CN106318920B - Flavones -6- hydroxylase and its application in scutellarin synthesis - Google Patents
Flavones -6- hydroxylase and its application in scutellarin synthesis Download PDFInfo
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- CN106318920B CN106318920B CN201610824470.9A CN201610824470A CN106318920B CN 106318920 B CN106318920 B CN 106318920B CN 201610824470 A CN201610824470 A CN 201610824470A CN 106318920 B CN106318920 B CN 106318920B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
Abstract
The present invention provides flavones -6- hydroxylase and its applications in scutellarin synthesis, and specifically, the present invention provides a kind of flavones -6- hydroxylases, it can be catalyzed apiolin 6 hydroxylatings, and can get scutellarin.In addition, utilizing glycosyltransferase and glycosyl donor, the method that scutellarin is made in scutellarin the present invention also provides a kind of.
Description
Technical field
The present invention relates to field of biotechnology, in particular it relates to flavones -6- hydroxylase and its be closed in scutellarin
Application in.
Background technique
Fleabane flower is the drying of compositae plant Erigeron breviscapus [Erigeron breviscapus (Vant.) Hand-Mazz]
Herb, cold in nature, slight bitter, Gan Wenxin have effects that slightly cold removing toxic substances, dispelling wind and eliminating dampness, activating microcirculation and removing stasis medicinal, clearing and activating the channels and collaterals, anti-inflammatory analgetic.
The application effect of fleabane flower similar drug clinically is significant, is listed in the herbal variety and the Chinese traditional treatment heart of state key development
The clinical indispensable first-aid medicine of cranial vascular disease.Fleabane injection clinically in addition to being mainly used for diseases of cardiovascular and cerebrovascular systems,
Diabetes, nephrosis, cervical vertigo, geriatric disease treatment on also have a better effect.The units such as Yunnan institute of materia medica
The research of chemistry and pharmacology is carried out to fleabane flower, and separation identifies a variety of chemical components, chief active from erigeron breviscapus
Ingredient includes scutellarin and a small amount of oil lamp A prime (wherein the content of scutellarin accounts for 90% or more).
The natural route of synthesis of scutellarin is still unintelligible at present, and 2013, Anna Berim and David R.Gang existed
Have been found that one kind can be catalyzed in sweet basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.)
7 methylation flavones carry out 6 hydroxylated P450 enzymes (CYP82D), but are not reported in scutellarin synthesis and have function
Energy.F6H at present in scutellarin synthesis process is unclear.
It there is no the relevant report of 6 hydroxylases of flavones in fleabane flower source at present, therefore there is an urgent need in the art to develop one
The method of the natural synthesis scutellarin and scutellarin of kind.
Summary of the invention
The purpose of the present invention is to provide the methods of a kind of natural synthesis scutellarin and scutellarin.
First aspect present invention provides a kind of for being catalyzed apiolin 6 hydroxylated flavones -6- hydroxylases, the Huang
Ketone -6- hydroxylase is selected from the group:
(a) albumen with amino acid sequence shown in SEQ ID NOs.:1；
(b) albumen of amino acid sequence shown in SEQ ID NOs.:1 is (preferable by one or more amino acid residues
Ground, 1-50, more preferably, 1-30, more preferably, 1-10, most preferably, 1-6) replace, miss or add and formed,
Or derived protein formed after addition signal peptide sequence and with catalysis 6 hydroxylation activities of apiolin；
(c) in sequence containing (a) or (b) described in protein sequence derived protein；
(d) amino acid sequence shown in amino acid sequence and SEQ ID NOs.:1 homology >=65% (preferably >=
80%, more preferably >=90%), and there is the derived protein of catalysis 6 hydroxylation activities of apiolin.
In another preferred example, the sequence (c) is served as reasons (a) or (b) is added to sequence label, signal sequence or secretion
Fusion protein is formed by after signal sequence.
In another preferred example, the flavones -6- hydroxylase comes from composite family, preferably, coming from fleabane flower.
In another preferred example, amino acid sequence shown in the SEQ ID NOs.:1 can be merged with compound, wherein described
Compound is the compound for extending the protein half-life.
In another preferred example, the compound includes polyethylene glycol.
In another preferred example, the albumen is the albumen of amino acid sequence shown in SEQ ID NOs.:1.
Second aspect of the present invention provides a kind of isolated nucleotide, and the nucleotide is selected from the group:
(a) nucleotide sequence of the albumen as shown in SEQ ID NOs.:1 is encoded；
(b) nucleotide sequence as shown in SEQ ID NOs.:2；
(c) with homology >=70% (preferably >=80%, more preferably >=90%) of sequence shown in SEQ ID NOs.:2
Nucleotide sequence；
(d) 5 ' ends and/or 3 ' ends of the nucleotide sequence shown in SEQ ID NOs.:2 truncate or addition 1-60 is a (preferably
Ground 1-30, more preferably 1-10) nucleotide is formed by nucleotide sequence；
(e) with the nucleotide sequence of any nucleotide sequence complementary (preferably complete complementary) of (a)-(d).
In another preferred example, the sequence of the nucleotide is as shown in SEQ ID NOs.:2.
In another preferred example, sequence polynucleotide encoding amino acid sequence such as SEQ as shown in SEQ ID NOs.:2
Polypeptide shown in ID NOs.:1.
Third aspect present invention provides a kind of carrier, and the carrier contains multicore glycosides described in second aspect of the present invention
Acid.
In another preferred example, the carrier is selected from the group: expression vector, shuttle vector, integration vector, or combinations thereof.
In another preferred example, the carrier is selected from the group: bacterial plasmid, bacteriophage, yeast plasmid, plant cell disease
Poison, zooblast virus, retrovirus, or combinations thereof.
In another preferred example, the carrier includes the carrier expressed in yeast, as YCp serial carrier, YEp series carry
Body, YIp serial carrier, pCS serial carrier, pRS serial carrier.
