JP2022535409A - Method for improving ability of Lactobacillus brevis to synthesize γ-aminobutyric acid and use thereof - Google Patents

Abstract

To provide a method for achieving knockout of the glnR gene by a method of homologous recombination, obtaining a glnR knockout strain of Lactobacillus brevis, and improving the ability of Lactobacillus brevis to synthesize γ-aminobutyric acid and the ability to withstand an acidic environment, and the use thereof do. Lactobacillus brevis glnR knockout strain was fermented by acid control and material fed-batch supplementation method, the cumulative concentration of GABA was 301.5 g/L, the production efficiency reached the highest 5.76 g/L/h, and the production efficiency of GABA was It can be used for large-scale production, reduces the production cost of GABA, and is used for fermentation of acidic foods, etc. [Selection figure] None

Description

The present invention relates to a method for improving the ability of Lactobacillus brevis to synthesize γ-aminobutyric acid and its use, and belongs to the field of microorganisms.

γ-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter that has various physiological functions in mammals, such as stabilizing the mind, lowering blood pressure, and improving sleep quality. As an active substance, it can be widely applied in fields such as food, medicine, feed, and chemical industry. Food grade GABA is often biosynthesized by microorganisms. Microorganisms mainly exert the action of the glutamate decarboxylase (GAD) system (including reversed glutamate/GABA vectors and glutamate decarboxylase), and extracellular glutamate is transferred into cells by reversed glutamate/GABA vectors. Transporting and consuming H ^{2 +} results in catalytic decarboxylation of the glutamate substrate by intracellular glutamate decarboxylase to synthesize GABA. Synthesized GABA also exports GABA from cells through glutamate/GABA antiport, achieves accumulation of GABA, maintains intracellular pH stability, and helps strains resist external environmental stress. .

Among the reported GABA-synthesizing strains, lactic acid bacteria, especially Lactobacillus brevis, have a clear preponderance. In previous research, our laboratory screened a strain of Lactobacillus brevis D17 that efficiently synthesizes GABA from the white sake brewing system. The production efficiency (3.76 g/L/h) of the strain can be improved by controlling the fermentation pH.

Conventional methods to improve GABA yield are mainly performed by screening for high-yielding GABA strains, optimizing GABA-producing fermentation conditions and media, and overexpressing the glutamate decarboxylase system. However, in order to increase its industrial use and continue to improve its production efficiency, there is a need to directionally adjust its metabolic pathways relative to conventional GABA high- or low-producing strains.

In order to solve the above problems, the present invention provides a method for improving the ability of Lactobacillus brevis strains to synthesize γ-aminobutyric acid.

In the course of the present study, the inventors discovered that the type strain Lactobacillus brevis ATCC 367 was associated with coding genes for the glutamate decarboxylation system of Lactobacillus brevis D17 (glutamate/GABA antiport, glutamate decarboxylase (GAD)). gene), but the GABA yield of Lactobacillus brevis ATCC 367 (10 g/L) is much higher than that of Lactobacillus brevis D17 (17.8 g/L) when the strain is fermented with 5% sodium glutamate. and the GABA yields of Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17 did not increase with the addition of monosodium glutamate, both limiting GABA yields.

Glutamate (sodium) is not only the sole substrate for GABA synthesis, but also one of the microbial nitrogen sources. Its metabolism is controlled by several regulatory factors such as TnrA, GlnR and CodY. GlnR (glutamine synthase repressor) is an important global nitrogen source regulator widely present in Gram-positive bacteria. It has a regulatory effect on certain genes of nitrogen source metabolism, especially in Streptomyces, Bacillus. In Bacillus, GlnR represses transcription of the glnRA operon and reduces nitrogen source utilization when the nitrogen source is sufficient. In the genus Streptomyces, GlnR not only regulates nitrogen metabolism, but also controls carbon source metabolism, phosphorus metabolism, antibiotic synthesis, strain resistance (osmotic pressure, acid resistance, oxidation), and the like. The GAD system, which regulates the synthesis of GABA, is an acid-tolerant system that allows microorganisms to resist acid stress and may be negatively regulated by GlnR when the glutamate nitrogen source is sufficient. In addition, at present, no glnR gene knockout strain of lactic acid bacteria has been reported.

The present invention knocks out the glnR gene of Lactobacillus brevis and eliminates the restriction of GABA synthesis of Lactobacillus brevis, thereby improving the yield of GABA, shortening the time for GABA synthesis, and industrial production of GABA. improve sexuality.

A first object of the present invention is to provide a method for improving the ability of a Lactobacillus brevis strain to synthesize γ-aminobutyric acid. ) is knocked out.

In one embodiment, the amino acid sequence expressed by the glnR gene is as shown in SEQ ID NO:1.

In one embodiment, the nucleotide sequence encoding said glnR gene is as shown in SEQ ID NO:2.

In one embodiment, the Lactobacillus brevis strain is any Lactobacillus brevis that is capable of synthesizing GABA.

In one embodiment, the Lactobacillus brevis strain is Lactobacillus brevis D17 (accession number CGMCC NO. 14385) or the type strain Lactobacillus brevis ATCC 367, Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306, Lactobacillus brevis NPS -QW-145, etc. In one embodiment, the knockout is performed using any one known knockout method, such as homologous recombination, two types of intron (Targetron or Clostron) gene insertion inactivation, CRISPR-Cas gene editing, etc. That is.

In one embodiment, said knockout is a markerless knockout, in particular a markerless gene knockout based on homologous recombination.

