Methyltransferase mutant high-throughput screening method, methyltransferase mutant and application

Abstract

The invention belongs to the technical field of biology, and particularly discloses a methyltransferase mutant high-throughput screening method, a methyltransferase mutant and application. The method can be used for high-throughput screening of methyltransferase mutants, the transformation efficiency of methyltransferase is improved, and high-activity methyltransferase mutants are obtained; the methyltransferase mutant provided by the invention can obviously improve the activity of methyltransferase, and is used for producing Nacetyl-5-methoxytryptamine to improve the yield. The activity of the methyltransferase can be remarkably improved by using the engineering bacterium expressing the methyltransferase mutant, the engineering bacterium is used for producing Nacetyl-5-methoxytryptamine to improve the yield, and the yield of the Nacetyl-5-methoxytryptamine is up to 22.7 mM.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a high-throughput screening method for a methyltransferase mutant, a methyltransferase mutant and an application thereof. Background Art

[0002] Methylation is the process of transferring a methyl group (-CH3) from one compound to another, which usually also causes significant changes in the biological activity and bioavailability of the substance. Currently, chemical-based methylation uses toxic reagents, produces toxic waste, and does not have regioselectivity. In contrast, enzymatic methylation has extremely high specificity, is environmentally friendly, and is safer to use. Methylases, also known as methyltransferases, are a large family with a wide range of biological functions, usually using SAM (S-adenosylmethionine) as a methylation donor. They play an important role in the methylation reactions of lipids, proteins, polysaccharides or nucleotides, and these modifications produce a variety of important biological functions, such as epigenetic phenomena and the generation of numerous secondary metabolites. Among them, oxygen-methyltransferases are used in the biosynthesis of natural products such as ferulic acid, sakura, rhamnetin, melatonin, vanillin and pterostilbene, and have extremely high bioeconomic value.

[0003] It is a common strategy to rationally modify methyltransferases to enhance enzyme activity and selectivity, thereby improving the production efficiency of the target. However, there is currently no universal and convenient analytical method to quickly detect methyltransferase activity for the evaluation and screening of methyltransferase modifications. Most methyltransferase activity assays are based on radioactive labeling or fluorescence detection, which is time-consuming and labor-intensive, and has high requirements for instruments and equipment. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a high-throughput screening method for methyltransferase mutants, methyltransferase mutants and applications in view of the deficiencies of the prior art. The present invention can be used for high-throughput screening of methyltransferase mutants, improve the efficiency of methyltransferase transformation, and obtain highly active methyltransferase mutants; the methyltransferase mutant provided by the present invention can significantly improve the activity of methyltransferase, and is used to produce N-acetyl-5-methoxytryptamine to increase the yield.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for high-throughput screening of methyltransferase mutants, comprising the following steps:

[0007] (a) activating, culturing, and inducing expression of a single clone in a methyltransferase mutant library;

[0008] (b) adding a substrate to the system of step (a), and catalyzing the substrate to undergo a methyltransferase-catalyzed reaction using SAM as a methylation donor;

[0009] (c) disrupting the cells and adding a thiol detection reagent, the thiol detection reagent reacts with the thiol compound produced in the methylation reaction catalyzed by the methylation donor SAM in the system to generate a colored product, measuring the absorbance value, and screening the methyltransferase mutant based on the absorbance value. A high absorbance value indicates that the corresponding methyltransferase mutant has a high activity.

[0010] According to the above scheme, the methyltransferase catalyzed reaction uses SAM as the methylation donor. After the reaction, SAM is converted into SAH, which is then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​(Hcys) with a thiol group (-SH). Based on the characteristic color development reaction between the thiol detection reagent and the thiol compound homocysteine ​​(Hcys) with a thiol group (-SH), the absorbance value of the system is determined, and the methyltransferase mutants are screened based on the absorbance value.

[0011] According to the above scheme, the thiol detection reagent is DTNB reagent, and the absorbance value is measured by utilizing the characteristic color development reaction of the thiol compound produced in the methylation reaction catalyzed by DTNB and the methylation donor SAM in the system.

[0012] According to the above scheme, the vector for constructing the plasmid and the methyltransferase gene are used as templates to amplify the linearized vector and the methyltransferase gene fragment, the methyltransferase gene fragment is connected to the linearized vector to obtain a recombinant plasmid, and the recombinant plasmid is used as a template to design primers for plasmid amplification and mutagenesis to obtain a linearized plasmid vector with base mutations, which is then transformed into Escherichia coli to construct a methyltransferase mutant library.

[0013] According to the above scheme, the above recombinant plasmid also contains MetK, Mtn, and LuxS gene fragments. The adenosylmethionine synthetase (Metk) gene catalyzes the reaction of methionine (Met) and ATP to generate SAM (S-adenosylmethionine), which is used as a methylation donor. After the methylation reaction of the catalyzed substrate is completed, SAM is converted into SAH, and then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​(Hcys) with a sulfhydryl group (-SH).

[0014] According to the above scheme, the monoclones with absorbance values ​​in the top 8-12% are selected for sequencing, and the methyltransferase mutants are screened. Preferably, the monoclones with absorbance values ​​in the top 8-12% are sequenced, which can achieve rapid screening and ensure better screening effect. Further, the activity of the methyltransferase mutants obtained by the above screening and sequencing for catalyzing the methylation reaction of the substrate methyltransferase is measured, and the methyltransferase mutants with high activity are further screened according to the reaction activity.

