A high-throughput screening method for methyltransferase mutants, methyltransferase mutants and their applications

Abstract

This invention belongs to the field of biotechnology, specifically disclosing a high-throughput screening method for methyltransferase mutants, methyltransferase mutants, and their applications. This invention enables high-throughput screening of methyltransferase mutants, improving the efficiency of methyltransferase modification and obtaining highly active methyltransferase mutants. The methyltransferase mutants provided by this invention significantly enhance methyltransferase activity, increasing the yield of N-acetyl-5-methoxytryptamine. Using engineered bacteria expressing methyltransferase mutants can significantly enhance methyltransferase activity, increasing the yield of N-acetyl-5-methoxytryptamine to as high as 22.7 mM.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a high-throughput screening method for methyltransferase mutants, methyltransferase mutants and their applications. Background Technology

[0002] Methylation is the process of transferring a methyl group (-CH3) from one compound to another, often resulting in significant alterations to the bioactivity and bioavailability of the substance. Currently, chemical-based methylation methods use toxic reagents, generate toxic waste, and lack regioselectivity. In contrast, enzymatic methylation exhibits high specificity, is environmentally friendly, and is safer to use. Methyltransferases, also known as methyltransferases, are a large family with a wide range of biological functions, typically using SAM (S-adenosylmethionine) as a methylation donor. They play a crucial role in the methylation of lipids, proteins, polysaccharides, and nucleotides, leading to various important biological functions such as epigenetic phenomena and the generation of numerous secondary metabolites. Among these, oxygen-methyltransferases are used in the biosynthesis of natural products such as ferulic acid, safflower extract, rhamnine, melatonin, vanillin, and pterostilbene, demonstrating significant bioeconomic value.

[0003] Modifying methyltransferases to enhance their activity and selectivity, thereby improving the production efficiency of target substances, is a common strategy. However, there is currently no universal and convenient analytical method for rapidly detecting methyltransferase activity for the evaluation and screening of methyltransferase modifications. Most methyltransferase activity assays are based on radiolabeling or fluorescence detection, which are time-consuming, labor-intensive, and require sophisticated equipment. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a high-throughput screening method for methyltransferase mutants, methyltransferase mutants, and their applications, addressing the shortcomings of existing technologies. This invention can be used for high-throughput screening of methyltransferase mutants, improving the efficiency of methyltransferase modification and obtaining highly active methyltransferase mutants. The methyltransferase mutants provided by this invention can significantly improve methyltransferase activity, thereby increasing the yield of N-acetyl-5-methoxytryptamine.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

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

[0007] (a) Activate and culture single clones from the methyltransferase mutant library to induce expression;

[0008] (b) Add the substrate to the system of step (a) and use SAM as a methylation donor to catalyze the methyltransferase-catalyzed reaction of the substrate;

[0009] (c) The cells were broken up and a thiol detection reagent was added. The thiol detection reagent reacted with the thiol compound produced in the methylation reaction catalyzed by the methylation donor SAM in the system to generate a colored product. The absorbance value was measured, and methyltransferase mutants were screened based on the absorbance value. The higher the absorbance value, the higher the activity of the corresponding methyltransferase mutant.

[0010] According to the above scheme, the methyltransferase catalytic reaction uses SAM as the methylation donor. After the reaction, SAM is converted into SAH, and then formed homocysteine ​​(Hcys) with a thiol group (-SH) through two consecutive Mtn-LuxS catalysis steps. Based on the characteristic colorimetric reaction between the thiol detection reagent and the thiol compound and the homocysteine ​​(Hcys) with a thiol group (-SH), the absorbance value of the system is measured, and 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 determined by utilizing the characteristic colorimetric reaction of DTNB with the thiol compound generated in the methylation reaction catalyzed by the methylation donor SAM in the system.

[0012] According to the above scheme, using the vector for constructing the plasmid and the methyltransferase gene as templates, a linearized vector and methyltransferase gene fragments are amplified. The methyltransferase gene fragments are then ligated to the linearized vector to obtain a recombinant plasmid. Using the recombinant plasmid as a template, primers are designed to amplify and induced the plasmid to obtain a linearized plasmid vector with base mutations. This vector is then transformed into E. coli to construct a methyltransferase mutant library.

[0013] According to the above scheme, the recombinant plasmid also contains MetK, Mtn, and LuxS gene fragments. The adenosylmethionine synthase (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 catalytic substrate is completed, SAM is converted into SAH, and then catalyzed by Mtn-LuxS in two consecutive steps to form homocysteine ​​(Hcys) with a thiol group (-SH).

