CN109321590A - Utilize the genetic engineering bacterium and its construction method of acetic acid production Pfansteihl and application - Google Patents

Abstract

The invention discloses the genetic engineering bacterium for utilizing acetic acid production Pfansteihl and its construction method and applications.Genetic engineering bacterium disclosed by the invention using acetic acid production Pfansteihl is obtained by carrying out following transformation to recipient bacterium: knocking out poxB gene, pflB gene, aceEF gene, ldhA gene, the lldD gene of recipient bacterium；Increase the content of acs gene, pta gene, ackA gene, aceA gene, aceK gene, aceB gene, maeA gene, maeB gene and ldh2 DNA encoding the protein in recipient bacterium.The engineering bacteria that the present invention obtains can reach desirable level using the Pfansteihl yield that acetic acid obtains in shaking flask culture, avoid the use of the carbon sources such as glucose, facilitate the cost of material for reducing Pfansteihl fermentation, with good application prospect.

Description

Utilize the genetic engineering bacterium and its construction method of acetic acid production Pfansteihl and application

Technical field

The invention belongs to biotechnology, genetic engineering and field of fermentation engineering, and in particular to a kind of to utilize acetic acid production L- The genetic engineering bacterium and its construction method of lactic acid and application.

Background technique

Human society consumes the fossil resources such as a large amount of oil and coal every year, thus discharges a large amount of greenhouse gases and causes The worry of climate change.Therefore, the heat that the development and utilization of renewable biomass resources become contemporary scientific circle and industry is paid close attention to One of point.Currently, being microorganism in fermentation industry from agricultural and glucose, xylose, starch and the cellulose etc. of planting industry The carbon source and energy substance generally utilized.China is a biofermentation great industry nation, and the yield of many products occupies the world One, a large amount of grain resource is consumed every year.Therefore, food shortage and agricultural and sideline product price continuous rise, have become restriction The key factor of biofermentation industry development.To solve the problems, such as " strive grain with people, strive ground with grain ", it is wide that there is an urgent need to develop sources General, cheap novel fermentation carbon source.Methanol, acetic acid and synthesis gas etc. are increasingly becoming the research in industrial biotechnology field Hot spot.

Acetic acid also known as acetic acid are the main components of the simple two carbon carboxylic acid of one kind and vinegar.Acetic acid divides in nature Cloth is extensive, is mainly present in fruit and vegetable oil with esters.In the tissue of animal, in excreta and blood, acetic acid with The form of free acid exists.Many microorganisms can convert acetic acid for organic matter by anaerobic fermentation.It can be made using acetic acid There are bacterium, fungi and yeast etc., such as Escherichia coli, saccharomyces cerevisiae, bacillus subtilis and paddy for the microorganism of carbon source for growth Propylhomoserin bar bacterium etc..

Lactic acid also known as Lactic acid are a kind of important organic acids.There is an asymmetric carbon atom in lactic acid molecules, there is D- Two kinds of configurations of lactic acid and Pfansteihl.Compared with D-ALPHA-Hydroxypropionic acid, Pfansteihl can be degraded by human body, be widely used in food, beverage, The fields such as medicine and cosmetics.In addition, lactic acid can be polymerized to polylactic acid by chemical method, can be used as biodegradable material makes With.2015, global lactic acid production was about 500,000 tons, and the market demand is based on Pfansteihl, for accounting for for food and beverage field Than close to 50%, the accounting for polylactic acid field is more than 30%.

The common preparation method of lactic acid has chemical method and microbe fermentation method.Chemical method production lactic acid main path include Lactonitrile method and acrylonitrile method, based on lactonitrile method.This method synthesizes lactonitrile through base catalysis using acetaldehyde and hydrogen cyanide as raw material, Lactic acid is generated using distillation purifying and with Concentrated acid hydrolysis, obtains different grades of product finally by esterification purification.Chemical method The raw material acetaldehyde and hydrogen cyanide utilized has stronger toxicity, and product is the mixture of D-ALPHA-Hydroxypropionic acid and Pfansteihl, due to D- Lactic acid is in the intracorporal metabolic problems of people, it is difficult to be applied to the fields such as food and beverage.

Microbe fermentation method has many advantages, such as that production cost is low, raw material sources are wide, product optical purity is high, is industrial life The main method of lactic acid producing.The strain that fermentation method uses has lactobacillus, bacillus and Rhizopus oryzae etc..Although using cellulose, The non-grain cheap raw material fermenting lactic acid such as cassava, molasses and jerusalem artichoke has several researchs, but cornstarch is still big at present Thus primary raw material used in scale fermentation causes the Pfansteihl market price higher, limit it in biodegradable material With the extensive use in other fields.

Summary of the invention

The technical problem to be solved by the present invention is to how using this potential large fermenting carbon source of acetic acid prepare L- Lactic acid.

In order to solve the above technical problems, present invention firstly provides the construction method of recombinant bacterium, the method includes to by Body bacterium carries out the transformation of following A 1-A14, obtains the recombinant bacterium；

A1, knock out the recipient bacterium acetonic acid oxidase gene (poxB gene, No. GeneBank: NC_000913.3, Gene ID:946132) or inhibit the expression of the poxB gene or inhibit the activity of the protein of the poxB gene coding；

A2, knock out the recipient bacterium pyruvate formate-lyase gene (pflB gene, No. GeneBank: NC_ 000913.3, Gene ID:945514) or inhibit the expression of the pflB gene or inhibit the albumen of the pflB gene coding The activity of matter；

A3, knocking out the pyruvate dehydrogenase gene of the recipient bacterium, (aceE gene and aceF gene, are denoted as aceEF gene； AceE gene, No. GeneBank: NC_000913.3, Gene ID:944834；AceF gene, No. GeneBank: NC_ 000913.3, Gene ID:944794) or inhibit the expression of the aceEF gene or inhibit the egg of the aceEF gene coding The activity of white matter；

A4, knock out the recipient bacterium D-lactic acid dehydrogenase gene (ldhA gene, No. GeneBank: NC_000913.3, Gene ID:946315) or inhibit the expression of the ldhA gene or inhibit the activity of the protein of the ldhA gene coding；

A5, knock out the recipient bacterium l-lactate dehydrogenase gene (lldD gene, No. GeneBank: NC_000913.3, Gene ID:948121) or inhibit the expression of the lldD gene or inhibit the activity of the protein of the lldD gene coding；

A6, the content or enhancing for increasing acetyl coenzyme A synthetase gene (acs gene) coding protein in the recipient bacterium The activity of the acs DNA encoding the protein；

A7, increase the content of phosphotransacetylase enzyme gene (pta gene) coding protein or enhancing institute in the recipient bacterium State the activity of pta DNA encoding the protein；

A8, increase in the recipient bacterium described in the content or enhancing of Acetokinase gene (ackA gene) coding protein The activity of ackA DNA encoding the protein；

A9, the content or enhancing for increasing isocitrate lyase gene (aceA gene) coding protein in the recipient bacterium The activity of the aceA DNA encoding the protein；

A10, increase isocitric dehydrogenase kinases/phosphatase gene (aceK gene) coding protein in the recipient bacterium Content or the enhancing aceK DNA encoding the protein activity；

A11, the content or enhancing for increasing malate synthetase gene (aceB gene) coding protein in the recipient bacterium The activity of the aceB DNA encoding the protein；

A12, increase in the recipient bacterium content of NAD- dependent form malic enzyme gene maeA DNA encoding the protein or Enhance the activity of the maeA DNA encoding the protein；

A13, increase in the recipient bacterium content of NADP- dependent form malic enzyme gene maeB DNA encoding the protein or Enhance the activity of the maeB DNA encoding the protein；

A14, the content or enhancing for increasing l-lactate dehydrogenase gene (ldh2 gene) coding protein in the recipient bacterium The activity of the ldh2 DNA encoding the protein；

The recipient bacterium is to contain the poxB gene, the pflB gene, the aceEF gene, the ldhA gene With the bacterium or fungi of the lldD gene.

In the above method, the recipient bacterium can be for 1) or 2):

1) Escherichia coli；

2) Escherichia coli MG1655.

In the above method, protein of the acs DNA encoding the protein for following a1) or a2):

A1) protein shown in sequence 12 in sequence table；

A2) amino acid sequence of sequence 12 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the ackA DNA encoding the protein for following b1) or b2):

B1) protein shown in sequence 13 in sequence table；

B2) amino acid sequence of sequence 13 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the pta DNA encoding the protein for following c1) or c2):

C1) protein shown in sequence 14 in sequence table；

C2) amino acid sequence of sequence 14 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the aceA DNA encoding the protein for following d1) or d2):

D1) protein shown in sequence 15 in sequence table；

D2) amino acid sequence of sequence 15 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the aceK DNA encoding the protein for following e1) or e2):

E1) protein shown in sequence 16 in sequence table；

E2) amino acid sequence of sequence 16 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the aceB DNA encoding the protein for following f1) or f2):

F1) protein shown in sequence 17 in sequence table；

F2) amino acid sequence of sequence 17 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the maeA DNA encoding the protein for following g1) or g2):

G1) protein shown in sequence 18 in sequence table；

G2) amino acid sequence of sequence 18 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the maeB DNA encoding the protein for following h1) or h2):

H1) protein shown in sequence 19 in sequence table；

H2) amino acid sequence of sequence 19 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function；

And/or protein of the ldh2 DNA encoding the protein for following i1) or i2):

I1) protein shown in sequence 20 in sequence table；

I2) amino acid sequence of sequence 20 in sequence table by the substitution of one or several amino acid residues and/or is lacked Mistake and/or addition and protein with the same function.

In the above method, the substitution and/or deletion and/or addition of one or several amino acid residues can for 1,2,3, 4, the substitution and/or deletion and/or addition of 5,6,7,8,9 or 10 amino acid residues.

In the above method, A1 is by importing the DNA for containing the poxB gene upstream and downstream homology arm into the recipient bacterium Segment is realized；

And/or A2 is real by importing the DNA fragmentation containing the pflB gene upstream and downstream homology arm into the recipient bacterium It is existing；

And/or A3 is by importing the DNA fragmentation for containing the aceEF gene upstream and downstream homology arm into the recipient bacterium It realizes；

And/or A4 is real by importing the DNA fragmentation containing the ldhA gene upstream and downstream homology arm into the recipient bacterium It is existing；

And/or A5 is real by importing the DNA fragmentation containing the lldD gene upstream and downstream homology arm into the recipient bacterium It is existing；

And/or A6 is realized by importing the acs expression casette containing the acs gene into the recipient bacterium；

And/or A7 is realized by importing the pta expression casette containing the pta gene into the recipient bacterium；

And/or A8 is realized by importing the ackA expression casette containing the ackA gene into the recipient bacterium；

And/or A9 is realized by importing the aceA expression casette containing the aceA gene into the recipient bacterium；

And/or A10 is realized by importing the aceK expression casette containing the aceK gene into the recipient bacterium；

And/or A11 is realized by importing the aceB expression casette containing the aceB gene into the recipient bacterium；

And/or A12 is realized by importing the maeA expression casette containing the maeA gene into the recipient bacterium；

And/or A13 is realized by importing the maeB expression casette containing the maeB gene into the recipient bacterium；

And/or A14 is realized by importing the ldh2 expression casette containing the ldh2 gene into the recipient bacterium.

Each expression cassette is imported into the recipient bacterium can be by that will contain described in the expression vector importing of corresponding expression cassette It is realized in recipient bacterium.

The expression vector can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid concretely plasmid PBBR1MCS-2 or plasmid pUC19.

In the above method, the knockout of each gene can be realized by λ-red homologous recombination mode.

In the above method, the DNA fragmentation containing the poxB gene upstream and downstream homology arm is 8 institute of sequence in sequence table The DNA molecular shown；

The DNA fragmentation containing the pflB gene upstream and downstream homology arm is DNA shown in sequence 9 points in sequence table Son；

The DNA fragmentation containing the aceEF gene upstream and downstream homology arm is DNA shown in sequence 7 points in sequence table Son；

The DNA fragmentation containing the ldhA gene upstream and downstream homology arm is DNA shown in sequence 10 points in sequence table Son；

The DNA fragmentation containing the lldD gene upstream and downstream homology arm is DNA shown in sequence 11 points in sequence table Son；

And/or the acs expression casette, the pta expression casette, the ackA expression casette, the aceA Expression casette, the aceK expression casette, the aceB expression casette, the maeA expression casette, the maeB Promoter is following j1 in expression casette and the ldh2 expression casette) or j2):

J1) DNA molecular shown in 9-51 of sequence 1 in sequence table；

J2 the nucleotide sequence) and j1) limited has 75% or 75% or more identity, and with promoter function DNA molecular；

And/or acs gene described in the acs expression casette is following k1) or k2):

K1) DNA molecular shown in 70-2028 of sequence 1 in sequence table；

K2 the nucleotide sequence) and k1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or pta gene described in the pta expression casette is following l1) or l2):

L1) DNA molecular shown in 1347-3491 of sequence 2 in sequence table；

L2 the nucleotide sequence) and l1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or ackA gene described in the ackA expression casette is following m1) or m2):

M1) DNA molecular shown in 70-1272 of sequence 2 in sequence table；

M2 the nucleotide sequence) and m1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or aceA gene described in the aceA expression casette is following n1) or n2):

N1) DNA molecular shown in 70-1374 of sequence 3 in sequence table；

N2 the nucleotide sequence) and n1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or aceK gene described in the aceK expression casette is following o1) or o2):

O1) DNA molecular shown in 1436-3172 of sequence 3 in sequence table；

O2 the nucleotide sequence) and o1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or aceB gene described in the aceB expression casette is following p1) or p2):

P1) DNA molecular shown in 70-1671 of sequence 4 in sequence table；

P2 the nucleotide sequence) and p1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or maeA gene described in the maeA expression casette is following q1) or q2):

Q1) DNA molecular shown in 70-1767 of sequence 5 in sequence table；

Q2 the nucleotide sequence) and q1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or maeB gene described in the maeB expression casette is following r1) or r2):

R1) DNA molecular shown in 1791-4070 of sequence 5 in sequence table；

R2 the nucleotide sequence) and r1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

And/or ldh2 gene described in the ldh2 expression casette is following s1) or s2):

S1) DNA molecular shown in 70-1050 of sequence 6 in sequence table；

S2 the nucleotide sequence) and s1) limited has 75% or 75% or more identity, and DNA with the same function Molecule；

In the above method, described 75% or 75% or more identity can for 80%, 85%, 90% or 95% or more it is same One property.

In the above method, the acs expression casette is DNA molecular shown in 9-2028 of sequence 1 in sequence table；

The pta expression casette and the ackA expression casette are shown in 9-3491 of sequence 2 in sequence DNA molecular；

The aceA expression casette and the aceK expression casette are shown in 9-3172 of sequence 3 in sequence table DNA molecular；

The aceB expression casette is DNA molecular shown in 9-1671 of sequence 4 in sequence table；

The maeA expression casette and the maeB expression casette are shown in 9-4070 of sequence 5 in sequence table DNA molecular；

The ldh2 expression casette is DNA molecular shown in 9-1050 of sequence 6 in sequence table.

The present invention also provides the recombinant bacteriums using the method preparation.

The present invention also provides a kind of reagent set, the reagent set is by described same containing the poxB gene upstream and downstream The DNA fragmentation of source arm, described contains the aceEF gene at the DNA fragmentation containing the pflB gene upstream and downstream homology arm It is the DNA fragmentation of upstream and downstream homology arm, the DNA fragmentation containing the ldhA gene upstream and downstream homology arm, described containing described DNA fragmentation, the acs expression casette, the pta expression casette, the ackA base of lldD gene upstream and downstream homology arm Because of expression cassette, the aceA expression casette, the aceK expression casette, the aceB expression casette, the maeA base Because expression cassette, the maeB expression casette and the ldh2 expression casette form.

The reagent set can be used for producing Pfansteihl and/or degradation acetic acid.

