CN102286504B - Lycopene cyclase gene in sphingomonas sp. and use thereof - Google Patents

Abstract

The invention provides the DNA sequence of a lycopene cyclase gene (crtY) in sphingomonas sp., a recombinant strain lacking lycopene cyclase and use thereof. The nucleotide sequence of the crtY gene is represented by SEQ ID NO.1. The DNA sequence of the lycopene cyclase gene (crtY) in sphingomonas sp., which is provided by the invention, lays a foundation for the genetic modification of a sphingomonas sp. carotenoid biological synthesis means. In addition, compared with a wild strain, the sphingomonas sp. recombinant strain lacking the lycopene cyclase gene, which is constructed by a gene knockout method, has the advantages that: the gellengum yield is basically unchanged; and lycopene can be produced in the production of gellengum by fermentation. And the strain can be used for further gene knockout or metabolic engineering modification.

Description

Lycopene cyclase gene and the application thereof of sphingomonas paucimobilis

(1) technical field

The present invention relates to sphingomonas paucimobilis lycopene cyclase gene (crtY), lack restructuring sphingomonas paucimobilis and the application thereof of this gene.

(2) background technology

Gelling gum is by β-1,3-D-glucose, β-Isosorbide-5-Nitrae-D-Glucose aldehydic acid, β-1, and 3-D-glucose, α-Isosorbide-5-Nitrae-L-rhamnosyl connect the Microbial exopolysaccharides that polymerization consists of.Compared with similar products, it has the lot of superiority energy, and is few such as consumption; Zero pour, fusing point etc. are adjustable; Acidproof, alkaline-resisting, thermostability is high; Good water-soluble, be flavor property, moisture retention, film-forming properties, slow-releasing etc.In recent years, gelling gum is used as New-type emulsifier, suspension agent, thickening material, stablizer, gelifying agent, sustained release dosage, film forming material etc. and is applied to the industrial circles such as food, medicine, chemical industry increasingly extensively.In addition, gelling gum can with the composite use of other foodstuff glues, make food can obtain best sense organ, matter structure and stability requirement.Gelling gum has a extensive future, and applies yet its higher production cost has limited.

Gelling gum is by the sphingomonas paucimobilis fermentative production.Sphingomonas paucimobilis is a kind of Gram-negative, and aerobic bacillus and end are given birth to flagellum, produces yellow carotenoid when gelling gum is produced in its fermentation.The biosynthetic pathway of carotenoid has been widely studied and has obtained huge progress, and a large amount of key genes obtain the clone.All carotenoid is all synthetic by isoprenoid compounds or terpenoid approach.IPP (isopentenylpyrophosphate) is the precursor substance of this route of synthesis.In most of bacterium, IPP is synthetic through MEP (2-C-methyl D-erythritol) approach by pyruvic acid and glyceraldehyde 3-phosphate.IPP generates DMAPP (dimethyl propylene thiazolinyl bisphosphate) under the effect of IPP isomerase, and then generates GGPP (geranyl geranyl tetra-sodium) with 3 IPP condensations.Two molecule GGPP form first colourless carotenoid-phytoene under the effect of phytoene synthetase, route of synthesis forms a lot of branches afterwards.In most bacterium, phytoene is forming red Lyeopene after through 4 continuous dehydrogenation steps under the effect of phytoene dehydrogenase.Lyeopene is again through forming various types of carotene under the effect of a series of different enzymes such as cyclase, hydroxylase, ketonize enzyme.

Lyeopene is a kind of of carotenoid, by 11 conjugation and 2 straight chain type hydrocarbon polymers that non-conjugated carbon-to-carbon double bond forms.Lyeopene has very strong oxidation-resistance, and efficiently singlet-oxygen quenching is removed interior free yl, has the anticancer effect that presses down cancer, preventing cardiovascular disease, enhancing human immune system and delay senility.As functional natural colorants, Lyeopene is increasingly extensive in the application of the aspects such as food, healthcare products, makeup and medicine, and demand is increasing, and market outlook are wide.

Thereby sphingomonas paucimobilis lycopene cyclase gene order is not yet arranged and utilize gene Knockout to knock out the report of sphingomonas paucimobilis lycopene cyclase gene gellan gum fermentation coproduction Lyeopene at present.