Fourth aspect present invention provides a kind of host cell, and the host cell contains described in third aspect present invention
Carrier or its genome in integrate polynucleotides described in second aspect of the present invention.
In another preferred example, the host cell is prokaryotic cell or eukaryocyte.
In another preferred example, the host cell is selected from the group: bacterium, yeast, higher plant, insect or mammal
Cell.
In another preferred example, the host cell is low eukaryocyte, such as yeast cells.
In another preferred example, the host cell is higher eucaryotic cells, such as mammalian cell.
In another preferred example, the host cell is prokaryotic cell, such as bacterial cell, preferably, Escherichia coli.
In another preferred example, the host cell is selected from the group: saccharomyces cerevisiae, Escherichia coli, or combinations thereof.
In another preferred example, the host cell is brewing yeast cell.
Fifth aspect present invention provides a kind of preparation method of flavones -6- hydroxylase, which comprises
(a) under conditions suitable for the expression, the host cell is cultivated；
(b) flavones -6- hydroxylase is isolated from culture.
Sixth aspect present invention provides described in a kind of flavones -6- hydroxylase or its derived protein or third aspect present invention
The purposes of host cell described in carrier or fourth aspect present invention, for being catalyzed 6 hydroxylatings of apiolin.
In another preferred example, the flavones -6- hydroxylase is selected from the group: any bar institute in SEQ ID NO.:1,3,4,5
Show the albumen of amino acid sequence.
In another preferred example, the source of the flavones -6- hydroxylase one or more plants selected from the group below: oil lamp
Flower, globe artichoke, sweet basil, peppermint.
Seventh aspect present invention provides a kind of 6 method for hydroxylation for being catalyzed apiolin, comprising steps of
In the presence of flavones -6- hydroxylase or its derived protein, apiolin 6 hydroxylated catalysis reactions are carried out.
In another preferred example, the flavones -6- hydroxylase is selected from the group: any bar institute in SEQ ID NO.:1,3,4,5
Show the albumen of amino acid sequence.
In another preferred example, the source of the flavones -6- hydroxylase one or more plants selected from the group below: oil lamp
Flower, globe artichoke, sweet basil, peppermint.
Eighth aspect present invention provides a kind of method for preparing scutellarin, comprising steps of
In the presence of cytochrome reductase (CPR), using flavones -6- hydroxylase catalysis apiolin, to obtain wild Huang
A kind of reed mentioned in ancient books element；
In another preferred example, the method also includes preparing flavones -6- hydroxylase.
In another preferred example, the preparation step of the flavones -6- hydroxylase is as described below:
(a) by SEQ ID NO.:2,6,7,8 any bar institute shown in nucleotide sequence or contain the nucleotide sequence
Recombinant expression carrier import host cell, cultivate the host cell；With
(b) flavones -6- hydroxylase is isolated from culture.
Ninth aspect present invention provides a kind of method for preparing scutellarin, comprising steps of
(i) in the presence of cytochrome reductase (CPR), using flavones -6- hydroxylase catalysis apiolin, to obtain open country
Baicalein；
With
(ii) glycosylation is carried out to the scutellarin that step (i) obtains, to obtain scutellarin；
In another preferred example, the step (ii) existing for the glycosyl transferase and glycosyl donor under the conditions of carry out.
In another preferred example, the glycosyl transferase is selected from the group: flavones -7- glycosyl transferase F7GAT, flavones -7-
Glycosyl transferase UGT73C6, flavones -7- glycosyl transferase UGT73B1, flavones -7- glycosyl transferase UBGAT, or combinations thereof.
In another preferred example, the source of the glycosyl transferase one or more plants selected from the group below: fleabane flower is intended
Southern mustard, radix scutellariae, or combinations thereof.
In another preferred example, the glycosyl donor is selected from the group: UDP-glucose aldehydic acid, UDP-glucose or its
Combination.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is the evolutionary analysis figure of EbF6H, CcsF6H, ObF6H and MpF6H.
Fig. 2 is EbF6H, CcsF6H, ObF6H and MpF6H encoding gene PCR electrophoretogram.
Fig. 3 is recombinant plasmid Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-
The restriction enzyme digestion and electrophoresis figure of MpF6H SpeI.
Fig. 4 is the plasmid map schematic diagram of recombinant plasmid Y22-ATR2-EbF6H.
Fig. 5 is the plasmid map schematic diagram of recombinant plasmid Y22-ATR2-CcsF6H.
Fig. 6 is the plasmid map schematic diagram of recombinant plasmid Y22-ATR2-ObF6H.
Fig. 7 is the plasmid map schematic diagram of recombinant plasmid Y22-ATR2-MpF6H.
Fig. 8 is the HPLC-MS analysis chart that EbF6H is catalyzed that 6 hydroxylatings of apiolin synthesize scutellarin.
Fig. 9 is that EbF6H is catalyzed 6 hydroxylatings synthesis scutellarins of apiolin, and scutellarin is by glycosylation later
HPLC-MS analysis chart of the modification plus UDP-glucose aldehydic acid production scutellarin.
Figure 10 is the HPLC-MS analysis chart that CcsF6H is catalyzed that 6 hydroxylatings of apiolin synthesize scutellarin.
Figure 11 is that CcsF6H is catalyzed 6 hydroxylatings synthesis scutellarins of apiolin, and scutellarin passes through glycosyl later
Change modification plus the HPLC-MS analysis chart of UDP-glucose aldehydic acid production scutellarin.
Figure 12 is the HPLC-MS analysis chart that ObF6H is catalyzed that 6 hydroxylatings of apiolin synthesize scutellarin.
Figure 13 is that ObF6H is catalyzed 6 hydroxylatings synthesis scutellarins of apiolin, and scutellarin is by glycosylation later
HPLC-MS analysis chart of the modification plus UDP-glucose aldehydic acid production scutellarin.
Figure 14 is the HPLC-MS analysis chart that MpF6H is catalyzed that 6 hydroxylatings of apiolin synthesize scutellarin.
