A genetically engineered bacterium of Clostridium tyrobutyricum capable of synthesizing GABA and its construction method and application

Abstract

The invention discloses a Clostridium tyrobutyricum genetic engineering bacterium capable of synthesizing GABA, and a construction method and application thereof. The Clostridium tyrobutyricum genetic engineering bacterium is a Clostridium tyrobutyricum genetic engineering bacterium that overexpresses glutamate decarboxylase and Class I heat shock protein GroESL. The strain can catalyze sodium glutamate to efficiently synthesize GABA under anaerobic conditions, and can produce 122.34 g / L GABA when glucose and sodium glutamate are used as substrates, and no expensive coenzyme pyridoxal phosphate needs to be added during the fermentation process. The invention provides an effective method for Clostridium tyrobutyricum to produce GABA, and lays a foundation for the industrialization of metabolic engineering transformation of Clostridium tyrobutyricum to produce GABA.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Clostridium tyrobutyricum genetic engineering bacterium capable of synthesizing GABA, and a construction method and application thereof. Background Art

[0002] γ-Aminobutyric acid (GABA) is a four-carbon, non-protein amino acid that is widely found in vertebrates, plants, and microorganisms. It is an important inhibitory neurotransmitter in the central nervous system of mammals (CUI Y, MIAO K, NIYAPHORN S, et al. Production of Gamma-Aminobutyric Acid from Lactic Acid Bacteria: A Systematic Review [J]. Int J Mol Sci, 2020, 21 (3)). GABA can improve brain cell metabolism, enhance immunity, reduce anxiety, treat epilepsy, improve liver and kidney function, lower blood pressure, and prevent diabetes. GABA can also improve sleep. Related products have been launched and are known as the "third generation of sleeping pills." At present, most Chinese and Western medicine formulas for the treatment of brain diseases contain GABA, which has few side effects and is safe and effective. GABA has become a new functional factor in modern society and is gradually being widely used in medicine, food, health care, chemical industry, agriculture and other industries. With the deepening of relevant research, scholars have gradually enhanced their understanding of the physiological functions of GABA, its safety has been recognized, and it has broad prospects in the field of food processing.

[0003] GABA production methods include chemical synthesis, plant enrichment and biological fermentation (SARASA SB, MAHENDRAN R, MUTHUSAMY G, et al. A Brief Review on the Non-protein Amino Acid, Gamma-amino Butyric Acid (GABA): Its Production and Role in Microbes [J]. Curr Microbiol, 2020, 77 (4): 534-44). Chemical synthesis is the traditional production method of GABA, which mainly includes the following three methods: γ-chlorobutyric acid method, using potassium phthalimide and γ-chlorobutyric acid as raw materials to react under strong conditions, and the resulting product is reacted with concentrated sulfuric acid and then hydrolyzed to obtain the product GABA; γ-butyrolactone and thionyl chloride method, using 1-butyrolactone and thionyl chloride as raw materials to open the ring and chlorinate at normal pressure to obtain 4-chlorobutyryl chloride, and then esterify with methanol to obtain 4-chlorobutyric acid methyl ester, and then aminize and hydrolyze with ammonia solution under the action of a catalyst to obtain GABA after separation and purification; under alkaline conditions, 2-pyrrolidone undergoes a ring opening reaction, and then decolorizes, recrystallizes, and purifies to obtain GABA. The GABA produced by the above processes has high purity, but the production process has high energy consumption, strict temperature requirements, environmental pollution, poor safety, high cost, and does not conform to the concept of green production.

[0004] The plant enrichment method is to enrich GABA through stress metabolism of plant tissues. After enrichment, grains such as rice, beans, wheat and other economic crops can be directly processed into food, functional beverages and livestock feed, which is a potential field for deep processing of grains. However, the content of GABA enriched in plants is usually low, and it is difficult to separate and purify. For example, the mass fraction of GABA in the enriched GABA tea is only 0.401%, and the mass fraction of GABA in germinated brown rice is only 0.042%. Therefore, the plant enrichment method is not suitable for large-scale production of GABA.

[0005] The biological fermentation method is to convert glutamate or glutamate salt into GABA under the catalysis of glutamate decarboxylase (GAD). This method has the advantages of high specificity, simple equipment, environmental protection, low cost, diverse production strains and easy availability, and is suitable for large-scale industrial production (LI H, QIU T, GAO D, et al. Medium optimization for production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 [J]. Amino Acids, 2010, 38 (5): 1439-45).

