CN107574174B - Construction method of plasmid expression vector for improving yield of rhodobacter sphaeroides coenzyme Q10 - Google Patents

Abstract

The invention discloses a coenzyme Q for increasing rhodobacter sphaeroides₁₀A method for constructing a plasmid expression vector for yield. ubiG, ubiE and dxsA are Rhodobacter sphaeroides coenzyme Q₁₀Three important catalytic enzyme genes in the synthetic pathway encode 3-demethylubiquinone-93-methyltransferase, menaquinone methyltransferase and 1-deoxy-D-xylulose-5-phosphate synthase, respectively. Amplification of rhodobacter sphaeroides coenzyme Q by PCR technology₁₀Three important catalytic enzyme genes ubiG, ubiE and dxsA in the biosynthesis pathway, and then the three genes were cloned into a plasmid DNA expression vector pRKgluf to obtain an expression vector pRKgluf-ubiE-ubiG-dxsA which simultaneously overexpresses ubiG, ubiE and dxsA. Transforming the expression vector into rhodobacter sphaeroides through escherichia coli S17-1 to obtain engineering bacteria for simultaneously over-expressing ubiG, ubiE and dxsA genes, and finally fermenting and producing coenzyme Q by utilizing the engineering bacteria₁₀. The invention provides a method for increasing coenzyme Q₁₀Method for producing coenzyme Q₁₀The yield of the coenzyme Q is improved by about 80 percent, and the coenzyme Q is suitable₁₀Large-scale industrial production.

Description

Construction method of plasmid expression vector for improving yield of rhodobacter sphaeroides coenzyme Q10

Technical Field

The invention relates to a biological expression vector, in particular to a method for improving rhodobacter sphaeroides coenzyme Q₁₀Construction of plasmid expression vectors for yield.

Background

Rhodobacter sphaeroides is widely distributed in different ecological environments such as fresh water, seawater, polar regions or hot springs (including high-heat water bodies), high-salt and high-organic-matter content and the like, and is a microorganism which performs photosynthesis without oxygen production and has a complex metabolic function.

Coenzyme Q₁₀(CoQ₁₀) Is thatA good biochemical medicine has the functions of improving human immunity, enhancing oxidation resistance and delaying senility, and the application of the biochemical medicine is expanded to the fields of cosmetics and health care products, and the market prospect is very wide. The invention utilizes PCR technology to amplify Rhodobacter sphaeroides coenzyme Q₁₀Key genes of the genes ubiG, ubiE and dxsA in the synthetic pathway were synthesized, and then three genes were cloned into a plasmid DNA expression vector pRKgluf to obtain an expression vector pRKgluf-ubiE-ubiG-dxsA which simultaneously overexpresses ubiG, ubiE and dxsA. And transforming the plasmid DNA expression vector into rhodobacter sphaeroides by using a conjugative transfer technology to obtain the engineering bacteria for simultaneously over-expressing ubiG, ubiE and dxsA.

The invention aims at the production of coenzyme Q by a microbial fermentation method₁₀The defects of lower yield, higher production cost and the like are overcome, and the coenzyme Q can be obviously improved₁₀Coenzyme Q in yield₁₀Engineering bacteria and a construction method thereof. By using the method, the production of coenzyme Q by using a microbial fermentation method can be improved₁₀While reducing the production of coenzyme Q₁₀The cost of (a).

Disclosure of Invention

The invention aims to improve rhodobacter sphaeroides coenzyme Q₁₀A method for constructing a plasmid expression vector for yield.

In order to achieve the purpose, the invention adopts the technical scheme that: coenzyme Q for increasing rhodobacter sphaeroides₁₀A method for constructing a plasmid expression vector for yield, comprising the steps of:

(1) extracting genome DNA from rhodobacter sphaeroides;

(2) key genes ubiG, ubiE and dxsA are amplified by using a PCR technology;

(3) connecting the ubiG, ubiE and dxsA to an expression vector to obtain an expression vector pRKgluf-ubiE-ubiG-dxsA for simultaneously over-expressing the ubiG, ubiE and dxsA;

(4) and transforming the expression vector into rhodobacter sphaeroides by utilizing a conjugative transfer technology to obtain the engineering bacteria for simultaneously over-expressing the ubiE, ubiG and dxsA genes.

