WO2020244380A1 - 提高短乳杆菌γ-氨基丁酸合成能力的方法及其应用 - Google Patents

提高短乳杆菌γ-氨基丁酸合成能力的方法及其应用 Download PDF

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WO2020244380A1
WO2020244380A1 PCT/CN2020/091049 CN2020091049W WO2020244380A1 WO 2020244380 A1 WO2020244380 A1 WO 2020244380A1 CN 2020091049 W CN2020091049 W CN 2020091049W WO 2020244380 A1 WO2020244380 A1 WO 2020244380A1
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lactobacillus brevis
glnr
fermentation
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glutamate
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徐岩
任聪
宫璐婵
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/121—Brevis

Definitions

  • the invention relates to a method for improving the synthesis ability of Lactobacillus brevis ⁇ -aminobutyric acid and its application, belonging to the field of microorganisms.
  • GABA Gamma-aminobutyric acid
  • GABA is an important inhibitory neurotransmitter. It has many physiological functions such as calming the nerves, lowering blood pressure and improving sleep in mammals. It can be widely used as a biologically active substance in food, medicine, and feed. , Chemical industry and other fields.
  • Food-grade GABA is mostly biosynthesized by microorganisms. Microorganisms are mainly caused by the glutamate decarboxylase (GAD) system (including glutamate/GABA reverse transporter and glutamate decarboxylase). The extracellular glutamine is removed by the glutamate/GABA reverse transporter.
  • GAD glutamate decarboxylase
  • the acid is transported into the cell, and by consuming H + , the intracellular glutamate decarboxylase catalyzes the decarboxylation of glutamate substrate to synthesize GABA.
  • the synthesized GABA then transports GABA out of the cell through the glutamate/GABA reverse transport body to achieve the accumulation of GABA, while maintaining the stability of the intracellular pH, helping the strain resist the stress of the acidic environment.
  • lactic acid bacteria have an absolute advantage, especially Lactobacillus brevis.
  • our laboratory selected a strain of Lactobacillus brevis D17 that efficiently synthesizes GABA from the liquor brewing system. The growth of this strain is coupled with the synthesis of GABA, and the production efficiency of the strain can be improved through optimization of fermentation strategies (controlling fermentation pH) (3.76g/L/h).
  • the existing methods for increasing GABA production are mainly aimed at the screening of high-producing GABA strains, the optimization of GABA production fermentation conditions and medium, and the overexpression of the glutamate decarboxylase system.
  • the existing high- or low-producing GABA bacteria if it is to increase its industrial application and continue to improve its production efficiency, it is necessary to specifically regulate its metabolic pathways.
  • the present invention provides a method for improving the synthesis ability of ⁇ -aminobutyric acid of Lactobacillus brevis strain.
  • the inventor found that although the model strain Lactobacillus brevis ATCC 367 and the related coding genes of the glutamate decarboxylation system of Lactobacillus brevis D17 (glutamate/GABA reverse carrier, glutamate
  • GABA glutamate/GABA reverse carrier, glutamate
  • the amino acid sequence of the gene of decarboxylase (GAD) is the same, but when the strain is fermented with 5% sodium glutamate, the GABA production (10g/L) of Lactobacillus brevis ATCC 367 is much lower than that of Lactobacillus brevis D17 (17.8g). /L), and the GABA production of Lactobacillus brevis ATCC 367 and Lactobacillus brevis D17 did not continue to increase with the addition of sodium glutamate, and the GABA production of both were restricted.
  • Glutamate sodium
  • GlnR Glutamine Synthetase Inhibitor
  • GlnR not only regulates nitrogen metabolism but also controls carbon source metabolism, phosphorus metabolism, antibiotic synthesis and strain tolerance (osmotic pressure, acid resistance, oxidation) and so on.
  • the GAD system that controls the synthesis of GABA is an acid-tolerant system for microorganisms to resist acid stress. In the case of sufficient glutamate nitrogen source, it may be regulated by the repression of GlnR. And there are no reports about the glnR gene knockout strains of lactic acid bacteria.
  • the present invention By knocking out the glnR gene of Lactobacillus brevis, the present invention further relieves the restriction of Lactobacillus brevis GABA synthesis, increases GABA output, shortens GABA synthesis time, and further improves the industrial production efficiency of GABA.
  • the first object of the present invention is to provide a method for improving the ⁇ -aminobutyric acid synthesis ability of a Lactobacillus brevis strain by knocking out the glnR gene (glutamine synthetase inhibitor gene) in the Lactobacillus brevis strain .
  • amino acid sequence expressed by the glnR gene is shown in SEQ ID NO:1.
  • nucleotide sequence encoding the glnR gene is shown in SEQ ID NO: 2.
  • the Lactobacillus brevis strain is any Lactobacillus brevis capable of synthesizing GABA.
  • the Lactobacillus brevis strain is Lactobacillus brevis D17 (the deposit number is CGMCC NO.14385) or the model strain Lactobacillus brevis ATCC367, Lactobacillus brevis NCL912, Lactobacillus brevis CGMCC1306, Lactobacillus brevis NPS -QW-145 and so on.
  • the knockout is performed using any known knockout method, such as homologous recombination, type II intron (Targetron or Clostron) gene insertion and inactivation, CRISPR-Cas gene editing, etc. .
  • the knockout is a scarless knockout, especially a marker-free gene knockout based on homologous recombination.
  • the knockout is specifically: the 1000 bp upstream and downstream homologous fragments of the glnR gene to be knocked out (including the front and rear ends of the glnR gene 21 bp) are respectively linked to the integration plasmid pGID023 of the lactic acid bacteria in a certain direction,
  • the knockout plasmid pGID023- ⁇ glnR of the target gene is obtained; the knockout plasmid is transformed into Lactobacillus brevis by electroporation, and the recombinant strain containing the knockout plasmid is obtained by screening on the erythromycin resistant plate.
  • the single exchanger was selected for the first homologous recombination to integrate the plasmid into the chromosome of the recipient bacteria; then the single exchanger was subcultured in liquid GYP medium without erythromycin. Spread the passaged bacterial liquid on a GYP plate without antibiotics, and select a single clone until the second homologous recombination has no erythromycin resistance strain to achieve the target gene knockout.
  • the above-mentioned pGID023 plasmid carries a replication origin, a multiple cloning site MCS, and erythromycin resistance genes that can replicate in E. coli.
  • the above-mentioned knock-out plasmid is connected to the multiple cloning site of pGID023 by digesting the homologous sequence fragments about 1000 bp upstream and downstream of the target gene to be knocked out on the chromosome of Lactobacillus brevis respectively according to the gene arrangement direction.
  • the method of electrotransformation of the above-mentioned knockout plasmid into Lactobacillus brevis to obtain a strain containing the knockout plasmid mix 1 ⁇ g of the knockout plasmid with competent cells of Lactobacillus brevis, and use appropriate electrotransformation conditions to electrotransform the plasmid into short milk Bacillus.
  • the pre-chilled MRS medium was quickly added and resuscitated in the 37°C incubator. The supernatant was removed by centrifugation, and the cells were resuspended and spread on an MRS plate containing erythromycin and cultured at 37°C until transformants were grown. Pick transformants for bacterial liquid PCR verification.
  • the method for verifying the single-exchanger for the first homologous recombination to integrate the plasmid into the chromosome of the recipient bacteria is to inoculate the transformant of Lactobacillus brevis containing the knock-out plasmid into MRS medium containing erythromycin 37 Incubate at °C for 24h.
  • the cultured bacteria solution was inoculated into the above-mentioned MRS liquid medium containing erythromycin, cultured at 37°C for 24 hours, and then subcultured according to this procedure. Dilute the passaged bacterial solution 10 -3 to 10 -6 times and spread it on an MRS plate containing erythromycin and cultivate at 37° C. until a single colony grows.
  • the method for screening the strains that undergo the second homologous recombination to achieve the target gene knockout is: inoculate the single exchanger into liquid MRS medium without erythromycin, and cultivate it at 37°C for 24 hours. After passaged without anti MRS medium, were diluted 10 - 3 to 10 -6 times the free MRS plates coated with erythromycin at 37 °C, grow until single colonies. Pick a single colony, inoculate it in a non-erythromycin-resistant MRS liquid medium, culture it for 24h at 37°C, and perform PCR verification of the bacterial solution to obtain a potential mutant strain of glnR gene knockout.
  • the second object of the present invention is to provide a Lactobacillus brevis with improved ⁇ -aminobutyric acid synthesis ability, which knocks out the glnR gene on the basis of the original strain.
  • amino acid sequence expressed by the glnR gene is shown in SEQ ID NO:1.
  • nucleotide sequence encoding the glnR gene is shown in SEQ ID NO: 2.
  • the starting strain is any Lactobacillus brevis capable of synthesizing GABA, optionally, Lactobacillus brevis D17 (the deposit number is CGMCC NO.14385), the model strain Lactobacillus brevis ATCC 367, Bacillus brevis NCL912, Lactobacillus brevis CGMCC1306 or Lactobacillus brevis NPS-QW-145, etc.
  • any known knockout methods can be used for the knockout, such as homologous recombination, type II intron (Targetron or Clostron) gene insertion and inactivation, CRISPR-Cas gene editing, etc. .
  • the third object of the present invention is to provide a method for synthesizing ⁇ -aminobutyric acid, which is to use Lactobacillus brevis whose glnR gene has been knocked out for fermentation production.
