CN113717912A - Escherichia coli engineering bacterium constructed by membrane engineering and used for producing lycopene, construction method and application thereof - Google Patents

Abstract

The invention discloses an escherichia coli engineering bacterium for producing lycopene, which is constructed by membrane engineering, and a construction method and application thereof. The Escherichia coli engineering bacteria are lycopene operon gene segments from rhodopseudomonas palustrislycAnd gene fragments encoding cell membrane proteinscav1、plsB、plsCAnd gene fragments of the MVA pathwaymvaE、mvaSAnd FPP/GPP Synthesis-related Gene fragmentsIspAIs obtained by the steps of amplification, connection, transformation, screening and the like. The invention not only realizes the production of lycopene by escherichia coli and improves the synthesis efficiency of lycopene, but also improves cell membrane pair by optimizing membrane engineeringThe storage capacity of the lycopene further improves the yield of the lycopene.

Description

Escherichia coli engineering bacterium constructed by membrane engineering and used for producing lycopene, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to an escherichia coli engineering bacterium for producing lycopene, which is constructed by membrane engineering, and a construction method and application thereof.

Background

The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to an escherichia coli engineering bacterium for producing lycopene, which is constructed by membrane engineering, and a construction method and application thereof.

Disclosure of Invention

The invention provides an escherichia coli engineering bacterium for producing lycopene, which is constructed by using membrane engineering, and a construction method and application thereof. The present invention first overexpresses the lycopene operon by plasmidslycAnd then the storage capacity of the cell membrane for lycopene is improved through membrane engineering, so that the capacity of synthesizing lycopene by using escherichia coli is gradually improved.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides an escherichia coli engineering bacterium for producing lycopene, which is constructed by membrane engineering and is obtained by constructing an expression vector containing a lycopene operon gene segment and an expression vector containing a gene segment of a coding cell membrane protein.

Further, the gene segment of the coding cell membrane protein comprises a gene segment of which the nucleotide sequence is shown as SEQ ID NO.7cav1The gene segment shown as SEQ ID NO.11plsBAnd the gene segment shown as SEQ ID NO.15plsC。

Further, the gene fragmentcav1FromHomo sapiensIt can code caveolin with the amino acid sequence shown in SEQ ID No. 8.

Further, the gene fragmentplsBAnd gene fragmentsplsCBoth of which are derived from Escherichia coli and can respectively encode glycerol phosphate acyltransferase with an amino acid sequence shown as SEQ ID NO.12 and acylglycerol acyltransferase with an amino acid sequence shown as SEQ ID NO. 16.

Further, said tomato redThe gene fragment of the biotin operon is a gene fragment with a nucleotide sequence shown as SEQ ID NO.1lycIt is derived from Rhodopseudomonas palustris.

Further, thelycThe gene segments comprise genes respectively encoding geranylgeranyl diphosphate synthase, phytoene synthase and phytoene desaturasecrtE、crtBAndcrtIa gene.

Further, the expression vector also comprises a gene segment for coding acetyl-CoA acyltransferase/hydroxymethyl glutaryl-CoA reductasemvaEGene fragment encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthasemvaSAnd gene fragments encoding farnesyl/geranyl pyrophosphate synthaseIspA。

The invention also provides a construction method of the escherichia coli engineering bacteria, which specifically comprises the following steps:

(1) amplification of lycopene operon gene fragmentlycThen, and throughHindІ II andEcoRІ connecting the pET28a plasmid fragments subjected to double enzyme digestion, converting the connection product into competent cells, and obtaining a recombinant plasmid pET-RPlyc through resistance screening and positive cloning;

(2) amplification of a Gene fragment encoding a cell Membrane proteincav1The channel of Chinese and Western medicineHindІ II andXhoi, connecting the double-restriction enzyme digestion recombinant plasmid pET-Rplyc fragments, converting the connection product into competent cells, and obtaining the recombinant plasmid pET-RPlyc-cav1 through resistance screening and positive cloning;

or amplifying a gene fragment encoding a cell membrane proteinplsBAndplsCthe two are overlapped and connected through overlap PCR and then are connected with each otherXhoI, connecting pACYC-MvaE-MvaS-IspA plasmid fragments subjected to single enzyme digestion, converting the connecting products into competent cells, and obtaining a recombinant plasmid pACYC-MvaE-MvaS-IspA-plsB-plsC through resistance screening and positive cloning;

(3) transforming the recombinant plasmid pET-RPlyc into an escherichia coli competent cell to obtain an escherichia coli engineering bacterium ELCXH 01;

or the recombinant plasmid pET-RPlyc and the plasmid pACYC-MvaE-MvaS-IspA are jointly transformed into an escherichia coli competent cell containing the pTrc-low plasmid to obtain an escherichia coli engineering bacterium ELCXH 02;

or the recombinant plasmid pET-RPlyc and the recombinant plasmid pACYC-MvaE-MvaS-IspA-plsB-plsC are jointly transformed into an escherichia coli competent cell containing the pTrc-low plasmid to obtain an escherichia coli engineering bacterium ELCXH 03;

or the recombinant plasmid pET-RPlyc-cav1 and the plasmid pACYC-MvaE-MvaS-IspA are jointly transformed into an Escherichia coli competent cell containing the pTrc-low plasmid to obtain the Escherichia coli engineering bacterium ELCXH 04.

Further, the Escherichia coli competent cells containing the pTrc-low plasmid areE. coli BL21(DE3):: Trc-low。

Further, the concentration ratio of the plasmid fragment after enzyme digestion to the gene fragment after amplification is 1: 2-4.

The invention provides application of the escherichia coli engineering bacteria in synthesizing lycopene and/or improving the yield of lycopene.

Further, the step of synthesizing lycopene by using the escherichia coli engineering bacteria is as follows: activating the engineering bacteria of Escherichia coli, selecting positive monoclonal antibody, transferring to LB culture medium containing antibiotic, culturing at 37 deg.C overnight, transferring the obtained seed liquid to M9 culture medium, culturing at 37 deg.C to OD₆₀₀=0.6-0.8, adding inducer IPTG, shaking culture at 30 ℃ for 48 h, centrifuging, taking supernatant, adding extractant, shaking culture, and breaking the precipitate with needle tip every 5min until the cell precipitate turns white; centrifuging to obtain supernatant containing lycopene, and detecting lycopene yield.

