CN111647575B - SAM-dependent methyltransferase DmtMT1 and application - Google Patents
SAM-dependent methyltransferase DmtMT1 and application Download PDFInfo
- Publication number
- CN111647575B CN111647575B CN202010571360.2A CN202010571360A CN111647575B CN 111647575 B CN111647575 B CN 111647575B CN 202010571360 A CN202010571360 A CN 202010571360A CN 111647575 B CN111647575 B CN 111647575B
- Authority
- CN
- China
- Prior art keywords
- methyltransferase
- dmts
- dmtmt1
- methylation
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
In order to solve the technical blank that the DMTs adopts enzyme reaction for methylation modification in the prior art, the invention provides methyltransferase (derived from marine streptomyces)Streptomyces youssoufiensisOUC6819), designated SAM-dependent methyltransferase DmtMT 1. Also provides the amino acid sequence and the nucleotide sequence of the methyltransferase; and provides a cloning and expression method of the methyltransferase. The methyltransferase DmtMT1 can take a plurality of cyclic dipeptides as substrates to generate methylated cyclic dipeptides, participate in methylation of pre-DMTs, and sweep key obstacles for modifying DMTs by enzymatic methylation. In addition, methylation of DMTs at position N15 has a significant effect on the biological activity of DMTs; therefore, methylation modification of DMTs is realized through an enzyme method, a new path is provided for drug development of diketopiperazine compounds, and the method has important application value and social significance.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and biological pharmacy, and particularly relates to methyltransferase and application thereof in methylation modification of DMTs.
Background
SAM-dependent methyltransferases are enzymes that catalyze the transmethylation reaction with SAM (S-adenosyl-L-methionine) as a direct donor for methyl groups. Studies have shown that methylation by SAM-dependent methyltransferases catalysis can greatly improve the properties of natural products. Therefore, SAM-dependent methyltransferases play an important role in natural product biosynthesis. The invention patent application 201310552458.3 discloses the application of "L11 MT protein as methyltransferase". The methyltransferase described in this application is capable of methylation modification of at least one of the following substrate proteins: sul7d protein, Cren7 protein, RPL11 protein, Rrp4 protein, Csl4 protein and Rrp42 protein. The methyltransferase in this application effects the transfer of a methyl group from S-adenosylmethionine to a substrate protein. It can be seen that the substrate targeted by the methyltransferase in this application is a macromolecular protein.
Drimentines (DMTs) are a class of prenylated diketopiperazines, and show significant antibacterial, antitumor, insecticidal and parasiticidal activities. First isolated from actinomycetes MST-8651 by Lacey Ernest et al, and subsequently submitted to a patent application for DMT A-E by Novartis Animal Health Australia and Microbial Screening Technologies (WO 98/09968). In 2012, India madder et al isolated and identified Streptomyces CHQ64(Genbank No: JQ405211) from the rhizosphere soil of Phragmites communis in the protected area of mangrove forest in Guangdong, and isolated the fermentation products to identify Indotertine A, B and DMT F, G, H and C, and filed a patent application (CN 102276613A). At present, the biosynthesis of the DMTs backbone has been elucidated. Taking DMT G as an example: first, cyclic dipeptide synthase synthesizes cyclo (L-Trp-L-Val) (cWV); then, isopentenyl transferase loads the farnesyl group to cWV to obtain pre-DMT G (the structure is shown as a formula (2)); finally, under the action of terpene cyclase, DMT G is obtained.
In addition, according to literature reports, DMTs are obtained by direct fermentation and chemical total synthesis of strains in the prior art, but the preparation of DMTs by an enzymatic reaction is not seen. Among them, the methylation modification at position N15 in DMTs is common, and DMT F is exemplified by the structure shown in formula (1), but the methylation mechanism has not been elucidated. Because methylation at position N15 has an adverse effect on the biological activity of the presently disclosed DMTs; and the content of the compounds in the fermentation liquor is low, the yield is limited, and the industrial application prospect of the compounds is greatly restricted. Therefore, methylation modification of DMTs is clarified by an enzyme method, a new path is provided for drug development of diketopiperazine compounds, and the method has important application value and social significance.
