A Bacillus subtilis strain with high glucose isomerase productivity and its application

Abstract

The present invention relates to the technical field of genetic engineering, and specifically provides a method for producing a protein and a Bacillus subtilis engineering strain that highly expresses glucose isomerase. The applicant first overexpressed the artificially synthesized glucose isomerase gene in a Bacillus subtilis host to construct a recombinant expression strain; then, using this strain as the starting strain, ultraviolet mutagenesis was carried out, and a mutant strain Bacillus subtilis JKX204 that can greatly increase the expression level of glucose isomerase was screened and obtained. The preservation number is CCTCC NO: M20221172, and its fermentation enzyme activity is as high as 925.3 U / ml, which is about 58.52% higher than that of the starting strain. The mutant strain can be widely used in the production of glucose isomerase, which is beneficial to reducing the production cost of this enzyme and accelerating the popularization and application of glucose isomerase.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to a Bacillus subtilis strain with high yield of glucose isomerase and its application. Background Art

[0002] Glucose isomerase (GI), also known as xylose isomerase (EC5.3.1.5), can catalyze the isomerization of glucose into fructose, and at the same time can also catalyze the isomerization of xylose into xylulose. Since fructose is the main sweetener in beverages and foods, glucose isomerase is one of the most important industrial enzymes. In addition, pentoses and hexoses are also good substrates for glucose isomerase, including D-ribose, L-arabinose, L-rhamnose and D-allose. GI is also used to produce some rare sugars, which have important applications in the pharmaceutical field. For example, GI can efficiently catalyze L-arabinose into L-ribose, and L-ribose is an important component of nucleoside-based, glycosyl complex and oligonucleotide antiviral and anticancer drugs.

[0003] In the industrial aspect, the most important use of GI is to utilize its activity of catalyzing glucose into fructose for the production of high fructose corn syrup (HFCS). Fructose is a natural ketohexose and is the known monosaccharide with the highest sweetness. It is rich in honey and fruits. Fructose is predicted to be a new type of functional sugar source replacing sucrose and glucose globally in the 21st century. Fructose can bypass the rate-limiting enzyme (phosphofructokinase) in glycolysis, and the decomposition rate of fructose in the liver is faster than that of glucose. The metabolic intensity of fructose depends on the fructose concentration and is not affected by insulin. Therefore, the human body's intake of fructose will not cause serious postprandial blood sugar peaks and hypoglycemia and other symptoms that are easily caused by the intake of glucose and sucrose, eliminating the potential factors that induce diabetes. Therefore, it is a healthy sugar that diabetics can eat. Due to the characteristics of fructose such as low calorific value, water retention property, antifreeze property, antiseptic property, flavor enhancement property and affinity, many countries use fructose to manufacture low-energy foods, baby foods, foods for the sick and other nutritional foods and therapeutic foods. In European and American countries, sucrose is basically not used in candies and beverages, but fructose is used. For example, the Canadian law stipulates that all beverages must use high fructose corn syrup as a sweetener. According to statistics, the consumption of high fructose corn syrup in 2013 was 1.07 million tons / year, which is the product with the largest production of industrial enzymes. Therefore, the dosage of glucose isomerase is also huge.

[0004] Currently, the production of glucose isomerase globally is mainly monopolized by two companies, DuPont in the United States and Novozymes in Denmark. The production strains are mostly Streptomyces rubiginosus and Bacillus coagulans.