In one embodiment, specifically, the knockout is carried out by knocking out 1000 bp of homologous regions upstream and downstream of the glnR gene to be knocked out (including 21 bp each at the front and rear ends of the glnR gene) along a certain direction. Ligation with the lactic acid bacteria integrative plasmid pGID023 to obtain the target gene knockout plasmid pGID023-ΔglnR, electrotransformation of the knockout plasmid into Lactobacillus brevis, screening on erythromycin-resistant plates to recombination containing the knockout plasmid Obtain a strain. After that, the correct recombinant strain is selected, subcultured in an erythromycin-containing GYP liquid medium, further subcultured in a GYP liquid medium that does not have erythromycin resistance, and the bacterial solution after subculture is erythromycin-containing. Plated on resistance plates and screened to generate a single crossover that has undergone a first round of homologous recombination to integrate the plasmid into the recipient strain chromosome, the single crossover was then treated with liquid GYP in the absence of erythromycin. Subculture in a medium, spread the subcultured bacterial solution on an antibiotic-free GYP plate, and cause the second homologous recombination. Strains that have achieved target gene knockout without erythromycin resistance are screened. Select monoclonals up to

The pGID023 plasmid has a replication origin capable of replicating in E. coli, a multicloning site MCS, and an erythromycin resistance gene.

The knockout plasmid is obtained by digesting homologous sequence fragments of about 1000 bp in size upstream and downstream of the target gene to be knocked out in the Lactobacillus brevis chromosome with an enzyme along the direction of the gene sequence, respectively, into the multicloning site of pGID023. It is concatenated.

The knockout plasmid is electrotransformed into the Lactobacillus brevis strain to obtain a strain containing the knockout plasmid. Conditions are adopted and the plasmid is electrotransformed into Lactobacillus brevis. Immediately add to pre-chilled MRS medium after electroshock (electroporation) and recover in 37° C. incubator. The supernatant is removed by centrifugation, and the cells are resuspended, plated on MRS plates containing erythromycin, and incubated at 37° C. to produce transformants. Transformants are selected for bacterial solution PCR verification.

As a method for verifying the single crossover in which the plasmid was integrated into the chromosome of the recipient strain by generating the first homologous recombination, the transformant of Lactobacillus brevis containing the knockout plasmid was seeded on MRS medium containing erythromycin, and heated at 37 ° C. 24 hours of static culture. The cultured bacterial solution is seeded in the erythromycin-containing MRS liquid medium, cultured at 37° C. for 24 hours, and subcultured according to this step. After the passaged bacterial solution is diluted 10 ⁻³ to 10 ⁻⁶ times, it is plated on an erythromycin-containing MRS plate and cultured at 37° C. until a single colony grows. A single colony was picked and inoculated on erythromycin-containing MRS medium, cultured at 37° C. for 24 h, subjected to bacterial liquid PCR verification, and screened to generate the first round of homologous recombination to integrate the plasmid onto the chromosome of the recipient strain. A potential single crossover is obtained, and total genomic DNA is extracted from the potential single crossover and verified by PCR and sequencing to ensure that a mutant with the correct glnR gene knockout is obtained.

As the above method for screening strains that generate the second homologous recombination to achieve target gene knockout, the single crossover is inoculated in liquid MRS medium without erythromycin and cultured at 37° C. for 24 h. . After subculturing on antibiotic-free MRS medium, the bacterial solution is diluted 10 ⁻³ to 10 ⁻⁶ times, plated on an erythromycin-free MRS plate, and cultured at 37° C. until a single colony grows. . A single colony is selected, inoculated on MRS liquid medium without erythromycin resistance, cultured at 37° C. for 24 h, and subjected to bacterial liquid PCR verification, thereby obtaining a potential mutant strain in which the glnR gene is knocked out. In addition, total genomic DNA is extracted from potential knockout strains and verified by PCR and sequencing to ensure that mutant strains with the correct glnR gene knockout are obtained. If no glnR gene knockout strain is obtained, continue to pass and screen and repeat the above steps until a glnR gene knockout strain is obtained.

A second object of the present invention is to provide Lactobacillus brevis that improves the ability to synthesize γ-aminobutyric acid, and the Lactobacillus brevis is obtained by knocking out the glnR gene from the starting strain.

In one embodiment, the amino acid sequence expressed by the glnR gene is as shown in SEQ ID NO:1.

In one embodiment, the nucleotide sequence encoding said glnR gene is as shown in SEQ ID NO:2.

In one embodiment, the starting strain is any Lactobacillus brevis capable of synthesizing GABA, optionally Lactobacillus brevis D17 (accession number CGMCC NO. 14385), the type strain Lactobacillus Brevis ATCC 367, Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306 or Lactobacillus brevis NPS-QW-145.

In one embodiment, the knockout is performed using any one known knockout method, such as homologous recombination, two types of intron (Targetron or Clostron) gene insertion inactivation, CRISPR-Cas gene editing, etc. That is.

A third object of the present invention is to provide a method for synthesizing γ-aminobutyric acid, wherein the method is fermentatively produced using glnR gene-knocked out Lactobacillus brevis.

In one embodiment, the fermentative production is carried out by inoculating Lactobacillus brevis in a medium containing glutamic acid or sodium glutamate.

In one embodiment, the fermentation production may be carried out using any of the following methods.
(1) A Lactobacillus brevis glnR knockout strain is seeded in a glutamic acid (sodium)-containing liquid fermentation medium and cultured for fermentation.
(2) In the fermentation process, the pH is adjusted and the materials are replenished in batches for fermentation (fed-batch fermentation is performed). The Lactobacillus brevis glnR knockout strain is inoculated into GYP fermentation medium containing glutamate (sodium), adjusted to pH 4.8-5.5, fermented under agitation and no aeration conditions, fermented for 6-24 h to ferment glutamate. Replenish (sodium) substrate. Preferably, glutamic acid (sodium) substrate is added every 6 h.
(3) Fed-batch fermentation is performed by adjusting the pH and feeding glucose in the fermentation process. The Lactobacillus brevis glnR knockout strain is inoculated into GYP fermentation medium containing glutamate (sodium), adjusted to pH 4.8-5.5, fermented under agitation and no aeration conditions, fermented for 6-36 h to ferment glutamate. Replenish (sodium) substrate. Preferably, the glutamic acid (sodium) substrate is added every 6 h and the glucose carbon source is fed from 6 to 36 h.

