CN104611354B

CN104611354B - Fusion protein Nt4CL5aPcSTS relevant biological material and its application

Info

Publication number: CN104611354B
Application number: CN201510061199.3A
Authority: CN
Inventors: 郭辉力; 马兰青; 张红; 薛飞燕; 马雅迪; 杨亚东; 罗在柒; 刘文彬; 冯静; 刘欢; 姜英英
Original assignee: Beijing University of Agriculture
Current assignee: Beijing University of Agriculture
Priority date: 2015-02-05
Filing date: 2015-02-05
Publication date: 2017-09-05
Anticipated expiration: 2035-02-05
Also published as: CN104611354A

Abstract

The invention discloses fusion protein Nt4CL5aPcSTS relevant biological material and its application.The fusion protein Nt4CL5aPcSTS relevant biological materials that the present invention is provided include fusion protein Nt4CL5aPcSTS, the fusion protein Nt4CL5aPcSTS is obtained by protein N t4CL and protein PcSTS by three amino acid residue connections, and the protein N t4CL is that amino acid sequence is the protein that SEQ ID No.1 are constituted from the amino acids residue of N-terminal the 1st 542；The protein PcSTS is that amino acid sequence is protein shown in SEQ ID No.1 from the amino acids residue of N-terminal the 548th 935.Fusion protein Nt4CL5aPcSTS relevant biological material can be catalyzed the resveratrol of 4 coumaric acids and malonyl coenzyme A generation with bioactivity.

Description

Fusion protein Nt4CL5aPcSTS relevant biological material and its application

Technical field

The present invention relates to fusion protein Nt4CL5aPcSTS relevant biological material and its application in biological technical field.

Background technology

Resveratrol (Resveratrol, abbreviation Res), entitled 3,4', 5- trihydroxies -1, the 2- talan of chemistry (trans-3,4', 5-trihydroxystilbene), also known as resvertrol, are a kind of non-flavonoids polyphenol containing stilbene class formation Compound, molecular weight is 228.2, water insoluble, is dissolved in the organic solvents such as ethanol.Natural resveratrol is deposited in many plants Such as giant knotweed, peanut and grape.

Resveratrol is that plant meets with a kind of plant protection sharply increased when ultraviolet irradiation, mechanical damage and fungal infection Element, research shows that resveratrol has anticancer, protects cardiovascular, anti-oxidant, anti-aging, immunological regulation, neuroprotection and plant Estrogen etc. is acted on, and can be widely used in the fields such as medicine, health products, food, cosmetics.Resveratrol is that grape wine is most heavy The health-care components wanted, Frenchman is under higher fatty acid, high protein, high-energy eating habit, and the incidence of disease of angiocardiopathy is far below Other American-European countries, just have benefited from often drinking the red wine containing resveratrol.

Resveratrol sources are in phenylpropyl alcohol alkane metabolic pathway (Phenylpropanoid pathway).Its Biometabolic pathway For：Phenylalanine is cracked into trans under phenylalanine lyase (Phenylalanine ammonia-lyase, PAL) effect Cinnamic acid, cinnamic acid is generated under the catalytic action of cinnamic acid -4- hydroxylases (Cinnamate-4-hydroxylase, C4H) 4- coumaric acids, 4- coumaric acids are closed in the presence of 4- coumaroyl A ligases (4-Coumarate-CoA ligase, 4CL) Into 4- coumaroyl A, finally, stilbene synthase (Stilbene synthase, STS) is catalyzed the 4- coumaroyls A and 3 of 1 molecule The malonyl coenzyme A generation resveratrol (Fig. 1) of molecule.

Resveratrol biosynthesis pathway rate-limiting step is more in vivo, although a certain key enzyme of simple overexpression Content to resveratrol may produce certain facilitation, but due to the limitation of other steps, the high level of resveratrol Accumulation is extremely difficult.

The content of the invention

The technical problems to be solved by the invention are how to improve the yield of resveratrol.

In order to solve the above technical problems, the invention provides fusion protein Nt4CL5aPcSTS relevant biological material.

Fusion protein Nt4CL5aPcSTS provided by the present invention relevant biological material, is following B1) to B20) in It is any：

B1) encoding fusion protein Nt4CL5aPcSTS nucleic acid molecules；

B2 B1) is contained) expression cassettes of the nucleic acid molecules；

B3 B1) is contained) recombinant vectors of the nucleic acid molecules；

B4 B2) is contained) recombinant vector of the expression cassette；

B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；

B6 B2) is contained) recombinant microorganism of the expression cassette；

B7 B3) is contained) recombinant microorganism of the recombinant vector；

B8 B4) is contained) recombinant microorganism of the recombinant vector；

B9 B1) is contained) the transgenic plant cells systems of the nucleic acid molecules；

B10 B2) is contained) the transgenic plant cells system of the expression cassette；

B11 B3) is contained) the transgenic plant cells system of the recombinant vector；

B12 B4) is contained) the transgenic plant cells system of the recombinant vector；

B13 B1) is contained) Transgenic plant tissues of the nucleic acid molecules；

B14 B2) is contained) Transgenic plant tissue of the expression cassette；

B15 B3) is contained) Transgenic plant tissue of the recombinant vector；

B16 B4) is contained) Transgenic plant tissue of the recombinant vector；

B17 B1) is contained) the genetically modified plants organs of the nucleic acid molecules；

B18 B2) is contained) the genetically modified plants organ of the expression cassette；

B19 B3) is contained) the genetically modified plants organ of the recombinant vector；

B20 B4) is contained) the genetically modified plants organ of the recombinant vector；

The fusion protein Nt4CL5aPcSTS is following protein (1) or (2)：

(1) the fusion protein obtained by Nt4CL albumen and PcSTS protein fusions：

The Nt4CL albumen be it is following a) or b)：

A) amino acid sequence is the protein of SEQ ID No.1 1-542 amino acids residues；

B) by substitution a) by one or several amino acid residues and/or missing and/or addition and with identical function By its derivative protein；

The PcSTS albumen is following A or B：

A, amino acid sequence are the protein shown in SEQ ID No.1 548-935 amino acids residues；

B, by A by the substitution and/or missing and/or addition of one or several amino acid residues and with identical function By its derivative protein.

(2) the tape label protein obtained N-terminal (1) or C-terminal connection label.

In the fusion protein, the protein N t4CL and protein PcSTS are connected by five amino acid residue.Institute The sequence of five amino acid residue is stated as shown in SEQ ID No.1 543-547, the amino acid sequence of the fusion protein is SEQ ID No.1。

In order that the fusion protein Nt4CL5aPcSTS is easy to purifying, can be the fusion protein Nt4CL5aPcSTS's N-terminal and/or the upper label as shown in table 1 of C-terminal connection.The N-terminal and C-terminal of protein as shown in SEQ ID No.1 connect respectively Connect the fusion protein that a 6*His label (HHHHHH) obtains.

The sequence of the label of table 1.

Label	Residue	Sequence
			Poly-Arg	5-6 (being usually 5)	RRRRR
Poly-His	2-10 (being usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned fusion protein, the substitution of one or several amino acid residues and/or missing and/or be added to is no more than The substitution and/or missing and/or addition of 10 amino acid residues.