[0005] Glucose isomerase genes are widespread in prokaryotes, and glucose isomerase genes have been found in many microorganisms such as Escherichia coli, Streptomyces, and Lactobacillus. In the previous stage, in order to improve the expression of glucose isomerase, the method of optimizing the culture medium was mainly used. Previous studies found that adding D-xylose to the fermentation medium could improve the expression of glucose isomerase, but the addition of D-xylose increased the industrial production cost. Later, it was found that adding starch, glucose, sorbitol, glycerol, etc. could achieve 75% of the effect of adding D-xylose. Later, Bejar S et al. found that strong promoters could improve the expression level of glucose isomerase. By replacing the original promoter of the glucose isomerase gene with a strong promoter (P1), it was found that the expression level of glucose isomerase in this strain was increased by 7 times compared with the wild strain induced by xylose. However, the expression level of glucose isomerase was still low and could not meet the needs of industrial production. Therefore, it is urgent to construct a high-yield strain of glucose isomerase with independent intellectual property rights, reduce production costs, and achieve industrial production. Summary of the Invention

[0006] The object of the present invention is to provide a Bacillus subtilis strain with high yield of glucose isomerase, and to provide its application in the production of glucose isomerase. The applicant first constructed a Bacillus subtilis strain recombinantly expressing the glucose isomerase gene, and then further performed ultraviolet mutagenesis on it to screen for mutant strains with significantly increased glucose isomerase production, which is beneficial to reducing the production cost of this enzyme and promoting the wide application of glucose isomerase.

[0007] On the one hand, the present invention provides a Bacillus subtilis engineering strain carrying a recombinant plasmid expressing glucose isomerase.

[0008] The glucose isomerase gene sequence is SEQ ID NO:1, and the amino acid sequence encoded by it is SEQ ID NO:2.

[0009] On the one hand, the present invention provides a mutant strain Bacillus subtilis JKX204 ( Bacillus subtilis JKX204), which was deposited on July 25, 2022 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20221172.

[0010] On the one hand, the present invention provides the application of the above-mentioned Bacillus subtilis in the production of glucose isomerase.

[0011] The present invention also provides a method for producing glucose isomerase, using the above-mentioned Bacillus subtilis as the fermentation strain.

[0012] The present invention also provides a glucose isomerase obtained by fermenting the above-mentioned Bacillus subtilis.

[0013] Beneficial effects

[0014] First, the glucose isomerase gene was expressed in the Bacillus subtilis host to construct the engineering strain Bacillus subtilis JKX203 that recombinantly expresses this enzyme. The glucose isomerase activities in the supernatant of the shake-flask fermentation and 15-L tank fermentation of this strain reached 70.2 U / mL and 672.8 U / mL, respectively.

[0015] To improve the yield of glucose isomerase, the applicant used Bacillus subtilis JKX203 as the starting strain and further screened a mutant strain, Bacillus subtilis JKX204, by ultraviolet mutagenesis. The glucose isomerase activity in the supernatant of the shake-flask fermentation of this mutant strain was as high as 110.6 U / mL, which was 57.54% higher than that of the starting strain; the glucose isomerase activity in the crude enzyme solution of its 15-L tank fermentation was as high as 1020.5 U / mL, which was 51.68% higher than that of the starting strain, achieving an unexpected technical effect. The mutant strain can be widely used in the production of glucose isomerase, which is beneficial to reducing the production cost of glucose isomerase and promoting the application of this enzyme. Description of the drawings

[0016] Figure 1 It is an SDS-PAGE electrophoresis analysis diagram of the fermentation supernatant of Bacillus subtilis JKX203.

[0017] Biological material preservation information

[0018] The mutant strain Bacillus subtilis JKX204 ( Bacillus subtilis JKX204), with the preservation number of CCTCC NO: M20221172, was preserved in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, zip code: 430072, and the preservation time was July 25, 2022. Detailed implementation manners

[0019] The method of the present invention will be further described below in combination with examples. For the experimental methods without specific conditions noted in the examples, conventional conditions can be used, such as the conditions described in "Molecular Cloning: A Laboratory Manual" written by J. Sambrook et al., or the conditions recommended by the manufacturer. Those skilled in the relevant art can better understand and master the present invention with the help of the examples. However, the method for implementing the present invention should not be limited to the specific method steps recorded in the examples of the present invention.

