CN116426499B

CN116426499B - Methyltransferase mutant, biological material and application

Info

Publication number: CN116426499B
Application number: CN202310603871.1A
Authority: CN
Inventors: 张君丽; 穆事成; 娄双颜; 张一鸣; 陶文文; 王瑞妍
Original assignee: Beijing Huaxi Rongxi Biotechnology Research Co ltd
Current assignee: Beijing Huaxi Rongxi Biotechnology Research Co ltd
Priority date: 2023-05-26
Filing date: 2023-05-26
Publication date: 2023-09-22
Anticipated expiration: 2043-05-26
Also published as: CN116426499A

Abstract

The application provides a methyltransferase mutant, a biological material and application, and relates to the field of mutation or genetic engineering. In particular, the application provides a methyltransferase mutant comprising one or more mutations at the following positions relative to a reference sequence: position 58 and position 203, wherein the amino acid position numbers are defined by a reference sequence, and the reference sequence is shown as SEQ ID No. 1. According to the application, egtD enzyme from Mycobacterium smegmatis is used as an initial strain, relevant sites are screened from active center sites by utilizing enzyme crystal structure analysis, 16 mutant strains are constructed, and finally, key amino acid sites with improved EgtD enzyme activity and engineering bacteria capable of efficiently synthesizing ergothioneine are obtained by screening, wherein the mutation can improve the EgtD enzyme activity by 39% and improve the ergothioneine yield by 10%.

Description

Methyltransferase mutant, biological material and application

Technical Field

The application relates to the field of mutation or genetic engineering, in particular to a methyltransferase mutant, a biological material and application.

Background

Methyltransferase (EgtD) is a key enzyme in ergothioneine synthesis. In 2010, seebeck F P was derived from mycobacterium smegmatisMycobacterium smegmatis) The gene of the enzyme egtABCDE and the like is recombined and expressed in the escherichia coli, and the synthesis of ergothioneine in the escherichia coli body is realized (Florian P.Seebeck, JACS,2010,132,6632-6633). The EgtD enzyme is responsible for transferring the methyl group of S-adenosylmethionine to histidine, producing histidine betaine, which is the first step in ergothioneine synthesis. In 2015, vit et al characterized EgtD, the conversion number of the enzyme to the substrate histidine was only 0.58 s ^-1 (Misson, laetitia, chembiochem: A European journal of chemical biology, 2015.) is 2.3% of the most active EgtE enzyme activity in the metabolic pathway. EgtD is therefore a key rate limiting step in ergothioneine synthesis. In 2015, vit et al performed structural analysis (PDB 4 PIN) (Misson, laetitia, chembiochem: A European journal of chemical biology, 2015) of the key enzyme EgtD derived from the synthesis of ergothioneine by Mycobacterium smegmatis, obtained structural complexes of EgtD and its substrate, engineered the EgtD enzyme active center, mutants E285A, E252A/E285A, none of which improved histidine methyltransferase activity.

So far, there is no case of increasing the EgtD enzyme activity by changing amino acids. Therefore, there is a need for EgtD with high enzyme activity to solve the problems that methyltransferase has low enzyme activity and does not meet the industrial production requirements.

Disclosure of Invention

The first step in the synthesis of ergothioneine is the synthesis of histidine betaine by histidine under the catalysis of methyltransferase, which is an important rate limiting step in the synthesis of ergothioneine. The methyltransferase reported in the current literature and patent has lower enzyme activity, which does not meet the requirement of industrial production.

Therefore, the methyltransferase mutant site and the enzyme mutant with improved enzyme activity are obtained for the first time after screening by carrying out site-directed mutation on methyltransferase and in-vitro enzyme activity measurement, so that the methyltransferase activity and the yield of ergothioneine are effectively improved, and a novel methyltransferase mutant and engineering bacteria are provided for industrial production of ergothioneine.

In one aspect, the application provides a methyltransferase mutant comprising any one of the following A1) -A3):

a1 A protein comprising one or more mutations relative to a reference sequence at the following positions: position 58 and position 203, wherein the amino acid position numbers are defined by a reference sequence, and the reference sequence is shown as SEQ ID No. 1;

a2 A protein which has 90% or more identity with the protein of A1) and has the same function, and is obtained by substitution and/or deletion and/or addition of amino acid residues in the amino acid sequence of the protein of A1);

a3 A) is a fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

Preferably, the protein of A2) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% more sequence identity with the amino acid sequence defined in A1).