In one embodiment, the fermentative production is carried out using any of the following methods.
(1) Lactobacillus brevis glnR knockout strains are each inoculated on 10 g/L glucose carbon source, added to 50 g/L glutamic acid (sodium) GYP liquid fermentation medium, and fermented under culture conditions of 37° C.×200 rpm.
(2) Fed-batch fermentation is performed by adjusting the pH in the fermentation process. Using a bioreactor, Lactobacillus brevis ATCC 367ΔglnR or D17ΔglnR mutants are inoculated into GYP fermentation medium containing glutamate (sodium). The pH of the fermentation liquid is adjusted to 5.0 using sulfuric acid having a concentration of 5 mol/L, and the mixture is stirred at 37° C.×200 rpm and fermented without aeration. Glutamate (sodium) substrate is supplemented from 6 to 24 h to avoid the suppression of high concentrations of glutamate (sodium) on cell growth. Preferably, glutamic acid (sodium) substrate is added every 6 h. After fermenting for 60 hours, the cumulative concentration of ATCC 367ΔglnR GABA is 233.9 g/L, after fermenting for 36 hours, the maximum production efficiency reaches 4.88 g/L/h, after fermenting for 48 hours, the cumulative concentration of GABA for D17ΔglnR is 148.0 g/L, fermented for 18 hours, the highest production efficiency reaches 5.73 g/L/h.
(3) Fed-batch fermentation is performed by adjusting the pH and feeding glucose in the fermentation process. Using a bioreactor, Lactobacillus brevis ATCC 367ΔglnR or D17ΔglnR mutants are inoculated into GYP fermentation medium containing glutamate (sodium). The pH of the fermentation liquid is adjusted to 5.0 using sulfuric acid having a concentration of 5 mol/L, and the mixture is stirred at 37° C.×200 rpm and fermented without aeration. Glutamate (sodium) substrate is supplemented from 6 to 36 h to avoid the suppression of high concentrations of glutamate (sodium) on cell growth. Preferably, in the fermentation process of ATCC 367ΔglnR, a glutamic acid (sodium) substrate is added every 6 h, and a glucose carbon source is fed from 12 to 36 h, fermented for 60 h, and the cumulative concentration of GABA of ATCC 367 ΔglnR is 282.80 g. /L, fermented for 36h, the production efficiency reaches the highest 4.93g/L/h, preferably, during the fermentation process of D17ΔglnR, glutamic acid (sodium) substrate is added every 6h, and from 6 to 30h Glucose carbon source was fed, fermented for 60h, the accumulated concentration of GABA in D17ΔglnR was 301.5g/L, and fermented for 36h, the production efficiency reached 5.76g/L/h at the highest.

A fourth object of the present invention is to provide a method for improving the acid tolerance of γ-aminobutyric acid-producing Lactobacillus brevis, said method comprising knocking out the Lactobacillus brevis glnR gene.

A fifth object of the present invention is to provide the use of the recombinant bacterium or the method in the fields of food, drug production, feed, chemical industry and the like.

In one embodiment, the food is an acidic fermented food, such as yellow wine or sourdough.

A sixth object of the present invention is the use of said recombinant bacterium or said method in the manufacture of a drug or skin care product having a calming, blood pressure reducing and sleep quality improving effect on mammals. provide use.

Biological material:
Lactobacillus brevis D17 is disclosed in patent application number 2017112754179, publication number CN108034599A, publication date May 15, 2018, and its taxonomic designation is Lactobacillus brevis, dated July 6, 2017. It was deposited with the General Microbiology Center of the China Microbial Species Conservation and Management Commission, and the accession number is CGMCC NO. 14385.

The type strain Lactobacillus brevis ATCC 367 was purchased from China Microbiology Collection Center with number CGMCC1.2028 and may be purchased from American Type Culture Collection with number ATCC 367.

Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306 and Lactobacillus brevis-QW-145 according to the present invention have been disclosed in the literature prior to the filing of this patent (Li, H.X., Gao, D.D., et al. al.A high γ-aminobutylic acid-producing Lactobacillus brevis isolated from Chinese traditional paocai [J].2008, Annals of Microbiology, 58(4):649-653. ．Ａ Ｔｗｏ－ｓｔａｇｅ ｐＨ ａｎｄ Ｔｅｍｐｅｒａｔｕｒｅ Ｃｏｎｔｒｏｌ ｗｉｔｈ Ｓｕｂｓｔｒａｔｅ Ｆｅｅｄｉｎｇ Ｓｔｒａｔｅｇｙ ｆｏｒ Ｐｒｏｄｕｃｔｉｏｎ ｏｆ Ｇａｍｍａ－ａｍｉｎｏｂｕｔｙｒｉｃ Ａｃｉｄ ｂｙ Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ ＣＧＭＣＣ １３０６ ［Ｊ］．Ｃｈｉｎｅｓｅ Ｊｏｕｒｎａｌ ｏｆ Ｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ、２１（１０）：１１９０－１１９４．Ｗｕ、Ｑ．、Ｌａｗ 、Ｙ．Ｓ．、ｅｔ ａｌ．Ｄａｉｒｙ Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｔｈｅｒｍｏｐｈｉｌｕｓ ｉｍｐｒｏｖｅｓ ｃｅｌｌ ｖｉａｂｉｌｉｔｙ ｏｆ Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ ＮＰＳ－ＱＷ－１４５ ａｎｄ ｉｔｓ ｇａｍｍａ－ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ ｂｉｏｓｙｎｔｈｅｓｉｓ ａｂｉｌｉｔｙ ｉｎ ｍｉｌｋ ［Ｊ］．Ｓｃｉｅｎｔｉｆｉｃ Ｒｅｐｏｒｔ、２０１５、５（１２８８５）：１－１２ .).

The integrative plasmid pGID023 has already been disclosed in 1994, as shown in FIG. 1 (Pascal Hols, Thierry Ferain, Dominique Garmyn, et al. in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression. Applied Environmental Microbiology 1994, 60: 1401-1413.).

