CN108624572B - Methylation transferase gene of glycoside compounds - Google Patents

Abstract

The invention relates to a methyltransferase gene of a glycoside compound, which can carry out methylation modification on 4-hydroxyl of glycosyl in the glycoside compound, can act synergistically with glycosyltransferase, and can convert different active substances including polyketone, flavonoid, anthraquinone and the like through combined biosynthesis, thereby obtaining a novel active compound different from the structures of natural products, realizing the directional biotransformation of the compound and enhancing the stability of the compound.

Description

Methylation transferase gene of glycoside compounds

The technical field is as follows:

the invention relates to a gene capable of methylating glucose 4-hydroxy in glycosylated substances, wherein the glycosylated substances specifically comprise glycosylated derivatives of polyphenol compounds such as polyketones, flavonoids, anthraquinones and the like.

Background art:

various natural and non-natural glycosylated substances are easily affected by various factors from organisms and the outside, so that the substances are degraded and the activity is reduced. It is advantageous to increase the stability of these materials if the hydroxyl group at the 4-position of the sugar group is alkylated.

Fungal biotransformation involves the structural modification and engineering of exogenous compounds using specific enzymes within the cell to obtain more valuable metabolic reactions.

The invention content is as follows:

the invention aims to obtain an O-methyltransferase gene which can participate in the methylation of the 4-hydroxyl of glycosyl in a glycosyl compound, realize the methylation modification of the glycosyl compound, obtain a natural product with a novel structure and activity and enhance the stability of the natural product.

Use of a methyltransferase in the methylation modification of a glycoside compound, the methyltransferase having the secondary structure:

starting from the N-terminus, 3 (α + β tandem) + β sheet +3 (α + β tandem) + β sheet;

and their amino acid sequences are selected from SEQ ID NO.2, 4, 6, 8 or 10.

Specifically, the following five specific O-methyltransferases, namely BbFkbM, CpOMT, MrOMT, IfOMT and CmOMT, were found from Beauveria bassiana, Isaria fumosorosea, Metarhizium robertsii, Claviceps purpurea and Cordyceps militaris CM 01. All five proteins have the same motif and arrangement (see FIGS. 30-32), and the amino acid sequences of the active centers are highly consistent and consistent in polarity.

Respectively amplifying glucose O-methyltransferase gene BbFkbM by cDNA, obtaining CpOMT, MrOMT, IfOMT and CmOMT by DNA synthesis, respectively constructing recombinant plasmids capable of being expressed in yeast, co-transferring the recombinant plasmids and glucosyltransferase heterologous expression vectors existing in laboratories into yeast, and performing heterologous expression and transformation in the yeast.

By adding different glycosylation derivatives from outside sources or carrying out combined biosynthesis with glucosyltransferase, a sugar methylation product with a structure different from that of the original compound is extracted from a fermentation product.

Experiments prove that the five O-methyltransferase genes can carry out methylation modification on glycosylation derivatives of various flavonoids, polyketones and anthraquinones through heterologous expression, the obtained product has enhanced hydrolytic stability compared with the product without modification, the degradation effect of glycoside hydrolase in yeast can be counteracted, and the stability in organisms is enhanced. Moreover, the homologous genes not only have the same function, but also can be combined with different glycosyltransferases for biosynthesis, so that different natural active products are modified, the transformation efficiency is improved, and a new research method and a new way are provided for discovery of lead compounds and directed transformation of medicaments.

Taking BbFkbM as an example, the following specific research process is illustrated:

1. obtaining BbFkbM Gene

The glycosyltransferase is structurally analyzed through genome sequencing and related prediction software, primers are designed, and a beauveria bassiana O-methyltransferase gene BbFkbM is obtained through cDNA amplification. Wherein, the nucleotide sequence of BbFkbM is shown in SEQ ID NO. 1.

2. Construction of yeast expression vector for glucosyltransferase BbFkbM and yeast transformation

The DNA fragment containing the complete BbFkbM is connected to PXW06F vector to construct a constitutive expression plasmid PXW06F-BbFkbM, and simultaneously the constitutive expression plasmid and a glucosyltransferase heterologous expression vector PRS425m-BbUGT86 are transferred into an auxotrophic yeast acceptor BJ5464 for heterologous expression.

3. Screening of recombinants

Transferring the expression vector into a deletion yeast incapable of synthesizing leucine and tryptophan, and screening a recombinant by utilizing leucine and tryptophan auxotroph markers.

4. Polyketide feeding conversion test and HPLC liquid phase analysis of conversion product

And respectively adding a polyketone compound Desmethyl-Lasiodipodin (hereinafter referred to as a compound 1) in the culture process of the recombinant strain, extracting a fermentation transformation product by using an organic solvent, and carrying out HPLC (high performance liquid chromatography) liquid phase analysis and nuclear magnetic resonance analysis on the product structure.