It will be appreciated by those skilled in the art that the protein coding rules of the present application follow the usual rules, taking the 58 th mutation as an example, which means counting the 58 th amino acid positions sequentially from the N-terminal to the C-terminal of the reference sequence.

Further, the proline residue at position 58 is mutated to a leucine residue; and/or, the valine residue at position 203 is mutated to an asparagine residue.

Further, the methyltransferase mutant comprises an amino acid sequence shown as SEQ ID NO.3 and/or an amino acid sequence shown as SEQ ID NO. 5.

In another aspect, the present application also provides a biomaterial comprising any one of the following B1) -B6):

b1 A nucleic acid molecule encoding a methyltransferase mutant as described above;

b2 An expression cassette comprising B1) the nucleic acid molecule;

b3 A recombinant vector comprising B1) said nucleic acid molecule and/or B2) said expression cassette;

b4 A recombinant microorganism comprising B1) the nucleic acid molecule, B2) the expression cassette, and/or B3) the recombinant vector;

b5 A recombinant cell comprising B1) the nucleic acid molecule, B2) the expression cassette, and/or B3) the recombinant vector;

b6 A whole cell catalyst comprising B1) the nucleic acid molecule, B2) the expression cassette, B3) the recombinant vector, B4) the recombinant microorganism, and/or B5) the recombinant cell.

The expression cassette may further comprise functional elements such as promoters, terminators, marker genes, etc., and those skilled in the art may choose conventionally according to the actual situation, as long as the expression of the nucleic acid molecule according to B1) can be accomplished, and the structure and composition of the expression cassette are not excessively limited.

The vectors described herein refer to vectors capable of carrying exogenous DNA or genes of interest into host cells for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), or viral vectors. Specifically, the vector pET30a and/or pACYC-PSC101-Ptac plasmid may be used.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria are derived from the genus EscherichiaEscherichia sp.) Genus ErwiniaErwinia sp.) Genus AgrobacteriumAgrobacterium sp.) The genus FlavobacteriumFlavobacterium sp.) Genus AlcaligenesAlcaligenes sp.) Genus PseudomonasPseudomonas sp.) Bacillus genusBacillus sp.) Genus BrevibacteriumBrevibacterium sp.) Genus CorynebacteriumCorynebacterium sp.) The genus AerobacterAerobacter sp.) The enterobacter genusEnterobacteria sp.) Micrococcus genusMicrococcus sp.) Serratia genusSerratia sp.) Salmonella genusSalmonella sp.) Streptomyces genusStreptomyces sp.) Provedsia species @Providencia sp.) And the like, but is not limited thereto.

The cell described herein may be a plant cell or an animal cell, and the cell may be any biological cell that can synthesize ergothioneine of interest.

It will be appreciated that the skilled artisan can select an appropriate gene editing system and method to accomplish the mutational alteration, depending on the circumstances.

Further, the nucleic acid molecule of B1) includes any one of the following C1) -C5):

c1 Nucleic acid molecules encoding the above methyltransferase mutants;

c2 A nucleic acid molecule encoding the amino acid sequence of SEQ ID No.3 or SEQ ID No. 5;

c3 A nucleic acid molecule with a nucleotide sequence shown as SEQ ID No.4 or SEQ ID No. 6;

c4 A nucleic acid molecule which has 98% or more identity to the nucleic acid molecule of any one of C1) to C3) and which encodes the above methyltransferase mutant;

c5 A genomic DNA molecule which hybridizes under stringent conditions with any of the nucleic acid molecules C1) to C3) and which codes for a methyltransferase mutant as described above.

Preferably, the nucleic acid molecule of C4) has at least 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or more than 99.9% identity with any of the nucleic acid molecules of C1) -C3).

Further, the recombinant microorganism is one or more of corynebacterium glutamicum, bacillus subtilis, escherichia coli and saccharomyces cerevisiae.

In a preferred embodiment, the recombinant microorganism (host) is EGT33 and BL21 (DE 3).

In another aspect, the application also provides the use of a methyltransferase mutant as described above or a biological material as described above for increasing methyltransferase enzymatic activity.