Above-mentioned fusion protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtains.It is above-mentioned to melt The encoding gene of hop protein can be by will lack one or several amino acid residues in the DNA sequence dna shown in SEQ ID No.2 Codon, and/or the missense mutation of one or several base-pairs of progress are obtained.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.

In above-mentioned biomaterial, B1) nucleic acid molecules for it is following 1) or 2) or 3) or 4) shown in nucleic acid molecules, i.e., Nt4CL5aPcSTS gene：

1) coded sequence is the cDNA molecules or DNA molecular of SEQ ID No.2 1-2808 nucleotides；

2) nucleotide sequence is SEQ ID No.2 cDNA molecule or DNA molecular；

1) or 2) 3) there is 75% or more than 75% homogeneity, and encoding said fusion protein with the nucleotide sequence that limits CDNA molecules or genomic DNA molecule；

4) under strict conditions with 1) or 2) nucleotide sequence hybridization limited, and cDNA points of encoding said fusion protein Son or genomic DNA molecule.

Wherein, SEQ ID No.2 are made up of 2808 nucleotides, SEQ ID No.2 nucleotide coding SEQ ID No.1 Shown amino acid sequence.

Those of ordinary skill in the art can be easily using known method, such as side of orthogenesis and point mutation Method, is mutated to the coding Nt4CL5aPcSTS of present invention nucleotide sequence.Those by manually modified, with this The artificial synthesized Nt4CL5aPcSTS of invention nucleotide sequence 75% or the nucleotides of higher homogeneity, as long as coding Nt4CL5aPcSTS and the protein with identical function are the nucleotide sequences derived from the present invention and are equal to this hair Bright sequence.

Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair Shown in bright coding SEQ ID No.1 amino acid sequence composition protein nucleotide sequence have 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or calculate Machine software is evaluated.Using computer software, the homogeneity between two or more sequences can be represented with percentage (%), It can be for the homogeneity between evaluation correlated series.

In above-mentioned biomaterial, the stringent condition is, in 2 × SSC, in 0.1%SDS solution, to hybridize simultaneously at 68 DEG C Wash film 2 times, each 5min, and in 0.5 × SSC, 0.1%SDS solution, hybridize at 68 DEG C and wash film 2 times, every time 15min。

Above-mentioned 75% or more than 75% homogeneity, can be 80%, 85%, 90% or more than 95% homogeneity.

In above-mentioned biomaterial, B2) described in the nucleic acid molecules containing coding Nt4CL5aPcSTS expression cassette (Nt4CL5aPcSTS expression casettes), is the DNA for referring to express Nt4CL5aPcSTS in host cell, the DNA is not only It may include the promoter for starting Nt4CL5aPcSTS genetic transcriptions, may also include the termination for terminating Nt4CL5aPcSTS genetic transcriptions Son.Further, the expression cassette may also include enhancer sequence.

In above-mentioned biomaterial, the Nt4CL5aPcSTS expression casettes can be contained with existing expression vector establishment Recombinant vector.

In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vector.

In above-mentioned biomaterial, described microorganism can be prokaryotic micro-organisms or eukaryotic microorganisms.The prokaryotic micro-organisms It can be bacterium.The bacterium can be Escherichia bacteria.The Escherichia bacteria can be Escherichia coli.The eucaryon is micro- Biology can be algae, fungi, protozoan.The fungi can be unicellular fungi.The unicellular fungi can be saccharomycete.Institute It can be yeast strain WAT11 to state saccharomycete.

In above-mentioned biomaterial, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are equal Do not include propagating materials.

Above-mentioned recombinant vector can be that the Nt4CL5aPcSTS genes are inserted to the restructuring table constituted in pET30a (+) plasmid Up to carrier；The recombinant bacterium can be recombination bacillus coli.

Above-mentioned recombinant vector can be that the Nt4CL5aPcSTS genes are inserted to the restructuring table constituted in pESC-Trp plasmids Up to carrier；The recombinant bacterium can be recombinant yeast.

Biological material related to fusion protein Nt4CL5aPcSTS fusion protein Nt4CL5aPcSTS or above-mentioned of the invention Expect that the application in production resveratrol is fallen within protection scope of the present invention.

Applications of the fusion protein Nt4CL5aPcSTS in as 4- coumarate CoA-ligases and stilbene synthase also belongs to Within protection scope of the present invention.

In order to solve the above technical problems, present invention also offers a kind of method for producing resveratrol.

A kind of method for producing resveratrol provided by the present invention, is included in the culture medium containing 4- coumaric acids and cultivates Fusion protein relevant biological material, obtains resveratrol；

Any of the fusion protein relevant biological material is following C1) to C12)：

C1) the recombinant microorganism containing nucleic acid molecules；Fusion protein described in the nucleic acid molecule encoding；

C2) the recombinant microorganism containing expression cassette；The expression cassette contains the nucleic acid molecules；

C3) the recombinant microorganism containing recombinant vector；The recombinant vector contains the nucleic acid molecules or the expression cassette；

C4 the transgenic plant cells system of the nucleic acid molecules) is contained；

C5 the transgenic plant cells system of the expression cassette) is contained；

C6 the transgenic plant cells system of the recombinant vector) is contained；

C7 the Transgenic plant tissue of the nucleic acid molecules) is contained；

C8 the Transgenic plant tissue of the expression cassette) is contained；

C9 the Transgenic plant tissue of the recombinant vector) is contained；

C10 the genetically modified plants organ of the nucleic acid molecules) is contained；

C11 the genetically modified plants organ of the expression cassette) is contained；

C12 the genetically modified plants organ of the recombinant vector) is contained.

In the method for above-mentioned production resveratrol, the microorganism can be prokaryotic micro-organisms or eukaryotic microorganisms.The original Core microorganism can be bacterium.The bacterium can be Escherichia bacteria.The Escherichia bacteria can be Escherichia coli.Institute It can be fungi to state eukaryotic microorganisms.The fungi can be saccharomycete.The saccharomycete can be yeast strain WAT11.The carrier Can be cloning vector and expression vector.The expression vector can be procaryotic cell expression carrier or eukaryotic expression vector.Its Its described fusion protein relevant biological material and the biomaterial related with the fusion protein Nt4CL5aPcSTS above It is identical.

Present invention also offers a kind of preparation method of fusion protein, including the fusion protein encoding gene in biology In cell express obtaining the fusion protein.

In above-mentioned preparation method, the encoding gene of the fusion protein is following 1. or 2. or 3. or 4. described DNA points Son：

1. coded sequence is the DNA molecular or cDNA molecules of SEQ ID No.2 1-2808 nucleotides；

2. nucleotide sequence is SEQ ID No.2 DNA molecular or cDNA molecules；

3. there is more than 75% homogeneity and code for said proteins with the DNA molecular or cDNA molecules that 1. or 2. limit DNA molecular or cDNA molecules；

4. under strict conditions with DNA molecular or cDNA the molecules hybridization 1. or 2. limited and code for said proteins DNA molecular or cDNA molecules.