Disclosure of Invention
In order to solve the technical blank that DMTs adopt enzyme reaction for methylation modification in the prior art, the invention provides methyltransferase (derived from Streptomyces youssuoufiensis OUC6819) which is named SAM dependent methyltransferase DmtMT 1. The methyltransferase DmtMT1 can take a plurality of cyclic dipeptides as substrates to generate methylated cyclic dipeptides, participate in methylation of pre-DMTs, and sweep out key obstacles for enzymatic methylation modification of DMTs.
The technical scheme of the invention is as follows: SAM-dependent methyltransferase DmtMT1, which catalyzes the transfer of a methyl group from S-adenosylmethionine to a cyclic dipeptide substrate; the amino acid sequence of the methyltransferase DmtMT1 is selected from the following (1), (2) or (3):
(1) 1, as shown in SEQ ID NO;
(2) 1, and has an amino acid sequence which has the activity of catalyzing a methyl donor to be combined with the cyclodipeptide alpha-N through substituting, deleting or adding one or more amino acids;
(3) the homology of the protein and the amino acid sequence shown in SEQ ID NO. 1 is more than or equal to 90 percent, and the expressed protein has the amino acid sequence which catalyzes the methyl donor to be combined with the activity of the cyclic dipeptide alpha-N.
The nucleotide sequence for coding the SAM dependent methyltransferase DmtMT1 is selected from the following (1), (2), (3) or (4):
(1) a nucleotide sequence shown as SEQ ID NO. 2;
(2) a nucleotide sequence which is different from the nucleotide sequence shown in SEQ ID NO. 2 but encodes the amino acid sequence shown in SEQ ID NO. 1;
(3) the homology of the protein and the nucleotide sequence shown in SEQ ID NO. 2 is more than or equal to 85 percent, and the expressed protein has the nucleotide sequence which catalyzes the methyl donor to be combined with the activity of the cyclodipeptide alpha-N;
(4) a nucleotide sequence complementary to the nucleotide sequence of any one of (1), (2) or (3).
An expression vector comprising a nucleotide sequence encoding said SAM-dependent methyltransferase DmtMT 1. The expression vector is a vector suitable for expression in Escherichia coli.
The expression vector is applied to expressing the SAM dependent methyltransferase DmtMT 1.
The clone expression method of the methyltransferase DmtMT1 comprises the following steps: cloning a nucleotide sequence coding the methyltransferase DmtMT1 into an expression vector to construct an expression vector; then transferring the expression vector into an expression system for protein expression; finally, the methyltransferase DmtMT1 is obtained after purification.
The application of the methyltransferase DmtMT1 in preparing methylated cyclic dipeptide. The application is that the methyltransferase DmtMT1 binds a methyl donor to the cyclic dipeptide alpha-N. Wherein the methyl donor is S-adenosylmethionine. The cyclic dipeptide is cyclo (L-Tyr-L-Val), cyclo (D-Trp-L-Val) and cyclo (L-Trp-L-Xaa), wherein Xaa is Val, Pro, Leu, Ile, Ala, Thr, Trp, Phe or Tyr.
A method for preparing pre-DMTs by using the methyltransferase DmtMT1 comprises the following steps: (1) synthesizing to obtain cyclo (L-Trp-L-Val) under the action of cyclic dipeptide synthase; (2) methylated cyclo (L-Trp-L-Val), Me-cWV, was obtained under the action of methyltransferase DmtMT 1; (3) methylated pre-DMTs were prepared from Me-cWV.
Wherein, the step (3) is specifically as follows: Me-cWV was used to obtain N15 methylated pre-DMTs by isopentenyl transferase DmtC 1.
