CN101285076A - Preparation method of lycopene - Google Patents

Abstract

The invention discloses a method for preparing lycopene. The crtI gene of Erwinia h erbicola is amplified through the PCR technology and is connected with an expression vector pRKR5 containing strong promoters Puc to construct an expression plasmid pRKR5-crtI which is inducted into a mutant TC72 of photosynthetic bacteria Rhodobacter s phaeroides in the mode of conjugal transfer. Due to the high-efficiency expression of the promoters Puc under the anaerobic condition, the adjustment and control on oxygen concentration can induct engineering bacteria to accumulate red pigment. After HPLC and absorption spectral analysis, the pigment synthesized in the engineering bacteria is the lycopene, the biomass (dry weight) of the engineering bacteria is 2.36gDCW/L, and the lycopene content is 1.52mg/gDCW. The method which lays the preliminary foundation for realizing industrial production of lycopene has potential application value in the production practice.

Description

A kind of preparation method of Lyeopene

Technical field

The invention belongs to bioengineering field, particularly a kind of biosynthesizing lycopene method.

Background technology

Lyeopene is a kind of important carotenoid, and molecular formula is C ₄₀H ₅₆, molecular weight is 536.85.Nearest studies show that the antioxidant property of Lyeopene is the strongest in the natural carotenoid, and its special physiological properties more and more causes people's attention: efficient quencher singlet oxygen of energy and removing peroxylradicals; The activity by influencing human body cell and the absorption and the circulation of nutritive substance, the growth and the metabolism thereof of control cell; Suppress cholesterol biosynthesis enzyme as a kind of low cholesterol agent, the cholesterol regulating metabolism prevents and treats cardiovascular disorder.Thereby Lyeopene has important use at food, makeup and field of medicaments.

Lyeopene can obtain by chemosynthesis, natural extract and 3 kinds of methods of microbial fermentation at present.Above-mentioned three kinds of production methods are done one relatively: the natural extract method is because the plantation of tomato is seasonal product, and output and content are subjected to multifactor control, and Lyeopene content instability in tomato, and extraction process is loaded down with trivial details tediously long, the cost costliness; Although and the chemical synthesis cost is lower, because contaminate environment and product activity are lower, the product application scope is restricted; Adopt the Production by Microorganism Fermentation Lyeopene, its quality product is fully identical with the natural extract product with physiologically active, and adopts fermentation method to reduce cost, and pollutes less relatively.Therefore, we as can be seen microbe fermentation method be the production method of potentialization.

Though Lyeopene all has distribution in a lot of plants and microorganism, because wherein the complicated component of contained pigment causes the cost that extracts one-component higher.Along with the clone in succession of different biological species carotene synthetic genes, provide possibility for utilizing microbial fermentation to obtain Lyeopene.CrtI genes encoding phytoene dehydrogenase, the effect of this desaturase is different in different species.Studies show that, Erwiniaherbicola is a kind of Gram-negative bacteria, do not belong to photosynthetic bacterium, its crt gene cluster produces carotenoid in the following order, at first phytoene forms Lyeopene through 4 dehydrogenations, produces β-Hu Luobusu, beta-cryptoxanthin, zeaxanthin then successively.And the phytoene of photosynthetic bacterium Rhodobactersphaeroides is equally forming neurosporene by following of the phytoene dehydrogenase effect of crtI genes encoding through three dehydrogenations, then then generate hydroxylation neurosporene, the spherical alkene of demethyl, spheroidene and spherical rhzomorph, do not form Lyeopene.The mutant strain TC72 of R.sphaeroides inserts transposon Tn5 and gets in the crtI of original strain R.sphaeroides gene.Studies show that though the crt gene cluster is complete in the mutant strain TC72, because the insertion of Tn5 makes its carotenoid route of synthesis rest on phytoene, crtI and later expression of gene all are interrupted (as shown in Figure 1).If can utilize engineered method that the crtI gene of E.herbicola is expressed in photosynthetic bacterium mutant strain TC72, make the phytoene biosynthesizing Lyeopene that accumulates among the TC72.Simultaneously, owing to do not have crtY and crtZ albumen among the TC72, make biosynthetic Lyeopene do not continued to be metabolized to other carotenoid, thereby make crtI protein biology synthetic Lyeopene a large amount of accumulation and the separate easily purifying in cell that utilize E.herbicola among the photosynthetic bacterium mutant strain TC72.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of Lyeopene.The inventive method is with the crtI gene clone of E.herbicola and be connected on the expression vector pRKR5 that contains strong promoter Puc, make up engineering bacteria in conjunction with transferring among the mutant strain TC72, promotor Puc under anaerobic efficiently expresses, and the regulation and control by oxygen concn make engineering bacteria production purpose product Lyeopene.Lay a good foundation for realization suitability for industrialized production Lyeopene, in production practice, have the potential using value, have production cost low, be beneficial to the advantage of environmental protection.

The inventive method may further comprise the steps:

(1) extracts the E.herbicola genomic dna, clone the crtI full length gene by PCR method;

(2) the SacI-XbaI enzyme is cut the pMD18-crtI plasmid, and the crtI fragment of downcutting is connected on the expression vector pRKR5 that contains strong promoter Puc construction of expression vector pRKR5-crtI;

(3) mode by conjugal transfer imports expression vector pRKR5-crtI among the photosynthetic bacterium Rhodobacter sphaeroides mutant strain TC72;

(4) promotor Puc under anaerobic efficiently expresses, and the regulation and control oxygen concn can be induced engineering bacteria TC72-pRKR5-crtI accumulation red pigments.

Through HPLC and absorption spectroanalysis, the synthetic pigment is a Lyeopene among the engineering bacteria TC72-pRKR5-crtI, and the biomass of engineering bacteria (dry weight) is 2.36gDCW/L, and content of lycopene can reach 1.52mg/gDCW.

Advantage of the present invention is: with the heterogenous expression in the photosynthetic bacterium R.sphaeroides mutant strain TC72 of the crtI gene transformation among the E.herbicola, make the R.sphaeroides that does not produce Lyeopene originally produce Lyeopene through transforming the back, and the Lyeopene that makes production does not continue to be metabolized to other carotenoid, and accumulation in a large number in cell helps the raising and the separation and purification of yield of lycopene.In addition, photosynthetic bacterium R.sphaeroides very easily cultivates, and also can be used for purifying high concentrated organic wastewater, and the production cost of Lyeopene is reduced greatly, and has great environment protection significance.

For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.

Description of drawings

Fig. 1 is R.sphaeroides, E.herbicola and R.sphaeroides mutant strain TC72 carotenoid pathways metabolism;

Fig. 2 is the pcr amplification electrophorogram of crtI full length gene among the E.herbicola of the present invention, among the figure, and the pcr amplification result of 1:crtI gene; 2: negative control; 3:2000bp DNA ladder;

Fig. 3 is the structure synoptic diagram of expression vector pRKR5 of the present invention;

Fig. 4 is the structure synoptic diagram of expression vector pRKR5-crtI of the present invention

Fig. 5 is the restriction analysis electrophorogram of expression vector pRKR5-crtI of the present invention, among the figure, and 1: the expression vector pRKR5-crtI that cuts of enzyme not; 2: the SacI of expression vector pRKR5-crtI and XbaI double digestion result; The pcr amplification result of 3:crtI full length gene;

Fig. 6 is the UV spectrophotometer measuring figure of Pure Lycopene of the present invention, TC72, TC72-pRKR5 and engineering bacteria TC72-pRKR5-crtI, among the figure, and 1: Pure Lycopene; 2: pigment that engineering bacteria produces; Pigment that 3:TC72 produces; Pigment that 4:TC72-pRKR5 produces;

Fig. 7 is that the HPLC of Pure Lycopene of the present invention, TC72, TC72-pRKR5 and engineering bacteria TC72-pRKR5-crtI detects figure, among the figure, (1): Pure Lycopene; (2): pigment that engineering bacteria produces; (3): pigment that TC72 produces; (4): pigment that TC72-pRKR5 produces;

Fig. 8 is preparation method's of the present invention schematic flow sheet.