In above-mentioned preparation method, the encoding gene of the fusion protein is carried out into expression in biological cell is included by described in The encoding gene of fusion protein imports the biological cell.

In above-mentioned preparation method, the encoding gene of the fusion protein passes through the encoding gene containing the fusion protein Recombinant expression carrier imports the recombinant cell that the biological cell obtains expressing the fusion protein；The biological cell can be micro- Biological cell, plant cell or non-human animal cell, the microbial cell can be yeast, bacterium, algae or fungi.In the present invention One embodiment in, the biological cell concretely bacterium and saccharomycete.

In above-mentioned preparation method, including the culture recombinant cell obtains the fusion protein.

In above-mentioned preparation method, the recombinant expression carrier can be by pET30a (+) EcoR I recognition sequences and Xho I DNA between recognition sequence replaces with the DNA molecular that nucleotide sequence is SEQ ID No.2, keeps pET30a (+) other sequences Constant obtained Nt4CL5aPcSTS expression vectors, its entitled pET30a-Nt4CL5aPcSTS.

In above-mentioned preparation method, the Escherichia coli can be to import recombinant expression plasmid pET30a-Nt4CL5aPcSTS The recombination bacillus coli that recipient E. coli is obtained.The recipient E. coli can be Escherichia coli Rosetta.

In above-mentioned preparation method, the recombinant expression carrier can be by pESC-Trp EcoR I recognition sequences and Bgl II DNA between recognition sequence replaces with the DNA molecular that nucleotide sequence is SEQ ID No.2, keeps pESC-Trp other sequences It is constant to obtain Nt4CL5aPcSTS expression vectors, its entitled pESC-Nt4CL5aPcSTS.

In above-mentioned preparation method, the recombinant yeast can be to obtain pESC-Nt4CL5aPcSTS importing recipient yeast bacterium The recombinant yeast arrived.The recipient yeast bacterium concretely saccharomycete WAT11.

In above-mentioned preparation method, the culture is included in 25 DEG C, and 2h is induced in the culture medium of the IPTG containing 0.2mmol/L Express the fusion protein Nt4CL5aPcSTS.

External enzymatic reaction it is demonstrated experimentally that the fusion protein Nt4CL5aPcSTS that the present invention is provided can be catalyzed 4- tonka-beans Acid and malonyl coenzyme A generate the resveratrol with bioactivity, it was demonstrated that fusion protein Nt4CL5aPcSTS has 4- tonka-beans The activity of acyl coenzyme A ligases and stilbene synthase.Recombination bacillus coli containing fusion protein Nt4CL5aPcSTS genes Rosetta/pET30a-Nt4CL5aPcSTS produces resveratrol in the culture medium containing 4- coumaric acids, and its yield is 26.5 μ g/mL；Recombination bacillus coli Rosetta/pET30a-PcSTS containing PcSTS genes is in the above-mentioned culture containing 4- coumaric acids Resveratrol is not produced in base.Show that white lamb's-quarters can be improved using fusion protein Nt4CL5aPcSTS in escherichia expression system The yield of reed alcohol.Recombinant yeast WAT11/pESC-Nt4CL5aPcSTS containing fusion protein Nt4CL5aPcSTS genes exists Resveratrol is produced in the culture medium of the coumaric acid containing 4-, its yield is 10.2 μ g/mL zymotic fluids；Recombinant yeast WAT11/ PESC, WAT11/pESC-Nt4CL and WAT11/pESC-PcSTS do not produce resveratrol in the cultivating system of the coumaric acid containing 4-；Weight Group saccharomycete WAT11/pESC-Nt4CLPPPcSTS is 1.3 μ g/ in the yield of the resveratrol of the cultivating system of the coumaric acid containing 4- ML zymotic fluids.Show that the yield of resveratrol can be improved using fusion protein Nt4CL5aPcSTS in yeast expression system.

Brief description of the drawings

Fig. 1 is resveratrol biosynthesis pathway figure.

Fig. 2 is the poly- propionamide gel electrophoresis figure of protein PcSTS induced expressions.Wherein, M1 is protein molecular weight Marker；Swimming lane 1 is recombinant bacterium Rosetta/pET30a-PcSTS bacterial cell disruption liquid；Swimming lane 2 is recombinant bacterium Rosetta/ PET30a-PcSTS cellular lysate supernatants；Swimming lane 3 precipitates for recombinant bacterium Rosetta/pET30a-PcSTS cellular lysates；Swimming lane 4 be the supernatant after Ni-NTA resins are incubated；Swimming lane 5 is to wash resin trap liquid；6 and 7 be through Ni-NTA agarose affinity chromatography posts Protein PcSTS after purification.

Fig. 3 is poly- propionamide gel electrophoresis figures of the protein PcSTS after PD-10 post desalinations.Swimming lane 1-7 is protein Eluent after PcSTS sample desalinations.

Fig. 4 is the poly- propionamide gel electrophoresis figure of fusion protein Nt4CL5aPcSTS induced expressions.Wherein, M1 is protein Molecular weight Marker；Swimming lane 1 is recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS bacterial cell disruption liquid；Swimming lane 2 is recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatants；Swimming lane 3 is recombinant bacterium Rosetta/pET30a- Nt4CL5aPcSTS cellular lysates are precipitated；Swimming lane 4 is the supernatant after Ni-NTA resins are incubated；Swimming lane 5 is to wash resin trap liquid；6 For the fusion protein Nt4CL5aPcSTS through Ni-NTA agarose affinity chromatographies post after purification.

Fig. 5 is poly- propionamide gel electrophoresis figures of the fusion protein Nt4CL5aPcSTS after PD-10 post desalinations.Swimming lane 1-4 For the eluent after fusion protein Nt4CL5aPcSTS sample desalinations.

Fig. 6 is high performance liquid chromatography detection collection of illustrative plates.Wherein, A is resveratrol standard items high-efficient liquid phase chromatogram；B is The high-efficient liquid phase chromatogram of the external enzymatic reaction products of PcSTS；C is resveratrol standard items and the external enzymatic reaction productions of PcSTS The cochromatograph figure of thing.Abscissa is retention time, and unit is minute.

Fig. 7 is fusion protein Nt4CL5aPcSTS high performance liquid chromatography detection collection of illustrative plates.Wherein, A is Nt4CL5aPcSTS bodies The high-efficient liquid phase chromatogram of outer enzymatic reaction product；B is resveratrol standard items and the external enzymatic reaction productions of Nt4CL5aPcSTS The cochromatograph figure of thing.Abscissa is retention time, and unit is minute.

Fig. 8 is HPLC-s of the recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS using coumaric acid as the tunning of substrate MS testing result.Wherein A is the HPLC-MS of resveratrol standard items；B is hair of the recombination bacillus coli using coumaric acid as substrate The HPLC-MS of ferment product.

Fig. 9 is HPLCs of the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS using coumaric acid as the tunning of substrate Testing result.Wherein A is tunnings of the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS using coumaric acid as substrate HPLC；B is tunnings of the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS using coumaric acid as substrate and resveratrol mark The cochromatograph figure of quasi- product.Abscissa is retention time, and unit is minute.

