CN106480035B

CN106480035B - A kind of 5 '-UTR elements and its application in production

Info

Publication number: CN106480035B
Application number: CN201610987080.3A
Authority: CN
Inventors: 刘树文; 温廷益; 商秀玲; 张芸
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2016-10-27
Filing date: 2016-11-09
Publication date: 2019-01-11
Anticipated expiration: 2036-11-09
Also published as: CN106480035A

Abstract

Application the invention discloses a kind of 5 '-UTR elements and its in production, the especially application in l-Alanine fermenting and producing.Present invention firstly provides a kind of DNA molecular first, and as shown in the 294th to n1 nucleotide of sequence 14 of sequence table, n1 is 310 or more 606 natural numbers below.The present invention also protects DNA molecular first as controlling element, is promoting the application in destination gene expression.The present invention also protects a kind of DNA molecular third, successively includes following element from upstream to downstream: the gene of DNA molecular first, encoding alanine dehydrogenase.The present invention also protects the recombinant bacterium third containing DNA molecular third.The present invention also protects application of the recombinant bacterium third in production l-Alanine.The present invention obtains the nucleic acid sequence of efficiently enhancing gene expression, constructs the bacterial strain of production alanine, provides new method to improve the fermenting and producing of alanine.

Description

A kind of 5 '-UTR elements and its application in production

Technical field

The invention belongs to field of biotechnology, it is related to a kind of 5 '-UTR elements and its application in production, especially exists Application in l-Alanine fermenting and producing.

Background technique

Express express target protein in the bioreactor, usually by the encoding gene with promoter and destination protein DNA molecular imports bioreactor, by the expression of promoter starting encoding gene, obtains destination protein.Therefore, increase purpose egg White ability to express has very big application prospect.

L-Alanine is human body nonessential amino acid, with sweet, and soluble easily in water, application field is extensive.In food industry The sense of taste and protein utilization of food can be improved in middle l-Alanine.In field of medicaments, l-Alanine is often used as amino acids Nutritional drugs, while l-Alanine is also to manufacture the important source material of the organic compounds such as aminopropanol and vitamin B6.In recent years, L- Alanine is also used for the engineering plastics such as synthesizing polyamides, polyesteramide.Especially conjunction of the l-Alanine as surfactant At precursor, application in washing industry substantially increases the market demand of l-Alanine, has expanded the application of l-Alanine Field.

Currently, l-Alanine mainly passes through Production by Microorganism Fermentation.Microbe fermentation method is with renewable carbons such as glucose Source is raw material, produces l-Alanine by bacterium.It is the key that production l-Alanine that alanine dehydrogenase is overexpressed in bacterial strain. L-Alanine is produced in the prior art in fermentation method, and the general copy number for increasing alanine dehydrogenase gene by plasmid mentions Expression quantity (Appl Microbiol Biotechnol (2004) 65:56-of high exogenous alanine dehydrogenase in Escherichia coli 60；Biotechnol Lett(2006)28:1695–1700；Appl Biochem Biotechnol(2016)178:324– 337).However, only by plasmid copy number improve alanine dehydrogenase expression quantity there are stability differences and metabolic burden weight The shortcomings that, it therefore, needs to develop efficient gene expression element, improves the alanine dehydrogenase expression of identical gene copy number Amount promotes the performance of engineering bacterium fermentation production l-Alanine.

Summary of the invention

Application the object of the present invention is to provide a kind of 5 '-UTR elements and its in production.

Present invention firstly provides a kind of DNA molecular first, as shown in the 294th to n1 nucleotide of sequence 14 of sequence table, N1 is 310 or more 606 natural numbers below.N1 is 310 or more 336 natural numbers below.N1 concretely 311 or 336 or 606。

The present invention also protects the DNA molecular first as controlling element, is promoting the application in destination gene expression.It is described In, the DNA molecular first is located between the initiation codon of the target gene and the promoter of the target gene.Institute State the target gene concretely gene of encoding alanine dehydrogenase, lacZ gene or gfp gene.

