A recombinant bacillus subtilis with high yield of polyglutamic acid, and a construction method and application thereof

Abstract

The application discloses a recombinant bacillus subtilis with high polyglutamic acid yield and a construction method and application thereof, and belongs to the technical field of molecular biology.The recombinant bacillus subtilis with high polyglutamic acid yield is prepared from the bacillus subtilis zju-7 as a starting strain, and expresses the recombinant plasmid pHT43-S3G1-PgdS.The synthesis of gamma-PGA and the enzymatic degradation process of gamma-PGA hydrolase are combined, and finally the improvement of the gamma-PGA yield in the bacillus subtilis is successfully realized.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to a recombinant Bacillus subtilis strain that produces high levels of polyglutamic acid, its construction method, and its applications. Background Technology

[0002] γ-polyglutamic acid (γ-PGA) is an anionic polypeptide polymer synthesized by microorganisms. It is a high-molecular-weight polymer formed by the condensation of D-glutamic acid and / or L-glutamic acid monomers through amide bonds formed by the condensation of α-amino and γ-carboxylic acid groups. As a novel multifunctional bioproduct, γ-PGA possesses excellent properties such as water solubility, non-toxicity, edibility, easy degradation, and water retention, making it a promising candidate material for various production applications. γ-PGA is a natural biopolymer with a molecular weight ranging from 10 to 10,000 kDa. Its properties are closely related to its molecular weight; therefore, γ-PGA with different molecular weights has different application areas.

[0003] Microbial fermentation is the most cost-effective, environmentally friendly, and yields the highest purity γ-PGA, making it the most suitable process for industrial production. The microbial synthesis process is relatively complex, involving the synthesis of γ-PGA by bacterial cells followed by secretion. Based on whether glutamate is added to the culture medium during γ-PGA production, γ-PGA-producing strains are divided into two main categories: glutamate-dependent strains and non-glutamate-dependent strains. Glutamate-dependent strains cannot synthesize γ-PGA without glutamate in the culture medium, and their yield increases with increasing glutamate levels within a certain range. Currently, the yields of non-glutamate-dependent strains are generally lower. The γ-PGA-producing strain *Bacillus subtilis* zju-7 is a glutamate-dependent strain.

[0004] γ-PGA polymerizes intracellularly in a non-ribosome-dependent manner, forming a polypeptide distinct from proteins and resistant to proteolytic hydrolysis. Currently, various polyglutamate hydrolases have been identified, including monomeric PGA degradative enzymes (PghP) from bacteriophage PhiNIT1; carboxypeptidase G, also known as glutamate carboxypeptidase, an extracellular carboxypeptidase widely found in animal and plant tissues and organs, which acts on γ-PGA containing the C-terminus of an IV-acylated substrate, releasing glutamate; exo-gamma-glutamyl transferase (GGT), endo-gamma-polyglutamate hydrolase (PgdS), and CapD PGA depolymerase, all of which possess γ-PGA degrading activity. Unlike PgdS, GGT degrades γ-PGA, releasing glutamate monomers; therefore, GGT-treated polymers not only have a lower molecular weight but also a reduced polymer yield.

[0005] Therefore, providing a recombinant Bacillus subtilis strain that produces high levels of polyglutamic acid, along with its construction method and applications, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, in order to solve the problem that the viscosity of the fermentation broth continuously increases as the product polyglutamic acid accumulates during the fermentation process, leading to a decrease in dissolved oxygen and thus limiting the final yield, the present invention provides a recombinant Bacillus subtilis strain for high polyglutamic acid production, its construction method, and its application. The invention introduces polyglutamic acid hydrolase PgdS into the fermentation production of polyglutamic acid and selects a secretory signal peptide suitable for the target protein. This one-step fermentation couples polyglutamic acid production with the hydrolysis process of the polyglutamic acid hydrolase, ultimately achieving efficient production of polyglutamic acid.

[0007] In the fermentation production of γ-PGA, the γ-PGA hydrolase PgdS gene (GenBank: AAU42651.1) from Bacillus licheniformis ATCC14580 was introduced, and a suitable secretion signal peptide S3G1 (GenBank: KIX84181.1) was selected. Specifically, the recombinant plasmid pHT43-S3G1-PgdS containing the secretion signal peptide was introduced into the γ-PGA producing strain Bacillus subtilis zju-7, whose accession number is CGMCC No. 1250 (see patent 200610155277.7). This invention features low raw material consumption, high yield, and low energy consumption during fermentation, making it suitable for large-scale industrial applications of polyglutamic acid products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A recombinant plasmid pHT43-S3G1-PgdS contains S3G1-PgdS; the nucleotide sequence of said S3G1-PgdS is shown in SEQ ID NO.7.

