CN109957555B

CN109957555B - Glycosyl transferase mutant and application thereof in catalyzing biosynthesis of gastrodin

Info

Publication number: CN109957555B
Application number: CN201711485080.4A
Authority: CN
Inventors: 刘涛; 殷华; 庄以彬; 马延和
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-04-02
Anticipated expiration: 2037-12-29
Also published as: CN109957555A

Abstract

The invention provides a glycosyltransferase mutant and application thereof in catalyzing biosynthesis of gastrodin, belonging to the technical field of genetic engineering. The glycosyltransferase mutant has an amino acid sequence shown as SEQ ID No.1 in a sequence table. The invention takes a mutant UGT73B6MK of glycosyltransferase UGT73B6 in rhodiola sachalinensis as a parent, and mutates the site of phenylalanine Phe (Phe) (F) at position 389 into alanine Ala (A), thereby improving the enzyme activity for catalyzing and synthesizing gastrodin. The in vitro enzyme activity of the modified glycosyltransferase mutant is parent UGT73B6^MK4.9 times of that of the parent UGT73B6, and the in vivo conversion rate is higher than that of the parent UGT73B6^MK3.8 times of the amount of the gastrodin, has better catalytic activity and is more suitable for the industrial production of gastrodin.

Description

Glycosyl transferase mutant and application thereof in catalyzing biosynthesis of gastrodin

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a glycosyltransferase mutant and application thereof in catalyzing biosynthesis of gastrodin.

Background

Glycosyltransferases (GT, EC 2.4.x.y) are capable of catalyzing the attachment of an activated sugar, such as UDP-glucose, to different acceptor molecules, such as proteins, nucleic acids, oligosaccharides, lipids and small molecules. Glycosylation can alter the activity of the receptor molecule, increase the water solubility, secretion, stability, etc., of the receptor molecule, and in particular, determines the biological activity of some compounds. Many important active ingredients in plant-derived natural products are glycosylation products, such as plant-derived medicine Gastrodin (Gastrodin), which is synthesized by a glucosylation reaction of phenol hydroxyl of hydroxybenzyl alcohol as a substrate under the catalytic action of glycosyltransferase.

Gastrodin (GAS) has the following characteristics: the chemical name is 4- (hydroxymethyl) phenyl beta-D-glucopyranoside, and the molecular formula is C₁₃H₁₈O₇Molecular weight is 286.1053, CAS number is 62499-27-8, and structural formula is:

the gastrodin aglycone p-Hydroxybenzyl alcohol (4-Hydroxybenzyl alcohol) has the following characteristics: the chemical name is p-Hydroxybenzyl alcohol, and the molecular formula is C₇H₈O₂Molecular weight 124.0524, CAS number 623-05-2, structural formula:

gastrodine has effects of tranquilizing mind, relieving convulsion, resisting inflammation, relieving pain, dilating blood vessel, resisting oxidation, enhancing organism immunity and resisting senile dementia, and can be widely used for clinically adjuvant treatment of neurasthenia, neurasthenia syndrome, giddiness, headache and epilepsia carbuncle. Domestic medical and health products taking gastrodine as the effective component, such as gastrodine injection, gastrodine capsule, dispersible tablet, etc., are on the market in large quantities or obtain clinical monographs. At present, gastrodin is mainly produced by chemical synthesis or extraction of gastrodia elata plants, but the chemical synthesis has the problems of high by-product, serious pollution and the like, and the plant extraction has resource limitation and the like. The production of gastrodin by microbial cell direct transformation and tissue culture is only in the laboratory research stage, and has the problems of long period, low yield and the like. The development of a green, efficient and large-scale production mode of gastrodin is urgently needed.

Disclosure of Invention

In view of the above, the present invention aims to provide a glycosyltransferase mutant and an application thereof in catalyzing gastrodin biosynthesis, wherein the glycosyltransferase mutant has a high enzyme activity, and is beneficial to the production of gastrodin by an industrial biological method.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a glycosyltransferase mutant which has an amino acid sequence shown as SEQ ID No.1 in a sequence table.

The invention provides a glycosyltransferase mutant coding gene which has a nucleotide sequence shown as SEQ ID No.2 in a sequence table.