  • the fermentation production is to inoculate Lactobacillus brevis in a medium containing glutamate or sodium glutamate for fermentation production.
  • the fermentation production can be performed by any of the following methods:
  • Fermentation process is pH-adjusted fed-batch fermentation with glucose in batches: Lactobacillus brevis glnR knockout strain is inoculated into GYP fermentation medium containing glutamate (sodium), and the pH is controlled to 4.8-5.5, Fermentation is carried out under the condition of aeration without stirring; the glutamate (sodium) substrate is supplemented during the fermentation period of 6 to 36 hours.
  • the glutamate (sodium) substrate can be added every 6 hours, and the glucose carbon source can be added in 6-36 hours.
  • the fermentation production is performed by any one of the following methods:
  • Lactobacillus brevis glnR knockout strains were respectively inoculated into 10g/L glucose carbon source and 50g/L glutamate (sodium) added to GYP liquid fermentation medium, and fermentation was carried out at 37°C ⁇ 200rpm.
  • the cumulative concentration of GABA of ATCC 367 ⁇ glnR was 233.9g/L, and the production efficiency was up to 4.88g/L/h after 36 hours of fermentation; the cumulative concentration of GABA of D17 ⁇ glnR was 148.0g/L after 48 hours of fermentation, and the production efficiency was 18 hours. The highest is 5.73g/L/h.
  • Fermentation process is pH-adjusted fed-batch fermentation with glucose: using a bioreactor, inoculate Lactobacillus brevis ATCC 367 ⁇ glnR or D17 ⁇ glnR mutant strains in a GYP fermentation medium containing glutamate (sodium).
  • the pH of the fermentation broth was controlled to 5.0 with sulfuric acid with a concentration of 5mol/L, and the fermentation was carried out at 37°C ⁇ 200rpm without aeration.
  • glutamate (sodium) substrate between 6 and 36 hours.
  • glutamate (sodium) substrate can be added every 6h, and glucose carbon source can be added at 12-36h at the same time, and the accumulated concentration of GABA of ATCC367 ⁇ glnR is 282.80g/L. Fermentation is 36h, and the production efficiency is up to 4.93g/L/h; preferably, during the fermentation process of D17 ⁇ glnR, glutamate (sodium) substrate can be added every 6h, and glucose carbon source can be added at 6-30h for 60h fermentation. , The cumulative concentration of GABA of D17 ⁇ glnR is 301.5g/L, the fermentation is 36h, the production efficiency is up to 5.76g/L/h.
  • the fourth object of the present invention is to provide a method for improving the acid tolerance of ⁇ -aminobutyric acid-producing Lactobacillus brevis by knocking out the glnR gene of Lactobacillus brevis.
  • the fifth objective of the present invention is to provide applications of the recombinant bacteria or the method in the fields of food, medicine preparation, feed, and chemical industry.
  • the food is an acid fermented food; such as rice wine or sourdough production.
  • the sixth object of the present invention is to provide the use of the recombinant bacteria or the method in the preparation of drugs or health products that can calm the nerves, lower blood pressure, and improve sleep in mammals.
  • Lactobacillus brevis D17 has been published in the patent application 2017112754179 with publication number CN 108034599A and the publication date of May 15, 2018; its taxonomic name is Lactobacillus brevis, and it has been deposited in Chinese microorganisms on July 6, 2017 The General Microbiology Center of the Culture Collection Management Committee, the deposit number is CGMCC NO.14385.
  • the type strain Lactobacillus brevis ATCC 367 purchased from the China Collection of Microorganisms under the number CGMCC1.2028, or can be purchased from the American Type Culture Institute under the number ATCC 367.
  • Lactobacillus brevis NCL912 Lactobacillus brevis CGMCC1306, and Lactobacillus brevis NPS-QW-145 involved in the present invention have been disclosed in the literature before this patent application (Li, HX, Gao, DD, et al.
  • the integration plasmid pGID023, as shown in Figure 1, has been published in the 1994 literature (Pascal Hols, Thierry Ferain, Anthony Garmyn, et al. Use of homologous expression-secretion signals and vector-free stable chromosomal integration in plant engineering for alpha-amylase and levanase expression. Applied Environmental Microbiology 1994, 60: 1401-1413.).
  • the present invention finds for the first time that the deletion of the glnR gene can significantly improve the GABA synthesis ability of Lactobacillus brevis, and through the obtained glnR gene knockout strain ATCC 367 ⁇ glnR and Lactobacillus brevis D17 ⁇ glnR of Lactobacillus brevis, the method is found to be versatile and suitable in principle. All Lactobacillus brevis.
  • the present invention utilizes the strain that knocks out the glnR gene to produce GABA, which can greatly improve the production efficiency or yield of Lactobacillus brevis GABA, shorten the fermentation cycle, and can significantly improve the survival ability of the strain in an acidic environment.
  • the strain is also suitable for rice wine brewing and acidification. Fermentation of acidic foods such as dough production.
  • Figure 1 The pGID023 plasmid used for gene knockout
  • FIG. 1 The construction of knockout plasmid pGID023- ⁇ glnR; the upper picture shows the amplification of the upstream and downstream homology arms of glnR gene, and the bottom picture shows the knockout plasmid pGID023- ⁇ glnR;
  • FIG. 1 Schematic diagram of the process of Lactobacillus brevis glnR gene knockout
  • the markers in Figure 4A are: M: 500bp Marker; 6: Amplification of Lactobacillus brevis D17 genome using primer pairs F-glnR and R-glnR; 9: Amplification of Lactobacillus brevis using primer pairs F-glnR and R-glnR ATCC 367 genome; 1 ⁇ 5: Extract the genome of the glnR knockout strain of Lactobacillus brevis D17, and amplify it with the primer pair F-glnR and R-glnR; 7 ⁇ 8: Extract the glnR knockout of Lactobacillus brevis ATCC 367 The genome of the strain was amplified using primer pairs F-up-glnR-BamH and R-down-glnR-Hind3.
  • the markers in Figure 4B are: M: 5kb Marker; 1: Using primer pair F-up-glnR-BamH and R-down-glnR-Hind3 to amplify the genome of Lactobacillus brevis D17; 7.
  • primer pair F-up- glnR-BamH and R-down-glnR-Hind3 amplify the genome of Lactobacillus brevis ATCC 367
  • 2-6 extract the genome of the glnR knockout strain of potential Lactobacillus brevis D17, and use the primer pair F-up-glnR-BamH and R -down-glnR-Hind3 amplification
  • 8-9 Extract the genome of the glnR knockout strain of Lactobacillus brevis ATCC 367, and use the primer pair F-up-glnR-BamH and R-down-glnR-Hind3 to amplify.
  • Figure 6 The effect of glnR gene deletion on GABA synthesis of Lactobacillus; the label in Figure 6A is: 367 represents Lactobacillus brevis ATCC 367 wild bacteria; ⁇ glnR represents Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain; the label in Figure 6B is : 367 represents Lactobacillus brevis D17 wild bacteria; ⁇ glnR represents Lactobacillus brevis D17 ⁇ glnR mutant strain.
  • Figure 7 Survival of Lactobacillus brevis glnR knockout bacteria in an extremely acidic environment; the label in Figure 7A is: 367 represents Lactobacillus brevis ATCC 367 wild bacteria; ⁇ glnR represents Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain; Figure 7B Marked as: 367 represents Lactobacillus brevis D17 wild strain; ⁇ glnR represents Lactobacillus brevis D17 ⁇ glnR mutant strain.
  • Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain adopts pH-adjusted batch fed-feed fermentation to synthesize GABA; the labels in Figure 8 are: 367 represents Lactobacillus brevis ATCC 367 wild bacteria; ⁇ glnR represents Lactobacillus brevis ATCC367 ⁇ glnR mutant strain.
  • Lactobacillus brevis ATCC 367 ⁇ glnR mutant uses pH-adjusted fed-batch fermentation of glucose to synthesize GABA; the label in Figure 9 is: 367 represents Lactobacillus brevis ATCC 367 wild bacteria; ⁇ glnR represents Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain.
  • Lactobacillus brevis D17 ⁇ glnR mutant strain adopts pH-adjusted batch fed-feed fermentation to synthesize GABA; the labels in Figure 10 are: D17 represents Lactobacillus brevis D17 wild bacteria; ⁇ glnR represents Lactobacillus brevis D17 ⁇ glnR mutant strain.
  • Lactobacillus brevis D17 ⁇ glnR mutant strain uses pH-adjusted fed-batch fermentation of glucose to synthesize GABA; the labels in Figure 11 are: D17 represents Lactobacillus brevis D17 wild bacteria; ⁇ glnR represents Lactobacillus brevis D17 ⁇ glnR mutant strains .
  • MRS medium glucose 2%, yeast extract 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% and 0.05% manganese sulfate, both are mass-volume fractions, adjusted to pH 6.2; solid medium is added with 2% agar.
  • MRS liquid medium containing erythromycin Add erythromycin with a final concentration of 1 ⁇ g/mL on the basis of MRS medium.
  • MRS plate containing erythromycin add erythromycin with a final concentration of 4 ⁇ g/mL on the basis of MRS medium.
  • Resuscitation solution 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 %, 0.05% manganese sulfate, 10.26% sucrose, all are mass-volume fractions.
  • LB medium liquid: yeast extract 5, peptone 10, sodium chloride 10; solid medium with 2% agar added.