Almost complete conversion of compound 1 was found. The liquid phase results showed that another peak was obtained in addition to the peak of compound 1. Further structural analysis of the separated product proves that the polyketide compound 2 with a structure different from that of the natural product compound 1 is obtained, and high-resolution mass spectrum and nuclear magnetic resonance analysis show that the compound 2 is formed by adding glucose to the hydroxyl of the 5-position carbon atom of the original product compound 1 and methylating the hydroxyl on the 4-position of the glucose. The results show that the O-methyltransferase gene can be used for modifying the structure of polyketide glycosylation derivatives of hydroquinone lactone and analogues thereof, is very suitable for modifying the molecular structure of various active compounds, and increases the diversity of polyketide libraries. The chromatogram and product structure of the recombinant strain are shown in FIG. 3.

5. Feeding transformation test and HPLC liquid phase analysis of transformation products of flavonoids compounds

Adding different flavonoids compounds in the culture process of the recombinant strain, extracting the fermentation conversion product with an organic solvent, performing HPLC liquid phase analysis, and analyzing the product components by high-resolution mass spectrometry.

6. Comparing the stability of the sugar methylation modification product compound 2 and the glycosylation derivative of the compound before modification, respectively adding the purified sugar methylation modification product compound 2 and the glycosylation derivative of the compound before modification into wild yeast fermentation liquor, culturing for the same time, extracting a crude extract, performing HPLC liquid phase analysis, and detecting the stability of the two substances under the action of a self enzyme system in a yeast body.

7. Influence of Yeast glycoside hydrolase EXG1 on stability of sugar methylation modification product Compound 2 and glycosylation derivative of Pre-modification Compound purified glycosylation modification product Compound 2 and glycosylation derivative of Pre-modification compound are added into yeast fermentation broth of wild type and knockout glycoside hydrolase EXG1 respectively, cultured for the same time, and subjected to HPLC liquid phase analysis by extracting crude extract to detect stability of two substances under action of Yeast self glycoside hydrolase.

8. Functional verification of homologous genes in entomogenous fungi

98 homologous protein sequences with amino acid sequence similarity of more than 30% and coverage rate of more than 90% are obtained from different entomogenous fungi by comparing the obtained amino acid sequences of glycosyltransferase and O-methyltransferase of beauveria bassiana with related bioinformatics software (partial sequences are shown in Table 1). 4O-methyltransferase genes in Isaria fumososea, Metarhizium robertsii, Claviceps purpureus purpurea and Cordyceps militaris are selected from sequences with amino acid sequence identity of 50%, similarity of 70% and coverage rate of more than 99% for heterologous expression (see underlined contents in Table 1), and the sequence numbers in NCBI are XP _007822447.1, XP _018701797.1, CCE31609.1 and XP _006674755.1 respectively. Through a substrate feeding test, glycosylated derivatives of polyketone compounds 1, flavonoid compounds F-4 and the like are successfully converted, and the similar functions of homologous O-methyltransferases in fungi are verified. Meanwhile, different conversion efficiencies can be achieved by combining different OMTs.

TABLE 1 list of homologous genes of the O-methyltransferase gene BbFkbM

Description of the drawings:

FIG. 1 shows the result of amplification of a BbFkbM gene fragment;

FIG. 2 is a physical map of recombinant vector PXW 06F-BbFkbM;

FIG. 3 is a liquid chromatogram before modification of Compound 1;

FIG. 4 is an HPLC liquid chromatogram of Compound 2 modified to produce a sugar methylation of Compound 1.

FIGS. 5-14 are chromatogram and mass spectrum results of sugar methylation products after conversion of the substrate flavone sample tested, wherein:

FIG. 5 shows the results of chromatogram and mass spectrum after conversion of F-4 compound;

FIG. 6 shows the results of chromatogram and mass spectrum after conversion of F-5 compound;

FIG. 7 shows the results of chromatogram and mass spectrum after conversion of F-6 compound;

FIG. 8 shows the results of chromatogram and mass spectrum after conversion of F-7 compound;

FIG. 9 shows the results of chromatogram and mass spectrum after conversion of F-8 compound;

FIG. 10 shows the results of chromatogram and mass spectrum after conversion of F-11 compound;

FIG. 11 shows the results of chromatogram and mass spectrum after conversion of F-12 compound;

FIG. 12 shows the results of chromatogram and mass spectrum after conversion of F-15 compound;

FIG. 13 shows the results of chromatogram and mass spectrum after conversion of F-16 compound;

FIG. 14 shows the results of chromatogram and mass spectrum after conversion of F-20 compound;

FIGS. 15 and 16 are liquid chromatograms of hydrolysates of Compound 3 after 24h and 48h of incubation, respectively;

FIGS. 17 and 18 are liquid chromatograms of hydrolysates of Compound 2 after 24h and 48h of incubation, respectively;

FIG. 19 shows the results of PCR-verified electrophoresis of the EXG1 knockout mutant;

FIGS. 20 and 21 are liquid chromatograms of hydrolysates of Compound 3 during culture of wild-type and mutant yeasts, respectively;

FIGS. 22 and 23 are liquid chromatograms of hydrolysates of Compound 2 during culture of wild-type and mutant yeasts, respectively;

FIGS. 24 to 27 are physical maps of recombinant vectors PXW06F-IfOMT, PXW06F-MrOMT, PXW06F-CpOMT and PXW06F-CmOMT, respectively.