[0014] Following the above scheme, single clones with absorbance values ​​in the top 8-12% were selected for sequencing to screen for methyltransferase mutants. Prioritizing sequencing of single clones with absorbance values ​​in the top 8-12% allows for rapid screening and ensures better screening results. Furthermore, the activity of the methyltransferase mutants obtained through the above screening and sequencing in catalyzing the methylation reaction of the substrate methyltransferase was determined. Based on the reactivity, methyltransferase mutants with high activity were further screened.

[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℃, shaking speed 300-1000rpm, and culture time 6-20h.

[0017] According to the above scheme, in step (a), the induction of expression is achieved by adding IPTG to induce protein expression, the concentration of the inducer is 0.05-2 mM, and the induction temperature is 18-30℃.

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

[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 synthesize N-acetyl-5-methoxytryptamine through a methyltransferase-catalyzed reaction.

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

[0022] Secondly, taking the substrate N-acetyl-5-hydroxytryptamine as an example, this invention provides a high-throughput screening method for methyltransferase mutants in the screening of methyltransferase mutants synthesized from N-acetyl-5-methoxytryptamine using methyltransferase catalysis.

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

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

[0025] Or (b) the methyltransferase has a sequence similarity of more than 90%, preferably more than 95%, more preferably more than 99%, to the amino acid sequence of (a), and has the function of the protein of (a), wherein the amino acids corresponding to the amino acid sequence shown in SEQ ID NO:2 at positions 128, 160, 321, 322 and 324 have the same mutation mode as the amino acid sequence of (a).

[0026] Or (c) the methyltransferase is formed by adding or deleting 1-20 amino acids, 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 corresponding to positions 128, 160, 321, 322, and 324 of 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 amino acids, preferably 1-10, more preferably 1-5, or it can be a protein tag sequence such as His, Flag, MBP, SUMO, GST, or Trx.

[0027] Preferably, 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.

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

[0029] Fifthly, the present invention provides an expression vector containing a gene encoding the above-mentioned methyltransferase mutant, which 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 contains the above-mentioned expression vector or its genome integrated with a gene encoding a methyltransferase mutant.

[0031] The recombinant genetically engineered bacteria can be obtained by transferring the above expression vector into host cells or by integrating genes encoding methyltransferase mutants into the genome.

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

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

[0034] According to the above scheme, the conversion 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 a shaking table oscillation with a rotation speed of 100-300 rpm.

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

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

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

[0040] The methyltransferase mutant screening method provided by this invention can be used for high-throughput screening of methyltransferase mutants, improving the efficiency of methyltransferase modification and obtaining highly active methyltransferase mutants. Specifically, in the high-throughput screening of this invention, SAM is used as a methylation donor, combined with the overexpression of Mtn and LuxS genes. Through two consecutive catalytic steps of Mtn-LuxS, the SAH obtained by the methylation donor SAM during the methylation process generates homocysteine ​​(Hcys) with a thiol group (-SH). Then, based on the characteristic colorimetric reaction between the thiol detection reagent and the thiol compound homocysteine ​​(Hcys) with a thiol group (-SH), it is possible to achieve high-throughput screening of methyltransferase mutants by measuring the absorbance value of the system.

[0041] This 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. This method improves the screening efficiency of methyltransferase modification and lays the foundation for industrial application.

[0042] This invention obtained a highly active methyltransferase mutant by saturating the original methyltransferase sequence shown in SEQ ID NO:2 and combining it with high-throughput screening technology. This mutant, used to generate N-acetyl-5-methoxytryptamine, significantly improves catalytic efficiency, reduces the amount of microbial cells or enzymes required in the catalytic process, and lowers production costs. Its maximum catalytic yield of N-acetyl-5-methoxytryptamine is 22.7 mM, with catalytic activity 5.5 times that of the wild type and a substrate conversion rate as high as 98.8%. Attached Figure Description

[0043] Figure 1 The image shows the pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid.

[0044] Figure 2 The image shows the screening results of the COMT mutant library.

[0045] Figure 3 The graph shows the results of the COMT single mutant catalytic activity assay for N-acetyl-5-hydroxytryptamine.

[0046] Figure 4 The figure shows the results of the COMT multiple mutant catalytic activity assay for N-acetyl-5-hydroxytryptamine. Detailed Implementation

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] Example 1: Construction of wild-type methyltransferase engineered 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 was a commercially available vector purchased from Novagen. The sequence of primer pRSF-F is shown in SEQ ID No. 9, and the sequence of primer pRSF-R is shown in SEQ ID No. 10. After recovery of the PCR product, the linearized vector pRSFDuet-1 was obtained, and the linearized vector fragment size was 3764 bp.

