CN106544348A - Isopentenyl diphosphate isomerase gene and its application - Google Patents
Isopentenyl diphosphate isomerase gene and its application Download PDFInfo
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Abstract
The present invention relates to a kind of Isopentenyl diphosphate isomerase gene and its application, the inefficient technical problem of utilizing works bacterium synthesis terpenoid is which solved, the invention provides the preparation method and application of a kind of Isopentenyl diphosphate isomerase gene, the protein of its expression, the prokaryotic expression carrier containing Isopentenyl diphosphate isomerase gene and product terpenoid engineering bacteria.The method can be widely used in terpenoid preparation field.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of isopentenyl pyrophosphate isomery
Enzyme gene and its application.
Background technology
Terpenoid is referred to isoprene structures unit (C5H8) n formulas compound and its
The derivant of oxygen-containing and different degree of saturation, terpenoid are the precursor isopentene groups by them
Pyrophosphoric acid (IPP) and its isomer dimethylallylpyrophosphate (DMAPP) condensation are formed.
Terpenoid is widespread in nature, higher plant, funguses, microorganism, insecticide
And marine organisms, terpenoid can be synthesized.Lycopene, astaxanthin, carotene,
Vitamin A, Ubidecarenone, paclitaxel and arteannuin etc. are all terpenoids.Terpenoid has
Many physiologically actives, such as eliminating the phlegm, cough-relieving, wind dispelling, diaphoresis, anthelmintic, analgesia etc., is medium-height grass
The important effective ingredient of a class in medicine；Simultaneously they can also be used as important natural of a class
Spice, is cosmetics and the indispensable raw material of food industry；Terpenoid or important work
Industry raw material, is the important source material of auto industry and airplane industry, such as isoprene long-chain compound rubber
Glue.
Traditional production method of terpenoid is extracted from such as plant, microorganism and animal,
But yield is extremely low.Reason is as follows：First, most of terpenoids are only a small amount of in nature
Accumulation；Secondly, donor organism is often unsuitable for large-scale culture, and large-scale culture is big rule
Necessary to mould commodity production terpenoid；3rd, the extraction needs of terpenoid are poisonous molten
Agent, the special process step of this needs, therefore the commodity production of terpenoid becomes more complicated.
From the C of n=15H8(the C of (isoprene) to n=n5H8)nNatural rubber, terpenoid
The route of synthesis of thing has two stages, generation rank of the first stage for isoprene unit IPP
Section, second stage are that terpenoid is generated and modifies the stage.Deposit in first stage nature
In two kinds of different approach, eukaryote relies primarily on mevalonic acid (MVA) approach, and acetyl is auxiliary
Enzyme A (acetyl-CoA) is converted into IPP, and then IPP is by Isopentenyl diphosphate isomerase (IDI)
DMAPP is isomerized to, prokaryote typically relies on deoxy-D-xylulose sugar -5- phosphoric acid (MEP) approach next life
Into IPP, then IPP is isomerized to into DMAPP by IDI, plant then i.e. can be using MVA approach again
MEP approach (Mendoza-Poudereux I et al.Plant Physiol can be utilized
Biochem.201595:113-20).It can be seen that no matter which kind of route of synthesis, be required for one it is identical
Enzyme, i.e. IPP tautomerizes to DMAPP into terpenoid by Isopentenyl diphosphate isomerase
Therefore generation phase, finds an efficient Isopentenyl diphosphate isomerase to strengthening terpenoid
Thing route of synthesis has huge help.
It is reported that, isoprene of the annual Plant emission in air reaches 5Tg, and isoprene is terpene
The synthesis unit of class compound, it is inferred that in plant, have very strong terpenoid route of synthesis,
Perhaps, the plant for selecting burst size very high, can be that an Isopentenyl diphosphate isomerase gene comes very well
Source.