  • LB medium with erythromycin add erythromycin at a final concentration of 200 ⁇ g/mL on the basis of LB medium.
  • GYP medium glucose 1%, yeast extract 1%, peptone 0.5%, sodium acetate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.01%, ferrous sulfate 0.01%, sodium chloride 0.01%, all are mass-volume Fraction: 2% concentration of agar is added to the solid medium, and different concentrations of sodium glutamate are added to the fermentation medium.
  • Acid tolerance test Lactobacillus brevis wild bacteria and glnR knockout strain were respectively inoculated in a fermentation medium containing 10g/L glucose carbon source and 10g/L glutamic acid (sodium), and fermented at 37°C for 10 hours. The cells were collected by centrifugation and washed once with potassium phosphate buffer at pH 7.0. Resuspend the bacteria in pH 7.0 potassium phosphate buffer with or without 10mM glutamate (sodium) to make an OD 600 of 1.0.
  • Example 1 Traceless knockout of Lactobacillus brevis glnR gene
  • the upstream and downstream of the glnR gene are reserved for 21bp each (to prevent the complete resection of the gene from affecting the expression of the upstream and downstream homology arms), and the primer pair F-up is designed -glnR- BamHI and R-up, F-down and R-down-glnR-HindIII.
  • primer pairs F-up-glnR-BamHI and R-up and F-down and R-down-glnR-HindIII were used to amplify the upstream and downstream homology of the glnR gene. Sequence up-glnR (sequence shown in SEQ ID NO: 3), down-glnR (sequence shown in SEQ ID NO: 4).
  • PCR conditions were pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min.
  • the Omega gel recovery kit was used to recover 1000 bp of the upstream and downstream homology arms. Take equimolar up-glnR and down-glnR fragments and add water to make up to 5 ⁇ L, then add 5 ⁇ L of Takara PrimeSTAR Max DNA Polymerase, PCR conditions are 98°C pre-denaturation 30s; 98°C denaturation 10s, 55°C annealing 2min, 72°C extension 2min , 15 cycles; extension 10min after 72°C. Using the obtained PCR product as a template, the PCR was continued using primers F-up-glnR-BamHI and R-down-glnR-HindIII.
  • PCR conditions were pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 10 min.
  • the overlapped PCR method is used to obtain the spliced fragment up-down-glnR of the upstream and downstream homology arms (sequence is shown in SEQ ID NO: 5).
  • the up-down-glnR fragment and plasmid pGID023 were double digested with BamHI and HindIII, and the digested product was ligated at 16°C for 16 hours, and then transformed into E. coli JM109 competent.
  • the positive strains were identified by bacterial liquid PCR, restriction enzyme digestion and sequencing, and then the gene knockout plasmid pGID023- ⁇ glnR (as shown in Figure 2) was obtained by cloning the upstream and downstream homologous sequences of the glnR gene into pGID023.
  • the constructed knockout plasmid pGID023- ⁇ glnR was electrotransformed into competent cells of Lactobacillus brevis ATCC 367 or Lactobacillus brevis D17, and the transformation conditions were electric shock at a voltage of 2000V ⁇ 2500V for 5ms, and the bacterial solution was coated on the erythromycin (Final concentration is 4 ⁇ g/ ⁇ L) MRS plates were incubated at 37°C until a single clone was grown. Pick a single clone and use primers to verify F-GID and R-GID by PCR. Select the correct recombinant strain that was transferred into the knockout plasmid.
  • MRS liquid medium containing erythromycin resistance for passage to promote the occurrence of single exchange integration.
  • Dilute the passaged bacterial solution apply it to the MRS plate containing erythromycin (final concentration of 4 ⁇ g/mL), pick out the resistant single colonies, and then connect them to erythromycin (final concentration of 1 ⁇ g/mL).
  • MRS medium was cultured at 37°C for 24h. Centrifuge 200 ⁇ L of bacteria to remove the fermentation supernatant, add 20 ⁇ L of deionized water to resuspend, and add 0.1g of glass powder (0.1mm), crushed with a bead mill for 2min, centrifuge to take the supernatant for PCR verification of bacteria.
  • F-GID and R-GID, F-367-0092 and R-GID, F-GID and R -367-0090 verified that the PCR conditions were 98°C pre-denaturation for 30s; 98°C denaturation for 10s, 55°C annealing for 15s, 72°C extension for 2min, 30 cycles; 72°C after extension for 10min. Make sure to screen the correct single exchange (the single exchange method is shown in Figure 3).
  • the single exchanger is inoculated into a liquid MRS medium without resistance, and passaged in the absence of resistance to promote recombination in the chromosome. Dilute the bacterial solution after continuous passage and spread it on a non-resistant plate. A single colony grown on the plate was picked and inoculated into MRS liquid medium at 37°C for 24 hours, the cells were disrupted by the above-mentioned method, and the supernatant was taken for bacterial liquid PCR verification.
  • the primer pair is F-up-glnR-BamHI and R-down-glnR-HindIII
  • PCR conditions are 98°C pre-denaturation 30s; 98°C denaturation 10s, 55°C annealing 15s, 72°C extension 2min, 30 cycles; after 72°C Extend for 10 minutes.
  • the PCR product cannot be amplified by the genome of the ⁇ glnR mutant, indicating that the glnR is missing, and the wild bacteria genome can be used to amplify a fragment with a size of about 300 bp.
  • the glnR gene of wild bacteria and the ⁇ glnR mutant strain was sequenced, and the glnR deletion fragments are shown in Figure 5.
  • Example 2 Application of Lactobacillus brevis glnR knockout bacteria to synthesize GABA
  • Lactobacillus brevis ATCC 367 wild bacteria, ATCC 367 ⁇ glnR mutant, Lactobacillus brevis D17 wild bacteria, and D17 ⁇ glnR mutants stored at -80°C, and streak them on the GYP solid plate, and let stand at 37°C to cultivate.
  • a 250mL Erlenmeyer flask was filled with 100mL of GYP fermentation medium supplemented with 10g/L glucose carbon source and 50g/L glutamate (sodium), and the fermented seeds cultured for 12h were inoculated at 10% of the inoculum amount, at 37°C ⁇ Culture at 200rpm for 48h.
  • the fermentation supernatant was tested for GABA content by high performance liquid chromatography.
  • the GABA synthesis capacity of the Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain is 18.0g/L, which is an increase of 52.5% compared to the 11.8g/L of the wild strain ATCC 367; the fermentation of the Lactobacillus brevis ATCC367 ⁇ glnR mutant strain 24h, GABA reached the highest yield, which was 100% shorter than the 48h of the wild strain ATCC 367; after 24h of fermentation, the production efficiency of the Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain was 0.72g/L/h, compared to 0.19 of the wild strain ATCC367 g/L/h, an increase of 280%.
  • the GABA synthesis capacity of the Lactobacillus brevis D17 ⁇ glnR mutant strain was 19.3g/L, which was only 4.9% higher than the 18.4g/L of the wild strain D17; but the Lactobacillus brevis D17 ⁇ glnR mutant strain was fermented for 24h, and GABA reached The highest yield is reduced to 50% compared with the 36h of the wild strain D17; the production efficiency of the Lactobacillus brevis D17 ⁇ glnR mutant strain is 0.76g/L/h after 24h fermentation, which is higher than the 0.55g/L/h of the wild strain D17 38.2%.
  • GlnR has a regulatory effect on the GABA synthesis ability of Lactobacillus brevis, and glnR knockout can significantly improve the GABA production efficiency of glnR knockout mutants.
  • Example 3 Application of Lactobacillus brevis glnR knockout bacteria to improve acid resistance
  • the activated Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain and the D17 ⁇ glnR mutant strain were respectively inoculated into GYP liquid medium and incubated at 37°C for 24 hours to prepare a first-level seed culture solution.
  • the first-level seed solution was inoculated into fresh GYP medium, and cultivated at 37°C ⁇ 200 rpm for 15 hours to prepare the second-level seed culture solution.
  • the secondary seed culture solution was inoculated into a GYP fermentation medium with 10 g/L glucose as a carbon source and 10 g/L glutamate (sodium) substrate at a 10% inoculum amount, and cultured at 37° C. ⁇ 200 rpm for 10 hours.
  • the cells were collected by centrifugation at 9000 rpm and 4°C for 10 min. After resuspending the bacteria in pH 7.0 potassium phosphate buffer, the supernatant was removed after centrifugation. Resuspend the bacteria in potassium phosphate buffer pH 7.0 containing 10 mM glutamate (sodium) to make the OD 600 of 1.0. When incubating at 37°C for 0h, 1h, 2h, 2.5h, immediately add 9 times the volume Potassium phosphate buffer containing 10 mM glutamate (sodium) pH 2.5 was placed in the bacterial suspension of different culture time, and the static culture was continued until the end of 3 hours.
  • Lactobacillus brevis D17 wild bacteria and D17 ⁇ glnR mutant strains were stimulated for 0.5h in an acid buffer without sodium glutamate, and there was no live bacteria at all.