Further, the method for detecting the yield of lycopene is HPLC, and the conditions are as follows: HPLC detector is thermo DioNex UltiMate 3000, column 4.6X 250 mm, 5 μm ZORBAX Eclipse Plus C₁₈A column; mobile phase: methanol/ethyl acetate (85: 15); the flow rate is 1 mL/min; sample introduction volume: 10 μ L, column temperature: 25^oC; UV：475 nm。

Further, the volume ratio of the extracting agent is 1: 1 of a mixed solution of methanol and acetone.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention selects a lycopene operon gene segment from rhodopseudomonas palustrislycAnd by overexpressing a gene segment encoding an acetyl-CoA acyltransferase/hydroxymethylglutaryl-CoA reductasemvaEGene fragment encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthasemvaSAnd gene fragments encoding farnesyl/geranyl pyrophosphate synthaseIspAOn the basis, the invention also optimizes the membrane engineering gene of the gene segment of the coding cell membrane protein, improves the storage capacity of the cell membrane to the lycopene, not only enables the engineering bacteria to synthesize the lycopene, but also further improves the yield of the lycopene, further increases the way of synthesizing the lycopene in vitro, has high yield and has practical application significance.

Drawings

FIG. 1 is a plasmid map of the constructed vector pET-RPlyc.

FIG. 2 is a plasmid map of the constructed vector pET-RPlyc-cav 1.

FIG. 3 is a plasmid map of the constructed vector pACYC-mvaE-mvaS-IspA-plsB-plsC.

FIG. 4 shows the lycopene yields (mg/L) of the constructed engineered strains ELCXH01, ELCXH02, ELCXH03 and ELCXH04 after 24 h and 48 h fermentation.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

The examples do not show the specific techniques or conditions, and the techniques described in the literature in the field or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.

Example 1: construction of pET-RPlyc expression vector

From Rhodopseudomonas palustris (Rhodopseudomonas palustris) The lycopene operon has a nucleotide sequence shown in SEQ ID NO.1, and comprises genes respectively encoding geranylgeranyl diphosphate synthase, phytoene synthase and phytoene desaturasecrtE、crtBAndcrtIthe amino acid sequences of the encoded CrtE, CrtB and CrtI are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

Taking the genome of the rhodopseudomonas palustris as a template, and carrying out Polymerase Chain Reaction (PCR) by using a primer crtI-PET-F and a primer crtE-PET-R, wherein a PCR amplification system is shown as follows:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 55 ℃ C15 s, 72 ℃ C1 min); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

crtE-PET-R:

5’-TCGAGTGCGGCCGCAAGCTTCTCAGGCCGCAGTCGCGGC-3’；

crtI-PET-F:

5’-TGGGTCGCGGATCCGAATTCATGCTCGATCCTGGCCCCAATC-3’ 。

the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).

Using restriction enzyme 1HindII І (TaKaRa, cat 1615) and restriction enzyme 2EcoRІ (TaKaRa, cat 1611) double digestion of pET28a plasmid, the digestion system is:

plasmid or PCR product	3 μg
		10 ×Q.Cut Buffer	10 μL
Restriction enzyme 1	5 μL
		Restriction enzyme 2	5 μL
Ultrapure water	Make up to 100 mu L

And (3) placing the enzyme digestion system at 37 ℃ for incubation for 1 h, and performing gel recovery and purification.

The PCR products were ligated by means of a seamless cloning method using 2 XClon Express Mix (Vazyme, cat # C115) as follows:

restriction fragment of vector	0.03 pmol
		PCR fragment having inserted therein each gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. The product was competent for transformation, spread on LB solid plate containing 50 mg/L kanamycin, positive clones were screened by PCR, and recombinant plasmid pET-RPlyc (FIG. 1) was extracted from the positive clones, and identified by restriction enzyme digestion and sequencing.

Example 2: construction of pET-RPlyc-cav1 expression vector

FromHomo sapiensIs/are as followscav1The nucleotide sequence is shown as SEQ ID NO.7, and the nucleotide sequence is chemically synthesized on a pUC-57 vector by Jinwei Zhi company to obtain a pUC-cav1 vector;cav1can code caveolin, and the amino acid sequence obtained by coding is shown in SEQ ID NO. 8.

The PCR amplification system was shown below by performing Polymerase Chain Reaction (PCR) using pUC-cav1 as a template, the primer cav1-F and the primer cav 1-R:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 55 ℃ C15 s, 72 ℃ C1 min); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

cav1-F:

5’-CCGTTTAGGAAGCTTGGTACCAAGGAGATATACATATGTCTGGGGGCAAATACGT-3’；

cav1-R:

5’-TGGTGGTGGTGGTGCTCGAGTTATATTTCTTTCTGCAAGTTGATGCGGA-3’。

the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).

Using restriction enzyme 1XhoІ (TaKaRa, cat No. 1635) and restriction enzyme 2Hind II І (TaKaRa, cat 1615) double digestion pET-RPlyc plasmid, its digestion system is:

plasmid or PCR product	3 μg
		10 ×Q.Cut Buffer	10 μL
Restriction enzyme 1	5 μL
		Restriction enzyme 2	5 μL
Ultrapure water	Make up to 100 mu L

And (3) placing the enzyme digestion system at 37 ℃ for incubation for 1 h, and performing gel recovery and purification.

The PCR products were ligated by means of a seamless cloning method using 2 XClon Express Mix (Vazyme, cat # C115) as follows:

p enzyme fragment of vector	0.03 pmol
		PCR fragment having inserted therein each gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. The product was competent for transformation, spread on LB solid plate containing 50 mg/L kanamycin, positive clones were screened by PCR, and the recombinant plasmid pET-RPlyc-cav1 (FIG. 2) was extracted from the positive clones and identified by restriction enzyme digestion and sequencing.

Example 3: construction of pACYC-mvaE-mvaS-IspA-plsBC expression vector

Using restriction enzyme 1 XhoІ (TaKaRa, cat No. 1618) digesting pACYC-MvaE-MvaS-IspA plasmid (cited from the patent application No. 2021107551170) capable of exogenously expressing acetyl-CoA acyltransferase/hydroxymethylglutaryl-CoA reductase genemvaE3-hydroxy-3-methylglutaryl coenzyme A synthase genemvaSAnd farnesyl/geranyl pyrophosphate synthasesIspAThe enzyme cutting system is as follows:

plasmid or PCR product	3 μg
		10 ×Q.Cut Buffer	10 μL
Restriction enzyme 1	10 μL
		Ultrapure water	Make up to 100 mu L

And (3) placing the enzyme digestion system at 37 ℃ for incubation for 1 h, and performing gel recovery and purification.

