Recombinant escherichia coli for efficiently producing gamma-aminobutyric acid as well as construction method and application of recombinant escherichia coli

Abstract

The invention discloses recombinant escherichia coli for efficiently producing gamma-aminobutyric acid as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of gene engineering or biology. The recombinant escherichia coli capable of efficiently producing gamma-aminobutyric acid is obtained by heterologous expression of a mutant glutamate decarboxylase gene gadB C11 in E.coli BL21 (DE3) and heterologous expression of a GABA / Glu reverse transport channel gene gadC C41 connected in series with weak RBS and a pyridoxal phosphokinase gene pdxY connected in series with strong RBS under the action of an araBAD promoter. The gamma-aminobutyric acid is inoculated into an LB liquid culture medium containing kanamycin and ampicillin to be cultured until OD600 is equal to 0.6, and IPTG, 1-arabinose and 1-glutamic acid are added for conversion, so that the gamma-aminobutyric acid can be obtained. According to the method, the pH value does not need to be adjusted, the gamma-aminobutyric acid can be synthesized by constructing a reverse transport path and an endogenous PLP refilling path in escherichia coli and directionally modifying a rate-limiting enzyme, and the problems that the GABA production cost is high, the enzyme activity is low, and the optimal pH value of GAD is incompatible with one-step production of GABA are solved.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering or biotechnology, and specifically relates to a recombinant Escherichia coli capable of efficiently producing gamma-aminobutyric acid, a construction method and an application thereof. Background Art

[0002] γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid that plays a variety of physiological functions in microorganisms, plants and animals. GABA is widely used in the field of medicine and food. Foods rich in GABA help improve sleep quality, promote metabolism, and restore brain cell vitality. It is mainly used clinically to treat diseases such as hypertension, epilepsy, arrhythmia, sleep disorders, anxiety and depression. In agriculture and animal husbandry, GABA can be used as a food additive to improve animal immunity and appetite and improve economic benefits. In the chemical industry, GABA can be used to synthesize 2-pyrrolidone and the precursor of the biodegradable material polyamide nylon 4.

[0003] At present, the main methods for producing GABA include chemical synthesis, plant enrichment and biosynthesis. Chemical synthesis has a fast reaction speed and high yield, but it has problems such as high raw material toxicity, strict reaction environment, high equipment cost, easy production of toxic and harmful by-products, and environmental pollution. Compared with chemical synthesis, enriching GABA from plants through low temperature or high salt stress treatment is simple, safe, and environmentally friendly, but it has the limitations of low enrichment yield and high cost. Biosynthesis is the use of microorganisms to convert substrates into GABA. It has the advantages of being environmentally friendly, mild in reaction, and green. It has also attracted widespread attention because microorganisms have the characteristics of short reproduction cycle, fast growth and strong adaptability. The biosynthetic pathway of GABA is mainly pyridoxal-5'-phosphate (Pyridoxal-5'-phosphate; PLP)-dependent glutamate decarboxylase (Glµtamate decarboxylase, GAD) catalyzing the decarboxylation of l-Glu to form γ-aminobutyric acid, and the reaction consumes H +And produce CO2. Most GAD is only active at low pH values, and almost completely loses its activity when the pH value is higher than 6. Through the analysis of GAD crystal structure under different conditions, it is found that the carboxyl end of GAD is mostly open at acidic pH values, but when it is close to neutral conditions, it will form a structure similar to a "plug", which prevents the substrate glutamate from binding to the active site at neutral pH values. Therefore, the existing GABA biosynthesis pathway has the following problems: 1) Exogenous addition of expensive PLP will lead to high GABA production costs; 2) If insufficient exogenous PLP is added, the enzyme activity will be low; 3) GAD has low adaptability to pH values; 4) There is a lack of efficient transport pathways. Although it has been reported that the activity of GAD in a neutral environment can be improved by expressing mutant glutamate decarboxylase from Escherichia coli (Glu89Gln / ΔC452-462) or lactic acid bacteria glutamate mutant (ΔC11 / K17T / D294G / E312S / Q346H), these methods still have problems such as insufficient coenzyme factors and lack of efficient transport. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid and a construction method and application thereof, so as to solve the technical problems of high cost of existing GABA production, low enzyme activity and incompatibility of the optimal pH value of GAD with the one-step production of GABA.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses a method for constructing a recombinant Escherichia coli that efficiently produces γ-aminobutyric acid. E.coli BL21 (DE3) was used as the host bacteria to heterologously express the gene encoding the mutant glutamate decarboxylase under the action of the T7 promoter. gadB △C11 Then, the gene with tandem weak RBS was heterologously expressed under the action of araBAD promoter. gadC △C41 and genes with strong tandem RBS pdxY , and obtain a recombinant Escherichia coli that efficiently produces γ-aminobutyric acid; Among them, the nucleotide sequence of the weak RBS is shown in SEQ ID NO.14, and the nucleotide sequence of the strong RBS is shown in SEQ ID NO.27.