[0006] The production strains of GABA mainly include Escherichia coli, lactic acid bacteria and Corynebacterium glutamicum, and usually need to pass a large amount of oxygen during the fermentation process. Compared with aerobic fermentation, anaerobic fermentation technology has the advantages of easy technical operation, low operating cost and stable process. At present, there are few reports on the production of GABA under anaerobic conditions, and there is no research on the use of Clostridium to synthesize GABA. Clostridium tyrobutyricum (Clostridium tyrobutyricum) is a new type of strict anaerobic microbial cell factory. It can accumulate a large amount of biomass under anaerobic conditions, has few by-products, and has a clear genetic background. It belongs to non-pathogenic bacteria. The natural Clostridium tyrobutyricum genome lacks GABA synthesis metabolic pathways and glutamate decarboxylase, and cannot synthesize GABA. Therefore, Clostridium tyrobutyricum was transformed through metabolic engineering to catalyze monosodium glutamate (MSG) to produce GABA with high added value; further overexpression of Class 1 heat shock protein GroESL improved the strain's MSG tolerance and GABA production. This bacterium has a wide range of application value in the industrial production of GABA and is of great significance to improving the market competitiveness of bio-based GABA. Summary of the invention

[0007] Currently, there are few reports on the synthesis of GABA by obligate anaerobic microorganisms. The primary purpose of the present invention is to provide a genetically engineered bacterium of Clostridium tyrobutyricum that can synthesize GABA, which overexpresses glutamate decarboxylase GAD and Class 1 heat shock protein GroESL.

[0008] Another object of the present invention is to provide a method for constructing the above-mentioned Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA.

[0009] Another object of the present invention is to provide the application of the above-mentioned Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA.

[0010] The object of the present invention is achieved through the following technical scheme: a genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA is Clostridium tyrobutyricum (Clostridium tyrobutyricum) overexpressing glutamate decarboxylase (GAD) and Class I heat shock protein GroESL.

[0011] The Clostridium tyrobutyricum is preferably Clostridium tyrobutyricum ATCC 25755, Clostridium tyrobutyricum L319, Clostridium tyrobutyricum CCTCC W428 or Clostridium tyrobutyricum Cirm BIA2237.

[0012] The amino acid sequence of the glutamate decarboxylase is shown in SEQ ID NO.1, and is also shown below: MLYGKENRDEAEFLEPIFGSESEQVDLPKYKLAQQSIEPRVAYQLVQDEMLDEGNARLNLATFCQTYMEPEAVKLMSQTLEKNAIDKSEYPRTTEIENRCVNMIADLWNASEKEKFMGTSTIGSSEACMLGGMAMKFSWRKRAEKLGLDINAKKPNLVISSGYQVCWEKFCVYWDIEMREVPMDREHMSINLDKVMDYVDEYTIGVVGIMGITYTGRYDDIKALDNL IEEYNKQTDYKVYIHVDAASGGLYAPFVEPELEWDFRLKNVISINTSGHKYGLVYPGVGWVLWRDKKYLPEELIFKVSYLGGELPTMAINFSHSASQLIGQYYNFVRYGFDGYKAIHERT HKVAMFLAKEIEKTGLFEIMNDGSQLPIVCYKLKEDSNRDWNLYDLADRLLMKGWQVPAYPLPKNLENEIIQRIVIRADFGMNMAFNYVQDMQEAIEALNKAHILYHEEPENKTYGFTH*.

[0013]

[0014] The Class I heat shock protein GroESL is the collective name for GroES protein and GroEL protein.

[0015] The amino acid sequence of the GroES protein is shown in SEQ ID NO.3, and is also shown below:

[0016] MNIRPLGDRVVIKKIEAEETTKSGIVLPEAAKEKPQEAEVVAVGLGGIVDGKEVRIEVKVGDRVLFSKYGGNEVKIDGVEYTILRQDDILAVIEK*.

[0017] The amino acid sequence of the GroEL protein is shown in SEQ ID NO.4, and is also shown below:

[0018] *.

[0019] The nucleic acid sequence of the gene encoding the Class I heat shock protein GroESL can be obtained according to the codon rule, preferably as shown in SEQ ID NO.5, wherein 289-332 are connection sequences and are not translated into protein.

[0020] atgaatattagaccacttggagacagagttgtaataaaaaaaatagaagcagaagaaacaacaaaagtggaatagttttaccagaagctgctaaagaaaaaccacaggaagcggaagttgtagcagttggactaggcggaattgttgatggaaaagaagtaagaattgaagttaaggtaggagatagggttctattctccaaatatggtggaaatgaagtaaagattgatggtgtagagtatactattttaagacaagatgatattttggctgtaattgaaaaataa aattagat ttaataatggtttgaaaattgtaaggaggttttttt

[0021] The overexpression is achieved by constructing the coding gene of glutamate decarboxylase and the coding gene of Class I heat shock protein GroESL downstream of the Clostridium tyrobutyricum promoter and then transferring them into Clostridium tyrobutyricum.

[0022] The Clostridium tyrobutyricum promoter is preferably the Clostridium tyrobutyricum cat1 gene promoter Pcat1.

[0023] The nucleic acid sequence of the promoter Pcat1 is shown in SEQ ID NO:6, and is also shown below:

[0024] GTAGACTTTAAGGATGGAACCTTTGAAATTAAGTAGAGAGCCCAAATCTTTGAAAATAATGTTCTTTCTTTGTATAGAAAGGACATTATTTTTTTATAGTTGTTTTGTAAACATAGCATTGTTAAGTTATTTTCAGCTACAGCTATTATTTTAATAATAACATTGATGTAATTATGTTAT TTTAACCAAAAGAAAATCATATTAATTTTGAATAAATGGATATATTATAATATAATATTAAAAGGAAGTTCAGGTTGTATATTATACAACATCTATTTTTTACTCATAATTGTAGTTTTTTAACAATCATAATGGAAGTTAATTATTAAATTTTATAATTTATGAAAGGGTGGTTTTTT.

