Recombinant Corynebacterium glutamicum heterologously expressing cspB gene for lysine production by biofilm immobilized fermentation
By heterologously expressing the cspB gene in Corynebacterium glutamicum, biofilm formation is promoted, and the problem of weak film-forming ability is solved, and the recycling of cells in immobilized fermentation and the increase in L-lysine yield is achieved, reducing costs.
Patent Information
- Application Number
- CN202210819917.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-13
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2042-07-13
AI Technical Summary
In the prior art, the film-forming ability of Corynebacterium glutamate is weak, which makes it difficult to achieve in immobilized continuous fermentation, increasing operating costs and reducing the productivity of L-lysine.
By heterologously expressing the cspB gene in Corynebacterium glutamicum, the formation of biofilms is promoted, and the recombinant Corynebacterium glutamicum is constructed to improve its efficiency in immobilized fermentation.
The recyclable use of cells is achieved, the fermentation cycle is shortened, and the production and production efficiency of L-lysine is improved, thus reducing costs.
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Figure CN116064341B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the biological field, and in particular to a recombinant Corynebacterium glutamicum capable of heterologously expressing a cspB gene to produce lysine through biofilm immobilization fermentation, and a construction method and application thereof. Background Art
[0002] L-lysine belongs to the aspartic acid family of amino acids. It is one of the essential amino acids for humans and animals that cannot be synthesized by themselves. It is one of the eight essential amino acids for humans and animals and the second most abundant amino acid in the world (after glutamate). It is widely used in feed additives, food fortifiers, and pharmaceutical products, with over 90% of lysine products used as feed additives. Because the lysine content in cereal foods is very low and is easily destroyed and deficient during processing, it is also known as the first limiting amino acid.
[0003] Biofilms are widely present in nature. During the biofilm formation process, the extracellular polymers (EPS) secreted by bacteria themselves are the material basis for biofilm formation. Biofilms can be regarded as a protective umbrella for microorganisms to resist the harsh environment around them. It can protect the bacteria from the invasion of harmful substances in the surrounding environment to a certain extent, thereby greatly enhancing the survival ability of microorganisms. At the same time, it also plays a key role in the adhesion and aggregation characteristics of bacteria.
[0004] At present, microbial fermentation has been widely used in industry to produce L-Lysine with Corynebacterium glutamicum as the best candidate strain. However, L-Lysine fermentation has been carried out in single batch free fermentation and can not be reused after fermentation. Batch fermentation and disposable cells can increase operating costs and reduce productivity. Meanwhile, the free cells dispersed in the fermentation medium are challenged by stress conditions (such as shearing force) during aerobic fermentation, resulting in a reduction in cell viability during fermentation. And fixed fermentation based on biofilm has higher metabolic activity and cell reuse rate can reach the purpose of saving cost and increasing output.
[0005] Immobilized continuous fermentation technology has been put into the middle of production nowadays, and wherein carrying out immobilized continuous fermentation with the mode of biofilm has been initially effective.Yet, in Corynebacterium glutamicum, immobilized continuous fermentation based on the mode of biofilm is seldom reported.Corynebacterium glutamicum is as a kind of safe industrial production bacterial strain, and its film forming ability is very weak, is difficult to continuous fermentation, therefore the present invention carries out molecular transformation to this bacterial strain, makes its film forming effect strengthen, to realize later stage immobilized continuous fermentation. Summary of the Invention
[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant Corynebacterium glutamicum that heterologously expresses the cspB gene in response to the deficiencies of the prior art.
[0007] The technical problem that the present invention also aims to solve is to provide a method for constructing the above-mentioned recombinant Corynebacterium glutamicum.
[0008] The technical problem to be solved by the present invention is to provide the application of the above-mentioned recombinant Corynebacterium glutamicum in the production of lysine by immobilized fermentation.
[0009] Invention concept: To increase the L-lysine production of Corynebacterium glutamicum ATCC13032, its genome was sequenced and compared with the genomes of multiple other Corynebacterium glutamicum strains. It was found that the mutant strain did not contain the target gene encoding the cspB protein. The cspB protein has a promoting effect on biofilm formation, which can promote the attachment of the strain to the carrier during immobilized fermentation, thereby improving the efficiency of immobilized fermentation and shortening the fermentation cycle to achieve the purpose of increasing L-lysine.
[0010] In order to solve the first technical problem mentioned above, the present invention discloses a recombinant Corynebacterium glutamicum, in which the cspB gene is heterologously expressed in the original Corynebacterium glutamicum.
[0011] Wherein, the original Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032.
[0012] Wherein, the nucleotide sequence of the cspB gene is shown as SEQ ID NO.1.
