A histidine methyltransferase mutant and its application

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a histidine methyltransferase mutant and an application thereof. The catalytic activity of the mutant protein is significantly improved compared with that of the parent protein, thereby promoting the production of ergothioneine catalyzed by histidine methyltransferase, and providing a new synthetic pathway and engineering bacteria for the production of ergothioneine.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a histidine methyltransferase mutant and application thereof. Background Art

[0002] Ergothioneine, also known as thiol histidine trimethyl salt (EGT), has a molecular formula of C9H 15 N₃O₂S was first discovered in Thiobacillus ergotii in 1909 and is named after it. Ergothioneine is a natural antioxidant that can scavenge reactive free radicals within cells, inhibit the activity of certain oxidases, and inhibit many intracellular oxidative reactions. Therefore, ergothioneine is widely used in food, medicine, cosmetics, health products, and biotechnology. Currently, ergothioneine can be synthesized chemically or extracted from natural products such as mushrooms, but both methods have disadvantages such as low yield, high impurities, and high costs. Microbial fermentation is used to produce ergothioneine, which has low raw material costs, is environmentally friendly, simple to operate, has a short cycle time, and is suitable for industrial production, making it a hot topic in current ergothioneine production research.

[0003] Natural microbial production of ergothioneine has low yields and long growth cycles, which cannot meet the needs of industrial production. The first step in the ergothioneine biosynthesis pathway is the SAM (S-adenosylmethionine)-dependent histidine trimethylase (EgtD), which catalyzes the conversion of histidine into histidine trimethyl inner salt (Hercynine, abbreviated as HER). This step plays an important role in the synthesis of ergothioneine. Without this step, ergothioneine cannot be synthesized. In 2015, Vit et al. characterized EgtD and found that the enzyme's conversion time for the substrate histidine was only 0.58 s. -1(Misson, Laetitia, Chembiochem: A European journal of chemical biology, 2015.), which is 2.3% of the activity of the most active enzyme in the metabolic pathway, EgtE. Therefore, EgtD is the key rate-limiting step in ergothioneine synthesis. At present, studies have reported the crystal structure of EgtD (e.g., PDB: 4UY5, 4UY7, and 4UY6; JEONG JH, CHA HJ, HAS C, et al. Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis[J]. Biochem Biophys Res Commun, 2014, 452(4): 1098-1103) and studied the related enzymatic properties. Existing studies have found that EgtD-catalyzed histidine trimethylation is a continuous process, and its catalytic activity is strictly regulated by the substrate. There is an urgent need for an EgtD with high enzyme activity to solve the problem of low methyltransferase activity that does not meet the requirements of industrial production. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a histidine methyltransferase (EgtD) mutant and its application. The catalytic activity of the mutant protein is significantly improved compared with the parent enzyme, thereby promoting the production of ergothioneine catalyzed by EgtD.

[0005] The technical solutions of the present invention are as follows:

[0006] The present invention provides a histidine methyltransferase EgtD mutant, wherein the substitution relative to the parent histidine methyltransferase is selected from one of the following: S70V, S70N, S70K, S70W; each position corresponds to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO: 1.

[0007] The present invention also provides a gene encoding the above-mentioned EgtD mutant.

[0008] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above-mentioned encoding gene.

[0009] The present invention also provides a host cell comprising the above encoding gene or recombinant vector.

[0010] The present invention also provides a use of the EgtD mutant, wherein the mutant protein is used to catalyze the conversion of histidine into histidine trimethylolate (HER).

[0011] The present invention also provides a method for preparing the above-mentioned EgtD mutant, which comprises performing gene recombination and expression using the coding gene of the EgtD mutant of the present invention or an expression vector comprising the coding gene.

[0012] The present invention also provides a method for producing ergothioneine (L-EGT), which comprises the steps of introducing the EgtD mutant into a microbial expression system containing EgtA, EgtB, EgtC and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

[0013] The present invention also provides a method for improving the enzymatic activity of histidine methyltransferase, which is by introducing the following substitutions into the parent histidine methyltransferase: S70V, S70N, S70K or S70W; each position corresponds to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO: 1.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. Modify the EgtD protein through directed evolution of active sites, gene mutation, and other means to screen mutants with improved activity.

[0016] 2. The activity-enhanced EgtD protein is combined with other enzymes in the ergothioneine synthesis pathway (e.g., EgtA, EgtB, EgtC, EgtE) for the biosynthesis of ergothioneine, thereby improving the microbial fermentation level of ergothioneine.

[0017] 3. The present invention has obtained for the first time the key amino acid site for improving EgtD enzyme activity, which is the 70th active site of the EgtD enzyme derived from Chlorella. The above key amino acid site can effectively improve the activity of EgtD enzyme after mutation, providing a key target for further improving the subsequent enzyme activity of the enzyme.