[0025] The method for constructing the genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA comprises the following steps:

[0026] (1) obtaining a gene encoding glutamate decarboxylase;

[0027] (2) Obtain the gene encoding the Class I heat shock protein GroESL;

[0028] (3) connecting the gene encoding glutamate decarboxylase and the gene encoding Class I heat shock protein GroESL to the downstream of the Clostridium tyrobutyricum promoter to obtain a recombinant gene;

[0029] (4) The recombinant gene obtained in step (3) is transferred into Clostridium tyrobutyricum to obtain a genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA.

[0030] The acquisition described in step (1) is obtained by gene synthesis or cloned using the genome of Lactococcus lactis as a template.

[0031] The nucleic acid sequence of the gene encoding glutamate decarboxylase described in step (1) is shown in SEQ ID NO.2.

[0032] The nucleic acid sequence of the gene encoding the Class I heat shock protein GroESL described in step (2) is shown in SEQ ID NO.5.

[0033] The acquisition described in step (2) is obtained by gene synthesis, or cloned using the genome of Clostridium tyrobutyricum as a template.

[0034] Step (3) is preferably as follows: In a plasmid vector, the coding gene for glutamate decarboxylase and the coding gene for Class I heat shock protein GroESL are constructed downstream of the Clostridium tyrobutyricum promoter to obtain a recombinant gene.

[0035] The forms of the construction described in step (3) include method A and method B:

[0036] Method A: The coding gene of glutamate decarboxylase and the coding gene of Class I heat shock protein GroESL are constructed together in the downstream of the promoter of Clostridium tyrobutyricum in no particular order; a linker sequence is selectively added between the coding gene of glutamate decarboxylase and the coding gene of Class I heat shock protein GroESL, and the linker sequence is used to ensure that the conformations of glutamate decarboxylase and Class I heat shock protein GroESL are not affected; the selective addition refers to adding or not adding;

[0037] Method B: The gene encoding glutamate decarboxylase and the gene encoding Class I heat shock protein GroESL are constructed downstream of the Clostridium tyrobutyricum promoter respectively.

[0038] The plasmid vector is preferably pMTL82151.

[0039] The promoter described in step (3) is preferably the Clostridium tyrobutyricum cat1 gene promoter Pcat1.

[0040] The transfer described in step (4) is preferably carried out by bacterial conjugation.

[0041] The Clostridium tyrobutyricum described in step (4) is preferably Clostridium tyrobutyricum ATCC 25755, Clostridium tyrobutyricum L319, Clostridium tyrobutyricum CCTCC W428 or Clostridium tyrobutyricum Cirm BIA2237.

[0042] The application of the Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA in the preparation of GABA.

[0043] A method for preparing GABA comprises the following steps: inoculating the Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA into a fermentation medium for fermentation to obtain GABA.

[0044] The Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA is preferably in a logarithmic growth phase or has just entered a stable phase when inoculated into a fermentation medium.

[0045] When the fermentation is carried out in a fermentation tank, the fermentation process comprises the following steps:

[0046] 1) Selectively activate the strains, expand the culture, and obtain seed liquid;

[0047] 2) inoculating the obtained seed liquid into a fermentation medium for fermentation;

[0048] 3) During the fermentation process, feed-batch fermentation was performed and the pH was controlled at 4-5.

[0049] The activation step described in step 1) is optional, and whether to perform activation depends on the status of the strain.

[0050] The activated culture medium in step 1) is preferably a reinforced Clostridium medium (RCM medium).

[0051] The composition of the enhanced Clostridium culture medium is as follows: 5g / L glucose, 5g / L NaCl, 10g / L peptone, 10g / L beef powder, 3g / L yeast extract, 3g / L anhydrous sodium acetate, 1g / L soluble starch, 0.5g / L cysteine-HCl; the solvent is water; and 10-20g / L agar is added to the solid culture medium.

[0052] The activation process described in step 1) is preferably as follows: the preserved bacterial strain is inoculated into RCM medium and cultured overnight at 37° C. for activation.

[0053] The expanded culture in step 1) is prepared by preparing seed liquid according to the scale of fermentation, and the seed liquid is calculated as 1-10% of the fermentation volume.

[0054] The culture medium for the expanded culture in step 1) is liquid RCM culture medium.

[0055] The carbon source of the fermentation medium is preferably glucose; the nitrogen source is preferably at least one of peptone and yeast extract.

[0056] The fermentation medium preferably further contains MSG.

[0057] The concentration of MSG in the fermentation medium is preferably 10-40 g / L.

[0058] The composition of the fermentation medium is preferably as follows: 3-5 g / L peptone, 1-3 g / L yeast extract, 0.5-1.5 g / L K2HPO4﹒3H2O, 0.3-0.6 g / L KH2PO4, 1-3 g / L (NH4)2SO4, 0.05-0.15 g / L MgSO4﹒7H2O, 20-60 g / L glucose, 10-40 g / L sodium glutamate, and 0.09-0.11% (v / v) of trace element mother solution; more preferably as follows: 4 g / L peptone, 2 g / L yeast extract, 1 g / L K2HPO4﹒3H2O, 0.5 g / L KH2PO4, 2g / L (NH4)2SO4, 0.1g / LMgSO4﹒7H2O, 20-60g / L glucose, 10-40g / L sodium glutamate, 0.1% (v / v) trace element mother solution. The solvent is water, preferably deionized water.