Using technique for gene engineering carry out Isopentenyl diphosphate isomerase gene research achieved with
Some progress, research worker isolation identification have obtained a small amount of Isopentenyl diphosphate isomerase gene,
But it is concentrated mainly on antibacterial (Hamano Y et al.Biosci Biotechnol Biochem.2001
65(7):1627-35), (Wu FL et al.Int J Med Mushrooms.2013 in funguses
15(3):223-32), less in plant (Tong Y et al.Biotechnol Appl
Biochem.2015Aug 2.doi:10.1002/bab.1427), research is particularly had no in Cortex Populi dividianae
Report, also no Cortex Populi dividianae Isopentenyl diphosphate isomerase gene in gene bank.
The content of the invention
The present invention is exactly to ask to solve the inefficient technology of utilizing works bacterium synthesis terpenoid
Topic, there is provided a kind of engineering bacteria with stronger terpenoid route of synthesis and application.
To reach above-mentioned purpose, a kind of Isopentenyl diphosphate isomerase gene, which is following (a)
Or the gene of (b)：The SEQ ID No.1 of the nucleotide sequence such as sequence table of (a) described gene cDNA
It is shown；B () described gene is the gene for encoding following protein：The SEQ ID No.2 of sequence table
Shown aminoacid sequence is through replacing, lacking or add one or several aminoacid and have isoamyl
Aminoacid sequence shown in the SEQ ID No.2 by sequence table of thiazolinyl pyrophosphoric acid isomerase activity is constituted
Protein derived protein.
Present invention simultaneously provides a kind of protein of Isopentenyl diphosphate isomerase gene expression, its
It is the protein of following (a) or (b)：The aminoacid sequence of (a) by shown in the sequence 2 of sequence table
The protein of row composition；Through replacing, lacking or add in the aminoacid sequence of (b) in (a)
One or several aminoacid and with Isopentenyl diphosphate isomerase activity by egg derived from (a)
White matter；The protein of the aminoacid sequence composition shown in the sequence 2 of sequence table is by the sequence of sequence table
Base sequence shown in row 1 is encoded.
The present invention also provides the prokaryotic expression carrier of Isopentenyl diphosphate isomerase gene.
The present invention also provides the product isoamyl two of Isopentenyl diphosphate isomerase prokaryotic expression vector
Alkene engineering bacteria.
Present invention simultaneously provides producing application of the isoprene engineering bacteria in isoprene is prepared.
The present invention also provides the terpenoid of Isopentenyl diphosphate isomerase prokaryotic expression vector
Thing is particularly lycopene engineering bacteria.
Present invention simultaneously provides producing application of the lycopene engineering bacteria in lycopene is prepared.
Beneficial effects of the present invention：According to plant nature isoprene rate of release, this
The bright higher willow Isopentenyl diphosphate isomerase gene of burst size that have selected has carried out isolation identification
And clone, isoprene production bacterial strain and terpenoid production bacterial strain are successfully built, is bioanalysises
Production terpenoid searches out an efficient Isopentenyl diphosphate isomerase.The present invention is utilized
Genetic engineering means, clone have obtained white poplar gene Paidi, are applied in escherichia coli, large intestine
Bacillus produces isoprene and the ability of lycopene is significantly increased than original strain, and the present invention is right
It is efficiently effective there is provided one in terpenoid large-scale industrial production is carried out using microorganism
Enzyme.
Description of the drawings
Fig. 1 is the sepharose electrophoresis result of white poplar total serum IgE；
Fig. 2 is the gas chromatograph results of isoprene standard substance；
Fig. 3 is the gas phase that white poplar Paidi genes are applied in escherichia coli isoprene engineering bacteria
Testing result；
Fig. 4 is the full wavelength scanner result of Pure Lycopene；
Fig. 5 is that white poplar Paidi genes convert the complete of 8h in escherichia coli lycopene engineering bacteria
Length scanning result；
Fig. 6 is that white poplar Paidi genes convert 24h in escherichia coli lycopene engineering bacteria
Full wavelength scanner result.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the reality for being given
Example is applied only for illustrating the present invention, rather than in order to limit the scope of the present invention.In following embodiments
Experimental technique, if no special instructions, be conventional method.Material used in following embodiments,
Reagent etc., if no special instructions, commercially obtains.
