A gene cluster replacing γ-PGA synthase from PgsB and a method for synthesizing polyglutamic acid using the same

Abstract

The present invention discloses a gene cluster for replacing a γ-PGA synthase sourced from PgsA and a method for synthesizing polyglutamic acid therefrom. The γ-polyglutamic acid synthase gene cluster pgsBCA of Bacillus licheniformis is cloned into a Corynebacterium glutamicum F343 strain with high glutamate production for exogenous expression. On this basis, pgsB is replaced with pgsB from other strains, namely Bacillus subtilis, Bacillus methylotrophicus, Bacillus amyloliquefaciens, and Bacillus anthracis, to obtain recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA. The yields of γ-PGA produced by B′(BS)CA, B′(BM)CA, and B′(BAM)CA are increased by 48.00%, 107.87%, and 98.43%, respectively, while the yield of γ-PGA produced by B′(BAM)CA is significantly decreased. Therefore, replacing the source of PgsB in a specific PgsBCA multiprotein complex is an effective way to further improve γ-PGA production.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology and fermentation engineering, specifically to a method for synthesizing polyglutamic acid by replacing the source of PgsB in the PgsBCA multiprotein complex and promoting the synthesis of γ-PGA, particularly a gene cluster of γ-PGA synthase that replaces the source of PgsB and a method for synthesizing polyglutamic acid. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is a biopolymer composed of L-glutamic acid and D-glutamic acid monomers. It exhibits high water solubility, good biodegradability, strong thickening properties, and excellent absorption and binding capacity for metal ions. In recent years, γ-PGA has been widely used in food, cosmetics, biomedicine, environmental protection, and other fields.

[0003] Currently, microbial fermentation is the primary method for commercially producing γ-PGA due to its advantages, including low raw material costs, minimal environmental pollution, and high purity of the natural product. The main γ-PGA-producing strain is a Bacillus sp. Depending on whether glutamate is added as a precursor during production, γ-PGA-producing strains can be divided into two types: glutamate-dependent and glutamate-independent. Some researchers have successfully synthesized γ-PGA without the need for glutamate by heterologously expressing γ-PGA synthase using E. coli and C. glutamicum as chassis cells, but yields have been limited. For example, patent CN103146630A describes a recombinant Corynebacterium glutamicum for producing γ-polyglutamic acid, as well as its construction method and uses. The engineered strain is derived from wild-type C. glutamicum ATCC13869, which is transformed with a recombinant expression plasmid containing the pgsBCA gene, a gene encoding the γ-PGA synthase complex.

[0004] Currently, some scholars have proposed methods for increasing γ-PGA production by heterologously expressing γ-PGA synthase. For example, patent CN113234764A proposes a method for heterologous expression of γ-polyglutamic acid, in which varying concentrations of D-Glu are exogenously added to the fermentation culture of the C. glutamicum F343pZM1-capBCA strain to synthesize γ-polyglutamic acid with varying D / L monomer ratios. Another example is patent CN112175982A, which proposes a recombinant strain for a γ-PGA polymerase gene, its construction method, and its application. Based on the tandem expression of the polyglutamate synthase gene cluster capBCA, gene expression regulatory elements are used to independently regulate the expression levels of individual genes, thereby constructing a recombinant strain for individually regulated polymerase genes. However, these methods have limited yield improvements.

[0005] How to more efficiently and simply increase the yield of γ-PGA in the heterologous expression of γ-PGA synthase has become a key research direction in this field and has important market value and application prospects. Summary of the Invention

[0006] Purpose of the invention: In response to the above-mentioned defects of the prior art, the present invention provides a gene cluster of γ-PGA synthase that replaces the source of PgsB and a method for synthesizing polyglutamic acid therefrom. By using Corynebacterium glutamicum, which has a high L-glutamic acid production, as a chassis microorganism, the γ-polyglutamate synthase gene cluster pgsBCA from Bacillus licheniformis is exogenously expressed. On this basis, the source of pgsB is replaced alone to obtain a new gene cluster of γ-PGA synthase, which is used to promote the synthesis of γ-PGA.

[0007] The first object of the present invention is to provide a gene cluster pgsB′CA of γ-PGA synthase, wherein the sequence of the synthase gene pgsB′ in the gene cluster pgsB′CA is any one of SEQ ID NO.4 to SEQ ID NO.6.

[0008] Optionally, in one embodiment of the present invention, the synthetase gene pgsB′ is derived from any one of Bacillus subtilis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens.