[0059] The composition of the trace element mother solution is as follows: 15g / L FeSO4﹒7H2O, 15g / L CaCl2﹒2H2O, 10g / L MnSO4﹒H2O, 20g / L CoCl2﹒6H2O, 20g / L ZnSO4﹒7H2O; the solvent is water, preferably deionized water.

[0060] The inoculation amount is 1-10% by volume.

[0061] The fermentation conditions are preferably 37±5°C and 150±100rpm.

[0062] The fermentation time is preferably at least 36 hours, preferably 48 to 84 hours.

[0063] The process of the fed-batch fermentation in step 3) is as follows: when the culture reaches the 36th hour, glucose and MSG are supplemented; then the culture is continued for 12 hours and MSG is supplemented or the culture is continued for 24 hours and MSG is supplemented.

[0064] The supplementary amount of glucose is preferably 60 g / L fermentation volume.

[0065] The supplementary amount of MSG is preferably 100-130 g / L fermentation volume; more preferably 110-120 g / L fermentation volume.

[0066] The MSG is preferably added in the form of MSG mother solution during feeding.

[0067] The MSG mother liquor is obtained by dissolving 140 g of MSG in 200 mL of deionized water.

[0068] The pH control described in step 3) is preferably performed when the feed is started.

[0069] The solvent for controlling pH is preferably H2SO4 solution; more preferably H2SO4 solution with a concentration of 3 mol / L.

[0070] The pH is preferably 4.4 to 4.6.

[0071] The Clostridium tyrobutyricum is an anaerobic bacterium, and the culture and fermentation conditions are all anaerobic conditions; the anaerobic conditions can be set according to the conventional settings in the art, including nitrogen filling and deoxygenation in the culture medium, and culturing in an anaerobic box.

[0072] Glutamate decarboxylase can directly use MSG as a substrate to catalyze the production of GABA. Clostridium tyrobutyricum itself contains a metabolic pathway for the coenzyme pyridoxal phosphate, so there is no need to add pyridoxal phosphate. Therefore, the present invention selects Clostridium tyrobutyricum as a starting strain, and its fermentation products include acetic acid and butyric acid. By overexpressing glutamate decarboxylase and adding MSG to the culture medium, the synthesis of GABA can be achieved; on the basis of this recombinant strain, overexpressing the Class 1 heat shock protein GroESL can further improve MSG tolerance and yield.

[0073] Compared with the prior art, the present invention has the following advantages and effects:

[0074] (1) The present invention studies the expression of Class I heat shock protein genes under MSG stress and finds that the expression levels of groES and groEL genes are the most prominent. The present invention obtains an engineered bacterium capable of synthesizing GABA for the first time by overexpressing glutamate decarboxylase and Class I heat shock protein GroESL in Clostridium tyrobutyricum, and provides a method for efficiently producing GABA.

[0075] (2) The engineered bacteria used in the present invention do not produce endotoxins, and the product has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is a diagram of the fermentation status of Ct-GAD engineered bacteria in a 50mL serum bottle.

[0077] Figure 2 This is a graph showing the expression changes of the Class I heat shock protein gene of Ct-GAD after 15 minutes of MSG stress.

[0078] Figure 3 This is a graph showing the expression changes of the Class I heat shock protein gene of Ct-GAD after 30 minutes of MSG stress.

[0079] Figure 4It is the recombinant plasmid map of the engineered bacteria.

[0080] Figure 5 This is a comparison of the specific growth rates of Ct-GAD and Ct-GAD-GroESL engineered bacteria at different MSG concentrations.

[0081] Figure 6 This is a comparison of the GABA production of Ct-GAD and Ct-GAD-GroESL engineered bacteria under different MSG concentrations.

[0082] Figure 7 This is a graph showing the fermentation results of the engineered bacteria in a 5L fermenter with pH-free batch feeding.

[0083] Figure 8 This is a graph showing the fermentation results of the engineered bacteria in a 5L fermenter with pH-control and batch feeding. DETAILED DESCRIPTION

[0084] In order to make those skilled in the art more clearly understand the core technology of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but the embodiments of the present invention are not limited thereto. It should be understood that the experimental methods used in the following implementation cases are all conventional methods unless otherwise specified.

[0085] Example 1. Construction of engineered strains Ct-GAD and Ct-GAD-GroESL

[0086] (1) Construction of recombinant plasmid pMTL82151-GAD

[0087] The genome of Clostridium tyrobutyricum ATCC 25755 was used as a template, and the cat1 gene promoter 1Pcat1 of GAD was amplified using primers lcgadPcat1-F and lcgadPcat1-R. The genome of Lactococcus lactis JN01 screened in the laboratory was used as a template, and the GAD gene was amplified using primers lcgad-F and lcgad-R. The sequence of the GAD coding gene obtained is shown in SEQ ID NO.2, and those skilled in the art can obtain the sequence by gene synthesis.