FIG. 28 is an HPLC liquid chromatogram of Compound 3 modified with five fungal OMTs, resulting in a single sugar methylated Compound 2;

FIG. 29 is an HPLC liquid chromatogram of a glycosylation product of compound F-4 modified by five fungal OMTs, resulting in a sugar methylation product;

FIG. 30 shows the alignment of BbFkbM with CpOMT, MrOMT, IfOMT, CmOMT protein sequences, the straight line marking the region as the active center of the protein, the bar graph showing the sequence identity and the gray scale of the letter indicating the polarity of amino acids.

FIG. 31 shows the results of motif analysis of protein sequences of BbFkbM and CpOMT, MrOMT, IfOMT, and CmOMT.

FIG. 32 shows the protein secondary structures of BbFkbM with CpOMT, MrOMT, IfOMT, and CmOMT.

Description of sequence listing

A nucleotide sequence of SEQ ID No.1 BbFkbM;

SEQ ID NO.2 the amino acid sequence encoded by BbFkbM deduced from SEQ ID NO. 1.

A nucleotide sequence of SEQ ID No.3 IfOMT;

SEQ ID NO.4 the amino acid sequence encoded by IfOMT deduced from SEQ ID NO. 3.

A nucleotide sequence of SEQ ID No.5 MrOMT;

SEQ ID NO.6 the amino acid sequence encoded by MrOMT deduced from SEQ ID NO. 5.

The nucleotide sequence of SEQ ID No.7 CpOMT;

SEQ ID NO.8 the amino acid sequence encoded by CpOMT deduced from SEQ ID NO. 7.

The nucleotide sequence of SEQ ID No.9 CmOMT;

SEQ ID NO.10 the amino acid sequence encoded by CmOMT deduced from SEQ ID NO. 9.

Detailed Description

The experimental materials used in the following experiments are illustrated and derived as follows:

the following strains and vectors: beauveria bassiana ATCC7159, Isaria fumosorosea ACCC37775, Metarhizium robertii ARSEF 23, Claviceps purpurea ACCC337002, Cordyceps militaris CM01, yeast S.cerevisiae BJ5464-NpgA, yeast expression vectors PRS425m-BbUGT86 and PXW06F, which are all from the institute of biotechnology of Chinese academy of agricultural sciences, were stored by the laboratory of the present inventors.

Enzyme and kit:

restriction enzymes, T4DNA ligase were purchased from NEB;

RNA reverse transcription kit purchased from TAKARA;

Quick-Fusion Cloning Kit was purchased from Biotool;

the hot start high fidelity DNA amplification kit is purchased from Biotool company;

DNA polymerase and DNA marker were purchased from Beijing Quanjin Biopsis;

the plasmid mini-extraction kit and the universal DNA purification gel recovery kit are purchased from Tiangen company;

the frozen yeast transformation kit was purchased from YMO RESEARCH Biometrics;

coli DH5 α was obtained from Kangwei century; other reagents are all domestic analytical pure products.

Culture medium:

the E.coli medium was LB medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). SC-^-Leu deficient medium (1% glucose, 6.7% Difco)^TMYeast Nitrogen Base w/o Amino Acids, -Leu/-Trp DO Supplement). YPD medium (1% yeast extract, 2% Peptone, 2% glucose). YPD low-sugar medium (1% yeast extract, 2% Peptone, 1% glucose.) if solid medium is prepared, 2% agar powder is added.

Other experimental procedures not specifically indicated for the conditions in the examples were carried out according to conventional methods, molecular cloning being carried out according to the conditions described in the Laboratory manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions recommended by the manufacturer.

Compound 1: Desmethyl-Lasiodipodin

Compound 2: methylation product of hydroxyl at 4-glycosyl of compound 1

Compound 3: compound 1 glycosylation product

Example 1 Synthesis of O-methyltransferase Gene BbFkbM and construction of Yeast expression vector to obtain Yeast transformant

The genome sequence of the strain is obtained by carrying out genome sequencing on the obtained beauveria bassiana, and an O-methyltransferase gene is discovered through comparison in the process of analyzing glycosyltransferase through related bioinformatics software, and is named as BbFkbM. The nucleotide sequence of the BbFkbM is subjected to prediction analysis by various software to remove intron splicing to obtain the coding region sequence of the BbFkbM. Extracting RNA of beauveria bassiana to obtain cDNA through reverse transcription, amplifying a coding gene of BbFkbM by using a designed primer, recovering a target fragment, and delivering the target fragment to a sequencing company for sequencing comparison to obtain a glycosyltransferase gene BbFkbM shown in SEQ ID NO. 1.