Further, in step (2), the ubiG, ubiE and dxsA amplification designs three pairs of PCR primers ubiG-F and ubiG-R, ubiG-F and ubiG-R and dxsA-F and dxsA-R:

the 5 'end of ubiG-F contains an enzyme cutting site XbaI, the 5' end of ubiG-R contains an enzyme cutting site BamHI, and the primer sequence is as follows: ubiG-F: 5'-GCTCTAGAGAATCGTCCAGCACCATCGACC-3', ubiG-R: 5'-CGGGATCCTCAGCTGCGCCGCACGC-3', respectively;

the 5 'end of ubiE-F contains an enzyme cutting site BamHI, the 5' end of ubiE-R contains an enzyme cutting site Kpn I, and the primer sequence is as follows: ubiE-F: 5'-CGGGATCCATGAGCGACGAAACTTCC-3', ubiE-R: 5'-GGGGTACCTCAGATCTTCCAGCCGG-3', respectively;

the 5 'end of dxsA-F contains an enzyme cutting site Kpn I, the 5' end of ubiE-R contains an enzyme cutting site EcoRI, and the primer sequences are as follows: dxsA-F: 5'-GGGGTACCATGACCAATCCCACCCCGC-3', dxsA-R: 5'-GGAATTCTCAGACCGCCCGCGGCT-3' are provided.

Further, in the step (2), the ubiG gene sequence is shown in SEQ ID NO.1, the ubiE gene sequence is shown in SEQ ID NO.2, and the dxsA gene sequence is shown in SEQ ID NO. 3.

The beneficial technical effects of the invention are as follows: the purpose of the present invention is to perform PCR amplification using the genomic DNA of rhodobacter sphaeroides as a template. The ubiG, ubiE and dxsA obtained by amplification and recovery are respectively connected to a pMD18-T vector by using ligase and then subjected to sequencing analysis. Sequentially connecting the fragments of ubiG, ubiE and dxsA which are correctly sequenced to an expression vector pRKgluf to obtain a plasmid expression vector pRKgluf-ubiG-ubiE-dxsA which simultaneously over-expresses ubiG, ubiE and dxsA, then transforming the plasmid expression vector into Escherichia coli S17-1, finally transforming the plasmid expression vector into rhodobacter sphaeroides by utilizing a conjugative transfer technology to obtain engineering bacteria which simultaneously over-expresses ubiG, ubiE and dxsA, and producing coenzyme Q by microbial fermentation of the invention₁₀In the process of (1), coenzyme Q₁₀The yield is improved by about 80 percent, and the method has higher application value and industrial practicability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a diagram showing the amplification of ubiG, ubiE and dxsA using rhodobacter sphaeroides as a genomic DNA as a template;

FIG. 2 is a colony PCR validation chart of pRKgluf-ubiG;

FIG. 3 is a diagram showing the colony PCR verification of pRKgluf-ubiG-ubiE;

FIG. 4 is a diagram showing the colony PCR verification of pRKgluf-ubiG-ubiE-dxsA;

FIG. 5 shows the coenzyme Q extracted from the original rhodobacter sphaeroides, the transformant containing pRKgluf only, and the engineered bacterium containing pRKgluf-ubiG-ubiE-dxsA gene₁₀Yield map of (a);

FIG. 6 is a schematic diagram illustrating the steps of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Materials:

1.PrimeSTAR^TMHS DNA polymerase (product of Takara corporation, Chinese Dalian)

2. Various restriction enzymes (product of Takara company, Chinese Dalian)

3. Ligation Kit DNA Ligation Kit Ver.2.0 (product of Takara Co., Chinese Dalian)

DNA purification kit, centrifugation column type ordinary agarose gel DNA recovery kit (Tian Shi company products, China Beijing)

DNA Extraction Kit Universal Genomic DNA Extraction Kit Ver.3.0 (product of Takara, Dalian China)

6. Escherichia coli S17-1 (laboratory preservation)

7. Escherichia coli DH5 alpha (laboratory preservation)

8. Rhodobacter sphaeroides (laboratory preservation)

Note: the following reagents are all commercially available products.