[0088] lcgadPcat1-F:5'-gaaacagctatgacc gcggccgc GTAGACTTTAAGGATGGAACCTTTGA-3′ (the underline indicates the Not I restriction enzyme site);

[0089] lcgadPcat1-R: 5'-cgtataacatAAAAACCACCCTTTCATAAATTATATAAA-3';

[0090] lcgad-F: 5'-ggtggtttttATGTTATACGGAAAAGAAAATCGCG-3';

[0091] lcgad-R: 5'-gacgtcgactctaga ggatcc TTAGTGAGTAAAGCCATAAGTTTTATTTTC-3' (the underline indicates the BamHI restriction enzyme site).

[0092] The DNA polymerase used for PCR amplification was purchased from Novazon Biotech Co., Ltd. The PCR amplification system is as follows:

[0093]

[0094] PCR reaction program: 98°C, 30 sec; 98°C 10 sec, 60°C 5 sec, 72°C 15 sec, 32 cycles; 72°C, 5 min.

[0095] First, the shuttle plasmid pMTL82151 (purchased from BioVector Plasmid Vector Bacteria Cell Protein Antibody Gene Collection Center-NTCC Type Culture Collection Center) was double-digested with NotⅠ and BamHI (restriction endonucleases were purchased from ThermoFisher Scientific Company), and the reaction system was as follows:

[0096]

[0097] The reaction conditions were: 37°C, water bath for 60 min.

[0098] The above enzyme digestion products were purified and recovered, and the linearized vector pMTL82151 was connected with the promoter 1Pcat1 and GAD. The connection system was:

[0099]

[0100] The reaction conditions were: 50°C, 20 min. After the reaction was completed, the centrifuge tube was immediately placed on ice to cool for 2 min.

[0101] The ligation product was transformed into Escherichia coli DH5α and incubated at 37°C for 24 h. The positive clones were picked for PCR verification using primers Pro-F and Pro-R, and sent to Bio-Tech for sequencing verification. The plasmid DNA was extracted, purified and recovered (the plasmid extraction kit was purchased from Novazonic Biotech Co., Ltd.) and named pMTL82151-GAD.

[0102] Pro-F: 5'-TGAAGTACATCACCGACGAGCAAG-3';

[0103] Pro-R: 5'-TGCTGCAAGGCGATTAAGTTTGGGT-3'.

[0104] (2) Construction of engineered strain Ct-GAD

[0105] The above recombinant plasmid pMTL82151-GAD was transferred into C. tyrobutyricum ATCC 25755 (American Type Culture Collection) by bacterial conjugation. The specific steps are as follows:

[0106] The recombinant plasmid was transformed into the E. coli conjugative strain E. coli CA434, and the positive clones were screened using a double-resistance LB plate (25 μg / mL chloramphenicol + 50 μg / mL kanamycin). The positive clones were verified by PCR using primers Pro-F and Pro-R. E. coli CA434 containing the recombinant plasmid was cultured in a double-resistance LB medium (25 μg / mL chloramphenicol + 50 μg / mL kanamycin) at 37°C and 150 rpm shaking until the OD 600 1.5-2.0, collect 3 mL of E. coli in a 1.5 mL sterile centrifuge tube, wash once with 1× sterile PBS, centrifuge at 4000 rpm for 2 min, and then culture with 300-500 μL in an anaerobic incubator until OD 600 2.0-3.0 of Clostridium tyrobutyricum ATCC 25755 were mixed evenly, spread on RCM non-antibiotic plates, and cultured anaerobically at 37°C for 24 hours. The colonies were washed with 0.5 mL of RCM medium, spread on RCM screening plates (25 μg / mL thiamphenicol and 250 μg / mL D-cycloserine), and cultured anaerobically at 37°C for more than 30 hours until a single colony appeared. A single colony was picked and cultured in RCM medium (25 μg / mL thiamphenicol and 250 μg / mL D-cycloserine), and PCR verification was performed with primers Pro-F and Pro-R to obtain the engineered bacteria Ct-GAD.

[0107] (3) Shake flask fermentation experiment of engineered strain Ct-GAD

[0108] Preparation of RCM medium: 5g / L glucose, 5g / L NaCl, 10g / L peptone, 10g / L beef powder, 3g / L yeast extract, 3g / L anhydrous sodium acetate, 1g / L soluble starch, 0.5g / L cysteine-HCl. The solvent is water, preferably deionized water.

[0109] Preparation of CGM medium: 4g / L peptone, 2g / L yeast extract, 1g / L K2HPO4﹒3H2O, 0.5g / LKH2PO4, 2g / L(NH4)2SO4, 0.1g / L MgSO4﹒7H2O, trace elements 1:1000 (v / v); trace element mother solution: 15g / L FeSO4﹒7H2O, 15g / L CaCl2﹒2H2O, 10g / L MnSO4﹒ H2O, 20g / L CoCl2﹒6H2O, 20g / L ZnSO4﹒7H2O. The solvent is water, preferably deionized water.

[0110] 50 mL of culture medium was filled into explosion-proof serum bottles, filled with 0.05 MP high-purity nitrogen, and sterilized by high-pressure steam at 115°C for 20 min (the carbon source and culture medium were sterilized separately).