[0010] Furthermore, a recombinant Bacillus subtilis strain that produces high levels of polyglutamic acid, using Bacillus subtilis zju-7 as the starting strain, expresses the recombinant plasmid pHT43-S3G1-PgdS.

[0011] Furthermore, the preservation number of the Bacillus subtilis zju-7 is: CGMCC No.1250.

[0012] Furthermore, the method for constructing a recombinant Bacillus subtilis strain that produces high levels of polyglutamic acid comprises the following steps:

[0013] S3G1-PgdS was inserted into the pHT43 vector to construct the recombinant plasmid pHT43-S3G1-PgdS; the obtained recombinant plasmid was introduced into Bacillus subtilis zju-7 to obtain recombinant Bacillus subtilis.

[0014] Specifically, the method for constructing recombinant Bacillus subtilis involves using the polyglutamic acid-producing strain Bacillus subtilis zju-7 as the host bacterium; designing specific primers and inserting the polyglutamic acid hydrolase gene PgdS containing the signal peptide S3G1 (GenBank: KIX84181.1) into the pHT43 vector via a seamless cloning reaction, thereby constructing the recombinant plasmid pHT43-S3G1-PgdS; and introducing the obtained recombinant plasmid into the host bacterium to obtain recombinant Bacillus subtilis.

[0015] Bacillus subtilis zju-7 can synthesize polyglutamic acid using glutamic acid as a raw material.

[0016] Furthermore, the application of the recombinant plasmid pHT43-S3G1-PgdS or the recombinant Bacillus subtilis in high-yield polyglutamic acid production.

[0017] As can be seen from the above technical solution, compared with the prior art, this invention discloses a recombinant Bacillus subtilis with high polyglutamate production, its construction method, and its application. The optimal secretion signal peptide for polyglutamate hydrolase in Bacillus subtilis is screened, achieving efficient secretory expression of γ-PGA hydrolase in Bacillus subtilis. This invention provides a new research approach, combining the synthesis and production of γ-PGA with the enzymatic degradation process of γ-PGA hydrolase, ultimately successfully increasing the yield of γ-PGA in Bacillus subtilis. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The polyglutamate hydrolase gene derived from B. licheniformis ATCC 14580 (GenBank: AAU42651.1) was selected. The nucleotide sequence of the polyglutamate hydrolase gene is shown in SEQ ID NO.1. The amino acid sequence of the PgdS gene is shown in SEQ ID NO.2.

[0020] The nucleotide sequence of the PgdS gene is as follows:

[0021]

[0022] The amino acid sequence of the PgdS gene is as follows:

[0023] MIKKAANKKLVLFCGIAVLWMSLFLTNHNDVRADTIGEKIAETARQLEGAKYSYGGEKPKTGFDSSGFVQYVFQSLDITLPRTVKEQSTLGSSVGRQQLEKGDLVFFKNAELESDGPTHVAIYLGNDQIIHSTKSNGVVVTKLEGSSYWSSGYFKAKRITKEPEISMDPVVQKAKSYVGVPYVFGGNSPDLGFDCSGLTQYVFREVLGVYLPRSAEQQWAVGQKVKLEDIRPGDVLFFSNTYKPGISHNGIYAGGGRFIHASRSNKVTISYLSASYWQKKFTGVRRFDNMSLPKNPIVSEAIRHIGEVGYQKGGTSPKEGFDTAGFIQYVYKTAAGVELPRYADKQYSTGKKITKQELEPGDIVFFKGTTVMNPAIYIGNGQVVLVTLSAGVTTADMETSAYWKDKYAGSVRIE; SEQ ID NO.2.

[0024] The nucleotide sequence of S3G1-PgdS is as follows: ATGTTTCGATTGTTTCACAATCAGCAAGGGCGAAGACGAAA CTGAAAGTTCTGCTTATCTTTCAGCTTTCAGTCATTTTCAGTCTGACTGCCGCAATATGCTTACAATTTTCCGATG ATACAAGCGCT

[0025] In SEQ ID NO.7, the sequence of signal peptide S3G1 is from 1 to 129 bp, and the sequence of polyglutamate hydrolase gene is from 130 to 1374 bp.