(3) summary of the invention

The purpose of this invention is to provide the dna sequence dna of lycopene cyclase gene of sphingomonas paucimobilis and recombinant bacterial strain and the application thereof of this enzyme disappearance.

The technical solution used in the present invention is:

The lycopene cyclase gene (crtY) of sphingomonas paucimobilis (Sphingomonas sp.), nucleotide sequence is shown in SEQ ID NO.1.This sequence length is 1158bp, the protein that is comprised of 385 amino acid of encoding.

The invention still further relates to a kind of restructuring sphingomonas paucimobilis of crtY genetically deficient, remove the crtY gene order shown in the SEQ ID NO.1 by sphingomonas paucimobilis through gene knockout and obtain.

Gene knockout is this area routine techniques, and in the known gene order situation that is knocked, those of ordinary skills can select ordinary method to carry out the gene knockout operation according to general knowledge.Specifically can be performed as follows:

1, take the sphingomonas paucimobilis genome as template, utilize the upstream and downstream segment of PCR method amplification crtY gene, respectively the upstream and downstream segment is inserted conventional knockout carrier pLO3 and made up crtY gene knockout carrier pLO3-△ Y;

2, crtY gene knockout carrier pLO3-△ Y is imported in the sphingomonas paucimobilis by three close method of joining;

3, for the first time wholely during homologous recombination knock out homologous site, the tetracyclin resistance screening that plasmid inserts in the genome and obtain positive recombinant, and with PCR method amplification sacB gene identification.The homologous recombination that screening is obtained exchanges for the first time positive recombinant and inoculates and go down to posterity in the preculture substratum three times, then is coated on the screening flat board that contains 8% sucrose with screening homologous recombination permutoid for the second time.The homologous recombination body second time that screening obtains is xeroxed and is further identified to the resistant panel that contains tsiklomitsin and with PCR method, thereby obtains the sphingomonas paucimobilis of crtY genetically deficient.

Among the present invention, described gene knockout can be undertaken by the nucleotide fragment shown in the SEQ ID NO.2.Specific as follows: as respectively SEQ ID NO.2 middle and upper reaches (SEQ ID NO.2 the 396th～773 bit base) downstreams (SEQ ID NO.2 the 1542nd～2018 bit base) segment to be inserted knockout carrier pLO3 and made up crtY gene knockout carrier pLO3-△ Y; Then crtY gene knockout carrier pLO3-△ Y is imported in the sphingomonas paucimobilis by three close method of joining, obtain the restructuring sphingomonas paucimobilis of crtY genetically deficient through screening.The dna sequence dna of SEQ ID NO.2 comprises altogether 2018bp of crtY gene, obtain by the following method: according to the partial sequence of sphingomonas paucimobilis ATCC31461crtY (the NCBI accession number: HQ202920) design gene-specific primer, and then by the SiteFinding-PCR technology obtain to comprise the crtY gene complete sequence and with the NCBI accession number be that the sequence assembly of HQ202920 obtains the required part flanking sequence of gene knockout.

The invention still further relates to described restructuring sphingomonas paucimobilis and prepare application in gelling gum and the Lyeopene at microbial fermentation.

Beneficial effect of the present invention is mainly reflected in: the dna sequence dna of sphingomonas paucimobilis lycopene cyclase gene provided by the invention (crtY) is that the sphingomonas paucimobilis Carotenoid biosynthetic pathway carries out genetic modification and lays the foundation.In addition, the present invention compares with wild type strain by the sphingomonas paucimobilis recombinant bacterial strain of the lycopene cyclase disappearance of the method structure of gene knockout, the substantially constant and product Lyeopene of gelling gum output, thereby can gellan gum fermentation coproduction Lyeopene, simultaneously, this bacterial strain can also be applied to further metabolic engineering.

