CN108624572A

CN108624572A - The methylated transferase gene of glucoside compound

Info

Publication number: CN108624572A
Application number: CN201810417402.XA
Authority: CN
Inventors: 张礼文; 徐玉泉; 谢李楠; 王辰; 王晓婧
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2017-08-11
Filing date: 2018-05-04
Publication date: 2018-10-09
Anticipated expiration: 2038-05-04
Also published as: CN108624572B; CN107674877A

Abstract

The present invention relates to the methyl transferase genes of a kind of glucoside compound, can 4 hydroxyls of glycosyl in glucoside compound be subjected to the modification that methylates, it can act synergistically simultaneously with glucosyl transferase, the different activities substance including polyketone class, flavonoids, Anthraquinones etc. is converted by Combinatorial biosynthesis, to obtain the novel active compound for being different from these Structures of Natural Products, the orientation bioconversion for realizing compound, enhances the stability of compound.

Description

The methylated transferase gene of glucoside compound

Technical field：

The present invention relates to it is a kind of can be by the gene of 4 hydroxymethylations of glucose in glycosylated materials, the glycosylation Substance specifically includes the glycosylated derivative of the polyphenol compounds such as polyketone class, flavonoids, Anthraquinones.

Background technology：

Various natural and non-natural glycosylated materials are easy to be influenced by from many factors of organism and the external world, Auto-degradation, activity is caused to reduce.If by 4 hydroxy alkylateds of glycosyl in these substances, be conducive to enhance its stability.

Fungal organism conversion is to carry out structural modification and transformation to xenobiontics using intracellular specific enzyme, to obtain Obtain more valuable metabolic response.

Invention content:

The purpose of the present invention is obtain the O- transmethylases for 4 hydroxymethylations that may participate in glycosyl in glycosyl compound Base gene realizes the modification that methylates of glycosylated compound, obtains new structure and active natural products, enhances its stabilization Property.

A kind of application of transmethylase in glucoside compound methylates modification, the transmethylase has as follows Secondary structure：

Since N-terminal, 3 (alpha+beta series connection)+β-pleated sheet+3 (alpha+beta series connection)+β-pleated sheets；

And their amino acid sequence is selected from SEQ ID NO.2,4,6,8 or 10.

Specifically, the present invention is from beauveria bassiana, fumosorosea Isaria fumosorosea, Robert green muscardine fungus Metarhizium robertsii, purple ergot Claviceps purpurea and Cordyceps militaris Cordyceps Following five special O- transmethylases are found that in militarisCM01, be respectively designated as BbFkbM, CpOMT, MrOMT, IfOMT and CmOMT.This five albumen all have identical motif and arrangement mode (see Figure 30-32), and the ammonia in activated centre Base acid sequence consistency is very high, and polarity is consistent.

Expand glucose O- methyl transferase genes BbFkbM respectively by cDNA, synthesize to obtain CpOMT by DNA, MrOMT, IfOMT and CmOMT build the recombinant plasmid that can be expressed in saccharomycete respectively, turn with the existing glucityl in laboratory It moves enzyme heterologous expression vector corotation and enters yeast, and carry out heterogenous expression conversion in yeast.

Different glycosylation derivative is added by external source, or biosynthesis is combined with glucosyltransferase, from fermentation Extraction has obtained the sugared methylate different from former compound structure in product.

It is verified by experiments, of the invention five O- methyl transferase genes, can be to a variety of flavonoids, poly- by heterogenous expression Ketone, Anthraquinones glycosylated derivative carry out the modification that methylates, the obtained more unmodified preceding hydrolytic stability enhancing of product, The degradation that glycoside hydrolase in yeast can be offset enhances stability in vivo.Moreover, these homologous genes are not Function only having the same can also be combined biosynthesis from different glycosyl transferases, modify different natural activity productions Object, and transformation efficiency is improved, provide new research method and approach for the discovery of lead compound and the orientation conversion of drug.

By taking BbFkbM as an example, it is described as follows specific research process：

1, BbFkbM genes are obtained

Structural analysis by gene order-checking and correlation predictive software to glycosyl transferase, design primer are expanded by cDNA Increase and obtains ball spore deadlock O- methyl transferase genes BbFkbM in vain.Wherein, BbFkbM nucleotide sequences are as shown in SEQ ID NO.1.

2, glucosyltransferase BbFkbM Yeast expression carriers structure and yeast conversion

DNA fragmentation containing complete BbFkbM is connected on PXW06F carriers, constitutive expression plasmid is constructed PXW06F-BbFkbM, while being transferred to auxotrophic yeast with glucosyltransferase heterologous expression vector PRS425m-BbUGT86 Receptor BJ5464 carries out heterogenous expression.

3, the screening of recon is common

Expression vector is transferred in the missing saccharomycete that cannot synthesize leucine, tryptophan, is sought using leucine, tryptophan Support deficiency label screening recon.

4, polyketides feeding conversion test and converted product HPLC liquid phase analysis

Polyketides Desmethyl-Lasiodiplodin is added respectively (hereinafter referred to as in recombinant bacterial strain incubation For compound 1), microbe conversion product is extracted with organic solvent, and carries out HPLC liquid phase analysis, nuclear magnetic resonance spectroscopy product structure.

It was found that 1 almost all of compound converts.Liquid phase result is shown is also obtained another other than the peak of compound 1 Peak.Separation product, which carries out further structural analysis, to be proved to have obtained the polyketone class with 1 different structure of natural products compound Object 2 is closed, finds that compound 2 is on the hydroxyl of 5 carbon atoms for originating in compounds 1 through high resolution mass spectrum and nuclear magnetic resonance spectroscopy Add a glucose, while the hydroxymethylation on glucose 4.O- methyl transferase genes are demonstrated to can be used for benzene The structure of the polyketone glycosylated derivative of diphenol lactone and the like is modified, and is well suited for carrying out various reactive compounds Molecular structure alteration increases the diversity in polyketide library.Recombinant bacterial strain tunning chromatogram and product structure are shown in Fig. 3.

5, the feeding conversion test of flavone compound and converted product HPLC liquid phase analysis

Different flavone compounds are added in recombinant bacterial strain incubation respectively, microbe conversion product is carried with organic solvent It takes, and carries out HPLC liquid phase analysis, product component is analyzed through high resolution mass spectrum.

6. sugar methylates, modified outcome compound 2 will purify compared with compound glycosylated derivative before modification is in stability Sugar afterwards methylate modified outcome compound 2 with modification before compound glycosylated derivative be respectively added to wild type yeast send out In zymotic fluid, cultivate the identical time, by extract crude extract carry out HPLC liquid phase analysis, detection two kinds of substances in yeast from Stability under the effect of body enzyme system.

7. yeast glycoside hydrolase EXG1 to sugar methylate modified outcome compound 2 with modification before compound glycosylation derive The influence of object stability adds glycosylation modified product Compound 2 after purification with compound glycosylated derivative before modification respectively Be added to wild type and knock out in the yeast fermentation broth of glycoside hydrolase EXG1, cultivate the identical time, by extract crude extract into Row HPLC liquid phase analysis, stability of the two kinds of substances of detection under yeast itself glycoside hydrolysis enzyme effect.