LB Medium

Yeast extract 5g

Tryptone 10g

NaCl 10g

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

Dissolved in 1000mL of deionized water, adjusted to pH 7.0 with 1mol/L NaOH, and autoclaved.

10.

Culture medium (1L)

1L of ultrapure H₂Dissolution of O

Note that: adjusting pH to 6.90 with NaOH, performing wet heat sterilization at 121 deg.C for 15min, and mixing with 20ml of 50xPHOSPHAte-Solution and 8ml of vitamin-Solution.

Trace Metal Solution

1L of ultrapure H₂Dissolution of O

Note that: weighing ferric citrate into a conical flask containing a proper amount of ultrapure water, heating and dissolving the ferric citrate in a microwave oven, cooling to room temperature, and adding other trace elements. Storing at 4 deg.C in the dark.

50x Phosphate Solution

K₂HPO₄45g

KH₂PO₄30g

1L of ultrapure H₂Dissolution of O

Note that: prepared according to the dosage, sterilized for 15min at 121 ℃.

Vitamine Solution

1L of ultrapure H₂Dissolution of O

Note that: filtering, sterilizing, and storing at 4 deg.C. Agar powder is added into the solid culture medium according to the proportion of 16 g/L.

Example 1 PCR amplification and ligation of rhodobacter sphaeroides ubiG, ubiE and dxsA

The rhodobacter sphaeroides genomic DNA was extracted according to the protocol of the DNA extraction kit, and three pairs of PCR primers ubiG-F and ubiG-R, ubiG-F and ubiG-R and dxsA-F and dxsA-R were designed based on the rhodobacter sphaeroides gene sequences reported on GenBank. The 5 'end of ubiG-F contains the XbaI cleavage site, and the 5' end of ubiG-R contains the BamHI cleavage site. The 5 'end of ubiE-F contains the cleavage site BamHI, and the 5' end of ubiE-R contains the cleavage site Kpn I. The 5 'end of dxsA-F contains a restriction site Kpn I, and the 5' end of ubiE-R contains a restriction site EcoRI. The primer sequences are as follows:

ubiG-F：5'-GCTCTAGAGAATCGTCCAGCACCATCGACC-3'

ubiG-R：5'-CGGGATCCTCAGCTGCGCCGCACGC-3'

ubiE-F：5'-CGGGATCCATGAGCGACGAAACTTCC-3'

ubiE-R：5'-GGGGTACCTCAGATCTTCCAGCCGG-3'

dxsA-F：5'-GGGGTACCATGACCAATCCCACCCCGC-3'

dxsA-R：5'-GGAATTCTCAGACCGCCCGCGGCT-3'

the reaction system for PCR was 50. mu.L, wherein that for ubiG was 1. mu.L each of the primers ubiG-F and ubiG-R, 4. mu.L of dNTP, 1. mu.L of genomic DNA, 2 × PrimeSTAR^TMReaction buffer 25. mu.L, high fidelity PrimeStar^TMHS DNA polymerase 0.5. mu.L. The reaction conditions of PCR were: a first stage of pre-denaturation at 98 ℃ for 10 seconds; the second stage annealing at 68 deg.c for 5 sec, extension at 72 deg.c for 45 sec, and 30 cycles. The obtained PCR product was subjected to 1% agarose electrophoresis to detect a band of about 750bp in size (FIG. 1), and the PCR product was recoveredThe reaction system for ubiE was 1. mu.L, 4. mu.L dNTP, 1. mu.L genomic DNA, 2 × PrimeSTAR for the primers ubiE-F and ubiE-R, respectively^TMReaction buffer 25. mu.L, high fidelity PrimeStar^TMHS DNA polymerase 0.5. mu.L.PCR reaction conditions of first stage 98 ℃ pre-denaturation 10 seconds, second stage 63 ℃ annealing 5 seconds, 72 ℃ extension 45 seconds, cycle 30 times, PCR product obtained by 1% agarose electrophoresis detection of about 750bp size electrophoresis band (figure 1), PCR product recovery, dxsA reaction system of primers dxsA-F and dxsA-R of 1. mu.L, 4. mu.L dNTP, 1. mu.L genomic DNA, 2 × PrimeSTAR^TMReaction buffer 25. mu.L, high fidelity PrimeStar^TMHS DNA polymerase 0.5. mu.L. The reaction conditions of PCR were: a first stage of pre-denaturation at 98 ℃ for 10 seconds; the second stage is annealed at 64.5 ℃ for 5 seconds, extended at 72 ℃ for 90 seconds, and cycled 30 times. The resulting PCR product was recovered by detecting a band of about 1500bp in size by 1% agarose electrophoresis (FIG. 1).