Polymerase Chain Reaction (PCR) amplification was performed using E.coli genome as template, primers plsB-F and plsB-RplsBThe nucleotide sequence of the gene fragment, plsB, is shown in SEQ ID NO.11, and can encode glycerophosphate acyltransferase, encoded ammoniaThe sequence of the gene is shown in SEQ ID NO.12, and the PCR amplification system is shown as follows:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 55 ℃ C15 s, 72 ℃ C1 min); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

plsB-F:

5’-GTAATAAATAAAGATCTCGAGAAGGAGCATCGTTTATGTCCGGCTGGCCA-3’；

plsB-R: 5’-CTCTCCTTCTCTCTGATTACCCTTCGCC-3’。

polymerase Chain Reaction (PCR) amplification was performed using E.coli genome as template, primer plsC-F and primer plsC-RplsCThe nucleotide sequence of the gene fragment, plsC, is shown in SEQ ID NO.15, which can encode acylglycerol acyltransferase, and the amino acid sequence of the gene fragment, which is shown in SEQ ID NO.16, is the same as the PCR amplification system of plsB.

The primer sequences are shown below:

plsC-F:

5’-GGTAATCAGAGAGAAGGAGAGGGTGTTATGCTATATATCTTTCG-3’；

plsC-R:

5’-GTTTCTTTACCAGACTCGAGTTGCCGACTTAAACTTTTCCGGC-3’。

the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat # DC 301-01).

The purified plsB and plsC were subjected to Overlap PCR with primers plsB-F and plsC-R to obtain a plsB-plsC fragment, the PCR system of which is shown in the following figure:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 55 ℃ C15 s, 72 ℃ C2 min); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The PCR products were ligated by means of a seamless cloning method using 2 XClon Express Mix (Vazyme, cat # C115) as follows:

restriction fragment of vector	0.03 pmol
		PCR fragment of inserted gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. The product was competent for transformation, coated on LB solid plate containing 34 mg/L chloramphenicol, positive clones were screened by PCR, and the recombinant plasmid pACYC-mvaE-mvaS-IspA-plsB-plsC (FIG. 3) was extracted from the positive clones, and identified by restriction enzyme digestion and sequencing.

Example 4: construction of engineered Strain

Transformation of pET-RPlyc plasmidE. coli BL21(DE3) competent cells were plated on LB solid plate containing 50 mg/L kanamycin to obtain positive clones, whereby the engineered strain ELCXH01 was obtained.

The pET-RPlyc plasmid and the pACYC-MvaE-MvaS-IspA plasmid are co-transformedE. coli BL21(DE3):: Trc-lowCompetent cells (cited from Cheng Tao, Guang Zhao, Mo Xian, Congxia Xie. Improved cis-Abienol production through increasing creating precorsor supplied in)Escherichia coliScientific Reports, 2020, 10, 16791), respectivelyPositive clones were obtained on LB solid plates containing 50 mg/L kanamycin and 34 mg/L chloramphenicol, whereby the engineered strain ELCXH02 was obtained.

The pET-RPlyc plasmid and pACYC-mvaE-mvaS-IspA-plsB-plsC plasmid were co-transformedE. coli BL21(DE3)::Trc-lowCompetent cells were plated on LB solid plates containing 50 mg/L kanamycin and 34 mg/L chloramphenicol, respectively, to obtain positive clones, whereby the engineered strain ELCXH03 was obtained.

The pET-RPlyc-cav1 plasmid and pACYC-MvaE-MvaS-IspA plasmid were co-transformedE. coli BL21(DE3)::Trc-lowCompetent cells were plated on LB solid plates containing 50 mg/L kanamycin and 34 mg/L chloramphenicol, respectively, to obtain positive clones, whereby the engineered strain ELCXH04 was obtained.

Example 5: production of lycopene by fermentation of escherichia coli

(1) The engineered strains ELCXH01, ELCXH02, ELCXH03 and ELCXH04 prepared in example 4 were activated by LB solid plate.

(2) First-order seed culture: positive single clones were picked, transferred to 10 mL antibiotic + LB medium and cultured overnight at 37 ℃.

(3) Shake cultivation: adding 3.3 mL of 60% glucose, 100. mu.L of 1M magnesium sulfate stock solution, 2 mL of seed culture solution, 100. mu.L of antibiotic and 100. mu.L of trace elements into M9 culture medium, and shake culturing at 37 ℃ to OD₆₀₀=0.6-0.8。

(4) Induction: shaking-culturing at 37 deg.C until OD600=0.6-0.8 for about 6 hr, adding 50 μ L IPTG, shaking-culturing at 30 deg.C, taking two 2 mL bacterial samples 24 hr and 48 hr after induction, measuring OD600, washing the bacteria with PBS buffer, discarding supernatant, and storing in refrigerator at-80 deg.C.

Example 6: extraction and detection HPLC of lycopene

(1) The extraction method of the lycopene comprises the following steps: adding 1 mL of extractant (methanol: acetone = 1: 1) into the bacterial liquid centrifuged and discarded supernatant, operating in a fume hood, wrapping a layer of tin foil paper to prevent degradation of lycopene, placing the mixture in a cell desktop culture instrument, shaking, and pricking the precipitate with a needle tip every 5min or so until the cell precipitate turns white. After centrifugation, the supernatant was taken at more than 500. mu.L for filter sterilization into a brown color chromatographic bottle and the lycopene production was measured by HPLC.

(2) Quantitative analysis of lycopene is carried out by HPLC, and the finally determined HPLC detection conditions of lycopene are as follows: the HPLC detector was thermo DioNex ultiMate 3000 and the column chromatography was ZORBAX Eclipse Plus C18 column (4.6X 250 mm, 5 μm, Agilent); mobile phase: methanol/ethyl acetate (85: 15); the flow rate is 1 mL/min; sample introduction volume: 10 μ L, column temperature: 25oC, UV: 475 nm.