The invention provides a glycosyltransferase recombinant plasmid, which comprises a glycosyltransferase mutant coding gene.

The invention provides a recombinant strain of glycosyltransferase, which is an expression vector transformed with the recombinant plasmid.

The invention provides application of the glycosyltransferase mutant or the glycosyltransferase recombinant strain in synthesis of gastrodin by catalyzing a substrate.

Preferably, the catalytic substrate is p-hydroxybenzyl alcohol and/or UDP-glucose.

Preferably, the temperature of the catalysis is 28-35 ℃.

Preferably, in the process of synthesizing gastrodin by catalyzing a substrate by the glycosyltransferase recombinant strain, the glycosyltransferase recombinant strain is induced for 15-25 h under the condition of 0.1-1 mmol/L isopropyl-beta-D-thiogalactopyranoside solution.

The invention provides a glycosyltransferase mutant which has an amino acid sequence shown as SEQ ID No.1 in a sequence table. The invention uses a mutant UGT73B6 derived from glycosyltransferase UGT73B6 in rhodiola sachalinensis^MKAs a parent, site mutation of phenylalanine Phe (F) at position 389 is mutated into alanine Ala (A, glycosyltransferase mutant shown in SEQ ID No. 1), so that the enzyme activity of catalytic synthesis of gastrodin is improved. The invention provides application of the glycosyltransferase mutant or the glycosyltransferase recombinant strain in catalysis of biosynthesis of gastrodin. Modified glycosyltransferase mutant UGT73B6 shown in SEQ ID No.1^MK+FAThe activity of catalyzing the p-hydroxybenzyl alcohol to be converted into the gastrodin is greatly improved, the in vitro enzyme activity is 4.9 times of that of the parent UGT73B6MK, the in vivo conversion rate is 3.8 times of that of the parent UGT73B6MK, and the catalytic activity is better and is more suitable for industrial production of the gastrodin.

Drawings

FIG. 1 shows the mutant UGT73B6 of example 2^MK+FAAnd parent UGT73B6^MKPurification of protein SDS-PAGE electrophorograms, in which the arrows indicate purified proteins; wherein Lane1 is protein Marker, Lane2 is purified parent UGT73B6^MKLane3 is mutant UGT73B6^MK+FA；

FIG. 2 is a comparison of the yields of the mutant recombinant strain and the parent recombinant strain in example 4; wherein FIG. 2-a is parental recombinant strain BL21(DE3) pET28a-ugt73b6^MKFIG. 2-b shows mutant recombinant bacteria BL21(DE3) pET28a-ugt73b6^MK ^+FAFIG. 2-c shows mutant recombinant bacteria BL21(DE3) pET28a-ugt73b6^MK+FS；

FIG. 3 is the HPLC and LC-MS identification spectra of the mutant recombinant strain and the parent recombinant strain transformed into gastrodin in example 3; wherein, FIG. 3-a shows mutant recombinant bacterium BL21(DE3) pET28a-ugt73b6^MK+FAHPLC (high performance liquid chromatography) spectrum of fermentation liquor; FIG. 3-b is the parental recombinant strain BL21(DE3) pET28a-ugt73b6^MKHPLC (high performance liquid chromatography) spectrum of fermentation liquor; FIG. 3-c shows HPLC of gastrodin standard; FIG. 3-d is an LC-MS spectrum of peak I; FIG. 3-e is an LC-MS spectrum of peak II.

Detailed Description

In the present invention, a method for producing a gene encoding a glycosyltransferase mutant preferably comprises the steps of: is a mutant UGT73B6 derived from glycosyltransferase UGT73B6 in rhodiola sachalinensis^MKAs a parent, a mutant coding gene is obtained by using a site-directed mutagenesis method, specifically, the site of the phenylalanine Phe (F) at the 389 site is mutated into the alanine Ala (A), and the formed mutant has great improvement on the enzyme activity for catalyzing and synthesizing the gastrodin. In the present invention, the method of site-directed mutagenesis comprises the following steps:

1) designing a site-directed mutagenesis primer;

2) to contain parent UGT73B6^MKGene expression plasmid pET28a-ugt73B6^MKUsing the site-directed mutagenesis primer in the step 1) as a template to carry out PCR amplification,obtaining a PCR product;

3) the resulting PCR product was digested to remove pET28a-ugt73B6^MKA template to obtain a digested PCR product;

4) and transforming the digested PCR product into escherichia coli DH5 alpha, selecting a transformant, culturing, extracting plasmids, sequencing DNA, and analyzing a sequence to obtain a mutant coding gene with correct mutation.