[0009] The γ-PGA synthase, PgsBCA, is encoded by the pgsB, pgsC, and pgsA genes and catalyzes the synthesis of γ-PGA from glutamate. The ability to synthesize γ-PGA is influenced by the source of the γ-PGA synthase, PgsBCA. Within the PgsBCA multienzyme complex, PgsB and PgsC are primarily responsible for catalysis, while PgsA is responsible for γ-PGA chain elongation and transport. Therefore, γ-PGA synthesis can be enhanced by replacing the synthase with alternative sources.

[0010] Optionally, in one embodiment of the present invention, the synthase genes pgsA and pgsC are derived from the gene cluster pgsBCA of γ-PGA synthase of Bacillus licheniformis.

[0011] In one embodiment of the present invention, the Bacillus licheniformis is purchased from ATCC, and the strain number is ATCC9945a.

[0012] In one embodiment of the present invention, the sequences of pgsB, pgsC, and pgsA derived from Bacillus licheniformis are shown as SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0013] The second object of the present invention is to provide a recombinant plasmid constructed based on the above gene cluster pgsB′CA.

[0014] Optionally, in one embodiment of the present invention, the recombinant plasmid is pZM1-B′(BS)CA, pZM1-B′(BM)CA, or pZM1-B′(BAM)CA, wherein BS, BM, and BAM in the recombinant plasmid represent pgsB′ derived from Bacillus subtilis, methylotrophic Bacillus, and Bacillus amyloliquefaciens, respectively.

[0015] Optionally, in one embodiment of the present invention, the recombinant plasmid is constructed using the same tail enzyme ligation technology, which is a new modular synthetic biology tool ePathBrick that is directly used for pathways.

[0016] The third object of the present invention is to provide a polymerase gene recombinant strain constructed by transformation based on the above-mentioned recombinant plasmid.

[0017] Optionally, in one embodiment of the present invention, the above-mentioned recombinant strain uses C. glutamicum F343 as a chassis.

[0018] In one embodiment of the present invention, the recombinant strains include B'(BS)CA, B'(BM)CA, and B'(BAM)CA.

[0019] The fourth object of the present invention is to provide a method for synthesizing γ-PGA, which utilizes the recombinant strain for fermentation culture to produce polyglutamic acid and increase the yield of γ-PGA.

[0020] A fifth object of the present invention is to provide a method for increasing polyglutamic acid production by replacing the source of PgsB, using C. glutamicum F343 as a chassis to heterologously express the gene cluster pgsB′CA of the γ-PGA synthase as described above, and to ferment and culture the resulting recombinant strain.

[0021] Optionally, in one embodiment of the present invention, the steps of fermentation culture of the recombinant strain are: inoculating the seed liquid of the recombinant strain into the fermentation medium, first culturing at 32±2°C for 1 to 2 hours, adding IPTG to induce for 1 to 2 hours, and finally culturing at 37±2°C for 48±5 hours.

[0022] Optionally, in one embodiment of the present invention, the fermentation medium comprises: corn steep liquor 10 g·L -1 , glucose 120g·L -1 , K2HPO4 1.0g·L -1 , MgSO4 0.6g·L -1 , FeSO4·7H2O 0.002g·L -1 , MnCl2·4H2O0.002g·L -1, urea 7.0g·L -1 , pH 6.8-7.0.

[0023] Optionally, in one embodiment of the present invention, the method of replacing the source of the pgsB gene alone includes: respectively synthesizing the pgsB genes derived from Bacillus subtilis, methylotrophic Bacillus, and Bacillus amyloliquefaciens, respectively amplifying pgsC and pgsA derived from Bacillus licheniformis by PCR, and expressing them in tandem with pgsB′ from the three sources, using Corynebacterium glutamicum C. glutamicum as the competent cell, and constructing a recombinant strain that replaces pgsB.

[0024] The present invention uses Corynebacterium glutamicum, a high-yield L-glutamic acid microorganism, as a chassis microorganism to express pgsB' derived from Bacillus subtilis, methylotrophic Bacillus, Bacillus amyloliquefaciens, and Bacillus anthracis in tandem with pgsC and pgsA derived from Bacillus licheniformis. Compared with a recombinant Corynebacterium glutamicum (F343-BCA) expressing pgsBCA derived from Bacillus licheniformis, it was found that the γ-PGA yield produced by the recombinant strains replaced with Bacillus subtilis, methylotrophic Bacillus, and Bacillus amyloliquefaciens pgsB' increased by 48.00%, 107.87%, and 98.43%, respectively, while the γ-PGA yield produced by the recombinant strain replaced with Bacillus anthracis pgsB' alone decreased significantly. Therefore, the solution provided by the present invention is of general significance for research on high-yield γ-PGA using Corynebacterium glutamicum.