Figure 10 is tunnings of the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS using coumaric acid as substrate HPLC-MS testing result.Wherein A is the HPLC-MS of resveratrol standard items；B is recombinant yeast WAT11/pESC- HPLC-MSs of the Nt4CL5aPcSTS using coumaric acid as the tunning of substrate.

Embodiment

Experimental method in following embodiments, is conventional method unless otherwise specified.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

PESC-Trp in following embodiments is Agilent Products, and catalog number is 217453.

Yeast strain WAT11 (Zhang Y, Li SZ, Li J, et al.Using unnatural in following embodiments protein fusions to engineer resveratrol biosynthesis in yeast and mammalian cells[J].Journal of the American Chemical Society,2006,128(40):13030-13031.) Presented for Wuhan Botanical Garden, Chinese Acadmey of Sciences teacher Zhang Yansheng.The biomaterial is only attached most importance to used in the related experiment of duplicate invention, It can not be used as other purposes.

Culture medium used is as follows in following embodiments：

Trp auxotrophy fluid nutrient mediums are prepared as follows：By Trp Minus Media (the general Jino sections in Beijing Skill Co., Ltd product) add in distilled water, the content to Trp Minus Media is 8g/L；PH after autoclaving naturally, obtain To Trp auxotrophy fluid nutrient mediums.

Trp auxotrophy solid mediums：It is 20g/L to add agar to its content into Trp auxotrophy fluid nutrient mediums Obtained culture medium.

Embodiment 1, fusion protein Nt4CL5aPcSTS prokaryotic expression and purifying

First, the structure of Nt4CL5aPcSTS gene expression plasmids

DNA molecular (Nt4CL5aPcSTS genes) shown in artificial synthesized SEQ ID No.2, coding SEQ ID No.1 institutes The fusion protein Nt4CL5aPcSTS shown.

DNA between pET30a (+) (Novagen Products) EcoR I recognition sequences and Xho I recognition sequences is replaced The DNA molecular that nucleotide sequence is SEQ ID No.2 is changed to, keeps pET30a (+) other sequences are constant to obtain Nt4CL5aPcSTS expression vectors, its entitled pET30a-Nt4CL5aPcSTS.PET30a-Nt4CL5aPcSTS is expressed Fusion protein Nt4CL5aPcSTS shown in SEQ ID No.1.

DNA between pET30a (+) EcoR I recognition sequences and Xho I recognition sequences is replaced with into nucleotide sequence is The DNA molecular of 1642-2808 in SEQ ID No.2, keeps pET30a (+) other sequences are constant to obtain PcSTS genes Expression vector, its entitled pET30a-PcSTS.Egg shown in pET30a-PcSTS expression SEQ ID No.1 548-935 White matter PcSTS.

Fusion protein Nt4CL5aPcSTS shown in above-mentioned SEQ ID No.1 includes protein N t4CL, linker and albumen Matter PcSTS.In SEQ ID No.1,1-542 amino acids residue is protein N t4CL amino acid sequence, 543-547 Amino acids residue is linker amino acid sequence, and 548-935 amino acids residue is protein PcSTS amino acid sequence Row.In SEQ ID No.2, the DNA molecular encoding proteins matter Nt4CL shown in 1-1626；Shown in 1627-1641 DNA molecular encodes linker；DNA molecular encoding proteins matter PcSTS shown in 1642-2808.

2nd, fusion protein Nt4CL5aPcSTS expression and purifying

Plasmid pET30a-PcSTS is imported into Escherichia coli Rosetta, (contains 50 μ g/mL kanamycins through LB solid plates With 25 μ g/mL chloramphenicol) screening, the recombinant bacterium Escherichia coli containing pET30a-PcSTS are obtained, the recombination bacillus coli is ordered Entitled Rosetta/pET30a-PcSTS, is used as control bacterium.

Recombinant expression plasmid pET30a-Nt4CL5aPcSTS is imported into Escherichia coli Rosetta, (contained through LB solid plates 50 μ g/mL kanamycins and 25 μ g/mL chloramphenicol) screening, obtain the restructuring large intestine bar containing pET30a-Nt4CL5aPcSTS Bacterium, Rosetta/pET30a-Nt4CL5aPcSTS is named as by the recombination bacillus coli.

Plasmid pET30a (+) is imported into Escherichia coli Rosetta, (contains 50 μ g/mL kanamycins and 25 through LB solid plates μ g/mL chloramphenicol) screening, the recombination bacillus coli containing pET30a (+) is obtained, the recombination bacillus coli is named as Rosetta/pET30a, is used as empty vector control bacterium.

By Rosetta/pET30a-PcSTS, Rosetta/pET30a-Nt4CL5aPcSTS and Rosetta/pET30a points LB fluid nutrient mediums are not inoculated in, and 37 DEG C of shaken cultivations, which are stayed overnight, to be obtained cultivating bacterium solution 1, is respectively designated as Rosetta/pET30a- PcSTS cultures bacterium solution 1, Rosetta/pET30a-Nt4CL5aPcSTS culture bacterium solutions 1 and Rosetta/pET30a culture bacterium solutions 1； By above-mentioned culture bacterium solution 1 respectively with 1:100 (volume ratios) are inoculated into LB fluid nutrient mediums and (contain 50 μ g/mL kanamycins and 25 μ g/ ML chloramphenicol) in, shaken cultivation to OD₆₀₀Value 0.6 obtains cultivating bacterium solution 2, is respectively designated as Rosetta/pET30a-PcSTS trainings Bacteria liquid 2, Rosetta/pET30a-Nt4CL5aPcSTS culture bacterium solutions 2 and Rosetta/pET30a culture bacterium solutions 2；To above-mentioned Rosetta/pET30a-PcSTS is cultivated to be separately added into bacterium solution 2 and Rosetta/pET30a-Nt4CL5aPcSTS culture bacterium solutions 2 IPTG, makes concentration of the IPTG in system be respectively 0.2mmol/L, 0.5mmol/L, 0.8mmol/L and 1.0mmol/L, 25 DEG C, 2h, 4h, 6h, 8h, 10h and 22h are induced respectively, collect equivalent thalline under each inductive condition.Above-mentioned each thalline sequentially adds SDS samples Buffer solution cracking is savored, centrifugation, obtained supernatant respectively takes 10 μ L to carry out 10%SDS- polyacrylamide gel electrophoresises (SDS- poly- third Acrylamide gel electrophoresis abbreviation SDS-PAGE below), electrophoresis knot is scanned with gel image analyser after coomassie brilliant blue staining Really.By the difference of expression quantity under relatively different IPTG concentration and different induction times, as a result show, protein PcSTS is most suitable Induced expression condition is：Concentration of the IPTG in system is 0.8mmol/L, 25 DEG C of induction 6h；Fusion protein Nt4CL5aPcSTS Most suitable induced expression condition is：Concentration of the IPTG in system is 0.2mmol/L, 25 DEG C of induction 2h.