The present invention also protects a kind of DNA molecular second, successively includes following element from upstream to downstream: promoter, the DNA Molecule first, target gene.The DNA molecular second from upstream to downstream successively include following element: promoter, the DNA molecular First, catenation sequence " GGTTCTGGTTCTGGTTCT ", target gene.The DNA molecular second is from upstream to downstream successively by as follows Element composition: promoter, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the DNA molecular first, catenation sequence " GGTTCTGGTTCTGGTTCT ", target gene.The target gene concretely lacZ gene or gfp gene.

Recombinant plasmid second containing the DNA molecular second also belongs to protection scope of the present invention.The recombinant plasmid second tool Body can be inserted into the recombinant plasmid that the DNA molecular second obtains for the multiple cloning sites in the plasmid that sets out.The plasmid that sets out is low Copy, middle copy or high copy number plasmid, such as pSC101, pACYC184, pBR322 or pTrc99a.The plasmid tool that sets out Body can be plasmid pACYC184.The recombinant plasmid second more specifically can be in I digestion of Xba I and BamH of plasmid pACYC184 The recombinant plasmid that the DNA molecular second obtains is inserted between site.

The present invention also protects the recombinant bacterium second containing any description above DNA molecular second.The recombinant bacterium second is concretely The recombinant plasmid second is imported into out the recombinant bacterium that bacterium germination obtains.

The present invention also protects a kind of DNA molecular third, successively includes following element from upstream to downstream: the DNA molecular first, The gene of encoding alanine dehydrogenase.The DNA molecular third is successively made of following element from upstream to downstream: the DNA points The gene of sub- first, encoding alanine dehydrogenase.The DNA molecular third is specific as shown in the sequence 18 of sequence table.

Recombinant plasmid third containing the DNA molecular third also belongs to protection scope of the present invention.The recombinant plasmid third has Body can be inserted into the recombinant plasmid that the DNA molecular third obtains for the multiple cloning sites in the plasmid that sets out.The plasmid that sets out is low Copy, middle copy or high copy number plasmid, such as pSC101, pACYC184, pBR322 or pTrc99a.The plasmid tool that sets out Body can be plasmid pACYC184.The recombinant plasmid third more specifically can be in the I digestion position Xba I and Sph of plasmid pACYC184 The recombinant plasmid that the DNA molecular third obtains is inserted between point.

The present invention also protects the recombinant bacterium third containing any description above DNA molecular third.The recombinant bacterium third is concretely The recombinant plasmid third is imported into out the recombinant bacterium that bacterium germination obtains.

The present invention also protects a kind of DNA molecular fourth, successively includes following element from upstream to downstream: promoter, DNA molecular Third.The DNA molecular fourth is successively made of following element from upstream to downstream: the enzyme of promoter, restriction enzyme BamH I Cut identification sequence, DNA molecular third.

Recombinant plasmid fourth containing the DNA molecular fourth also belongs to protection scope of the present invention.The recombinant plasmid fourth tool Body can be inserted into the recombinant plasmid that the DNA molecular fourth obtains for the multiple cloning sites in the plasmid that sets out.The plasmid that sets out is low Copy, middle copy or high copy number plasmid, such as pSC101, pACYC184, pBR322 or pTrc99a.The plasmid tool that sets out Body can be plasmid pACYC184.The recombinant plasmid fourth more specifically can be in the I digestion position Xba I and Sph of plasmid pACYC184 The recombinant plasmid that the DNA molecular fourth obtains is inserted between point.

The present invention also protects the recombinant bacterium fourth containing any description above DNA molecular fourth.The recombinant bacterium fourth is concretely The recombinant plasmid fourth is imported into out the recombinant bacterium that bacterium germination obtains.

Any description above alanine dehydrogenase concretely derives from the alanine dehydrogenase of bacillus subtilis W168.

Any description above alanine dehydrogenase is concretely following (a1) or (a2):

(a1) protein that the amino acid sequence shown in sequence 19 in sequence table forms；

(a2) by the amino acid sequence of sequence 19 by one or several amino acid residues substitution and/or missing and/or Addition and the protein as derived from sequence 19 with alanine dehydrogenase function.