Reaction mechanism/mechanism: (1) the methyltransferase DmtMT1 takes SAM (S-adenosylmethionine) as a methyl donor to realize the methylation of a plurality of cyclic dipeptides alpha-N and obtain methylated cyclic dipeptides; (2) the prenyltransferase DmtC1, which has a broad substrate spectrum, binds farnesyl to methylated cyclic dipeptides, resulting in a series of pre-DMTs methylated at the N15 position; (3) preparing a plurality of N15 methylated DMTs from N15 methylated pre-DMTs; the technical problem of preparing DMTs by an enzyme method is not overcome, so the step is realized by direct fermentation of strains and chemical total synthesis at present.
The invention has the beneficial effects that:
(1) the SAM-dependent methyltransferase DmtMT1 disclosed by the invention has the function of catalyzing the transfer of methyl from S-adenosylmethionine to a cyclodipeptide substrate, fills the technical blank of carrying out N15 methylation modification on pre-DMTs by an enzyme method, and has milestone significance for the technical development of synthesizing DMTs by the enzyme method.
(2) The SAM dependent methyltransferase DmtMT1 can take a plurality of cyclic dipeptides as substrates to generate a plurality of single alpha-N methylated cyclic dipeptides; due to the broad substrate spectrum, a plurality of pre-DMTs products can be obtained; therefore, the method has important position in the enzymatic synthesis of DMTs.
(3) The SAM dependent methyltransferase DmtMT1 provided by the invention methylates DMTs at the N15 position, thereby having important influence on the biological activity of the DMTs; therefore, methylation modification of DMTs is realized through an enzyme method, a new path is provided for drug development of diketopiperazine compounds, and the method has important application value and social significance.
Drawings
FIG. 1: SDS-PAGE analysis of the DmtMT1 purified methyltransferase of the invention;
FIG. 2: high Performance Liquid Chromatography (HPLC) profiles of DmtMT1 and cWV/cWL in the present invention;
FIG. 3: in the invention, the high resolution secondary mass spectrum (HR-MS/MS) spectrum of the reaction product of DmtMT1 and cWV;
FIG. 4: in the invention, the high resolution secondary mass spectrum (HR-MS/MS) spectrum of the reaction product of DmtMT1 and cWL;
FIG. 5: in the present invention, a pre-DMT F High Performance Liquid Chromatography (HPLC) chart was generated using Me-cWV as a substrate.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: cloning and in vitro expression of methyltransferase DmtMT1 gene
1. Extraction of genomic DNA
Inoculating Streptomyces youssuoufiensis OUC6819 into TSBY liquid culture medium, culturing at 30 deg.C, centrifuging to collect thallus, and washing with 1mL STE buffer; adding 3-5 mg/ml lysozyme solution prepared by 500uL STE buffer, fully suspending the thalli, and carrying out water bath at 37 ℃ for 30min until the cells become semitransparent; adding 250uL of 6% SDS, gently mixing up and down, and continuing the water bath at 37 ℃ until the mixture is clear; 1/10 volumes of 3M NaAc (pH 4.8) was added, followed by 200uL of phenol, chloroform, isoamyl alcohol (25:24: 1; v/v/v), mixed, and centrifuged at 12000 rpm; transferring supernatant, repeatedly extracting with phenol, chloroform and isoamylol until the middle layer has no protein impurity, transferring supernatant, adding isopropanol of the same volume, and mixing until white flocculent DNA precipitates; picking out flocculent precipitate, washing with 70% ethanol for 1 time; after drying at room temperature, genomic DNA was dissolved in an appropriate amount of TE for further use.
2. Construction of recombinant vectors
Performing PCR by using 10 times of diluted T-DNA of the Streptomyces youssuoensis OUC6819 as a template, and designing a primer pair:
DmtMT 1-FP: 5'-GGAATTCCATATGGGAAGTAAGCAGTACGAC-3'/DmtMT 1-RP: 5'-CCGCTCGAGCTTCACCGCCTCCAGGAC-3' are provided. Primer pair DmtMT1-FP/RP was used to amplify a functional gene for the methyltransferase DmtMT 1.
And (3) PCR reaction system:
mu.l each of primer pair DmtMT1-FP/RP (50pmol), template 5. mu.l, 10 × Reaction Buffer 10. mu.l, 2.5mM dNTP 10. mu.l, 25mM MgCl 26. mu.l, pfu DNA Polymerase 1. mu.l (5U/. mu.l), and ddH2O to 100. mu.l.