The Rosetta/pET30a-PcSTS culture bacterium solutions 2 obtained into above-mentioned steps add IPTG, make IPTG in system In concentration be 0.8mmol/L, 25 DEG C, induce 6h, collect thalline after ultrasonication, obtain bacterial cell disruption liquid (entitled Rosetta/pET30a-PcSTS bacterial cell disruptions liquid).Bacterial cell disruption liquid 12000rpm centrifugations 10min is obtained into supernatant (name Referred to as Rosetta/pET30a-PcSTS cellular lysates supernatant) and precipitation (entitled Rosetta/pET30a-PcSTS thalline Cracking precipitation).

The Rosetta/pET30a-Nt4CL5aPcSTS culture bacterium solutions 2 obtained into above-mentioned steps add IPTG, make IPTG Concentration in system is 0.2mmol/L, 25 DEG C, induces 2h, collects ultrasonication after thalline, obtain bacterial cell disruption liquid (title For Rosetta/pET30a-Nt4CL5aPcSTS bacterial cell disruptions liquid).Bacterial cell disruption liquid 12000rpm centrifugations 10min is obtained Supernatant (entitled Rosetta/pET30a-Nt4CL5aPcSTS cellular lysates supernatant) and precipitation (entitled Rosetta/ PET30a-Nt4CL5aPcSTS cellular lysates are precipitated).

By Rosetta/pET30a-PcSTS bacterial cell disruptions liquid, Rosetta/pET30a-PcSTS cellular lysates supernatant, Rosetta/pET30a-PcSTS cellular lysates precipitation, Rosetta/pET30a-Nt4CL5aPcSTS bacterial cell disruptions liquid, Rosetta/pET30a-Nt4CL5aPcSTS cellular lysates supernatant and Rosetta/pET30a-Nt4CL5aPcSTS thalline split Solution precipitation carries out SDS-PAGE.Using Ni-NTA agarose affinity chromatographies post from cellular lysate supernatant purifying protein, then Utilize PD-10 post desalinations.

Experimental result is shown in such as Fig. 2, Fig. 3, Fig. 4 and Fig. 5.As a result show, protein PcSTS is primarily present in Rosetta/ In pET30a-PcSTS cellular lysate supernatants, molecular size range is 43kDa；Fusion protein Nt4CL5aPcSTS is primarily present in In Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatants, molecular size range is 103kDa.Utilize Ni-NTA agar Sugared affinity column is purified from Rosetta/pET30a-PcSTS cellular lysate supernatants obtains the protein with His labels PcSTS, purifying obtains the fusion with His labels from Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatants Albumen Nt4CL5aPcSTS.After the filter membrane concentration of molecular cut off (MWCO) 10000, obtained with the dissolving of protein storage solution Concentration is 1mg/mL protein PcSTS and fusion protein Nt4CL5aPcSTS solution.Protein storage solution：Solute and its dense Spend and be：20mM Tris (pH7.5), 150mM sodium chloride, 0.5mM EDTA, 25% (v/v) glycerine, when protein storage solution is used Add after 1mM DTT are mixed and use；Solvent is distilled water；PH is natural.

Embodiment 2, the HPLC external enzymatic reactions of detection fusion albumen Nt4CL5aPcSTS and product

2.1st, resveratrol standard items HPLC is detected：Accurately weigh resveratrol standard items (Sigma Products) 1.0mg In 10mL volumetric flasks, dissolved with chromatogram methanol, be settled to 10mL, be configured to 100 μ g/mL resveratrol mother liquor.Through 0.22 μ M membrane filtrations, with methanol dilution obtain concentration be respectively 2.5,5,12.5,25,50 μ g/mL standard liquid, concentration from it is low to High sample introduction successively.Using equipped with Sunfire^TMThe Waters HPLC analyses of C18 reversed-phase columns (5.0 μm, 4.6mm × 250mm).Stream Dynamic to be made up of methanol (A) and water (B), flow velocity is 0.8mL/min, uses following condition of gradient elution：In 0-30min, stream The volumn concentration of methanol at the uniform velocity increases to 70% by 10% in dynamic phase, and the volumn concentration of water is at the uniform velocity down to by 90% 30%, carry out linear gradient elution；Again with mobile phase (by first alcohol and water according to 7:The solution that 3 volume ratio is mixed to get) elution 10min.Detection wavelength is 306nm.Resveratrol standard items high-efficient liquid phase chromatogram is as shown in A in Fig. 6.

2.2nd, the external enzymatic reactions of albumen PcSTS and product HPLC are detected：Prepare the 300 external enzymatic reactants of μ L PcSTS System, the pH of the external enzymatic reaction system is 7.5, solvent be 0.1mol/L kaliumphosphate buffers (solute be potassium dihydrogen phosphate with Dipotassium hydrogen phosphate, solvent is water), the true protein that solute is 4- coumaroyls A, malonyl coenzyme A, embodiment 1 are obtained PcSTS.In the 300 external enzymatic reaction systems of μ L PcSTS, 4- coumaroyls A concentration is 150 μm of ol/L, malcryscoa A concentration is 280 μm of ol/L, and the pure protein PcSTS of embodiment 1 concentration is 0.1 μm of ol/L.Above-mentioned reaction system is placed in 35 DEG C Lower reaction 30min, adds acetic acid, and it is 5% (volumn concentration) to make concentration of the acetic acid in system, then with 300 μ L second Acetoacetic ester is extracted, 10000r/min centrifugations 10min.Take upper strata ethyl acetate layer to be dried in vacuo, obtain PcSTS enzymatic reactions Product, 50 μ L 50% (volumn concentration) methanol aqueous solutions are added into PcSTS enzymatic reaction products, PcSTS enzymatics are obtained Reaction product testing sample.Using equipped with Sunfire^TMThe Waters HPLC of C18 reversed-phase columns (5.0 μm, 4.6mm × 250mm) Analyze PcSTS enzymatic reaction product testing samples.Mobile phase is made up of methanol (A) and water (B), and flow velocity is 0.8mL/min, is used Following condition of gradient elution：In 0-30min, the volumn concentration of methanol at the uniform velocity increases to 70%, water by 10% in mobile phase Volumn concentration be at the uniform velocity down to 30% by 90%, carry out linear gradient elution；Again with mobile phase (by first alcohol and water according to 7: The solution that 3 volume ratio is mixed to get) elution 10min.Detection wavelength is 306nm.With resveratrol (Sigma companies, article No. R5010 quantitative analysis resveratrol) is carried out using calibration curve method (external standard method) for standard items.Experiment is in triplicate, heavy every time 10 reaction systems are set again.

