Bacillus subtilis knockout strain for producing gamma-PGA by using glutamic acid waste liquid as well as construction method and application of bacillus subtilis knockout strain

Abstract

The invention discloses a bacillus subtilis knockout strain for producing gamma-PGA by utilizing glutamic acid waste liquid as well as a construction method and application of the bacillus subtilis knockout strain, and belongs to the technical field of microbial fermentation and genetic engineering. In order to solve the technical problem that gamma-PGA synthesis efficiency is low due to metabolic inhibition of natural strains in GWL, key genes of gamma-PGA synthesis pathways are mined through transcriptomics comparison, inhibition factors are analyzed, and through key gene knockout and fermentation verification, it is found that a bacillus subtilis gene knockout strain KH2deltares1 deltares2 is used as an original strain, and a bacillus subtilis gene knockout strain KH2deltares1 deltares2 is used as an original strain. The engineering bacterium with the gudB gene and the rocG gene knocked out can well grow in GWL and can efficiently produce gamma-PGA, and the engineering bacterium is expected to be used for industrial production of gamma-PGA.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation and genetic engineering, and particularly relates to a knockout strain of Bacillus subtilis for producing gamma-PGA by utilizing glutamic acid waste liquid, and a construction method and application thereof. Background Art

[0002] Poly-γ-glutamic acid (γ-PGA) is a biodegradable polymer widely used in food, medicine, agriculture, and environmental protection. Currently, γ-PGA production relies primarily on microbial fermentation, but the high cost of traditional fermentation media limits its industrial production. Glutamate waste liquid (GWL) generated during MSG production has high concentrations of biological oxygen demand (BOD) and chemical oxygen demand (COD), making it difficult to treat using conventional chemical or biological processes. Improper treatment can result in severe environmental pollution. GWL contains a certain amount of unreacted glutamate and other organic and inorganic components, which can serve as a substrate for γ-PGA production, addressing the challenges of GWL treatment and the risk of environmental pollution. However, natural strains often exhibit low γ-PGA synthesis efficiency in GWL due to metabolic inhibition. High glutamate concentrations increase the osmotic pressure of the solution, causing osmotic stress on microbial cells, hindering normal physiological metabolism and thus inhibiting their growth. Existing technologies have not systematically elucidated the mechanism by which GWL inhibits γ-PGA synthesis, nor have targeted strain engineering strategies. Therefore, studying the inhibitory mechanism of GWL on γ-PGA synthesis and constructing an engineered bacterium for producing γ-PGA that is adaptable to GWL are of great significance for realizing the industrial production of γ-PGA.

[0003] Bacillus subtilis ( Bacillus subtilis ) KH2 is deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection with the deposit number CGMCC No. 12426. It is a natural γ-PGA-producing bacterium that can produce γ-PGA extracellularly. In previous studies, the inventors developed a conjugation-based genetic operating system in this undomesticated bacterium and constructed a gene knockout strain KH2Δres1Δres2 with higher transformation efficiency (S. Chen, J. Fu, B. Yu, L. Wang, Development of a conjugation-based genome editing system in an undomesticated bacterium). Bacillus subtilisStrain for poly-γ-glutamic acid production with diverse molecular masses, Journal of Agricultural and Food Chemistry 71(20) (2023) 7734-7743, http: / / doi.org / 10.1021 / acs.jafc.3c01505. However, there is no report on the modification of the gene knockout strain KH2Δres1Δres2 as a starting strain and the production of γ-PGA in GWL with high glutamic acid content. SUMMARY

[0004] In order to solve the technical problem that the natural strain has low γ-PGA synthesis efficiency in GWL with high glutamic acid content due to metabolic inhibition, the present application mines the key genes of the γ-PGA synthesis pathway through transcriptome comparison, analyzes the inhibitors, and verifies through overexpression of the key genes and fermentation, finds that the engineering bacteria obtained by knocking out the gudB gene and the rocG gene of the Bacillus subtilis gene knockout strain KH2Δres1Δres2 as a starting strain can grow well in GWL with high glutamic acid content and can efficiently produce γ-PGA, which is expected to be used for industrial production of γ-PGA.

[0005] In order to solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a Bacillus subtilis knockout strain for producing γ-PGA using glutamic acid waste liquid, which is obtained by knocking out the gudB gene and the rocG gene from the Bacillus subtilis KH2Δres1Δres2 as a starting strain; the nucleotide sequences of the gudB gene and the rocG gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0006] The second object of the present application is to provide a construction method of the above-mentioned Bacillus subtilis knockout strain, which is to knock out the gudB gene and the rocG gene from the Bacillus subtilis KH2Δres1Δres2 as a starting strain.