[0020] The formula of the culture medium involved in the examples of the present invention is as follows:

[0021] The preparation method of GM I is as follows: 96 mL of 1× minimum salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, 1 mL of 10% yeast extract; the preparation method of 1× minimum salt solution is as follows: 14 g / L of K2HPO4, 6 g / L of KH2PO4, 2 g / L of (NH4)2SO4, 1 g / L of trisodium citrate, 0.2 g / L of MgSO4•7H2O, which are dissolved in distilled water in sequence;

[0022] The preparation method of GM II is as follows: 97 mL of 1× minimum salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast extract, 0.25 mL of 1 M MgCl2, 0.05 mL of 1 M CaCl2;

[0023] LB plate: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1.5% agar powder;

[0024] Skim milk powder plate: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% skim milk powder, 1.5% agar powder;

[0025] Seed medium: 0.5% yeast extract powder, 0.5% tryptone, 1% glucose, 1.8% K2HPO4, 5 μg / mL of chloramphenicol;

[0026] Fermentation medium: 1 - 2% yeast powder, 2 - 5% soybean cake powder, 5 - 10% maltodextrin, 0.1 - 0.5% sodium citrate, 0.1 - 0.5% CaCl2, 0.1 - 0.5% MgSO4, 0.5 - 2% K2HPO4.

[0027] Example 1 Glucose isomerase integrated expression plasmid pBE2R - GI and expression

[0028] The amino acid sequence of glucose isomerase is SEQ ID NO:2. According to the amino acid sequence of SEQ ID NO:2, its optimized nucleic acid sequence SEQ ID NO:1 was obtained, and SEQ ID NO:1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. SEQ ID NO:1 was ligated to the pBE2R vector using the Gibson Assembly method, and was transformed into Escherichia coli DH5α by heat shock method. The plasmid was extracted and sequenced to confirm, and the glucose isomerase recombinant plasmid pBE2R - gi .

[0029] The correctly sequenced recombinant plasmid pBE2R-GI was transferred into competent cells WB600. The specific transformation process was as follows: A single colony of WB600 growing on the LB plate was picked with a pipette tip and inoculated into 2 mL of GMⅠ, and cultured for 12 h; The overnight cultured bacterial solution was added to 98 mL of GMⅠ, and cultured at 37 °C and 200 rpm for about 4 h; 10 mL of the bacterial solution was taken and added to 90 mL of GMII, and cultured at 37 °C and 200 rpm for about 1.5 h; The cells were placed in an ice-water bath for 30 min, centrifuged at 4000 rpm and 4 °C for 30 min, and the supernatant was removed; 10 mL of GMⅢ was added and mixed evenly to obtain competent cells WB600. Then, 5 μL of pBE2R-GI plasmid was added to 500 μL of competent cells, and the competent cells were directly placed on a shaker at 37 °C and 200 rpm for 1.5 h, centrifuged at low speed for 3 min, and part of the supernatant was discarded, and evenly spread on a skim milk medium plate containing 40 μg / mL kanamycin, and cultured in a constant temperature incubator at 37 °C for 12 h. The single colonies on the plate the next day were the recombinant strain WB600 / pBE2R-GI containing the glucose isomerase gene. The recombinant Bacillus subtilis engineering bacteria expressing glucose isomerase were inoculated into 5 mL of LB liquid medium (1% peptone, 1% NaCl, 0.5% yeast powder), and cultured at 37 °C and 200 rpm with shaking for 12 h. The bacterial solution was transferred to the fermentation medium for enzyme production at an inoculation amount of 2% respectively, and cultured at 37 °C and 200 rpm with shaking for 72 h. Centrifuge at 4000 rpm for 10 min to obtain the supernatant; The glucose isomerase activity of the fermentation supernatant of the above strains was measured respectively by the method for the determination of glucose isomerase activity of the National Standard of the People's Republic of China (GB23533-2009).

[0030] The results showed that the glucose isomerase activity in the fermentation supernatant of the recombinant bacteria was as high as 10618 U / mL. The applicant named this strain Bacillus subtilis JKX203 ( Bacillus subtilis JKX203). The fermentation supernatant of this strain was analyzed by SDS-PAGE electrophoresis. The results were as Figure 1 shown, and obvious glucose isomerase expression bands could be seen.