The present invention also provides following any applications of the reagent set or the recombinant bacterium:

X1, production Pfansteihl；

X2, preparation production Pfansteihl product；

X3, degradation acetic acid；

X4, preparation degradation acetic acid product.

The present invention also provides the preparation methods of Pfansteihl, which comprises using acetic acid as carbon source, utilizes the recombination Bacterium carries out bioconversion, and Pfansteihl is prepared.

Wherein, carrying out the bioconversion can carry out in MM fluid nutrient medium.

The composition of the MM fluid nutrient medium is as follows: every liter of culture medium acetic acid containing 8g, 2g NH₄Cl、5g(NH₄)₂SO₄、6g KH₂PO₄, 8g 3- N-morpholinyl, 0.5g NaCl, 1mL trace element solution, surplus is water.

The composition of the trace element solution is as follows: every liter of trace element solution FeCl containing 3.6g₂·4H₂O、5g CaCl₂·2H₂O、1.3g MnCl₂·2H₂O、0.38g CuCl₂·2H₂O、0.5g CoCl₂·6H₂O、0.94g ZnCl₂、 0.03g H₃BO₃、0.4g Na₂EDTA·2H₂O, 1g thiamine-HCl, remaining is 0.5M HCl.

The condition for carrying out the bioconversion can are as follows: 30-37 DEG C of bottle shake culture 24-72h (such as 37 DEG C, 48h).Shaking flask Revolving speed can be 50-200rpm.

Beneficial effects of the present invention: the present invention is by the relevant gene of 9 metabolic pathways of expression in escherichia coli and striking Except 6 endogenous genes, obtaining can ferment using acetic acid as carbon source and obtain the engineered strain of Pfansteihl, and recombinant bacterium is shaking The yield of Pfansteihl in bottle culture can reach higher level, avoid the use of the carbon sources such as glucose, help to reduce L- cream The cost of material of acid fermentation, with good application prospect.

Detailed description of the invention

Fig. 1 is pMCS-LA1 Vector map.

Fig. 2 is pMCS-LA2 Vector map.

Fig. 3 is pUC19-LA1 Vector map.

Fig. 4 is pUC19-LA2 Vector map.

Fig. 5 is pUC19-LA3 Vector map.

Fig. 6 is pUC19-LA4 Vector map.

Fig. 7 is pUC19-LA24 Vector map.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified. Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.In following embodiments, such as without special Illustrate, the 1st of each nucleotide sequence is the 5 ' terminal nucleotides of corresponding DNA in sequence table, and last bit is the 3 ' of corresponding DNA Terminal nucleotide.

Enzyme used in molecular biology manipulations involved in following embodiments, be NEB (New England Biolabs, Http:// www.neb-china.com/) Products；The kit that plasmid extracts and DNA fragmentation recycling is used, is Beijing Bo Maide gene technology Co., Ltd (http://www.biomed168.com/) product；DNA involved in embodiment synthesis and Examining order is completed by Beijing Bo Maide gene technology Co., Ltd.

E.coli MG1655: E.coli Genetic Resources at Yale CGSC, The Coli are derived from Genetic Stock Center (http://cgsc2.biology.yale.edu/), number CGSC#6300.

E.coli NEB 5-alpha: it derives from NEB (New England Biolabs), article No. C2987I.

Plasmid pKD13: E.coli Genetic Resources at Yale CGSC, The Coli are derived from Genetic Stock Center, number CGSC#7633.

Plasmid pKD46: E.coli Genetic Resources at Yale CGSC, The Coli are derived from Genetic Stock Center, number CGSC#7739.

Plasmid pCP20: E.coli Genetic Resources at Yale CGSC, The Coli are derived from Genetic Stock Center, number CGSC#7637.

Plasmid pBBR1MCS-2: the public can voluntarily submit according to the sequence of NCBI accession number U23751.1 Gene chemical synthesis company obtains, which records in the following literature: Four new derivatives of the broad- host-range cloning vector pBBR1MCS,carrying different antibiotic-resistance cassettes,1995,Gene,166:175-176。

Plasmid pUC19: NEB (New England Biolabs) company, article No. N3041S are derived from.

The composition of MM fluid nutrient medium is as follows: every liter of culture medium acetic acid containing 8g, 2g NH₄Cl、5g(NH₄)₂SO₄、6g KH₂PO₄, 8g 3- N-morpholinyl, 0.5g NaCl, 1mL trace element solution, surplus is water.

The composition of trace element solution is as follows: every liter of trace element solution FeCl containing 3.6g₂·4H₂O、5g CaCl₂· 2H₂O、1.3g MnCl₂·2H₂O、0.38g CuCl₂·2H₂O、0.5g CoCl₂·6H₂O、0.94g ZnCl₂、0.03g H₃BO₃、0.4g Na₂EDTA·2H₂O, 1g thiamine-HCl, remaining is 0.5M HCl.

Embodiment 1, building recombinant bacterium E.coli LA5 (pMCS-LA2+pUC19-LA4)

One, the building of recombinant expression carrier pMCS2-LA1 and pMCS2-LA2

1, DNA shown in sequence 1 in artificial synthesized sequence table, containing acs expression cassette, upstream is the site SacI, and downstream is The site XbaI, wherein 9-51 nucleotide are promoter sequence, 70-2028 nucleotide are acs gene order.

2, DNA shown in sequence 2 in artificial synthesized sequence table, containing ackA and pta expression cassette, upstream is the site XbaI, Downstream is the site EcoRI, wherein 9-51 nucleotide are promoter sequence, 70-1272 nucleotide are ackA gene sequence Column, 1347-3491 nucleotide are pta gene order.

3, with the DNA sequence dna synthesized in SacI and XbaI double digestion sequence 1, the DNA fragmentation that size is about 2022bp is recycled； With SacI and XbaI double digestion plasmid pBBR1MCS-2, the DNA fragmentation that size is about 5124bp is recycled；By above-mentioned about 2022bp and Two DNA fragmentations of 5124bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB5-alpha In, and it is coated on LB solid medium containing kanamycin, 37 DEG C of culture 16h obtain transformant.Extract the matter of transformant Grain carries out digestion verification with SacI and XbaI, and digestion products size is about 2022bp and the plasmid of 5124bp is positive plasmid, will This recombinant plasmid is named as recombinant plasmid pMCS-LA1.Recombinant plasmid pMCS-LA1 is sequenced, the result shows that: pMCS-LA1 For the SacI of plasmid pBBR1MCS-2 and XbaI being identified to, the DNA fragmentation between sequence replaces with the 9- of sequence 1 in sequence table The recombinant plasmid that acs expression cassette obtains shown in 2028.

4, with the DNA sequence dna synthesized in XbaI and EcoRI double digestion sequence 2, the DNA piece that size is about 3489bp is recycled Section；With XbaI and EcoRI double digestion plasmid pMCS-LA1, the DNA molecular that size is about 7116bp is recycled；By above-mentioned about 3489bp It is connected with two DNA fragmentations of 7116bp, obtains connection product by the method for chemical conversion and imported into E.coli NEB5- In alpha, and it is coated on LB solid medium containing kanamycin, 37 DEG C of culture 16h obtain transformant.Extract transformant Plasmid, carry out digestion verification with XbaI and EcoRI, digestion products size is about 3489bp and the plasmid of 7116bp is positive matter Grain, is named as recombinant plasmid pMCS-LA2 for this recombinant plasmid.Recombinant plasmid pMCS-LA2 is sequenced, the result shows that: PMCS-LA2 is that the XbaI of plasmid pMCS-LA1 and EcoRI is identified that the DNA fragmentation between sequence replaces with sequence 2 in sequence table The recombinant plasmid that ackA and pta expression cassette obtains shown in 9-3491.

Two, the building of recombinant expression carrier pUC19-LA1 and pUC19-LA2

1, DNA shown in sequence 3 in artificial synthesized sequence table, containing aceA and aceK expression cassette, upstream is the site SacI, Downstream is the site SpeI and KpnI, wherein 9-51 nucleotide are promoter sequence, 70-1374 nucleotide are aceA base Because of sequence, 1436-3172 nucleotide are aceK gene order.

2, DNA shown in sequence 4 in artificial synthesized sequence table, containing aceB expression cassette, upstream is the site SpeI, and downstream is The site XhoI and KpnI, wherein 9-51 nucleotide are promoter sequence, 70-1671 nucleotide are aceB gene sequence Column.

3, with the DNA sequence dna synthesized in SacI and KpnI double digestion step 1, the DNA fragmentation that size is about 3179bp is recycled； With SacI and KpnI double digestion plasmid pUC19, the DNA fragmentation that size is about 2680bp is recycled；By above-mentioned about 3179bp and Two DNA fragmentations of 2680bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB 5- In alpha, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of culture 16h obtain transformant.Extract conversion The plasmid of son carries out digestion verification with SacI and KpnI, and digestion products size is about 3179bp and the plasmid of 2680bp is the positive This recombinant plasmid is named as pUC19-LA1 by plasmid.Obtained recombinant plasmid pUC19-LA1 is subjected to sequence verification, as a result table Bright: pUC19-LA1 is that the SacI of plasmid pUC19 and KpnI is identified that the DNA fragmentation between sequence replaces with sequence 3 in sequence table The recombinant plasmid that DNA sequence dna shown in 9-3181 obtains, the recombinant plasmid contain shown in 9-3172 of sequence 3 AceA and aceK expression cassette.

4, with the DNA sequence dna synthesized in SpeI and KpnI double digestion step 2, the DNA fragmentation that size is about 1682bp is recycled； With SpeI and KpnI double digestion plasmid pUC19-LA1, the DNA fragmentation that size is about 5846bp is recycled；By above-mentioned about 1682bp and Two DNA fragmentations of 5846bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB 5- In alpha, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of culture 16h obtain transformant.Extract conversion The plasmid of son carries out digestion verification with SpeI and KpnI, and digestion products size is about 1682bp and the plasmid of 5846bp is the positive This recombinant plasmid is named as pUC19-LA2 by plasmid.Obtained recombinant plasmid pUC19-LA2 is subjected to sequence verification, as a result table Bright: pUC19-LA2 is to replace with the DNA fragmentation between the identification sequence of the SpeI of plasmid pUC19-LA1 and KpnI in sequence table The recombinant plasmid that DNA sequence dna shown in 9-1680 of sequence 4 obtains, the recombinant plasmid contain 9-1671 of sequence 4 Shown in aceB expression cassette.

Three, the building of recombinant expression carrier pUC19-LA3

1, DNA shown in sequence 5 in artificial synthesized sequence table, containing maeA and maeB expression cassette, upstream is the site XhoI, Downstream is the site KpnI, wherein 9-51 nucleotide are promoter sequence, 70-1767 nucleotide are maeA gene sequence Column, 1791-4070 nucleotide are maeB gene order.

2, with the DNA sequence dna synthesized in XhoI and KpnI double digestion step 1, the DNA fragmentation that size is about 4072bp is recycled； With XhoI and KpnI double digestion plasmid pUC19-LA2, the DNA fragmentation that size is about 7515bp is recycled；By above-mentioned about 4072bp and Two DNA fragmentations of 7515bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB 5- In alpha, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of culture 16h obtain transformant.Extract conversion The plasmid of son carries out digestion verification with XhoI and KpnI, and digestion products size is about 4072bp and the plasmid of 7515bp is the positive This recombinant plasmid is named as pUC19-LA3 by plasmid.Obtained recombinant plasmid pUC19-LA3 is subjected to sequence verification, as a result table Bright: pUC19-LA3 is that the XhoI of plasmid pUC19-LA2 and KpnI is identified that the DNA fragmentation between sequence replaces with sequence in sequence table The recombinant plasmid that maeA and maeB expression cassette shown in 9-4070 of column 5 obtains.

Four, the building of recombinant expression carrier pUC19-LA4

1, DNA shown in sequence 6 in artificial synthesized sequence table, containing ldh2 expression cassette, upstream is the site KpnI, and downstream is The site XbaI, wherein 9-51 nucleotide are promoter sequence, 70-1050 nucleotide are ldh2 gene order.

2, with the DNA sequence dna synthesized in KpnI and XbaI double digestion step 1, the DNA fragmentation that size is about 1044bp is recycled； With KpnI and XbaI double digestion plasmid pUC19-LA3, the DNA fragmentation that size is about 11576bp is recycled；By above-mentioned about 1044bp and Two DNA fragmentations of 11576bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB5- In alpha, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of culture 16h obtain transformant.Extract conversion The plasmid of son carries out digestion verification with KpnI and XbaI, and digestion products size is about 1044bp and the plasmid of 11576bp is the positive This recombinant plasmid is named as pUC19-LA4 by plasmid.Obtained recombinant plasmid pUC19-LA4 is subjected to sequence verification, as a result table Bright: pUC19-LA4 is to replace with the DNA fragmentation between the identification sequence of the KpnI of plasmid pUC19-LA3 and XbaI in sequence table The recombinant plasmid that ldh2 expression cassette shown in 9-1050 of sequence 6 obtains.

Five, the building of recombinant expression carrier pUC19-LA24

1, DNA shown in sequence 6 in artificial synthesized sequence table, containing ldh2 expression cassette, upstream is the site KpnI, and downstream is The site XbaI, wherein 9-51 nucleotide are promoter sequence, 70-1050 nucleotide are ldh2 gene order.

2, with the DNA sequence dna synthesized in KpnI and XbaI double digestion step 1, the DNA fragmentation that size is about 1044bp is recycled； With KpnI and XbaI double digestion plasmid pUC19-LA2, the DNA fragmentation that size is about 7517bp is recycled；By above-mentioned 1044bp and Two DNA fragmentations of 7517bp connect, and obtain connection product by the method for chemical conversion and imported into E.coli NEB 5- In alpha, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of culture 16h obtain transformant.Extract conversion The plasmid of son carries out digestion verification with KpnI and XbaI, and digestion products size is about 1044bp and the plasmid of 7517bp is the positive This recombinant plasmid is named as pUC19-LA24 by plasmid.Obtained recombinant plasmid pUC19-LA24 is subjected to sequence verification, as a result Show: pUC19-LA24 is that the KpnI of plasmid pUC19-LA2 and XbaI is identified that the DNA fragmentation between sequence replaces in sequence table The recombinant plasmid that ldh2 expression cassette shown in 9-1050 of sequence 6 obtains.

Six, the building of E. coli LA5

1, poxB gene is knocked out

1) primer poxBF and poxBR used in poxB gene knockout are synthesized, sequence is as follows:

PoxBF:

5’-ATGAAACAAACGGTTGCAGCTTATATCGCCAAAACACTCGAATCGGCAGGGGTGAAACGCGTGTA GGCTGGAGC TGCTTCG-3'；

PoxBR:

5’-GGGCGTCGCGGTAATCTTCCAGCGCTTTATCCAGAAACTTGCGATCGGCTTTTTCTTCCAATTCC GGGGATCCG TCGACC-3’。

2) using plasmid pKD13 as template, PCR amplification is carried out using primer poxBF and poxBR and obtains 1500bp's or so DNA fragmentation, is named as poxB homologous recombination segment, and agarose gel electrophoresis purifies obtained DNA fragmentation.By surveying The nucleotides sequence of sequence, poxB homologous recombination segment is classified as sequence 8, wherein and 1-60 are poxB upstream region of gene homology arm, the 61-1364 are FRT sequence and Kan resistant gene, and 1365-1424 are poxB downstream of gene homology arm.

3) pKD46 is converted into F-strain E.coli MG1655 using the method for electrotransformation, and be coated on containing ammonia The LB solid medium of parasiticin, 30 DEG C of cultures for 24 hours, obtain transformant, and the verifying of upgrading grain obtains the recombination containing pKD46 Bacterium is denoted as E.coli MG1655 (pKD46).