[0025] In one embodiment of the present invention, the method for synthesizing γ-PGA comprises aspirating 2-5 μL of bacterial solution of B′(BS)CA, B′(BM)CA, B′(BAM)CA, or B′(BAN)CA from a cryopreserved tube, streaking onto an LB-Glu plate (containing 25 mg / L kanamycin), and incubating for 24 hours at 30°C. A single colony is then transferred to a seed culture medium and incubated at 32°C, 120 rpm, for 12 hours. A 5% inoculum is then inoculated into a fermentation medium, incubated at 32°C, 120 rpm for 2 hours, and induced with IPTG for 1 hour before the temperature is adjusted to 37°C and incubated for 48 hours.

[0026] Beneficial effects of the present invention:

[0027] (1) The present invention proposes a new gene cluster pgsB′CA for γ-PGA synthase, which promotes the synthesis of γ-PGA by replacing the synthase gene pgsB from other sources;

[0028] (2) The present invention successfully constructed an exogenous synthesis pathway for γ-PGA and found that replacing the source of pgsB with pgsA′ of Bacillus subtilis, Bacillus methylotrophicus, or Bacillus amyloliquefaciens is beneficial to the synthesis of γ-PGA. Compared with the unreplaced gene cluster pgsBCA, the γ-PGA yield increased by 48.00%, 107.87%, and 98.43%, respectively, providing an effective method for further increasing the γ-PGA yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of colony PCR verification of recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA;

[0030] Figure 2 : Schematic diagram of the fermentation performance of the recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] The present invention can be better understood based on the following examples. However, it is readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the present invention and should not and will not limit the present invention described in detail in the claims.

[0033] In the embodiment of the present invention, the method for replacing the source of the pgsB gene alone is to synthesize the pgsB genes from Bacillus subtilis, methylotrophic Bacillus, Bacillus amyloliquefaciens, and Bacillus anthracis, respectively, and PCR amplify pgsC and pgsA from Bacillus licheniformis, respectively. The pgsB from the four sources are expressed in tandem with the pgsC and pgsA of Bacillus licheniformis, and Corynebacterium glutamicum C. glutamicum is used as a competent cell to construct a recombinant strain replacing pgsB, and the recombinant strain is applied to fermentation culture to produce polyglutamic acid, and the yield of polyglutamic acid is investigated.

[0034] The assay methods or culture methods involved in the following examples are as follows:

[0035] Seed culture medium: corn steep liquor 35g·L -1 , glucose 25g·L -1 , K2HPO4 1.5g·L -1 , MgSO40.6g·L -1 , FeSO4·7H2O 0.005g·L -1 , MnCl2·4H2O 0.005g·L-1 , urea 2.5g·L -1 (Separate sterilization), pH 6.8-7.0, 25 mL of liquid in each 250 mL Erlenmeyer flask, sterilize at 121°C for 20 min.

[0036] Fermentation medium: corn steep liquor 10 g·L -1 , glucose 120g·L -1 , K2HPO4 1.0g·L -1 , MgSO4 0.6g·L -1 , FeSO4·7H2O 0.002g·L -1 , MnCl2·4H2O 0.002g·L -1 , urea 7.0g·L -1 (Separate sterilization), pH 6.8-7.0, 50 mL of liquid per 500 mL triangular bottle, sterilize at 121℃ for 20 min.

[0037] Biomass determination method (UV-visible photometer): dilute the sample at each sampling point to an appropriate multiple to OD 600 The value is 0.2-0.8, and the absorbance is measured at a wavelength of 600nm.

[0038] Glucose and glutamate content detection: Fermentation broth was collected at different time points and the fermentation temperature was 12000 r·min -1 After centrifugation for 20 min, the supernatant was diluted to a concentration of glucose and L-glutamic acid between 0 and 1.0 g·L. -1 Within the specified range, the contents of glucose and L-glutamic acid were determined respectively using a biochemical analyzer.

[0039] Polyglutamic acid yield determination method: Sample preparation: Centrifuge the fermentation broth at 12,000 rpm for 15 minutes. Remove the supernatant, dilute it appropriately, filter it through a 0.45 μm filter membrane, and transfer 500 μL into a 2 mL injection vial for analysis. Gel permeation chromatography columns: TSKgelsuperAw 4000 or TSKgelsuperAw 5000. Column temperature: 40°C. Injection volume: 50 μL. Mobile phase: 0.2 M Na₂SO₄, pH adjusted to approximately 4.0 with glacial acetic acid. Detector: Waters liquid phase RID differential detector.