Advantageous effects of the present invention are as follows.
The present invention is the first to discover that deletion of the glnR gene can significantly improve the GABA synthesis ability of Lactobacillus brevis, and the resulting Lactobacillus brevis glnR gene knockout strains ATCC 367ΔglnR and Lactobacillus brevis D17ΔglnR find that the method is versatile and suitable in principle for all Lactobacillus brevis. The present invention utilizes a glnR gene knockout strain to produce GABA, significantly improves the production efficiency or yield of Lactobacillus brevis GABA, shortens the fermentation cycle, and is capable of The viability can be significantly improved, and the strain is also applied to acid food fermentation, such as yellow wine brewing, sourdough making.

pGID023 plasmid used for gene knockout. Construction of the knockout plasmid pGID023-ΔglnR, the upper diagram is the amplification of the upstream and downstream homologous arms of the glnR gene, and the lower diagram is the knockout plasmid pGID023-ΔglnR. FIG. 4 is a flow chart of Lactobacillus brevis glnR gene knockout. FIG. 10 is a PCR validation of Lactobacillus brevis ATCC 367 and D17glnR gene knockouts. For the marks in FIG. 4A, M: 500 bp Marker, 6: Lactobacillus brevis D17 genome amplified using primer pair F-glnR and R-glnR, 9: amplified using primer pair F-glnR and R-glnR. Genomes of potential Lactobacillus brevis ATCC 367 ATCC 367 genomes, 1-5: The genomes of potential Lactobacillus brevis D17 glnR knockout strains were extracted and amplified using primer pairs F-glnR and R-glnR, 7-8: The genome of a potential Lactobacillus brevis ATCC 367 glnR knockout strain was extracted and amplified using the primer pair F-up-glnR-BamH and R-down-glnR-Hind3. For the marks in FIG. 4B, M: 5 kb Marker, 1: Lactobacillus brevis D17 genome amplified with primer pair F-up-glnR-BamH and R-down-glnR-Hind3, 7: primer pair F-up - Lactobacillus brevis ATCC 367 genome amplified with glnR-BamH and R-down-glnR-Hind3, 2-6: Extract the genome of a potential Lactobacillus brevis D17 glnR knockout strain and use primer pair F- Amplified with up-glnR-BamH and R-down-glnR-Hind3, 8-9: The genome of a potential Lactobacillus brevis ATCC 367 glnR knockout strain was extracted and the primer pair F-up-glnR- Amplified with BamH and R-down-glnR-Hind3. Validation by sequencing of glnR gene knockouts of Lactobacillus brevis ATCC 367 and D17. Effect of deletion of the glnR gene on Lactobacillus brevis GABA synthesis, for the marks in FIG. 6A, 367 represents the wild strain of Lactobacillus brevis ATCC 367 and ΔglnR represents the Lactobacillus brevis ATCC 367 ΔglnR mutant strain. 6B, 367 represents Lactobacillus brevis D17 wild strain and ΔglnR represents Lactobacillus brevis D17 ΔglnR mutant strain. Survival of Lactobacillus brevis glnR knockout strains in an extremely acidic environment, 367 represents the wild strain of Lactobacillus brevis ATCC 367 and ΔglnR represents the Lactobacillus brevis ATCC 367 ΔglnR mutant for the marks in FIG. 7A. 7B, 367 represents Lactobacillus brevis D17 wild strain and ΔglnR represents Lactobacillus brevis D17 ΔglnR mutant strain. Lactobacillus brevis ATCC 367ΔglnR mutant synthesized GABA by pH-controlled fed-batch fermentation. For the marks in Figure 8, 367 represents Lactobacillus brevis ATCC 367 wild strain and ΔglnR represents Lactobacillus brevis ATCC 367 ΔglnR mutant strain. Lactobacillus brevis ATCC 367ΔglnR mutant synthesized GABA by fed-batch fermentation with adjusted pH and fed-batch glucose. For the marks in Figure 9, 367 represents Lactobacillus brevis ATCC 367 wild strain and ΔglnR represents Lactobacillus brevis ATCC 367 ΔglnR mutant strain. The Lactobacillus brevis D17ΔglnR mutant synthesized GABA by pH-controlled fed-batch fermentation. For the marks in FIG. 10, D17 represents the Lactobacillus brevis D17 wild strain and ΔglnR represents the Lactobacillus brevis D17 ΔglnR mutant strain. The Lactobacillus brevis D17ΔglnR mutant synthesized GABA by fed-batch fermentation with adjusted pH and fed-batch glucose. For the marks in FIG. 11, D17 represents the Lactobacillus brevis D17 wild strain and ΔglnR represents the Lactobacillus brevis D17 ΔglnR mutant strain.

For MRS medium, glucose 2%, yeast extract powder 0.4%, peptone 1%, beef extract 0.5%, Tween 80 0.1%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2% , triammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.05%, all in mass-volume fractions, adjusted to pH 6.2, and 2% in solid medium Concentrated agar was added.

For MRS liquid medium containing erythromycin, erythromycin was added to the MRS medium to a final concentration of 1 μg/mL.

For MRS plates containing erythromycin, add erythromycin to a final concentration of 4 μg/mL in MRS medium.

For resuscitation fluid: 2% glucose, 0.4% yeast extract powder, 1% peptone, 0.5% beef extract, 0.1% Tween 80, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate , triammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.05%, and sucrose 10.26%, all in mass-volume fractions.

For LB medium (g/L): liquid: yeast extract powder 5, peptone 10, sodium chloride 10, solid medium plus agar with a concentration of 2%.

For erythromycin-containing LB medium, erythromycin was added to the LB medium to a final concentration of 200 μg/mL.

For GYP medium, glucose 1%, yeast extract powder 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01%, Sodium chloride 0.01%, both mass-volume fractions, 2% concentration of agar was added to the solid medium, and different concentrations of monosodium glutamate were added to the fermentation medium.

For the test of acid tolerance, the wild strain and glnR knockout strain of Lactobacillus brevis were each inoculated into fermentation medium with 10 g/L glucose carbon source and 10 g/L glutamic acid (sodium) and fermented at 37° C. for 10 h. Cells were harvested by centrifugation and strains were washed once with potassium phosphate wash buffer pH 7.0. In addition, the cells were resuspended in a potassium phosphate buffer with a pH of 7.0 with or without 10 mM glutamic acid (sodium) to an OD ₆₀₀ of 1.0. Immediately after static culture at 37° C. for 0 h, 1 h, 2 h, and 2.5 h, respectively, 9 times the volume of potassium phosphate buffer with a pH of 2.5 containing or not containing 10 mM sodium glutamate was added to the bacterial suspension. In addition to the liquid, static culture was continued for 3 hours until the end of the test. Immediately after taking the sample, dilute it with PBS buffer pH 7.4 from 10 ⁻¹ to 10 ⁻⁵ , select the appropriate gradient and apply 100 μL of the dilution to the GYP plate and statically at 37° C. for 24 h. It was placed and cultured.