  • the Lactobacillus brevis D17 ⁇ glnR mutant strain was added to 10mM glutamate (sodium) buffer for 0.5h, the colony number was 9.6 ⁇ 10 9 cfu/mL, which was an increase of 37.1 compared to the wild strain D17's 7.0 ⁇ 10 9 cfu/mL %; Lactobacillus brevis D17 ⁇ glnR mutant strain added 10mM glutamate (sodium) buffer for 1h, the number of colonies was 7.6 ⁇ 10 9 cfu/mL, which was an increase of 105.4 compared with the wild strain D17's 3.7 ⁇ 10 9 cfu/mL %; Lactobacillus brevis D17 ⁇ glnR mutant strain added 10mM glutamate (so
  • GlnR can regulate the glutamate-dependent acid tolerance of strains, and glnR knockout can effectively improve the acid tolerance of glnR knockout mutants.
  • the obtained highly acid-tolerant mutant strain is suitable for acidic food starters, such as rice wine brewing and sourdough production.
  • Example 4 Application of Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain using pH-adjusted batch fed fermentation to synthesize GABA
  • the activated Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain was inoculated into GYP liquid medium and cultured at 37°C for 24 hours to prepare a first-grade seed culture solution.
  • the first-level seed solution was inoculated into fresh GYP medium, and cultivated at 37°C ⁇ 200 rpm for 15 hours to prepare the second-level seed culture solution.
  • the secondary seed culture solution was inoculated with a 10% inoculum amount in a GYP fermentation medium with a pH of 5, 30 g/L glucose as a carbon source and 74.8 g/L glutamate (sodium).
  • the bioreactor was used to control the temperature at 37°C, and the non-aeration fermentation was carried out with 200 rpm stirring, and the pH was controlled to 5.0 on-line with 5mol/L sulfuric acid.
  • 74.8g (100mL) of glutamate (sodium) was fed every 6h and fermented for 72h.
  • HPLC analysis was used to detect the GABA content of the fermentation supernatant.
  • the wild strain ATCC 367 uses the same method to cultivate and test the GABA content of the fermentation supernatant.
  • Example 5 Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain uses pH-adjusted fed-batch fermentation to synthesize GABA with glucose
  • the activated Lactobacillus brevis ATCC 367 ⁇ glnR mutant strain or wild bacteria ATCC 367 on the slope into GYP liquid medium and cultivate at 37°C for 24 hours to prepare a first-grade seed culture solution.
  • the first-level seed solution was inoculated into fresh GYP medium, and cultivated at 37°C ⁇ 200 rpm for 15 hours to prepare the second-level seed culture solution.
  • the secondary seed culture solution was inoculated with 10% inoculum and connected to GYP fermentation medium with pH 5, 20g/L glucose as carbon source and 74.8g/L glutamate (sodium), and the temperature was controlled by a bioreactor At 37°C, 200rpm stirring was carried out without aeration, and the pH was controlled to 5.0 on-line with 5mol/L sulfuric acid. Feeding (glutamate (sodium)) 74.8g (100mL) every 6h within 6-36h, while feeding 150g/L glucose during 12-36h, flow rate is 0.625g/h, fermentation is 72h. HPLC analysis was used to detect the GABA content of the fermentation supernatant.
  • Example 6 Application of Lactobacillus brevis D17 ⁇ glnR mutant strain using pH-adjusted batch fed-feed fermentation to synthesize GABA
  • the first-level seed solution was inoculated into fresh GYP medium, and cultivated at 37°C ⁇ 200 rpm for 15 hours to prepare the second-level seed culture solution.
  • the secondary seed culture solution was inoculated with a 10% inoculum amount and connected to a GYP fermentation medium with pH 5, 50 g/L glucose as a carbon source and 74.8 g/L glutamate (sodium).
  • the bioreactor was used to control the temperature at 37°C, and the non-aeration fermentation was carried out with 200 rpm stirring, and the pH was controlled to 5.0 on-line with 5mol/L sulfuric acid. Feed (glutamate (sodium)) 74.8g (100mL) every 6h within 6-24h, and ferment for 72h. HPLC analysis was used to detect the GABA content of the fermentation supernatant.
  • Example 7 Application of GABA synthesis by batch fed-feed fermentation of L. brevis D17 ⁇ glnR mutant strain with pH-adjusted feeding glucose
  • the activated Lactobacillus brevis D17 ⁇ glnR mutant strain or the wild Lactobacillus brevis D17 on the slant in a GYP liquid medium and cultivate at 37°C for 24 hours to prepare a first-grade seed culture solution.