[0013] In order to solve the above-mentioned second technical problem, the present invention discloses a method for constructing the above-mentioned recombinant Corynebacterium glutamicum, comprising the following steps:
[0014] (1) Amplify the target gene cspB;
[0015] (2) cloning the target gene cspB obtained in step (1) between the two restriction enzyme sites of XbaI enzyme and Kpn1 enzyme of plasmid pXMJ19 to obtain the recombinant plasmid pXMJ19-cspB;
[0016] (3) transforming the recombinant plasmid pXMJ19-cspB obtained in step (2) into competent Escherichia coli DH5α, screening Escherichia coli containing the recombinant plasmid pXMJ19-cspB, and then extracting the recombinant plasmid pXMJ19-cspB;
[0017] (4) The recombinant plasmid extracted in step (3) is transformed into a competent cell of Corynebacterium glutamicum to obtain recombinant Corynebacterium glutamicum.
[0018] In step (1), the primers for amplifying the target gene cspB are cspB-F and cspB-R, whose nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.
[0019] In step (2), the nucleotide sequence of the plasmid pXMJ19 is shown in SEQ ID NO.4.
[0020] In order to solve the third technical problem mentioned above, the present invention discloses the use of the above-mentioned recombinant Corynebacterium glutamicum in the production of lysine by immobilized fermentation.
[0021] The fermentation medium comprises 118-138 mL / L of 70% sugar, 35-55 g / L of ammonium sulfate, 0.2-2.2 g / L of magnesium sulfate, 5-25 mL / L of molasses, 5-25 mL / L of corn steep liquor, 0.1-2.1 g / L of potassium dihydrogen phosphate, 150-250 mg / L of ferrous sulfate, 100-200 mg / L of manganese sulfate, 50-70 mg / L of nicotinamide, 5-15 mg / L of calcium pantothenate, 5-15 mg / L of VB1, 0.5-1.5 mg / L of copper sulfate, 0.5-1.5 mg / L of zinc sulfate, 0.8-2.8 mg / L of biotin, and 20-40 g / L of calcium carbonate.
[0022] The seed liquid of the recombinant Corynebacterium glutamicum is added to the fermentation medium at a volume ratio of 5% to 15% for fermentation.
[0023] The dosage of the immobilized carrier is 0.5-4 g / 50 mL fermentation medium, preferably 0.5-2.5 g / 50 mL fermentation medium, and preferably 1.5 g / 50 mL fermentation medium.
[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0025] The present invention constructs a recombinant Corynebacterium glutamicum strain that heterologously expresses the cspB extracellular protein gene for lysine production. Experimental results demonstrate that heterologous expression of the cspB gene promotes biofilm formation in the recombinant bacteria, thereby promoting L-lysine fermentation. Furthermore, by combining biofilm with immobilized fermentation technology, the fermentation cycle is shortened, cell recycling is achieved, and costs are reduced. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.
[0027] Figure 1 This is the gel image of cspB gene PCR.
[0028] Figure 2 The results of restriction enzyme digestion of pXMJ19 plasmid are shown.
[0029] Figure 3 Schematic diagram of the construction of recombinant expression plasmid.
[0030] Figure 4 This is the result of the recombinant plasmid pXMJ19-cspB in Escherichia coli.
[0031] Figure 5 This is a gel run of the pXMJ19-cspB plasmid.
[0032] Figure 6 This is the result of electroporation of pXMJ19-cspB plasmid into Corynebacterium glutamicum.
[0033] Figure 7 This is the result of crystal violet staining.
[0034] Figure 8 is the biofilm absorbance.
[0035] Figure 9 is the L-lysine yield.
[0036] Figure 10 For comparison of fermentation cycles. DETAILED DESCRIPTION
[0037] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
[0038] Example 1 Molecular transformation of target strains
[0039] 1. Amplify the target gene cspB. The cspB gene sequence from Corynebacterium glutamicum SCgG2 was retrieved from NCBI, and the cspB gene was obtained through a synthetic gene from General Corporation. The specific synthetic amplification method is as follows: Using cspB-F and cspB-R in Table 1, PCR was performed under the following reaction conditions: denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 2 minutes for 35 cycles: amplify a 1518 bp gene fragment including the two restriction sites of XbaI and KpnI. Figure 1 As shown, the first lane is a 2000 bp DNA marker, and the length of cspB including the double enzyme cutting site is 1518 bp, which shows that the cspB gene was successfully amplified.
[0040] cspB-F has a restriction enzyme recognition site of XbaI, and cspB-R has a restriction enzyme recognition site of KpnI.