Example 1: Markerless Knockout of Lactobacillus brevis glnR Gene Based on the DNA sequences upstream and downstream of the glnR gene in the Lactobacillus brevis type strain ATCC 367 or D17, 21 bp were left upstream and downstream of the glnR gene (upstream and downstream homology prevent the effect of complete gene excision on the expression of the arm gene), the primer pairs F-up-glnR-BamHI and R-up, F-down and R-down-glnR-HindIII were designed. Using the genomic DNA of Lactobacillus brevis ATCC 367 or D17 as a template, the primer pairs F-up-glnR-BamHI and R-up and F-down and R-down-glnR-HindIII were used to construct homologous sequences up- and downstream of the glnR gene. glnR (sequence is as shown in SEQ ID NO:3), down-glnR (sequence is as shown in SEQ ID NO:4) were amplified. The PCR conditions were 30 cycles of pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, and extension at 72°C for 10 min. 1000 bp each of the upstream and downstream homologous arms were recovered using the Omega gel recovery kit. Equimolar up-glnR, down-glnR fragments were supplemented with water to 5 μL, and 5 μL of Takara's PrimeSTAR Max DNA Polymerase was added. 15 cycles of denaturation at 55° C. for 10 s, annealing at 55° C. for 2 min, extension at 72° C. for 2 min, and extension at 72° C. for 10 min. Using the resulting PCR product as a template, PCR was continued using primers F-up-glnR-BamHI and R-down-glnR-HindIII. The PCR conditions were 30 cycles of pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 2 min, and extension at 72°C for 10 min. A junction fragment up-down-glnR (sequence shown in SEQ ID NO: 5) of upstream and downstream homologous arms was obtained by overlapping PCR method.

The up-down-glnR fragment and plasmid pGID023 were double digested with BamHI and HindIII, after which the enzymatic digestion products were ligated at 16° C. for 16 h before transformation into E. coli JM109 competent cells. A positive strain was obtained by identification of bacterial solution PCR, enzymatic digestion and sequencing, and a gene knockout plasmid pGID023-ΔglnR was obtained by cloning the upstream and downstream homologous sequences of the glnR gene into pGID023 (as shown in FIG. 2). .

(1) Screening for single-crossover strains in which the first homologous recombination occurred Electrically transforming the constructed gene knockout plasmid pGID023-ΔglnR into Lactobacillus brevis ATCC 367 or Lactobacillus brevis D17 competent cells, The transformation conditions were electric shock at a voltage of 2000 V to 2500 V for 5 ms, the bacterial solution was plated on an MRS plate containing erythromycin (final concentration: 4 μg/μL), and cultured at 37° C. until monoclones grew. Monoclones were selected and subjected to PCR verification using the primer pair F-GID and R-GID. Correct recombinants transformed with the knockout plasmid were screened. Single colonies of recombinant bacteria were plated and passaged in erythromycin-resistant MRS liquid medium to promote the development of single-crossover integrations. After diluting the bacterial solution after subculture, it was applied to an MRS plate containing erythromycin (final concentration is 4 μg/mL), and grown resistant single colonies were selected, and erythromycin (final concentration is 1 μg/mL). It was seeded in the containing MRS medium and cultured at 37°C for 24 hours. 200 μL of the bacterial suspension was centrifuged to remove the fermentation supernatant, resuspended by adding 20 μL of deionized water, and 0.1 g of glass powder (0.1 mm) was added and crushed with a bead mill for 2 min. After that, it was centrifuged and the supernatant was subjected to PCR verification of the bacterial solution. Multiple primer pairs F-367-0092 and R-367-0090 (primers designed on both ends of homologous arms), F-GID and R-GID, F-367-0092 and R-GID, F-GID and R -367-0090, PCR conditions were 30 cycles of pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 2 min, and It was extended for 10 min. It was ensured that the correct single crossover (single crossover method is as shown in Figure 3) was screened.

(2) Screening of gene knockout strains in which the second homologous recombination occurred Intrachromosomal recombination was promoted by inoculating single crossovers in a liquid MRS medium with no resistance and subculturing when there was no resistance. . Serially passaged bacterial suspensions were diluted and plated on non-resistant plates. A single colony grown on the plate was selected, inoculated in MRS liquid medium, cultured at 37° C. for 24 hours, the cells were disrupted by the above method, and the supernatant was subjected to bacterial solution PCR verification. The primer pair was F-up-glnR-BamHI and R-down-glnR-HindIII, PCR conditions were predenaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 55°C for 15s, 30 cycles of 2 min extension at 72°C and 10 min extension at 72°C. After screening potential knockout strains, genomic DNA of potential knockout strains was extracted and primer pairs F-up-glnR-BamHI and R-down-glnR-HindIII and F-glnR and R-glnR (glnR gene PCR amplification was performed (as shown in Figure 4) and verified by sequencing (as shown in Figure 5).

In the double crossover in which the second round of homologous recombination occurred, one was reverted to wild type and the other was a glnR gene knockout strain (Fig. 3).

As shown in FIG. 4A, a PCR product (up-glnR and down-glnR), but were able to amplify a fragment approximately 2300 bp in size using the wild-type genome, indicating that glnR was deleted in the ΔglnR mutant. As shown in FIG. 4B, employing the primer pair F-glnR and R-glnR, the genome of the ΔglnR mutant failed to amplify the PCR product, indicating a deletion of glnR, but the wild-type genome. can be used to amplify fragments approximately 300 bp in size.

The glnR genes of the wild type and the ΔglnR mutant were sequenced, and the glnR deletion fragments are shown in FIG.

Lactobacillus brevis ATCC 367ΔglnR mutant strain and Lactobacillus brevis D17ΔglnR mutant strain in which the correct glnR gene was knocked out were obtained by the method.