PCR conditions were as follows:
functional gene amplification condition, denaturation at 98 ℃ for 2 min; at 95 ℃ for 10s, at 66.3 ℃ for 15s and at 72 ℃ for 10s, and circulating for 25 seconds; 5min at 72 ℃. It was cloned into expression vector pET28a to construct recombinant plasmid pET28a-dmtMT 1.
Expression and purification of dmtMT1 in E.coli
The constructed recombinant vectors were introduced into E.coli BL21(DE3), respectively, and the overnight inoculum was inoculated into 1L of a culture containing 50. mu.g mL-1Carnacin LB broth, cultured at 37 ℃ to OD600 of about 0.8, was added with 0.05mM isopropyl thiogalactoside (IPTG), and the culture was continued at 16 ℃ for 16 hours. Centrifuging to collect thallus, re-dissolving thallus with buffer (0.05M Tris-HCl,0.5M NaCl, pH 7.5, stabilizing cOmpletet (TM) protease inhibitor cocktail), ultra-breaking thallus, purifying with nickel column, detecting with SDS-PAGE (shown in figure 1) to obtain DmtMT1 with 29.7kDa, concentrating and replacing enzyme with high purity to buffer (0.025M Tr)is-HCl,0.02M NaCl, and 10% glycerol, pH 7.5) and stored at-80 ℃ until use.
Example 2: DmtMT1 in vitro enzyme activity detection
In vitro enzyme activity reaction system:
DmtMT1 reaction system: 50mM Tris-HCl (pH 8.0) buffer, 0.5mM SAM, 10. mu.M DmtMT1,0.5mM cWV/cWL (cyclo (L-Trp-L-Leu)).
Reaction conditions are as follows: adding methanol with the same volume as the reaction system to stop the reaction after the reaction is finished at 30 ℃ for 12h, and centrifuging at 17,000Xg for 30min to remove protein in the reaction system. The obtained supernatant was analyzed by HPLC.
HPLC detection conditions: using a reversed phase YMC-Pack ODS-AQ C18 column (specification: 50 mm. times.4.6 mm,5 μm,) (ii) a The column temperature is 30 ℃; mobile phase a (acetonitrile + 0.1% formic acid) and B (ddH)2O + 0.1% formic acid); DmtMT1 reaction elution conditions: 0-5min 10% of phase A and 90% of phase B; gradient eluting for 5-25min, with 10% -50% of phase A and 90% -50% of phase B, detection wavelength of 280nm, and flow rate of 1mL min-1. As can be seen from the HPLC-obtained spectrum (fig. 2), fig. 2ii) shows a new absorption peak at 20min compared to fig. 2 i); fig. 2iv) compared to fig. 2iii), a new absorption peak appeared at 21min, indicating that DmtMT1 can accurately recognize cyclic dipeptides cWV and cWL and an enzymatic reaction occurs.
The cWV reaction product obtained, Me-cWV, was analyzed by high resolution secondary mass spectrometry (HR-MS/MS). From the HR-MS/MS spectrum (FIG. 3), the product [ M + H ] in FIG. 2ii)]+300.1726, with Me-cWV compound molecular weight ([ M + H)]+Theoretical 300.1712), and from the appearance of characteristic ion peak 86.0964, it can be concluded that its methylation is located at Val-derived α -N. The cWL reaction product obtained was subjected to high resolution secondary mass spectrometry (HR-MS/MS) (FIG. 4). From the HR-MS/MS spectrum, the product [ M + H ] in FIG. 2iv)]+314.1856, with Me-cWL compound molecular weight ([ M + H)]+Theoretical 318.1469), it can be concluded from the appearance of characteristic ion peak 100.1113 that its methylation is located at the alpha-N derived from Leu.