(4) description of drawings

Fig. 1 is the structural representation of pLO3 plasmid;

Fig. 2 is the electrophorogram that the PCR method is identified the crtY gene knockout: 1 take the sphingomonas paucimobilis genome of the crY genetically deficient that makes up as template (1107bp), and 2 compare (1869bp) take wild-type sphingomonas paucimobilis genome as template.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: the acquisition of sphingomonas paucimobilis crtY gene DNA sequence

According to the partial sequence of sphingomonas paucimobilis ATCC 31461 crtY (the NCBI accession number: HQ202920) design upstream gene Auele Specific Primer, by the SiteFinding-PCR technology obtain to comprise the crtY gene complete sequence and with the NCBI accession number be that the sequence assembly of HQ202920 obtains the required part flanking sequence of gene knockout.Used upstream gene Auele Specific Primer, SiteFinder, SFP primer sequence are as follows:

The upstream sequence Auele Specific Primer:

UP1：5′-GCTGTAATAGGTGTCCTCGACGA-3′

UP2：5′-TGAACGGCAGGCAATAGACGAAG-3′

SiteFinder：

SiteFl：

5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNCATGG-3′

SiteF2：

5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNCATGC-3′

SiteF3：

5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNGCCT-3′

SiteF4：

5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNGCCACG-3′

The SFP primer:

SFP1：5′-CACGACACGCTACTCAACAC-3′

SFP2：5′-ACTCAACACACCACCACGCACAGC-3′

Extraction genomic dna from sphingomonas paucimobilis Sphingomonas paucimobilis ATCC 31461 (buying from ATCC) (using AxyPrep bacterial genomes DNA to prepare in a small amount test kit) is connected on pMD19-T (Takara) carrier after reclaiming (use AxyPrep dna gel reclaims test kit) purifying as the target dna segment rubber tapping that pcr template utilizes the amplification of SiteFing-PCR technology to obtain, transform intestinal bacteria Top10 competent cell, picking ammonia benzyl resistance clone, bacterium colony PCR identifies the PCR fragment of whether inserting recovery among the pMD19-T.To positive colony order-checking (Invitrogen company mensuration), and make BLAST and analyze, the upstream target dna segment that discovery obtains comprises initiator codon, be to be used in the sequence of HQ202920 knocking out obtaining a segment length behind the sequence assembly of crtY gene and being the dna sequence dna of 2018bp with above-mentioned sequence and NCBI accession number, shown in SEQ ID No.2, the crtY gene that wherein comprises (SEQ ID No.1) total length is 1158bp (SEQ ID No.2 the 621st bit base～the 1778th bit base).

Embodiment 2: the structure of the sphingomonas paucimobilis recombinant bacterial strain of lycopene cyclase disappearance

1, the structure of gene knockout carrier pLO3-△ Y

According to the dna sequence dna design primer that obtains.From sphingomonas paucimobilis Sphingonmonas paucimobilis ATCC 31461 (ATCC purchase), extract genomic dna (using AxyPrep bacterial genomes DNA to prepare in a small amount test kit) as pcr template.Y1, Y2 are primer Taq enzyme (Takara) amplification upstream homologous sequence, and the PCR response procedures is that 95 ℃ of denaturation 5min enter working cycle; 94 ℃ of sex change 30sec, 62 ℃ of annealing 30sec, 72 ℃ are extended 30sec, and 30 circulations are extended 10min with 72 ℃ at last.Y3, Y4 are primer Taq enzyme (Takara) amplification downstream homologous sequence, and the PCR response procedures is: 95 ℃ of denaturation 5min enter working cycle; 94 ℃ of sex change 30sec, 63 ℃ of annealing 30sec, 72 ℃ are extended 40s, and 30 circulations are extended 10min with 72 ℃ at last.Above-mentioned primer sequence is as follows:

Y1:5 '-AT GAGCTCCAACCTGTTCAACACCAC-3 ' underscore is the SacI restriction enzyme site

Y2:5 '-GCG TCTAGAGAACGACCAGAGGTGATT-3 ' underscore is the XbaI enzyme cutting site

Y3:5 '-CG TCTAGATATTCCCGTGGGCTCTGG-3 ' underscore is the XbaI enzyme cutting site

Y4:5 '-AC GTTTAAACTCAGCGTCACATCTTCCG-3 ' underscore is the PmeI restriction enzyme site

Then PCR product 1% agarose gel electrophoresis reclaims the test kit rubber tapping with the AxyPrep dna gel and reclaims.