4) E.coli MG1655 (pKD46) is inoculated into the LB liquid medium containing ampicillin, 30 DEG C of cultures 1h is added arabinose to final concentration 5g/L, continues to cultivate 1.5h, next prepare the impression of E.coli MG1655 (pKD46) The DNA fragmentation obtained in step 2) is transferred in the competent cell of E.coli MG1655 (pKD46), and is coated on by state cell LB solid medium containing kanamycin, 37 DEG C of cultures for 24 hours, obtain transformant.

5) method for utilizing bacterium colony PCR carries out PCR by primer of poxBF and poxBR, will survey after PCR product purifying Sequence screens the clone that correct poxB gene has been replaced by Kan resistant gene, is denoted as E.coli MG1655poxB-K (pKD46)。

6) E.coli MG1655poxB-K (pKD46) is inoculated in LB liquid medium, 42 DEG C of subcultures three times, PKD46 plasmid is removed, E.coli MG1655poxB-K is obtained；E.coli MG1655poxB-K is poxB gene by Kan gene The E.coli MG1655 being substituted.

7) E.coli MG1655poxB-K is inoculated into LB liquid medium containing kanamycin, 37 DEG C of cultures For 24 hours, it is transferred in LB liquid medium containing kanamycin, 37 DEG C of culture 3h, preparation E.coli MG1655poxB-K impression State cell.

8) plasmid pCP20 is transformed into E.coli MG1655poxB-K competent cell using the method for electrotransformation, and It is coated on the LB solid medium containing ampicillin and chloramphenicol, 30 DEG C of culture 48h obtain transformant.Utilize bacterium colony PCR Method validation transformant, using poxBF and poxBR as primer, obtaining 202bp segment is positive colony.By the positive colony Sequencing is sent to, to knock out the bacterium that the poxB gene on E.coli MG1655 genome obtains, which is inoculated into the training of LB liquid It supports in base, 42 DEG C of passages three times, remove pCP20, are mutant E.coli LA1 by obtained Strain Designation.

2, pflB gene is knocked out

It is essentially identical with the method for above-mentioned steps 1, the difference is that as follows:

PflB gene knockout primer sequence is as follows:

PflBF:

5’-

ATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTTTTACCAAAGGTGACTGGGTGTAGGC TGGAGCTGCTTCG-3'；

PflBR:

5’

-TTACATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGCTGTTCTTTAGTCAGCGAATTCCGG GGATCCGTCGACC-3'；

The nucleotides sequence of the pflB homologous recombination segment obtained using pflB gene knockout primer is classified as sequence 9, wherein the 1-60 are pflB upstream region of gene homology arm, and 61-1364 are FRT sequence and Kan resistant gene, and 1365-1424 are PflB downstream of gene homology arm.Transformation receptor bacterial strain used is the mutant E.coli LA1 that above-mentioned steps 1 obtain.Utilize bacterium colony The method validation transformant of PCR obtains the segment positive colony of 202bp using pflBF and pflBR as primer.By the positive colony Sequencing is sent to, to knock out the bacterium that the pflB gene on E.coli LA1 genome obtains, which is inoculated into LB Liquid Culture In base, 42 DEG C of passages three times, remove pCP20, are mutant E.coli LA2 by obtained Strain Designation.

3, aceEF gene is knocked out

It is essentially identical with the method for above-mentioned steps 1, the difference is that as follows:

AceEF gene knockout primer sequence is as follows:

AceEFF:

5’-ATGTCAGAACGTTTCCCAAATGACGTGGATCCGATCGAAACTCGCGACTGGCTCCAGGCGGTGTA GGCTGGA GCTGCTTCG-3'；

AceEFR:

5’-TTACATCACCAGACGGCGAATGTCAGACAGCGTGTTGTTAATGATGGTAATGAAACGGGCATTCC GGGGATC CGTCGACC-3'；

The nucleotides sequence of the aceEF homologous recombination segment obtained using aceEF gene knockout primer is classified as sequence 7, wherein 1-60 be aceEF upstream region of gene homology arm, 61-1364 be FRT sequence and Kan resistant gene, 1365-1424 For aceEF downstream of gene homology arm.Transformation receptor bacterial strain is the mutant E.coli LA2 that above-mentioned steps 2 obtain.Utilize bacterium colony The method validation transformant of PCR obtains the segment positive colony of 202bp using aceEFF and aceEFR as primer.By the positive gram It is grand to send to sequencing, to knock out the bacterium that the aceEF gene on E.coli LA2 genome obtains, which is inoculated into the training of LB liquid It supports in base, 42 DEG C of passages three times, remove pCP20, are mutant E.coli LA3 by obtained Strain Designation.

4, ldhA gene is knocked out

It is essentially identical with the method for above-mentioned steps 1, the difference is that as follows:

LdhA gene knockout primer sequence is as follows:

LdhAF:

5’-

ATGAAACTCGCCGTTTATAGCACAAAACAGTACGACAAGAAGTACCTGCAACAGGTGAACGTGTAGGC TGGAGCTGCTTCG-3'；

LdhAR:

5’-

TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTTAAGTTTTGCAGCGTATTCCGGG GATCCGTCGACC-3'；

The nucleotides sequence of the ldhA homologous recombination segment obtained using ldhA gene knockout primer is classified as sequence 10, wherein 1-60 be ldhA upstream region of gene homology arm, 61-1364 be FRT sequence and Kan resistant gene, 1365-1424 For ldhA downstream of gene homology arm.Transformation receptor bacterial strain is the mutant E.coli LA3 that above-mentioned steps 3 obtain.Utilize bacterium colony The method validation transformant of PCR obtains the segment positive colony of 202bp using ldhAF and ldhAR as primer.By the positive colony Sequencing is sent to, to knock out the bacterium that the ldhA gene on E.coli LA3 genome obtains, which is inoculated into LB Liquid Culture In base, 42 DEG C of passages three times, remove pCP20, are mutant E.coli LA4 by obtained Strain Designation.

5, lldD gene is knocked out

It is essentially identical with the method for above-mentioned steps 1, the difference is that as follows:

LldD gene knockout primer sequence is as follows:

LldDF:

5’-

ATGATTATTTCCGCAGCCAGCGATTATCGCGCCGCAGCGCAACGCATTCTGCCGCCGTTCGTGTAGGC TGGAGCTGCTTCG-3'；

LldDR:

5’-

CTATGCCGCATTCCCTTTCGCCATGGGAGCCAGTGCCGCAGGCAACTCTTTACCCAGCCCATTCCGGG GATCCGTCGACC-3'；

The nucleotides sequence of the lldD homologous recombination segment obtained using lldD gene knockout primer is classified as sequence 11, wherein 1-60 be lldD upstream region of gene homology arm, 61-1364 be FRT sequence and Kan resistant gene, 1365-1424 For lldD downstream of gene homology arm.Transformation receptor bacterial strain is the mutant E.coli LA4 that above-mentioned steps 4 obtain.Utilize bacterium colony The method validation transformant of PCR obtains the segment positive colony of 202bp using lldDF and lldDR as primer.By the positive colony Sequencing is sent to, to knock out the bacterium that the lldD gene on E.coli LA4 genome obtains, which is inoculated into LB Liquid Culture In base, 42 DEG C of passages three times, remove pCP20, are mutant E.coli LA5 by obtained Strain Designation.

Seven, the building of recombinant bacterium E.coli LA5 (pMCS-LA2+pUC19-LA4)

1, by the recombinant plasmid pMCS-LA2 that above-mentioned steps one the obtain and recombinant plasmid pUC19- that above-mentioned steps four obtain LA4 is transformed into the E. coli LA5 that above-mentioned steps six obtain by the method for electrotransformation, and is coated on containing ammonia benzyl The LB solid medium of penicillin and kanamycins, 37 DEG C of cultures are for 24 hours.

2, picking monoclonal is in the LB liquid medium containing ampicillin and kanamycins, and 37 DEG C of cultures are for 24 hours.

3, the plasmid for extracting transformant, verifies the correctness of conversion, obtains containing there are two plasmid (pMCS-LA2 and pUC19- LA4 recombinant bacterium) is denoted as E.coli LA5 (pMCS-LA2+pUC19-LA4).

E.coli LA5 (pMCS-LA2+pUC19-LA4) be knock out E.coli MG1655 chromosomal gene poxB, pflB, AceEF, ldhA and lldD, and it is outer containing gene acs, pta, ackA, aceA, aceK, aceB, maeA, maeB and ldh2 The recombinant bacterium of source expression cassette.

Eight, the building of bacterium E.coli LA5 (pUC19-LA4) is compareed

1, the recombinant plasmid pUC19-LA4 that above-mentioned steps four obtain is transformed into above-mentioned steps six by the method for electrotransformation Obtained E. coli LA5, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of cultures are for 24 hours.

2, picking monoclonal is in the LB liquid medium containing ampicillin, and 37 DEG C of cultures are for 24 hours.

3, the plasmid for extracting transformant, verifies the correctness of conversion, obtains being denoted as containing the recombinant bacterium of plasmid pUC19-LA4 E.coli LA5(pUC19-LA4)。

E.coli LA5 (pUC19-LA4) be knock out E.coli MG1655 chromosomal gene poxB, pflB, aceEF, LdhA and lldD, and the recombinant bacterium of the heterogenous expression box containing gene aceA, aceK, aceB, maeA, maeB and ldh2.

Nine, the building of bacterium E.coli LA5 (pUC19-LA24) is compareed

1, the recombinant plasmid pUC19-LA24 that above-mentioned steps five obtain is transformed into above-mentioned steps by the method for electrotransformation Six obtained E. coli LA5, and it is coated on the LB solid medium containing ampicillin, 37 DEG C of cultures are for 24 hours.

2, picking monoclonal is in the LB liquid medium containing ampicillin, and 37 DEG C of cultures are for 24 hours.

3, the plasmid for extracting transformant, verifies the correctness of conversion, obtains the recombinant bacterium note containing plasmid pUC19-LA24 For E.coli LA5 (pUC19-LA24).

E.coli LA5 (pUC19-LA24) be knock out E.coli MG1655 chromosomal gene poxB, pflB, aceEF, LdhA and lldD, and the recombinant bacterium of the heterogenous expression box containing gene aceA, aceK, aceB and ldh2.

The application of embodiment 2, recombinant bacterium E.coli LA5 (pMCS-LA2+pUC19-LA4) in production Pfansteihl

One, the shaking flask culture experiment of recombinant bacterium E.coli LA5 (pMCS-LA2+pUC19-LA4)

1, the E.coli LA5 (pMCS-LA2+pUC19-LA4) for preparing step 7 in embodiment 1 is containing ammonia benzyl mould In the LB liquid medium of element and kanamycins, 16h is cultivated under the conditions of 37 DEG C, revolving speed 200rpm, as seed liquor.

2, seed liquor, is inoculated into the MM liquid containing ampicillin and kanamycins by 4% inoculum concentration by volume In culture medium, liquid amount is 50ml in 250ml shaking flask, is cultivated for 24 hours under the conditions of 37 DEG C, revolving speed 100rpm, respectively in culture 12h Fermentation liquid is collected for 24 hours.

3, quantitative detection is carried out by acetic acid of the high performance liquid chromatography to the genetic engineering bacterium Pfansteihl generated and consumption.Tool Concrete conditions in the establishment of a specific crime is as follows:

Instrument: Shimadzu Corporation's Essentia LC series HPLC instrument is furnished with DGU-20A degasser, LC-16 liquid-feeding pump, SIL- 16 type autosamplers, RID-20A detector.

Chromatographic condition: Bio-RadHPX-87H(7.8×300mm)；Flow velocity 0.50mL/min；55 DEG C of column temperature；Stream Dynamic is mutually 7mM aqueous sulfuric acid.

Detection method:

Taking Pfansteihl concentration is respectively Pfansteihl standard items aqueous solution (Pfansteihl, the Sigma- of 0,1,2,3,4,5g/L Aldrich, product number L1875), with 0.22 μm of filtering with microporous membrane, 10 μ L of sample introduction carries out HPLC detection, uses various concentration Pfansteihl standard solution chromatographic peak area be ordinate, various concentration is abscissa, draw standard curve.Take acetic acid concentration The acetic acid standard items aqueous solution (acetic acid, Sigma-Aldrich, product number A6283) of respectively 0,2,4,6,8,10g/L are used 0.22 μm of filtering with microporous membrane, 10 μ L of sample introduction carry out HPLC detection, with the chromatographic peak area of the acetic acid standard solution of various concentration For ordinate, the concentration of different material is abscissa, draws standard curve.

Take the fermentation liquid of 2mL, first with ultraviolet specrophotometer measurement bacterium solution 600nm OD value, then at 12000rpm from Its fermented supernatant fluid is transferred in new centrifuge tube by heart 10min, and with 0.22 μm of filtering with microporous membrane, 10 μ L of sample introduction is carried out HPLC detection.

The Pfansteihl chromatographic peak area of sample to be tested fermented supernatant fluid is substituted into above-mentioned standard curve, is calculated to be measured The L-lactic acid of sample fermented supernatant fluid.It is bent that the acetic acid chromatographic peak area of sample to be tested fermented supernatant fluid is substituted into above-mentioned standard In line, the remaining acetic acid content of sample to be tested fermented supernatant fluid is calculated, acetic acid content initial after inoculation is subtracted into fermentation Remaining acetic acid content in supernatant obtains the acetic acid amount of recombinant bacterium consumption.

Pfansteihl yield and acetic acid consumption such as table 1 of the E.coli LA5 (pMCS-LA2+pUC19-LA4) in shaking flask culture It is shown:

The Pfansteihl yield and acetic acid of 1 E.coli LA5 (pMCS-LA2+pUC19-LA4) of table consumes

Note: data are the resulting mean value of three repeated experiments in table.

As seen from Table 1, the recombinant bacterial strain E.coli LA5 (pMCS-LA2+pUC19- that the present invention constructs LA4) can be in the culture medium that addition acetic acid is carbon source, fermentation obtains Pfansteihl, and final Pfansteihl yield is 2.46g/L, obtains Rate is 0.40g/g, about the 53% of theoretical yield.

Two, the shaking flask culture experiment of bacterium E.coli LA5 (pUC19-LA4) is compareed

1, the E.coli LA5 (pUC19-LA4) for preparing step 8 in embodiment 1 is in the LB liquid containing ampicillin In body culture medium, 16h is cultivated under the conditions of 37 DEG C, revolving speed 200rpm, as seed liquor.

2,4% inoculum concentration by volume, seed liquor is inoculated into the MM fluid nutrient medium containing ampicillin, Liquid amount is 50ml in 250ml shaking flask, is cultivated for 24 hours under the conditions of 37 DEG C, revolving speed 100rpm, is collected respectively in culture 12h and for 24 hours Fermentation liquid.

3, quantitative detection, tool are carried out by acetic acid of the high performance liquid chromatography to the genetic engineering bacterium Pfansteihl generated and consumption In concrete conditions in the establishment of a specific crime above-mentioned steps one described in step 3, Pfansteihl yield and second of the E.coli LA5 (pUC19-LA4) in shaking flask culture Acid consumption is as shown in table 2:

The Pfansteihl yield and acetic acid of 2 E.coli LA5 (pUC19-LA4) of table consumes

Note: data are the resulting mean value of three repeated experiments in table.

Compared with E.coli LA5 (pMCS-LA2+pUC19-LA4), control bacterium E.coli LA5 (pUC19-LA4) does not have It is overexpressed acetic acid and utilizes relevant acs, pta and ackA gene, although what E.coli LA5 (pUC19-LA4) process was fermented for 24 hours Pfansteihl yield is 1.69g/L, but Pfansteihl yield and acetic acid consume opposite E.coli LA5 (pMCS-LA2+pUC19-LA4) It is lower.It can be seen that the overexpression of gene acs, pta and ackA have weight for the acetic acid utilization and Pfansteihl synthesis that increase bacterium It acts on.

Three, the shaking flask culture experiment of bacterium E.coli LA5 (pUC19-LA24) is compareed

1, the E.coli LA5 (pUC19-LA24) for preparing step 9 in embodiment 1 is in the LB liquid containing ampicillin In body culture medium, 16h is cultivated under the conditions of 37 DEG C, revolving speed 200rpm, as seed liquor.

2,4% inoculum concentration by volume, seed liquor is inoculated into the MM fluid nutrient medium containing ampicillin, Liquid amount is 50ml in 250ml shaking flask, is cultivated for 24 hours under the conditions of 37 DEG C, revolving speed 100rpm, is collected respectively in culture 12h and for 24 hours Fermentation liquid.

3, quantitative detection, tool are carried out by acetic acid of the high performance liquid chromatography to the genetic engineering bacterium Pfansteihl generated and consumption In concrete conditions in the establishment of a specific crime above-mentioned steps one described in step 3, Pfansteihl yield of the E.coli LA5 (pUC19-LA24) in shaking flask culture and Acetic acid consumption is as shown in table 3:

The Pfansteihl yield and acetic acid of 3 E.coli LA5 (pUC19-LA24) of table consumes

Note: data are the resulting mean value of three repeated experiments in table.Compared with E.coli LA5 (pUC19-LA4), control Bacterium E.coli LA5 (pUC19-LA24) is not overexpressed NAD- dependent form malic enzyme gene maeA and NADP- dependent form apple Phytase gene maeB can not accumulate Pfansteihl as tunning although bacterium can consume acetic acid and grow.Thus may be used To see, the overexpression of gene maeA and maeB can promote Malic Metabolism slime flux in tricarboxylic acid cycle to go out to synthesize pyruvic acid, Pfansteihl is catalyzed and synthesized by l-lactate dehydrogenase again, gene maeA and maeB plays a key effect for the synthesis of Pfansteihl.

<110>Beijing University of Chemical Technology

<120>genetic engineering bacterium of acetic acid production Pfansteihl and its construction method and application are utilized

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 2036

<212> DNA

<213>artificial sequence

<400> 1

aagagctctt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tgagccaaat tcacaaacac accattcctg ccaacatcgc agaccgttgc 120

ctgataaacc ctcagcagta cgaggcgatg tatcaacaat ctattaacgt acctgatacc 180

ttctggggcg aacagggaaa aattcttgac tggatcaaac cttaccagaa ggtgaaaaac 240

acctcctttg cccccggtaa tgtgtccatt aaatggtacg aggacggcac gctgaatctg 300

gcggcaaact gccttgaccg ccatctgcaa gaaaacggcg atcgtaccgc catcatctgg 360

gaaggcgacg acgccagcca gagcaaacat atcagctata aagagctgca ccgcgacgtc 420

tgccgcttcg ccaataccct gctcgagctg ggcattaaaa aaggtgatgt ggtggcgatt 480

tatatgccga tggtgccgga agccgcggtt gcgatgctgg cctgcgcccg cattggcgcg 540

gtgcattcgg tgattttcgg cggcttctcg ccggaagccg ttgccgggcg cattattgat 600

tccaactcac gactggtgat cacttccgac gaaggtgtgc gtgccgggcg cagtattccg 660

ctgaagaaaa acgttgatga cgcgctgaaa aacccgaacg tcaccagcgt agagcatgtg 720

gtggtactga agcgtactgg cgggaaaatt gactggcagg aagggcgcga cctgtggtgg 780

cacgacctgg ttgagcaagc gagcgatcag caccaggcgg aagagatgaa cgccgaagat 840

ccgctgttta ttctctacac ctccggttct accggtaagc caaaaggtgt gctgcatact 900

accggcggtt atctggtgta cgcggcgctg acctttaaat atgtctttga ttatcatccg 960

ggtgatatct actggtgcac cgccgatgtg ggctgggtga ccggacacag ttacttgctg 1020

tacggcccgc tggcctgcgg tgcgaccacg ctgatgtttg aaggcgtacc caactggccg 1080

acgcctgccc gtatggcgca ggtggtggac aagcatcagg tcaatattct ctataccgca 1140

cccacggcga tccgcgcgct gatggcggaa ggcgataaag cgatcgaagg caccgaccgt 1200

tcgtcgctgc gcattctcgg ttccgtgggc gagccaatta acccggaagc gtgggagtgg 1260

tactggaaaa aaatcggcaa cgagaaatgt ccggtggtcg atacctggtg gcagaccgaa 1320

accggcggtt tcatgatcac cccgctgcct ggcgctaccg agctgaaagc cggttcggca 1380

acacgtccgt tcttcggcgt gcaaccggcg ctggtcgata acgaaggtaa cccgctggag 1440

ggggccaccg aaggtagcct ggtaatcacc gactcctggc cgggtcaggc gcgtacgctg 1500

tttggcgatc acgaacgttt tgaacagacc tacttctcca ccttcaaaaa tatgtatttc 1560

agcggcgacg gcgcgcgtcg cgatgaagat ggctattact ggataaccgg gcgtgtggac 1620

gacgtgctga acgtctccgg tcaccgtctg gggacggcag agattgagtc ggcgctggtg 1680

gcgcatccga agattgccga agccgccgta gtaggtattc cgcacaatat taaaggtcag 1740

gcgatctacg cctacgtcac gcttaatcac ggggaggaac cgtcaccaga actgtacgca 1800

gaagtccgca actgggtgcg taaagagatt ggcccgctgg cgacgccaga cgtgctgcac 1860

tggaccgact ccctgcctaa aacccgctcc ggcaaaatta tgcgccgtat tctgcgcaaa 1920

attgcggcgg gcgataccag caacctgggc gatacctcga cgcttgccga tcctggcgta 1980

gtcgagaagc tgcttgaaga gaagcaggct atcgcgatgc catcgtaatc tagaaa 2036

<210> 2

<211> 3499

<212> DNA

<213>artificial sequence

<400> 2

aatctagatt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tgtcgagtaa gttagtactg gttctgaact gcggtagttc ttcactgaaa 120

tttgccatca tcgatgcagt aaatggtgaa gagtaccttt ctggtttagc cgaatgtttc 180

cacctgcccg aagcacgtat caaatggaaa atggacggca ataaacagga agcggcttta 240

ggtgcaggcg ccgctcacag cgaagcgctc aactttatcg ttaatactat tctggcacaa 300

aaaccagaac tgtctgcgca gctgactgct atcggtcacc gtatcgtaca cggcggcgaa 360

aagtatacca gctccgtagt gatcgatgag tctgttattc agggtatcaa agatgcagct 420

tcttttgcac cgctgcacaa cccggctcac ctgatcggta tcgaagaagc tctgaaatct 480

ttcccacagc tgaaagacaa aaacgttgct gtatttgaca ccgcgttcca ccagactatg 540

ccggaagagt cttacctcta cgccctgcct tacaacctgt acaaagagca cggcatccgt 600

cgttacggcg cgcacggcac cagccacttc tatgtaaccc aggaagcggc aaaaatgctg 660

aacaaaccgg tagaagaact gaacatcatc acctgccacc tgggcaacgg tggttccgtt 720

tctgctatcc gcaacggtaa atgcgttgac acctctatgg gcctgacccc gctggaaggt 780

ctggtcatgg gtacacgttc tggtgatatc gatccggcga tcatcttcca cctgcacgac 840

accctgggca tgagcgttga cgcaatcaac aaactgctga ccaaagagtc tggcctgctg 900

ggtctgaccg aagtgaccag cgactgccgc tatgttgaag acaactacgc gacgaaagaa 960

gacgcgaagc gcgcaatgga cgtttactgc caccgcctgg cgaaatacat cggtgcctac 1020

actgcgctga tggatggtcg tctggacgct gttgtattca ctggtggtat cggtgaaaat 1080

gccgcaatgg ttcgtgaact gtctctgggc aaactgggcg tgctgggctt tgaagttgat 1140

catgaacgca acctggctgc acgtttcggc aaatctggtt tcatcaacaa agaaggaacc 1200

cgtcctgcgg tggttatccc aaccaacgaa gaactggtta tcgcgcaaga cgcgagccgc 1260

ctgactgcct gatttcacac cgccagctca gctggcggtg ctgttttgta acccgccaaa 1320

tcggcggtaa cgaaagagga taaaccgtgt cccgtattat tatgctgatc cctaccggaa 1380

ccagcgtcgg tctgaccagc gtcagccttg gcgtgatccg tgcaatggaa cgcaaaggcg 1440

ttcgtctgag cgttttcaaa cctatcgctc agccgcgtac cggtggcgat gcgcccgatc 1500

agactacgac tatcgtgcgt gcgaactctt ccaccacgac ggccgctgaa ccgctgaaaa 1560

tgagctacgt tgaaggtctg ctttccagca atcagaaaga tgtgctgatg gaagagatcg 1620

tcgcaaacta ccacgctaac accaaagacg ctgaagtcgt tctggttgaa ggtctggtcc 1680

cgacacgtaa gcaccagttt gcccagtctc tgaactacga aatcgctaaa acgctgaatg 1740

cggaaatcgt cttcgttatg tctcagggca ctgacacccc ggaacagctg aaagagcgta 1800

tcgaactgac ccgcaacagc ttcggcggtg ccaaaaacac caacatcacc ggcgttatcg 1860

ttaacaaact gaacgcaccg gttgatgaac agggtcgtac tcgcccggat ctgtccgaga 1920

ttttcgacga ctcttccaaa gctaaagtaa acaatgttga tccggcgaag ctgcaagaat 1980

ccagcccgct gccggttctc ggcgctgtgc cgtggagctt tgacctgatc gcgactcgtg 2040

cgatcgatat ggctcgccac ctgaatgcga ccatcatcaa cgaaggcgac atcaatactc 2100

gccgcgttaa atccgtcact ttctgcgcac gcagcattcc gcacatgctg gagcacttcc 2160

gtgccggttc tctgctggtg acttccgcag accgtcctga cgtgctggtg gccgcttgcc 2220

tggcagccat gaacggcgta gaaatcggtg ccctgctgct gactggcggt tacgaaatgg 2280

acgcgcgcat ttctaaactg tgcgaacgtg ctttcgctac cggcctgccg gtatttatgg 2340

tgaacaccaa cacctggcag acctctctga gcctgcagag cttcaacctg gaagttccgg 2400

ttgacgatca cgaacgtatc gagaaagttc aggaatacgt tgctaactac atcaacgctg 2460

actggatcga atctctgact gccacttctg agcgcagccg tcgtctgtct ccgcctgcgt 2520

tccgttatca gctgactgaa cttgcgcgca aagcgggcaa acgtatcgta ctgccggaag 2580

gtgacgaacc gcgtaccgtt aaagcagccg ctatctgtgc tgaacgtggt atcgcaactt 2640

gcgtactgct gggtaatccg gcagagatca accgtgttgc agcgtctcag ggtgtagaac 2700

tgggtgcagg gattgaaatc gttgatccag aagtggttcg cgaaagctat gttggtcgtc 2760

tggtcgaact gcgtaagaac aaaggcatga ccgaaaccgt tgcccgcgaa cagctggaag 2820

acaacgtggt gctcggtacg ctgatgctgg aacaggatga agttgatggt ctggtttccg 2880

gtgctgttca cactaccgca aacaccatcc gtccgccgct gcagctgatc aaaactgcac 2940

cgggcagctc cctggtatct tccgtgttct tcatgctgct gccggaacag gtttacgttt 3000

acggtgactg tgcgatcaac ccggatccga ccgctgaaca gctggcagaa atcgcgattc 3060

agtccgctga ttccgctgcg gccttcggta tcgaaccgcg cgttgctatg ctctcctact 3120

ccaccggtac ttctggtgca ggtagcgacg tagaaaaagt tcgcgaagca actcgtctgg 3180

cgcaggaaaa acgtcctgac ctgatgatcg acggtccgct gcagtacgac gctgcggtaa 3240

tggctgacgt tgcgaaatcc aaagcgccga actctccggt tgcaggtcgc gctaccgtgt 3300

tcatcttccc ggatctgaac accggtaaca ccacctacaa agcggtacag cgttctgccg 3360

acctgatctc catcgggccg atgctgcagg gtatgcgcaa gccggttaac gacctgtccc 3420

gtggcgcact ggttgacgat atcgtctaca ccatcgcgct gactgcgatt cagtctgcac 3480

agcagcagta agaattctt 3499

<210> 3

<211> 3189

<212> DNA

<213>artificial sequence

<400> 3

aagagctctt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tgaaaacccg tacacaacaa attgaagaat tacagaaaga gtggactcaa 120

ccgcgttggg aaggcattac tcgcccatac agtgcggaag atgtggtgaa attacgcggt 180

tcagtcaatc ctgaatgcac gctggcgcaa ctgggcgcag cgaaaatgtg gcgtctgctg 240

cacggtgagt cgaaaaaagg ctacatcaac agcctcggcg cactgactgg cggtcaggcg 300

ctgcaacagg cgaaagcggg tattgaagca gtctatctgt cgggatggca ggtagcggcg 360

gacgctaacc tggcggccag catgtatccg gatcagtcgc tctatccggc aaactcggtg 420

ccagctgtgg tggagcggat caacaacacc ttccgtcgtg ccgatcagat ccaatggtcc 480

gcgggcattg agccgggcga tccgcgctat gtcgattact tcctgccgat cgttgccgat 540

gcggaagccg gttttggcgg tgtcctgaat gcctttgaac tgatgaaagc gatgattgaa 600

gccggtgcag cggcagttca cttcgaagat cagctggcgt cagtgaagaa atgcggtcac 660

atgggcggca aagttttagt gccaactcag gaagctattc agaaactggt cgcggcgcgt 720

ctggcagctg acgtgacggg cgttccaacc ctgctggttg cccgtaccga tgctgatgcg 780

gcggatctga tcacctccga ttgcgacccg tatgacagcg aatttattac cggcgagcgt 840

accagtgaag gcttcttccg tactcatgcg ggcattgagc aagcgatcag ccgtggcctg 900

gcgtatgcgc catatgctga cctggtctgg tgtgaaacct ccacgccgga tctggaactg 960

gcgcgtcgct ttgcacaagc tatccacgcg aaatatccgg gcaaactgct ggcttataac 1020

tgctcgccgt cgttcaactg gcagaaaaac ctcgacgaca aaactattgc cagcttccag 1080

cagcagctgt cggatatggg ctacaagttc cagttcatca ccctggcagg tatccacagc 1140

atgtggttca acatgtttga cctggcaaac gcctatgccc agggcgaggg tatgaagcac 1200

tacgttgaga aagtgcagca gccggaattt gccgccgcga aagatggcta taccttcgta 1260

tctcaccagc aggaagtggg tacaggttac ttcgataaag tgacgactat tattcagggc 1320

ggcacgtctt cagtcaccgc gctgaccggc tccactgaag aatcgcagtt ctaattgaca 1380

gctagctcag tcctaggtat aatgctagct actagagaaa gaggagaaat ataccatgcc 1440

gcgtggcctg gaattattga ttgctcaaac cattttgcaa ggcttcgatg ctcagtatgg 1500

tcgattcctc gaagtgacct ccggtgcgca gcagcgtttc gaacaggccg actggcatgc 1560

tgtccagcag gcgatgaaaa accgtatcca tctttacgat catcacgttg gtctggtcgt 1620

ggagcaactg cgctgcatta ctaacggcca aagtacggac gcggcatttt tactacgtgt 1680

taaagagcat tacacccggc tgttgccgga ttacccgcgc ttcgagattg cggagagctt 1740

ttttaactcc gtgtactgtc ggttatttga ccaccgctcg cttactcccg agcggctttt 1800

tatctttagc tctcagccag agcgccgctt tcgtaccatt ccccgcccgc tggcgaaaga 1860

ctttcacccc gatcacggct gggaatctct actgatgcgc gttatcagcg acctaccgct 1920

gcgcctgcgc tggcagaata aaagccgtga catccattac attattcgcc atctgacgga 1980

aacgctgggg acagacaacc tcgcggaaag tcatttacag gtggcgaacg aactgtttta 2040

ccgcaataaa gccgcctggc tggtaggcaa actgatcaca ccttccggca cattgccatt 2100

tttgctgccg atccaccaga cggacgacgg cgagttattt attgatacct gcctgacgac 2160

gaccgccgaa gcgagcattg tttttggctt tgcgcgttct tattttatgg tttatgcgcc 2220

gctgcccgca gcgctggtcg agtggctacg ggaaattctg ccaggtaaaa ccaccgctga 2280

attgtatatg gctatcggct gccagaagca cgccaaaacc gaaagctacc gcgaatatct 2340

cgtttatcta cagggctgta atgagcagtt cattgaagcg ccgggtattc gtggaatggt 2400

gatgttggtg tttacgctgc cgggctttga tcgggtattc aaagtcatca aagacaggtt 2460

cgcgccgcag aaagagatgt ctgccgctca cgttcgtgcc tgctatcaac tggtgaaaga 2520

gcacgatcgc gtgggccgaa tggcggacac ccaggagttt gaaaactttg tgctggagaa 2580

gcggcatatt tccccggcat taatggaatt actgcttcag gaagcagcgg aaaaaatcac 2640

cgatctcggc gaacaaattg tgattcgcca tctttatatt gagcggcgga tggtgccgct 2700

caatatctgg ctggaacaag tggaaggtca gcagttgcgc gacgccattg aagaatacgg 2760

taacgctatt cgccagcttg ccgctgctaa cattttccct ggcgacatgc tgtttaaaaa 2820

cttcggtgtc acccgtcacg ggcgtgtggt tttttatgat tacgatgaaa tttgctacat 2880

gacggaagtg aatttccgcg acatcccgcc gccgcgctat ccggaagacg aacttgccag 2940

cgaaccgtgg tacagcgtct cgccgggcga tgttttcccg gaagagtttc gccactggct 3000

atgcgccgac ccgcgtattg gtccgctgtt tgaagagatg cacgccgacc tgttccgcgc 3060

tgattactgg cgcgcactac aaaaccgcat acgtgaaggg catgtggaag atgtttatgc 3120

gtatcggcgc aggcaaagat ttagcgtacg gtatggggag atgctttttt gaactagtaa 3180

aggtaccaa 3189

<210> 4

<211> 1688

<212> DNA

<213>artificial sequence

<400> 4

aaactagttt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tgactgaaca ggcaacaaca accgatgaac tggctttcac aaggccgtat 120