[0007] In an embodiment of the present application, the construction method comprises the following steps: The upstream and downstream sequences of the target gene gudB and the upstream and downstream sequences of rocG are respectively amplified by taking the genomic DNA of Bacillus subtilis KH2 as a template; the upstream and downstream sequences of the target gene gudB and the upstream and downstream sequences of rocG are respectively connected to the linearized vector pKVMK7-2 which is digested by DpnI enzyme to construct a recombination plasmid, the recombination plasmid is transformed into E. coli, and the obtained transformant is used as a donor strain; Bacillus subtilis KH2Δres1Δres2 is used as a receptor strain; the donor strain and the receptor strain are mixed and then cultured, and the knockout strain is obtained through screening.

[0008] In an embodiment of the present application, the E. coli is E.coli S17-1.

[0009] A third object of the present application is to provide the application of the above-mentioned Bacillus subtilis knockout strain in the production of gamma-PGA by using glutamic acid waste liquid, wherein the glutamic acid waste liquid is waste liquid generated in the production of monosodium glutamate.

[0010] In an embodiment of the present application, the components of the glutamic acid waste liquid include 300 g / L of glutamic acid, 0.7 g / L of (NH4)2SO4, and 0.15 g / L of ammonia nitrogen, and the pH value of the glutamic acid waste liquid is 8.3.

[0011] In an embodiment of the present application, the application is that the Bacillus subtilis knockout strain is inoculated into a seed culture medium, and then cultured at 37℃ for 16 h to obtain a seed liquid, and then the seed liquid is inoculated into a fermentation culture medium containing glutamic acid waste liquid, and then cultured at 37℃ for 24-50 h.

[0012] In an embodiment of the present application, the inoculation amount of the inoculation is 10%.

[0013] In an embodiment of the present application, the composition of the seed culture medium is 20.00 g / L of glucose, 20.00 g / L of L-glutamic acid sodium, 5.00 g / L of yeast extract, 2.00 g / L of K2HPO4, 0.25 g / L of MgSO4·7H2O, and the rest is water, and the pH value is adjusted to 7.00.

[0014] In an embodiment of the present application, the composition of the fermentation culture medium is 30.00 g / L of glucose, 2.00 g / L of K2HPO4, 0.25 g / L of MgSO4·7H2O, 5.00 g / L of (NH4)2SO4, and the glutamic acid waste liquid is added to make the final concentration of glutamic acid be 30.00 g / L, and the pH value is adjusted to 7.00.

[0015] The present application has the following beneficial effects: The present application early utilizes GWL fermentation medium and glutamic acid fermentation medium respectively to carry out fermentation on strain KH2Δres1Δres2, finds that GWL fermentation medium can obviously inhibit the growth of strain KH2Δres1Δres2 and the production of γ-PGA. Further, by transcriptome comparison, the key genes of γ-PGA synthesis pathway are found, and the transcription level of two genes (gudB and rocG) participating in the γ-PGA synthesis pathway in strain KH2Δres1Δres2 is found to change significantly. The gudB gene and the rocG gene in strain KH2Δres1Δres2 are knocked out, and a gene knockout strain is constructed, and it is found through fermentation verification that: in the GWL fermentation system, the knockout strain KH2△res1△res2△gudB△rocG, compared with the control strain KH2△res1△res2, the four knockout strains keep the glutamic acid consumption basically unchanged, and the γ-PGA yield reaches 32.24 g / L, which is increased by 0.28 times.