Example 3: role of DmtMT1 in DMTs biosynthesis
The pre-DMTs compound was synthesized using the reaction product of DmtMT1 and CWV of example 2 as a substrate. Reaction system: 50mM Tris-HCl (pH 8.0) buffer, 1.0mM Me-cWV,0.2mM FPP, 10. mu.M DmtC1,0.25mM Mg2Cl; reaction conditions are as follows: adding methanol with the same volume to stop the reaction after the reaction is finished at 30 ℃ for 1h, and centrifuging at 17,000Xg for 30min to remove protein in the reaction system. The obtained supernatant was analyzed by HPLC.
HPLC detection conditions: using a reversed phase YMC-Pack ODS-AQ C18 column (specification: 50 mm. times.4.6 mm,5 μm,) (ii) a The column temperature is 30 ℃; mobile phase a (acetonitrile + 0.1% formic acid) and B (ddH)2O + 0.1% formic acid); elution conditions: 0-5min 10% of phase A and 50% of phase B; gradient eluting for 5-15min, 10% -50% of phase A and 90% -50% of phase B, gradient eluting for 15-25min, detecting wavelength of 80% -100% of phase A and 20% -0% of phase B at 300nm, and flow rate of 1mL min-1。
From the spectrum obtained by HPLC (FIG. 5), the reaction set of FIG. 5ii) shows a new absorption peak at 25min, compared to the control of FIG. 5i), indicating the occurrence of the enzymatic reaction. The reaction product was separated and purified, and subjected to nuclear magnetic resonance detection, and the data of the carbon spectrum and the hydrogen spectrum thereof are shown in table 1. The obtained compound is identified to be a precursor compound pre-DMT F of DMT F (the structure is shown as a formula (4)).
TABLE 1 Nuclear magnetic hydrogen (600MHz) and carbon (500MHz) spectra data of pre-DMTF (solvent DMSO-d6)
In conclusion, the SAM dependent methyltransferase DmtMT1 realizes the transfer of methyl from S-adenosylmethionine to cyclodipeptide substrate, and further obtains pre-DMTs methylated at N15 position through isopentenyl transferase DmtC 1; the preparation of various N15 methylated DMTs by the enzyme method is cleared. In addition, because SAM dependent methyltransferase DmtMT1 has broad substrate spectrum, various pre-DMTs can be obtained by using various cyclic dipeptides as substrates. Therefore, the SAM-dependent methyltransferase DmtMT1 fills the technical blank of N15 methylation modification of pre-DMTs by an enzymatic method, overcomes the technical problem of low yield of a strain direct-extension method, and has milestone significance for the industrial application prospect of enzymatic synthesis of DMTs.
Sequence listing
<110> China oceanic university
<120> SAM-dependent methyltransferase DmtMT1 and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 249
<212> PRT
<213> Marine Streptomyces (Streptomyces yosussufield OUC6819)
<400> 1
Val Gly Ser Lys Gln Tyr Asp Glu Thr Ala Asp Val Tyr Asp Asp Thr
1 5 10 15
Leu Asn Val Leu Thr Tyr Ala Arg His Ala Glu Glu Pro Thr Phe Arg
20 25 30
Ala Val Met Gly Asp Val Arg Gly Leu Asp Val Leu Asp Leu Gly Ala
35 40 45
Gly Thr Gly Ile Trp Thr Arg Arg Ile Lys Gln Ala Gly Ala Gly Arg