In following embodiments, escherichia coli BW25113 (Baba T et al.Mol Syst Biol.
2006；2:2006.0008. it is) one plant of nonpathogenic bacteria, genetic background understands, generation time is short, hold
Easily cultivate and culture medium raw material is cheap.The escherichia coli BW25113 public can be from the micro- life of the Chinese Academy of Sciences
Thing institute obtain, the above biomaterial only attach most importance to duplicate invention related experiment used by, no
Can use as other purposes.
Embodiment 1：The acquisition of genetic fragment
1. Cortex Populi dividianae blade total serum IgE is extracted
Collection Cortex Populi dividianae blade, is extracted using RNeasy Plant Mini Kit (Qiagen companies)
Poplar leaf total serum IgE, is carried out according to test kit illustration method, carries out electrophoresis (Fig. 1) checking RNA
Extract quality, it is seen that RNA integrity is good, can carry out subsequent experimental.
The preparation of 2.RACE-Ready cDNA
The reverse transcription system of the first chains of RACE-Ready cDNA is as follows：
The method for obtaining cDNA total lengths is SMARTer-RACE, is usedPCR cDNA
Synthesis Kit (Clontech companies) are carried out, and primer used below and reagent remove GSP
It isThere is provided in PCR cDNA Synthesis Kit, according to test kit illustration method
Carry out.
The reverse transcription system of the first chains of RACE-Ready cDNA is as follows：
3. the design of gene-specific primer：
According to the plant such as the microorganisms such as escherichia coli, saccharomyces cerevisiae and gum, cocoa tree, Amygdalus communis Lamarck
IDI aminoacid sequences conserved region, with reference to all known idi genes nucleotide sequence design base
Because of specific primer (GSP), RACE-Ready cDNA do masterplate, GSP and universal primer
(Universal Primer Mix, UPM) does primer and is expanded, and can obtain 3 '-RACE cDNA
Fragment and 5 '-RACE cDNA fragments.
Totally 5 GSP sequences, such as following table：
Embodiment 2：The acquisition of Paidi gene coding regions total length
1. the acquisition of 3 '-RACE cDNA end sequences
The use of the 3 '-RACE-Ready cDNA of white poplar is masterplate, UPM entered for primer with GSP
Row amplification
Reaction system：
Reaction condition：
The agarose gel detection of 3 '-RACE can see there is a single bright white poplar DNA
Amplified band, connects carrier T, and transformed competence colibacillus cell selects positive colony Sanger sequencings,
Obtain 3 ' end cDNA sequences.
2. the acquisition of 5 '-RACE cDNA end sequences
The use of the 5 '-RACE-Ready cDNA of white poplar is masterplate, UPM entered for primer with GSP
Row amplification.
Reaction system：
Reaction condition：
The agarose gel detection of 5 '-RACE obtains single bright amplified band, selects one to connect
Carrier T is connect, transformed competence colibacillus cell selects positive colony Sanger sequencings, obtains 5 ' end cDNA
Sequence.
3. the acquisition of full length sequence
According to 3 '-RACE and 5 '-RACE sequencing results, sequence alignment is carried out, obtain the gene
CDNA full length sequences (sequence shown in SEQ ID No.1), are analyzed to DNA, aminoacid sequence：
The gene has 705bp, encodes 234 aminoacid, has ATG initiation codon and TGA to terminate close
Numeral, illustrates the integrity of the gene；Homologous comparison is carried out in NCBI using BLAST softwares,
As a result show that the gene is Nudix_hydrolase superfamily members, functional areas are defined as
Idi, with Populus euphratica (Populus euphratica) isopentenyl-diphosphate
Delta-isomerase I homologys reach 98%；With comospore poplar (Populus trichocarpa)
IPP isomerase family protein have 98% homology, with rubber tree (Hevea
Brasiliensis) the isopentenyl-diphosphate of (Populus trichocarpa)
Delta-isomerase has 92% homology, and illustrate that we obtain is that isopentenyl pyrophosphate is different
Structure enzyme gene, is abbreviated as Paidi genes, has obtained encoding the aminoacid sequence (SEQ of PAIDI albumen
Sequence shown in ID No.2).
