Construction method and application of engineering bacteria for efficiently biosynthesizing gamma-polyglutamic acid

Abstract

The invention belongs to the technical field of synthetic biology, and particularly relates to a construction method and application of efficient biosynthesis gamma-polyglutamic acid engineering bacteria. The method comprises the following steps: carrying out point mutation on three proteins PgsB, PgsC and PgsA of gamma-polyglutamic acid synthetase, artificially synthesizing a key gene and a promoter for induced expression of gamma-polyglutamic acid synthetase, constructing a gamma-polyglutamic acid synthetase gene induced expression vector pET-P20-pgsBCAm, introducing the induced expression vector into Escherichia coli to obtain an engineering bacterium, and purifying the engineering bacterium to obtain the gamma-polyglutamic acid synthetase gene. The invention also provides a method for biologically synthesizing gamma-polyglutamic acid by using the engineering bacteria. According to the engineering bacterium, glucose is used as an initial growth carbon source, glutamic acid is used as an inducer, and high-level expression of gamma-polyglutamic acid synthetase is induced in the later stage of fermentation, so that high-purity and high-yield gamma-polyglutamic acid is obtained. Compared with the existing polyglutamic acid synthesis technology, the catalytic activity of the polyglutamic acid synthetase mutant is high, gamma-polyglutamic acid is synthesized at a high level after induction, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and particularly relates to a method for constructing and applying an engineering bacterium for highly efficient biosynthesis of γ-polyglutamic acid. The present invention mainly relates to the construction of an engineering bacterium for highly efficient synthesis of γ-polyglutamic acid. The present invention artificially synthesizes the key genes and promoters for the induced expression of γ-polyglutamic acid synthase, constructs an induced expression vector for the γ-polyglutamic acid synthase gene, then introduces the induced expression vector into the bacterial cells to obtain an engineering bacterium, and uses the engineering bacterium to obtain a method for biosynthesizing γ-polyglutamic acid. Background Art

[0002] Polyglutamic acid is a high molecular polymer formed by the polymerization of glutamic acid monomers through amide bonds. Due to different polymerization methods, polyglutamic acid mainly has two configurations, namely: α-polyglutamic acid polymerized through α-amide bonds and γ-polyglutamic acid polymerized through γ-amide bonds, with a molecular weight ranging from 10 to 10000 kDa.

[0003] γ-Polyglutamic acid (γ-PGA) is a high molecular polymer formed by the catalytic connection of glutamic acid monomers through γ-amino acid bonds by a polyglutamic acid synthase system in vivo, with the molecular formula (C5H8NO4) n (The structure is as Figure 1 shown). γ-Polyglutamic acid is not only non-toxic to the human body but also environmentally friendly, showing excellent biocompatibility and biodegradability. In the living body, it can be degraded into glutamic acid and directly absorbed, which makes it have potential applications in biomedical materials. At the same time, it can also be used as a food thickener, food antifreeze and preservative, and cosmetic moisturizer, and is widely used in multiple fields. γ-Polyglutamic acid not only has extremely strong water solubility and can form a tasteless, clean and transparent solution, but more remarkably, it has amazing hygroscopicity. It can absorb more than 4000 times its own weight of water, which makes it easy to crosslink and then form a hydrogel with excellent properties. In the cosmetic field, γ-polyglutamic acid is widely used as a moisturizer, bringing long-lasting moisturizing effects to products such as facial masks, moisturizing creams and moisturizing lotions. In addition, it is widely used in water purification treatment, and can effectively remove heavy metals and radioactive metal ions in water. At the same time, γ-polyglutamic acid can replace polyacrylamide in sewage treatment projects and play an excellent flocculation role. In the pharmaceutical industry, it is often used as a pharmaceutically carrier with good biocompatibility. In addition, it can also be seen as a superabsorbent in agricultural and sanitary ware products. When γ-polyglutamic acid is applied as a new type of biological fertilizer additive in agriculture, it has the following advantages: improving the utilization rate of fertilizers, increasing soil water retention, chelating heavy metal elements, buffering salinity and pH, isolating harmful substances, and improving the disease resistance of plants, etc.

[0004] At present, the main methods for synthesizing polyglutamic acid are: chemical synthesis method and biological synthesis method. The products synthesized by chemical methods are mostly α-polyglutamic acid, mainly including (1) glutamate linkage method, that is, glutamate monomers are connected in series, or first connected into sheets and then the fragments are connected into polymers. (2) Dimer polycondensation method: First, glutamate reacts to form glutamate dimer: α-methylglutamate; then N,N-dimethylformamide is added to make it condense into polyglutamate methyl ester, and finally it becomes α-polyglutamic acid through alkaline hydrolysis. However, the chemical synthesis method has complex processes, many by-products and high costs, etc., and cannot be industrially applied.

