Recombinant bacillus subtilis with high yield of polyglutamic acid as well as construction method and application of recombinant bacillus subtilis

Abstract

The invention discloses recombinant bacillus subtilis for high-yield polyglutamic acid as well as a construction method and application of the recombinant bacillus subtilis, and belongs to the technical field of molecular biology. According to the recombinant bacillus subtilis for high-yield polyglutamic acid disclosed by the invention, bacillus subtilis zju-7 is used as a starting strain, and a recombinant plasmid pHT43-S3G1-PgdS is expressed. According to the invention, synthesis production of gamma-PGA is combined with an enzymatic degradation process of gamma-PGA hydrolase, and finally, the yield of gamma-PGA in bacillus subtilis is successfully improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and more specifically, to a recombinant Bacillus subtilis with high yield of polyglutamic acid, its construction method and application. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is an anionic polypeptide polymer synthesized by microorganisms. It is a high molecular polymer formed by connecting D-glutamic acid and / or L-glutamic acid monomers through amide bonds condensed between α-amino and γ-carboxylic acid groups. As a new type of multifunctional biological product, γ-PGA has excellent properties such as water solubility, non-toxicity, edibility, easy degradability and water retention. Therefore, it is a good candidate material for various production applications. γ-PGA is a natural biopolymer with a molecular weight range of 10-10,000 kDa, and its properties are closely related to the size of its molecular weight. Therefore, γ-PGA with different molecular weights has different application fields.

[0003] Synthesizing γ-PGA by microbial fermentation method is the most cost-effective, has the least environmental pollution and the synthesized product has a relatively high purity, and is also the most suitable preparation process for industrial production. Its microbial synthesis process is relatively complex and is secreted extracellularly after being synthesized by bacterial cells. According to whether glutamate needs to be added to the culture medium during the production of γ-PGA by bacterial cells, the production strains of γ-PGA are divided into two major categories: glutamate-dependent strains and non-glutamate-dependent strains. If glutamate is not added to the culture medium, glutamate-dependent strains cannot synthesize γ-PGA, and the γ-PGA yield of glutamate-dependent strains increases within a certain range with the increase of glutamate addition amount; the yields of currently discovered non-glutamate-dependent strains are generally low. The γ-PGA production strain Bacillus subtilis zju-7 is a glutamate-dependent strain.

[0004] γ-PGA polymerizes in a non-ribosome-dependent manner in cells and is a polypeptide substance different from proteins and is resistant to protease hydrolysis. Currently, polyglutamic acid hydrolases from various sources have been discovered, such as the monomeric PGA degrading enzyme (PghP) from phage PhiNIT1; carboxypeptidase G in extracellular carboxypeptidases widely present in animal and plant tissues and organs, also known as glutamate carboxypeptidase, acts on γ-PGA containing the C-terminal of Ⅳ-acylated substrate to release glutamate; exo-hydrolytic enzyme γ-glutamyl transpeptidase (GGT), endo-hydrolytic enzyme γ-polyglutamic acid hydrolase (PgdS), CapD PGA depolymerase, etc. also have the activity of degrading γ-PGA. Different from PgdS, GGT degrades γ-PGA to release glutamate monomers. Therefore, not only the molecular weight of the polymer is reduced after GGT treatment, but also the yield of the polymer is correspondingly reduced.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a recombinant Bacillus subtilis with high-yield polyglutamic acid, as well as its construction method and application. Summary of the Invention

[0006] In view of this, in order to solve the problem that during the fermentation production process, as the product polyglutamic acid accumulates, the viscosity of the fermentation broth continuously increases, resulting in poor dissolved oxygen during the fermentation process and limited final yield, the present invention provides a recombinant Bacillus subtilis with high-yield polyglutamic acid, as well as its construction method and application. The polyglutamic acid hydrolase PgdS is introduced in the fermentation production of polyglutamic acid, and a secretory signal peptide suitable for the target protein is selected. The coupling of polyglutamic acid production and the hydrolysis process of polyglutamic acid hydrolase is realized through one-step fermentation, and finally the efficient production of polyglutamic acid is achieved.