[0006] Preferably, the gene encoding the mutant glutamate decarboxylase gadB △C11 Derived from Lactobacillus plantarum.

[0007] Preferably, the gene gadC △C41 From E.coliDH5α.

[0008] Preferably, the gene pdxY From E.coli DH5α.

[0009] Preferably, the gene encoding the mutant glutamate decarboxylase is heterologously expressed gadB △C11 The expression vector used was pET28a.

[0010] Preferably, the heterologously expressed gene gadC △C41 and genes pdxY The expression vector used was pBAD18.

[0011] The second aspect of the present invention discloses a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid obtained by the above construction method.

[0012] The third aspect of the present invention discloses the use of the above-mentioned recombinant Escherichia coli capable of efficiently producing γ-aminobutyric acid in the production of γ-aminobutyric acid.

[0013] The fourth aspect of the present invention discloses a method for whole-cell transformation to produce γ-aminobutyric acid, wherein the recombinant Escherichia coli that efficiently produces γ-aminobutyric acid is inoculated into an LB liquid medium containing kanamycin and ampicillin and cultured until the OD 600 =0.6, IPTG, l-arabinose and vitamin B6 were added to induce, and the cells were collected; substrates were added to the cells for conversion to obtain γ-aminobutyric acid; Among them, each liter of LB liquid culture medium containing kanamycin and ampicillin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 10 mM vitamin B6, 0.1 g kanamycin and 0.1 g ampicillin, and the balance is water.

[0014] Preferably, the final concentration of IPTG is 0.5 mM.

[0015] Preferably, the final concentration of l-arabinose is 20-100 µM.

[0016] Preferably, the final concentration of vitamin B6 is 10 mM.

[0017] Preferably, the induction is carried out at 16°C, 200 rpm for 16 h.

[0018] Preferably, during transformation, the bacterial concentration is 5-20 OD 600 .

[0019] Preferably, the substrate is l-glutamic acid or sodium l-glutamate.

[0020] Preferably, the concentration of the substrate is 600-1200 mg / mL.

[0021] Preferably, the conversion takes 1 to 8 h.

[0022] Preferably, the recombinant Escherichia coli efficiently producing γ-aminobutyric acid obtained by the above construction method is inoculated into LB liquid culture medium containing kanamycin and ampicillin and cultured until OD 600 =0.6, add IPTG with a final concentration of 0.5 mM, l-arabinose with a final concentration of 60 µM, and vitamin B6 with a final concentration of 10 mM to induce, and collect the bacteria; at a concentration of 15 OD 600 Add l-glutamate at a concentration of 1200 mg / mL to the bacteria and convert it for 7 hours to obtain γ-aminobutyric acid.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The recombinant Escherichia coli for efficiently producing γ-aminobutyric acid provided by the present invention is Escherichia coli BL21 (DE3) (abbreviated as E.coli BL21 (DE3)) was used as the host bacteria. E.coli BL21 (DE3) has the characteristics of integrating the bacteriophage T7 RNA polymerase gene. E.coli BL21 (DE3) was combined with the expression vector pET28a containing the strong inducible promoter T7 to heterologously express the gene encoding the mutant glutamate decarboxylase B (glutamate decarboxylase B, gadB △C11 ), the response range of recombinant Escherichia coli to pH was expanded to neutral. By using a weak inducible arabinose promoter (araBAD), a weak ribosome binding site sequence (RBS) and a strong RBS to express a mutant γ-aminobutyric acid / glutamate antiporter gene (Glu / GABA antiporter, gadC △C41 ) and pyridoxal 5'-phosphate kinase encoding gene (pyridoxal kinase, pdxY ), through genes gadC △C41 To increase the production of γ-aminobutyric acid and relieve intracellular metabolic pressure; pdxY To catalyze cheap vitamin B6 to generate PLP, an endogenous PLP replenishment pathway was constructed in E. coli, which not only met the demand of glutamate decarboxylase for the coenzyme factor PLP, but also significantly reduced the production cost; by combining weak RBS and strong RBS for regulation, overexpression can be prevented gadC △C41This method can not only meet the coenzyme requirements of GAD, but also greatly improve the synthesis efficiency of the product GABA, and can eliminate the dependence of microorganisms on exogenous expensive cofactor PLP in the process of converting substrates to produce γ-aminobutyric acid.