[0031] Example 2 Fermentation verification of Bacillus subtilis JKX203 in a 15L tank

[0032] Bacillus subtilis JKX203 was inoculated into 500 mL of seed medium and cultured at 37 °C and 220 rpm with shaking for about 12 h.

[0033] Transfer all the seed liquid into a 15-L fermenter (the components of the fermenter medium are as follows: 3% corn starch, 1% glucose, 3% soybean cake powder, 3% wheat bran, 0.78% Na2HPO4, 0.05% KH2PO4, and the volume after sterilization in the fermenter is 8 L); control the temperature at 37 °C, the initial pH of fermentation at 7.2, and use ammonia water to control the pH not lower than 7.0 during the fermentation process; the air volume is 1 - 1.5 vvm, the rotation speed is 300 - 1000 rpm, and control the DO not lower than 10% during the fermentation process; start to feed 50% glucose after 3 - 4 h, and the feeding rate is 3 g / L·h; stop culturing after 25 - 30 h of fermentation when both DO and pH recover. Collect the supernatant of the fermentation broth, and determine the glucose isomerase activity of the fermentation supernatant by the method for the determination of glucose isomerase activity in the national standard of the People's Republic of China (GB 1886.174 - 2016).

[0034] The results showed that the enzyme activity of the fermentation supernatant of Bacillus subtilis strain JKX203 was as high as 96882 U / mL. This indicates that the recombinant engineering strain Bacillus subtilis JKX203 constructed in the present invention can highly express the exogenous glucose isomerase gene. gi .

[0035] Example 3 Mutagenesis screening of high-yield glucose isomerase strains

[0036] The mutations caused by ultraviolet mutagenesis are highly random, and the effects of the mutations are also random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, which involves a large amount of screening work and there is a possibility of not obtaining effective positive mutations. However, because ultraviolet mutagenesis requires simple equipment, low cost, and a large number of mutants can be obtained in a short time, it is still a commonly used mutagenesis and breeding method at present.

[0037] The applicant used the Bacillus subtilis JKX203 constructed in Example 1 as the starting strain and carried out genetic transformation on it by ultraviolet mutagenesis to further improve the yield of its glucose isomerase.

[0038] 4.1 Preparation of bacterial suspension

[0039] Inoculate the starting strain Bacillus subtilis JKX203 on an LB slant by streaking and culture it at 37 °C for 24 h; add 5 mL of 0.85% sterile normal saline, wash all the bacteria on the slant, transfer them into a sterile test tube containing glass beads, vortex for 10 min to completely break them into single-cell bacteria; transfer all the bacterial suspension into a 15-mL centrifuge tube, centrifuge at 6000 rpm for 3 min to collect the bacteria, take the supernatant, and suspend the bacteria with 10 mL of normal saline; wash the cells twice, and finally adjust the cell concentration to 10 8 cells / mL.

[0040] 4.2 UV mutagenesis treatment and determination of mutagenic dose

[0041] Turn on the 9 W UV lamp switch and preheat for about 30 min; Take a sterile petri dish with a diameter of 9 cm, add 10 mL of the above-mentioned bacterial suspension with a cell concentration of 10 8 CFU / mL, add a sterile magnetic stirring rotor, turn on the magnetic stirrer, open the petri dish lid, and stir and irradiate at a vertical distance of 15 cm for 0.5 min, 1 min, 1.5 min, 2 min, 2.5 min, and 3 min respectively; Cover the petri dish lid, turn off the UV lamp, and incubate in the dark for 30 min.

[0042] Dilute the irradiated bacterial suspension with 0.85% normal saline in gradient to 10 -1 ~10 -6 ; Take 100 μL of the bacterial suspensions with three dilution factors of 10 -4 , 10 -5 , and 10 -6 respectively, coat them on LB plates, and coat three plates for each dilution factor; Take the non-UV-irradiated bacterial solution for dilution and plating as a control in the same operation. Wrap the above-mentioned evenly coated plates with black cloth or newspaper and place them in an incubator at 37°C overnight.