Embodiment 3：Escherichia coli isoprene produces the structure of bacterial strain
1. coli expression carrier p2-0 builds
Total length primer sequence is as follows：
The use of MB-F and MB-R is primer, p2 plasmids are masterplate, and genetic fragment MB of acquisition carries out
SalI and PstI (TAKARA companies) double digestion, and by pSB1c expression vector (this laboratory
Build, arabinose inducible promoter, sequence sequence as shown in SEQ ID No.3) carry out XhoI
With PstI double digestions, MB genetic fragments have obtained p2-0 after being connected to pSB1c carriers, convert to
Trans5 α competent cells, choose positive colony and are sequenced, and the nucleotide sequence of p2-0 is SEQ
ID No.4。
2. coli expression carrier p2-paidi builds
Total length primer PAFa and PARa sequence is as follows：
PAFa：5'ATCGGctgcagATGGGTGACGCTCCTGATGC 3'
PARa：5'ATCCGctgcagTCAAGTCAGCTTGTGAATCGC 3'
The Paidi genetic fragments obtained using primer PAFa and PARa are carried out PstI, and (TAKARA is public
Department) single endonuclease digestion, and by p2-0 expression vector PstI single endonuclease digestions, Paidi genetic fragments are connected to bat
P2-paidi is obtained after -0 carrier, has been converted to trans5 α competent cells, chosen positive gram
Grand to be sequenced, the nucleotide sequence of p2-paidi is SEQ ID No.5.
3. isoprene produces the structure of bacterial strain MV/paidi
By the p2-paidi for building and plasmid p1 and pBAD-SbispS cotransformation to BW25113
Host obtains isoprene production bacterial strain MV/paidi.
And control strain MV/ecidi is built, method is by p2 and plasmid p1 and pBAD-SbispS
Corotation obtains escherichia coli idi genetic contrast bacterial strain MV/ecidi to BW25113 host.
In the construction method of above-mentioned isoprene production bacterium, p1, p2 comprising isoprene route of synthesis-
Mevalonic acid (MVA) pathway gene.Wherein p1 is by MvaE (S-acetyl-coenzyme-A Acetylase)
Gene, MvaS (HMG- acetyl-CoA-synthetases) genes and MVK (E.C. 2.7.1.36) gene
Composition, the albumen that aminoacid sequence of the MvaE gene codes shown in SEQ ID No.6 is constituted
Matter；The protein that aminoacid sequence of the MvaS gene codes shown in SEQ ID No.7 is constituted；
The protein that aminoacid sequence of the mvk gene coding shown in SEQ ID No.8 is constituted.p2
By PMK (phosphomevalonate kinase) gene, MVD (pyrophosphoric acid mevalonic acid decarboxylase) gene
And idi (Isoprenoid isomerase) genomic constitution, the PMK gene codes are by SEQ ID
The protein of the aminoacid sequence composition shown in No.9；The MVD gene codes are by SEQ ID No.10
The protein of shown aminoacid sequence composition；The idi gene codes are by SEQ ID No.11 institutes
The protein of the aminoacid sequence composition for showing.