[0005] The products synthesized by biological methods are mostly γ-polyglutamic acid, which is catalyzed and produced by γ-polyglutamic acid synthase. The synthesis route includes (1) in vitro enzymatic method: that is, a certain amount of γ-polyglutamic acid synthase is added to the substrate glutamate, adjusted to the optimal reaction temperature of the enzyme, and γ-polyglutamic acid is directly synthesized in vitro; the advantage of the enzymatic reaction method is that the reaction speed is fast, avoiding the process of feedback regulation, so that γ-polyglutamic acid can accumulate to a relatively high level. However, due to the property of γ-polyglutamic acid synthase to bind to the cell membrane, it is difficult to mass-produce this kind of enzyme, and it has no application value at present; (2) microbial fermentation method: Some microorganisms, especially Bacillus, can efficiently synthesize γ-polyglutamic acid in a medium rich in glutamate and glucose. The microbial fermentation method has the advantages of low cost, large yield, controllable molecular weight, high extraction rate, and almost no environmental damage, and has good industrial application value.

[0006] According to whether glutamate is required in the medium, γ-polyglutamic acid-producing bacteria can be divided into glutamate-dependent strains and non-glutamate-dependent strains. The former mainly synthesizes γ-polyglutamic acid through exogenous glutamate, while the latter converts non-glutamate substrates such as citric acid and glucose into γ-polyglutamic acid through an intracellular synthesis pathway. Both types of microorganisms contain the γ-polyglutamic acid synthase-encoding gene pgsBCA. Among them, PgsB is a hydrophilic and unstable protein, anchored to the plasma membrane by a myristoyl hook, and its catalytic action requires binding to ATP to provide energy. PgsC is a hydrophobic and stable protein, located in the plasma membrane through 4 transmembrane regions and multiple myristoyl hooks, and has an amidation site. PgsA is a hydrophilic and stable protein, bound to the plasma membrane through a transmembrane region at the N-terminus and a myristoyl hook, and has multiple phosphorylation sites. The γ-polyglutamic acid synthase forms a complex composed of 3 protein components and is located on the plasma membrane. Among them, PgsB catalyzes the synthesis of γ-polyglutamic acid in the cell, PgsC is fixed on the plasma membrane, connecting the PgsB and PgsA components, and PgsA is responsible for the transport of γ-polyglutamic acid outside the cell. At present, by using genetic engineering technology, the γ-polyglutamic acid synthase-encoding gene pgsBCA is transferred into model microorganisms such as Escherichia coli and Saccharomyces cerevisiae, and the goal of efficiently synthesizing γ-polyglutamic acid using glucose or glutamate as raw materials can be achieved.

[0007] In summary, γ-polyglutamic acid is a type of amino acid polymer with important application value. Currently, its synthesis can be achieved using natural microorganisms or genetically engineered strains, but there are still many technical bottlenecks in production. For example, (1) the metabolic regulation of γ-polyglutamic acid is complex, and it is difficult to obtain highly productive strains; (2) the synthesis of γ-polyglutamic acid increases the viscosity of the fermentation broth, and the high-viscosity fermentation system hinders the mass transfer of dissolved oxygen and nutrient delivery, and inhibits the synthesis of γ-polyglutamic acid, etc. Therefore, there is an urgent need for a method that can efficiently synthesize γ-polyglutamic acid to promote the industrial application of γ-polyglutamic acid. Summary of the Invention

[0008] The purpose of this application is to provide a method for constructing and applying an engineering bacterium for the efficient biosynthesis of γ-polyglutamic acid. Based on the deep learning algorithm HDMLF (Hierarchical Dual-core Multitask Learning Framework, https: / / ecrecer.biodesign.ac.cn / ), point mutations are made on three proteins, PgsB, PgsC, and PgsA, of γ-polyglutamic acid synthase, and the key gene and promoter for the induced expression of γ-polyglutamic acid synthase (γ-polyglutamic acid synthase mutant gene cluster pgsBCAm and glutamate-inducible strong promoter P 20 ) are artificially synthesized, and an induced expression vector pET-P 20 -pgsBCAm of γ-polyglutamic acid synthase gene is constructed. Then, the induced expression vector is introduced into Escherichia coli to construct an engineering bacterium for the biosynthesis of γ-polyglutamic acid, which is used for the production and application of γ-polyglutamic acid.