[0111] The wild type of Clostridium tyrobutyricum (ATCC 25755) and the engineered bacteria Ct-GAD were taken out from a -80°C refrigerator, inoculated into RCM seed medium, and cultured overnight at 37°C for activation. The activated seed liquid was inoculated into RCM seed medium again for activation, and then the seed liquid was inoculated into CGM fermentation medium at a 5% (v / v) inoculation amount, supplemented with 20 g / L glucose, and cultured at 37°C. Samples were taken every 12 hours for HPLC detection of product formation.

[0112] The results are as follows Figure 1 As shown in the figure, after overexpression of GAD, GABA was detected in the fermentation supernatant of the engineered bacteria. Ct-GAD produced 5.37 g / L of GABA after 72 h of fermentation, indicating that GAD was successfully expressed in Clostridium tyrobutyricum and had a decarboxylation function, which could convert MSG into GABA. No GABA was detected in the fermentation supernatant of the wild type Clostridium tyrobutyricum due to the lack of glutamate decarboxylase.

[0113] (4) Screening of heat shock proteins and construction of the engineered strain Ct-GAD-GroESL

[0114] Fluorescence quantitative PCR was used to detect the expression changes of Class I heat shock protein genes (grpE, dnaK, dnaJ, groEL, groES and htpG) of the engineered bacteria Ct-GAD under MSG stress; then the Class I heat shock protein genes whose expression levels increased significantly under MSG stress were overexpressed in the engineered bacteria Ct-GAD.

[0115] The engineered bacteria Ct-GAD was cultured in RCM and waited until its OD 600When the concentration reached about 1.5, MSG was added at a final concentration of 10 g / L and 40 g / L for stress test, and then samples were taken and total RNA was extracted after 15 min and 30 min of treatment. Fluorescence quantitative PCR was performed using cDNA reverse transcribed from RNA as a template to analyze the expression changes of Class I heat shock protein genes under MSG stress. The housekeeping gene GTP-binding gene was used as an internal reference.

[0116] First, RNA was extracted using the Tiangen Bacteria Total RNA Extraction Kit, and then RNA was reverse transcribed using ABScript III RT MasterMix for qPCR with gDNA remover to obtain cDNA. Finally, cDNA was used as a template using TaKaRa Premix Ex Taq TM II was used for fluorescence quantitative PCR detection, and the reaction was carried out using Light Cycler (Roche, Switzerland). After the reaction, the data were analyzed using LightCycler96SW1.1 software. The specific operation was consistent with the product manual. The primers used were as follows:

[0117] grpE-QF: 5'-GATGTAGAAGAAATTCCTGCA-3';

[0118] grpE-QR:5'-ATTTGCCACTTTAACCATACTGT-3';

[0119] dnaK-QF: 5'-AGGGGGTTAATCCAGATGAGTGTG-3';

[0120] dnaK-QR:5'-CAAGAGTAAGTGGTGTAACATCAAG-3';

[0121] dnaJ-QF: 5'-GAAGCCTATCAGGTTTTATCGG-3';

[0122] dnaJ-QR:5'-GCTCCACCAGCACCACTAAAATCAG-3';

[0123] groES-QF: 5'-GCTGCTAAAGAAAAACCACAGGAAG-3';

[0124] groES-QR:5'-CTCTACACCATCAATCTTTAC-3';

[0125] groEL-QF: 5'-GGCAAAGAGCATTTTATTCGGTG-3';

[0126] groEL-QR:5'-CTTCCCTTTGGTCCAAGTGTAA-3';

[0127] htpG-QF: 5'-AAGCATTTCAGGAGGATAAGG-3';

[0128] htpG-QR:5'-CATCATTGGCAAATTGCACTGG-3';

[0129] GTP-binding gene-QF: 5'-CAATAATTGCCCATGTAGATCACGG-3';

[0130] GTP-binding gene-QR: 5'-GAGTCCATAACCCTCTCTTGAAC-3'.

[0131] The results are as follows Figure 2 , Figure 3 As shown in the figure, when treated with 10g / L MSG, the expression levels of grpE, dnaK, dnaJ, groEL, groES and htpG increased by more than 1.5 times, and as the MSG concentration continued to increase to 40g / L, the expression levels of the six detected genes increased further, especially grpE, groES, groEL and htpG. As the treatment time increased, the expression levels of grpE, dnaK, dnaJ, groEL, groES and htpG all slowly decreased. This result showed that when under MSG stress, the expression levels of Class I heat shock protein genes would be greatly upregulated to help the bacteria better cope with MSG stress, and when the bacteria adapted to MSG stress, the expression levels of these genes would gradually return to normal levels. Since the expression levels of groES and groEL were prominent under 10g / L and 40g / L MSG stress, the groES and groEL genes were overexpressed on the basis of the engineered strain Ct-GAD.

[0132] Using the genome of Clostridium tyrobutyricum ATCC 25755 as a template, the cat1 gene promoter Pcat1 of GroESL was amplified with primers groESLPcat1-F and groESLPcat1-R, and the GroESL gene was amplified with primers groESL-F and groESL-R.