ubiG, ubiE and dxsA were cloned into pMD18-T vector, respectively. The connecting system is as follows: mu.L of pMD18-T vector, 4.3. mu.L of PCR-recovered product, 5. mu.L of Solution I, were ligated overnight in a thermostatted water bath at 16 ℃. Ligation product through CaCl₂Transformation method transformation of DH5 α Escherichia coli competent cells (refer to molecular cloning instructions: preparation with calcium chloride and transformation of competent Escherichia coli), colony PCR and plasmid DNA extraction, respectively, and sequencing confirmation.

Example 2 construction of pRKgluf-ubiG expression vector

The pMD18-T-ubiG plasmid with correct sequencing is taken, and is subjected to double digestion with BamHI and XbaI, and then is cut into gel and recovered (refer to the kit description), wherein the digestion system is as follows: mu.L of 10 XK buffer, 2. mu.L of BamHI enzyme solution, 2. mu.L of XbaI enzyme solution, and appropriate amount of plasmid DNA, sterilized double distilled water was added to 50. mu.L, and the mixture was digested at 37 ℃ for 4 hours. Meanwhile, the expression vector pRKkuf plasmid DNA was digested with BamHI and XbaI in the above digestion system, and then purified by column chromatography (see kit).

The appropriate amount of the pRKgluf fragment and the ubiG fragment obtained by digestion with BamHI and XbaI were ligated according to the instructions of the ligation kit. Ligation product through CaCl₂Transformation method transformation of DH5 α E.coli competent cells (reference molecule)Cloning experimental guidelines: preparing and transforming competent escherichia coli by using calcium chloride), and carrying out high-fidelity enzyme colony PCR verification (high GC content of a target fragment) after screening the tetracycline resistance of 5 mg/mL. The reaction conditions of PCR were: a first stage of pre-denaturation at 98 ℃ for 10 seconds; the second stage is annealed at 55 ℃ for 5 seconds, extended at 72 ℃ for 80 seconds, and cycled 30 times. The obtained PCR product was detected by 1% agarose electrophoresis to show an electrophoretic band of about 1200bp in size, and the electrophoretogram is shown in FIG. 2. A single colony with an electrophoretic band of about 1200bp was placed in LB liquid medium containing 5mg/mL tetracycline at 37 ℃ and 200rpm and cultured overnight with shaking, and the plasmid DNA was extracted the next day and confirmed by sequencing, and named pRKgluf-ubiG.

Example 3 construction of pRKgluf-ubiG-ubiE expression vector

Taking pMD18-T-ubiE plasmid with correct enzyme digestion verification, carrying out double enzyme digestion by BamHI and KpnI, cutting gel and recycling (refer to the description of a kit), wherein the enzyme digestion system is as follows: mu.L 10 XK buffer, 2. mu.L BamHI enzyme solution, 2. mu.L KpnI enzyme solution, appropriate amount of pMD18-T-ubiE, sterile double distilled water to 50. mu.L, and digestion at 37 ℃ for 4 h. Meanwhile, the pRKgluf-ubiG plasmid DNA was digested with BamHI and KpnI and purified by column chromatography (see kit instructions).