The enzyme cutting site is introduced during synthesis. An NdeI restriction site is added in front of a promoter, a PmeI restriction site is added behind a terminator, and the length of a fragment is 0.75 Kb. The BbFkbM amplified fragment is shown in FIG. 1.

Shuttle vector PXW06F was double digested with NdeI and pmeI and a 6114bp sized fragment was recovered. The two fragments were ligated using a seamless ligation cloning kit.

Heat shock method transformed into Escherichia coli competent DH5 alpha. Amp^rResistance screening, plasmid extraction, and enzyme digestion identification of ScaI and Nde I. The map of the recombinant plasmid PXW06F-BbFkbM is shown in FIG. 2.

Saccharomyces cerevisiae BJ5464-NpgA is inoculated into YPD culture medium, cultured at 30 ℃ and 200r/min until the O.D. value is 0.8-1.0, and Saccharomyces cerevisiae competent cells are prepared according to the instructions provided by ZYMO company and recombinant plasmid transformation is carried out. PRS425m-BbUGT86 containing the glycosylation transferase gene and PXW06F-BbFkbM recombinant plasmid containing O-methyltransferase are co-transformed into saccharomyces cerevisiae, spread on a leucine and tryptophan deficient SC solid defective culture medium, and cultured for about 3 days at 30 ℃. The obtained yeast transformant was streaked and cultured to a novel SC-^-Leu/^-Culturing in a 30 ℃ incubator for about 2 days on a Trp defect culture medium;

example 2 modification of glycosylated derivative of polyketide Desmethyl-Lasiodipodin (Compound 1) with recombinant vector PXW 06-06F-BbFkbM for O-methyltransferase

1. Purpose of experiment

The metabolites modified by glucosyltransferase and O-methyltransferase BbFkbM were obtained by HPLC high performance liquid separation and their molecular structures were analyzed.

2. The experimental method comprises the following steps:

1) fermentation culture

Adopting a two-step fermentation technology, firstly inoculating a proper amount of yeast transformant thalli to corresponding 25-mL^-Leu/^-Culturing in Trp liquid-deficient culture medium at 30 deg.C for about 16h at 200r min-1, adding YPD low-sugar culture medium 25ml, and simultaneously adding 5mg Desmethyl-Lasiodipodin pure product respectively, and culturing for 48 h; extracting the fermentation product with ethyl acetate at a ratio of ethyl acetate to fermentation broth of 1:1, namely extracting the fermentation product with 50ml of ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

2) High Performance Liquid Chromatography (HPLC) detection:

and (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: performing gradient elution on a chromatographic column Kromasil 100-5-C18 by using acetonitrile-H2O as a mobile phase, wherein the gradient elution conditions comprise that the gradient elution conditions are 0-5 min and the acetonitrile is 10%; 5-15 min, acetonitrile from 10% → 95%; 15-25 min, and the acetonitrile is 95%; 25-28 min, acetonitrile from 95% → 10%; 28-31 min, and acetonitrile is 10%. The flow rate was 0.8mL min-1, and the detection wavelength was 300 nm.

3. Experimental results and analysis:

HPLC analysis shows that the peak of the natural polyketide 1 (Desmethyl-Lasiodipodin) almost disappears, and another peak is obtained, which indicates that the natural product compound 1 is almost completely converted.

Mass spectrometry and NMR analysis of the isolated product demonstrated that most of Compound 1 was converted to Compound 2 by modification with glucosyltransferase and O-methyltransferase.

The approximate structural formulas of the compounds 1 and 2 are respectively obtained through mass spectrometry and C spectrum and H spectrum results of nuclear magnetic resonance, the relative molecular mass of the compound 2 is found to be larger than that of the compound 1 through comparison of the relative molecular masses of the two compounds, and the compound 2 can be determined to have one more methylated glucose molecule through combination of the molecular structure.

Further analysis of the C and H spectra by the software revealed that Compound 2 was glucose with a 4-hydroxymethylated hydroxyl group added to the 5-carbon of Compound 1 as the original product.

The results prove that the O-methyltransferase can be used for carrying out methylation modification on the polyketide glycosylation derivatives, and increasing the diversity of the polyketide library.

The chromatogram and product structure of the recombinant strain are shown in FIGS. 3 and 4.

The NMR C and H spectra of Compound 1 and Compound 2 are shown in Table 2.

TABLE 2NMR C and H spectra of Compound 1 and Compound 2

Example 3 modification of Flavonoids and anthraquinones Using O-methyltransferase recombinant vector PXW06F-BbFkbM

1. Purpose of experiment

And (3) obtaining metabolites modified by glucosyltransferase and O-methyltransferase by HPLC high performance liquid separation and analyzing the molecular structures of the metabolites.