[0133] groESLPcat1-F: 5'-taaggatcc tctagagtcgacGTAGACTTTAAGGATGGAACCTTTGA-3′ (the underline indicates the XbaⅠ restriction enzyme site);

[0134] groESLPcat1-R: 5'-ttcatAAAAACCACCCTTTCATAAATTATATAAA-3';

[0135] groESL-F: 5'-gaaagggtggtttttATGAATATTAGACCACTTGGAGACAGA-3';

[0136] groESL-R: 5'-caggcctcgagatct ccatgg TTAATACATTCCTTCCATTCCGC-3' (the underline indicates the Nco I restriction enzyme site).

[0137] Then pMTL82151-GAD was double-digested with SalⅠ and NcoⅠ (restriction endonucleases purchased from ThermoFisher Scientific), and the reaction system was:

[0138]

[0139] The reaction conditions were: 37°C, water bath for 60 min.

[0140] The above enzyme digestion products were purified and recovered, and the linearized vector pMTL82151-GAD was connected with the promoter Pcat1 and GroESL. The connection system was:

[0141]

[0142] The reaction conditions were: 50°C, 20 min. After the reaction was completed, the centrifuge tube was immediately placed on ice to cool for 2 min.

[0143] The ligation product was transformed into Escherichia coli DH5α and incubated at 37°C for 24 h. The positive clones were selected for PCR verification using primers Pro-F and Pro-R, and sent to Bio-Tech for sequencing verification. The plasmid DNA was extracted and purified (the plasmid extraction kit was purchased from Novozyme Biotech Co., Ltd.) and named pMTL82151-GAD-GroESL (as Figure 4 Then, the engineered strain Ct-GAD-GroESL was obtained by bacterial conjugation, and the method was consistent with the above.

[0144] Example 2: Shake flask fermentation experiment of engineered strain Ct-GAD-GroESL

[0145] The shake flask fermentation experimental method refers to step (3) of Example 1.

[0146] The specific growth rate is calculated by the following formula: Where N and N0 represent the OD of bacteria at t and t0 during the logarithmic growth phase. 600 Numeric value.

[0147]

[0148] The results are as follows Figure 5 As shown, further tandem overexpression of GroESL improved the tolerance of Clostridium tyrobutyricum to MSG. At 0 g / L, the growth rate of Ct-GAD-GroESL was slightly lower than that of Ct-GAD, while it was higher than Ct-GAD at 10-40 g / L, and increased by 41.4% at 30 g / L.

[0149] The results are as follows Figure 6 As shown, tandem overexpression of GroESL not only improved the MSG tolerance of the engineered bacteria, but also kept the GABA production of the two strains equal at 10 g / L MSG. With the increase of concentration, the GABA production of Ct-GAD-GroESL increased by 17.02%, 22.78% and 20.75%, respectively.

[0150] Example 3: The engineered strain Ct-GAD-GroESL was subjected to pH-free and pH-control batch fermentation using glucose and MSG as substrates in a 5 L fermenter, and the fermentation system was 1 L. The fermenter was sterilized at 121° C. for 20 min, and then CGM medium was added and sterilized at 121° C. for 20 min. After cooling, sterile nitrogen was introduced to replace the air in the tank to achieve an anaerobic environment.

[0151] High concentration of MSG stress is not conducive to the growth of Clostridium tyrobutyricum, so the batch fed fermentation mode is used to increase the production of GABA. Clostridium tyrobutyricum Ct-GAD-GroESL was taken out from the -80℃ refrigerator, inoculated in RCM seed culture medium, and cultured at 37℃ overnight for activation. The activated seed liquid was inoculated in RCM seed culture medium again for activation, and then the seed liquid was inoculated into CGM fermentation medium according to 5% (v / v) inoculation amount, supplemented with 60g / L glucose, and cultured at 37℃. Samples were taken regularly for HPLC detection of product generation; MSG mother liquor was added in time during the fermentation process, and pH was adjusted according to the situation.

[0152] MSG mother solution: 30 g of sodium glutamate was added to 50 mL of ddH2O in the pH-free group; 140 g of sodium glutamate was added to 200 mL of ddH2O in the pH-control group.

[0153] H2SO4 mother solution: 3mol / L H2SO4.

[0154] In pH-free fed-batch fermentation, glucose was supplemented at 36 h (60 g / L), and MSG was supplemented to the mother liquor once at 36 h and 60 h, respectively.

[0155] In pH-control fed-batch fermentation, glucose was supplemented at 36 h (60 g / L), and MSG was supplemented to the mother liquor once at 36 h and 48 h, respectively.

[0156] The results are as follows Figure 7 As shown in the figure, the pH-free group did not need to be manually adjusted during the reaction. Through batch-fed fermentation, when the bacteria grew to the early stage of the stable phase (36h), MSG mother liquor and 60g / L glucose were added to further increase the GABA production. The GABA production reached 35.57g / L at 84h. In pH-free, the synthesis of GABA consumes hydrogen ions, causing the pH to rise, and gradually cannot meet the acidic conditions required for GABA synthesis. Therefore, a two-stage pH-control strategy was used to perform batch-fed fermentation to increase the yield. The results are shown in the figure. Figure 8 As shown, the reaction process of pH-control group from 0 to 36h was the same as that of pH-free group. When the bacteria grew to the early stable phase (36h), MSG mother solution and 60g / L glucose were added and the pH was maintained at 4.5 by adding H2SO4 mother solution. The GABA production of pH-control group reached 122.34g / L at 84h.