[0026] Example 1

[0027] 1) Construction of expression plasmids

[0028] The required base sequence was synthesized by a professional biotechnology company using chemical methods.

[0029] pHT43-S3G1-PgdS-F:

[0030] aattaaaggaggaaggatcaATGTTTCGATTGTTTCACAA; SEQ ID NO.3.

[0031] pHT43-S3G1-PgdS-R:

[0032] ccaggtaaggtataaactttTTATTCAATGCGCACGCTGC; SEQ ID NO.4.

[0033] pHT43-F:

[0034] aaagtttataccttacctgg; SEQ ID NO.5.

[0035] pHT43-R:

[0036] tgatccttcctcctttaatt; SEQ ID NO. 6.

[0037] Using the gene sequence of S3G1-PgdS synthesized by the company (as shown in SEQ ID NO.7) as a template, PCR amplification was performed using primers pHT43-S3G1-PgdS-F / R, and the DNA fragment was recovered using a DNA Gel Extraction Kit to obtain the S3G1-PgdS DNA fragment. Simultaneously, using plasmid pHT43 as a template, PCR amplification was performed using primers pHT43-F / R to obtain the linearized vector pHT43. Then, the S3G1-PgdS DNA fragment was ligated into the linearized vector pHT43 using Gibson Assembly technology to obtain the plasmid pHT43-S3G1-PgdS.

[0038] PCR reaction system: PrimerSTART Max DNA Polymerase 25µl, upstream primer 2µl, downstream primer 2µl, template 0.5µl, ddH2O 20.5µl, total 50µl.

[0039] PCR reaction program: 98℃ 3 min; 98℃ 10 s, 55℃ 15 s, 72℃ 16 s (S3G1-PgdS DNA fragment) / 56 s (linearized vector pHT43), 34 cycles; 72℃ 10 min; 4℃ Forever.

[0040] 2) Construction of recombinant strains

[0041] Bacillus subtilis zju-7 competent cells were prepared using the Spizizen method and introduced into the recombinant plasmid pHT43-S3G1-PgdS to obtain recombinant Bacillus subtilis. After resuscitation culture at 37℃ and 200rpm, the bacterial culture was spread on LB plates containing Kana resistance and cultured overnight. After sequencing, positive clones were screened for preservation or subsequent experiments.

[0042] 3) Experimental Results

[0043] (1) PCR amplification yielded the target gene S3G1-PgdS containing the signal peptide and the linearized vector pHT43. The theoretical size of the target gene and the linearized vector were consistent with the electrophoresis results.

[0044] (2) The recombinant plasmid was transformed into Bacillus subtilis zju-7 competent cells to obtain plate colonies. Single colonies were picked and cultured overnight, and the plasmid was extracted and sent for sequencing for further verification. The sequencing results were correct. Recombinant Bacillus subtilis zju-7-pHT43-S3G1-PgdS containing secreted signal peptide was obtained.

[0045] Example 2: Application of polyglutamate hydrolase in polyglutamate shake-flask fermentation

[0046] Recombinant Bacillus subtilis zju-7-pHT43-S3G1-PgdS (recombinant strain) and glycerol-preserved strain of wild-type Bacillus subtilis zju-7 were inoculated into seed culture medium at a ratio of 2‰, and then cultured in a constant temperature shaker at 37℃ for 24h to obtain seed culture solution.

[0047] Seed culture medium: sodium glutamate 20 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.01 g / L, yeast extract 10 g / L, corn steep liquor 1 g / L, citric acid 3 g / L, glucose 30 g / L.

[0048] The above seed culture solution (OD≈0.65) was inoculated into the fermentation medium at an inoculation ratio of 2%, and then γ-polyglutamic acid was produced by shake-flask fermentation in a constant temperature shaker at 37℃ for 80 hours. Samples were taken and feed was added every eight hours (250g / L of glucose was added each time at a replenishment ratio of 1%).

[0049] Fermentation medium: monosodium glutamate 85 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.05 g / L, manganese sulfate 0.01 g / L, yeast extract 10 g / L, corn steep liquor 10 g / L, citric acid 15 g / L, glucose 30 g / L. Adjust the pH to 7.0 with sodium hydroxide.

[0050] After fermentation, the viscosity of the fermentation broth was measured using an SNB-1 digital viscometer. The viscosity of the fermentation broth of the recombinant strain was 1544±128 mPa·S, while that of the wild-type zju-7 strain was 5038±169 mPa·S. The results showed that the viscosity of the fermentation broth decreased significantly after the introduction of the exogenous plasmid.