[0041] Table 1 Experimental primer sequences used for amplification
[0042]
[0043] 2. Construction of recombinant plasmid
[0044] 2.1 Plasmid extraction
[0045] (1) Inoculate E. coli DH5α glycerol strain (containing plasmid pXMJ19) into liquid LB (chloramphenicol resistance concentration: 50 μg / mL) and culture at 37°C for 12 hours.
[0046] (2) Collect the cells cultured in the previous step using a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 2 min, and remove the supernatant.
[0047] (3) Plasmid pXMJ19 was extracted using the AxyPrep plasmid extraction kit from Corning Life Sciences, Inc.
[0048] 2.2 Vector linearization
[0049] In order to connect the target gene cspB to the plasmid pXMJ19, the vector needs to be enzymatically digested. The double enzyme digestion reaction system is (20μL) 0.1% BSA 2μL, 1*M buffer 2μL, 0.5μL each of XbaI enzyme and Kpnl enzyme, 10μL of plasmid pXMJ19, and 5μL of sterile water. The enzymatic digestion reaction is conditioned at 37℃ for 2h. After enzymatic digestion, gel recovery is performed for subsequent experiments. The results of the pXMJ19 plasmid enzymatic digestion are shown in the figure. Figure 2 As shown, the first and second lanes are double-digested pXMJ19 plasmids, 6586 bp, and the third lane is a 10000 bp DNA marker, proving that the pXMJ19 plasmid was successfully linearized.
[0050] 2.3 Construction of recombinant plasmid pXMJ19-cspB
[0051] The cspB fragment was combined with the purified linear vector pXMJ19 according to Vazyme II one-step cloning kit instructions to obtain the recombinant plasmid pXMJ19-cspB. The one-step cloning reaction system is shown in Table 2. After 30 minutes in a 37°C water bath, immediately ice bath for 5 minutes and store at -20°C. The schematic diagram of constructing the recombinant expression plasmid is shown in Figure 3 shown.
[0052] Table 2 One-step cloning reaction system
[0053]
[0054] 3. Transformation and Screening of Recombinant Plasmids
[0055] Thaw competent E. coli DH5α cells on ice until liquid. Add competent cells and recombinant plasmid pXMJ19-cspB at a ratio of 10:1 to a pre-chilled centrifuge tube. Place on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds. Cool in an ice box for 3 minutes, add 1 mL of LB, and incubate on a shaker at 37°C, 200 rpm, for 1 hour. Centrifuge, remove most of the supernatant, resuspend the culture, and plate onto chloramphenicol-resistant plates.
[0056] Pick up a single point of bacteria P on the plate, the bacteria P result is as follows Figure 4 As shown, the first lane is a 2000bp DNA marker, and the second to ninth lanes are the results of the construction of the pXMJ19-cspB plasmid in E. coli. The 2000bp band is correct. Select the correct colony and inoculate it into a 50mL centrifuge tube (50μg / mL chloramphenicol), culture it in a 37℃ incubator overnight, and then extract the plasmid pXMJ19-cspB as shown in the figure. Figure 5 The sequencing primers were designed as shown in the following table.
[0057] Table 3 Experimental primer sequences used for sequencing
[0058]
[0059] After successful sequencing, the E. coli DH5α-pXMJ19+cspB strain was obtained.
[0060] 4. Construction of Corynebacterium glutamicum-pXMJ19+cspB strain
[0061] 4.1 Preparation of competent cells of Corynebacterium glutamicum
[0062] Corynebacterium glutamicum liquid culture medium: peptone 10 g / L, yeast powder 5 g / L, beef powder 10 g / L, urea 2 g / L, sucrose 10 g / L, sodium chloride 2.5 g / L;
[0063] Corynebacterium glutamicum solid culture medium: peptone 10 g / L, yeast powder 5 g / L, beef powder 10 g / L, urea 2 g / L, sucrose 10 g / L, sodium chloride 2.5 g / L, agar 15 g / L;
[0064] Corynebacterium glutamicum competent cell culture medium: peptone 10 g / L, yeast powder 5 g / L, beef powder 10 g / L, urea 2 g / L, sucrose 10 g / L, sodium chloride 2.5 g / L, agar 15 g / L, 0.1% Tween 80, glycine 30 g / L;
[0065] Take 1-2 mL of overnight cultured Corynebacterium glutamicum ATCC13032 bacteria and add it to 100 mL of Corynebacterium glutamicum competent cell culture medium. Culture in a 500 mL shake flask at 30°C, 220 rpm for 4-8 hours, until the OD600 reaches between 0.6 and 1.2. Let the cultured competent cells stand on ice for 20 minutes, then divide evenly into sterile 50 mL centrifuge tubes in a clean bench and centrifuge at 4°C, 4000 rpm for 10 minutes.