Figure 15 is that MpF6H is catalyzed 6 hydroxylatings synthesis scutellarins of apiolin, and scutellarin is by glycosylation later
HPLC-MS analysis chart of the modification plus UDP-glucose aldehydic acid production scutellarin.
Specific embodiment
The present inventor after extensive and in-depth study, is found surprisingly that for the first time, from fleabane flower, globe artichoke, pale reddish brown
Sweet basil or the flavones -6- hydroxylase of peppermint can be catalyzed apiolin 6 hydroxylatings, to obtain scutellarin.This
Outside, the present inventors have additionally discovered that, in glycosyl transferase (such as UDP-glucose aldehydic acid synzyme (UDPGDH)) and glycosyl donor is (such as
UDP-glucose aldehydic acid) in the presence of, glycosylation is carried out to the position C7 of scutellarin, can get scutellarin.It is basic herein
On, the present inventor completes the present invention.
Flavones -6- hydroxylase
As used herein, term " albumen of the invention ", " polypeptide of the invention ", " flavones -6- hydroxylase ", " present invention
Enzyme " be used interchangeably, refer both to catalysis apiolin C6 hydroxylase.In the present invention, the flavones -6- hydroxylase is SEQ
Albumen shown in ID NO.:1,3,4,5 or its derived protein, the flavones -6- hydroxylase are respectively derived from fleabane flower, Korea
Ji, sweet basil, peppermint Four Plants, are respectively designated as EbF6H, CcsF6H, ObF6H, MpF6H, and pass through homologous sequence
It compares, it is found that the similarity of albumen shown in protein sequence shown in SEQ ID NO:1 and SEQ ID NO:3 is 69%.Oil lamp
The sequence analysis the result is shown in Figure 1 of the flavones -6- hydroxylase of the flavones -6- hydroxylase and other species in flower source.
The term as used herein " separation " refers to that substance is separated from its primal environment (if it is natural object
Matter, primal environment are natural surroundings).As under the native state in active somatic cell polynucleotide and polypeptide be not separate
Purifying, but same polynucleotide or polypeptide in native state in other substances with existing for such as from separating, then is separation
Purifying.Therefore, the term as used herein " isolated flavones -6- hydroxylase " refer to the albumen substantially free of naturally and its
Relevant other albumen, lipid, carbohydrate or other materials.Those skilled in the art can be pure with the purified technology of protein of standard
Change flavones -6- hydroxylase of the invention.Substantially pure albumen can generate single item in non-reducing polyacrylamide gel
Band.However, those skilled in the art are also to be understood that " flavones -6- hydroxylase " also in view of the teachings of the present invention and the prior art
It should include the variant form of the albumen, the variant form has same or similar with " flavones -6- hydroxylase of the invention "
Function, but amino acid sequence shown in its amino acid sequence and SEQ ID NO:1,3,4 or 5 has a small amount of difference.These variation shapes
Formula includes but is not limited to: one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10
A, also more preferably such as 1-8,1-6) missing, insertion and/or the substitution of amino acid, and in C-terminal and/or N-terminal addition one
A or multiple (usually 20 within, be more preferably within 6 within preferably 10) amino acid.For example, this field skill
Art personnel are known, when being substituted with similar nature or similar amino acid, do not usually change the function of protein.Compare again
Such as, the function of protein will not be changed by adding one or several amino acid generally also in C-terminal and/or N-terminal.The term also wraps
Include the active fragment and reactive derivative of flavones -6- hydroxylase protein.
The variant form of polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induces and dash forward
Variant, the DNA that can hybridize with the coding DNA of " flavones -6- hydroxylase of the invention " under high or low stringency are compiled
The albumen of code.The invention also includes other polypeptides, such as the fusion egg comprising " flavones -6- hydroxylase of the invention " or its segment
It is white.Other than the almost polypeptide of overall length, the present invention should also include the active fragment of " flavones -6- hydroxylase of the invention ".In general,
The segment has at least about 10 continuous amino acids of the amino acid sequence of " flavones -6- hydroxylase of the invention ", typically at least
About 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably
At least about 100 continuous amino acids.
The present invention also provides the analogs of " flavones -6- hydroxylase ".These analogs and natural " flavones -6- of the invention
The difference of hydroxylase " can be the difference on amino acid sequence, be also possible to not influence the difference on the modified forms of sequence, or
Person haves both at the same time.These polypeptides include natural or induction genetic variant.Induction variant can be obtained by various technologies,
Random mutagenesis such as is generated by radiating or being exposed to mutagens, can also pass through site-directed mutagenesis or other known molecular biology
Technology.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and is had
The analog of non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid).It should be understood that albumen of the invention is not limited to
Enumerated representativeness albumen.
Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external polypeptide
Change or carboxylated.Modification further includes glycosylation.Modified forms further include with phosphorylated amino acid residue (such as phosphotyrosine,
Phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimize molten
Solve the albumen of performance.
In the present invention, the conservative variation's polypeptides of " flavones -6- hydroxylase " refer to shown in SEQ ID NO:1,3,4 or 5
Amino acid sequence is compared, and has at most 20, and preferably at most 10, more preferably at most 5, most preferably at most 3 amino acid quilts
Amino acid with similar or analogous properties is replaced and forms polypeptide, but the conservative variation's polypeptides still have and amino acid sequence
Arrange the same or similar activity of the albumen as shown in SEQ ID NO:1,3,4 or 5, that is, catalysis 6 hydroxylated activity of apiolin.
Therefore, in view of the teachings of the present invention and the prior art, those skilled in the art can basis, such as carried out shown in following table
Amino acid substitution and the mutant for generating conservative variation.
Therefore, " containing " used herein, " having " or " comprising " include "comprising", " mainly by ... constitute ", " base
On this by ... constitute " and " by ... constitute "；" mainly by ... constitute ", " substantially by ... constitute " and " by ... structure
At " belong to the subordinate concept of " containing ", " having " or " comprising ".