According to the application, two key amino acid sites for improving EgtD enzyme activity are obtained for the first time, namely the 58 th active site and the 203 th active site of EgtD enzyme derived from mycobacterium smegmatis, specifically, the 58 th active site is mutated from proline to leucine, the 203 th active site is mutated from valine residue to asparagine residue, the EgtD enzyme activity can be effectively improved after the two key amino acid sites are mutated, and compared with the non-mutated EgtD enzyme, the EgtD enzyme activity after mutation is respectively improved by 39% and 17%, so that a key target point is provided for further improvement of the subsequent enzyme activity of the enzyme.

In another aspect, the application also provides the use of a methyltransferase mutant as described above or a biomaterial as described above in any one of the following D1) to D3):

d1 Construction of recombinant microorganisms for the production of ergothioneine;

d2 Preparation of ergothioneine;

d3 Regulating and controlling the yield of the ergothioneine produced by the recombinant microorganism.

In the application, an escherichia coli expression system is taken as an example, engineering bacteria containing the mutation are obtained, and the engineering bacteria are applied to ergothioneine production. The mutation is found to be capable of effectively improving the yield of the ergothioneine, and compared with engineering bacteria without mutation, the engineering bacteria after mutation can improve the yield of the ergothioneine by 10 percent, and the maximum yield of the ergothioneine can reach 228.28 +/-11.53 mg/L. The mutation effectively improves the synthesis efficiency of the ergothioneine, and provides a new synthesis way and engineering bacteria for producing the ergothioneine.

In another aspect, the application also provides the use of a methyltransferase mutant as described above or a biological material as described above in the preparation of a drug or cosmetic containing ergothioneine.

Alternatively, the pharmaceutical or cosmetic dosage form includes, but is not limited to, powders, tablets, capsules, gels, or topical wipes such as lotions, emulsions, creams, masks, etc., or daily necessities such as shampoos, conditioners, hair films, etc.

In another aspect, the present application also provides a method for producing ergothioneine, the method comprising the steps of:

step one, constructing to obtain the whole cell catalyst;

culturing the whole cell catalyst to obtain the ergothioneine.

In a preferred embodiment, the whole cell catalyst comprises a recombinant microorganism.

The production method of ergothioneine comprises the following steps: the recombinant microorganism is transferred to a resistant fermentation medium at 37 ℃ and 200 rpm under the condition of the inoculation amount of 10% to obtain seed liquid overnight, 0.5 h is cultivated at 37 ℃ and 200 rpm, 0.2 mM IPTG is added to carry out induction cultivation at 37 ℃ for 10-12 h, and ergothioneine is produced.

The application has the following beneficial effects:

1. the application adopts a high-efficiency escherichia coli expression system, takes EgtD enzyme from mycobacterium smegmatis with highest activity reported at present as an initial strain, utilizes the analysis of the crystal structure of the enzyme to screen relevant sites from active center sites, totally constructs 16 mutant strains, and finally screens and obtains engineering bacteria capable of synthesizing ergothioneine with high efficiency;

2. according to the application, the key amino acid site for improving the EgtD enzyme activity is obtained for the first time, the EgtD enzyme from mycobacterium smegmatis is taken as an initial sequence, and the 58-site active site is mutated from proline to leucine, so that the EgtD enzyme activity can be effectively improved, compared with the unmutated EgtD enzyme, the mutated EgtD enzyme can improve the EgtD enzyme activity by 39%, and a key target point is provided for further improvement of the subsequent enzyme activity of the enzyme;

3. the engineering bacteria and the EgtD enzyme with high enzyme activity obtained by the application can be used in the production process of the ergothioneine, compared with the engineering bacteria without mutation, the engineering bacteria after mutation can improve the yield of the ergothioneine by 10%, and the maximum yield of the ergothioneine can reach 228.28 +/-11.53 mg/L.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:

FIG. 1 is a pET30a-E0 vector construction map;

FIG. 2 shows the SAH standard curve of the enzyme coupling assay.

Detailed Description

Technical terms:

expression: the term "expression" includes any step involving the production of a methyltransferase or mutant thereof.

Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in a molecular evolution study.

Recombination: in a broad sense, any process of gene communication that causes a genotype change is called recombination.

Expression cassette: an expression cassette refers to a set of DNA sequences consisting of a promoter, a target gene, a reporter gene, etc., which are expressed in a specific tissue and are easily detected.