[0015] According to the above scheme, the multi-well plate used for activation and culture in step (a) is a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate; preferably, the multi-well plate is a 96-well plate.

[0016] According to the above scheme, the activation and culture conditions are: temperature 25-37°C, shaking speed 300-1000rpm, and culture time 6-20h.

[0017] According to the above scheme, in step (a), the induced expression is to add IPTG to induce protein expression, the inducer concentration is 0.05-2mM, and the induction temperature is 18-30°C.

[0018] According to the above scheme, the concentration of the thiol detection reagent is 0.01-5mM, further 0.05-1mM.

[0019] According to the above scheme, the crushing method is lysozyme reaction, liquid nitrogen quick freezing, acid and alkali treatment, surfactant treatment, ultrasonic treatment and quartz sand vibration grinding; preferably, it is lysozyme reaction.

[0020] According to the above scheme, in the high-throughput screening method for methyltransferase mutants, N-acetyl 5-hydroxytryptamine is used as a substrate to perform a methyltransferase-catalyzed reaction to synthesize N-acetyl-5-methoxytryptamine.

[0021] According to the above scheme, the N-acetyl 5-hydroxytryptamine substrate concentration is 0.1-10 g / L, the reaction pH is 5.0-9.0, and the catalytic reaction temperature is 20-40°C.

[0022] In the second aspect, the present invention takes the substrate N-acetyl 5-hydroxytryptamine as an example and provides a high-throughput screening method for methyltransferase mutants for use in screening methyltransferase mutants that synthesize N-acetyl-5-methoxytryptamine through a methyltransferase-catalyzed reaction using N-acetyl 5-hydroxytryptamine as a substrate.

[0023] In a third aspect, the present invention provides a methyltransferase mutant having an amino acid sequence as shown in any one of the following:

[0024] (a) the amino acid sequence is obtained by mutation of one or more amino acid residue positions selected from the group consisting of: 128, 160, 321, 322 and 324 of the sequence shown in SEQ ID NO: 2, i.e., the above positions can be selected from any one of the amino acids other than the original amino acids;

[0025] or (b) the methyltransferase has a similarity of more than 90%, preferably more than 95%, more preferably more than 99% with the amino acid sequence of (a), and has the function of the protein of (a), wherein the amino acids at positions 128, 160, 321, 322 and 324 corresponding to the amino acid sequence of SEQ ID NO: 2 are mutated in the same manner as the amino acid sequence of (a);

[0026] Or (c) the methyltransferase is formed by adding multiple amino acids or deleting 1-20, preferably 1-10, more preferably 1-5 amino acids at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the protein described in (a), wherein the amino acids at positions 128, 160, 321, 322 and 324 corresponding to the amino acid sequence shown in SEQ ID NO: 2 are mutated in the same manner as the amino acid sequence described in (a). Specifically, the added amino acid sequence can be 1-20, preferably 1-10, more preferably 1-5 amino acids, or a protein tag sequence such as His, Flag, MBP, SUMO, GST, Trx.

[0027] Preferably, the mutation at position 128 is to leucine, the mutation at position 160 is to serine or valine, the mutation at position 321 is to leucine, the mutation at position 322 is to leucine or phenylalanine, and the mutation at position 324 is to aspartic acid.

[0028] In a fourth aspect, the present invention also provides a gene encoding the above-mentioned methyltransferase mutant.

[0029] In a fifth aspect, the present invention provides an expression vector, which comprises a gene encoding the above-mentioned methyltransferase mutant and is capable of expressing the above-mentioned methyltransferase mutant.

[0030] In a sixth aspect, the present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium comprises the above-mentioned expression vector or a gene encoding a methyltransferase mutant is integrated into its genome.

[0031] The recombinant genetic engineering bacteria can be obtained by transferring the above expression vector into a host cell or by integrating a gene encoding a methyltransferase mutant into the genome.

[0032] In a specific embodiment, the host cell is a bacterium or a yeast; preferably, the host cell is Escherichia coli (E. coli), Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica; more preferably, the host cell is Escherichia coli, Saccharomyces cerevisiae; most preferably, the host cell is Escherichia coli.

[0033] In a seventh aspect, the present invention provides the use of the above-mentioned methyltransferase mutant or expression vector or genetically engineered bacteria in the production of N-acetyl-5-methoxytryptamine. Specifically, the genetically engineered bacteria are induced to express proteins, and then the cells are collected by centrifugation, and then added to a conversion liquid containing the substrate N-acetyl-5-hydroxytryptamine for reaction, and finally N-acetyl-5-methoxytryptamine is separated from the conversion liquid.

[0034] According to the above scheme, the transformation solution is a phosphate buffer containing 0.5-100 mM N-acetyl-5-hydroxytryptamine and 0.5-200 mM S-adenosylmethionine (SAM).

[0035] According to the above scheme, the reaction temperature is 20-40°C.

[0036] According to the above scheme, the reaction process is shaken on a shaking table with a rotation speed of 100 to 300 rpm.

[0037] According to the above scheme, the reaction time is 3 to 24 hours.