Wherein, p1 be streptomycin resistance arabinose-inducible expression vector, the nucleotide sequence of p1
It is SEQ ID No.12, comprising MVA upstream pathway expression casettes, MVA upstream pathway gene tables
Up to box nucleotide sequence be SEQ ID No.12 1307-5821 positions, SEQ ID No.12's
89-964 positions are Arabinose promoter, and the 5930-6087 positions of SEQ ID No.12 are TrrnB
Terminator, the 1307-3729 positions of SEQ ID No.12 are the coded sequences of MvaE genes, SEQ
The 3730-4904 positions of ID No.12 are the coded sequences of MvaS genes, SEQ ID No.12's
4905-5821 positions are the coded sequences of mvk gene.
P2 is chlorampenicol resistant arabinose-inducible expression vector, and the nucleotide sequence of p2 is SEQ
ID No.13, comprising MVA downstream pathway expression casettes, the core of MVA downstream pathway expression casettes
Nucleotide sequence is the 1309-4442 positions of SEQ ID No.13, the 89-964 of SEQ ID No.13
Position is Arabinose promoter, and the 4569-4726 positions of SEQ ID No.13 terminate for TrrnB
Son, the 1309-2661 positions of SEQ ID No.13 are the coded sequences of PMK genes, SEQ ID No.13
2677-3864 positions be MVD genes coded sequence, the 3894-4442 of SEQ ID No.13
Position is the coded sequence of idi genes.
PBAD-SbispS be ammonia benzyl resistance arabinose inducible expression's carrier, its sequence such as SEQ ID
Shown in No.20, comprising Salix babylonica L. isoprenoid synthase gene, escherichia coli are made to possess production isoamyl two
The ability of alkene.
Embodiment 4：Application of the Paidi genes in escherichia coli isoprene engineering bacteria
1st, the detection of Escherichia coli fermentation product
Above-mentioned colibacillus engineering MV/paidi and the MV/ecidi fermentations of control bacterium, method are as follows：
By engineering bacteria with centesimal inoculum concentration be transferred to 30mL (500mL triangular flasks) containing streptomycin,
In arabinose self-induction culture medium (ZYM) of chloromycetin and ammonia benzyl resistance, 30 DEG C, 280rpm trainings
After foster 20h.4 DEG C, 4000rpm is collected by centrifugation bacterium solution, with the M9 culture medium containing 4% glucose
It is resuspended to take 1mL resuspended bacterium solutions and be placed in 20mL ml headspace bottles to 60OD cell concentrations, 37 DEG C, 280rpm
Concussion and cultivate 30h.
Self-induction culture medium self-induction culture medium ZYM formula containing streptomycin, chloromycetin and ammonia benzyl
It is as follows：100mL A+2mL B+2mL C+200 μ L D+100 μ L E (are quality percentage below
Specific concentration)；
A.ZY：1% tryptone, 0.5% yeast powder；
B.50×M：1.25M Na2HPO4,1.25M KH2PO4,2.5M NH4Cl and 0.25M Na2SO4；
C.50×5052：25% glycerol, 2.5% glucose, 10% Lactose；
D.1M MgSO4；
E.1000 × trace element：50Mm FeCl3,20mM CaCl2,10mM MnCl2,10mM ZnSO4,
The each 2mM of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3；
Streptomycin:Final concentration 50mg/L, chloromycetin final concentration 34mg/L, ammonia benzyl final concentration 100mg/L.
M9 culture medium prescriptions are as shown in Molecular Cloning:A Laboratory guide (Science Press) third edition page 1595.
After reaction terminates, gas chromatogram (GC) analysis is carried out, the gas chromatographicanalyzer for using is
Agilent 7890A GC Sysytem and Agilent7697A headspace Sampler head spaces enter
Sample device, gas phase detached dowel are HP-5.Head-space sampling method is as follows, Time：GC cycle time 20min,
Vial equib time 6min；Temperature(℃)：Oven 51,Loop/Valve 55,
Transfer line 60.GC methods are as follows：Flow velocity：2mL/min, 0min～50 DEG C of 4min,
280 DEG C of 50～280 DEG C of 4min～8.5min, 8.5min～10.6min.
Under the method, isoprene standard substance (Sigma companies) appearance time is 1.75min (Fig. 2),
The GC chromatograms (Fig. 3) of colibacillus engineering MV/paidi and negative control bacterium MV/ecidi,
It can be seen that MV/paidi yield lifts 3.71 times than the yield of MV/ecidi, reach 7.8g/L's
Yield.
