Glutamate decarboxylase mutants and uses thereof

Abstract

The application discloses a glutamate decarboxylase mutant, wherein the glutamate decarboxylase mutant has one or more mutations in the 62th, 68th, 218th and 366th amino acids relative to the amino acid sequence of a wild-type glutamate decarboxylase, and the wild-type glutamate decarboxylase is a glutamate decarboxylase derived from Escherichia coli. The glutamate decarboxylase mutant provided by the application can be used to catalyze preparation of gamma-aminobutyric acid from glutamic acid in a wide pH value range, and the catalytic reaction does not need to use an acid to adjust the pH value, so that material consumption is reduced, generation of inorganic salts is avoided, and a post-extraction process is simplified.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of genetic engineering, in particular, to a glutamate decarboxylase mutant and its application in producing gamma-aminobutyric acid. BACKGROUND

[0002] Gamma-aminobutyric acid is usually prepared from glutamic acid catalyzed by glutamate decarboxylase due to the simple process, easy purification, green and non-pollution characteristics of the enzyme catalytic method. In microorganisms, the generation of gamma-aminobutyric acid is directly related to the acid tolerance mechanism of the bacteria. In addition, it also has a promoting effect on the growth of spores of eukaryotic microorganisms. In the process of plant growth, gamma-aminobutyric acid is directly related to the metabolism and storage of carbon and nitrogen sources, ethylene synthesis and intracellular pH balance, and plays an important role in resisting adverse external environments. Gamma-aminobutyric acid also has physiological activity in the animal body, mainly manifested as: regulation of blood pressure, treatment of epilepsy, treatment of asthma, liver and kidney protection, sedation, regulation of hormone secretion in the body and anti-aging, etc. In addition to the above physiological functions, gamma-aminobutyric acid also has other functions such as resisting skin oxidative aging, regulating lipid metabolism, promoting reproduction and inhibiting cancer cell growth. In recent years, gamma-aminobutyric acid plays a strong role as a secretagogue in normal pancreas, and may inhibit diabetes complications related to the nervous system by inhibiting Fas-dependent and mitochondria-dependent apoptosis pathways in the cerebral cortex.

[0003] At present, the wild-type glutamate decarboxylase of Escherichia coli has an optimal pH of 3.8-4.5, and the enzyme molecule is easily inactivated when the reaction pH is greater than or equal to 6.0, which limits its industrial application. Therefore, it is necessary to develop a glutamate decarboxylase that can work under a wide range of pH conditions. SUMMARY

[0004] The purpose of the present disclosure is to provide a glutamate decarboxylase mutant, and to use the mutant to catalyze the preparation of gamma-aminobutyric acid from glutamic acid in a wide range of pH values. The catalytic reaction does not require the use of acid to adjust the pH value, which reduces material consumption and avoids the generation of inorganic salts, and simplifies the post-extraction process.

[0005] One aspect of the present disclosure provides a glutamate decarboxylase mutant, which has a mutation in one or more of the 62nd, 68th, 218th and 366th amino acids relative to the amino acid sequence of a wild-type glutamate decarboxylase, wherein the wild-type glutamate decarboxylase is a glutamate decarboxylase derived from Escherichia coli.

[0006] In some embodiments, the wild-type glutamate decarboxylase is a glutamate decarboxylase derived from Escherichia coli elppa8 strain.

[0007] In some embodiments, the amino acid sequence of the wild-type glutamate decarboxylase is shown as SEQ ID NO: 2.

[0008] In some embodiments, the mutant amino acid site of the glutamate decarboxylase mutant comprises one or more mutations in T62S, D68H, E218K and I366S.

[0009] In some embodiments, the glutamate decarboxylase mutant has an amino acid sequence shown as SEQ ID NO: 6, or an amino acid sequence with equivalent function formed by one or more amino acid additions, deletions, substitutions or modifications to the amino acid sequence shown as SEQ ID NO: 6.

[0010] Another aspect of the present disclosure provides an isolated nucleic acid molecule encoding the glutamate decarboxylase mutant of the present disclosure.

[0011] In some embodiments, the nucleic acid molecule has a nucleotide sequence shown as SEQ ID NO: 5, or a sequence complementary to the nucleotide sequence shown as SEQ ID NO: 5, or a sequence having 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5, 99.8% or 99.9% or more homology to the nucleotide sequence shown as SEQ ID NO: 5.