[0066] Discard the supernatant in a clean hood, resuspend the bacterial slurry in 25 mL of pre-chilled 10% glycerol, and centrifuge again. Repeat this process once more (combine the bacterial suspension into the same tube).
[0067] The supernatant was discarded, 500 μL of pre-cooled 10% glycerol was added, the bacterial slurry was resuspended, and each 90 μL was aliquoted into one electroporation competent cell (the electroporation competent cells of Corynebacterium glutamicum need to be prepared and used immediately).
[0068] 4.2 Construction and screening of recombinant Corynebacterium glutamicum
[0069] The plasmid to be electroporated (about 1 μg of plasmid) was mixed with 90 μL of Corynebacterium glutamicum competent cells and placed on ice for 10 min.
[0070] The competent cells with the plasmid were transferred into a pre-cooled electroporation cuvette and electroporated twice continuously (1800 V, 25 μF, 200 Ω, 2 mm).
[0071] Immediately after electroporation, add 1 mL of preheated culture medium to the cuvette. Transfer the competent cells to a 1.5 mL centrifuge tube and heat shock at 46°C for 6 minutes. Then, recover and culture at 30°C, 170 rpm for 2-4 hours. Plate 150 μL of the bacterial solution on a 25 μg / mL plate. After 2-3 days of incubation, perform colony PCR verification. Colony PCR primers are listed in Table 4.
[0072] Table 4
[0073]
[0074]
[0075] The results of bacteria p are as follows Figure 6 As shown, the first lane is a 2000bp DNA marker, and the second to thirteenth lanes are the results of bacterial p after electroporation of Corynebacterium glutamicum. Among them, the bacterial p results in the second and tenth lanes are correct, and the colony PCR correctly screened out lysine-producing Corynebacterium glutamicum carrying the pXMJ19-cspB plasmid.
[0076] Example 2 Characterization experiment of biofilm
[0077] This experiment used crystal violet staining to compare the relative expression of biofilms of the original and modified strains. Therefore, in this example, the biofilm formation of these two strains was verified in a 96-well plate.
[0078] (1) Cultivate the two strains at 30°C and 220 rpm until they reach the logarithmic phase (OD600 is approximately 0.6).
[0079] (2) The bacterial solution was diluted to an OD600 of 0.1, 180 μL of liquid culture medium of Corynebacterium glutamicum was added to a 96-well plate, and then 20 μL of the diluted bacterial solution was inoculated into the culture medium. The culture was incubated at 37°C for 12 h, 24 h, 36 h, 48 h, and 72 h, respectively, to allow Corynebacterium glutamicum to form a film at the bottom of the 96-well plate.
[0080] (3) Pour off the liquid culture medium, rinse with PBS 2-3 times, fix the biofilm with methanol for 15 minutes, pour off the methanol and dry it, and finally add 0.1% crystal violet to stain for 20 minutes.
[0081] (4) Pour off the crystal violet, rinse with PBS, add 30% glacial acetic acid and shake gently on an oscillator for 30 minutes to decolorize and dissolve the crystal violet.
[0082] (5) Read the result at OD570 using a microplate reader.
[0083] (6) Repeat the above experimental process and observe the differences in film formation between the two strains in the fermentation medium.
[0084] Crystal violet staining and absorbance comparison results are as follows Figure 7 、 8 shown
[0085] Example 3 Comparison of L-lysine production by fermentation of original bacteria and Corynebacterium glutamicum expressing the cspB gene heterologously
[0086] The activation medium components are as follows: peptone 10 g / L, yeast powder 5 g / L, beef powder 10 g / L, urea 2 g / L, sucrose 10 g / L, sodium chloride 2.5 g / L.
[0087] Seed culture medium: 20 g / L sucrose, 10 g / L peptone, 5 g / L yeast extract, 3.5 g / L urea, 5 g / L ammonium sulfate, 4 g / L potassium dihydrogen phosphate, 10.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 0.5 g / L cysteine, 20 mL / L seed stock, adjust pH to 7.0 with 20% KOH solution, and sterilize at 121°C for 30 min. Seed stock preparation: 0.1 g / L calcium pantothenate, 0.15 g / L nicotinamide, 0.075 g / L VB1, and 7.5 mg / L biotin.
