CN109609424A - For producing the Escherichia coli of farnesene - Google Patents

Abstract

The present invention constructs a kind of β-farnesene production bacterium by genetic engineering, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.16709.β of the invention-farnesene production bacterium can be realized the effective accumulation of β-farnesene in fermentation liquid by fermentation, and β-farnesene yield is up to 250mg/L, has industrial prospect.

Description

For producing the Escherichia coli of farnesene

Technical field

The invention belongs to genetic engineering fields, specifically, being related to a kind of genetic engineering production bacterium of high yield β-farnesene.

Background technique

Aphid secrets out of a kind of " alarm pheromones " in the Shi Kecong cornical that gets a fright, so that surrounding other aphids perception And escape rapidly, to stop the infringement to crop.Bowers is equal to separation for the first time in 1972 and identifies Aphid Alarm Pheromone Trans-beta-farnesene (E- β-farnesene, abbreviation E β F or EBF), E β F is a kind of sesquiterpenoids substance.The discovery such as Francis EBF be 16 kinds of Aphid Alarm Pheromones main or sole component (Bowers et al., 1972；Francis et al., 2005).The parapheromone is applied to field, can make the control of a certain range of aphid insect density within thresholding, at this time worm, Influence of the disease to crop is little.E β F be not only present in aphid alarm hormone in and many Secondary metabolites it One.E β F ingredient is rich in many plants, purifying by classification can get high concentration essential oil, such as Labiatae Hemizygia E β F component content is up to 70% or more (Bruce et al., 2005) in petiolata plant；Umbelliferae Pimpinella E β F ingredient also has higher content (Askari et al., 2013) in the stem of khorasanica plant, flower and seed.In addition to Except alarm activity, EBF also has other multi-biological activity, such as has the function of similar juvenile hormone III to certain insects Can, have the function of regulation aphid offspring in alatae and wingless aphid ratio, high dose using when have obvious poisoning to aphid Effect etc..Therefore, prevention and control of aphids is carried out using the biological characteristics of natural EBF, has dosage few, specificity is strong, to non-target The advantages that biology and Environmental security, cause the favor of plant protection expert and agricultural producer.Meanwhile based on natural EBF Structure and transformation are carried out, EBF analog is made to develop hypotoxicity or avirulent novel pesticide is the research of field of agricultural sciences One of hot spot.

In addition, it has been found that β-farnesene is other than it can be applied to pesticide field, also in chemical field and medicine neck Domain has many purposes, for example for synthesising complex E etc., has a vast market value, receive the wide of people in recent years General concern.

Content of the farnesene in plant is very low, can not be prepared in a manner of extracting natural products；Chemical synthesis Cost it is high, and purity is lower；Thus seem that becoming people utilizes this native compound using production in microorganisms Buddhist nun's alkene Best mode.The maximum bottleneck of microbe fermentation method is, the expression of enzyme relevant to its biosynthesis in microbial cells It is horizontal lower.Therefore, it is to move towards work that building, which high-level can synthesize β-farnesene and be suitable for the engineering bacteria of industrial applications, The key of industry.

Summary of the invention

In order to overcome the horizontal low defect of existing β-farnesene production strain fermentation, the present invention using technique for gene engineering come Transformation produces the Escherichia coli of isoprene, by enhancing gene relevant to β-farnesene generation, replacement isoprene branch generation It thanks to approach, obtains a plant height and produce β-farnesene production bacterial strain.

Therefore, the first purpose of this invention is to provide a kind of β-farnesene production bacterium.

Second object of the present invention is to provide the β-farnesene production bacterium for producing the application of β-farnesene.

Third object of the present invention, which is to provide, a kind of produces β-farnesene method.