ggcgagcagg agaagcaaat tcttactgcc gaagcggtag aatttctgac tgagctggtg 180

acgcatttta cgccacaacg caataaactt ctggcagcgc gcattcagca gcagcaagat 240

attgataacg gaacgttgcc tgattttatt tcggaaacag cttccattcg cgatgctgat 300

tggaaaattc gcgggattcc tgcggactta gaagaccgcc gcgtagagat aactggcccg 360

gtagagcgca agatggtgat caacgcgctc aacgccaatg tgaaagtctt tatggccgat 420

ttcgaagatt cactggcacc agactggaac aaagtgatcg acgggcaaat taacctgcgt 480

gatgcggtta acggcaccat cagttacacc aatgaagcag gcaaaattta ccagctcaag 540

cccaatccag cggttttgat ttgtcgggta cgcggtctgc acttgccgga aaaacatgtc 600

acctggcgtg gtgaggcaat ccccggcagc ctgtttgatt ttgcgctcta tttcttccac 660

aactatcagg cactgttggc aaagggcagt ggtccctatt tctatctgcc gaaaacccag 720

tcctggcagg aagcggcctg gtggagcgaa gtcttcagct atgcagaaga tcgctttaat 780

ctgccgcgcg gcaccatcaa ggcgacgttg ctgattgaaa cgctgcccgc cgtgttccag 840

atggatgaaa tccttcacgc gctgcgtgac catattgttg gtctgaactg cggtcgttgg 900

gattacatct tcagctatat caaaacgttg aaaaactatc ccgatcgcgt cctgccagac 960

agacaggcag tgacgatgga taaaccattc ctgaatgctt actcacgcct gttgattaaa 1020

acctgccata aacgcggtgc ttttgcgatg ggcggcatgg cggcgtttat tccgagcaaa 1080

gatgaagagc acaataacca ggtgctcaac aaagtaaaag cggataaatc gctggaagcc 1140

aataacggtc acgatggcac atggatcgct cacccaggcc ttgcggacac ggcaatggcg 1200

gtattcaacg acattctcgg ctcccgtaaa aatcagcttg aagtgatgcg cgaacaagac 1260

gcgccgatta ctgccgatca gctgctggca ccttgtgatg gtgaacgcac cgaagaaggt 1320

atgcgcgcca acattcgcgt ggctgtgcag tacatcgaag cgtggatctc tggcaacggc 1380

tgtgtgccga tttatggcct gatggaagat gcggcgacgg ctgaaatttc ccgtacctcg 1440

atctggcagt ggatccatca tcaaaaaacg ttgagcaatg gcaaaccggt gaccaaagcc 1500

ttgttccgcc agatgctggg cgaagagatg aaagtcattg ccagcgaact gggcgaagaa 1560

cgtttctccc aggggcgttt tgacgatgcc gcacgcttga tggaacagat caccacttcc 1620

gatgagttaa ttgatttcct gaccctgcca ggctaccgcc tgttagcgta actcgagaaa 1680

ggtaccaa 1688

<210> 5

<211> 4078

<212> DNA

<213>artificial sequence

<400> 5

aactcgagtt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tggaaccaaa aacaaaaaaa cagcgttcgc tttatatccc ttacgctggc 120

cctgtactgc tggaatttcc gttgttgaat aaaggcagtg ccttcagcat ggaagaacgc 180

cgtaacttca acctgctggg gttactgccg gaagtggtcg aaaccatcga agaacaagcg 240

gaacgagcat ggatccagta tcagggattc aaaaccgaaa tcgacaaaca catctacctg 300

cgtaacatcc aggacactaa cgaaaccctc ttctaccgtc tggtaaacaa tcatcttgat 360

gagatgatgc ctgttattta taccccaacc gtcggcgcag cctgtgagcg tttttctgag 420

atctaccgcc gttcacgcgg cgtgtttatc tcttaccaga accggcacaa tatggacgat 480

attctgcaaa acgtgccgaa ccataatatt aaagtgattg tggtgactga cggtgaacgc 540

attctggggc ttggtgacca gggcatcggc gggatgggca ttccgatcgg taaactgtcg 600

ctctataccg cctgtggcgg catcagcccg gcgtataccc ttccggtggt gctggatgtc 660

ggaacgaaca accaacagct gcttaacgat ccgctgtata tgggctggcg taatccgcgt 720

atcactgacg acgaatacta tgaattcgtt gatgaattta tccaggctgt gaaacaacgc 780

tggccagacg tgctgttgca gtttgaagac tttgctcaaa aaaatgcgat gccgttactt 840

aaccgctatc gcaatgaaat ttgttctttt aacgatgaca ttcagggcac tgcggcggta 900

acagtcggca cactgatcgc agcaagccgc gcggcaggtg gtcagttaag cgagaaaaaa 960

atcgtcttcc ttggcgcagg ttcagcggga tgcggcattg ccgaaatgat catctcccag 1020

acccagcgcg aaggattaag cgaggaagcg gcgcggcaga aagtctttat ggtcgatcgc 1080

tttggcttgc tgactgacaa gatgccgaac ctgctgcctt tccagaccaa actggtgcag 1140

aagcgcgaaa acctcagtga ctgggatacc gacagcgatg tgctgtcact gctggatgtg 1200

gtgcgcaatg taaaaccaga tattctgatt ggcgtctcag gacagaccgg gctgtttacg 1260

gaagagatca tccgtgagat gcataaacac tgtccgcgtc cgatcgtgat gccgctgtct 1320

aacccgacgt cacgcgtgga agccacaccg caggacatta tcgcctggac cgaaggtaac 1380

gcgctggtcg ccacgggcag cccgtttaat ccagtggtat ggaaagataa aatctaccct 1440

atcgcccagt gtaacaacgc ctttattttc ccgggcatcg gcctgggtgt tattgcttcc 1500

ggcgcgtcac gtatcaccga tgagatgctg atgtcggcaa gtgaaacgct ggcgcagtat 1560

tcaccattgg tgctgaacgg cgaaggtatg gtactgccgg aactgaaaga tattcagaaa 1620

gtctcccgcg caattgcgtt tgcggttggc aaaatggcgc agcagcaagg cgtggcggtg 1680

aaaacctctg ccgaagccct gcaacaggcc attgacgata atttctggca agccgaatac 1740

cgcgactacc gccgtacctc catctaatag agaaagagga gaaatatacc atggatgacc 1800

agttaaaaca aagtgcactt gatttccatg aatttccagt tccagggaaa atccaggttt 1860

ctccaaccaa gcctctggca acacagcgcg atctggcgct ggcctactca ccaggcgttg 1920

ccgcaccttg tcttgaaatc gaaaaagacc cgttaaaagc ctacaaatat accgcccgag 1980

gtaacctggt ggcggtgatc tctaacggta cggcggtgct ggggttaggc aacattggcg 2040

cgctggcagg caaaccggtg atggaaggca agggcgttct gtttaagaaa ttcgccggga 2100

ttgatgtatt tgacattgaa gttgacgaac tcgacccgga caaatttatt gaagttgtcg 2160

ccgcgctcga accaaccttc ggcggcatca acctcgaaga cattaaagcg ccagaatgtt 2220

tctatattga acagaaactg cgcgagcgga tgaatattcc ggtattccac gacgatcagc 2280

acggcacggc aattatcagc actgccgcca tcctcaacgg cttgcgcgtg gtggagaaaa 2340

acatctccga cgtgcggatg gtggtttccg gcgcgggtgc cgcagcaatc gcctgtatga 2400

acctgctggt agcgctgggt ctgcaaaaac ataacatcgt ggtttgcgat tcaaaaggcg 2460

ttatctatca gggccgtgag ccaaacatgg cggaaaccaa agccgcatat gcggtggtgg 2520

atgacggcaa acgtaccctc gatgatgtga ttgaaggcgc ggatattttc ctgggctgtt 2580

ccggcccgaa agtgctgacc caggaaatgg tgaagaaaat ggctcgtgcg ccaatgatcc 2640

tggcgctggc gaacccggaa ccggaaattc tgccgccgct ggcgaaagaa gtgcgtccgg 2700

atgccatcat ttgcaccggt cgttctgact atccgaacca ggtgaacaac gtcctgtgct 2760

tcccgttcat cttccgtggc gcgctggacg ttggcgcaac cgccatcaac gaagagatga 2820

aactggcggc ggtacgtgcg attgcagaac tcgcccatgc ggaacagagc gaagtggtgg 2880

cttcagcgta tggcgatcag gatctgagct ttggtccgga atacatcatt ccaaaaccgt 2940

ttgatccgcg cttgatcgtt aagatcgctc ctgcggtcgc taaagccgcg atggagtcgg 3000

gcgtggcgac tcgtccgatt gctgatttcg acgtctacat cgacaagctg actgagttcg 3060

tttacaaaac caacctgttt atgaagccga ttttctccca ggctcgcaaa gcgccgaagc 3120

gcgttgttct gccggaaggg gaagaggcgc gcgttctgca tgccactcag gaactggtaa 3180

cgctgggact ggcgaaaccg atccttatcg gtcgtccgaa cgtgatcgaa atgcgcattc 3240

agaaactggg cttgcagatc aaagcgggcg ttgattttga gatcgtcaat aacgaatccg 3300

atccgcgctt taaagagtac tggaccgaat acttccagat catgaagcgt cgcggcgtca 3360

ctcaggaaca ggcgcagcgg gcgctgatca gtaacccgac agtgatcggc gcgatcatgg 3420

ttcagcgtgg ggaagccgat gcaatgattt gcggtacggt gggtgattat catgaacatt 3480

ttagcgtggt gaaaaatgtc tttggttatc gcgatggcgt tcacaccgca ggtgccatga 3540

acgcgctgct gctgccgagt ggtaacacct ttattgccga tacatatgtt aatgatgaac 3600

cggatgcaga agagctggcg gagatcacct tgatggcggc agaaactgtc cgtcgttttg 3660

gtattgagcc gcgcgttgct ttgttgtcgc actccaactt tggttcttct gactgcccgt 3720

cgtcgagcaa aatgcgtcag gcgctggaac tggtcaggga acgtgcacca gaactgatga 3780

ttgatggtga aatgcacggc gatgcagcgc tggtggaagc gattcgcaac gaccgtatgc 3840

cggacagctc tttgaaaggt tccgccaata ttctggtgat gccgaacatg gaagctgccc 3900

gcattagtta caacttactg cgtgtttcca gctcggaagg tgtgactgtc ggcccggtgc 3960

tgatgggtgt ggcgaaaccg gttcacgtgt taacgccgat cgcatcggtg cgtcgtatcg 4020

tcaacatggt ggcgctggcc gtggtagaag cgcaaaccca accgctgtaa ggtaccaa 4078

<210> 6

<211> 1058

<212> DNA

<213>artificial sequence

<400> 6

aaggtacctt gacagctagc tcagtcctag gtataatgct agctactaga gaaagaggag 60

aaatatacca tggcaagtat tacggataag gatcaccaaa aagttattct cgttggtgat 120

ggcgccgttg gttcaagcta tgcctatgca atggttttgc aaggtatcgc tcaagaaatc 180

gggattgttg acattttcaa ggacaagacg aaaggtgacg caattgattt aagcaacgcg 240

ctcccattca ccagtcctaa gaagatttat tctgctgaat acagcgatgc caaggatgct 300

gatctggttg ttatcactgc tggtgctcct caaaagcctg gcgaaactcg cttggatctg 360

gttaacaaga acttgaagat tttgaagtcc attgttgatc cgatcgtgga ttctggcttt 420

aacggtatct tcttggttgc tgctaaccca gttgatatct tgacttatgc tacttggaag 480

ctttccggat tcccgaagag ccgggttgtt ggttcaggta cttctctgga caccgctcgt 540

ttccgtcaat ccattgctga aatggttaac gttgatgctc gttccgtcca cgcatatatc 600

atgggtgaac acggcgacac agaattccct gtatggtccc acgctaacat tggtggcgtt 660

accatcgctg aatgggttaa agcacatcca gaaatcaaag aagacaaact tgttaagatg 720

tttgaagacg ttcgtgacgc tgcttacgaa atcatcaaac tcaagggtgc aaccttctac 780

ggtatcgcaa ctgctttggc acggatttcc aaagcaattc ttaacgatga aaatgcggta 840

ctcccattgt ccgtttacat ggacggccaa tatggcttga acgacatcta cattggtaca 900

cctgctgtga tcaaccgcaa tggtattcag aacattctgg aaatcccatt gaccgaccac 960

gaagaagaat ccatgcagaa gtcggcttca caattgaaga aagttctgac cgatgcgttt 1020

gctaagaacg acatcgaaac acgtcagtaa tctagaaa 1058

<210> 7

<211> 1424

<212> DNA

<213>artificial sequence

<400> 7

atgtcagaac gtttcccaaa tgacgtggat ccgatcgaaa ctcgcgactg gctccaggcg 60

gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata 120

ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 180

tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 240

agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 300

agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 360

caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 420

gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 480

agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 540

gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 600

ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 660

agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 720

gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 780

tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 840

gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 900

ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 960

tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 1020

actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1080

gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1140

tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1200

ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1260

cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1320

agagaatagg aacttcgaac tgcaggtcga cggatccccg gaatgcccgt ttcattacca 1380

tcattaacaa cacgctgtct gacattcgcc gtctggtgat gtaa 1424

<210> 8

<211> 1424

<212> DNA

<213>artificial sequence

<400> 8

atgaaacaaa cggttgcagc ttatatcgcc aaaacactcg aatcggcagg ggtgaaacgc 60

gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata 120

ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 180

tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 240

agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 300

agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 360

caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 420

gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 480

agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 540

gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 600

ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 660

agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 720

gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 780

tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 840

gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 900

ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 960

tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 1020

actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1080

gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1140

tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1200

ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1260

cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1320

agagaatagg aacttcgaac tgcaggtcga cggatccccg gaattggaag aaaaagccga 1380

tcgcaagttt ctggataaag cgctggaaga ttaccgcgac gccc 1424

<210> 9

<211> 1424

<212> DNA

<213>artificial sequence

<400> 9

atgtccgagc ttaatgaaaa gttagccaca gcctgggaag gttttaccaa aggtgactgg 60

gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata 120

ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 180

tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 240

agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 300

agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 360

caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 420

gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 480

agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 540

gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 600

ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 660

agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 720

gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 780

tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 840

gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 900

ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 960

tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 1020

actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1080

gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1140

tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1200

ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1260

cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1320

agagaatagg aacttcgaac tgcaggtcga cggatccccg gaattcgctg actaaagaac 1380

agcagcagga cgttattact cgtaccttca ctcaatctat gtaa 1424

<210> 10

<211> 1424

<212> DNA

<213>artificial sequence

<400> 10

atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60

gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata 120

ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 180

tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 240

agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 300

agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 360

caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 420

gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 480

agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 540

gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 600

ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 660

agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 720

gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 780

tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 840

gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 900

ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 960

tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 1020

actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1080

gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1140

tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1200

ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1260

cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1320

agagaatagg aacttcgaac tgcaggtcga cggatccccg gaatacgctg caaaacttaa 1380

gcaatctgga aaaaggcgaa acctgcccga acgaactggt ttaa 1424

<210> 11

<211> 1424

<212> DNA

<213>artificial sequence

<400> 11

atgattattt ccgcagccag cgattatcgc gccgcagcgc aacgcattct gccgccgttc 60

gtgtaggctg gagctgcttc gaagttccta tactttctag agaataggaa cttcggaata 120

ggaacttcaa gatcccctta ttagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc 180

tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca 240

agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc 300

agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag 360

caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg 420

gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca 480

agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat 540

gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact 600

ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc 660

agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc 720

gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg 780

tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca 840

gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc 900

ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct 960

tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt 1020

actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt 1080

gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc 1140

tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg 1200

ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg 1260

cgccctgagt gcttgcggca gcgtgagctt caaaagcgct ctgaagttcc tatactttct 1320

agagaatagg aacttcgaac tgcaggtcga cggatccccg gaatgggctg ggtaaagagt 1380

tgcctgcggc actggctccc atggcgaaag ggaatgcggc atag 1424

<210> 12

<211> 652

<212> PRT

<213>artificial sequence

<400> 12

Met Ser Gln Ile His Lys His Thr Ile Pro Ala Asn Ile Ala Asp Arg

1 5 10 15

Cys Leu Ile Asn Pro Gln Gln Tyr Glu Ala Met Tyr Gln Gln Ser Ile

20 25 30

Asn Val Pro Asp Thr Phe Trp Gly Glu Gln Gly Lys Ile Leu Asp Trp

35 40 45

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

50 55 60

Val Ser Ile Lys Trp Tyr Glu Asp Gly Thr Leu Asn Leu Ala Ala Asn

65 70 75 80

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

85 90 95

Trp Glu Gly Asp Asp Ala Ser Gln Ser Lys His Ile Ser Tyr Lys Glu

100 105 110

Leu His Arg Asp Val Cys Arg Phe Ala Asn Thr Leu Leu Glu Leu Gly

115 120 125

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

130 135 140

Ala Ala Val Ala Met Leu Ala Cys Ala Arg Ile Gly Ala Val His Ser

145 150 155 160

Val Ile Phe Gly Gly Phe Ser Pro Glu Ala Val Ala Gly Arg Ile Ile

165 170 175

Asp Ser Asn Ser Arg Leu Val Ile Thr Ser Asp Glu Gly Val Arg Ala

180 185 190

Gly Arg Ser Ile Pro Leu Lys Lys Asn Val Asp Asp Ala Leu Lys Asn

195 200 205

Pro Asn Val Thr Ser Val Glu His Val Val Val Leu Lys Arg Thr Gly

210 215 220

Gly Lys Ile Asp Trp Gln Glu Gly Arg Asp Leu Trp Trp His Asp Leu

225 230 235 240

Val Glu Gln Ala Ser Asp Gln His Gln Ala Glu Glu Met Asn Ala Glu

245 250 255

Asp Pro Leu Phe Ile Leu Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys

260 265 270

Gly Val Leu His Thr Thr Gly Gly Tyr Leu Val Tyr Ala Ala Leu Thr

275 280 285

Phe Lys Tyr Val Phe Asp Tyr His Pro Gly Asp Ile Tyr Trp Cys Thr

290 295 300

Ala Asp Val Gly Trp Val Thr Gly His Ser Tyr Leu Leu Tyr Gly Pro

305 310 315 320

Leu Ala Cys Gly Ala Thr Thr Leu Met Phe Glu Gly Val Pro Asn Trp

325 330 335

Pro Thr Pro Ala Arg Met Ala Gln Val Val Asp Lys His Gln Val Asn

340 345 350

Ile Leu Tyr Thr Ala Pro Thr Ala Ile Arg Ala Leu Met Ala Glu Gly

355 360 365

Asp Lys Ala Ile Glu Gly Thr Asp Arg Ser Ser Leu Arg Ile Leu Gly

370 375 380

Ser Val Gly Glu Pro Ile Asn Pro Glu Ala Trp Glu Trp Tyr Trp Lys

385 390 395 400

Lys Ile Gly Asn Glu Lys Cys Pro Val Val Asp Thr Trp Trp Gln Thr

405 410 415

Glu Thr Gly Gly Phe Met Ile Thr Pro Leu Pro Gly Ala Thr Glu Leu

420 425 430

Lys Ala Gly Ser Ala Thr Arg Pro Phe Phe Gly Val Gln Pro Ala Leu

435 440 445

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

450 455 460

Val Ile Thr Asp Ser Trp Pro Gly Gln Ala Arg Thr Leu Phe Gly Asp

465 470 475 480

His Glu Arg Phe Glu Gln Thr Tyr Phe Ser Thr Phe Lys Asn Met Tyr

485 490 495

Phe Ser Gly Asp Gly Ala Arg Arg Asp Glu Asp Gly Tyr Tyr Trp Ile

500 505 510

Thr Gly Arg Val Asp Asp Val Leu Asn Val Ser Gly His Arg Leu Gly

515 520 525

Thr Ala Glu Ile Glu Ser Ala Leu Val Ala His Pro Lys Ile Ala Glu

530 535 540

Ala Ala Val Val Gly Ile Pro His Asn Ile Lys Gly Gln Ala Ile Tyr

545 550 555 560

Ala Tyr Val Thr Leu Asn His Gly Glu Glu Pro Ser Pro Glu Leu Tyr

565 570 575

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

580 585 590

Pro Asp Val Leu His Trp Thr Asp Ser Leu Pro Lys Thr Arg Ser Gly

595 600 605

Lys Ile Met Arg Arg Ile Leu Arg Lys Ile Ala Ala Gly Asp Thr Ser

610 615 620

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

625 630 635 640

Leu Leu Glu Glu Lys Gln Ala Ile Ala Met Pro Ser

645 650

<210> 13

<211> 400

<212> PRT

<213>artificial sequence

<400> 13

Met Ser Ser Lys Leu Val Leu Val Leu Asn Cys Gly Ser Ser Ser Leu

1 5 10 15

Lys Phe Ala Ile Ile Asp Ala Val Asn Gly Glu Glu Tyr Leu Ser Gly

20 25 30

Leu Ala Glu Cys Phe His Leu Pro Glu Ala Arg Ile Lys Trp Lys Met

35 40 45

Asp Gly Asn Lys Gln Glu Ala Ala Leu Gly Ala Gly Ala Ala His Ser

50 55 60

Glu Ala Leu Asn Phe Ile Val Asn Thr Ile Leu Ala Gln Lys Pro Glu

65 70 75 80

Leu Ser Ala Gln Leu Thr Ala Ile Gly His Arg Ile Val His Gly Gly

85 90 95

Glu Lys Tyr Thr Ser Ser Val Val Ile Asp Glu Ser Val Ile Gln Gly

100 105 110

Ile Lys Asp Ala Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu

115 120 125

Ile Gly Ile Glu Glu Ala Leu Lys Ser Phe Pro Gln Leu Lys Asp Lys

130 135 140

Asn Val Ala Val Phe Asp Thr Ala Phe His Gln Thr Met Pro Glu Glu

145 150 155 160

Ser Tyr Leu Tyr Ala Leu Pro Tyr Asn Leu Tyr Lys Glu His Gly Ile

165 170 175

Arg Arg Tyr Gly Ala His Gly Thr Ser His Phe Tyr Val Thr Gln Glu

180 185 190

Ala Ala Lys Met Leu Asn Lys Pro Val Glu Glu Leu Asn Ile Ile Thr

195 200 205

Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala Ile Arg Asn Gly Lys

210 215 220

Cys Val Asp Thr Ser Met Gly Leu Thr Pro Leu Glu Gly Leu Val Met

225 230 235 240

Gly Thr Arg Ser Gly Asp Ile Asp Pro Ala Ile Ile Phe His Leu His

245 250 255

Asp Thr Leu Gly Met Ser Val Asp Ala Ile Asn Lys Leu Leu Thr Lys

260 265 270

Glu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr

275 280 285

Val Glu Asp Asn Tyr Ala Thr Lys Glu Asp Ala Lys Arg Ala Met Asp

290 295 300

Val Tyr Cys His Arg Leu Ala Lys Tyr Ile Gly Ala Tyr Thr Ala Leu

305 310 315 320

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

325 330 335

Asn Ala Ala Met Val Arg Glu Leu Ser Leu Gly Lys Leu Gly Val Leu

340 345 350

Gly Phe Glu Val Asp His Glu Arg Asn Leu Ala Ala Arg Phe Gly Lys

355 360 365

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

370 375 380

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

385 390 395 400

<210> 14

<211> 714

<212> PRT

<213>artificial sequence

<400> 14

Val Ser Arg Ile Ile Met Leu Ile Pro Thr Gly Thr Ser Val Gly Leu

1 5 10 15

Thr Ser Val Ser Leu Gly Val Ile Arg Ala Met Glu Arg Lys Gly Val

20 25 30

Arg Leu Ser Val Phe Lys Pro Ile Ala Gln Pro Arg Thr Gly Gly Asp

35 40 45

Ala Pro Asp Gln Thr Thr Thr Ile Val Arg Ala Asn Ser Ser Thr Thr

50 55 60

Thr Ala Ala Glu Pro Leu Lys Met Ser Tyr Val Glu Gly Leu Leu Ser

65 70 75 80

Ser Asn Gln Lys Asp Val Leu Met Glu Glu Ile Val Ala Asn Tyr His

85 90 95

Ala Asn Thr Lys Asp Ala Glu Val Val Leu Val Glu Gly Leu Val Pro

100 105 110

Thr Arg Lys His Gln Phe Ala Gln Ser Leu Asn Tyr Glu Ile Ala Lys

115 120 125

Thr Leu Asn Ala Glu Ile Val Phe Val Met Ser Gln Gly Thr Asp Thr

130 135 140

Pro Glu Gln Leu Lys Glu Arg Ile Glu Leu Thr Arg Asn Ser Phe Gly

145 150 155 160

Gly Ala Lys Asn Thr Asn Ile Thr Gly Val Ile Val Asn Lys Leu Asn

165 170 175

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

180 185 190

Phe Asp Asp Ser Ser Lys Ala Lys Val Asn Asn Val Asp Pro Ala Lys

195 200 205

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

210 215 220

Phe Asp Leu Ile Ala Thr Arg Ala Ile Asp Met Ala Arg His Leu Asn

225 230 235 240

Ala Thr Ile Ile Asn Glu Gly Asp Ile Asn Thr Arg Arg Val Lys Ser

245 250 255

Val Thr Phe Cys Ala Arg Ser Ile Pro His Met Leu Glu His Phe Arg

260 265 270

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

275 280 285

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

290 295 300

Leu Thr Gly Gly Tyr Glu Met Asp Ala Arg Ile Ser Lys Leu Cys Glu

305 310 315 320

Arg Ala Phe Ala Thr Gly Leu Pro Val Phe Met Val Asn Thr Asn Thr

325 330 335

Trp Gln Thr Ser Leu Ser Leu Gln Ser Phe Asn Leu Glu Val Pro Val

340 345 350

Asp Asp His Glu Arg Ile Glu Lys Val Gln Glu Tyr Val Ala Asn Tyr

355 360 365

Ile Asn Ala Asp Trp Ile Glu Ser Leu Thr Ala Thr Ser Glu Arg Ser

370 375 380

Arg Arg Leu Ser Pro Pro Ala Phe Arg Tyr Gln Leu Thr Glu Leu Ala

385 390 395 400

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

405 410 415

Thr Val Lys Ala Ala Ala Ile Cys Ala Glu Arg Gly Ile Ala Thr Cys

420 425 430

Val Leu Leu Gly Asn Pro Ala Glu Ile Asn Arg Val Ala Ala Ser Gln

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Gly Thr Leu Met Leu Glu Gln Asp Glu Val Asp Gly Leu Val Ser Gly