[0157] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium Clostridium tyrobutyricum capable of synthesizing GABA, characterized in that: Clostridium tyrobutyricum Clostridium tyrobutyricum ) overexpressed glutamate decarboxylase and class I heat shock protein GroESL; The Class I heat shock protein GroESL is the collective name of GroES protein and GroEL protein; The glutamate decarboxylase is derived from Lactococcus lactis; The Class I heat shock protein GroESL is derived from Clostridium tyrobutyricum.

2. The genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA according to claim 1, characterized in that: The amino acid sequence of the glutamate decarboxylase is shown in SEQ ID NO.1; The amino acid sequence of the GroES protein is shown in SEQ ID NO.3; The amino acid sequence of the GroEL protein is shown in SEQ ID NO.

4.

3. The genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA according to claim 1, characterized in that: The Clostridium tyrobutyricum is Clostridium tyrobutyricum ATCC 25755, Clostridium tyrobutyricum L319, Clostridium tyrobutyricum CCTCCW428 or Clostridium tyrobutyricum Cirm BIA 2237.

4. The method for constructing the genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA according to any one of claims 1 to 3, characterized in that The steps include: (1) Obtaining the gene encoding glutamate decarboxylase; (2) Obtain the gene encoding the Class I heat shock protein GroESL; (3) Connecting the gene encoding glutamate decarboxylase and the gene encoding Class I heat shock protein GroESL to the downstream of the Clostridium tyrobutyricum promoter to obtain a recombinant gene; (4) The recombinant gene obtained in step (3) is transferred into Clostridium tyrobutyricum to obtain a genetically engineered Clostridium tyrobutyricum capable of synthesizing GABA.

5. The method for constructing a Clostridium tyrobutyricum genetically engineered bacterium capable of synthesizing GABA according to claim 4, characterized in that: Step (3) is as follows: in a plasmid vector, a gene encoding glutamate decarboxylase and a gene encoding Class I heat shock protein GroESL are constructed downstream of a Clostridium tyrobutyricum promoter to obtain a recombinant gene; The construction described in step (3) includes method A and method B: Method A: The coding gene of glutamate decarboxylase and the coding gene of Class I heat shock protein GroESL are constructed in the downstream of the promoter of Clostridium tyrobutyricum in no particular order; a linker sequence is selectively added between the coding gene of glutamate decarboxylase and the coding gene of Class I heat shock protein GroESL, and the linker sequence is used to ensure that the conformations of glutamate decarboxylase and Class I heat shock protein GroESL are not affected; the selective addition refers to adding or not adding; Method B: The gene encoding glutamate decarboxylase and the gene encoding Class I heat shock protein GroESL are constructed downstream of the Clostridium tyrobutyricum promoter respectively.

6. The method for constructing the Clostridium tyrobutyricum genetically engineered bacteria capable of synthesizing GABA according to claim 4 or 5, characterized in that: The nucleic acid sequence of the gene encoding glutamate decarboxylase in step (1) is shown in SEQ ID NO.2; The nucleic acid sequence of the gene encoding the Class I heat shock protein GroESL described in step (2) is shown in SEQ ID NO.5; The promoter in step (3) is Clostridium tyrobutyricum cat1 Gene promoter Pcat1.

7. Use of the genetically engineered bacterium Clostridium tyrobutyricum capable of synthesizing GABA according to any one of claims 1 to 3 in the preparation of GABA.

8. A method for preparing GABA, characterized in that The method comprises the following steps: inoculating the Clostridium tyrobutyricum genetic engineering bacteria capable of synthesizing GABA according to any one of claims 1 to 3 into a fermentation medium for fermentation to obtain GABA.

9. The method for preparing GABA according to claim 8, characterized in that: The fermentation is carried out using a fermentation tank, and the fermentation process includes the following steps: 1) Selectively activate the strains, expand the culture, and obtain the seed solution; 2) inoculating the obtained seed liquid into a fermentation medium for fermentation; 3) During the fermentation process, feed the fermentation and control the pH at 4-5.

10. The method for preparing GABA according to claim 9, characterized in that: The activated culture medium described in step 1) is a fortified Clostridium culture medium; The culture medium for the expanded culture in step 1) is a liquid fortified Clostridium culture medium; The carbon source of the fermentation medium in step 2) is glucose; The nitrogen source of the fermentation medium in step 2) is at least one of peptone and yeast extract; The inoculation amount in step 2) is 1 to 10% by volume; The fermentation conditions in step 2) are 37±5°C and 150±100 rpm; The process of the fed-batch fermentation in step 3) is as follows: when the culture reaches the 36th hour, glucose and MSG are supplemented; then the culture is continued for 12 hours to supplement MSG or the culture is continued for 24 hours to supplement MSG; The pH control described in step 3) is performed when the feed is started.

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