  • the first-level seed solution was inoculated into fresh GYP medium, and cultivated at 37°C ⁇ 200 rpm for 15 hours to prepare the second-level seed culture solution.
  • the secondary seed culture solution was inoculated with 10% inoculum and connected to GYP fermentation medium with pH 5, 30g/L glucose as carbon source and 74.8g/L glutamate (sodium), and the temperature was controlled by a bioreactor 37°C, 200 rpm agitated non-aeration fermentation, with 5 mol/L sulfuric acid to online control pH to 5.0. Feed 74.8g (100mL) of glutamate (sodium) every 6h within 6 ⁇ 36h, and feed 400g/L glucose at 12 ⁇ 36h at the same time, the flow rate is 2.58g/h, and the fermentation is 72h. HPLC analysis was used to detect the GABA content of the fermentation supernatant.

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Abstract

提供了一种通过同源重组的方式实现glnR基因的敲除，获得短乳杆菌glnR敲除株以提高短乳杆菌γ-氨基丁酸合成能力和高耐受酸性环境能力的方法及其应用。采用控酸流加补料法发酵短乳杆菌glnR敲除株，GABA的累计浓度为301.5g/L，生产效率最高达5.76g/L/h，可用于GABA的规模化生产，降低GABA的生产成本，用于酸性食品的发酵等。

Description

提高短乳杆菌γ-氨基丁酸合成能力的方法及其应用 技术领域
本发明涉及提高短乳杆菌γ-氨基丁酸合成能力的方法及其应用，属于微生物领域。
背景技术
γ-氨基丁酸(GABA)是作为一种重要的抑制性神经递质，对哺乳动物具有安神、降低血压、改善睡眠等多种生理功能，可作为生物活性物质广泛应用于食品、医药、饲料、化工等领域。食品级的GABA多由微生物采用生物法合成。微生物主要是谷氨酸脱羧酶(GAD)系统(包括谷氨酸/GABA的反转运体、谷氨酸脱羧酶)的作用，通过谷氨酸/GABA反转运体将胞外的谷氨酸运输至胞内，通过消耗H +，由胞内的谷氨酸脱羧酶对谷氨酸底物进行催化脱羧合成GABA。合成的GABA再通过谷氨酸/GABA反转运体将GABA运出胞内，实现GABA的积累，同时维持胞内pH的稳定，帮助菌株抵抗酸性环境的胁迫。
已报道的GABA合成菌株中，乳酸菌具有绝对的优势，尤其是短乳杆菌。前期研究中本研究室从白酒酿造体系中筛选出一株高效合成GABA的短乳杆菌D17，该菌株的生长与GABA的合成耦联，通过发酵策略优化(控制发酵pH)可提高菌株的生产效率(3.76g/L/h)。
现有提高GABA产量的方法主要针对高产GABA菌株的筛选，GABA生产发酵条件及培养基的优化，谷氨酸脱羧酶系统的过表达。但对于已有的GABA高产菌或低产菌而言，若要增加其工业化应用，继续提高其生产效率，需要有针对的调控其代谢途径。
发明内容
为了解决上述问题，本发明提供了一种提高短乳杆菌菌株γ-氨基丁酸合成能力的方法。
在本发明的研究过程中，发明人发现，虽然模式菌株短乳杆菌ATCC 367与短乳杆菌D17的谷氨酸脱羧系统的相关编码基因(谷氨酸/GABA的反转运体、谷氨酸脱羧酶(GAD)的基因)的氨基酸序列一致，但当菌株在5％的谷氨酸钠发酵时，短乳杆菌ATCC 367的GABA产量(10g/L)远低于短乳杆菌D17(17.8g/L)，且短乳杆菌ATCC 367与短乳杆菌D17的GABA产量并不随着谷氨酸钠的添加而继续增加，二者的GABA产量均受到限制。
谷氨酸(钠)不仅是GABA合成的唯一底物，同时也是微生物的氮源之一，它的代谢会受到一些调控子的控制，如TnrA，GlnR及CodY等。GlnR(谷氨酰胺合成酶抑制子)是广泛存在于革兰氏阳性菌中的一个重要的全局的氮源调控因子。它对氮源代谢的某些基因具有调控作用，尤其在链霉菌属、芽孢杆菌属中。在芽孢杆菌属中，当氮源充足时，GlnR会抑制glnRA操纵子的转录，降低氮源的利用。在链霉菌属中，GlnR不仅调控氮代谢且控制碳源代谢、磷 代谢，抗生素合成与菌株的耐受性(渗透压，耐酸、氧化)等。控制GABA的合成的GAD系统是微生物抵抗酸胁迫的耐酸系统，在谷氨酸氮源充足的情况下，可能受GlnR的阻遏调控。而且目前有关于乳酸菌的glnR基因敲除菌株未有报道。
本发明通过对短乳杆菌的glnR基因进行敲除，进一步解除了短乳杆菌GABA合成的限制，提高GABA产量，缩短GABA合成时间，进一步提高GABA的工业化生产效率。
本发明的第一个目的是提供一种提高短乳杆菌菌株γ-氨基丁酸合成能力的方法，所述方法是敲除短乳杆菌菌株中的glnR基因(谷氨酰胺合成酶抑制子基因)。
在一种实施方式中，所述glnR基因表达的氨基酸序列如SEQ ID NO:1所示。
在一种实施方式中，编码所述glnR基因的核苷酸序列如SEQ ID NO:2所示。
在一种实施方式中，所述短乳杆菌菌株为任意能够合成GABA的短乳杆菌。
在一种实施方式中，所述短乳杆菌菌株为短乳杆菌D17(保藏编号为CGMCC NO.14385)或者模式菌株短乳杆菌ATCC 367、短乳杆菌NCL912、短乳杆菌CGMCC1306、短乳杆菌NPS-QW-145等。在一种实施方式中，所述敲除，是使用任意一种已知的敲除方法，比如同源重组，二类内含子(Targetron或Clostron)基因插入失活、CRISPR-Cas基因编辑等。
在一种实施方式中，所述敲除是使用无痕敲除，尤其是基于同源重组的无标记基因敲除。
在一种实施方式中，所述敲除，具体是：将要敲除的glnR基因上、下游同源片段1000bp(包含glnR基因前后端各21bp)分别按一定方向连接到乳酸菌的整合质粒pGID023上，获得靶基因的敲除质粒pGID023-ΔglnR；将敲除质粒通过电转化进入短乳杆菌中，通过在红霉素抗性平板上筛选得到含敲除质粒的重组菌株。随后选择正确的重组菌株，在含红霉素的GYP液体培养基中，进行传代，再在无红霉素抗性的GYP液体培养基中传代，将传代后的菌液涂于含红霉素的抗性平板上，筛选获得发生第一次同源重组使质粒整合到受体菌染色体上的单交换子；然后将单交换子在无红霉素条件下，液体GYP培养基中传代培养，将传代后的菌液涂于无抗生素的GYP平板上，挑选单克隆直至筛选得到发生第二次同源重组的无红霉素抗性实现靶基因敲除的菌株。
上述pGID023质粒携带能在大肠杆菌中复制的复制原点、多克隆位点MCS、红霉素抗性基因。
上述敲除质粒是通过将短乳杆菌染色体上要敲除的靶基因上游和下游、大小约为1000bp的同源序列片段分别按基因排列方向酶切连接至pGID023的多克隆位点上。
上述敲除质粒通过电转化进入短乳杆菌中得到含敲除质粒的菌株的方法：将1μg敲除质粒与短乳杆菌感受态细胞混合，采用合适的电转化条件，将质粒电转化至短乳杆菌中。电击 后迅速加入预冷MRS培养基，于37℃培养箱中复苏。离心去除上清，细胞重悬后涂布含红霉素的MRS平板，37℃培养至长出转化子。挑取转化子进行菌液PCR验证。
上述发生第一次同源重组使质粒整合到受体菌染色体上的单交换子的验证方法是：将含敲除质粒的短乳杆菌的转化子接种至含有红霉素的MRS培养基中37℃静置培养24h。取培养后的菌液接种于上述的含红霉素的MRS液体培养基中，37℃培养24h后，按照此步骤进行传代培养。将传代后的菌液稀释10 -3～10 -6倍后涂布含有红霉素的MRS平板37℃培养，直至长出单菌落。挑取单菌落接种于含有红霉素的MRS培养基中，37℃培养24h，进行菌液PCR验证，筛选获得发生第一次同源重组使质粒整合到受体菌染色体上的潜在单交换子，再将潜在单交换子提取全基因组DNA，采用PCR及测序验证，确保获得正确敲除glnR基因的突变株。
上述筛选发生第二次同源重组实现靶基因敲除的菌株的方法是：将单交换子接种到不含红霉素的液体MRS培养基中，37℃培养24h。在无抗的MRS培养基中传代后，菌液稀释10 - 3～10 -6倍后涂布不含红霉素的MRS平板37℃培养，直至长出单菌落。挑取单菌落，接种于无红霉素抗性的MRS液体培养基中37℃培养24h，进行菌液PCR验证，从而得到glnR基因敲除的潜在突变株。再将潜在敲除株提取全基因组DNA，采用PCR及测序验证，确保获得正确敲除glnR基因的突变株。若未获得glnR基因敲除株，可继续传代，重复上述步骤，直至筛选获得glnR基因敲除株。
本发明的第二个目的是提供一种γ-氨基丁酸合成能力提高的短乳杆菌，所述短乳杆菌在出发菌株的基础上敲除了glnR基因。
在一种实施方式中，所述glnR基因表达的氨基酸序列如SEQ ID NO:1所示。
在一种实施方式中，编码所述glnR基因的核苷酸序列如SEQ ID NO:2所示。
在一种实施方式中，所述出发菌株为任意能够合成GABA的短乳杆菌，可选地，为短乳杆菌D17(保藏编号为CGMCC NO.14385)、模式菌株短乳杆菌ATCC 367、短乳杆菌NCL912、短乳杆菌CGMCC1306或者短乳杆菌NPS-QW-145等。
在一种实施方式中，所述敲除，可以使用任意一种已知的敲除方法，比如同源重组，二类内含子(Targetron或Clostron)基因插入失活、CRISPR-Cas基因编辑等。
本发明的第三个目的是提供一种合成γ-氨基丁酸的方法，所述方法是利用敲除了glnR基因的短乳杆菌进行发酵生产。
在一种实施方式中，所述发酵生产是将短乳杆菌接种于含有谷氨酸或谷氨酸钠的培养基中进行发酵生产。
在一种实施方式中，所述发酵生产可以是采用如下任意一种方法进行：