The gene of any description above encoding alanine dehydrogenase is following (a3) or (a4) or (a5) or (a6):

(a3) code area DNA molecular as shown in 44-1180 nucleotide of sequence 18 in sequence table；

(a4) DNA molecular shown in the 44-1262 nucleotide of sequence 18 in sequence table；

(a5) hybridize and the DNA of encoding alanine dehydrogenase with (a3) or (a4) DNA sequence dna limited under strict conditions Molecule；

(a6) DNA sequence dna limited with (a3) or (a4) has 90% or more homology and encoding alanine dehydrogenase DNA molecular.

Any description above lacZ gene is following (b1) or (b2) or (b3) or (b4):

(b1) code area DNA molecular as shown in 1-3075 nucleotide of sequence 15 in sequence table；

(b2) DNA molecular shown in sequence 15 in sequence table；

(b3) hybridize and the DNA of coding beta-galactosidase with (b1) or (b2) DNA sequence dna limited under strict conditions Molecule；

(b4) DNA sequence dna limited with (b1) or (b2) has 90% or more homology and coding beta-galactosidase DNA molecular.

Any description above gfp gene is following (c1) or (c2) or (c3):

(c1) code area DNA molecular as shown in sequence 16 in sequence table；

(c2) hybridize and the DNA molecular of encoding green fluorescent protein with (c1) DNA sequence dna limited under strict conditions；

(c3) DNA sequence dna limited with (c1) or (c2) has 90% or more homology and encoding green fluorescent protein DNA molecular.

Any description above promoter concretely strong promoter, such as L promoter, trc promoter, T5 promoter, lac Promoter, tac promoter or T7 promoter.

Any description above promoter concretely promoter P_PL.Promoter P_PLFor as follows (d1) or (d2) or (d3):

(d1) DNA molecular shown in sequence 13 in sequence table；

(d2) hybridize under strict conditions with (d1) DNA sequence dna limited and there is the DNA molecular of promoter function；

(d3) DNA sequence dna limited with (d1) is with 90% or more homology and with the DNA molecular of promoter function.

Any description above stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS, in DNA Or hybridizes at 65 DEG C in RNA hybrid experiment and wash film.

Any description above, which goes out bacterium germination, to be bacterium, can be further Escherichia bacteria, more specifically can be large intestine bar Bacterium.

It more specifically can be e. coli k12 W3110 that any description above, which goes out bacterium germination,.

It can be to inhibit metA base using e. coli k12 W3110 as starting strain that any description above, which goes out bacterium germination more specifically, The bacterial strain that cause, ilvA gene, lysA gene, tdh gene, tdcC gene and sstT gene expression obtain.

The metA gene is the gene of encoded homoserine succinyltransferase (MetA albumen).The MetA albumen is As follows (g1) or (g2):

(g1) protein that the amino acid sequence shown in sequence 2 in sequence table forms；

(g2) amino acid sequence of sequence 2 by the substitution of one or several amino acid residues and/or missing and/or is added Add and the protein with the same function as derived from sequence 2.

The metA gene is following (g3) or (g4) or (g5) or (g6):

(g3) code area DNA molecular as shown in 752-1681 nucleotide of sequence 1 in sequence table；

(g4) DNA molecular shown in sequence 1 in sequence table；

(g5) hybridize under strict conditions with (g3) or (g4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(g6) DNA sequence dna limited with (g3) or (g4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

The ilvA gene is the gene of encoding Thr deaminase (IlvA albumen).The IlvA albumen is following (h1) Or (h2):

(h1) protein that the amino acid sequence shown in sequence 4 in sequence table forms；

(h2) amino acid sequence of sequence 4 by the substitution of one or several amino acid residues and/or missing and/or is added Add and the protein with the same function as derived from sequence 4.

The ilvA gene is following (h3) or (h4) or (h5) or (h6):

(h3) code area DNA molecular as shown in 638-2182 nucleotide of sequence 3 in sequence table；

(h4) DNA molecular shown in sequence 3 in sequence table；

(h5) hybridize under strict conditions with (h3) or (h4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(h6) DNA sequence dna limited with (h3) or (h4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

The lysA gene is the gene for encoding diaminapimelate decarboxylase (LysA albumen).The LysA albumen is such as Under (i1) or (i2):

(i1) protein that the amino acid sequence shown in sequence 6 in sequence table forms；

(i2) amino acid sequence of sequence 6 by the substitution of one or several amino acid residues and/or missing and/or is added Add and the protein with the same function as derived from sequence 6.