2. The experimental method comprises the following steps:

1) fermentation culture

Adopts a two-step fermentation technology, firstly, a proper amount of yeast transformant thalli is inoculated to corresponding 25ml^-Leu/^-Culturing in Trp liquid-deficient culture medium at 30 deg.C for about 16 hr at 200r min-1, adding YPD low-sugar culture medium 25ml, and adding 5mg pure product respectively, and culturing for 48 hr to obtain 20 kinds of flavonoids; extracting the fermentation product with ethyl acetate at a ratio of ethyl acetate to fermentation broth of 1:1, namely extracting the fermentation product with 50ml of ethyl acetate; recovering ethyl acetate dry extract by rotary evaporator, and re-dissolving and extracting with 1ml methanolAnd (6) taking the object.

2) High Performance Liquid Chromatography (HPLC) detection:

and (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: 31min conditions: performing gradient elution on a chromatographic column Kromasil 100-5-C18 by using acetonitrile-H2O as a mobile phase, wherein the gradient elution conditions comprise that the gradient elution conditions are 0-5 min and the acetonitrile is 10%; 5-15 min, acetonitrile from 10% → 95%; 15-25 min, and the acetonitrile is 95%; 25-28 min, acetonitrile from 95% → 10%; 28-31 min, and acetonitrile is 10%. The flow rate was 0.8mL min-1, and the detection wavelength was 300 nm.

13min conditions: performing gradient elution on a chromatographic column RRHD Eclipse Plus C18,4.6x100mm by using acetonitrile-H2O as a mobile phase, wherein the gradient elution condition is that the gradient elution condition is 0-5 min, and the acetonitrile content is from 10% → 50%; 5-10 min, acetonitrile from 50% → 95%; 10-12 min, and the acetonitrile is 95%; 12-12.5 min, acetonitrile 95% → 10%; 12.5-13 min, and acetonitrile is 10%. The flow rate was 0.5 mL/min-1, and the detection wavelength was 300 nm.

2. Experimental results and analysis:

HPLC analysis revealed that a total of 10 samples of the 20 added substrates were transformed, numbered F-4, F-5, F-6, F-7, F-8, F-11, F-12, F-15, F-16, F-20, respectively. In the chromatogram of the fermentation product, in addition to the peak of the added starting substrate and the peak of the glycosylation product, another peak of the compound was obtained, confirming that the added starting flavone sample was transformed.

The relative molecular weight of the converted product is respectively obtained through mass spectrum result analysis, the relative molecular weight of the two compounds is compared to find that the converted product is 176 greater than that of the initial substrate, and the methylated glucose molecule is added on the converted compound molecule by combining the molecular structure.

Further analysis by the software found that methylation occurred at the hydroxyl group at the 4-position of the glucose, the glycosylation product of the starting substrate. The verification proves that the O-methyltransferase gene can be used for modifying the structure of the glycosylation derivative of the flavonoid, is very suitable for modifying the molecular structure of various active compounds and increasing the diversity of a flavonoid glycoside compound library.

The chromatogram of the fermentation product of the recombinant strain is shown in FIGS. 5 to 14,

the chemical formulas, structures and molecular weights of the compounds before and after the conversion are shown in Table 3.

TABLE 3 before and after modification of various flavonoid compounds

Example 4 comparison of the stability of the sugar methylation modification product Compound 2 with that of the glycoside Compound 3 before methylation modification

1. Purpose of experiment

The hydrolytic stability of the sugar methylation modification product compound 2 was compared with that of the glycoside compound 3 before methylation modification under the same conditions in the presence of the yeast enzyme system.

2. The experimental method comprises the following steps:

to 50mL of yeast BJ5464 cultured in YPD, 2mg of sugar methylation modification product compound 2 and glycoside compound 3 before methylation modification were added, respectively, and the samples were dissolved in 100. mu.L of chromatographic methanol with the blank control being equal volume of chromatographic methanol. After culturing for 24h and 48h at 30 ℃ and 200r min < -1 >, respectively, extracting a fermentation product by using ethyl acetate, wherein the ratio of the ethyl acetate to the fermentation liquid is 1:1, namely extracting the fermentation product by using 50ml of ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

And (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: performing gradient elution on a chromatographic column Kromasil 100-5-C18 by using acetonitrile-H2O as a mobile phase, wherein the gradient elution conditions comprise that the gradient elution conditions are 0-5 min and the acetonitrile is 10%; 5-15 min, acetonitrile from 10% → 95%; 15-25 min, and the acetonitrile is 95%; 25-28 min, acetonitrile from 95% → 10%; 28-31 min, and acetonitrile is 10%. The flow rate was 0.8mL min-1, and the detection wavelength was 300 nm.