Embodiment

Material:

1.PrimeSTAR ^TMHS archaeal dna polymerase and various restriction enzyme (Takara company product, DaLian, China)

2. connect test kit DNA Ligation Kit Ver.2.0 (Takara company product, DaLian, China)

3.DNA purification kit, centrifugal column type plain agar sugar gel DNA reclaim test kit (sky is Time Inc.'s product, the BeiJing, China)

4.DNA extract test kit Universal Genomic DNA Extraction Kit Ver.3.0 (Takara company product, DaLian, China)

5. escherichia coli jm109 competent cell (Takara company product, DaLian, China)

6.Erwinia herbicola069 bacterial strain (available from the Chinese Academy of Agricultural Sciences Institute of Plant Protection)

7. intestinal bacteria S _17-1(U.S. representative microbial DSMZ, preservation registration number is: ATCC No.47055, preservation time: 1960)

8. (DSMZ of Britain country, preservation registration number is the red bacterium RS-1 of class ball: UKNCC No.8253, preservation time: 1956)

Annotate: following reagent is the commercially available prod.

9.LB substratum

Yeast extract 5g

Tryptones 10g

NaCl 10g

Solid medium: add 2% (w/v) agar powder

Be dissolved in the 1000ml deionized water, and regulate pH value to 7.0, high pressure steam sterilization with the NaOH of 1mol/L.

10. Jin Shi substratum

Peptone 5g,

Yeast extract paste 3g,

Glucose 2.5g,

Solid medium: add 1.5% (w/v) agar powder

Be dissolved in the 1000ml deionized water, and regulate pH value to 7.2, high pressure steam sterilization with the NaOH of 1mol/L

11.M22+ substratum

With photosynthetic bacteria culture medium called after M22+ substratum, its prescription is as follows:

M22+ solid medium: in 100ml 10 * M22 storing solution, add 900ml deionized water and 15g agar powder, mixing, high pressure steam sterilization.Make the solid medium dissolving before using, add 1000 * vitamin mixture of 1ml filtration sterilization when cooling to 60 ℃.

Wherein: the preparation of 10 * M22 storing solution

KH ₂PO ₄ 30.6g

K ₂HPO ₄.3H ₂O 30g

Sodium.alpha.-hydroxypropionate 25g

(NH4) ₂SO ₄ 5g

NaCl 5g

Sodium succinate 43.425g

Sodium Glutamate 2.7g

Aspartic acid 0.4g

Solution?C 200ml

The deionized water constant volume is regulated pH value to 6.8, high pressure steam sterilization to 1000ml with NaOH.

Wherein: the preparation of Solution C

MgCl ₂.6H ₂O 24g

CaCl ₂ 3.34g

EDTA.Na ₂.2H ₂O 0.125g

ZnCl ₂ 0.261g

FeCl ₂.4H ₂O 0.25g

MnCl ₂.4H ₂O 0.09g

(NH ₄) ₆Mo ₇O ₂₄4H ₂O 0.009g

CuCl ₂.2H ₂O 0.008g

Co(NO ₃) ₂.6H ₂O 0.0124g

Boric acid 0.0057g

Nitrilotriacetic acid 10g

The deionized water constant volume is to 1000ml, and-20 ℃ of preservations need not sterilization.

Wherein: the preparation of 1000 * vitamin mixture

Nicotinic acid 1g

VitB1 0.5g

Para-amino benzoic acid 0.1g

Vitamin H 0.01g

The deionized water constant volume is to 1000ml, filtration sterilization ,-20 ℃ of preservations.

The M22+ liquid nutrient medium: on the Bechtop to the 10 * M22 storing solution that in the 1L culturing bottle of crossing high pressure steam sterilization, adds the 100ml high pressure steam sterilization respectively, 1000 * vitamin mixture of 1ml filtration sterilization, the acid hydrolysis casein of 20ml 5% high pressure steam sterilization and 879ml sterilization deionized water, standby behind the mixing in 4 ℃ of preservations.

Wherein: the preparation of 5% acid hydrolysis casein

Take by weighing 5g acid hydrolysis casein, use the deionized water dissolving constant volume to 100ml, high pressure steam sterilization ,-20 ℃ of preservations.

The clone of crtI gene among the embodiment one Erwinia herbicola

Operation instructions according to the DNA extraction test kit, from Erwinia herbicola069, extract genomic dna, according to crtI gene order (GenBank accession number: AB076662) among the Erwinia herbicola pv.Milletiae that has reported, the design primer is to S1/R1 (SEQ ID NO:8 and SEQ ID NO:9), draw the SacI restriction enzyme site at 5 ' of S1 respectively, draw the restriction enzyme site of XbaI at 5 ' of R1, primer sequence is as follows:

S1：5’-GCGCGAGCTCCATATGAATAGAACTACAGTAATTGG-3’；

R1：5’-GCGCTCTAGATCAAGCCAGATCCTCCAGCATCAA-3’。

With the E.herbicola069 genomic dna is template, sets up following PCR reaction system: reaction system is 25 μ l, wherein contains each 1 μ l of primer S1 and R1, the dNTP of 0.5 μ l, 0.5 μ l genomic dna, 5 * PrimeSTAR ^TMReaction buffer 2.5 μ l, PrimesTAR ^TMHS archaeal dna polymerase 0.2 μ l.The reaction conditions of PCR is: fs 94 ℃ of pre-sex change 4 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 56 ℃, and 72 ℃ were extended 90 seconds, and circulated 35 times; Phase III 72 ℃ of extensions 10 minutes.Gained PCR product detects visible about 1500bp size electrophoresis band through agarose electrophoresis, as shown in Figure 2.To check order behind the recovery of PCR product, the purifying.Pcr amplification goes out the dna segment of the about 1500bp of size, will check order behind the recovery of PCR product, the purifying.Sequential analysis shows that this segment is big or small consistent with the crtI gene of having reported; It is translated into aminoacid sequence, with Erwinia herbicola pv.Milletiae (GenBank accession number: AB076662) aminoacid sequence comparative analysis, found that the two amino acid sequence homology is 95.8%, proof institute clone gene is a crtI gene among the Erwinia herbicola069, and login NCBI, accession number is DQ408590.