The lysA gene is following (i3) or (i4) or (i5) or (i6):

(i3) code area DNA molecular as shown in 639-1901 nucleotide of sequence 5 in sequence table；

(i4) DNA molecular shown in sequence 5 in sequence table；

(i5) hybridize under strict conditions with (i3) or (i4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(i6) DNA sequence dna limited with (i3) or (i4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

The tdh gene is the gene of encoding Thr dehydratase (Tdh albumen).The Tdh albumen be following (j1) or (j2):

(j1) protein that the amino acid sequence shown in sequence 8 in sequence table forms；

(j2) amino acid sequence of sequence 8 by the substitution of one or several amino acid residues and/or missing and/or is added Add and the protein with the same function as derived from sequence 8.

The tdh gene is following (j3) or (j4) or (j5) or (j6):

(j3) code area DNA molecular as shown in 753-1778 nucleotide of sequence 7 in sequence table；

(j4) DNA molecular shown in sequence 7 in sequence table；

(j5) hybridize under strict conditions with (j3) or (j4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(j6) DNA sequence dna limited with (j3) or (j4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

The tdcC gene is the gene of encoding Thr absorption and transport albumen (TdcC albumen).The TdcC albumen is such as Under (k1) or (k2):

(k1) protein that the amino acid sequence shown in sequence 10 in sequence table forms；

(k2) by the amino acid sequence of sequence 10 by one or several amino acid residues substitution and/or missing and/or Addition and the protein with the same function as derived from sequence 10.

The tdcC gene is following (k3) or (k4) or (k5) or (k6):

(k3) code area DNA molecular as shown in 701-2032 nucleotide of sequence 9 in sequence table；

(k4) DNA molecular shown in sequence 9 in sequence table；

(k5) hybridize under strict conditions with (k3) or (k4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(k6) DNA sequence dna limited with (k3) or (k4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

The sstT gene is the gene of encoding Thr absorption and transport albumen (SstT albumen).The SstT albumen is such as Under (m1) or (m2):

(m1) protein that the amino acid sequence shown in sequence 12 in sequence table forms；

(m2) by the amino acid sequence of sequence 12 by one or several amino acid residues substitution and/or missing and/or Addition and the protein with the same function as derived from sequence 12.

The sstT gene is following (m3) or (m4) or (m5) or (m6):

(m3) code area DNA molecular as shown in 701-1945 nucleotide of sequence 11 in sequence table；

(m4) DNA molecular shown in sequence 11 in sequence table；

(m5) hybridize under strict conditions with (m3) or (m4) DNA sequence dna limited and encode albumen with the same function The DNA molecular of matter；

(m6) DNA sequence dna limited with (m3) or (m4) has 90% or more homology and coding egg with the same function The DNA molecular of white matter.

It can be to knock out following six using e. coli k12 W3110 as starting strain that any description above, which goes out bacterium germination more specifically, The bacterial strain that a constant gene segment C obtains:

The open reading frame (752-1681 nucleotide of sequence 1 of sequence table) of metA gene；

The open reading frame (638-2182 nucleotide of sequence 3 of sequence table) of ilvA gene；

The open reading frame (639-1901 nucleotide of sequence 5 of sequence table) of lysA gene；

The open reading frame (753-1778 nucleotide of sequence 7 of sequence table) of tdh gene；

TdcC gene such as lower curtate: 701-1852 nucleotide in sequence 9；

SstT gene such as lower curtate: 697-1759 nucleotide in sequence 11.

The open reading frame of knockout metA gene is realized particular by importing interference fragment I or interference plasmid I.Interference Segment I is from the successively upstream zone as shown in the 245-751 nucleotide of the sequence of sequence table 1 and the sequence table of upstream to downstream The composition of downstream section shown in the 1682-2154 nucleotide of sequence 1.Interference plasmid I is the recombination matter with interference fragment I Grain.Interference plasmid I is concretely inserted at the multiple cloning sites of pKOV plasmid (such as between Sal I and Not I restriction enzyme site) The recombinant plasmid that interference fragment I obtains.