[0043] Count the number of single colonies grown on the plates at each dilution factor under different irradiation times. If the number of single colonies grown at a certain dilution factor is between 30 and 300, then this dilution factor is considered appropriate. Calculate the average value of the number of single colonies grown on the three plates at this dilution factor, and calculate the concentration of the bacterial suspension according to the following formula:

[0044] Concentration of bacterial suspension (CFU / mL) = average number of colonies at a certain dilution factor × dilution multiple × 10

[0045] Calculate the lethality at a certain UV treatment dose according to the following formula:

[0046] Lethality (%) = (1 - concentration of bacterial suspension after treatment at a certain dose / concentration of bacterial suspension before treatment) × 100%

[0047] After calculation, the lethality of Bacillus subtilis JKX203 under different UV mutagenic doses is shown in Table 1.

[0048] Table 1 UV mutagenic lethality of Bacillus subtilis JKX203

[0049] Time / min 0.5 1 1.5 2 2.5 3 Lethality rate / % 85.3 95.7 98.6 99.8 99.9 99.9

[0050] It can be seen from Table 1 that the lethality reaches over 95% after the bacterial suspension is irradiated with UV for 1 min. Therefore, the final mutagenesis time is determined to be 1 min.

[0051] 4.3 Primary screening in shake flasks

[0052] The irradiated bacterial suspension was serially diluted 10-fold with 0.85% normal saline to 10 -1 ~10 -6 ; 100 μL of the bacterial suspensions at three dilution factors of 10 -4 , 10 -5 , and 10 -6 were each taken and spread on nutrient agar plates. Three plates were spread for each dilution factor, and a sterile glass rod was used to evenly coat the entire surface of the plate. The above-mentioned evenly spread plates were wrapped with black cloth or newspaper and incubated overnight at 37°C.

[0053] Single colonies grown on the plates were streaked and purified on nutrient agar plates containing 50 μg / mL chloramphenicol; single colonies were picked and streaked and inoculated on nutrient agar slopes containing 50 μg / mL chloramphenicol for preservation; a total of 50 mutant strains were enriched and screened, and were named GI-1, GI2, GI-3... GI-50 respectively.

[0054] The 50 mutant strains obtained from the above screening were respectively inoculated into 50 mL of shake flask fermentation medium and fermented at 37°C and 220 rpm for 72 h. After centrifugation, the supernatant was taken, and the glucose isomerase activity in the fermentation supernatant was measured. At the same time, the original strain was used as a control, and mutant strains with a shake flask fermentation enzyme activity increased by more than 15% compared with the original strain were selected for the second round of UV mutagenesis screening.

[0055] The applicant continued the above method for 8 rounds of UV mutagenesis screening, and finally obtained 1 mutant strain with a significantly higher glucose isomerase yield than the original strain, named Bacillus subtilis JKX204 ( Bacillus subtilis JKX204). After this strain was fermented in 50 mL of shake flask fermentation medium at 37°C and 220 rpm for 72 h, the supernatant was taken after centrifugation. The glucose isomerase activity in the supernatant was as high as 110.6 U / mL, which was 57.54% higher than that of the original strain; the glucose isomerase activity in the crude enzyme solution of 15 L tank fermentation was as high as 1020.5 U / mL, which was 51.68% higher than that of the original strain, achieving an unexpected technical effect.

[0056] The applicant deposited the mutant strain Bacillus subtilis JKX204 ( Bacillus subtilis JKX204) at the China Center for Type Culture Collection of Wuhan University, Wuhan, China on July 25, 2022, with the deposit number CCTCC NO: M20221172.

Claims

1. A mutant Bacillus subtilis, characterized in that, The preservation number of the described Bacillus subtilis mutant strain is CCTCC NO: M20221172.

2. Use of the mutant Bacillus subtilis according to claim 1 in the production of high fructose syrup.

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