[0016] The Bacillus subtilis knockout strain KH2△res1△res2△gudB△rocG obtained by the present application can utilize the fermentation medium containing high glutamic acid content GWL to efficiently synthesize γ-PGA, and is expected to be used for industrial production of γ-PGA, and realize rationalization of recycling of monosodium glutamate production waste liquid. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The statistical result graphs of the components of the fermentation medium and the growth status of the strain KH2Δres1Δres2 at different fermentation time points in the GWL-containing fermentation medium and the glutamic acid-containing fermentation medium are shown in FIGS. 1 and 2, respectively. Figure 1 A in FIG. 1 is a detection result graph of the L-glutamic acid content in the fermentation medium at different time points, Figure 1 B in FIG. 1 is a detection result graph of the glucose content in the fermentation medium at different time points, Figure 1 C in FIG. 1 is an OD 600 value detection result graph of the fermentation liquid at different time points. Figure 2 The detection result graph of the corresponding γ-PGA yield of the Bacillus subtilis knockout strain constructed by the present application and the control strain in the GWL fermentation medium at different fermentation times is shown in FIG. 3. Figure 3 The detection result graph of the corresponding L-glutamic acid content in the fermentation medium of the Bacillus subtilis knockout strain constructed by the present application and the control strain in the GWL fermentation medium at different fermentation times is shown in FIG. 4. Figure 4 The detection result graph of the corresponding glucose content in the fermentation medium of the Bacillus subtilis knockout strain constructed by the present application and the control strain in the GWL fermentation medium at different fermentation times is shown in FIG. 5. Figure 5 The OD values of the fermentation broth obtained by fermenting the B. subtilis knockout strain and the control strain in GWL fermentation medium for different fermentation times 600 The detection results of the OD values of the fermentation broth obtained by fermenting the B. subtilis knockout strain and the control strain in GWL fermentation medium for different fermentation times DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be noted that the following examples are only used to explain the present application and are not used to limit the scope of the present application. The following examples are only a part of the embodiments of the present application, not all the embodiments. Other skilled persons in the art can obtain the embodiments without making creative efforts, and the embodiments are protected by the present application.

[0019] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art unless otherwise specified, and can be obtained by commercial channels by skilled persons in the art. The molecular biology experimental operations such as PCR amplification, enzyme digestion and ligation, transformation involved in the present application are conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.

[0020] The starting strain for strain modification of the present application B. subtilis KH2Δres1Δres2 is disclosed in the following article: S. Chen, J. Fu, B. Yu, L. Wang, Development of a conjugation-based genome editing system in an undomesticated Bacillus subtilis strain for poly-γ-glutamic acid production with diverse molecular masses, Journal of Agricultural and Food Chemistry 71(20) (2023) 7734-7743, http: / / doi.org / 10.1021 / acs.jafc.3c01505. The plasmid pKVMK7-2 used in the present invention was constructed by the laboratory in the early stage and disclosed in the following article: S. Chen, J. Fu, B. Yu, L. Wang, Development of a conjugation-based genome editing system in an undomesticated Bacillus subtilis strain for poly-γ-glutamic acid production with diverse molecular masses, Journal of Agricultural and FoodChemistry 71(20) (2023) 7734-7743, http: / / doi.org / 10.1021 / acs.jafc.3c01505. The GWL used in this paper refers to the wastewater generated by the glutamate fermentation and subsequent separation, neutralization, and separation steps of MSG production. It was collected from Ningxia Yipin Biotechnology Co., Ltd. and stored at 4°C. The main components of GWL are 300 g / L glutamate, 0.7 g / L (NH4)2SO4, and 0.15 g / L ammonia nitrogen. The pH value of the original GWL is 8.3.

[0021] The composition of the seed culture medium was as follows: glucose 20.00 g / L, sodium L-glutamate 20.00 g / L, yeast extract 5.00 g / L, K2HPO4 2.00 g / L, MgSO4·7H2O 0.25 g / L, and the pH was adjusted to 7.00.

[0022] The composition of GWL fermentation medium was as follows: glucose 30.00 g / L, K2HPO4 2.00 g / L, MgSO4·7H2O 0.25 g / L, (NH4)2SO4 5.00 g / L, GWL was added to make the final concentration of glutamate 30.00 g / L, and the pH was adjusted to 7.00.

[0023] The composition of glutamate fermentation medium: glucose 30.00 g / L, K2HPO4 2.00 g / L, MgSO4·7H2O 0.25 g / L, (NH4)2SO4 5.00 g / L, sodium L-glutamate 30.00 g / L, pH adjusted to 7.00.

[0024] Example 1: Screening of genes affecting the production of γ-PGA by Bacillus subtilis using GWL The present application previously utilizes GWL fermentation medium and glutamic acid fermentation medium to respectively ferment the strain KH2Δres1Δres2, respectively detects the L-glutamic acid content and glucose content in the fermentation medium at 0 h, 24 h, 30 h and 48 h of fermentation, and detects the OD 600 value of the fermentation liquor, and it is found that 54.22% of glutamate is consumed for γ-PGA synthesis when there is no GWL for 48 h of fermentation, and only 22.99% of glutamate is converted into γ-PGA when GWL replaces L-glutamic acid sodium, which can be seen that GWL has obvious inhibitory effect on the production of γ-PGA; the detection of the OD 600 value of the fermentation liquor can also show that GWL inhibits the growth of the strain KH2Δres1Δres2 (see Figure 1 ).