[0038] According to the above scheme, the reaction pH is 5.0-9.0.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The screening method for methyltransferase mutants provided by the present invention can be used for high-throughput screening of methyltransferase mutants, improve the efficiency of methyltransferase modification, and obtain highly active methyltransferase mutants; specifically, in the high-throughput screening of the present invention, SAM is used as a methylation donor, and the overexpression of Mtn and LuxS genes is coordinated, and SAH obtained in the methylation transfer process of the methylation donor SAM is promoted by Mtn-LuxS in two consecutive steps of catalysis to produce homocysteine ​​(Hcys) with a sulfhydryl group (-SH), and then based on the characteristic color development reaction of the sulfhydryl detection reagent and the homocysteine ​​(Hcys) with a sulfhydryl group (-SH) in the sulfhydryl compound, it is helpful to achieve high-throughput screening of methyltransferase mutants based on the absorbance value of the measurement system.

[0041] The present invention takes the enzyme-catalyzed production of melatonin N-acetyl-5-methoxytryptamine as an example, and proposes a rapid high-throughput screening method for methyltransferase mutants, which is used for the high-throughput screening of methyltransferase mutants, improves the screening efficiency of methyltransferase modification, and lays a foundation for industrial application.

[0042] The present invention obtains a highly active methyltransferase mutant by saturation mutation of the original sequence of the methyltransferase shown in SEQ ID NO: 2 and combining it with high-throughput screening technology. It is used to generate N-acetyl-5-methoxytryptamine, which can significantly improve the catalytic efficiency, reduce the amount of microbial cells or enzymes used in the catalytic process, and reduce production costs. The maximum catalytic production of N-acetyl-5-methoxytryptamine is 22.7mM, the catalytic activity is 5.5 times that of the wild type, and the substrate conversion rate is as high as 98.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the plasmid map of pRSFDuet1-COMT-MetK-Mtn-LuxS.

[0044] Figure 2 This is a diagram showing the results of screening the COMT mutant library.

[0045] Figure 3 This is a graph showing the results of assaying the activity of COMT single mutants in catalyzing N-acetyl-5-hydroxytryptamine.

[0046] Figure 4 This is a graph showing the results of assaying the activity of COMT multiple mutants in catalyzing N-acetyl-5-hydroxytryptamine. DETAILED DESCRIPTION

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0048] Example 1 Construction of wild-type methyltransferase engineering strain

[0049] (1) Using the pRSFDuet-1 vector as a template, PCR amplification was performed using primers pRSF-F and pRSF-R. The pRSFDuet-1 vector is a commercial vector purchased from Novagen. The sequence of the primer pRSF-F is shown in SEQ ID No.9, and the sequence of the primer pRSF-R is shown in SEQ ID No.10. After the PCR product was recovered, a linearized vector pRSFDuet-1 was obtained, and the size of the linearized vector fragment was 3764 bp;

[0050] (2) Using the artificially synthesized Arabidopsis thaliana methyltransferase COMT gene after codon optimization as a template, PCR amplification was performed using primers COMT-F / R, and the amplified product was recovered to obtain the COMT gene target fragment, with a fragment size of 1092 bp. The nucleotide sequence of the COMT gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The primer COMT-F sequence is shown in SEQ ID No.11, and the COMT-R sequence is shown in SEQ ID No.12;

[0051] (3) Using Escherichia coli genomic DNA as a template, PCR amplification was performed using primers MetK-F / R, Mtn-F / R, and LuxS-F / R, respectively. After the amplification products were recovered, the target fragments of the MetK, Mtn, and LuxS genes were obtained, and the fragment sizes were 1155 bp, 699 bp, and 516 bp, respectively. The nucleotide sequence of the MetK gene is shown in SEQ ID NO.3, the amino acid sequence is shown in SEQ ID NO.4, the primer MetK-F sequence is shown in SEQ ID No.13, and the MetK-R sequence is shown in SEQ ID No.14; the nucleotide sequence of the Mtn gene is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6, the primer Mtn-F sequence is shown in SEQ ID No.15, and the Mtn-R sequence is shown in SEQ ID No.16; the nucleotide sequence of the LuxS gene is shown in SEQ ID NO.7, the amino acid sequence is shown in SEQ ID NO.8, the primer LuxS-F sequence is shown in SEQ ID No.17, and the LuxS-R sequence is shown in SEQ ID No.18;

[0052] (4) The PCR reaction system is as follows: 1 μL template, 2 μL upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double distilled water.

[0053] (5) The PCR amplification program was as follows: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 50 s, 30 cycles, and 72°C extension for 7 min.

[0054] (6) Using the multi-fragment one-step cloning kit (Hieff Plus Multi One Step Cloning Kit) was used to connect the target fragments COMT, MetK, Mtn, and LuxS with the linearized vector pRSFDuet-1 to obtain a recombinant plasmid, which was named pRSFDuet1-COMT-MetK-Mtn-LuxS. The recombinant plasmid was successfully constructed and verified to be correct by sequencing. The plasmid map is shown in the figure. Figure 1 shown.

[0055] (7) The pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid was transformed into the Escherichia coli expression host BL21 (DE3) by electroporation and spread onto LB solid medium containing kanamycin. The LB plate was cultured at 37°C until transformants grew, and the positive transformants were picked to obtain the wild-type methyltransferase engineered bacteria (WT).