The method of designing the site-directed mutagenesis primer of the present invention is not particularly limited, and a primer design method known to those skilled in the art may be used. In the present invention, the site of site-directed mutation is the 389 th amino acid residue of glycosyltransferase. In the present invention, the 389 Phe codon TTC was mutated to Ala high frequency codon GCG.

In the present invention, the site-directed mutagenesis primer used for the mutation to Ala is preferably F389A-PS, F389A-PA. The F389A-PS has a nucleotide sequence shown as SEQ ID No.3 in a sequence table. The F389A-PA has a nucleotide sequence shown as SEQ ID No.4 in the sequence table.

SEQ ID Nos. 3 to 4 are shown in Table 1, wherein the base after mutation is underlined.

TABLE 1 mutant primers

Primer name	Primer sequences
		F389A-PS	5’-GTTACTTGGCCGGTGGCGGCTGAACAGTTCTAC-3’(SEQ ID No.3)
F389A-PA	5’-GTAGAACTGTTCAGCCGCCACCGGCCAAGTAAC-3’(SEQ ID No.4)

After obtaining the site-directed mutagenesis primer, the invention uses the gene containing parent UGT73B6^MKGene expression plasmid pET28a-ugt73B6^MKAnd (3) taking the site-directed mutagenesis primer as a template, and carrying out PCR amplification to obtain a PCR product.

In the invention, the parent UGT73B6^MKGene expression plasmid pET28a-ugt73B6^MKDisclosed in chinese patent No. 201510160496.3.

In the present invention, the site-directed mutagenesis PCR reaction system is preferably as follows:

reaction components	Volume (μ L)
		5 XQ 5 buffer solution	10
dNTPs(2mM)	5
		Upstream primer (25. mu.M)	1
Downstream primer (25. mu.M)	1
		Stencil (20 ng/. mu.L)	4
Q5DNA polymerase (2.5U/. mu.L)	0.5
		ddH₂O	50

In the present invention, the site-directed mutagenesis PCR amplification conditions are preferably as follows: 30s at 98 ℃; 10s at 98 ℃, 15s at 60 ℃, 2min at 72 ℃ for 30s, and 30 cycles; 5min at 72 ℃.

After obtaining the PCR product, the invention digests the PCR product to remove pET28a-ugt73B6^MKAnd (5) template obtaining a digested PCR product.

In the present invention, the enzyme for digestion is preferably DpnI enzyme. The preferable digestion conditions of the DpnI enzyme are as follows: 20. mu.L of the amplified PCR product was digested overnight at 37 ℃ with 1. mu.L of LDpnI enzyme.

Obtaining a digested PCR product, transforming the digested PCR product into escherichia coli DH5 alpha, culturing and selecting a transformant, extracting a plasmid, carrying out DNA sequencing of Jinwei Zhi company, and obtaining a high-frequency codon GCG mutant with 389 th Phe codon TTC mutated into Ala through sequence analysis.

In the present invention, the plasmid pET28a was purchased from Novagen under the accession number 69864.

The present invention is not limited to any particular connection method, and may be any connection method known to those skilled in the art.

The method of transformation is not particularly limited in the present invention, and a transformation method known to those skilled in the art may be used. The high frequency codon GCG obtained by mutating the Phe codon at 389 to Ala is named ugt73B6^MK+FAAnd the plasmid containing the mutant gene is pET28a-ugt73B6^MK+FA。

In the present invention, the kind of the expression vector is not particularly limited, and eukaryotic expression vectors and prokaryotic expression vectors known in the art may be used. The prokaryotic expression vector is preferably Escherichia coli DH5 alpha. The origin of the Escherichia coli DH 5. alpha. is not particularly limited, and a commercially available product known in the art may be used. The E.coli DH 5. alpha. was used for cloning all the genes of the present invention. The eukaryotic expression vector is preferably Saccharomyces cerevisiae. The source of the Saccharomyces cerevisiae is not particularly limited, and commercially available products known to those skilled in the art may be used.

