A mutant strain of Streptomyces parvum and its application in producing glutathione

Abstract

The present invention discloses a mutant strain of Streptomyces parvum and its application in producing glutathione, belonging to the field of genetic engineering. The present invention successfully constructs a mutant strain of Streptomyces parvum by overexpressing the glnA gene in Streptomyces parvum; wherein the nucleotide sequence of the glnA gene is shown in SEQ ID NO: 1. Experiments have shown that compared with the wild-type Streptomyces parvum, the mutant strain of Streptomyces parvum can significantly increase the content of glutathione and glucose in the fermentation product, and can also improve the utilization efficiency of ammonia nitrogen during the fermentation process. The present invention provides new strain resources and methods for mass production of bacterial-derived glutathione, and provides new agricultural antibiotic resources and strategies for preventing and controlling a variety of plant diseases.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to a mutant strain of Streptomyces parvum and application thereof in producing glutathione. Background Art

[0002] Gougerotin is a cytosine nucleoside peptide antibiotic that has a strong inhibitory effect on both Gram-positive and Gram-negative bacteria, and also has biological activities such as anti-tumor, anti-pathogenic fungi, and anti-virus. The mechanism of action of gougerotin is to inhibit the function of peptidyl transferase in ribosomes to hinder the extension of polypeptide chains, thereby inhibiting protein synthesis. Studies have shown that the main active components of many agricultural antibiotics are gougerotin or its analogs. Ningnanmycin is mainly used to control rice stripe leaf blight, tomato virus disease, etc. Its chemical structure is a stereoisomer of glutathione, with only the serine configuration being different, glutathione being D-type and Ningnanmycin being L-type. Ningnanmycin is an intermediate in the biosynthesis of glutathione or produced by the racemization of glutathione; Blasticidin S successfully replaced mercury preparations to control rice blast disease, and its chemical structure has a similar nucleoside skeleton to glutathione, both of which are cytosine glucuronic acid (CGA) derivatives; Wuyimycin is used to control cucumber powdery mildew, tomato gray mold, tomato leaf mold, etc., and its water-soluble active ingredient contains glutathione. It can be seen that glutathione has shown important value in the field of agricultural plant protection, has good control effects on a variety of fungal diseases, can be developed as an agricultural antibiotic, and has huge biocontrol potential and market application prospects.

[0003] In order to maintain the nitrogen source needed for survival, bacteria can use many nitrogen-containing compounds in nature, such as simple inorganic compounds such as N2 and nitrate; complex macromolecular organic substances such as histidine and arginine. Different nitrogen sources are assimilated to form two amino acids, glutamine (Glutamin, Gln) and glutamate (Glutamic acid, Glu), which are important nitrogen sources in cells. Glutamine is a key metabolite in the nitrogen assimilation process. It is not only one of the constituent amino acids for protein synthesis, but also a nitrogen source donor for nearly 25% of nitrogen-containing compounds. Glutamine is formed by glutamine synthetase (GS) catalyzing glutamate, adenosine triphosphate (ATP) and ammonia. As a structural gene of the nitrogen metabolism pathway, glnA can regulate the biosynthesis of glutamine synthetase. Glutamine synthetase is an ancient and widely present enzyme in organisms. Its gene family is one of the oldest existing families. It can participate in nitrogen metabolism and carbon metabolism of organisms. It plays an important role in life and is widely used in production and life. Therefore, it is of great significance to clarify the role of GlnA in the bacterium Streptomyces albulus CK-15. Summary of the invention

[0004] The purpose of the present invention is to provide a mutant strain of Streptomyces parvum and its application in producing glutathione to solve the problems existing in the above-mentioned prior art. By overexpressing glnA in Streptomyces parvum to construct a mutant strain of Streptomyces parvum, the glutathione content in the fermentation product can be significantly increased.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a strain of Streptomyces albulus, the deposit number of which is CGMCC No. 31251, the deposit time is July 10, 2024, the deposit unit is the General Microbiology Center of China Culture Collection Administration, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a mutant strain of Streptomyces parvum, in which the glnA gene is overexpressed, and the nucleotide sequence of the glnA gene is shown in SEQ ID NO:1.