Embodiment 5：Escherichia coli lycopene produces the structure of bacterial strain
1. the structure of coli expression carrier pSB1s-paidi
Total length primer PAFa and PARa sequence is as follows：
PAFa：5'atCGGctgcagATGGGTGACGCTCCTGATGCT 3'
PARa：5'atCCGctgcagTCAAGTCAGCTTGTGAATCGC 3'
The use of primer PAFa and PARa Cortex Populi dividianae cDNA is the Paidi genes that masterplate is obtained, will obtain
Fragment carry out PstI (TAKARA companies) single endonuclease digestion, and pSB1s expression vectors are carried out into PstI
Single endonuclease digestion, Paidi genetic fragments have obtained pSB1s-paidi after being connected to pSB1s carriers, conversion
To trans5 α competent cells, choose positive colony and be sequenced, the nucleoside of pSB1s-paidi
Acid sequence is SEQ ID No.14.
2. the structure of coli expression carrier pSB1s-ecidi
Total length primer ECFa and ECRa sequence is as follows：
ECFa：5'ATCGGCTGCAGAAGGAGATATAATGCAAACGG 3'
ECRa：5'ATCCGCTGCAGTTATTTAAGCTGGGTAAATG 3'
The use of ECFa and ECRa is primer, escherichia coli BW25113 bacterium solutions are masterplate, and amplification obtains
The fragment of acquisition is carried out PstI (TAKARA companies) single endonuclease digestion by the Ecidi genes for obtaining, and will
PSB1s expression vectors carry out PstI single endonuclease digestions, after Paidi genetic fragments are connected to pSB1s carriers
PSB1s-ecidi is obtained, has been converted to trans5 α competent cells, choosing positive colony has been carried out
Sequencing, the nucleotide sequence of pSB1s-ecidi is SEQ ID No.15.
3. lycopene produces the structure of bacterial strain BW/paidi
The pSB1s-paidi for building is obtained to BW25113 host with plasmid pL10-2 cotransformations
Lycopene produces bacterial strain BW/paidi10.
And control strain BW/ecidi is built, method is by pSB1s-ecidi and plasmid pL10-2
Corotation obtains escherichia coli idi control strain BW/ecidi10 to BW25113 host.
In the construction method of above-mentioned lycopene production bacterium, pL10-2 nucleotide sequences such as SEQ ID
Shown in No.19.Gene comprising lycopene route of synthesis.The lycopene is synthesis related
Reason crtE (window cattle window cattle base pyrophosphate synthetase) gene, crtI (phytoenes
Dehydrogenase) gene, crtB (phytoene synthetase) genomic constitution；The crtE genes
The protein that aminoacid sequence of the coding shown in SEQ ID No.16 is constituted；The crtI genes
The protein that aminoacid sequence of the coding shown in SEQ ID No.17 is constituted；The crtB genes
The protein that aminoacid sequence of the coding shown in SEQ ID No.18 is constituted.
In the construction method of above-mentioned product neurosporene recombinant bacterium, the coded sequence of the crtE genes is
The 1995-2906 positions of SEQ ID No.19；The coded sequence of the crtI genes is SEQ ID
The 2924-4402 positions of No.19；The coded sequence of the crtB genes is SEQ ID No.19
4420-5349 positions.
