CN104611354A - Fusion protein Nt4CL5aPcSTS related biomaterial and application thereof - Google Patents
Fusion protein Nt4CL5aPcSTS related biomaterial and application thereof Download PDFInfo
- Publication number
- CN104611354A CN104611354A CN201510061199.3A CN201510061199A CN104611354A CN 104611354 A CN104611354 A CN 104611354A CN 201510061199 A CN201510061199 A CN 201510061199A CN 104611354 A CN104611354 A CN 104611354A
- Authority
- CN
- China
- Prior art keywords
- protein
- transgenic plant
- nucleic acid
- pcsts
- resveratrol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention discloses a fusion protein Nt4CL5aPcSTS related biomaterial and application thereof. The fusion protein Nt4CL5aPcSTS related biomaterial provided by the invention comprises a fusion protein Nt4CL5aPcSTS, wherein the fusion protein Nt4CL5aPcSTS is obtained in a way that a protein Nt4CL and a protein PcSTS are connected through three amino acid residues, the protein Nt4CL comprises amino acid residues at the 1st-542th sites of an amino acid sequence shown in SEQ ID No.1 from the N terminal and the protein PcSTS comprises amino acid residues at the 548-935th sites of an amino acid sequence shown in SEQ ID No.1 from the N terminal. The fusion protein Nt4CL5aPcSTS related biomaterial can be used for catalyzing 4-cumaric acid and Malonyl Coenzyme A to generate resveratrol with bioactivity.
Description
Technical field
The present invention relates to relevant biological material and the application thereof of fusion rotein Nt4CL5aPcSTS in biological technical field.
Background technology
Trans-resveratrol (Resveratrol is called for short Res), chemistry by name 3,4', 5-trihydroxy--stilbene (trans-3,4', 5-trihydroxystilbene), also known as resvertrol, be a kind of non-flavonoid polyphenolic substance containing stilbene class formation, molecular weight is 228.2, water insoluble, be dissolved in the organic solvents such as ethanol.Natural trans-resveratrol exists in a lot of plant, such as giant knotweed, peanut and grape etc.
Trans-resveratrol be plant meet with uviolizing, physical abuse and fungi infestation time a kind of plant protecting chemical of sharply increasing; research display trans-resveratrol have anticancer, protect the effects such as cardiovascular, anti-oxidant, anti-ageing, immunomodulatory, neuroprotective and phytoestrogen, can be widely used in the fields such as medicine, healthcare products, food, makeup.Trans-resveratrol is the most important health-care components of grape wine, and Frenchman is under higher fatty acid, high protein, high-energy food habits, and the sickness rate of cardiovascular disorder, far below other American-European countries, just has benefited from often drinking the red wine containing trans-resveratrol.
Resveratrol sources is in phenylpropyl alcohol alkane pathways metabolism (Phenylpropanoid pathway).Its Biometabolic pathway is: phenylalanine is at phenylalanine ammonia lyase (Phenylalanine ammonia-lyase, PAL) trans-cinnamic acid is cracked under effect, styracin is at styracin-4-hydroxylase (Cinnamate-4-hydroxylase, C4H) 4-coumaric acid is generated under katalysis, 4-coumaric acid is at 4-coumaroyl A ligase enzyme (4-Coumarate-CoAligase, 4-coumaroyl A is synthesized under effect 4CL), finally, stilbene synthase (Stilbene synthase, STS) the 4-coumaroyl A of catalysis 1 molecule and the malonyl coenzyme A of 3 molecules generate trans-resveratrol (Fig. 1).
Trans-resveratrol biosynthetic pathway rate-limiting step is more in vivo, although a certain key enzyme of simple overexpression may produce certain promoter action to the content of trans-resveratrol, because other step limits, the high level accumulation of trans-resveratrol is very difficult.
Summary of the invention
Technical problem to be solved by this invention how to improve the output of trans-resveratrol.
For solving the problems of the technologies described above, the invention provides the relevant biological material of fusion rotein Nt4CL5aPcSTS.
The relevant biological material of fusion rotein Nt4CL5aPcSTS provided by the present invention is following B1) to B20) in any one:
B1) nucleic acid molecule of encoding fusion protein Nt4CL5aPcSTS;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) containing B1) the transgenic plant cells system of described nucleic acid molecule;
B10) containing B2) the transgenic plant cells system of described expression cassette;
B11) containing B3) the transgenic plant cells system of described recombinant vectors;
B12) containing B4) the transgenic plant cells system of described recombinant vectors;
B13) containing B1) Transgenic plant tissue of described nucleic acid molecule;
B14) containing B2) Transgenic plant tissue of described expression cassette;
B15) containing B3) Transgenic plant tissue of described recombinant vectors;
B16) containing B4) Transgenic plant tissue of described recombinant vectors;
B17) containing B1) the transgenic plant organ of described nucleic acid molecule;
B18) containing B2) the transgenic plant organ of described expression cassette;
B19) containing B3) the transgenic plant organ of described recombinant vectors;
B20) containing B4) the transgenic plant organ of described recombinant vectors;
Described fusion rotein Nt4CL5aPcSTS is following protein (1) or (2):
(1) the fusion rotein obtained by Nt4CL albumen and PcSTS protein fusion:
Described Nt4CL albumen be following a) or b):
A) aminoacid sequence is the protein of SEQ ID No.1 1-542 amino acids residue;
B) by a) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein;
Described PcSTS albumen is following A or B:
A, aminoacid sequence are the protein shown in SEQ ID No.1 548-935 amino acids residue;
B, by A through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein.
(2) N end (1) or C end are connected the tape label protein that label obtains.
In described fusion rotein, described protein N t4CL is connected by five amino acid residue with protein PcSTS.The sequence of described five amino acid residue is as shown in SEQ ID No.1 543-547 position, and the aminoacid sequence of described fusion rotein is SEQ ID No.1.
In order to make described fusion rotein Nt4CL5aPcSTS be convenient to purifying, label as shown in table 1 can be connected at the N end of described fusion rotein Nt4CL5aPcSTS and/or C end.N end as the protein shown in SEQ ID No.1 holds with C the fusion rotein being connected a 6*His label (HHHHHH) respectively and obtaining.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned fusion rotein, the replacement of one or several amino-acid residue described and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
Above-mentioned fusion rotein can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of above-mentioned fusion rotein is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in SEQ ID No.2, and/or the missense mutation carrying out one or several base pair obtains.
Wherein, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.
In above-mentioned biomaterial, B1) described nucleic acid molecule is following 1) or 2) or 3) or 4) shown in nucleic acid molecule, i.e. the gene of Nt4CL5aPcSTS:
1) encoding sequence is cDNA molecule or the DNA molecular of the 1-2808 position Nucleotide of SEQ ID No.2;
2) nucleotide sequence is cDNA molecule or the DNA molecular of SEQ ID No.2;
3) with 1) or 2) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of encoding said fusion protein or genomic DNA molecule;
4) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of encoding said fusion protein or genomic DNA molecule.
Wherein, SEQ ID No.2 is made up of 2808 Nucleotide, the aminoacid sequence shown in nucleotide coding SEQ ID No.1 of SEQ ID No.2.
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of coding Nt4CL5aPcSTS of the present invention.Those are through manually modified, there is the Nucleotide of nucleotide sequence 75% with the Nt4CL5aPcSTS of synthetic of the present invention or higher identity, as long as coding Nt4CL5aPcSTS and there is the protein of identical function, be all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention.
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid.The nucleotide sequence that " identity " comprises the protein formed with the aminoacid sequence shown in the SEQ of coding ID No.1 of the present invention has 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.
In above-mentioned biomaterial, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C.
More than above-mentioned 75% or 75% identity, can be the identity of more than 80%, 85%, 90% or 95%.