The appropriate amount of the pRKpdu-ubiG fragment and ubiE fragment obtained by digestion with BamHI and KpnI were ligated together according to the instructions of the ligation kit. Ligation product through CaCl₂DH5 α colibacillus competent cells were transformed by transformation (refer to molecular cloning guidelines: preparation and transformation of competent colibacillus with calcium chloride), after 5mg/mL tetracycline resistance selection, high fidelity enzyme colony PCR validation (GC content of target gene) was performed, PCR reaction conditions were first 98 ℃ pre-denaturation for 10 seconds, second 55 ℃ annealing for 5 seconds, 72 ℃ extension for 120 seconds, 30 cycles, 1% agarose electrophoresis detection of the obtained PCR product revealed an electrophoretic band of about 2000bp size, the electrophoretic pattern is shown in FIG. 3. A single colony with an electrophoretic band of about 2000bp was placed in LB liquid medium containing 5mg/mL tetracycline, 37 ℃ 200rpm, shaking culture overnight, the next day plasmid DNA was extracted for confirmation, and the plasmid was named pRKpouf-ubiG-ubiE.

Example 4 construction of pRKgluf-ubiG-ubiE-dxsA expression vector

Taking a pMD18-T-dxsA plasmid with correct enzyme digestion verification, carrying out double enzyme digestion by using EcoRI and KpnI, cutting gel and recycling (refer to the description of a kit), wherein the enzyme digestion system is as follows: mu.L of 10 XM buffer, 2. mu.L of EcoRI enzyme solution, 2. mu.L of KpnI enzyme solution, an appropriate amount of plasmid DNA, sterile double distilled water to 50. mu.L, and digestion at 37 ℃ for 4 hours. Meanwhile, EcoRI and KpnI double digestion and column purification were carried out on the successfully constructed expression vector pRKgluf-ubiG-ubiE plasmid DNA (refer to kit instructions).

The appropriate amount of the pRKgluf-ubiG-ubiE fragment and dxsA fragment cleaved with BamHI and KpnI were ligated according to the instructions of the ligation kit. Ligation product through CaCl₂DH5 α E.coli competent cells were transformed by transformation (refer to molecular cloning guidelines: preparation and transformation of competent E.coli with calcium chloride), and after 5mg/mL tetracycline resistance selection, high fidelity enzyme colony PCR was performed (GC content of the target gene). The reaction conditions of PCR were first 98 ℃ pre-denaturation for 10 seconds, second 55 ℃ annealing for 5 seconds, 72 ℃ extension for 240 seconds, and 30 cycles.1% agarose electrophoresis detection of the PCR product resulted in about 3500bp electrophoresis band, the electrophoresis pattern is shown in FIG. 4. A single colony with the electrophoresis band at about 3500bp was placed in LB liquid medium containing 5mg/mL tetracycline, 37 ℃ 200rpm, shaking culture overnight, and the following day plasmid DNA was extracted for confirmation, and named pRKpouf-ubiG-ubiE-dxsA.

EXAMPLE 5 engineering bacteria obtained by transformation of expression vectors into rhodobacter sphaeroides by conjugation transfer

The expression vector pRKgluf-ubiG-ubiE-dxsA was first transformed into E.coli S17-1 (reference molecular cloning protocols: preparation and transformation of competent E.coli with calcium chloride). Inoculating rhodobacter sphaeroides to 40mL

In the medium, 30 ℃ and 150 rpm. Meanwhile, the S17-1 transformant was cultured on an LB (tetracycline-containing) plate. 1.5mL of OD was taken₆₆₀The rhodobacter sphaeroides culture medium of 0.4-0.8 is centrifuged at 5000rpm for 5min at room temperature. The rhodobacter sphaeroides was resuspended with 100. mu.L of LB liquid medium. The S17-1 transformed bacteria were scraped off with a yellow tip and resuspended in 100. mu.L of rhodobacter sphaeroidesIn the liquid. The control was a rhodobacter sphaeroides resuspension without the addition of S17-1 transformants. A re-suspension of rhodobacter sphaeroides and S17-1 was carefully dropped onto the filter on LB medium (without antibiotics). Then, the mixture is placed at 30 ℃ for not less than 8 hours and can be placed overnight. Controls were also dropped onto the filters. The whole thallus on the filter membrane is scraped by a gun head and is resuspended by 500 mu L

In the culture medium, 20. mu.L and 100. mu.L were applied to each of the cells

(working concentration of tetracycline 1.5. mu.g/mL) solid medium, and cultured at 30 ℃ for 3-4 days. Randomly picking three single colonies and culturing the colonies in

(working concentration of tetracycline is 1.5. mu.g/ml) in the culture medium, culturing at 30 ℃ for 1-2 days, and storing the strain at-80 ℃.