Embodiment two has the structure of the expression vector pRKR5 of puc strong promoter

Utilize the DNA extraction test kit to extract the red bacterium RS-1 of class ball genomic dna, according to GenBank go up report the puc operon sequence (the GenBank accession number: X68796) design two couples of PCR primer: S2/R2 (SEQ ID NO:4 and SEQ ID NO:5) and S3/R3 (SEQ ID NO:6 and SEQ ID NO:7); wherein 5 ' of upstream primer S2 end contains protection base and restriction enzyme site EcoRI; 5 ' the end of downstream primer R2 contains protection base and restriction enzyme site SacI; 5 ' the end of upstream primer S3 contains protection base and restriction enzyme site XbaI; 5 ' the end of downstream primer R3 contains protection base and restriction enzyme site PstI, and primer sequence is as follows:

S2：5’-CTCTGAATTC?GCCTCGGA?CACCCTCG-3’

R2：5’-CTCTGAGCTC?TGTGTCGTCT?CCCAACTGG-3’

S3：5’-ATATTCTAGA?CGAATCACCA?CGCCCTGCG-3’

R3：5’-ATATCTGCAG?CAGTGGCAGG?CAG-3’

With above-mentioned S2/R2 is that primer can amplify puc promotor and SD sequence, is that primer can amplify puc terminator sequence with above-mentioned S 3/R3.The reaction system of PCR is 100 μ l, wherein contains each 20pmol of primer S2 and R2, the dNTP of 200 μ mol, 1 μ l RS-1 genomic dna, 5 * PrimeSTAR ^TMReaction buffer 20 μ l, the PrimeSTAR of high-fidelity ^TMHS archaeal dna polymerase 5U.The reaction conditions of PCR is: fs 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change of subordinate phase 30 seconds were annealed 30 seconds for 60 ℃, and 72 ℃ were extended 30 seconds, and circulated 30 times; Phase III 72 ℃ of extensions 5 minutes.Gained PCR product (nucleotides sequence of puc promotor and SD is classified SEQ ID NO:1 as) detects visible about 360bp size electrophoresis band through 1.5% agarose electrophoresis, with the recovery of PCR product, purifying.With above-mentioned PCR reaction system and condition, be template with the RS-1 genomic dna, with primer S3/R3 is carried out pcr amplification, the PCR product (nucleotides sequence of puc terminator is classified SEQ ID NO:2 as) of recovery, purifying one about 479bp.

The PCR product of getting the about 360bp of an amount of purifying carries out EcoRI and SacI substep enzyme is cut, and utilizes the DNA purification kit to cross the column purification enzyme then and cuts product.Simultaneously, according to above-mentioned enzyme cut that strategy carries out with wide host's property expression vector pRK415 plasmid DNA that EcoRI and SacI substep enzyme is cut and purifying (the pRK415 plasmid dna sequence can be consulted from NCBI, GenBank accession number: EF437940, pRK415 plasmid source: Keen, N.T., S.Tamaki, D.Kobayashi, and D.Trollinger.1988.Improved broad-host-rangeplasmids for DNA cloning in Gram-negative bacteria.Gene 70:191-197.).

PRK415 plasmid DNA of will be in right amount cutting through EcoRI and SacI enzyme according to the operation instructions that connects test kit and the PCR product of about 360bp are connected.Connect product and pass through CaCl ₂Conversion method transforms the JM109 competent escherichia coli cell, screens positive recombinant plasmid pRKP through 10 μ g/ml tetracyclin resistances, and sequence verification.

The PCR product of getting the about 479bp of an amount of purifying carries out XbaI and PstI substep enzyme is cut, and utilizes the DNA purification kit to cross the column purification enzyme then and cuts product.Simultaneously, recombinant plasmid pRKP being carried out EcoRI and SacI substep enzyme cuts and purifying.PRKP plasmid DNA of will be in right amount cutting through XbaI and PstI enzyme according to the operation instructions that connects test kit and the PCR product of about 479bp are connected.Connect product and pass through CaCl ₂Conversion method transforms the JM109 competent escherichia coli cell, screens positive recombinant plasmid pRKR5 through 10 μ g/ml tetracyclin resistances, and sequence verification.The nucleotides sequence of pRKR5 is classified SEQ ID NO:3 as.The construction strategy of expression vector pRKR5 is seen Fig. 3.

The structure of embodiment three expression vector pRKR5-crtI

Expression vector pRKR5 and cloning vector pMD18-crtI are carried out double digestion with SacI and XbaI respectively, its enzyme system of cutting is: 4 μ l, 10 * T damping fluid, 1.5 μ l SacI and XbaI enzyme liquid, 4 μ l%BSA, add the sterilization distilled water to 40 μ l, 37 ℃ of enzymes were cut 2 hours, and the DNA purification kit is crossed column purification SacI-XbaI double digestion product.

According to the operation instructions that connects test kit an amount of pRKR5 plasmid DNA through SacI and XbaI enzyme cutting is connected with the crtI segment.Connect product and pass through CaCl ₂Conversion method transforms the JM109 competent escherichia coli cell, after 10 μ g/ml tetracyclin resistances screenings, the white colony on the picking LB flat board in the LB liquid nutrient medium that contains 10 μ g/ml tsiklomitsins, 37 ℃, 250rpm, shaking culture is spent the night, and extracts plasmid DNA next day, carries out double digestion with SacI and XbaI and identifies, it is the same that enzyme is cut system, restriction enzyme mapping as shown in Figure 5, enzyme downcuts the segment of about 1500bp, and is big or small consistent with the crtI gene.Cut correct expression vector for enzyme and check order, sequencing result shows that the crtI gene has been connected on the expression vector pRKR5, this expression vector called after pRKR5-crtI (the vector construction strategy is seen Fig. 4).

Embodiment four expression vector transformed into escherichia coli S _17-1

(1) S _17-1The preparation of competent cell

Get the intestinal bacteria S of-80 ℃ of preservations _17-1Line on the LB flat board 37 ℃ of incubated overnight; Choose single colony inoculation in 10ml LB nutrient solution, 37 ℃, 250rpm, shaking culture is spent the night; Get the 1ml overnight culture and add in the 100ml LB nutrient solution, 37 ℃, 250rpm, shaking culture 2 hours; Place frozen water to make bacterium be cooled to 0 ℃ in 10 minutes the 100ml culture; Then in 4 ℃, 2500rpm, centrifugal 7 minutes; Abandon supernatant, with the 0.1M CaCl of 10ml ice bath precooling ₂Resuspended; 4 ℃, 2000rpm, centrifugal 5 minutes; Abandon supernatant, with the 0.1M CaCl of 2ml ice bath precooling ₂Resuspended, leave standstill in the frozen water 2 hours standby.

(2) transform

Get the S of 100 μ l ice baths _17-1Competent cell is added in the 1.5ml EP pipe of precooling, adds the expression vector pRKR5-crtI plasmid DNA that 1 μ l builds again, mixes lightly, places 30 minutes in the frozen water; In 42 ℃ of heat shocks 90 seconds, place frozen water rapidly then, placed 2 minutes; Add 500 μ l LB nutrient solutions, 37 ℃, 150rpm, shaking culture 1 hour; Get 50 μ l bacterium liquid and evenly coat on the LB flat board that contains 10 μ g/ml tsiklomitsins, 37 ℃, be inverted overnight incubation.Next day, that normal growth is S on the tetracyclin resistance flat board _17-1Transformed bacteria.Simultaneously, change the pRKR5 plasmid DNA over to S according to above-mentioned method for transformation _17-1As contrast.

Embodiment five expression vectors arrive R.sphaeroides mutant strain TC72 through conjugal transfer

According to (Coomber SA such as Coomber, _ Chaudhri M, Connor A, Britton G, Hunter CN.Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster ofRhodobacter sphaeroides.Mol Microbiol.1990; 4 (6): 977-989) reported method, transposon Tn5 is inserted acquisition Rhodobacter sphaeroides mutant strain TC72 in the red bacterium RS-1 of the class ball crtI gene.Though the crt gene cluster is complete in the mutant strain TC72, owing to the insertion of Tn5 makes its carotenoid route of synthesis rest on phytoene, the expression of this pathways metabolism crtI gene and downstream gene all is interrupted.