8. the functional verification of homologous gene in entomogenous fungi

By the glycosyl transferase of the beauveria bassiana of acquisition related life is utilized to the amino acid sequence of O- transmethylases Object informatics software be compared obtained from different entomogenous fungis amino acid sequence similarity more than 30%, coverage rate it is big In 90% 98 homologous protein sequences (partial sequence is shown in Table 1).From consensus amino acid sequence degree 50%, similarity 70%, cover Fumosorosea Isaria fumosorosea, Robert green muscardine fungus are picked in the sequence of 99% or more lid rate Metarhizium robertsii and purple ergot Claviceps purpurea and Cordyceps militaris Cordyceps 4 O- methyl transferase genes in militaris carry out heterogenous expression (content for being shown in Table underscore in 1), the sequence in NCBI Number be respectively XP_007822447.1, XP_018701797.1, CCE31609.1 and XP_006674755.1.It is raised by substrate Feed experiment, the glycosylated derivative of equal successful conversion polyketides 1 and flavone compound F-4 etc. demonstrate fungi In homologous O- transmethylases identity function.Meanwhile by combining different OMT, different transformation efficiencies can be reached.

The homologous gene list of 1 O- methyl transferase genes BbFkbM of table

Description of the drawings：

Fig. 1 is the amplification of BbFkbM genetic fragments；

Fig. 2 is the physical map of recombinant vector PXW06F-BbFkbM；

Fig. 3 is the liquid chromatogram before compound 1 is modified；

Fig. 4 is after compound 1 is modified, to produce the HPLC liquid chromatograms for the compound 2 that a sugar methylates.

Fig. 5-Figure 14 for tested substrate flavones sample conversion after sugared methylate chromatogram and mass spectral results, Wherein：

Fig. 5 is the chromatogram and mass spectral results after the conversion of F-4 compounds；

Fig. 6 is the chromatogram and mass spectral results after the conversion of F-5 compounds；

Fig. 7 is the chromatogram and mass spectral results after the conversion of F-6 compounds；

Fig. 8 is the chromatogram and mass spectral results after the conversion of F-7 compounds；

Fig. 9 is the chromatogram and mass spectral results after the conversion of F-8 compounds；

Figure 10 is the chromatogram and mass spectral results after the conversion of F-11 compounds；

Figure 11 is the chromatogram and mass spectral results after the conversion of F-12 compounds；

Figure 12 is the chromatogram and mass spectral results after the conversion of F-15 compounds；

Figure 13 is the chromatogram and mass spectral results after the conversion of F-16 compounds；

Figure 14 is the chromatogram and mass spectral results after the conversion of F-20 compounds；

Figure 15 and Figure 16 is respectively that compound 3 is cultivating the liquid chromatogram with the hydrolysate after 48h for 24 hours；

Figure 17 and Figure 18 is respectively that compound 2 is cultivating the liquid chromatogram with the hydrolysate after 48h for 24 hours；

Figure 19 is the electrophoresis result of EXG1 knockout mutant strains PCR verifications；

Figure 20 and Figure 21 is respectively the liquid phase of hydrolysate of the compound 3 in wild type and mutant yeast incubation Chromatogram；

Figure 22 and Figure 23 is respectively the liquid phase of hydrolysate of the compound 2 in wild type and mutant yeast incubation Chromatogram；

Figure 24 to Figure 27 be respectively recombinant vector PXW06F-IfOMT, PXW06F-MrOMT, PXW06F-CpOMT and The physical map of PXW06F-CmOMT.

Figure 28 is compound 3 after five fungi OMT modifications, produces the HPLC liquid for the compound 2 that a sugar methylates Phase chromatogram；

The glycation product that Figure 29 is compound F-4 produces a sugared methylate after five fungi OMT modifications HPLC liquid chromatograms；

Figure 30 is the protein sequence comparison result of BbFkbM and CpOMT, MrOMT, IfOMT, CmOMT, and straight line marks region For the protein active center, bar chart indicates that sequence consistent degree, alphabetical gray scale indicate amino acid polarity.

Figure 31 is the protein sequence motif analysis results of BbFkbM and CpOMT, MrOMT, IfOMT, CmOMT.

Figure 32 is the Protein secondary structure of BbFkbM and CpOMT, MrOMT, IfOMT, CmOMT.

Sequence table explanation

The nucleotide sequence of SEQ ID NO.1 BbFkbM；

The amino acid sequence that SEQ ID NO.2 are encoded by the BbFkbM that SEQ ID NO.1 are derived.

The nucleotide sequence of SEQ ID NO.3 IfOMT；

The amino acid sequence that SEQ ID NO.4 are encoded by the IfOMT that SEQ ID NO.3 are derived.

The nucleotide sequence of SEQ ID NO.5 MrOMT；

The amino acid sequence that SEQ ID NO.6 are encoded by the MrOMT that SEQ ID NO.5 are derived.

The nucleotide sequence of SEQ ID NO.7 CpOMT；

The amino acid sequence that SEQ ID NO.8 are encoded by the CpOMT that SEQ ID NO.7 are derived.

The nucleotide sequence of SEQ ID NO.9 CmOMT；

The amino acid sequence that SEQ ID NO.10 are encoded by the CmOMT that SEQ ID NO.9 are derived.

Specific implementation mode

Experiment material explanation and source used in experiment are as follows below：

Following bacterial strain and carrier：Beauveria bassiana Beauveria bassiana ATCC7159, fumosorosea bacterium Isaria fumosorosea ACCC37775, Robert green muscardine fungus Metarhizium robertsii ARSEF 23, purple Ergot Claviceps purpurea ACCC337002, Cordyceps militaris Cordyceps militarisCM01 and saccharomycete S.cerevisiae BJ5464-NpgA, Yeast expression carrier PRS425m-BbUGT86 and PXW06F, derive from Chinese agriculture Biotechnology institute of the academy of sciences, is voluntarily preserved by the present inventor laboratory.

Enzyme and kit：

Restriction enzyme, T4DNA ligases are purchased from NEB companies；

RNA reverse transcription reagent box is purchased from TAKARA companies；

Quick-Fusion Cloning Kit are purchased from Biotool companies；

Thermal starting high-fidelity DNA amplification kit is purchased from Biotool companies；

Archaeal dna polymerase and DNA marker are purchased from Beijing Quan Shijin biotech firms；

The small extraction reagent kit of plasmid, universal DNA purifying plastic recovery kits are purchased from Tiangeng company；

Frozen yeast conversion reagent box is purchased from YMO RESEARCH biotech firms；

Bacillus coli DH 5 alpha competence is purchased from Kang Wei ShiJi Co., Ltd；Other reagents are domestic analysis net product.

Culture medium：

Escherichia coli culture medium is LB culture mediums (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0). SC-^-Leu defects culture medium (1% glucose, 6.7%Difco^TM Yeast Nitrogen Base w/o Amino Acids、- Leu/-Trp DO Supplement).YPD culture mediums (1% yeast extract, 2%Peptone peptones, 2% glucose).YPD is low 2% agar powder is added if solid medium processed in sugar culture-medium (1% yeast extract, 2%Peptone peptones, 1% glucose).