In order to achieve the above object, the present invention carries out gene relevant to β-farnesene metabolic pathway in Escherichia coli Systematic research and screening, and

Design the construction of genetic engineering and screening scheme included the following steps:

A. plasmid PHGFH is constructed, it includes petH, petF, ispG, ispH, duplicate field p15A genes；

Plasmid PAGEs is constructed, it includes mvaE, mvaS, fldA, ispG, replicon repA genes；

Plasmid pAKF is constructed, it includes the ispA of Escherichia coli, the mvaK (base in geneva sarcina methanica source Because being had been described in document Chen Yang, et al., Metabolic Engineering.37 (2016) 79-91.), sequence Codon optimization bfs (β-farnesene synthase gene, β-Farmesene synthase gene), duplication for SEQ ID NO:1 Sub- ColE1 gene；

B. it will enter Escherichia coli to plasmid PHGFH, PAGEs and pAKF corotation constructed in step A, can preferably produce isoamyl In the Escherichia coli of diene, the positive colony comprising gene mvaE, mvaS, mk, pmk, pmd, idi, ispA and bfs is obtained, The middle Escherichia coli for producing isoprene include gene mk, pmk, pmd and idi of mevalonate pathway；

C. from positive colony constructed in step B, production β-farnesene bacterial strain is filtered out.

Preferably, the Escherichia coli for isoprene being produced described in step B are document (Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.Chen Yang,et al.,Metabolic Engineering,37 (2016) 79-91) bacterial strain CIBTS1758 described in.

It is constructed by said gene engineering, filters out a kind of bacterial strain of high yield β-farnesene, be now preserved in China Microbiological Culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.16709.

The genetic engineering bacterium CGMCC No.16709 of building include related gene mvaE, mvaS of mevalonate pathway, mk, Bfs (β-the farnesene that pmk, pmd, idi and farnesene synthesis related gene ispA and sequence are SEQ ID NO:1 Synthase gene, β-Farmesene synthase gene), wherein the Escherichia coli for producing isoprene are substituted in gene ispA Original ispS gene in CIBTS1758.

According to the second aspect of the invention, said gene engineering bacteria CGMCC No.16709 is provided in β-farnesene Application in production.

In one embodiment, β-farnesene is produced by being fermented to above-mentioned β-farnesene production bacterium.

Wherein, culture medium used when fermentation can be any culture medium suitable for Escherichia coli Growth fermentation.

In a preferred embodiment, include when above-mentioned β-farnesene production bacterium is fermented seed growth phase and Thallus fermentation stage (production fermentation stage).The two stages use seed culture medium and production fermentation medium respectively, can be with It is not identical.

Preferably, seed culture medium is, for example, LB culture medium, may include 100 μ g/ml ampicillins, 34 μ g/ml Chloramphenicol and 100 μ g/ml spectinomycins.It is, for example, V7S culture medium that seed, which expands culture medium, may include 100 μ g/ml ammonia benzyls Penicillin, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins.Producing fermentation medium is, for example, V7E culture medium, be can wrap Containing 100 μ g/ml ampicillins, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins and 0.1mM IPTG.

The V7S culture medium composition is as follows: glucose 10g/L, yeast powder 5g/L, MOPS 0.1M/L, (NH₄)₂SO₄ 5g/ L, Na₂HPO₄1g/L, KH₂PO₄0.4g/L, MgSO₄0.5g/L, CaCl₂·2H₂O 0.1g/L, vitamin B12 mg/L, 100 Times trace element solution (Citric Acid Mono 10g/L, MnSO₄·7H₂O 2g/L,FeSO₄·7H₂O 0.5g/L,ZnSO₄·7H₂O 0.2g/L,CuSO₄·5H₂O 0.05g/L,0.05g CoCl₂·6H₂O,Na₂MoO₄·2H₂O 0.05g/L)。

The V7E culture medium composition is as follows: glucose 5g/L, yeast powder 1g/L, MOPS 0.1M/L, (NH₄)₂SO₄ 5g/ L, Na₂HPO₄1g/L, KH₂PO₄0.4g/L, MgSO₄0.5g/L, CaCl₂·2H₂O 0.1g/L, vitamin B1 2mg/L, 100 Times trace element solution (Citric Acid Mono 10g/L, MnSO₄·7H₂O 2g/L,FeSO₄·7H₂O 0.5g/L,ZnSO₄·7H₂O 0.2g/L,CuSO₄·5H₂O 0.05g/L,0.05g CoCl₂·6H₂O,Na₂MoO₄·2H₂O 0.05g/L).Institute's structure of the present invention It builds β-farnesene production bacterium CGMCC No.16709 and the effective accumulation of β-farnesene in fermentation liquid, β-method can be realized by fermentation Buddhist nun's alkene yield is up to 10mg/L, has industrial applications prospect.

