 37 deg.C for 2 days, adding IPTG, culturing at 30 deg.C for 3-5 days, and culturing to obtain mutant strain vitamin K₂The yield is 21.6-34.2mg/L, which is improved by 1.2-1.9 times compared with BS-13.

The invention adopts an overexpression mode, regulates and controls the self-agglutination capability and the mycoderm characteristic of the bacillus natto by regulating the overexpression level of the temperature control hbs and the protein overexpression amount of the nonspecific DNA binding protein (HBsu) (shown in attached figures 1 and 2), and finally influences the synthesis of vitamin K by the bacillus natto₂The ability of the cell to perform.

The present invention has been described above by way of example. It is to be understood that the specific implementations of the invention are not limited in this respect. Various insubstantial improvements are made by adopting the method conception and the technical scheme of the invention; the present invention is not limited to the above embodiments, and can be modified in various ways.

SEQUENCE LISTING

<110> university of Anhui engineering

<120> a method for regulating the self-aggregation capability and the mycoderm characteristic of bacillus natto

<130> 1

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 279

<212> DNA

<213> Bacillus natto hbs

<400> 1

atgaacaaaa cagaacttat caatgcggtt gcagaagcaa gcgaattgtc taaaaaagac 60

gctacaaaag cagttgactc tgtttttgat acgatcttag atgcacttaa aaacggtgat 120

aaaatccaac tgatcggttt tggtaacttc gaggtgcgtg aacgttctgc acgtaaagga 180

cgcaaccctc aaacaggtga agaaatcgaa attccagcga gcaaagtacc tgctttcaaa 240

ccaggtaaag cgcttaaaga cgcagttgcc ggaaaataa 279

<210> 2

<211> 35

<212> DNA

<213> Artificial sequence

<400> 2

gcgtcgacat gaacaaaaca gaacttatca atgcg 35

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence

<400> 3

gaattccgtt attttccggc aactgcgtct ttaag 35

Claims

1. a method for regulating Bacillus natto self-aggregation ability and biofilm characteristic, is characterized in that, comprises the steps:

a. Bacillus natto is cultivated in LB liquid medium;

b. Extract the whole genome of Bacillus natto;

c. Design primer pairs with the non-specific DNA binding protein HBsu encoding gene as the target gene to overexpress, and carry out PCR amplification of the target gene fragment for the whole genome of Bacillus natto;

Both sides of the target gene fragment are added with restriction endonuclease enzyme cleavage sites and protective base sequences;

The HBsu encoding gene is the base sequence shown in SEQ ID No: 1;

The primer pair is the nucleotide sequence shown in SEQ ID No: 2 and SEQ ID No: 3;

d. Linking the target gene fragment with the vector to construct a pHY-P43 enhancing vector for enhancing the activity of HBsu;

E, enhance the vector transformation Bacillus natto, and utilize the resistance gene on the carrier to screen transformants, and transfer the screened transformants to the slant medium;

The selection of resistance gene selection is ampicillin and tetracycline resistance;

F, pick colony from slant medium and carry out culture transformant on seed medium;

g. Bacillus natto and the transformant were respectively inserted into the fermentation medium, and the expression level and protein expression of the non-specific DNA-binding protein HBsu encoding gene were changed by adjusting the temperature during IPTG culture, so as to regulate the self-agglutination ability and the biofilm of the transformant. characteristics, thereby changing the ability of Bacillus natto to synthesize vitamin K ₂ ;

The method for regulating the self-aggregation ability of the transformant is as follows: firstly insert the transformant into the fermentation medium for cultivation, then add IPTG at 15-30°C to cultivate to OD600=3.0, and then perform centrifugation, washing and resuspension in sequence, and control OD600= 2.5. Finally, after standing the resuspended liquid for 24 hours, measure the OD600 of the bacterial liquid at 1 cm below each culture solution after self-agglutination, and calculate the self-agglutination rate. The calculation formula of the self-agglutination rate is:

AAg (%)=(OD600 of upper bacterial solution after self-agglutination-OD600 before self-agglutination)/OD600 before self-agglutination ×100%;

The method of regulating the biofilm characteristics is as follows: firstly culture the transformant in LB medium to OD600=0.02, then transfer to fermentation medium and cultivate to OD600=2.0, add IPTG for shaking culture at 15-30℃, and then remove the culture The solution was washed, dyed, left to stand, washed again until no purple color, and finally added acetic acid for treatment, and the absorbance of the obtained solution was measured under visible light 597 nm, which was recorded as the biofilm formation rate;

Wherein, when IPTG was added and transferred to 20°C for culture, the vitamin K output of the transformant was _50.4-77.4 mg/L, which was 2.8-4.3 times higher than that of the original strain.