[0050] (2) Using the Arabidopsis-derived methyltransferase COMT gene, synthesized with optimized codons, as a template, PCR amplification was performed using primers COMT-F / R. The amplified product was recovered to obtain the target fragment of the COMT gene, with a fragment size of 1092 bp. The nucleotide sequence of the COMT gene is shown in SEQ ID NO.1, 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. The amplification products were recovered to obtain the target fragments of the MetK, Mtn, and LuxS genes, with fragment sizes of 1155bp, 699bp, and 516bp, respectively. The nucleotide sequence of the MetK gene is shown in SEQ ID NO.3, and 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, and 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, and 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 above PCR reaction system consists of 1 μL template, 2 μL each of upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double-distilled water.

[0053] (5) The above PCR amplification program is: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, 30 cycles, 72℃ extension for 7 min.

[0054] (6) Using the multi-fragment one-step cloning kit (Hieff) The Plus Multi One Step Cloning Kit was used to ligate the target fragments COMT, MetK, Mtn, and LuxS with the linearized vector pRSFDuet-1 to obtain a recombinant plasmid, named pRSFDuet1-COMT-MetK-Mtn-LuxS. Sequencing confirmed its correct construction, and the recombinant plasmid was successfully constructed. The plasmid map is shown below. Figure 1 As shown.

[0055] (7) The pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid was transformed into the Escherichia coli expression host strain BL21(DE3) by electroporation and plated onto LB solid medium containing kanamycin. The LB plates were cultured at 37°C until transformants grew. Positive transformants were picked to obtain 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 to amplify and induced mutagenesis of the plasmid, resulting in a linearized plasmid vector with base mutations. This vector was then transformed into E. coli BL21(DE3), and after in vivo repair and circularization, the plasmid with base mutations was obtained.

[0146] (1) Using the constructed pRSFDuet1-COMT-MetK-Mtn-LuxS plasmid as a template, PCR amplification was performed using 10 pairs of primers. The primer sequences are shown in Table 2. Ten mutant library sequences were obtained, with the following mutation methods: point saturation mutations were performed on 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 resulting mutant expression libraries were named COMT(M1)~COMT(M10).

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

[0148]

[0149]

[0150] (2) The above PCR reaction system consists of 1 μL template, 2 μL each of upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double-distilled water.

[0151] (3) The above PCR amplification program is: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min 30 s, 30 cycles, 72℃ extension for 7 min.

[0152] (4) After the reaction, purify the PCR product using a PCR product purification kit. Take 8.5 μL of the purified PCR product, add 0.5 μL of Dpn I restriction endonuclease and 1 μL of 10×CutSmart, and incubate at 37℃ for 4 h.

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

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

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

[0156] The specific method is as follows:

[0157] (1) Use a sterile bamboo stick to pick up the monoclonal antibody from Example 2 into a 96-well plate, each well containing 600 μL of LB liquid medium with kan antibiotic, and incubate overnight at 37°C and 600 rpm. This type of 96-well plate is called "Level 1 plate".

[0158] (2) Add 200 μL of LB liquid medium (containing antibiotics) to a new 96-well plate. Take 20 μL of bacterial culture from the primary plate and add it to the corresponding well. This type of 96-well plate is called a "secondary plate". Incubate at 600 rpm and 37°C for about 3-4 hours until the OD reaches the target. 600 It is 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 until the final IPTG concentration is 0.5 mM, and induce for 16 h at 600 rpm and 25 ℃.

[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 is complete, add 30 μL of lysozyme solution to each well and react at 37°C and 600 rpm for 1 h.

[0162] After centrifugation, 10 μL of the supernatant was transferred to an ELISA plate, and 200 μL of 0.1 mM DTNB analytical solution was added. After standing for 10 min, the absorbance (A) was measured at 412 nm using an ELISA reader. Figure 2 As shown, based on the measurement results, single clones with high absorbance values ​​(top 10%) were selected for sequencing. The following mutants were obtained: M128L, L134F, F152W, A160S, A160V, F170W, I317A, T314S, H321L, N322L, N322F, and G324D. The activities of the mutants initially screened using the above high-throughput screening method were all enhanced compared to the wild type.

[0163] Example 4: Activity assay of methyltransferase mutant

[0164] (1) The COMT mutant strain obtained 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 (mass percentage) consisted of 1% tryptone, 1% sodium chloride and 0.5% yeast extract. The culture conditions for the seed solution were 37°C and 220 rpm.