[0056] SEQ ID NO.1

[0057] ATGGGATCAACGGCTGAAACACAACTAACTCCGGTTCAGGTCACCGACGATGAGGCC

[0058] GCATTGTTTGCAATGCAGCTGGCGTCTGCGTCGGTGCTGCCGATGGCTTTAAAGAGCG

[0059] CGCTTGAACTGGACCTGTTGGAGATTATGGCAAAGAACGGCAGCCCGATGAGTCCGA

[0060] CCGAGATCGCGAGCAAGCTGCCAACGAAGAACCCGGAGGCGCCAGTGATGTTGGATC

[0061] GTATCTTGCGCCTGTTGACTTCATATAGCGTTCTGACCTGTAGCAACCGTAAACTGAG

[0062] CGGTGACGGCGTTGAACGTATTTACGGCCTGGGTCCGGTCTGCAAATACTTGACCAAG

[0063] AACGAAGACGGCGTGAGCATTGCGGCGCTTTGCCTGATGAACCAAGATAAAGTGTTA

[0064] ATGGAGAGCTGGTATCACCTGAAGGACGCGATCCTGGACGGCGGCATACCGTTCAAC

[0065] AAAGCGTACGGTATGTCTGCCTTCGAATATCACGGAACCGATCCGCGTTTTAATAAGG

[0066] TTTTCAACAACGGTATGTCCAATCATAGCACCATTACCATGAAAAAGATCCTCGAAAC

[0067] GTACAAAGGCTTTGAGGGTCTGACTTCGCTCGTGGACGTTGGTGGTGGGATCGGCGCA

[0068] ACCCTGAAGATGATCGTGTCCAAGTACCCGAATCTAAAGGGCATCAACTTTGATCTGC

[0069] CTCATGTGATCGAGGACGCTCCGAGCCATCCGGGTATCGAGCACGTGGGCGGCGACA

[0070] TGTTTGTTTCCGTTCCGAAAGGTGATGCAATTTTTATGAAATGGATCTGCCACGATTGG

[0071] TCAGATGAACACTGCGTTAAATTCCTGAAGAACTGCTATGAAAGCCTGCCCGAGGAC

[0072] GGTAAGGTGATTCTGGCTGAATGTATCTTGCCGGAAACCCCGGATAGCAGCCTGTCTA

[0073] CGAAGCAAGTTGTTCACGTTGACTGTATTATGCTGGCGCATAATCCGGGTGGCAAAGA

[0074] GCGCACCGAAAAAGAGTTCGAGGCTCTCGCGAAAGCCTCCGGTTTCAAAGGTATTAA

[0075] AGTCGTGTGCGATGCGTTCGGTGTAAATCTGATTGAACTGTTGAAGAAACTGTAASEQ ID NO.2

[0076] MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPMSPTEI

[0077] ASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNEDGVS

[0078] IAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMSAFEYHGTDPRFNKVFNNGMS

[0079] NHSTITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAPSHP

[0080] GIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECILPET

[0081] PDSSLSTKQVVHVDCIMLAHNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLK

[0082] KL

[0083] SEQ ID NO.3

[0084] ATGGCAAAACACCTTTTTACGTCCGAGTCCGTCTCTGAAGGGCATCCTGACAAAATTG

[0085] CTGACCAAATTTCTGATGCCGTTTTAGACGCGATCCTCGAACAGGATCCGAAAGCACG

[0086] CGTTGCTTGCGAAACCTACGTAAAAACCGGCATGGTTTTAGTTGGCGGCGAAATCACC

[0087] ACCAGCGCCTGGGTAGACATCGAAGAGATCACCCGTAACACCGTTCGCGAAATTGGC

[0088] TATGTGCATTCCGACATGGGCTTTGACGCTAACTCCTGTGCGGTTCTGAGCGCTATCG

[0089] GCAAACAGTCTCCTGACATCAACCAGGGCGTTGACCGTGCCGATCCGCTGGAACAGG

[0090] GCGCGGGTGACCAGGGTCTGATGTTTGGCTACGCAACTAATGAAACCGACGTGCTGAT

[0091] GCCAGCACCTATCACCTATGCACACCGTCTGGTACAGCGTCAGGCTGAAGTGCGTAAA

[0092] AACGGCACTCTGCCGTGGCTGCGCCCGGACGCGAAAAGCCAGGTGACTTTTCAGTATG

[0093] ACGACGGCAAAATCGTTGGTATCGATGCTGTCGTGCTTTCCACTCAGCACTCTGAAGA

[0094] GATCGACCAGAAATCGCTGCAAGAAGCGGTAATGGAAGAGATCATCAAGCCAATTCT

[0095] GCCCGCTGAATGGCTGACTTCTGCCACCAAATTCTTCATCAACCCGACCGGTCGTTTC

[0096] GTTATCGGTGGCCCAATGGGTGACTGCGGTCTGACTGGTCGTAAAATTATCGTTGATA

[0097] CCTACGGCGGCATGGCGCGTCACGGTGGCGGTGCATTCTCTGGTAAAGATCCATCAAA

[0098] AGTGGACCGTTCCGCAGCCTACGCAGCACGTTATGTCGCGAAAAACATCGTTGCTGCT

[0099] GGCCTGGCCGATCGTTGTGAAATTCAGGTTTCCTACGCAATCGGCGTGGCTGAACCGA

[0100] CCTCCATCATGGTAGAAACTTTCGGTACTGAGAAAGTGCCTTCTGAACAACTGACCCT

[0101] GCTGGTACGTGAGTTCTTCGACCTGCGCCCATACGGTCTGATTCAGATGCTGGATCTG

[0102] CTGCACCCGATCTACAAAGAAACCGCAGCATACGGTCACTTTGGTCGTGAACATTTCC

[0103] CGTGGGAAAAAACCGACAAAGCGCAGCTGCTGCGCGATGCTGCCGGTCTGAAGTAASEQ ID NO.4

[0104] MAKHLFTSESVSEGHPDKIADQISDAVLDAILEQDPKARVACETYVKTGMVLVGGEITTS

[0105] AWVDIEEITRNTVREIGYVHSDMGFDANSCAVLSAIGKQSPDINQGVDRADPLEQGAGDQ

[0106] GLMFGYATNETDVLMPAPITYAHRLVQRQAEVRKNGTLPWLRPDAKSQVTFQYDDGKIV

[0107] GIDAVVLSTQHSEEIDQKSLQEAVMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGL

[0108] TGRKIIVDTYGGMARHGGGAFSGKDPSKVDRSAAYAARYVAKNIVAAGLADRCEIQVSY

[0109] AIGVAEPTSIMVETFGTEKVPSEQLTLLVREFFDLRPYGLIQMLDLLHPIYKETAAYGHFGR

[0110] EHFPWEKTDKAQLLRDAAGLK

[0111] SEQ ID NO.5

[0112] ATGAAAATCGGCATCATTGGTGCAATGGAAGAAGAAGTTACGCTGCTGCGTGACAAA

[0113] ATCGAAAACCGTCAAACTATCAGTCTCGGCGGTTGCGAAATCTATACCGGCCAACTGA

[0114] ATGGAACCGAGGTTGCGCTTCTGAAATCGGGCATCGGTAAAGTCGCTGCGGCGCTGG

[0115] GTGCCACTTTGCTGTTGGAACACTGCAAGCCAGATGTGATTATTAACACCGGTTCTGC

[0116] CGGTGGCCTGGCACCAACGTTGAAAGTGGGCGATATCGTTGTCTCGGACGAAGCACG

[0117] TTATCACGACGCGGATGTCACGGCATTTGGTTATGAATACGGTCAGTTACCAGGCTGT

[0118] CCGGCAGGCTTTAAAGCTGACGATAAACTGATCGCTGCCGCTGAGGCCTGCATTGCCG

[0119] AACTGAATCTTAACGCTGTACGTGGCCTGATTGTTAGCGGCGACGCTTTCATCAACGG

[0120] TTCTGTTGGTCTGGCGAAAATCCGCCACAACTTCCCACAGGCCATTGCTGTAGAGATG

[0121] GAAGCGACGGCAATCGCCCATGTCTGCCACAATTTCAACGTCCCGTTTGTTGTCGTAC

[0122] GCGCCATCTCCGACGTGGCCGATCAACAGTCTCATCTTAGCTTCGATGAGTTCCTGGC

[0123] TGTTGCCGCTAAACAGTCCAGCCTGATGGTTGAGTCACTGGTGCAGAAACTTGCACAT

[0124] GGCTAA

[0125] SEQ ID NO.6

[0126] MKIGIIGAMEEEVTLLRDKIENRQTISLGGCEIYTGQLNGTEVALLKSGIGKVAAALGATLL

[0127] LEHCKPDVIINTGSAGGLAPTLKVGDIVVSDEARYHDADVTAFGYEYGQLPGCPAGFKAD

[0128] DKLIAAAEACIAELNLNAVRGLIVSGDAFINGSVGLAKIRHNFPQAIAVEMEATAIAHVCH

[0129] NFNVPFVVVRAISDVADQQSHLSFDEFLAVAAKQSSLMVESLVQKLAHGSEQ ID NO.7

[0130] ATGCCGTTGTTAGATAGCTTCACAGTCGATCATACCCGGATGGAAGCGCCTGCAGTTC

[0131] GGGTGGCGAAAACAATGAACACCCCGCATGGCGACGCAATCACCGTGTTCGATCTGC

[0132] GCTTCTGCGTGCCGAACAAAGAAGTGATGCCAGAAAGAGGGATCCATACCCTGGAGC

[0133] ACCTGTTTGCTGGTTTTATGCGTAACCATCTTAACGGTAATGGTGTAGAGATTATCGAT

[0134] ATCTCGCCAATGGGCTGCCGCACCGGTTTTTATATGAGTCTGATTGGTACGCCAGATG

[0135] AGCAGCGTGTTGCTGATGCCTGGAAAGCGGCAATGGAAGACGTGCTGAAAGTGCAGG

[0136] ATCAGAATCAGATCCCGGAACTGAACGTCTACCAGTGTGGCACTTACCAGATGCACTC

[0137] GTTGCAGGAAGCGCAGGATATTGCGCGTAGCATTCTGGAACGTGACGTACGCATCAA

[0138] CAGCAACGAAGAACTGGCACTGCCGAAAGAGAAGTTGCAGGAACTGCACATCTAGSEQ ID NO.8

[0139] MPLLDSFTVDHTRMEAPAVRVAKTMNTPHGDAITVFDLRFCVPNKEVMPERGIHTLEHLF

[0140] AGFMRNHLNGNGVEIIDISPMGCRTGFYMSLIGTPDEQRVADAWKAAMEDVLKVQDQN

[0141] QIPELNVYQCGTYQMHSLQEAQDIARSILERDVRINSNEELALPKEKLQELHI

[0142] Table 1. Primer sequences for COMT plasmid construction

[0143]