[0009] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0010] A method for constructing an engineering bacterium for the efficient biosynthesis of γ-polyglutamic acid, comprising the following steps:

[0011] (1) Design of an induced expression cassette for γ-polyglutamic acid synthase mutant gene

[0012] 1) The induced expression cassette for γ-polyglutamic acid synthase mutant gene is an induced expression cassette composed of a glutamate-inducible strong promoter P 20 and the γ-polyglutamic acid synthase mutant gene cluster pgsBCAm;

[0013] The γ-polyglutamic acid synthase mutant gene cluster pgsBCAm is composed of three mutant genes, pgsBm, pgsCm, and pgsAm, corresponding to three mutant proteins, PgsBm, PgsCm, and PgsAm, of polyglutamic acid synthase, which are sequentially connected in series;

[0014] The induced expression cassette is promoter P20 It is ligated with three mutant genes pgsBm - pgsCm - pgsAm through the ribosome - binding site (RBS) to form a linear DNA fragment;

[0015] (II) Construction of the γ - polyglutamic acid synthase inducible expression vector

[0016] 2) According to the nucleic acid sequence of the γ - polyglutamic acid synthase mutant gene cluster pgsBCAm and the promoter P 20 nucleic acid sequence, an inducible expression cassette DNA fragment containing the glutamate - inducible strong promoter P 20 and the γ - polyglutamic acid synthase mutant gene cluster pgsBCAm is artificially synthesized in vitro;

[0017] 3) Using the pET - 28a plasmid as a vector, the pET28a plasmid DNA and the inducible expression cassette DNA fragment in step 2) are double - digested respectively, and the double - digested products are ligated, so as to insert the artificially synthesized inducible expression cassette into the pET - 28a plasmid, obtaining the γ - polyglutamic acid synthase gene inducible expression vector pET - P 20 -pgsBCAm;

[0018] (III) Transformation, screening and inducible expression

[0019] 4) The inducible expression vector pET - P 20 -pgsBCAm constructed in step 3) is transformed into Escherichia coli competent cells, cultured at 35 - 37 °C for 1 - 2 h, spread on a solid medium containing resistance and cultured for a certain time, and then single colonies are selected and verified by double - digestion, thus realizing the construction of engineering bacteria carrying the inducible expression vector pET - P 20 -pgsBCAm.

[0020] Furthermore, in step 1), the glutamate - inducible strong promoter P 20 and the γ - polyglutamic acid synthase mutant gene cluster pgsBCAm both come from the metagenomic DNA sequence library of sewage treatment sludge samples in a glutamate fermentation factory.

[0021] Furthermore, in step 1), the glutamate - inducible strong promoter P 20 can highly initiate the expression of the γ - polyglutamic acid synthase gene under the condition of adding an inducer; the nucleotide sequence of the promoter P 20 is shown in SEQ ID No.1.

[0022] Further, in step 1), the point mutation sites of the mutant gene pgsBm encoding the protein PgsBm are as follows: based on the amino acid sequence of the original PgsB protein, the asparagine residue at the 34th position is mutated to a leucine residue, the serine residue at the 42nd position is mutated to a glutamic acid residue, and the asparagine residue at the 89th position is mutated to an arginine residue. The amino acid sequence and mutation sites of the mutant protein PgsBm are shown in SEQ ID No.5.

[0023] Further, in step 1), the point mutation sites of the mutant gene pgsCm encoding the protein PgsCm are as follows: based on the amino acid sequence of the original PgsC protein, the leucine residue at the 15th position is mutated to an isoleucine residue, the isoleucine residue at the 26th position is mutated to a methionine residue, and the glutamic acid residue at the 104th position is mutated to an aspartic acid residue. The amino acid sequence and mutation sites of the mutant protein PgsCm are shown in SEQ ID No.6.

[0024] Further, in step 1), the point mutation sites of the mutant gene pgsAm encoding the protein PgsAm are as follows: based on the amino acid sequence of the original PgsA protein, the leucine residue at the 24th position is mutated to a tryptophan residue, the isoleucine residue at the 32nd position is mutated to a proline residue, and the lysine residue at the 90th position is mutated to a threonine residue. The amino acid sequence and mutation sites of the mutant protein PgsAm are shown in SEQ ID No.7.

[0025] Further, in step 1), the nucleotide sequence of the mutant gene pgsBm is shown in SEQ ID No.2; the nucleotide sequence of the mutant gene pgsCm is shown in SEQ ID No.3; the nucleotide sequence of the mutant gene pgsAm is shown in SEQ ID No.4.

[0026] Further, in step 1), the induction expression cassette includes two restriction enzyme sites, XhoI and NcoI.

[0027] Further, in step 3), double digestion is performed using the two restriction enzyme sites XhoI and NcoI.

[0028] Further, in step 4), the Escherichia coli is BL21(DE3).

[0029] Further, based on a general inventive concept, the present invention also provides an engineered bacterium carrying the induction expression vector pET-P 20 -pgsBCAm constructed by the above method, and it is named PM20.

[0030] Further, based on a general inventive concept, the present invention also provides the said induction expression vector pET-P 20- Application of pgsBCAm engineered bacteria in the preparation of γ-polyglutamic acid, which is used to prepare γ-polyglutamic acid by biological fermentation method.