TC72 is lined on the M22+ substratum that contains 20 μ g/ml Xin Meisus, and 34 ℃, dark is inverted and was cultivated 3 days; The single colony inoculation of picking TC72 contains in the M22+ nutrient solution of 20 μ g/ml Xin Meisus in 10ml, and 34 ℃, 250rpm, dark shaking culture is spent the night; Next day, 4 ℃, 6000rpm, centrifugal 10 minutes collection thalline, the resuspended thalline of 1ml LB nutrient solution; Scrape and get S _17-1Transformed bacteria list bacterium colony adds in the resuspended liquid of 100 μ l LB bacteriums, abundant mixing, drip to then on the LB substratum of thorough drying, 34 ℃, cultivated 6h-8 hour, scrape from the LB substratum and to get bacterium, the resuspended thalline of 1ml M22+ is got the resuspended bacterium liquid of 100 μ l and is coated on the M22+ substratum and (to contain tsiklomitsin: 2 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, can see red bacterium colony about dark culturing 3-4 days; Owing to contain tetracycline resistance gene on expression vector pRKR5-crtI and the pRKR5, contain neomycin resistance gene in the Rhodobacter sphaeroides mutant strain TC72 genome, only contain the red bacterium of class ball of expression vector pRKR5-crtI or pRKR5 plasmid DNA could be in having these two kinds of antibiotic substratum normal growth, so these red bacterium colonies are the red bacterium of class ball that contains expression vector pRKR5-crtI or pRKR5 plasmid DNA; Picking list colony inoculation to 10ml M22+ nutrient solution (tsiklomitsin: 2 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, 250rpm, dark shaking culture is spent the night; Next day ,-80 ℃ of culture presevation.

The expression of embodiment six regulation and control oxygen concn inducible expression carriers

To contain the engineering bacteria TC72-pRKR5-crtI of expression vector pRKR5-crtI streak culture in the M22+ substratum (tsiklomitsin: 2 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, the dark inversion cultivated 4 days; Picking list colony inoculation to 10ml M22+ nutrient solution (tsiklomitsin: 2 μ g/ml, Xin Meisu: 20 μ g/ml), 34 ℃, 220rpm, shaking culture is spent the night under the dark condition; Get the 1ml overnight culture be inoculated in the 100mlM22+ nutrient solution (tsiklomitsin: 2 μ g/ml, Xin Meisu: 20 μ g/ml, nutrient solution volume be container 20%), 34 ℃, 220rpm, shaking culture is 20 hours under the dark condition; The M22+ liquid nutrient medium that adds identical antibiotic concentration is 60%, 34 ℃ until substratum and culturing bottle volume ratio, rotating speed 170rpm, inducing culture 20h.Simultaneously, the TC72 that cultivates TC72 (not adding tsiklomitsin in the M22+ substratum) according to above-mentioned condition and comprise expression vector pRKR5 is as negative control.

The engineering bacteria TC72-pRKR5-crtI that contains expression vector pRKR5-crtI is after the regulation and control oxygen concn is induced, thalline takes on a red color, and colour-change does not take place with the TC72 that comprises pRKR5 in TC72 after inducing, remain green, crtI gene in this explanation engineering bacteria has been realized expression in TC72, the recombinase biologically active of expressing makes a new pathways metabolism build up in engineering bacteria, and this approach has been finished the synthetic of a kind of red natural pigment.

The extraction and the evaluation of embodiment seven Lyeopenes

(1) extraction of Lyeopene

Lyeopene is soluble in organic solvents such as chloroform, benzene, dissolves in acetone, ethyl acetate, ether, sherwood oil, hexane, is insoluble in methyl alcohol, ethanol, and is water insoluble.Choose acetone and from photosynthetic bacterium broken wall cell, extract Lyeopene.Lyeopene is very easy oxidized in addition, and therefore after experience fragmentation and the extraction several engineering, its loss is more serious, and in preserving engineering, also will adopt necessary anti-oxidation measure.In the organic solvent of extraction, add suitable antioxidant, can the Lyeopene in the extraction liquid be played a protective role, delay its oxidized time.Thereby be chosen in the extraction liquid and select to add 1% butylated hydroxytoluene (BHT).

With 4 ℃ of cultured bacterium liquid, the centrifugal 15min of 6000rpm collects thalline.Use the aqua sterilisa washed twice, the thalline that obtains adds phosphoric acid buffer in 1: 5 ratio and carries out ultrasonic disruption (60 watts, broken 15s, 5s works 20 times at interval).Bacterium liquid after the fragmentation adds the acetone that contains BHT1% according to certain proportioning and extracts, and constantly vibration makes it thorough mixing, leaves standstill behind the 10min 4 ℃, and 6000rpm is centrifugal, and 15min makes it layering, gets supernatant liquor and is the pigment solution that extracts.

(2) evaluation of Lyeopene

1) ultraviolet spectrophotometer is identified

For Lyeopene, it has the characteristic peak appearance with UV spectrophotometer measuring, wherein at the 472nm place the last one absorption peak is arranged.According to this character, we are contrast with acetone with the pigment solution that obtains, pigment solution that will extract from engineering bacteria TC72-pRKR5-crtI, TC72 and TC72-pRKR5 and Pure Lycopene use spectrophotometer (UV-2450 day island proper Tianjin) at the interscan of 380～600nm wavelength region, the absorption spectrum that obtains such as Fig. 6 respectively.Among the figure, 1 is Pure Lycopene, and 2 are produced pigment by engineering bacteria, and 3 are produced pigment by TC72, and 4 are produced pigment by TC72-pRKR5.The absorption peak of engineering bacteria and Pure Lycopene is basic identical as seen from the figure, the three finger-type charateristic avsorption bands that all possess Lyeopene, 3 absorption peaks are respectively 446nm, 472nm, 508nm, maximum absorption band is all at the 472nm place, and TC72 and TC72-pRKR5 be not owing to produce Lyeopene thereby do not possess its three finger-types charateristic avsorption band.The light absorption value of working sample under 472nm, the sample content of carotenoid is calculated according to following formula: light absorption value * 10 ÷s 2500 of Lyeopene (mg)=mixeding liquid volume * mixed solution under 472nm.Calculating content of lycopene according to above formula is 1.52mg/gDCW, and every liter of fermented liquid can get the 2.36g dry mycelium.

2) HPLC identifies

At first accurately take by weighing the 5mg Pure Lycopene, be settled to 25ml with after the methylene dichloride dissolving, making its mass concentration is 0.2mg/ml, after HPLC measures as standard reference.

Get the centrifugal 5min of broken thalline extraction supernatant liquor 12000rpm, get supernatant liquor once more, through the aseptic membrane filtration degerming in 0.45 μ m aperture, sample size is 20 μ l before the upper prop, and be made as 20min the analysis time of each sample, and the HPLC that obtains pigment analyzes collection of illustrative plates.Chromatographic condition is: and Diamonsil C18 chromatographic column (5 μ m, 250mm * 4.6mm); Moving phase is methyl alcohol: acetonitrile: and methylene dichloride (40: 30: 30, V/V), detect wavelength 472nm, column temperature is 25 ℃, and flow velocity is 1ml/min, and sample size is 20 μ l.Measurement result as shown in Figure 7, among the figure, (1) is Pure Lycopene, (2) are produced pigment by engineering bacteria, (3) are produced pigment by TC72, (4) are produced pigment by TC72-pRKR5.As can be seen from the figure the retention time of pigment that engineering bacteria produces is 8.741min, close with the retention time 9.178 of Pure Lycopene, and TC72-pRKR5 and pigment that TC72 produces do not have characteristic peak in this retention time, can infer that from absorption spectrum and HPLC analysis the natural pigment of synthetic redness is a Lyeopene in the engineering bacteria.