[0040] Transformation method of Corynebacterium glutamicum (plasmid): (1) Take a single colony and inoculate it into seed culture medium, culture it at 32℃, 120rpm overnight; inoculate an appropriate amount of seed culture into competent culture medium, make the initial OD 600 =0.3; culture at 30°C, 120 rpm until OD 600=0.7-0.8, about 4h; place the culture medium on ice for 10min, divide the bacterial solution into centrifuge tubes, centrifuge for 10min, 4000rpm, obtain the strain; wash 4 times with 25ml ice bath 10% glycerol; suspend with 2mL 10% glycerol, divide into 1.5mL Eppendorf tubes after ice bath to obtain competent cells. (2) Place the electroporation cup on the clean bench for cleaning in advance and place in the refrigerator; (3) Take the competent cells and place them on ice to melt, add 3-5μL DNA to mix, add to the electroporation cup, 1.8kV, 5mS electroporation, and immediately add 1mL BHIS to the electrode cup for suspension; (4) After suspension, transfer to a 1.5mL EP tube, 46℃, incubate for 6min; (5) After incubation, culture at 30℃ for 2h to allow the cells to recover and express resistance; (6) After incubation, centrifuge at 12000rpm for 1min, and spread on a plate containing 50μg / mL Kan + Resistant LBHIS plates for 1-2 days.

[0041] Example 1: Construction of a recombinant strain with a single replacement of the PgsB source

[0042] NCBI was used to search for the γ-PGA polymerase genes pgsB, pgsC, and pgsA from B. licheniformis ATCC9945a strain, and the sequences were shown in SEQ ID NO.1-SEQ ID NO.3, with lengths of 1182 bp, 450 bp, and 1170 bp, respectively; the pgsB′ genes from B. subtilis NX-2, B. methylotrophicus SK19.001, B. amyloliquefaciens LL3, and B. anthracis str. BF1 strains were shown in SEQ ID NO.4-SEQ ID NO.7, with lengths of 1182 bp, 1182 bp, 1182 bp, and 1395 bp, respectively.

[0043] Amplification primers containing Nde I and BamH I restriction sites were designed (see Table 1). For the construction of the recombinant strain B′(BS)CA, the pgsC and pgsA gene fragments were amplified using the genomes of B. licheniformis and rmis ATCC9945a as templates. The PCR products were then digested with Nde I and BamH I and ligated with the inducible vector pZM(Ptac) to generate the recombinant plasmids pZM1-pgsC and pZM1-pgsA. Plasmid pZM1-pgsB′(BS) was obtained by gene synthesis. pZM1-pgsB′(BS) was double-digested with Nhe I and Sal I, and pZM1-pgsC was double-digested with Avr II and Sal I. Taking advantage of the fact that Nhe I and Avr II are a pair of homotypic enzymes, resulting in identical sticky ends after digestion, the two digested fragments were ligated by T4, and the Nhe I and Avr II restriction sites at the junction disappeared, resulting in the recombinant plasmid pZM1-pgsB′(BS)-pgsC. pZM1-pgsA was double-digested with Nhe I and Sal I, and Avr II and Sal I were double-digested. The two digested products were then ligated by T4 to obtain the recombinant plasmid pZM1-B′(BS)CA.

[0044] The other recombinant plasmids pZM1-B′(BM)CA, pZM1-B′(BAM)CA, and pZM1-B′(BAN)CA were constructed using the same method.

[0045] The recombinant plasmids pZM1-B′(BM)CA, pZM1-B′(BAM)CA, and pZM1-B′(BAN)CA were transformed into C. glutamicum F343, respectively, and screened in a medium containing 25 μg / L Kan. Transformants were picked for colony PCR to verify correctness (e.g. Figure 1 ), the recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA were successfully constructed.

[0046] Figure 1 In the figure, M: DNA Marker; 1: recombinant bacteria B′(BS)CA; 2: recombinant bacteria B′(BM)CA; 3: recombinant bacteria B′(BAM)CA; 4: recombinant bacteria B′(BAN)CA. The recombinant bacteria have a clear band at around 3800 bp, which is consistent with the total length of multiple fragments in each recombinant plasmid, indicating that the recombinant strain with the sole replacement of PgsB source in C. glutamicum F343 was successfully constructed.

[0047] Table 1 Primers used in Example 1

[0048]

[0049] Example 2: Shake flask fermentation performance test of recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA

[0050] The recombinant strains B′(BS)CA, B′(BM)CA, B′(BAM)CA, and B′(BAN)CA were fermented in shake flasks, and the recombinant strain F343-BCA was used as a control to evaluate the effect of replacing the PgsB source alone on γ-PGA synthesis.