[0144] Example 2 Construction of a methyltransferase mutant library

[0145] Using the constructed pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid as a template, primers were designed for plasmid amplification and mutagenesis to obtain a linearized plasmid vector with base mutations, which was then transformed into Escherichia coli BL21 (DE3) and repaired and cyclized in vivo to obtain a plasmid with base mutations, specifically:

[0146] (1) Using the constructed pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid as a template, 10 pairs of primers were used for PCR amplification. The primer sequences are shown in Table 2. Ten mutant library sequences were obtained. The mutation methods were: point saturation mutation of methionine at position 128, leucine at position 134, phenylalanine at position 152, alanine at position 160, phenylalanine at position 170, valine at position 314, isoleucine at position 317, histidine at position 321, asparagine at position 322, and glycine at position 324 of COMT. The obtained mutant expression libraries were named COMT (M1) to COMT (M10).

[0147] Table 2. Primer sequences for mutant library construction

[0148]

[0149]

[0150] (2) The PCR reaction system is as follows: 1 μL template, 2 μL upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double distilled water;

[0151] (3) The PCR amplification program was as follows: 98°C pre-denaturation for 5 min, 98°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min 30 s, 30 cycles, and 72°C extension for 7 min;

[0152] (4) After the reaction is completed, purify it using a PCR product purification kit. Take 8.5 μL of the purified PCR product, add 0.5 μL of Dpn I restriction endonuclease, 1 μL of 10×CutSmart, and incubate at 37°C in a water bath for 4 hours;

[0153] (5) The 10 linearized plasmids treated with Dpn I were transformed into the Escherichia coli expression host BL21 (DE3) by electroporation and plated on LB solid medium containing kanamycin. The LB plates were cultured at 37°C until transformants grew, thereby obtaining the methyltransferase mutant libraries M1 to M10.

[0154] Example 3 High-throughput screening of mutant libraries

[0155] The reaction catalyzed by COMT requires SAM as a methylation donor (specifically, it can be generated by the reaction of methionine (Met) and ATP catalyzed by adenosylmethionine synthetase (Metk)) to catalyze the substrate for methylation reaction. After the reaction, SAM is converted into SAH, which is then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​(Hcys) with a thiol group (-SH). In the presence of thiol compounds, colorless DTNB will be converted into yellow 5-mercapto-2-nitrobenzoic acid. 5-Mercapto-2-nitrobenzoic acid has a maximum absorption at 412nm and can be used for colorimetric determination. Specifically, by measuring the absorbance value, methyltransferase mutants can be screened based on the absorbance value. The higher the absorbance value, the higher the activity of the corresponding methyltransferase mutant. The catalytic efficiency of COMT can be indirectly detected by DTNB reagent.

[0156] The specific method is:

[0157] (1) Using a sterile bamboo stick, pick up the monoclonal clone of Example 2 and place it in a 96-well plate. Add 600 μL of LB liquid culture medium containing kan antibiotics to each well and culture overnight at 600 rpm and 37° C. This type of 96-well plate is referred to as a “primary plate”.

[0158] (2) Add 200 μL LB liquid culture medium (containing antibiotics) to a new 96-well plate, take 20 μL of bacterial solution from the primary plate and add it to the corresponding wells. This type of 96-well plate is recorded as the "secondary plate" and cultured at 600 rpm and 37°C for about 3-4 h until the OD 600 0.8-1.0;

[0159] (3) Add 200 μL of LB liquid medium (containing antibiotics) mixed with IPTG inducer to each well of the secondary plate to a final IPTG concentration of 0.5 mM, and induce at 600 rpm and 25°C for 16 h;

[0160] (4) After induction, add 200 μL of reaction solution (containing 2 g / L methionine, 2 g / L N-acetyl-5-hydroxytryptamine, and 5 g / L glucose) to each well and react at 37°C and 600 rpm for 3 h;

[0161] (5) After the reaction, add 30 μL of lysozyme solution to each well and react at 37°C and 600 rpm for 1 h;

[0162] After centrifugation, take 10 μL of the supernatant after the reaction and place it in the ELISA plate. Add 200 μL of DTNB analysis solution (concentration 0.1 mM). After standing for 10 minutes, place the plate in an ELISA reader and measure the absorbance value (A) at 412 nm. Figure 2 As shown, according to the measurement results, monoclones with high absorbance values ​​(absorbance values ​​in the top 10%) were selected for sequencing. The following mutants were obtained: M128L, L134F, F152W, A160S, A160V, F170W, I317A, T314S, H321L, N322L, N322F, G324D. The activities of the mutants initially screened based on the above high-throughput screening method were improved compared to the wild type.