As a result show the high-efficient liquid phase chromatogram of the external enzymatic reaction products of PcSTS as shown in B in Fig. 6.Resveratrol mark The cochromatograph figure of the quasi- external enzymatic reaction product of product and PcSTS is respectively as shown in A in Fig. 6 and C, and standard items resveratrol is in the color Retention time under spectral condition is 23.253 minutes (see A in Fig. 6)；The external enzymatic reaction products of PcSTS are under the chromatographic condition Contain chromatographic peak (in Fig. 6 B) of the retention time for the resveratrol of 23.232 minutes.Treated to above-mentioned PcSTS enzymatic reaction products Above-mentioned resveratrol standard items are added in test sample product and obtain above-mentioned resveratrol standard items in cochromatograph sample, cochromatograph sample Mass content be one times of PcSTS mass contents in cochromatograph sample.Complete PcSTS enzymatic reaction product testing samples HPLC analysis detection after 10 minutes in, under identical chromatographic condition to cochromatograph sample carry out HPLC analyses.Cochromatograph The chromatographic peak, containing the chromatographic peak that retention time is 23.250 minutes, is named as cochromatograph peak by sample under the chromatographic condition (B in Fig. 7), the peak height at the cochromatograph peak is twice of PcSTS chromatographic peaks, illustrates the chromatogram that PcSTS chromatographic peaks are resveratrol Peak.

2.3rd, the external enzymatic reactions of fusion protein Nt4CL5aPcSTS and product HPLC are detected：Except by albumen PcSTS in 2.2 External enzymatic reaction system during external enzymatic reaction is detected with product HPLC is replaced by 300 μ L Nt4CL5aPcSTS vitro enzymes Promote outside reaction system, remaining is identical with 2.2.The pH of the 300 external enzymatic reaction systems of μ L Nt4CL5aPcSTS is 7.5, solvent For 0.1mol/L kaliumphosphate buffers (solute is potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and solvent is water), solute and its concentration are： 150 μm of ol/L 4- coumaric acids, 280 μm of ol/L malonyl coenzyme As, 250 μm of ol/L ATP, 100 μm of ol/L CoA, 0.1 μm of ol/L The pure fusion protein Nt4CL5aPcSTS of embodiment 1.Experiment repeats to set 10 reaction systems in triplicate, every time.

Above-mentioned reaction system, which is placed at 35 DEG C, reacts 30min, adds acetic acid, and it is 5% (body to make concentration of the acetic acid in system Product percentage composition), then extracted with 300 μ L ethyl acetate, 10000r/min centrifugations 10min.Upper strata ethyl acetate layer is taken to enter Row vacuum drying, obtains Nt4CL5aPcSTS enzymatic reaction products, 50 μ L is added into Nt4CL5aPcSTS enzymatic reaction products 50% (volumn concentration) methanol aqueous solution, obtains Nt4CL5aPcSTS enzymatic reaction product testing samples.According to 2.2 side Method carries out HPLC analysis Nt4CL5aPcSTS enzymatic reaction product testing samples.As a result show that the external enzymatics of Nt4CL5aPcSTS are anti- Product is answered, containing the chromatographic peak that retention time is 21.876 minutes, the chromatographic peak to be named as under the chromatographic condition Nt4CL5aPcSTS chromatographic peaks (A in Fig. 7).Added into above-mentioned Nt4CL5aPcSTS enzymatic reaction products testing sample above-mentioned white The mass content that veratryl alcohol standard items obtain the above-mentioned resveratrol standard items in cochromatograph sample, cochromatograph sample is cochromatograph Four times of Nt4CL5aPcSTS mass contents in sample.Complete Nt4CL5aPcSTS enzymatic reaction product testing samples In 10 minutes after HPLC analysis detections, HPLC analyses are carried out to cochromatograph sample under identical chromatographic condition.Cochromatograph sample The chromatographic peak, containing the chromatographic peak that retention time is 21.903 minutes, is named as cochromatograph peak (Fig. 7 by product under the chromatographic condition Middle B), the peak height at the cochromatograph peak is five times of Nt4CL5aPcSTS chromatographic peaks, and it is white lamb's-quarters to illustrate Nt4CL5aPcSTS chromatographic peaks The chromatographic peak of reed alcohol.

As a result show, the external enzymatic reaction products of protein PcSTS are resveratrol, in vitro egg in enzymatic reaction system White matter PcSTS can be catalyzed the resveratrol of 4- coumaroyls A and malonyl coenzyme A formation with bioactivity, and yield is 4.9kg resveratrols/mol protein PcSTS；The external enzymatic reaction products of fusion protein Nt4CL5aPcSTS are resveratrol, Fusion protein Nt4CL5aPcSTS can be catalyzed the resveratrol of 4- coumaric acids and malonyl coenzyme A generation with bioactivity, Yield is 8.0kg resveratrols/mol fusion proteins Nt4CL5aPcSTS.

Embodiment 3, utilize recombination bacillus coli production resveratrol

3.1st, recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS produces resveratrol：By in embodiment 1 Recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS is inoculated in LB fluid nutrient mediums, and 37 DEG C of shaken cultivations are stayed overnight To culture bacterium solution 1, by above-mentioned culture bacterium solution 1 with 1:100 (volume ratios) are inoculated into (contains 50 μ equipped with 100mL LB fluid nutrient mediums G/mL kanamycins and 25 μ g/mL chloramphenicol) triangular flask in, shaken cultivation to OD₆₀₀Obtain cultivating bacterium between value 0.4-0.5 Liquid 2, IPTG is added into culture bacterium solution 2, and obtained system is referred to as to the system for cultivating bacterium solution 2, makes IPTG in culture bacterium solution 2 Concentration in system is 0.2mM, and 25 DEG C, 150rpm Fiber differentiations 2h, 6000rpm centrifugation 2min abandons supernatant, contains 50 μ with 100mL Culture medium (culture containing 50 μ g/mL kanamycins and 25 μ g/mL chloramphenicol of g/mL kanamycins and 25 μ g/mL chloramphenicol Base is that the fluid nutrient medium that kanamycins and chloramphenicol are obtained is added into M9 culture mediums, and kanamycins is in the fluid nutrient medium Concentration be 50 μ g/mL, concentration of the chloramphenicol in the fluid nutrient medium be 25 μ g/mL) be resuspended bacterial sediment obtain re-suspension liquid. Into re-suspension liquid add IPTG and 4- coumaric acids, by obtained system be referred to as the coumaric acid containing 4- cultivating system, make IPTG containing Concentration in the cultivating system of 4- coumaric acids is 0.2mM, make concentration of the 4- coumaric acids in the cultivating system of the coumaric acid containing 4- be 12 μ g/mL, 25 DEG C, 150rpm cultures 48h obtains fermentation culture.The above-mentioned fermentation culture 12000rpm centrifugations 2min of 1mL are taken, Supernatant is taken, is extracted with 1mL ethyl acetate, vortex oscillation 40s, repetition is extracted twice.Centrifuging and taking upper strata ethyl acetate layer, volatilization After drying, 50 μ L 50% (volumn concentration) methanol aqueous solutions are added, recombination bacillus coli Rosetta/pET30a- is obtained Nt4CL5aPcSTS sample solution, carries out HPLC-MS detection.Using equipped with Sunfire^TMC18 reversed-phase columns (5.0 μm, 4.6mm × 250mm) Waters HPLC detected.Mobile phase is made up of methanol (A) and water (B), and flow velocity is 0.8mL/ Min, uses following condition of gradient elution：In 0-30min, the volumn concentration of methanol is at the uniform velocity increased to by 10% in mobile phase 70%, the volumn concentration of water is at the uniform velocity down to 30% by 90%, carries out linear gradient elution；Again with mobile phase (by methanol and Water is according to 7:The solution that 3 volume ratio is mixed to get) elution 10min.Detection wavelength is 306nm.With resveratrol, (Sigma is public Department, article No. R5010) quantitative analysis resveratrol is carried out using calibration curve method (external standard method) for standard items.Mass Spectrometer Method condition For：Scanning of the mass spectrum scope 150-1000 (m/z)；Sheath gas：Nitrogen, sheath gas：40mL/min, secondary air speed：3mL/min, Capillary temperature：300 DEG C, capillary voltage：-35V.Experiment repeats to set 10 triangular flasks in triplicate, every time.