[0031] Furthermore, based on a general inventive concept, the present invention also provides a method for producing γ-polyglutamic acid using the said engineered bacteria, comprising the following steps:

[0032] (1) Fermentation culture and induction of γ-polyglutamic acid production

[0033] Inoculate the engineered bacteria carrying the inducible expression vector pET-P 20 -pgsBCAm into a liquid medium, culture at 35 - 37 °C for 10 - 12 h until the logarithmic phase (OD 600 is about 2), as the seed liquid, inoculate it into a liquid medium containing glucose, grow at 35 - 37 °C for 18 - 22 hours, then add an inducer and continue to induce growth for 8 - 12 hours to obtain a fermentation broth containing crude γ-polyglutamic acid;

[0034] (2) Purification of the product (γ-polyglutamic acid)

[0035] Dilute the fermentation broth in step (1), centrifuge, take the supernatant, add an extractant and let it stand overnight (12 h) for extraction, collect the precipitate, wash it, then dissolve it in water and dialyze for a certain time, then centrifuge the dialyzate and lyophilize the supernatant to obtain purified γ-polyglutamic acid.

[0036] Specifically, in step (1), when inoculating into the liquid medium containing glucose, the inoculation amount is 1 - 5% (volume fraction).

[0037] Specifically, the medium in step (1) is LB medium.

[0038] Specifically, in step (1), in the liquid medium containing glucose, the glucose concentration is 12 - 20 g / L.

[0039] Specifically, in step (1), the inducer is glutamic acid, and the addition amount is 2 - 8 g / L.

[0040] Specifically, in step (2), the extractant is absolute ethanol.

[0041] Specifically, in step (2), the specific operation during extraction is to dilute 100 - 120 mL of the fermentation broth in step (1) by 1 - 2 times, centrifuge, take the supernatant, and add 2 - 4 times the volume of the extractant and let it stand overnight (12 h) for extraction.

[0042] Specifically, in step (2), the dialysis time is 18 - 24.

[0043] Further, based on a general inventive concept, the present invention also provides γ-polyglutamic acid prepared by using the said method.

[0044] Compared with the prior art, the advantages of the present invention are as follows:

[0045] By using genetic engineering technology, the present invention performs point mutations on three proteins, namely PgsB, PgsC, and PgsA of γ-polyglutamic acid synthase, synthesizes artificially the mutant gene cluster pgsBCAm of γ-polyglutamic acid synthase derived from the metagenomic DNA library of sewage treatment sludge samples from a glutamic acid fermentation plant and the glutamate-inducible strong promoter P 20 adopts the glutamate-inducible strong promoter P20 to improve the catalytic efficiency of γ-polyglutamic acid synthase, constructs the γ-polyglutamic acid synthase gene inducible expression vector pET-P 20 -pgsBCAm, then introduces the pET-P 20 -pgsBCAm inducible expression vector into Escherichia coli to construct an engineering strain for highly efficient synthesis of γ-polyglutamic acid. Using 15 g / L glucose as the starting growth carbon source and 5 g / L glutamate as the inducer, high-level expression of γ-polyglutamic acid synthase is induced in the later stage of fermentation, thereby obtaining high-purity and high-yield γ-polyglutamic acid.

[0046] Compared with the existing polyglutamic acid synthesis technology, the present invention has a high catalytic activity of the polyglutamic acid synthase mutant, high-level synthesis of γ-polyglutamic acid after induction, and significantly reduced production costs. γ-Polyglutamic acid has excellent water solubility, super strong adsorption, and biodegradability. The degradation product is non-polluting glutamic acid, which can be used as a fertilizer synergist, water retention agent, soil water retention agent, food thickener, sewage flocculant, heavy metal adsorbent, drug or fertilizer sustained release agent, and drug carrier, etc., and has good application prospects in the fields of agricultural production, food processing, medicine, and cosmetics production. Description of the Drawings

[0047] Figure 1 is the structural formula of γ-polyglutamic acid;

[0048] Figure 2 is the schematic structural diagram of the inducible expression cassette composed of the glutamate-inducible strong promoter P 20 and the mutant gene cluster pgsBCAm of γ-polyglutamic acid synthase;

[0049] Figure 3 is the construction flow chart of the pET-P20-pgsBCAm inducible expression vector;

[0050] Figure 4 is the NMR spectrum analysis of the fermentation and purification product of the engineering strain, where A is the 1 1H-NMR spectrum, and B is the 13 13C-NMR spectrum. Detailed implementation manners

[0051] The following examples will further illustrate the present invention in conjunction with the accompanying drawings. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0052] For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions. The raw materials and reagents used are all commercially available products without special instructions.

[0053] Experimental strains, reagents, culture media and materials:

[0054] 1.1 The pET28a plasmid was purchased from Miaoling Biology, and the product number is P0048.

[0055] 1.2 The Escherichia coli chassis strain DH5α was purchased from Nanjing Novozymes Biotech Co., Ltd., and the product number is C502-02.