The sequencing method is not particularly limited, and can be performed by a sequencing mode known to those skilled in the art. In the examples of the present invention, the sequencing was performed by Jinzhi corporation.

The invention provides a glycosyltransferase recombinant plasmid, which comprises a glycosyltransferase mutant coding gene. The plasmid is not particularly limited, and any plasmid known in the art can be used.

The invention provides a recombinant strain of glycosyltransferase, wherein the recombinant strain is escherichia coli transformed with the recombinant plasmid.

In the invention, the expression vector of the glycosyltransferase recombinant strain is escherichia coli. The species of E.coli is preferably strain BL21(DE 3). The source of the strain BL21(DE3) is not particularly limited in the present invention, and a strain BL21(DE3) known to those skilled in the art can be used.

The invention provides application of the glycosyltransferase mutant or the glycosyltransferase recombinant strain in catalysis of biosynthesis of gastrodin.

In the invention, the glycosyltransferase mutant UGT73B6^MK+FACatalysis is used in the form of whole cells of engineering bacteria or glycosyltransferase mutant UGT73B6^MK+FAThe crude enzyme is used either in partially purified or fully purified form. In the present invention, the enzyme comprises the glycosyltransferase mutant UGT73B6 of the present invention immobilized by the art^MK+FAImmobilized enzymes are prepared in the form of immobilized enzymes or immobilized cells.

In the present invention, the method for separating and extracting the crude enzyme or the partially purified or completely purified enzyme is not particularly limited, and a crude enzyme extraction protocol well known in the art may be used.

In the present invention, the catalytic substrate is preferably p-hydroxybenzyl alcohol and/or UDP-glucose.

In the invention, the concentration of the catalyzed substrate is preferably 2-5 mmol/L.

In the invention, the temperature of catalysis is preferably 28-35 ℃, and more preferably 30 ℃.

In the present invention, the concentration of the glycosyltransferase mutant is preferably 80 to 100. mu.g/. mu.L. In the present invention, glycosyltransferase mutants are catalyzed by Mg²⁺The process is carried out under participation; the Mg²⁺The concentration of (b) is 4-6 mmol/L. In the present invention, the culture conditions for the catalysis of the recombinant strain are as follows: culturing at 37 deg.C; the culture time is preferably 0.6 to 0.8 OD value of the culture medium, and the substrate is added. The glycosyltransferase recombinant strain is induced for 15-25 h under the condition of 0.1-1 mmol/L isopropyl-beta-D-thiogalactopyranoside solution.

The glycosyltransferase mutant and its application in catalyzing the biosynthesis of gastrodin will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction of mutant UGT73B6 by site-directed mutagenesis^MK+FA. Design site-directed mutagenesis primers to contain the parent UGT73B6^MKGene expression plasmid pET28a-ugt73B6^MK(patent No. 201510160496.3) was used as a template for PCR amplification. Site-directed mutagenesis PCR reaction system:

reaction components	Volume (μ L)
		5 XQ 5 buffer solution	10
dNTPs(2mM)	5
		Upstream primer (25. mu.M)	1
Downstream primer (25. mu.M)	1
		Stencil (20 ng/. mu.L)	4
Q5DNA polymerase (2.5U/. mu.L)	0.5
		ddH₂O	to50

PCR amplification conditions: 30s at 98 ℃; 10s at 98 ℃, 15s at 60 ℃, 2min at 72 ℃ for 30s, and 30 cycles; 5min at 72 ℃.

The mutation of Phe to Ala at position 389 was performed using F389A-PS, F389A-PA as shown in Table 1, with the base after mutation underlined.

TABLE 1 mutant primers

Primer name	Primer sequences
		F389A-PS	5’-GTTACTTGGCCGGTGGCGGCTGAACAGTTCTAC-3’
F389A-PA	5’-GTAGAACTGTTCAGCCGCCACCGGCCAAGTAAC-3’

Directly digesting the amplified PCR product by using DpnI enzyme to remove pET28a-ugt73B6^MKAnd (5) template. The DpnI enzyme digestion conditions are as follows: 20. mu.L of the amplified PCR product was digested overnight at 37 ℃ with 1. mu.L of DpnI enzyme.