The preparation method's of Lyeopene of the present invention schematic flow sheet as shown in Figure 8, experimental result of the present invention shows, utilize round pcr to amplify the crtI gene of Erwinia herbicola and be connected on the expression vector pRKR5 that contains strong promoter Puc, construction expression plasmid pRKR5-crtI, mode by conjugal transfer imports it among photosynthetic bacterium Rhodobacter sphaeroides mutant strain TC72, because promotor Puc under anaerobic efficiently expresses, the regulation and control oxygen concn can be induced engineering bacteria accumulation red pigments, through HPLC and absorption spectroanalysis, the synthetic pigment is a Lyeopene in the engineering bacteria, the biomass of engineering bacteria (dry weight) is 2.36gDCW/L, content of lycopene can reach 1.52mg/gDCW, this invention has been established preliminary basis for realizing the suitability for industrialized production Lyeopene, has the potential using value in production practice.

Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.

Sequence table

＜110〉University Of Chongqing

＜120〉a kind of preparation method of Lyeopene

<130>

<160>3

<170>Patent?In?version?3.2

<210>1

<211>360

<212>DNA

＜213〉with S2/R2 be the nucleotide sequence that primer amplification goes out the sharp SD of puc promotor

<400>1

ctctgaattc?gcctcggaca?ccctcgtttt?tgcagcagcg?agaggctgcg?ggacggccct 60

gtggggccgg?gacaggcagc?gtcaatttcc?cgcgcgcctg?cggcaaaatt?gtcccttttc 120

aagccgttac?gcaggattcc?cggccgatct?ggcggccaat?aagtcgcacc?caaaacggcc 180

ttgtcagcca?acactgacat?tgaatctgtc?agcgcaatgt?gacacccata?atgcgagccg 240

gggcggatca?gaaatcgccg?acaaggtgat?ccaggtctct?ccggtctcgt?cgaagcccgc 300

gtgcaggccc?tacacgcaaa?ccgtcgattt?accagttggg?agacgacaca?gagctcagag 360

<210>2

<211>479

<212>DNA

＜213〉with S3/R3 be the nucleotide sequence that primer amplification goes out the puc terminator