The results are shown in fig. 4, with higher lycopene production of ELCXH02, ELCXH03 and ELCXH04 compared to ELCXH01 in the same fermentation time. In addition, the lycopene production by ELCXH01 did not change significantly with increasing fermentation time, but increased significantly in ELCXH02, ELCXH03 and ELCXH 04. Among the 3 engineering strains with high lycopene yield, the lycopene yield of ELCXH04 is the highest.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Val Thr Asn Arg Ile Glu Arg Ala Leu Thr Glu Ala Leu Asn

1 5 10 15

Gln Ala Asp Met Ala Gly Cys Pro Pro Arg Leu Ala Ala Ala Met Arg

20 25 30

Ser Ala Val Phe Pro Arg Gly Ala Arg Val Arg Pro Arg Leu Cys His

35 40 45

Ser Val Ala Leu Ala Cys Gly Glu Asp Asn Pro Gly Ile Thr Glu Ala

50 55 60

Ala Gly Ala Ala Leu Glu Leu Met His Cys Ala Ser Leu Ile His Asp

65 70 75 80

Asp Leu Pro Cys Phe Asp Gly Ala Asp Leu Arg Arg Gly Arg Pro Ser

85 90 95

Val His Lys Ala Phe Gly Glu Pro Leu Ala Val Leu Ala Gly Asp Ala

100 105 110

Met Ile Val Leu Ala Phe Gln Thr Ile Ala Arg Ala Glu Gly Gln Ala

115 120 125

Asp Arg Leu Leu Lys Leu Thr Gln Ile Ile Gly Gly Ala Val Gly Val

130 135 140

Pro Phe Gly Ile Val Ala Gly Gln Ala Trp Glu Cys Glu Thr Glu Ile

145 150 155 160

Asn Leu Ala His Tyr His Arg Ser Lys Thr Gly Ala Leu Phe Val Ala

165 170 175

Ala Thr Ala Met Gly Ala Ala Ser Ala Gly Ser Asp Pro Glu Pro Trp

180 185 190

Arg Met Val Gly Glu Lys Ile Gly Glu Ala Tyr Gln Val Ala Asp Asp

195 200 205

Leu Arg Asp Ala Ala Ala Asp Val Glu Glu Ile Gly Lys Pro Val Gly

210 215 220

Gln Asp Val Ala His Asp Arg Pro Ser Ala Val Arg Glu Met Gly Ile

225 230 235 240

Asp Gly Ala Ile Tyr His Leu Lys Ser Leu Val Arg Gly Ala Val Glu

245 250 255

Ala Met Pro Glu Cys Thr Asn Gly Asn Glu Leu Arg Ser Leu Ile Met

260 265 270

Ser Glu Ala Thr Arg Leu Val Pro Ala Lys Leu Ala Ala Thr Ala Ala

275 280 285

<210> 3

<211> 355

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Leu Gln Ser Asp Met Leu Ala Gln Ser Asp Met Leu Ala Cys

1 5 10 15

Arg Glu Met Ile Lys Glu Gly Ser His Thr Phe His Ala Ala Ser Lys

20 25 30

Val Leu Pro Arg Arg Ile Ser Asp Pro Ala Ile Ala Leu Tyr Ala Phe

35 40 45

Cys Arg Val Ala Asp Asp Ala Val Asp Leu Gly Leu Asp Arg Ala Ala

50 55 60

Ala Val Glu Leu Leu Lys Asp Arg Leu Asp Arg Ala Cys Arg Gly Val

65 70 75 80

Pro Arg Ala Tyr Pro Ser Asp Arg Ala Phe Ala Asp Val Val Ala Arg

85 90 95

Phe Ser Ile Pro Pro Ala Ile Pro Glu Ala Leu Ile Glu Gly Leu Glu

100 105 110

Trp Asp Ala Gln Gly Arg Arg Phe Glu Thr Leu Ser Asp Leu Tyr Ser

115 120 125

Tyr Cys Ala Arg Val Ala Gly Thr Val Gly Val Met Met Thr Leu Val

130 135 140

Met Gly Gln Arg Lys Pro Asp Ile Val Ala Arg Ala Cys Asp Leu Gly

145 150 155 160

Cys Ala Met Gln Leu Thr Asn Ile Ala Arg Asp Ile Gly Glu Asp Ala

165 170 175

Arg Asn Gly Arg Ile Tyr Met Pro Leu Ser Trp Met Arg Glu Ala Gly

180 185 190

Leu Asp Pro Glu Thr Trp Leu Ala Asn Pro Lys Phe Thr Pro Glu Ile

195 200 205

Ala Ser Ile Val Lys Arg Leu Ile Asp Thr Ala Asp Ala Leu Tyr Asp

210 215 220

Arg Ala Thr Leu Gly Ile Ala Asn Leu Pro Arg Ser Cys Arg Pro Gly

225 230 235 240

Ile Phe Ala Ala Arg Ala Leu Tyr Ala Glu Ile Gly Arg Glu Val Glu

245 250 255

Arg Ser Gly Leu Asp Ser Val Ser Ser Arg Ala Val Val Ser Thr Gly

260 265 270

Arg Lys Leu Ala Val Leu Ala Arg Leu Leu Ala Phe Gln Glu Thr Glu

275 280 285

Trp Ala Pro Ala Lys Tyr Leu Pro Ala Lys Phe Gly Asp Met Glu Glu

290 295 300

Thr Lys Phe Leu Val Asp Ala Val Ile Ala His Pro Val Arg Glu Leu

305 310 315 320

Pro Ala Arg Gln Lys Val Lys Pro Ile Glu Gln Lys Val Ala Trp Leu

325 330 335

Val Asp Leu Phe Thr Arg Leu Glu Arg Arg Asp Gln Met Leu Gln Arg

340 345 350

Ser Arg Val

355

<210> 4

<211> 511

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Leu Asp Pro Gly Pro Asn Pro Ala Pro Arg Pro Ser Arg Asp Arg