Recombinant vector: the recombinant vector is a vector which is transferred into a target gene on the basis of the basic skeleton of the cloning vector so as to enable the target gene to be expressed.

Whole cell catalyst: whole-cell biocatalysis refers to a process of chemical transformation using whole biological organisms (i.e., whole cells, tissues, or even individuals) as catalysts, and the whole biological organisms involved in the catalytic process are the whole-cell catalysts accordingly.

Engineering strain (recombinant microorganism): by using a genetic engineering method, the exogenous gene can be obtained into a fungus cell line with high expression efficiency.

Recombinant cells: the term "recombinant cell" means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present application. The term "recombinant cell" encompasses any parent cell progeny that are not identical to the parent cell due to mutations that occur during replication.

Enzyme activity: also referred to as enzyme activity, refers to the ability of an enzyme to catalyze certain chemical reactions. The amount of enzyme activity can be expressed in terms of the rate of conversion of a chemical reaction that it catalyzes under certain conditions.

In order to more clearly illustrate the general concept of the present application, the following detailed description is given by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the application may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the application.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.

The plasmids, endonucleases, PCR enzymes, column type DNA extraction kits, DNA gel recovery kits and the like used in the following examples are commercially available products, and specific operations are performed according to the kit instructions.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present application are all performed according to Molecular Cloning: A Laboratory Manual (fourths Edition) using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and conventional techniques in the relevant arts, which are conventional in the art.

The construction method of the escherichia coli EGT33 comprises the following steps:

the construction method of E.coli EGT33 is disclosed in the patent document with application number 202211546541.5. The genetically engineered bacterium takes escherichia coli as a host, and is subjected to heterologous overexpressionMicrocoleus sp.The egtD gene derived from PCC 7113,Methylobacterium pseudosasicolathe egtB gene of origin,Neurospora crassathe egt2 gene of origin,Corynebacterium glutamicumthe ATP-transphosphorylation ribosylase mutant encoding gene hisG; and overexpressing the hisdbcha gene cluster, the serine transacetylase mutant CysE (M201R) encoding gene, the phosphoglycerate dehydrogenase mutant SerA (H344A/N364A) encoding gene, the adenosylmethionine synthase mutant MetK (I303V) encoding gene; and does not express the tryptophan enzyme gene tnaA. The strain systematically modifies the ergothioneine synthesis module, precursor histidine, cysteine and adenosylmethionine synthesis module, and realizes the efficient and stable production of ergothioneine in escherichia coli.

EXAMPLE 1 construction of mutants

The inventor obtains the protein crystal structure (PDB: 4 PIN) of EgtD gene from mycobacterium smegmatis through UniProt database, further analyzes the relevant site of the active center, and constructs 16 mutants.

The construction method comprises the following steps: selecting EgtD gene (amino acid sequence is shown as SEQ ID NO.1, nucleotide sequence is shown as SEQ ID NO. 2) derived from Mycobacterium smegmatis, removing enzyme cutting sites (NdeI and XhoI) in the base sequence of the gene, inserting NdeI site at the N end of the gene, connecting 6 XHis tag, stop codon and XhoI site at the C end, and connecting into a vector pET30a to obtain recombinant plasmid pET30a-E0, wherein the construction map of the recombinant plasmid is shown as figure 1. Then transferring the plasmid into colibacillusE. coliBL21 (DE 3) (Optimaceae) host to obtain genetically engineered bacteriaPlant strainE. coliBL21 (DE 3)/pET 30a-E0 (D0 strain).

The PCR products were detected by agarose gel electrophoresis using Phanta Max Super-Fidelity DNA Polymerase (available from vazyme) as an amplification enzyme and plasmid pET30a-E0 as a template, and the primers shown in Table 1 below. The band of interest was excised from the agarose gel and subjected to DNA purification recovery using a gel recovery kit (Omega). Transferring the recovered fragment into E.coliE. coliIn DH5 alpha, after single colony sequencing is picked up and verified to be correct, plasmid extraction is carried out by using a plasmid extraction kit (Omega) to obtain each mutant plasmid, and the mutant plasmids are transferred into escherichia coliE. coliBL21 (DE 3) and mutants D1-D16 were obtained, and specific engineering strain information is shown in Table 2.