[0024] The present invention provides a method for producing γ-aminobutyric acid through whole-cell transformation, which utilizes constructed recombinant Escherichia coli and adopts whole-cell catalysis to induce glutamate decarboxylase gene expression by adding isopropyl β-D-Thiogalactoside (IPTG), and induces glutamate decarboxylase gene expression by adding l-arabinose. pdxY and gadC △C41 Gene expression, by adding vitamin B6 to provide the coenzyme factor PLP for GAD, there is no need to adjust the pH value during the fermentation process. 747.8 mg / mL of GABA can be obtained by converting 1200 mg / mL of l-glutamate in 7 h, and the molar conversion rate reaches 88.93%, realizing the one-step efficient biosynthesis of GABA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 SDS-PAGE verification of LpgadB and LpgadB of the present invention △C11 Result diagram; from left to right are Marker, CK control group, LpgadB group and LpgadB △C11 Group; Figure 2 The chromatogram and standard curve of GABA of the present invention; wherein A is the chromatogram and B is the standard curve; Figure 3 This is a graph showing the effect of the induction concentration of l-arabinose on the yield of γ-aminobutyric acid of the present invention; Figure 4 The figure is an optimization diagram of the conditions for efficiently synthesizing γ-aminobutyric acid by the recombinant Escherichia coli of the present invention; wherein A is the effect of the substrate l-glutamate / sodium l-glutamate on GABA synthesis, B is the optimization of the l-glutamate substrate concentration, C is the optimization of the bacterial concentration, and D is the optimization of the reaction time. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific words used in the text are the common meanings understood by those skilled in the art for the present invention. When there is a conflict, the definition in this specification shall prevail. The theories or mechanisms described and disclosed herein, whether right or wrong, should not limit the scope of the present invention in any way, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0027] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0028] Herein, under the assay conditions, the enzyme activity required to generate 1 µmol of GABA per minute was defined as 1 unit (U / mL).

[0029] In this article, the culture medium formula involved is as follows: Each liter of LB liquid medium containing kanamycin and ampicillin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 10 mM vitamin B6, 0.1 g kanamycin and 0.1 g ampicillin, and the balance is water.

[0030] Each liter of LB solid medium containing kanamycin and ampicillin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 0.1 g kanamycin, 0.1 g ampicillin and 15 g agar powder, and the balance is water.

[0031] Each liter of LB solid medium containing kanamycin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 0.1 g kanamycin and 15 g agar powder, and the balance is water.

[0032] Each liter of LB liquid culture medium containing kanamycin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl and 0.1 g kanamycin, and the balance is water.

[0033] Per liter of LB solid medium containing ampicillin: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 0.1 g ampicillin and 15 g agar powder, the balance is water.

[0034] Per liter of LB liquid medium containing ampicillin: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 0.1 g ampicillin, and the balance is water.

[0035] The present invention provides a method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid, comprising the following steps: 1. Construction of Escherichia coli chassis cells with γ-aminobutyric acid synthesis ability exist E.coli The strong inducible promoter T7 in the expression vector pET28a was used in BL21 (DE3) to heterologously express the gene encoding the mutant glutamate decarboxylase from Lactobacillus plantarum gadB △C11 , construct Escherichia coli chassis cells with the ability to synthesize γ-aminobutyric acid.

[0036] 2. Construction of recombinant Escherichia coli capable of efficiently producing γ-aminobutyric acid (1) Use a strong RBS to tandemly link the gene encoding pyridoxal 5'-phosphate kinase pdxY ; (2) Using weak RBS in tandem with mutant γ-aminobutyric acid / glutamate reverse transporter gene gadC △C41 ; (3) In the E. coli chassis cells with γ-aminobutyric acid synthesis ability obtained in step 1, the weakly inducible arabinose promoter araBAD in the expression vector pBAD18 was used to express E. coli DH5α ( E.coli DH5α) derived tandem weak RBS gene gadC △C41 and genes with strong tandem RBS pdxY Heterologous expression was performed to construct the endogenous PLP synthesis pathway and transport channel, and recombinant Escherichia coli that efficiently produced γ-aminobutyric acid was obtained.

[0037] The present invention provides a method for producing γ-aminobutyric acid using the above-mentioned recombinant Escherichia coli, comprising the following steps: 1. Inoculate the recombinant Escherichia coli that efficiently produces γ-aminobutyric acid into 5 mL of LB liquid medium containing kanamycin and ampicillin at a 1% inoculation rate, and culture overnight at 37°C and 200 rpm to obtain an activated bacterial solution; 2. Inoculate the activated bacterial solution into 15 mL of LB liquid medium containing kanamycin and ampicillin at a 2% inoculum volume, and culture at 37°C and 200 rpm until the OD 600=0.6, add IPTG with a final concentration of 0.5 mM for induction, then add l-arabinose with a final concentration of 20~100 µM and vitamin B6 with a final concentration of 10 mM, and induce at 16°C, 200 rpm for 16 h. Centrifuge the induced bacterial solution at 6000 rpm for 5 min, remove the supernatant, collect the bacteria, and dilute the bacteria with pure water to OD 600 =5-20, add substrate at a concentration of 600-1200 mg / mL, convert for 1-8 h, and obtain γ-aminobutyric acid; The concentration of l-arabinose is preferably 60 µM; the substrate is l-glutamic acid or sodium l-glutamate, the substrate is preferably l-glutamic acid, and the concentration of l-glutamic acid is preferably 1200 mg / mL; the bacterial concentration is preferably 15 OD 600 ; The conversion time is preferably 7 h.