The β that the present invention constructs-farnesene high-yield genetic engineering bacterium Classification system is Escherichia coli, Chinese name Title is escherichia coli, that is, Escherichia coli have been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, preservation date are on November 05th, 2018, and preservation address is the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Institute of microbiology, deposit number are CGMCC No.16709.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of plasmid pAKF.

Fig. 2 is the metabolic pathway variation schematic diagram for the Escherichia coli CIBTS2509C that the present invention constructs.Original strain CIBTS1758 is isoprene producing strains, comprising being overexpressed MEP approach, relative to more original E. coli BL21 For, which introduce Eukaryotic MVA approach.On the basis of isoprene producing strains CIBTS1758, through base of the invention After engineered, farnesyl pyrophosphate synthase IspA is replaced into isoprene synthase IspS, and introduce β-Farmesene synthase BFS (its encoding gene be sequence be SEQ ID NO:1 bfs), enhance gene relevant to β-farnesene generation, be substituted it is different Pentadiene branched metabolic pathway, to construct farnesene synthesis bacterial strain.

Specific embodiment

The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for The bright present invention is not for limiting the scope of the invention.

Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.

Unlike the prior art, in order to provide a kind of high yield β-farnesene, be effectively reduced β-farnesene production cost, It is suitable for the engineering bacteria of industrial fermentation, it to be bacterium germination with the Escherichia coli of high yield isoprene that the present invention is based on metabolic engineerings, By blocking the synthesis of isoprene, and farnesyl pyrophosphate synthase IspA and β-Farmesene synthase gene of high efficient expression, obtain The genetic engineering bacterium obtained can realize the effective accumulation of β-farnesene in low-cost fermentation culture medium.Specifically, the present invention with Isoprene producing strains CIBTS1758 is bacterium germination, and introduce high efficient expression comes from Eukaryotic mevalonate pathway (MVA approach), while phosphoric acid methyl erythrite approach (MEP approach) in host cell is enhanced, it is closed for increasing β-farnesene At the supply of precursor I PP (isovaleryl pyrophosphoric acid), DMAPP (dimethylallyl pyrophosphoric acid) and FPP (farnesyl pyrophosphate), and By a large amount of screenings to positive colony bacterial strain, satisfactory bacterial strain CIBTS2509C has been obtained.

Herein, for the present invention, term " β-farnesene genetic engineering produces bacterium ", " β-farnesene genetic engineering Bacterium ", " farnesene produces bacterium ", " Escherichia coli CIBTS2509C ", " CIBTS2509C bacterial strain " indicate identical meaning, all refer to β-farnesene produces bacterium CGMCC No.16709.

Herein, term " Escherichia coli CIBTS1758 ", " bacterial strain CIBTS1758 ", " CIBTS1758 " indicate identical Meaning, all refer to as farnesene production bacterium CGMCC No.16709 building basis original strain CIBTS1758, by Shanghai The building of industrial biotechnology research and development centre and preservation, in document Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Have in Escherichia coli.Chen Yang, et al., Metabolic Engineering 37 (2016) 79-91. in detail Thin description.Bacterial strain CIBTS1758 is to be constructed and obtained, genotype BL21, glmS- based on E. coli BL21 pstS::P_L*MK_MM PMK_SC PMD_SC idi_SC,Δidi::PGI*idi_SC,P_L**dxs,PGI*dxr。

The present invention is by entering plasmid PHGFH, PAGEs and pAKF corotation in Escherichia coli CIBTS1758, by acquisition Positive clone strain CIBTS1758/PHGFH/PAGEs/pAKF screened, be named as CIBTS2509C, it is raw to obtain farnesene Produce bacterium CGMCC No.16709.It includes gene mvaE, mvaS, mk, pmk, pmd, idi, ispA and bfs.Wherein gene mvaE, MvaS is to be transformed into host CIBTS1758 to obtain by plasmid PHGFH and PAGEs, and gene mk, pmk, pmd, idi are The mevalonate pathway related gene that host CIBTS1758 includes, gene ispA and bfs are to be transformed into host by plasmid pAKF It is obtained in CIBTS1758.