The upstream segment (SEQ ID NO.2 the 396th～773 bit base) that reclaims, conventional gene knockout plasmid pLO3 (from Oliver doctor Lenz of Germany) are after 37 ℃ of double digestions of SacI, XbaI spend the night, segment PCR cleaning in upstream reclaims (using AxyPrep PCR cleaning to reclaim test kit), 4 ℃ of connections of T4DNA ligase were spent the night after plasmid 1% agarose gel electrophoresis and rubber tapping were reclaimed, and transformed intestinal bacteria S17-1 competent cell.Dull and stereotyped (adding the tsiklomitsin of the 25 μ g/ml) screening positive clone of LB.Screening gained positive colony bacterium colony PCR checking is extracted the further enzyme of plasmid and is cut checking and order-checking.Clone by checking extracts plasmid, called after pLO3-Y1.

PLO3-Y1 (SEQ ID NO.2 the 1542nd～2018 bit base) similarly clones and verifies after XbaI, a PmeI37 ℃ double digestion spends the night, the gene knockout carrier called after pLO3-△ Y that builds.

2, gene knockout carrier pLO3-△ Y transforms

Gene knockout carrier pLO3-△ Y adopts three close method of joining to import among sphingomonas paucimobilis ATCC 31461 and the DSM 6314.Three close engaging processes need three kinds of bacteriums: the intestinal bacteria donor bacterium S17-1/pLO3-△ Y (pressing preceding method obtains) that 1. contains recombinant plasmid pLO3-△ Y;

2. the intestinal bacteria " assistance " that contain conventional helper plasmid pRK2013 are bacterium HB101/pRK2013 (from Institute of Genetics, Academia Sinica) (helper); 3. sphingomonas paucimobilis ATCC 31461 or DSM 6314 (from the DSM of DSMZ) (recipient bacterium).When three kinds of bacterium mix, assist plasmid pRK2013 to move about and enter in the intestinal bacteria, provide and move about (mob) and shift (tra) function, the recombinant plasmid pLO3-△ Y of donor transfer is entered in the sphingomonas paucimobilis.Transfer vector plasmid need to be with a specific starting point (oirT) that shifts in this system, and it works driven transfer so that assist the tra of plasmid and mob gene pairs.The pLO3 plasmid is tetracyclin resistance, contains the reverse selection markers sacB gene of sucrose, pBR322_origin replication orgin and oriT_RP4 and shifts starting point.This plasmid can copy in intestinal bacteria, but can not in sphingomonas paucimobilis, copy, so the pLO3 plasmid can only be integrated into karyomit(e) on the same group through homology when entering sphingomonas paucimobilis, copy together with karyomit(e), and can not be present in outside the karyomit(e) with free form.Utilize these characteristics of pLO3 plasmid, thereby the homology segment of wanting to knock out the gene both sides is cloned into the integration site of pLO3 plasmid location pLO3 plasmid.The principle of utilizing the homology dna segment to recombinate makes up the genetically deficient bacterial strain.It is as described below that three parents engage concrete grammar:

Sphingomonas paucimobilis is 30 ℃, 200rpm overnight incubation in the preculture substratum.Cultivate 8h with 30 ℃ of 10% inoculum size switching preculture substratum, 200rpm.S17-1/pLO3-△ Y, HB101/pRK2013 be 37 ℃ of overnight incubation in adding corresponding antibiotic LB liquid nutrient medium (microbiotic addition: tsiklomitsin 25 μ g/ml, kantlex 50 μ g/ml).Get S17-1/pLO3-△ Y, the centrifugal collection thalline of each 2ml of HB101/pRK2013 bacterium liquid (3000rpm, 5min); Get sphingomonas paucimobilis 5ml, the centrifugal 5min of 6000rpm abandons supernatant, uses respectively the aseptic deionized water washed twice, and is centrifugal.Above-mentioned 3 kinds of bacterium are merged, with behind the resuspended mixing of 5ml aseptic deionized water with the filter membrane suction filtration of 0.45 μ m aperture diameter 5cm.The filter membrane thalline is attached in the LB flat board up, and 37 ℃ leave standstill and with the 5ml aseptic deionized water thalline on the filter membrane washed after cultivating 7h, and gradient dilution coating to the suitable concn contains the YM flat board of 25 μ g/ml Streptomycin sulphates, 5 μ g/ml tsiklomitsins.Used substratum is composed as follows:

The preculture substratum: yeast extract paste 0.2%, extractum carnis 0.3%, peptone 0.5%, NaCl 0.1%, and glucose 0.5%, solvent are distilled water, pH7.2;

The YM solid medium: yeast powder 0.3%, malt meal 0.3%, peptone 0.5%, glucose 1%, agar powder 1.5%, solvent are distilled water, pH7.2;

Annotate: substratum concentration all refers to the quality concentration of volume percent among the present invention, contains this material of 1g in certain concentration of component 1% expression 100mL substratum.

