CN107058253A - A kind of albumen of AePGT 6 for participating in alkannin biosynthesis and its encoding gene and application - Google Patents
A kind of albumen of AePGT 6 for participating in alkannin biosynthesis and its encoding gene and application Download PDFInfo
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- CN107058253A CN107058253A CN201710023493.4A CN201710023493A CN107058253A CN 107058253 A CN107058253 A CN 107058253A CN 201710023493 A CN201710023493 A CN 201710023493A CN 107058253 A CN107058253 A CN 107058253A
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Abstract
The invention discloses a kind of albumen of AePGT 6 for participating in alkannin biosynthesis and its encoding gene and application.The invention provides protein, obtained from lithospermum euchromum Royle, be named as the albumen of AePGT 6, be following (a1) or (a2)：(a1) protein being made up of the amino acid sequence shown in sequence in sequence table 1；(a2) substitution by the amino acid sequence of sequence 1 by one or several amino acid residues and/or missing and/or addition and the protein as derived from sequence 1 with identical function.The albumen of AePGT 6 that the present invention is provided and the metabolism of alkannin class compound synthesis are closely related, can be catalyzed and combine to form GBA to PHBA and GPP.The content that the present invention improves naphthoquinones active component alkannin class in lithospermum euchromum Royle for regulation production plant naphthoquinone compound and by biotechnology has important theoretical and practical significance.
Description
Technical field
The present invention relates to a kind of AePGT-6 albumen for participating in alkannin biosynthesis and its encoding gene and application.
Background technology
Medicinal plant active component is typically growth and development of plants and adapts to the class that secondary metabolism is produced in environmental process
Small molecular organic compounds.The parsing of active ingredients Biogenic route of synthesis is in the core of medicinal plant secondary metabolite research
Hold.As the extensive of plant functional genomics research is with deeply, showing unique characteristics and having the medicinal plant of broad prospect of application secondary
The research of metabolism synthesis correlation function gene is increasingly becoming the focus of research, and the clone of these genes will have to annotate medicinal plant
The biosynthesis pathway and its regulatory mechanism of composition are imitated, is that the formation of medical material quanlity is provided fundamental basis, while to utilize biology
Technology improves target component content or directly production active ingredient or intermediate bring wide application space.
Lithospermum euchromum Royle Arnebia euchroma (Royle) Johns is traditional Chinese medicine Asian puccoon Arnebiae Radix
Important sources, have been listed in the class of natural crude drugs animal and plant resource three protection kind.The active chemical of lithospermum euchromum Royle mainly divides
For two major classes, a class is fat-soluble naphthoquinone compound, Asian puccoon chlorins compound；Another kind of is water soluble ingredient, mainly
Polysaccharide and phenolic acid class.Modern pharmacology research find Asian puccoon chlorins compound have antitumor, HIV-resistant activity, regulation it is immune,
The various clinical effects such as antifertility, antithrombotic, calmness, promotion wound healing, are widely used in pharmaceuticals industry.In addition, alkannin
Even more important natural dye is referred to as " king of natural red colouring matter " in the world, with very high economic value.Alkannin is naphthalene
Quinones, passes through phenylalanine (Phenylalanine pathway, PP)-mevalonic acid in plant
(Mevalonate pathway, MVA)/deoxidation xylulose phosphate (2-C-methyl-D-erythritol 4-phosphate,
MEP) approach composite pathway is synthesized, by a molecule p-hydroxy Benzoic Acid (4-Hydroxybenzoic acid, PHBA) and one point
Sub- geranyl pyrophosphate (Geranylpyrophosphate, GPP) combines, and forms basic framework --- the spiceleaf alkenyl of alkannin
P-hydroxybenzoic acid (3-Geranyl-4-hydroxybenzoic acid, GBA), GBA is to pass through a series of decarboxylation again, add
Oxygen, dehydrogenation generation alkannin.P-hydroxy Benzoic Acid myrcene based transferase (4-hydroxybenzoate
Geranyltransferase, PGT) two approach are connected, the peculiar enzymatic for belonging to alkannin biosynthetic process is anti-
Should, GPP and PHBA can be catalyzed and combine generation GBA.
P-hydroxybenzoic acid myrcene based transferase (PGT) is one of alkannin derivant biosynthesis in paniculatum cell
Crucial and important metabolic enzyme, the GPP that it forms mevalonic acid metabolic pathway myrcene group-transfer is to coming from Phenylpropanoid Glycosides class
Spiceleaf alkenyl P-hydroxybenzoic acid (GBA), end reaction generation alkannin and its derivative are formed on the PHBA of metabolic pathway.
PGT is located on the endoplasmic reticulum of paniculatum cell, and the activity for increasing or decreasing it directly influences the generation of alkannin, therefore in Asian puccoon
Element plays an important role during being formed.In Asian puccoon culture cell, the expression pattern of PGT genes and its Enzyme activities
With uniformity of the formation with height of alkannin and its derivative, it is seen that the expression of PGT genes to regulation and control alkannin and
The importance of its derivative formation.
The content of the invention
It is an object of the invention to provide a kind of AePGT-6 albumen and its encoding gene for participating in alkannin biosynthesis with answering
With.
The invention provides a kind of protein, obtained from lithospermum euchromum Royle, be named as AePGT-6 albumen, be following (a1) or
(a2)：
(a1) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(a2) by the amino acid sequence of sequence 1 is by the substitution of one or several amino acid residues and/or missing and/or adds
Plus and the protein as derived from sequence 1 with identical function.
In order that the protein in (a1) is easy to purify and detected, amino acid sequence that can be in as sequence table shown in sequence 1
Arrange the amino terminal or the upper label as shown in table 1 of carboxyl terminal connection of the protein of composition.
The sequence of the label of table 1
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (a2) can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtains.
The gene (AePGT-6 genes) of the present invention also protection coding AePGT-6 albumen.
The AePGT-6 genes are any described DNA molecular in following (b1)-(b3)：
(b1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(b2) protein DNA described in the DNA sequence dna hybridization limited under strict conditions with (b1) and coding claim 1
Molecule；
(b3) DNA sequence dna limited with (b1) or (b2) has egg described in more than 90% homology and coding claim 1
The DNA molecular of white matter.
Above-mentioned stringent condition can be that 0.1%SDS solution is miscellaneous in DNA or RNA with 0.1 × SSPE (or 0.1 × SSC)
Hand over and hybridize in experiment at 65 DEG C and wash film.
The present invention also protects the application of AePGT-6 albumen, is following (c1) and/or (c2)：
(c1) as P-hydroxybenzoic acid myrcene based transferase；
(c2) catalysis P-hydroxybenzoic acid and geranyl pyrophosphate reaction produce spiceleaf alkenyl-P-hydroxybenzoic acid.
The present invention also protects a kind of recombinant bacterium (restructuring fungus beetle), will be obtained in AePGT-6 channel genes Host Strains；Institute
Geranyl pyrophosphate can not be produced by stating Host Strains.
The present invention also protects a kind of recombinant bacterium (recombinant bacterium second), will be obtained in AePGT-6 channel genes Host Strains；Institute
Geranyl pyrophosphate can be produced by stating Host Strains.
The Host Strains that geranyl pyrophosphate can be produced concretely saccharomyces cerevisiae K197G.
The AePGT-6 genes can import Host Strains by the recombinant expression carrier containing AePGT-6 genes and be recombinated
Bacterium.
The recombinant expression carrier of AePGT-6 genes can be contained with existing expression vector establishment.Use AePGT-6 gene structures
When building recombinant expression carrier, can before its transcription initiation nucleotides plus any enhanced, composing type, organizing specific type or
Inducible promoter；In addition, during recombinant expression carrier gene constructed using AePGT-6, enhancer is it is also possible to use, including translation increases
Hadron or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must
Need to be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.The translation control signal and initiation codon
The source of son is extensive, can be natural or synthesis.Translation initiation region can come from transcription initiation region
Or structural gene.
The recombinant expression carrier is concretely by the small pieces between pESC-His plasmid BamH I and Apa I restriction enzyme sites
Section is substituted by the recombinant expression carrier that the DNA molecular shown in the sequence 2 from 5 ' the 1-924 nucleotides in end of sequence table is obtained.
The present invention also protects a kind of preparation method of P-hydroxybenzoic acid myrcene based transferase, comprises the following steps：Training
Recombinant bacterium described in supporting any of the above, obtains P-hydroxybenzoic acid myrcene based transferase from recombinant bacterium.
Recombinant bacterium described in the culture any of the above comprises the following steps：
(a1) recombinant bacterium is seeded in SD-His-Ura culture mediums and cultivated, obtain bacterium solution；
(a2) precipitation is collected by centrifugation in the bacterium solution after step (a1) is cultivated；
(a3) bacterium that step (a2) is obtained is resuspended using the SD-His-Ura culture mediums containing 2% (mass percent) galactolipin
Body, continues to cultivate.
It is described to cultivate concretely 30 DEG C, 200r in step (a1).min-1Culture.
In step (a1), the bacterium solution OD600nm=0.6.
In step (a2), concretely 3000g is centrifuged for the centrifugation.
It is described to cultivate concretely 30 DEG C, 200r in step (a3).min-1Culture.
In step (a3), the incubation time concretely 48h.
Recombinant bacterium described in the culture any of the above also includes completing after step (a3), collects cultivating system, is collected by centrifugation
Bacterial sediment.
Concretely 3000g is centrifuged for the centrifugation.
The method that P-hydroxybenzoic acid myrcene based transferase is obtained from recombinant bacterium is concretely from the thalline
Yeast microsomes albumen is extracted in precipitation, P-hydroxybenzoic acid myrcene based transferase is obtained.
The extraction yeast microsomes albumen can specifically use glass bead method.
The present invention also protects recombinant bacterium described in any of the above in P-hydroxybenzoic acid myrcene based transferase product is prepared
Application.
The present invention also protects the preparation method of spiceleaf alkenyl P-hydroxybenzoic acid, is method first or method second.
Methods described first comprises the following steps：The restructuring fungus beetle is cultivated, while adding P-hydroxybenzoic acid and pyrophosphoric acid
Geraniol ester, the isolated spiceleaf alkenyl P-hydroxybenzoic acid from cultured products.
Methods described second comprises the following steps：Recombinant bacterium second described in any of the above is cultivated, while P-hydroxybenzoic acid is added,
Isolated spiceleaf alkenyl-P-hydroxybenzoic acid from cultured products.
In method first or method second, the recombinant bacterium that can produce geranyl pyrophosphate is concretely seeded to by the culture
Cultivated in SD-His-Ura culture mediums containing 2% (mass percent) galactolipin.
The cultivating system initial OD of the culture600nm=0.5.
It is described to cultivate concretely 30 DEG C, 200r.min-1Culture.
The incubation time can be 36h-72h.
The incubation time concretely 36h.
The incubation time concretely 72h.
The incubation time concretely 48h.
In method first or method second, the concentration of P-hydroxybenzoic acid is 0.05mM-1mM.
The concentration of P-hydroxybenzoic acid concretely 0.05mM.
The concentration of P-hydroxybenzoic acid concretely 0.1mM.
The concentration of P-hydroxybenzoic acid concretely 0.5mM.
The concentration of P-hydroxybenzoic acid concretely 1mM.
In method first or method second, the spiceleaf alkenyl-P-hydroxybenzoic acid isolated from cultured products specifically may be used
Realization is isolated and purified by HPLC.
The spiceleaf alkenyl P-hydroxybenzoic acid isolated from cultured products specifically includes following steps：
(b1) cultured products are used into ethyl acetate ultrasonic extraction, collects supernatant (organic phase)；
(b2) supernatant (organic phase) for extracting step (b1) is dissolved after drying up ethyl acetate with nitrogen with methanol, will be molten
Isolated and purified after the filtering of liquid product by HPLC.
In step (b1), the volume proportion of the cultured products and ethyl acetate is 1: 1.
It is described to be filtered into 0.22 μM of filtering with microporous membrane in step (b2).
The parameter that the HPLC is isolated and purified is as follows：Chromatographic column is 1.8 μm of 2.1 × 100mm T3HHS of Waters C18；
Mobile phase is made up of solution A and B；Solution A is acetonitrile；Solution B is 0.1% (volume fraction) aqueous formic acid；Flow velocity：0.5mL/
min；Elution program is：0min：10% solution A；4min：35% solution A；4.3min：60% solution A；8min；72% solution A：
8.5min, 98% solution A；10.5min：98% solution A；11min：10% solution A；13min：10% solution A；When collecting reservation
Between for 5.79min occur the corresponding eluent of chromatographic peak；40 DEG C of column temperature；PDA detector full wavelength scanners.
The present invention also protects kit first, and the kit first includes the restructuring fungus beetle, P-hydroxybenzoic acid and burnt phosphorus
Sour geraniol ester.
The present invention also protects kit second, and the kit second includes recombinant bacterium second described in any of the above and para hydroxybenzene first
Acid.
The composition first or composition second also include SD-His-Ura culture mediums.
The purposes of the composition first or composition second is production myrcene base-P-hydroxybenzoic acid.
SD-His-Ura culture mediums are made up of solute and solvent described in any of the above；The solute and its in SD-His-Ura
Concentration in culture medium is：Without amino yeast nitrogen 0.67g/100ml, glucose 2g/100ml, iron ammonium sulfate 0.4g/
100ml, agar 2g/100ml, adenine 20mg/100ml, Valine 150mg/100ml, ILE 30mg/100ml,
L-arginine 20mg/100ml, L-Leu 100mg/100ml, 1B 30mg/100ml, METHIONINE 20mg/
100ml, L-phenylalanine 50mg/100ml, L-threonine 200mg/100ml, L-Trp 20mg/100ml, TYR
30mg/100ml, L-Aspartic acid 10mg/100ml, Serine 40mg/100ml, Pidolidone 10mg/100ml.
The AePGT-6 albumen that the present invention is provided and the metabolism of alkannin class compound synthesis are closely related, can be catalyzed to hydroxyl
Benzoic acid (PHBA) and geranyl pyrophosphate (GPP) combine to form GBA.The present invention is for regulation production plant naphthoquinone compound
Have with the content that naphthoquinones active component alkannin class in lithospermum euchromum Royle is improved by biotechnology important theoretical and actual
Meaning.
Brief description of the drawings
Fig. 1 is liquid chromatographic detection result in embodiment 2.
Fig. 2 is proton nmr spectra (HNMR) figure in embodiment 2.
Fig. 3 is carbon-13 nmr spectra (CNMR) figure in embodiment 2.
Fig. 4 is HMBC figures in embodiment 2.
Fig. 5 is high resolution mass spectrum figure in embodiment 2.
Fig. 6 is new product structure formula in embodiment 2.
Fig. 7 is PHBA and GPP reaction generations GBA course of reaction.
Fig. 8 is GBA output statistics result figures in embodiment 3.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
PESC-His plasmids：Agilent Technologies (China) Co., Ltd, article No.：217451.
Saccharomyces cerevisiae K197G：Bibliography：Fischer M J, Meyer S, Claudel P, et al.Metabolic
engineering of monoterpene synthesis in yeast.[J].Biotechnology and
Bioengineering, 2011,108 (8)：1883-92.；Entitled " K197G " of saccharomyces cerevisiae K197G in the literature, the public
It can be obtained from Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences.
SD-His-Ura culture mediums：The general Jino Science and Technology Ltd. in Beijing, article No.：YGM003A-13.
SD-His-Ura culture mediums are made up of solute and solvent；The solute and its dense in SD-His-Ura culture mediums
Spend and be：Without amino yeast nitrogen 0.67g/100ml, glucose 2g/100ml, iron ammonium sulfate 0.4g/100ml, agar 2g/
100ml, adenine 20mg/100ml, Valine 150mg/100ml, ILE 30mg/100ml, L-arginine 20mg/
100ml, L-Leu 100mg/100ml, 1B 30mg/100ml, METHIONINE 20mg/100ml, L-phenylalanine
50mg/100ml, L-threonine 200mg/100ml, L-Trp 20mg/100ml, TYR 30mg/100ml, L- asparagus fern ammonia
Sour 10mg/100ml, Serine 40mg/100ml, Pidolidone 10mg/100ml.
Without amino yeast nitrogen：Sigma companies, article No.：Y0626.
PHBA：Upper Hiroad standing grain medical sci-tech Development Co., Ltd, article No.：131109.PHBA molecular formula is C7H6O3,
No. CAS is：99-96-7, structural formula is as follows：
GPP：Sigma-Aldrich, article No.：76532-10MG.GPP molecular formula is C10H20O7P2, No. CAS
For：21141-43-5, structural formula is as follows：
The acquisition of embodiment 1, AePGT-6 albumen and its encoding gene
By carrying out a large amount of sequence analyses, expression analysis and functional verification to lithospermum euchromum Royle, it was found that a DNA encoding
Sequence, as shown in the sequence 2 of sequence table, the protein that it is encoded is as shown in the sequence 1 of sequence table.
Protein shown in the sequence 1 of sequence table is named as AePGT-6 albumen, is made up of 307 amino acid residues.Will
The unnamed gene for encoding AePGT-6 albumen is AePGT-6 genes, and the sequence 2 of its ORFs such as sequence table is from 5 ' ends the
Shown in 1-924 nucleotides.
Embodiment 2, AePGT-6 protein-function assays
First, the structure of recombinant bacterial strain
1st, construction recombination plasmid pESC-AePGT-6.According to sequencing result, recombinant plasmid pESC-AePGT-6 is tied
Structure is described as follows：Using pESC-His plasmids as the carrier that sets out, by between pESC-His plasmid BamH I and Apa I restriction enzyme sites
Small fragment is substituted by the DNA molecular shown in the sequence 2 from 5 ' the 1-924 nucleotides in end of sequence table.
2nd, the recombinant plasmid pESC-AePGT-6 transformed saccharomyces cerevisiae K197G for preparing step 1, obtain recombinant bacterial strain
K197G-AePGT-6；By pESC-His plasmid transformed saccharomyces cerevisiae engineered strain K197G, recombinant bacterial strain K197G-pESC- is obtained
His (turns empty carrier bacterial strain).
2nd, external enzymatic experiment
Strain to be tested is：Recombinant bacterial strain K197G-AePGT-6 or recombinant bacterial strain K197G-pESC-His that step one is obtained
(turning empty carrier bacterial strain).
1st, the single bacterium colony of strain to be tested is inoculated in 5mLSD-His-Ura culture mediums, 30 DEG C, 200r.min-1Culture
24h；Then 1mL bacterium solutions are taken to be seeded in 50mL SD-His-Ura culture mediums, 30 DEG C, 200r.min-1Cultivate to bacterium solution OD600nm
=0.6,3000g centrifuge cultivating system, abandon supernatant, are washed with deionized after bacterial sediment, contain 2% (quality hundred using 50mL
Point ratio) galactolipin SD-His-Ura culture mediums be resuspended thalline, 30 DEG C, 200r.min-1Cultivate 48h, 3000g centrifugation culture bodies
System, abandons supernatant, collects thalline.
2nd, the thalline that step 1 is obtained is taken, yeast microsomes albumen is extracted using glass bead method, grasped in accordance with the following steps successively
Obtain yeast microsomes protein solution：
(1) thalline that step 1 is collected is resuspended using 5mL TEK buffer solutions (0.1M KCl+TE buffer solutions), room temperature is placed
5min, is collected by centrifugation thalline.
(2) thalline is resuspended with the TESB buffer solutions (0.6M sorbierite+TE buffer solutions) of 500 μ L ice baths, obtains bacteria suspension.
(3) bead is carefully added into bacteria suspension, it is just contacted bacteria suspension surface, 4 DEG C of concussion 30min that are vortexed are broken
Broken thalline, adds 1mL TESB buffer solutions, reclaims supernatant.
(4) TESB repeated in (3) is cleaned 3 times, merges supernatant.By 4 DEG C of the supernatant of recovery, 12000rpm centrifuges 15min,
Collect supernatant.
(5) the precipitation buffering liquid (TESB buffer solution+0.225M NaCl+0.15g/ml of 2 times of volumes are added into supernatant
PEG4000), ice bath 15min, 4 DEG C, 12000rpm centrifugation 15min abandon supernatant and collect precipitation.
(6) precipitation is dissolved using 400 μ L TEG buffer solutions (glycerine of TE buffer solutions+20%).
3rd, the yeast microsomes protein solution for obtaining step 2 carries out protein quantification, and protein concentration is 20mg/mL.
4th, the yeast microsomes protein solution after taking step 3 quantitative, carries out external enzymatic reaction, obtains reaction product.
Reaction system is：Microsomal protein solution (protein content is 500 μ g), Tris-HCl (pH 7.8) 50mM, PHBA
200nmol, GPP100nmol, MgCl210 μm of ol, supplement distilled water to 250 μ L.
Reaction condition is：30 DEG C, 150rpm reactions 1h.Reaction adds 1 μ L formic acid terminating reactions after terminating.
5th, the reaction product obtained using high performance liquid chromatography detection step 4,250 μ L acetic acid second are added into reaction product
Ester, vortex 1min, at room temperature 10000g centrifuge 2min, careful absorption supernatant (organic phase), N2Drying, adds 250 μ L methanol
Dissolving, 0.22 filtering with microporous membrane, for detecting；
HPLC detections use Waters ACQUITY-UPLC-PDA systems.Detect that parameter is as follows：
Chromatographic column：Waters C18 1.8μm 2.1×100mm T3HHS；
Eluent：Eluent is made up of A liquid (50%) and B liquid (50%), and A liquid is acetonitrile, and B liquid is 0.1% (volume integral
Number) aqueous formic acid.
Flow velocity：0.5mL/min；
40 DEG C of column temperature；
PDA detector full wavelength scanners.
As a result it is as shown in Figure 1.In Fig. 1, abscissa is the time (min), and ordinate is ultraviolet absorptivity.Fig. 1 results show,
Compared with turning empty carrier bacterial strain, new product is occurred in that in recombinant bacterial strain K197G-AePGT-6 external enzymatic reaction product.
The corresponding eluting peak of new product is collected, Characterization of The Products is further verified by nuclear-magnetism and mass spectrum.
Proton nmr spectra (HNMR) figure is shown in Fig. 2.Carbon-13 nmr spectra (CNMR) figure is shown in Fig. 3.HMBC figures are shown in Fig. 4, high score
Distinguish that mass spectrogram is shown in Fig. 5.
Nuclear-magnetism 1H-NMR, 13C-NMR characterization result analysis in table 2.
Table 2 nuclear-magnetism 1H-NMR, 13C-NMR characterization result is analyzed
Analysis result shows：PHBA and GPP is under AePGT-6 catalysis, and it is the new of 274.16 (m/z) to generate molecular weight
Product, it is believed that the product (structural formula is shown in Fig. 6) is that GBA, PHBA and GPP reaction generation GBA course of reaction are shown in Fig. 7.
GBA products are subjected to purity detecting through efficient liquid phase, purity is more than 98%.
Embodiment 3, recombinant bacterium K197G-AePGT-6 fermenting and producings GBA and its volume analysis
1st, by recombinant bacterial strain K197G-AePGT-6 prepared by embodiment 2 be inoculated in 10mL contain various concentrations PHBA (0mM,
0.05mM, 0.1mM, 0.5mM, 1mM) the SD-His-Ura culture mediums containing 2% (mass percent) galactolipin in (initial bacterium solution
OD600nm=0.5), 30 DEG C, 200r.min-1Different time (36h, 72h) is cultivated, bacterium solution is obtained.
2nd, the bacterium solution for taking 5mL steps 1 to obtain, adds 5mL ethyl acetate, and supernatant organic phase is collected by centrifugation in ultrasonic extraction 2h,
Retain lower floor's bacterium solution.
3rd, isometric ethyl acetate is added in the lower floor's bacterium solution for taking step 2 again, ultrasonic extraction 2h, supernatant, which is collected by centrifugation, to be had
Machine phase.
4th, the supernatant organic phase that combining step 2 and step 3 are obtained, dries up ethyl acetate with nitrogen, obtains mixture.
5th, the mixture obtained with 1.5mL methanol dissolving step 4,0.22 μM of filtering with microporous membrane, for the inspection of UPLC contents
Survey.
HPLC detections use Waters ACQUITY-UPLC-PDA systems.Detect that parameter is as follows：
Chromatographic column：Waters C18 1.8μm 2.1×100mm T3HHS；
Eluent is made up of solution A and solution B：Solution A is acetonitrile, and solution B is that 0.1% (volume fraction) formic acid is water-soluble
Liquid.
Flow velocity：0.5mL/min；
By the way of gradient elution：0min：10% solution A；4min：35% solution A；4.3min：60% solution A；
8min；72% solution A：8.5min, 98% solution A；10.5min：98% solution A；11min：10% solution A；13min：10%
Solution A.
40 DEG C of column temperature；
PDA detector full wavelength scanners.
Standard items are used as using the GBA products of embodiment 2 after purification (purity is more than 98%).
The appearance time of standard items is 5.79min, collects the GBA products in same time appearance.
GBA yield is calculated according to testing result.
As a result it is as shown in Figure 8.In Fig. 8,1-5 is culture 36h tunning testing result, the fermentation production that 6-10 is 72h
Analyte detection result.The PHBA concentration that 1-5 is followed successively by culture medium is 0mM, 0.05mM, 0.1mM, 0.5mM, 1mM tunning
Testing result, 6-10 is followed successively by the PHBA concentration in culture medium and examined for 0mM, 0.05mM, 0.1mM, 0.5mM, 1mM tunning
Survey result.
As a result show, GBA yield increases and increased with PHBA concentration, in the culture medium containing 1mM PHBA, lures
72h is led, GBA yield can reach 71.69mg/L cultivating systems.
Embodiment 4, the experiment of homologous recombination strain control
1st, construction recombination plasmid pESC-AePGT.According to sequencing result, structure is carried out to recombinant plasmid pESC-AePGT and retouched
State as follows：Using pESC-His plasmids as the carrier that sets out, by the small pieces between pESC-His plasmid BamH I and Apa I restriction enzyme sites
Section substitution is for the DNA molecular shown in the sequence 4 from 5 ' the 1-921 nucleotides in end of sequence table.The coded sequence 3 of sequence 4
Shown protein (protein shown in sequence 3 is the homologous protein of AePGT-6 albumen).
2nd, the recombinant plasmid pESC-AePGT transformed saccharomyces cerevisiae K197G for preparing step 1, obtain recombinant bacterial strain K197G-
AePGT。
Recombinant bacterial strain K197G- prepared by the recombinant bacterial strain K197G-AePGT-6 and step 2 that the 3, Example 2 is built
AePGT, is proceeded as follows：
(1) recombinant bacterial strain is inoculated in the SD-His- containing 2% (mass percent) galactolipin that 10mL contains 1mM PHBA
(initial bacterium solution OD in Ura culture mediums600nm=0.5), 30 DEG C, 200r.min-148h is cultivated, bacterium solution is obtained.
(2) bacterium solution for taking 5mL steps (1) to obtain, adds 5mL ethyl acetate, and it is organic that supernatant is collected by centrifugation in ultrasonic extraction 2h
Phase.
(3) the supernatant organic phase for taking step (2) to obtain, adds isometric ethyl acetate, and ultrasonic extraction 2h is collected by centrifugation
Clear organic phase.
(4) the supernatant organic phase for taking step (3) to obtain, dries up ethyl acetate with nitrogen, obtains mixture.
(5) mixture obtained with 1.5mL methanol dissolving step 4,0.22 μM of filtering with microporous membrane, for the inspection of UPLC contents
Survey.HPLC detects that parameter and method are identical with the step 5 of embodiment 3.
Standard items are used as using the GBA products of embodiment 2 after purification (purity is more than 98%).Calculated according to testing result
GBA yield.
By calculating, recombinant bacterial strain K197G-AePGT-6 tunning GBA yield is 70.96mg/L, recombinant bacterial strain
K197G-AePGT tunning GBA yield is 20.86mg/L.
<110>Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences
<120>A kind of AePGT-6 albumen for participating in Asian puccoon biosynthesis and its encoding gene and application
<160> 4
<210> 1
<211> 307
<212> PRT
<213>Asian puccoon（Arnebia euchroma）
<400> 1
Met Thr Tyr Lys Gln Ser Leu Lys Lys Gln Asn Lys Arg Pro Ser Trp
1 5 10 15
Ile Asp Thr Asn Leu Pro Ile Thr Phe Gln Pro Tyr Ala His Leu Ala
20 25 30
Arg Leu Asp Lys Pro Ile Gly Ser Trp Leu Leu Ala Trp Pro Ala Phe
35 40 45
Trp Ser Val Ala Leu Ala Ala Asp Asp Leu Gly Ser Leu Pro Lys Met
50 55 60
Leu Ala Ile Phe Gly Trp Trp Ala Ile Trp Ile Arg Gly Ala Gly Cys
65 70 75 80
Thr Ile Asn Asp Tyr Phe Asp Arg Asp Phe Asp Lys Lys Val Glu Arg
85 90 95
Thr Lys Ser Arg Pro Leu Ala Ser Gly Ala Leu Ser Pro Ala Gln Gly
100 105 110
Leu Trp Trp Leu Ala Ile Gln Leu Phe Met Gly Leu Gly Val Leu Tyr
115 120 125
Gln Leu Asn Met Leu Thr Leu Val Leu Ala Ile Leu His Val Pro Leu
130 135 140
Val Phe Ala Tyr Pro Leu Met Lys Arg Ile Thr Tyr Trp Pro Gln Ala
145 150 155 160
Phe Leu Gly Val Met Ile Ser Trp Gly Ala Leu Leu Gly Pro Ala Ala
165 170 175
Leu Glu Gly Thr Ile Asp Pro Lys Ile Ala Cys Pro Leu Tyr Val Ser
180 185 190
Ser Phe Phe Trp Thr Leu Val Tyr Asp Thr Ile Tyr Ala His Gln Asp
195 200 205
Lys Glu Asp Asp Ala Lys Ala Gly Ile Lys Ser Thr Thr Leu Arg Phe
210 215 220
Gly Asp Leu Thr Lys Val Trp Val Gly Gly Phe Gly Val Ala Cys Thr
225 230 235 240
Leu Ala Leu Leu Leu Gly Gly Phe Val Ile Asn Ile Gly Leu Pro Tyr
245 250 255
Tyr Val Val Leu Thr Val Ala Thr Cys Gln Leu Ala Trp Gln Ile Val
260 265 270
Thr Val Asp Leu Ser Ser Pro Met Asp Cys Gly Arg Lys Phe Val Ser
275 280 285
Asn Gln Trp Tyr Gly Ala Ile Ile Phe Ser Ala Ile Leu Val Gly Lys
290 295 300
Leu Ile Ser
305
<210> 2
<211> 924
<212> DNA
<213>Asian puccoon（Arnebia euchroma）
<400> 2