In above-mentioned biomaterial, the expression cassette (Nt4CL5aPcSTS expression casette) of the nucleic acid molecule containing coding Nt4CL5aPcSTS B2), refer to the DNA that can express Nt4CL5aPcSTS in host cell, this DNA not only can comprise the promotor starting Nt4CL5aPcSTS genetic transcription, also can comprise the terminator stopping Nt4CL5aPcSTS genetic transcription.Further, described expression cassette also can comprise enhancer sequence.
In above-mentioned biomaterial, available existing expression vector establishment contains the recombinant vectors of described Nt4CL5aPcSTS expression casette.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or virus vector.
In above-mentioned biomaterial, described microorganism can be prokaryotic micro-organisms or eukaryotic microorganisms.Described prokaryotic micro-organisms can be bacterium.Described bacterium can be Escherichia bacteria.Described Escherichia bacteria can be intestinal bacteria.Described eukaryotic microorganisms can be algae, fungi, protozoon.Described fungi can be unicellular fungi.Described unicellular fungi can be yeast.Described yeast can be yeast strain WAT11.
In above-mentioned biomaterial, described transgenic plant cells system, Transgenic plant tissue and transgenic plant organ all do not comprise reproductive material.
Above-mentioned recombinant vectors can be and described Nt4CL5aPcSTS gene is inserted the recombinant expression vector formed in pET30a (+) plasmid; Described recombinant bacterium can be recombination bacillus coli.
Above-mentioned recombinant vectors can be and described Nt4CL5aPcSTS gene is inserted the recombinant expression vector formed in pESC-Trp plasmid; Described recombinant bacterium can be recombination microzyme.
Fusion rotein Nt4CL5aPcSTS of the present invention or the above-mentioned biomaterial relevant to fusion rotein Nt4CL5aPcSTS also belong within protection scope of the present invention producing the application in trans-resveratrol.
Described fusion rotein Nt4CL5aPcSTS is also belonging within protection scope of the present invention as the application in 4-coumarate CoA-ligase and stilbene synthase.
For solving the problems of the technologies described above, present invention also offers a kind of method of producing trans-resveratrol.
A kind of method of producing trans-resveratrol provided by the present invention, is included in the substratum containing 4-coumaric acid and cultivates fusion rotein relevant biological material, obtain trans-resveratrol;
Described fusion rotein relevant biological material is following C1) to C12) in any one:
C1) recombinant microorganism containing nucleic acid molecule; Fusion rotein described in described nucleic acid molecule encoding;
C2) recombinant microorganism containing expression cassette; Described expression cassette contains described nucleic acid molecule;
C3) recombinant microorganism containing recombinant vectors; Described recombinant vectors contains described nucleic acid molecule or described expression cassette;
C4) the transgenic plant cells system containing described nucleic acid molecule;
C5) the transgenic plant cells system containing described expression cassette;
C6) the transgenic plant cells system containing described recombinant vectors;
C7) Transgenic plant tissue containing described nucleic acid molecule;
C8) Transgenic plant tissue containing described expression cassette;
C9) Transgenic plant tissue containing described recombinant vectors;
C10) the transgenic plant organ containing described nucleic acid molecule;
C11) the transgenic plant organ containing described expression cassette;
C12) the transgenic plant organ containing described recombinant vectors.
In the method for above-mentioned production trans-resveratrol, described microorganism can be prokaryotic micro-organisms or eukaryotic microorganisms.Described prokaryotic micro-organisms can be bacterium.Described bacterium can be Escherichia bacteria.Described Escherichia bacteria can be intestinal bacteria.Described eukaryotic microorganisms can be fungi.Described fungi can be yeast.Described yeast can be yeast strain WAT11.Described carrier can be cloning vector and expression vector.Described expression vector can be procaryotic cell expression carrier or eukaryotic expression vector.Fusion rotein relevant biological material described in other is identical with the biomaterial relevant with described fusion rotein Nt4CL5aPcSTS above.
Present invention also offers a kind of preparation method of fusion rotein, the encoding gene comprising described fusion rotein carries out expression and obtains described fusion rotein in biomass cells.
In above-mentioned preparation method, the encoding gene of described fusion rotein be following 1. or 2. or 3. or 4. described DNA molecular:
1. encoding sequence is DNA molecular or the cDNA molecule of the 1-2808 position Nucleotide of SEQ ID No.2;
2. nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.2;
3. there is the identity of more than 75% and the DNA molecular of code for said proteins or cDNA molecule with the DNA molecular 1. or 2. limited or cDNA molecule;
4. under strict conditions with the DNA molecular 1. or 2. limited or cDNA molecular hybridization and the DNA molecular of code for said proteins or cDNA molecule.
In above-mentioned preparation method, the encoding gene of described fusion rotein is carried out in biomass cells express to comprise the encoding gene of described fusion rotein is imported described biomass cells.
In above-mentioned preparation method, the encoding gene of described fusion rotein imports by the recombinant expression vector of the encoding gene containing described fusion rotein the reconstitution cell that described biomass cells obtains expressing described fusion rotein; Described biomass cells can be microorganism cells, vegetable cell or non-human animal cell, and described microorganism cells can be yeast, bacterium, algae or fungi.In one embodiment of the invention, described biomass cells specifically can be bacterium and yeast.
In above-mentioned preparation method, comprise the described reconstitution cell of cultivation and obtain described fusion rotein.
In above-mentioned preparation method, described recombinant expression vector can be and the DNA between the EcoR I recognition sequence of pET30a (+) and XhoI recognition sequence is replaced with the DNA molecular that nucleotide sequence is SEQ ID No.2, keep the constant Nt4CL5aPcSTS expression vector obtained of other sequence of pET30a (+), its name is called pET30a-Nt4CL5aPcSTS.
In above-mentioned preparation method, described intestinal bacteria can be the recombination bacillus coli obtained by recombinant expression plasmid pET30a-Nt4CL5aPcSTS importing recipient E. coli.Described recipient E. coli can be intestinal bacteria Rosetta.
In above-mentioned preparation method, described recombinant expression vector can be and the DNA between the EcoR I recognition sequence of pESC-Trp and Bgl II recognition sequence is replaced with the DNA molecular that nucleotide sequence is SEQ ID No.2, other sequence of maintenance pESC-Trp is constant obtains Nt4CL5aPcSTS expression vector, and its name is called pESC-Nt4CL5aPcSTS.
In above-mentioned preparation method, described recombination microzyme can be recombination microzyme pESC-Nt4CL5aPcSTS being imported recipient yeast bacterium and obtain.Described recipient yeast bacterium specifically can be yeast WAT11.
In above-mentioned preparation method, described cultivation is included in 25 DEG C, induces 2h to express described fusion rotein Nt4CL5aPcSTS in the substratum of the IPTG containing 0.2mmol/L.
The experiment of external enzymatic reaction proves, fusion rotein Nt4CL5aPcSTS provided by the invention catalysis 4-coumaric acid and malonyl coenzyme A can generate and have bioactive trans-resveratrol, proves that fusion rotein Nt4CL5aPcSTS has the activity of 4-coumaroyl A ligase enzyme and stilbene synthase.Recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS containing fusion rotein Nt4CL5aPcSTS gene produces trans-resveratrol in the substratum containing 4-coumaric acid, and its output is 26.5 μ g/mL; Recombination bacillus coli Rosetta/pET30a-PcSTS containing PcSTS gene does not produce trans-resveratrol in above-mentioned containing in the substratum of 4-coumaric acid.Show in escherichia expression system, utilize fusion rotein Nt4CL5aPcSTS can improve the output of trans-resveratrol.Recombination microzyme WAT11/pESC-Nt4CL5aPcSTS containing fusion rotein Nt4CL5aPcSTS gene produces trans-resveratrol in containing the substratum of 4-coumaric acid, and its output is 10.2 μ g/mL fermented liquids; Recombination microzyme WAT11/pESC, WAT11/pESC-Nt4CL and WAT11/pESC-PcSTS do not produce trans-resveratrol at the culture system containing 4-coumaric acid; Recombination microzyme WAT11/pESC-Nt4CLPPPcSTS is 1.3 μ g/mL fermented liquids in the output of the trans-resveratrol of the culture system containing 4-coumaric acid.Show in yeast expression system, utilize fusion rotein Nt4CL5aPcSTS can improve the output of trans-resveratrol.