Example 2 Use of Lactobacillus brevis glnR Gene Knockout Strains in the Synthesis of GABA The D17ΔglnR mutant strain was taken out, streaked on a GYP solid plate, and statically cultured at 37°C.

After single colony growth, Lactobacillus brevis ATCC 367 wild strain, ATCC 367ΔglnR mutant and D17 wild strain, D17ΔglnR mutant single colonies were picked from the activated GYP solid plates and inoculated onto GYP seed medium. C. for 24 hours. A seed culture was taken, seeded in a new GYP seed medium at a seeding rate of 10%, and statically cultured at 37° C. for 12 hours, after which the culture was used as a fermentation seed. Place 100 mL of GYP fermentation medium containing 10 g/L of glucose carbon source and 50 g/L of glutamic acid (sodium) in a 250 mL Erlenmeyer flask, seed the fermentation seeds cultured for 12 h at a seeding rate of 10%, and heat at 37°C. Cultured at 200 rpm for 48 h. The GABA content of the fermentation supernatant was detected by high performance liquid chromatography.

As can be seen from the results, the GABA synthesis capacity of the Lactobacillus brevis ATCC 367ΔglnR mutant strain was 18.0 g/L, compared to 11.8 g/L for the wild type ATCC 367, as shown in FIG. 5% improvement. Fermented with Lactobacillus brevis ATCC 367ΔglnR mutant for 24 h, GABA reached the highest yield and was shortened by 100% compared to wild type ATCC 367 for 48 h. After fermenting for 24 h, the production efficiency of the Lactobacillus brevis ATCC 367ΔglnR mutant was 0.72 g/L/h, which was 280% higher than that of the wild type ATCC 367, 0.19 g/L/h.

As shown in FIG. 6B, the GABA synthesis ability of the Lactobacillus brevis D17ΔglnR mutant was 19.3 g/L, which was improved by only 4.9% compared to 18.4 g/L of the wild type D17. , fermented with the Lactobacillus brevis D17ΔglnR mutant strain for 24 h, GABA reached the highest yield and was shortened by 50% compared to wild type D17 for 36 h. After fermenting for 24 hours, the production efficiency of the Lactobacillus brevis D17ΔglnR mutant was 0.76 g/L/h, which was 38.2% higher than that of the wild strain D17 (0.55 g/L/h).

In summary, it has a regulating effect on the GABA synthesis ability of GlnR against Lactobacillus brevis, and can remarkably improve the GABA production efficiency of the glnR knockout mutant after glnR is knocked out.

Example 3: Use for Improving Acid Tolerance of Lactobacillus brevis glnR Gene Knockout Bacteria Slant-activated Lactobacillus brevis ATCC 367ΔglnR and D17ΔglnR mutants were each seeded in GYP liquid medium and incubated at 37°C. After stationary culture for 24 hours, a primary seed culture was prepared. The primary seed liquid was seeded in a fresh GYP medium at a seeding rate of 10% and cultured at 37°C x 200 rpm for 15 h to prepare a secondary seed culture. The secondary seed culture was inoculated into GYP fermentation medium with 10 g/L glucose as carbon source and 10 g/L glutamic acid (sodium) substrate at 10% seeding rate and incubated at 37° C.×200 rpm for 10 h. cultured. Cells were harvested by centrifugation at 9000 rpm for 10 min at 4°C. After resuspending the cells in a potassium phosphate buffer of pH 7.0, the suspension was centrifuged to remove the supernatant. Cells were resuspended in potassium phosphate buffer pH 7.0 containing 10 mM glutamic acid (sodium) to an OD ₆₀₀ of 1.0. Immediately after static culture at 37° C. for 0 h, 1 h, 2 h, and 2.5 h, bacteria with different culture times were added to 9 times the volume of potassium phosphate buffer containing 10 mM glutamic acid (sodium) and pH 2.5. In addition to the suspension, static culture was continued for 3 h until the end of the test. Similarly, the cells were resuspended in a potassium phosphate buffer containing no glutamic acid (sodium) and having a pH of 7.0, and the OD ₆₀₀ thereof was adjusted to 1.0. Immediately after static culture at 37° C. for 0 h, 1 h, 2 h, and 2.5 h, bacterial suspensions with different culture times were added to 9 times the volume of potassium phosphate buffer containing no glutamic acid (sodium) and having a pH of 2.5. In addition to the liquid, static culture was continued for 3 hours until the end of the test. Immediately after removing the acid-stimulated samples, dilute them with PBS buffer pH 7.4 in a gradient of 10 ⁻¹ to 10 ⁻⁵ , select the appropriate gradient and apply 100 μL of the dilutions to the GYP plate. Then, the plate was cultured statically at 37° C. for 24 hours, and the number of colonies on the plate was recorded.

As can be seen from the results, as shown in FIG. 7A, when the wild-type strain of Lactobacillus brevis ATCC 367 and the ATCC 367ΔglnR mutant strain were placed in an acidic buffer without sodium glutamate for 0.5 h, no viable bacteria were present. became. After placing the Lactobacillus brevis ATCC 367ΔglnR mutant in a buffer containing 10 mM glutamic acid (sodium) for 0.5 h, the number of colonies reached 6.8×10 ⁹ cfu/mL, compared with 4.4×10 of the wild type ATCC 367. 54.5% improvement compared to ⁹ cfu/mL. After placing the Lactobacillus brevis ATCC 367ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium) for 1 h, the number of colonies was 4.9×10 ⁹ cfu/mL, compared to 2.2×10 ⁹ cfu of wild type ATCC 367. /mL, it was improved by 122.7%. After placing the Lactobacillus brevis ATCC 367ΔglnR mutant in a buffer containing 10 mM glutamic acid (sodium) for 2 h, the number of colonies was 2.4×10 ⁹ cfu/mL, compared to 1.2×10 ⁹ cfu of the wild type ATCC 367. /mL, improved by 100%. After placing the Lactobacillus brevis ATCC 367ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium) for 3 h, the number of colonies was 1.5×10 ⁹ cfu/mL, compared to 2.8×10 ⁸ cfu of wild type ATCC 367. /mL, it was improved by 435.7%. It was shown that the acid tolerance of the Lactobacillus brevis ATCC 367ΔglnR mutant was significantly higher than that of the wild type ATCC 367 by adding glutamic acid (sodium) to the buffer.