TABLE 1 primers for PCR amplification

Note that: taking the mutation site "P58L" as an example, "P58L" means that the 58 th amino acid site is mutated from a proline (P) residue to a leucine (L) residue, counting from the N-terminus of the reference sequence SEQ ID NO.1 toward the C-terminus.

TABLE 2 engineering strain information

Example 2 high throughput enzyme Activity assay

In this example, the wild type (D0) and 16 mutant strains (D1-D16) of example 1 were used for enzyme activity detection, and the high-throughput enzyme activity detection was carried out according to the method for measuring methyltransferase activity described in the literature (ref. Gu Jinsong, 2012, university journal of chemistry, 3, 512-525).

The method comprises the following steps: synthesis of the S-adenosyl-L-homocysteine ribonuclease SAHN (EC 3.2.2.9) and S-ribosyl Gao Banguang Aminoenzyme SRHH (EC 4.4.1.21) genes, 1 hexahistidine tag inserted into the N-terminus of the SAHN gene and 1 hexahistidine tag inserted into the C-terminus of the SRHH geneHistidine tag, recombinant plasmids pET30-SAHN and pET30-SRHH are obtained, and the plasmids are transferred intoE. coliBL21 (DE 3) host to obtain genetically engineered strainE. coliBL21 (DE 3)/pET 30-SAHN andE. coliBL21(DE3)/pET30-SRHH。

respectively picking from the flat plateE. coliBL21 (DE 3)/pET 30-SAHN andE. coliBL21 (DE 3)/pET 30-SRHH single colonies were inoculated into LB medium (containing 50. Mu.g/mL Kan) of 5 mL at 37℃and cultured overnight at 220 rpm. Transferring the culture solution 1:100 into LB medium (containing 50 μg/mL Kan) of 50 mL, culturing at 37deg.C and 220 rpm, and OD ₆₀₀ When reaching 0.6-0.8, 0.2. 0.2 mM isopropyl thiogalactoside (IPTG) was added and incubated at 16℃and 220 rpm for 16 h. Collect 120 OD ₆₀₀ Suspending thallus with 1 mL buffer (100 mM Tris-HCl, pH=8.0), ultrasonic crushing thallus cells, centrifuging at 12000 rpm/min for 60 min to remove precipitate, collecting supernatant, purifying enzyme protein, adding Ni into supernatant ²⁺ Agarose affinity chromatography column, using Binding buffer (50 mmol/L Tris-HCl, 50mmol/L NaCl and 5 mmol/L imidazole, pH=8.0) washing 3-4 column volumes, finally using the solution of the solution buffer (50 mmol/L Tris-HCl, 150mmol/L NaCl and 250 mmol/L imidazole, pH=8.0) Elution, finally obtaining pure enzyme protein, using Nanodrop to carry out protein concentration determination, SAHN enzyme solution dilution 100 times for standby.

Enzyme coupling analysis and detection method: S-adenosyl-L-homocysteine (AdoHcy) was used as a standard, the concentration of the standard was in the range of 0 to 200. Mu. Mol/L, an enzyme reaction mixture (0.1. Mu. Mol/L SAHN, 10. Mu. Mol/L SRHH and 100 mmol/L Hepes buffer (pH=8.0)) was added, the reaction was carried out at 37℃for 30 minutes, and then 4 volumes of 5,5 '-dithio-2, 2' -dinitrobenzoic acid (DTNB) (133. Mu. Mol/L) -guanidine hydrochloride (8 mol/L) solution was added to terminate the reaction, and the amount of TNB produced was measured by colorimetry at 412 and nm. The blank was a mixture of 4 volumes of DTNB-guanidine hydrochloride and an enzyme reaction system without SAHN. The measured calibration curve is shown in fig. 2.