[0008] The present invention also provides a method for constructing a mutant strain of Streptomyces parvum, comprising the following steps:

[0009] Cloning the glnA gene, and constructing a recombinant plasmid with the glnA gene and an expression vector; wherein the nucleotide sequence of the glnA gene is shown in SEQ ID NO: 1;

[0010] The recombinant plasmid is transferred to competent Escherichia coli, and then transferred into the Streptomyces parvum by conjugation transfer method to overexpress the glnA gene to obtain a mutant strain of Streptomyces parvum.

[0011] The present invention also provides an application of the Streptomyces parvum or the mutant strain of the Streptomyces parvum in producing glutathione.

[0012] The invention also provides an application of the mutant strain of Streptomyces parvum in improving the content of glutathione in the fermentation product of Streptomyces parvum.

[0013] The invention also provides an application of the mutant strain of Streptomyces parvum in improving the glucose content in the fermentation product of Streptomyces parvum.

[0014] The present invention also provides an application of the mutant strain of Streptomyces parvum in improving the utilization efficiency of ammonia nitrogen in the fermentation process of Streptomyces parvum.

[0015] The present invention also provides an application of the Streptomyces parvum or the mutant of the Streptomyces parvum in inhibiting pathogens, characterized in that the pathogens include tomato gray mold (Botrytis cinerea), wheat fusarium rust (Gibberella sanbinetti), apple rot (Valsa mail) and rice blast (Magnaportheoryzae).

[0016] The present invention also provides a method for producing glutathione, which comprises fermenting the Streptomyces parvum or the mutant strain of Streptomyces parvum and separating and extracting glutathione from the fermentation liquid.

[0017] Preferably, the culture medium for fermentation is: soybean cake powder 15.0-25.0 g / L, (NH4)2SO4 3.0-8.0 g / L, glucose 20.0-25.0 g / L, corn flour 25.0-35.0 g / L, CaCO3 1.0-5.0 g / L, and the fermentation conditions are 220 rpm, 28°C for 70-80 h.

[0018] The present invention discloses the following technical effects:

[0019] The invention constructs a mutant strain of Streptomyces parvum by overexpressing the glnA gene in Streptomyces parvum CK-15. Compared with the wild-type Streptomyces parvum CK-15, the mutant strain of Streptomyces parvum can significantly accelerate the spore production rate of the strain, increase the amount of hyphae produced, and promote the growth of the strain; it can also promote the increase of the content of glutathione and the content of glucose in the fermentation product, and accelerate the utilization efficiency of the strain to the ammonia nitrogen source during the fermentation process. In addition, compared with the wild-type Streptomyces parvum CK-15, the mutant strain of Streptomyces parvum has improved antibacterial effects on tomato gray mold, wheat fusarium, apple rot pathogen (Valsa mail) and rice blast pathogen. The invention transfers the glnA gene into Streptomyces parvum through genetic engineering technology to construct a mutant strain, which can be used to produce glutathione, provides new strain resources and methods for large-scale production of bacterial-derived glutathione, and provides new agricultural antibiotic resources and strategies for preventing and controlling various plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The electrophoresis results of PCR amplification of glnA gene fragment; M is the standard DNA molecule, glnA is the amplified product;

[0022] Figure 2 The pSETC plasmid electrophoresis results are shown in Figure 1. M is the standard DNA molecule, 1 is the undigested plasmid, and 2 is the digested plasmid.