3.2nd, recombinant bacterium Rosetta/pET30a-PcSTS produces resveratrol：Except by the recombination bacillus coli in 3.1 Rosetta/pET30a-Nt4CL5aPcSTS cultures bacterium solution 2 replaces with the Rosetta/pET30a-PcSTS culture bacterium of embodiment 1 Liquid 2, remaining all same.

3.3rd, recombinant bacterium Rosetta/pET30a produces resveratrol：Except by 3.1 pET30a-Nt4CL5aPcSTS train Bacteria liquid 2 replaces with the Rosetta/pET30a culture bacterium solutions 2 of embodiment 1, remaining all same.

The white lamb's-quarters that recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS is produced in the cultivating system of the above-mentioned coumaric acid containing 4- The HPLC-MS of reed alcohol testing result is as shown in Figure 8.As a result show, recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS exists The yield of the resveratrol of the cultivating system of the above-mentioned coumaric acid containing 4- is 26.5 μ g/mL zymotic fluids；Recombinant bacterium Rosetta/ PET30a-PcSTS does not produce resveratrol in the cultivating system of the above-mentioned coumaric acid containing 4-；Recombinant bacterium Rosetta/pET30a is above-mentioned Resveratrol is not produced in the cultivating system of the coumaric acid containing 4-.Show to utilize fusion protein in escherichia expression system Nt4CL5aPcSTS can improve the yield of resveratrol.

Embodiment 4, utilize recombinant yeast production resveratrol

1st, the structure of recombinant yeast expression vector and conversion

By pESC-Trp, (Agilent Products, catalog number is EcoR I recognition sequences and Bgl 217453) DNA between II recognition sequences replaces with the DNA molecular that nucleotide sequence is SEQ ID No.2, keeps pESC-Trp other sequences Row are constant to obtain Nt4CL5aPcSTS expression vectors, its entitled pESC-Nt4CL5aPcSTS.pESC- Fusion protein Nt4CL5aPcSTS shown in Nt4CL5aPcSTS expression SEQ ID No.1.

It is SEQ that DNA between pESC-Trp EcoR I recognition sequences and Pac I recognition sequences is replaced with into nucleotide sequence The DNA molecular of 1-1626 in ID No.2, by the DNA between pESC-Trp BamH I recognition sequences and Xho I recognition sequences The DNA molecular that nucleotide sequence is 1642-2808 in SEQ ID No.2 is replaced with, pESC-Trp other sequences are kept It is constant to obtain expression vector, its entitled pESC-Nt4CLPPPcSTS.PESC-Nt4CLPPPcSTS expression SEQ ID Protein N t4CL and SEQ ID No.1 548-935 amino acids residues institute shown in No.1 1-542 amino acids residues The protein PcSTS shown.

DNA between pESC-Trp EcoR I recognition sequences and Bgl II recognition sequences is replaced with into nucleotide sequence is The DNA molecular of 1-1626 in SEQ ID No.2, keeps pESC-Trp other sequences are constant to obtain Nt4CL gene expressions Carrier, its entitled pESC-Nt4CL.Protein N t4CL shown in pESC-Nt4CL expression SEQ ID No.1 1-542.

DNA between pESC-Trp EcoR I recognition sequences and Bgl II recognition sequences is replaced with into nucleotide sequence is The DNA molecular of 1642-2808 in SEQ ID No.2, keeps pESC-Trp other sequences are constant to obtain PcSTS gene tables Up to carrier, its entitled pESC-PcSTS.Protein shown in pESC-PcSTS expression SEQ ID No.1 548-935 PcSTS。

Fusion protein Nt4CL5aPcSTS shown in above-mentioned SEQ ID No.1 includes protein N t4CL, linker and albumen Matter PcSTS.In SEQ ID No.1,1-542 amino acids residue is protein N t4CL amino acid sequence, 543-547 Amino acids residue is linker amino acid sequence, 548-935 amino acids residues proteins PcSTS amino acid sequence Row.In SEQ ID No.2, the DNA molecular encoding proteins matter Nt4CL shown in 1-1626；Shown in 1627-1641 DNA molecular encodes linker；DNA molecular encoding proteins matter PcSTS shown in 1642-2808.

The solid plate that Trp auxotrophy solid mediums are made, is named as Trp auxotrophy flat boards.Ferment will be recombinated Female expression plasmid pESC-Nt4CL5aPcSTS is transferred to yeast strain WAT11, then screens, obtains on Trp auxotrophy flat boards Recombinant yeast containing pESC-Nt4CL5aPcSTS, WAT11/pESC- is named as by the recombinant yeast Nt4CL5aPcSTS。

Plasmid pESC-Trp is imported into yeast strain WAT11, then screens, is contained on Trp auxotrophy flat boards PESC-Trp recombinant yeast, WAT11/pESC is named as by the recombinant yeast, is used as empty vector control bacterium.

Expression of recombinant yeast plasmid pESC-Nt4CLPPPcSTS is imported into yeast strain WAT11, then lacked in Trp nutrition Fall on flat board and screen, obtain the recombinant yeast containing pESC-Nt4CLPPPcSTS, the recombinant yeast is named as WAT11/ pESC-Nt4CLPPPcSTS。

Expression of recombinant yeast plasmid pESC-Nt4CL is imported into yeast strain WAT11, then on Trp auxotrophy flat boards Screening, obtains the recombinant yeast containing pESC-Nt4CL, the recombinant yeast is named as into WAT11/pESC-Nt4CL.

Expression of recombinant yeast plasmid pESC-PcSTS is imported into yeast strain WAT11, then on Trp auxotrophy flat boards Screening, obtains the recombinant yeast containing pESC-PcSTS, the recombinant yeast is named as into WAT11/pESC-PcSTS.

2nd, resveratrol is produced using recombinant yeast

20% (mass percent concentration) sterile dextrose solution (solute is glucose, and solvent is sterilized water) is added Trp auxotrophy fluid nutrient mediums, so as to get culture medium in glucose concentration be 2%, the culture medium is named as grape Sugar-Trp defect fluid nutrient mediums；By 20% (mass percent concentration) sterile dextrose solution, (solute is glucose, and solvent is Sterilized water) add Trp auxotrophy solid mediums, so as to get solid medium in glucose concentration be 2%, this is trained Support the solid plate being made and be named as glucose-Trp defect flat boards；By 20% (mass percent concentration) sterile galactose solution (solute is galactolipin, and solvent is sterilized water) adds Trp auxotrophy fluid nutrient mediums, so as to get culture medium in galactolipin Concentration is 2%, and the culture medium is named as into galactolipin-Trp defect fluid nutrient mediums.