[0051] (1) Biomass comparison

[0052] The results are as follows Figure 2 As shown in (a), compared with the control strain F343-BCA, the growth of strains B′(BS)CA and B′(BAM)CA was good, and the biomass at 24 h increased by 18.40% and 15.60%, respectively; the growth of strain B′(BM)CA was similar to that of F343-BCA, with the highest biomass OD 600 was about 20.0; however, the growth of strain B′(BAN)CA was significantly inhibited, with the biomass OD 600 The highest was only 13.57, and then gradually declined.

[0053] (2) Comparison of glucose consumption

[0054] The results are as follows Figure 2 As shown in (b), the glucose utilization of the recombinant strains was similar, with rapid glucose consumption in the first 12 h and then slow glucose consumption after 12 h.

[0055] (3) Comparison of glutamate content

[0056] The results are as follows Figure 2 As shown in (c), C. glutamicum F343 is a strain that can produce high glutamate, and can produce 11.05 g·L under shake flask fermentation conditions. -1 glutamate is produced by the fermentation of C. glutamicum F343, so no exogenous glutamate addition is required. Using C. glutamicum F343 as a host to express the γ-PGA synthase gene, glutamate can be efficiently polymerized to produce γ-PGA. During the fermentation process, the glutamate accumulation of F343-BCA gradually increased, reaching a glutamate content of 4.50 g·L at 48 hours. -1 In contrast, strains B′(BM)CA and B′(BAM)CA showed almost no glutamate accumulation. The glutamate accumulation of strain B′(BS)CA was lower than that of F343-BCA, with the highest glutamate content at 36 h reaching 2.77 g·L -1 The glutamic acid content of B′(BAN)CA was the lowest, and after 24 hours, the glutamic acid content was basically maintained at 7.90 g·L -1 .

[0057] (4) Comparison of γ-PGA production

[0058] The results are as follows Figure 2 As shown in (d), γ-PGA synthesis and glutamate accumulation are inversely proportional. Compared with F343-BCA, the γ-PGA synthases of B′(BS)CA, B′(BM)CA, and B′(BAM)CA efficiently polymerize glutamate, resulting in γ-PGA yield increases of 48.00%, 107.87%, and 98.43%, respectively. However, the yield of B′(BAN)CA was significantly lower than that of F343-BCA, which is partially related to the growth inhibition of the strain. Therefore, it is inferred that replacing the source of PgsB in specific PgsBCA multiprotein complexes is an effective method to further increase γ-PGA production.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene cluster encoding γ-PGA synthase pgsB ' CA , characterized in that, The gene cluster pgsB ' CA synthase genes in pgsB ' is any one of the sequences shown in SEQ ID NO.4-SEQ ID NO.6, the gene cluster pgsB' CA synthase genes in pgsC The sequence of the synthase gene is shown in SEQ ID NO.

2. pgsA The sequence is shown in SEQ ID NO.

3.

2. The gene cluster of γ-PGA synthase according to claim 1 pgsB'CA , characterized in that, The synthetase gene pgsB' Derived from any one of Bacillus subtilis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens.

3. The gene cluster of γ-PGA synthase according to claim 1 pgsB ' CA , characterized in that, The synthetase gene pgsB 、 pgsC Gene cluster encoding γ-PGA synthase from Bacillus licheniformis pgsBCA .

4. A recombinant plasmid comprising the gene cluster according to any one of claims 1 to 3 pgsB ' CA .

5. A polymerase gene recombinant strain comprising the recombinant plasmid according to claim 4, C. glutamicum F343 is the chassis.

6. A method for synthesizing polyglutamic acid, characterized in that: The recombinant strain according to claim 5 is used for fermentation and culture.

7. A method for increasing polyglutamic acid production by replacing the source of PgsB, characterized in that: by C. glutamicum F343 is a chassis that heterologously expresses the gene cluster of γ-PGA synthase as described in any one of claims 1 to 3 pgsB ' CA , and the obtained recombinant strain was fermented and cultured.

8. The method according to claim 6 or 7, characterized in that The steps of fermentation culture of the recombinant strain are: inoculating the seed liquid of the recombinant strain into the fermentation medium, first culturing at 32±2°C for 1-2 hours, adding IPTG for induction for 1-2 hours, and finally culturing at 37±2°C for 48±5 hours.

9. The method according to claim 8, characterized in that The fermentation medium includes: corn steep liquor 10 g·L -1 , glucose 120 g·L -1 , K2HPO4 1.0 g·L -1 , MgSO4 0.6 g·L -1 , FeSO4·7H2O 0.002 g·L -1 , MnCl2·4H2O 0.002 g·L -1 , urea 7.0g·L -1 , pH 6.8-7.0.

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