Control strain as the comparison of farnesene production capacity is by entering greatly plasmid PHGFH, PAGEs and pAK corotation The bacterial strain CIBTS1758/PHGFH/PAGEs/pAK obtained in enterobacteria CIBTS1758.Since plasmid pAKF includes and method Buddhist nun Alkene synthesizes relevant gene ispA and sequence is the bfs of SEQ ID NO:1, and plasmid pAK includes a kind of and farnesyl pyrophosphate It synthesizes relevant gene ispA but does not include bfs gene, this gene difference makes Escherichia coli CIBTS2509C's to have β- Farnesene production capacity, and control strain does not have this ability.

Embodiment

Material and method

Full genome synthesis, primer synthesis and sequencing herein is all completed by Nanjing Genscript Biotechnology Co., Ltd..

Molecular biology experiment herein include plasmid construction, digestion, competent cell preparation, conversion etc. referring especially to " Molecular Cloning:A Laboratory guide " (third edition), J. Pehanorm Brooker, D.W. Russell (beauty) write, and Huang Peitang etc. is translated, and science goes out Version society, Beijing, 2002) it carries out.For example competent cell method for transformation and competence preparation method are referring to " Molecular Cloning: A Laboratory Guide " (third edition) the 1st chapter page 96 progress.Specific experiment condition can be determined by simple experiment when necessary.

Plasmid PHGFH is provided by Shanghai Research and Development Center of Industrial Biotechnology, it includes petH, petF, ispG, ispH, is answered P15A gene in area processed.

Plasmid PAGEs is provided by Shanghai Research and Development Center of Industrial Biotechnology, it includes mvaE, mvaS, fldA, ispG, is answered System repA gene.

Plasmid pSK is provided by Shanghai Research and Development Center of Industrial Biotechnology, and it includes ispS and mvaK genes.

Plasmid pUC57-150-1 is synthesized by Nanjing Genscript Biotechnology Co., Ltd., is SEQ ID it includes sequence The bfs gene of NO:1, the sequence are the Escherichia coli Preference codon of optimization.

Escherichia coli CIBTS1758 is constructed and is provided by Shanghai Research and Development Center of Industrial Biotechnology.

Plasmid PHGFH, plasmid PAGEs and Escherichia coli CIBTS1758 are in document Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.Chen Yang,et al.,Metabolic Engineering 37 It is disclosed in (2016) 79-91..

Main medium:

Seed culture medium is, for example, LB culture medium, may include 100 μ g/ml ampicillins, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins.Seed expand culture medium be, for example, V7S culture medium, may include 100 μ g/ml ampicillins, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins.Producing fermentation medium is, for example, V7E culture medium, may include 100 μ G/ml ampicillin, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins and 0.1mM IPTG.

The V7S culture medium composition is as follows: glucose 10g/L, yeast powder 5g/L, MOPS 0.1M/L, (NH₄)₂SO₄ 5g/ L, Na₂HPO₄1g/L, KH₂PO₄0.4g/L, MgSO₄0.5g/L, CaCl₂·2H₂O 0.1g/L, vitamin B1 2mg/L, 100 Times trace element solution (Citric Acid Mono 10g/L, MnSO₄·7H₂O 2g/L,FeSO₄·7H₂O 0.5g/L,ZnSO₄·7H₂O 0.2g/L,CuSO₄·5H₂O 0.05g/L,0.05g CoCl₂·6H₂O,Na₂MoO₄·2H₂O 0.05g/L)。

The V7E culture medium composition is as follows: glucose 5g/L, yeast powder 1g/L, MOPS 0.1M/L, (NH₄)₂SO₄ 5g/ L, Na₂HPO₄1g/L, KH₂PO₄0.4g/L, MgSO₄0.5g/L, CaCl₂·2H₂O 0.1g/L, vitamin B1 2mg/L, 100 Times trace element solution (Citric Acid Mono 10g/L, MnSO₄·7H₂O 2g/L,FeSO₄·7H₂O 0.5g/L,ZnSO₄·7H₂O 0.2g/L,CuSO₄·5H₂O 0.05g/L,0.05g CoCl₂·6H₂O,Na₂MoO₄·2H₂O 0.05g/L)。

Embodiment 1: the building of plasmid pAKF

Primer sequence information used in plasmid pAKF building is as shown in table 1.