Accompanying drawing explanation
Fig. 1 is trans-resveratrol biosynthetic pathway figure.
Fig. 2 is the poly-propionic acid amide gel electrophoresis figure of protein PcSTS abduction delivering.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 supernatant liquor; Swimming lane 3 is recombinant bacterium Rosetta/pET30a-PcSTS cellular lysate precipitation; Swimming lane 4 for Ni-NTA resin hatch after supernatant; Swimming lane 5 is for washing resin trap liquid; 6 and 7 is the protein PcSTS after Ni-NTA agarose affinity chromatography column purification.
Fig. 3 is the poly-propionic acid amide gel electrophoresis figure of protein PcSTS after the desalination of PD-10 post.Swimming lane 1-7 is the elutriant after the desalination of protein PcSTS sample.
Fig. 4 is the poly-propionic acid amide gel electrophoresis figure of fusion rotein Nt4CL5aPcSTS abduction delivering.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 supernatant liquor; Swimming lane 3 is recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate precipitation; Swimming lane 4 for Ni-NTA resin hatch after supernatant; Swimming lane 5 is for washing resin trap liquid; 6 is the fusion rotein Nt4CL5aPcSTS after Ni-NTA agarose affinity chromatography column purification.
Fig. 5 is the poly-propionic acid amide gel electrophoresis figure of fusion rotein Nt4CL5aPcSTS after the desalination of PD-10 post.Swimming lane 1-4 is the elutriant after the desalination of fusion rotein Nt4CL5aPcSTS sample.
Fig. 6 is that high performance liquid chromatography detects collection of illustrative plates.Wherein, A is trans-resveratrol standard substance high-efficient liquid phase chromatograms; B is the high-efficient liquid phase chromatogram of the external enzymatic reaction product of PcSTS; C is the cochromatograph figure of trans-resveratrol standard substance and the external enzymatic reaction product of PcSTS.X-coordinate is retention time, and unit is minute.
Fig. 7 is that fusion rotein Nt4CL5aPcSTS high performance liquid chromatography detects collection of illustrative plates.Wherein, A is the high-efficient liquid phase chromatogram of the external enzymatic reaction product of Nt4CL5aPcSTS; B is the cochromatograph figure of trans-resveratrol standard substance and the external enzymatic reaction product of Nt4CL5aPcSTS.X-coordinate is retention time, and unit is minute.
The detected result of Fig. 8 to be recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS with coumaric acid be HPLC-MS of the tunning of substrate.Wherein A is the HPLC-MS of trans-resveratrol standard substance; The HPLC-MS of B to be recombination bacillus coli with coumaric acid the be tunning of substrate.
The detected result of Fig. 9 to be recombination microzyme WAT11/pESC-Nt4CL5aPcSTS with coumaric acid be HPLC of the tunning of substrate.The wherein HPLC of A to be recombination microzyme WAT11/pESC-Nt4CL5aPcSTS with coumaric acid the be tunning of substrate; B is recombination microzyme WAT11/pESC-Nt4CL5aPcSTS take coumaric acid as the tunning of substrate and the cochromatograph figure of trans-resveratrol standard substance.X-coordinate is retention time, and unit is minute.
The detected result of Figure 10 to be recombination microzyme WAT11/pESC-Nt4CL5aPcSTS with coumaric acid be HPLC-MS of the tunning of substrate.Wherein A is the HPLC-MS of trans-resveratrol standard substance; The HPLC-MS of B to be recombination microzyme WAT11/pESC-Nt4CL5aPcSTS with coumaric acid the be tunning of substrate.
Embodiment
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
PESC-Trp in following embodiment is Agilent Products, and catalog number is 217453.
Yeast strain WAT11 (Zhang Y in following embodiment, Li SZ, Li J, et al.Using unnaturalprotein fusions to engineer resveratrol biosynthesis in yeast and mammaliancells [J] .Journal of the American Chemical Society, 2006,128 (40): 13030-13031.) be Wuhan Botanical Garden, Chinese Acadmey of Sciences teacher Zhang Yansheng present.This biomaterial related experiment of the present invention of only attaching most importance to again is used, not can be used as other purposes and uses.
Substratum used in following embodiment is as follows:
Trp auxotrophy liquid nutrient medium is prepared as follows: add in distilled water by Trp Minus Media (the general Jino Science and Technology Ltd. in Beijing product), the content to Trp Minus Media is 8g/L; PH nature, obtains Trp auxotrophy liquid nutrient medium after autoclaving.
Trp auxotrophy solid medium: adding agar to its content in Trp auxotrophy liquid nutrient medium is the substratum that 20g/L obtains.
The prokaryotic expression of embodiment 1, fusion rotein Nt4CL5aPcSTS and purifying
One, the structure of Nt4CL5aPcSTS gene expression plasmid
DNA molecular (Nt4CL5aPcSTS gene) shown in synthetic SEQ ID No.2, the fusion rotein Nt4CL5aPcSTS shown in coding SEQ ID No.1.
DNA between the EcoR I recognition sequence of pET30a (+) (Novagen Products) and Xho I recognition sequence is replaced with the DNA molecular that nucleotide sequence is SEQ ID No.2, other sequence of maintenance pET30a (+) is constant obtains Nt4CL5aPcSTS expression vector, and its name is called pET30a-Nt4CL5aPcSTS.PET30a-Nt4CL5aPcSTS expresses the fusion rotein Nt4CL5aPcSTS shown in SEQ ID No.1.
DNA between the EcoR I recognition sequence of pET30a (+) and Xho I recognition sequence is replaced with the DNA molecular that nucleotide sequence is 1642-2808 position in SEQ ID No.2, other sequence of maintenance pET30a (+) is constant obtains PcSTS expression vector, and its name is called pET30a-PcSTS.PET30a-PcSTS expresses the protein PcSTS shown in SEQ ID No.1 548-935 position.
Fusion rotein Nt4CL5aPcSTS shown in above-mentioned SEQ ID No.1 comprises protein N t4CL, linker and protein PcSTS.In SEQ ID No.1,1-542 amino acids residue is the aminoacid sequence of protein N t4CL, and 543-547 amino acids residue is the aminoacid sequence of linker, and 548-935 amino acids residue is the aminoacid sequence of protein PcSTS.In SEQ ID No.2, the DNA molecule encode protein N t4CL shown in 1-1626 position; DNA molecule encode linker shown in 1627-1641 position; DNA molecule encode protein PcSTS shown in 1642-2808 position.
Two, the expression and purification of fusion rotein Nt4CL5aPcSTS
Plasmid pET30a-PcSTS is imported intestinal bacteria Rosetta, screen through LB solid plate (containing 50 μ g/mL kantlex and 25 μ g/mL paraxin), obtain the recombinant bacterium intestinal bacteria containing pET30a-PcSTS, by this recombination bacillus coli called after Rosetta/pET30a-PcSTS, bacterium in contrast.
Recombinant expression plasmid pET30a-Nt4CL5aPcSTS is imported intestinal bacteria Rosetta, screen through LB solid plate (containing 50 μ g/mL kantlex and 25 μ g/mL paraxin), obtain the recombination bacillus coli containing pET30a-Nt4CL5aPcSTS, by this recombination bacillus coli called after Rosetta/pET30a-Nt4CL5aPcSTS.
