CN103243065B - Bacterial strain for producing farnesene and application of bacterial strain - Google Patents

Abstract

The invention discloses a bacterial strain for producing farnesene and an application of the bacterial strain, belonging to the field of synthetic biology. The bacterial strain for producing the farnesene contains related genes for synthesizing the farnesene through a mevalonic acid pathway and codon optimization; and the sequence of a farnesene synthetic gene afs optimized by codons is as shown by SEQ ID NO.1. The bacterial strain can be used for producing the farnesene, and a seed solution of the bacterial strain is inoculated into a culture medium containing a carbon source to carry out prokaryotic expression to obtain the farnesene. The gene for synthesizing the farnesene is subjected to codon optimization or the farnesene is synthesized by using the mevalonic acid pathway to ensure that each protein is closer to a ratio of AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:Idi:IspA:AFS=1:10:2:5:5:2:5:2:2, so that the production of the farnesene can be further promoted. By adopting the bacterial strain, the output of the farnesene is greatly improved to be more than 1g/L.

Description

A kind of bacterial strain and application thereof of producing farnesene

Technical field

The invention belongs to synthetic biology field, relate to a kind of bacterial strain and application thereof of producing farnesene.

Background technology

Farnesene the earliest from apple tree separation obtain, aspect plant defense, play an important role.Because it is a kind of important industrial chemicals, for example can be used for asynthetic rubber, the more important thing is that farnesene is a kind of precursor of good bioenergy method Buddhist nun alkane, there is wide marketable value, be subject in recent years people's extensive concern.

The content of farnesene in plant is very low, cannot come for Chemical market to extract the mode of natural product, thereby utilize production in microorganisms Buddhist nun alkene to seem to become the best mode that people utilize this compound.The microorganism maximum bottleneck that ferments is how to realize industrialization, and industrialized prerequisite is its output, need to meet can industrialized level.But be only cloned into now a few Farmesene synthase, and its protein expression situation in microorganism is all undesirable, utilizes production in microorganisms Buddhist nun alkene slowly to move ahead always, fail to carry out suitability for industrialized production.Existing report (Wang, C., et al., 2011.Metabolic engineering of Escherichia coli for α-farnesene production.Metabolic Engineering.13,648-655) production peak of farnesene is only 0.38g/L.

Summary of the invention

Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of bacterial strain of producing farnesene.

Another object of the present invention is to provide the application of the bacterial strain of above-mentioned production farnesene.

A further object of the present invention is to provide a kind of method of utilizing above-mentioned bacterial strains to produce farnesene.

The present invention also aims to provide a kind of method that prokaryotic organism produce farnesene ability that improves.

Produce a bacterial strain for farnesene, contain the synthetic genes involved of farnesene that mevalonate pathway (MVA approach) and part or all of codon are optimized, the genes involved of described mevalonate pathway comprises the gene atoB that (1) is acetoacetyl-CoA by acetyl-CoA condensation, (2) the gene erg13 that is HMG-CoA by acetyl-CoA and acetoacetyl-CoA condensation, (3) HMG-CoA is reduced to the gene thmg1 of mevalonic acid, (4) mevalonic acid phosphoric acid is turned to the gene erg12 of mevalonic acid-5-phosphoric acid, (5) mevalonic acid-5-monophosphate monophosphate is turned to the gene erg8 of mevalonate-5-pyrophosphate, (6) gene mvd1 and (7) of mevalonate-5-pyrophosphate decarboxylation generation isopentenylpyrophosphate are tautomerized to isopentenylpyrophosphate to the gene idi of dimethylallylpyrophosphate, the synthetic genes involved of described farnesene comprises that (1) is converted into farnesyl tetra-sodium the gene afs of farnesene by isopentenylpyrophosphate molecule and dimethylallylpyrophosphate molecular combinations with gene ispA and (2) of forming method Thessaloniki tetra-sodium.

The genes involved of described mevalonate pathway is preferably the atoB(CP001509.3:2216470-2217654 that derives from e. coli bl21 (DE3)), idi(CP001509.3:2863926-2864474) and derive from the erg13(329138949:19060-20535 of yeast saccharomyces cerevisiae INVSC1), tHMG1(329138949:115734-117239), erg12(329138949:684467-685798), erg8(329138949:712316-713671), mvd1(329138953:701895-703085).

Described ispA gene is preferably the ispA(CP001509.3:405603-406502 that derives from e. coli bl21 (DE3))

Described afs gene is preferably to derive from the afs gene (Q84LB2) of Malus x domestica (apple) of Malus for carrying out codon optimized obtaining in basis, and its sequence is as shown in SEQ ID NO.1.

Preferably, the bacterial strain of described production farnesene is the intestinal bacteria that contain plasmid pMH1, plasmid pFZ81 and plasmid pFZ38; Described plasmid pMH1 be take pBBR1MCS as skeleton carrier, promotor are as lac promotor, replicon replace with p15A replicon, comprises atoB, erg13 and thmg1 gene, and the sequence of pMH1 (not containing skeleton carrier sequence) is as shown in SEQ ID NO.2; Described plasmid pFZ81 be take the pBBR1MCS replicon that pBBR1MCS-2 carries as plasmid as lac promotor, replicon as skeleton carrier, promotor, comprise erg12, erg8, mvd1 and idi gene, the sequence of pFZ81 (not containing skeleton carrier sequence) is as shown in SEQ ID NO.3; Described plasmid pFZ38 be take pET21a (+) as skeleton carrier, promotor are as T7 promotor, the high copy of replicon pBBR322 replicon, comprises afs and ispA gene, and the sequence of pFZ38 (not containing skeleton carrier sequence) is as shown in SEQ ID NO.4.

Preferred, the bacterial strain of described production farnesene is the intestinal bacteria that contain plasmid pMH1, plasmid pFZ81 and plasmid pFZ71; Described plasmid pFZ71 take pET21a (+) as skeleton carrier, promotor be T7 promotor, the high copy of replicon pBBR322 replicon, comprise afs, ispA and idi gene, the sequence of pFZ71 (not containing skeleton carrier sequence) is as shown in SEQ ID NO.5.

The application of the bacterial strain of above-mentioned production farnesene in producing farnesene.

The method of utilizing the bacterial strain of above-mentioned production farnesene to produce farnesene, comprises following steps: by the bacterial strain seed liquor access carbonaceous sources of above-mentioned production farnesene substratum in carry out prokaryotic expression and obtain farnesene.

During prokaryotic expression, add after inductor IPTG2-4 hour and add again n-decane to carry out biphasic fermentation.

During prokaryotic expression, the ratio of synthetic each albumen of farnesene of MVA approach is preferably AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1-56: 2-16: 2-30: 1-5: 1-5: 2-20: 4-600: 20-40: 2-11; Preferred, AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: 2: 2.

Improve the method that prokaryotic organism produce farnesene ability, codon optimized by the synthetic genes involved of farnesene is carried out, comprise afs, erg8, the genes such as erg12 and erg13; Or make more to approach optimized proportion AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1 by the ratio of synthetic each albumen of farnesene of MVA approach: 10: 2: 5: 5: 2: 5: the further transformation of 2: 2; Or cross one or more gene of appointing of expressing in idi, erg8, mvd1, erg13, erg12 and thmg1 and can realize.