500 505 510

Ala Val His Thr Thr Ala Asn Thr Ile Arg Pro Pro Leu Gln Leu Ile

515 520 525

Lys Thr Ala Pro Gly Ser Ser Leu Val Ser Ser Val Phe Phe Met Leu

530 535 540

Leu Pro Glu Gln Val Tyr Val Tyr Gly Asp Cys Ala Ile Asn Pro Asp

545 550 555 560

Pro Thr Ala Glu Gln Leu Ala Glu Ile Ala Ile Gln Ser Ala Asp Ser

565 570 575

Ala Ala Ala Phe Gly Ile Glu Pro Arg Val Ala Met Leu Ser Tyr Ser

580 585 590

Thr Gly Thr Ser Gly Ala Gly Ser Asp Val Glu Lys Val Arg Glu Ala

595 600 605

Thr Arg Leu Ala Gln Glu Lys Arg Pro Asp Leu Met Ile Asp Gly Pro

610 615 620

Leu Gln Tyr Asp Ala Ala Val Met Ala Asp Val Ala Lys Ser Lys Ala

625 630 635 640

Pro Asn Ser Pro Val Ala Gly Arg Ala Thr Val Phe Ile Phe Pro Asp

645 650 655

Leu Asn Thr Gly Asn Thr Thr Tyr Lys Ala Val Gln Arg Ser Ala Asp

660 665 670

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

675 680 685

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

690 695 700

Leu Thr Ala Ile Gln Ser Ala Gln Gln Gln

705 710

<210> 15

<211> 434

<212> PRT

<213>artificial sequence

<400> 15

Met Lys Thr Arg Thr Gln Gln Ile Glu Glu Leu Gln Lys Glu Trp Thr

1 5 10 15

Gln Pro Arg Trp Glu Gly Ile Thr Arg Pro Tyr Ser Ala Glu Asp Val

20 25 30

Val Lys Leu Arg Gly Ser Val Asn Pro Glu Cys Thr Leu Ala Gln Leu

35 40 45

Gly Ala Ala Lys Met Trp Arg Leu Leu His Gly Glu Ser Lys Lys Gly

50 55 60

Tyr Ile Asn Ser Leu Gly Ala Leu Thr Gly Gly Gln Ala Leu Gln Gln

65 70 75 80

Ala Lys Ala Gly Ile Glu Ala Val Tyr Leu Ser Gly Trp Gln Val Ala

85 90 95

Ala Asp Ala Asn Leu Ala Ala Ser Met Tyr Pro Asp Gln Ser Leu Tyr

100 105 110

Pro Ala Asn Ser Val Pro Ala Val Val Glu Arg Ile Asn Asn Thr Phe

115 120 125

Arg Arg Ala Asp Gln Ile Gln Trp Ser Ala Gly Ile Glu Pro Gly Asp

130 135 140

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

145 150 155 160

Gly Phe Gly Gly Val Leu Asn Ala Phe Glu Leu Met Lys Ala Met Ile

165 170 175

Glu Ala Gly Ala Ala Ala Val His Phe Glu Asp Gln Leu Ala Ser Val

180 185 190

Lys Lys Cys Gly His Met Gly Gly Lys Val Leu Val Pro Thr Gln Glu

195 200 205

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

210 215 220

Val Pro Thr Leu Leu Val Ala Arg Thr Asp Ala Asp Ala Ala Asp Leu

225 230 235 240

Ile Thr Ser Asp Cys Asp Pro Tyr Asp Ser Glu Phe Ile Thr Gly Glu

245 250 255

Arg Thr Ser Glu Gly Phe Phe Arg Thr His Ala Gly Ile Glu Gln Ala

260 265 270

Ile Ser Arg Gly Leu Ala Tyr Ala Pro Tyr Ala Asp Leu Val Trp Cys

275 280 285

Glu Thr Ser Thr Pro Asp Leu Glu Leu Ala Arg Arg Phe Ala Gln Ala

290 295 300

Ile His Ala Lys Tyr Pro Gly Lys Leu Leu Ala Tyr Asn Cys Ser Pro

305 310 315 320

Ser Phe Asn Trp Gln Lys Asn Leu Asp Asp Lys Thr Ile Ala Ser Phe

325 330 335

Gln Gln Gln Leu Ser Asp Met Gly Tyr Lys Phe Gln Phe Ile Thr Leu

340 345 350

Ala Gly Ile His Ser Met Trp Phe Asn Met Phe Asp Leu Ala Asn Ala

355 360 365

Tyr Ala Gln Gly Glu Gly Met Lys His Tyr Val Glu Lys Val Gln Gln

370 375 380

Pro Glu Phe Ala Ala Ala Lys Asp Gly Tyr Thr Phe Val Ser His Gln

385 390 395 400

Gln Glu Val Gly Thr Gly Tyr Phe Asp Lys Val Thr Thr Ile Ile Gln

405 410 415

Gly Gly Thr Ser Ser Val Thr Ala Leu Thr Gly Ser Thr Glu Glu Ser

420 425 430

Gln Phe

<210> 16

<211> 578

<212> PRT

<213>artificial sequence

<400> 16

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

1 5 10 15

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

20 25 30

Gln Arg Phe Glu Gln Ala Asp Trp His Ala Val Gln Gln Ala Met Lys

35 40 45

Asn Arg Ile His Leu Tyr Asp His His Val Gly Leu Val Val Glu Gln

50 55 60

Leu Arg Cys Ile Thr Asn Gly Gln Ser Thr Asp Ala Ala Phe Leu Leu

65 70 75 80

Arg Val Lys Glu His Tyr Thr Arg Leu Leu Pro Asp Tyr Pro Arg Phe

85 90 95

Glu Ile Ala Glu Ser Phe Phe Asn Ser Val Tyr Cys Arg Leu Phe Asp

100 105 110

His Arg Ser Leu Thr Pro Glu Arg Leu Phe Ile Phe Ser Ser Gln Pro

115 120 125

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

130 135 140

Pro Asp His Gly Trp Glu Ser Leu Leu Met Arg Val Ile Ser Asp Leu

145 150 155 160

Pro Leu Arg Leu Arg Trp Gln Asn Lys Ser Arg Asp Ile His Tyr Ile

165 170 175

Ile Arg His Leu Thr Glu Thr Leu Gly Thr Asp Asn Leu Ala Glu Ser

180 185 190

His Leu Gln Val Ala Asn Glu Leu Phe Tyr Arg Asn Lys Ala Ala Trp

195 200 205

Leu Val Gly Lys Leu Ile Thr Pro Ser Gly Thr Leu Pro Phe Leu Leu

210 215 220

Pro Ile His Gln Thr Asp Asp Gly Glu Leu Phe Ile Asp Thr Cys Leu

225 230 235 240

Thr Thr Thr Ala Glu Ala Ser Ile Val Phe Gly Phe Ala Arg Ser Tyr

245 250 255

Phe Met Val Tyr Ala Pro Leu Pro Ala Ala Leu Val Glu Trp Leu Arg

260 265 270

Glu Ile Leu Pro Gly Lys Thr Thr Ala Glu Leu Tyr Met Ala Ile Gly

275 280 285

Cys Gln Lys His Ala Lys Thr Glu Ser Tyr Arg Glu Tyr Leu Val Tyr

290 295 300

Leu Gln Gly Cys Asn Glu Gln Phe Ile Glu Ala Pro Gly Ile Arg Gly

305 310 315 320

Met Val Met Leu Val Phe Thr Leu Pro Gly Phe Asp Arg Val Phe Lys

325 330 335

Val Ile Lys Asp Arg Phe Ala Pro Gln Lys Glu Met Ser Ala Ala His

340 345 350

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

355 360 365

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

370 375 380

Ile Ser Pro Ala Leu Met Glu Leu Leu Leu Gln Glu Ala Ala Glu Lys

385 390 395 400

Ile Thr Asp Leu Gly Glu Gln Ile Val Ile Arg His Leu Tyr Ile Glu

405 410 415

Arg Arg Met Val Pro Leu Asn Ile Trp Leu Glu Gln Val Glu Gly Gln

420 425 430

Gln Leu Arg Asp Ala Ile Glu Glu Tyr Gly Asn Ala Ile Arg Gln Leu

435 440 445

Ala Ala Ala Asn Ile Phe Pro Gly Asp Met Leu Phe Lys Asn Phe Gly

450 455 460

Val Thr Arg His Gly Arg Val Val Phe Tyr Asp Tyr Asp Glu Ile Cys

465 470 475 480

Tyr Met Thr Glu Val Asn Phe Arg Asp Ile Pro Pro Pro Arg Tyr Pro

485 490 495

Glu Asp Glu Leu Ala Ser Glu Pro Trp Tyr Ser Val Ser Pro Gly Asp

500 505 510

Val Phe Pro Glu Glu Phe Arg His Trp Leu Cys Ala Asp Pro Arg Ile

515 520 525

Gly Pro Leu Phe Glu Glu Met His Ala Asp Leu Phe Arg Ala Asp Tyr

530 535 540

Trp Arg Ala Leu Gln Asn Arg Ile Arg Glu Gly His Val Glu Asp Val

545 550 555 560

Tyr Ala Tyr Arg Arg Arg Gln Arg Phe Ser Val Arg Tyr Gly Glu Met

565 570 575

Leu Phe

<210> 17

<211> 533

<212> PRT

<213>artificial sequence

<400> 17

Met Thr Glu Gln Ala Thr Thr Thr Asp Glu Leu Ala Phe Thr Arg Pro

1 5 10 15

Tyr Gly Glu Gln Glu Lys Gln Ile Leu Thr Ala Glu Ala Val Glu Phe

20 25 30

Leu Thr Glu Leu Val Thr His Phe Thr Pro Gln Arg Asn Lys Leu Leu

35 40 45

Ala Ala Arg Ile Gln Gln Gln Gln Asp Ile Asp Asn Gly Thr Leu Pro

50 55 60

Asp Phe Ile Ser Glu Thr Ala Ser Ile Arg Asp Ala Asp Trp Lys Ile

65 70 75 80

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

85 90 95

Pro Val Glu Arg Lys Met Val Ile Asn Ala Leu Asn Ala Asn Val Lys

100 105 110

Val Phe Met Ala Asp Phe Glu Asp Ser Leu Ala Pro Asp Trp Asn Lys

115 120 125

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

130 135 140

Ser Tyr Thr Asn Glu Ala Gly Lys Ile Tyr Gln Leu Lys Pro Asn Pro

145 150 155 160

Ala Val Leu Ile Cys Arg Val Arg Gly Leu His Leu Pro Glu Lys His

165 170 175

Val Thr Trp Arg Gly Glu Ala Ile Pro Gly Ser Leu Phe Asp Phe Ala

180 185 190

Leu Tyr Phe Phe His Asn Tyr Gln Ala Leu Leu Ala Lys Gly Ser Gly

195 200 205

Pro Tyr Phe Tyr Leu Pro Lys Thr Gln Ser Trp Gln Glu Ala Ala Trp

210 215 220

Trp Ser Glu Val Phe Ser Tyr Ala Glu Asp Arg Phe Asn Leu Pro Arg

225 230 235 240

Gly Thr Ile Lys Ala Thr Leu Leu Ile Glu Thr Leu Pro Ala Val Phe

245 250 255

Gln Met Asp Glu Ile Leu His Ala Leu Arg Asp His Ile Val Gly Leu

260 265 270

Asn Cys Gly Arg Trp Asp Tyr Ile Phe Ser Tyr Ile Lys Thr Leu Lys

275 280 285

Asn Tyr Pro Asp Arg Val Leu Pro Asp Arg Gln Ala Val Thr Met Asp

290 295 300

Lys Pro Phe Leu Asn Ala Tyr Ser Arg Leu Leu Ile Lys Thr Cys His

305 310 315 320

Lys Arg Gly Ala Phe Ala Met Gly Gly Met Ala Ala Phe Ile Pro Ser

325 330 335

Lys Asp Glu Glu His Asn Asn Gln Val Leu Asn Lys Val Lys Ala Asp

340 345 350

Lys Ser Leu Glu Ala Asn Asn Gly His Asp Gly Thr Trp Ile Ala His

355 360 365

Pro Gly Leu Ala Asp Thr Ala Met Ala Val Phe Asn Asp Ile Leu Gly

370 375 380

Ser Arg Lys Asn Gln Leu Glu Val Met Arg Glu Gln Asp Ala Pro Ile

385 390 395 400

Thr Ala Asp Gln Leu Leu Ala Pro Cys Asp Gly Glu Arg Thr Glu Glu

405 410 415

Gly Met Arg Ala Asn Ile Arg Val Ala Val Gln Tyr Ile Glu Ala Trp

420 425 430

Ile Ser Gly Asn Gly Cys Val Pro Ile Tyr Gly Leu Met Glu Asp Ala

435 440 445

Ala Thr Ala Glu Ile Ser Arg Thr Ser Ile Trp Gln Trp Ile His His

450 455 460

Gln Lys Thr Leu Ser Asn Gly Lys Pro Val Thr Lys Ala Leu Phe Arg

465 470 475 480

Gln Met Leu Gly Glu Glu Met Lys Val Ile Ala Ser Glu Leu Gly Glu

485 490 495

Glu Arg Phe Ser Gln Gly Arg Phe Asp Asp Ala Ala Arg Leu Met Glu

500 505 510

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

515 520 525

Tyr Arg Leu Leu Ala

530

<210> 18

<211> 565

<212> PRT

<213>artificial sequence

<400> 18

Met Glu Pro Lys Thr Lys Lys Gln Arg Ser Leu Tyr Ile Pro Tyr Ala

1 5 10 15

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

20 25 30

Ser Met Glu Glu Arg Arg Asn Phe Asn Leu Leu Gly Leu Leu Pro Glu

35 40 45

Val Val Glu Thr Ile Glu Glu Gln Ala Glu Arg Ala Trp Ile Gln Tyr

50 55 60

Gln Gly Phe Lys Thr Glu Ile Asp Lys His Ile Tyr Leu Arg Asn Ile

65 70 75 80

Gln Asp Thr Asn Glu Thr Leu Phe Tyr Arg Leu Val Asn Asn His Leu

85 90 95

Asp Glu Met Met Pro Val Ile Tyr Thr Pro Thr Val Gly Ala Ala Cys

100 105 110

Glu Arg Phe Ser Glu Ile Tyr Arg Arg Ser Arg Gly Val Phe Ile Ser

115 120 125

Tyr Gln Asn Arg His Asn Met Asp Asp Ile Leu Gln Asn Val Pro Asn

130 135 140

His Asn Ile Lys Val Ile Val Val Thr Asp Gly Glu Arg Ile Leu Gly

145 150 155 160

Leu Gly Asp Gln Gly Ile Gly Gly Met Gly Ile Pro Ile Gly Lys Leu

165 170 175

Ser Leu Tyr Thr Ala Cys Gly Gly Ile Ser Pro Ala Tyr Thr Leu Pro

180 185 190

Val Val Leu Asp Val Gly Thr Asn Asn Gln Gln Leu Leu Asn Asp Pro

195 200 205

Leu Tyr Met Gly Trp Arg Asn Pro Arg Ile Thr Asp Asp Glu Tyr Tyr

210 215 220

Glu Phe Val Asp Glu Phe Ile Gln Ala Val Lys Gln Arg Trp Pro Asp

225 230 235 240

Val Leu Leu Gln Phe Glu Asp Phe Ala Gln Lys Asn Ala Met Pro Leu

245 250 255

Leu Asn Arg Tyr Arg Asn Glu Ile Cys Ser Phe Asn Asp Asp Ile Gln

260 265 270

Gly Thr Ala Ala Val Thr Val Gly Thr Leu Ile Ala Ala Ser Arg Ala

275 280 285

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

290 295 300

Ser Ala Gly Cys Gly Ile Ala Glu Met Ile Ile Ser Gln Thr Gln Arg

305 310 315 320

Glu Gly Leu Ser Glu Glu Ala Ala Arg Gln Lys Val Phe Met Val Asp

325 330 335

Arg Phe Gly Leu Leu Thr Asp Lys Met Pro Asn Leu Leu Pro Phe Gln

340 345 350

Thr Lys Leu Val Gln Lys Arg Glu Asn Leu Ser Asp Trp Asp Thr Asp

355 360 365

Ser Asp Val Leu Ser Leu Leu Asp Val Val Arg Asn Val Lys Pro Asp

370 375 380

Ile Leu Ile Gly Val Ser Gly Gln Thr Gly Leu Phe Thr Glu Glu Ile

385 390 395 400

Ile Arg Glu Met His Lys His Cys Pro Arg Pro Ile Val Met Pro Leu

405 410 415

Ser Asn Pro Thr Ser Arg Val Glu Ala Thr Pro Gln Asp Ile Ile Ala

420 425 430

Trp Thr Glu Gly Asn Ala Leu Val Ala Thr Gly Ser Pro Phe Asn Pro

435 440 445

Val Val Trp Lys Asp Lys Ile Tyr Pro Ile Ala Gln Cys Asn Asn Ala

450 455 460

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

465 470 475 480

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

485 490 495

Tyr Ser Pro Leu Val Leu Asn Gly Glu Gly Met Val Leu Pro Glu Leu

500 505 510

Lys Asp Ile Gln Lys Val Ser Arg Ala Ile Ala Phe Ala Val Gly Lys

515 520 525

Met Ala Gln Gln Gln Gly Val Ala Val Lys Thr Ser Ala Glu Ala Leu

530 535 540

Gln Gln Ala Ile Asp Asp Asn Phe Trp Gln Ala Glu Tyr Arg Asp Tyr

545 550 555 560

Arg Arg Thr Ser Ile

565

<210> 19

<211> 759

<212> PRT

<213>artificial sequence

<400> 19

Met Asp Asp Gln Leu Lys Gln Ser Ala Leu Asp Phe His Glu Phe Pro

1 5 10 15

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

20 25 30

Arg Asp Leu Ala Leu Ala Tyr Ser Pro Gly Val Ala Ala Pro Cys Leu

35 40 45

Glu Ile Glu Lys Asp Pro Leu Lys Ala Tyr Lys Tyr Thr Ala Arg Gly

50 55 60

Asn Leu Val Ala Val Ile Ser Asn Gly Thr Ala Val Leu Gly Leu Gly

65 70 75 80

Asn Ile Gly Ala Leu Ala Gly Lys Pro Val Met Glu Gly Lys Gly Val

85 90 95

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

100 105 110

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

115 120 125

Thr Phe Gly Gly Ile Asn Leu Glu Asp Ile Lys Ala Pro Glu Cys Phe

130 135 140

Tyr Ile Glu Gln Lys Leu Arg Glu Arg Met Asn Ile Pro Val Phe His

145 150 155 160

Asp Asp Gln His Gly Thr Ala Ile Ile Ser Thr Ala Ala Ile Leu Asn

165 170 175

Gly Leu Arg Val Val Glu Lys Asn Ile Ser Asp Val Arg Met Val Val

180 185 190

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

195 200 205

Leu Gly Leu Gln Lys His Asn Ile Val Val Cys Asp Ser Lys Gly Val

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Met Val Lys Lys Met Ala Arg Ala Pro Met Ile Leu Ala Leu Ala Asn