Other test method without specific conditions in embodiment, conventionally carry out, molecular cloning is pressed (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in laboratory manual, or press According to the condition proposed by manufacturer.

Compound 1：Desmethyl-Lasiodiplodin

Compound 2：The methylate of 14 hydroxyls of glycosyl of compound

Compound 3：1 glycation product of compound

Embodiment 1 synthesizes O- methyl transferase gene BbFkbM, and builds Yeast expression carrier and obtain yeast transformant

The genome sequence that gene order-checking obtains the bacterial strain is carried out to one plant of beauveria bassiana of acquisition, passes through correlation During bioinformatics software analyzes glycosyl transferase, an O- methyl transferase gene is found that by comparing, we It is named as BbFkbM.The nucleotide sequence of BbFkbM is got rid of introne by various software forecast analysis to splice to obtain The coding region sequence of BbFkbM.The RNA for extracting beauveria bassiana obtains cDNA by reverse transcription, utilizes the primer amplification of design The encoding gene of BbFkbM, recycling target fragment are given sequencing company sequencing and are compared, and obtain glycosyl shown in SEQ ID NO.1 and turn Move enzyme gene BbFkbM.

Restriction enzyme site is introduced when synthesis.NdeI restriction enzyme sites are added before promoter, PmeI digestions are added after terminator Site, fragment length 0.75Kb.BbFkbM amplified fragments are shown in Fig. 1.

With NdeI and I double digestion shuttle vector PXW06F of Pme and the segment for recycling 6114bp sizes.Utilize seamless connection gram Grand kit connects two segments.

Heat shock method is transformed into E. coli competent DH5 α.Amp^rResistance screening extracts plasmid, ScaI and I digestions of Nde mirror It is fixed.Recombinant plasmid PXW06F-BbFkbM collection of illustrative plates is shown in Fig. 2.

Saccharomyces Cerevisiae in S .cerevisiae BJ5464-NpgA are seeded in YPD culture mediums, 30 DEG C, 200r/min cultures To O.D. value 0.8-1.0, the specification provided according to ZYMO companies prepares Saccharomyces cerevisiae competent cell and carries out recombinant plasmid Conversion.By the PRS425m-BbUGT86 containing glycosyltransferase gene with the PXW06F- containing O- transmethylases BbFkbM recombinant plasmid corotation dissolves into saccharomyces cerevisiae, is coated on the SC solid defects culture mediums of leucine, tryptophan depletion, and 30 DEG C culture 3 days or so.The yeast transformant of acquisition, scribing line culture to new SC-^-Leu/^-On Trp defect culture mediums, in 30 DEG C of trainings It supports and is cultivated 2 days or so in case；

The application O- transmethylase recombinant vectors of embodiment 2 PXW06F-BbFkbM is realized to polyketides The modification of Desmethyl-Lasiodiplodin (compound 1) glycosylated derivative

1. experiment purpose

Pass through the HPLC efficient liquid phases isolated generation after glucosyltransferase and the BbFkbM modifications of O- transmethylases It thanks to product and analyzes its molecular structure.

2. experimental method：

1) fermented and cultured

Two-step fermentation technology is taken, suitable yeast transformant thalline is seeded to corresponding 25-mL first^-Leu/^- In Trp liquid defect culture mediums, 30 DEG C, 200rmin-1 culture 16h or so add YPD low sugar culture medium 25ml, simultaneously 5mg Desmethyl-Lasiodiplodin sterlings are separately added into continue to cultivate 48h；It is extracted with ethyl acetate tunning, second The ratio of acetoacetic ester and zymotic fluid is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle acetic acid second Ester dry extract, 1ml methanol redissolve extract.

2) efficient liquid phase (HPLC) chromatography detects：

Above-mentioned gained tunning is used into high performance liquid chromatography detection after high speed centrifugation.

HPLC testing conditions are as follows：Chromatographic column Kromasil 100-5-C18 use acetonitrile-H2O to carry out ladder for mobile phase Degree elution, condition of gradient elution:Condition of gradient elution:0~5min, acetonitrile 10%；5~15min, acetonitrile is from 10% → 95%； 15~25min, acetonitrile 95%；25~28min, acetonitrile is from 95% → 10%；28~31min, acetonitrile 10%.Flow velocity 0.8mLmin-1, Detection wavelength 300nm.

3. experimental result and analysis：

It is found by HPLC analyses, the peak of natural polyketides 1 (Desmethyl-Lasiodiplodin) almost disappears It loses, has obtained another peak, illustrated that 1 almost all of natural products compound is converted.

Separation product, which carries out mass spectrum and nuclear magnetic resonance spectroscopy, proves that majority of compounds 1 passes through glucosyltransferase and O- The modification of transmethylase converts for compound 2.

The general configuration formula that compound 1,2 has been respectively obtained through the C of mass spectral analysis and nuclear magnetic resonance spectrums and H spectrum results, leads to It crosses and compares the relative molecular masses of two kinds of compounds and find that compound 2 is bigger than the relative molecular mass of compound 1, binding molecule knot Structure, which can determine, has had more a glucose molecule to methylate on 2 molecule of compound.

C spectrums and H spectrums are further analyzed by software, it is found that compound 2 is in 5 carbon atoms for originating in compounds 1 The glucose of 4 hydroxymethylation is added on hydroxyl.

Result above proves that O- transmethylases of the present invention can be used for carrying out methyl to polyketone glycosylated derivative Change modification, increases the diversity in polyketide library.

Recombinant bacterial strain tunning chromatogram and product structure are shown in Fig. 3 and Fig. 4.

The C of compound 1 and compound 2NMR are composed and H spectrums are shown in Table 2.

The C of 2 compound 1 of table and compound 2NMR are composed and H spectrums

Embodiment 3 repaiies flavonoids, anthraquinone analog compound using O- transmethylase recombinant vector PXW06F-BbFkbM Decorations

1. experiment purpose

Pass through the isolated metabolite after glucosyltransferase and the modification of O- transmethylases of HPLC efficient liquid phases And analyze its molecular structure.

2. experimental method：

1) fermented and cultured

Two-step fermentation technology is taken, suitable yeast transformant thalline is seeded to corresponding 25ml first^-Leu/^-Trp In liquid defect culture medium, 30 DEG C, 200rmin-1 culture 16h or so add YPD low sugar culture medium 25ml, simultaneously respectively 5mg sterlings are added to continue to cultivate 48h, totally 20 kinds of flavonoids sample used；Be extracted with ethyl acetate tunning, ethyl acetate with The ratio of zymotic fluid is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle ethyl acetate drying extraction Object, 1ml methanol is taken to redissolve extract.

2) efficient liquid phase (HPLC) chromatography detects：

HPLC testing conditions are as follows：31min conditions：Chromatographic column Kromasil 100-5-C18 use acetonitrile-H2O for stream It is dynamic mutually to carry out gradient elution, condition of gradient elution:Condition of gradient elution:0~5min, acetonitrile 10%；5~15min, acetonitrile from 10% → 95%；15~25min, acetonitrile 95%；25~28min, acetonitrile is from 95% → 10%；28~31min, acetonitrile are 10%.Flow velocity 0.8mLmin-1, Detection wavelength 300nm.