The present invention has the following advantages and effect with respect to prior art tool:

In metabolic pathway, each albumen is not to be only the most efficient according to mole situation of grade, but should maximize according to a certain percentage speed of reaction, the present invention utilizes reconstruction in vitro architectural study to determine that the optimum proportion of synthetic each albumen of farnesene of MVA approach is AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: 2: 2, then pass through the synthetic biology means transformation of rationality more and by MVA approach, produce the ratio of each albumen of farnesene, by controlling the ratio of these albumen, within the shortest time, can improve fast the output of farnesene, the method utilizes the expressing quantity on randomly changing metabolic pathway to have more targeted than traditional genetically engineered, thereby test rationality more, fast, effectively.The present invention simultaneously, by codon optimized synthetic farnesene synthesis related gene afs, can more easily express these genes in heterologous host.By these technology, make more than farnesene shaking flask output reaches 1g/L, to there is good industrial applicability prospect.

Accompanying drawing explanation

Fig. 1 is mevalonate pathway schematic diagram.

Fig. 2 is farnesene route of synthesis schematic diagram.

Fig. 3 is that Idi is beneficial to terpenoid production schematic diagram.

Fig. 4 is the affect figure of synthetic each albumen optimized proportion of farnesene of MVA approach on speed of reaction.

Fig. 5 is plasmid pMH1 schematic diagram.

Fig. 6 is plasmid pFZ81 schematic diagram.

Fig. 7 is plasmid pFZ35 schematic diagram.

Fig. 8 is plasmid pFZ38 schematic diagram.

Fig. 9 is plasmid pFZ71 schematic diagram.

Figure 10 is farnesene producing bacterial strain of the present invention (F0, F1 and F5) schematic diagram, and F0 produces farnesene by MEP approach, and F1 and F5 produce farnesene by MEP and MVA approach.

Figure 11 is that farnesene producing bacterial strain of the present invention (F0, F1 and F5) shake flask fermentation is produced farnesene result figure.

Embodiment

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.

By the corresponding gene that increases, built the mevalonate pathway for escherichia coli expression from intestinal bacteria and genes of brewing yeast group DNA.With producing the path of farnesene by codon optimized and synthetic the structure from Isoprenoid (IPP) and dimethylallyl tetra-sodium (DMAPP) of gene, and realized recombination bacillus coli High-efficient Production farnesene.Correlated response approach is shown in Fig. 1 and Fig. 2.

In embodiment, primer used is as shown in table 1:

The list of table 1 primer

Embodiment 1 reconstruction in vitro system optimization mevalonate pathway

Method (the Yu that reconstruction in vitro system experimental technique is set up with people such as Yu in 2011, X., et al., 2011.In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli.Proceedings of the National Academy of Sciences.108, 18643-18648.), by the research of each albumen of mevalonate pathway is shown to increase ERG13, ERG8, IspA and Idi produce and have promoter action terpenoid, the effect that particularly increases Idi is (Fig. 3) more obviously, the optimized proportion of synthetic each albumen of farnesene of MVA approach that reconstruction in vitro experiment obtains is AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: 2: 2, compare with equimolar each albumen, speed of reaction be significantly improved (Fig. 4).

Embodiment 2 mevalonate pathway plasmid constructions

With the Blood and Cell Culture DNA Mini Kit of Qiagen company, purify and obtain e. coli bl21 (DE3) genomic dna and yeast saccharomyces cerevisiae INVSC1 genomic dna.

Plasmid pMH1 contains first three gene of mevalonate pathway: the atoB gene (acetoacetyl-CoA thioesterase) that derives from e. coli bl21 (DE3), derive from the erg13(HMG-CoA synthase of yeast saccharomyces cerevisiae INVSC1,3-methylol-glutaryl coenzyme A synthase) and thmg1(HMG-CoA reductase enzyme, 3-methylol-glutaryl CoA-reductase, the cross-film region of having deleted HMG1).Plasmid pFZ81 contains four genes after mevalonate pathway: the erg12(Mevalonic kinase that derives from yeast saccharomyces cerevisiae INVSC1), erg8(mevalonic acid-5-phosphokinase) and mvd1(mevalonate-5-pyrophosphate kinases), derive from the idi(isopentenylpyrophosphate isomerase of e. coli bl21 (DE3)) gene.All genes all obtain by pcr amplification, and the primer is in Table 1.

Concrete construction process is as follows:

(1) structure of plasmid pMH1

First utilize simple cloning method (You, C., et al., 2012.Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis.Applied and environmental microbiology.78,1593-1595.), the replicon of pBBR1MCS plasmid is replaced with to the p15A replicon that derives from pMSD15 plasmid.The plasmid pBBR1MCS of take increases and obtains pBBR1MCS fragment with primer pBBR1MCS1-p15A-f and pBBR1MCS1-p15A-r as template, p15A replicon obtains p15A fragment with primer p15A-f and p15A-r amplification (primer sequence is in Table 1) simultaneously, after PCR product purification, with Nanodrop, measure DNA concentration, then the p15A fragment of 20ng pcr amplification and equimolar pBBR1MCS fragment are mixed, through one, take turns pcr amplification, amplification condition is: 98 ℃, 2min denaturation, then 30 PCR circulate 98 ℃, 20s; 60 ℃, 20s; 72 ℃, 6min, last 72 ℃ are fully extended 10min.Transform subsequently E.colistrain XL1 blue and obtain plasmid pBBR1MCS/p15A.

With primer pBBR1MCS1-f and pBBR1MCS1-r, take pBBR1MCS/p15A as template amplification pMH1 plasmid skeleton, use AtoB-f, AtoB-r simultaneously; ERG13-f, ERG13-r; THMG1-f, tHMG1-r is primer amplification corresponding gene.After PCR product purification, get 50ng pMH1 plasmid skeleton and equimolar each gene amplification product mixing, and adjust volume to 5 μ L with deionized water, be added to subsequently the Gibson damping fluid (Gibson of 15 μ L, D.G., 2011.Enzymatic assembly of overlapping DNA fragments.Methods Enzymol.498, 349-361.) in mix, after 50 ℃ of reaction 1h, transform E.colistrain XL1 blue, picking clone, and positive colony order-checking is obtained to plasmid pMH1, this plasmid schematic diagram as shown in Figure 5, sequence (not containing skeleton carrier sequence) is as shown in SEQ ID NO.2.What this plasmid controlling gene was expressed is the lac promotor of medium tenacity, and plasmid replicon is p15A replicon.

(2) structure of plasmid pFZ81

With primer pBBR1MCS2-f and pBBR1MCS2-r, take pBBR1MCS-2 as template amplification pFZ81 plasmid skeleton, use ERG12-f, ERG12-r simultaneously; ERG8-f, ERG8-r; MVD1-f, MVD1-r; Idi-f, Idi-r is primer amplification corresponding gene.After PCR product purification, get 50ng pFZ81 plasmid skeleton and equimolar each gene amplification product mixing, and adjust volume to 5 μ L with deionized water, be added to subsequently in the Gibson damping fluid of 15 μ L and mix, after 50 ℃ of reaction 1h, transform E.colistrain XL1 blue, picking clone, and positive colony order-checking is obtained to plasmid pFZ81, as shown in Figure 6, sequence (not containing skeleton carrier sequence) is as shown in SEQ ID NO.3 for this plasmid schematic diagram.What this plasmid controlling gene was expressed is the lac promotor of medium tenacity, and plasmid replicon is pBBR1MCS replicon.

Embodiment 3 farnesenes produce pathways metabolism plasmid construction

The afs gene that derives from Malus Malus x domestica carries out gene synthetic (sequence is as shown in SEQ ID NO.1) after Nanjing Genscript Biotechnology Co., Ltd. is codon optimized, by NdeI and XhoI restriction enzyme site, be inserted in plasmid pET21a (+) subsequently, acquisition can be produced plasmid pFZ35(Fig. 7 of farnesene).

After IspA gene increase from genome of E.coli DNA with primer I spA-BamHI and IspA-EcoRI-SpeI, after cutting, BamHI and EcoRI enzyme be inserted into acquisition pFZ32 in plasmid pET28a (+).Then by deriving from XbaI-XhoI fragment that pFZ32 contains ispA and be inserted in the SpeI-XhoI fragment of pFZ35, obtain plasmid pFZ38, as shown in Figure 8, sequence (containing skeleton carrier sequence) is as shown in SEQ ID NO.4 for this plasmid schematic diagram.

Idi gene be take BL21 (DE3) genomic dna and is passed through pcr amplification (primer I di-NdeI, Idi-XhoI is in Table 1) as template, and then, by NdeI, XhoI enzyme is inserted into the pET28a(+ cutting through same enzyme after cutting) on plasmid, obtain plasmid pET28-Idi.By deriving from pET28-Idi plasmid XbaI-XhoI fragment containing Idi gene, be inserted in pFZ38 and obtain plasmid pFZ71, as shown in Figure 9, sequence (containing skeleton carrier sequence) is as shown in SEQ ID NO.5 for this plasmid schematic diagram.PFZ35, pFZ38, pFZ71 all be take pET21a(+) and be plasmid skeleton, promotor is T7 strong promoter, replicon is the high copy of pBBR322 replicon.

Embodiment 4 farnesenes produce the structure of bacterium

Directly plasmid pFZ35 is proceeded in e. coli bl21 (DE3), obtain bacterial strain BL21 (DE3)/pFZ35, called after bacterial strain F0.

Utilizing bacterial strain background MEP approach (non-mevalonate pathway) to produce farnesene proceeds to two plasmid pMH1 of mevalonate pathway and pFZ81 simultaneously and in e. coli bl21 (DE3), obtains BL21 (DE3)/pMH1/pFZ81, called after PS(parent strain).PFZ38 and pFZ71 are transformed respectively and entered in bacterial strain PS, obtain bacterial strain F1 and F5.The schematic diagram of bacterial strain F0, F1 and F5 as shown in figure 10.

Embodiment 5 shake flask fermentations are produced farnesene

Respectively single bacterium colony of picking ten F0, F1 and F5 bacterial strain is to containing 2%(v/v) in 2 * TY substratum of glycerine (containing 100 μ g/mL penbritins, 50 μ g/mL kantlex and 34 μ g/mL paraxin) simultaneously, in 30 ℃, 220rpm incubated overnight, subsequently by 1%(v/v) inoculum size is inoculated in fresh same substratum 30 ℃, and 220rpm continues to be cultured to OD ₆₀₀be about at 0.6～0.8 o'clock, adding final concentration is that the IPTG of 0.1mM carries out abduction delivering, adds inductor after 3 hours, adds the n-decane of 1/5 culture volume to carry out biphasic fermentation.After abduction delivering, according to the time point of setting, sample, be sampled as n-decane phase 1mL at every turn, substratum 5mL, collects supernatant after the centrifugal 3min of n-decane phase 8000g and carries out GC-MS analysis, calculates percentage extraction with β-farnesene (sigma) simultaneously.GC-MS analysis condition is as follows: GC-MS is that Agilent 7890A gas-chromatography is equipped with 5975MS, gas chromatographic column is HP-5 post (30m*0.32mm*25 μ m), each sample introduction 1 μ L that analyzes, GC condition is 80 ℃ and maintains 1 minute, the speed of 10 ℃/min is warmed up to 160 ℃, then is warmed up to 260 ℃ with the speed of 20 ℃/min.β-the farnesene of usining carries out quantitatively as standard substance drawing standard curve, result is as shown in figure 11: the generation of farnesene in bacterial strain F0 fermented liquid, can be detected, but underproduce 1mg/L, and F1 and F5 output obviously improve, induce latter 48 hours farnesene output to be respectively 0.2g/L, 1.1g/L is follow-up still to be continued to increase.

Conclusion:

(1) four strain bacterium all can produce farnesene, proves that the recombinant bacterial strain that utilizes mevalonate pathway and non-mevalonate pathway (MEP approach) to produce farnesene that we build is feasible.

(2) F1 bacterial strain output is the more than 400 times of F0 bacterial strain output, proves that the mevalonate pathway that we build is relatively efficient.

(3) F5 bacterial strain output is 50 times of F1 bacterial strain output, proves that overexpression Idi albumen can significantly improve the amount of farnesene.

Embodiment 6 shake flask fermentations are produced each protein quantification of farnesene by MVA approach

It is the same that F1 and F5 cultivate inductive condition, and 100mL bacterium liquid was as a child got in IPTG induction 20, centrifugal collection thalline, and thalline is resuspended in 100mM NH ₄hCO ₃in solution, with Ultrasonic Cell Disruptor smudge cells (pulse 5s, stops 8s, and ultrasonic time is 8min).4 ℃ of centrifugal 30min of 10000g of broken liquid, draw 4 ℃ of supernatants, and 200000g ultracentrifugation 2h carefully draws supernatant to new 1.5mL centrifuge tube, every pipe packing 100 μ L, and after liquid nitrogen flash freezer ,-80 ℃ save backup.With the mass spectrum that utilizes of Keasling group report, carry out protein quantification method (Redding-Johanson subsequently, A.M., et al., 2011.Targeted proteomics for metabolic pathway optimization:application to terpene production.Metabolic Engineering.13,194-203.) to each protein quantification in MVA approach.

Protein quantification result shows that in the synthetic farnesene of MVA approach, each albumen ratio is:

AtoB in F1 bacterial strain: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=10: 16: 2: 1.6: 1: 19: 4: 25: 11;

AtoB in F5 bacterial strain: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=56: 2: 30: 2: 1: 2: 600: 40: 3;

The optimized proportion AtoB that this and reconstruction in vitro system obtain: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: also have certain distance at 2: 2, illustrate that the bacterial strain building does not also reach perfect condition, also have very large production potential to have to be developed, can towards albumen optimized proportion, transform by further transformation.

Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan University

<120> bacterial strain and application thereof of producing farnesene

<130> 1

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 1745

<212> DNA

<213> Artificial Sequence

<220>

<223> AFS(opti) sequence

<400> 1

catatggaat ttcgtgtcca cctgcaagcc gataatgaac aaaaaatctt tcaaaaccaa 60

atgaagccgg aaccggaagc ctcctacctg attaatcaac gtcgctccgc gaactacaag 120

ccgaatattt ggaagaacga ttttctggac cagtccctga tctcaaaata tgatggtgac 180

gaataccgta aactgtcgga aaagctgatc gaagaagtga aaatttacat cagcgcggaa 240

accatggatc tggttgccaa actggaactg attgacagcg tccgtaagct gggcctggca 300

aacctgtttg aaaaagaaat caaggaagct ctggattcga ttgcggccat cgaaagcgac 360

aacctgggta cccgcgatga cctgtatggc acggcgctgc attttaaaat tctgcgtcaa 420

cacggttaca aggtttcgca ggatatcttt ggtcgcttca tggacgaaaa aggcaccctg 480

gaaaatcatc acttcgcgca tctgaagggc atgctggaac tgtttgaagc cagcaacctg 540

ggtttcgaag gcgaagatat tctggacgaa gcgaaagcct ctctgacgct ggccctgcgt 600

gatagtggtc acatctgcta tccggattcg aatctgagcc gcgacgtggt tcattccctg 660

gaactgccgt cacaccgtcg cgttcaatgg tttgatgtca aatggcagat taatgcatac 720

gaaaaggata tctgtcgtgt gaacgcaacc ctgctggaac tggctaaact gaacttcaat 780

gtcgtgcagg ctcaactgca gaagaacctg cgtgaagcga gccgttggtg ggctaatctg 840

ggtattgcgg ataacctgaa atttgcacgt gaccgcctgg ttgaatgctt cgcatgtgct 900

gttggcgtcg cttttgaacc ggaacatagc tctttccgca tttgcctgac caaagtcatc 960

aatctggtgc tgattatcga tgacgtctat gatatttacg gttctgaaga agaactgaaa 1020

cactttacca acgcagtgga tcgttgggac agtcgcgaaa cggaacaact gccggaatgc 1080

atgaaaatgt gtttccaggt gctgtataac accacgtgtg aaattgcccg tgaaatcgaa 1140

gaagaaaacg gctggaatca agtcctgccg cagctgacca aagtgtgggc cgatttttgc 1200

aaggcactgc tggttgaagc tgaatggtat aataaatccc atattccgac gctggaagaa 1260

tacctgcgca acggttgtat cagttcctca gtgtcagttc tgctggtcca tagctttttc 1320

agcattaccc acgaaggcac gaaagaaatg gcggatttcc tgcacaagaa tgaagacctg 1380

ctgtataaca tttctctgat cgtgcgtctg aacaatgatc tgggtacctc cgcagctgaa 1440

caggaacgcg gcgattcacc gtcgagcatc gtgtgctaca tgcgtgaagt taatgcgagt 1500

gaagaaacgg cccgcaaaaa cattaagggt atgatcgata atgcgtggaa aaaggtgaac 1560

ggcaaatgtt ttaccacgaa tcaggttccg tttctgtcta gtttcatgaa caatgccacc 1620

aacatggcgc gcgttgccca tagcctgtat aaagatggcg acggtttcgg cgatcaagaa 1680

aaaggtccgc gtacccacat cctgagcctg ctgttccaac cgctggtcaa ctaactagtc 1740

tcgag 1745

<210> 2

<211> 4515

<212> DNA

<213> Artificial Sequence

<220>

<223> pMH1 sequence (not containing skeleton carrier sequence)

<400> 2

gcgcgtaata cgactcacta tagggcgaat tggagctcca ccgcggtggc ggccgctcta 60

gaactagtgg atccttagga tttaatgcag gtgacggacc catctttcaa acgatttata 120

tcagtggcgt ccaaattgtt aggttttgtt ggttcagcag gtttcctgtt gtgggtcata 180

tgactttgaa ccaaatggcc ggctgctagg gcagcacata aggataattc acctgccaag 240

acggcacagg caactattct tgctaattga cgtgcgttgg taccaggagc ggtagcatgc 300

gggcctctta cacctaataa gtccaacatg gcaccttgtg gttctagaac agtaccacca 360

ccgatggtac ctacttcgat ggatggcatg gatacggaaa ttctcaaatc accgtccact 420

tctttcatca atgttataca gttggaactt tcaacatttt gtgcaggatc ttgtcctaat 480

gccaagaaaa cagctgtcac taaattagct gcatgtgcgt taaatccacc aacagaccca 540

gccattgcag atccaaccaa attcttagca atgttcaact caaccaatgc ggaaacatca 600

ctttttaaca cttttctgac aacatcacca ggaatagtag cttctgcgac gacactctta 660

ccacgacctt cgatccagtt gatggcagct ggtttttttg tcggtacagt agttaccaga 720

aacggagaca acctccatat cttcccagcc atactcttct accatttgct ttaatgagta 780

ttcgacacct ttagaaatca tattcatacc cattgcgtca ccagtagttg ttctaaatct 840

catgaagagt aaatctcctg ctagacaagt ttgaatatgt tgcagacgtg caaatcttga 900

tgtagagtta aaagcttttt taattgcgtt ttgtccctct tctgagtcta accatatctt 960

acaggcacca gatcttttca aagttgggaa acggactact gggcctcttg tcataccatc 1020

cttagttaaa acagttgttg caccaccgcc agcattgatt gccttacagc cacgcatggc 1080

agaagctacc aaacaaccct ctgtagttgc cattggtata tgataagatg taccatcgat 1140

aaccaagggg cctataacac caacgggcaa aggcatgtaa cctataacat tttcacaaca 1200

agcgccaaat acgcggtcgt agtcataatt tttatatggt aaacgatcag atgctaatac 1260

aggagcttct gccaaaattg aaagagcctt cctacgtacc gcaaccgctc tcgtagtatc 1320

acctaatttt ttctccaaag cgtacaaagg taacttaccg tgaataacca aggcagcgac 1380

ctctttgttc ttcaattgtt ttgtatttcc actacttaat aatgcttcta attcttctaa 1440

aggacgtatt ttcttatcca agctttcaat atcgcgggaa tcatcttcct cactagatga 1500

tgaaggtcct gatgagctcg attgcgcaga tgataaactt ttgactttcg atccagaaat 1560

gactgtttta ttggttaaaa cgaattcgga tccgcgaccc atttgctgtc caccagtcat 1620

gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct 1680

gctgcccata gtgtaatcct ccttattttt taacatcgta agatcttcta aatttgtcat 1740

cgatgttggt caagtagtaa acaccacttt gcaaatgctc aatggaacct tgaggtttga 1800

agttcttctt caaatgggca ttttctctca attcgatggc agcttcgtaa tcctttggag 1860

tttcggtgat tctcttggct aatttgttag taatatctaa ttccttgata atatgttgga 1920

cgtcaccaac aattttgcaa gaatatagag atgcagctaa accggaaccg taagaaaata 1980

aaccaacacg cttgccttgt aagtcgtcag atccaacata gtttaataga gatgcaaagg 2040

cggcataaac agatgcggtg tacatgttac ctgtgtttgt tggaacaatc aaagattggg 2100

caactctctc tttgtggaat ggcttagcaa cattaacaaa agttttttca atgttcttat 2160

cggttaaaga ttcgtcataa tcgcgagtag ctaattcggc gtcaacttct gggaacaatt 2220

gaggattggc tctgaaatcg ttatatagta atctaccgta tgattttgtg accaatttac 2280

aggttggaac atggaaaacg ttgtagtcga aatatttcaa aacgttcaaa gcatccgaac 2340

cagcgggatc gctaaccaac cctttagaaa tagccttctt ggaataactc ttgtaaactt 2400

gatcaagagc cttgacgtaa caagttaatg aaaaatgacc atcgacgtaa ggatattcgc 2460

tggtgaaatc tggcttgtaa aaatcgtagg cgtgttccat gtaagaagct cttacagagt 2520

caaatacaat tggagcatca ggaccgatcc acatagcaac agtaccggca ccaccggttg 2580

gtcttgcggc acccttatcg tagatggcaa tatcaccgca aactacaatg gcgtctctac 2640

catcccatgc gttagattca atccagttca aagagttgaa caacgcgttg gtaccaccgt 2700

aacaggcatt aagcgtgtca ataccttcga cgtcagtgtt ttcaccaaac aattgcatca 2760

agacagactt gacagacttg gacttgtcaa tcagagtttc agtaccgact tctaatctac 2820

caattttgtt ggtgtcgatg ttgtaactct tgatcaactt agacaaaaca gttagggaca 2880

tcgagtagat atcttctctg tcattgacaa aagacatgtt ggtttggccc agaccaattg 2940

tgtatttacc ttgagaaacg ccatcaaatt tctctagctc agattggttg acacattgag 3000

ttgggatgta aatttggata cctttaatac cgacattttg aggtctggtt ttttgttcag 3060

cggtcttttg tttttttagt tcagtcattt gcaagtttgt attgtgtaat tgttgttgct 3120

tttgcggcct aagtcttcct ttaataccac accaacaaag tttagttgag agtttcattt 3180

agctgtcctc cttaattcaa ccgttcaatc accatcgcaa ttccctgacc gccgccaatg 3240

cacagtgttg ccagccccag cgttttatcg cgtgcctgca tggcatgtaa tagtgtgacc 3300

agaatacgag caccactggc accgatagga tgcccgagcg cgatggcccc gccgttgaca 3360

ttcactttct cagaatcaaa gcccaggttt ttcccaacgg caaggaactg tgcagcaaat 3420

gcttcattag cctcaatgag atcaatatcc gccagttgca gccccgccag ttgtaacgct 3480

ttttgcgtgg caggtactgg ccccataccc atcaatgcgg ggggcacgcc accgctggca 3540

taacttttaa tgcgagccag gggggtaagg cctgctgcca gcgccgcaga ttcttccata 3600

atcaccagag cggcagcacc gtcgttaata ccagacgcgt tcccagcggt gactgttcct 3660

gctttatcga aggccgggcg caatgcacct aacgcttcag ccgttgaatt cgctttcggg 3720

aattcgtctt gactgaagac gaaggttttc tttcgagtga caacatttac cgggacgatt 3780

tcggctgtaa aagcaccgga ctcaattgcg gctgccgctt tacgctgtga atgtagcgcc 3840

agttcatcct gcatttcacg ggtaattccg tactctttag ccacgttttc ggcggtaatc 3900

cccatatgat aaccatgggt ggcgcacatc aggccatcgc gcaggattac gtcataaacc 3960

tgtccgtctc caagacgata accagagcgt gcttttgcat cgagtaagta gggggctaaa 4020

ctcatatttt ccataccccc cgccacaatg ctctgcgcct gacctgcctg aatggcctgg 4080

gcggcaagcg ccacactttt aagacccgaa ccacatactt tattgaccgt gaatccgcac 4140

accgtttctg ccagcccgct ttttaacagt gcctgacgcg ccggattttg ccccagcccg 4200

gcttgtaaca cgttacccat aatcacttca tcaacgtgtt gtgaatcgat ttttgcacgt 4260

tcaatggcgg ctttaattac tgtcgccccc aggtcgatgg cgctggtgga agcgagtgaa 4320

ccgttaaaac taccgatagc agtacgtacc gcactgacga tgacacaatt tttcatttta 4380

tattcctcct agtcgactct agaggatccc cgggctgcag gaattcgata tcaagcttat 4440

cgataccgtc gacctcgagg gggggcccgg tacccagctt ttgttccctt tagtgagggt 4500

taattgcgcg ctggg 4515

<210> 3

<211> 4667

<212> DNA

<213> Artificial Sequence

<220>

<223> pFZ81 sequence (not containing skeleton carrier sequence)