Albumen of the invention can be recombinant protein, native protein, synthetic proteins, preferably recombinant protein.Egg of the invention
It is white to can be native purified product or chemically synthesized product, or use recombinant technique from protokaryon or eucaryon host (example
Such as, bacterium, yeast, higher plant, insect and mammalian cell) in generate.According to host used in recombinant production scheme, originally
The albumen of invention can be glycosylated, or can be nonglycosylated.Albumen of the invention may also include or not include starting
Methionine residues.
It will be understood by those skilled in the art that " flavones -6- hydroxylase " of the invention further includes the piece of " flavones -6- hydroxylase "
Section, derivative and analogue.As used herein, term " segment ", " derivative " and " analog ", which refers to, is kept substantially this hair
Bright " flavones -6- hydroxylase " identical biological function or active polypeptide.Polypeptide fragment of the invention, derivative or similar
Object, which can be (i), has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid) substituted
Polypeptide, and such substituted amino acid residue can be and may not be by genetic code encoding, or (ii) at one or
With the polypeptide of substituent group in more amino acid, or (iii) mature polypeptide and another compound (for example extend polypeptide
The compound of half-life period, such as polyethylene glycol) fusion is formed by polypeptide, or to be fused to this more for (iv) additional amino acid sequence
Peptide sequence and formed polypeptide (such as leader sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or
Fusion protein).Belong to model well known to those skilled in the art according to the definition of this paper these segments, derivative and analogue
It encloses.
In view of state of the art and the teachings of the present invention, those skilled in the art are not difficult to obtain flavones -6- of the present invention
The active fragment of hydroxylase.Therefore, the bioactive fragment of any " flavones -6- hydroxylase " can be applied to this hair
It is bright.Herein, the bioactive fragment of " flavones -6- hydroxylase " refers to the segment of " flavones -6- hydroxylase ", but it still can
Keep all or part of function of overall length " flavones -6- hydroxylase ".Under normal conditions, the bioactive fragment is at least kept
50% activity of overall length " flavones -6- hydroxylase ".Under still more preferential conditions, it is " yellow to be able to maintain overall length for the active fragment
60%, 70%, 80%, 90%, 95%, 99% or 100% activity of ketone -6- hydroxylase ".
Based on the teachings of the present invention and the prior art, those skilled in the art are to be further understood that can be by Huang of the invention
The other utilizations form such as immobilised enzymes is made in ketone -6- hydroxylase.
The present invention also provides the polynucleotides sequences for encoding " flavones -6- hydroxylase " of the invention or its conservative variation's polypeptides
Column.
Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people
The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide
Coding region sequence can variant identical as coding region sequence shown in SEQ ID NO.:2,6,7 or 8 or degeneracy.
As used herein, " variant of degeneracy " refers to that coding has albumen shown in SEQ ID NO.:1,3,4 or 5 in the present invention
Matter, but with the differentiated nucleic acid sequence of coding region sequence shown in SEQ ID NO.:2,6,7 or 8.
The polynucleotides for encoding mature polypeptide shown in SEQ ID NO.:1,3,4 or 5 include: an encoding mature polypeptide
Coded sequence；The coded sequence of mature polypeptide and various additional coding sequences；The coded sequence of mature polypeptide is (and optional additional
Coded sequence) and non-coding sequence.
Term " polynucleotides of coding polypeptide " can be the polynucleotides including coding said polypeptide, be also possible to also wrap
Include the polynucleotides of additional code and/or non-coding sequence.
The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention
The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or
The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this
Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution,
Missing or insertion, but not from substantially change its encode polypeptide function.
The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least
70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more
The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature
Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization
Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with
On, more preferably 95% or more when, just hybridizes.Also, the polypeptide of interfertile polynucleotide encoding and SEQ ID NO:1,3,
Mature polypeptide shown in 4 or 5 has identical biological function and activity.
The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence.As used herein, the length of " nucleic acid fragment " is extremely
Contain 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably at least 100 nucleosides less
It is more than acid.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid to determine and/or separate coding " flavones -6- hydroxylase "
Polynucleotide.
" flavones -6- hydroxylase " nucleotide full length sequence of the invention or its segment can usually use PCR amplification method, recombination
Method or artificial synthesized method obtain.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, especially
It is that open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses conventional method institute well known by persons skilled in the art
The library cDNA of preparation expands as template and obtains related sequence.
Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, by first synthesizing
Then multiple small fragments are attached the very long segment of available sequence again.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely
Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and
In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
In a preferred embodiment, described " flavones -6- hydroxylase " is: (a) having the institute of SEQ ID NO.:1,3,4 or 5
Show the albumen of amino acid sequence；Or (b) amino acid sequence as shown in SEQ ID NO.:1,3,4 or 5 passes through one or several amino
Sour replacing, missing or adding for residue and formed and with " flavones -6- hydroxylase " function the albumen as derived from (a)；Or
(c) amino acid sequence as shown in SEQ ID NO.:1,3,4 or 5 is by one or several, and preferably 1-50, more preferable 1-30,
More typically 1-10, missing or the addition of most preferably 1-6 amino acid residue and formed and with (a) described protein function
Derived protein；Or (d) C-terminal of the amino acid sequence shown in SEQ ID NO.:1,3,4 or 5 and/or N-terminal addition or scarce
Lose one or several, preferably 1-50, more preferable 1-30, more typically 1-10, most preferably 1-6 amino acid residue and shape
At and with (a) described protein function derived protein.
Correspondingly, the encoding gene of described " flavones -6- hydroxylase " is:
(a) coding nucleotide sequence of amino acid sequence albumen as shown in SEQ ID NO:1,3,4 or 5；Or
(b) amino acid sequence as shown in SEQ ID NO.:1,3,4 or 5 by one or several amino acid residues substitution,
Derived protein lacking or add and formed and with amino acid sequence protein function as shown in SEQ ID NO.:1,3,4 or 5
Coding nucleotide sequence；Or
(c) amino acid sequence as shown in SEQ ID NO.:1,3,4 or 5 is preferably 1-50, more excellent by one or several
Select 1-30, more typically 1-10, missing or the addition of most preferably 1-6 amino acid residue and formed and with (a) institute
State the coded sequence of the derived protein of protein function；Or
(d) C-terminal of the amino acid sequence shown in SEQ ID NO:1,3,4 or 5 and/or N-terminal addition or missing one
Or several, preferably 1-50, more preferable 1-30, more typically 1-10, most preferably 1-6 amino acid residue and formed and
The coded sequence of derived protein with protein function described in (a).