3. Experimental results and analysis:

the chromatogram of the extraction products of compound 3 and compound 2 at the same incubation time was analyzed. The extraction product chromatograms of compound 3 after 24h and 48h of incubation are shown in fig. 15 and fig. 16, respectively: the chromatogram of the extraction product of compound 2 after 24h and 48h incubation is shown in fig. 17 and fig. 18, respectively.

The chromatogram shows that the modified glycosylated derivative starts to hydrolyze after 24 hours, and the higher the degree of hydrolysis along with the increase of the culture time; however, the sugar methylation product 2 is relatively stable in the yeast culture process, and no obvious hydrolysis phenomenon occurs, which indicates that methylation on glucose can increase the stability of the glycosylation product and prevent the compound from being degraded by enzyme.

Example 5 Effect of glycoside hydrolase EXG1 in Yeast on the sugar methylation modification product Compound 2 and the glycoside Compound 3 before methylation modification in the culture Process

1. Purpose of experiment

The stability of the sugar methylation modification product compound 2 and the glycoside compound 3 before methylation modification under the same culture conditions under the condition of knocking out yeast glycoside hydrolase EXG1 is compared, and the influence of methylation on the stability of the glycosylation product is determined.

2. The experimental method comprises the following steps:

1) construction of mutant Yeast strains deficient in glycoside hydrolase EXG1

Two sequences of about 500bp are selected from a glycoside hydrolase EXG1 gene of saccharomyces cerevisiae S.cerevisiae as upstream and downstream homologous arms, uracil synthetic gene Ura is used as a resistance marker, primers are designed, and a homologous exchange fragment for gene knockout is constructed by fusion PCR, wherein the primer list is shown in the specification.

TABLE 4 synthetic primers and sequences

When the upstream and downstream homologous arms of the EXG1 gene are amplified, the S.cerevisiae BJ5464-NpgA genome is used as a template, and when the uracil synthetic gene Ura gene is amplified, the PXK-30F plasmid is used as a template. PCR amplification is carried out by a Biotool hot start high-fidelity DNA amplification kit, the size of a target band is detected by a PCR product through agarose gel electrophoresis, the target band is recovered by a Tiangen DNA purification kit, a recovered fragment is used as a template, EXG1-UP-F and EXG1-Down-R are used as primers to carry out fusion PCR amplification of a second round, the size of the target band is detected by the PCR product through agarose gel electrophoresis, and the total length of the fusion fragment is 1941 bp. And recovering the fusion fragment by using a Tiangen DNA purification kit, and using the fusion fragment for gene knockout of yeast.

Saccharomyces cerevisiae BJ5464-NpgA is inoculated into YPD culture medium, cultured at 30 ℃ and 200r/min until the O.D. value is 0.8-1.0, and Saccharomyces cerevisiae competent cells are prepared according to the instructions provided by ZYMO company and recombinant fragments are transformed. The fusion fragment is transformed into saccharomyces cerevisiae, coated on an SC solid defect culture medium with uracil deletion, and cultured for about 3 days at 30 ℃. The obtained yeast transformant was streaked and cultured to a novel SC-^-Culturing in a 30 ℃ incubator for about 2 days on a Ura defect culture medium; simultaneously selecting 8 yeast transformants for colony PCR, verifying the correctness of the knockout result and verifying the primer to be CTGTGTTTACAGTGCGGTGCACACG; CGTTTGGATGAGGACCCACTTGAAACAAT, respectively; the PCR product should have 2.4kb of wild type band and 1.9kb of mutant band. The electrophoresis result of the PCR product is shown in figure 19, 7 strains of yeast successfully realize gene knockout in 8 yeast transformants, and the transformation efficiency is up to 87.5%.

2mg of sugar methylation modification product compound 2 and glycoside compound 3 before methylation modification were added to 50mL of Δ EXG1 yeast BJ5464 cultured in YPD, and the samples were dissolved in 100 μ L of chromatographic methanol, except for the case where the control was a wild-type strain BJ5464, and the conditions were not changed. Respectively culturing at 30 deg.C and 200r/min for 48 hr, extracting with ethyl acetate at a ratio of ethyl acetate to fermentation liquid of 1:1, namely extracting with 50ml ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

And (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: performing gradient elution on a chromatographic column Kromasil 100-5-C18 by using acetonitrile-H2O as a mobile phase, wherein the gradient elution conditions comprise that the gradient elution conditions are 0-5 min and the acetonitrile is 10%; 5-15 min, acetonitrile from 10% → 95%; 15-25 min, and the acetonitrile is 95%; 25-28 min, acetonitrile from 95% → 10%; 28-31 min, and acetonitrile is 10%. The flow rate was 0.8mL min-1, and the detection wavelength was 300 nm.