50 55 60
Val Glu Gly Leu Glu Ile Ser Ala Ser Met Val Glu Thr Ala Arg Glu
65 70 75 80
Arg Glu Ala Gly Glu Arg Leu Gly Ile Thr Tyr His Val Gly Asp Val
85 90 95
Ala Arg Gly His Ala Ser Ile Glu Ala Val Ala Phe Asp Val Val Thr
100 105 110
Gly Val Asn Val Leu His Tyr Ser Ala Ser Arg Asp Glu Leu Val Ala
115 120 125
Met Cys Arg Thr Ala Ser Arg Ala Leu Arg Pro Gly Gly Arg Leu Val
130 135 140
Ala Asn Cys Ala Asn Phe His Met Ala Ala Asp Leu Asp Tyr Tyr Gly
145 150 155 160
Pro Phe Gly Ile Thr Ala Thr Val Pro Gln Pro Arg Val Glu Gly Ser
165 170 175
Met Val Arg Ala His Thr Thr Met Gly Gly Arg Pro Val Asp Ile Glu
180 185 190
Phe Tyr Leu Trp Leu Ala Ser Thr Tyr Glu Ser Ala Leu Arg Glu Ala
195 200 205
Gly Phe Thr Gln Val Arg Trp His Pro Trp Arg Ile Ser Glu Glu Gly
210 215 220
Ile Ala Gln Tyr Gly Gln Asp Tyr Trp Gln Arg Tyr Thr Glu Arg Pro
225 230 235 240
Pro Thr Leu Val Leu Glu Ala Val Lys
245
<210> 2
<211> 750
<212> DNA
<213> Marine Streptomyces (Streptomyces yosussufield OUC6819)
<400> 2
gtgggaagta agcagtacga cgagacggcc gatgtgtacg acgacacctt gaacgtcctc 60
acctatgcgc ggcacgcgga ggagcccacc ttccgtgcgg tgatggggga cgtgcgcggg 120
ctggacgtgc tggacctagg ggcggggacg gggatctgga cgcgccggat caagcaggcg 180
ggggcgggcc gggtggaggg cctggaaatc tcggcgtcca tggtggagac ggcccgcgag 240
cgcgaggcgg gcgaacggct tggcatcacg taccacgtcg gcgatgtggc ccgcgggcac 300
gcatccatcg aggcggtcgc cttcgacgtg gtgaccggcg tcaacgtcct gcactacagc 360
gccagcaggg acgagttggt ggccatgtgt cgcaccgcga gtcgggcgct acggccgggc 420
gggcgcctgg tggccaactg tgccaacttc cacatggccg ccgatctgga ctactacggc 480
ccgttcggta tcacggccac cgtgccccag cctcgcgtcg aggggtcgat ggtgcgcgcg 540
cacaccacga tgggcggacg cccggtcgac atcgagttct acctgtggct ggccagcacc 600
tacgagtccg cactgcgcga ggccggcttc acgcaggtgc gctggcaccc gtggcggatc 660
tcggaggagg gcatcgccca gtacggccag gactactggc agcgctacac cgagcgcccg 720
cccacgctgg tcctggaggc ggtgaagtga 750
Claims (4)
- Use of a SAM-dependent methyltransferase DmtMT1, said methyltransferase DmtMT1 having the amino acid sequence shown in SEQ ID No. 1; the method is characterized in that: the application of the methyltransferase DmtMT1 in preparing methylated cyclic dipeptide; said use is the binding of a methyl donor to the cyclic dipeptide alpha-N by said methyltransferase DmtMT 1; the methyl donor is S-adenosylmethionine; the cyclic dipeptide is cyclo (L-Trp-L-Val), and the alpha-N is N-13.
- 2. Use according to claim 1, characterized in that: the nucleotide sequence of the SAM dependent methyltransferase DmtMT1 is shown as SEQ ID NO. 2.
- 3. A method of making pre-DMTs using methyltransferase DmtMT1, characterized in that: the method comprises the following steps: (1) synthesizing to obtain cyclo (L-Trp-L-Val) under the action of cyclic dipeptide synthase; (2) methylated cyclo (L-Trp-L-Val) is obtained under the action of methyltransferase DmtMT1, namely Me-cWV; the amino acid sequence of the methyltransferase DmtMT1 is shown as SEQ ID NO. 1; (3) Pre-DMTs were prepared from Me-cWV.