[0023] Figure 3 This is the growth of single spores after the overexpression recombinant plasmid pSETC-glnA was transferred by conjugation with CK-15;

[0024] Figure 4 The results of PCR amplification and electrophoresis of the amp fragment of the glnA overexpression conjugate; M is the standard DNA molecule, 1 is the glnA overexpression conjugate, and 2 is CK-15;

[0025] Figure 5 The growth of the oglnA strain at different time points in the plate; the left side of the plate is the CK-15 wild-type strain, and the right side is the oglnA overexpression mutant strain;

[0026] Figure 6 The curves of mycelial dry weight changes of CK-15 and oglnA at different time periods;

[0027] Figure 7 Results of determination of glutathione content in fermentation products of CK-15 and oglnA overexpression mutant strain; A: glutathione standard product, B: CK-15, C: oglnA overexpression mutant strain;

[0028] Figure 8 Results of determination of toyocamycin content in fermentation products of CK-15 and oglnA overexpression mutant strain; A: toyocamycin standard, B: CK-15, C: oglnA overexpression mutant strain;

[0029] Fig. 9 The antibacterial effects of the fermentation products of CK-15 and oglnA overexpression mutant on four pathogens;

[0030] Fig.10 The results of glucose content determination in fermentation products of CK-15 and oglnA overexpression mutant at different time periods;

[0031] Fig.11 These are the results of determining ammonia nitrogen content in the fermentation products of CK-15 and oglnA overexpression mutants at different time periods. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1 Isolation and Identification of Streptomyces parvum CK-15

[0038] Streptomyces albulus CK-15 was isolated and purified from soil samples collected from Wuyi Mountain area in Fujian Province. Based on routine morphological observations, physiological and biochemical tests, and whole genome sequencing and comparison, it was identified as Streptomyces albulus and named Streptomyces albulus CK-15.

[0039] The Streptomyces albulus CK-15 isolated above was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 10, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31251.

[0040] Example 2 Construction of overexpression mutants

[0041] 1. Construction of recombinant plasmid pSETC-glnA of overexpression mutant

[0042] The glnA gene fragment was cloned according to the designed primers, and the plasmid pSETC was double-digested with BamH I endonuclease and Spe I endonuclease. The glnA fragment amplification product and the double-digested pSETC plasmid were assembled together by Gibson Assembly one-step isothermal method. After the fragment sequencing was correct, the overexpression recombinant plasmid was obtained and named pSETC-glnA.

[0043] The recombinant plasmid pSETC-glnA was transformed into DH5α by heat shock method, and the single colony was verified as positive by colony PCR and sent to the company for sequencing. The sequencing results fed back by the company were compared with the target gene sequence, and the comparison was consistent. The recombinant plasmid for overexpression of glnA gene was successfully constructed.

[0044] Primers used for cloning:

[0045] glnA-F: 5'-TCAATACTCGATTAGGGATCCATGTTCCAGAACGCCGACG-3', (SEQ ID NO: 2);

[0046] glnA-R: 5'-CACGTCTTAAGACGTACTAGTCTAGAGATCGAAGTAAAGCTCGAACTC-3', (SEQ ID NO: 3).

[0047] The amplification reaction system was: S.albulus CK-15DNA 1 μL, upstream primer (final concentration 10 μM) 2 μL, downstream primer (final concentration 10 μM) 2 μL, 2×PhantaFlash Master Mix 25 μL, and ddH2O was added to a total volume of 50 μL.

[0048] The amplification reaction program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 61.5°C for 5 s, extension at 72°C for 7 s, 35 cycles; final extension at 72°C for 1 min, and storage at 4°C.

[0049] The results of electrophoresis detection of amplified products are as follows Figure 1 The results show that a 1410 bp glnA gene fragment (SEQ ID NO: 1) can be amplified, which is consistent with the expected result. Figure 1 , the results showed that the enzyme digestion was successful.

[0050] The sequence of the glnA gene is as follows:

[0051]

[0052] 2. Overexpression recombinant plasmid combined with CK-15 strain transfer

[0053] The overexpression recombinant plasmid pSETC-glnA was introduced into ET12567 (pUZ8002) competent cells to obtain ETZ bacterial solution. The overexpression recombinant plasmid introduced into ET12567 (pUZ8002) competent cells was transferred into the CK-15 strain by conjugation transfer. The specific steps of conjugation transfer are as follows:

[0054] (1) The correctly sequenced glnA gene overexpressing ETZ bacterial solution was inoculated into the resistant LB liquid medium and cultured at 37°C, 220 rpm until OD 600 Approximately between 0.4-0.6 (close to 0.6).