(1)将短乳杆菌glnR敲除株，接种于含有谷氨酸(钠)的液体发酵培养基中，进行发酵培养；
(2)发酵过程进行酸碱度调节的批次补料发酵：将短乳杆菌glnR敲除株接种于含谷氨酸(钠)的GYP发酵培养基中，pH控制为4.8-5.5，搅拌不通气条件下进行发酵；发酵6～24h间补充谷氨酸(钠)底物。优选地，每6h可进行谷氨酸(钠)底物添加；
(3)发酵过程进行酸碱度调节的流加葡萄糖的批次补料发酵：将短乳杆菌glnR敲除株接种于含谷氨酸(钠)的GYP发酵培养基中，pH控制为4.8-5.5，搅拌不通气条件下进行发酵；发酵6～36h间补充谷氨酸(钠)底物。优选地，每6h可进行谷氨酸(钠)底物添加，同时在6～36h流加葡萄糖碳源。
在一种实施方式中，所述发酵生产是采用如下任意一种方法进行：
(1)将短乳杆菌glnR敲除株分别接种于10g/L葡萄糖碳源，添加50g/L谷氨酸(钠)的GYP液体发酵培养基中，于37℃×200rpm培养条件下进行发酵。
(2)发酵过程进行酸碱度调节的批次补料发酵：采用生物反应器，将短乳杆菌ATCC367ΔglnR或D17ΔglnR突变株接种于含谷氨酸(钠)的GYP发酵培养基中。采用5mol/L浓度的硫酸将发酵液的pH控制为5.0，于37℃×200rpm搅拌不通气条件下进行发酵。为避免高浓度谷氨酸(钠)对菌体生长的抑制，于6～24h间补充谷氨酸(钠)底物。优选地，每6h可进行谷氨酸(钠)底物添加。发酵60h，ATCC 367ΔglnR的GABA的累计浓度为233.9g/L，发酵36h，生产效率最高达4.88g/L/h；发酵48h，D17ΔglnR的GABA的累计浓度为148.0g/L，发酵18h，生产效率最高达5.73g/L/h。
(3)发酵过程进行酸碱度调节的流加葡萄糖的批次补料发酵：采用生物反应器，将短乳杆菌ATCC 367ΔglnR或D17ΔglnR突变株接种于含谷氨酸(钠)的GYP发酵培养基中。采用5mol/L浓度的硫酸将发酵液的pH控制为5.0，于37℃×200rpm搅拌不通气条件下进行发酵。为避免高浓度谷氨酸(钠)对菌体生长的抑制，于6～36h间补充谷氨酸(钠)底物。优选地，ATCC367ΔglnR的发酵过程中，每6h可进行谷氨酸(钠)底物添加，同时在12～36h流加葡萄糖碳源，发酵60h，ATCC 367ΔglnR的GABA的累计浓度为282.80g/L，发酵36h，生产效率最高达4.93g/L/h；优选地，D17ΔglnR的发酵过程中，每6h可进行谷氨酸(钠)底物添加，同时在6～30h流加葡萄糖碳源，发酵60h，D17ΔglnR的GABA的累计浓度为301.5g/L，发酵36h，生产效率最高达5.76g/L/h。
本发明的第四个目的是提供一种提高产γ-氨基丁酸的短乳杆菌的耐酸能力的方法，所述 方法是敲除短乳杆的菌glnR基因。
本发明的第五个目的是提供所述重组菌或者所述方法，在食品、制备药物、饲料、化工等领域的应用。
在一种实施方式中，所述食品为酸性发酵食品；比如黄酒或者酸面团生产。
本发明的第六个目的是提供所述重组菌或者所述方法，在制备对哺乳动物具有安神、降低血压、改善睡眠的药物或者保健品方面的应用。
生物材料：
短乳杆菌D17，已经在公布号CN 108034599A、公开日为2018年05月15日的专利申请2017112754179中公开；其分类学命名为短乳杆菌Lactobacillus brevis，于2017年7月6日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.14385。
模式菌株短乳杆菌ATCC 367，购自中国微生物保藏中心编号CGMCC1.2028，也可以从美国模式培养物研究所编号ATCC 367购买得到。
本发明涉及的短乳杆菌NCL912、短乳杆菌CGMCC1306及短乳杆菌NPS-QW-145已在本专利申请之前的文献中公开(Li,H.X.,Gao,D.D.,et al.A highγ-aminobutyric acid-producing Lactobacillus brevis isolated from Chinese traditional paocai[J].2008,Annals of Microbiology,58(4):649-653.Peng,C.L.,Huang,J.,et al.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.)。
整合质粒pGID023，如图1所示，已经在1994年的文献中公开(Pascal Hols,Thierry Ferain,Dominique Garmyn,et al.Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression.Applied Environmental Microbiology 1994，60:1401-1413.)。
本发明的有益效果：
本发明首次发现glnR基因的缺失可显著提高短乳杆菌的GABA合成能力，且通过获得的短乳杆菌的glnR基因敲除菌株ATCC 367ΔglnR与短乳杆菌D17ΔglnR，发现该方法具有通用性，原则上适合所有的短乳杆菌。本发明利用敲除了glnR基因的菌株生产GABA，可以大幅度提高短乳杆菌GABA生产效率或产量、缩短发酵周期，同时可显著提高菌株在酸性环境 中的存活能力，菌株也适用于黄酒酿造、酸面团生产等酸性食品的发酵。
附图说明
图1.用于基因敲除的pGID023质粒；
图2.敲除质粒pGID023-ΔglnR的构建；上图为glnR基因上下游同源臂扩增，下图为敲除质粒pGID023-ΔglnR；
图3.短乳杆菌glnR基因敲除的流程示意图；
图4.短乳杆菌ATCC 367及D17glnR基因敲除的PCR验证图；
附图4A中的标记为：M:500bp Marker；6:采用引物对F-glnR与R-glnR扩增短乳杆菌D17基因组；9:采用引物对F-glnR与R-glnR扩增短乳杆菌ATCC 367基因组；1～5:提取潜在短乳杆菌D17的glnR敲除菌株的基因组，采用引物对F-glnR与R-glnR扩增；7～8:提取潜在短乳杆菌ATCC 367的glnR敲除菌株的基因组，采用引物对F-up-glnR-BamH与R-down-glnR-Hind3扩增。
附图4B中的标记为：M:5kb Marker；1:采用引物对F-up-glnR-BamH与R-down-glnR-Hind3扩增短乳杆菌D17基因组；7.采用引物对F-up-glnR-BamH与R-down-glnR-Hind3扩增短乳杆菌ATCC 367基因组；2～6:提取潜在短乳杆菌D17的glnR敲除菌株的基因组，采用引物对F-up-glnR-BamH与R-down-glnR-Hind3扩增；8～9:提取潜在短乳杆菌ATCC 367的glnR敲除菌株的基因组，采用引物对F-up-glnR-BamH与R-down-glnR-Hind3扩增。
图5.短乳杆菌ATCC 367及D17的glnR基因敲除的测序验证；
图6.glnR基因的缺失对乳杆菌GABA合成的影响；附图6A中的标记为：367代表短乳杆菌ATCC 367野生菌；ΔglnR代表短乳杆菌ATCC 367ΔglnR突变株；附图6B中的标记为：367代表短乳杆菌D17野生菌；ΔglnR代表短乳杆菌D17ΔglnR突变株。
图7.短乳杆菌glnR敲除菌在极端酸性环境的存活；附图7A中的标记为：367代表短乳杆菌ATCC 367野生菌；ΔglnR代表短乳杆菌ATCC 367ΔglnR突变株；附图7B中的标记为：367代表短乳杆菌D17野生菌；ΔglnR代表短乳杆菌D17ΔglnR突变株。
图8.短乳杆菌ATCC 367ΔglnR突变株采用酸碱度调节的批次补料发酵合成GABA；附图8中的标记为：367代表短乳杆菌ATCC 367野生菌；ΔglnR代表短乳杆菌ATCC 367ΔglnR突变株。
图9.短乳杆菌ATCC 367ΔglnR突变株采用酸碱度调节的流加葡萄糖的批次补料发酵合成GABA；附图9中的标记为：367代表短乳杆菌ATCC 367野生菌；ΔglnR代表短乳杆菌ATCC 367ΔglnR突变株。
图10.短乳杆菌D17ΔglnR突变株采用酸碱度调节的批次补料发酵合成GABA；附图10中的标记为：D17代表短乳杆菌D17野生菌；ΔglnR代表短乳杆菌D17ΔglnR突变株。
图11.短乳杆菌D17ΔglnR突变株采用酸碱度调节的流加葡萄糖的批次补料发酵合成GABA；附图11中的标记为：D17代表短乳杆菌D17野生菌；ΔglnR代表短乳杆菌D17ΔglnR突变株。
具体实施方式
MRS培养基：葡萄糖2％，酵母浸粉0.4％，蛋白胨1％，牛肉膏0.5％，吐温80 0.1％，乙酸钠0.5％，磷酸氢二钾0.2％，柠檬酸三铵0.2％，硫酸镁0.02％，硫酸锰0.05％，均为质量-体积分数，调节pH为6.2；固体培养基中添加2％浓度的琼脂。
含红霉素的MRS液体培养基：在MRS培养基基础上加入终浓度为1μg/mL的红霉素。
含红霉素的MRS平板：在MRS培养基基础上加入终浓度为4μg/mL的红霉素。
复苏液：葡萄糖2％，酵母浸粉0.4％，蛋白胨1％，牛肉膏0.5％，吐温80 0.1％，乙酸钠0.5％，磷酸氢二钾0.2％，柠檬酸三铵0.2％，硫酸镁0.02％，硫酸锰0.05％，蔗糖10.26％，均为质量-体积分数。
LB培养基(g/L)：液体：酵母浸粉5，蛋白胨10，氯化钠10；固体培养基中添加2％浓度的琼脂。
含红霉素的LB培养基：在LB培养基基础上加入终浓度200μg/mL的红霉素。
GYP培养基：葡萄糖1％，酵母浸粉1％，蛋白胨0.5％，乙酸钠0.2％，硫酸镁0.02％，硫酸锰0.01％，硫酸亚铁0.01％，氯化钠0.01％，均为质量-体积分数；固体培养基中添加2％浓度的琼脂，发酵培养基中添加不同浓度谷氨酸钠。
耐酸能力测试：将短乳杆菌野生菌及glnR敲除株分别接种于10g/L葡萄糖碳源，10g/L谷氨酸(钠)的发酵培养基中，37℃发酵10h。离心收集细胞，采用pH 7.0的磷酸钾缓冲洗菌1次。并用含有或不含10mM谷氨酸(钠)的pH 7.0的磷酸钾缓冲重悬菌体，使其OD 600为1.0。37℃分别静置培养0h，1h，2h，2.5h时，立即加入9倍体积含有或不含10mM谷氨酸钠pH 2.5的磷酸钾缓冲于菌悬液中，继续静置培养至3h结束。取出样品立即用pH 7.4的PBS缓冲按10 -1～10 -5稀释，选择合适的梯度取100μL稀释液涂于GYP平板上，37℃静置培养24h。