- 4. A method of making pre-DMTs as claimed in claim 3 wherein: the step (3) is specifically as follows: Me-cWV gave pre-DMTs with a methylation modification at N15 by the action of the prenyltransferase DmtC 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010571360.2A CN111647575B (en) | 2020-06-22 | 2020-06-22 | SAM-dependent methyltransferase DmtMT1 and application |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010571360.2A CN111647575B (en) | 2020-06-22 | 2020-06-22 | SAM-dependent methyltransferase DmtMT1 and application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111647575A CN111647575A (en) | 2020-09-11 |
CN111647575B true CN111647575B (en) | 2022-05-17 |
Family
ID=72349797
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010571360.2A Active CN111647575B (en) | 2020-06-22 | 2020-06-22 | SAM-dependent methyltransferase DmtMT1 and application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111647575B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108913672A (en) * | 2018-07-26 | 2018-11-30 | 中国海洋大学 | A kind of novel prenyltransferase and its application |
-
2020
- 2020-06-22 CN CN202010571360.2A patent/CN111647575B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108913672A (en) * | 2018-07-26 | 2018-11-30 | 中国海洋大学 | A kind of novel prenyltransferase and its application |
Non-Patent Citations (4)
Title |
---|
Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery;Tingting Yao et al;《NATURE COMMUNICATIONS》;20181231;1-12 * |
Streptomyces youssoufiensis strain OUC6819 SAM-dependent methyltransferase (MT) gene, complete cds;Yao T et al;《GenBank: MK894429.1》;20200601;1 * |
Yao T et al.Streptomyces youssoufiensis strain OUC6819 SAM-dependent methyltransferase (MT) gene, complete cds.《GenBank: MK894429.1》.2020, * |
环二肽合酶生物合成途径研究进展;张京星等;《有机化学》;20191231;328-338 * |
Also Published As
Publication number | Publication date |
---|---|
CN111647575A (en) | 2020-09-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108913672B (en) | Novel isopentene transferase and application thereof | |
JP5632370B2 (en) | Recombinant bacteria producing deoxyviolacein and use thereof | |
CN114350727B (en) | Method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system | |
WO2021221418A1 (en) | Allulose epimerase variant, method for producing same, and method for producing allulose using same | |
CN110777155B (en) | Minimal mycin biosynthesis gene cluster, recombinant bacterium and application thereof | |
CN101613712A (en) | Improve the method for Avrmectin and/or ivermectin output and produce bacterial strain | |
CN111575260B (en) | Application of SAM dependent methyltransferase DmtMT2-1 | |
CN113265433A (en) | Bifunctional carbon glycoside glycosyl transferase and application thereof | |
CN111647575B (en) | SAM-dependent methyltransferase DmtMT1 and application | |
CN116445519B (en) | Glycosyltransferase and application thereof in biosynthesis of eugenol glucoside | |
CN108220260B (en) | Fusion enzyme for catalyzing naringenin to generate kaempferol and application thereof | |
CN114561369B (en) | Glycosyltransferase for biosynthesis of paris polyphylla saponin, encoding gene and application thereof | |
CN116355820A (en) | Engineering strain for high yield of ergothioneine and method for producing ergothioneine by same | |
CN112553175B (en) | Preparation and application of glycosyltransferase UGT76G1 mutant | |
CN115992109A (en) | Gelidine glycosyltransferase protein, and coding gene and application thereof | |
KR100679759B1 (en) | Transformant producing secondary metabolite modified with functional group and novel biosynthesis genes | |
CN116656641A (en) | Caffeic acid O-methyltransferase mutant and application thereof | |
CN108359652A (en) | Glycosyl transferase and its application | |
KR102232837B1 (en) | Novel polypeptides having glucosylglycerol productivity and a method for producing glucosylglycerol using the same | |
CN111635894B (en) | Piericidin glycosyltransferase sGT1 and application thereof | |
CN111662891B (en) | Piericidin glycosyltransferase sbmGT1 and application thereof | |
KR102080160B1 (en) | A Novel Thermostable Amylosucrase and Enzymatic Preparation Method of Amylose using the same | |
WO2022131130A1 (en) | Prenylflavonoid glucosidase, polynucleotide encoding same, and method for producing prenylflavonoid glycoside | |
CN117737029B (en) | Glycosyltransferase mutant and application thereof in synthesis of collaterals plug | |
CN107699581A (en) | 3,7 dihydroxy Zhuo phenolic ketone biological synthesis gene clusters and its application |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |