CN108753744A - Sesquiterpene cyclase and its preparation and application, 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane synthetic method - Google Patents
Sesquiterpene cyclase and its preparation and application, 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane synthetic method Download PDFInfo
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Abstract
The present invention discloses a kind of sesquiterpene cyclase and its preparation and application, 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane synthetic method.Amino acid sequence present invention firstly provides sesquiterpene cyclase BcABA3 is amino acid sequence and its derived peptides shown in sequence 2.The enzyme has the function of catalysis farnesyl pyrophosphate Cyclization 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane.The gene order of encoding sesquiterpene cyclisation enzyme amino acid sequence is provided simultaneously.The recombinant protein for including above-mentioned amino acid sequence is provided simultaneously.Sesquiterpene cyclase is provided simultaneously, recombinant protein is synthesizing 2Z, the application process in 4E- α-root of Dahurian angelica perfume (or spice) ethane, abscisic acid by substrate of farnesyl pyrophosphate.Recombinant expression carrier containing sesquiterpene cyclase encoding gene, transformant and its application are provided simultaneously.2Z is provided simultaneously, and the synthetic method of 4E- α-root of Dahurian angelica perfume (or spice) ethane is using farnesyl pyrophosphate as substrate, by sesquiterpene cyclase or recombination sesquiterpene cyclase catalysis.
Description
Technical field
The present invention relates to a kind of sesquiterpene cyclase, its encoding gene, its application, its preparations, are related to farnesyl coke phosphorus
Acid is that substrate synthesizes 2Z, the method for 4E- α-root of Dahurian angelica perfume (or spice) ethane.Belong to chemistry of micro-organisms, field of natural product chemistry.
Background technology
Abscisic acid (abscisic acid, ABA) is a kind of plant signal transduction molecule with sequiterpene structure, is state
Generally acknowledged one of the five major class endogenous growth regulators matter of plant in border, there are two types of optical isomers for tool.The day existed in plant
Right ABA is substantially dextroform (+)-ABA (Fig. 1).
Natural A BA the fields such as cell engineering and agricultural production, ecological construction, afforestation have it is huge basis and
Application value, but since its content in plant is very low, thus artificial synthesized is its important sources.The prior art is closed through chemistry
At be that the mixture of two kinds of optical isomer (±)-ABA extracted wherein biologically active (+)-ABA yields are relatively low
Journey is more complex, and cost is high.Thus the efficiency of final artificial synthesized natural A BA is low, of high cost.
Invention content
The purpose of the present invention aiming at the deficiencies in the prior art, provide a kind of sesquiterpene cyclase, its encoding gene, its
Using, its preparation, which can catalyze and synthesize acid precursors 2Z, the 4E- α-that falls off using farnesyl pyrophosphate as substrate
Root of Dahurian angelica perfume (or spice) ethane (2Z, 4E- α-ionylideneehane, 2Z, 4E-5- (2 ', 6 ', 6 '-trimethyl-2 '-cyclohexene-
1 '-yl) -3-methyl-2,4-pentadiene, 2Z, 4E-5- (2 ', 6 ', 6 '--2 '-cyclohexene -1 of methyl '-yl) -3- first
Base -2,4- pentadiene, Fig. 2), it is greatly improved the efficiency of artificial synthesized natural A BA, reduces cost.
To achieve the above object, present invention firstly provides a kind of sesquiterpene cyclase and its encoding gene, technical solutions
It is as follows：
A kind of sesquiterpene cyclase BcABA3, it is characterised in that：Amino acid sequence is following alternative one：
One, as shown in SEQ ID NO.2；
Two, polypeptide derived from amino acid sequence shown in SEQ ID NO.2.
Above-mentioned sesquiterpene cyclase is using high throughput sequencing technologies to a kind of ash for capableing of a large amount of synthesis of natural abscisic acids
Botrytis sp bacterium genetic improvement bacterial strain Botrytis cinerea TBC-20 (culture presevation CGMCC No.4645) carry out base
Because of a group sequencing, and after the progress bioinformatic analysis of the genomic dna sequence to being obtained and coding protein function verification, find
One section of gene homologous with gene bcaba3 is that a new sesquiterpene cyclase encoding gene (is named as bcaba3, gene sequence
Arrange SEQ ID NO.1).The gene code polypeptide (amino acid sequence SEQ ID NO.2) and the Botrytis cinerea reported
Polypeptide BcABA3 in B.cinerea B05.10 and T4 is respectively provided with 95% and 99% homology (Fig. 3), therefore also that this is more
Peptide is named as BcABA3.Activity determination and functional study are carried out to polypeptide BcABA3, it is found that it has catalysis farnesyl pyrophosphate
The sesquiterpene cyclase function of (farnesyl diphosphate, FPP, C15) Cyclization 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane, therefore
Think that BcABA3 is a new sesquiterpene cyclase.Present invention discover that the amino acid sequence of sesquiterpene cyclase BcABA3 is simultaneously
It can be the derived peptides of amino acid sequence shown in SEQ ID NO.2, i.e., pass through amino acid residue sequence shown in SEQ ID NO.2
Cross the substitution of one or more amino acid residues and/or lack and or add and with it is identical as SEQ ID NO.2 it is active by
Amino acid sequence derived from SEQ ID NO.2.Involved substitution and/or the amino acid residue lacked and ored add are located at SEQ
Any position or both ends in amino acid sequence shown in ID NO.2.
A kind of recombinant protein, it is characterised in that：It is following alternative one：
One, include amino acid sequence SEQ ID NO.2；
Two, include the derived peptides of amino acid sequence shown in SEQ ID NO.2.
In upper recombinant protein, the derived peptides of amino acid sequence shown in SEQ ID NO.2 refer to will be shown in SEQ ID NO.2
Amino acid residue sequence by one or more amino acid residues substitution and/or lack and or add and have and SEQ ID
The identical active sequences derived from SEQ ID NO.2 of NO.2.
Further, above-mentioned recombinant protein also separately includes protein tag.Present invention experiment is further discovered that external through DNA
The recombinant protein that recombinant technique obtains equally has above-mentioned when it includes amino acid sequence SEQ ID NO.2 and protein tag
The function of sesquiterpene cyclase BcABA3.The recombinant protein can also be spreading out comprising amino acid sequence shown in SEQ ID NO.2
Raw polypeptide and protein tag.Usually, protein tag can be purification tag and soluble flag.There are commonly had by 6~10
Six histidine residues composition fusion tag His-tag, GST (glutathione-S-transferase label), FLAG, STREP-II,
MBP, CPB, CBD, Halo, NusA, Trx, SUMO, GB1, SET/SEP, EGFP, HA, IgG, c-Myc or Profinity eXact
Deng.
Encode the gene order of above-mentioned sesquiterpene cyclase BcABA3 amino acid sequences, it is characterised in that：It is both following
One of：
One, coding region gene sequence nucleotide sequence as shown in SEQ ID NO.1；
Two, there is 90% or more homology with SEQ ID NO.1 sequences, and encode the nucleotides sequence of identical function protein
Row.
Present invention discover that encoding the gene order of above-mentioned sesquiterpene cyclase amino acid sequence as shown in SEQ ID NO.1
Polynucleotides can also be to have 90% or more homology with SEQ ID NO.1 sequences, and encode the core of identical function protein
Nucleotide sequence.The present invention tests it has furthermore been found that adding the nucleosides of protein tag on coding region gene sequence SEQ ID NO.1
When sour sequence label, protein of the coding with above-mentioned sesquiterpene cyclase BcABA3 functions can be also instructed.Usually, albumen mark
Label can be purification tag and soluble flag etc..There are commonly the fusion tag His- being made of 6~10 histidine residues
Tag, GST (glutathione-S-transferase label), FLAG, STREP-II, MBP, CPB, CBD, Halo, NusA, Trx, SUMO,
GB1, SET/SEP, EGFP, HA, IgG, c-Myc or Profinity eXact etc..
By the FPP in the source mevalonic acid (mevalonic acid, MVA) initial terpene carbon is generated through being cyclized and/or recombinating
Skeleton step is to determine the committed step of abscisic acid structure, and the 2Z of Cyclization, 4E- α-root of Dahurian angelica perfume (or spice) ethane are the precursors of abscisic acid.13C flag experiment speculates that FPP passes through C-1 reduction, and C-4 desaturations, C-2 isomeries and cyclization process form bone
Frame product 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane.Root of Dahurian angelica perfume (or spice) ethane undergoes a series of oxidative modification reactions and ultimately forms ABA.18O2With13C flag
Experiment speculates that the O of this series of oxidation reaction derives from O2, intermediate product includes root of Dahurian angelica perfume (or spice) ethyl alcohol, root of Dahurian angelica perfume (or spice) acetic acid, 1', and 4'- is trans--
ABA- glycol etc..
Accordingly, present invention simultaneously provides with the relevant following technical schemes of sesquiterpene cyclase BcABA3 of the present invention：
Above-mentioned sesquiterpene cyclase BcABA3, recombinant protein are synthesizing 2Z by substrate of farnesyl pyrophosphate, and 4E- α-root of Dahurian angelica is fragrant
Application process in ethane.
Above-mentioned sesquiterpene cyclase BcABA3, recombinant protein are synthesizing dextroform abscisic acid by substrate of farnesyl pyrophosphate
In application process.
The present invention also provides recombinant expression carrier, transformant containing above-mentioned sesquiterpene cyclase BcABA3 encoding genes,
Specific technical solution is：
A kind of recombinant expression carrier, it is characterised in that：It is that said gene sequence is cloned on expression vector to obtain.
Above-mentioned recombinant expression carrier is plasmid, colibacillus expression plasmid is preferably commercialized, further preferably
PET28a (+) plasmid.The recombinant expression carrier inserts base sequence in multiple cloning sites area：One, coding region gene sequence is such as
Nucleotide sequence shown in SEQ ID NO.1 or two, with SEQ ID NO.1 sequences have 90% or more homology, and encode
The nucleotide sequence of identical function protein.
A kind of transformant, it is characterised in that：It is to convert above-mentioned recombinant expression carrier to obtain into recipient cell.
The recipient cell of above-mentioned transformant can be prokaryotic cell or eukaryocyte, the former is such as coli strain, the latter
Such as yeast.The transformant includes the expression plasmid of gene order SEQ ID NO.1, can largely stablize expression under inductive condition
Sesquiterpene cyclase of the present invention.
Present invention simultaneously provides the preparation method of above-mentioned sesquiterpene cyclase BcABA3, this method is to utilize to contain gene sequence
The recombinant expression carrier of row SEQ ID NO.1 is converted into microbial host cell, and acquisition can express sequiterpene cyclisation of the present invention
The recombinant microorganism cell of enzyme, then sesquiterpene cyclase BcABA3, technical side are isolated from the culture of recombinant microorganism
Case is as follows：
A kind of preparation method of sesquiterpene cyclase BcABA3, it is characterised in that：Implement according to following steps：
Step S1, sesquiterpene cyclase BcABA3 coding gene sequences, base sequence such as SEQ ID NO.1 institutes are obtained
Show；
Step S2, above-mentioned encoding gene SEQ ID NO.1 are cloned on expression plasmid, obtain recombinant expression carrier；
Step S3, recombinant expression carrier is converted to recipient cell, recombination engineering is built, to total egg of acquisition after expression
It is isolated and purified in vain, obtains the sesquiterpene cyclase of recombination.
In the preparation method of above-mentioned sesquiterpene cyclase BcABA3, sesquiterpene cyclase BcABA3 encoding genes in step S1
Sequence can by with DNA, cDNA of Botrytis cinerea B.cinerea TBC-20 (culture presevation CGMCC No.4645) or
RNA is that template amplification clones to obtain, and can also be obtained by synthesizing.
Compared with prior art, the beneficial effects of the invention are as follows：(1) provide a kind of sesquiterpene cyclase BcABA3 and its
Preparation method, sesquiterpene cyclase BcABA3 have times of catalysis farnesyl pyrophosphate Cyclization 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane
Hemiterpene cyclase function.(2) coding gene sequence of sesquiterpene cyclase BcABA3 is provided.(3) it provides comprising sequiterpene
Recombinant expression carrier, the transformant of cyclase BcABA3 coding gene sequences.(4) it provides and utilizes sesquiterpene cyclase of the present invention
What BcABA3 was realized synthesizes 2Z by substrate of farnesyl pyrophosphate, and the method for 4E- α-root of Dahurian angelica perfume (or spice) ethane synthesizes dextroform abscisic acid
Method.(5) tests prove that, sesquiterpene cyclase BcABA3 provided by the invention has laboratory and industrialization purposes.
Description of the drawings
Fig. 1 is abscisic acid [(+)-S-ABA] molecular structure.
Fig. 2 is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane molecule structure chart.
Fig. 3 is the BcABA3 protein amino acid sequence comparison charts of B.cinerea TBC-20 and B05.10 and T4.
Fig. 4 is the cDNA PCR amplification result electrophoretograms of B.cinerea TBC-20 bacterium sesquiterpene cyclase encoding genes
Figure.
Fig. 5 is the SDS-PAGE analysis charts of the BcABA3 recombinant proteins with His labels.
Fig. 6 is the GC-MS result figures of BcABA3 reaction product n-hexane extracts.
Fig. 7 is that metal ion synthesizes 2Z, the influence diagram of 4E- α-root of Dahurian angelica perfume (or spice) ethane to BcABA3 conversions FPP.
Fig. 8 is that pH synthesizes 2Z, the influence diagram of 4E- α-root of Dahurian angelica perfume (or spice) ethane to BcABA3 conversions FPP.
Fig. 9 is that temperature synthesizes 2Z, the influence diagram of 4E- α-root of Dahurian angelica perfume (or spice) ethane to BcABA3 conversions FPP.
The SDS-PAGE analysis charts of BcABA3 recombinant proteins of the Figure 10 with GST labels.
Specific implementation mode
Below in conjunction with the accompanying drawings, technical scheme of the present invention will be further described.
Embodiment one
Sesquiterpene cyclase BcABA3 encoding genes are cloned.
1, the culture of B.cinerea TBC-20 bacterium
Taking one, TBC-20 single bacterium colonies inclined-plane, (the high about 8cm in inclined-plane, 26 DEG C, PDA culture medium culture 7d is placed on 4 DEG C of guarantors
Deposit), under drawing about 5ml aqua sterilisas with dropper and drawing the spore on inclined-plane, blow and beat several times by the mixed liquor mixing of spore, with drop
Pipe is drawn in 2ml to the 250ml triangular flasks equipped with 50ml culture mediums, and 26 DEG C of shaking table 180rpm cultures are placed in.50h or so is cultivated,
Seed liquor is inoculated into the 250ml triangular flasks equipped with 50ml culture mediums in the ratio of 5% inoculum concentration with 5ml pipettors, 26 DEG C,
180rpm is cultivated.Culture medium is by 6.0g/L glucose, 10g/L yeast extracts, 3.0g/L soluble starches, 1.0g/L soybean
Powder, 2.0g/L sucrose, 0.5g/L NH4NO3With 1.0g/L KH2PO4Composition.
2, the extraction of B.cinerea TBC-20 bacterium genomic DNA
1) B.cinerea is seeded on PDA solid mediums, 26 DEG C of culture 5d~7d.
2) mycelium is scraped into mortar, is transferred in 10mL centrifuge tubes at powder with liquid nitrogen grinding rapidly, be added one
Quantitative DNA extracts (0.1M Tris-HCl pH 8.0,10mM EDTA, 2%SDS, 100 μ g/mL Proteinase K,
1% beta -mercaptoethanol adds 500 μ L extracts per 50mg thalline), Vortex vibrates mixing, 55 DEG C of incubation 60min.
3) the NaCl final concentrations of extract are adjusted to 1.4M with 5M NaCl, the 10%CTAB that 1/10 volume is then added is molten
Liquid, 65 DEG C of incubation 10min after mixing.Isometric chloroform/isoamyl alcohol is added, overturns ice bath 30min after mixing.4 DEG C,
15000r/min centrifuges 10min.
4) supernatant is transferred in another centrifuge tube, is added isometric isopropanol, ice bath 30min after mixing.
5) 4 DEG C, 10000r/min, 10min is centrifuged, supernatant is abandoned, precipitation is washed twice with 70% ethyl alcohol, is placed in air
Middle drying ultimately joins suitable TE or ddH2O dissolving DNAs precipitate.
3, the extraction of B.cinerea TBC-20 bacterium RNA
It is respectively drawn from 3 bottles of B.cinerea TBC-20 culture shaking flasks of specific incubation time point for measuring dry cell weight
10ml culture solutions after sample is mixed, collected by suction whole thalline is simultaneously residual with being drained as possible with filter paper after sterile water washing 3 times
The water stayed is fully ground rapidly rapidly in the mortar of input Liquid nitrogen precooler, and Total RNAs extraction presses OMEGA kits Fungal
RNA Kit (article No. R6840-01) product description operates.By OMEGA kit RNase-Free DNase I Set (article No.s
E1091 it) carries out DNAase I digestion on column and removes residual DNA.It is monitored by agarose gel electrophoresis after the completion of RNA sample extraction
RNA mass detects RNA sample by NanoDrop spectro-photometer and with Agilent 2100Bioanalyzer
Quality and concentration.The absolute ethyl alcohol mixing of NaAc (pH=4.8) and three times volume that 1/10 volume is added are placed on -80 DEG C of jellies
It deposits.
4, sesquiterpene cyclase BcABA3 encoding genes are cloned
Using Homology-based cloning according to designing degenerate primer SC3-F, SC3-R (table 1) in conserved sequence from Botrytis cinerea
Cloned in the RNA samples of mould B.cinerea TBC-20 (culture presevation CGMCC No.4645) gene bcaba3 cDNA
(Fig. 4, wherein M are DNA marker：Trans2K@Plus DNA Marker, 1 is with primer SC3-F and SC3-R amplification
CDNA segments, 3 DNA fragmentation to be expanded with primer ABA3-F and ABA3-R).Total RNAs extraction presses OMEGA kits Fungal
RNA Kit (article No. R6840-01) product description operates, and carries out DNAase I digestion on column and remove residual DNA.RNA sample
RNA mass is monitored by electrophoresis after the completion of extraction, TOYOBO high efficiency Reverse Transcriptase kit ReverTra Ace- α-are pressed in reverse transcription
(article No. FSK-100) specification operates.
Sequence needed for the gene cloning of 1 sesquiterpene cyclase BcABA3 of table
PCR reaction conditions are：94℃4min；94 DEG C of 30s, 56 DEG C of 30s, 68 DEG C of 1min/kb, 30 cycles；68℃5min.
Sequencing finds gene bcaba3 gene cDNA sequences and consensus dna sequence, and base sequence is as shown in SEQ ID No.1.
Embodiment two
The structure of the expression vector of sesquiterpene cyclase BcABA3.
Determine the digestion of restriction endonuclease contained on the sesquiterpene cyclase BcABA3 base sequences obtained by embodiment one
Site NcoI, XhoI.
It is the carrier expressed needed for sesquiterpene cyclase BcABA3 to select pET28a (+) plasmid (laboratory preservation).It will be again
Hemiterpene cyclase BcABA3 corresponds to the restriction enzyme site of restriction enzyme site contained on base sequence and multiple cloning sites area on the carrier
It is compared, selects to contain in expression vector multiple cloning sites areas and does not have on sesquiterpene cyclase BcABA3 base sequences
Restriction enzyme site of the sites NcoI and XhoI as structure plasmid.Primer of the design containing restriction enzyme site is shown in Table 2.Use KOD
Plus-neo enzymes (spinning company purchased from Japan) carry out PCR amplification.
Sequence needed for the expression vector structure of 2 sesquiterpene cyclase BcABA3 of table
PCR reaction conditions are：94℃4min；94 DEG C of 30s, 56 DEG C of 30s, 68 DEG C of 1min/kb, 30 cycles；68℃5min.
Purpose base sequence segment is connected to T4 ligases (being purchased from TaKaRa) in carrier, with OMEGA small amount plasmids
Extracts kit extracts plasmid, obtains recombinant expression pET-28 (a)-SC3.
Embodiment three
The linearisation of colibacillus expression plasmid and the conversion of Escherichia coli, obtain and contain base sequence such as SEQ ID
The transformant of gene shown in No.1.
Embodiment two gained recombinant plasmid pET-28 (a)-SC3 is transformed into E. coli BL21 (DE3) bacterial strain
In the competent cell (preparing competent cell using Inoue methods) of (laboratory preservation).Concrete operations are as follows：
1) it takes a small amount of Transgene for being stored in -80 DEG C to freeze E.coli DH5 α thalline with Tip choicests, is not then adding
The flat lining outs of LB of added with antibiotic, tablet are inverted in bacteriological incubator and cultivate 16h.
2) the E.coli DH5 α single bacterium colonies (2-3mm in diameter) newly activated from picking on LB tablets, are inoculated in
3mL SOB fluid nutrient mediums are (with preceding plus MgCl2) in, 37 DEG C, 250rpm shaken cultivations 7 hours record OD values.
3) bacteria suspension in logarithmic growth later stage will be grown to 1:100 ratio is inoculated in 25mL SOB fluid nutrient mediums
In, room temperature 250rpm oscillations about 5h is cultivated to OD600It (is measured once per 1h for 0.4-0.5, pays attention to the centrifugation of half an hour precooling in advance
Machine and pipette tips, pipette, centrifuge tube).Culture solution is transferred in 2 15mL centrifuge tubes, places 10min on ice, then in 4 DEG C,
2500g centrifuges 5min, discards supernatant, and remaining culture medium is blotted with pipette tips.
4) with TB (pH 5.6~7.0) solution of precooling (new to prepare, pH is surveyed in timing) 8mL, gently suspension cell is (at a slow speed
Vortax), after placing 10min on ice, 4 DEG C, 2500g centrifuges 10min, precooling 1.5mL EP pipes.Supernatant is abandoned, is blotted with pipette tips residual
Remaining culture medium, it is each that 0.5mL TB are added, then it is each be drop by drop added 37.6 μ L precoolings DMSO (final concentration of 7%), at a slow speed
Vortax gently suspension cells, place 10min on ice.After 100 μ L packing -80 DEG C of refrigerators are stored in liquid nitrogen flash freezer.
5) the E.coliDH5 α competent cells of -80 DEG C of preservations is taken to be placed on ice；Add while competent cell dissolves
Enter the Plasmid DNA being pre-chilled in right amount, is uniformly mixed and is placed on 10min on ice；42 DEG C of water-bath heat shock 30s, are immediately placed on cooled on ice
10min；The fresh SOC culture mediums of 900 μ L are added, after mixing 37 DEG C of culture 30min；It will be abandoned after bacterium solution 1000g centrifugations 1min
Fall most of supernatant, leaves and takes after thalline is resuspended 100 μ L culture mediums and be spread evenly across on the LB tablets containing corresponding antibiotic, 37 DEG C
It is inverted overnight incubation.
Obtain the transformant containing base sequence gene as shown in SEQ ID No.1.
Example IV
The Escherichia coli positive colony screening of height expression purpose sesquiterpene cyclase BcABA3.
The method that the screening and identification of positive clone molecule uses bacterium solution PCR.Single bacterium in picking resistant panel is fallen within containing 3mL
37 DEG C in the test tube of the LB liquid medium containing corresponding antibiotic, 200rpm is incubated overnight, and draws the bacterium solution conduct that 2 μ L are incubated overnight
The template of PCR reactions.Designed for identifying the special primer of target gene, positive clone molecule is carried out using the PCR system of 50 μ L
Identification.PCR reaction systems are：2.5U Easy Taq DNA Polymerase (Transgene), 5 μ L10 × Easy Taq
Buffer, 2 μ L bacterium solutions, 0.25mM dNTP, 0.2 μM of primer and 2.5 μ L of DMSO.
PCR programs are：94℃20min；94 DEG C of 30s, 55 DEG C~60 DEG C 30s, 72 DEG C of 1min/kb, 30 cycles；72℃
10min。
PCR product sequence verification.
Embodiment five
Recombinate induced expressions of the sesquiterpene cyclase BcABA3 in Escherichia coli.
Filter out positive colony LB liquid medium (Tryptone10.0g/L, Yeast extract5.0g/L, NaCl
10.0g/L separately adds 15g/L agar, pH 7.0 when preparing LB solid mediums) 37 DEG C of shaking tables expand culture.Work as OD600Reach
When 0.4~0.6 or so, IPTG (concentration 1mol/L), which is added, makes its final concentration reach 0.5mmol/L, continues to cultivate 4h~6h progress
Induced expression.
Induced expression concrete operations are as follows：
1) this laboratory early period is turned using the good pET-28a-aba3 plasmids of pET-28a (+) Prokaryotic expression vector construction
Change E.coli BL21 (DE3) host's bacterium competence cell, picking single bacterium drops down onto LB on the LB tablets containing 50 μ g/mL Kana
Be incubated overnight in fluid nutrient medium and extract plasmid carry out sequencing identification obtain correct transformant.
2) picking transformant single bacterium drops down onto in the LB liquid medium that 5mL contains kana resistances, and 37 DEG C, 200rpm is trained overnight
It supports, by 1:100 ratio is transferred in 50mL fresh LBs, is transferred to 28 DEG C, and 200rpm, which expands to cultivate to OD600, is
0.4~0.6, isopropylthiogalactoside (IPTG) is added to final concentration 1mM, proceeds by recombinant protein induced expression,
The separately sampled 1mL cultures of 2h, 4h, 6h are analyzing the Best Times of induced expression.
3) the 1mL culture 12000rpm that each induction time takes out centrifuge 2min, and 1mL 100mM are added after abandoning supernatant
KH2PO4-KHPO4Buffer solution (PBK) (pH=7.0) washing thalline, 12000rpm, centrifuges 2min, is added after abandoning supernatant again
300mL PBK carry out ultrasonication after cell is resuspended (operating condition is shown in embodiment six).4 DEG C, 12000rpm centrifuges 10min, point
Albumen sample-loading buffer is not added in supernatant precipitation, SDS-PAGE analyzes distribution of the recombinant protein in supernatant precipitation,
The final Best Times for determining IPTG induction recombinant protein expression.If there is apparent purpose band in supernatant, judge that this is heavy
Histone is solubility expression, can amplify and cultivate and then purify destination protein.
4) soluble protein determines carries out induced expression after the best induced expression time according to above-mentioned induced expression condition,
By the culture 12000rpm after induction, 2min is centrifuged, supernatant is abandoned and collects thalline and with 100mM PBK buffer solutions (pH=7.0)
Washing thalline is twice.
5) 12000rpm centrifuges 2min, abandon after supernatant by thalline lysate (50mM PBNa buffer solutions, pH=8.0,
300mM NaCl, 1mM PMSF and 100 μ g/mL lysozymes) it suspends.
Embodiment six
Recombinate preparation, purifying, the sequencing of the thick enzymes of sesquiterpene cyclase BcABA3.
Thalline were collected by centrifugation for the cultured zymotic fluid of induced expression, primary using broken born of the same parents' buffer solution washing thalline, recycles
Broken born of the same parents' buffer solution is resuspended after thalline under condition of ice bath that (condition is using ultrasonic disruption：Ultrasonic 3s stops 5s, and 40 times are one week
Phase, amplitude transformer 6mm, power are 25% (about 150w).Ultrasound to bacterium solution becomes clear, until no longer sticky), it is clear to bacterium solution to break born of the same parents
Clearly, 10000rpm centrifuges 10min, collects supernatant.Supernatant metallic nickel affinity chromatography purifies destination protein.SDS-PAGE
Electrophoresis detection purification effect, (Fig. 5 is from left to right that 1M IPTG inductions 4h is complete respectively to the thick enzymes of final acquisition destination protein BcABA3
Bacterium sample；1M IPTG induce the full bacterium samples of 2h；Supernatant samples after 1M IPTG induction 4h thalline ultrasonic disruptions；Full bacterium before induction
Sample；Arrow meaning is the BcABA3 albumen that affinity purification obtains).
Embodiment seven
The Function Identification of sesquiterpene cyclase BcABA3, whether the specific recombinant protein for identifying that embodiment six obtains, which has, is urged
Change FPP and synthesizes 2Z, the biological activity of 4E- α-root of Dahurian angelica perfume (or spice) ethane.
Concentrate BcABA3 recombinant proteins：Purified recombinant protein BcABA3 solution 1ml~10ml μ L, concentration 2pmol/
uL。
1 μM~5 μM of concentration BcABA3 recombinant proteins are added in enzyme activity reaction system.Enzyme reaction system ingredient is：150mM
Tris-Cl (pH=7.0~8.0), 100mM NaCl, 5%glycerol, 30mM MgCl2, 5mM beta -mercaptoethanols, 20 μ g/mL
FPP.It is covered with the n-hexane of isometric chromatographically pure after mixing, 0.5h~4h is reacted under 30 DEG C~35 DEG C water bath conditions.
1mL reaction solutions 200 μ L are concentrated to three times, after extract liquor mixing with isometric n-hexane extraction after reaction to use
It is detected in GC/MS.Testing conditions are：1 μ L Splitless injecting samples, 60 DEG C of initial temperature keep 2min；10 DEG C/min rises to 200
℃；20 DEG C/min rises to 280 DEG C, keeps 10min.Molecular weight scanning range 30~450.Detect the product of molecular weight with
Mass Finder databases are compared.The detection of GC-MS finds that product is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane (Fig. 6), can detect
By-product 2Z, 4E, 6E- α-allofarnesene and 2E, 4E, 6E- α-allofarnesene (Fig. 6).
Embodiment eight
The Function Identification of sesquiterpene cyclase BcABA3, whether the specific recombinant protein for identifying that embodiment six obtains, which has, is urged
Change FPP and synthesizes 2Z, the biological activity of 4E- α-root of Dahurian angelica perfume (or spice) ethane.It is not repeated that difference exists with seven something in common of embodiment
In：1 μM~5 μM sesquiterpene cyclases are added in reaction system and are added without 30mM MgCl2。
Enzyme reaction system ingredient is as follows：150mM Tris-Cl (pH=7.0~8.0), 100mM NaCl, 5%
Glycerol, 5mM beta -mercaptoethanol, 60 μm of ol/mL FPP.It is covered with the n-hexane of isometric chromatographically pure after mixing, 30 DEG C~
0.5h~4h is reacted under 35 DEG C of water bath conditions.
By reaction solution, with isometric n-hexane extraction, being used for GC/MS detects (inspection three times, after extract liquor mixing after reaction
Operation is surveyed with embodiment seven), discovery principal product is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane.It is not repeated with seven something in common of embodiment.
Embodiment seven, eight is not the results show that Mg is added2+Enzyme reaction system in root of Dahurian angelica perfume (or spice) ethane combined coefficient be added
Mg2+50%~60% (Fig. 8) of combined coefficient afterwards.
Fig. 7 is to select the result of different metal ions and control group to show in same reaction system.Fig. 7 shows, Mg2+、Ca2 +It can promote synthesis, Al3+、Zn2+、Mn2+、Cu2+、Fe2+It can inhibit to synthesize.
Embodiment nine
The Function Identification of sesquiterpene cyclase BcABA3, whether the specific recombinant protein for identifying that embodiment six obtains, which has, is urged
Change FPP and synthesizes 2Z, the biological activity of 4E- α-root of Dahurian angelica perfume (or spice) ethane.It is not repeated with seven something in common of embodiment.
1 μM~5 μM of concentration BcABA3 recombinant proteins are added in enzyme activity reaction system.Enzyme reaction system ingredient is：150mM
Tris-Cl (pH=8.0~9.0), 100mM NaCl, 5%glycerol, 30mM MgCl2, 5mM beta -mercaptoethanols, 20 μ g/mL
FPP.It is covered with the n-hexane of isometric chromatographically pure after mixing, 1h~4h is reacted under 30 DEG C~35 DEG C water bath conditions.Reaction terminates
Afterwards three times by isometric n-hexane extraction, it is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane to be concentrated in vacuo after extract liquor mixing and obtain product.
Embodiment seven, nine is the results show that be added root of Dahurian angelica perfume (or spice) ethane in the enzyme reaction system of Tris-Cl (pH=8.5~9.5)
Combined coefficient is that 50%~70% (Fig. 8) of Tris-Cl (pH=7.0~8.0) combined coefficient is added.
Fig. 8 is the test result of same reaction system selection condition of different pH.Fig. 8 shows that pH=7.0~8.0 are more excellent
Condition, but 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane can be effectively synthesized under the conditions of pH=5.0~10.0.
Embodiment ten
The Function Identification of sesquiterpene cyclase BcABA3, whether the specific recombinant protein for identifying that embodiment six obtains, which has, is urged
Change FPP and synthesizes 2Z, the biological activity of 4E- α-root of Dahurian angelica perfume (or spice) ethane.It is not repeated with seven something in common of embodiment.
1 μM~5 μM of concentration BcABA3 recombinant proteins are added in enzyme activity reaction system.Enzyme reaction system ingredient is：150mM
Tris-Cl (pH=7.0~8.0), 100mM NaCl, 5%glycerol, 30mM MgCl2, 5mM beta -mercaptoethanols, 20 μ g/mL
FPP.After mixing with the n-hexane of isometric chromatographically pure cover, 25 DEG C after reaction by isometric n-hexane extraction three times,
It is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane to be concentrated in vacuo after extract liquor mixing and obtain product.
Embodiment seven, ten are the results show that the efficiency of 25 DEG C of reaction synthesis root of Dahurian angelica perfume (or spice) ethane is under 30 DEG C~35 DEG C water bath conditions
React 50% or so (Fig. 9) of combined coefficient.
Fig. 9 is the test result that differential responses temperature is selected under same reaction system.Fig. 9 shows, 30 DEG C~35 DEG C be compared with
Excellent condition, but 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane can be effectively synthesized under the conditions of 15 DEG C~50 DEG C.
Embodiment 11
It is prepared by the sesquiterpene cyclase BcABA3 with GST protein tags.
It is the carrier expressed needed for sesquiterpene cyclase BcABA3 to select pGEX-6P-1 plasmids (laboratory preservation).Design
Primer is shown in Table 3.PCR amplification is carried out using KOD plus-neo enzymes (company is spun by source Japan).
Sequence needed for the expression vector structure of 3 sesquiterpene cyclase BcABA3 of table
PCR reaction conditions are：94℃4min；94 DEG C of 30s, 56 DEG C of 30s, 68 DEG C of 1min/kb, 30 cycles；68℃5min.
Purpose base sequence segment is connected in carrier, plasmid is extracted with OMEGA small amount plasmid extraction kits, obtains
Recombinant expression pGEX-SC3.Recombinant plasmid pGEX-SC3 is transformed into E. coli Rosetta (DE3)
In the competent cell of (Cat.Nos.CD801-03, Transgen Biotech Co., Ltd., China).Amp resistances LB is flat
Plate screening positive clone is identified using bacterium solution PCR method.
PGEX-SC3 monoclonals (37 DEG C, cultivate 12h) are incubated overnight, are transferred in LB shaking flasks (100ml~200ml), 37
IPTG inductions (1 ‰, 37 DEG C of 20h of final concentration~for 24 hours) are added in DEG C culture 4h (OD600=0.6~0.8) afterwards, and thalline were collected by centrifugation
(4 DEG C of 8000rpm 10min) is added 1 × PBS of 15ml, carries out ultrasonication (operating condition is shown in embodiment six), with GST parents
Obtaining recombinant protein GST-BcABA3 with Purification Kit, (Figure 10, M are albumen Marker；1 is pGEX-SC3/Rosetta
Induce the full bacterium samples of 0h；2 induce the full bacterium samples of 20h for pGEX-SC3/Rosetta IPTG；3 be ABA3 protein purifications sample).
Embodiment 12
The sesquiterpene cyclase BcABA3 Function Identifications of tape label, whether the specific recombinant protein for identifying the acquisition of embodiment nine
2Z, the biological activity of 4E- α-root of Dahurian angelica perfume (or spice) ethane are synthesized with catalysis FPP.
Ten gained recombinant protein GST-BcABA3 of embodiment (prescission of GE companies after label is cut off
protease；Or the thrombin of sigma companies), according to the method validation vigor of embodiment seven.Concentration BcABA3 is recombinated into egg
White 1 μM is added in enzyme activity reaction system.Enzyme reaction system ingredient is as follows：150mM Tris-Cl (pH=7.0~8.0), 100mM
NaCl, 5%glycerol, 30mM MgCl2, 5mM beta -mercaptoethanols, 60 μm of ol/mL FPP.With isometric chromatographically pure after mixing
N-hexane covering, react 2h under 35 DEG C of water bath conditions.
After reaction by reaction solution with isometric n-hexane extraction three times, extract liquor mixing after concentration carry out GC/MS inspections
It surveys, discovery principal product is 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane.
The combined coefficient of embodiment seven, 11 the results show that GST purifying proteins is his label purifying protein combined coefficienies
90%~100%.
Sequence table
SEQ ID NO.1
<212>DNA
<213>Botrytis cinerea (Botrytis cinerea TBC-20)
atgcagcaagttattactcaaacgcttgtcgatgatagattcattcaaatctcagattcgaagaagagcgaaggcct
agccaccgacagtaccaagcgtcaaagtcaagagcagcctattcatgataaagaccccattaaagcagcaacggcag
caatggctaccaccccccttgtcaaagagcatcaggatacctggtactaccctcctgacattgcgaacgatttgcaa
tctatcaaccttccggccgaattgaagggagaaatctttgcgtgcgcatgggaatacacacgctgtgttattcctaa
ctacacaaactggaatagatatgtggccttcatgcgtattatcatcatgggtatcatcgccgagtttcgaggcgaga
tggtggatgtcactgccagcaacaacctcctcggttacgatttggacgctactcttgcggctttatttgagggaacc
ccagggcacaaggaaatggcacgagagtacaagacttttctgttaatcacggcggacaaggcgagtgaaagacgaga
tggggagcttttcagacgttatgtaaacgctctcgcgcaatcaccgagacactggttccgcatgcgggattgtgatg
cgctggccagattcacgattgcctcggctctagcatgcaacgatctcgacgacatctggttcaccgaagatcaattt
gaaattttgaccgaaattggagacactctgtatgatgcagtggctttctacaaacatcgtgctgaaggtgagacaaa
cagcacatttgcctacatgccggaggacttgcgcatcaaagcctacagcgagtgtcgcgagatactttgggcccttg
acgccgcctgggcacgaaatcctaagctggcaaatgttattaatttcgtgcgcttctttggtggacctatacacatg
atgatgcgccgttaccggttcgttgaagagaatttgacaattggcaagtcagagactgacaaagtcgttgatcaaac
ccggaagaacttcaagctctggaatcgagtcgatgccaacaaaaggtctgttcttaacacacagcggtacaaggccc
tcatcgctcgtagtgaagagcttatgttcccggggctttctgagttccttgaaatgggcggtgatgggatttgtgac
aaatgcaaatatcgcgagtcctacggtgcagaattgtcacaccagtttggtggtgttgaactatgcagcgaatgcag
actatcgtggagaaagtacctagaatgttttgtagagcgtgcgacaaaggtttttcctgagctgaaaacacattttg
aggttccagtttga
SEQ ID NO.2
<212>PRT
<213>Botrytis cinerea (Botrytis cinerea TBC-20)
MQQVITQTLVDDRFIQISDSKKSEGLATDSTKRQSQEQPIHDKDPIKAATAAMATTPLVKEHQDTWYYP
PDIANDLQSINLPAELKGEIFACAWEYTRCVIPNYTNWNRYVAFMRIIIMGIIAEFRGEMVDVTASNNLLGYDLDAT
LAALFEGTPGHKEMAREYKTFLLITADKASERRDGELFRRYVNALAQSPRHWFRMRDCDALARFTIASALACNDLDD
IWFTEDQFEILTEIGDTLYDAVAFYKHRAEGETNSTFAYMPEDLRIKAYSECREILWALDAAWARNPKLANVINFVR
FFGGPIHMMMRRYRFVEENLTIGKSETDKVVDQTRKNFKLWNRVDANKRSVLNTQRYKALIARSEELMFPGLSEFLE
MGGDGICDKCKYRESYGAELSHQFGGVELCSECRLSWRKYLECFVERATKVFPELKTHFEVPV
Sequence table
<110>Chengdu Inst. of Biology, Chinese Academy of Sciences
<120>Sesquiterpene cyclase and its preparation and application, 2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane synthetic method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1323
<212> DNA
<213>Botrytis cinerea TBC-20 (Botrytis cinerea TBC-20)
<400> 1
atgcagcaag ttattactca aacgcttgtc gatgatagat tcattcaaat ctcagattcg 60
aagaagagcg aaggcctagc caccgacagt accaagcgtc aaagtcaaga gcagcctatt 120
catgataaag accccattaa agcagcaacg gcagcaatgg ctaccacccc ccttgtcaaa 180
gagcatcagg atacctggta ctaccctcct gacattgcga acgatttgca atctatcaac 240
cttccggccg aattgaaggg agaaatcttt gcgtgcgcat gggaatacac acgctgtgtt 300
attcctaact acacaaactg gaatagatat gtggccttca tgcgtattat catcatgggt 360
atcatcgccg agtttcgagg cgagatggtg gatgtcactg ccagcaacaa cctcctcggt 420
tacgatttgg acgctactct tgcggcttta tttgagggaa ccccagggca caaggaaatg 480
gcacgagagt acaagacttt tctgttaatc acggcggaca aggcgagtga aagacgagat 540
ggggagcttt tcagacgtta tgtaaacgct ctcgcgcaat caccgagaca ctggttccgc 600
atgcgggatt gtgatgcgct ggccagattc acgattgcct cggctctagc atgcaacgat 660
ctcgacgaca tctggttcac cgaagatcaa tttgaaattt tgaccgaaat tggagacact 720
ctgtatgatg cagtggcttt ctacaaacat cgtgctgaag gtgagacaaa cagcacattt 780
gcctacatgc cggaggactt gcgcatcaaa gcctacagcg agtgtcgcga gatactttgg 840
gcccttgacg ccgcctgggc acgaaatcct aagctggcaa atgttattaa tttcgtgcgc 900
ttctttggtg gacctataca catgatgatg cgccgttacc ggttcgttga agagaatttg 960
acaattggca agtcagagac tgacaaagtc gttgatcaaa cccggaagaa cttcaagctc 1020
tggaatcgag tcgatgccaa caaaaggtct gttcttaaca cacagcggta caaggccctc 1080
atcgctcgta gtgaagagct tatgttcccg gggctttctg agttccttga aatgggcggt 1140
gatgggattt gtgacaaatg caaatatcgc gagtcctacg gtgcagaatt gtcacaccag 1200
tttggtggtg ttgaactatg cagcgaatgc agactatcgt ggagaaagta cctagaatgt 1260
tttgtagagc gtgcgacaaa ggtttttcct gagctgaaaa cacattttga ggttccagtt 1320
tga 1323
<210> 2
<211> 440
<212> PRT
<213>Botrytis cinerea TBC-20 (Botrytis cinerea TBC-20)
<400> 2
Met Gln Gln Val Ile Thr Gln Thr Leu Val Asp Asp Arg Phe Ile Gln
1 5 10 15
Ile Ser Asp Ser Lys Lys Ser Glu Gly Leu Ala Thr Asp Ser Thr Lys
20 25 30
Arg Gln Ser Gln Glu Gln Pro Ile His Asp Lys Asp Pro Ile Lys Ala
35 40 45
Ala Thr Ala Ala Met Ala Thr Thr Pro Leu Val Lys Glu His Gln Asp
50 55 60
Thr Trp Tyr Tyr Pro Pro Asp Ile Ala Asn Asp Leu Gln Ser Ile Asn
65 70 75 80
Leu Pro Ala Glu Leu Lys Gly Glu Ile Phe Ala Cys Ala Trp Glu Tyr
85 90 95
Thr Arg Cys Val Ile Pro Asn Tyr Thr Asn Trp Asn Arg Tyr Val Ala
100 105 110
Phe Met Arg Ile Ile Ile Met Gly Ile Ile Ala Glu Phe Arg Gly Glu
115 120 125
Met Val Asp Val Thr Ala Ser Asn Asn Leu Leu Gly Tyr Asp Leu Asp
130 135 140
Ala Thr Leu Ala Ala Leu Phe Glu Gly Thr Pro Gly His Lys Glu Met
145 150 155 160
Ala Arg Glu Tyr Lys Thr Phe Leu Leu Ile Thr Ala Asp Lys Ala Ser
165 170 175
Glu Arg Arg Asp Gly Glu Leu Phe Arg Arg Tyr Val Asn Ala Leu Ala
180 185 190
Gln Ser Pro Arg His Trp Phe Arg Met Arg Asp Cys Asp Ala Leu Ala
195 200 205
Arg Phe Thr Ile Ala Ser Ala Leu Ala Cys Asn Asp Leu Asp Asp Ile
210 215 220
Trp Phe Thr Glu Asp Gln Phe Glu Ile Leu Thr Glu Ile Gly Asp Thr
225 230 235 240
Leu Tyr Asp Ala Val Ala Phe Tyr Lys His Arg Ala Glu Gly Glu Thr
245 250 255
Asn Ser Thr Phe Ala Tyr Met Pro Glu Asp Leu Arg Ile Lys Ala Tyr
260 265 270
Ser Glu Cys Arg Glu Ile Leu Trp Ala Leu Asp Ala Ala Trp Ala Arg
275 280 285
Asn Pro Lys Leu Ala Asn Val Ile Asn Phe Val Arg Phe Phe Gly Gly
290 295 300
Pro Ile His Met Met Met Arg Arg Tyr Arg Phe Val Glu Glu Asn Leu
305 310 315 320
Thr Ile Gly Lys Ser Glu Thr Asp Lys Val Val Asp Gln Thr Arg Lys
325 330 335
Asn Phe Lys Leu Trp Asn Arg Val Asp Ala Asn Lys Arg Ser Val Leu
340 345 350
Asn Thr Gln Arg Tyr Lys Ala Leu Ile Ala Arg Ser Glu Glu Leu Met
355 360 365
Phe Pro Gly Leu Ser Glu Phe Leu Glu Met Gly Gly Asp Gly Ile Cys
370 375 380
Asp Lys Cys Lys Tyr Arg Glu Ser Tyr Gly Ala Glu Leu Ser His Gln
385 390 395 400
Phe Gly Gly Val Glu Leu Cys Ser Glu Cys Arg Leu Ser Trp Arg Lys
405 410 415
Tyr Leu Glu Cys Phe Val Glu Arg Ala Thr Lys Val Phe Pro Glu Leu
420 425 430
Lys Thr His Phe Glu Val Pro Val
435 440
Claims (10)
1. sesquiterpene cyclase BcABA3, it is characterised in that：Amino acid sequence is following alternative one：
One, as shown in SEQ ID NO.2；
Two, polypeptide derived from amino acid sequence shown in SEQ ID NO.2.
2. the gene order of coding sesquiterpene cyclase BcABA3 amino acid sequences described in claim 1, it is characterised in that：It is
Following alternative one：
One, coding region gene sequence nucleotide sequence as shown in SEQ ID NO.1；
Two, there is 90% or more homology with SEQ ID NO.1 sequences, and encode the nucleotide sequence of identical function protein.
3. recombinant protein, it is characterised in that：It is under following the two：
One, include amino acid sequence SEQ ID NO.2；
Two, include polypeptide derived from amino acid sequence shown in SEQ ID NO.2.
4. the recombinant protein described in sesquiterpene cyclase BcABA3 described in claim 1, claim 3 is with farnesyl coke phosphorus
Acid is that substrate synthesizes 2Z, the application process in 4E- α-root of Dahurian angelica perfume (or spice) ethane or abscisic acid.
5. the application process described in claim 4, it is characterised in that：The abscisic acid is dextroform abscisic acid.
6. recombinant expression carrier, it is characterised in that：It is that the gene order described in claim 2 is cloned on expression vector to obtain
?.
7. transformant, it is characterised in that：It is to convert the recombinant expression carrier that claim 6 is stated to obtain into recipient cell.
8. the transformant described in recombinant expression carrier, claim 7 described in claim 6 is in preparing sesquiterpene cyclase
Application process.
The synthetic method of 9.2Z, 4E- α-root of Dahurian angelica perfume (or spice) ethane, it is characterised in that：Using farnesyl pyrophosphate as substrate, it is cyclized by sequiterpene
Enzyme BcABA3 or the BcABA3 catalysis of recombination sesquiterpene cyclase；15 DEG C~50 DEG C of enzymatic condition, pH5.0~10.0.
10. synthetic method according to claim 9, it is characterised in that：Mg is added in reaction system2+And/or Ca2+Activation times
Al is not contained in hemiterpene cyclase BcABA3 activity or reaction system3+And/or Zn2+And/or Mn2+And/or Cu2+And/or Fe2 +。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157720A (en) * | 2019-06-20 | 2019-08-23 | 重庆医科大学 | Sesquiterpene cyclase gene peniA and its method that heterogenous expression synthesizes silphinene in yeast |
| CN113278657A (en) * | 2020-09-21 | 2021-08-20 | 中国科学院成都生物研究所 | Fermentation method for preparing 1',4' -trans-ABA-diol |
| WO2023012111A2 (en) | 2021-08-02 | 2023-02-09 | Basf Se | Novel production of aroma compounds with ionylideneethane synthases |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041837A (en) * | 2007-01-24 | 2007-09-26 | 中国科学院成都生物研究所 | Preparation method of new natural abscisic acid |
| US20080295196A1 (en) * | 2006-12-06 | 2008-11-27 | Abad Mark S | Genes and uses for plant improvement |
| CN101688191A (en) * | 2007-04-03 | 2010-03-31 | 菲力普莫里斯产品股份公司 | Genes encoding z,z-farnesyl diphosphate synthase and a sesquiterpene synthase with multiple products and uses thereof |
| CN102399827A (en) * | 2011-11-18 | 2012-04-04 | 中国科学院成都生物研究所 | Method for efficiently preparing natural abscisic acid |
| CN104093836A (en) * | 2012-02-27 | 2014-10-08 | 丰田自动车株式会社 | Hydrocarbon synthase gene, and use thereor |
-
2018
- 2018-06-27 CN CN201810678989.XA patent/CN108753744B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080295196A1 (en) * | 2006-12-06 | 2008-11-27 | Abad Mark S | Genes and uses for plant improvement |
| CN101041837A (en) * | 2007-01-24 | 2007-09-26 | 中国科学院成都生物研究所 | Preparation method of new natural abscisic acid |
| CN101688191A (en) * | 2007-04-03 | 2010-03-31 | 菲力普莫里斯产品股份公司 | Genes encoding z,z-farnesyl diphosphate synthase and a sesquiterpene synthase with multiple products and uses thereof |
| CN102399827A (en) * | 2011-11-18 | 2012-04-04 | 中国科学院成都生物研究所 | Method for efficiently preparing natural abscisic acid |
| CN104093836A (en) * | 2012-02-27 | 2014-10-08 | 丰田自动车株式会社 | Hydrocarbon synthase gene, and use thereor |
Non-Patent Citations (16)
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157720A (en) * | 2019-06-20 | 2019-08-23 | 重庆医科大学 | Sesquiterpene cyclase gene peniA and its method that heterogenous expression synthesizes silphinene in yeast |
| CN110157720B (en) * | 2019-06-20 | 2020-08-28 | 重庆医科大学 | Sesquiterpene cyclase gene peniA and method for synthesizing silphine through heterologous expression in yeast |
| CN113278657A (en) * | 2020-09-21 | 2021-08-20 | 中国科学院成都生物研究所 | Fermentation method for preparing 1',4' -trans-ABA-diol |
| CN113278657B (en) * | 2020-09-21 | 2023-07-07 | 中国科学院成都生物研究所 | Fermentation method for preparing 1',4' -trans-ABA-diol |
| WO2023012111A2 (en) | 2021-08-02 | 2023-02-09 | Basf Se | Novel production of aroma compounds with ionylideneethane synthases |
| WO2023012111A3 (en) * | 2021-08-02 | 2023-07-06 | Basf Se | Novel production of aroma compounds with ionylideneethane synthases |
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