[0055] (2) Wash fresh spores of CK-15 strain with 2×YT, filter with sterile cotton wool, take 500 μL of the solution and dispense it into 1.5 mL EP tubes. Heat shock at 50°C for 10 min, and pre-germinate at 37°C, 220 rpm for 3 h before use.

[0056] (3) Collect 4-5 mL of ETZ cells in a 1.5 mL EP tube, centrifuge and discard the supernatant in a clean bench. Wash the cells three times with antibiotic-free LB liquid medium and resuspend them in 500 μL antibiotic-free liquid LB for later use.

[0057] (4) After mixing 500 μL of spore suspension with 500 μL of ETZ bacterial solution, centrifuge at 4000 rpm for 5 min, remove 600 μL of supernatant, resuspend the remaining precipitate, take 200 μL and spread it on an MS plate containing MgCl2. After drying, invert and culture at 28°C.

[0058] (5) After 17 hours, observe the plates and apply antibiotics.

[0059] (6) The conjugates will grow after culturing at 28°C for 2-3 days.

[0060] The results are as follows Figure 3 As shown, the results showed that the single colony could still grow normally after being covered with antibiotics, thus indicating that the recombinant plasmid was successfully introduced.

[0061] 3. Screening and verification of overexpression mutants

[0062] pSETC is a single-copy integrative plasmid and contains an apra resistance gene, so strains containing the pSETC-glnA recombinant plasmid can grow on MS plates containing apramycin at a final concentration of 50 μg / mL. The strains that can grow on MS plates containing apramycin resistance were purified and cultured, and single colonies were picked for liquid culture, and the genome was extracted for PCR verification. Apra-F / R primers were designed: upstream primer 5'-GAGTGCAATGTCGTGCAATACGA-3', downstream primer 5'-GCATTCTTCGCATCCCGCCT-3'. If the Apra gene fragment is amplified in the screening strain, it proves that the strain is a glnA gene overexpression mutant, named oglnA.

[0063] The glnA overexpression conjugate DNA was extracted and PCR amplified using primers Apra-F / R and then subjected to agarose gel electrophoresis. Figure 4 As shown, the glnA gene overexpression conjugate can amplify the Amp resistance gene fragment (777 bp), while the CK-15 wild-type strain cannot amplify a fragment of the corresponding length. Therefore, the glnA gene overexpression strain oglnA was successfully constructed.

[0064] Example 3 Analysis of culture characteristics of overexpression mutants

[0065] The CK-15 wild-type strain and the overexpression mutant oglnA were streaked into MS medium, cultured at 28°C, and observed every 24 hours for a total of 168 hours.

[0066] The results are as follows Figure 5 As shown, from 48h onwards, the oglnA mutant strain had a tendency to produce spores, while the CK-15 wild-type strain had almost no gray spores on its surface. Therefore, overexpression of the glnA gene can accelerate the spore production rate of the strain.

[0067] Example 4 Overexpression mutant cell growth analysis

[0068] The CK-15 wild strain and the overexpression mutant oglnA were inoculated into 100 mL seed culture medium (glucose 20.0 g / L, yeast powder 6.0 g / L, NaCl 10.0 g / L), and the shaker parameters were set to 28 ° C, 220 rpm. The OD value of the seed solution was measured after 1 day of culture. 2 mL of CK-15 seed solution was added to 100 mL of M3G medium, and the overexpression strain seed solution was added in the corresponding proportion. Each treatment was repeated 3 times. Samples were taken every 6 hours, for a total of 16 times (90 hours in total). The filter paper dried to a constant weight was first used for filtration, and then the filter paper containing the mycelium was placed in an oven at 54 ° C for drying to make its weight constant. The difference in weight was the dry weight of the mycelium.