[0012] Yet another aspect of the present disclosure provides a recombinant vector containing the nucleic acid molecule of the present disclosure.

[0013] Yet another aspect of the present disclosure provides a host cell containing the nucleic acid molecule or the recombinant expression vector of the present disclosure, or expressing the glutamate decarboxylase mutant of the present disclosure.

[0014] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0015] In some embodiments, the host cell is a bacterium or a fungus.

[0016] In some embodiments, the host cell is a genetically engineered bacterium.

[0017] In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, etc., such as Escherichia coli BL21, T7E, C41, Arctic, etc. In some specific embodiments, the host cell is Escherichia coli. Preferably, the host cell is Escherichia coli BL21 (DE3) strain.

[0018] Yet another aspect of the present disclosure provides a method for preparing the glutamate decarboxylase mutant of the present disclosure, wherein the method comprises the steps of:

[0019] culturing the host cell of the present disclosure; and

[0020] optionally isolating the glutamate decarboxylase mutant from the host cell or from the growth medium or supernatant.

[0021] Yet another aspect of the present disclosure provides the use of the glutamate decarboxylase mutant of the present disclosure, the nucleic acid molecule, the recombinant expression vector, the host cell or the glutamate decarboxylase mutant obtained by the method in the production or catalyzing the production of gamma-aminobutyric acid.

[0022] Yet another aspect of the present disclosure provides a method for producing gamma-aminobutyric acid, which comprises contacting a mixture comprising glutamic acid and pyridoxal phosphate with the glutamate decarboxylase mutant of the present disclosure or with the host cell expressing the glutamate decarboxylase mutant to perform a catalytic reaction to produce gamma-aminobutyric acid.

[0023] In some embodiments, the pH of the mixture is 3.0-8.5, preferably 3.8-7.7. In some specific embodiments, the pH of the mixture is 3.0, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0, 5.2, 5.5, 6.8, 7.0, 7.2, 7.5, 7.7, 8.0, 8.2, 8.5 or any value therebetween.

[0024] In some embodiments, the concentration of glutamic acid in the mixture is 0.2-10 M, preferably 0.5-3 M.

[0025] In some embodiments, the concentration of pyridoxal phosphate in the mixture is 50-500 μM, preferably 100-200 μM.

[0026] In some embodiments, the mass volume ratio of the glutamate decarboxylase mutant is 0.02-5%, preferably 0.05-0.5%.

[0027] In some embodiments, the temperature of the catalytic reaction is 30-50°C, preferably 35-40°C. In some specific embodiments, the temperature of the catalytic reaction is 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, 45°C, 48°C, 50°C or any value therebetween.

[0028] In some embodiments, the time for the catalytic reaction is 0.2-10h, for example, 0.2h, 0.5h, 1h, 2h, 4h, 5h, 6h, 8h, 10h, or any value between them.

[0029] In some embodiments, the host cell is Escherichia Coli. Preferably, the host cell is Escherichia Coli BL21(DE3) strain.

[0030] The glutamate decarboxylase mutant of the present disclosure can improve the yield of catalytic production of gamma-aminobutyric acid when used for the production of gamma-aminobutyric acid, and also expand the pH value range of the catalytic reaction. DETAILED DESCRIPTION

[0031] In order to make the objects, technical solutions and advantages of the present disclosure clearer, the following further describes the present disclosure in combination with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation on the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0032] Definitions

[0033] Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as commonly used in the art to which the present disclosure belongs. For the purpose of interpreting the present specification, the following definitions will apply, and the singular form will include the plural form and vice versa, where appropriate, as appropriate.

[0034] Unless the context clearly indicates otherwise, as used herein, the expressions "a" and "an" include plural referents. For example, reference to "a cell" includes a plurality of such cells, and equivalents thereof known to those skilled in the art, and so on.

[0035] As used herein, the term "about" means a range of ±20% of the numerical value that follows. In some embodiments, the term "about" means a range of ±10% of the numerical value that follows. In some embodiments, the term "about" means a range of ±5% of the numerical value that follows.

[0036] As used herein, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, "T62S" means that the amino acid T at position 62 is mutated to S, and so on.

[0037] As used herein, the term "wild-type glutamate decarboxylase" refers to a naturally occurring, artificially unmodified glutamate decarboxylase, the nucleotide of which can be obtained by genetic engineering techniques such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence of which can be deduced from the nucleotide sequence.

[0038] As used herein, the terms "mutant", "mutant protein", "glutamate decarboxylase mutant" are used interchangeably and all refer to a non-naturally occurring glutamate decarboxylase mutant protein, and the mutant is an artificially modified protein based on the wild-type glutamate decarboxylase shown in SEQ ID NO: 2, and the mutant has the enzyme activity of catalyzing glutamic acid to generate gamma-aminobutyric acid under a wide range of pH conditions.

[0039] As used herein in reference to amino acids, the term "substitution" refers to the replacement of at least one amino acid residue in a sequence with another, different "replacement" amino acid residue. As used herein in reference to amino acids, the term "insertion" refers to the incorporation of at least one additional amino acid into a sequence. While an insertion will typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" can also be made, for example, insertions of about 3 to 5, or even up to about 10, 15, or 20 amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. As used herein in reference to amino acids, the term "deletion" refers to the removal of at least one amino acid residue from a sequence.

[0040] A mutant of the present disclosure, or a fragment thereof, can comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in the present context, an essential or non-essential amino acid residue in a mutant is preferably replaced with another amino acid residue from the same side chain family.

[0041] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions in the comparison window, after aligning the two sequences for optimal comparison purposes (i.e., adding or subtracting gaps in either sequence as necessary to achieve the best alignment). A match position is any position wherein the same nucleotide or amino acid is present in both the target and reference sequences. Gaps are not counted as nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted as are nucleotides or amino acids from the reference sequence.

[0042] The percent sequence identity can be calculated by determining the number of positions at which the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software programs commonly available to those skilled in the art for online use and download. Suitable software programs are available from various sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparison of nucleic acid sequences, while BLASTP is used for comparison of amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0043] The following examples are provided to aid understanding of the present disclosure. It should be understood that these examples are for illustration only and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present disclosure.

[0044] Unless specifically indicated otherwise, the technical means used in the examples are conventional means known to those skilled in the art and commonly used instruments and reagents available on the market, which can be referred to Molecular Cloning: A Laboratory Manual (3rd Edition) (Science Press), Microbiology Experiments (4th Edition) (Higher Education Press), and the manufacturer's instructions of the corresponding instruments and reagents, etc.

[0045] Example 1 Obtaining Escherichia coli glutamate decarboxylase (gad) gene

[0046] The synthetic E. coli elppa8 strain gad gene sequence (SEQ ID NO: 1, synthesized by General Biosystems (Anhui) Co., Ltd., encoding the wild-type gad shown in SEQ ID NO: 2), using the gad gene sequence as the PCR reaction template, and performing PCR amplification according to the primer sequences shown below;

[0047] gad-F: 5'-C CAT ATG ATGGATAAGAAGCAAGTAAC-3' (SEQ ID NO: 3),

[0048] gad-R: 5'-C CTC GAG TCAGGTATGTTTAAAGCTGT-3' (SEQ ID NO: 4);

[0049] Wherein, the italicized letter parts are enzyme digestion sites Nde I and Xho I, respectively.

[0050] The PCR reaction was performed in a 50 μL system, and the reaction conditions were as follows: denaturation at 95 °C for 3 min, denaturation at 95 °C for 50 s, annealing at 58 °C for 1 min, extension at 72 °C for 3 min, for a total of 35 cycles; extension at 72 °C for 10 min, and 3 μL of the PCR amplification product was taken for agarose gel electrophoresis verification; 100 μL of the PCR product was taken for agarose gel electrophoresis, and the target fragment was recovered according to the instructions of the gel recovery kit.

[0051] Example 2 Construction of a gad gene expression vector

[0052] The PCR product in Example 1 was digested with restriction endonucleases Nde I and Xho I, and then subjected to a ligation reaction with a pET-42a plasmid (purchased from Novagen Co.) digested with Nde I and Xho I endonucleases, and the constructed vector was named pET42a-gad, and then the ligation product pET42a-gad was used to transform an E. coli BL21 (DE3) strain (purchased from promega Co.).