In further preferred embodiment, the encoding gene of " flavones -6- hydroxylase " is: (i) has SEQ
The polynucleotides of sequence shown in ID NO.:2,6,7,8；Or (ii) have it is complementary with sequence shown in SEQ ID NO.:2,6,7,8
Polynucleotides.
Cytochrome reductase (CPR)
Cytochrome reductase (CPR) is the essential electron donor that P450 enzyme plays a role.In the present invention, it is catalyzed celery
Plain 6 hydroxylated enzymes (that is, flavones -6- hydroxylase) are a kind of P450 enzymes, can with CPR collective effect, to be catalyzed apiolin,
Obtain scutellarin.
Expression vector
The present invention also relates to the expression vectors comprising coded sequence of the present invention, and with expression vector of the invention or " yellow
The genetically engineered host cell of ketone -6- hydroxylase " coded sequence, and polypeptide of the present invention is generated through recombinant technique
Method.
By the recombinant dna technology of routine (Science, 1984；224:1431), using polynucleotide of the invention
Sequence come express or produce recombination " flavones -6- hydroxylase ".In general there are following steps:
1. with the polynucleotides (or its variant) of coding " flavones -6- hydroxylase " of the invention, or with containing the multicore
The recombinant expression carrier of thuja acid converts or suitable host cell of transduceing；
2. the host cell cultivated in suitable culture medium；
3. being separated from culture medium or cell, protein purification.
In the present invention, the encoding polynucleotide sequence of " flavones -6- hydroxylase " can be inserted into recombinant expression carrier or genome.
Term " recombinant expression carrier " refers to that bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, lactation are dynamic
Object cell virus or other carriers.As long as any plasmid and carrier can be used in short, can replicate and stablize in host.
One important feature of expression vector is to usually contain replication orgin, promoter, marker gene and translation control element.
Those skilled in the art can be used well known method and can be used to construct containing " flavones -6- hydroxylase " coding DNA sequence
The expression vector of column and suitable transcription/translation control signal, including recombinant DNA technology in vi, DNA synthetic technology, in vivo weight
Group technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Table
It further include the ribosome bind site and transcription terminator of translation initiation up to carrier.
In addition, expression vector preferably comprises one or more selected markers, to provide the place for selecting conversion
The phenotypic character of chief cell, such as the dihyrofolate reductase, neomycin resistance and green fluorescent protein of eukaryotic culture
(GFP), or for Escherichia coli kanamycins or amicillin resistance.
Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable
When host cell, allow it to expression protein.
Host cell as described herein includes comprising incorporating the present invention " flavones -6- hydroxylation on expression vector or genome
The host cell of enzyme " coded sequence.Host cell or bacterial strain of the invention can high efficient expression have apiolin C6 hydroxylating
Flavones -6- the hydroxylase of energy, host cell of the invention can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte,
Such as yeast cells.In a particular embodiment, the bacterial strain includes but is not limited to: saccharomyces cerevisiae or Escherichia coli.Preferred
Embodiment in, the bacterial strain be saccharomyces cerevisiae.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is using MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..When host is saccharomyces cerevisiae, method that lithium acetate transformation can be selected
The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used
Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
In view of the teachings of the present invention and the prior art, it will be appreciated by those skilled in the art that flavones -6- hydroxyl of the invention
Change enzyme and its coded sequence, expression vector, host cell can be used for being catalyzed the C6 hydroxylating of apiolin.
The preparation method of scutellarin
The present invention also provides a kind of preparation methods of scutellarin, comprising steps of
(i) in the presence of cytochrome reductase (CPR), using flavones -6- hydroxylase catalysis apiolin, to obtain open country
Baicalein；
With
(ii) glycosylation is carried out to the scutellarin that step (i) obtains, to obtain scutellarin；
Main advantages of the present invention include:
(1) present invention firstly discovers that flavones -6- hydroxylase can carry out hydroxylated catalysis reaction to the C6 of apiolin, thus
Obtain scutellarin.
(2) present invention provides the flavones -6- hydroxylase in fleabane flower source for the first time, and finds the flavones-in fleabane flower source
The C6 that 6- hydroxylase can be catalyzed apiolin carries out hydroxylated catalysis reaction, so that scutellarin is obtained, in addition, the present invention is also
For the first time disclose globe artichoke, sweet basil, peppermint Four Plants source flavones -6- hydroxylase also have catalysis apiolin C6 into
The function of the hydroxylated catalysis reaction of row.
(3) present invention provides a kind of new way for preparing scutellarin for the first time, by flavones -6- hydroxylase by apiolin
Catalysis is that the scutellarin of acquisition is further converted to by scutellarin later in the presence of glycosyl transferase and glycosyl donor
Scutellarin.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
If not otherwise specified, material used in embodiment is commercial product.
Gene order SEQ ID NO:6 used in the present invention is synthesized by Suzhou Hong Xun Biotechnology Co., Ltd.Remaining
Gene order synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd, primer is closed by Tianjin Institute of Industrial Biotechnology
At.
In the present invention, fleabane flower, globe artichoke, sweet basil, peppermint Four Plants source have chromocor compound 6
The polypeptide of hydroxylase function is respectively designated as EbF6H, CcsF6H, ObF6H, MpF6H, respectively SEQ ID NO:1, SEQ respectively
ID NO:3, SEQ ID NO:4, the composition of amino acid sequence shown in SEQ ID NO:5 polypeptide.