[0025] Further, the present application mines the key genes of the γ-PGA synthesis pathway through transcriptomic comparison, finds that the transcription levels of 2 genes (gudB and rocG) involved in the γ-PGA synthesis pathway in KH2Δres1Δres2 significantly change in the fermentation process of glutamic acid fermentation medium and GWL fermentation medium. Therefore, taking the strain KH2Δres1Δres2 as the starting strain, the 2 genes are simultaneously knocked out, and the obtained engineering bacteria are subjected to fermentation verification.

[0026] Example 2: Construction of γ-PGA high-yield Bacillus subtilis knockout strain using GWL 1. Construction of recombinant expression vector The recombinant expression vector is constructed based on the plasmid pKVMK7-2: first, the plasmid pKVMK7-2 is linearized by reverse PCR technology using primers pKVMK7-2-F and pKVMK7-2-R; then, the target gene gudB upstream sequence is obtained by PCR amplification using the genomic DNA of Bacillus subtilis KH2 as the template and using primers gudBup pKVMK7-2-F and gudBup-R. At the same time, the target gene gudB downstream sequence is obtained by PCR amplification using the genomic DNA of Bacillus subtilis KH2 as the template and using primers gudBdown-F and gudBdown pKVMK7-2-R. Finally, the Gibson assembly method is used to connect the upstream and downstream fragments of the target gene gudB to the DpnI enzyme-digested linearized pKVMK7-2 vector. The construction of the recombinant plasmid pKVMK7-2-ΔgudB is confirmed by agarose gel electrophoresis identification and DNA sequencing. The construction method of the recombinant plasmid pKVMK7-2-ΔrocG is the same as above, and the only difference is that different primers are used, and the specific primer information is shown in Table 1.

[0027] The nucleotide sequences of the gudB gene and the rocG gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0028] Table 1 Information of primers used in construction of recombinant expression vector

[0029] 2. Construction of recombinant engineering bacteria Introducing the target plasmid into E. coli by chemical transformation E.coli In S17-1, the transformants obtained by kanamycin (25 μg / mL) screening according to the plasmid properties were used as the donor strain. The gene knockout strain Bacillus subtilis KH2Δres1Δres2 with higher transformation efficiency was used as the recipient strain in the present application. Both the donor bacteria and the recipient bacteria were cultured to OD 600 The value was 1.0, after washing twice with fresh LB medium without antibiotics, the two strains were mixed and inoculated on LB solid plate without antibiotics. Incubated at 30°C for 6 h, scraped a single colony and resuspended in 1 mL LB liquid medium, 200 μL of bacterial solution was spread on LB solid plate containing kanamycin (10 μg / mL) and polymyxin B (20 μg / mL), and after 22 h incubation at 30°C, colony PCR was performed for verification. After screening, the transformants were picked and incubated on LB plate containing 1% xylose and 30 μg / mL kanamycin and LB plate containing only 30 μg / mL kanamycin at 45°C, and colony PCR was used to screen the homologous recombinant strain. It was inoculated into LB liquid medium and incubated at 37°C for 4 h. It was streaked on LB plate without antibiotics and incubated at 45°C for 17 h to screen the second homologous recombinant strain. It was streaked on LB plate containing 30 μg / mL kanamycin and LB plate without antibiotics and incubated at 37°C to test the resistance. Single colonies that could only grow on antibiotic-free plates were picked and subjected to colony PCR verification using primers △gudB-seq-F / △gudB-seq-R or △rocG-seq-F / △rocG-seq-R. If the verification result was positive, the knockout was successful, and the Bacillus subtilis knockout strain KH2Δres1Δres2△gudB△rocG was obtained.

[0030] Example 3: Application of Bacillus subtilis knockout strain with high yield of γ-PGA by GWL Take 200 μL of bacterial liquid of strain KH2Δres1Δres2△gudB△rocG to inoculate into LB culture medium, and culture at 37°C and 200 r / min for 24 h. Then inoculate into seed culture medium at 10% inoculation amount, and continue to culture at 37°C for 16 h to prepare seed liquid. Inoculate the seed liquid into GWL fermentation culture medium at 10% inoculation amount, and continue to culture at 37°C for 48 h. At 0, 24 and 48 h of fermentation, take samples to determine the biomass, glucose concentration, glutamic acid concentration and γ-PGA concentration. Meanwhile, take KH2Δres1Δres2 as a control.

[0031] The biomass is determined by optical density method to obtain OD 600 value.