Plasmid pET30a (+) is imported intestinal bacteria Rosetta, screen through LB solid plate (containing 50 μ g/mL kantlex and 25 μ g/mL paraxin), obtain the recombination bacillus coli containing pET30a (+), by this recombination bacillus coli called after Rosetta/pET30a, as empty vector control bacterium.
Rosetta/pET30a-PcSTS, Rosetta/pET30a-Nt4CL5aPcSTS and Rosetta/pET30a are inoculated in LB liquid nutrient medium respectively, 37 DEG C of shaking culture are spent the night and are obtained cultivating bacterium liquid 1, and called after Rosetta/pET30a-PcSTS cultivates bacterium liquid 1 respectively, Rosetta/pET30a-Nt4CL5aPcSTS cultivates bacterium liquid 1 and Rosetta/pET30a cultivates bacterium liquid 1; Be inoculated in LB liquid nutrient medium (containing 50 μ g/mL kantlex and 25 μ g/mL paraxin) with 1:100 (volume ratio) respectively by above-mentioned cultivation bacterium liquid 1, shaking culture is to OD
600value 0.6 obtains cultivating bacterium liquid 2, and called after Rosetta/pET30a-PcSTS cultivates bacterium liquid 2 respectively, Rosetta/pET30a-Nt4CL5aPcSTS cultivates bacterium liquid 2 and Rosetta/pET30a cultivates bacterium liquid 2; Cultivate in bacterium liquid 2 and Rosetta/pET30a-Nt4CL5aPcSTS cultivation bacterium liquid 2 to above-mentioned Rosetta/pET30a-PcSTS and add IPTG respectively, the concentration of IPTG in system is made to be respectively 0.2mmol/L, 0.5mmol/L, 0.8mmol/L and 1.0mmol/L, 25 DEG C, induce 2h, 4h, 6h, 8h, 10h and 22h respectively, collect equivalent thalline under each inductive condition.Above-mentioned each thalline adds the cracking of SDS sample buffer successively, centrifugal, the supernatant liquor obtained respectively is got 10 μ L and is carried out 10%SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis is at hereinafter referred SDS-PAGE), scans electrophoresis result after coomassie brilliant blue staining with gel image analyser.By comparing the difference of expression amount under different IP TG concentration and different induction time, result shows, the suitableeest abduction delivering condition of protein PcSTS is: the concentration of IPTG in system is 0.8mmol/L, 25 DEG C of induction 6h; The suitableeest abduction delivering condition of fusion rotein Nt4CL5aPcSTS is: the concentration of IPTG in system is 0.2mmol/L, 25 DEG C of induction 2h.
The Rosetta/pET30a-PcSTS obtained in above-mentioned steps cultivates bacterium liquid 2 and adds IPTG, the concentration of IPTG in system is made to be 0.8mmol/L, 25 DEG C, induction 6h, ultrasonication after collection thalline, obtains bacterial cell disruption liquid (name is called Rosetta/pET30a-PcSTS bacterial cell disruption liquid).The centrifugal 10min of this bacterial cell disruption liquid 12000rpm is obtained supernatant liquor (name is called Rosetta/pET30a-PcSTS cellular lysate supernatant liquor) and precipitation (name is called that Rosetta/pET30a-PcSTS cellular lysate precipitates).
The Rosetta/pET30a-Nt4CL5aPcSTS obtained in above-mentioned steps cultivates bacterium liquid 2 and adds IPTG, the concentration of IPTG in system is made to be 0.2mmol/L, 25 DEG C, induction 2h, ultrasonication after collection thalline, obtains bacterial cell disruption liquid (name is called Rosetta/pET30a-Nt4CL5aPcSTS bacterial cell disruption liquid).The centrifugal 10min of this bacterial cell disruption liquid 12000rpm is obtained supernatant liquor (name is called Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatant liquor) and precipitation (name is called that Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate precipitates).
Rosetta/pET30a-PcSTS bacterial cell disruption liquid, Rosetta/pET30a-PcSTS cellular lysate supernatant liquor, Rosetta/pET30a-PcSTS cellular lysate precipitation, Rosetta/pET30a-Nt4CL5aPcSTS bacterial cell disruption liquid, Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatant liquor and Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate precipitation are carried out SDS-PAGE.Utilize Ni-NTA agarose affinity chromatography post purifying protein from cellular lysate supernatant liquor, then utilize the desalination of PD-10 post.
Experimental result is shown in as Fig. 2, Fig. 3, Fig. 4 and Fig. 5.Result shows, protein PcSTS is mainly present in Rosetta/pET30a-PcSTS cellular lysate supernatant liquor, and molecular size range is 43kDa; Fusion rotein Nt4CL5aPcSTS is mainly present in Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatant liquor, and molecular size range is 103kDa.Utilize Ni-NTA agarose affinity chromatography post purifying from Rosetta/pET30a-PcSTS cellular lysate supernatant liquor to obtain the protein PcSTS with His label, from Rosetta/pET30a-Nt4CL5aPcSTS cellular lysate supernatant liquor, purifying obtains the fusion rotein Nt4CL5aPcSTS with His label.After the filter membrane of molecular weight cut-off (MWCO) 10000 is concentrated, obtain with protein storage solubilize protein PcSTS and the fusion rotein Nt4CL5aPcSTS solution that concentration is 1mg/mL.Protein storage solution: solute and concentration thereof are: 20mM Tris (pH7.5), 150mM sodium-chlor, 0.5mM EDTA, 25% (v/v) glycerine, adds when protein storage solution uses after 1mM DTT mixes and uses; Solvent is distilled water; PH nature.
Embodiment 2, the external enzymatic reaction of HPLC detection fusion albumen Nt4CL5aPcSTS and product
2.1, trans-resveratrol standard substance HPLC detects: accurately take trans-resveratrol standard substance (Sigma Products) 1.0mg and, in 10mL volumetric flask, with chromatogram dissolve with methanol, be settled to 10mL, be configured to the trans-resveratrol mother liquor of 100 μ g/mL.Through 0.22 μm of membrane filtration, obtain concentration with methanol dilution and be respectively the standardized solution of 2.5,5,12.5,25,50 μ g/mL, concentration sample introduction successively from low to high.Use is furnished with Sunfire
tMthe Waters HPLC of C18 reversed-phase column (5.0 μm, 4.6mm × 250mm) analyzes.Moving phase is made up of methyl alcohol (A) and water (B), flow velocity is 0.8mL/min, use following condition of gradient elution: in 0-30min, in moving phase, the volumn concentration of methyl alcohol at the uniform velocity increases to 70% by 10%, the volumn concentration of water is at the uniform velocity down to 30% by 90%, carries out linear gradient elution; Use moving phase (solution be mixed to get according to the volume ratio of 7:3 by first alcohol and water) wash-out 10min again.Determined wavelength is 306nm.Trans-resveratrol standard substance high-efficient liquid phase chromatogram is as shown in A in Fig. 6.