<400>2

atattctaga?cgaatcacca?cgccctgcgt?gcccgcttcc?cggaggtgca?aaaggctact 60

cctttggggg?tctttgaaat?ctcagcctgc?aggttcgcgc?cctcggcgcg?cttcgccgca 120

gggttgaacc?aggacaaccc?tgacggcgag?attccgcgca?cgtcccccac?tcgccgccga 180

ggcgaaaccc?ttggttaaca?caacgttaac?aagaccttcc?ctgccccgac?ttgtccacaa 240

cctgaccatt?tcggcagggc?acagggtagc?ctcccccttt?ttcctgctat?aacagacctt 300

tgcggcatca?ttggctgcat?gctaaagtat?ggaaatcgac?aaaactttga?agagtaggat 360

ttagccgggt?ttgcgtccca?aacacaaagt?ggacagattg?tgatgaatca?ttcgaccgac 420

tctttttcga?aatctttgaa?taccctaggc?agcgacctgc?ctgccactgc?tgcagatat 479

<210>3

<211>11489

<212>DNA

＜213〉expression vector pRKR5 plasmid dna sequence

<400>3

ctgccatttt?tggggtgagg?ccgttcgcgg?ccgaggggcg?cagcccctgg?ggggatggga 60

ggcccgcgtt?agcgggccgg?gagggttcga?gaaggggggg?cacccccctt?cggcgtgcgc 120

ggtcacgcgc?acagggcgca?gccctggtta?aaaacaaggt?ttataaatat?tggtttaaaa 180

gcaggttaaa?agacaggtta?gcggtggccg?aaaaacgggc?ggaaaccctt?gcaaatgctg 240

gattttctgc?ctgtggacag?cccctcaaat?gtcaataggt?gcgcccctca?tctgtcagca 300

ctctgcccct?caagtgtcaa?ggatcgcgcc?cctcatctgt?cagtagtcgc?gcccctcaag 360

tgtcaatacc?gcagggcact?tatccccagg?cttgtccaca?tcatctgtgg?gaaactcgcg 420

taaaatcagg?cgttttcgcc?gatttgcgag?gctggccagc?tccacgtcgc?cggccgaaat 480

cgagcctgcc?cctcatctgt?caacgccgcg?ccgggtgagt?cggcccctca?agtgtcaacg 540

tccgcccctc?atctgtcagt?gagggccaag?ttttccgcga?ggtatccaca?acgccggcgg 600

ccgcggtgtc?tcgcacacgg?cttcgacggc?gtttctggcg?cgtttgcagg?gccatagacg 660

gccgccagcc?cagcggcgag?ggcaaccagc?ccggtgagcg?tcggaaaggc?gctcttccgc 720

ttcctcgctc?actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca 780

ctcaaaggcg?gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg 840

agcaaaaggc?cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca 900

taggctccgc?ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa 960

cccgacagga?ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc 1020

tgttccgacc?ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc 1080

gccattcgcc?attcaggctg?cgcaactgtt?gggaagggcg?atcggtgcgg?gcctcttcgc 1140

tattacgcca?gctggcgaaa?gggggatgtg?ctgcaaggcg?attaagttgg?gtaacgccag 1200

ggttttccca?gtcacgacgt?tgtaaaacga?cggccagtga?attcgcctcg?gacaccctcg 1260

tttttgcagc?agcgagaggc?tgcgggacgg?ccctgtgggg?ccgggacagg?cagcgtcaat 1320

ttcccgcgcg?cctgcggcaa?aattgtccct?tttcaagccg?ttacgcagga?ttcccggccg 1380

atctggcggc?caataagtcg?cacccaaaac?ggccttgtca?gccaacactg?acattgaatc 1440

tgtcagcgca?atgtgacacc?cataatgcga?gccggggcgg?atcagaaatc?gccgacaagg 1500

tgatccaggt?ctctccggtc?tcgtcgaagc?ccgcgtgcag?gccctacacg?caaaccgtcg 1560

atttaccagt?tgggagacga?cacagagctc?ggtacccggg?gatcctctag?acgaatcacc 1620

acgccctgcg?tgcccgcttc?ccggaggtgc?aaaaggctac?tcctttgggg?gtctttgaaa 1680

tctcagcctg?caggttcgcg?ccctcggcgc?gcttcgccgc?agggttgaac?caggacaacc 1740

ctgacggcga?gattccgcgc?acgtccccca?ctcgccgccg?aggcgaaacc?cttggttaac 1800

acaacgttaa?caagaccttc?cctgccccga?cttgtccaca?acctgaccat?ttcggcaggg 1860

cacagggtag?cctccccctt?tttcctgcta?taacagacct?ttgcggcatc?attggctgca 1920

tgctaaagta?tggaaatcga?caaaactttg?aagagtagga?tttagccggg?tttgcgtccc 1980

aaacacaaag?tggacagatt?gtgatgaatc?attcgaccga?ctctttttcg?aaatctttga 2040

ataccctagg?cagcgacctg?cctgccactg?gtcgacctgc?aggcatgcaa?gcttggcgta 2100

atcatggtca?tagctgtttc?ctgtgtgaaa?ttgttatccg?ctcacaattc?cacacaacat 2160

acgagccgga?agcataaagt?gtaaagcctg?gggtgcctaa?tgagtgagct?aactcacatt 2220

aattgcgttg?cgctcactgc?ccgctttcca?gtcgggaaac?ctgtcgtgcc?agctgcatta 2280

atgaatcggc?caacgcgcgg?ggagaggcgg?tttgcgtatt?gggcgctcgg?tcttgccttg 2340

ctcgtcggtg?atgtacttca?ccagctccgc?gaagtcgctc?ttcttgatgg?agcgcatggg 2400

gacgtgcttg?gcaatcacgc?gcaccccccg?gccgttttag?cggctaaaaa?agtcatggct 2460

ctgccctcgg?gcggaccacg?cccatcatga?ccttgccaag?ctcgtcctgc?ttctcttcga 2520

tcttcgccag?cagggcgagg?atcgtggcat?caccgaaccg?cgccgtgcgc?gggtcgtcgg 2580

tgagccagag?tttcagcagg?ccgcccaggc?ggcccaggtc?gccattgatg?cgggccagct 2640

cgcggacgtg?ctcatagtcc?acgacgcccg?tgattttgta?gccctggccg?acggccagca 2700

ggtaggccga?caggctcatg?ccggccgccg?ccgccttttc?ctcaatcgct?cttcgttcgt 2760

ctggaaggca?gtacaccttg?ataggtgggc?tgcccttcct?ggttggcttg?gtttcatcag 2820

ccatccgctt?gccctcatct?gttacgccgg?cggtagccgg?ccagcctcgc?agagcaggat 2880

tcccgttgag?caccgccagg?tgcgaataag?ggacagtgaa?gaaggaacac?ccgctcgcgg 2940

gtgggcctac?ttcacctatc?ctgcccggct?gacgccgttg?gatacaccaa?ggaaagtcta 3000

cacgaaccct?ttggcaaaat?cctgtatatc?gtgcgaaaaa?ggatggatat?accgaaaaaa 3060

tcgctataat?gaccccgaag?cagggttatg?cagcggaaaa?gcgccacgct?tcccgaaggg 3120

agaaaggcgg?acaggtatcc?ggtaagcggc?agggtcggaa?caggagagcg?cacgagggag 3180

cttccagggg?gaaacgcctg?gtatctttat?agtcctgtcg?ggtttcgcca?cctctgactt 3240

gagcgtcgat?ttttgtgatg?ctcgtcaggg?gggcggagcc?tatggaaaaa?cgccagcaac 3300

gcggcctttt?tacggttcct?ggccttttgc?tggccttttg?ctcacatgtt?ctttcctgcg 3360

ttatcccctg?attctgtgga?taaccgtatt?accgcctttg?agtgagctga?taccgctcgc 3420

cgcagccgaa?cgaccgagcg?cagcgagtca?gtgagcgagg?aagcggaaga?gcgccagaag 3480

gccgccagag?aggccgagcg?cggccgtgag?gcttggacgc?tagggcaggg?catgaaaaag 3540

cccgtagcgg?gctgctacgg?gcgtctgacg?cggtggaaag?ggggagggga?tgttgtctac 3600

atggctctgc?tgtagtgagt?gggttgcgct?ccggcagcgg?tcctgatcaa?tcgtcaccct 3660

ttctcggtcc?ttcaacgttc?ctgacaacga?gcctcctttt?cgccaatcca?tcgacaatca 3720

ccgcgagtcc?ctgctcgaac?gctgcgtccg?gaccggcttc?gtcgaaggcg?tctatcgcgg 3780

cccgcaacag?cggcgagagc?ggagcctgtt?caacggtgcc?gccgcgctcg?ccggcatcgc 3840

tgtcgccggc?ctgctcctca?agcacggccc?caacagtgaa?gtagctgatt?gtcatcagcg 3900

cattgacggc?gtccccggcc?gaaaaacccg?cctcgcagag?gaagcgaagc?tgcgcgtcgg 3960

ccgtttccat?ctgcggtgcg?cccggtcgcg?tgccggcatg?gatgcgcgcg?ccatcgcggt 4020