[0051] The yield of polyglutamic acid in the fermentation broth was determined by using high performance liquid chromatography with an ultraviolet detector and a standard working curve plotted with γ-polyglutamic acid standards.

[0052] result:

[0053] Using gel permeation chromatography (GPC), two columns (Waters Ultrahydrogel 250 and Waters Ultrahydrogel 2000) were connected in series, with a differential refractive index detector (DRC). A standard curve was plotted using dextran standards of different molecular weights to determine the molecular weight of polyglutamic acid in the fermentation broth. The molecular weight of γ-PGA produced by the recombinant strain was determined to be 208.47 ± 20.2 kDa, while the molecular weight of γ-PGA produced by the wild-type zju-7 strain was 474.92 ± 30.2 kDa. This shows that the molecular weight of the produced γ-PGA significantly decreased after the introduction of the exogenous plasmid.

[0054] In addition, at the shake-flask fermentation level, the γ-PGA yield in the wild-type strain was 16.09±1.17 g / L, and the γ-PGA yield in the recombinant strain was 39.04±1.29 g / L, which was 142.7% higher than that in the wild-type strain. At the same time, compared with the wild-type strain, the molecular weight of γ-PGA produced by the recombinant strain and the viscosity of the fermentation broth were also reduced to some extent.

[0055] Example 3: Application of polyglutamic acid hydrolase in the production of polyglutamic acid via fermentation.

[0056] The recombinant Bacillus subtilis zju-7-pHT43-S3G1-PgdS strain and the wild-type Bacillus subtilis zju-7 strain were fermented separately in a 1.5L small-scale test to verify the high-yield effect of the recombinant strain in the small-scale scale-up.

[0057] After fermentation in seed culture medium at 37℃ and 200 rpm for 24 h (OD≈0.65), the culture medium was inoculated at a rate of 3%. During the process, ammonium sulfate and phosphoric acid were used to maintain the pH of the fermentation broth at 7.0. The fermentation was carried out at 37℃ and 600 rpm with an aeration rate of 1.2 vvm in the fermenter. Soybean oil was used as an antifoaming agent at a concentration of 20 g / L.

[0058] Samples were taken every 12 hours to measure the yield of γ-PGA during the process. Fermentation was stopped after 48 hours and the product was transferred to the tank.

[0059] The results are shown in Table 1.

[0060] Table 1. γ-PGA yield of recombinant and wild-type strains during fermentation.

[0061]

[0062] Table 1 shows that, at the 1.5L fermenter level, after 48 hours of fermentation, the γ-PGA yield in the wild-type strain was 25.26 g / L, and the γ-PGA yield in the recombinant strain was 116.52 g / L, which was 361.28% higher than that in the wild-type strain.

[0063] This invention discloses a method for constructing a high-polyglutamic acid-producing recombinant Bacillus subtilis and its application. The high-polyglutamic acid-producing recombinant Bacillus subtilis contains exogenous recombinant plasmids, which can simultaneously secrete and express γ-PGA hydrolase while efficiently synthesizing and producing γ-PGA in the bacterial cells, thereby reducing the viscosity of the fermentation broth and the molecular weight of γ-PGA, thus achieving efficient production of γ-PGA in Bacillus subtilis.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of recombinant Bacillus subtilis in improving production of polyglutamic acid, characterized in that, The recombinant Bacillus subtilis is fermented in a fermentation medium, the pH of the fermentation liquid is maintained at 7.0, the fermentation is carried out at 37℃ and 600 rpm, the aeration rate of the fermenter is 1.2vvm, soybean oil is used as an antifoaming agent, and the addition amount is 20 g / L; The fermentation medium comprises 85 g / L of sodium glutamate, 5 g / L of ammonium sulfate, 2 g / L of potassium phosphate dibasic, 0.05 g / L of magnesium sulfate, 0.01 g / L of manganese sulfate, 10 g / L of yeast powder, 10 g / L of corn syrup, 15 g / L of citric acid and 30 g / L of glucose; and the pH is adjusted to 7.0 by sodium hydroxide; The recombinant Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis zju-7 was the starting strain, expressing the recombinant plasmid pHT43-S3G1-PgdS; The preservation number of the Bacillus subtilis zju-7 is CGMCC No. 1250; The recombinant plasmid pHT43-S3G1-PgdS contains S3G1-PgdS; the nucleotide sequence of the S3G1-PgdS is shown as SEQ ID NO. 7.

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