1 5 10 15

Ala Pro His Ala Val Val Ile Gly Ser Gly Phe Gly Gly Leu Ala Ala

20 25 30

Ala Val Arg Leu Gly Ala Lys Gly Tyr Arg Val Thr Val Leu Glu Lys

35 40 45

Leu Asp Lys Ala Gly Gly Arg Ala Tyr Val His Lys Gln Asp Gly Phe

50 55 60

Ser Phe Asp Ala Gly Pro Thr Ile Val Thr Ala Pro Tyr Leu Phe Glu

65 70 75 80

Glu Leu Trp Lys Leu Cys Gly Lys Arg Met Ser Asp Asp Ile Thr Leu

85 90 95

Lys Pro Met Ser Pro Phe Tyr Arg Ile Arg Phe Asp Asp Gly Thr His

100 105 110

Phe Asp Tyr Ser Asp Asp Arg Asp Ala Val Leu Asp Gln Ile Ala Lys

115 120 125

Phe Cys Pro Asp Asp Val Pro Ala Tyr Asp Arg Phe Met Ala Ala Ser

130 135 140

His Glu Ile Phe Lys Val Gly Phe Glu Gln Leu Gly Asp Gln Pro Phe

145 150 155 160

Ser His Phe Thr Asp Met Leu Lys Ile Ala Pro Ala Met Ile Lys Leu

165 170 175

Glu Ser Tyr Arg Ser Val Tyr Gly Leu Val Ala Lys His Phe Lys Asp

180 185 190

Pro Lys Leu Arg Gln Val Phe Ser Phe His Pro Leu Leu Ile Gly Gly

195 200 205

Asn Pro Phe Met Ser Ser Ser Val Tyr Cys Leu Ile Thr Tyr Leu Glu

210 215 220

Lys Gln Trp Gly Val His Ser Ala Met Gly Gly Thr Gly Ala Leu Val

225 230 235 240

Thr Gly Leu Val Asn Leu Ile Glu Gly Gln Gly Asn Thr Ile Arg Tyr

245 250 255

Asn Gln Asp Val Arg Gln Ile Val Val Glu Asn Gly Thr Ala Cys Gly

260 265 270

Val Lys Leu Ala Asp Gly Glu Val Ile Lys Ala Asp Ile Val Val Ser

275 280 285

Asn Ala Asp Ser Ala Ser Thr Tyr Arg Tyr Leu Leu Pro Pro Glu Thr

290 295 300

Arg Lys Arg Trp Thr Asp Ala Lys Ile Glu Lys Ser Arg Tyr Ser Met

305 310 315 320

Ser Leu Phe Val Trp Tyr Phe Gly Thr Lys Arg Arg Tyr Glu Asp Val

325 330 335

Lys His His Thr Ile Leu Leu Gly Pro Arg Tyr Lys Glu Leu Ile Ser

340 345 350

Asp Ile Phe Ser Arg Lys Val Val Ala Glu Asp Phe Ser Leu Tyr Leu

355 360 365

His Arg Pro Thr Ala Thr Asp Pro Ser Leu Ala Pro Gln Gly Cys Asp

370 375 380

Thr Phe Tyr Val Leu Ser Pro Val Pro Asn Leu Leu Gly Asp Thr Asp

385 390 395 400

Trp His Thr Lys Ala Glu Thr Tyr Arg Ala Ser Ile Ala Lys Met Leu

405 410 415

Gly Ala Thr Val Leu Pro Asp Leu Glu Asn Gln Ile Ala Thr Ser Lys

420 425 430

Ile Thr Thr Pro Ile Asp Phe Gln Asp Arg Leu Ser Ser Phe Arg Gly

435 440 445

Ala Ala Phe Gly Leu Glu Pro Val Leu Trp Gln Ser Ala Trp Phe Arg

450 455 460

Pro His Asn Gln Ser Glu Asp Val Lys Arg Leu Tyr Leu Val Gly Ala

465 470 475 480

Gly Thr His Pro Gly Ala Gly Leu Pro Gly Val Leu Ser Ser Ala Arg

485 490 495

Val Leu Asp Ala Leu Val Pro Glu Ala Asp Ser Leu Val Thr Ser

500 505 510

<210> 5

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcgagtgcgg ccgcaagctt ctcaggccgc agtcgcggc 39

<210> 6

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgggtcgcgg atccgaattc atgctcgatc ctggccccaa tc 42

<210> 7

<211> 537

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgtctgggg gcaaatacgt agactcggag ggacatctct acaccgttcc catccgggaa 60

cagggcaaca tctacaagcc caacaacaag gccatggcag acgagctgag cgagaagcaa 120

gtgtacgacg cgcacaccaa ggagatcgac ctggtcaacc gcgaccctaa acacctcaac 180

gatgacgtgg tcaagattga ctttgaagat gtgattgcag aaccagaagg gacacacagt 240

tttcacggca tttggaaggc cagcttcacc accttcactg tgacgaaata ctggttttac 300

cgcttgctgt ctgccctctt tggcatcccg atggcactca tctggggcat ttacttcgcc 360

attctctctt tcctgcacat ctgggcagtt gtaccatgca ttaagagctt cctgattgag 420

attcagtgca ccagccgtgt ctattccatc tacgtccaca ccgtctgtga cccactcttt 480

gaagctgttg ggaaaatatt cagcaatgtc cgcatcaact tgcagaaaga aatataa 537

<210> 8

<211> 178

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ser Gly Gly Lys Tyr Val Asp Ser Glu Gly His Leu Tyr Thr Val

1 5 10 15

Pro Ile Arg Glu Gln Gly Asn Ile Tyr Lys Pro Asn Asn Lys Ala Met

20 25 30

Ala Asp Glu Leu Ser Glu Lys Gln Val Tyr Asp Ala His Thr Lys Glu

35 40 45

Ile Asp Leu Val Asn Arg Asp Pro Lys His Leu Asn Asp Asp Val Val

50 55 60

Lys Ile Asp Phe Glu Asp Val Ile Ala Glu Pro Glu Gly Thr His Ser

65 70 75 80

Phe His Gly Ile Trp Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys

85 90 95

Tyr Trp Phe Tyr Arg Leu Leu Ser Ala Leu Phe Gly Ile Pro Met Ala

100 105 110

Leu Ile Trp Gly Ile Tyr Phe Ala Ile Leu Ser Phe Leu His Ile Trp

115 120 125

Ala Val Val Pro Cys Ile Lys Ser Phe Leu Ile Glu Ile Gln Cys Thr

130 135 140

Ser Arg Val Tyr Ser Ile Tyr Val His Thr Val Cys Asp Pro Leu Phe

145 150 155 160

Glu Ala Val Gly Lys Ile Phe Ser Asn Val Arg Ile Asn Leu Gln Lys

165 170 175

Glu Ile

<210> 9

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgtttagga agcttggtac caaggagata tacatatgtc tgggggcaaa tacgt 55

<210> 10

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tggtggtggt ggtgctcgag ttatatttct ttctgcaagt tgatgcgga 49

