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

Abstract

The present invention discloses a gene cluster for replacing the γ-PGA synthase sourced from PgsA and a method for synthesizing polyglutamic acid therefrom. The present invention clones the γ-polyglutamic acid synthase gene cluster pgsBCA from Bacillus licheniformis into a high-glutamic acid-producing strain, Corynebacterium glutamicum F343, for exogenous expression. On this basis, pgsA is replaced with pgsA from other strains, namely Bacillus subtilis, Bacillus methylotrophicus, Bacillus amyloliquefaciens, and Bacillus anthracis, to obtain recombinant strains BCA'(BS), BCA'(BM), BCA'(BAM), and BCA'(BAN). The γ-PGA yields produced by BCA'(BS), BCA'(BM), and BCA'(BAM) increased by 54.73%, 34.87%, and 25.10%, respectively, while the γ-PGA yield produced by BCA'(BAN) decreased by 24.09%. Therefore, replacing the source of PgsA 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 replacing the source of PgsA in a PgsBCA multiprotein complex to promote the synthesis of γ-PGA, and more particularly to a gene cluster of γ-PGA synthase that replaces the source of PgsA 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 primary 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 glutamate addition 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, its construction method, and its use. The engineered strain is derived from wild-type C. glutamicum ATCC 13869, 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 PgsA 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 pgsA 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 pgsBCA′ of γ-PGA synthase, wherein the sequence of the synthase gene pgsA′ in the gene cluster pgsBCA′ 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 pgsA′ 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 pgsB and pgsC are derived from the gene cluster pgsBCA of γ-PGA synthase of Bacillus licheniformis.

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

[0012] Optionally, 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 pgsBCA′.

[0014] Optionally, in one embodiment of the present invention, the recombinant plasmids are pZM1-BCA′(BS), pZM1-BCA′(BM), and pZM1-BCA′(BAM), and BS, BM, and BAM in the recombinant plasmids represent pgsA′ 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] Optionally, in one embodiment of the present invention, the recombinant strains include BCA′(BS), BCA′(BM), and BCA′(BAM).

[0019] The fourth object of the present invention is to provide a method for synthesizing polyglutamic acid, which utilizes the above-mentioned 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 PgsA, using C. glutamicum F343 as a chassis to heterologously express the gene cluster pgsBCA′ 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 , K2HPO41.0g·L -1 , MgSO40.6g·L -1 , FeSO4·7H2O0.002g·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 pgsA gene alone includes: respectively synthesizing the pgsA genes derived from Bacillus subtilis, methylotrophic Bacillus, and Bacillus amyloliquefaciens, PCR amplifying pgsB and pgsC derived from Bacillus licheniformis, expressing them in tandem with pgsA from the three sources, and using Corynebacterium glutamicum C. glutamicum as the competent cell to construct a recombinant strain that replaces pgsA.

[0024] The present invention uses Corynebacterium glutamicum, a high-yield L-glutamic acid microorganism, as a chassis microorganism, and expresses pgsB and pgsC derived from Bacillus licheniformis in tandem with pgsA' derived from Bacillus subtilis, methylotrophic Bacillus, Bacillus amyloliquefaciens, and Bacillus anthracis, respectively. 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 in which the pgsA' source was replaced with Bacillus subtilis, methylotrophic Bacillus, and Bacillus amyloliquefaciens alone increased by 54.73%, 34.87%, and 25.10%, respectively. In contrast, the γ-PGA yield produced by the recombinant strain in which the pgsA' source was replaced with Bacillus anthracis alone decreased by 24.09%. 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 is to aspirate 2-5 μL of BCA′(BS), BCA′(BM), or BCA′(BAM) from a cryopreserved tube, streak the plate onto an LB-Glu (containing 25 mg / L kanamycin) plate, and incubate for 24 hours at 30°C. A single colony is then placed in a seed culture medium, incubated at 32°C, 120 rpm, and then inoculated into a fermentation medium at a 5% inoculum concentration, incubated at 32°C, 120 rpm, for 2 hours, induced with IPTG for 1 hour, and then incubated at 37°C for 48 hours.

[0026] Beneficial effects of the present invention:

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

[0028] (2) The present invention successfully constructed an exogenous synthesis pathway for γ-PGA and found that replacing the pgsA′ source with Bacillus subtilis, Bacillus methylotrophicus, or Bacillus amyloliquefaciens alone was beneficial to the synthesis of γ-PGA. Compared with the unreplaced gene cluster pgsBCA, the γ-PGA yield increased by 54.73%, 34.87%, and 25.10%, 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 BCA′(BS), BCA′(BM), BCA′(BAM), and BCA′(BAN);