[0163] Example 4 Activity determination of methyltransferase mutants

[0164] (1) The COMT mutant strain screened in Example 3 was cultured overnight in a seed culture medium containing 50 μg / mL kanamycin to obtain a seed solution. The seed culture medium (by mass percentage) was 1% tryptone, 1% sodium chloride and 0.5% yeast extract. The culture conditions of the seed solution were 37°C and 220 rpm.

[0165] (2) The above seed solution was inoculated at 1% into a protein expression medium containing 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 0.231% KH2PO4 and 1.254% K2HPO4 (mass percentage) at 37°C and 220 rpm.

[0166] (3) After culturing for 3 h, IPTG was added at a final concentration of 0.5 mM to induce expression. The induction expression conditions were 25°C, 220 rpm, and the induction time was 16 h.

[0167] (4) After protein induction, the cells were collected by centrifugation at 4000 rpm for 20 min at 4°C and resuspended in 0.9% saline until the OD of the resuspended liquid was 600 10. Take 5 mL of the cell resuspension, centrifuge again, discard the supernatant, and use liquid nitrogen to quickly freeze the bacterial pellet three times to fully disrupt the cells;

[0168] (5) Use 5 mL of transformation solution to suspend the cells and perform whole-cell catalysis. The transformation solution contains 23 mM N-acetyl-5-hydroxytryptamine and 46 mM SAM in 0.2 M phosphate buffer at pH 7.8. The whole-cell catalytic reaction conditions are 37°C, 150 rpm, and the reaction time is 6 h.

[0169] (6) High performance liquid chromatography detection method: the chromatographic column is C18 (250 mm*4.6 mm, 5 μm) or an equivalent chromatographic column, the mobile phase is 40 wt% methanol and 60 wt% pure water, the flow rate is 1 mL / min, the injection volume is 10 μL, the column oven temperature is 35°C, N-acetyl-5-methoxytryptamine is detected at a wavelength of 275 nm, and the content is determined by the external standard method.

[0170] The results are as follows Figure 3 As shown, compared with the wild-type COMT strain, the activities of the mutants (M128L, L134F, F152W, A160S, A160V, F170W, I317A, T314S, H321L, N322L, N322F, G324D) screened based on the above high-throughput screening method have been improved compared with the wild-type. Among them: the activities of mutants M128L, A160S, A160V, H321L, N322L, N322F, and G324D are significantly improved. The production of N-acetyl-5-methoxytryptamine in mutant N322F reaches 9.4mM, which is 2.3 times that of the wild-type.

[0171] Example 5 Construction and activity determination of multiple mutant methyltransferase engineering bacteria

[0172] Based on the above-mentioned single mutant methyltransferase engineering bacteria, multiple mutant methyltransferase engineering bacteria were further constructed. Multiple mutant methyltransferase engineering bacteria were constructed by combining the 6 most active single point mutations (A160S, A160V, H321L, N322L, N322F, G324D). The specific results are shown below, and the construction method is the same as Example 2.

[0173] Based on the single mutant plasmid COMT (H321L), primers N322L-F and N322L-R were used to construct the double mutant plasmid COMT (H321L / N322L); primers N322F-F and N322F-R were used to construct the double mutant plasmid COMT (H321L / N322F). Based on the double mutant plasmid COMT (H321L / N322F), primers A160S-F and A160S-R were used to construct the triple mutant plasmid COMT (A160S / H321L / N322F); primers A160V-F and A160V-R were used to construct the triple mutant plasmid COMT (A160V / H321L / N322F). Based on the above two triple mutant plasmids, primers G324D-F and G324D-R were used to construct quadruple mutant plasmids COMT (A160S / H321L / N322F / G324D) and

[0174] COMT (A160V / H321L / N322F / G324D). The sequences of primers N322L-F, N322L-R, N322F-F, N322F-R, A160S-F, A160S-R, A160V-F, A160V-R, G324D-F and G324D-R are shown in Table 3.

[0175] Table 3. Primer sequences for multiple mutant construction

[0176]

[0177] The catalytic activity of each strain on the substrate N-acetyl-5-hydroxytryptamine was determined using the multiple mutant engineered bacteria constructed above, and the specific method was the same as in Example 4. The results are shown in FIG. Figure 4 As shown, compared with the wild-type COMT strain, the catalytic activity of the multiple mutant strains was further improved, among which the quadruple mutant strain A160V / H321L / N322F / G324D catalyzed the production of N-acetyl-5-methoxytryptamine up to 22.7 mM, which was 5.5 times that of the wild type, and the substrate conversion rate was 98.8%, and the substrate conversion rate = (1-(remaining substrate / input substrate))*100%.

[0178] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A high-throughput screening method for methyltransferase mutants, characterized in that: The following steps are involved: (a) activating, culturing, and inducing expression of a single clone in a methyltransferase mutant library; (b) adding a substrate to the system of step (a), and using SAM as a methylation donor to catalyze the substrate to perform a methyltransferase-catalyzed reaction; (c) disrupting the cells and adding a thiol detection reagent, the thiol detection reagent reacts with the thiol compound produced in the methylation reaction catalyzed by the methylation donor SAM in the system to generate a colored product, measuring the absorbance value, and screening the methyltransferase mutant based on the absorbance value. A high absorbance value indicates that the corresponding methyltransferase mutant has a high activity.