13min conditions：Chromatographic column RRHD Eclipse Plus C18,4.6x100mm, use acetonitrile-H2O for mobile phase Carry out gradient elution, condition of gradient elution:Condition of gradient elution:0~5min, acetonitrile is from 10% → 50%；5~10min, acetonitrile From 50% → 95%；10~12min, acetonitrile 95%；12~12.5min, acetonitrile is from 95% → 10%；12.5~13min, second Nitrile is 10%.Flow velocity 0.5mLmin-1, Detection wavelength 300nm.

2. experimental result and analysis：

It is found by HPLC analyses, sharing 10 kinds of samples in 20 kinds of substrates of addition is converted, respectively number F- 4, F-5, F-6, F-7, F-8, F-11, F-12, F-15, F-16, F-20.In addition to the starting of addition in the chromatogram of tunning The peak of another compound is also obtained in the peak of substrate and the peak of glycation product, determines the flavones sample of the starting of addition Converted.

It is analyzed through mass spectral results, has respectively obtained the relative molecular weight of product after conversion, by comparing two kinds of compounds Product is bigger than the relative molecular mass of starting material by 176 after relative molecular mass finds conversion, and binding molecule structure can determine A glucose molecule to methylate has been had more after conversion on chemical molecules.

4 hydroxyls being happened in the glycation product glucose of starting material that methylate are found by the further analysis of software On base.O- methyl transferase genes are demonstrated to can be used for, to the structural modification of flavonoids glycosylated derivative, being well suited for various Reactive compound carries out molecular structure alteration, increases the diversity in flavonoid glycoside compound library.

Recombinant bacterial strain tunning chromatogram is shown in Fig. 5 to Figure 14,

The chemical formula of the front and back compound of conversion, structure and molecular weight are shown in Table 3.

3 all kinds of flavone compounds of table modify cross-reference

4 sugar of embodiment methylates modified outcome compound 2 compared with the stability of the glycoside compounds 3 before the modification that methylates

1. experiment purpose

Under the action of yeast itself enzyme system, the sugar before more sugared methylate modified outcome compound 2 and the modification that methylates The hydrolytic stability of glycoside compound 3 under the same conditions.

2. experimental method：

It adds 2mg sugar respectively in the 50mL yeast BJ5464 cultivated with YPD to methylate modified outcome compound 2, with first Glycoside compounds 3 before baseization modification, sample are dissolved in 100 μ L chromatography methanol, and blank control is isometric chromatography methanol.30 DEG C, 200rmin-1 cultivate respectively for 24 hours with after 48h, be extracted with ethyl acetate tunning, the ratio of ethyl acetate and zymotic fluid Example is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle ethyl acetate dry extract, 1ml first Alcohol redissolves extract.

3. experimental result and analysis：

The extraction product chromatogram of analysis of compounds 3 and compound 2 under same incubation time.Compound 3 is being cultivated for 24 hours It is as shown in Figure 15 and Figure 16 with the extraction product chromatogram difference after 48h：Compound 2 is produced with the extraction after 48h for 24 hours in culture Look for spectrogram difference as shown in Figure 17 and Figure 18.

By chromatogram it can be seen that glycosylated derivative starts to hydrolyze after 24h after modification, with the increasing of incubation time Long, hydrolysis degree is higher；But sugared methylate 2 is relatively stable during Yeast Cultivation, and apparent hydrolysis phenomena does not occur, Methylating for illustrating to occur on glucose can increase the stability of glycation product, and compound is prevented to be degraded by enzymes.

Before glycoside hydrolase EXG1 is to sugared methylate modified outcome compound 2 and the modification that methylates in 5 yeast of embodiment Influence of the glycoside compounds 3 in incubation

1. experiment purpose

Under conditions of knocking out yeast glycoside hydrolase EXG1, it is more sugared methylate modified outcome compound 2 with methylate Stability of the glycoside compounds 3 under same culture conditions before modification, determines methylation to glycation product stability Influence.

2. experimental method：

1) yeast mutant of structure glycoside hydrolase EXG1 missings

The sequence that two sections of 500bp or so are chosen in the glycoside hydrolase EXG1 genes of Saccharomyces Cerevisiae in S .cerevisiae is made For upstream and downstream homology arm, using uracil synthetic gene Ura as resistance marker, design primer builds gene knockout by fusion DNA vaccine Homo~logous exchange segment used, list of primers are shown in.

4 synthetic primer of table and sequence

When expanding the upstream and downstream homology arm of EXG1 genes, using S.cerevisiae BJ5464-NpgA genomes as template, When expanding uracil synthetic gene Ura genes, using PXK-30F plasmids as template.Expanded by Biotool thermal starting high-fidelity DNAs Increase kit and carry out PCR amplification, PCR product passes through agarose gel electrophoresis testing goal stripe size, Tiangeng DNA purified reagents Box recycles purpose band, and to recycle segment as template, EXG1-UP-F, EXG1-Down-R are the fusion that primer carries out the second wheel PCR amplification, for same PCR product by agarose gel electrophoresis testing goal stripe size, fusion segment overall length is 1941bp.It Root DNA purification kits recycling fusion segment, is used for the gene knockout of yeast.

Saccharomyces Cerevisiae in S .cerevisiae BJ5464-NpgA are seeded in YPD culture mediums, 30 DEG C, 200r/min cultures To O.D. value 0.8-1.0, the specification provided according to ZYMO companies prepares Saccharomyces cerevisiae competent cell and carries out recombinant fragment Conversion.Fusion segment is transformed into saccharomyces cerevisiae, the SC solid defects culture mediums of uracil missing, 30 DEG C of cultures are coated on 3 days or so.The yeast transformant of acquisition, scribing line culture to new SC-^-On Ura defect culture mediums, cultivated in 30 DEG C of incubators 2 days or so；Bacterium colony PCR is carried out with 8 yeast transformants of picking, verification knocks out the correctness of result, and verification primer is CTGTGTTTACAGTGCGGTGCACACG；CGTTTGGATGAGGACCCACTTGAAACAAT；PCR product wild type band should be 2.4kb, mutant strain 1.9kb.PCR product electrophoresis result is shown in Figure 19, has 7 plants of yeast successfully to realize in 8 yeast transformants Gene knockout, transformation efficiency is up to 87.5%.

It is methylated modified outcome compound 2 with 2mg sugar is added respectively in the 50mL △ EXG1 yeast BJ5464 of YPD cultures, With the glycoside compounds 3 before the modification that methylates, sample is dissolved in 100 μ L chromatography methanol, compares as except bacterial strain is wild type Outside BJ5464, other conditions are constant.30 DEG C, after 200r/min cultivates 48h respectively, be extracted with ethyl acetate tunning, second The ratio of acetoacetic ester and zymotic fluid is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle acetic acid second Ester dry extract, 1ml methanol redissolve extract.

3. experimental result and analysis：

Extraction product chromatogram such as Figure 20 and 21 institute of the compound 3 under wild type and mutant yeast same culture conditions Show；Extraction product chromatogram of the compound 2 under wild type and mutant yeast same culture conditions is as depicted in figures 22 and 23.