[0053] Example 3 Error-prone PCR amplification of an E. coli gad gene

[0054] The frequency of random mutation was controlled by using the property of Taq DNA polymerase without 3'-5' proofreading function, under high magnesium ion concentration (8 mmol / L) and different concentrations of dNTP (1.5 mmol / L for dATP and dGTP, and 3.0 mmol / L for dTTP and dCTP), to introduce random mutation into the target gene, to construct a mutation library, the template concentration A260 value was 1000 ng / mL, the enzyme concentration was 5 U / μL, and the primer concentration was 100 μM, and the optimal mutation rate in the experiment was about 0.6%.

[0055] The error-prone PCR reaction system (100 μL) is shown in Table 1 below.

[0056] Table 1

[0057]

[0058]

[0059] The PCR program was as follows: 95 °C pre-denaturation for 3 min; 94 °C denaturation for 1 min, 56 °C annealing for 1 min, 75 °C extension for 3 min, 45 cycles; finally 75 °C extension for 15 min, and the PCR product was recovered by gel recovery method, 5 μL of the product was subjected to 1% agarose gel electrophoresis for examination, and was stored at -20 °C for standby use.

[0060] Example 4 Construction of Glutamate Decarboxylase Gene Mutant Library

[0061] After the error-prone PCR product of Example 3 was digested by restriction endonucleases Nde I and Xho I, a ligation reaction was performed with the pET-42a plasmid digested by Nde I and Xho I endonucleases, to construct the vector library pET42a-gadM, and then the pET42a-gadM was transformed into the E. coli BL21 (DE3) strain, to construct the expression mutant library.

[0062] Example 5 Construction of Expression Mutant Library and Screening of Mutants

[0063] The mutant strains obtained in Example 4 were randomly picked and inoculated into 6-well plates containing LB medium with 60 μg / mL kanamycin, and were cultured at 37 °C and 150 rpm, until the OD600 value reached 0.6-0.8, then IPTG was added (final concentration 0.1 mmol / L), and the culture was continued at 25 °C for 12 h; the bacterial bodies were collected by centrifugation, suspended in 50 mmol / L, pH 8.0 Tris-HCl buffer containing 1 mmol / L imidazole (the ratio of wet bacterial bodies to buffer was 1 g wet bacterial bodies:5 mL buffer), and the bacterial bodies were broken by ultrasonic wave in an ice bath, and the supernatant was collected after centrifugation, and subjected to protein purification and enzyme activity determination.

[0064] The protein purification method is shown as follows:

[0065] Take Ni 2+ -NTA agarose 5 mL is loaded into a chromatographic column, washed with 25 mL of water, and repeated twice, then the chromatographic column is equilibrated with 25 mL of NAT-0 buffer (20 mM Tris-HCl pH 7.9, 0.5 M NaCl), and the supernatant obtained after centrifugation of the above-mentioned broken bacteria is added to the Ni 2+ -NTA chromatographic column, and after repeated addition of 2-3 times, the medium is washed with 25 mL of NAT-1 buffer (i.e. NAT-0 buffer containing 80 mmol / L imidazole) to remove impurities, and finally the target protein is eluted with the above-mentioned buffer containing 300 mmol / L imidazole, and SDS-PAGE analysis shows that the specific protein band of the expected size is obtained, and the protein concentration is determined by the Coomassie brilliant blue method;

[0066] The enzyme activity determination method is shown as follows:

[0067] To 10 mL of a mixed reaction solution containing 0.5 M glutamic acid at pH 6.0 (containing 150 μM pyridoxyl phosphate, solvent is water), 0.1% (mass / volume ratio) purified protein is added and mixed, and reacted at 37°C for 30 min, and the conversion rate is calculated by determining the content of glutamic acid and γ-aminobutyric acid by liquid chromatography to determine the enzyme activity. The conditions of liquid chromatography include: the chromatographic column is a C18 reversed-phase chromatographic column (4.6*150 mm, 5 μm), the mobile phase is methanol: water (20:80), the flow rate is 1 mL / min, the column temperature is 35°C, the detection wavelength is 210 nm, and the injection volume is 20 μL.

[0068] After calculation, the conversion rate of the mixed solution containing glutamic acid and pyridoxyl phosphate of the wild-type strain after purification is 7.53%, and the conversion rate of the mutant strain M1 (GADM1) is the highest, reaching 99.38%.