As shown in FIG. 7B, no viable bacteria were present after stimulation of Lactobacillus brevis D17 wild type strain and D17ΔglnR mutant strain in acidic buffer without sodium glutamate for 0.5 h. After placing the Lactobacillus brevis D17ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium) for 0.5 h, the number of colonies was 9.6×10 ⁹ cfu/mL, compared to 7.0×10 ⁹ cfu of the wild strain D17. /mL, it was improved by 37.1%. After 1 h of immersing the Lactobacillus brevis D17ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium), the number of colonies was 7.6×10 ⁹ cfu/mL, compared with 3.7×10 ⁹ cfu/mL of wild strain D17. compared to 105.4% improved. After placing the Lactobacillus brevis D17ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium) for 2 h, the number of colonies was 4.9×10 ⁹ cfu/mL, compared with 2.2×10 ⁹ cfu/mL for the wild strain D17. compared to 122.7% improved. After placing the Lactobacillus brevis D17ΔglnR mutant strain in a buffer containing 10 mM glutamic acid (sodium) for 3 h, the number of colonies was 2.6×10 ⁹ cfu/mL, compared with 9.0×10 ⁸ cfu/mL for the wild strain D17. compared to 188.9% improved. The addition of 10 mM glutamic acid (sodium) showed that the ability of the Lactobacillus brevis D17ΔglnR mutant to withstand an extremely acidic environment was significantly higher than that of the wild type D17.

In other words, GlnR can modulate the glutamate-dependent acid tolerance of strains, and can effectively improve the acid tolerance of glnR knockout mutants after glnR knockout. The resulting highly acid-tolerant mutants are applied to fermenting agents for acidic foods, such as yellow wine brewing and sourdough production.

Example 4: Use of Lactobacillus brevis ATCC 367ΔglnR Mutant Strain in the Synthesis of GABA by pH-Controlled Fed-Batch Fermentation to prepare a primary seed culture medium. The primary seed liquid was seeded in a fresh GYP medium at a seeding rate of 10% and cultured at 37°C x 200 rpm for 15 h to prepare a secondary seed culture. The secondary seed culture was inoculated into a GYP fermentation medium with a seed rate of 10%, a pH of 5, a carbon source of 30 g/L glucose and containing 74.8 g/L glutamic acid (sodium). A bioreactor was used to control the temperature at 37° C., stir at 200 rpm, ferment without aeration, and adjust the pH to 5.0 online with 5 mol/L sulfuric acid. Within 6-24 h, 74.8 g (100 mL) of material (glutamic acid (sodium)) was supplemented every 6 h and fermented for 72 h. GABA content of the fermentation supernatant was detected by HPLC analysis. Wild strain ATCC 367 was cultured in a similar manner and the GABA content of the fermentation supernatant was measured.

As can be seen from the results, as shown in FIG. 2193% improvement compared to 10.2 g/L. When fermented for 36 h, the maximum production efficiency of the Lactobacillus brevis ATCC 367ΔglnR mutant strain was 4.88 g/L/h, an improvement of 2340% compared to 0.20 g/L/h of wild type ATCC 367.

Example 5: Use of Lactobacillus brevis ATCC 367ΔglnR Mutant in Synthesis of GABA by Fed-Batch Fermentation of Glucose with pH Adjustment The cells were seeded in a liquid medium and cultured at 37°C for 24 hours to prepare a primary seed culture. The primary seed liquid was seeded in a fresh GYP medium at a seeding rate of 10% and cultured at 37°C x 200 rpm for 15 h to prepare a secondary seed culture. The secondary seed culture was inoculated into a GYP fermentation medium containing 74.8 g/L glutamic acid (sodium) with 20 g/L glucose as a carbon source at a seeding rate of 10%, pH of 5, and using a bioreactor. The temperature was controlled at 37° C. with 200 rpm and fermentation was performed without aeration, and the pH was adjusted to 5.0 with 5 mol/L sulfuric acid online. Within 6 to 36 h, 74.8 g (100 mL) of material (glutamic acid (sodium)) was supplemented every 6 h, and 150 g / L of glucose was fed from 12 to 36 h, and the flow rate was 0.625 g / h. Yes, fermented for 72 h. GABA content of the fermentation supernatant was detected by HPLC analysis.

As can be seen from the results, as shown in FIG. 9, the Lactobacillus brevis ATCC 367ΔglnR mutant had the highest GABA synthesis ability and yield of 282.8 g/L after 60 h of fermentation, and the yield of the wild type ATCC 367 was 282.8 g/L. 1906% improvement compared to 14.1 g/L. When fermented for 36 h, the maximum production efficiency of the Lactobacillus brevis ATCC 367ΔglnR mutant strain was 4.93 g/L/h, an improvement of 2248% compared to 0.21 g/L/h of wild type ATCC 367.

Example 6: Use of Lactobacillus brevis D17ΔglnR Mutants in Synthesis of GABA by pH-Controlled Fed-Batch Fermentation and cultured at 37° C. for 24 hours to prepare a primary seed culture. The primary seed liquid was seeded in a fresh GYP medium at a seeding rate of 10% and cultured at 37°C x 200 rpm for 15 h to prepare a secondary seed culture. The secondary seed culture was inoculated into a GYP fermentation medium with a seed rate of 10%, a pH of 5, a carbon source of 50 g/L glucose, and containing 74.8 g/L glutamic acid (sodium). A bioreactor was used to control the temperature at 37° C., stir at 200 rpm, ferment without aeration, and adjust the pH to 5.0 online with 5 mol/L sulfuric acid. Within 6-24 h, 74.8 g (100 mL) of material (glutamic acid (sodium)) was supplemented every 6 h and fermented for 72 h. GABA content of the fermentation supernatant was detected by HPLC analysis.