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0039] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art. The strains used in the following examples are: E.coli DH 5α and E.coli BL21 (DE3) can be purchased, the restriction endonucleases used are purchased from TAKARA Pharmaceuticals, ClonExpress Ultra One Step Cloning Kit V2 is purchased from Novozymes, catalog number: C116, and the vectors used are shown in Table 1.

[0040] Table 1 Vectors used in the experiment

[0041] one, E.coli BL21(DE3) / pET28a-LpgadB △C11 Construction of chassis strains 1. Control chassis strain E.coli Construction of BL21(DE3) / pET28a-LpgadB 1) Comparing the gene sequences of Lactobacillus plantarum glutamate decarboxylase reported in NCBI and literature, the wild-type GAD coding gene from Lactobacillus plantarum was synthesized by GenScript gadB (GENE ID: 9784727), designed primers LpgadB-F and LpgadB-R (sequences are shown in SEQ ID NO.1 and SEQ ID NO.2). gadB As a template, LpgadB-F and LpgadB-R were used as upstream and downstream primers, and the gene was obtained by PCR amplification. gadB ,purification.

[0042] 2) The expression vector pET28a linearized with restriction endonucleases Nde I and Xho I was combined with the gene purified in step 1) gadB The mixture was mixed and connected by homologous recombination to obtain the expression vector pET28a-LpgadB. pET28a-LpgadB was transformed into E.coli DH5α, and then evenly spread the transformed cell suspension on LB solid medium containing kanamycin and culture overnight.

[0043] 3) The single clones formed by overnight culture in step 2) were picked into LB liquid culture medium containing kanamycin, cultured at 37°C, 200 rpm, and cultured for 4 h. Colony PCR verification was performed using universal primers T7 and T7t (sequences as shown in SEQ ID NO.3 and SEQ ID NO.4), and the strains with the correct fragment size were sequenced.

[0044] 4) The plasmid was extracted from the colonies with the correct sequencing using a plasmid extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd., catalog number: DP103), and then transferred into E.coli BL21 (DE3), spread on LB solid medium containing kanamycin, culture overnight, screen positive transformants, and obtain E.coli BL21(DE3) / pET28a-LpgadB.

[0045] 2. Chassis strain E.coli BL21(DE3) / pET28a-LpgadB △C11 Construction 1) The coding gene of wild-type GAD from Lactobacillus plantarum synthesized in step 1 gadB As a template, design primer LpgadB △C11 -F and LpgadB △C11 -R (sequences as shown in SEQ ID NO.5 and SEQ ID NO.6), and the coding gene of mutant GAD was obtained by PCR amplification. gadB △C11(Sequence shown in SEQ ID NO.7), and purified.

[0046] 2) Linearize pET28a with Nco I / Nde I and the purified gene from step 1) gadB △C11 Mix and connect by homologous recombination to obtain the expression vector pET28a-LpgadB △C11 pET28a-LpgadB was transformed into △C11 Convert to E.coli DH5α, and then spread the transformed cell suspension evenly on LB solid medium containing kanamycin and culture overnight.

[0047] 3) The single clones formed by overnight culture in step 2) were picked into LB liquid culture medium containing kanamycin, and cultured at 37°C and 200 rpm for 4 h. Colony PCR verification was performed using universal primers T7 and T7t (sequences as shown in SEQ ID NO.3 and SEQ ID NO.4), and the strains with the correct fragment size were sequenced.

[0048] 4) Use a plasmid extraction kit to extract the plasmid from the colonies that have been sequenced correctly, and use the heat shock method to transform E.coli BL21 (DE3), spread on LB solid medium containing kanamycin, culture overnight, screen positive transformants, and obtain E.coli BL21(DE3) / pET28a-LpgadB △C11 .

[0049] 3. Contrast gadB and gadB △C11 Enzyme activity and GABA production in a neutral environment 1) Verification of GAD protein expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) a. Protein induction expression. Transform the expression vector pET28a into E.coli DH5α, and then evenly spread the transformed cell suspension on LB solid medium containing kanamycin, culture overnight, and transform to E.coli BL21 (DE3), spread on LB solid medium containing kanamycin, culture overnight, screen positive transformants, and obtain E.coli BL21 (DE3) / pET28a. E.coli BL21 (DE3) / pET28a was used as CK control, and CK control E.coli BL21 (DE3) / pET28a, obtained in step 1 E.coli BL21 (DE3) / pET28a-LpgadB and obtained in step 2E.coli BL21(DE3) / pET28a-LpgadB △C11 Inoculate 1% of the inoculum into 5 mL of LB liquid medium containing kanamycin and culture at 37°C, 200 rpm overnight. Inoculate the activated bacterial solution into 15 mL of LB liquid medium containing kanamycin at a 2% inoculum and culture at 37°C, 200 rpm until OD 600 =0.6, add IPTG to a final concentration of 0.5 mM, and induce for 16 h at 16°C and 200 rpm.