实施例1：短乳杆菌glnR基因的无痕敲除
根据短乳杆菌的模式菌株ATCC 367或D17中glnR基因的上下游DNA序列，glnR基因的上游与下游保留各21bp(防止基因全切除影响上下游同源臂基因表达)，设计引物对F-up-glnR- BamHI与R-up，F-down与R-down-glnR-HindIII。以短乳杆菌ATCC 367或D17的基因组DNA为模板，分别用引物对F-up-glnR-BamHI与R-up和F-down与R-down-glnR-HindIII扩增glnR基因的上下游同源序列up-glnR(序列如SEQ ID NO:3所示)、down-glnR(序列如SEQ ID NO:4所示)。PCR条件为98℃预变性30s；98℃变性10s，55℃退火15s，72℃延伸1min，30个循环；72℃后延伸10min。采用Omega胶回收试剂盒回收上下游同源臂各1000bp。取等摩尔up-glnR、down-glnR片段加水补足为5μL，再加入5μL的Takara的PrimeSTAR Max DNA Polymerase，PCR条件为98℃预变性30s；98℃变性10s，55℃退火2min，72℃延伸2min，15个循环；72℃后延伸10min。以得到的PCR产物为模板，采用引物F-up-glnR-BamHI、R-down-glnR-HindIII继续进行PCR。PCR条件为98℃预变性30s；98℃变性10s，55℃退火15s，72℃延伸2min，30个循环；72℃后延伸10min。采用重叠PCR方法得到上下游同源臂的拼接片段up-down-glnR(序列如SEQ ID NO:5所示)。
利用BamHI和HindIII双酶切up-down-glnR片段和质粒pGID023，之后将酶切产物于16℃连接16h后，转化至大肠杆菌JM109感受态中。经菌液PCR、酶切及测序鉴定获得阳性菌株，进而得到将glnR基因上下游同源序列克隆至pGID023的基因敲除质粒pGID023-ΔglnR(如图2所示)。
(1)发生第一次同源重组的单交换菌株的筛选
将构建好的基因敲除质粒pGID023-ΔglnR电转化入短乳杆菌ATCC 367或短乳杆菌D17的感受态细胞，其转化条件为电压2000V～2500V电击5ms，将菌液涂布在含红霉素(终浓度为4μg/μL)的MRS平板上37℃培养，直至长出单克隆。挑选单克隆，用引物对F-GID与R-GID进行PCR验证。挑选转入敲除质粒的正确重组菌。将重组菌单菌落接种至含有红霉素抗性的MRS液体培养基中传代，促进单交换整合的发生。将传代后的菌液稀释后，涂于含红霉素(终浓度为4μg/mL)的MRS平板上，挑取长出的抗性单菌落，接至含红霉素(终浓度为1μg/mL)的MRS培养基中37℃培养24h。取200μL菌液离心去除发酵上清，加入20μL去离子水重悬，并加入0.1g的玻璃粉(0.1mm)，采用珠磨仪破碎2min后，离心取上清进行菌液PCR验证。采用多对引物对F-367-0092与R-367-0090(同源臂两端设计的引物)、F-GID与R-GID、F-367-0092与R-GID、F-GID与R-367-0090验证，PCR条件为98℃预变性30s；98℃变性10s，55℃退火15s，72℃延伸2min，30个循环；72℃后延伸10min。确定筛选到正确的单交换子(单交换方式如图3所示)。
(2)发生第二次同源重组的基因敲除菌株的筛选
将单交换子接种到不含抗性的液体MRS培养基中，在无抗的情况下通过传代，促进染色 体内的重组。将连续传代后的菌液稀释后，涂布于无抗性的平板。挑取平板上长出的单菌落接种至MRS液体培养基中37℃培养24h，采用上述的方法破碎细胞，取上清进行菌液PCR验证。引物对为F-up-glnR-BamHI与R-down-glnR-HindIII，PCR条件为98℃预变性30s；98℃变性10s，55℃退火15s，72℃延伸2min，30个循环；72℃后延伸10min。筛选出潜在的敲除菌株后，提取潜在敲除菌株的基因组DNA，用引物对F-up-glnR-BamHI与R-down-glnR-HindIII和F-glnR与R-glnR(根据glnR基因内部序列设计引物)进行PCR扩增(如图4所示)并测序验证(如图5所示)。
在发生第二次同源重组的双交换子中，一种是回复突变成野生型，另一种就是glnR基因敲除株(图3)。
如图4A所示，采用引物对F-up-glnR-BamHI与R-down-glnR-HindIII，利用ΔglnR突变株的基因组只能扩增出约2000bp大小的PCR产物(含up-glnR和down-glnR)，而利用野生菌基因组可扩增出约2300bp大小的片段，说明在ΔglnR突变株中缺失glnR。如图4B所示采用引物对F-glnR与R-glnR，利用ΔglnR突变株的基因组无法扩增出PCR产物，说明glnR缺失，而利用野生菌基因组可扩增出约300bp大小的片段。
对野生菌与ΔglnR突变株的glnR基因进行测序，glnR缺失片段如图5所示。
通过该方法得到的正确的glnR基因敲除的短乳杆菌ATCC 367ΔglnR突变株及短乳杆菌D17ΔglnR突变株。
表1引物
Figure PCTCN2020091049-appb-000001
实施例2：短乳杆菌glnR基因敲除菌合成GABA的应用
在无菌条件下，将-80℃保存的短乳杆菌ATCC 367野生菌、ATCC 367ΔglnR突变株、短乳杆菌D17野生菌、D17ΔglnR突变株取出，分别划线于GYP固体平板上，37℃静置培养。
待单菌落长出后从活化的GYP固体平板上挑取短乳杆菌ATCC 367野生菌、ATCC 367ΔglnR突变株及D17野生菌、D17ΔglnR突变株单菌落，接种于GYP种子培养基中，37℃静置培养24h。取种子培养液，按10％的接种量接种于新的GYP种子培养基中，37℃静置培养12h后，培养液作为发酵种子。在250mL三角瓶中装入100mL的添加10g/L葡萄糖碳源和50g/L谷氨酸(钠)的GYP发酵培养基，取培养12h的发酵种子按10％的接种量接种，于37℃×200rpm培养48h。发酵上清液经高效液相色谱检测GABA含量。
结果显示：如图6A所示，短乳杆菌ATCC 367ΔglnR突变株的GABA合成能力18.0g/L，相比野生菌ATCC 367的11.8g/L，提高了52.5％；短乳杆菌ATCC 367ΔglnR突变株发酵24h,GABA达到最高产量，相比野生菌ATCC 367的48h，缩短了100％；发酵24h，短乳杆菌ATCC 367ΔglnR突变株的生产效率为0.72g/L/h，相比野生菌ATCC 367的0.19g/L/h，提高了280％。
如图6B所示，短乳杆菌D17ΔglnR突变株的GABA合成能力19.3g/L，相比野生菌D17的18.4g/L，仅提高了4.9％；但短乳杆菌D17ΔglnR突变株发酵24h,GABA达到最高产量，相比野生菌D17的36h，缩短到了50％；发酵24h，短乳杆菌D17ΔglnR突变株的生产效率为0.76g/L/h，相比野生菌D17的0.55g/L/h，提高了38.2％。
综上所述，GlnR对短乳杆菌的GABA合成能力有调控作用，glnR敲除后可显著提高glnR敲除突变株的GABA生产效率。
实施例3：短乳杆菌glnR基因敲除菌提高耐酸性的应用
将斜面上活化的短乳杆菌ATCC 367ΔglnR突变株与D17ΔglnR突变株，分别接种于GYP液体培养基中，37℃静置培养24h，制成一级种子培养液。按10％的接种量，将一级种子液接种于新鲜的GYP培养基中，37℃×200rpm培养15h，制成二级种子培养液。将二级种子培养液按10％接种量，接种于10g/L葡萄糖为碳源，10g/L谷氨酸(钠)底物的GYP的发酵培养基中，37℃×200rpm培养10h。9000rpm，4℃离心10min收集细胞。采用pH 7.0的磷酸钾缓冲液重悬菌体后，离心后去除上清。用含有10mM谷氨酸(钠)的pH 7.0的磷酸钾缓冲液重悬菌体，使其OD 600为1.0。37℃静置培养0h，1h，2h，2.5h时，立即分别加入9倍体积含 有10mM谷氨酸(钠)pH 2.5的磷酸钾缓冲液于不同培养时间的菌悬液中，继续静置培养至3h结束。同样的，用不含谷氨酸(钠)的pH 7.0的磷酸钾缓冲液重悬菌体，使其OD 600为1.0。37℃静置培养0h，1h，2h，2.5h时，立即分别加入9倍体积不含谷氨酸(钠)pH 2.5的磷酸钾缓冲液于不同培养时间的菌悬液中，继续静置培养至3h结束。取出所以酸刺激的样品立即用pH 7.4的PBS缓冲液按10 -1～10 -5梯度稀释，选择合适的梯度，取100μL稀释液涂于GYP平板上，37℃静置培养24h，记录平板上的菌落数。
结果显示：如图7A所示，短乳杆菌ATCC 367野生菌与ATCC 367ΔglnR突变株，在未添加谷氨酸钠的酸性缓冲中0.5h后，完全没有活菌存在。而短乳杆菌ATCC 367ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中0.5h后，菌落数为6.8×10 9cfu/mL，相比野生菌ATCC 367的4.4×10 9cfu/mL，提高了54.5％；短乳杆菌ATCC 367ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中1h后，菌落数为4.9×10 9cfu/mL，相比野生菌ATCC 367的2.2×10 9cfu/mL，提高了122.7％；短乳杆菌ATCC 367ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中2h后，菌落数为2.4×10 9cfu/mL，相比野生菌ATCC 367的1.2×10 9cfu/mL，提高了100％；短乳杆菌ATCC 367ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中3h后，菌落数为1.5×10 9cfu/mL，相比野生菌ATCC 367的2.8×10 8cfu/mL，提高了435.7％；说明通过在缓冲中添加谷氨酸(钠)，短乳杆菌ATCC 367ΔglnR突变株的耐酸能力显著高于野生菌ATCC 367。