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

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

[0167] (4) After protein induction expression is completed, the bacterial cells are collected by centrifugation at 4000 rpm for 20 min at 4℃, and resuspended in 0.9% physiological saline until the OD of the resuspended solution is reached. 600 10. Take 5 mL of cell resuspended solution, centrifuge again, discard the supernatant, and then flash-freeze the bacterial cell pellet three times with liquid nitrogen to fully disrupt the cells;

[0168] (5) The bacterial cells were suspended in 5 mL of transformation buffer for whole-cell catalysis. The transformation buffer consisted of 23 mM N-acetyl-5-hydroxytryptamine and 46 mM SAM, prepared with 0.2 M, pH 7.8 phosphate buffer. The whole-cell catalysis reaction conditions were 37 °C, 150 rpm, and 6 h.

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

[0170] The results are as follows Figure 3 As shown, compared with the wild-type COMT strain, the mutants (M128L, L134F, F152W, A160S, A160V, F170W, I317A, T314S, H321L, N322L, N322F, G324D) screened using the above high-throughput screening method all showed enhanced activity. Specifically, mutants M128L, A160S, A160V, H321L, N322L, N322F, and G324D exhibited significantly enhanced activity. The mutant N322F produced 9.4 mM of N-acetyl-5-methoxytryptamine, which is 2.3 times that of the wild-type.

[0171] Example 5: Construction and Activity Assay of Engineered Bacteria with Multiple Mutant Methyltransferases

[0172] Based on the above-mentioned single-mutant methyltransferase engineered bacteria, a multiple mutant methyltransferase engineered bacteria was further constructed. The multiple mutant methyltransferase engineered bacteria constructed using a combination of the six single-point mutations with the highest activity (A160S, A160V, H321L, N322L, N322F, G324D) are shown below in detail. The construction method is the same as in Example 2.

[0173] Based on the single mutant plasmid COMT(H321L), the double mutant plasmid COMT(H321L / N322L) was constructed using primers N322L-F and N322L-R; the double mutant plasmid COMT(H321L / N322F) was constructed using primers N322F-F and N322F-R. Based on the double mutant plasmid COMT(H321L / N322F), the triple mutant plasmid COMT(A160S / H321L / N322F) was constructed using primers A160S-F and A160S-R; the triple mutant plasmid COMT(A160V / H321L / N322F) was constructed using primers A160V-F and A160V-R. Based on the two types of triple mutant plasmids mentioned above, quadruple mutant plasmids COMT(A160S / H321L / N322F / G324D) were constructed using primers G324D-F and G324D-R.

[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 constructing multiple mutants

[0176]

[0177] Using the multiple mutant engineered bacteria constructed above, the catalytic activity of each strain for the substrate N-acetyl-5-hydroxytryptamine was determined, using the same method as in Example 4. The results are as follows: Figure 4 As shown, compared with the wild-type COMT strain, the multiple mutant strains showed further enhanced catalytic activity. Among them, the four mutant strains A160V / H321L / N322F / G324D produced N-acetyl-5-methoxytryptamine at a rate of up to 22.7 mM, which is 5.5 times that of the wild type. The substrate conversion rate was 98.8%, and the substrate conversion rate was calculated as (1 - (remaining substrate / input substrate)) * 100%.

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

Claims

1. A methyltransferase mutant, characterized in that: The amino acid sequence is shown in any of the following: (a) Its amino acid sequence is obtained by mutating the sequence shown in SEQ ID NO:2 at positions 160, 321, 322 and 324, wherein position 160 is mutated to valine, position 321 to leucine, position 322 to phenylalanine and position 324 to aspartic acid.

2. The gene encoding the methyltransferase mutant of claim 1.

3. An expression vector, characterized in that: The expression vector contains the gene encoding the methyltransferase mutant as described in claim 2, and is capable of expressing the methyltransferase mutant.

4. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria contain the expression vector of claim 3 or the genome of which the gene encoding the methyltransferase mutant of claim 2 is integrated.

5. The application of the methyltransferase mutant of claim 1, the expression vector of claim 3, or the recombinant genetically engineered bacteria of claim 4 in the production of N-acetyl-5-methoxytryptamine, characterized in that; The recombinant genetically engineered bacteria were induced to express protein, and then the cells were collected by centrifugation. The cells were then added to a transformation solution containing the substrate N-acetyl-5-hydroxytryptamine and reacted. Finally, N-acetyl-5-methoxytryptamine was separated from the transformation solution.

Images