Example 7 inducible expression of the engineered bacteria

Taking the transformed bacteria preserved at-80 ℃ for streak culture

Culturing in the dark for 3 days at 30 ℃ on a culture medium (tetracycline: 1.5. mu.g/mL); 3 single colonies were picked and inoculated into 40mL of RA medium (tetracycline: 1.5. mu.g/mL), cultured at 30 ℃ at 150rpm under shaking in the dark for about 36 hours at OD₆₆₀0.6; 1.6mL of overnight culture was inoculated into 80

In a culture medium (tetracycline: 1.5. mu.g/ml, culture medium volume 80% of the vessel), shaking-culturing was carried out at 30 ℃ and 150rpm in the dark for 48 hours. Meanwhile, under the same conditions, the original rhodobacter sphaeroides and the rhodobacter sphaeroides transformed with only the pRKpuf vector were cultured as negative controls.

EXAMPLE 8 coenzyme Q of engineering bacteria₁₀Detection of yield

The fermentation broth was centrifuged at 40mL to collect the cells. Adding wet bacteria into 5mL of acetoneIn vivo, cells were disrupted with a glass homogenizer. Transferring the crushed bacterial suspension into a round-bottom flask, adding 10mL of acidic water with the pH value of 3, uniformly stirring, refluxing at 90 ℃ for 1h, slowly adding 15mL of 10% NaOH, refluxing at 90 ℃ for 1h, and rapidly cooling. Adding 25mL of n-hexane, extracting for 2-3 times, mixing extractive solutions, washing with ultrapure water to neutrality, dehydrating with anhydrous sodium sulfate to clarify, vacuum rotary evaporating for concentration, and dissolving with 10mL of anhydrous ethanol to obtain coenzyme Q₁₀The extract of (3). Measuring absorbance at 275nm with spectrophotometer, and calculating coenzyme Q according to standard curve drawn by standard₁₀Yield of coenzyme Q₁₀The yields are shown in FIG. 5. By comparison of coenzyme Q₁₀Screening of high-yield coenzyme Q₁₀The high-yield gene engineering bacteria.

The construction method of the expression vector and the step schematic diagram for proving the effectiveness thereof provided by the invention are shown in FIG. 6, the expression vector is constructed by a genetic engineering method, and high-yield coenzyme Q is screened out₁₀The strain of (1).

Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.

Sequence listing

<110> Sichuan institute of technology and technology

<120> construction method of plasmid expression vector for improving yield of rhodobacter sphaeroides coenzyme Q10

<130> claims, specification

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>744

<212>DNA

<213> Gene sequence (ubiG)

<400>1

atggaatcgt ccagcaccat cgacccggcc gaggttgcca agttcgaggc catggcagcc 60

gaatggtgga acccgcacgg gaaattcaag ccgctgcacc agatgaaccc ctgccggctg 120

gattacatca cccagcagat cgccgccgag ttcgaccgcg acctctccgc ccccctgccc 180

ttcgaggggc tgcggctcct cgacatcggc tgcggcggcg ggcttctgtc cgagccgatg 240

gcgcgtctgg gggccgaagt gatcggcgcc gacgccgcac cgcgcaacat tccggtggca 300

aagctccatg ccgagcagtc gggcctcacc atcgactatc gcaacacgac ggccgaggcc 360

ctcgccgccg cgggcgaacg gttcgacgtg gtgctgaaca tggaggtggt cgagcatgtg 420

gccgatccgc tgacctatct gacggcctgc cgcgagcttc tgaagccggg cggcctgatg 480

atctgctcga cgctgaaccg caatcccaag agcttcgcca tggccatcgt gggcgccgaa 540

tgggtgatgc gctggctgcc caagggcacg cacgactggt cgaaattcat cacgcccgac 600

gagctttacg atctgatccg caaggcgggc ctcgatccgg tcgaccgcaa gggcatggtg 660

ttcaatccgg tcagctggag ctggagcctg tctgcccgcg acctgtcggt gaactacgtt 720

accgcgagcg tgcggcgcag ctga 744

<210>2

<211>753

<212>DNA

<213> Gene sequence (ubiE)