Table 1, primer sequence

In table 1, "-F " in title represents forward direction；"-R " represents reversed.

1.1 using Escherichia coli MG1655 genome as template, using ispA-FG/ispAL-R2 as primer, PCR amplification ispAL Segment, about 969bp (amplification homology arm+ispA+linker).PCR condition: 95 DEG C of denaturation 5min, 95 DEG C of denaturation 30s, 58 DEG C are moved back 32 circulations, 68 DEG C of heat preservations 10min, last 16 DEG C of heat preservations 10min are arranged in fiery 30s, 68 DEG C of extension 1min.

Glue recycles ispAL segment, using ispAL segment as template, using ispA-FG/ispA-RG as primer, and PCR amplification IspALG segment, about 1004bp (amplification homology arm+ispA+linker+ homology arm).PCR condition: 95 DEG C of denaturation 5min, 95 DEG C It is denaturalized 30s, 58 DEG C of annealing 30s, 68 DEG C of extension 1min, 32 circulations, 68 DEG C of heat preservation 10min, last 16 DEG C of heat preservations are set 10min。

PSK plasmid utilizes NcoI/NruI digestion, and glue recycles about 5.4kb segment.IspALG segment using Gibson clone with Above-mentioned 5.4kb recycling segment connection.Junction fragment is transferred to bacillus coli DH 5 alpha competent cell, and it is flat to be coated on ammonia benzyl antibiotic Plate, 37 DEG C of cultures.Picking positive colony, and by positive colony sequencing, obtain plasmid pAK.IspA gene is completed for ispS base The replacement of cause.

1.2 plasmid pUC57-150-1 BamHI/HindIII digestions, the band that recycling size is 1.7kb, and through identical The plasmid pAK segment connection of digestion recycling, junction fragment is transferred to DH5 α competent cell, is coated on ammonia benzyl antibiotic plate, and 37 DEG C culture.Picking positive colony, and by positive colony sequencing, plasmid pAKF is obtained, structure is as shown in Figure 1.

The building of embodiment 2: β-farnesene producing strains

PAKF corotation in plasmid PHGFH, PAGEs, embodiment 1 is directly entered Escherichia coli using electrotransformation method by 2.1 In CIBTS1758, bacterial strain CIBTS1758/PHGFH/PAGEs/pAKF is obtained, CIBTS2509C is named as.

PAK corotation in plasmid PHGFH, PAGEs, embodiment 1 is entered Escherichia coli using electrotransformation method by 2.2 In CIBTS1758, bacterial strain CIBTS1758/PHGFH/PAGEs/pAK is obtained, as control strain.

Embodiment 3: strain fermentation produces β-farnesene

3.1 bacterial strain CIBTS2509C cultivation and fermentations

Choose CIBTS2509 single bacterium drop down onto it is grand containing 100 μ g/ml ampicillins, 34 μ g/ml chloramphenicol and 100 μ g/ml In the LB culture medium of mycin, 37 DEG C, 220rpm is incubated overnight；It is then inoculated by the inoculum concentration of 8%v/v anti-containing above-mentioned three kinds In the V7S culture medium of raw element, 37 DEG C, 220rpm continues to cultivate 8-12h；Then it is inoculated by the inoculum concentration of 8%v/v containing above-mentioned In the V7E culture medium of three kinds of antibiotic and 0.1mM IPTG, 37 DEG C, 220rpm continue cultivate 8-12h；The last of the ten Heavenly stems of 1/5 volume is added Alkane continues to cultivate 4h.12000rpm is centrifuged 10min, collects decane phase, GC-MS is sent to analyze.