Single colonies of the wild type (D0) and 16 mutant strains (D1-D16) were picked from the plates and inoculated into LB medium (containing 50. Mu.g/mL Kan) of 5 mL at 37℃overnight at 220 rpm for cultivation. The culture solution 1:100 was transferred to LB medium (containing 50Mu g/mL Kan), 37℃and 220 rpm, OD ₆₀₀ When reaching 0.6-0.8, 0.2 mM isopropyl thiogalactoside (IPTG) was added and incubated at 16℃and 220 rpm for 20 h. Collect 120 OD ₆₀₀ Suspending with 1 mL buffer (100 mM Tris-HCl, pH=8.0), then ultrasonic disrupting the bacterial cells, centrifuging at 12000 rpm/min for 60 min to remove precipitate, collecting supernatant, purifying enzyme protein, collecting supernatant, and collecting supernatant on Ni ²⁺ Agarose affinity column, washing 3-4 column volumes with Binding buffer (50 mmol/L Tris-HCl, 50mmol/L NaCl and 5 mmol/L imidazole, pH=8.0), eluting with solution containing Elutation buffer (50 mmol/L Tris-HCl, 150mmol/L NaCl and 250 mmol/L imidazole, pH=8.0), and finally obtaining pure enzyme protein, measuring protein concentration by Nanodrop, and diluting enzyme solution 50 times for later use.

Enzyme activity detection system and method: to ensure accurate detection of enzyme activity, the final system for enzyme activity detection was 200. Mu.L, wherein the final concentration of L-histidine and S-adenosylmethionine was 400. Mu. Mol/L, SAHN was added in an amount of 0.1. Mu. Mol/L, SRHH was added in an amount of 10. Mu. Mol/L according to the method for enzyme activity detection, methyltransferase (egtD) was added in an amount of 5. Mu.g/mL, hepes buffer (pH=8.0) was added in a system for supplementing, and after 30 min reaction at 37℃the reaction was completed, 4-fold volume of DTNB-guanidine hydrochloride solution was added to terminate the reaction, the amount of TNB produced was measured by colorimetry at 412 and nm, relative enzyme activity was calculated by a standard curve, D0 was used as a control, and the change in enzyme activity was compared, and the specific results are shown in Table 3.

TABLE 3 construction of mutant numbering, mutation sites and relative Activity comparison in example 1

From Table 3, egtD mutant strains D4 and D12 with improved methyltransferase activity are successfully screened, wherein the methyltransferase mutant in D12 has 58 active sites mutated from proline to leucine, the amino acid sequence is shown as SEQ ID NO.3, the nucleotide sequence is shown as SEQ ID NO.4, the methyltransferase mutant in D4 has 203 active sites mutated from valine to asparagine, the amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6. Wherein, the relative activity of the methyltransferase mutant in D12 is highest and can reach 139%, and the final enzyme activity is improved by 39%.

Example 3 application of engineering Strain in production of ergothioneine

Construction of ergothioneine genetically engineered bacteria

Using PrimeSTAR ^® Max DNA Polymerase (available from takara, R045A) was used as an amplification enzyme, and the primers used for PCR amplification of Ms-F and Ms-R were as shown in Table 4 below, using the plasmids pET30a-E0 and pET30a-E12 (P58L) described in example 1 as templates, respectively.

TABLE 4 primers for PCR amplification

Primer name	Nucleotide sequence
		Ms-F	CTTTAAGAAGGAGATATACCATGGGCATGACGCTCTCACTGGCCAAC
Ms-R	CGGTTTCTTTACCAGACTCGAGTTAGTGGTGGTGGTGGTGGTGACCAGAAGACCGCACCGCCAGCGACAAC
		Ms1-F	CTTTAAGAAGGAGATATACCATGGGCTTGATCGCACGCGAGACAC
Ms1-R	CGGTTTCTTTACCAGACTCGAGTTTTCAGGGCGCCTCACGC

After the amplification is completed, agarose gel electrophoresis is performed to detect the PCR products. The band of interest was excised from the agarose gel and subjected to DNA purification recovery using a gel recovery kit (Omega, D2500-02). The recovered fragment was digested with NcoI-HF (from NEB, R3193S) and XhoI (from NEB, R0146V), ligated into the vector pACYC-PSC101-Ptac (construction method was that the T7-1 promoter in plasmid pACYCDuet-1 (Ubao organism) was replaced with Ptac promoter, the replicon was replaced with PSC101 to obtain plasmid pACYC-PSC 101-Ptac), and after the plasmids pACYC-PSC101-Ptac-E0, pACYC-PSC101-Ptac-E12 were correctly sequenced, E.coli EGT33 producing ergothioneine was transformed to obtain engineering strains F0, F12.