Glycosylated derivative is in yeast glucosides before can be seen that modification by the chromatogram of wild type and △ EXG1 saltant types Apparent glucose occurs under the hydrolysis of hydrolase and falls phenomenon, the starting material being re-converted into before glycosylation, then strike This hydrolysis degree reduces after removing EXG1 genes, and it is glycosylated to illustrate that the glycoside hydrolase EXG1 in yeast may act on Hydrolysis phenomena occurs for compound, and this hydrolysis is obvious；And sugared methylate 2 is either in wild type Or it is all relatively stable during the Yeast Cultivation of saltant type, apparent hydrolysis phenomena does not occur, illustrates the first occurred on glucose Baseization can increase the stability of glycation product, and compound is prevented to be hydrolyzed enzyme degradation.

The functional verification of O- transmethylase homologous genes in 6 entomogenous fungi of embodiment

1. experiment purpose

It is found that the O- transmethylases in beauveria bassiana are given birth in other worms using the analysis of relevant biological information software There are a large amount of homologous gene (tables 1) in fungi carries out the substrate feeding of polyketide by the heterologous expression system of yeast Experiment, determines the function of these homologous genes, and compares it and be directed to the transformation efficiency of compound 3.

2. experimental method：

1. building Yeast expression carrier, yeast transformant is obtained

It is compared using relevant biological information software and obtains beauveria bassiana O- methyl from different entomogenous fungis The Homologous gene sequences of transferase.Fumosorosea is picked from the gene order that these similarities are up to 50% or more Isaria fumosorosea, Robert green muscardine fungus Metarhiziumrobertsii, Cordyceps militaris Cordyceps militaris Sequence analysis is carried out with the O- methyl transferase genes of 4 plants of bacterium of purple ergot Claviceps purpurea, is predicted It translates protein sequence and secondary structure height is approximate, motif (motif) (Figure 30-32) having the same.It is pre- by various software Survey analysis and get rid of coding region sequence and synthetic gene that introne splices to obtain O- methyl transferase genes, obtain IfOMT, The sequence fragment of MrOMT, CmOMT and CpOMT.Gene order is successively respectively such as SEQ ID NO.3, SEQ IDNO.5, SEQ ID Shown in NO.7, SEQ ID NO.9.Restriction enzyme site is introduced when synthesis respectively.NdeI restriction enzyme sites are added before promoter, are terminating PmeI restriction enzyme sites are added after son.With NdeI and I double digestion shuttle vector PXW06F of Pme and the segment for recycling 6114bp sizes. Genetic fragment is connect with carrier respectively using Cloning Kit is seamlessly connected.

Heat shock method is transformed into E. coli competent DH5 α.Amp^rResistance screening extracts plasmid, digestion identification.It obtains Recombinant plasmid PXW06F-IfOMT, PXW06F-MrOMT, PXW06F-CmOMT and PXW06F-CpOMT plasmid map is shown in figure successively 24, Figure 25, Figure 26 and Figure 27.Saccharomyces Cerevisiae in S .cerevisiae BJ5464-NpgA are seeded in YPD culture mediums, 30 DEG C, 200r/min is cultivated to O.D. value 0.8-1.0, and the specification provided according to ZYMO companies prepares Saccharomyces cerevisiae competent cell simultaneously Carry out the conversion of recombinant plasmid.Recombinant plasmid transformed is entered in saccharomyces cerevisiae, the SC solid defects training of tryptophan depletion is coated on Base is supported, 30 DEG C are cultivated 3 days or so.The yeast transformant of acquisition, scribing line culture to new SC-^-On Trp defect culture mediums, in 30 It is cultivated 2 days or so in DEG C incubator.

2) fermented and cultured

Two-step fermentation technology is taken, suitable yeast transformant thalline is seeded to corresponding 25ml first^-Trp liquid In defect culture medium, 30 DEG C, 200rmin-1 culture 16h or so add YPD low sugar culture medium 25ml, are separately added into simultaneously The glycosylated derivative compound 3 of 5mg compounds 1 continues to cultivate 48h, is extracted with ethyl acetate tunning, ethyl acetate with The ratio of zymotic fluid is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle ethyl acetate drying extraction Object, 1ml methanol is taken to redissolve extract.

3) efficient liquid phase (HPLC) chromatography detects：

HPLC testing conditions are as follows：Chromatographic column RRHD Eclipse Plus C18,4.6x100mm, use acetonitrile-H2O for Mobile phase carries out gradient elution, condition of gradient elution:Condition of gradient elution:0~5min, acetonitrile is from 10% → 50%；5~ 10min, acetonitrile is from 50% → 95%；10~12min, acetonitrile 95%；12~12.5min, acetonitrile is from 95% → 10%；12.5 ~13min, acetonitrile 10%.Flow velocity 0.5mLmin-1, Detection wavelength 300nm.

3. experimental result and analysis：

It is found by HPLC analyses, the O- transmethylases of separate sources can be by glycosylated compound by heterogenous expression 3 conversions have obtained the peak of another sugared methylate, and in identical retention time.This illustrates in different entomogenous fungis O- transmethylase homologous genes rhetorical function having the same, can successfully realize to the glycosylations such as polyketide derive Methylating to achieve the purpose that improve glycosylated compound stability on 4 hydroxyls of object glucose.CpOMT、MrOMT、CmOMT The chromatogram of tunning is fed with the recombinant bacterial strain substrate of IfOMT and sees Figure 28.

Embodiment 7 is realized using O- transmethylases BbFkbM, CpOMT, MrOMT, CmOMT and IfOMT in entomogenous fungi Modification to flavone compound

1. experiment purpose

The metabolite after the modification of O- transmethylases is detected by HPLC efficient liquid phases and calculates its molecular weight, is verified The function of CpOMT, MrOMT, CmOMT and IfOMT, and compare it and be directed to the transformation efficiency of different substrates.

2. experimental method：

1) fermented and cultured

Two-step fermentation technology is taken, suitable yeast transformant thalline is seeded to corresponding 25ml first^-Trp liquid In defect culture medium, 30 DEG C, 200rmin-1 culture 16h or so add YPD low sugar culture medium 25ml, are separately added into simultaneously The glycosylated derivative of 5mg flavone compounds F-4 continues to cultivate 48h, is extracted with ethyl acetate tunning, ethyl acetate with The ratio of zymotic fluid is 1:1, that is, use the ethyl acetate of 50ml to extract tunning；Rotary Evaporators recycle ethyl acetate drying extraction Object, 1ml methanol is taken to redissolve extract.

2) efficient liquid phase (HPLC) chromatography detects：

3. experimental result and analysis：

It is found by HPLC analyses, the O- transmethylases of separate sources can be by flavone compound by heterogenous expression The glycosylated compound of F-4 converts, and has obtained the peak of other sugared methylate.This illustrates the O- first in different entomogenous fungis Based transferase homologous gene has similar substrate spectrum, can successfully realize the glycosylated derivative Portugal to flavone compound Methylating on grape 4 hydroxyls of sugar, to achieve the purpose that improve glycosylated compound stability, there are the consistency of function. The recombinant bacterial strain substrate of CpOMT, MrOMT, CmOMT and IfOMT feed the chromatogram of tunning and see Figure 29.