[0050] b. Centrifuge the induced bacterial solution at 6000 rpm for 5 min, remove the supernatant, collect the bacteria, wash three times with pure water, add PBS buffer at a ratio of 1 / 10 of the volume of the bacterial solution, fully suspend, and ultrasonically disrupt in an ice bath (ultrasonic power: 300 W; ultrasound on for 3 s, rest for 5 s; total running time 10 min, power ratio: 60%), and centrifuge at 3000 rpm at 4°C to collect the supernatant.

[0051] c. SDS-PAGE. Prepare polyacrylamide gel according to the formula shown in Table 2, load the sample, set the voltage to 60 V for separation for 30 min, then use 120 V for separation for 1 h, stain and decolorize to obtain protein bands.

[0052] Table 2 Polyacrylamide gel formula

[0053] Protein band results are as follows Figure 1 As shown, LpgadB and LpgadB △C11 Obvious bands appeared at about 55 kDa and 50 kDa, respectively, which were consistent with the expected size, indicating that LpgadB and LpgadB △C11 All were successfully expressed in BL21(DE3).

[0054] 2) GAD enzyme activity assay SDS-PAGE experimental step 1) after induction of bacterial solution (BL21 (DE3) / Lp gadB, BL21(DE3) / Ec gadBm) enzyme activity was determined. The specific operation was as follows: 100 µL of bacterial solution was centrifuged to collect the cells, 100 µL of substrate solution (0.2 M, pH 4 acetate buffer, containing 0.1 mmol / L PLP, 100 mmol / L l-MSG·H2O) was added, the reaction was carried out at 37°C for 60 min, high temperature inactivation was performed, and the GABA content was detected by HPLC.

[0055] Test results such as Figure 2As shown in A, it can be seen from the figure that the peak time of GABA is around 19 min, and the peak shape is good. The standard curve equation of GABA is: y=49.1638x+1, R 2 >0.9994, in the range of GABA concentration 0~2 mg / mL, indicating that this detection method has a good linear relationship. Lp The enzyme activity after gadB induction was 20.36 U / mL, BL21(DE3) / Ec The enzyme activity after gadBm induction was 19.32 U / mL. Comparison of the activities of wild-type GAD and mutant GAD showed that at pH 4, the enzyme activity of mutant GAD retained 94.8% compared with that of wild-type GAD at pH 4.

[0056] 3) Comparison of GABA production between wild-type GAD and mutant GAD at different pH values The obtained results in step 1 are E.coli BL21 (DE3) / pET28a-LpgadB and the one obtained in step 2 E.coli BL21(DE3) / pET28a-LpgadB △C11 Inoculate 1% of the inoculum into 5 mL of LB liquid medium containing kanamycin and culture overnight at 37°C and 200 rpm. Inoculate the activated bacterial solution into 15 mL of LB liquid medium containing kanamycin at a 2% inoculum and culture at 37°C and 200 rpm until OD 600 =0.6, add IPTG to a final concentration of 0.5 mM, and induce at 16°C, 200 rpm for 16 h. Centrifuge the induced bacterial solution at 6000 rpm for 5 min, remove the supernatant, collect the bacteria, wash three times with pure water, and dilute to OD with acetate buffer with initial pH values ​​of 3, 4, 5, 6, and 7, respectively. 600 =10, add 40 mg / mL l-glutamate, transform for 12 h, and detect the production of GABA.

[0057] Test results such as Figure 2 As shown in B, BL21 (DE3) / pET28a- Lp The catalytic activity of gadB ranges from pH 3 to 5. When the pH is greater than 5, the enzyme is rapidly inactivated. △C11 The activity range was extended to neutral. This result suggests that the mutant GAD is more suitable for GABA production.

[0058] two, E.coli BL21(DE3) / pET28a-LpgadB △C11 -gadC △C41Construction of chassis strains 1. E.coli DH 5α genome was used as template and primers were used Ec gadBm-F1 / Ec gadBm-R1 (sequences shown in SEQ ID NO.8 and SEQ ID NO.9) and Ec gadBm-F2 / Ec gadBm-R2 (sequences shown in SEQ ID NO.10 and SEQ ID NO.11) were amplified respectively Ec The two fragments of gadBm were purified and recovered by gel excision. Then, they were seamlessly cloned by in vitro multi-fragment homologous recombination with the expression vector pET28a linearized by Nde I and Xho I to construct the recombinant expression vector pET28a- Ec gadBm.

[0059] 2. E.coli DH5α genomic DNA was used as a template and primer gadC △C41 -F1 / gadC △C41 -R1 (sequences shown in SEQ ID NO.12 and SEQ ID NO.13) completed the first round of amplification. △C41 A weak RBS (sequence shown in SEQ ID NO.14) was added to the 5' end of the amplified product; the primer gadC △C41 -F2 / gadC △C41 -R2 (sequences shown in SEQ ID NO.15 and SEQ ID NO.16) added homology arms, and the purified amplified product was combined with pET28a- Ec After in vitro homologous recombination, the plasmid pET28a- Ec gadBm-gadC △C41 .