3, the screening of the sphingomonas paucimobilis recombinant bacterial strain of crtY genetically deficient

For the first time during homologous recombination, plasmid integration enters genome.The clone that the tetracyclin resistance screening obtains extracts genome, sacB primer PCR amplification sacB gene identification positive recombinant.The PCR response procedures is: 95 ℃ of denaturation 5min enter working cycle; 94 ℃ of sex change 30sec, 50 ℃ of annealing 30sec, 72 ℃ are extended 90sec, and 30 circulations are extended 10min with 72 ℃ at last.The PCR product is the positive recon of 1151bp segment.

Above-mentioned checking primer sequence is as follows:

sacBsense：5′-CGAACCAAAAGCCATATAAG-3′

sacBanti：5′-AGCGAAGTGTGAGTAAGTAA-3′

The homologous recombination plasmid breaks away from genomic dna for the second time, produces two kinds of switch types: deletion mutantion bacterial strain (redness) and wild type strain (yellow).The positive colony that the first time, the homologous recombination screening obtained is inoculated and is gone down to posterity in the preculture substratum three times, and it is dull and stereotyped upper to screen for the second time homologous recombination permutoid then to be coated on the screening of 8% sucrose.Because it is Polylevulosan that the sucrose levanase of sacB genes encoding can make sucrose inversion.Polylevulosan is toxic to cell, only loses the sacB gene and could grow in the presence of high concentration sucrose.Therefore can utilize high concentration sucrose oppositely to screen and obtain removing wild type strain or the deletion mutantion bacterial strain that integration knocks out plasmid.The permutoid second time that screening obtains is xeroxed the YM plate culture medium that contains 5 μ g/ml tsiklomitsins, exchanges resistance marker for the second time with further affirmation and loses.

Selecting at random for the second time, the homologous recombination phenotype is that primer PrimeSTAR HS DNA Polymerase enzyme (Takara) PCR confirms further whether the crtY gene knocks out for red permutoid with Y5, Y6.The PCR response procedures is: 95 ℃ of denaturation 5min enter working cycle; 98 ℃ of sex change 10sec, 59 ℃ of annealing 15sec, 72 ℃ are extended 2min, 30 circulations.Do simultaneously the PCR contrast take the wild type strain genome as template, electrophoresis result as shown in Figure 2, PCR product take the wild type strain genome as template is 1869bp, and the PCR product take the sphingomonas paucimobilis genome of crtY genetically deficient as template is as 1107bp.Experiment showed, that through this phenotype is the red crtY gene deletion mutants that is really.

Y5：5′-CCAAGATCACCCAGGACC-3′

Y6：5′-CAGCGTCACATCTTCCGA-3′

The sphingomonas paucimobilis bacterial strain of embodiment 3:crtY genetically deficient produces the mensuration of glue ability

1, sphingomonas paucimobilis (the △ Y1～△ Y7 of wild type strain (Sphingomonas paucimobilis ATCC 31461) and crtY genetically deficient, wherein △ Y1～△ Y4 derives from sphingomonas paucimobilis ATCC 31461, △ Y5～△ Y7 then derives from sphingomonas paucimobilis DSM 6314) connect on the YM medium slant, cultivate 72h for 30 ℃;

2, first order seed is cultivated: with inclined-plane seed access 50ml first order seed substratum (being contained in the 250ml triangular flask), in 30 ℃, 200rpm shaking culture 24h is primary seed solution respectively;

3, secondary seed is cultivated: with the inoculum size access 100ml secondary seed medium (be contained in 500mL triangular flask) of primary seed solution with 5% volume ratio, in 30 ℃, 200rpm shaking culture 12h is secondary seed solution respectively;

4, fermentation: the inoculum size of secondary seed solution with 5% volume ratio accessed in the 100mL level fermention medium (being contained in the 500mL triangular flask), in 30 ℃, 200rpm oscillation and fermentation 48h;

5, measure respectively viscosity and the rate of gum output of the fermented liquid of the sphingomonas paucimobilis (△ Y1～△ Y7) that contrasts (wild type strain) and crtY genetically deficient, result such as table 1:

Table 1, wild strain and mutant strain fermentation 48h rate of gum output, viscosity

Compared with the control, △ Y bacterial strain rate of gum output and viscosity significantly do not change, and rate of gum output is in same level with respect to control strain, therefore can select △ Y as producing bacterial strain, and gelling gum output can not reduce, and can gellan gum fermentation coproduction Lyeopene.Used medium consists of:

First order seed substratum: yeast extract paste 0.20%; Extractum carnis 0.30%; Peptone 0.50%; Repone K 0.10%, solvent are distilled water, pH7.2;

Secondary seed medium: glucose 1.50%; Yeast extract paste .0.50%; Peptone 0.50%; Potassium primary phosphate 0.06%; Dipotassium hydrogen phosphate 0.06%; Sal epsom 0.06%, solvent are distilled water, pH7.2;

Fermention medium: glucose 3.00%; Yeast extract paste 0.05%; Peptone 0.30%; Potassium primary phosphate 0.06%; Dipotassium hydrogen phosphate 0.10%; Sal epsom 0.06%; Solvent is distilled water, pH7.2.Embodiment 4: the preparation technology of lycopene cyclase deletion mycopremna high acyl gellan gum and the extraction process of Lyeopene

1, fermentation liquor pretreatment: fermented liquid 100L, the HCl accent pH6.0 with 10% (v/v) is warming up to 60 ℃, insulation 1h;

2, albumen Impurity removal: when being cooled to 40 ℃, transfer pH7.0 with 10% (w/v) NaOH, add the N,O-Diacetylmuramidase (200,000 U/g, Pang Bo is biological) of 50g and the Sumizyme MP (20,000 U/g, Pang Bo is biological) of 100g, insulation 2h;

3, gelling gum flocculation sediment separates and Lyeopene extracts: add 40 ℃ of lucifuges of 50L ethanol acetone soln (v/v=2: 1～1: 2, preferred 1: 1) in the fermented liquid after processing through pre-treatment and albumen impurity elimination and stir and extract 2h.Filter press after extracting 4 times is collected filtrate, adds an amount of BHT.

4, dry, the pulverizing of gelling gum: pulverize behind 90 ℃ of dry 2h of press filtration gained fiber material, make the high acyl gellan gum finished product, be off-white powder, nitrogen content is 0.05～0.30%.

5, filtrate with Rotary Evaporators 35 ℃ of vacuum concentration to 500mL, the absorption with macroporous adsorbent resin Lyeopene, with acetone to the macroporous resin that adsorbed Lyeopene wash-out at normal temperatures.

6, the elutriant that obtains is concentrated into certain volume, it is dry to put into 45 ℃ of vacuum drying ovens, obtains lycopene crystal.

7, the lycopene crystal that obtains is dissolved in proper amount of acetone, filtration and recrystallization obtains the 0.85g lycopene crystal.It is 97% that high performance liquid chromatography detects Lyeopene purity.

SEQUENCE LISTING

＜110〉Zhejiang University

＜120〉lycopene cyclase gene and the application thereof of sphingomonas paucimobilis

<130>

<160> 14

<170> PatentIn version 3.4

<210> 1

<211> 1158

<212> DNA

<213> Sphingomonas sp.

<400> 1

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gcgcggatcg gcggcaatca cctctggtcg ttcttcgccg acgacgtggc gcccgagcat 180

cgctggctgg ccgccccgct gatcggccat ggctggaacg gctatgacgt cgccttcccc 240

ggccatttcc gtaccctgcc cgcccgctat tattcggtcg aatccgatca tttcgatcgc 300

gtcgtgcgcg gggcgctggg cgagaagagt ctgctgctcg aacgcgaggc ggcggacatc 360

actcccaccg ccgtcgcgct cgcggacggc gacctggtcg aggccgaggg cgtgatcgac 420

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gaactggcgc tcagcgacct gcacgacgtc acccgcccga tcatcatgga cggcacggtg 540

ccgcagatcg acggctatcg cttcgtctat tgcctgccgt tcagcccgac gcggatgttc 600

gtcgaggaca cctattacag cgacacgccg gcgctcgaca tcctcgccac cggcgaccgg 660

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ggcagcctgc ccgtcgcgat gggcggcgac catgccgcct attgggcggc cggcgggcgc 780