[0056] 1.3 Restriction endonucleases XhoI, NcoI and T4 DNA ligase were purchased from Sangon Biotech Co., Ltd., and the product numbers are B600236, B600747 and B600511 respectively.

[0057] 1.4 pET-T7-pgsBCAm (constitutive T7 promoter drives the expression of mutant pgsBCAm) and pET-P 20 -pgsBCA (inducible -P 20 promoter drives the expression of wild-type pgsBCA) and their transformed strains were constructed and preserved by the laboratory where the inventor is located as control strains. The construction methods of the two control strains can be found in the literature (The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifKmRNA in Pseudomonas stutzeri A1501. Proc. Natl. Acad. Sci. USA. 2016. 30: 4348 - 4356), where the expression vectors were constructed using the pET28a plasmid and transformed into Escherichia coli DH5α.

[0058] 1.5 γ-polyglutamic acid standard product (specification: L874930-100mg, CAS#: 25513-46-6, purity: HPLC>99%) was purchased from Macklin Inc.

[0059] 1.6 Culture media:

[0060] LB solid medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, 18.0 g of agar powder, add deionized water to 1.0 L; pH 7.0 - 7.2, sterilize at 121 °C for 20 min.

[0061] LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, add deionized water to 1.0 L; pH 7.0 - 7.2, sterilize at 121 °C for 20 min.

[0062] Example 1

[0063] Example 1 provides a method for constructing a γ - polyglutamic acid synthase inducible expression vector pET - P 20 -pgsBCAm, and the specific steps are as follows:

[0064] (1) Design of the inducible expression cassette of the γ - polyglutamic acid synthase mutant gene:

[0065] The present invention first designs an inducible expression cassette of a γ - polyglutamic acid synthase mutant gene, and the inducible expression cassette is an inducible expression cassette composed of a glutamate - inducible strong promoter P 20 and the γ - polyglutamic acid synthase mutant gene cluster pgsBCAm (as Figure 2 shown).

[0066] The glutamate - inducible strong promoter P 20 and the γ - polyglutamic acid synthase mutant gene cluster pgsBCAm both come from the metagenomic DNA sequence library of sewage treatment sludge samples from a glutamate fermentation plant. The construction method of the metagenomic DNA sequence library can be referred to the literature (Identification of a New Gene Encoding EPSPS with High GlyphosateResistance from the Metagenomic Library.Curr Microbiol,2007,55:350 - 355), that is, using the cosmid SuperCos1 Cosmid Vector Kit (purchased from Stratagene) to construct a cosmid library of total community DNA, and obtaining it through deep sequencing.

[0067] Among them, the glutamate - inducible strong promoter P 20 can highly initiate the expression of the γ - polyglutamic acid synthase gene under the condition of adding an inducer; the nucleotide sequence of the promoter P 20 is shown in SEQ ID No.1.

[0068] The γ-polyglutamic acid synthase mutant gene cluster pgsBCAm is composed of three mutant genes pgsBm, pgsCm, and pgsAm corresponding to three mutant proteins PgsBm, PgsCm, and PgsAm of polyglutamic acid synthase in series. Its mutation sites are designed based on the deep learning algorithm HDMLF (Hierarchical Dual-core Multitask Learning Framework, https: / / ecrecer.biodesign.ac.cn / ).

[0069] The point mutation sites of the mutant gene pgsBm encoding the protein PgsBm are as follows: on the basis of the amino acid sequence of the original PgsB protein, the 34th asparagine residue is mutated into a leucine residue, the 42nd serine residue is mutated into a glutamic acid residue, and the 89th asparagine residue is mutated into an arginine residue. The amino acid sequence and mutation sites of the mutant protein PgsBm are shown in SEQ ID No. 5.

[0070] The point mutation sites of the mutant gene pgsCm encoding the protein PgsCm are as follows: on the basis of the amino acid sequence of the original PgsC protein, the 15th leucine residue is mutated into an isoleucine residue, the 26th isoleucine residue is mutated into a methionine residue, and the 104th glutamic acid residue is mutated into an aspartic acid residue. The amino acid sequence and mutation sites of the mutant protein PgsCm are shown in SEQ ID No. 6.

[0071] The point mutation sites of the mutant gene pgsAm encoding the protein PgsAm are as follows: on the basis of the amino acid sequence of the original PgsA protein, the 24th leucine residue is mutated into a tryptophan residue, the 32nd isoleucine residue is mutated into a proline residue, and the 90th lysine residue is mutated into a threonine residue. The amino acid sequence and mutation sites of the mutant protein PgsAm are shown in SEQ ID No. 7.

[0072] The nucleotide sequence of the mutant gene pgsBm is shown in SEQ ID No. 2; the nucleotide sequence of the mutant gene pgsCm is shown in SEQ ID No. 3; the nucleotide sequence of the mutant gene pgsAm is shown in SEQ ID No. 4.