As can be seen from the results, as shown in FIG. 10, after fermenting for 60 h, the Lactobacillus brevis D17ΔglnR mutant has the highest ability to synthesize GABA, with a yield of 148.0 g/L, compared to 116.1 g of the wild strain D17. /L, improved by 27.4%. After fermentation for 18 h, the maximum production efficiency of the Lactobacillus brevis D17ΔglnR mutant strain was 5.73 g/L/h, an improvement of 70.0% compared to 3.39 g/L/h of the wild strain D17.

Example 7 Use of Lactobacillus brevis D17ΔglnR Mutants in the Synthesis of GABA by Fed-Batch Fermentation of Glucose with pH Adjustment The cells were seeded in a GYP liquid medium and cultured at 37° C. for 24 hours to prepare a primary seed culture. The primary seed liquid was seeded in a fresh GYP medium at a seeding rate of 10% and cultured at 37°C x 200 rpm for 15 h to prepare a secondary seed culture. The secondary seed culture was inoculated into GYP fermentation medium containing 74.8 g/L glutamic acid (sodium) with 30 g/L glucose as a carbon source at a seeding rate of 10% at a pH of 5, and the bioreactor was used to The temperature was controlled at 37° C., stirring was performed at 200 rpm, fermentation was performed without aeration, and the pH was adjusted to 5.0 online with 5 mol/L sulfuric acid. Within 6 to 36 h, 74.8 g (100 mL) of material (glutamic acid (sodium)) was supplemented every 6 h, and 400 g / L glucose was fed from 12 to 36 h, and the flow rate was 2.58 g / h. Yes, fermented for 72 h. GABA content of the fermentation supernatant was detected by HPLC analysis.

As can be seen from the results, as shown in FIG. 11, after fermenting for 60 h, the Lactobacillus brevis D17ΔglnR mutant strain had the highest GABA synthesis ability with a yield of 301.5 g/L, compared to 180.7 g of the wild strain D17. /L, improved by 66.8%. After fermentation for 36 h, the maximum production efficiency of the Lactobacillus brevis D17ΔglnR mutant strain was 5.76 g/L/h, which was 17.0% higher than that of the wild strain D17 of 4.93 g/L/h.

Claims (20)

Lactobacillus brevis with improved ability to synthesize γ-aminobutyric acid, wherein the glnR gene is knocked out from the starting strain, and the amino acid sequence expressed by the glnR gene is shown in SEQ ID NO: 1. is as shown in SEQ ID NO:2, wherein the starting strain is Lactobacillus brevis D17, the type strain Lactobacillus brevis ATCC 367, Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306 or Lactobacillus brevis Lactobacillus brevis, characterized as being NPS-QW-145. Lactobacillus brevis having an improved ability to synthesize γ-aminobutyric acid, wherein the glnR gene is knocked out from a starting strain. 3. The Lactobacillus brevis according to claim 2, wherein the amino acid sequence expressed by the glnR gene is as shown in SEQ ID NO:1. 4. Lactobacillus brevis according to claim 3, characterized in that the nucleotide sequence encoding the glnR gene is as shown in SEQ ID NO:2. Lactobacillus brevis according to claim 2, characterized in that the starting strain is any Lactobacillus brevis capable of synthesizing GABA. The Lactobacillus brevis capable of synthesizing GABA is Lactobacillus brevis D17, the type strain Lactobacillus brevis ATCC 367, Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306 or Lactobacillus brevis NPS-QW-145. , Lactobacillus brevis according to claim 5. The knockout is performed using any one known knockout method, such as homologous recombination, two intron (Targetron or Clostron) gene insertion inactivation, CRISPR-Cas gene editing, etc., optionally Lactobacillus brevis according to any one of claims 1-6, characterized in that said knockout is a markerless knockout, in particular a markerless gene knockout based on homologous recombination. A method for improving the ability of a Lactobacillus brevis strain to synthesize γ-aminobutyric acid, the method comprising knocking out the glnR gene in the Lactobacillus brevis strain. 9. The method of claim 8, wherein the amino acid sequence expressed by the glnR gene is as shown in SEQ ID NO:1. A method for synthesizing γ-aminobutyric acid, characterized by fermentative production using the glnR gene-knocked out Lactobacillus brevis according to any one of claims 1 to 6. 11. The method according to claim 10, wherein the fermentative production is carried out by seeding Lactobacillus brevis in a medium containing glutamic acid or sodium glutamate. 11. The method according to claim 10, wherein the Lactobacillus brevis glnR knockout strain is inoculated in a liquid fermentation medium containing glutamic acid (sodium) and cultured for fermentation. During the fermentation process, the Lactobacillus brevis glnR knockout strain was inoculated into GYP fermentation medium containing glutamic acid (sodium), pH adjusted to 4.8-5.5 and fermented under agitated but no aeration conditions for 6-24 h. 11. Process according to claim 10, characterized in that a pH-adjusted fed-batch fermentation is carried out in which the glutamic acid (sodium) substrate is supplemented during fermentation, preferably every 6 h. During the fermentation process, the Lactobacillus brevis glnR knockout strain was inoculated into GYP fermentation medium containing glutamate (sodium), pH adjusted to 4.8-5.5 and fermented under agitated but no aeration conditions for 6-36 h. 11. Process according to claim 10, characterized in that a glucose fed-batch fermentation is carried out with pH adjustment to replenish the glutamic acid (sodium) substrate during fermentation. Glutamic acid (sodium) substrate may be added every 6 h, and the glucose carbon source is fed from 6 to 36 h, and the flow rate is 0.6 to 3 g/h. described method. A method for improving the acid tolerance of Lactobacillus brevis that has produced γ-aminobutyric acid, the method comprising knocking out the glnR gene of Lactobacillus brevis. Use of Lactobacillus brevis according to any one of claims 1-6 or the method according to any one of claims 7-16 in the fields of food, skin care products, drug production, feed, chemical industry etc. . 18. Use according to claim 17, characterized in that the food is an acid fermented food. 19. Use according to claim 18, characterized in that the acidic fermented food comprises yellow wine or sourdough. 18. Use according to claim 17, characterized in that said drug is a tranquilizing, blood pressure reducing and sleep quality improving drug for mammals.

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