[0007] In the fermentation production of γ-PGA, the γ-PGA hydrolase PgdS gene (GenBank: AAU42651.1) from Bacillus licheniformis ATCC14580 is introduced, and a suitable secretory signal peptide S3G1 (GenBank: KIX84181.1) is selected. That is, the recombinant plasmid pHT43-S3G1-PgdS containing the secretory signal peptide is introduced into the γ-PGA producing strain Bacillus subtilis zju-7. The preservation number of Bacillus subtilis zju-7 is: CGMCC No. 1250 (see Patent 200610155277.7). The present invention has low raw material consumption, high yield and low energy consumption during the fermentation process, and is suitable for large-scale industrial application of polyglutamic acid products.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: A recombinant plasmid pHT43-S3G1-PgdS, containing S3G1-PgdS; the nucleotide sequence of S3G1-PgdS is as shown in SEQ ID NO.7.

[0009] Furthermore, a recombinant Bacillus subtilis with high-yield polyglutamic acid uses Bacillus subtilis zju-7 as the starting strain and expresses the recombinant plasmid pHT43-S3G1-PgdS.

[0010] Furthermore, the preservation number of Bacillus subtilis zju-7 is: CGMCC No. 1250.

[0011] Furthermore, the construction method of the recombinant Bacillus subtilis with high-yield polyglutamic acid is as follows: Insert S3G1-PgdS into the pHT43 vector to construct the recombinant plasmid pHT43-S3G1-PgdS; introduce the obtained recombinant plasmid into Bacillus subtilis zju-7 to obtain recombinant Bacillus subtilis.

[0012] Specifically, for the construction method of the recombinant Bacillus subtilis, the polyglutamic acid-producing strain Bacillus subtilis zju-7 is used as the host bacterium; specific primers are designed, and the polyglutamic acid hydrolase gene PgdS containing the signal peptide S3G1 (GenBank: KIX84181.1) is inserted into the pHT43 vector through seamless cloning reaction, that is, the recombinant plasmid pHT43-S3G1-PgdS is constructed; the obtained recombinant plasmid is introduced into the host bacterium to obtain the recombinant Bacillus subtilis.

[0013] Bacillus subtilis zju-7 can use glutamate as a raw material to synthesize polyglutamic acid.

[0014] Furthermore, the application of the recombinant plasmid pHT43-S3G1-PgdS or the recombinant Bacillus subtilis in high-yield polyglutamic acid production.

[0015] According to the above technical solutions, compared with the prior art, the present invention discloses a recombinant Bacillus subtilis with high-yield polyglutamic acid, its construction method and application, screens the optimal secretion signal peptide of polyglutamic acid hydrolase in Bacillus subtilis, and realizes the efficient secretion and expression of γ-PGA hydrolase in Bacillus subtilis. The present invention provides a new research idea, combines the synthesis and production of γ-PGA with the enzymatic degradation process of γ-PGA hydrolase, and finally successfully realizes the increase of γ-PGA production in Bacillus subtilis. Detailed implementation mode

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0017] Select the polyglutamic acid hydrolase gene (GenBank: AAU42651.1) from B. licheniformis ATCC 14580. The nucleotide sequence of the polyglutamic acid hydrolase gene is shown in SEQ ID NO.1. The amino acid sequence of the PgdS gene is shown in SEQ ID NO.2.

[0018] The nucleotide sequence of the PgdS gene is as follows:

[0019] The amino acid sequence of the PgdS gene is as follows: MIKKAANKKLVLFCGIAVLWMSLFLTNHNDVRADTIGEKIAETARQLEGAKYSYGGEKPKTGFDSSGFVQYVFQSLDITLPRTVKEQSTLGSSVGRQQLEKGDLVFFKNAELESDGPTHVAIYLGNDQIIHSTKSNGVVVTKLEGSSYWSSGYFKAKRITKEPEISMDPVVQKAKSYVGVPYVFGGNSPDLGFDCSGLTQYVFREVLGVYLPRSAEQQWAVGQKVKLEDIRPGDVLFFSNTYKPGISHNGIYAGGGRFIHASRSNKVTISYLSASYWQKKFTGVRRFDNMSLPKNPIVSEAIRHIGEVGYQKGGTSPKEGFDTAGFIQYVYKTAAGVELPRYADKQYSTGKKITKQELEPGDIVFFKGTTVMNPAIYIGNGQVVLVTLSAGVTTADMETSAYWKDKYAGSVRIE; SEQ ID NO.2.

[0020] The nucleotide sequence of S3G1-PgdS is as follows: ATGTTTCGATTGTTTCACAATCAGCAAAAGGCGAAGACGAAA CTGAAAGTTCTGCTTATCTTTCAGCTTTCAGTCATTTTCAGTCTGACTGCCGCAATATGCTTACAATTTTCCGATG ATACAAGCGCT

[0021] In SEQ ID NO.7, bp 1 - 129 is the sequence of signal peptide S3G1, and bp 130 - 1374 is the polyglutamate hydrolase gene sequence.