[0060] 3. Use the plasmid pET28a- Ec gadBm-gadC △C41 As a template, design primer gadC △C41 F and gadC △C41 R (sequences shown in SEQ ID NO.17 and SEQ ID NO.18), and the mutant reverse transport channel protein gene was obtained by PCR amplification. gadC △C41 (The sequence is shown in SEQ ID NO.19) and purified.

[0061] 4. Construct the expression vector pET28a-LpgadB according to step 1 △C11Then, the expression vector pET28a-LpgadB was cleaved with EcoRI. △C11 After linearization, the purified gene gadC △C41 Mix and connect by homologous recombination to obtain the expression vector pET28a-LpgadB △C11 -gadC △C41 The expression vector pET28a-LpgadB was transformed into △C11 -gadC △C41 Convert to E.coli DH5α, and then spread the transformed cell suspension evenly on LB solid medium containing kanamycin and culture overnight.

[0062] 5. The single clones formed by overnight culture in step 4 were picked into LB liquid culture medium containing kanamycin, and cultured at 37°C and 200 rpm for 4 h. Then, colony PCR verification was performed using universal primers T7 and T7t (sequences as shown in SEQ ID NO.3 and SEQ ID NO.4), and the strains with correct fragment size were sequenced.

[0063] 6. Extract the plasmid from the strain with the correct sequencing using a plasmid extraction kit and introduce it into the strain using the heat shock method. E. coli BL21 (DE3), spread on LB solid medium containing kanamycin, culture overnight, screen positive transformants, and obtain E.coli BL21(DE3) / pET28a-LpgadB △C11 -gadC △C41 .

[0064] three, E.coli BL21(DE3) / pET28a-LpgadB △C11 -gadC △C41 Construction of -pdxY chassis strains 1. E.coli The genomic DNA of DH5α was used as a template and amplified using primers 3C-F1 / 3C-R1 (sequences shown in SEQ ID NO.20 and SEQ ID NO.21). gadC △C41 A weak RBS (sequence shown in SEQ ID NO.14) was added upstream, and upstream and downstream homology arms were added to the product of one round using primers 3C-F2 / 3C-R2 (sequence shown in SEQ ID NO.22 and SEQ ID NO.23) to obtain the amplified product. gadC △C41 ; Amplified by primers 3P-F1 / 3P-R1 (sequences shown in SEQ ID NO.24 and SEQ ID NO.25), pdxY(sequence as shown in SEQ ID NO.26) add a strong RBS sequence upstream (sequence as shown in SEQ ID NO.27), and add upstream and downstream homology arms to the product of one round through primers 3P-F2 / 3P-R2 (sequence as shown in SEQ ID NO.28 and SEQ ID NO.29) to obtain the amplified product. pdxY ; The purified amplified product gadC △C41 , pdxY with pET28a-linearized with Xho I Ec gadBm (preparation method refers to step 1 in step 2) was subjected to in vitro multi-fragment homologous recombination to express in tandem gadBm, gadC △C41 and pdxY , and obtain plasmid pET28a- Ec gadBm-gadC △C41 -pdxY.

[0065] 2. Using plasmid pET28a- Ec gadBm-gadC △C41 -pdxY was used as a template, and primers CmY-F and CmY-R (sequences shown in SEQ ID NO.30 and SEQ ID NO.31) were designed. By PCR amplification, gadC △C41 -pdxY, purified.

[0066] 3. Construct the expression vector pET28a-LpgadB according to step 1 △C11 Then, the expression vector pET28a-LpgadB was cleaved with EcoRI. △C11 After linearization, the purified gene gadC △C41 -pdxY were mixed and connected by homologous recombination to obtain the expression vector pET28a-LpgadB △C11 -gadC △C41 -pdxY. The expression vector pET28a-LpgadB was transformed into △C11 -gadC △C41 -pdxY transfer in E.coli DH5α, and then spread the transformed cell suspension evenly on LB solid medium containing kanamycin and culture overnight.

[0067] 4. The single clones formed by overnight culture in step 3 were picked into LB liquid culture medium containing kanamycin, and cultured at 37°C and 200 rpm for 4 h. Then, colony PCR verification was performed using universal primers T7 and T7t (sequences as shown in SEQ ID NO.3 and SEQ ID NO.4), and the strains with correct fragment size were sequenced.

[0068] 5. Use a plasmid extraction kit to extract the plasmid from the strain with the correct sequencing, and use the heat shock method to transform E. coli BL21 (DE3), spread on LB solid medium containing kanamycin, culture overnight, screen positive transformants, and obtain E.coli BL21(DE3) / pET28a-LpgadB △C11 -gadC △C41 -pdxY.