The GC analysis method of farnesene is as follows: Aglient 7890A gas chromatograph, is equipped with 5975MS；Chromatographic column Agilent HP-1(30mx0.25mm,0.25μm)；Sample volume: 0.5 μ l；Injector temperature: 280 DEG C, split ratio 10:1；gas saver:20ml/min；Column flow rate: 1ml/min；Carrier gas: high pure nitrogen is pressed before column: 11.724psi, Mean Speed: 26.511cm/sec；Column temperature: (1), 60 DEG C, 0min is kept；(2), 10 DEG C/min is warming up to 150 DEG C, keeps 0min；(3),30 DEG C/min, 280 DEG C are warming up to, 2min is kept.Total run time 15.33min.Detector (FID): temperature: 300 DEG C, hydrogen stream Speed: 40ml/min；Air velocity: 400ml/min；Tail blows 25ml/min；Target peak appearance time: 11.07min.

Confirm through GC-MS, target product β-farnesene yield is 250mg/L.

3.2 control strain CIBTS1758/PHGFH/PAGEs/pAK cultivation and fermentations

According to method identical with above-mentioned steps 3.1, culture hair is carried out to bacterial strain CIBTS1758/PHGFH/PAGEs/pAK Ferment, and detect β-farnesene yield.Confirm through GC-MS, control strain does not produce β-farnesene.

It is demonstrated experimentally that bacterial strain CIBTS2509C has β-farnesene production capacity, β-farnesene in fermentation liquid can be realized High concentration accumulation, have industrial applications prospect.

Sequence table

<110>Zhejiang medicine limited liability company

<120>for producing the Escherichia coli of farnesene

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<170> SIPOSequenceListing 1.0

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<211> 1725

<212> DNA

<213>artificial sequence ()