[0022] Example 1 1) Construction of expression plasmid Entrust a professional biotechnology company to chemically synthesize the required base sequences: pHT43 - S3G1 - PgdS - F: aattaaaggaggaaggatcaATGTTTCGATTGTTTCACAA; SEQ ID NO.3.

[0023] pHT43 - S3G1 - PgdS - R: ccaggtaaggtataaactttTTATTCAATGCGCACGCTGC; SEQ ID NO.4.

[0024] pHT43 - F: aaagtttataccttacctgg; SEQ ID NO.5.

[0025] pHT43 - R: tgatccttcctcctttaatt; SEQ ID NO.6.

[0026] Using the gene sequence of S3G1 - PgdS synthesized by the company (as shown in SEQ ID NO.7) as a template, perform PCR amplification with primers pHT43 - S3G1 - PgdS - F / R, and recover with the DNA Gel Extraction Kit to obtain the S3G1 - PgdS DNA fragment; at the same time, use plasmid pHT43 as a template, perform PCR amplification with primers pHT43 - F / R to obtain the linearized vector pHT43; then connect the S3G1 - PgdS DNA fragment to the linearized vector pHT43 through Gibson Assembly technology to obtain plasmid pHT43 - S3G1 - PgdS.

[0027] PCR reaction system: PrimerSTART Max DNA Polymerase 25µl, upstream primer 2µl, downstream primer 2µl, template 0.5µl, ddH2O 20.5µl, a total of 50µl.

[0028] PCR reaction program: 98°C for 3 min; 98°C for 10 s, 55°C for 15 s, 72°C for 16 s (S3G1-PgdS DNA fragment) / 56 s (linearized vector pHT43), 34 cycles; 72°C for 10 min; 4°C forever.

[0029] 2) Construction of recombinant strains Competent cells of Bacillus subtilis zju-7 were prepared by the Spizizen method and transformed with the recombinant plasmid pHT43-S3G1-PgdS to obtain recombinant Bacillus subtilis. After resuscitation culture at 37°C and 200 rpm, the bacterial solution was spread on an LB plate containing kana resistance and cultured overnight. Positive clones were screened by sequencing for preservation or subsequent experiments.

[0030] 3) Experimental results (1) The target gene S3G1-PgdS containing the signal peptide and the linearized vector pHT43 were obtained by PCR amplification. The theoretical sizes of the target gene and the linearized vector were consistent with the electrophoresis results.

[0031] (2) The recombinant plasmid was transferred into the competent cells of Bacillus subtilis zju-7 to obtain plate colonies. Single colonies were picked and cultured overnight, and then the plasmids were extracted and sent for sequencing for further verification. The sequencing results were correctly aligned. The recombinant Bacillus subtilis Bacillus subtilis zju-7-pHT43-S3G1-PgdS containing the secretion signal peptide was obtained.

[0032] Example 2 Application of polyglutamate hydrolase in polyglutamate shake flask fermentation The glycerol-preserved bacteria of the recombinant Bacillus subtilis Bacillus subtilis zju-7-pHT43-S3G1-PgdS (recombinant strain) and the wild-type strain Bacillus subtilis zju-7 were inoculated into the seed medium at a ratio of 2‰, and then cultured in a constant temperature shaker at 37°C for 24 h to obtain seed culture broth.

[0033] Seed medium: 20 g / L sodium glutamate, 5 g / L ammonium sulfate, 2 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 10 g / L yeast powder, 1 g / L corn steep liquor, 3 g / L citric acid, 30 g / L glucose.

[0034] Inoculate the above seed culture solution (OD≈0.65) into the fermentation medium at an inoculation ratio of 2%, and then perform shake-flask fermentation for 80 h simultaneously in a constant-temperature shaker at 37°C to produce γ-polyglutamic acid. Sample and replenish the medium every eight hours (each time replenish 250 g / L of glucose at a replenishment ratio of 1%).

[0035] Fermentation medium: 85 g / L of sodium glutamate, 5 g / L of ammonium sulfate, 2 g / L of dipotassium hydrogen phosphate, 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 steep liquor, 15 g / L of citric acid, 30 g / L of glucose. Adjust the pH to 7.0 with sodium hydroxide.

[0036] After the fermentation is completed, use an SNB-1 digital viscometer to measure the viscosity of the fermentation broth respectively. It is found that the viscosity of the fermentation broth of the recombinant strain is 1544±128 mPa·S, while the viscosity of the fermentation broth of the wild-type zju-7 strain is 5038±169 mPa·S. The results show that the viscosity of the fermentation broth decreases significantly after introducing the exogenous plasmid.