[0069] Four, E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 Construction of -pdxY chassis strains 1. Using pET28a- Ec gadBm-gadC △C41 -pdxY (preparation method refers to step 1 in step 3) as a template, design primers 2CmY-F and 2CmY-R (sequences shown in SEQ ID NO.32 and SEQ ID NO.33), and obtain gadC by PCR amplification. △C41 -pdxY, purified.

[0070] 2. Linearize pBAD18 with EcoRI / SphI and gadC purified in step 1. △C41 -pdxY were mixed and connected through in vitro homologous recombination to obtain the expression vector pBAD18-gadC △C41 pBAD18-gadC △C41 -pdxY conversion to E.coli DH5α, then spread the transformed cell suspension evenly on LB solid medium containing ampicillin and culture overnight.

[0071] 3. The single clones formed by overnight culture in step 2 were picked into LB liquid culture medium containing ampicillin, and cultured at 37°C and 200 rpm for 4 h. Colony PCR verification was performed using universal primers pBAD18-F and pBAD18-R (sequences as shown in SEQ ID NO.34 and SEQ ID NO.35), and the strains with correct fragment size were sequenced.

[0072] 4. Use a plasmid extraction kit to extract the plasmid from the colonies with correct sequencing, and construct the expression vector pET28a-LpgadB according to the method in step 1. △C11 The expression vector pET28a-LpgadB was transformed into △C11and the expression vector pBAD18-gadC obtained in step 2 △C41 -pdxY together with E.coli BL21 (DE3), spread on LB solid medium containing kanamycin and ampicillin, culture overnight, screen positive transformants, and obtain E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY.

[0073] five, E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 Optimization of conditions for the production of γ-aminobutyric acid by whole-cell transformation of -pdxY 1. Optimization of l-arabinose induction concentration The obtained step 4 E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY was inoculated into 5 mL of LB liquid medium containing kanamycin and ampicillin at a 1% inoculum and cultured at 37°C, 200 rpm overnight. The activated bacterial solution was inoculated into 15 mL of LB liquid medium containing kanamycin and ampicillin at a 2% inoculum and cultured at 37°C, 200 rpm until OD 600 =0.6, IPTG was added to a final concentration of 0.5 mM for induction, and different concentrations of l-arabinose (20 µM, 40 µM, 60 µM, 80 µM, 100 µM) and 10 mM vitamin B6 were added, and the cells were induced at 16°C and 200 rpm for 16 h.

[0074] The induced bacterial liquid E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY was centrifuged at 6000 rpm for 5 min, the supernatant was removed, the cells were collected, and the cells were diluted to OD 0. 600 =10, 500 mg / mL substrate l-glutamate was added, the conversion was carried out for 12 h, and the production of GABA was detected.

[0075] Test results such as Figure 3As shown in the figure, when the induction concentration of l-arabinose was 60 µM, the yield of GABA was the highest, which was 323.68 mg / mL, and the molar conversion rate was 92.3%. After that, with the increase of the inducer concentration, the yield of GABA did not increase. Therefore, the optimal induction concentration of l-arabinose was determined to be 60 µM.

[0076] 2. Effect of substrate l-glutamate / sodium l-glutamate on GABA synthesis Referring to the method in step 1, the effects of different substrates on GABA synthesis were investigated at a l-arabinose concentration of 60 µM.

[0077] The steps are as follows: E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY was centrifuged at 6000 rpm for 5 min, the supernatant was removed, the cells were collected, and the cells were diluted to OD 600 =10, 100 mg / mL of substrate l-glutamate and l-glutamic acid were added respectively, and the conversion was carried out for 12 h, and the production of GABA was detected.

[0078] Test results such as Figure 4 As shown in Figure A, when 100 mg / mL of l-glutamate sodium was used as the substrate, the yield of GABA was 24.3 mg / mL, and the conversion rate was 44.17%; when 100 mg / mL of l-glutamic acid was used as the substrate, the yield of GABA was 64.8 mg / mL, and the conversion rate was 92.4%. This result shows that l-glutamic acid is more suitable as a substrate for GABA biosynthesis, which is related to the fact that l-glutamic acid can stably provide an acidic environment.

[0079] 3. Optimization of l-glutamate substrate concentration Referring to the method in step 1, under the conditions of 60 µM l-arabinose concentration and l-glutamate as substrate, the effects of different substrate concentrations on GABA synthesis were investigated.

[0080] The steps are as follows: E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY was centrifuged at 6000 rpm for 5 min, the supernatant was removed, the cells were collected, and the cells were diluted with pure water to OD 600 =10, 600, 800, 1000, and 1200 mg / mL of l-glutamate were added, the conversion was carried out for 12 h, and the production of GABA was detected.