<400> 3

cgcgcgtaat acgactcact atagggcgaa ttggagctct tatttaagct gggtaaatgc 60

agataatcgt tttctggctt cgcgatttgt cgcctgcatc accatccacg gactgaacgc 120

ccacggcgtg gcatcaatac cgtgtaatac atctgctaaa tcacaccatt gataatccat 180

cacttcatca tcattgatct gtaacgcact agtggtgcgt gcggcaaata ccggacacac 240

ttcattttcc acaatgccac tcggatcggt ggcgcggtag cgaaagtcag gatagataga 300

ttcaggaggc gtaatttcca cgccaagctc ataacggcaa cggcggatca ctgcgtcttc 360

gttgctttct cccagttgtg ggtgcccaca aaccgagtta gtccacacgc caggccatgc 420

ttttttgctc agtgcgcggc gggtaactaa taattgtcct ttggcattaa acagccaact 480

ggagaacgcg agatgtaagc gggtgtctgc cgtgtgtgcg gcatactttt ccagcgtacc 540

cgtgggaact ccctgtgcat tcaataaaat gacgtgttcc gtttgcatat ggctgccgcg 600

cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatat agtaatcctc 660

ctcccgggct gcagttattc ctttggtaga ccagtctttg cgtcaatcaa agattcgttt 720

gtttcttgtg ggcctgaacc gacttgagtt aaaatcactc tggcaacatc cttttgcaac 780

tcaagatcca attcacgtgc agtaaagtta gatgattcaa attgatggtt gaaagcctca 840

agctgctcag tagtaaattt cttgtcccat ccaggaacag agccaaacaa tttatagata 900

aatgcaaaga gtttcgactc attttcagct aagtagtaca acacagcatt tggacctgca 960

tcaaacgtgt atgcaacgat tgtttctccg taaaactgat taatggtgtg gcaccaactg 1020

atgatacgct tggaagtgtc attcatgtag aatattggag ggaaagagtc caaacatgtg 1080

gcatggaaag agttggaatc catcattgtt tcctttgcaa aggtggcgaa atctttttca 1140

acaatggctt tacgcatgac ttcaaatctc tttggtacga catgttcaat tctttcttta 1200

aatagttcgg aggttgccac ggtcaattgc ataccctgag tggaactcac atccttttta 1260

atatcgctga caactaggac acaagctttc atctgaggcc agtcagagct gtctgcgatt 1320

tgtactgcca tggaatcatg accatcttca gcttttccca tttcccaggc cacgtatccg 1380

ccaaacaacg atctacaagc tgaaccagac ccctttcttg ctattctaga tatttctgaa 1440

gttgactgtg gtaattggta taacttagca attgcagaga ccaatgcagc aaagccagca 1500

gcggaggaag ctaaaccagc tgctgtagga aagttatttt cggagacaat gtggagtttc 1560

cattgagata atgtgggcaa tgaggcgtcc ttcgattcca tttcctttct taattggcgt 1620

aggtcgcgca gacaattttg agttctttca ttgtcgatgc tgtgtggttc tccatttaac 1680

cacaaagtgt cgcgttcaaa ctcaggtgca gtagccgcag aggtcaacgt tctgaggtca 1740

tcttgcgata aagtcactga tatggacgaa ttggtgggca gattcaactt cgtgtccctt 1800

ttcccccaat acttaagggt tgcgatgttg acgggtgcgg taacggatgc tgtgtaaacg 1860

gtcatgagta ttacctccta tttatcaaga taagtttccg gatctttttc tttcctaaca 1920

ccccagtcag cctgagttac atccagccat tgaaccttag aaaatctttt gtcattagcg 1980

gtttgagccc taagatcaac atcttgctta gtaatcactg caatggcgtc ataaccacca 2040

gcaccaggta ttaagcaagt aagaactcct tttaaggtct ggcaatcatc caataagcta 2100

gtttgtacgg gaggttcgat atcggcacca gattctttag ttatttttct aaaggaacgt 2160

ctaattgtgg caactgcatc tctaacttct gtgatttcag gatacttttg acaggtacag 2220

tcattcctct caagagactc aaatatctga tcgctgtaat cgtcatgagt ctcgtgtaag 2280

cgatctagtt tagatagtcc atccataaat ctagaatttg catgatcgag ttctgtatat 2340

attttcaagc tttctggcat atgcgaatca taccaatttt ttaccttctg gaccagtttt 2400

actgtttctg aaccattctt aatatcgccc atccataaag ttaatcccga aggtaaatgg 2460

ttacttttaa tcgtaatatt ccagtcttct tcatcaacca aatgcgccag tttactgccg 2520

taagtagcac ttccaatatc tggcaaatta gagattaatg cgggtgggaa tcttctatat 2580

ctgatagatc catatgctgc cgccgctaca tcaaacccgc ttccaatttt accctgagct 2640

tgacaatgag caacttgtgc taaattatga ataacttctc tatatttgtc tacattattt 2700

tccaggtccg atacaaaaaa ggaggccaaa gctgtagtta aaactgtgac taaacctgcc 2760

gaggagccca gccctgtttt gggaacttct tcaattctgt gcgaatgaaa actcaatctt 2820

ctgttgccac gatgttcggt aacgctatcc tcctgagaat ggtaggcatc atcagagaaa 2880

atatcaataa cgaacaagtt tctattgcag tagtcgtcca tgttaggttt aaagtagcta 2940

aatacgttag cgataacttt ttcaatgaaa gggttcttag atccgcctat cgaaacagga 3000

atgaagccac ttttaggact tatatggtac agccactccc catctttaaa ttgtttactt 3060

ttcacacgca cttcaaactt atcagaccct tgcaatgaac cgtaaggatg ggctacagca 3120

tgcattcttg ccgataatcc gactacaaat gcttcatatt ttgtatctaa aactaaatat 3180

ccaccagcta gtaacgcttt ccctggggca ctgaaggctc tcaactctga catttgatct 3240

gcctcctatg aagtccatgg taaattcgtg tttcctggca ataatagatc gtcaatttgt 3300

tgctttgtgg tagttttatt ttcaaataat tggaatacta gggatttgat tttaggatct 3360

ttattcaaat tttttgcgct taacaaacag cagccagtcc cacccaagtc tgtttcaaat 3420

gtctcgtaac taaaatcatc ttgcaatttc tttttgaaac tgtcaatttg ctcttgagta 3480

atgtctcttc gtaacaaagt caaagagcaa ccgccgccac cagcaccggt aagttttgtg 3540

gagccaattc tcaaatcatc gctcagattt ttaataagtt ctaatccagg atgagaaaca 3600

ccgattgaga caagcagtcc atgatttatt cttatcaatt ccaatagttg ttcatacagt 3660

tcattattag tttctacggc ctcgtcatcg gtgcctttac atttacttaa ctttgtcatg 3720

atctctaagc cttgtagggc acattcaccc atggcatcta gaattggctt cataacttca 3780

ggaaatttct cggtgaccaa cacacgaacg cgagcaacaa gatcttttgt agaccttgga 3840

attctagtat aggttaggat cattggaatg gctgggaaat catctaagaa cttaaaattg 3900

tttgtattta ttgttccatt atgtgagtct ttttcaaata gcagggcatt accataagtg 3960

gccacagcgt tatctattcc tgaaggggta ccgtgaatac acttttcacc tatgaaggcc 4020

cattgattca ctatatgctt atcgttttct gacagctttt ccaagtcatt agatcctatt 4080

aaccccccca agtaggccat agctaaggcc agtgatacag aaatagaggc gcttgagccc 4140

aacccagcac cgatgggtaa agtagacttt aaagaaaact taatattctt ggcatggggg 4200

cataggcaaa caaacatata caggaaacaa aacgctgcat ggtagtggaa ggattcggat 4260

agttgagcta acaacggatc caaaagacta acgagttcct gagacaagcc atcggtggct 4320

tgttgagcct tggccaattt ttgggagttt acttgatcct cggtgatggc attgaaatca 4380

ttgatggacc acttatgatt aaagctaatg tccgggaagt ccaattcaat agtatctggt 4440

gcagatgact cgcttattag caggtaggtt ctcaacgcag acacactagc agcgacggca 4500

ggcttgttgt acacagcaga gtgttcacca aaaataataa cctttcccgg tgcagaagtt 4560

aagaacggta atgacatggt taattcctcc tactgcagga attcgatatc aagcttatcg 4620

ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccc 4667

<210> 4

<211> 2773

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid pFZ38 sequence (not containing skeleton carrier sequence)