<400>2

atgagcgacg aaacttccaa cacgacacat ttcggcttcc ggacggttcc ggaaggcgag 60

aaggcgggga tggttcatgg ggtcttcacc cgcgtggcct cgaaatacga catcatgaac 120

gacctgatgt cgggcggcgt gcaccggctc tggaaggacg cgatgatgga ctggctcgcg 180

ccgcggccgg gccagaagct cctcgatgtg gcgggcggca cgggtgacat ctcgttccgc 240

ttcctcaagc gcgcgccggg cgccgaggcg accgtctgcg acatgaccga gtcgatgctg 300

gtcgagggcc ggcaacgcgc cgacgcggcc cagatggccg accggctcga ctgggtggtg 360

ggcgatgcga tggccctgcc cttcgcctcg aacacgttcg acgtctatac gatcagcttc 420

ggcatccgga acgtgacccg cgtgcaggac gcgctgaacg aggcctaccg tgtgctgaag 480

ccgggcggcc ggctcatggt gctggagttc agccagctgc ccaacccgat gatgcaatgg 540

gcctatgacc gttattcctt caacgtgatc ccggtgatgg ggcagatcgt ggcgaacgac 600

cgcgacagct atcaatatct tgtggaatcc atccgcaagt tcccggatca ggagacgttc 660

gcggacatga tccgcaaggc cggcttcggt ctggtgaaat accgcaacct ctccctgggc 720

atcgccgcgc tgcattccgg ctggaagatc tga 753

<210>3

<211>1947

<212>DNA

<213> Gene sequence (dxsA)