ggcatcgcca aggccgggat gaaggccggg ctgttccacc cgatgaccgg ctattccttc 840

cccgatgcga tccgcaccgc cgcgctgatc gccgacgcgc gcgactattc gggcgaggcg 900

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tcagcccgac gcggatgttc gtcgaggaca cctattacag cgacacgccg gcgctcgaca 1260

tcctcgccac cggcgaccgg atcgaagcct atgccgatgc cgcgggctgg aaggtcgacc 1320

gcgtgctgcg cgaggaagtg ggcagcctgc ccgtcgcgat gggcggcgac catgccgcct 1380

attgggcggc cggcgggcgc ggcatcgcca aggccgggat gaaggccggg ctgttccacc 1440

cgatgaccgg ctattccttc cccgatgcga tccgcaccgc cgcgctgatc gccgacgcgc 1500

gcgactattc gggcgaggcg ctgcaccaaa tgctgtatgg ctattcccgt gggctctggc 1560

gcaagcgcgg cttctatcgc atgctcgccg cgatgctgtt caaggcggcc gaacccgagg 1620

agcgctatcg cgtgctggaa cgtttctatc gtctggatgc aggtctgatc cagcgcttct 1680

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cggtggggcg cgccctttcc gcgatctgga gccgctgatg cgccgcgcag tcgtgatcgg 1800

cgccggtttc ggcggtctcg ctctcgccat ccgcctgcag tccgcggggg tcgacaccac 1860

cgtgatcgag gcccgggaca agcccggcgg ccgcgcctat tattgggagc gcgacggctt 1920

caccttcgac ggcggcccga cggtgatcac cgatccggac gcgctgaagg agctgtggcg 1980

cctctcgggc cacgacatct cggaagatgt gacgctga 2018

<210> 3

<211> 23

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 3

gctgtaatag gtgtcctcga cga 23

<210> 4

<211> 23

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 4

tgaacggcag gcaatagacg aag 23

<210> 5

<211> 54

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<220>

<221> misc_feature

<222> (44)..(49)

<223> n is a, c, g, or t

<400> 5

cacgacacgc tactcaacac accaccacgc acagcgtcct caannnnnnc atgg 54

<210> 6

<211> 54

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<220>

<221> misc_feature

<222> (44)..(49)

<223> n is a, c, g, or t

<400> 6

cacgacacgc tactcaacac accaccacgc acagcgtcct caannnnnnc atgc 54

<210> 7

<211> 53

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<220>

<221> misc_feature

<222> (44)..(49)

<223> n is a, c, g, or t

<400> 7

cacgacacgc tactcaacac accaccacgc acagcgtcct caannnnnng cct 53

<210> 8

<211> 55

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<220>

<221> misc_feature

<222> (44)..(49)

<223> n is a, c, g, or t

<400> 8

cacgacacgc tactcaacac accaccacgc acagcgtcct caannnnnng ccacg 55

<210> 9

<211> 20

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 9

cacgacacgc tactcaacac 20

<210> 10

<211> 24

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 10

actcaacaca ccaccacgca cagc 24

<210> 11

<211> 26

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 11

atgagctcca acctgttcaa caccac 26

<210> 12

<211> 27

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 12

gcgtctagag aacgaccaga ggtgatt 27

<210> 13

<211> 26

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 13

cgtctagata ttcccgtggg ctctgg 26

<210> 14

<211> 28

<212> DNA

<213> Unknown

<220>

＜223〉artificial sequence

<400> 14

acgtttaaac tcagcgtcac atcttccg 28

Claims (3)

1. Sphingomonas paucimobilis ( Sphingomonas sp.) the lycopene cyclase gene, nucleotide sequence is shown in SEQ ID NO.1.
2. 2. the restructuring sphingomonas paucimobilis of a lycopene cyclase genetically deficient, remove the lycopene cyclase gene order shown in the SEQ ID NO.1 by sphingomonas paucimobilis ATCC 31461 or DSM 6314 through gene knockout and obtain, described gene knockout is undertaken by the nucleotide fragment shown in the SEQ ID NO.2.
3. 3. restructuring sphingomonas paucimobilis as claimed in claim 2 prepares application in gelling gum and the Lyeopene at microbial fermentation.

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