atgacttaca aacaatcact aaagaaacag aataagagac catcttggat agacacaaat 60
ctgccaataa catttcagcc ttatgcacat cttgcaaggt tagataagcc tataggtagt 120
tggttgctag cttggccagc tttctggtcc gtcgcattag cagcggacga tcttggaagc 180
ctgcctaaaa tgttggcaat attcggatgg tgggcaattt ggattcgtgg tgcaggatgt 240
actatcaatg attattttga tagagatttt gataagaagg ttgaacgtac aaagtctagg 300
ccacttgcta gtggcgcttt gtccccggct caaggtcttt ggtggcttgc cattcaattg 360
ttcatgggtc taggtgttct ttaccaattg aatatgttga ctcttgtatt agctattctt 420
catgtacctt tggtgtttgc ttatcccctc atgaaaagga tcacatattg gcctcaagct 480
tttctcggag tgatgattag ttggggagct ttattgggac cggccgctct tgaaggaacc 540
attgatccca aaattgcttg tccactttat gtttctagtt tcttttggac gcttgtttat 600
gatacaatat atgcacatca agataaagaa gatgatgcca aggctggaat taaatcaaca 660
actttgaggt ttggagattt aaccaaagta tgggttggag gttttggagt ggcatgcacc 720
cttgctttgc ttcttggtgg gtttgttatc aatattggat taccttacta tgtagttttg 780
actgttgcaa cttgtcaatt agcatggcaa attgttacag ttgacctatc ttcaccgatg 840
gattgtggta gaaaatttgt ttctaaccaa tggtatggtg ctattatctt tagcgccatc 900
ctagtaggaa aattgatttc ttaa 924
<210> 3
<211> 306
<212> PRT
<213>Asian puccoon（Arnebia euchroma）
<400> 3
Met Thr Ser Lys Gln Ala Gln Gln Lys Lys Gly Lys Gln Pro Ser Trp
1 5 10 15
Ile Glu Leu Tyr Leu Pro Lys Glu Val Arg Pro Tyr Ala His Leu Ala
20 25 30
Arg Leu Asp Lys Pro Ile Gly Ser Trp Leu Leu Ala Trp Pro Ala Phe
35 40 45
Trp Ser Val Ala Leu Val Ala Asp Phe Gly Ser Leu Pro Lys Met Leu
50 55 60
Ala Ile Phe Gly Trp Trp Ala Val Trp Ile Arg Gly Ala Gly Cys Thr
65 70 75 80
Ile Asn Asp Tyr Phe Asp Arg Asp Phe Asp Lys Lys Val Glu Arg Thr
85 90 95
Lys Ser Arg Pro Leu Ala Ser Gly Ala Val Ser Pro Ser Gln Gly Leu
100 105 110
Trp Trp Leu Ala Phe Gln Leu Phe Ile Gly Leu Gly Val Leu Tyr Gln
115 120 125
Phe Asn Ile Leu Thr Leu Ala Leu Ala Ile Leu His Val Pro Leu Val
130 135 140
Phe Ala Tyr Pro Leu Met Lys Arg Ile Thr Tyr Trp Pro Gln Ala Phe
145 150 155 160
Leu Gly Val Met Ile Ser Trp Gly Ala Leu Leu Gly Ser Ser Ala Leu
165 170 175
Lys Gly Ser Val Val Pro Ser Ile Ala Tyr Pro Leu Tyr Ile Ser Ser
180 185 190
Phe Phe Trp Thr Leu Val Tyr Asp Thr Ile Tyr Ala His Gln Asp Lys
195 200 205
Val Asp Asp Ala Lys Ala Gly Ile Lys Ser Thr Ala Leu Arg Phe Gly
210 215 220
Asp Ala Thr Lys Ile Trp Ile Ser Trp Phe Gly Val Gly Cys Ile Ala
225 230 235 240
Ala Leu Val Ile Gly Gly Leu Ile Val Asn Ile Gly Phe Pro Tyr Tyr
245 250 255
Val Phe Val Ala Ile Ala Thr Gly Gln Leu Ala Trp Gln Ile Val Thr
260 265 270
Val Asp Leu Ser Ser Pro Met Asp Cys Gly Arg Lys Phe Val Ser Asn
275 280 285
Gln Trp Phe Gly Ala Ile Ile Phe Thr Gly Ile Leu Leu Gly Arg Leu
290 295 300
Phe Thr
305
<210> 4
<211> 921
<212> DNA
<213>Asian puccoon（Arnebia euchroma）
<400> 4
atgacgtcca agcaagcaca gcagaagaaa ggcaagcaac catcttggat tgagctgtat 60
ttgcccaaag aggttcgacc atatgcacac cttgcaaggt tagacaagcc tataggcagc 120
tggctactag cttggccggc tttctggtcc gttgcattgg ttgcagattt tggaagtcta 180
ccaaaaatgt tagccatatt tggatggtgg gcagtttgga ttcgaggtgc tggatgtacc 240
atcaacgatt actttgatcg cgatttcgat aagaaagtgg aacgtacaaa atctagacca 300
cttgctagtg gcgctgtctc accttcccaa ggattgtggt ggcttgcatt tcagctgttc 360
attggcttgg gtgttcttta ccaattcaac atcttgactc ttgcattggc tatcttgcat 420
gttccccttg tttttgctta tcctctcatg aaaagaatta cctattggcc tcaagctttt 480
cttggagtaa tgatcagttg gggagctctc ttaggctcct ctgctcttaa aggaagtgtt 540
gttcccagta tcgcctaccc gctttacatt tcgagctttt tctggactct tgtttatgat 600
actatctatg cacatcaaga caaggtagac gatgcaaaag cagggattaa atccactgct 660
ctaagatttg gagatgcaac caagatatgg attagttggt tcggagtagg atgcattgct 720
gctctagtta ttggggggct cattgtgaac attgggtttc cttattatgt atttgtggca 780
atcgcaactg gtcaattggc ttggcaaatt gtcacagttg atttatcatc tcctatggat 840
tgtggtcgga aattcgtttc taaccaatgg tttggtgcta taatcttcac cggaatctta 900
cttggaagat tgtttactta g 921
Claims (10)
1. a kind of protein, is following (a1) or (a2)：
(a1) protein being made up of the amino acid sequence shown in sequence in sequence table 1；
(a2) by the amino acid sequence of sequence 1 by the substitution and/or missing and/or addition of one or several amino acid residues and
With identical function as derived from sequence 1 protein.