<400>3

atgaccaatc ccaccccgcg acccgaaacc ccgcttttgg atcgcgtctg ctgcccggcc 60

gacatgaagg cgctgagtga cgccgaactg gagcggctgg ccgacgaagt gcgttccgag 120

gtgatttcgg tcgttgccga gacgggagga catctggggt cctcgctggg ggtggttgag 180

ctgactgtcg cgctgcatgc ggtcttcaac acgcccaccg acaagctcgt ctgggacgtg 240

ggccaccagt gctaccccca caagatcctc accggccggc gcgagcagat gcgcaccctg 300

cgccagaagg gcggcctctc gggcttcacc aagcgctcgg aatccgccta cgacccgttc 360

ggcgcggctc attcctcgac ctcgatctcg gccgcgctcg gctttgccat gggtcgcgag 420

ctgggccagc ccgtgggcga cacgatcgcc gtgatcggcg acggctccat caccgcgggc 480

atggcctacg aggcactgaa ccacgcgggc catctgaaca agcgcctgtt cgtgatcctg 540

aacgacaatg acatgagcat cgcgccgccc gtgggggcgc ttgcgcgcta tctcgtgaat 600

ctctcctcga aggcgccctt cgccacgctg cgcgcggccg ccgacgggct cgaggcctcg 660

ctgccggggc cgctccgcga cggggcgcgc cgggcgcgcc agctcgtgac cgggatgccg 720

ggcgggggca cgctcttcga ggagctgggc ttcacctatg tcggccccat cgacggccac 780

gacatggagg cgctcctcca gacgctgcgc gcggcgcggg cccggaccac ggggccggtg 840

ctcatccatg tggtcacgaa gaagggcaag ggttacgccc ccgccgagaa tgcccccgac 900

aagtatcacg gggtgaacaa gttcgacccc gtcacgggcg agcagaagaa gtcggtggcc 960

aacgcgccga actacaccaa ggtcttcggc tccaccctga ccgaggaggc cgcgcgcgat 1020

ccgcgcatcg tggcgatcac cgccgctatg ccctcgggca ccggcgtcga catcatgcag 1080

aagcgtttcc cgaaccgcgt cttcgacgtg ggcatcgccg agcagcatgc cgtgaccttc 1140

gcggccggcc tcgccggggc cgggatgaag cccttctgcg cgatctattc ctcgttcctg 1200

caacggggtt acgaccagat cgcccatgac gtggcgctgc agaaccttcc cgtccgcttc 1260

gtgatcgacc gggcggggct cgtgggggcc gatggcgcga cccatgcggg ggccttcgac 1320

gttggcttca tcacttcgct gcccaacatg accgtgatgg ccgcggccga cgaggccgag 1380

ctcatccaca tgatcgccac cgccgtggcc ttcgacgagg gccccatcgc cttccgcttc 1440

ccgcggggcg agggggtggg cgtcgagatg cccgagcgcg ggacggtgct ggagcccggc 1500

cggggccgcg tggtgcgcga agggacggat gtcgcgatcc tctccttcgg cgcgcatctg 1560

cacgaggcct tgcaggcggc gaaacttctc gaggccgagg gggtgagcgt gaccgtggcc 1620

gacgcccgct tctcgcgccc gctcgacacg gggctcatcg accagctcgt gcgccatcac 1680

gcggcgctgg taacggtgga gcagggggcc atgggcggct tcggcgccca tgtcatgcac 1740

tatctcgcca attccggcgg cttcgacggg ggcctcgcgc tccgggtcat gacgctgccc 1800

gaccgcttca tcgagcaggc gagccccgag gacatgtatg ccgatgcggg gctgcgggcc 1860

gaggatatcg cggccaccgc gcggggcgcg ctcgcccggg ggcgcgtgat gccgctccgg 1920

cagacggcaa agccgcgggc ggtctga 1947

Claims (2)

1. Coenzyme Q for increasing rhodobacter sphaeroides₁₀A method for constructing a plasmid expression vector for yield, comprising the steps of:

(1) extracting genome DNA from rhodobacter sphaeroides;

(2) key genes ubiG, ubiE and dxsA are amplified by using a PCR technology; the ubiG gene sequence is shown as SEQ ID NO.1, the ubiE gene sequence is shown as SEQ ID NO.2, and the dxsA gene sequence is shown as SEQ ID NO. 3;

(3) connecting the ubiG, ubiE and dxsA to an expression vector to obtain an expression vector pRKgluf-ubiE-ubiG-dxsA for simultaneously over-expressing the ubiG, ubiE and dxsA;

(4) and transforming the expression vector into rhodobacter sphaeroides by utilizing a conjugative transfer technology to obtain the engineering bacteria for simultaneously over-expressing the ubiE, ubiG and dxsA genes.

2. The method of claim 1, wherein in step (2), the ubiG, ubiE, and dxsA amplifications are performed by designing three pairs of PCR primers ubiG-F and ubiG-R, ubiG-F and ubiG-R and dxsA-F and dxsA-R:

the 5 'end of ubiG-F contains an enzyme cutting site XbaI, the 5' end of ubiG-R contains an enzyme cutting site BamHI, and the primer sequence is as follows: ubiG-F: 5'-GCTCTAGAGAATCGTCCAGCACCATCGACC-3', ubiG-R: 5'-CGGGATCCTCAGCTGCGCCGCACGC-3', respectively;

the 5 'end of ubiE-F contains an enzyme cutting site BamHI, the 5' end of ubiE-R contains an enzyme cutting site KpnI, and the primer sequence is as follows: ubiE-F: 5'-CGGGATCCATGAGCGACGAAACTTCC-3', ubiE-R: 5'-GGGGTACCTCAGATCTTCCAGCCGG-3', respectively;

the 5 'end of dxsA-F contains an enzyme cutting site KpnI, the 5' end of ubiE-R contains an enzyme cutting site EcoRI, and the primer sequences are as follows: dxsA-F: 5'-GGGGTACCATGACCAATCCCACCCCGC-3', dxsA-R: 5'-GGAATTCTCAGACCGCCCGCGGCT-3' are provided.

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