[0069] Example 6 Test of mutant strain M1 (GADM1) catalyzing the preparation of γ-aminobutyric acid

[0070] To 20 mL of a mixed reaction solution containing 3 M glutamic acid (containing 150 μM pyridoxyl phosphate, solvent is water), 0.2% (mass / volume ratio) purified protein is added and mixed, and reacted at 37°C for 2 h, and the content of glutamic acid and γ-aminobutyric acid is determined according to the same method as in Example 5, and the conversion rate is calculated (see Table 2).

[0071] Table 2 Determination of glutamic acid conversion rate

[0072]

[0073] As can be seen from the results in Table 2, GADM1 can catalyze the preparation of gamma-aminobutyric acid from glutamic acid in a wide pH range (pH 3.8-7.7).

[0074] The corresponding clone of the picked mutant strain GADM1 was extracted, and the plasmid pET42a-gadM1 was subjected to PCR verification and sequencing identification. The determined nucleotide sequence of the glutamate decarboxylase mutant M1 is shown as SEQ ID NO: 5, and the corresponding amino acid sequence is shown as SEQ ID NO: 6. Compared with the wild-type glutamate decarboxylase shown as SEQ ID NO: 2, the amino acid at the 62nd position of GADM1 is mutated from T to S, the amino acid at the 68th position is mutated from D to H, the amino acid at the 218th position is mutated from E to K, and the amino acid at the 366th position is mutated from I to S.

[0075] The technical solutions of the present disclosure are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A glutamate decarboxylase mutant, characterized in that, The glutamate decarboxylase mutant has mutations at amino acids 62, 68, 218, and 366 relative to the amino acid sequence of a wild-type glutamate decarboxylase, wherein the wild-type glutamate decarboxylase is a glutamate decarboxylase derived from Escherichia coli (GenBank® Accession No. NP_418230.1) Escherichia coli , The amino acid sequence of the glutamate decarboxylase mutant is shown as SEQ ID NO:

6.

2. The glutamate decarboxylase mutant according to claim 1, characterized in that, The wild-type glutamate decarboxylase is derived from the Escherichia coli elppa8 strain.

3. The glutamate decarboxylase mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the wild-type glutamate decarboxylase is shown as SEQ ID NO:

2.

4. An isolated nucleic acid molecule encoding the glutamate decarboxylase mutant of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein, The nucleic acid molecule is shown as SEQ ID NO: 5, or is complementary to the nucleotide sequence shown as SEQ ID NO: 5, or has 95% or more homology to the nucleotide sequence shown as SEQ ID NO:

5.

6. A recombinant vector containing the nucleic acid molecule of claim 4 or 5.

7. A host cell containing the nucleic acid molecule of claim 4 or 5 or the recombinant vector of claim 6, or expressing the glutamate decarboxylase mutant of any one of claims 1-3.

8. The host cell of claim 7, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

9. The host cell of claim 7, wherein, The host cell is a bacterium or a fungus.

10. The host cell of claim 7, wherein, The host cell is Escherichia coli or Bacillus subtilis.

11. A method of preparing the glutamate decarboxylase mutant of any one of claims 1-3, wherein, The method comprises the following steps: culturing the host cell of any one of claims 7-10; and isolating the glutamate decarboxylase mutant from the host cell or from the growth medium or supernatant.

12. The glutamate decarboxylase mutant of any one of claims 1-3, the nucleic acid molecule of claim 4 or 5, the recombinant vector of claim 6, the host cell of any one of claims 7-10, or the glutamate decarboxylase mutant obtained by the method of claim 11, for use in producing or catalyzing the production of gamma-aminobutyric acid.

13. A method of producing gamma-aminobutyric acid, characterized by, The method comprises contacting a mixture containing glutamic acid and pyridoxal phosphate with the glutamate decarboxylase mutant of any one of claims 1-3, or with a host cell expressing the glutamate decarboxylase mutant, to perform a catalytic reaction to produce gamma-aminobutyric acid.

14. The method of claim 13, wherein, The pH of the mixture is 3.0-8.

5.

15. The method of claim 13, wherein, The pH of the mixture is 3.8-7.

7.

16. The method of claim 13, wherein, The temperature of the catalytic reaction is 30-50℃.

17. The method of claim 13, wherein, The temperature of the catalytic reaction is 35-40℃.

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