Directly transforming the digested PCR product into Escherichia coli DH5 alpha, culturing, selecting a transformant, extracting a plasmid, carrying out DNA sequencing by Jinzhi, and determining UGT73B6 by sequence analysis^MKThe 389 th Phe codon TTC of the gene is mutated into Ala high-frequency codon GCG, thus obtaining the gene ugt73B6 containing mutant^MK+FAOr plasmid pET28a-ugt73B6 containing mutant gene^MK+FA。

Example 2

Mutant UGT73B6^MK+FAAnd parent UGT73B6^MKRecombinant expression and purification of

Plasmid pET28a-ugt73B6^MK，pET28a-ugt73b6^MK+FAColi strain BL21(DE3) was transformed by chemical transformation. The construction method of the recombinant bacteria comprises the following steps: 100 μ L of competent Escherichia coli strain BL21(DE3) cells were placed on ice, 1 μ L of plasmid was added after 10 minutes, the mixture was gently mixed, placed on ice for 30 minutes, heat-shocked at 42 ℃ for 90 seconds, immediately taken out and placed on ice for 2 minutes, 800 μ L of non-resistant LB liquid medium was added, shaking-bed cultivation was performed at 37 ℃ and 200rpm for 40 minutes, and then the bacterial solution was spread on LB plate containing kanamycin. Screening a transformation strain carrying an expression vector by using kanamycin resistance, and performing enzyme digestion verification by extracting plasmids to obtain a recombinant strain BL21(DE3) pET28a-ugt73b6 containing a mutant^MK+FAAnd a parent recombinant strain BL21(DE3) pET28a-ugt73b6^MK。

Protein induced expression and purification: the mutant strain and the parent strain are selected and cloned, and cultured overnight at 37 ℃ in a liquid LB culture medium added with 50mg/L kanamycin, then inoculated into 1000mL of the liquid LB culture medium added with 50mg/L kanamycin at a ratio of 1:100, cultured at 37 ℃ until OD is 0.6-0.8, and added with 0.1mM IPTG (isopropyl-beta-D-thiogalactopyranoside) for induction culture at 16 ℃ for 20 hours. The cells were collected by centrifugation at 3000rpm for 10min at 4 ℃ and resuspended in 50mL of lysis buffer (50mM Tris/HCl, pH 8.0, 10mM imidazole, 10% glycerol). Resuspend the cells, crush them 3 times with French pressure cell crusher, centrifuge them at 10,000g for 30min at 4 ℃, mix the supernatant with 2mL of nickel column agarose resin (Ni-NTAresin, QIAGEN Valencia, Calif.) equilibrated with lysis buffer immediately, bind at 4 ℃ for 50min, and wash the column with 100mL of lysis buffer. The nickel column was then eluted with 3mL of elution buffer (50mM Tris/HCl, pH 8.0, 0.5M imidazole, 10% glycerol), the eluate was collected into dialysis bags and the imidazole was removed by equilibration with 50mM Tris/HCl (pH 8.0) containing 10% glycerol buffer. The purified protein was concentrated in a Milipore ultrafiltration tube (3 kDa). The Bradford protein assay kit measures the concentration of protein after concentration and dispenses and stores at-80 ℃.

The purified protein was subjected to SDS-PAGE, and as shown in FIG. 1, the protein size was about 54kDa, and the purified mutant UGT73B6^MK+FAAnd parent UGT73B6^MKThe protein concentration of (a) was about 20mg/mL each, and there was no significant difference in the expression amount of the mutant protein. The purified protein is subjected to in vitro enzyme activity determination.