[0088] Fermentation medium formula: 70% sugar (corn starch) 128 mL / L, ammonium sulfate 45 g / L, magnesium sulfate 1.2 g / L, molasses 15 mL / L, corn steep liquor 15 mL / L, potassium dihydrogen phosphate 1.1 g / L, ferrous sulfate 200 mg / L, manganese sulfate 150 mg / L, nicotinamide 60 mg / L, calcium pantothenate 9 mg / L, VB1 9 mg / L, copper sulfate 0.9 mg / L, zinc sulfate 1 mg / L, biotin 1.8 mg / L, calcium carbonate 30 g / L.
[0089] Pretreatment of cotton fiber carrier material: Cut the cotton fiber carrier into 5cm×5cm squares, wash with pure water, dry it, soak it in ethanol for 1h, wash it twice with pure water, boil it in a water bath for 20min, and then put it in an oven to dry. Weigh 1.5g, then put it into a shake flask with fermentation liquid and sterilize it together at 115℃ for 20min.
[0090] Add 5 mL of activation medium to each 50 mL centrifuge tube, inoculate the original bacteria and recombinant bacteria, and activate them at 30°C and 220 rpm for 20 h.
[0091] After activation, the cells were poured into 500 mL shaking flasks containing 50 mL seed culture medium and cultured at 30° C. and 220 rpm for 12 h to obtain seed solution.
[0092] Pour 50 mL of fermentation medium into each 500 mL shake flask, and place the pretreated cotton fiber carrier material into the fermentation medium and sterilize together at 115° C. for 20 min.
[0093] 5 mL of seed liquid was added to the fermentation medium and fermented at 30°C and 220 rpm for 72 h.
[0094] Fermentation results such as Figure 9 、 10 shown.
[0095] A series of experiments were conducted using the original bacteria and the recombinant bacteria constructed by the present invention. The crystal violet staining results showed that the biofilm volume of the recombinant bacteria increased by 83%. Finally, a continuous immobilized fermentation experiment was conducted. Figure 9 It can be seen that the immobilization yield of the modified strain is 23.3% higher than that of the original strain. Figure 10 It can be seen that the immobilized fermentation cycle of the modified strain is 23% shorter than that of the original strain.
[0096] The present invention provides a concept and method for producing lysine by biofilm-immobilized fermentation of recombinant Corynebacterium glutamicum heterologously expressing the cspB gene. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of the present invention. Any components not specified in this example may be implemented using existing technologies.
Claims
1. Use of a recombinant Corynebacterium glutamicum in the production of lysine by immobilized fermentation, characterized in that: The recombinant Corynebacterium glutamicum is a cspB gene heterologously expressed in the original Corynebacterium glutamicum ATCC13032 with a nucleotide sequence as shown in SEQ ID NO.1; The method for constructing the recombinant Corynebacterium glutamicum comprises the following steps: (1) Amplify the target gene cspB; (2) cloning the target gene cspB obtained in step (1) between the two restriction enzyme sites of XbaI enzyme and Kpn1 enzyme of plasmid pXMJ19 to obtain the recombinant plasmid pXMJ19-cspB; (3) transforming the recombinant plasmid pXMJ19-cspB obtained in step (2) into competent Escherichia coli DH5α, screening Escherichia coli containing the recombinant plasmid pXMJ19-cspB, and then extracting the recombinant plasmid pXMJ19-cspB; (4) transforming the recombinant plasmid extracted in step (3) into a competent cell of Corynebacterium glutamicum to obtain a recombinant Corynebacterium glutamicum; The composition of the fermentation medium is 70% sugar 118-138 mL / L, ammonium sulfate 35-55 g / L, magnesium sulfate 0.2-2.2 g / L, molasses 5-25 mL / L, corn steep liquor 5-25 mL / L, potassium dihydrogen phosphate 0.1-2.1 g / L, ferrous sulfate 150-250 mg / L, manganese sulfate 100-200 mg / L, nicotinamide 50-70 mg / L, calcium pantothenate 5-15 mg / L, VB1 5-15 mg / L, copper sulfate 0.5-1.5 mg / L, zinc sulfate 0.5-1.5 mg / L, biotin 0.8-2.8 mg / L, and calcium carbonate 20-40 g / L.
2. The application according to claim 1, characterized in that In step (1), the primers for amplifying the target gene cspB are cspB-F and cspB-R, whose nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.
3. The application according to claim 1, characterized in that In step (2), the nucleotide sequence of the plasmid pXMJ19 is shown in SEQ ID NO.
4.
4. The application according to claim 1, characterized in that The seed liquid of the recombinant Corynebacterium glutamicum is added to the fermentation medium at a volume ratio of 5% to 15% for fermentation.
5. The application according to claim 1, characterized in that: The dosage of the immobilized carrier is 0.5-4 g / 50 mL of fermentation medium.