2.2, the external enzymatic reaction of albumen PcSTS and product HPLC detect: prepare the external enzymatic reaction system of 300 μ L PcSTS, the pH of this external enzymatic reaction system is 7.5, solvent is that (solute is potassium primary phosphate and dipotassium hydrogen phosphate to 0.1mol/L potassium phosphate buffer, solvent is water), the true protein PcSTS that solute is 4-coumaroyl A, malonyl coenzyme A, embodiment 1 obtain.In the external enzymatic reaction system of 300 μ L PcSTS, the concentration of 4-coumaroyl A is 150 μm of ol/L, and the concentration of malonyl coenzyme A is 280 μm of ol/L, and the concentration of the pure protein PcSTS of embodiment 1 is 0.1 μm of ol/L.Above-mentioned reaction system reacts 30min at being placed in 35 DEG C, adds acetic acid, makes the concentration of acetic acid in system be 5% (volumn concentration), then uses the extraction into ethyl acetate of 300 μ L, the centrifugal 10min of 10000r/min.Get upper strata ethyl acetate layer and carry out vacuum-drying, obtain PcSTS enzymatic reaction product, in PcSTS enzymatic reaction product, add 50 μ L 50% (volumn concentration) methanol aqueous solutions, obtain PcSTS enzymatic reaction product testing sample.Use is furnished with Sunfire
tMthe Waters HPLC of C18 reversed-phase column (5.0 μm, 4.6mm × 250mm) analyzes PcSTS enzymatic reaction product testing sample.Moving phase is made up of methyl alcohol (A) and water (B), flow velocity is 0.8mL/min, use following condition of gradient elution: in 0-30min, in moving phase, the volumn concentration of methyl alcohol at the uniform velocity increases to 70% by 10%, the volumn concentration of water is at the uniform velocity down to 30% by 90%, carries out linear gradient elution; Use moving phase (solution be mixed to get according to the volume ratio of 7:3 by first alcohol and water) wash-out 10min again.Determined wavelength is 306nm.With trans-resveratrol (Sigma company, article No. R5010) for standard substance adopt calibration curve method (external standard method) to carry out quantitative analysis trans-resveratrol.Experiment in triplicate, repeats to establish 10 reaction systems at every turn.
Result shows that the high-efficient liquid phase chromatogram of the external enzymatic reaction product of PcSTS is as shown in B in Fig. 6.The cochromatograph figure of trans-resveratrol standard substance and the external enzymatic reaction product of PcSTS is respectively as shown in A and C in Fig. 6, and the retention time of standard substance trans-resveratrol under this chromatographic condition is 23.253 minutes (in see Fig. 6 A); The external enzymatic reaction product of PcSTS is the chromatographic peak (in Fig. 6 B) of the trans-resveratrol of 23.232 minutes containing retention time under this chromatographic condition.In above-mentioned PcSTS enzymatic reaction product testing sample, add above-mentioned trans-resveratrol standard substance obtain cochromatograph sample, the mass content of the above-mentioned trans-resveratrol standard substance in cochromatograph sample is one times of the PcSTS mass content in cochromatograph sample.In 10 minutes after the HPLC analyzing and testing completing PcSTS enzymatic reaction product testing sample, under identical chromatographic condition, HPLC analysis is carried out to cochromatograph sample.Cochromatograph sample is the chromatographic peak of 23.250 minutes containing retention time under this chromatographic condition, by this chromatographic peak called after cochromatograph peak (in Fig. 7 B), the peak height at this cochromatograph peak is the twice of PcSTS chromatographic peak, illustrates that PcSTS chromatographic peak is the chromatographic peak of trans-resveratrol.
2.3, the external enzymatic reaction of fusion rotein Nt4CL5aPcSTS and product HPLC detect: except the external enzymatic reaction system in the external enzymatic reaction of albumen PcSTS and product HPLC in 2.2 being detected is replaced by the external enzymatic reaction system of 300 μ L Nt4CL5aPcSTS, all the other are all identical with 2.2.The pH of the external enzymatic reaction system of 300 μ L Nt4CL5aPcSTS is 7.5, solvent is that (solute is potassium primary phosphate and dipotassium hydrogen phosphate to 0.1mol/L potassium phosphate buffer, solvent is water), solute and concentration thereof are: the pure fusion rotein Nt4CL5aPcSTS of 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 embodiment 1.Experiment in triplicate, repeats to establish 10 reaction systems at every turn.
Above-mentioned reaction system reacts 30min at being placed in 35 DEG C, adds acetic acid, makes the concentration of acetic acid in system be 5% (volumn concentration), then uses the extraction into ethyl acetate of 300 μ L, the centrifugal 10min of 10000r/min.Get upper strata ethyl acetate layer and carry out vacuum-drying, obtain Nt4CL5aPcSTS enzymatic reaction product, in Nt4CL5aPcSTS enzymatic reaction product, add 50 μ L 50% (volumn concentration) methanol aqueous solutions, obtain Nt4CL5aPcSTS enzymatic reaction product testing sample.Method according to 2.2 is carried out HPLC and is analyzed Nt4CL5aPcSTS enzymatic reaction product testing sample.Result shows that the external enzymatic reaction product of Nt4CL5aPcSTS is the chromatographic peak of 21.876 minutes containing retention time under this chromatographic condition, by this chromatographic peak called after Nt4CL5aPcSTS chromatographic peak (in Fig. 7 A).In above-mentioned Nt4CL5aPcSTS enzymatic reaction product testing sample, add above-mentioned trans-resveratrol standard substance obtain cochromatograph sample, the mass content of the above-mentioned trans-resveratrol standard substance in cochromatograph sample is four times of the Nt4CL5aPcSTS mass content in cochromatograph sample.In 10 minutes after the HPLC analyzing and testing completing Nt4CL5aPcSTS enzymatic reaction product testing sample, under identical chromatographic condition, HPLC analysis is carried out to cochromatograph sample.Cochromatograph sample is the chromatographic peak of 21.903 minutes containing retention time under this chromatographic condition, by this chromatographic peak called after cochromatograph peak (in Fig. 7 B), the peak height at this cochromatograph peak is five times of Nt4CL5aPcSTS chromatographic peak, illustrates that Nt4CL5aPcSTS chromatographic peak is the chromatographic peak of trans-resveratrol.
Result shows, the external enzymatic reaction product of protein PcSTS is trans-resveratrol, in enzymatic reaction system, protein PcSTS catalysis 4-coumaroyl A and malonyl coenzyme A can be formed and have bioactive trans-resveratrol in vitro, and output is 4.9kg trans-resveratrol/mol protein PcSTS; The external enzymatic reaction product of fusion rotein Nt4CL5aPcSTS is trans-resveratrol, fusion rotein Nt4CL5aPcSTS catalysis 4-coumaric acid and malonyl coenzyme A can generate and have bioactive trans-resveratrol, and output is 8.0kg trans-resveratrol/mol fusion rotein Nt4CL5aPcSTS.