Sequence table

<110>Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>The methylated transferase gene of glucoside compound

<130>

<160> 10

<170> PatentIn version 3.1

<210> 1

<211> 750

<212> DNA

<213>Beauveria bassiana (Beauveria bassiana)

<400> 1

ATGGCTCTCG TCGAAAAAAT TCAGTTGACA GACGATTTTT CTGTCTATGC AAATCCAGCC 60

GCTAAGCTTG AAGTGGAATT TATTCACAAA GAAATCTTCA TCGACAAGTG CTATGATGTC 120

GCGCCATTTC CCGACGATAG CTTCATAGTC GATGCTGGTG GCAACATTGG CATGTTCACC 180

CTGTACATGA AGAGAAAATA TCCACAATCA ACCATTCTCG CTTTTGAGCC TGCTCCGGCT 240

ACGTTTTCTA CTTTTCAGCG CAATATGGAA TTACACAACG TTTCCGGTGT ACAAGCCCAT 300

CAATGTGGGC TCGGCAGGGA AGATGCAAGT CTGGCCTTGA CGTTCTACCC GCAGATGCCA 360

GGCAACTCGA CGCTGTATGC CGAGGACAAA ACGAACCAAA TGAAGTCTGT GGACCAAAAT 420

CACCCTATCG CCAAGCTTAT GCAAGAGACG CATGAGGTGC AAGTTGATGT GAAACGGCTG 480

TCGGATTTCC TTGGCGAGGT CCCCAATCTG AAACGCGTCA ACCTGCTCAA GGTAGACGTG 540

GAGGGCGCCG AGATGGATGT GTTACGAGGT TTAGATGACG AGCATTGGGA TCTGATTGAC 600

AATGTTGTAG TCGAGCTTTG CGACAGCAAA GGAGACTTTG CCACGGCCAA GACTCTGCTG 660

GAATCCAAGG GATTTGCGGT TGCTGTAGAG AGGCCCGACT GGGCACCACC AGATCTAAAG 720

ATGTACATGT TGATCGCAAA AAGAAACTGA 750

<210> 2

<211> 249

<212> PRT

<213>Beauveria bassiana (Beauveria bassiana)