<210> 11

<211> 2424

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgtccggct ggccacgaat ttactacaaa ttactgaatt taccattaag catcctggta 60

aaaagcaagt ctattccggc agatcctgcc ccggaactgg ggctggatac ctctcgtcca 120

attatgtacg ttttaccgta caactcgaaa gcagatttgc tgacgttgcg cgcccagtgt 180

ctggcacatg acttgcctga cccgttagag ccgctggaaa tcgacggcac gctactgccg 240

cgctatgtgt tcattcacgg cgggccgcgt gtgttcacct attacacgcc gaaagaagag 300

tctattaagc tgttccacga ctatctcgat ttgcaccgta gcaacccaaa tctggatgtg 360

cagatggtgc cagtgtcggt gatgtttggt cgcgcgccgg ggcgtgaaaa aggcgaagtg 420

aacccgccgc tgcgtatgct taacggcgta cagaaatttt tcgctgtact gtggctcggt 480

cgcgacagtt ttgtgcgttt ctcgccgtca gtttcgctgc gccgtatggc ggatgaacac 540

ggcacggata aaactatcgc tcagaaactg gcgcgcgtgg cgcgtatgca ctttgcccgt 600

caacgtctgg ctgccgtagg cccacgtttg cctgctcgtc aggatctgtt taataagctg 660

ctcgcctccc gcgccattgc caaagcggta gaagatgaag cgcgcagcaa aaaaatctcc 720

catgaaaaag cgcagcagaa cgcgattgca ctgatggaag agattgcggc gaatttctct 780

tacgagatga ttcgcctgac tgaccgtatt ctgggcttca cctggaaccg actttaccag 840

ggcatcaacg tccataacgc tgagcgcgtt cgccagctgg cccacgacgg tcatgagctg 900

gtatatgtgc cttgccaccg cagtcacatg gactacctgc tgctttctta cgtgctgtat 960

caccaggggc tggtgccgcc gcatatcgcc gccgggatca acctgaattt ctggcctgcc 1020

gggccgattt tccgccgtct gggggcgttc tttattcgcc gtacgtttaa aggcaataaa 1080

ctttattcca ccgttttccg ggagtatctc ggcgaactgt tcagccgtgg ttattccgtc 1140

gagtacttcg tggaaggcgg tcgttcccgt acggggcgtt tgctggatcc gaaaactggt 1200

acgctgtcga tgaccattca ggcgatgctg cgtggcggca cgcgtccgat tacgctgatt 1260

ccgatctata tcggttatga gcacgtcatg gaagtgggta cttacgccaa agaactgcgc 1320

ggcgcgacga aagagaaaga gagcctgccg cagatgctgc gcggtttaag caagctgcgt 1380

aatctcggtc agggttacgt caacttcggt gaaccaatgc cgttgatgac ctaccttaac 1440

cagcatgtac ctgactggcg tgaatctatc gatcccatcg aagcggtgcg tccggcatgg 1500

ttaacgccga cggtcaataa tattgctgcc gatctgatgg tacgcattaa caacgcaggc 1560

gcggcaaacg ccatgaacct gtgctgtact gcgctactgg catcacgtca gcgctcactc 1620

acccgcgagc agttaaccga gcaactcaac tgctacctgg atctgatgcg caacgtgccc 1680

tactccacgg actctaccgt tccttcagcc agcgccagcg agcttatcga tcacgcgctg 1740

caaatgaaca agtttgaagt cgagaaagac acaatcggcg acatcatcat tctgccgcgc 1800

gagcaagcgg tgctgatgac ctactatcgc aacaacattg cgcatatgtt ggtgctgcct 1860

tcgctgatgg cggcaatcgt cacccagcat cgccacatct cccgcgacgt attgatggag 1920

cacgtcaatg tgctttaccc aatgctgaaa gcggagctgt tcctgcgctg ggatcgcgac 1980

gagttgccgg acgttattga tgcgctggca aatgagatgc aacgtcaggg gctgattacc 2040

ctgcaagatg atgagttgca tatcaacccg gcgcattctc gcacgctaca gctgctggcc 2100

gcaggcgcgc gcgaaacgct gcaacgttat gccatcacct tctggttgtt gagtgccaac 2160

ccgtcgatca accgcggtac gctggagaaa gagagccgca ccgtcgcgca acgtctctcc 2220

gtgctgcacg gcatcaacgc gccggagttc ttcgacaagg cggtgttcag ttctctggtg 2280

ctgacactgc gtgatgaagg gtatatcagc gatagcggcg atgccgaacc ggcagaaacg 2340

atgaaggttt atcagttgct ggcggagttg attacatcag acgtgcgttt gacgattgag 2400

agtgcgacgc agggcgaagg gtaa 2424

<210> 12

<211> 807

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Ser Gly Trp Pro Arg Ile Tyr Tyr Lys Leu Leu Asn Leu Pro Leu