[0073] The inducible expression cassette is the inducible promoter P 20 is connected to the three mutant genes pgsBm - pgsCm - pgsAm through the ribosome binding site RBS, and a linear DNA fragment is obtained through in vitro artificial synthesis.

[0074] The inducible expression cassette includes two restriction enzyme sites, XhoI and NcoI.

[0075] (2) Synthesis of the inducible expression cassette DNA fragment:

[0076] According to the nucleic acid sequences of the γ-polyglutamate synthase mutant gene cluster pgsBCAm (SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4) and the promoter P 20 nucleic acid sequence (SEQ ID NO.1), Sangon Biotech Co., Ltd. was commissioned to artificially synthesize an inducible strong promoter P 20 and a 2.8 kb inducible expression cassette DNA fragment containing the γ-polyglutamate synthase mutant gene cluster pgsBCAm (as Figure 2 shown).

[0077] (3) Construction of the inducible expression vector

[0078] Using the pET-28a plasmid as the vector, XhoI and NcoI were used to double-digest the 2.8 kb inducible expression cassette DNA fragment and the 5.2 kb pET28a plasmid DNA respectively. Catalyzed by T4 DNA ligase, the artificially synthesized inducible expression cassette was inserted into the pET-28a plasmid to obtain the γ-polyglutamate synthase gene inducible expression vector pET-P 20 -pgsBCAm (see the construction flowchart in Figure 3 ).

[0079] Example 2

[0080] For the inducible expression vector constructed in Example 1, transformation, screening and inducible expression were carried out. The specific steps are as follows:

[0081] (I) Transformation and screening

[0082] The inducible expression vector pET-P 20 -pgsBCAm constructed in Example 1 was transformed into Escherichia coli BL21(DE3) competent cells. The specific operation is as follows:

[0083] 1) After thawing the BL21(DE3) competent cells on ice, add the inducible expression vector and mix well, then place it on ice for 30 min;

[0084] 2) Subsequently, heat shock the above mixed bacterial solution in a 42 °C water bath for 90 s; after the heat shock, quickly transfer the centrifuge tube containing the bacterial solution to ice and incubate on ice for about 5 min;

[0085] 3) Then, add 800 μL of LB liquid medium to the centrifuge tube and incubate it in a shaker at 37 °C and 200 r / min for 1 h;

[0086] 4) After the cultivation, centrifuge at 4000×g for 5 min, pour out the supernatant. After the remaining approximately 200 μL of bacterial liquid is resuspended by oscillation, it is evenly spread on the LB solid medium containing 0.1% kanamycin. After incubating upside down at 37°C for about 12 h, pick the transformed engineering strains for colony PCR verification. Use NcoI and XhoI to perform double digestion verification on the plasmid of the engineering strains, and send the plasmid extracted from the engineering bacteria to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0087] The results showed that after agarose gel electrophoresis, two bands of approximately 2.8 kb and 5.2 kb were shown. The sequencing results were completely consistent with the actual sequence, indicating that the engineering strain carrying the inducible expression vector pET-P 20 -pgsBCAm had been successfully constructed and named PM20.

[0088] (II) Inducible expression

[0089] Pick a single colony on the LB solid medium in step (I) (i.e., the engineering strain PM20), inoculate it into 100 mL of LB liquid medium, and culture it at 37°C for 12 h until the OD 600 is about 2, which is used as the seed liquid. Inoculate it into 5000 mL of LB liquid medium containing glucose (glucose concentration is 15 g / L) at an inoculation amount of 5% (volume fraction), and culture it with shaking at 220 rpm at 37°C for 20 hours (i.e., the growth stage), then add 5 g / L of inducer glutamate and continue to induce growth for 10 hours (i.e., the induction stage), and then stop fermentation.

[0090] At the same time, the transformed strain of pET-T7-pgsBCAm (constitutive T7 promoter drives the expression of mutant pgsBCAm) (named control strain 1 in the present invention) and pET-P 20 -pgsBCA (inducible -P 20 promoter drives the expression of wild-type pgsBCA) transformed strain (named control strain 2 in the present invention) are used as the control group, and a control experiment is carried out simultaneously. The γ-polyglutamic acid yields and fermentation broth viscosities of the engineering strain and the control strains are shown in Table 1.

[0091] At the end of the growth stage and the end of the induction stage, 10 ml of fermentation broth is taken for 6M HCl hydrolysis respectively. Use a Shimadzu high-performance liquid chromatograph (model LC-16, Shimadzu Corporation, Japan) to measure the content of γ-polyglutamic acid in the fermentation broth. The content of γ-polyglutamic acid is expressed as the glutamate equivalent of the difference between the hydrolyzed sample and the unhydrolyzed sample. Use a digital viscometer SNB-2 (Nirun, Shanghai, China) to measure the viscosity (×100 cP) of the culture broth of the engineering strain at 25°C.