[0030] Figure 2 : Schematic diagram of the fermentation performance of recombinant strains BCA′(BS), BCA″(BM), BCA′(BAM), and BCA′(BAN). 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 pgsA gene alone is to synthesize the pgsA genes from Bacillus subtilis, methylotrophic Bacillus, Bacillus amyloliquefaciens, and Bacillus anthracis, respectively, and PCR amplify pgsB and pgsC from Bacillus licheniformis, respectively. The pgsA from the four sources is expressed in tandem with the pgsC and pgsB of Bacillus licheniformis, and Corynebacterium glutamicum C glutamicum is used as a competent cell to construct a recombinant strain replacing pgsA, 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 , K2HPO41.5g·L -1 , MgSO40.6g·L -1 , FeSO4·7H2O0.005g·L -1 , MnCl2·4H2O0.005g·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 , K2HPO41.0g·L -1 , MgSO40.6g·L -1 , FeSO4·7H2O0.002g·L -1 , MnCl2·4H2O0.002g·L -1 , urea 7.0g·L -1 (Separate sterilization), pH 6.8-7.0, 50 mL of liquid per 500 mL Erlenmeyer flask, sterilize at 121°C 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 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 to a 2 mL injection vial for analysis. Gel permeation chromatography columns: TSKgelsuper Aw 4000 or TSKgel super Aw 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 chromatography differential detection (RID).

[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 and incubate at 46℃ 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 PgsA source

[0042] The γ-PGA polymerase genes pgsB, pgsC, and pgsA from B. licheniformis ATCC9945a strain were searched using NCBI, 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 pgsA 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 1143 bp, 1149 bp, and 1149 bp, 1236 bp, respectively.

[0043] Amplification primers containing Nde I and BamH I restriction sites were designed (see Table 1). Using the recombinant strain BCA′(BS) as an example, the pgsB and pgsC gene fragments were amplified using the B. licheniformis ATCC9945a genome as a template. The PCR products and the inducible vector pZM(Ptac) were double-digested with Nde I and BamH I, and then ligated to obtain the recombinant plasmids pZM1-pgsB and pZM1-pgsC. pZM1-pgsB was double-digested with Nhe I and Sal I, while 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 homotyping enzymes, resulting in identical cohesive ends after digestion, the two digested fragments were ligated using T4 ligation, eliminating the Nhe I and Avr II restriction sites at the ligation site, resulting in the recombinant plasmid pZM1-pgsB-pgsC. Plasmid pZM1-pgsA′(BS) was obtained by gene synthesis. pZM1-pgsB-pgsC was double-digested with Nhe I and Sal I, and pZM1-pgsA′(BS) was double-digested with Avr II and Sal I. The two digestion products were ligated by T4 to obtain the recombinant plasmid pZM1-BCA′(BS).

[0044] The other recombinant plasmids pZM1-BCA′(BM), pZM1-BCA′(BAM) and pZM1-BCA′(BAN) were constructed in the same way.

[0045] The recombinant plasmids pZM1-BCA′(BS), pZM1-BCA′(BM), pZM1-BCA′(BAM), and pZM1-BCA′(BAN) 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 BCA′(BS), BCA′(BM), BCA′(BAM), and BCA′(BAN) were successfully constructed.

[0046] Figure 1 In the figure, M: DNA Marker; 1: recombinant bacteria BCA′ (BS); 2: recombinant bacteria BCA′ (BM); 3: recombinant bacteria BCA′ (BAM); 4: recombinant bacteria BCA′ (BAN). There is 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 PgsA 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 BCA′(BS), BCA′(BM), BCA′(BAM), and BCA′(BAN)

[0050] The recombinant strains BCA′(BS), BCA′(BM), BCA′(BAM), and BCA′(BAN) were fermented in shake flasks, and the recombinant strain F343-BCA was used as a control to evaluate the effect of replacing the PgsA 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 BCA′(BS), BCA′(BM), BCA′(BAM), and BCA′(BAN) was inhibited to varying degrees. The overall growth of strains BCA′(BS), BCA′(BM), and BCA′(BAM) was poor. They were in the logarithmic growth phase in the first 12 hours and in the stationary phase from 12 to 36 hours. The biomass OD 600 The highest values were 10.76, 9.28 and 10.36 respectively, and then the biomass slowly decreased. BCA′(BAN) grew well in the first 12h, and the biomass OD 600 It reached 14.20, after which the strain quickly died out.

[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, the glutamate content of strains BCA′(BS), BCA′(BM), and BCA′(BAM) was significantly reduced, maintaining at 0.20 g·L -1The glutamic acid content of BCA (BAN) is about 1.50 g·L -1 .

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

[0058] The results are as follows Figure 2 As shown in (d), compared with F343-BCA, the γ-PGA yields of BCA′(BS), BCA′(BM), and BCA′(BAM) increased by 54.73%, 34.87%, and 25.10%, respectively. The highest yield of BCA′(BS) could reach 13.70 g·L -1 The γ-PGA yield of BCA′(BAN) decreased by 24.09% compared to F343-BCA, which is largely related to bacterial viability. Therefore, it can be inferred that replacing the source of PgsA in the specific PgsBCA multiprotein complex 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 pgsBCA' , characterized in that, The gene cluster pgsBCA' synthase genes in pgsA' The sequence is any one of SEQ ID NO.4-SEQ ID NO.6, the gene cluster pgsBCA' synthase genes in pgsB The sequence of the synthase gene is shown in SEQ ID NO.

1. pgsC The sequence is shown in SEQ ID NO.

2.

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

3. The gene cluster of γ-PGA synthase according to claim 1 pgsBCA' , 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 pgsBCA' .

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 PgsA, 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 pgsBCA' , 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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