The acquisition of 1 EbF6H coding nucleotide of embodiment
The nucleotide sequence information (SEQ ID NO.:2) provided according to the present invention, passes through the RNA reverse transcription to fleabane flower
Group amplification or isolated.Specifically, conventional method can be used or tried using Easy spin plus plant RNA rapidly extracting
RNA is extracted from plants in agent box (being purchased from Beijing Ai Delai Biotechnology Co., Ltd), then using the RNA of acquisition as template, benefit
It is obtained with First Strand cDNA synthetic agent box (Thermo, USA) according to the operating procedure reverse transcription on kit
CDNA utilizes routine using primer EbF6H-F1 (SEQ ID NO:9) and EbF6H-R1 (SEQ ID NO:10) using it as template
PCR method, the coding nucleotide of specific amplification EbF6H.Pcr amplified fragment carries out agarose gel electrophoresis, as a result such as Fig. 2 institute
Show.
The acquisition of the coding nucleotide of 2 CcsF6H of embodiment
Using sequence alignment is carried out in the amino acid sequence and other Also Asteraceae species of fleabane flower EbF6H, discovery is current
In another Also Asteraceae species globe artichoke of gene order-checking there are one with EbF6H similarity about 69% amino acid sequence,
Its Genbank number is KVH90146.1.Identified, inferring the gene also has the function of 6 hydroxylatings of similar flavonoids, and
It is named as CcsF6H.
According to amino acid sequence SEQ ID NO:3 for target host cell (saccharomyces cerevisiae) to nucleotide sequence codon
It carries out artificial synthesized obtaining sequence shown in SEQ ID NO:6 after optimization.With CcsF6H-F1 (SEQ ID NO:11) and
CcsF6H-R1 (SEQ ID NO:12) is primer, utilizes conventional PCR method, the coding nucleotide of specific amplification CcsF6H.
Pcr amplified fragment carries out agarose gel electrophoresis, as a result as shown in Figure 2.
The acquisition of the coding nucleotide of embodiment 3 ObF6H and MpF6H
According to the information of sequence SEQ ID NO:7 and SEQ ID NO:8, to gene ObF6H, (Genbank is numbered
AGF30364.1) and the coding nucleotide progress of MpF6H (Genbank number is AGF30366.1) is artificial synthesized, can also root
It is close to nucleotide sequence for target host cell (saccharomyces cerevisiae) according to amino acid sequence SEQ ID NO:4 or SEQ ID NO:5
It carries out artificial synthesized obtaining sequence shown in SEQ ID NO.:7 and 8 after numeral optimization.Respectively with ObF6H-F1 (SEQ ID NO:
13) and ObF6H-R1 (SEQ ID NO:14), MpF6H-F1 (SEQ ID NO:15) and MpF6H-R1 (SEQ ID NO:16) are
Primer utilizes conventional PCR method, the coding nucleotide of specific amplification ObF6H and MpF6H.Pcr amplified fragment carries out agarose
Gel electrophoresis, as a result as shown in Figure 2.The F6H evolutionary relationship in Four Plants source is as shown in Figure 1.
Embodiment 4 constructs EbF6H, CcsF6H, ObF6H, MpF6H table using Golden Gate clone's construction method respectively
Up to carrier
For the difference of expression EbF6H, CcsF6H, ObF6H, MpF6H host cell, chooses shuttle vector and carry out
YCPlacz-22 clone's building.
Amplification, the purifying of 4.1 genetic fragments
With EbF6H-F1 (SEQ ID NO:12) and EbF6H-R1 (SEQ ID NO:13) for primer, to be compiled comprising EbF6H
The cDNA of code nucleotide sequence is that template carries out Standard PCR, obtains the coding nucleotide of EbF6H, and by resulting amplified fragments
It carries out cutting glue purification；
Respectively with CcsF6H-F1 (SEQ ID NO:14) and CcsF6H-R1 (SEQ ID NO:15), ObF6H-F1 (SEQ
ID NO:16) and ObF6H-R1 (SEQ ID NO:17), MpF6H-F1 (SEQ ID NO:18) and MpF6H-R1 (SEQ ID NO:
It 19) is primer, using the coding nucleotide sequence of CcsF6H, ObF6H, MpF6H after artificial synthesized codon optimization as template,
Standard PCR is carried out, the coding nucleotide sequence segment (Fig. 2) of CcsF6H, ObF6H, MpF6H for cloning building are obtained, and
Resulting amplified fragments are carried out to cut glue purification；
According to p450 reductase (ATR2) gene order in the source arabidopsis (Arabidopsi s thaliana)
(No. Genbank: 145361355,2139bp, SEQ ID NO:9) design primer ATR2-F (SEQ ID NO:20) and ATR2-R
(SEQ ID NO:21) carries out Standard PCR as template using arabidopsis cDNA and obtains the coding nucleotide of ATR2, and by resulting expansion
Increase segment to carry out cutting glue purification；
According to the nucleotide of the artificial synthesized bidirectional promoter PGK1+TDH3 of information of sequence SEQ ID NO:10.As
Template utilizes Standard PCR side using primer PGK1+TDH-F (SEQ ID NO:22) and PGK1+TDH-R (SEQ ID NO:23)
Method, the nucleotide of specific amplification bidirectional promoter PGK1+TDH3, and resulting amplified fragments are carried out to cut glue purification.
According to the artificial synthesized empty carrier YCplac22 of information (No. GenBank: X75455.1) of sequence SEQ ID NO:11
Nucleotide.As template, primer Y22-F (SEQ ID NO:24) and Y22-R (SEQ ID NO:25), carrier are used
The nucleotide of YCplac22, and resulting amplified fragments are carried out to cut glue purification.