[0092] The test results are shown in Table 1. It can be seen that during the growth stage of the engineered strain (PM20) constructed in the present invention with glucose as the carbon source, the viscosity of the fermentation broth remained at a relatively low level, approximately 2 - 4 (×100 cP), and the synthesis level of γ-polyglutamic acid was low. However, during the induction stage with the addition of glutamic acid, due to the induced synthesis of a large amount of γ-polyglutamic acid, the viscosity of its culture broth increased rapidly, with a maximum value of approximately 25 (×100 cP).

[0093] In contrast, the pET-T7-pgsBCAm transformed strain began to synthesize γ-polyglutamic acid during the growth stage, and the viscosity of its fermentation broth continued to increase, with a maximum value of approximately 10 (×100 cP), and the maximum value during the induction stage was approximately 15 (×100 cP), indicating that the continuous increase in the viscosity of the fermentation broth led to a decrease in the synthesis amount of γ-polyglutamic acid. pET-P 20 The change in the viscosity of the fermentation broth of the pET-P

[0094] -pgsBCA transformed strain was consistent with that of the engineered strain (PM20), but the synthesis amount of γ-polyglutamic acid during the induction stage was significantly lower than that of the engineered strain (PM20), indicating that the catalytic enzyme activity of the mutant polyglutamic acid synthase expressed by the engineered strain (PM20) was significantly improved.

[0095]

[0096] Note: Control strain 1 is: the pET-T7-pgsBCAm (constitutive T7 promoter drives the expression of mutant pgsBCAm) transformed strain; Control strain 2 is: pET-P 20 -pgsBCA (inducible -P 20 promoter drives the expression of wild-type pgsBCA) transformed strain.

[0097] Example 3

[0098] Purification, structural characterization, and molecular weight determination of the γ-polyglutamic acid prepared in Example 2

[0099] (I) Collection and purification

[0100] Dilute 100 ml of the fermentation broth obtained after fermenting the engineered bacteria PM20 in Example 2 by 2 times, and centrifuge at 10,000×g for 25 minutes. Pour the supernatant into 2 - 4 times the volume of absolute ethanol, gently stir and let stand overnight (12 h). Collect the obtained precipitate and wash it with absolute ethanol, then dissolve it in deionized water and dialyze for 24 hours, changing the water three times. Then centrifuge the dialysate, and lyophilize the supernatant to obtain the purified γ-polyglutamic acid product.

[0101] (II) Structural characterization

[0102] Using a quadrupole time-of-flight liquid chromatography-mass spectrometry (model 1290 Infinity Ⅱ - G6530B, Agilent Technologies, USA) and a nuclear magnetic resonance spectrometer (model DD2 600, Agilent Technologies, USA), the γ-polyglutamic acid pure sample obtained in step (i) was 1 1H NMR and 13 13C NMR structural characterization. The target compound was collected using semi-preparative liquid chromatography with a flow rate set at 2.5 mL / min and detection wavelengths set at 210 and 254 nm. After the collected sample was concentrated to dryness by rotary evaporation and dissolved in methanol, the purity was examined by liquid chromatography. If the sample purity was greater than 98%, nuclear magnetic resonance tests were performed. HRESIMS / MS test conditions and parameters: The ion source was a Dual AJS ESI source; the scanning mode was auto MS / MS positive ion mode, with the mass-to-charge ratio m / z scanning range for the first-stage mass spectrometry being 100 - 3000 a.m.u and the mass-to-charge ratio m / z scanning range for the second-stage mass spectrometry being 50 - 3000 a.m.u; the collision energy was 30 - 70 eV. Before each batch of injections, a standard solution in the range of m / z 150 - 1900 a.m.u was used to calibrate the mass axis. The mass spectrometry graph was recorded as a bar graph, with the first-stage mass spectrometry peak height threshold (Intensity) set at 1000 (absolute value) and the second-stage mass spectrometry peak height threshold set at 100 (absolute value).

[0103] The results showed (as Figure 4 shown): Through 1H NMR and 13C NMR spectral analysis and identification, it was proven that the synthetic product of the engineering strain PM20 was γ-polyglutamic acid.

[0104] (iii) Molecular weight determination

[0105] The molecular weight of the γ-polyglutamic acid synthesized by the engineering strain was determined using a fully automatic GPC gel chromatography instrument (Waters 600 system controller, Waters, USA). The system was equipped with an Ultrahydrogel linear chromatographic column and a 2410 differential refractive index detector, and was calibrated with a Dextran HDXTKIT-1 standard (2.5, 21.4, 133.8, 2000, and 5900 kDa).

[0106] The determination results showed that the molecular weight of the γ-polyglutamic acid synthesized by the engineering strain was between 12,601 - 14,972 kDa.