The genome of Mycobacterium smegmatis (purchased from CGMCC, no. 1.562) is used as a template, the MsEgtBCDE gene cluster amplified by plasmids Ms1-F and Ms1-R is connected into NcoI and XhoI sites of a vector pETDuet-1 (purchased from Youbao organism, VT1237, amp resistance) by using a primer to obtain a plasmid pETDuet-MsEgtBCDE, and the plasmid pETDuet-MsEgtBCDE and plasmids pET-30a-E0 and pET-30a-E12 are respectively transferred into BL21 (DE 3) hosts to obtain engineering strains G0 and G12, and the engineering strains are specifically shown in a table 5.

TABLE 5 engineering bacteria information

Engineering bacteria shake flask culture and ergothioneine yield

Engineering strains F0, F12 were streaked on LB plates containing 30. Mu.g/mL Cm resistance, and G0, G12 were streaked on LB plates containing 100. Mu.g/mL Amp resistance and 50. Mu.g/mL Kan resistance, and cultured overnight in an incubator at 37 ℃.3 single clones of each engineering bacterium are respectively selected and inoculated into LB culture medium containing corresponding antibiotics of 4 mL, and the engineering bacterium is cultured overnight at 37 ℃ and 200 rpm. Respectively adding the above seed solution 3 mL into 500 mL triangular flask containing 30 mL corresponding resistant fermentation medium (specific medium formula shown in Table 6), culturing at 37deg.C and 200 rpm for 0.5 h, adding inducer isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.2 mM, and adding into the flask at 37Inducing at the temperature of DEG C to synthesize the target product ergothioneine. During the culture, ammonia water was used to control pH to maintain pH around 7.0. After shaking culture 10-12 and h, fermenting supernatant was collected by centrifugation at 12000 rpm for 2 min, and biomass OD was measured ₆₀₀ And ergothioneine yields, the test results are shown in Table 7. Specifically, the concentration of ergothioneine in the fermentation broth is detected by a high performance liquid chromatography method.

Detection conditions: the column was Kromasil C18 (250 mm X4.60 mm,5 μm); the mobile phase is acetonitrile: water = 2:98 (volume ratio); the flow rate is 0.7 mL/min; column temperature is 30 ℃; ultraviolet detection wavelength 257 nm.

TABLE 6 fermentation Medium formulation

TABLE 7 production of ergothioneine by engineering strains

From the results in Table 7, it can be seen that the activity of engineering bacteria and the yield of ergothioneine can be effectively improved after the 58 th active site of the methyltransferase from Mycobacterium smegmatis is mutated from proline to leucine. Specifically, the maximum yield of the ergothioneine of the F12 strain can reach 228.28 +/-11.53 mg/L, and compared with the F0 strain, the yield of the ergothioneine of the F12 strain is improved by 10 percent. In BL21 (DE 3) host, the yield of G12 strain reaches 23.45+/-1.02 mg/L, which is 23% higher than that of control G0 strain.

Claims

1. A methyltransferase mutant, comprising the following A1) or A2):

a1 An amino acid sequence shown as SEQ ID NO.3 or an amino acid sequence shown as SEQ ID NO. 5;

a2 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1).

2. A biomaterial, characterized in that it comprises any one of the following B1) -B6):

b1 A nucleic acid molecule encoding the methyltransferase mutant of claim 1;

b2 An expression cassette comprising B1) the nucleic acid molecule;

3. The biomaterial according to claim 2, wherein the nucleic acid molecule of B1) is a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No.4 or SEQ ID No. 6.

4. The biomaterial of claim 2, wherein the recombinant microorganism is one or more of corynebacterium glutamicum, bacillus subtilis, escherichia coli, saccharomyces cerevisiae.

5. Use of a methyltransferase mutant according to claim 1 or a biological material according to any one of claims 2-4 for increasing methyltransferase activity.

6. Use of a methyltransferase mutant according to claim 1 or a biomaterial according to any one of claims 2 to 4 in any one of the following D1) to D3):

d2 Preparation of ergothioneine;

7. Use of a methyltransferase mutant according to claim 1 or a biomaterial according to any one of claims 2-4 for the preparation of a ergothioneine-containing pharmaceutical or cosmetic product.

8. A process for the production of ergothioneine, comprising the steps of:

step one, constructing to obtain the biological material according to claim 2, wherein the biological material is a recombinant microorganism or a recombinant cell;

culturing the recombinant microorganism or recombinant cell to obtain the ergothioneine.