2. The high-throughput screening method according to claim 1, characterized in that: In step (a), a linearized vector and a methyltransferase gene fragment are obtained by amplification using a vector for constructing a plasmid and a methyltransferase gene as a template, the methyltransferase gene fragment is connected to the linearized vector to obtain a recombinant plasmid, the recombinant plasmid is used as a template, primers are designed to carry out plasmid amplification and mutagenesis, a linearized plasmid vector with base mutations is obtained, and the linearized plasmid vector is transformed into Escherichia coli to construct a methyltransferase mutant library; The thiol detection reagent described in step (c) is a DTNB reagent, and the absorbance value is measured by utilizing the characteristic color development reaction of the thiol compound produced in the methylation reaction catalyzed by DTNB and the methylation donor SAM in the system.

3. The high-throughput screening method according to claim 1, characterized in that: The methyltransferase catalyzed reaction uses SAM as a methylation donor. After the reaction, SAM is converted into SAH, which is then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​with a thiol group. Based on the characteristic color development reaction between the thiol detection reagent and the thiol compound homocysteine ​​with a thiol group, the absorbance value of the system is determined, and the methyltransferase mutants are screened based on the absorbance value. The single clones with absorbance values ​​in the top 8-12% were selected for sequencing to screen and obtain methyltransferase mutants.

4. The high-throughput screening method according to claim 3, characterized in that: The recombinant plasmid also contains MetK, Mtn, and LuxS gene fragments. The adenosylmethionine synthetase gene catalyzes the reaction of methionine and ATP to generate SAM, which is used as a methylation donor. After the methylation reaction of the catalyzed substrate is completed, SAM is converted into SAH, which is then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​with a thiol group. The methyltransferase mutants obtained by the above screening and sequencing are used to test the activity of catalyzing the methylation reaction of the substrate methyltransferase, and the methyltransferase mutants with high activity are further screened according to the reaction activity.

5. The high-throughput screening method according to claim 1, characterized in that: The multi-well plate used for activation and culture in step (a) is a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate; The activation and culture conditions are: temperature 25-37°C, shaking speed 300-1000rpm, and culture time 6-20h; The induced expression is to add IPTG to induce protein expression, the inducing agent concentration is 0.05-2mM, and the induction temperature is 18-30°C; The concentration of the thiol detection reagent is 0.01-5 mM. The crushing method comprises lysozyme reaction, liquid nitrogen quick freezing, acid-base treatment, surfactant treatment, ultrasonic treatment and quartz sand vibration grinding.

6. Application of the high-throughput screening method for methyltransferase mutants according to claim 1 in screening methyltransferase mutants that synthesize N-acetyl-5-methoxytryptamine by methyltransferase-catalyzed reaction using N-acetyl-5-hydroxytryptamine as substrate.

7. A methyltransferase mutant, characterized in that: The amino acid sequence shown in any of the following: (a) the amino acid sequence is obtained by mutation of one or more amino acid residue positions selected from the group consisting of: 128, 160, 321, 322 and 324 of the sequence shown in SEQ ID NO: 2, i.e., the above positions can be selected from any one of the amino acids other than the original amino acids; or (b) the methyltransferase has a similarity of more than 90%, preferably more than 95%, more preferably more than 99% with the amino acid sequence of (a), and has the function of the protein of (a), wherein the amino acids at positions 128, 160, 321, 322 and 324 corresponding to the amino acid sequence of SEQ ID NO: 2 are mutated in the same manner as the amino acid sequence of (a); Or (c) the methyltransferase is formed by adding multiple amino acids or deleting 1-20, preferably 1-10, more preferably 1-5 amino acids at the C-terminus and / or N-terminus of the amino acid sequence described in (a), and has the function of the protein described in (a), wherein the amino acids at positions 128, 160, 321, 322 and 324 corresponding to the amino acid sequence shown in SEQ ID NO: 2 are mutated in the same manner as the amino acid sequence described in (a).

8. The methyltransferase mutant according to claim 7, characterized in that: The mutation at position 128 is a mutation to leucine, the mutation at position 160 is a mutation to serine or valine, the mutation at position 321 is a mutation to leucine, the mutation at position 322 is a mutation to leucine or phenylalanine, and the mutation at position 324 is a mutation to aspartic acid.

9. A gene encoding the methyltransferase mutant according to claim 7. 10 . An expression vector, comprising the gene encoding the methyltransferase mutant according to claim 9 , and capable of expressing the methyltransferase mutant.

11. A recombinant genetically engineered bacterium, comprising the expression vector according to claim 10 or a gene encoding the methyltransferase mutant according to claim 9 integrated into its genome.

12. Use of the methyltransferase mutant according to claim 7, the expression vector according to claim 10, or the recombinant genetically engineered bacteria according to claim 11 in producing N-acetyl-5-methoxytryptamine, inducing protein expression in the genetically engineered bacteria, then collecting the cells by centrifugation, and then adding them to a conversion solution containing a substrate N-acetyl-5-hydroxytryptamine to react, and finally separating N-acetyl-5-methoxytryptamine from the conversion solution.

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