1 5 10 15

Ser Ile Leu Val Lys Ser Lys Ser Ile Pro Ala Asp Pro Ala Pro Glu

20 25 30

Leu Gly Leu Asp Thr Ser Arg Pro Ile Met Tyr Val Leu Pro Tyr Asn

35 40 45

Ser Lys Ala Asp Leu Leu Thr Leu Arg Ala Gln Cys Leu Ala His Asp

50 55 60

Leu Pro Asp Pro Leu Glu Pro Leu Glu Ile Asp Gly Thr Leu Leu Pro

65 70 75 80

Arg Tyr Val Phe Ile His Gly Gly Pro Arg Val Phe Thr Tyr Tyr Thr

85 90 95

Pro Lys Glu Glu Ser Ile Lys Leu Phe His Asp Tyr Leu Asp Leu His

100 105 110

Arg Ser Asn Pro Asn Leu Asp Val Gln Met Val Pro Val Ser Val Met

115 120 125

Phe Gly Arg Ala Pro Gly Arg Glu Lys Gly Glu Val Asn Pro Pro Leu

130 135 140

Arg Met Leu Asn Gly Val Gln Lys Phe Phe Ala Val Leu Trp Leu Gly

145 150 155 160

Arg Asp Ser Phe Val Arg Phe Ser Pro Ser Val Ser Leu Arg Arg Met

165 170 175

Ala Asp Glu His Gly Thr Asp Lys Thr Ile Ala Gln Lys Leu Ala Arg

180 185 190

Val Ala Arg Met His Phe Ala Arg Gln Arg Leu Ala Ala Val Gly Pro

195 200 205

Arg Leu Pro Ala Arg Gln Asp Leu Phe Asn Lys Leu Leu Ala Ser Arg

210 215 220

Ala Ile Ala Lys Ala Val Glu Asp Glu Ala Arg Ser Lys Lys Ile Ser

225 230 235 240

His Glu Lys Ala Gln Gln Asn Ala Ile Ala Leu Met Glu Glu Ile Ala

245 250 255

Ala Asn Phe Ser Tyr Glu Met Ile Arg Leu Thr Asp Arg Ile Leu Gly

260 265 270

Phe Thr Trp Asn Arg Leu Tyr Gln Gly Ile Asn Val His Asn Ala Glu

275 280 285

Arg Val Arg Gln Leu Ala His Asp Gly His Glu Leu Val Tyr Val Pro

290 295 300

Cys His Arg Ser His Met Asp Tyr Leu Leu Leu Ser Tyr Val Leu Tyr

305 310 315 320

His Gln Gly Leu Val Pro Pro His Ile Ala Ala Gly Ile Asn Leu Asn

325 330 335

Phe Trp Pro Ala Gly Pro Ile Phe Arg Arg Leu Gly Ala Phe Phe Ile

340 345 350

Arg Arg Thr Phe Lys Gly Asn Lys Leu Tyr Ser Thr Val Phe Arg Glu

355 360 365

Tyr Leu Gly Glu Leu Phe Ser Arg Gly Tyr Ser Val Glu Tyr Phe Val

370 375 380

Glu Gly Gly Arg Ser Arg Thr Gly Arg Leu Leu Asp Pro Lys Thr Gly

385 390 395 400

Thr Leu Ser Met Thr Ile Gln Ala Met Leu Arg Gly Gly Thr Arg Pro

405 410 415

Ile Thr Leu Ile Pro Ile Tyr Ile Gly Tyr Glu His Val Met Glu Val

420 425 430

Gly Thr Tyr Ala Lys Glu Leu Arg Gly Ala Thr Lys Glu Lys Glu Ser

435 440 445

Leu Pro Gln Met Leu Arg Gly Leu Ser Lys Leu Arg Asn Leu Gly Gln

450 455 460

Gly Tyr Val Asn Phe Gly Glu Pro Met Pro Leu Met Thr Tyr Leu Asn

465 470 475 480

Gln His Val Pro Asp Trp Arg Glu Ser Ile Asp Pro Ile Glu Ala Val

485 490 495

Arg Pro Ala Trp Leu Thr Pro Thr Val Asn Asn Ile Ala Ala Asp Leu

500 505 510

Met Val Arg Ile Asn Asn Ala Gly Ala Ala Asn Ala Met Asn Leu Cys

515 520 525

Cys Thr Ala Leu Leu Ala Ser Arg Gln Arg Ser Leu Thr Arg Glu Gln

530 535 540

Leu Thr Glu Gln Leu Asn Cys Tyr Leu Asp Leu Met Arg Asn Val Pro

545 550 555 560

Tyr Ser Thr Asp Ser Thr Val Pro Ser Ala Ser Ala Ser Glu Leu Ile

565 570 575

Asp His Ala Leu Gln Met Asn Lys Phe Glu Val Glu Lys Asp Thr Ile

580 585 590

Gly Asp Ile Ile Ile Leu Pro Arg Glu Gln Ala Val Leu Met Thr Tyr

595 600 605

Tyr Arg Asn Asn Ile Ala His Met Leu Val Leu Pro Ser Leu Met Ala

610 615 620

Ala Ile Val Thr Gln His Arg His Ile Ser Arg Asp Val Leu Met Glu

625 630 635 640

His Val Asn Val Leu Tyr Pro Met Leu Lys Ala Glu Leu Phe Leu Arg

645 650 655

Trp Asp Arg Asp Glu Leu Pro Asp Val Ile Asp Ala Leu Ala Asn Glu

660 665 670

Met Gln Arg Gln Gly Leu Ile Thr Leu Gln Asp Asp Glu Leu His Ile

675 680 685

Asn Pro Ala His Ser Arg Thr Leu Gln Leu Leu Ala Ala Gly Ala Arg

690 695 700

Glu Thr Leu Gln Arg Tyr Ala Ile Thr Phe Trp Leu Leu Ser Ala Asn

705 710 715 720

Pro Ser Ile Asn Arg Gly Thr Leu Glu Lys Glu Ser Arg Thr Val Ala

725 730 735

Gln Arg Leu Ser Val Leu His Gly Ile Asn Ala Pro Glu Phe Phe Asp

740 745 750

Lys Ala Val Phe Ser Ser Leu Val Leu Thr Leu Arg Asp Glu Gly Tyr

755 760 765

Ile Ser Asp Ser Gly Asp Ala Glu Pro Ala Glu Thr Met Lys Val Tyr

770 775 780

Gln Leu Leu Ala Glu Leu Ile Thr Ser Asp Val Arg Leu Thr Ile Glu

785 790 795 800

Ser Ala Thr Gln Gly Glu Gly

805

<210> 14

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtaataaata aagatctcga gaaggagcat cgtttatgtc cggctggcca 50

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctctccttct ctctgattac ccttcgcc 28

<210> 15

<211> 738

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgctatata tctttcgtct tattattacc gtgatttaca gcatcttagt ctgtgtattc 60

ggctccattt actgcctttt cagcccgcgt aacccgaaac atgtggccac ctttgggcat 120

atgtttggcc gtcttgcgcc gctgtttggc ctgaaagttg agtgccgtaa acctacagac 180

gctgaaagct acggcaatgc tatctatatc gctaaccacc agaacaacta tgacatggtg 240

acagcatcga acatcgtgca accgccgacg gtgacggtag gtaaaaagag cttgctgtgg 300

atccccttct tcgggcagtt gtactggtta accggcaact tattgatcga cagaaacaat 360

cgcactaaag ctcacggcac cattgcggaa gtagtgaatc acttcaaaaa acgccgtatt 420

tccatctgga tgttcccgga aggaacccgc agccgtggtc gcggcctgct accgttcaag 480

actggagcat ttcacgcggc aattgcggcg ggcgtcccga ttattcccgt gtgcgtctct 540

acaacttcga ataagattaa tcttaatcga ctgcacaacg gtctggtgat tgtcgaaatg 600

ctgccgccaa ttgacgtcag tcagtatggc aaagatcagg ttcgtgagct ggctgcccat 660

tgtcgttcga taatggaaca aaaaatcgcc gagctcgata aagaagtcgc agaacgcgaa 720

gccgccggaa aagtttaa 738

<210> 16

<211> 245

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Leu Tyr Ile Phe Arg Leu Ile Ile Thr Val Ile Tyr Ser Ile Leu