[0069] The results are as follows Figure 6 As shown in the figure, the mycelial dry weight of the oglnA mutant strain was not significantly different from that of the CK-15 wild-type strain between 6 and 24 hours, but from 24 hours onwards, the mycelial dry weight of the oglnA mutant strain was almost always higher than that of the CK-15 wild-type strain, and the maximum mycelial dry weight of the oglnA strain reached 0.84 g. The above results show that overexpression of the glnA gene can increase the amount of mycelial production and promote the growth of the strain.

[0070] Example 5 Analysis of the level of glutathione or toyocamycin produced by overexpression mutants

[0071] 1. Determination of glutathione content in fermentation products of overexpression mutants

[0072] The wild strain of CK-15 and the bacterial blocks of the overexpressed mutant strain oglnA were inoculated into 100 mL of the above seed culture medium, respectively, and cultured at 220 rpm and 28°C for 24 h. The OD value was measured after the seed solution became turbid. The control group (wild strain) was inoculated into 100 mL of glutathione fermentation medium (soybean cake powder 20.0 g / L, (NH4)2SO4 4.0 g / L, glucose 20.0 g / L, corn flour 30.0 g / L, CaCO3 3.0 g / L) at an inoculum of 1%, and the experimental group (mutant strain) was inoculated into the fermentation medium at the same proportion, and cultured at 220 rpm and 28°C for 72 h. The fermentation product was treated with a 0.22 μm bacterial filter, and the content of glutathione in the fermentation product of the strain was detected by HPLC under the following conditions.

[0073] The test conditions are shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] The results of the determination of glutathione content in the fermentation products of CK-15 and oglnA overexpression mutants are as follows Figure 7 As shown in the figure, the peak time of glutathione standard is about 8 minutes, and the detection results of CK-15 wild strain and oglnA overexpression mutant strain both have obvious peaks at about 8 minutes. The content of glutathione in the fermentation product of CK-15 is 930.96 mg / L, and the content of glutathione in the oglnA overexpression mutant strain is 1177.61 mg / L. Compared with the wild strain CK-15, the content of glutathione in the fermentation product of the oglnA overexpression mutant strain increased by about 26.49%.

[0078] 2. Determination of Toyocamycin Content in Fermentation Products of Overexpressed Mutant Strain

[0079] The wild strain of CK-15 and the bacterial blocks of the overexpressed mutant strain oglnA were inoculated into 100 mL of the above seed culture medium, respectively, and cultured at 220 rpm and 28°C for 24 h. The OD value was measured after the seed solution became turbid. The control group (wild strain) was inoculated into 100 mL of Fengjiamycin fermentation medium (glucose 5.0 g / L, yeast extract 5.0 g / L, beef extract 5.0 g / L, corn flour 4.0 g / L, soybean cake powder 10.0 g / L, starch 20.0 g / L, calcium carbonate 4.0 g / L) at an inoculum of 1%, and the experimental group (mutant strain) was inoculated into the fermentation medium in proportion, and cultured at 220 rpm and 28°C for 72 h. The fermentation product was treated with a 0.22 μm bacterial filter, and the content of Fengjiamycin in the fermentation product of the strain was detected by HPLC under the following conditions.

[0080] The test conditions are shown in Table 2.

[0081] Table 2

[0082] Mobile phase Milli-Q grade water Stationary Phase HPLC grade methanol solution Flow rate 1mL / min Detection wavelength 279nm Injection volume 10μL Column temperature 30℃

[0083] The results are as follows Figure 8 As shown in the figure, the peak time of the standard product of Toyocamycin is about 3.5 minutes, and the detection results of the wild-type strain of CK-15 and the oglnA overexpression mutant strain both have obvious peaks at about 3.5 minutes. The peak area of ​​the fermentation product of oglnA mutant strain Toyocamycin is 6930784mV, and the concentration is 339.03mg / L, which is 4.96% lower than that of the fermentation product of the wild-type strain of CK-15.