<400> 2

Met Ala Leu Val Glu Lys Ile Gln Leu Thr Asp Asp Phe Ser Val Tyr

1 5 10 15

Ala Asn Pro Ala Ala Lys Leu Glu Val Glu Phe Ile His Lys Glu Ile

20 25 30

Phe Ile Asp Lys Cys Tyr Asp Val Ala Pro Phe Pro Asp Asp Ser Phe

35 40 45

Ile Val Asp Ala Gly Gly Asn Ile Gly Met Phe Thr Leu Tyr Met Lys

50 55 60

Arg Lys Tyr Pro Gln Ser Thr Ile Leu Ala Phe Glu Pro Ala Pro Ala

65 70 75 80

Thr Phe Ser Thr Phe Gln Arg Asn Met Glu Leu His Asn Val Ser Gly

85 90 95

Val Gln Ala His Gln Cys Gly Leu Gly Arg Glu Asp Ala Ser Leu Ala

100 105 110

Leu Thr Phe Tyr Pro Gln Met Pro Gly Asn Ser Thr Leu Tyr Ala Glu

115 120 125

Asp Lys Thr Asn Gln Met Lys Ser Val Asp Gln Asn His Pro Ile Ala

130 135 140

Lys Leu Met Gln Glu Thr His Glu Val Gln Val Asp Val Lys Arg Leu

145 150 155 160

Ser Asp Phe Leu Gly Glu Val Pro Asn Leu Lys Arg Val Asn Leu Leu

165 170 175

Lys Val Asp Val Glu Gly Ala Glu Met Asp Val Leu Arg Gly Leu Asp

180 185 190

Asp Glu His Trp Asp Leu Ile Asp Asn Val Val Val Glu Leu Cys Asp

195 200 205

Ser Lys Gly Asp Phe Ala Thr Ala Lys Thr Leu Leu Glu Ser Lys Gly

210 215 220

Phe Ala Val Ala Val Glu Arg Pro Asp Trp Ala Pro Pro Asp Leu Lys

225 230 235 240

Met Tyr Met Leu Ile Ala Lys Arg Asn

245

<210> 3

<211> 750

<212> DNA

<213>Fumosorosea（Isaria fumosorosea）

<400> 3

ATGGCTGCCG TGCAAAAAAT CCAACTTGCA GATGACTTTT CCGTCTACGC AAACCCCGCA 60

GCAAAGCTCG AGGTCGAGTT CATCTACAAG GAAATCTTCG TCGATGGGTG CTACAACAAC 120

GCGTCGATCC CCGACGACGC CTTCATCGTC GACGCCGGAG GCAACATTGG CATGTTCAGC 180

CTTTTCATGA AGAAGAAATA TCCGCAATCG ACTATCCTCG CTTTTGAGCC TGCGCCGGCT 240

ACCTTTTCCA CCTTTCAGCG CAACATGGAG CTGCACGGTG TTTCTGGGGT GCAGGCCCAT 300

CAATGCGGGC TCGGCAAGGA GAACGCCAGC ATGGCCCTGA CCTTTTACCC GCAGATGCCT 360

GGCAACTCGA CGCTATACCT AGAGGACAAG AAGAACCAGA TGAAGTCTAT CGACAAGGAG 420

CACCCCATCG CCAAGCTCAT GCAGGAGACG GAAGAGGTGC AGGTGGACGT GAAGCGGCTG 480

TCCGAATTCC TCGACCGCGT GCCGGACCTG AAACGCGTTG ACCTGCTCAA GATAGACGTC 540

GAGGGTGCTG AGCTGGACGT GCTGAAGGGT CTGGACGACA AGCACTGGAA CCTGATTAAC 600

AATATTGTGA TCGAACTTTG CGACAGCAAG AGCGAGTTCG CCATCACCAA GGCACTGCTG 660

GAATCGAAAG GGTTCACGGT TGCGATAGAA CGGCCTGACT GGGCACCCGA AGACCTCAAG 720

ATGTACATGT TGATCGCGAA CAGACGCTAA 750

<210> 4

<211> 249

<212> PRT

<213>Fumosorosea（Isaria fumosorosea）

<400> 4

Met Ala Ala Val Gln Lys Ile Gln Leu Ala Asp Asp Phe Ser Val Tyr

1 5 10 15

Ala Asn Pro Ala Ala Lys Leu Glu Val Glu Phe Ile Tyr Lys Glu Ile

20 25 30

Phe Val Asp Gly Cys Tyr Asn Asn Ala Ser Ile Pro Asp Asp Ala Phe

35 40 45

Ile Val Asp Ala Gly Gly Asn Ile Gly Met Phe Ser Leu Phe Met Lys

50 55 60

Lys Lys Tyr Pro Gln Ser Thr Ile Leu Ala Phe Glu Pro Ala Pro Ala

65 70 75 80

Thr Phe Ser Thr Phe Gln Arg Asn Met Glu Leu His Gly Val Ser Gly

85 90 95

Val Gln Ala His Gln Cys Gly Leu Gly Lys Glu Asn Ala Ser Met Ala

100 105 110

Leu Thr Phe Tyr Pro Gln Met Pro Gly Asn Ser Thr Leu Tyr Leu Glu

115 120 125

Asp Lys Lys Asn Gln Met Lys Ser Ile Asp Lys Glu His Pro Ile Ala

130 135 140

Lys Leu Met Gln Glu Thr Glu Glu Val Gln Val Asp Val Lys Arg Leu

145 150 155 160

Ser Glu Phe Leu Asp Arg Val Pro Asp Leu Lys Arg Val Asp Leu Leu

165 170 175

Lys Ile Asp Val Glu Gly Ala Glu Leu Asp Val Leu Lys Gly Leu Asp

180 185 190

Asp Lys His Trp Asn Leu Ile Asn Asn Ile Val Ile Glu Leu Cys Asp

195 200 205

Ser Lys Ser Glu Phe Ala Ile Thr Lys Ala Leu Leu Glu Ser Lys Gly

210 215 220

Phe Thr Val Ala Ile Glu Arg Pro Asp Trp Ala Pro Glu Asp Leu Lys

225 230 235 240

Met Tyr Met Leu Ile Ala Asn Arg Arg

245

<210> 5

<211> 759

<212> DNA

<213>Robert green muscardine fungus（Metarhizium robertsii）

<400> 5

ATGGCGGCCG AAATACAGAA ACTAGAAATG TCGGACGGTT TTCTCGTCTA CGCCAACCCC 60

AAGGCAGCCA TGGAGACGCA ATTCATCCAC AAGGAAATCT TTCAAGACAA ATGTTATGAT 120

GTCGCACCCT TCCCCGAGGA CGCCTTCATG ATTGATGCGG GAGGCAACAT TGGCATGTTC 180

AGCCTGTACA TGAAGAAGAA ATACCCGGCT GCCACAATCC TAGCATTCGA ACCCGCACCG 240

ACGACTTTCA ACACATTCAA GAAGAACATG GAACTGCACA ACATTTCAGG CGTGCAGGTC 300

TACCAGTGCG GGCTGGGTCG TGAGAATTCC AACGAGACGT TGACTTTTTA TCCCAACATG 360

CCCGGCAATT CGACCTTGCA TGGTGGCGAA AAAGAAGAGT TTATCAAGAC GGCAGATTCT 420

GAACACCCTG TTATTAAGTT GCTAAGCGAG GTCGAGCAGG TTCAGGTCGA CGTCAAGCGA 480

CTATCGGGGT TTCTAAACGA TCTTCCCGAC CTGAAGCGGA TTGACTTGCT CAAGATTGAT 540

GTGGAAGGTG CGGAGCTGGA CATCTTCCGC GGACTGGACA ATGTACACTG GGACTTGATT 600

GAAAACATTG TCTTGGAGAT TTGTGACCAC AATGGAGCAT TGGAAGAAGC TGAAGCGCTT 660

TTGCGAGAGA AGGGATTTGA GACTTCCAAG GAGTTGGCGG ACTGGGCGCC GAAAGAGATG 720

CCGATGTACA TGATGGTAGC TAAACGGGCT CATCACTAG 759

<210> 6

<211> 252

<212> PRT

<213>Robert green muscardine fungus（Metarhizium robertsii）

<400> 6

Met Ala Ala Glu Ile Gln Lys Leu Glu Met Ser Asp Gly Phe Leu Val

1 5 10 15

Tyr Ala Asn Pro Lys Ala Ala Met Glu Thr Gln Phe Ile His Lys Glu

20 25 30

Ile Phe Gln Asp Lys Cys Tyr Asp Val Ala Pro Phe Pro Glu Asp Ala

35 40 45

Phe Met Ile Asp Ala Gly Gly Asn Ile Gly Met Phe Ser Leu Tyr Met

50 55 60

Lys Lys Lys Tyr Pro Ala Ala Thr Ile Leu Ala Phe Glu Pro Ala Pro

65 70 75 80

Thr Thr Phe Asn Thr Phe Lys Lys Asn Met Glu Leu His Asn Ile Ser

85 90 95

Gly Val Gln Val Tyr Gln Cys Gly Leu Gly Arg Glu Asn Ser Asn Glu

100 105 110

Thr Leu Thr Phe Tyr Pro Asn Met Pro Gly Asn Ser Thr Leu His Gly

115 120 125

Gly Glu Lys Glu Glu Phe Ile Lys Thr Ala Asp Ser Glu His Pro Val

130 135 140

Ile Lys Leu Leu Ser Glu Val Glu Gln Val Gln Val Asp Val Lys Arg

145 150 155 160

Leu Ser Gly Phe Leu Asn Asp Leu Pro Asp Leu Lys Arg Ile Asp Leu

165 170 175

Leu Lys Ile Asp Val Glu Gly Ala Glu Leu Asp Ile Phe Arg Gly Leu

180 185 190

Asp Asn Val His Trp Asp Leu Ile Glu Asn Ile Val Leu Glu Ile Cys

195 200 205

Asp His Asn Gly Ala Leu Glu Glu Ala Glu Ala Leu Leu Arg Glu Lys

210 215 220

Gly Phe Glu Thr Ser Lys Glu Leu Ala Asp Trp Ala Pro Lys Glu Met