[0032] The glucose and glutamic acid concentrations are determined by using SBA-40D biological sensor analyzer.

[0033] The detection method of γ-PGA concentration is as follows: take the bacterial liquid to be tested, centrifuge at 4°C and 10000×g for 20 min to remove the bacterial body, add 4 times the volume of pre-cooled anhydrous ethanol to the supernatant, and store at 4°C overnight, then centrifuge at 4°C and 10000×g for 20 min to remove the anhydrous ethanol, obtain the γ-PGA precipitate, and add a certain volume of distilled water to re-dissolve and dilute to an appropriate multiple. Prepare 5g / L CTAB solution with 2% NaOH solution, accurately take 100 μL of standard solution and the liquid to be tested, and place them in a 96-well plate, add 100 μL of CTAB solution, react for 3 min, and detect the absorbance value at 240 nm. Calculate the γ-PGA concentration of the liquid to be tested according to the standard curve.

[0034] As shown in Figure 2 , Figure 3 , Figure 4 and Figure 5 , in the GWL fermentation system, the four-knockout strain KH2△res1△res2△gudB△rocG has a γ-PGA yield of 32.24 g / L, which is increased by 0.28 times compared with the control strain KH2△res1△res2, while maintaining the glutamic acid consumption basically unchanged.

[0035] SEQ ID NO.1: SEQ ID NO. 2: Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is to be understood that the application is not limited to those precise embodiments, and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope or spirit of the application. Therefore, the scope of the present application should be limited only by the appended claims.

Claims

1. A Bacillus subtilis knockout strain for producing γ-PGA using glutamic acid waste liquid, characterized in that, The Bacillus subtilis knockout strain is obtained by knocking out gudB gene and rocG gene from Bacillus subtilis KH2Δres1Δres2, and the nucleotide sequences of the gudB gene and the rocG gene are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. The method for constructing the knock-out strain of Bacillus subtilis according to claim 1, characterized in that, The construction method is to knock out gudB gene and rocG gene from Bacillus subtilis KH2Δres1Δres2.

3. The construction method of claim 2, wherein, The method comprises the following steps: The upper and lower sequences of the target gene gudB and the upper and lower sequences of the target gene rocG are amplified from the genomic DNA of Bacillus subtilis KH2, and the Gibson assembly method is used to connect the upper and lower sequences of the target gene gudB and the upper and lower sequences of the target gene rocG to the DpnI enzyme-digested pKVMK7-2 linearized vector to construct a recombinant plasmid, the recombinant plasmid is transformed into E. coli, and the obtained transformant is used as a donor strain, Bacillus subtilis KH2Δres1Δres2 is used as a recipient strain, the donor strain and the recipient strain are mixed and then cultured, and the knockout strain is obtained through screening.

4. The construction method according to claim 3, characterized in that, The E. coli is E.coli S17-1.

5. Use of the B. subtilis knockout strain of claim 1 in the production of γ-PGA using glutamic acid waste liquid, characterized in that, The glutamic acid waste liquid is a waste liquid generated in the production of monosodium glutamate.

6. Use according to claim 5, characterized in that, The components of the glutamic acid waste liquid include 300 g / L of glutamic acid, 0.7 g / L of (NH4)2SO4, and 0.15 g / L of ammonia nitrogen, and the pH value of the glutamic acid waste liquid is 8.

3.

7. Use according to claim 6, characterized in that, The application is to inoculate the Bacillus subtilis knockout strain into a seed culture medium, cultivate at 37℃ for 16 h to obtain a seed liquid, and then inoculate the seed liquid into a fermentation culture medium containing the glutamic acid waste liquid, and cultivate at 37℃ for 24-50 h.

8. Use according to claim 7, characterized in that, The inoculation amount of the inoculation is 10%.

9. Use according to claim 7, characterized in that, The composition of the seed culture medium is 20.00 g / L of glucose, 20.00 g / L of L-glutamic acid sodium, 5.00 g / L of yeast extract, 2.00 g / L of K2HPO4, 0.25 g / L of MgSO4·7H2O, and the rest is water, and the pH value is adjusted to 7.

00.

10. Use according to claim 7, characterized in that, The composition of the fermentation culture medium is 30.00 g / L of glucose, 2.00 g / L of K2HPO4, 0.25 g / L of MgSO4·7H2O, 5.00 g / L of (NH4)2SO4, and the glutamic acid waste liquid is added to make the final concentration of glutamic acid 30.00 g / L, and the pH value is adjusted to 7.00.

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