<400> 4

atggaatttc gtgtccacct gcaagccgat aatgaacaaa aaatctttca aaaccaaatg 60

aagccggaac cggaagcctc ctacctgatt aatcaacgtc gctccgcgaa ctacaagccg 120

aatatttgga agaacgattt tctggaccag tccctgatct caaaatatga tggtgacgaa 180

taccgtaaac tgtcggaaaa gctgatcgaa gaagtgaaaa tttacatcag cgcggaaacc 240

atggatctgg ttgccaaact ggaactgatt gacagcgtcc gtaagctggg cctggcaaac 300

ctgtttgaaa aagaaatcaa ggaagctctg gattcgattg cggccatcga aagcgacaac 360

ctgggtaccc gcgatgacct gtatggcacg gcgctgcatt ttaaaattct gcgtcaacac 420

ggttacaagg tttcgcagga tatctttggt cgcttcatgg acgaaaaagg caccctggaa 480

aatcatcact tcgcgcatct gaagggcatg ctggaactgt ttgaagccag caacctgggt 540

ttcgaaggcg aagatattct ggacgaagcg aaagcctctc tgacgctggc cctgcgtgat 600

agtggtcaca tctgctatcc ggattcgaat ctgagccgcg acgtggttca ttccctggaa 660

ctgccgtcac accgtcgcgt tcaatggttt gatgtcaaat ggcagattaa tgcatacgaa 720

aaggatatct gtcgtgtgaa cgcaaccctg ctggaactgg ctaaactgaa cttcaatgtc 780

gtgcaggctc aactgcagaa gaacctgcgt gaagcgagcc gttggtgggc taatctgggt 840

attgcggata acctgaaatt tgcacgtgac cgcctggttg aatgcttcgc atgtgctgtt 900

ggcgtcgctt ttgaaccgga acatagctct ttccgcattt gcctgaccaa agtcatcaat 960

ctggtgctga ttatcgatga cgtctatgat atttacggtt ctgaagaaga actgaaacac 1020

tttaccaacg cagtggatcg ttgggacagt cgcgaaacgg aacaactgcc ggaatgcatg 1080

aaaatgtgtt tccaggtgct gtataacacc acgtgtgaaa ttgcccgtga aatcgaagaa 1140

gaaaacggct ggaatcaagt cctgccgcag ctgaccaaag tgtgggccga tttttgcaag 1200

gcactgctgg ttgaagctga atggtataat aaatcccata ttccgacgct ggaagaatac 1260

ctgcgcaacg gttgtatcag ttcctcagtg tcagttctgc tggtccatag ctttttcagc 1320

attacccacg aaggcacgaa agaaatggcg gatttcctgc acaagaatga agacctgctg 1380

tataacattt ctctgatcgt gcgtctgaac aatgatctgg gtacctccgc agctgaacag 1440

gaacgcggcg attcaccgtc gagcatcgtg tgctacatgc gtgaagttaa tgcgagtgaa 1500

gaaacggccc gcaaaaacat taagggtatg atcgataatg cgtggaaaaa ggtgaacggc 1560

aaatgtttta ccacgaatca ggttccgttt ctgtctagtt tcatgaacaa tgccaccaac 1620

atggcgcgcg ttgcccatag cctgtataaa gatggcgacg gtttcggcga tcaagaaaaa 1680

ggtccgcgta cccacatcct gagcctgctg ttccaaccgc tggtcaacta actagaaata 1740

attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca 1800

cagcagcggc ctggtgccgc gcggcagcca tatggctagc atgactggtg gacagcaaat 1860

gggtcgcgga tccatggact ttccgcagca actcgaagcc tgcgttaagc aggccaacca 1920

ggcgctgagc cgttttatcg ccccactgcc ctttcagaac actcccgtgg tcgaaaccat 1980

gcagtatggc gcattattag gtggtaagcg cctgcgacct ttcctggttt atgccaccgg 2040

tcatatgttc ggcgttagca caaacacgct ggacgcaccc gctgccgccg ttgagtgtat 2100

ccacgcttac tcattaattc atgatgattt accggcaatg gatgatgacg atctgcgtcg 2160

cggtttgcca acctgccatg tgaagtttgg cgaagcaaac gcgattctcg ctggcgacgc 2220

tttacaaacg ctggcgttct cgattttaag cgatgccgat atgccggaag tgtcggaccg 2280

cgacagaatt tcgatgattt ctgaactggc gagcgccagt ggtattgccg gaatgtgcgg 2340

tggtcaggca ttagatttag acgcggaagg caaacacgta cctctggacg cgcttgagcg 2400

tattcatcgt cataaaaccg gcgcattgat tcgcgccgcc gttcgccttg gtgcattaag 2460

cgccggagat aaaggacgtc gtgctctgcc ggtactcgac aagtatgcag agagcatcgg 2520

ccttgccttc caggttcagg atgacatcct ggatgtggtg ggagatactg caacgttggg 2580

aaaacgccag ggtgccgacc agcaacttgg taaaagtacc taccctgcac ttctgggtct 2640

tgagcaagcc cggaagaaag cccgggatct gatcgacgat gcccgtcagt cgctgaaaca 2700

actggctgaa cagtcactcg atacctcggc actggaagcg ctagcggact acatcatcca 2760

gcgtaataaa taa 2773

<210> 5

<211> 3422

<212> DNA

<213> Artificial Sequence

<220>

<223> plasmid pFZ71 sequence (not containing skeleton carrier sequence)