Embodiment 3, utilize recombination bacillus coli produce trans-resveratrol
3.1, recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS produces trans-resveratrol: the recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS in embodiment 1 is inoculated in LB liquid nutrient medium, 37 DEG C of shaking culture are spent the night and are obtained cultivating bacterium liquid 1, be inoculated into 1:100 (volume ratio) in the triangular flask that 100mL LB liquid nutrient medium (containing 50 μ g/mL kantlex and 25 μ g/mL paraxin) are housed by above-mentioned cultivation bacterium liquid 1, shaking culture is to OD
600obtain between value 0.4-0.5 cultivating bacterium liquid 2, IPTG is added in cultivation bacterium liquid 2, the system obtained is called the system of cultivating bacterium liquid 2, the concentration of IPTG in the system of cultivating bacterium liquid 2 is made to be 0.2mM, 25 DEG C, 150rpm inducing culture 2h, the centrifugal 2min of 6000rpm, abandon supernatant, (should be in M9 substratum, add the liquid nutrient medium that kantlex and paraxin obtains containing substratum of 50 μ g/mL kantlex and 25 μ g/mL paraxin with 100mL containing the substratum of 50 μ g/mL kantlex and 25 μ g/mL paraxin, the concentration of kantlex in this liquid nutrient medium is 50 μ g/mL, the concentration of paraxin in this liquid nutrient medium is 25 μ g/mL) resuspended bacterial sediment obtains re-suspension liquid.IPTG and 4-coumaric acid is added in re-suspension liquid, the system obtained is called the culture system containing 4-coumaric acid, IPTG is made to be 0.2mM containing the concentration in the culture system of 4-coumaric acid, to make 4-coumaric acid be 12 μ g/mL containing the concentration in the culture system of 4-coumaric acid, 25 DEG C, 150rpm cultivates 48h and obtains fermentation culture.Get the centrifugal 2min of 1mL above-mentioned fermentation culture 12000rpm, get supernatant liquor, use 1mL extraction into ethyl acetate, vortex oscillation 40s, re-extract twice.Centrifuging and taking upper strata ethyl acetate layer, after volatile dry, adds 50 μ L 50% (volumn concentration) methanol aqueous solutions, obtains the sample solution of recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS, carry out the detection of HPLC-MS.Use is furnished with Sunfire
tMthe Waters HPLC of C18 reversed-phase column (5.0 μm, 4.6mm × 250mm) detects.Moving phase is made up of methyl alcohol (A) and water (B), flow velocity is 0.8mL/min, use following condition of gradient elution: in 0-30min, in moving phase, the volumn concentration of methyl alcohol at the uniform velocity increases to 70% by 10%, the volumn concentration of water is at the uniform velocity down to 30% by 90%, carries out linear gradient elution; Use moving phase (solution be mixed to get according to the volume ratio of 7:3 by first alcohol and water) wash-out 10min again.Determined wavelength is 306nm.With trans-resveratrol (Sigma company, article No. R5010) for standard substance adopt calibration curve method (external standard method) to carry out quantitative analysis trans-resveratrol.Mass spectrometric detection condition is: scanning of the mass spectrum scope 150-1000 (m/z); Sheath gas: nitrogen, sheath gas velocity: 40mL/min, assisted gas flow velocity: 3mL/min, capillary temperature: 300 DEG C, capillary voltage :-35V.Experiment in triplicate, repeats to establish 10 triangular flasks at every turn.
3.2, recombinant bacterium Rosetta/pET30a-PcSTS produces trans-resveratrol: except the Rosetta/pET30a-PcSTS that the recombination bacillus coli Rosetta/pET30a-Nt4CL5aPcSTS cultivation bacterium liquid 2 in 3.1 is replaced with embodiment 1 cultivates bacterium liquid 2, all the other are all identical.
3.3, recombinant bacterium Rosetta/pET30a produces trans-resveratrol: except the Rosetta/pET30a that the pET30a-Nt4CL5aPcSTS cultivation bacterium liquid 2 in 3.1 is replaced with embodiment 1 cultivates bacterium liquid 2, all the other are all identical.
The detected result of the HPLC-MS of the trans-resveratrol that recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS produces at the above-mentioned culture system containing 4-coumaric acid as shown in Figure 8.Result shows, recombinant bacterium Rosetta/pET30a-Nt4CL5aPcSTS is 26.5 μ g/mL fermented liquids in the output of the trans-resveratrol of the above-mentioned culture system containing 4-coumaric acid; Recombinant bacterium Rosetta/pET30a-PcSTS does not produce trans-resveratrol at the above-mentioned culture system containing 4-coumaric acid; Recombinant bacterium Rosetta/pET30a does not produce trans-resveratrol in above-mentioned containing in the culture system of 4-coumaric acid.Show in escherichia expression system, utilize fusion rotein Nt4CL5aPcSTS can improve the output of trans-resveratrol.
Embodiment 4, utilize recombination microzyme produce trans-resveratrol
1, the structure of recombinant yeast expression vector and conversion
DNA molecular (Nt4CL5aPcSTS gene) shown in synthetic SEQ ID No.2, the fusion rotein Nt4CL5aPcSTS shown in coding SEQ ID No.1.
By pESC-Trp (Agilent Products, catalog number is 217453) EcoR I recognition sequence and Bgl II recognition sequence between DNA replace with the DNA molecular that nucleotide sequence is SEQ ID No.2, other sequence of maintenance pESC-Trp is constant obtains Nt4CL5aPcSTS expression vector, and its name is called pESC-Nt4CL5aPcSTS.PESC-Nt4CL5aPcSTS expresses the fusion rotein Nt4CL5aPcSTS shown in SEQ ID No.1.
DNA between the EcoR I recognition sequence of pESC-Trp and Pac I recognition sequence is replaced with the DNA molecular that nucleotide sequence is 1-1626 position in SEQ ID No.2, DNA between the BamH I recognition sequence of pESC-Trp and Xho I recognition sequence is replaced with the DNA molecular that nucleotide sequence is 1642-2808 position in SEQ ID No.2, other sequence of maintenance pESC-Trp is constant obtains expression vector, and its name is called pESC-Nt4CLPPPcSTS.PESC-Nt4CLPPPcSTS expresses the protein N t4CL shown in SEQ ID No.1 1-542 amino acids residue and the protein PcSTS shown in SEQ ID No.1 548-935 amino acids residue.
DNA between the EcoR I recognition sequence of pESC-Trp and Bgl II recognition sequence is replaced with the DNA molecular that nucleotide sequence is 1-1626 position in SEQ ID No.2, other sequence of maintenance pESC-Trp is constant obtains Nt4CL expression vector, and its name is called pESC-Nt4CL.PESC-Nt4CL expresses the protein N t4CL shown in SEQ ID No.1 1-542 position.
DNA between the EcoR I recognition sequence of pESC-Trp and Bgl II recognition sequence is replaced with the DNA molecular that nucleotide sequence is 1642-2808 position in SEQ ID No.2, other sequence of maintenance pESC-Trp is constant obtains PcSTS expression vector, and its name is called pESC-PcSTS.PESC-PcSTS expresses the protein PcSTS shown in SEQ ID No.1 548-935 position.
Fusion rotein Nt4CL5aPcSTS shown in above-mentioned SEQ ID No.1 comprises protein N t4CL, linker and protein PcSTS.In SEQ ID No.1,1-542 amino acids residue is the aminoacid sequence of protein N t4CL, and 543-547 amino acids residue is the aminoacid sequence of linker, the aminoacid sequence of 548-935 amino acids residues protein PcSTS.In SEQ ID No.2, the DNA molecule encode protein N t4CL shown in 1-1626 position; DNA molecule encode linker shown in 1627-1641 position; DNA molecule encode protein PcSTS shown in 1642-2808 position.
By the solid plate that Trp auxotrophy solid medium is made, called after Trp auxotrophy is dull and stereotyped.Expression of recombinant yeast plasmid pESC-Nt4CL5aPcSTS is proceeded to yeast strain WAT11, then screen on Trp auxotrophy flat board, obtain the recombination microzyme containing pESC-Nt4CL5aPcSTS, by this recombination microzyme called after WAT11/pESC-Nt4CL5aPcSTS.
Plasmid pESC-Trp is imported yeast strain WAT11, then screens on Trp auxotrophy flat board, obtain the recombination microzyme containing pESC-Trp, by this recombination microzyme called after WAT11/pESC, as empty vector control bacterium.
Expression of recombinant yeast plasmid pESC-Nt4CLPPPcSTS is imported yeast strain WAT11, then screen on Trp auxotrophy flat board, obtain the recombination microzyme containing pESC-Nt4CLPPPcSTS, by this recombination microzyme called after WAT11/pESC-Nt4CLPPPcSTS.
Expression of recombinant yeast plasmid pESC-Nt4CL is imported yeast strain WAT11, then screens on Trp auxotrophy flat board, obtain the recombination microzyme containing pESC-Nt4CL, by this recombination microzyme called after WAT11/pESC-Nt4CL.