225 230 235 240

Pro Met Tyr Met Met Val Ala Lys Arg Ala His His

245 250

<210> 7

<211> 759

<212> DNA

<213>Purple ergot（Claviceps purpurea）

<400> 7

ATGGCCACCG CAAATTTACA AAAAGTGCAA CTCGCTGATG ATTTAGCCGT CTACGCAAAC 60

TCAGGCGCCG AATTCGAGAC CCAGTTCCTC TACAGGGAAA TCTTCGGAGA CAAGTGCTAC 120

GACACAGGCC CTCTGCCCGA AGACGCAGTC ATCATCGACG CAGGCGCCAA CATCGGCATG 180

TTCAGCCTAT ACATCAAGCG GCAGTGTCCC GGGGCGCGCA TCACGGCCTT TGAGCCCGCG 240

CCCGATACGG CGGCGGCGCT GAGGCTCAAT CTGGCGCTGC ATAAGGTGCA TGGGGTCGAG 300

GTGCACGAGT GCGCGCTGGG AAGCCAGGAC TGTGAGATGA AGTTGACGTA CTTTCCCAAT 360

ATGCCGGGAA ACTCGACATT GCATGGCGAT GATGAACCGG CCATCTTTGC GGGAGAGGTC 420

GGGCGCGCGC ATCCGGTGGC GCGGCTGCGG GAGGAGCGGC GGGAGGTGCC GGTGCCGGTG 480

CGGCGGCTAT CGGATGTTTT GCGGCAGATG CCGGGACTAG AGCGCGTGGA TCTGCTCAAA 540

ATCGACGTCG AAGGCGCCGA ACTAGACGTC CTGCGGGGAC TAGACGATGA TCATTGGGAG 600

CTAGTGCGCC GTATCGTCAT GGAGGTGGGC GACGAACACG GCGATCTGGC GGCCGCCGAG 660

ACTCTGCTGC GGGGGCGCGG CTTCGAGGTC GTGAGCGAGC GCGCGGCGTG GGCGCCAGAG 720

ACGTTGCCCA TGTATACCTT GATGGCGCGA AGGTGA 759

<210> 8

<211> 251

<212> PRT

<213>Purple ergot（Claviceps purpurea）

<400> 8

Met Ala Thr Ala Asn Leu Gln Lys Val Gln Leu Ala Asp Asp Leu Ala

1 5 10 15

Val Tyr Ala Asn Ser Gly Ala Glu Phe Glu Thr Gln Phe Leu Tyr Arg

20 25 30

Glu Ile Phe Gly Asp Lys Cys Tyr Asp Thr Gly Pro Leu Pro Glu Asp

35 40 45

Ala Val Ile Ile Asp Ala Gly Ala Asn Ile Gly Met Phe Ser Leu Tyr

50 55 60

Ile Lys Arg Gln Cys Pro Gly Ala Arg Ile Thr Ala Phe Glu Pro Ala

65 70 75 80

Pro Asp Thr Ala Ala Ala Leu Arg Leu Asn Leu Ala Leu His Lys Val

85 90 95

His Gly Val Glu Val His Glu Cys Ala Leu Gly Ser Gln Asp Cys Glu

100 105 110

Met Lys Leu Thr Tyr Phe Pro Asn Met Pro Gly Asn Ser Thr Leu His

115 120 125

Gly Asp Asp Glu Pro Ala Ile Phe Ala Gly Glu Val Gly Arg Ala His

130 135 140

Pro Val Ala Arg Leu Arg Glu Glu Arg Arg Glu Val Pro Val Pro Val

145 150 155 160

Arg Arg Leu Ser Asp Val Leu Arg Gln Met Pro Gly Leu Glu Arg Val

165 170 175

Asp Leu Leu Lys Ile Asp Val Glu Gly Ala Glu Leu Asp Val Leu Arg

180 185 190

Gly Leu Asp Asp Asp His Trp Glu Leu Val Arg Arg Ile Val Met Glu

195 200 205

Val Gly Asp Glu His Gly Asp Leu Ala Ala Ala Glu Thr Leu Leu Arg

210 215 220

Gly Arg Gly Phe Glu Val Val Ser Glu Arg Ala Ala Trp Ala Pro Glu

225 230 235 240

Thr Leu Pro Met Tyr Thr Leu Met Ala Arg Arg

245 250

<210> 9

<211> 747

<212> DNA

<213>Cordyceps militaris（Cordyceps militaris）

<400> 9

ATGGCTTTGG AAAAAATACA GCTAGCAGAT GACTTTGCCG TCTATGCAAA CCCTGCGGCC 60

AAGCTCGAGG TCGAATTCAT CTACAAGGAA ATCTTCACCG ACAAGTGCTA CGAGATTGCG 120

TCGCTGCCCG ACGATGCCTT CATGGTCGAC GCCGGCGGCA ACATTGGCAT GTTCAGCCTC 180

TTCATGAAGA AGAAGTATCC GTCCTCGACC ATTCTCGCCT TTGAGCCTGC GCCGGCGACT 240

TTTTCCGCCT TTGAGCGCAA CATGGCGCTG CACGGCGTCT CGGGCGTGCA AGCGCATCAG 300

TGCGGACTCG GTCGGGAGAA TGCGACCATG GCCTTGACGT TTTACCCGCA GATGCCGGGC 360

AATTCGACCC TGTACTCGGA GGACAAGGCG AATCAGATGA AGTCTGTCGA CGAACATCAC 420

CCCGTTGCCA AACTCATGCA GGAAACGCAA GAAGTGCAGG TGGATGTCAA GCGACTTTCT 480

GATTTCCTCA ACCAGGTCCC GGCCCTCAAA CGAATTGACC TTGTCAAGGT GGATGTGGAA 540

GGCGCCGAGC TGGACGTGTT GCTGGGCCTG GACGACAGGC ACTGGGACAT GATTCAGAAT 600

ATTGCAGTCG AGCTCTGCGA CAGCAAGGGC GAGCTCGCCG AGGCCAAGGC GCTGCTAGAG 660

GCGAAAGGGT TTTCAGTTGT GACAGAGAGG CCTGACTGGG CACCGGAGAA CCTGAAGATG 720

TATATGCTAG TTGCAAAGAG AAACTAG 747

<210> 10

<211> 248

<212> PRT

<213>Cordyceps militaris（Cordyceps militaris）

<400> 10

Met Ala Leu Glu Lys Ile Gln Leu Ala Asp Asp Phe Ala Val Tyr Ala

1 5 10 15

Asn Pro Ala Ala Lys Leu Glu Val Glu Phe Ile Tyr Lys Glu Ile Phe

20 25 30

Thr Asp Lys Cys Tyr Glu Ile Ala Ser Leu Pro Asp Asp Ala Phe Met

35 40 45

Val Asp Ala Gly Gly Asn Ile Gly Met Phe Ser Leu Phe Met Lys Lys

50 55 60

Lys Tyr Pro Ser Ser Thr Ile Leu Ala Phe Glu Pro Ala Pro Ala Thr

65 70 75 80

Phe Ser Ala Phe Glu Arg Asn Met Ala Leu His Gly Val Ser Gly Val

85 90 95

Gln Ala His Gln Cys Gly Leu Gly Arg Glu Asn Ala Thr Met Ala Leu

100 105 110

Thr Phe Tyr Pro Gln Met Pro Gly Asn Ser Thr Leu Tyr Ser Glu Asp

115 120 125

Lys Ala Asn Gln Met Lys Ser Val Asp Glu His His Pro Val Ala Lys

130 135 140

Leu Met Gln Glu Thr Gln Glu Val Gln Val Asp Val Lys Arg Leu Ser

145 150 155 160

Asp Phe Leu Asn Gln Val Pro Ala Leu Lys Arg Ile Asp Leu Val Lys

165 170 175

Val Asp Val Glu Gly Ala Glu Leu Asp Val Leu Leu Gly Leu Asp Asp

180 185 190

Arg His Trp Asp Met Ile Gln Asn Ile Ala Val Glu Leu Cys Asp Ser

195 200 205

Lys Gly Glu Leu Ala Glu Ala Lys Ala Leu Leu Glu Ala Lys Gly Phe

210 215 220

Ser Val Val Thr Glu Arg Pro Asp Trp Ala Pro Glu Asn Leu Lys Met

225 230 235 240

Tyr Met Leu Val Ala Lys Arg Asn

245

Claims

1. a kind of application of transmethylase in glucoside compound methylates modification, the transmethylase have following two Level structure：

And their amino acid sequence is selected from SEQ ID NO.2,4,6,8 or 10.

2. application of the gene of transmethylase described in coding claim 1 in glucoside compound methylates modification, described The nucleotide sequence of gene is respectively as shown in SEQ ID NO.1,3,5,7 or 9.

3. application as claimed in claim 1 or 2, the glucoside compound methylates modification, is glycosyl in glucoside compound 4 hydroxymethylations modification.

4. the application described in claim 3, the glucoside compound is the glucosides of polyphenol compound, the Polyphenols chemical combination Object is selected from flavonoids, polyketone class or anthraquinone analog compound.

5. application as claimed in claim 1 or 2, the modification that methylates is external glycosylation modified.

6. in 4 hydroxymethylations modification of the glycosyl in glucoside compound of the recombinant plasmid containing gene described in claim 2 Application.