aggcgagcag?cgcctgcctg?aagctgcggg?cattcccgat?cagaaatgag?cgccagtcgt 4080

cgtcggctct?cggcaccgaa?tgcgtatgat?tctccgccag?catggcttcg?gccagtgcgt 4140

cgagcagcgc?ccgcttgttc?ctgaagtgcc?agtaaagcgc?cggctgctga?acccccaacc 4200

gttccgccag?tttgcgtgtc?gtcagaccgt?ctacgccgac?ctcgttcaac?aggtccaggg 4260

cggcacggat?cactgtattc?ggctgcaact?ttgtcatgct?tgacacttta?tcactgataa 4320

acataatatg?tccaccaact?tatcagtgat?aaagaatccg?cgcgttcaat?cggaccagcg 4380

gaggctggtc?cggaggccag?acatgaaacc?caacataccc?ctgatcgtaa?ttctgagcac 4440

tgtcgcgctc?gacgctgtcg?gcatcggcct?gattatgccg?gtgctgccgg?gcctcctgcg 4500

cgatctggtt?cactcgaacg?acgtcaccgc?ccactatggc?attctgctgg?cgctgtatgc 4560

gttggtgcaa?tttgcctgcg?cacctgtgct?gggcgcgctg?tcggatcgtt?tcgggcggcg 4620

gccaatcttg?ctcgtctcgc?tggccggcgc?cactgtcgac?tacgccatca?tggcgacagc 4680

gcctttcctt?tgggttctct?atatcgggcg?gatcgtggcc?ggcatcaccg?gggcgactgg 4740

ggcggtagcc?ggcgcttata?ttgccgatat?cactgatggc?gatgagcgcg?cgcggcactt 4800

cggcttcatg?agcgcctgtt?tcgggttcgg?gatggtcgcg?ggacctgtgc?tcggtgggct 4860

gatgggcggt?ttctcccccc?acgctccgtt?cttcgccgcg?gcagccttga?acggcctcaa 4920

tttcctgacg?ggctgtttcc?ttttgccgga?gtcgcacaaa?ggcgaacgcc?ggccgttacg 4980

ccgggaggct?ctcaacccgc?tcgcttcgtt?ccggtgggcc?cggggcatga?ccgtcgtcgc 5040

cgccctgatg?gcggtcttct?tcatcatgca?acttgtcgga?caggtgccgg?ccgcgctttg 5100

ggtcattttc?ggcgaggatc?gctttcactg?ggacgcgacc?acgatcggca?tttcgcttgc 5160

cgcatttggc?attctgcatt?cactcgccca?ggcaatgatc?accggccctg?tagccgcccg 5220

gctcggcgaa?aggcgggcac?tcatgctcgg?aatgattgcc?gacggcacag?gctacatcct 5280

gcttgccttc?gcgacacggg?gatggatggc?gttcccgatc?atggtcctgc?ttgcttcggg 5340

tggcatcgga?atgccggcgc?tgcaagcaat?gttgtccagg?caggtggatg?aggaacgtca 5400

ggggcagctg?caaggctcac?tggcggcgct?caccagcctg?acctcgatcg?tcggacccct 5460

cctcttcacg?gcgatctatg?cggcttctat?aacaacgtgg?aacgggtggg?catggattgc 5520

aggcgctgcc?ctctacttgc?tctgcctgcc?ggcgctgcgt?cgcgggcttt?ggagcggcgc 5580

agggcaacga?gccgatcgct?gatcgtggaa?acgataggcc?tatgccatgc?gggtcaaggc 5640

gacttccggc?aagctatacg?cgccctagga?gtgcggttgg?aacgttggcc?cagccagata 5700

ctcccgatca?cgagcaggac?gccgatgatt?tgaagcgcac?tcagcgtctg?atccaagaac 5760

aaccatccta?gcaacacggc?ggtccccggg?ctgagaaagc?ccagtaagga?aacaactgta 5820

ggttcgagtc?gcgagatccc?ccggaaccaa?aggaagtagg?ttaaacccgc?tccgatcagg 5880

ccgagccacg?ccaggccgag?aacattggtt?cctgtaggca?tcgggattgg?cggatcaaac 5940

actaaagcta?ctggaacgag?cagaagtcct?ccggccgcca?gttgccaggc?ggtaaaggtg 6000

agcagaggca?cgggaggttg?ccacttgcgg?gtcagcacgg?ttccgaacgc?catggaaacc 6060

gcccccgcca?ggcccgctgc?gacgccgaca?ggatctagcg?ctgcgtttgg?tgtcaacacc 6120

aacagcgcca?cgcccgcagt?tccgcaaata?gcccccagga?ccgccatcaa?tcgtatcggg 6180

ctacctagca?gagcggcaga?gatgaacacg?accatcagcg?gctgcacagc?gcctaccgtc 6240

gccgcgaccc?gcccggcagg?cggtagaccg?aaataaacaa?caagctccag?aatagcgaaa 6300

tattaagtgc?gccgaggatg?aagatgcgca?tccaccagat?tcccgttgga?atctgtcgga 6360

cgatcatcac?gagcaataaa?cccgccggca?acgcccgcag?cagcataccg?gcgacccctc 6420

ggcctcgctg?ttcgggctcc?acgaaaacgc?cggacagatg?cgccttgtga?gcgtccttgg 6480

ggccgtcctc?ctgtttgaag?accgacagcc?caatgatctc?gccgtcgatg?taggcgccga 6540

atgccacggc?atctcgcaac?cgttcagcga?acgcctccat?gggctttttc?tcctcgtgct 6600

cgtaaacgga?cccgaacatc?tctggagctt?tcttcagggc?cgacaatcgg?atctcgcgga 6660

aatcctgcac?gtcggccgct?ccaagccgtc?gaatctgagc?cttaatcaca?attgtcaatt 6720

ttaatcctct?gtttatcggc?agttcgtaga?gcgcgccgtg?cgcccgagcg?atactgagcg 6780

aagcaagtgc?gtcgagcagt?gcccgcttgt?tcctgaaatg?ccagtaaagc?gctggctgct 6840

gaacccccag?ccggaactga?ccccacaagg?ccctagcgtt?tgcaatgcac?caggtcatca 6900

ttgacccagg?cgtgttccac?caggccgctg?cctcgcaact?cttcgcaggc?ttcgccgacc 6960

tgctcgcgcc?acttcttcac?gcgggtggaa?tccgatccgc?acatgaggcg?gaaggtttcc 7020

agcttgagcg?ggtacggctc?ccggtgcgag?ctgaaatagt?cgaacatccg?tcgggccgtc 7080

ggcgacagct?tgcggtactt?ctcccatatg?aatttcgtgt?agtggtcgcc?agcaaacagc 7140

acgacgattt?cctcgtcgat?caggacctgg?caacgggacg?ttttcttgcc?acggtccagg 7200

acgcggaagc?ggtgcagcag?cgacaccgat?tccaggtgcc?caacgcggtc?ggacgtgaag 7260

cccatcgccg?tcgcctgtag?gcgcgacagg?cattcctcgg?ccttcgtgta?ataccggcca 7320

ttgatcgacc?agcccaggtc?ctggcaaagc?tcgtagaacg?tgaaggtgat?cggctcgccg 7380

ataggggtgc?gcttcgcgta?ctccaacacc?tgctgccaca?ccagttcgtc?atcgtcggcc 7440

cgcagctcga?cgccggtgta?ggtgatcttc?acgtccttgt?tgacgtggaa?aatgaccttg 7500

ttttgcagcg?cctcgcgcgg?gattttcttg?ttgcgcgtgg?tgaacagggc?agagcgggcc 7560

gtgtcgtttg?gcatcgctcg?catcgtgtcc?ggccacggcg?caatatcgaa?caaggaaagc 7620

tgcatttcct?tgatctgctg?cttcgtgtgt?ttcagcaacg?cggcctgctt?ggcctcgctg 7680

acctgttttg?ccaggtcctc?gccggcggtt?tttcgcttct?tggtcgtcat?agttcctcgc 7740

gtgtcgatgg?tcatcgactt?cgccaaacct?gccgcctcct?gttcgagacg?acgcgaacgc 7800

tccacggcgg?ccgatggcgc?gggcagggca?gggggagcca?gttgcacgct?gtcgcgctcg 7860

atcttggccg?tagcttgctg?gaccatcgag?ccgacggact?ggaaggtttc?gcggggcgca 7920

cgcatgacgg?tgcggcttgc?gatggtttcg?gcatcctcgg?cggaaaaccc?cgcgtcgatc 7980

agttcttgcc?tgtatgcctt?ccggtcaaac?gtccgattca?ttcaccctcc?ttgcgggatt 8040

gccccgactc?acgccggggc?aatgtgccct?tattcctgat?ttgacccgcc?tggtgccttg 8100

gtgtccagat?aatccacctt?atcggcaatg?aagtcggtcc?cgtagaccgt?ctggccgtcc 8160

ttctcgtact?tggtattccg?aatcttgccc?tgcacgaata?ccagcgaccc?cttgcccaaa 8220

tacttgccgt?gggcctcggc?ctgagagcca?aaacacttga?tgcggaagaa?gtcggtgcgc 8280

tcctgcttgt?cgccggcatc?gttgcgccac?tcttcattaa?ccgctatatc?gaaaattgct 8340

tgcggcttgt?tagaattgcc?atgacgtacc?tcggtgtcac?gggtaagatt?accgataaac 8400

tggaactgat?tatggctcat?atcgaaagtc?tccttgagaa?aggagactct?agtttagcta 8460

aacattggtt?ccgctgtcaa?gaactttagc?ggctaaaatt?ttgcgggccg?cgaccaaagg 8520

tgcgaggggc?ggcttccgct?gtgtacaacc?agatattttt?caccaacatc?cttcgtctgc 8580