[0018] 4th, the present invention is taking Escherichia coli expression system as an example, obtains the engineering bacteria comprising EgtD mutant, and engineering bacteria is applied to thioneine production.Found that this mutation can effectively improve thioneine output, compared with the engineering bacteria that does not mutate, the engineering bacteria containing mutant can improve thioneine fermentation output by 22~24 times.Mutant of the present invention effectively improves the synthesis efficiency of thioneine, for the production of thioneine provides a kind of new synthetic route and engineering bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : The amino acid sequence of the parent histidine methyltransferase of the present invention.

[0020] Figure 2 : The nucleotide sequence of the gene encoding the parent histidine methyltransferase of the present invention.

[0021] Figure 3 : The plasmid map for expressing histidine methyltransferase (EgtD) in the examples.

[0022] Figure 4 : Alanine mutagenesis strain shake flask fermentation test results in the embodiment.

[0023] Figure 5 : The results of shake flask fermentation of the saturated mutant strain S70 in the embodiment. DETAILED DESCRIPTION

[0024] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0025] The present invention provides a histidine methyltransferase EgtD mutant, wherein the substitution relative to the parent histidine methyltransferase is selected from one of the following: S70V, S70N, S70K, S70W; each position corresponds to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO: 1.

[0026] The present invention also provides a gene encoding the above-mentioned EgtD mutant.

[0027] The present invention also provides a vector, a recombinant vector or an expression vector comprising the above-mentioned encoding gene, for example, a recombinant vector composed of the vector plasmid pTrc99a and the encoding gene of the present invention.

[0028] The present invention also provides a host cell comprising the above encoding gene or recombinant vector. The host cell can be any host suitable for producing the EgtD mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems such as Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae.

[0029] The present invention also provides a use of the EgtD mutant, wherein the mutant protein is used to catalyze the conversion of histidine into histidine trimethyl inner salt (HER).

[0030] The EgtD mutants of the present invention have increased enzyme activity compared to their parent EgtD, for example, more than 5 times in some embodiments, more than 10 times in other embodiments, more than 20 times in other embodiments, and more than 30 times in other embodiments.

[0031] The present invention also provides a method for preparing the aforementioned EgtD mutant, comprising genetic recombination and expression using a gene encoding the EgtD mutant described herein or an expression vector comprising the gene encoding the EgtD mutant. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and a culture medium and culture conditions suitable for host expression can be selected. The method can also include a step of recovering the EgtD mutant, which may involve isolating or purifying the EgtD mutant from a host culture or expression product, and can be performed using any method known to those skilled in the art.

[0032] The present invention also provides a method for producing ergothioneine (L-EGT), which comprises the steps of introducing the EgtD mutant into a microbial expression system (such as an Escherichia coli expression system) containing EgtA, EgtB, EgtC and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

[0033] In the above-mentioned method for producing thioneine, the preferred culture medium composition is: glucose 20-40 g / L, yeast extract 1-4 g / L, peptone 2-5 g / L, sodium citrate 1-2 g / L, KH2PO41-2 g / L, MgSO4·7H2O 1-2 g / L, FeSO410-20 mg / L, V B·mix (1,3,5,12) 1-2 mg / L, V H 1-2 mg / L, V B6 1-10 mg / L, MnSO41-10 mg / L, the rest is water, pH 7.0-7.2.

[0034] The present invention also provides a method for improving the enzymatic activity of histidine methyltransferase, which is by introducing the following substitutions into the parent histidine methyltransferase: S70V, S70N, S70K or S70W; each position corresponds to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO: 1.

[0035] The following definitions are used in the present invention:

[0036] 1. Nomenclature of amino acid and DNA sequences

[0037] Amino acid residues are referred to using the generally accepted IUPAC nomenclature, using either three-letter abbreviations or single-letter symbols. DNA sequences are referred to using the generally accepted IUPAC nomenclature.

[0038] 2. Identification of EgtD mutants

[0039] The term "amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the EgtD mutant. For example, S70K indicates that the amino acid residue at position 70, starting from the N-terminus and moving toward the C-terminus, is substituted from serine (S) in the parent EgtD to lysine (K). The amino acid numbering scheme for EgtD in the present invention is based on the sequence set forth in SEQ ID NO: 1.

[0040] 3. The amino acid sequence of the parent histidine methyltransferase EgtD (SEQ ID NO: 1) is as follows Figure 1 The coding gene sequence (SEQ ID NO: 2) is shown in Figure 2 shown.

[0041] The present invention will be described in more detail below through specific examples. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] Example 1. Preparation method of mutants

[0043] 1.1 Primer design

[0044] Using the primer design software Primer5, primers were designed at the beginning and end of the target gene using the EgtD enzyme sequence as a template. Amplification primers containing the bases of the mutation site were also designed at the mutation site using the EgtD enzyme sequence as a template. Separate upstream and downstream primers were amplified by PCR, and recombinant fragments were prepared by overlapping PCR.