[0081] Test results such as Figure 4 As shown in B, when the substrate concentration was 1200 mg / mL, the concentration of GABA was 716.47 mg / mL and the molar conversion rate was 85.2%, which was the highest yield obtained. Therefore, 1200 mg / mL substrate concentration was selected as the optimal concentration.

[0082] 4. Optimization of bacterial concentration Referring to the method in step 1, the effects of different bacterial concentrations on GABA synthesis were investigated under the conditions of 60 µM l-arabinose concentration, l-glutamate as substrate, and 1200 mg / mL substrate concentration.

[0083] The steps are as follows: E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY were centrifuged at 6000 rpm for 5 min, the supernatant was removed, the cells were collected, and the cells were diluted with pure water to OD 600 =5, 10, 15 and 20, 1200 mg / mL l-glutamate was added, the conversion was carried out for 12 h, and the production of GABA was detected.

[0084] Test results such as Figure 4 As shown in C, when the bacterial concentration was 5~15 OD, the production of GABA gradually increased. When the bacterial concentration was 15 OD, the concentration of GABA was 756.5 mg / mL, and the molar conversion rate was 90%. Continuing to increase the bacterial concentration did not result in significant improvement. Therefore, 15 OD was selected as the optimal bacterial concentration and further optimized.

[0085] 5. Optimize response time Follow the method in step 1, with l-arabinose concentration of 60 µM, substrate l-glutamate, substrate concentration of 1200 mg / mL, and bacterial concentration of 15 OD 600 Under the conditions of , the effects of different conversion times on GABA synthesis were explored.

[0086] The steps are as follows: E.coli BL21(DE3) / pET28a-LpgadB △C11 &pBAD18-gadC △C41 -pdxY was centrifuged at 6000 rpm for 5 min, the supernatant was removed, the cells were collected, and the cells were diluted with pure water to OD 600 =15, 1200 mg / mL l-glutamic acid was added, and the conversion time was set to 1, 2, 3, 4, 5, 6, 7, and 8 h, respectively, and the production of GABA was detected.

[0087] Test results such asFigure 4 As shown in D, when the induction concentration of L-arabinose is 60 µM, the concentration of substrate l-glutamate is 1200 mg / mL, and the bacterial concentration is 15 OD, within 7 h, GABA gradually accumulates as the catalytic time increases, and 1200 mg / mL of substrate l-glutamate can be converted into 747.8 mg / mL of GABA, with a molar conversion rate of 88.93%. After that, the increase in GABA production is not obvious. Therefore, 7 h is selected as the optimal catalytic time.

[0088] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a recombinant Escherichia coli that efficiently produces γ-aminobutyric acid, characterized in that: by E. coli BL21 (DE3) was used as the host bacteria to heterologously express the gene encoding the mutant glutamate decarboxylase under the action of the T7 promoter. gadB △C11 Then, the gene with tandem weak RBS was heterologously expressed under the action of araBAD promoter. gadC △C41 and genes with strong tandem RBS ikB , and obtain a recombinant Escherichia coli that efficiently produces γ-aminobutyric acid; Among them, the nucleotide sequence of the weak RBS is shown in SEQ ID NO.14, and the nucleotide sequence of the strong RBS is shown in SEQ ID NO.

27.

2. The method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 1, characterized in that: Mutant glutamate decarboxylase encoding gene gadB △C11 Derived from Lactobacillus plantarum.

3. The method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 1, characterized in that: Gene gadC △C41 From E. coli DH5α.

4. The method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 1, characterized in that: Gene ikB From E. coli DH5α.

5. The method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 1, characterized in that: Heterologous expression of a gene encoding a mutant glutamate decarboxylase gadB △C11 The expression vector used was pET28a.

6. The method for constructing a recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 1, characterized in that: Heterologous expression of genes gadC △C41 and genes ikB The expression vector used was pBAD18.

7. A recombinant Escherichia coli that efficiently produces γ-aminobutyric acid obtained by the construction method according to any one of claims 1 to 6.

8. Use of the recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 7 in producing γ-aminobutyric acid.

9. A method for producing γ-aminobutyric acid by whole-cell transformation, characterized in that: The recombinant Escherichia coli for efficiently producing γ-aminobutyric acid according to claim 7 is inoculated into LB liquid culture medium containing kanamycin and ampicillin and cultured until OD 600 =0.6, IPTG, l-arabinose and vitamin B6 were added to induce, and the cells were collected; substrates were added to the cells for conversion to obtain γ-aminobutyric acid; Among them, each liter of LB liquid culture medium containing kanamycin and ampicillin includes: 10 g tryptone, 5 g yeast extract powder, 10 g NaCl, 10 mM vitamin B6, 0.1 g kanamycin and 0.1 g ampicillin, and the balance is water.

10. The method for producing γ-aminobutyric acid by whole-cell transformation according to claim 9, characterized in that: The final concentration of IPTG was 0.5 mM, the final concentration of l-arabinose was 20-100 µM, and the final concentration of vitamin B6 was 10 mM.

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