如图7B所示，短乳杆菌D17野生菌与D17ΔglnR突变株，在未添加谷氨酸钠的酸性缓冲中刺激0.5h后，完全没有活菌存在。而短乳杆菌D17ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中0.5h后，菌落数为9.6×10 9cfu/mL，相比野生菌D17的7.0×10 9cfu/mL，提高了37.1％；短乳杆菌D17ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中1h后，菌落数为7.6×10 9cfu/mL，相比野生菌D17的3.7×10 9cfu/mL，提高了105.4％；短乳杆菌D17ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中2h后，菌落数为4.9×10 9cfu/mL，相比野生菌D17的2.2×10 9cfu/mL，提高了122.7％；短乳杆菌D17ΔglnR突变株在添加10mM谷氨酸(钠)缓冲中3h后，菌落数为2.6×10 9cfu/mL，相比野生菌D17的9.0×10 8cfu/mL，提高了188.9％；说明通过添加10mM谷氨酸(钠)，短乳杆菌D17ΔglnR突变株的耐受极端酸性环境的能力显著高于野生菌D17。
综上说明GlnR可调控菌株的谷氨酸依赖的酸耐受性，glnR敲除后可有效提高glnR敲除突变株的耐酸性。获得的高耐酸的突变株适用于酸性的食品的发酵剂，如黄酒酿造，酸面团生产等。
实施例4：短乳杆菌ATCC 367ΔglnR突变株采用酸碱度调节的批次补料发酵合成GABA的应用
将斜面上活化的短乳杆菌ATCC 367ΔglnR突变株，接种于GYP液体培养基中，37℃静置培养24h，制成一级种子培养液。按10％的接种量，将一级种子液接种于新鲜的GYP培养基中，37℃×200rpm培养15h，制成二级种子培养液。将二级种子培养液按10％接种量，接于pH为5，30g/L葡萄糖为碳源及含有74.8g/L谷氨酸(钠)的GYP发酵培养基中。使用生物反应器控制温度37℃，进行200rpm搅拌不通气发酵，以5mol/L硫酸在线控制pH为5.0。在6～24h内，每6h补料(谷氨酸(钠))74.8g(100mL)，发酵72h。采用HPLC分析检测发酵上清液的GABA含量。野生菌ATCC 367采用同样的方法培养和测试发酵上清液的GABA含量。
结果显示，如附图8所示，发酵60h，短乳杆菌ATCC 367ΔglnR突变株的GABA合成能力产量最高，为233.9g/L，相比野生菌ATCC 367的10.2g/L，提高了2193％；发酵36h，短乳杆菌ATCC 367ΔglnR突变株的生产效率最高为4.88g/L/h，相比野生菌ATCC 367的0.20g/L/h，提高了2340％。
实施例5：短乳杆菌ATCC 367ΔglnR突变株采用酸碱度调节的流加葡萄糖的批次补料发酵合成GABA的应用
将斜面上活化的短乳杆菌ATCC 367ΔglnR突变株或野生菌ATCC 367，接种于GYP液体培养基中，37℃培养24h，制成一级种子培养液。按10％的接种量，将一级种子液接种于新鲜的GYP培养基中，37℃×200rpm培养15h，制成二级种子培养液。将二级种子培养液按10％接种量，接于pH为5，20g/L葡萄糖为碳源并含74.8g/L谷氨酸(钠)的GYP发酵培养基中，使用生物反应器控制温度37℃，进行200rpm搅拌不通气发酵，以5mol/L硫酸在线控制pH为5.0。在6～36h内每6h补料(谷氨酸(钠))74.8g(100mL)，同时在12～36h流加150g/L葡萄糖，流速为0.625g/h，发酵72h。采用HPLC分析检测发酵上清液的GABA含量。
结果显示，如附图9所示，发酵60h，短乳杆菌ATCC 367ΔglnR突变株的GABA合成能力产量最高，为282.8g/L，相比野生菌ATCC 367的14.1g/L，提高了1906％；发酵36h，短乳杆菌ATCC 367ΔglnR突变株的生产效率最高为4.93g/L/h，相比野生菌ATCC 367的0.21g/L/h，提高了2248％。
实施例6：短乳杆菌D17ΔglnR突变株采用酸碱度调节的批次补料发酵合成GABA的应用
将斜面上活化的短乳杆菌D17ΔglnR突变株或野生菌短乳杆菌D17，接种于GYP液体培养基中，37℃培养24h，制成一级种子培养液。按10％的接种量，将一级种子液接种于新鲜的GYP培养基中，37℃×200rpm培养15h，制成二级种子培养液。将二级种子培养液按10％接种量，接于pH为5，50g/L葡萄糖为碳源含74.8g/L谷氨酸(钠)的GYP发酵培养基中。使用生物反应器控制温度37℃，进行200rpm搅拌不通气发酵，以5mol/L硫酸在线控制pH为5.0。在6～24h内每6h补料(谷氨酸(钠))74.8g(100mL)，发酵72h。采用HPLC分析检测发酵上清液的GABA含量。
结果如附图10所示，发酵60h，短乳杆菌D17ΔglnR突变株的GABA合成能力产量最高，为148.0g/L，相比野生菌D17的116.1g/L，提高了27.4％；发酵18h，短乳杆菌D17ΔglnR突变株的生产效率最高为5.73g/L/h，相比野生菌D17的3.39g/L/h，提高了70.0％。
实施例7：短乳杆菌D17ΔglnR突变株采用酸碱度调节的流加葡萄糖的批次补料发酵合成GABA的应用
将斜面上活化的短乳杆菌D17ΔglnR突变株或野生菌短乳杆菌D17，接种于GYP液体培养基中，37℃培养24h，制成一级种子培养液。按10％的接种量，将一级种子液接种于新鲜的GYP培养基中，37℃×200rpm培养15h，制成二级种子培养液。将二级种子培养液按10％接种量，接于pH为5，30g/L葡萄糖为碳源并含74.8g/L谷氨酸(钠)的GYP发酵培养基中，使用生物反应器控制温度37℃，进行200rpm搅拌不通气发酵，以5mol/L硫酸在线控制pH为5.0。在6～36h内每6h补料(谷氨酸(钠))74.8g(100mL)，同时在12～36h流加400g/L葡萄糖，流速为2.58g/h，发酵72h。采用HPLC分析检测发酵上清液的GABA含量。
结果如附图11所示，发酵60h，短乳杆菌D17ΔglnR突变株的GABA合成能力产量最高，为301.5g/L，相比野生菌D17的180.7g/L，提高了66.8％；发酵36h，短乳杆菌D17ΔglnR突变株的生产效率最高为5.76g/L/h，相比野生菌D17的4.93g/L/h，提高了17.0％。

Claims (20)

  1. 一种γ-氨基丁酸合成能力提高的短乳杆菌，其特征在于，所述短乳杆菌在出发菌株的基础上敲除了glnR基因；所述glnR基因表达的氨基酸序列如SEQ ID NO:1所示；编码所述glnR基因的核苷酸序列如SEQ ID NO:2所示；所述出发菌株为短乳杆菌D17、模式菌株短乳杆菌ATCC 367、短乳杆菌NCL912、短乳杆菌CGMCC1306或短乳杆菌NPS-QW-145。
  2. 一种γ-氨基丁酸合成能力提高的短乳杆菌，其特征在于，所述短乳杆菌在出发菌株的基础上敲除了glnR基因。
  3. 根据权利要求2所述的短乳杆菌，其特征在于，所述glnR基因表达的氨基酸序列如SEQ ID NO:1所示。
  4. 根据权利要求3所述的短乳杆菌，其特征在于，编码所述glnR基因的核苷酸序列如SEQ ID NO:2所示。
  5. 根据权利要求2所述的短乳杆菌，其特征在于，所述出发菌株为任意能够合成GABA的短乳杆菌。
  6. 根据权利要求5所述的短乳杆菌，其特征在于，所述能够合成GABA的短乳杆菌为短乳杆菌D17、模式菌株短乳杆菌ATCC 367、短乳杆菌NCL912、短乳杆菌CGMCC1306或者短乳杆菌NPS-QW-145。
  7. 根据权利要求1-6任一所述的短乳杆菌，其特征在于，所述敲除，可以使用任意一种已知的敲除方法；比如同源重组，二类内含子(Targetron或Clostron)基因插入失活、CRISPR-Cas基因编辑等；可选地，所述敲除是使用无痕敲除，尤其是基于同源重组的无标记基因敲除。
  8. 一种提高短乳杆菌菌株γ-氨基丁酸合成能力的方法，其特征在于，所述方法是敲除短乳杆菌菌株中的glnR基因。
  9. 根据权利要求8所述的方法，其特征在于，所述glnR基因表达的氨基酸序列如SEQ ID NO:1所示。
  10. 一种合成γ-氨基丁酸的方法，其特征在于，所述方法是利用权利要求1-6任一所述的敲除了glnR基因的短乳杆菌进行发酵生产。
  11. 根据权利要求10所述的方法，其特征在于，所述发酵生产是将短乳杆菌接种于含有谷氨酸或谷氨酸钠的培养基中进行发酵生产。
  12. 根据权利要求10所述的方法，其特征在于，将短乳杆菌glnR敲除株，接种于含有谷氨酸(钠)的液体发酵培养基中，进行发酵培养。
  13. 根据权利要求10所述的方法，其特征在于，发酵过程进行酸碱度调节的批次补料发 酵：将短乳杆菌glnR敲除株接种于含谷氨酸(钠)的GYP发酵培养基中，pH控制为4.8-5.5，搅拌但不通气条件下进行发酵；发酵6～24h间补充谷氨酸(钠)底物。优选地，每6h可进行谷氨酸(钠)底物添加。
  14. 根据权利要求10所述的方法，其特征在于，发酵过程进行酸碱度调节的流加葡萄糖批次补料发酵：将短乳杆菌glnR敲除株接种于含谷氨酸(钠)的GYP发酵培养基中，pH控制为4.8-5.5，搅拌不通气条件下进行发酵；发酵6～36h间补充谷氨酸(钠)底物。
  15. 根据权利要求14所述的方法，其特征在于，每6h可进行谷氨酸(钠)底物添加，同时在6～36h流加葡萄糖碳源，流加速率0.6～3g/h。
  16. 一种提高产γ-氨基丁酸的短乳杆菌的耐酸能力的方法，其特征在于，所述方法是敲除短乳杆菌的glnR基因。
  17. 权利要求1-6任一所述短乳杆菌或者权利要求7-16任一所述方法，在食品、保健品、制备药物、饲料、化工等领域的应用。
  18. 根据权利要求17所述的应用，其特征在于，所述食品为酸性发酵食品。
  19. 根据权利要求18所述的应用，其特征在于，所述酸性发酵食品包括黄酒或者酸面团。
  20. 根据权利要求17所述的应用，其特征在于，所述药物为对哺乳动物具有安神、降低血压、改善睡眠的药物。
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