275 280 285

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

290 295 300

Ala Ile Ile Cys Thr Gly Arg Ser Asp Tyr Pro Asn Gln Val Asn Asn

305 310 315 320

Val Leu Cys Phe Pro Phe Ile Phe Arg Gly Ala Leu Asp Val Gly Ala

325 330 335

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

340 345 350

Glu Leu Ala His Ala Glu Gln Ser Glu Val Val Ala Ser Ala Tyr Gly

355 360 365

Asp Gln Asp Leu Ser Phe Gly Pro Glu Tyr Ile Ile Pro Lys Pro Phe

370 375 380

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

385 390 395 400

Met Glu Ser Gly Val Ala Thr Arg Pro Ile Ala Asp Phe Asp Val Tyr

405 410 415

Ile Asp Lys Leu Thr Glu Phe Val Tyr Lys Thr Asn Leu Phe Met Lys

420 425 430

Pro Ile Phe Ser Gln Ala Arg Lys Ala Pro Lys Arg Val Val Leu Pro

435 440 445

Glu Gly Glu Glu Ala Arg Val Leu His Ala Thr Gln Glu Leu Val Thr

450 455 460

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

465 470 475 480

Met Arg Ile Gln Lys Leu Gly Leu Gln Ile Lys Ala Gly Val Asp Phe

485 490 495

Glu Ile Val Asn Asn Glu Ser Asp Pro Arg Phe Lys Glu Tyr Trp Thr

500 505 510

Glu Tyr Phe Gln Ile Met Lys Arg Arg Gly Val Thr Gln Glu Gln Ala

515 520 525

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

530 535 540

Gln Arg Gly Glu Ala Asp Ala Met Ile Cys Gly Thr Val Gly Asp Tyr

545 550 555 560

His Glu His Phe Ser Val Val Lys Asn Val Phe Gly Tyr Arg Asp Gly

565 570 575

Val His Thr Ala Gly Ala Met Asn Ala Leu Leu Leu Pro Ser Gly Asn

580 585 590

Thr Phe Ile Ala Asp Thr Tyr Val Asn Asp Glu Pro Asp Ala Glu Glu

595 600 605

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

610 615 620

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

625 630 635 640

Asp Cys Pro Ser Ser Ser Lys Met Arg Gln Ala Leu Glu Leu Val Arg

645 650 655

Glu Arg Ala Pro Glu Leu Met Ile Asp Gly Glu Met His Gly Asp Ala

660 665 670

Ala Leu Val Glu Ala Ile Arg Asn Asp Arg Met Pro Asp Ser Ser Leu

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Ile Ala Ser Val Arg Arg Ile Val Asn Met Val Ala Leu Ala Val Val

740 745 750

Glu Ala Gln Thr Gln Pro Leu

755

<210> 20

<211> 326

<212> PRT

<213>artificial sequence

<400> 20

Met Ala Ser Ile Thr Asp Lys Asp His Gln Lys Val Ile Leu Val Gly

1 5 10 15

Asp Gly Ala Val Gly Ser Ser Tyr Ala Tyr Ala Met Val Leu Gln Gly

20 25 30

Ile Ala Gln Glu Ile Gly Ile Val Asp Ile Phe Lys Asp Lys Thr Lys

35 40 45

Gly Asp Ala Ile Asp Leu Ser Asn Ala Leu Pro Phe Thr Ser Pro Lys

50 55 60

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

65 70 75 80

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

85 90 95

Leu Val Asn Lys Asn Leu Lys Ile Leu Lys Ser Ile Val Asp Pro Ile

100 105 110

Val Asp Ser Gly Phe Asn Gly Ile Phe Leu Val Ala Ala Asn Pro Val

115 120 125

Asp Ile Leu Thr Tyr Ala Thr Trp Lys Leu Ser Gly Phe Pro Lys Ser

130 135 140

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

145 150 155 160

Ser Ile Ala Glu Met Val Asn Val Asp Ala Arg Ser Val His Ala Tyr

165 170 175

Ile Met Gly Glu His Gly Asp Thr Glu Phe Pro Val Trp Ser His Ala

180 185 190

Asn Ile Gly Gly Val Thr Ile Ala Glu Trp Val Lys Ala His Pro Glu

195 200 205

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

210 215 220

Ala Tyr Glu Ile Ile Lys Leu Lys Gly Ala Thr Phe Tyr Gly Ile Ala

225 230 235 240

Thr Ala Leu Ala Arg Ile Ser Lys Ala Ile Leu Asn Asp Glu Asn Ala

245 250 255

Val Leu Pro Leu Ser Val Tyr Met Asp Gly Gln Tyr Gly Leu Asn Asp

260 265 270

Ile Tyr Ile Gly Thr Pro Ala Val Ile Asn Arg Asn Gly Ile Gln Asn

275 280 285

Ile Leu Glu Ile Pro Leu Thr Asp His Glu Glu Glu Ser Met Gln Lys

290 295 300

Ser Ala Ser Gln Leu Lys Lys Val Leu Thr Asp Ala Phe Ala Lys Asn

305 310 315 320

Asp Ile Glu Thr Arg Gln

325

Claims (10)

1. the construction method of recombinant bacterium, the transformation including carrying out following A 1-A14 to recipient bacterium, obtain the recombinant bacterium；

A1, the poxB gene for knocking out the recipient bacterium or the expression for inhibiting the poxB gene inhibit the poxB gene coding Protein activity；

A2, the pflB gene for knocking out the recipient bacterium or the expression for inhibiting the pflB gene inhibit the pflB gene coding Protein activity；

A3, the aceEF gene for knocking out the recipient bacterium or the expression for inhibiting the aceEF gene inhibit the aceEF gene The activity of the protein of coding；

A4, the ldhA gene for knocking out the recipient bacterium or the expression for inhibiting the ldhA gene inhibit the ldhA gene coding Protein activity；

A5, the lldD gene for knocking out the recipient bacterium or the expression for inhibiting the lldD gene inhibit the lldD gene coding Protein activity；

A6, the work for increasing the content of acs DNA encoding the protein or the enhancing acs DNA encoding the protein in the recipient bacterium Property；

A7, the work for increasing the content of pta DNA encoding the protein or the enhancing pta DNA encoding the protein in the recipient bacterium Property；

A8, increase the content of ackA DNA encoding the protein in the recipient bacterium or enhance the ackA DNA encoding the protein Activity；

A9, increase the content of aceA DNA encoding the protein in the recipient bacterium or enhance the aceA DNA encoding the protein Activity；

A10, increase the content of aceK DNA encoding the protein or the enhancing aceK DNA encoding the protein in the recipient bacterium Activity；

A11, increase the content of aceB DNA encoding the protein or the enhancing aceB DNA encoding the protein in the recipient bacterium Activity；

A12, increase the content of maeA DNA encoding the protein or the enhancing maeA DNA encoding the protein in the recipient bacterium Activity；

A13, increase the content of maeB DNA encoding the protein or the enhancing maeB DNA encoding the protein in the recipient bacterium Activity；

A14, increase the content of ldh2 DNA encoding the protein or the enhancing ldh2 DNA encoding the protein in the recipient bacterium Activity；

The recipient bacterium is contains the poxB gene, the pflB gene, the aceEF gene, the ldhA gene and institute State the bacterium or fungi of lldD gene.

2. according to the method described in claim 1, it is characterized by: 1) or 2) recipient bacterium is:

1) Escherichia coli；

2) Escherichia coli MG1655.

3. method according to claim 1 or 2, it is characterised in that:

Protein of the acs DNA encoding the protein for following a1) or a2):

A1) protein shown in sequence 12 in sequence table；

A2) by the amino acid sequence of sequence 12 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the ackA DNA encoding the protein for following b1) or b2):

B1) protein shown in sequence 13 in sequence table；

B2) by the amino acid sequence of sequence 13 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the pta DNA encoding the protein for following c1) or c2):

C1) protein shown in sequence 14 in sequence table；

C2) by the amino acid sequence of sequence 14 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the aceA DNA encoding the protein for following d1) or d2):

D1) protein shown in sequence 15 in sequence table；

D2) by the amino acid sequence of sequence 15 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the aceK DNA encoding the protein for following e1) or e2):

E1) protein shown in sequence 16 in sequence table；

E2) by the amino acid sequence of sequence 16 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the aceB DNA encoding the protein for following f1) or f2):

F1) protein shown in sequence 17 in sequence table；

F2) by the amino acid sequence of sequence 17 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the maeA DNA encoding the protein for following g1) or g2):

G1) protein shown in sequence 18 in sequence table；

G2) by the amino acid sequence of sequence 18 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the maeB DNA encoding the protein for following h1) or h2):

H1) protein shown in sequence 19 in sequence table；

H2) by the amino acid sequence of sequence 19 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

And/or protein of the ldh2 DNA encoding the protein for following i1) or i2):

I1) protein shown in sequence 20 in sequence table；

I2) by the amino acid sequence of sequence 20 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function.

4. method according to claim 1 to 3, it is characterised in that:

A1 is realized by importing the DNA fragmentation containing the poxB gene upstream and downstream homology arm into the recipient bacterium；

And/or A2 is realized by importing the DNA fragmentation containing the pflB gene upstream and downstream homology arm into the recipient bacterium；

And/or A3 is realized by importing the DNA fragmentation containing the aceEF gene upstream and downstream homology arm into the recipient bacterium；

And/or A4 is realized by importing the DNA fragmentation containing the ldhA gene upstream and downstream homology arm into the recipient bacterium；

And/or A5 is realized by importing the DNA fragmentation containing the lldD gene upstream and downstream homology arm into the recipient bacterium；

And/or A6 is realized by importing the acs expression casette containing the acs gene into the recipient bacterium；

And/or A7 is realized by importing the pta expression casette containing the pta gene into the recipient bacterium；

And/or A8 is realized by importing the ackA expression casette containing the ackA gene into the recipient bacterium；

And/or A9 is realized by importing the aceA expression casette containing the aceA gene into the recipient bacterium；

And/or A10 is realized by importing the aceK expression casette containing the aceK gene into the recipient bacterium；

And/or A11 is realized by importing the aceB expression casette containing the aceB gene into the recipient bacterium；

And/or A12 is realized by importing the maeA expression casette containing the maeA gene into the recipient bacterium；

And/or A13 is realized by importing the maeB expression casette containing the maeB gene into the recipient bacterium；

And/or A14 is realized by importing the ldh2 expression casette containing the ldh2 gene into the recipient bacterium.

5. according to the method described in claim 4, it is characterized by:

The DNA fragmentation containing the poxB gene upstream and downstream homology arm is DNA molecular shown in sequence 8 in sequence table；

The DNA fragmentation containing the pflB gene upstream and downstream homology arm is DNA molecular shown in sequence 9 in sequence table；

The DNA fragmentation containing the aceEF gene upstream and downstream homology arm is DNA molecular shown in sequence 7 in sequence table；

The DNA fragmentation containing the ldhA gene upstream and downstream homology arm is DNA molecular shown in sequence 10 in sequence table；

The DNA fragmentation containing the lldD gene upstream and downstream homology arm is DNA molecular shown in sequence 11 in sequence table；

And/or the acs expression casette, the pta expression casette, the ackA expression casette, the aceA gene Expression cassette, the aceK expression casette, the aceB expression casette, the maeA expression casette, the maeB gene Promoter is following j1 in expression cassette and the ldh2 expression casette) or j2):

J1) DNA molecular shown in 9-51 of sequence 1 in sequence table；

J2 the nucleotide sequence) and j1) limited has 75% or 75% or more identity, and the DNA with promoter function points Son；

And/or acs gene described in the acs expression casette is following k1) or k2):

K1) DNA molecular shown in 70-2028 of sequence 1 in sequence table；

K2 the nucleotide sequence) and k1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or pta gene described in the pta expression casette is following l1) or l2):

L1) DNA molecular shown in 1347-3491 of sequence 2 in sequence table；

L2 the nucleotide sequence) and l1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or ackA gene described in the ackA expression casette is following m1) or m2):

M1) DNA molecular shown in 70-1272 of sequence 2 in sequence table；

M2 the nucleotide sequence) and m1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or aceA gene described in the aceA expression casette is following n1) or n2):

N1) DNA molecular shown in 70-1374 of sequence 3 in sequence table；

N2 the nucleotide sequence) and n1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or aceK gene described in the aceK expression casette is following o1) or o2):

O1) DNA molecular shown in 1436-3172 of sequence 3 in sequence table；

O2 the nucleotide sequence) and o1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or aceB gene described in the aceB expression casette is following p1) or p2):

P1) DNA molecular shown in 70-1671 of sequence 4 in sequence table；

P2 the nucleotide sequence) and p1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or maeA gene described in the maeA expression casette is following q1) or q2):

Q1) DNA molecular shown in 70-1767 of sequence 5 in sequence table；

Q2 the nucleotide sequence) and q1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or maeB gene described in the maeB expression casette is following r1) or r2):

R1) DNA molecular shown in 1791-4070 of sequence 5 in sequence table；

R2 the nucleotide sequence) and r1) limited has 75% or 75% or more identity, and DNA molecular with the same function；

And/or ldh2 gene described in the ldh2 expression casette is following s1) or s2):

S1) DNA molecular shown in 70-1050 of sequence 6 in sequence table；

S2 the nucleotide sequence) and s1) limited has 75% or 75% or more identity, and DNA molecular with the same function.

6. method according to claim 4 or 5, it is characterised in that:

The acs expression casette is DNA molecular shown in 9-2028 of sequence 1 in sequence table；

The pta expression casette and the ackA expression casette are DNA shown in 9-3491 of sequence 2 in sequence points Son；

The aceA expression casette and the aceK expression casette are shown in 9-3172 of sequence 3 in sequence table DNA molecular；

The aceB expression casette is DNA molecular shown in 9-1671 of sequence 4 in sequence table；

The maeA expression casette and the maeB expression casette are shown in 9-4070 of sequence 5 in sequence table DNA molecular；

The ldh2 expression casette is DNA molecular shown in 9-1050 of sequence 6 in sequence table.

7. utilizing the recombinant bacterium of the method preparation any in claim 1-6.

8. reagent set, by the DNA fragmentation containing the poxB gene upstream and downstream homology arm any in claim 4-6, It is the DNA fragmentation containing the pflB gene upstream and downstream homology arm, described containing the aceEF gene upstream and downstream homology arm DNA fragmentation, described contains the lldD gene upstream and downstream at the DNA fragmentation containing the ldhA gene upstream and downstream homology arm It is the DNA fragmentation of homology arm, the acs expression casette, the pta expression casette, the ackA expression casette, described It is aceA expression casette, the aceK expression casette, the aceB expression casette, the maeA expression casette, described MaeB expression casette and ldh2 expression casette composition.

9. reagent set described in claim 8 utilizes the following of the recombinant bacterium that in claim 1-6 prepared by any the method Any application:

X1, production Pfansteihl；

X2, preparation production Pfansteihl product；

X3, degradation acetic acid；

X4, preparation degradation acetic acid product.

The preparation method of 10.L- lactic acid, comprising: using acetic acid as carbon source, prepared using the method any in claim 1-6 Recombinant bacterium carry out bioconversion, Pfansteihl is prepared.