3. Experimental results and analysis:

chromatogram of the extraction product of compound 3 under the same culture conditions of wild type and mutant yeast are shown in FIGS. 20 and 21; chromatograms of the extract of compound 2 under the same culture conditions for wild-type and mutant yeast are shown in fig. 22 and 23.

The obvious glucose drop phenomenon of the modified pre-glycosylation derivative under the hydrolysis action of yeast glycoside hydrolase can be seen through chromatograms of a wild type and a delta EXG1 mutant, the modified pre-glycosylation derivative is converted into an initial substrate before glycosylation again, and the hydrolysis degree is reduced after the EXG1 gene is knocked out, so that the glycoside hydrolase EXG1 in yeast can act on a glycosylated compound to generate the hydrolysis phenomenon, and the hydrolysis effect is obvious; the sugar methylation product 2 is stable in the process of culturing the wild type yeast or the mutant type yeast, and no obvious hydrolysis phenomenon occurs, so that the methylation on glucose can increase the stability of the glycosylation product and prevent the compound from being degraded by hydrolase.

Example 6 functional verification of O-methyltransferase homologous genes in entomogenous fungi

1. Purpose of experiment

The analysis by using related bioinformatics software discovers that a large number of homologous genes of O-methyltransferase in beauveria bassiana exist in other entomogenous fungi (table 1), a substrate feeding test of polyketide is carried out through a yeast heterologous expression system, the functions of the homologous genes are determined, and the transformation efficiency of the homologous genes to the compound 3 is compared.

2. The experimental method comprises the following steps:

1. constructing yeast expression vector to obtain yeast transformant

And (3) comparing the homologous gene sequences of the beauveria bassiana O-methyltransferase from different entomogenous fungi by using related bioinformatics software. From these gene sequences with similarity as high as 50% or more, O-methyltransferase genes of 4 strains of Isaria fumosorosea, Metarhizium robertsi, Cordyceps militaris and Claviceps purpurea were selected and subjected to sequence analysis, and the predicted translated protein sequences were highly similar to the secondary structure and had the same motif (motif) (FIGS. 30-32). The coding region sequence of the O-methyltransferase gene is obtained by removing intron splicing through prediction analysis of various software, and the gene is synthesized, so that the sequence fragments of IfOMT, MrOMT, CmOMT and CpOMT are obtained. The gene sequences are respectively shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9 in sequence. The restriction sites are introduced during synthesis. An NdeI restriction site is added in front of a promoter, and a PmeI restriction site is added behind a terminator. Shuttle vector PXW06F was double digested with NdeI and pmeI and a 6114bp sized fragment was recovered. And respectively connecting the gene fragments with the vector by using a seamless connection cloning kit.

Heat shock method transformed into Escherichia coli competent DH5 alpha. Amp^rAnd (4) resistance screening, plasmid extraction and enzyme digestion identification. The obtained recombinant plasmids PXW06F-IfOMT, PXW06F-MrOMT, PXW06F-CmOMT and PXW06F-CpOMT have the plasmid maps shown in the sequence of FIG. 24, FIG. 25, FIG. 26 and FIG. 27. Saccharomyces cerevisiae BJ5464-NpgA is inoculated into YPD culture medium, cultured at 30 ℃ and 200r/min until the O.D. value is 0.8-1.0, and Saccharomyces cerevisiae competent cells are prepared according to the instructions provided by ZYMO company and recombinant plasmid transformation is carried out. The recombinant plasmid is transformed into saccharomyces cerevisiae, coated on an SC solid defect culture medium with tryptophan deletion, and cultured for about 3 days at 30 ℃. The resulting yeast transformants were streaked to new SC-^-Cultured on Trp-deficient medium in an incubator at 30 ℃ for about 2 days.

2) Fermentation culture

Adopts a two-step fermentation technology, firstly, a proper amount of yeast transformant thalli is inoculated to corresponding 25ml^-Culturing in Trp liquid-deficient culture medium at 30 deg.C for about 16 hr at 200r min-1, adding YPD low-sugar culture medium 25ml, adding 5mg of glycosylation derivative compound 3 of compound 1, culturing for 48 hr, and culturing with ethyl acetateExtracting the fermentation product by ethyl acetate, wherein the ratio of the ethyl acetate to the fermentation liquid is 1:1, namely extracting the fermentation product by 50ml of ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

3) High Performance Liquid Chromatography (HPLC) detection:

and (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: performing gradient elution on a chromatographic column RRHD Eclipse Plus C18,4.6x100mm by using acetonitrile-H2O as a mobile phase, wherein the gradient elution condition is that the gradient elution condition is 0-5 min, and the acetonitrile content is from 10% → 50%; 5-10 min, acetonitrile from 50% → 95%; 10-12 min, and the acetonitrile is 95%; 12-12.5 min, acetonitrile 95% → 10%; 12.5-13 min, and acetonitrile is 10%. The flow rate was 0.5 mL/min-1, and the detection wavelength was 300 nm.

3. Experimental results and analysis:

HPLC analysis shows that the glycosylated compound 3 can be transformed by O-methyltransferases from different sources through heterologous expression, and another peak of the sugar methylation product is obtained and has the same retention time. The result shows that O-methyltransferase homologous genes in different entomogenous fungi have the same modification function, and can successfully realize methylation on the 4-hydroxyl of glucose of glycosylated derivatives such as polyketide and the like so as to achieve the aim of improving the stability of the glycosylated compounds. The chromatograms of the fermentation products fed with recombinant strain substrates of CpOMT, MrOMT, CmOMT and IfOMT are shown in FIG. 28.

Example 7 modification of Flavonoids Using the O-methyltransferases BbFkbM, CpOMT, MrOMT, CmOMT and IfOMT in entomogenous fungi

1. Purpose of experiment

Detecting the metabolite modified by O-methyltransferase by HPLC high performance liquid phase and calculating the molecular weight, verifying the functions of CpOMT, MrOMT, CmOMT and IfOMT, and comparing the conversion efficiency of the CpOMT, the MrOMT, the CmOMT and the IfOMT to different substrates.

2. The experimental method comprises the following steps:

1) fermentation culture

Adopts a two-step fermentation technology, firstly, a proper amount of yeast transformant thalli is inoculated to the phase25ml of the solution^-Culturing in Trp liquid defect culture medium at 30 deg.C for about 16 hr at 200r min-1, adding YPD low sugar culture medium 25ml, simultaneously adding glycosylated derivatives of flavonoid F-4 5mg, culturing for 48 hr, extracting with ethyl acetate at a ratio of ethyl acetate to fermentation liquid of 1:1, namely extracting with 50ml ethyl acetate; the ethyl acetate dry extract was recovered by rotary evaporator and the extract was redissolved in 1ml of methanol.

2) High Performance Liquid Chromatography (HPLC) detection:

and (3) centrifuging the obtained fermentation product at a high speed, and detecting by using a high performance liquid chromatography.

The HPLC detection conditions were as follows: performing gradient elution on a chromatographic column RRHD Eclipse Plus C18,4.6x100mm by using acetonitrile-H2O as a mobile phase, wherein the gradient elution condition is that the gradient elution condition is 0-5 min, and the acetonitrile content is from 10% → 50%; 5-10 min, acetonitrile from 50% → 95%; 10-12 min, and the acetonitrile is 95%; 12-12.5 min, acetonitrile 95% → 10%; 12.5-13 min, and acetonitrile is 10%. The flow rate was 0.5 mL/min-1, and the detection wavelength was 300 nm.

3. Experimental results and analysis:

HPLC analysis shows that O-methyltransferases from different sources can convert the glycosylated compound of the flavonoid F-4 through heterologous expression, and an additional peak of the sugar methylation product is obtained. The results show that O-methyltransferase homologous genes in different entomogenous fungi have similar substrate ranges, and can successfully realize methylation on the 4-hydroxyl of the glycosylation derivative glucose of the flavonoid compound so as to achieve the purpose of improving the stability of the glycosylation compound and have functional consistency. Chromatograms of the fermentation products fed with recombinant strain substrates of CpOMT, MrOMT, CmOMT and IfOMT are shown in FIG. 29.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> methyltransferase gene of glycoside 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> Isaria 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> Isaria 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> Metarhizium robertsi

<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> Metarhizium robertsi

<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 Claviceps purpurea (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 Claviceps purpurea (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 (cordyces 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 (cordyces 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 (3)

1. The application of methyltransferase with the following secondary structure in methylation modification of glucoside compounds:

starting from the N end, 3 'alpha + beta tandem + beta sheet + 3' alpha + beta tandem + beta sheet;

and, the amino acid sequence of said methyltransferase is selected from SEQ ID number 2, 4, 6, 8, or 10;

the glucoside compound is glucoside of a polyphenol compound; the polyphenol compounds are selected from flavonoid compounds, polyketone compounds or anthraquinone compounds;

the methylation modification is the methylation modification of the hydroxyl at the 4-position of glycosyl in the glucoside compound.

2. The application of the gene with the nucleotide sequence shown as SEQ ID number 1, 3, 5, 7 or 9 in the methylation modification of glycoside compounds;

the glucoside compound is glucoside of a polyphenol compound; the polyphenol compounds are selected from flavonoid compounds, polyketone compounds or anthraquinone compounds;

the methylation modification is the methylation modification of the hydroxyl at the 4-position of glycosyl in the glucoside compound.

3. The application of recombinant plasmid containing gene with nucleotide sequence as shown in SEQ ID number 1, 3, 5, 7 or 9 in modifying 4-position hydroxyl methylation of glycosyl in glycoside compounds;

the glucoside compound is glucoside of a polyphenol compound; the polyphenol compounds are selected from flavonoid compounds, polyketone compounds or anthraquinone compounds.

Images