Example 3

Mutant UGT73B6^MK+FAAnd UGT73B6^MKComparison of enzyme Activity

(1) In vivo transformation experiments

Respectively picking up each strain clone, including recombinant strain BL21(DE3) pET28a-ugt73b6 containing mutant^MK+FAParent recombinant strain BL21(DE3) pET28a-ugt73b6^MKAnd the strain BL21(DE3) pET28a-ugt73b6 which is already applied for patent in the laboratory^FS+MK(patent No. 201510160497.8), cultured overnight at 37 ℃ in a liquid LB medium containing 50mg/L of kanamycin, then inoculated into 50mL of a liquid LB medium containing 50mg/L of kanamycin at a ratio of 1:100, cultured at 37 ℃ to an OD of 0.6 to 0.8, and subjected to induction culture at 16 ℃ for 20 hours with the addition of 0.1mM of IPTG (isopropyl-. beta. -D-thiogalactopyranoside). Centrifuging the induced bacteria liquid at 4000rpm for 10min, removing supernatant, collecting thallus, transferring to 50mL fresh M9Y culture medium, and feeding2mM substrate p-hydroxybenzyl alcohol was added. And then placing the mixture at 30 ℃ and 200rpm for shaking culture for 84 hours, taking fermentation liquor every 12 hours from 24 hours, centrifuging to take supernatant, carrying out gastrodin LC-MS identification, measuring gastrodin yield by HPLC, and comparing in vivo conversion rates of the mutant protein and the parent protein.

LC-MS identification results show that the peak I is gastrodin, and the peak II is added substrate p-hydroxybenzyl alcohol, as shown in figure 3-d and figure 3-e. The results of in vivo transformation experiments show that when 2mM substrate is fed in the process of shake flask culture, the gastrodin yield of each strain tends to be stable within 72 hours. 72 hour mutant UGT73B6^MK+FAThe yield of the transformed gastrodin is 574mg/L, and the reference parent UGT73B6^MKThe yield of the transformed gastrodin is 161mg/L, and the mutant UGT73B6 which is applied for patent is contrasted^FS+MKThe yield of the transformed gastrodin is 317mg/L as shown in figure 2. UGT73B6^MK+FAThe conversion rate of in vivo conversion of 2mM substrate to the synthesis of gastrodin by using hydroxybenzyl alcohol is parent UGT73B6^MK3.8 times of the mutant of the already applied patent ugt73b6^FS+MK1.7 times of the total weight of the powder. And substrate was added for 24 hours, mutant UGT73B6^MK+FA70% of the substrate had been converted and 2mM p-hydroxybenzyl alcohol had been completely converted for 72 hours in culture to synthesize gastrodin, as shown in FIG. 2.

(2) In vitro transformation experiments

The enzymatic reaction temperature was 30 ℃ and the reaction system was 100. mu.L containing 50mM Tris/HCl (pH 8.0), 5mM MgCl₂Mu.g of pure enzyme, p-hydroxybenzyl alcohol and UDP-glucose at 5mM each, reaction time 10min (determined by preliminary experiments), and termination of the enzyme reaction by addition of 4. mu.L of 10% TFA. Centrifuging 10,000g of reaction product for 10min, taking 50 mu L of supernatant for HPLC analysis, measuring the yield of the synthesized gastrodin, and calculating the initial speed of the enzymatic reaction.

The results show that the mutant UGT73B6^MK+FAThe initial reaction speed for converting gastrodin is 2.68 mu M/min, and the parent UGT73B6^MKThe initial reaction speed for converting gastrodin is 0.5 mu M/min, which indicates that the mutant UGT73B6^MK+FAThe enzyme activity is parent UGT73B6^MK4.9 times of the total weight of the powder.

Example 4

Detection of hydroxybenzyl alcohol and gastrodin

(1) HPLC detection

An Agilent liquid chromatograph, and the determination conditions are as follows: a C18 column (4.6X 250 mm); the detection wavelength is 224 nm; mobile phase a ═ water (containing 0.1% by volume of formic acid), B ═ methanol; the flow rate is 1 ml/min; gradient elution conditions: 0-20 min 10% volume B; the amount of sample was 50. mu.L.

(2) LC-MS analysis

Conditions for LC-MS analysis: a C18 column (4.6X 250 mm); the detection wavelength is 224 nm; mobile phase a ═ water (containing 0.1 vol% formic acid), B ═ methanol; the flow rate is 1 ml/min; elution conditions: 0-35 min 10% volume B; the sample volume is 20 mu L; ESI positive ion source, molecular weight scanning range 50 ~ 800.