[0037] By means of high-performance liquid chromatography, with an ultraviolet detector, draw a standard working curve with a γ-polyglutamic acid standard product to determine the yield of polyglutamic acid in the fermentation broth.

[0038] Results: Use a gel permeation chromatograph, with two chromatographic columns of Waters Ultrahydrogel 250 and Waters Ultrahydrogel 2000 connected in series, and a differential refractive index detector. Draw a standard working curve with dextran standard products of different molecular weights to determine the molecular weight of polyglutamic acid in the fermentation broth. After measurement, the molecular weight of γ-PGA produced by the recombinant strain is 208.47±20.2 KDa, while the molecular weight of γ-PGA produced by the wild-type zju-7 strain is 474.92±30.2 KDa. It can be seen that after introducing the exogenous plasmid, the molecular weight of the produced γ-PGA decreases significantly.

[0039] In addition, at the shake-flask fermentation level, the yield of γ-PGA in the wild-type strain is 16.09±1.17 g / L, and the yield of γ-PGA in the recombinant strain is 39.04±1.29 g / L, which is 142.7% higher than that of the wild-type strain; at the same time, compared with the wild-type strain, the molecular weight and viscosity of the fermentation broth of γ-PGA produced by the recombinant strain also decrease to a certain extent.

[0040] Example 3 Application of polyglutamic acid hydrolase in the production of polyglutamic acid by fed-batch fermentation The recombinant Bacillus subtilis Bacillus subtilis zju-7-pHT43-S3G1-PgdS (recombinant strain) and the wild-type strain Bacillus subtilis zju-7 were respectively subjected to tank fermentation for a 1.5 L pilot test to verify the high-yield effect of the recombinant strain during pilot-scale amplification.

[0041] In the seed medium, culture and ferment at 37 °C and 200 rpm for 24 h (OD≈0.65), and then inoculate the fermentation medium at an inoculation amount of 3%. During the process, maintain the pH of the fermentation broth at 7.0 with ammonium sulfate and phosphoric acid, culture at 37 °C and 600 rpm, the aeration rate of the fermenter is 1.2 vvm, and use soybean oil as the antifoaming agent with an addition amount of 20 g / L.

[0042] Sample once every 12 h to measure the yield of γ-PGA during the process. After 48 h, end the fermentation and empty the tank.

[0043] The results are shown in Table 1.

[0044] Table 1 γ-PGA yields of the recombinant strain and the wild-type strain during fermentation

[0045] The results in Table 1 show that at the 1.5 L fermenter level, after 48 h of fermentation, the γ-PGA yield in the wild-type strain is 25.26 g / L, and the γ-PGA yield in the recombinant strain is 116.52 g / L, which is 361.28% higher than that of the wild-type strain.

[0046] The construction method and application of a recombinant Bacillus subtilis with high γ-PGA production of the present invention. The recombinant Bacillus subtilis with high γ-PGA production contains an exogenous recombinant plasmid, which can secrete and express γ-PGA hydrolase while the bacteria efficiently synthesize and produce γ-PGA, reducing the viscosity of the fermentation broth and the molecular weight of γ-PGA, thereby realizing the efficient production of γ-PGA in Bacillus subtilis.

[0047] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant plasmid pHT43-S3G1-PgdS, characterized in that, Containing S3G1-PgdS; the nucleotide sequence of the S3G1-PgdS is as shown in SEQ ID NO.

7.

2. A recombinant Bacillus subtilis with high polyglutamic acid productivity, characterized in that, Using Bacillus subtilis zju-7 as the starting strain, expressing the recombinant plasmid pHT43-S3G1-PgdS described in claim 1.

3. The recombinant Bacillus subtilis with high yield of polyglutamic acid according to claim 2, characterized in that, The preservation number of the Bacillus subtilis zju-7 is: CGMCC No.1250.

4. A method for constructing a recombinant Bacillus subtilis with high polyglutamic acid yield according to claim 2 or 3, characterized in that, The steps are as follows: Insert S3G1-PgdS into the pHT43 vector to construct the recombinant plasmid pHT43-S3G1-PgdS; introduce the obtained recombinant plasmid into Bacillus subtilis zju-7 to obtain recombinant Bacillus subtilis.

5. Application of the recombinant plasmid pHT43-S3G1-PgdS described in claim 1 or the recombinant Bacillus subtilis described in any one of claims 2-3 in high-yield production of polyglutamic acid.

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