4.2 using Golden gate clone constructing technologies building respectively containing gene EbF6H, CcsF6H, ObF6H,
Recombinant vector Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2- of MpF6H and ATR2
MpF6H, Golden gate linked system and reaction condition are as follows:
Reaction system is as follows:
Carrier framework (terminating subcomponent) (200ng)
Promoter element and the equimolar amount of carrier framework
Gene 1 and the equimolar amount of carrier framework
Gene 2 and the equimolar amount of carrier framework
10XNEB T4buffer(NEB)1.5ul
100XBSA*(NEB)0.15ul
BsaI(NEB)1ul
NEB T4Ligase(NEB)1ul
DdH2O supplies 15ul
Total 15ul
Reaction condition is as follows:
Obtained product directly converts DH5 α Escherichia coli (purchased from Beijing CoWin Bioscience Co., Ltd.), culture
12-16h, picking single colonie digestion verification find out correct clone (Fig. 2) by agarose gel electrophoresis analysis.Gained is correct
Recombinant vector is respectively designated as Y22-ATR2-EbF6H, Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-
MpF6H, digestion result is as shown in figure 3, its physical map is distinguished shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7.
Embodiment 5 constructs recombinant vector Y33-UDPGDH-F7GAT using Golden Gate clone's construction method
Constructing technology is cloned using Golden gate described in embodiment 4 4.2, the glycosyl in fleabane flower source is shifted
The UDP-glucose aldehydic acid synthase gene UDPGDH in enzyme gene F7GAT and streptococcus pyogenes source and artificial synthesized two-way
Promoter ADH1+TDH3 is building up on artificial synthesized carrier YCplac33 (artificial synthesized No. Genbank: X75456.1), is obtained
To recombinant vector Y33-UDPGDH-F7GAT.
The building of the production apiolin Saccharomyces cerevisiae host bacterium of embodiment 6
By apiolin synthesis related gene PAL (phenylalanine deaminase gene), C4H (the cinnamic acid hydroxyl in fleabane flower source
Change enzyme gene), 4CL (coumaric acid CoA synthetase gene), CHS (chalcone synthase genes), CHI (enzyme, namely chalcone isomerase base
Cause), FSII (flavones synthetase II gene) by common molecular clone construction method be integrated into conventional saccharomyces cerevisiae wild mushroom
W303(Thomas,B.J.and Rothstein R(1989)Elevated recombination rates in
Transcriptionally active DNA.Cell, 56 (4): 619-663.) genome on, obtain histidine nutrition lack
The production apiolin Saccharomyces cerevisiae host bacterium SC1 of swaged.
Embodiment 7 produces scutellarin S. cervisiae and produces the building of scutellarin S. cervisiae
7.1 produce the building of scutellarin S. cervisiae
Using conventional lithium acetate transformation method respectively by the embodiment 4 4.2 recombinant vector Y22-ATR2-EbF6H,
Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H, which are transformed into, produces apiolin Saccharomyces cerevisiae host bacterium SC1
In, picking can histidine, tryptophan defect CM culture medium on the clone that grows, number be respectively SC223, SC251,
SC114,SC137；
7.2 produce the building of scutellarin S. cervisiae
Using conventional lithium acetate transformation method respectively by embodiment 4 4.2 recombinant vector Y22-ATR2-EbF6H,
Y22-ATR2-CcsF6H, Y22-ATR2-ObF6H, Y22-ATR2-MpF6H and Y33-UDPGDH-F7GAT corotation dissolve into embodiment
In 6 produce apiolin Saccharomyces cerevisiae host bacterium SC1 in, picking can histidine, tryptophan, uracil-deficient CM culture medium
The clone grown on upper culture medium, number are respectively SC225, SC254, SC125, SC145.
8 fermenting and producing scutellarin of embodiment, scutellarin
8.1 saccharomyces cerevisiae engineered yeast strain seed liquor cultures
Select CM culture medium (being purchased from Suo Laibao Biotechnology Co., Ltd) (formula: YNB w/o AA (0.67%), grape
Sugared (2g/L), Dropout powder (0.083%) wherein contain (mg/L) in Dropout powder: threonine 150, junket
Propylhomoserin 30, valine 150, lysine 30, glutamic acid 100, serine 150, aspartic acid 100, methionine 20, phenylalanine
50, isoleucine 30, arginase 12 0；Other nutritional ingredients (mg/L): adenine 50, uracil 50, histidine 100, leucine
100, tryptophan 100 (amino acid)), Liquid Culture keynote pH5.6, solid medium adds 1.5% agar powder (purchased from Suo Laibaosheng
Object Science and Technology Ltd.), adjust pH6.5.Respectively on plate picking monoclonal to the test tube containing 4mL sterilized culture medium
In, under the conditions of 30 DEG C, 200rpm to saccharomyces cerevisiae engineered yeast strain SC223, SC251, SC114, SC137, SC225, SC254,
SC125, SC145 are incubated overnight.
8.2. fermenting and producing
Seed liquor is inoculated into the conical flask containing 50mL sterilized culture medium with the inoculative proportion of 1:50,30 DEG C,
Fermented and cultured 5 days under the conditions of 200rpm.
The LC-MS of 9 reaction product of embodiment is identified
9.1 after fermentation, takes 900uL sample, and isometric anhydrous methanol is added, and carries out ultrasound with ultrasonic cleaning instrument
30min.12000rpm is centrifuged 10min, and supernatant is cooked high-efficient liquid phase analysis.
9.2 HPLC, LC-MS testing conditions
HPLC analysis:
Instrument: Shimadzu high performance liquid chromatograph 1200
Chromatographic column: Kinetex H15-168747 (4.6 × 250mm), UV detector, Detection wavelength 335nm.
Mobile phase: A phase is 0.1% formic acid；B phase is acetonitrile；C phase is methanol
Initial concentration A:75%B:22%C:5%
Flow velocity: 1mL/min
Column temperature: 30 DEG C
Detector: PDA detector
Gradient elution program: (concentration is B phase percentage)
MS analysis
Mass spectrograph: Bruker-micrOTOF-II:
ESI ion source, positive ion mode
Nucleocytoplasmic ratio (m/z): 50-1000
Nitrogen flow rate: 6.0 liters/min
Temperature: 180 DEG C
Nebulizer pressure: 1bar
Probe voltage: 14.5KV.