The results of the detection are shown in FIG. 3. The results of the detection are shown in FIG. 3. Indicating mutant UGT73B6^MK+FAConverting substrate p-hydroxybenzyl alcohol (peak II) to synthesize gastrodin (peak I), and converting 2mM substrate p-hydroxybenzyl alcohol in vivo to synthesize gastrodin with parent UGT73B6^MK3.8 times of the total weight of the powder.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> glycosyltransferase mutant and application thereof in catalyzing biosynthesis of gastrodin

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 480

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gly Ser Glu Thr Arg Pro Leu Ser Ile Phe Phe Phe Pro Phe Met

1 5 10 15

Ala His Gly His Met Ile Pro Met Val Asp Met Ala Arg Leu Phe Ala

20 25 30

Ser Gln Gly Val Arg Cys Thr Ile Val Thr Thr Pro Gly Asn Gln Pro

35 40 45

Leu Ile Ala Arg Ser Ile Gly Lys Val Gln Leu Leu Gly Phe Glu Ile

50 55 60

Gly Val Thr Thr Ile Pro Phe Arg Gly Thr Glu Phe Gly Leu Pro Asp

65 70 75 80

Gly Cys Glu Asn Leu Asp Ser Val Pro Ser Pro Gln His Val Phe His

85 90 95

Phe Phe Glu Ala Ala Gly Ser Leu Arg Glu Pro Phe Glu Gln Leu Leu

100 105 110

Glu Glu His Lys Pro Asp Cys Val Val Gly Asp Met Phe Phe Pro Trp

115 120 125

Ser Thr Asp Ser Ala Ala Lys Phe Gly Ile Pro Arg Leu Val Phe His

130 135 140

Gly Thr Ser Tyr Phe Ala Leu Cys Ala Gly Glu Ala Val Arg Ile His

145 150 155 160

Lys Pro Tyr Leu Ser Val Ser Ser Asp Asp Glu Pro Phe Val Ile Pro

165 170 175

Gly Leu Pro Asp Glu Ile Lys Leu Thr Lys Ser Gln Leu Pro Met His

180 185 190

Leu Leu Glu Gly Lys Lys Asp Ser Val Leu Ala Gln Leu Leu Asp Glu

195 200 205

Val Lys Glu Thr Glu Val Ser Ser Tyr Gly Val Ile Val Asn Ser Ile

210 215 220

Tyr Glu Leu Glu Pro Ala Tyr Ala Asp Tyr Phe Arg Asn Val Leu Lys

225 230 235 240

Arg Arg Ala Trp Glu Ile Gly Pro Leu Ser Leu Cys Asn Arg Asp Val

245 250 255

Glu Glu Lys Ala Met Arg Gly Lys Gln Ala Ala Ile Asp Gln His Glu

260 265 270

Cys Leu Lys Trp Leu Asp Ser Lys Glu Pro Asp Ser Val Val Tyr Val

275 280 285

Cys Phe Gly Ser Thr Cys Lys Phe Pro Asp Asp Gln Leu Ala Glu Ile

290 295 300

Ala Ser Gly Leu Glu Ala Ser Gly Gln Gln Phe Ile Trp Val Ile Arg

305 310 315 320

Arg Met Ser Asp Asp Ser Lys Glu Asp Tyr Leu Pro Lys Gly Phe Glu

325 330 335

Glu Arg Val Lys Asp Arg Ala Leu Leu Ile Arg Gly Trp Ala Pro Gln

340 345 350

Val Leu Ile Leu Asp His Gln Ser Val Gly Gly Phe Val Ser His Cys

355 360 365

Gly Trp Asn Ser Thr Leu Glu Gly Ile Ser Ala Gly Leu Pro Met Val

370 375 380

Thr Trp Pro Val Ala Ala Glu Gln Phe Tyr Asn Glu Lys Leu Leu Thr

385 390 395 400

Glu Val Leu Lys Ile Gly Val Ala Val Gly Ala Arg Lys Trp Arg Gln

405 410 415

Leu Val Gly Asp Phe Val His Lys Asp Ala Ile Gln Arg Ala Val Arg

420 425 430

Glu Ile Met Glu Gly Glu Glu Ala Glu Glu Arg Arg Ile Ile Ala Arg

435 440 445

Gln Met Gly Lys Met Ala Lys Arg Ala Val Glu Lys Asp Gly Ser Ser

450 455 460

Trp Thr Asn Leu Asn Asn Leu Leu Gln Glu Leu Lys Leu Lys Lys Val

465 470 475 480

<210> 2

<211> 1440

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggctctg aaactcgccc gctgagcatc ttcttttttc cgtttatggc gcatggccac 60