2.1st, recombinant yeast WAT11/pESC-Nt4CL5aPcSTS produces resveratrol

Recombinant yeast WAT11/pESC-Nt4CL5aPcSTS is lived in the flat lining out of glucose-Trp defects Change, picking single bacterium colony is seeded in glucose-Trp defect fluid nutrient mediums, 30 DEG C, 200rpm cultures 24h obtains cultivating bacterium solution. Supernatant is removed in above-mentioned culture bacterium solution centrifugation, and precipitation uses ddH₂O is washed 3 times, and the precipitation is recombinant yeast WAT11/pESC- Nt4CL5aPcSTS thalline, are then resuspended recombinant yeast WAT11/pESC- with galactolipin-Trp defects fluid nutrient medium Nt4CL5aPcSTS thalline obtain re-suspension liquid, and 4- coumaric acids are added into re-suspension liquid, and obtained system is referred to as into coumaric acid containing 4- Cultivating system, make after concentration of the 4- coumaric acids in the cultivating system of the coumaric acid containing 4- is 12 μ g/mL, 30 DEG C of culture 24h Obtain zymotic fluid.

The above-mentioned zymotic fluids of 1mL are taken, are added after the extraction of equivalent ethyl acetate, centrifuging and taking upper strata ethyl acetate layer, volatile dry, WAT11/pESC- is obtained after adding 50 μ L 50% (volumn concentration) methanol aqueous solutions, used membrane filtration film Nt4CL5aPcSTS sample solutions.The above-mentioned μ L of sample solution 10 are taken, HPLC-MS detection is carried out according to the method for embodiment 3.It is real Test in triplicate, repeat to set 10 triangular flasks every time.As a result show WAT11/pESC-Nt4CL5aPcSTS sample solutions in the color Containing the chromatographic peak that retention time is 23.249 minutes under spectral condition, the chromatographic peak is named as Nt4CL5aPcSTS chromatographic peaks (A in Fig. 9).Above-mentioned resveratrol standard items are added into above-mentioned WAT11/pESC-Nt4CL5aPcSTS sample solutions to be total to The mass content of above-mentioned resveratrol standard items in chromatographic sample, cochromatograph sample is in cochromatograph sample One times of Nt4CL5aPcSTS mass contents.Inspection is analyzed in the HPLC for completing WAT11/pESC-Nt4CL5aPcSTS sample solutions In 10 minutes after survey, HPLC analyses are carried out to cochromatograph sample under identical chromatographic condition.Cochromatograph sample is in the chromatogram Under the conditions of containing retention time be the chromatographic peak of 23.237 minutes, the chromatographic peak is named as cochromatograph peak (B in Fig. 7), this is total to The peak height of chromatographic peak is twice of Nt4CL5aPcSTS chromatographic peaks, illustrates the chromatogram that Nt4CL5aPcSTS chromatographic peaks are resveratrol Peak.

2.2nd, recombinant yeast WAT11/pESC produces resveratrol

Except the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with into recombinant yeast WAT11/ PESC, remaining all same.

2.3rd, recombinant yeast WAT11/pESC-Nt4CLPPPcSTS produces resveratrol

Except the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with into recombinant yeast WAT11/ PESC-Nt4CLPPPcSTS, remaining all same.

2.4th, recombinant yeast WAT11/pESC-Nt4CL produces resveratrol

Except the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with into recombinant yeast WAT11/ PESC-Nt4CL, remaining all same.

2.5th, recombinant yeast WAT11/pESC-PcSTS produces resveratrol

Except the recombinant yeast WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with into recombinant yeast WAT11/ PESC-PcSTS, remaining all same.

The white lamb's-quarters that recombinant yeast WAT11/pESC-Nt4CL5aPcSTS is produced in the cultivating system of the above-mentioned coumaric acid containing 4- The HPLC-MS of reed alcohol testing result is as shown in Figure 9, Figure 10.As a result show, contain fusion protein Nt4CL5aPcSTS genes Recombinant yeast WAT11/pESC-Nt4CL5aPcSTS produces resveratrol in the cultivating system of the coumaric acid containing 4-, its yield For 10.2 μ g/mL zymotic fluids；Recombinant yeast WAT11/pESC, WAT11/pESC-Nt4CL and WAT11/pESC-PcSTS containing Resveratrol is not produced in the cultivating system of 4- coumaric acids；Recombinant yeast WAT11/pESC-Nt4CLPPPcSTS is in tonka-bean containing 4- The yield of the resveratrol of the cultivating system of acid is 1.3 μ g/mL zymotic fluids.Show to utilize fusion protein in yeast expression system Nt4CL5aPcSTS can improve the yield of resveratrol.

Claims

Any of 1. encoding fusion protein Nt4CL5aPcSTS relevant biological material, is following B1) to B8)：

B1) encoding fusion protein Nt4CL5aPcSTS nucleic acid molecules；

B2 B1) is contained) expression cassettes of the nucleic acid molecules；

B3 B1) is contained) recombinant vectors of the nucleic acid molecules；

B4 B2) is contained) recombinant vector of the expression cassette；

B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；

B6 B2) is contained) recombinant microorganism of the expression cassette；

B7 B3) is contained) recombinant microorganism of the recombinant vector；

B8 B4) is contained) recombinant microorganism of the recombinant vector；

The amino acid sequence of the fusion protein is SEQ ID No.1.
2. relevant biological material as claimed in claim 1, it is characterised in that：B1) nucleic acid molecules are that coded sequence is SEQ The DNA molecular or cDNA molecules of ID No.2 1-2808 nucleotides.
3. relevant biological material as claimed in claim 2, it is characterised in that：B1) nucleic acid molecules are that nucleotide sequence is SEQ ID No.2 DNA molecular or cDNA molecules.
4. application of the relevant biological material in production resveratrol described in claim 1,2 or 3.
5. a kind of method for producing resveratrol, is included in the culture medium containing 4- coumaric acids and cultivates the institute of claim 1,2 or 3 The relevant biological material stated, obtains resveratrol；The fusion protein relevant biological material be following D1)-D3) and in any Kind：

D1) the recombinant microorganism containing nucleic acid molecules；Fusion protein described in the nucleic acid molecule encoding claim 1；

D2) the recombinant microorganism containing expression cassette；The expression cassette contains D1) described in nucleic acid molecules；

D3) the recombinant microorganism containing recombinant vector；The recombinant vector contains D1) described in nucleic acid molecules or D2) described in Expression cassette.
6. method according to claim 5, it is characterised in that：The microorganism is eukaryotic microorganisms.
7. method according to claim 6, it is characterised in that：The eukaryotic microorganisms are fungi.
8. method according to claim 7, it is characterised in that：The fungi is saccharomycete.