9.3 EbF6H activity identifications
Bacterial strain SC223 tunning, shows by HPLC testing result, and EbF6H can be catalyzed apiolin and generate one newly
Product, appearance time are 8.5min (Fig. 8 A) consistent with scutellarin mark condition, and mass spectral results show (Fig. 8 B) new product point
Son amount is (m/z, 287 [M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is scutellarin, shaking flask
Fermentation yield is 16.4mg/L.
In addition, the new product generates a new product with UDP-glucose aldehydic acid by glycosyl transferase catalysis, when appearance
Between be 4.5min (Fig. 9 A) consistent with scutellarin mark condition, mass spectral results show the molecular weight of (Fig. 9 B) the new product for (m/
z,463[M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is scutellarin, shake flask fermentation yield is
22.7mg/L。
9.4 CcsF6H activity identifications
Bacterial strain SC251 tunning, shows by HPLC testing result, and CcsF6H can be catalyzed apiolin and generate one newly
Product, appearance time are 8.5min (Figure 10 A) consistent with scutellarin mark condition, and mass spectral results show (attached drawing 10B) the new production
Object molecular weight is (m/z, 287 [M+H]+), this is consistent with the molecular weight of scutellarin mark product, determine that the substance is scutellarin,
Shake flask fermentation yield is 15.2mg/L.
In addition, the new product generates a new product with UDP-glucose aldehydic acid by glycosyl transferase catalysis, when appearance
Between be 4.5min (Figure 11 A) consistent with scutellarin mark condition, mass spectral results show that the molecular weight of (Figure 11 B) the new product is
(m/z,463[M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is scutellarin, shake flask fermentation produces
Amount is 22mg/L.
9.5 ObF6H activity identifications
Bacterial strain SC114 tunning, shows by HPLC testing result, and ObF6H can be catalyzed micro apiolin and generate
One new product, appearance time are 8.5min (Figure 12 A) consistent with scutellarin mark condition, and mass spectral results show that (Figure 12 B) should
New molecular weight of product is (m/z, 287 [M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is wild radix scutellariae
Element, shake flask fermentation yield are 0.5mg/L.
In addition, the new product generates a new product with UDP-glucose aldehydic acid by glycosyl transferase catalysis, when appearance
Between be 4.5min (Figure 13 A) consistent with scutellarin mark condition, mass spectral results show that the molecular weight of (Figure 13 B) the new product is
(m/z,463[M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is scutellarin, shake flask fermentation produces
Amount is 3.5mg/L.
9.6 MpF6H activity identifications
Bacterial strain SC137 tunning, shows by HPLC testing result, and MpF6H can be catalyzed micro apiolin and generate
One new product, appearance time are 8.5min (Figure 14 A) consistent with scutellarin mark condition, and mass spectral results show that (Figure 14 B) should
New molecular weight of product is (m/z, 287 [M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is wild radix scutellariae
Element, shake flask fermentation yield are 0.5mg/L.
In addition, the new product generates a new product with UDP-glucose aldehydic acid by glycosyl transferase catalysis, when appearance
Between be 4.5min (Figure 15 A) consistent with scutellarin mark condition, mass spectral results show that the molecular weight of (Figure 15 B) the new product is
(m/z,463[M+H]+), this is consistent with the molecular weight of scutellarin mark product, determines that the substance is scutellarin, shake flask fermentation produces
Amount is 3.5mg/L.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1. one kind is for being catalyzed apiolin 6 hydroxylated flavones -6- hydroxylases, which is characterized in that the flavones -6- hydroxylase
For
The albumen of the amino acid sequence as shown in SEQ ID NOs.:1.
2. a kind of isolated nucleotide, which is characterized in that the nucleotide is
(a) nucleotide sequence of the albumen as shown in SEQ ID NOs.:1 is encoded；
(b) nucleotide sequence as shown in SEQ ID NOs.:2；
(e) nucleotide sequence complementary with any nucleotide sequence of (a)-(b).
3. a kind of carrier, which is characterized in that the carrier contains polynucleotides as claimed in claim 2.
4. a kind of host cell, which is characterized in that the host cell contains carrier as claimed in claim 3 or its gene
Polynucleotides as claimed in claim 2 are integrated in group.
5. a kind of preparation method of flavones -6- hydroxylase described in claim 1, which is characterized in that the described method includes:
(a) under conditions suitable for the expression, the host cell is cultivated；
(b) flavones -6- hydroxylase is isolated from culture.
6. host cell described in carrier described in a kind of flavones -6- hydroxylase or its derived protein or claim 3 or claim 4
Purposes, which is characterized in that for being catalyzed 6 hydroxylatings of apiolin, the flavones -6- hydroxylase is selected from the group: SEQ ID
The albumen of amino acid sequence shown in NO.:1.
7. a kind of 6 method for hydroxylation for being catalyzed apiolin, which is characterized in that comprising steps of
In the presence of flavones -6- hydroxylase or its derived protein, apiolin 6 hydroxylated catalysis reactions, the flavones-are carried out
6- hydroxylase is selected from the group: the albumen of amino acid sequence shown in SEQ ID NO.:1.
8. a kind of method for preparing scutellarin, which is characterized in that comprising steps of
In the presence of cytochrome reductase (CPR), using flavones -6- hydroxylase catalysis apiolin, to obtain scutellarin；
Wherein, the flavones -6- hydroxylase is selected from the group: the albumen of amino acid sequence shown in SEQ ID NO.:1.
9. a kind of method for preparing scutellarin, which is characterized in that comprising steps of
(i) in the presence of cytochrome reductase (CPR), using flavones -6- hydroxylase catalysis apiolin, to obtain wild radix scutellariae
Element；
With
(ii) glycosylation is carried out to the scutellarin that step (i) obtains, to obtain scutellarin；
Wherein, the flavones -6- hydroxylase is selected from the group: the albumen of amino acid sequence shown in SEQ ID NO.:1.
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