atgattccga tggtggatat ggcacgtctg tttgcttctc agggtgtgcg ttgcaccatt 120

gttaccactc cgggtaacca gccgctgatt gctcgctcta tcggtaaggt tcagctgctg 180

ggttttgaaa ttggtgtgac tactatcccg ttccgcggta ctgagttcgg cctgccggat 240

ggctgtgaaa acctggatag cgtgccgagc ccgcagcatg tgtttcattt ctttgaggca 300

gcgggtagcc tgcgtgagcc gtttgaacag ctgctggaag agcacaaacc ggactgtgtt 360

gtgggcgata tgttctttcc gtggtctacc gactctgcgg ctaaattcgg tattccgcgc 420

ctggttttcc acggtacctc ctacttcgcg ctgtgcgctg gcgaagcagt gcgtattcat 480

aagccgtacc tgtctgtgtc ttctgatgat gaaccgttcg ttattccggg cctgccggac 540

gagatcaaac tgaccaagtc ccagctgccg atgcacctgc tggagggtaa gaaagactct 600

gttctggcac agctgctgga tgaggtgaaa gaaactgagg tttcctctta cggtgttatc 660

gttaactcta tctacgaact ggaaccggct tacgcagatt acttccgtaa cgttctgaag 720

cgccgtgcgt gggagatcgg tccgctgtct ctgtgtaacc gtgacgttga agagaaagcg 780

atgcgtggta agcaggctgc tatcgatcag catgaatgcc tgaaatggct ggattccaaa 840

gaaccggatt ccgttgttta cgtttgtttt ggtagcactt gcaaattccc ggatgatcag 900

ctggcggaaa tcgcgtctgg tctggaggca agcggccagc agttcatctg ggttattcgc 960

cgtatgtctg acgactctaa ggaagactac ctgccgaaag gtttcgaaga gcgtgttaag 1020

gaccgtgctc tgctgattcg cggttgggct ccgcaggttc tgatcctgga ccatcagtcc 1080

gttggcggtt ttgtttctca ctgtggttgg aactctaccc tggaaggcat cagcgcgggt 1140

ctgccgatgg ttacttggcc ggtggcggct gaacagttct acaacgaaaa actgctgacc 1200

gaggtgctga aaatcggtgt tgcagtgggt gctcgtaagt ggcgtcagct ggtgggtgac 1260

ttcgttcaca aagacgctat tcagcgtgcg gtgcgtgaaa ttatggaggg cgaagaggcg 1320

gaggaacgtc gtatcatcgc gcgtcagatg ggtaaaatgg cgaaacgcgc ggtggagaag 1380

gacggtagct cttggaccaa cctgaacaac ctgctgcagg aactgaagct gaagaaggtt 1440

<210> 3

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gttacttggc cggtggcggc tgaacagttc tac 33

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtagaactgt tcagccgcca ccggccaagt aac 33

Claims

1. A glycosyltransferase mutant is characterized in that the amino acid sequence of the glycosyltransferase mutant is shown as SEQ ID No.1 in a sequence table.

2. The glycosyltransferase mutant encoding gene of claim 1, wherein the nucleotide sequence of the gene is the nucleotide sequence shown as SEQ ID No.2 in the sequence listing.

3. A recombinant plasmid for glycosyltransferase, comprising the gene encoding the glycosyltransferase mutant of claim 2.

4. A recombinant strain of glycosyltransferase, comprising an expression vector for the recombinant plasmid of claim 3.

5. Use of the glycosyltransferase mutant of claim 1 or the recombinant strain of glycosyltransferase of claim 4 in catalyzing a substrate to synthesize gastrodin.

6. Use according to claim 5, wherein the substrate is p-hydroxybenzyl alcohol and/or UDP-glucose.

7. Use according to claim 5 or 6, wherein the temperature of the catalysis is between 28 and 35 ℃.

8. The use of claim 7, wherein in the process of synthesizing gastrodin by catalyzing a substrate with the glycosyltransferase recombinant strain, the glycosyltransferase recombinant strain is induced for 15-25 h under the condition of 0.1-1.0 mmol/L isopropyl-beta-D-thiogalactopyranoside solution.