tcgatgagcg?gggcatgacg?aaacatgagc?tgtcggagag?ggcaggggtt?tcaatttcgt 8640

ttttatcaga?cttaaccaac?ggtaaggcca?acccctcgtt?gaaggtgatg?gaggccattg 8700

ccgacgccct?ggaaactccc?ctacctcttc?tcctggagtc?caccgacctt?gaccgcgagg 8760

cactcgcgga?gattgcgggt?catcctttca?agagcagcgt?gccgcccgga?tacgaacgca 8820

tcagtgtggt?tttgccgtca?cataaggcgt?ttatcgtaaa?gaaatggggc?gacgacaccc 8880

gaaaaaagct?gcgtggaagg?ctctgacgcc?aagggttagg?gcttgcactt?ccttctttag 8940

ccgctaaaac?ggccccttct?ctgcgggccg?tcggctcgcg?catcatatcg?acatcctcaa 9000

cggaagccgt?gccgcgaatg?gcatcgggcg?ggtgcgcttt?gacagttgtt?ttctatcaga 9060

acccctacgt?cgtgcggttc?gattagctgt?ttgtcttgca?ggctaaacac?tttcggtata 9120

tcgtttgcct?gtgcgataat?gttgctaatg?atttgttgcg?taggggttac?tgaaaagtga 9180

gcgggaaaga?agagtttcag?accatcaagg?agcgggccaa?gcgcaagctg?gaacgcgaca 9240

tgggtgcgga?cctgttggcc?gcgctcaacg?acccgaaaac?cgttgaagtc?atgctcaacg 9300

cggacggcaa?ggtgtggcac?gaacgccttg?gcgagccgat?gcggtacatc?tgcgacatgc 9360

ggcccagcca?gtcgcaggcg?attatagaaa?cggtggccgg?attccacggc?aaagaggtca 9420

cgcggcattc?gcccatcctg?gaaggcgagt?tccccttgga?tggcagccgc?tttgccggcc 9480

aattgccgcc?ggtcgtggcc?gcgccaacct?ttgcgatccg?caagcgcgcg?gtcgccatct 9540

tcacgctgga?acagtacgtc?gaggcgggca?tcatgacccg?cgagcaatac?gaggtcatta 9600

aaagcgccgt?gattgatgat?atagcggccc?ggctgctcct?ggttctcgcg?caccgaaatg 9660

ggtgacttca?ccccgcgctc?tttgatcgtg?gcaccgattt?ccgcgatgct?ctccggggaa 9720

aagccggggt?tgtcggccgt?ccgcggctga?tgcggatctt?cgtcgatcag?gtccaggtcc 9780

agctcgatag?ggccggaacc?gccctgagac?gccgcaggag?cgtccaggag?gctcgacagg 9840

tcgccgatgc?tatccaaccc?caggccggac?ggctgcgccg?cgcctgcggc?ttcctgagcg 9900

gccgcagcgg?tgtttttctt?ggtggtcttg?gcttgagccg?cagtcattgg?gaaatctcca 9960

tcttcgtgaa?cacgtaatca?gccagggcgc?gaacctcttt?cgatgccttg?cgcgcggccg 10020

ttttcttgat?cttccagacc?ggcacaccgg?atgcgagggc?atcggcgatg?ctgctgcgca 10080

ggccaacggt?ggccggaatc?atcatcttgg?ggtacgcggc?cagcagctcg?gcttggtggc 10140

gcgcgtggcg?cggattccgc?gcatcgacct?tgctgggcac?catgccaagg?aattgcagct 10200

tggcgttctt?ctggcgcacg?ttcgcaatgg?tcgtgaccat?cttcttgatg?ccctggatgc 10260

tgtacgcctc?aagctcgatg?ggggacagca?catagtcggc?cgcgaagagg?gcggccgcca 10320

ggccgacgcc?aagggtcggg?gccgtgtcga?tcaggcacac?gtcgaagcct?tggttcgcca 10380

gggccttgat?gttcgccccg?aacagctcgc?gggcgtcgtc?cagcgacagc?cgttcggcgt 10440

tcgccagtac?cgggttggac?tcgatgaggg?cgaggcgcgc?ggcctggccg?tcgccggctg 10500

cgggtgcggt?ttcggtccag?ccgccggcag?ggacagcgcc?gaacagcttg?cttgcatgca 10560

ggccggtagc?aaagtccttg?agcgtgtagg?acgcattgcc?ctgggggtcc?aggtcgatca 10620

cggcaacccg?caagccgcgc?tcgaaaaagt?cgaaggcaag?atgcacaagg?gtcgaagtct 10680

tgccgacgcc?gcctttctgg?ttggccgtga?ccaaagtttt?catcgtttgg?tttcctgttt 10740

tttcttggcg?tccgcttccc?acttccggac?gatgtacgcc?tgatgttccg?gcagaaccgc 10800

cgttacccgc?gcgtacccct?cgggcaagtt?cttgtcctcg?aacgcggccc?acacgcgatg 10860

caccgcttgc?gacactgcgc?ccctggtcag?tcccagcgac?gttgcgaacg?tcgcctgtgg 10920

cttcccatcg?actaagacgc?cccgcgctat?ctcgatggtc?tgctgcccca?cttccagccc 10980

ctggatcgcc?tcctggaact?ggctttcggt?aagccgtttc?ttcatggata?acacccataa 11040

tttgctccgc?gccttggttg?aacatagcgg?tgacagccgc?cagcacatga?gagaagttta 11100

gctaaacatt?tctcgcacgt?caacaccttt?agccgctaaa?actcgtcctt?ggcgtaacaa 11160

aacaaaagcc?cggaaaccgg?gctttcgtct?cttgccgctt?atggctctgc?acccggctcc 11220

atcaccaaca?ggtcgcgcac?gcgcttcact?cggttgcgga?tcgacactgc?cagcccaaca 11280

aagccggttg?ccgccgccgc?caggatcgcg?ccgatgatgc?cggccacacc?ggccatcgcc 11340

caccaggtcg?ccgccctcac?tgcccggcac?ctggtcgctg?aatgtcgatg?ccagcacctg 11400

cggcacgtca?atgcttccgg?gcgtcgcgct?cgggctgatc?gcccatcccg?ttactgcccc 11460

gatcccggca?atggcaagga?ctgccagcg 11489

<210>4

<211>26

<212>DNA

＜213〉PCR primer S2

<400>4

ctctgaattc?gcctcggaca?ccctcg 26

<210>5

<211>29

<212>DNA

＜213〉PCR primer R2

<400>5

ctctgagctc?tgtgtcgtct?cccaactgg 29

<210>6

<211>29

<212>DNA

＜213〉PCR primer S3

<400>6

atattctaga?cgaatcacca?cgccctgcg 29

<210>7

<211>23

<212>DNA

＜213〉PCR primer R3

<400>7

atatctgcag?cagtggcagg?cag 23

<210>8

<211>36

<212>DNA

＜213〉sequence of crtI gene primer S1

<400>8

gcgcgagctc?catatgaata?gaactacagt?aattgg 36

<210>9

<211>34

<212>DNA

＜213〉sequence of crtI gene primer R1

<400>9

gcgctctaga?tcaagccaga?tcctccagca?tcaa 34

Claims (2)

1. the preparation method of a Lyeopene is characterized in that in steps:

(1) extracts the E.herbicola genomic dna, clone the crtI full length gene by PCR;

(2) make up the pRKR5 carrier;

(3) the crtI gene fragment is connected on the pRKR5 carrier construction of expression vector pRKR5-crtI;

(4) the pRKR5-crtI plasmid is imported structure engineering bacteria TC72-pRKR5-crtI among the photosynthetic bacterium Rhodobacter sphaeroides mutant strain TC72;

(5) the engineering bacteria TC72-pRKR5-crtI that step (4) is obtained regulates and control the oxygen concn abduction delivering, obtains the Lyeopene of cell inner accumulation red pigments.

2. the preparation method of Lyeopene according to claim 1 is characterized in that the following steps that are built with of described pRKR5 expression vector:

With S2/R2 is that primer amplification goes out puc promotor and SD sequence, and puc promotor and SD sequence have the nucleotide sequence shown in the SEQ ID NO:1, are that primer amplification goes out the puc terminator with S3/R3, and the puc terminator has the nucleotide sequence shown in the SEQ ID NO:2;

Described puc promoter gene and SD sequence are inserted between the EcoRI and SacI of pRK415, and puc terminator gene is inserted between the XbaI and PstI of pRK415, and construction strategy is seen Fig. 3.

Images