<400> 5

atggaatttc gtgtccacct gcaagccgat aatgaacaaa aaatctttca aaaccaaatg 60

aagccggaac cggaagcctc ctacctgatt aatcaacgtc gctccgcgaa ctacaagccg 120

aatatttgga agaacgattt tctggaccag tccctgatct caaaatatga tggtgacgaa 180

taccgtaaac tgtcggaaaa gctgatcgaa gaagtgaaaa tttacatcag cgcggaaacc 240

atggatctgg ttgccaaact ggaactgatt gacagcgtcc gtaagctggg cctggcaaac 300

ctgtttgaaa aagaaatcaa ggaagctctg gattcgattg cggccatcga aagcgacaac 360

ctgggtaccc gcgatgacct gtatggcacg gcgctgcatt ttaaaattct gcgtcaacac 420

ggttacaagg tttcgcagga tatctttggt cgcttcatgg acgaaaaagg caccctggaa 480

aatcatcact tcgcgcatct gaagggcatg ctggaactgt ttgaagccag caacctgggt 540

ttcgaaggcg aagatattct ggacgaagcg aaagcctctc tgacgctggc cctgcgtgat 600

agtggtcaca tctgctatcc ggattcgaat ctgagccgcg acgtggttca ttccctggaa 660

ctgccgtcac accgtcgcgt tcaatggttt gatgtcaaat ggcagattaa tgcatacgaa 720

aaggatatct gtcgtgtgaa cgcaaccctg ctggaactgg ctaaactgaa cttcaatgtc 780

gtgcaggctc aactgcagaa gaacctgcgt gaagcgagcc gttggtgggc taatctgggt 840

attgcggata acctgaaatt tgcacgtgac cgcctggttg aatgcttcgc atgtgctgtt 900

ggcgtcgctt ttgaaccgga acatagctct ttccgcattt gcctgaccaa agtcatcaat 960

ctggtgctga ttatcgatga cgtctatgat atttacggtt ctgaagaaga actgaaacac 1020

tttaccaacg cagtggatcg ttgggacagt cgcgaaacgg aacaactgcc ggaatgcatg 1080

aaaatgtgtt tccaggtgct gtataacacc acgtgtgaaa ttgcccgtga aatcgaagaa 1140

gaaaacggct ggaatcaagt cctgccgcag ctgaccaaag tgtgggccga tttttgcaag 1200

gcactgctgg ttgaagctga atggtataat aaatcccata ttccgacgct ggaagaatac 1260

ctgcgcaacg gttgtatcag ttcctcagtg tcagttctgc tggtccatag ctttttcagc 1320

attacccacg aaggcacgaa agaaatggcg gatttcctgc acaagaatga agacctgctg 1380

tataacattt ctctgatcgt gcgtctgaac aatgatctgg gtacctccgc agctgaacag 1440

gaacgcggcg attcaccgtc gagcatcgtg tgctacatgc gtgaagttaa tgcgagtgaa 1500

gaaacggccc gcaaaaacat taagggtatg atcgataatg cgtggaaaaa ggtgaacggc 1560

aaatgtttta ccacgaatca ggttccgttt ctgtctagtt tcatgaacaa tgccaccaac 1620

atggcgcgcg ttgcccatag cctgtataaa gatggcgacg gtttcggcga tcaagaaaaa 1680

ggtccgcgta cccacatcct gagcctgctg ttccaaccgc tggtcaacta actagaaata 1740

attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca 1800

cagcagcggc ctggtgccgc gcggcagcca tatggctagc atgactggtg gacagcaaat 1860

gggtcgcgga tccatggact ttccgcagca actcgaagcc tgcgttaagc aggccaacca 1920

ggcgctgagc cgttttatcg ccccactgcc ctttcagaac actcccgtgg tcgaaaccat 1980

gcagtatggc gcattattag gtggtaagcg cctgcgacct ttcctggttt atgccaccgg 2040

tcatatgttc ggcgttagca caaacacgct ggacgcaccc gctgccgccg ttgagtgtat 2100

ccacgcttac tcattaattc atgatgattt accggcaatg gatgatgacg atctgcgtcg 2160

cggtttgcca acctgccatg tgaagtttgg cgaagcaaac gcgattctcg ctggcgacgc 2220

tttacaaacg ctggcgttct cgattttaag cgatgccgat atgccggaag tgtcggaccg 2280

cgacagaatt tcgatgattt ctgaactggc gagcgccagt ggtattgccg gaatgtgcgg 2340

tggtcaggca ttagatttag acgcggaagg caaacacgta cctctggacg cgcttgagcg 2400

tattcatcgt cataaaaccg gcgcattgat tcgcgccgcc gttcgccttg gtgcattaag 2460

cgccggagat aaaggacgtc gtgctctgcc ggtactcgac aagtatgcag agagcatcgg 2520

ccttgccttc caggttcagg atgacatcct ggatgtggtg ggagatactg caacgttggg 2580

aaaacgccag ggtgccgacc agcaacttgg taaaagtacc taccctgcac ttctgggtct 2640

tgagcaagcc cggaagaaag cccgggatct gatcgacgat gcccgtcagt cgctgaaaca 2700

actggctgaa cagtcactcg atacctcggc actggaagcg ctagcggact acatcatcca 2760

gcgtaataaa taactagaaa taattttgtt taactttaag aaggagatat accatgggca 2820

gcagccatca tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatgcaaa 2880

cggaacacgt cattttattg aatgcacagg gagttcccac gggtacgctg gaaaagtatg 2940

ccgcacacac ggcagacacc cgcttacatc tcgcgttctc cagttggctg tttaatgcca 3000

aaggacaatt attagttacc cgccgcgcac tgagcaaaaa agcatggcct ggcgtgtgga 3060

ctaactcggt ttgtgggcac ccacaactgg gagaaagcaa cgaagacgca gtgatccgcc 3120

gttgccgtta tgagcttggc gtggaaatta cgcctcctga atctatctat cctgactttc 3180

gctaccgcgc caccgatccg agtggcattg tggaaaatga agtgtgtccg gtatttgccg 3240

cacgcaccac tagtgcgtta cagatcaatg atgatgaagt gatggattat caatggtgtg 3300

atttagcaga tgtattacac ggtattgatg ccacgccgtg ggcgttcagt ccgtggatgg 3360

tgatgcaggc gacaaatcgc gaagccagaa aacgattatc tgcatttacc cagcttaaat 3420

aa 3422

<210> 6

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> P15A-f

<400> 6

gcattgccgc cgtgggtttc tcgagcgggg cggagatttc ctggaagatg 50

<210> 7

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> P15A-r

<400> 7

ggggccactt tttgccggag ctcgagaaat attttatctg attaataaga tg 52

<210> 8

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS1-p15A-f

<400> 8

catcttatta atcagataaa atatttctcg agctccggca aaaagtggcc cc 52

<210> 9

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS1-p15A-r

<400> 9

catcttccag gaaatctccg ccccgctcga gaaacccacg gcggcaatgc 50

<210> 10

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS1-f

<400> 10

tttgaaagat gggtccgtca cctgcattaa atcctaagga tccactagtt ctaga 55

<210> 11

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS1-r

<400> 11

ttttatattc ctcctagtcg actctagagg atccccgggc tgcaggaatt cgatatcaa 59

<210> 12

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> atoB-f

<400> 12

cccggggatc ctctagagtc gactaggagg aatataaaat gaaaaattgt gtcatcgtc 59

<210> 13

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> atoB-r

<400> 13

gttgagagtt tcatttagct gtcctcctta attcaaccgt tcaatcacc 49

<210> 14

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG13-f

<400> 14

acggttgaat taaggaggac agctaaatga aactctcaac taaact 46

<210> 15

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG13-r

<400> 15

tggctgctgc ccatagtgta atcctcctta ttttttaaca tcgtaagat 49

<210> 16

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> tHMG1-f

<400> 16

atgttaaaaa ataaggagga ttacactatg ggcagcagcc atcatca 47

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> tHMG1-r

<400> 17

ttaggattta atgcaggtga cgg 23

<210> 18

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS2-f

<400> 18

cagaaaacga ttatctgcat ttacccagct taaataagag ctccaattcg ccctatagt 59

<210> 19

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> pBBR1MCS2-r

<400> 19

ttaagaacgg taatgacatg gttaattcct cctactgcag gaattcgata tcaag 55

<210> 20

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG12-f

<400> 20

ctgcagtagg aggaattaac catgtcatta ccgttcttaa ct 42

<210> 21

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG12-r

<400> 21

ctcaactctg acatttgatc tgcctcctat gaagtccatg gtaa 44

<210> 22

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG8-f

<400> 22

ttaccatgga cttcatagga ggcagatcaa atgtcagagt tgagagcctt c 51

<210> 23

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> ERG8-r

<400> 23

gatgctgtgt aaacggtcat gagtattacc tcctatttat caagataagt ttc 53

<210> 24

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> MVD1-f

<400> 24

atcttgataa ataggaggta atactcatga ccgtttacac agcatccgtt 50

<210> 25

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> MVD1-r

<400> 25

tgcccatata gtaatcctcc tcccgggctg cagttattcc tttggtagac cagtc 55

<210> 26

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> Idi-f

<400> 26

ggaataactg cagcccggga ggaggattac tatatgggca gcagccatca t 51

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> Idi-r

<400> 27

ttatttaagc tgggtaaatg caga 24

<210> 28

<211> 58

<212> DNA

<213> Artificial Sequence

<220>

<223> IspA-BamHI

<400> 28

gctagcatga ctggtggaca gcaaatgggt cgcggatcca tggactttcc gcagcaac 58

<210> 29

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> IspA-EcoRI-SpeI

<400> 29

gctagaattc actagttatt tattacgctg gatga 35

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Idi-NdeI

<400> 30

atatcatatg caaacggaac acgtc 25

<210> 31

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Idi-XhoI

<400> 31

tatactcgag ttatttaagc tgggtaaatg 30

Claims (9)

1. produce a bacterial strain for farnesene, it is characterized in that the synthetic genes involved of farnesene that contains mevalonate pathway and part or all of codon optimization; The genes involved of described mevalonate pathway comprises atoB, erg13, thmg1, erg12, erg8, mvd1 and idi gene; The synthetic genes involved of described farnesene comprises ispA and afs gene; Described afs gene codon is optimized, and its sequence is as shown in SEQ ID NO.1.

2. the bacterial strain of production farnesene according to claim 1, is characterized in that: the bacterial strain of producing farnesene is the intestinal bacteria that contain plasmid pMH1, plasmid pFZ81 and plasmid pFZ38 or the intestinal bacteria that contain plasmid pMH1, plasmid pFZ81 and plasmid pFZ71;

Described plasmid pMH1 take pBBR1MCS as skeleton carrier, replicon as p15A, promotor as lac promotor, comprise atoB, erg13 and thmg1 gene;

Described plasmid pFZ81 take pBBR1MCS-2 as skeleton carrier, replicon as pBBR1MCS replicon, promotor as lac promotor, comprise erg12, erg8, mvd1 and idi gene;

Described plasmid pFZ38 take pET21a (+) as skeleton carrier, replicon as pBBR322 high copy replicon, promotor as T7 strong promoter, comprise afs and ispA gene;

Described plasmid pFZ71 take pET21a (+) as skeleton carrier, replicon as pBBR322 high copy replicon, promotor as T7 strong promoter, comprise afs, ispA and idi gene.

3. the bacterial strain of production farnesene according to claim 2, is characterized in that: the sequence of described plasmid pMH1 is as shown in SEQ ID NO.2; The sequence of described plasmid pFZ81 is as shown in SEQ ID NO.3; The sequence of described plasmid pFZ38 is as shown in SEQ ID NO.4; The sequence of described plasmid pFZ71 is as shown in SEQ ID NO.5.

4. the application of the bacterial strain described in claim 1-3 any one in producing farnesene.

5. the method for utilizing the bacterial strain described in claim 1-3 any one to produce farnesene, is characterized in that comprising following steps: by carrying out prokaryotic expression in the substratum of the bacterial strain seed liquor access carbonaceous sources described in claim 1-3 any one, obtain farnesene.

6. the method for production farnesene according to claim 5, is characterized in that: during prokaryotic expression, add after inductor IPTG2-4 hour and add n-decane to carry out biphasic fermentation again.

7. the method for production farnesene according to claim 5, is characterized in that: during prokaryotic expression, each albumen ratio is AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1-56: 2-16: 2-30: 1-5: 1-5: 2-20: 4-600: 20-40: 2-11.

8. the method for production farnesene according to claim 5, is characterized in that: during prokaryotic expression, each albumen ratio is AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: 2: 2.

9. improve the method that prokaryotic organism produce farnesene, it is characterized in that comprising following method: the synthetic genes involved AFS of farnesene is carried out codon optimized, the sequence of the AFS gene after optimization is as shown in SEQ ID NO.1; Or make each albumen more approach AtoB: ERG13: tHMG1: ERG12: ERG8: MVD1: Idi: IspA: AFS=1: 10: 2: 5: 5: 2: 5: the further transformation of 2: 2.