1 5 10 15

Val Cys Val Phe Gly Ser Ile Tyr Cys Leu Phe Ser Pro Arg Asn Pro

20 25 30

Lys His Val Ala Thr Phe Gly His Met Phe Gly Arg Leu Ala Pro Leu

35 40 45

Phe Gly Leu Lys Val Glu Cys Arg Lys Pro Thr Asp Ala Glu Ser Tyr

50 55 60

Gly Asn Ala Ile Tyr Ile Ala Asn His Gln Asn Asn Tyr Asp Met Val

65 70 75 80

Thr Ala Ser Asn Ile Val Gln Pro Pro Thr Val Thr Val Gly Lys Lys

85 90 95

Ser Leu Leu Trp Ile Pro Phe Phe Gly Gln Leu Tyr Trp Leu Thr Gly

100 105 110

Asn Leu Leu Ile Asp Arg Asn Asn Arg Thr Lys Ala His Gly Thr Ile

115 120 125

Ala Glu Val Val Asn His Phe Lys Lys Arg Arg Ile Ser Ile Trp Met

130 135 140

Phe Pro Glu Gly Thr Arg Ser Arg Gly Arg Gly Leu Leu Pro Phe Lys

145 150 155 160

Thr Gly Ala Phe His Ala Ala Ile Ala Ala Gly Val Pro Ile Ile Pro

165 170 175

Val Cys Val Ser Thr Thr Ser Asn Lys Ile Asn Leu Asn Arg Leu His

180 185 190

Asn Gly Leu Val Ile Val Glu Met Leu Pro Pro Ile Asp Val Ser Gln

195 200 205

Tyr Gly Lys Asp Gln Val Arg Glu Leu Ala Ala His Cys Arg Ser Ile

210 215 220

Met Glu Gln Lys Ile Ala Glu Leu Asp Lys Glu Val Ala Glu Arg Glu

225 230 235 240

Ala Ala Gly Lys Val

245

<210> 18

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggtaatcaga gagaaggaga gggtgttatg ctatatatct ttcg 44

<210> 18

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gtttctttac cagactcgag ttgccgactt aaacttttcc ggc 43

Claims (10)

1. The colibacillus engineering bacteria for producing lycopene constructed by using membrane engineering is characterized by being constructed by expression vectors containing lycopene operon gene segments and expression vectors containing gene segments of coding cell membrane proteins.

2. The engineered Escherichia coli strain of claim 1, wherein the gene fragment encoding the cell membrane protein comprises a gene fragment having a nucleotide sequence shown in SEQ ID No.7cav1The gene segment shown as SEQ ID NO.11plsBAnd the gene segment shown as SEQ ID NO.15plsC。

3. The engineered Escherichia coli as claimed in claim 1, wherein the lycopene operon gene fragment is a gene fragment having a nucleotide sequence shown in SEQ ID NO.1lycIt is derived from Rhodopseudomonas palustris.

4. The engineered Escherichia coli strain of claim 2 or 3, wherein the expression vector further comprises a gene fragment encoding acetyl-CoA acyltransferase/hydroxymethylglutaryl-CoA reductasemvaEGene fragment encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthasemvaSAnd gene fragments encoding farnesyl/geranyl pyrophosphate synthaseIspA。

5. The method for constructing engineering bacteria of Escherichia coli as claimed in claim 4, which comprises the following steps:

(1) amplification of lycopene operon gene fragmentlycThen, the plasmid is connected with a pET28a plasmid fragment which is subjected to double enzyme digestion, a connecting product is converted into a competent cell, and a recombinant plasmid pET-RPlyc is obtained through resistance screening and positive cloning;

(2) amplification of encoded cell membranesGene fragment of proteincav1Connecting with a recombinant plasmid pET-Rplyc fragment subjected to double enzyme digestion, converting a connecting product into a competent cell, and obtaining a recombinant plasmid pET-RPlyc-cav1 through resistance screening and positive cloning;

or amplifying a gene fragment encoding a cell membrane proteinplsBAndplsCconnecting the two plasmids after overlapping connection with a pACYC-MvaE-MvaS-IspA plasmid fragment subjected to single enzyme digestion, converting a connecting product into a competent cell, and obtaining a recombinant plasmid pACYC-MvaE-MvaS-IspA-plsB-plsC through resistance screening and positive cloning;

(3) transforming the recombinant plasmid pET-RPlyc into an escherichia coli competent cell to obtain an escherichia coli engineering bacterium ELCXH 01;

or the recombinant plasmid pET-RPlyc and the plasmid pACYC-MvaE-MvaS-IspA are jointly transformed into an escherichia coli competent cell containing the pTrc-low plasmid to obtain an escherichia coli engineering bacterium ELCXH 02;

or the recombinant plasmid pET-RPlyc and the recombinant plasmid pACYC-MvaE-MvaS-IspA-plsB-plsC are jointly transformed into an escherichia coli competent cell containing the pTrc-low plasmid to obtain an escherichia coli engineering bacterium ELCXH 03;

or the recombinant plasmid pET-RPlyc-cav1 and the plasmid pACYC-MvaE-MvaS-IspA are jointly transformed into an Escherichia coli competent cell containing the pTrc-low plasmid to obtain the Escherichia coli engineering bacterium ELCXH 04.

6. The method according to claim 5, wherein the concentration ratio of the digested plasmid fragment to the amplified gene fragment is 1: 2-4.

7. Use of the engineered bacterium of Escherichia coli according to claim 4 for synthesizing lycopene and/or increasing lycopene production.

8. The use of claim 7, wherein the engineering bacteria of Escherichia coli comprises the following steps in lycopene synthesis: activating the engineering bacteria of Escherichia coli, selecting positive monoclonal antibody, transferring to LB culture medium containing antibiotic, culturing at 37 deg.C overnight, transferring the obtained seed liquid to M9Culturing in culture medium at 37 deg.C to OD with glucose as carbon source₆₀₀=0.6-0.8, adding inducer, shake culturing at 30 ℃ for 48-72 h, centrifuging, taking supernatant, adding extractant, shake culturing, and pricking with needle tip every 5-8 min until cell precipitate turns white; centrifuging to obtain supernatant containing lycopene, and detecting lycopene yield.

9. Use according to claim 8, characterized in that the method for detecting the production of lycopene is HPLC, with the conditions: HPLC detector is thermo DioNex UltiMate 3000, column 4.6X 250 mm, 5 μm ZORBAX Eclipse Plus C₁₈A column; mobile phase: methanol/ethyl acetate (85: 15); the flow rate is 1 mL/min; sample introduction volume: 10 μ L, column temperature: 25^oC; UV：475 nm。

10. The use according to claim 8, wherein the extractant is a mixture of 1: 1 of a mixed solution of methanol and acetone.

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