[0084] Example 6 Analysis of antibacterial activity of fermentation products of overexpressed mutants

[0085] Botrytis cinerea, Gibberella sanbinetti, Valsa mail, and Magnaporthe oryzae were selected as target pathogens, and the antibacterial activity of the fermentation products of the wild strain and the overexpression mutant oglnA of CK-15 was determined. The fermentation products of each strain were added to the PDA medium cooled to 37°C and mixed to make the medium contain 1.2% of the fermentation product. After the medium solidified, a sterile punch with a diameter of 0.6 cm was used to take the above pathogen cakes with the same growth conditions, and the side containing the hyphae was placed in the center of the PDA plate with the side facing down. After culturing at 25°C for 3-5 days, the colony growth was observed. PDA without fermentation products was used as the control, and each treatment was repeated 5 times. When the colonies of the control group filled the culture dish, the colony diameter was measured using the cross method, and the antibacterial rate of each treatment group was calculated.

[0086] Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%

[0087] like Fig. 9 As shown in Table 3, the fermentation products of the oglnA mutant had control effects of 39.20%, 80.45%, 39.04% and 47.76% on Botrytis cinerea, 80.45% on apple rot, 39.04% on rice blast and 47.76% on wheat fusarium sphaeroides, respectively, which were significantly higher than those of the fermentation products of the wild strain CK-15.

[0088] Table 3 Antibacterial effects of fermentation products of CK-15 and oglnA strains on four pathogenic fungi

[0089]

[0090] Note: Different letters indicate statistically significant differences.

[0091] Example 7 Determination of glucose content in fermentation products of overexpression mutants

[0092] Take 1 mL of CK-15 wild strain seed solution and oglnA overexpression mutant seed solution and inoculate them into the glutathione fermentation medium in equal volume ratio as the wild strain, culture at 28°C, 220rpm for 5 days, sample every 12 hours, a total of 10 samples, and set 3 replicates for each treatment. The glucose content in the fermentation products at different time periods was detected by the glucose content detection kit (Beijing Box Biotechnology). The detection method is as follows:

[0093] 1. Dilute the standard and draw a standard curve.

[0094] Table 4 Preparation of glucose standard curve solution

[0095]

[0096]

[0097] 2. Preparation of the detection working solution: Mix reagent 1 and reagent 2 in the kit in a volume ratio of 1:1. The principle of preparing the working solution before use should be followed.

[0098] 3. Take 20 μL of the fermentation product of each strain at different time periods and add it to a 96-well ELISA plate, then add 180 μL of the detection working solution. For the blank group, replace the fermentation product with distilled water. After thorough mixing, color development was carried out at 37°C for 15 minutes.

[0099] 4. Measure the absorbance at 505 nm using an enzyme-labeled instrument.

[0100] 5. Calculate the x value of the sample based on the standard curve and the measured absorbance value.

[0101] 6. Substitute x into the formula: glucose content (μg / mL) = 100 × x × dilution factor to obtain the glucose content of each sample.

[0102] The results are as follows Fig.10 As shown in the figure, since there is 2g / 100mL glucose in the fermentation medium of Wuyi mycin, the glucose content of the fermentation products of each strain is the maximum at 12h. During the fermentation process, the strain will use glucose for metabolism to produce secondary metabolites. Fig.10 It can be seen that as time goes by, the glucose content in the fermentation product of the wild strain CK-15 shows a trend of first decreasing, then increasing, and then decreasing, and the glucose content drops sharply at 60-72h. There is no significant difference in the change of glucose content between oglnA and CK-15 wild strains, but at 96-120h, the glucose content in the fermentation product of the wild strain CK-15 shows a trend of first increasing and then decreasing, while the glucose content in the fermentation product of the oglnA strain shows an increasing trend.

[0103] Example 8 Determination of ammonia nitrogen content in overexpression mutants

[0104] The sodium nitroprusside method was used to determine the ammonia nitrogen content in the fermentation products of the CK-15 wild strain and the overexpression mutant oglnA at different time periods:

[0105] (1) Preparation of the detection working solution: Dissolve 35 g of phenol and 0.2 g of sodium nitrosoferricyanide in distilled water, and dilute to 1 L. Mix well to obtain solution A. Dissolve 18 g of NaOH in distilled water, add 1.8 mL of 0.04 M sodium hypochlorite solution, and dilute to 1 L with distilled water. Mix well to obtain solution B. Solution B is easy to deteriorate and decompose, so it must be prepared before use.