<400> 1

atgagcaccc tgccgatcag cagcgtgagc agcagcagca gcaccagccc gattgtggtt 60

gacgataaag acagcaccaa gccggatgtt atccgtcaca ccaccaactt caacgcgagc 120

atttggggcg accagtttct gacctacgac gagccggaag atctggtgat gaagaaacaa 180

ctggttgagg aactgaaaga ggaagtgaag aaagagctga tcaccattaa aggtagcaac 240

gaaccgatgc agcacgtgaa gctgatcgag ctgattgacg cggttcaacg tctgggcatc 300

gcgtaccact tcgaggaaga gatcgaagag gcgctgcagc acattcacgt gacctatggc 360

gagcagtggg ttgataaaga aaacctgcaa agcattagcc tgtggttccg tctgctgcgt 420

cagcaaggtt ttaacgtgag cagcggcgtt ttcaaggact ttatggatga gaagggtaaa 480

tttaaggaaa gcctgtgcaa cgacgcgcag ggcatcctgg cgctgtacga agcggcgttc 540

atgcgtgtgg aggacgaaac cattctggat aacgcgctgg agtttagcaa agttcacctg 600

gatatcattg cgaaggaccc gagctgcgat agcagcctgc gtacccagat ccaccaagcg 660

ctgaagcaac cgctgcgtcg tcgtctggcg cgtatcgaag cgctgcacta catgccgatt 720

tatcagcaag agaccagcca cgacgaagtg ctgctgaaac tggcgaagct ggatttcagc 780

gttctgcaga gcatgcacaa gaaagagctg agccacatct gcaaatggtg gaaggacctg 840

gatctgcaaa acaaactgcc gttcgtgcgt gaccgtgtgg ttgaaggcta cttttggatt 900

ctgagcattt actatgagcc gcagcacgcg cgtacccgta tgtttctgat gaagagctgc 960

atgtggctgg ttgtgctgga cgataccttc gacaactacg gtacctatga agagctggag 1020

atctttaccc aagcggtgga aaaatggagc attagctgcc tggatatgct gccggaatac 1080

atgaagctga tctatcagga gctggtgaac ctgcacgttg aaatggaaga gagcctggag 1140

aaagaaggca aggcgtacca aattcactat gttaaagaga tggcgaagga actggtgcgt 1200

aactacctgg ttgaggcgcg ttggctgaaa gaaggttaca tgccgaccct ggaagagtat 1260

atgagcgtga gcatggttac cggtacctac ggcctgatga ccgcgcgtag ctatgtgggt 1320

cgtggcgaca tcgttaacga agataccttc aaatgggtta gcagctatcc gccgatcgtg 1380

aaggcgagct gcgttatcat tcgtctgatg gacgatattg tgagccacaa agaagagcag 1440

gagcgtggtc acgttgcgag cagcattgag tgctacagca aggaaagcgg cgcgagcgaa 1500

gaggaagcgt gcgaatatat cagccgtaaa gtggaagatg cgtggaaggt tattaaccgt 1560

gagagcctgc gtccgaccgc ggtgccgttc ccgctgctga tgccggcgat caacctggcg 1620

cgtatgtgcg aagtgctgta cagcgttaac gacggtttta cccacgcgga gggcgatatg 1680

aaaagctata tgaagagctt ctttgttcac ccgatggtgg tttaa 1725

<210> 2

<211> 53

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attaatgtat cgattaaata aggaggaata aaccatggac tttccgcagc aac 53

<210> 3

<211> 59

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aagcttgcgg atccgccgcc acccgagcca ccgccacctt tattacgctg gatgatgta 59

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<211> 57

<212> DNA

<213>artificial sequence ()

<400> 4

acaggatacc atgttttttt acctccttta tgcagaagct tgcggatccg ccgccac 57

<210> 5

<211> 22

<212> DNA

<213>artificial sequence ()

<400> 5

gcattaagcg ccggagataa ag 22

<210> 6

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 6

cgatcaaatc cgggtagctt tcg 23

Claims (10)

1. a kind of β-farnesene produces bacterium, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, is protected Hiding number is CGMCC No.16709.

2. a kind of construct β as described in claim 1-farnesene production bacterium method comprising following steps:

A. plasmid PHGFH is constructed, it includes petH, petF, ispG, ispH, duplicate field p15A genes；

Plasmid PAGEs is constructed, it includes mvaE, mvaS, fldA, ispG, replicon repA genes；

Plasmid pAKF is constructed, it includes the ispA of Escherichia coli, the mvaK in geneva sarcina methanica source, sequences to be Bfs, the replicon ColE1 gene of SEQ ID NO:1；

B. plasmid PHGFH, PAGEs and pAKF corotation constructed in step A will be entered in Escherichia coli, is obtained comprising gene The positive colony of mvaE, mvaS, mk, pmk, pmd, idi, ispA and bfs；

C. from positive colony constructed in step B, production β-farnesene bacterial strain is filtered out.

3. β as described in claim 1-farnesene production bacterium is for producing the application of β-farnesene.

4. application as claimed in claim 3, which is characterized in that produce the hair of bacterium by β as described in claim 1-farnesene Ferment produces β-farnesene.

5. application as claimed in claim 4, which is characterized in that fermentation includes seed growth phase and thallus fermentation stage.

6. application as claimed in claim 5, which is characterized in that seed culture medium, thallus fermentation medium be not identical.

7. application as claimed in claim 6, which is characterized in that fermentation medium composition is as follows: glucose 5g/L, yeast powder 1g/L, MOPS 0.1M/L, (NH₄)₂SO₄5g/L, Na₂HPO₄1g/L, KH₂PO₄0.4g/L, MgSO₄0.5g/L, CaCl₂· 2H₂O 0.1g/L, vitamin B1 2mg/L, 100 times of trace element solutions；The wherein trace element solution composition an are as follows: water Citric acid 10g/L, MnSO₄·7H₂O 2g/L,FeSO₄·7H₂O 0.5g/L,ZnSO₄·7H₂O 0.2g/L,CuSO₄·5H₂O 0.05g/L,0.05g CoCl₂·6H₂O,Na₂MoO₄·2H₂O 0.05g/L。

8. application as claimed in claim 6, which is characterized in that separately include 100 in seed culture medium, thallus fermentation medium μ g/ml ampicillin, 34 μ g/ml chloramphenicol and 100 μ g/ml spectinomycins.

9. application as claimed in claim 6, which is characterized in that include 0.1mM IPTG in thallus fermentation medium.

10. application as claimed in claim 5, which is characterized in that fermentation temperature is 37 DEG C.