Application of the 4.Paidi genes in the production of escherichia coli lycopene
(1) fermentation and conversion of lycopene engineering bacteria are produced
Above-mentioned colibacillus engineering BW/paidi10 and the BW/ecidi10 fermentations of control bacterium, method
It is as follows：Engineering bacteria is transferred to 30mL (500mL triangular flasks) with centesimal inoculum concentration to contain
In the arabinose self-induction culture medium (formula is ibid) of streptomycin and chloromycetin, 30 DEG C, 280rpm
After culture 20h.4 DEG C, 4000rpm is collected by centrifugation bacterium solution, is cultivated with the M9 containing 4% glucose
Base is resuspended to 1OD cell concentrations, takes 2mL resuspended bacterium solutions and is placed in 20mL triangular flasks, 37 DEG C, 280rpm
Concussion and cultivate 24h, samples respectively at 8h and 24h.
Sampling method is as follows：Take 1 × 108Cfu thalline add 1ml acetone to be extracted, 4 DEG C of placements
3 hours, centrifuging and taking supernatant obtained testing sample, carries out the length scanning of 474nm.Experiment
In triplicate, every kind of bacterium carries out above-mentioned glucose conversion in 3 20ml triangular flasks every time.
(2) produce the measure of the yield of lycopene of lycopene engineering bacteria
Carried out by standard substance of lycopene (Sigma companies) using standard curve method (external standard method)
The content of lycopene in quantitative analyses testing sample.After length scanning, standard substance go out at 474nm
Existing summit (Fig. 4), this two plants are produced lycopene engineering bacteria sample and highest occur at 474nm
Peak, Fig. 5 be convert 8h after lycopene full wavelength scanner result, Fig. 6 for conversion 24h after kind
The full wavelength scanner result of lycopene.
The colibacillary yield of lycopene of quantitative analyses, as shown in table 1, shows BW/paidi10
Apparently higher than control bacterium BW/ecidi10, the yield of 8 hours is the 3.48 of control to the yield of lycopene
Times, the yield of 24 hours is 3.06 times of control.
Table 1
Claims (7)
1. a kind of Isopentenyl diphosphate isomerase gene, is characterized in that following (a) or (b)
Gene：
A the nucleotide sequence of () described gene cDNA is as shown in the sequence 1 of sequence table；
B () described gene is the gene for encoding following protein：Ammonia shown in the sequence 2 of sequence table
Through replacing, lacking or add one or several aminoacid and have isopentene group burnt in base acid sequence
The protein derived that aminoacid sequence shown in the sequence 2 by sequence table of phosphate isomerase activity is constituted
Protein.
2. the protein of Isopentenyl diphosphate isomerase gene expression as claimed in claim 1,
It is characterized in that the protein of following (a) or (b)：
A protein that the aminoacid sequence of () shown in the sequence 2 of sequence table is constituted；
Through replacing, lacking or add one or several in the aminoacid sequence of (b) in (a)
Aminoacid and with Isopentenyl diphosphate isomerase activity by protein derived from (a)；
The protein of the aminoacid sequence composition shown in the sequence 2 of the sequence table is by sequence table
Base sequence shown in sequence 1 is encoded.
3. a kind of protokaryon table containing Isopentenyl diphosphate isomerase gene as claimed in claim 1
Up to carrier.
4. it is a kind of to contain Isopentenyl diphosphate isomerase gene prokaryotic as claimed in claim 3
The product isoprene engineering bacteria of carrier.
5. it is a kind of to contain Isopentenyl diphosphate isomerase gene prokaryotic as claimed in claim 3
The product lycopene engineering bacteria of carrier.
6. it is as claimed in claim 4 to produce application of the isoprene engineering bacteria in isoprene is prepared.
7. it is as claimed in claim 5 to produce application of the lycopene engineering bacteria in lycopene is prepared.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114173817A (en) * | 2019-05-31 | 2022-03-11 | 塔夫茨大学信托人 | Cultured meat product using genetically modified cells |
| CN115247182A (en) * | 2022-02-25 | 2022-10-28 | 江南大学 | Preparation method and application of isopentenyl pyrophosphate isomerase mutant |
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| CN115247182B (en) * | 2022-02-25 | 2024-03-15 | 江南大学 | Preparation method and application of isopentenyl pyrophosphoric acid isomerase mutant |
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