[0106] (2) Take 100 μL of each sample and dilute to 4 mL with distilled water.

[0107] (3) Take 1 mL of each diluted sample into a glass test tube, add 5 mL of solution A and 5 mL of solution B in sequence, mix well and place in a water bath at 37°C for 35 min. Set up 3 replicates for each treatment. Replace the diluted fermentation product with distilled water in the control group.

[0108] (4) Preheat the spectrophotometer for 30 minutes and adjust the wavelength to 625 nm. Use the control group to zero and measure the absorbance of each sample.

[0109] (5) Substitute the measured absorbance value into the formula to determine the ammonia nitrogen value contained in each sample.

[0110] Ammonia nitrogen value = (0.2004 × absorbance value - 0.0018) × sample dilution factor × 0.212

[0111] The sodium nitroprusside method was used to determine the ammonia nitrogen content in the fermentation products of each strain at different time periods. The results are as follows: Fig.11 As shown in the figure, with the increase of fermentation time, the ammonia nitrogen content in the fermentation products of each strain showed an overall downward trend. During the entire fermentation process, the ammonia nitrogen content in the fermentation products of the CK-15 wild strain showed a steady downward trend. The ammonia nitrogen content of the fermentation products of the oglnA mutant strain was not significantly different from that of CK-15 at 12-60h, and the ammonia nitrogen content in the fermentation products was almost lower than that of the CK-15 wild strain from 60h. Therefore, overexpression of the glnA gene can accelerate the utilization efficiency of the ammonia nitrogen source of the strain during the fermentation process.

[0112] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Streptomyces parvum ( Streptomyces albulus ), characterized in that, The deposit number of this strain is CGMCC No.31251.

2. A mutant strain of Streptomyces parvum, characterized in that: Overexpression in the Streptomyces parvus of claim 1 gL Gene, gL The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. A method for constructing a mutant strain of Streptomyces parvum, characterized in that: The following steps are involved: clone gL gene, and the gL Gene and expression vector to construct recombinant plasmid; wherein, gL The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The recombinant plasmid is transferred to the competent Escherichia coli, and then transferred into the Streptomyces parvum of claim 1 by conjugation transfer method to overexpress the gL gene, and obtained mutant strains of Streptomyces parvus.

4. Use of the Streptomyces parvum according to claim 1 or the mutant strain of Streptomyces parvum according to claim 2 in producing glutathione.

5. Use of the mutant strain of Streptomyces parvum as claimed in claim 2 in increasing the content of glutathione in a fermentation product of Streptomyces parvum.

6. Use of the mutant strain of Streptomyces parvum as claimed in claim 2 in improving the utilization efficiency of ammonia nitrogen in the fermentation process of Streptomyces parvum.

7. Use of the Streptomyces parvum according to claim 1 or the mutant strain of Streptomyces parvum according to claim 2 in inhibiting pathogenic bacteria, characterized in that: The pathogen is tomato gray mold ( Botrytis cinerea )、Triticum fusarium ( Gibberella sanbinetti )、Apple rot bacteria( Valsa mail ) and rice blast fungus ( Magnaporthe oryzae ).

8. A method for producing glutathione, characterized in that: The Streptomyces parvum described in claim 1 or the mutant strain of Streptomyces parvum described in claim 2 is fermented, and glutathione is separated and extracted from the fermentation liquid.

9. The method according to claim 8, characterized in that The fermentation medium is: soybean cake powder 15.0-25.0 g / L, (NH4)2SO4 3.0-8.0 g / L, glucose 20.0-25.0 g / L, corn flour 25.0-35.0 g / L, CaCO3 1.0-5.0 g / L, and the fermentation conditions are 220 rpm, 28°C and culturing for 70-80 h.

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