CN104611301B - Fusion protein Nt4CL5aPcSTS and preparation method and application - Google Patents
Fusion protein Nt4CL5aPcSTS and preparation method and application Download PDFInfo
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Abstract
The invention discloses fusion protein Nt4CL5aPcSTS and preparation method and application.Fusion protein Nt4CL5aPcSTS provided by the present invention, is the fusion protein obtained by protein N t4CL and protein PcSTS fusions：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 can be catalyzed the resveratrol of 4 coumaric acids and malonyl coenzyme A generation with bioactivity, it was demonstrated that fusion protein Nt4CL5aPcSTS has the activity of 4 coumarate CoA-ligases and stilbene synthase.
Description
Technical field
The present invention relates to fusion protein Nt4CL5aPcSTS and preparation method and 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.
Fusion protein provided by the present invention, entitled Nt4CL5aPcSTS is following protein (1) or (2)：
(1) the fusion protein obtained by protein N t4CL and protein PcSTS fusions：
The protein N t4CL 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 protein PcSTS 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.
In order to solve the above technical problems, present invention also offers the biology related to the fusion protein Nt4CL5aPcSTS
Material.
The biomaterial related to the fusion protein Nt4CL5aPcSTS provided by the present invention, is following B1) extremely
Any of B20)：
B1) encoding said 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.
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
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 XhoI
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 SunfireTMThe 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 SunfireTMThe 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 SunfireTMC18 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
DNA molecular (Nt4CL5aPcSTS genes) shown in artificial synthesized SEQ ID No.2, coding SEQ ID No.1 institutes
The fusion protein Nt4CL5aPcSTS shown.
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 (9)
1. a kind of fusion protein, the amino acid sequence of the fusion protein is SEQ ID No.1.
2. the preparation method of fusion protein described in claim 1, including by the encoding gene of the fusion protein in biological cell
Middle progress, which is expressed, obtains the fusion protein.
3. method according to claim 2, it is characterised in that：The encoding gene of the fusion protein enters in biological cell
Row expression includes the encoding gene of the fusion protein importing the biological cell.
4. according to the method in claim 2 or 3, it is characterised in that：The biological cell is microbial cell, non-human animal
Cell or plant cell.
5. method according to claim 4, it is characterised in that：The microbial cell is prokaryotic micro-organisms cell.
6. method according to claim 5, it is characterised in that：The prokaryotic micro-organisms cell is bacterium.
7. according to the method in claim 2 or 3, it is characterised in that：The encoding gene of the fusion protein be it is following 1. or
Or described DNA molecular 2.：
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.
8. a kind of method for producing resveratrol, is included in culture fusion protein associated biomolecule in the culture medium containing 4- coumaric acids
Material, obtains resveratrol；Any of the fusion protein relevant biological material is following C1) to C3)：
C1) the recombinant microorganism containing nucleic acid molecules；Fusion protein described in the nucleic acid molecule encoding claim 1；It is described micro-
Biology is prokaryotic micro-organisms；
C2) the recombinant microorganism containing expression cassette；The expression cassette contains the nucleic acid molecules；The microorganism is the micro- life of protokaryon
Thing；
C3) the recombinant microorganism containing recombinant vector；The recombinant vector is procaryotic cell expression carrier, and the recombinant vector contains
There are the nucleic acid molecules or the expression cassette；The microorganism is prokaryotic micro-organisms.
9. application of the fusion protein in production resveratrol described in claim 1.
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