Expression of recombinant yeast plasmid pESC-PcSTS is imported yeast strain WAT11, then screens on Trp auxotrophy flat board, obtain the recombination microzyme containing pESC-PcSTS, by this recombination microzyme called after WAT11/pESC-PcSTS.
2, recombination microzyme is utilized to produce trans-resveratrol
By 20% (mass percent concentration) sterile dextrose solution, (solute is glucose, solvent is sterilized water) add Trp auxotrophy liquid nutrient medium, the concentration of glucose in the substratum obtained is made to be 2%, by this substratum called after glucose-Trp defect liquid nutrient medium; By 20% (mass percent concentration) sterile dextrose solution, (solute is glucose, solvent is sterilized water) add Trp auxotrophy solid medium, make the concentration of glucose in the solid medium obtained be 2%, the solid plate called after glucose-Trp defect this cultivation made is dull and stereotyped; By 20% (mass percent concentration) aseptic galactose solution, (solute is semi-lactosi, solvent is sterilized water) add Trp auxotrophy liquid nutrient medium, the concentration of semi-lactosi in the substratum obtained is made to be 2%, by this substratum called after semi-lactosi-Trp defect liquid nutrient medium.
2.1, recombination microzyme WAT11/pESC-Nt4CL5aPcSTS produces trans-resveratrol
Activated at the flat lining out of glucose-Trp defect by recombination microzyme WAT11/pESC-Nt4CL5aPcSTS, picking list colony inoculation is in glucose-Trp defect liquid nutrient medium, and 30 DEG C, 200rpm cultivates 24h and obtains cultivating bacterium liquid.Above-mentioned cultivation bacterium liquid is centrifugal removes supernatant, precipitation ddH
2o washes 3 times, this precipitation is recombination microzyme WAT11/pESC-Nt4CL5aPcSTS thalline, then re-suspension liquid is obtained with semi-lactosi-Trp defect liquid nutrient medium resuspended recombination microzyme WAT11/pESC-Nt4CL5aPcSTS thalline, 4-coumaric acid is added in re-suspension liquid, the system obtained is called the culture system containing 4-coumaric acid, make the concentration of 4-coumaric acid in this culture system containing 4-coumaric acid be 12 μ g/mL, after 30 DEG C of cultivation 24h, obtain fermented liquid.
Get the above-mentioned fermented liquid of 1mL, add equivalent extraction into ethyl acetate, centrifuging and taking upper strata ethyl acetate layer, after volatile dry, add 50 μ L 50% (volumn concentration) methanol aqueous solutions, obtain WAT11/pESC-Nt4CL5aPcSTS sample solution with after filtering membrane filtering membrane.Get above-mentioned sample solution 10 μ L, carry out the detection of HPLC-MS according to the method for embodiment 3.Experiment in triplicate, repeats to establish 10 triangular flasks at every turn.Result shows that WAT11/pESC-Nt4CL5aPcSTS sample solution is the chromatographic peak of 23.249 minutes containing retention time under this chromatographic condition, by this chromatographic peak called after Nt4CL5aPcSTS chromatographic peak (in Fig. 9 A).In above-mentioned WAT11/pESC-Nt4CL5aPcSTS sample solution, add above-mentioned trans-resveratrol standard substance obtain cochromatograph sample, the mass content of the above-mentioned trans-resveratrol standard substance in cochromatograph sample is one times of the Nt4CL5aPcSTS mass content in cochromatograph sample.In 10 minutes after the HPLC analyzing and testing completing WAT11/pESC-Nt4CL5aPcSTS sample solution, under identical chromatographic condition, HPLC analysis is carried out to cochromatograph sample.Cochromatograph sample is the chromatographic peak of 23.237 minutes containing retention time under this chromatographic condition, by this chromatographic peak called after cochromatograph peak (in Fig. 7 B), the peak height at this cochromatograph peak is the twice of Nt4CL5aPcSTS chromatographic peak, illustrates that Nt4CL5aPcSTS chromatographic peak is the chromatographic peak of trans-resveratrol.
2.2, recombination microzyme WAT11/pESC produces trans-resveratrol
Except the recombination microzyme WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with recombination microzyme WAT11/pESC, all the other are all identical.
2.3, recombination microzyme WAT11/pESC-Nt4CLPPPcSTS produces trans-resveratrol
Except the recombination microzyme WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with recombination microzyme WAT11/pESC-Nt4CLPPPcSTS, all the other are all identical.
2.4, recombination microzyme WAT11/pESC-Nt4CL produces trans-resveratrol
Except the recombination microzyme WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with recombination microzyme WAT11/pESC-Nt4CL, all the other are all identical.
2.5, recombination microzyme WAT11/pESC-PcSTS produces trans-resveratrol
Except the recombination microzyme WAT11/pESC-Nt4CL5aPcSTS in 2.1 is replaced with recombination microzyme WAT11/pESC-PcSTS, all the other are all identical.
The detected result of the HPLC-MS of the trans-resveratrol that recombination microzyme WAT11/pESC-Nt4CL5aPcSTS produces at the above-mentioned culture system containing 4-coumaric acid as shown in Figure 9, Figure 10.Result shows, the recombination microzyme WAT11/pESC-Nt4CL5aPcSTS containing fusion rotein Nt4CL5aPcSTS gene produces trans-resveratrol in containing the culture system of 4-coumaric acid, and its output is 10.2 μ g/mL fermented liquids; Recombination microzyme WAT11/pESC, WAT11/pESC-Nt4CL and WAT11/pESC-PcSTS do not produce trans-resveratrol in containing the culture system of 4-coumaric acid; Recombination microzyme WAT11/pESC-Nt4CLPPPcSTS is 1.3 μ g/mL fermented liquids in the output of the trans-resveratrol of the culture system containing 4-coumaric acid.Show in yeast expression system, utilize fusion rotein Nt4CL5aPcSTS can improve the output of trans-resveratrol.
Claims (8)
1. the relevant biological material of encoding fusion protein Nt4CL5aPcSTS is following B1) to B20) in any one:
B1) nucleic acid molecule of encoding fusion protein Nt4CL5aPcSTS;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) containing B1) the transgenic plant cells system of described nucleic acid molecule;
B10) containing B2) the transgenic plant cells system of described expression cassette;
B11) containing B3) the transgenic plant cells system of described recombinant vectors;
B12) containing B4) the transgenic plant cells system of described recombinant vectors;
B13) containing B1) Transgenic plant tissue of described nucleic acid molecule;
B14) containing B2) Transgenic plant tissue of described expression cassette;
B15) containing B3) Transgenic plant tissue of described recombinant vectors;
B16) containing B4) Transgenic plant tissue of described recombinant vectors;
B17) containing B1) the transgenic plant organ of described nucleic acid molecule;
B18) containing B2) the transgenic plant organ of described expression cassette;
B19) containing B3) the transgenic plant organ of described recombinant vectors;
B20) containing B4) the transgenic plant organ of described recombinant vectors;
Described fusion rotein Nt4CL5aPcSTS is following protein (1) or (2):
(1) the fusion rotein obtained by Nt4CL albumen and PcSTS protein fusion:
Described Nt4CL albumen be following a) or b):
A) aminoacid sequence is the protein of SEQ ID No.1 1-542 amino acids residue;
B) by a) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein;
Described PcSTS albumen is following A or B:
A, aminoacid sequence are the protein shown in SEQ ID No.1 548-935 amino acids residue;
B, by A through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by its derivative protein.
Described protein N t4CL is connected by five amino acid residue with protein PcSTS;
(2) N end (1) or C end are connected the tape label protein that label obtains.
2. relevant biological material as claimed in claim 1, is characterized in that: the sequence of described five amino acid residue is as shown in SEQ ID No.1 543-547 position, and the aminoacid sequence of described fusion rotein is SEQ ID No.1.