[0045] The PCR amplification system is as follows:

[0046]

[0047] The overlapping PCR system is as follows:

[0048]

[0049] PCR reaction conditions (Bao Bio PrimeSTAR HS enzyme): pre-denaturation (95°C) for 5 min; then 30 cycles of denaturation (98°C) for 10 s, annealing ((Tm-3 / 5)°C) for 15 s, and extension at 72°C (this enzyme activity extends approximately 1 kb per 1 min); further extension at 72°C for 10 min; and holding (4°C).

[0050] 1.2 Preparation of linear vector: The vector was linearized by inverse PCR amplification.

[0051] 1.3 Plasmid Construction Method: Obtain the target fragment by PCR, amplify the plasmid by reverse PCR, and recover the target and vector fragments by gel extraction. After homologous recombination, transform them into competent E. coli DH5α cells and apply resistance plates. Pick and culture single colonies that are positive for plasmid extraction.

[0052] 1.4 Recombination reaction: All recombinases used were from the ClonExpress® II One Step Cloning Kit series. Recombination conditions were: 37°C, 30 min.

[0053] The recombinant system is as follows:

[0054]

[0055] 1.5 Plasmid Transformation: Add 10 μL of the reaction solution to 100 mL of DH5α competent cells. Gently mix, incubate on ice for 20 minutes, heat shock at 42°C for 45-90 seconds, and immediately incubate on ice for 2-3 minutes. Add 900 μL of SOC and resuspend the cells at 37°C for 1 hour. Centrifuge at 8000 rpm for 2 minutes, discard the supernatant, and resuspend the cells in approximately 200 μL. Spread the cells onto a plate containing 100 mg / L ampicillin. Invert the plate and incubate at 37°C overnight. Once a single colony has grown on the plate, identify the positive recombinants by colony PCR.

[0056] 1.6 Clone identification: PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight. The plasmids were extracted and identified by enzyme digestion.

[0057] Table 1 PCR amplification primers (S70 site saturation mutagenesis)

[0058]

[0059] Example 2. Construction of mutant strains and fermentation tests

[0060] 2.1 Construction of alanine-induced mutagenesis strain

[0061] The EgtD enzyme was molecularly docked with the substrates SAM and L-histidine using Discovery Studio software to simulate their potential interactions. Based on the results of molecular docking, the amino acid residue sites (Phe73, Ser115, Glu74, Ser70, Cys77, Lys118, Asp139, Tyr65, Ser312, Met280, Glu310, Gly193, Phe64, Tyr234, Phe244, Ile311, Asn245) were obtained. Primers were designed to replace the amino acids at the corresponding sites with alanine to obtain mutant genes. egtD F73A 、egtD S115A 、egtD E74A 、egD S70A 、egtD C77A 、egtD K118A 、egtD D139A 、egtD Y65A 、 egtD S312A 、egtD M280A 、egtD E310A 、egtD G193A 、egtD F64A 、egtD Y234A 、egtD F244A 、egtD I311A 、 egtD N245A , then the mutant fragment was combined with pTrc99a ( Figure 3 ) linearized vector ligation to obtain a recombinant plasmid, which was then transformed into DH5α competent cells. After plating and culturing, several clones from the recombination reaction transformation plate were selected for colony PCR identification. Colonies identified as positive by colony PCR were cultured and the plasmid was extracted. The plasmid was then transformed into an ergothioneine-producing strain to obtain an alanine-scanning mutagenesis strain.

[0062] 2.2 Selection of key sites for site-directed mutagenesis

[0063] Take 10 μL of glycerol bacteria (mutant strain) and inoculate it into 5 mL of LB. Incubate it at 37°C and 220 rpm for 12 h. Then transfer the seed solution to a shake flask containing 30 mL of LB medium at a 3% inoculation rate and incubate it at 37°C and 220 rpm for 12 h. Then transfer the seed solution to a fermentation bottle containing 30 mL of fermentation medium at a 10% inoculation rate and incubate it at 37°C and 220 rpm for 24 h. After the fermentation is completed, treat the fermentation broth according to the fermentation broth treatment method and perform high-performance liquid chromatography detection. The fermentation results are shown in Figure 2. Figure 4 As shown in Table 2, among all the mutants, the S70A mutant had the greatest increase in EGT production compared with the control strain, which was increased by 105%. Therefore, S70 was judged to be the key site for EgtD to bind to the substrate.

[0064] Table 2 Shake flask fermentation results of alanine-induced strains

[0065]

[0066] 2.3 Construction of saturated mutant strains

[0067] By designing primers (Table 1), serine at position 70 was mutated to the remaining 18 amino acids to obtain mutant genes. egtD S70F 、egtD S70L 、egtD S70I 、egtD S70M 、egtD S70V 、egtD S70W 、egtD S70P 、egtD S70T 、egtD S70Y 、egtD S70H 、 egtD S70Q 、egtD S70N 、egtD S70K 、egtD S70C 、egtD S70R 、egtD S70G 、egtD S70D 、egtD S70EThen, the mutant fragment was connected to the pTrc99a linearized vector to obtain a recombinant plasmid, and the recombinant plasmid was transformed into DH5α competent cells. After coating and culturing, several clones on the recombinant reaction transformation plate were picked for colony PCR identification. The colonies identified as positive by colony PCR were cultured and the plasmid was extracted. The plasmid was then transformed into the ergothioneine production strain SX180110 (MG1655 ∆lacIZ ::P xylF - T7RNAP,∆mlc :: mlc*,∆purR,∆tdcD ::P trc -hisG,∆yghX ::P trc -hisDCBHAFI,∆ilvG:: Ptrc-egtBCDE,∆mbhA ::P trc -gshA,∆tehB ::P trc -egtE ncr ) to obtain saturated mutant strains.

[0068] 2.4 Obtaining mutants with improved catalytic activity and thioneine-producing strains thereof

[0069] The saturated mutant strain was compared with the control strain SX220401 (SX180110 / pTrc99a- egtD mva ) to conduct shake flask fermentation experiments and test the results. Figure 5 As shown in Table 3, the ergothioneine production capacity of mutant strains such as S70N, S70K, S70W, and S70V was improved, and the EGT production was increased by 24 times, 23 times, 23 times, and 22 times respectively compared with the control strain, indicating that these four mutant enzymes have higher catalytic activity than the parent enzyme.

[0070] Table 3 Shake flask fermentation results of S70 site saturation mutant strains

[0071]

[0072] Shake flask fermentation: After activating the strain, prepare the seed solution, inoculate 10% of the inoculum into a 500 mL Erlenmeyer flask (final volume 30 mL), seal the flask with nine layers of gauze, and culture at 37°C, 200 rpm, with shaking. During the fermentation process, the pH was maintained at 7.0 by adding ammonia water; 60% (m / v) glucose solution was added to maintain the fermentation process.

[0073] The preferred medium composition is: glucose 20 g / L, yeast extract 4 g / L, peptone 5 g / L, sodium citrate 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 2 g / L, FeSO4 20 mg / L, V B·mix(1,3,5,12) 2 mg / L, V H 2 mg / L, V B6 10 mg / L, MnSO4 10 mg / L, the rest is water, pH 7.0-7.2.

[0074] Fermentation broth treatment method: Take 1 mL of fermentation broth, centrifuge at 13000 rpm for 3 min, take the supernatant and filter it with 0.22 μm filter membrane before HPLC analysis.

[0075] HPLC conditions: the mobile phase was 2% (v / v) acetonitrile, the flow rate was 0.7 mL / min, the chromatographic column was a Titank C18 column (250 mm × 4.60 mm, 5 μm), the column temperature was 30°C, the UV detection wavelength was 257 nm, and the injection volume was 10 μL.

[0076] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A histidine methyltransferase EgtD mutant, characterized in that: The substitution of the EgtD mutant relative to the parent histidine methyltransferase is selected from one of the following: S70V, S70N, S70K, S70W; each position corresponds to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO:

1.

2. The gene encoding the EgtD mutant according to claim 1.

3. A recombinant vector, characterized in that Contains the coding gene according to claim 2.

4. A host cell, characterized in that Comprising the coding gene according to claim 2 or the recombinant vector according to claim 3.

5. The host cell according to claim 4, wherein The host cell is Escherichia coli.

6. Use of the EgtD mutant according to claim 1, wherein the mutant protein is used to catalyze the conversion of histidine into histidine trimethyl inner salt (HER). 7 . A method for preparing the EgtD mutant according to claim 1 , comprising performing gene recombination and expression using a gene encoding the EgtD mutant or an expression vector comprising the gene encoding the EgtD mutant.

8. A method for producing ergothioneine (L-EGT), comprising the steps of introducing the EgtD mutant according to claim 1 into a microbial expression system containing EgtA, EgtB, EgtC, and EgtE, and preparing L-EGT under suitable conditions using L-His and L-Met as substrates.

9. The method according to claim 8, wherein The microbial expression system is Escherichia coli.

10. A method for increasing the enzymatic activity of a histidine methyltransferase, the method comprising introducing the following substitutions into a parent histidine methyltransferase: S70V, S70N, S70K, or S70W; each position corresponding to the amino acid sequence of the parent histidine methyltransferase shown in SEQ ID NO: 1.

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