[0107] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0108] In addition, it should be noted that, for each of the specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combinations.

[0109] In addition, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should equally be regarded as the content disclosed by the present invention.

Claims

1. A method for constructing an engineered bacterium for highly efficient biosynthesis of γ-polyglutamic acid, characterized in that, Including the following steps: (1) Design of the inducible expression cassette of the γ-polyglutamate synthase mutant gene 1) γ-Polyglutamic acid synthase mutant gene inducible expression cassette, which is an inducible expression cassette composed of a glutamate-inducible strong promoter P 20 and a γ-polyglutamic acid synthase mutant gene cluster pgsBCAm constituting the inducible expression cassette; The γ-polyglutamate synthase mutant gene cluster pgsBCAm is composed of three mutant genes corresponding to three mutant proteins PgsBm, PgsCm, and PgsAm of polyglutamate synthase pgsBm , pgsCm and pgsAm connected in series in sequence; The inducible expression cassette is a promoter P 20 and three mutant genes pgsBm - pgsCm - pgsAm are connected by a ribosome binding site RBS to form a linear DNA fragment; (2) Construction of the inducible expression vector of the γ-polyglutamate synthase 2) According to the γ-polyglutamic acid synthase mutant gene cluster pgsBCAm Nucleic acid sequence and promoter P 20 Nucleic acid sequence, in vitro artificial synthesis of a glutamate-inducible strong promoter P 20 and the γ-polyglutamic acid synthase mutant gene cluster pgsBCAm of the inducible expression cassette DNA fragment; 3) Using the pET-28a plasmid as a vector, double digest the pET28a plasmid DNA and the induced expression cassette DNA fragment in step 2), and ligate the double-digested products, thereby inserting the artificially synthesized induced expression cassette into the pET-28a plasmid to obtain the γ-polyglutamate synthase gene-induced expression vector pET- P 20 - pgsBCAm ; (3) Transformation, screening and inducible expression 4) Transform the inducible expression vector pET- P 20 - pgsBCAm constructed in step 3) into Escherichia coli competent cells, culture at 35-37 °C for 1-2 h, spread on a solid medium containing the resistance, and after culturing for a certain time, select single colonies and verify by double digestion, thus realizing the construction of the engineered bacteria carrying the inducible expression vector pET- P 20 - pgsBCAm .

2. The construction method according to claim 1, characterized in that, Glutamate-induced strong promoter P 20 The nucleotide sequence is shown in SEQ ID No.

1.

3. The construction method according to claim 1, characterized in that, In step (1), the amino acid sequence and mutation site of the mutant protein PgsBm are as shown in SEQ ID No. 5; In step (1), the amino acid sequence and mutation site of the mutant protein PgsCm are as shown in SEQ ID No. 6; In step (1), the amino acid sequence and mutation site of the mutant protein PgsAm are as shown in SEQ ID No.

7.

4. The construction method according to claim 1, characterized in that, The mutant gene pgsBm has a nucleotide sequence as shown in SEQ ID No. 2; the mutant gene pgsCm has a nucleotide sequence as shown in SEQ ID No. 3; the mutant gene pgsAm has a nucleotide sequence as shown in SEQ ID No.

4.

5. An engineered bacterium carrying an inducible expression vector pET- constructed by the method according to any one of claims 1 to 4 P 20 - pgsBCAm .

6. Use of the engineered bacterium carrying the inducible expression vector pET- P 20 - pgsBCAm in the preparation of γ-polyglutamic acid.

7. A method for producing γ-polyglutamic acid using the engineered bacteria carrying the inducible expression vector pET- P 20 - pgsBCAm comprises the following steps: (1) Fermentation culture and induction of γ-polyglutamate production The engineered bacteria carrying the inducible expression vector pET- P 20 - pgsBCAm were inoculated into a liquid medium and cultured at 35-37 °C for 10-12 h until the logarithmic phase was reached, which was used as the seed solution. Then, the seed solution was inoculated into a liquid medium containing glucose and grown at 35-37 °C for 18-22 h. Subsequently, an inducer was added and the growth was continued for 8-12 h to obtain a fermentation broth containing the crude product of γ-polyglutamic acid; (2) Product purification Dilute the fermentation broth in step (1), centrifuge, take the supernatant, add an extractant and let it stand overnight for extraction, collect the precipitate, wash it, then dissolve it in water and dialyze for a certain time, then centrifuge the dialysate, and lyophilize the supernatant to obtain purified γ-polyglutamic acid.

8. The method according to claim 7, wherein In step (1), when inoculating into the liquid medium containing glucose, the inoculation amount is 1-5%.

9. The method according to claim 7, wherein In step (1), in the liquid medium containing glucose, the glucose concentration is 12-20 g / L.

10. The method according to claim 7, characterized in that In step (1), the inducer is glutamic acid, and the addition amount is 2-8 g / L.

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