3. relevant biological material as claimed in claim 1 or 2, is characterized in that: B1) described nucleic acid molecule be following 1. or 2. or 3. or 4. described DNA molecular:
1. encoding sequence is DNA molecular or the cDNA molecule of the 1-2808 position Nucleotide of SEQ ID No.1;
2. nucleotide sequence is DNA molecular or the cDNA molecule of SEQ ID No.1;
3. there is the identity of more than 75% and the DNA molecular of code for said proteins or cDNA molecule with the DNA molecular 1. or 2. limited or cDNA molecule;
4. under strict conditions with the DNA molecular 1. or 2. limited or cDNA molecular hybridization and the DNA molecular of code for said proteins or cDNA molecule.
4. in claim 1-3, arbitrary described relevant biological material is producing the application in trans-resveratrol.
5. produce a method for trans-resveratrol, be included in the substratum containing 4-coumaric acid and cultivate arbitrary described relevant biological material in claim 1-3, obtain trans-resveratrol; Described fusion rotein relevant biological material is following D1)-D5) in any one:
D1) recombinant microorganism containing nucleic acid molecule; Fusion rotein described in described nucleic acid molecule encoding claim 1;
D2) recombinant microorganism containing expression cassette; Described expression cassette contains described nucleic acid molecule;
D3) recombinant microorganism containing recombinant vectors; Described recombinant vectors contains described nucleic acid molecule or described expression cassette;
D4) the transgenic plant cells system containing described recombinant vectors;
D5) the transgenic plant organ containing described recombinant vectors.
6. method according to claim 5, is characterized in that: described microorganism is eukaryotic microorganisms.
7. method according to claim 6, is characterized in that: described eukaryotic microorganisms is fungi.
8. the method according to claim 6 or 7, is characterized in that: described fungi is yeast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510061199.3A CN104611354B (en) | 2015-02-05 | 2015-02-05 | Fusion protein Nt4CL5aPcSTS relevant biological material and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510061199.3A CN104611354B (en) | 2015-02-05 | 2015-02-05 | Fusion protein Nt4CL5aPcSTS relevant biological material and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104611354A true CN104611354A (en) | 2015-05-13 |
| CN104611354B CN104611354B (en) | 2017-09-05 |
Family
ID=53146005
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510061199.3A Active CN104611354B (en) | 2015-02-05 | 2015-02-05 | Fusion protein Nt4CL5aPcSTS relevant biological material and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104611354B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101160393A (en) * | 2005-02-22 | 2008-04-09 | 弗卢克索姆科学公司 | Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof |
| WO2009124966A2 (en) * | 2008-04-11 | 2009-10-15 | Fluxome Sciences A/S | Conversion of stilbenoids |
| US20130130347A1 (en) * | 2011-10-06 | 2013-05-23 | Cornell University | Constructs and methods for the assembly of biological pathways |
| CN103540561A (en) * | 2013-10-22 | 2014-01-29 | 江南大学 | Genetically engineered bacterium for producing resveratrol and construction method thereof |
| CN103773704A (en) * | 2013-12-25 | 2014-05-07 | 无锡新和源发酵技术研究院有限公司 | Method for biologically synthesizing red sandalwood celery by utilizing O-transmethylase |
-
2015
- 2015-02-05 CN CN201510061199.3A patent/CN104611354B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101160393A (en) * | 2005-02-22 | 2008-04-09 | 弗卢克索姆科学公司 | Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof |
| WO2009124966A2 (en) * | 2008-04-11 | 2009-10-15 | Fluxome Sciences A/S | Conversion of stilbenoids |
| US20130130347A1 (en) * | 2011-10-06 | 2013-05-23 | Cornell University | Constructs and methods for the assembly of biological pathways |
| CN103540561A (en) * | 2013-10-22 | 2014-01-29 | 江南大学 | Genetically engineered bacterium for producing resveratrol and construction method thereof |
| CN103773704A (en) * | 2013-12-25 | 2014-05-07 | 无锡新和源发酵技术研究院有限公司 | Method for biologically synthesizing red sandalwood celery by utilizing O-transmethylase |
Non-Patent Citations (3)
| Title |
|---|
| YANSHENG ZHANG ET.AL.: "Using Unnatural Protein Fusions to Engineer Resveratrol Biosynthesis inYeast and Mammalian Cells", 《JACS》 * |
| 杨亚东等: "烟草4CL与虎杖STS融合基因的构建、表达及融合蛋白纯化", 《北京农业学院学报》 * |
| 王长松等: "微生物合成白藜芦醇的研究进展", 《微生物学通报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104611354B (en) | 2017-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Feduraev et al. | Phenylalanine and tyrosine as exogenous precursors of wheat (Triticum aestivum L.) secondary metabolism through PAL-associated pathways | |
| Miyamoto et al. | Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression | |
| Mei et al. | Biocatalysis and biotransformation of resveratrol in microorganisms | |
| Donnez et al. | Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms | |
| Zhu et al. | Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries | |
| Li et al. | De novo biosynthesis of p-coumaric acid in E. coli with a trans-cinnamic acid 4-hydroxylase from the Amaryllidaceae plant Lycoris aurea | |
| Huang et al. | AlgM4: A new salt-activated alginate lyase of the PL7 family with endolytic activity | |
| Sun et al. | Molecular Cloning and Characterization of a meta/para-O-Methyltransferase from Lycoris aurea | |
| CN104611355A (en) | Related biological materials of fusion protein Nt4CL3aPcSTS and application of related biological materials | |
| Zernova et al. | Regulation of plant immunity through modulation of phytoalexin synthesis | |
| Liu et al. | Characterization of a flavonoid 3’/5’/7-O-methyltransferase from Citrus reticulata and evaluation of the in vitro cytotoxicity of its methylated products | |
| Fiesel et al. | Molecular cloning and characterization of a xanthone prenyltransferase from Hypericum calycinum cell cultures | |
| CN108795960B (en) | Method for synthesizing various sesquiterpenes compounds by applying salvia miltiorrhiza SmTPS3 gene | |
| Liu et al. | The functional characterization of a site-specific apigenin 4′-O-methyltransferase synthesized by the liverwort species Plagiochasma appendiculatum | |
| CN104140959A (en) | Novel esterase as well as coding gene and application of esterase | |
| Park et al. | Identification and Biosynthesis of New Acyloins from the Thermophilic Bacterium Thermosporothrix hazakensis SK20‐1T | |
| Abbas et al. | Functional characterization of Hedychium coronarium J. Koenig MYB132 confers the potential role in floral aroma synthesis | |
| Park et al. | Biochemical characterization of a flavonoid O-methyltransferase from perilla leaves and its application in 7-methoxyflavonoid production | |
| Georgiev et al. | Recent progress in Amaryllidaceae biotechnology | |
| Wang et al. | Functional characteristics of caffeoyl shikimate esterase in Larix kaempferi and monolignol biosynthesis in gymnosperms | |
| Herrera et al. | Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene | |
| Zhu et al. | Characterization of Bioactivities and Biosynthesis of Angucycline/Angucyclinone Derivatives Derived from Gephyromycinifex aptenodytis gen. nov., sp. nov. | |
| Martín et al. | Comparative molecular mechanisms of biosynthesis of naringenin and related chalcones in actinobacteria and plants: Relevance for the obtention of potent bioactive metabolites | |
| Lackus et al. | The Sesquiterpene Synthase PtTPS5 Produces (1 S, 5 S, 7 R, 10 R)-Guaia-4 (15)-en-11-ol and (1 S, 7 R, 10 R)-Guaia-4-en-11-ol in Oomycete-Infected Poplar Roots | |
| Cheng et al. | Enzyme-catalyzed glycosylation of curcumin and its analogues by glycosyltransferases from Bacillus subtilis ATCC 6633 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |