Methyl transferase gene for catalyzing methylation of resveratrol and application thereof

Abstract

The invention relates to a methyltransferase gene for catalyzing methylation of resveratrol and application of the methyltransferase gene, and belongs to the technical field of biology. The invention provides a gene of methyltransferase capable of catalyzing resveratrol to generate pinostilbene and pterostilbene. The gene of the methyltransferase is a gene YC26, a gene SS15 or a gene HS97-5. The methyltransferase gene provided by the invention has the advantages of high substrate conversion rate, rich products, high activity and the like, the conversion rate of resveratrol can be increased and synthesis of pterostilbene can be promoted when the methyltransferase gene is combined with other methyltransferase genes to express in different strains, additional addition of expensive methyl donors is avoided, and the production cost is reduced. Only a small amount of substrate resveratrol needs to be added or no substrate resveratrol is added.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a methyltransferase gene for catalyzing the methylation of resveratrol and an application thereof. Background Art

[0002] Resveratrol is a phytoalexin produced when plants are infected by pathogens or when the environment deteriorates. It has antibacterial, anti-tumor, antioxidant, anti-free radical, immune regulation and other effects, and plays an important role in human health and disease. However, resveratrol has poor stability, is easily oxidized, and has low bioavailability, which reduces its bioavailability. Resveratrol can undergo glycosylation, methylation, hydroxylation, oligomerization, isoprenylation and other reactions to form derivatives with different physiological and pharmacological activities. Among them, methylated derivatives, especially the dimethyl derivative pterostilbene, have stronger chemical structure stability and better physiological activity than resveratrol, and are called second-generation resveratrol. Pterostilbene is only found in a small number of plants such as grapes, blueberries, peanuts and rosewood heartwood. It has better lipophilicity and oral absorption than resveratrol, stronger cell absorption ability and longer half-life, etc., which has attracted more and more attention and exploration, and the market demand in the fields of medicine, food, cosmetics and health products has surged. Pterostilbene is an intermediate product of the methylation of resveratrol to form pterostilbene, and is also a natural methylated derivative of resveratrol. It has been shown to have similar biological activity to pterostilbene.

[0003] The use of biotechnology to synthesize pterostilbene and increase its content plays a key role in the application and promotion of pterostilbene. However, few enzymes that have been identified and reported to catalyze the methylation derivatization reaction of resveratrol to produce pterostilbene / pinestilbene are currently available. The most commonly used methyltransferase for catalyzing the conversion of resveratrol to synthesize pterostilbene comes from grapes (VvOMT). In addition, methyltransferases from Arabidopsis thaliana (AtROMT) and sorghum (SbSOMT) have also been reported to catalyze the methylation derivatization of resveratrol. However, the catalytic efficiency of enzymes from these sources is low, the conversion rate of the substrate resveratrol is low, and the catalytic cost in vitro is high. There is an urgent need for a methyltransferase that can improve the efficiency of the methylation derivatization of resveratrol and is low in cost.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a methyltransferase gene for catalyzing the methylation of resveratrol and its application, so as to solve the defects in the prior art that there are few methyltransferases for catalyzing the methylation of resveratrol, the conversion rate of resveratrol catalyzed by the methyltransferase in the prior art is low, and the cost of methyltransferase in vitro catalysis is high.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a gene of a methyltransferase capable of catalyzing resveratrol to generate matstilbene and pterostilbene, wherein the gene of the methyltransferase is gene YC26, gene SS15 or gene HS97-5;

[0008] The nucleotide sequence of the gene YC26 is shown in SEQ ID NO: 1;

[0009] The nucleotide sequence of the gene SS15 is shown in SEQ ID NO: 2;

[0010] The nucleotide sequence of the gene HS97-5 is shown in SEQ ID NO: 3;

[0011] The nucleotide sequences of the primer pairs for amplifying gene YC26 are shown in SEQ ID NOs: 4-5;

[0012] The nucleotide sequences of the primer pairs for amplifying gene SS15 are shown in SEQ ID NOs: 6-7;

[0013] The nucleotide sequences of the primer pair for amplifying gene HS97-5 are shown in SEQ ID NOs: 8-9.

[0014] The present invention provides a methyltransferase capable of catalyzing resveratrol to generate matsostigmine and pterostilbene, wherein the methyltransferase is methyltransferase YC26, SS15 or HS97-5;

[0015] The amino acid sequence of the methyltransferase YC26 is shown in SEQ ID NO: 10;

[0016] The amino acid sequence of the methyltransferase SS15 is shown in SEQ ID NO: 11;

[0017] The amino acid sequence of the methyltransferase HS97-5 is shown in SEQ ID NO:12.

[0018] The invention provides a recombinant vector for expressing a methyltransferase gene. The recombinant vector comprises the methyltransferase gene and an empty vector; the empty vector is pGEX-4t-1.

[0019] The invention provides a recombinant bacterium expressing a methyltransferase gene. The recombinant bacterium comprises the methyltransferase gene or the recombinant vector and a host bacterium; the host bacterium is Escherichia coli BL21 (DE3).

[0020] The invention provides the use of the methyltransferase in catalyzing resveratrol to generate matsostigmine and pterostilbene.

[0021] The present invention provides the use of the recombinant vector or the recombinant bacteria in preparing a product for catalyzing resveratrol to generate matsostigmine and pterostilbene.

[0022] The present invention provides a method for catalyzing resveratrol to produce matsostigmine and pterostilbene, comprising the following steps:

[0023] (1) inoculating the methyltransferase into an induction medium, and culturing at a temperature of 35 to 39° C. for 1.5 to 2.5 hours to obtain a culture solution;

[0024] (2) adding resveratrol to the culture solution obtained in step (1), and then culturing at a temperature of 28 to 32° C.;

[0025] The methyltransferase is the methyltransferase described above or the methyltransferase expressed by the recombinant vector or the methyltransferase expressed by the recombinant bacteria;

[0026] The inoculation amount of the methyltransferase is 1 to 3% of the volume of the induction culture medium;

[0027] The induction medium uses water as solvent and includes the following components at final concentrations: 3-7 g / L yeast extract, 8-12 g / L tryptone, 0.3-0.7 g / L glucose, 1-5 g / L lactose, 3-7 g / L glycerol, 0.3-0.7 g / L sodium chloride, 0.5-1.5 g / L ammonium chloride, 9.7-13.7 g / L dipotassium hydrogen phosphate and 1.5-5.5 g / L potassium dihydrogen phosphate;

[0028] The final concentration of resveratrol in the culture medium is 0.5-1.5 mM.

[0029] The present invention provides a recombinant bacterium for catalyzing resveratrol to synthesize pterostilbene, wherein the genome of the recombinant bacterium includes a gene VvOMT and a gene HS97-5; the host bacterium of the recombinant bacterium is Saccharomyces cerevisiae W303-1A;

[0030] The gene HS97-5 is the gene HS97-5;

[0031] The gene VvOMT is a methyltransferase gene derived from grape.

[0032] The present invention provides a method for catalyzing resveratrol to synthesize pterostilbene, wherein the recombinant bacteria are inoculated into a YPD liquid culture medium, resveratrol is added, and the culture is carried out at a temperature of 28 to 32°C;

[0033] The recombinant bacteria are the recombinant bacteria described above;

[0034] The inoculation amount of the recombinant bacteria is 1-3% of the volume of the YPD liquid culture medium;

[0035] The final concentration of the resveratrol added to the liquid culture medium is 0.15-0.35 mM.

[0036] The present invention also provides a recombinant bacterium for realizing de novo synthesis of pterostilbene, wherein the genome of the recombinant bacterium includes gene VvOMT and gene HS97-5;

[0037] The host bacteria of the recombinant bacteria is strain BRT10;

[0038] The gene HS97-5 is the gene HS97-5;

[0039] The gene VvOMT is a methyltransferase gene VvOMT derived from grape;

[0040] The method for realizing de novo synthesis of pterostilbene by using the recombinant bacteria is as follows: the recombinant bacteria are inoculated into a YPD liquid culture medium, and cultured at a temperature of 28 to 32° C. and a shaking speed of 200 to 240 rpm;

[0041] The inoculation amount of the recombinant bacteria is 0.5% to 10% of the volume of the YPD liquid culture medium.

[0042] The present invention has the following technical effects and advantages:

[0043] 1. The present invention provides a methyltransferase gene for catalyzing the methylation of resveratrol and its application. The methyltransferase gene of the present invention makes up for the defects of the prior art, such as the small number of genes related to the methylation modification of resveratrol, limited sources, and low activity, and provides a new gene for the synthesis of pterostilbene and pinostilbene, which has the advantages of high substrate conversion rate, rich products, high activity, etc.

[0044] 2. The methyltransferase gene of the present invention combines the enzyme production and conversion processes in the Escherichia coli BL21 (DE3) expression system by using an automatic induction medium, thereby avoiding the additional addition of expensive methyl donors, and can effectively synthesize pterostilbene and pinostilbene, and significantly improve the conversion rate of the substrate resveratrol, enabling the pterostilbene yield to reach 150 mg / L, the pinostilbene yield to reach 92 mg / L, and the resveratrol conversion rate to reach 93%.

[0045] 3. The methyltransferase of the present invention can be expressed in Saccharomyces cerevisiae W303-1A and catalyze the complete conversion of low-concentration resveratrol into pterostilbene. The methyltransferase gene HS97-5 and the methyltransferase gene VvOMT are co-expressed in Saccharomyces cerevisiae W303-1A, which can further increase the yield of pterostilbene to 127 mg / L, providing more options for the synthesis of pterostilbene.

[0046] 4. The methyltransferase of the present invention is integrated and expressed in the genome of the recombinant resveratrol yeast BRT10, which can realize the de novo fermentation synthesis of pterostilbene and pinostilbene. When the methyltransferase gene HS97-5 and the methyltransferase gene VvOMT are integrated into the genome of the recombinant bacterium BRT10 and co-expressed, the production of pterostilbene reaches 117 mg / L, and there is no need to add the substrate resveratrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the HPLC spectrum of the methyltransferase catalyzed reaction from tobacco;

[0048] Figure 2 This is the HPLC spectrum of the methyltransferase catalyzed reaction from mulberry;

[0049] Figure 3 This is the HPLC spectrum of the methyltransferase catalyzed reaction from peanut;

[0050] Figure 4 The in vitro catalytic process curves of methyltransferases from different sources, wherein Figure A shows the change of the amount of pterostilbene produced with the reaction time, the horizontal axis is the reaction time, and the vertical axis is the content of pterostilbene; Figure B shows the change of the amount of pine stilbene produced with the reaction time, the horizontal axis is the reaction time, and the vertical axis is the yield of pine stilbene;

[0051] Figure 5 Figure 2 shows the biomass of the recombinant bacteria BL21 containing different genes and the changes in the contents of the substrate resveratrol, the product pterostilbene and pinostilbene. Figure A shows the changes in the production of pterostilbene with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the pterostilbene content; Figure B shows the changes in the production of pinostilbene with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the pinostilbene content; Figure C shows the changes in the consumption of resveratrol with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the resveratrol content; Figure D shows the changes in the biomass of the recombinant bacteria with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the OD of the recombinant bacteria 600 value;

[0052] Figure 6 A recombinant vector comprising gene VvOMT, gene HS97-5 and gene VvOMT&HS97-5;

[0053] Figure 7 The contents of resveratrol, pterostilbene and pinostilbene in the fermentation broth of recombinant bacteria W303-1A expressing different genes. In the figure, Pin represents pinostilbene, Pter represents pterostilbene, and Res represents the substrate resveratrol.

[0054] Figure 8The contents of resveratrol, pterostilbene and pinostilbene in the fermentation broth of the recombinant bacteria BRT10 expressing different genes. In the figure, Pin represents pinostilbene, Pter represents pterostilbene, and Res represents resveratrol. DETAILED DESCRIPTION

[0055] The present invention provides a gene of a methyltransferase capable of catalyzing resveratrol to generate matstilbene and pterostilbene, wherein the gene of the methyltransferase is gene YC26, gene SS15 or gene HS97-5;

[0056] The nucleotide sequence of the gene YC26 is shown in SEQ ID NO: 1;

[0057]

[0058] The nucleotide sequence of the gene SS15 is shown in SEQ ID NO: 2;

[0059]

[0060] The nucleotide sequence of the gene HS97-5 is shown in SEQ ID NO: 3;

[0061]

[0062] The nucleotide sequences of the primer pairs for amplifying gene YC26 are shown in SEQ ID NOs: 4-5;

[0063] The nucleotide sequence of SEQ ID NO: 4 is: GATCTGGTTCCGCGTGGATCCATGGGTTCAACAAGCGAGAGC;

[0064] The nucleotide sequence of SEQ ID NO:5 is: GTCACGATGCGGCCGCTCGAGTTACTTGTTGAATTCCATGACCCA;

[0065] The nucleotide sequences of the primer pairs for amplifying gene SS15 are shown in SEQ ID NOs: 6-7;

[0066] The nucleotide sequence of SEQ ID NO: 6 is: ATCTGGTTCCGCGTGGATCCATGTCTAGCAGTGATGAGCTGATCA;

[0067] The nucleotide sequence of SEQ ID NO:7 is: TCACGATGCGGCCGCTCGAGTTAAGGGTAAACCTCAATGACAGATC;

[0068] The nucleotide sequences of the primer pairs for amplifying gene HS97-5 are shown in SEQ ID NOs: 8-9;

[0069] The nucleotide sequence of SEQ ID NO: 8 is: ATCTGGTTCCGCGTGGATCCATGAGTTCCAGCAATAATAATTCCC;

[0070] The nucleotide sequence of SEQ ID NO:9 is: TCACGATGCGGCCGCTCGAGTTATTTGTAAAACTCCATCACTGCATC.

[0071] The present invention provides a methyltransferase capable of catalyzing resveratrol to generate matsostigmine and pterostilbene, wherein the methyltransferase is methyltransferase YC26, SS15 or HS97-5;

[0072] The amino acid sequence of the methyltransferase YC26 is shown in SEQ ID NO: 10;

[0073] The SEQ ID The amino acid sequence of NO:10 is: MGSTSESQNNSLTHTEDEDFLFAMHLCSASVLPMVLKSALELDLLELMAKAGPGAAISPSELAAQLSTKNPEAPVMLDRMLRLLATYSVLNCTLRTQPDGSVERLYSLTPACKYLTKNADGVSVAPLLLMNQDKVLMESWYHLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKV FNRGMSDHSTMSMKKILEDYKGFEGLNSIVDVGGGTGATVNMIVSKYPSIKGINFDLPHVIEDAPAYPGVEHIGGDMFASVPKADAIFMKWICHDWSDEHCLKFLKNCYEALPANGKVIVAECILPEAPDTSLATKNTVHIDIVMLAHNPGGKERTEKEFQALAKGAGFTGFHKACCAYNTWVMEFNK;

[0074] The amino acid sequence of the methyltransferase SS15 is shown in SEQ ID NO: 11;

[0075] The SEQ ID The amino acid sequence of NO:11 is: MAFSEEVMSSSELLQAHSLLWSTSVNYAKYVTVKCAIELGIPDIIHKHGKPITLSKLTNALPIHPSKTHCIYRIMRILVHSGFFSTGRQVEEEEESYSLTNASRLLLSEGSDFTKMKSFVLFHLLPEAAAPWHSMSTWLQSSDGSTFEHQKGKTFWDCLADEPSHNRLFNEAM ANDSQLIAKLTLTECKDVFEGLTTLVDVGGGTGTMAKAIASTFPHIKCTVFDLPQVVANLEGNDNLNFIGGNMFSDTIPPADAILLKWILHDWNDEESLTILKKCREAILKNEKGGKVIVIETVIDSKNTDEITGIQLCNDMIMMVSFSGKERNLREWEKLFFSAGFSHYKINRKLGVRSLIELYP;

[0076] The amino acid sequence of the methyltransferase HS97-5 is shown in SEQ ID NO: 12;

[0077] The SEQ ID The amino acid sequence of NO:12 is: MSSSNNNSQEEVDHDALTKASSVALAQVFSSVLEAAVDMNLFEIISKAESSLGMSASEIASKIPKQHSEMGSRLERMLPSLVAHSLLSCSIRTINEDGDTERVYAVSPVGQYFRRSHDQSENSLAALSALFYRGYRHVWKDTKDAILDANNHNHFQRMYGKMAFEYMETDRELGNLF GEAMSQAGPLGVKSILDAYKGGFDGISTLIDVGGGYGQVLKQILFQYPSIKGINFDLPHVVKNAPPHPGIEHIGGDMFESVPKGDAILIKHVCHN WGDEECVKFLRKCYEALPADGKVIVLEILVPEIPKSTSKDICAVDMDYSMFLVHGGKERTEKQYEKLCKRSGFSRFKVACNDSSAIDAVMEFYK.

[0078] The invention provides a recombinant vector for expressing a methyltransferase gene. The recombinant vector comprises the methyltransferase gene and an empty vector; the empty vector is pGEX-4t-1.

[0079] The invention provides a recombinant bacterium expressing a methyltransferase gene. The recombinant bacterium comprises the methyltransferase gene or the recombinant vector and a host bacterium; the host bacterium is Escherichia coli BL21 (DE3).

[0080] The invention provides the use of the methyltransferase in catalyzing resveratrol to generate matsostigmine and pterostilbene.

[0081] The present invention provides the use of the recombinant vector or the recombinant bacteria in preparing a product for catalyzing resveratrol to generate matsostigmine and pterostilbene.

[0082] The present invention provides a method for catalyzing resveratrol to produce matsostigmine and pterostilbene, comprising the following steps:

[0083] (1) inoculating the methyltransferase into an induction medium, culturing at a temperature of 35 to 39° C. for 1.5 to 2.5 hours to obtain a culture solution, wherein the temperature is preferably 37° C., and the culturing time is preferably 2 hours;

[0084] (2) adding resveratrol to the culture solution obtained in step (1), and then culturing at a temperature of 28 to 32° C., preferably 30° C.;

[0085] The methyltransferase is the methyltransferase described above or the methyltransferase expressed by the recombinant vector or the methyltransferase expressed by the recombinant bacteria;

[0086] The inoculation amount of the methyltransferase is 1-3% of the volume of the induction culture medium, preferably 2%;

[0087] The induction medium uses water as solvent and includes the following components in final concentrations: yeast extract 3-7 g / L, preferably 5 g / L; tryptone 8-12 g / L, preferably 10 g / L; glucose 0.3-0.7 g / L, preferably 0.5 g / L; lactose 1-5 g / L, preferably 3 g / L; glycerol 3-7 g / L, preferably 5 g / L; sodium chloride 0.3-0.7 g / L, preferably 0.5 g / L; ammonium chloride 0.5-1.5 g / L, preferably 1 g / L; dipotassium hydrogen phosphate 9.7-13.7 g / L, preferably 11.7 g / L, potassium dihydrogen phosphate 1.5-5.5 g / L, preferably 3.5 g / L;

[0088] The final concentration of resveratrol in the culture medium is 0.5-1.5 mM, preferably 1 mM.

[0089] The present invention provides a recombinant bacterium for catalyzing resveratrol to synthesize pterostilbene, wherein the genome of the recombinant bacterium includes a gene VvOMT and a gene HS97-5; the host bacterium of the recombinant bacterium is Saccharomyces cerevisiae W303-1A;

[0090] The gene HS97-5 is the gene HS97-5;

[0091] The gene VvOMT is a methyltransferase gene VvOMT derived from grape.

[0092] The present invention provides a method for catalyzing resveratrol to synthesize pterostilbene, wherein the recombinant bacteria are inoculated into a YPD liquid culture medium, resveratrol is added, and the culture is carried out at a temperature of 28 to 32° C., and the temperature is preferably 30° C.;

[0093] The recombinant bacteria are the recombinant bacteria described above;

[0094] The inoculation amount of the recombinant bacteria is 1-3% of the volume of the YPD liquid culture medium, preferably 2%;

[0095] The final concentration of resveratrol added to the liquid culture medium is 0.15-0.35 mM, preferably 0.25 mM.

[0096] The present invention also provides a recombinant bacterium for realizing de novo synthesis of pterostilbene, wherein the genome of the recombinant bacterium includes a gene VvOMT and a gene HS97-5;

[0097] The host bacteria of the recombinant bacteria is strain BRT10;

[0098] The gene VvOMT and the gene HS97-5 are located at 1414a on the genome of strain BRT10, and the specific recognition sequence of the site is shown in SEQ ID NO: 13;

[0099] The nucleotide sequence of SEQ ID NO: 13 is: GCTGTTGTCTTAGAAACCGG;

[0100] The gene HS97-5 is the gene HS97-5;

[0101] The gene VvOMT is a methyltransferase gene derived from grape;

[0102] The method for realizing de novo synthesis of pterostilbene by using the recombinant bacteria is as follows: the recombinant bacteria are inoculated into a YPD liquid culture medium, and cultured at a temperature of 28 to 32° C. and a shaking speed of 200 to 240 rpm, wherein the temperature is preferably 30° C. and the shaking speed is preferably 220 rpm;

[0103] The inoculation amount of the recombinant bacteria is 0.5% to 10% of the volume of the YPD liquid culture medium, preferably 5%.

[0104] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0105] Example 1: Acquisition of methyltransferase genes YC26, SS15, and HS97-5

[0106] Using the NCBI tblastn function, based on the known grape-derived methyltransferase VvOMT (UniProtKB: B6VJS4.2) as the query sequence, similar nucleotide sequences of tobacco (taxid: 4097), mulberry (taxid: 3497) and peanut (taxid: 3818) were retrieved respectively, and the obtained sequences were sorted according to the scores, and the nucleotide sequences YC1-YC38 from tobacco, SS1-SS41 from mulberry, and HS1-HS98 from peanut were screened. The screened sequences were further aligned to construct a phylogenetic tree, and sequences with extremely high similarity were eliminated. The potential methyltransferase gene sequences YC26, SS15 and HS97-5 were screened according to the branches of the phylogenetic tree. The nucleotide sequence of gene YC26 is shown in SEQ ID NO: 1, the nucleotide sequence of gene SS15 is shown in SEQ ID NO: 2, and the nucleotide sequence of gene HS97-5 is shown in SEQ ID NO: 3. Amplification primer pairs were designed for genes YC26, SS13 and HS97-5, respectively. The nucleotide sequences of primer pairs for amplification of different genes are shown in Table 1.

[0107] Table 1 Nucleotide sequences of primer pairs for amplification of different genes

[0108]

[0109]

[0110] Example 2: Construction of recombinant vector and recombinant bacteria expressing methyltransferase gene

[0111] Fresh leaves and rhizomes of tobacco, mulberry and peanut were collected, mixed and frozen in liquid nitrogen and ground into powder, and total RNA of tobacco, mulberry and peanut were extracted by Trizol method, and the total RNA of plants was reverse transcribed by TaKaRa reverse transcription kit (purchased from Baoriyi Biotechnology (Beijing) Co., Ltd. Code No. 6210A) to obtain cDNA. PCR amplification was performed using the cDNA of tobacco, mulberry and peanut as templates, primers designed in Example 1, and the amplified products were subjected to agarose gel electrophoresis, and bands between 1 and 2 kb were cut and recovered. The fragments were ligated to the BamH I and Xhol I restriction sites of the vector pGEX-4t-1 using a seamless cloning kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain recombinant vectors pGEX-4t-1-YC26, pGEX-4t-1-SS15 and pGEX-4t-1-HS97-5 that can express genes YC26, SS15 and HS97-5, respectively.

[0112] The recombinant vectors pGEX-4t-1-YC26, pGEX-4t-1-SS15 and pGEX-4t-1-HS97-5 were transformed into competent cells of Escherichia coli BL21 (DE3), recombinant bacteria were constructed, and single cell colonies were picked for PCR verification to verify whether the strain was correct. After the verification was correct, two different primers were selected to send samples of the corresponding recombinant bacteria for verification sequencing. The results showed that the recombinant bacteria could express genes YC26, SS15 and HS97-5, and the amino acid sequence of the methyltransferase YC26 expressed by the recombinant bacteria BL21 containing the gene YC26 was shown in SEQ ID NO: 10, the amino acid sequence of the methyltransferase SS15 expressed by the recombinant bacteria BL21 containing the gene SS15 was shown in SEQ ID NO: 11, and the amino acid sequence of the methyltransferase HS97-5 expressed by the recombinant bacteria BL21 containing the gene HS97-5 was shown in SEQ ID NO: 12.

[0113] Example 3: In vitro catalysis of methyltransferase

[0114] (1) Selecting genes YC1-2, YC3-3, YC3-4, YC20-1, YC20-2, YC32-1, YC32-4, YC8 and YC22 retrieved in Example 1, and using the method of Example 2 to construct recombinant bacteria containing genes of methyltransferases from different tobacco sources for later use;

[0115] The genes SS1, SS5, SS10, SS-15, SS31, SS7, SS8, SS19, SS23, SS28, SS25, SS30, SS37, SS37-2, SSS38, SS15, and SS34 retrieved in Example 1 were selected and the method of Example 2 was adopted to construct recombinant bacteria containing genes from different mulberry sources for use;

[0116] The genes HS3-1, HS5-3, HS3-1, HS910-4, HS10-2, HS15-1, HS15-4, HS23-1, HS7-1, HS9-2, HS10-3, HS15-5, HS23, HS25, HS33, HS34, HS51, HS70, HS27-1, HS27-3, HS85, HS89, HS91, and HS97-3 retrieved in Example 1 were selected and the method of Example 2 was adopted to construct recombinant bacteria containing genes from different peanut sources for use.

[0117] (2) The recombinant bacteria BL21 containing the gene YC26, the recombinant bacteria BL21 containing the gene SS15, and the recombinant bacteria BL21 containing the gene HS97-5 obtained in Example 2 and the recombinant bacteria obtained in step (1) were respectively transferred to LB liquid culture medium containing ampicillin and cultured overnight to obtain the corresponding seed liquid. Then, the corresponding seed liquid was inoculated into 50 mL of LB liquid culture medium at an inoculation ratio of 2% of the culture liquid volume, and cultured at a temperature of 37° C. with shaking until the OD of the bacterial liquid reached 0.05. 600 The value was 0.6-0.8, and then IPTG solution with a final concentration of 0.1 mM was added for induction. The culture was shaken at 16°C for 16 hours, and the corresponding bacteria were collected by centrifugation for later use.

[0118] (3) Add 100 mM Mg-containing solution with a pH of 8.0 to the corresponding bacterial cells obtained in step (1). 2+ The bacteria were resuspended in phosphate buffer solution and sonicated on ice for 5 min (the sonication program was 30 times, each time for 3 s) until the bacterial solution was no longer viscous, thereby obtaining the corresponding crude enzyme solution of methyltransferase.

[0119] (4) A qualitative reaction was performed in a 2 mL centrifuge tube. The reaction system was 300 μL, wherein the concentration of the substrate resveratrol was 0.1 mM, and the concentration of the methyl donor S-adenosylmethionine was 0.2 mM. 150 μL of crude enzyme solution was added to the reaction system, mixed well, and placed in a 30°C water bath for reaction for 12 h. Since the solubility of resveratrol in aqueous solution is very low and it is not completely dissolved in the system, after the reaction, 900 μL of anhydrous ethanol was added to the system to terminate the reaction. The reaction substrate and product were then extracted by vortexing. The supernatant was centrifuged and filtered to obtain the corresponding solutions, which were then tested by HPLC to compare the products and yields of resveratrol produced by methyltransferases from different sources. The HPLC spectrum of the methyltransferase-catalyzed reaction from tobacco is shown in FIG. Figure 1 As shown, the HPLC spectrum of the methyltransferase catalyzed reaction from mulberry is as follows Figure 2 As shown, the HPLC spectrum of the methyltransferase catalyzed reaction from peanut is as follows Figure 3 shown.

[0120] according to Figures 1 to 3 It can be seen that genes YC26, SS15 and HS97-5 are the methyltransferase genes with the highest substrate conversion rate and product generation among the methyltransferase genes from tobacco, mulberry and peanut, respectively.

[0121] Example 4: In vitro catalytic activity and reaction process of methyltransferase

[0122] 1. Catalytic Activity Assay

[0123] Genes YC22, YC32-1, and YC32-4 retrieved in Example 1 were selected and the method of Example 2 was used to construct recombinant bacteria containing genes of methyltransferases from different tobacco sources for later use;

[0124] The gene SS8 retrieved in Example 1 was selected and the method in Example 2 was adopted to construct a recombinant bacterium containing genes from different mulberry sources for use;

[0125] The genes HS25, HS91, and HS97-3 retrieved in Example 1 were selected and the method of Example 2 was adopted to construct recombinant bacteria containing genes from different peanut sources for use.

[0126] According to the method in Example 3, crude enzyme solutions of recombinant methyltransferases from different sources were prepared, and crude enzyme activity was determined in the same reaction system as in Example 3, and the methyltransferase VvOMT from grape disclosed in the prior art was used as a positive control. The timing was started when the enzyme solution was added, and 3 volumes of anhydrous ethanol were added after 15 minutes of reaction to terminate the reaction, followed by vortex oscillation to extract the reaction substrate and product, and the corresponding solutions were obtained after the supernatant was centrifuged and filtered, and the contents of pterostilbene and pinostilbene were detected by HPLC, and the results are shown in Table 1.

[0127] Table 1 Activity determination results of recombinant methyltransferases from different sources

[0128] Recombinant methyltransferase Pterostilbene production (mg / L) Matsutomonas production (mg / L) YC22 4.35 3.93 YC26 5.13 19.21 YC32-1 0.14 0.79 YC32-4 ND 5.22 SS8 ND 1.59 SS15 0.49 41.76 HS25 ND 1.43 HS91 ND 2.32 HS97-3 ND 4.01 HS97-5 1.81 26.30

[0129] According to Table 1, the methyltransferases YC26, SS15 and HS97-5 expressed by the recombinant bacteria BL21 containing the gene YC26, the recombinant bacteria BL21 containing the gene SS15 and the recombinant bacteria BL21 containing the gene HS97-5 are the enzymes with the highest activity from tobacco, mulberry and peanut.

[0130] 2. In vitro reaction process

[0131] The in vitro reaction process was monitored. The crude enzyme solutions of methyltransferase YC26, methyltransferase SS15, methyltransferase HS97-5 and methyltransferase VvOMT were diluted respectively to control the conversion rate of the reaction system. Anhydrous ethanol was added at different reaction times to terminate the reaction. The yields of the products pterostilbene and pinostilbene were detected. The in vitro catalytic process curves of methyltransferases from different sources were drawn. The results are shown in FIG. Figure 4 As shown, Figure A shows the change of the amount of pterostilbene produced with the reaction time, the horizontal axis is the reaction time, and the vertical axis is the content of pterostilbene; Figure B shows the change of the amount of pinostilbene produced with the reaction time, the horizontal axis is the reaction time, and the vertical axis is the yield of pinostilbene.

[0132] Depend on Figure 4It can be seen that the positive control VvOMT produces pterostilbene at the fastest speed and has the highest content in the final reaction system, and the yield of the intermediate product pine stilbene remains at a low level during the reaction; in contrast, the methyltransferases YC26, methyltransferases SS15 and methyltransferases HS97-5 expressed by the recombinant bacteria BL21 containing gene YC26, recombinant bacteria BL21 containing gene SS15 and recombinant bacteria BL21 containing gene HS97-5 produce pterostilbene at a slower speed, but the yield is also increasing, and the yield of the intermediate product pine stilbene is also much higher than that of VvOMT, which has its own advantages. Although the yield of pterostilbene is very low, the yield of pine stilbene by methyltransferase SS15 is the highest, and it can be used to catalyze the preparation of the monomethylated product pine stilbene; methyltransferase YC26 and methyltransferase HS97-5 catalyze the coexistence of pine stilbene and pterostilbene in the products, and the product ratios are different, so the reaction process and reaction products can be controlled according to needs.

[0133] Example 5: Catalysis of resveratrol to produce matsostigmine and pterostilbene

[0134] Weigh 5 g of yeast extract, 10 g of tryptone, 0.5 g of glucose, 3 g of lactose, 5 g of glycerol, 0.5 g of sodium chloride, 1 g of ammonium chloride, 11.7 g of dipotassium hydrogen phosphate and 3.5 g of potassium dihydrogen phosphate, dissolve in 1 L of water, stir and mix to obtain an automatic induction medium for use.

[0135] The recombinant bacteria BL21 containing gene YC26, the recombinant bacteria BL21 containing gene SS15, and the recombinant bacteria BL21 containing gene HS97-5 obtained in Example 2 were respectively transferred to LB liquid culture medium containing ampicillin and cultured overnight to obtain the corresponding seed solution. The seed solution of methyltransferase was inoculated into 50 mL of automatic induction medium at an inoculum size of 2%, and cultured at a temperature of 37 ° C for 2 h to ensure the rapid growth of the recombinant bacteria and consume the glucose in the culture medium. Subsequently, resveratrol was added at a final concentration of 1 mM, and the culture was continued at 30 ° C for 60 h. During the period, the fermentation broth was fully mixed and sampled, and the biomass of the recombinant bacteria BL21 containing different genes and the changes in the content of the substrate resveratrol, the product pterostilbene, and the pinostilbene were detected. The results are as follows: Figure 5 As shown, Figure A shows the change of pterostilbene production with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the pterostilbene content; Figure B shows the change of pinostilbene production with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the pinostilbene content; Figure C shows the change of resveratrol consumption with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the resveratrol content; Figure D shows the change of biomass of recombinant bacteria with fermentation time, in which the horizontal axis represents the fermentation time and the vertical axis represents the OD of the recombinant bacteria 600 value.

[0136] according to Figure 5It can be seen that after 60h of fermentation, the pterostilbene production of methyltransferase HS97-5 was basically equivalent to that of the positive control VvOMT, with a pterostilbene production of 150mg / L, and it could also produce 92mg / L of pinostelium, making the conversion rate of its substrate resveratrol reach 93%, which was much higher than that of other recombinant bacteria; the product yield of methyltransferase SS15 was extremely low, which may be due to the high fermentation temperature of 30℃, which induced the expressed enzyme to produce inclusion bodies, or because the intracellular SAM concentration of the microorganism was maintained at a low level, which was not conducive to the activity of SS15; in YC26, the conversion of resveratrol and the generation of products tended to be stable within the initial 15h of fermentation, and the fermentation time was short. In addition, the in vitro catalytic reaction of the methyltransferase requires the addition of a large amount of methyl donor SAM, which results in high reaction costs. The present application scheme inoculates the recombinant bacteria into the induction medium, which enables the methyltransferase expressed by the recombinant bacteria to fully utilize the SAM produced by the metabolic process of the recombinant bacteria themselves, thereby significantly reducing the cost of raw materials. At the same time, the microorganisms can metabolize the byproduct S-adenosylhomocysteine ​​produced in the methyl transfer reaction process, eliminating its inhibitory effect on the reaction.

[0137] Example 6: Catalysis of resveratrol to synthesize pterostilbene

[0138] The shuttle plasmids with expression cassettes inserted with the gene VvOMT, the gene HS97-5, and the gene VvOMT&HS97-5 expression cassettes were constructed in E. coli by seamless cloning technology, and the constructed recombinant vectors were verified by sequencing. Figure 6 shown.

[0139] The recombinant plasmids were transformed into competent cells of Saccharomyces cerevisiae W303-1A using the lithium acetate / polyethylene glycol 3350 method. The specific competent preparation method and transformation system were conventional methods, and the concentration of the transformed plasmid was 500ng-2000ng. The transformation product was coated on a YPD plate containing G418 resistance and cultured for 3 to 4 days. The recombinant bacteria were picked for colony PCR to verify that the plasmid had been introduced into Saccharomyces cerevisiae, and recombinant bacteria W303-1A that can express the gene VvOMT, recombinant bacteria W303-1A that can express the gene HS97-5, and recombinant bacteria W303-1A that can simultaneously express the gene VvOMT and the gene HS97-5 were obtained.

[0140] Single colonies of recombinant bacteria W303-1A expressing gene VvOMT, recombinant bacteria W303-1A expressing gene HS97-5, and recombinant bacteria W303-1A expressing both gene VvOMT and gene HS97-5 were picked from the plate and inoculated into 5 mL of YPD liquid medium containing G418 resistance, cultured for 16 h as seed solution, and then transferred to 50 mL of YPD liquid medium containing G418 resistance at a 2% inoculum, and resveratrol was added at final concentrations of 0.25 mM and 0.5 mM, respectively. After cultured at 30°C for 72 h, the contents of resveratrol, pterostilbene, and matsostigmine in the fermentation broth of recombinant bacteria W303-1A expressing different genes were detected by HPLC. The results are as follows: Figure 7 As shown, Pin represents pinostearyl, Pter represents pterostilbene, and Res represents the substrate resveratrol.

[0141] according to Figure 7 It can be seen that when the final concentration of the substrate resveratrol was 0.25mM, the three recombinant bacteria almost completely converted resveratrol into pterostilbene; when the addition amount was 0.5mM, the catalytic product of the recombinant bacteria that could express the gene HS97-5 was a mixture of pterostilbene and pinostilbene, and the total amount was higher than that of the recombinant bacteria that could express the gene VvOMT. The recombinant bacteria that could express the gene VvOMT and the gene HS97-5 at the same time completely converted resveratrol into pterostilbene, with a yield of 127mg / L, which was more significant than expressing one gene alone. This shows that the combination of the methyltransferase gene VvOMT and the gene HS97-5 to construct a recombinant bacterium can be applied to the methylation conversion of resveratrol, which can improve the conversion efficiency of the substrate.

[0142] Example 7: De novo synthesis of pterostilbene

[0143] The expression frames of gene VvOMT, gene HS97-5 and gene VvOMT&HS97-5 were respectively integrated into the genome of resveratrol production strain BRT10 by CRISPR-Cas9 gene editing method, the integration site was 1414a, and the specific recognition sequence of the site was as shown in SEQ ID NO: 13, and the nucleotide sequence of SEQ ID NO: 13 was: GCTGTTGTCTTAGAAACCGG. Recombinant bacteria BRT10 that can express gene VvOMT, recombinant bacteria BRT10 that can express gene HS97-5, and recombinant bacteria BRT10 that can express gene VvOMT and gene HS97-5 at the same time were constructed.

[0144] Recombinant bacteria BRT10 that can express gene VvOMT, recombinant bacteria BRT10 that can express gene HS97-5, and recombinant bacteria BRT10 that can simultaneously express gene VvOMT and gene HS97-5 were inoculated on YPD plates and streaked for 2 to 3 days, and a single colony was picked and inoculated into 5 mL of YPD liquid culture medium, and cultured in a shaking incubator at 30°C and 220 rpm for 24 hours to obtain seed liquid, and then the seed liquid was inoculated into YPD liquid culture medium at an inoculation rate of 5% of the volume of the culture medium, and fermented and cultured at 30°C and 220 rpm for 120 hours. After the fermentation, the contents of resveratrol, pterostilbene, and pinostilbene in the fermentation broth of recombinant bacteria BRT10 expressing different genes were detected, and the test results were as follows: Figure 8 As shown in the figure, Pin represents pinostearyl, Pter represents pterostilbene, and Res represents resveratrol.

[0145] according to Figure 8 It can be seen that the three recombinant bacteria do not need to add the substrate resveratrol. The product of the recombinant bacteria BRT10 that can express the gene VvOMT is mainly pterostilbene, but the yield is low and the conversion rate of resveratrol is low; the recombinant bacteria BRT10 that can express the gene HS97-5 (the BRT10 strain is constructed and preserved in this laboratory, and the specific construction process can be referred to the paper Meng et al. Microbial Cell Factories 2023, 22: 46) The product is a mixture of pterostilbene and pinostilbene, and the yield of pterostilbene is not much different from that of the recombinant bacteria BRT10 that can express the gene VvOMT, but it can produce pinostilbene; the product of the recombinant bacteria BRT10 that can simultaneously express the genes VvOMT and HS97-5 is a mixture of pterostilbene and pinostilbene, among which the pterostilbene yield is as high as 117 mg / L, which is significantly higher than the other two recombinant bacteria, and can achieve de novo synthesis of pterostilbene, and the conversion rate of resveratrol is higher than that of the other two.

[0146] From the above examples, it can be seen that the present invention provides a methyltransferase gene for catalyzing the methylation of resveratrol and its application. The methyltransferase gene for catalyzing the methylation of resveratrol provided by the present invention has the advantages of high substrate conversion rate, rich products, high activity and the like. The methyltransferase gene of the invention uses an automatic induction culture medium to produce enzymes and convert the results of the process in an Escherichia coli expression system, thereby avoiding the additional addition of expensive methyl donors, effectively synthesizing pterostilbene and pinostilbene, and significantly improving the conversion rate of substrate resveratrol, with the pterostilbene yield reaching 150 mg / L, the pinostilbene yield reaching 92 mg / L, and the resveratrol conversion rate reaching 93%; the methyltransferase gene HS97-5 of the invention and the methyltransferase gene VvOMT of the invention are co-expressed in saccharomyces cerevisiae W303-1A, and can catalyze the complete conversion of low-concentration resveratrol into pterostilbene, and can further increase the pterostilbene yield to 127 mg / L; the methyltransferase gene HS97-5 of the invention and the methyltransferase gene VvOMT of the invention are integrated and expressed on the genome of resveratrol saccharomyces cerevisiae gene recombinant bacteria BRT10, and can realize the de novo fermentation synthesis of pterostilbene and pinostilbene, with the pterostilbene yield reaching 117 mg / L, and no substrate resveratrol needs to be added.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene for a methyltransferase capable of catalyzing resveratrol to produce matstilbene and pterostilbene, characterized in that: The methyltransferase gene is gene YC26, gene SS15 or gene HS97-5; The nucleotide sequence of the gene YC26 is shown in SEQ ID NO: 1; The nucleotide sequence of the gene SS15 is shown in SEQ ID NO: 2; The nucleotide sequence of the gene HS97-5 is shown in SEQ ID NO: 3; The nucleotide sequences of the primer pairs for amplifying gene YC26 are shown in SEQ ID NOs: 4-5; The nucleotide sequences of the primer pairs for amplifying gene SS15 are shown in SEQ ID NOs: 6-7; The nucleotide sequences of the primer pair for amplifying gene HS97-5 are shown in SEQ ID NOs: 8-9.

2. A methyltransferase capable of catalyzing resveratrol to produce matstilbene and pterostilbene, characterized in that: The methyltransferase is methyltransferase YC26, SS15 or HS97-5; The amino acid sequence of the methyltransferase YC26 is shown in SEQ ID NO: 10; The amino acid sequence of the methyltransferase SS15 is shown in SEQ ID NO: 11; The amino acid sequence of the methyltransferase HS97-5 is shown in SEQ ID NO:

12.

3. A recombinant vector expressing a methyltransferase gene, characterized in that: The recombinant vector comprises the gene of the methyltransferase according to claim 1 and an empty vector; the empty vector is pGEX-4t-1.

4. A recombinant bacterium expressing a methyltransferase gene, characterized in that: The recombinant bacteria comprises the methyltransferase gene of claim 1 or the recombinant vector of claim 3 and a host bacteria; the host bacteria is Escherichia coli BL21 (DE3).

5. Use of the methyltransferase according to claim 2 in catalyzing resveratrol to produce matstilbene and pterostilbene.

6. Use of the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in preparing a product that catalyzes resveratrol to produce matsostigmine and pterostilbene.

7. A method for catalyzing resveratrol to produce matsostigmine and pterostilbene, characterized in that: The steps include: (1) inoculating the methyltransferase into an induction medium, and culturing at a temperature of 35 to 39° C. for 1.5 to 2.5 hours to obtain a culture solution; (2) adding resveratrol to the culture solution obtained in step (1), and then culturing at a temperature of 28 to 32° C.; The methyltransferase is the methyltransferase according to claim 2, or the methyltransferase expressed by the recombinant vector according to claim 3, or the methyltransferase expressed by the recombinant bacteria according to claim 4; The inoculation amount of the methyltransferase is 1 to 3% of the volume of the induction culture medium; The induction medium uses water as solvent and includes the following components at final concentrations: 3-7 g / L yeast extract, 8-12 g / L tryptone, 0.3-0.7 g / L glucose, 1-5 g / L lactose, 3-7 g / L glycerol, 0.3-0.7 g / L sodium chloride, 0.5-1.5 g / L ammonium chloride, 9.7-13.7 g / L dipotassium hydrogen phosphate and 1.5-5.5 g / L potassium dihydrogen phosphate; The final concentration of resveratrol in the culture medium is 0.5-1.5 mM.

8. A recombinant bacterium that catalyzes the synthesis of pterostilbene from resveratrol, characterized in that: The genome of the recombinant bacteria includes genes VvOMT and HS97-5; the host bacteria of the recombinant bacteria is Saccharomyces cerevisiae W303-1A; The gene HS97-5 is the gene HS97-5 described in claim 1; The gene VvOMT is a methyltransferase gene VvOMT derived from grape.

9. A method for catalyzing resveratrol to synthesize pterostilbene, characterized in that: The recombinant bacteria were inoculated into YPD liquid medium, resveratrol was added, and the culture was carried out at a temperature of 28-32°C; The recombinant bacteria is the recombinant bacteria according to claim 8; The inoculation amount of the recombinant bacteria is 1-3% of the volume of the YPD liquid culture medium; The final concentration of the resveratrol added to the liquid culture medium is 0.15-0.35 mM.

10. A recombinant bacterium for achieving de novo synthesis of pterostilbene, characterized in that: The genome of the recombinant bacteria includes gene VvOMT and gene HS97-5; The host bacteria of the recombinant bacteria is strain BRT10; The gene HS97-5 is the gene HS97-5 described in claim 1; The gene VvOMT is a methyltransferase gene derived from grape; The method for realizing de novo synthesis of pterostilbene by using the recombinant bacteria is as follows: the recombinant bacteria are inoculated into a YPD liquid culture medium, and cultured at a temperature of 28 to 32° C. and a shaking speed of 200 to 240 rpm; The inoculation amount of the recombinant bacteria is 0.5% to 10% of the volume of the YPD liquid culture medium.

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