2. encode the gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that：The gene is any described DNA in following (b1)-(b3)
Molecule：
(b1) DNA molecular of the code area as shown in sequence 2 in sequence table；
(b2) protein DNA point described in the DNA sequence dna hybridization limited under strict conditions with (b1) and coding claim 1
Son；
(b3) DNA sequence dna limited with (b1) or (b2) has protein described in more than 90% homology and coding claim 1
DNA molecular.
4. the application of the protein described in claim 1, is following (c1) and/or (c2)：
(c1) as P-hydroxybenzoic acid myrcene based transferase；
(c2) catalysis P-hydroxybenzoic acid and geranyl pyrophosphate reaction produce spiceleaf alkenyl-P-hydroxybenzoic acid.
5. a kind of recombinant bacterium, is that will be obtained in the channel genes Host Strains described in Claims 2 or 3；The Host Strains can not
Produce geranyl pyrophosphate.
6. a kind of recombinant bacterium, is that will be obtained in the channel genes Host Strains described in Claims 2 or 3；The Host Strains can be with
Produce geranyl pyrophosphate.
7. a kind of preparation method of P-hydroxybenzoic acid myrcene based transferase, comprises the following steps：Cultivate claim 5 or 6
Described recombinant bacterium, obtains P-hydroxybenzoic acid myrcene based transferase from recombinant bacterium.
8. the recombinant bacterium described in claim 5 or 6, or, the method described in claim 7, are preparing P-hydroxybenzoic acid spiceleaf
Application in alkenyl transfer enzyme product.
9. the preparation method of spiceleaf alkenyl-P-hydroxybenzoic acid, is method first or method second；
Methods described first comprises the following steps：The recombinant bacterium described in claim 5 is cultivated, while adding P-hydroxybenzoic acid and Jiao
Phosphoric acid geraniol ester, isolated spiceleaf alkenyl-P-hydroxybenzoic acid from cultured products；
Methods described second comprises the following steps：The recombinant bacterium described in claim 6 is cultivated, while P-hydroxybenzoic acid is added, from
Isolated spiceleaf alkenyl-P-hydroxybenzoic acid in cultured products.
10. kit first or kit second；
The kit first includes recombinant bacterium, P-hydroxybenzoic acid and the geranyl pyrophosphate described in claim 6；
Recombinant bacterium and P-hydroxybenzoic acid described in the kit second claim 7；
The purposes of the kit first or kit second is production myrcene base-P-hydroxybenzoic acid.
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| CN113755458A (en) * | 2021-01-22 | 2021-12-07 | 中国科学院天津工业生物技术研究所 | CYP82AR2 protein involved in alkannin and/or alkannin biosynthesis, coding gene thereof and application thereof |
| CN114058602A (en) * | 2020-07-30 | 2022-02-18 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyl transferase as well as coding gene and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060143A (en) * | 2018-02-13 | 2018-05-22 | 中国中医科学院中药研究所 | A kind of CYP76B74 albumen for participating in alkannin biosynthesis and its encoding gene and application |
| CN108060143B (en) * | 2018-02-13 | 2020-04-28 | 中国中医科学院中药研究所 | CYP76B74 protein involved in alkannin biosynthesis and coding gene and application thereof |
| CN114058602A (en) * | 2020-07-30 | 2022-02-18 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyl transferase as well as coding gene and application |
| CN114058602B (en) * | 2020-07-30 | 2023-08-22 | 中国中医科学院中药研究所 | Sinkiang lithospermum caffeic acid and rosmarinic acid glycosyltransferase, and coding gene and application thereof |
| CN113755458A (en) * | 2021-01-22 | 2021-12-07 | 中国科学院天津工业生物技术研究所 | CYP82AR2 protein involved in alkannin and/or alkannin biosynthesis, coding gene thereof and application thereof |
| CN113755458B (en) * | 2021-01-22 | 2023-04-21 | 中国科学院天津工业生物技术研究所 | CYP82AR2 protein involved in shikonin and/or acarnine biosynthesis, and encoding gene and application thereof |
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