The open reading frame of knockout ilvA gene is realized particular by importing interference fragment II or interference plasmid II.It is dry Segment II is disturbed from upstream to downstream successively upstream zone and sequence as shown in the 140-637 nucleotide of the sequence of sequence table 3 The composition of downstream section shown in 2183-2712 nucleotide of sequence 3 of table.Interference plasmid II is the weight with interference fragment II Group plasmid.Interference plasmid II concretely the multiple cloning sites of pKOV plasmid (such as Bam HI and Not I restriction enzyme site it Between) it is inserted into the recombinant plasmid that interference fragment II obtains.

The open reading frame of knockout lysA gene is realized particular by importing interference fragment III or interference plasmid III.It is dry Segment III is disturbed from upstream to downstream successively upstream zone and sequence as shown in the 132-638 nucleotide of the sequence of sequence table 5 The composition of downstream section shown in 1902-2445 nucleotide of sequence 5 of table.Interference plasmid III is the weight with interference fragment III Group plasmid.Interference plasmid III concretely the multiple cloning sites of pKOV plasmid (such as Bam HI and Not I restriction enzyme site it Between) it is inserted into the recombinant plasmid that interference fragment III obtains.

The open reading frame of knockout tdh gene is realized particular by importing interference fragment IV or interference plasmid IV.It is dry Segment IV is disturbed from upstream to downstream successively upstream zone and sequence as shown in the 227-752 nucleotide of the sequence of sequence table 7 The composition of downstream section shown in 1779-2271 nucleotide of sequence 7 of table.Interference plasmid IV is the weight with interference fragment IV Group plasmid.Interference plasmid IV concretely the multiple cloning sites of pKOV plasmid (such as Bam HI and Not I restriction enzyme site it Between) it is inserted into the recombinant plasmid that interference fragment IV obtains.

Knocking out " tdcC gene such as lower curtate: 701-1852 nucleotide in sequence 9 " is by importing interference fragment V or interference plasmid V realize.Interference fragment V is from upstream to downstream successively by the 176-700 nucleosides of the sequence of sequence table 9 The composition of downstream section shown in 1853-2388 nucleotide of sequence 9 of upstream zone and sequence table shown in acid.Interference plasmid V is the recombinant plasmid with interference fragment V.Interference plasmid V is concretely in the multiple cloning sites of pKOV plasmid (such as Bam Between HI and Not I restriction enzyme site) it is inserted into the recombinant plasmid that interference fragment V obtains.

Knocking out " sstT gene such as lower curtate: 697-1759 nucleotide in sequence 11 " is by importing interference fragment VI or interference plasmid VI realize.Interference fragment VI is from upstream to downstream successively by the 14-696 nucleosides of the sequence of sequence table 11 The composition of downstream section shown in 1760-2240 nucleotide of sequence 11 of upstream zone and sequence table shown in acid.Interfere matter Grain VI is the recombinant plasmid with interference fragment VI.Interference plasmid VI concretely the multiple cloning sites of pKOV plasmid (such as Between Bam HI and Not I restriction enzyme site) it is inserted into the recombinant plasmid that interference fragment VI obtains.

The present invention also protects application of the recombinant bacterium third in production l-Alanine.

The present invention also protects application of the recombinant bacterium fourth in production l-Alanine.

When producing l-Alanine using the recombinant bacterium, using glucose as carbon source.

When producing l-Alanine using the recombinant bacterium, using recombinant bacterium described in fermentation medium culture.

The fermentation medium can be rich medium, be also possible to minimal medium.Culture medium includes carbon source, nitrogen Source, inorganic ions, antibiotic and other trophic factors.As carbon source, the carbohydrates such as glucose, lactose, galactolipin can be used； It is also possible to the alcohols such as glycerol, mannitol；Also the organic acids such as gluconic acid, citric acid, succinic acid can be used.As nitrogen It is inorganic nitrogen-sourced that ammonium hydroxide, ammonium sulfate, ammonium phosphate, ammonium chloride etc. can be used in source；Also corn pulp, soybean meal hydrolysate, hair can be used The organic nitrogen sources such as hair powder, yeast extract, peptone.Inorganic ions is comprising in iron, calcium, magnesium, manganese, molybdenum, cobalt, copper, potassium plasma It is one or more.Other trophic factors further include the vitamins such as biotin, vitamin B1, pyridoxal.

Carbon source in the fermentation medium is glucose.

The fermentation medium is concretely: glucose 20.0g/L, yeast powder 2.0g/L, peptone 4g/L, ammonium sulfate 6.0g/L, potassium dihydrogen phosphate 2.0g/L, epsom salt 1.0g/L, glycine betaine 1.0g/L, calcium carbonate 15.0g/L, microelement Mixed liquor 1mL/L, surplus are water.

Micro-mixed liquor: FeSO₄·7H₂O10g/L、CaCl₂1.35g/L、ZnSO₄·7H₂O2.25g/L、MnSO₄· 4H₂O0.5g/L、CuSO₄·5H₂O1g/L、(NH₄)₆Mo₇O₂₄·4H₂O0.106g/L、Na₂B₄O₇·10H₂O0.23g/L、 CoCl₂·6H₂O0.48g/L, 35%HCl10mL/L, surplus are water.

The condition of culture is concretely: 37 DEG C, 220rpm shake culture 12-48h.

The condition of the culture is concretely: seed liquor is seeded in fermentation medium with 3% inoculum concentration, 37 DEG C, 220rpm shake culture 12-48h.Seed liquor the preparation method is as follows: recombinant bacterium is seeded in LB liquid medium, 37 DEG C, 220rpm shaken cultivation 12h, obtains seed liquor.

Following process control is carried out during the culture: in incubation, the pH value of reaction system is adjusted with ammonium hydroxide It is set to maintain 6.8-7.0；It is primary every 3-4h sampling in incubation, glucose content is detected, when the glucose in system When content is lower than 5g/L, adds glucose and the concentration of glucose in system is made to reach 10g/L.

The present invention provides the threonine attenuator mutant for releasing feedback repression and its application, acquisition significantly increases gene 5 '-UTR mutant of expression construct using mutant regulation alanine dehydrogenase expression and efficiently produce l-Alanine Bacterial strain provides new way to improve the fermenting and producing of l-Alanine.

The correctional effect that the present invention passes through the method validation threonine attenuator of translation fusion reporter gene.Pass through fusion The complete reading expression cassette of 5 ' terminal sequences of thrA gene and two kinds of reporter genes lacZ and gfp, then measures reporter protein The enzyme activity and GFP fluorescent value of LacZ calculates the gene expression amount under different threonine attenuator mutant regulations, verifies threonine The effect of attenuator mutant controlling gene expression.

The present invention provides the remodeling method for gradually truncating threonine attenuator functional sequence from 5 ' ends, screening is increased 5 ' non-translational regions (5 '-Untranslated Region, 5 '-UTR) Expression element of strong gene expression.Optimize using the present invention 5 ' obtained-UTR element regulating and expressing alanine dehydrogenase gene ald, can be improved the L- propionic acid propylhomoserin yield of engineering bacteria.This Invention obtains the nucleic acid sequence of efficiently enhancing gene expression, constructs the bacterial strain of production l-Alanine, to improve l-Alanine Fermenting and producing provides new method.

Detailed description of the invention

GFP expression intensity under the sub- mutant regulation of Fig. 1 flow cytometry analysis differential declines.

The l-Alanine yield of Fig. 2 engineering bacteria shake flask fermentation.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.As not specified in following embodiments, technological means used in embodiment is well known to those skilled in the art Conventional means and commercially available common instrument, reagent, reference can be made to " Molecular Cloning:A Laboratory guide (the 3rd edition) " (Science Press), " micro- Biological experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent the reference such as manufacturers instruction.

E. coli k12 W3110 (also known as E.coli K12 W3110): in day this technology evaluation study institute living resources The heart (NITE Biological Resource Center, NBRC).PKOV plasmid: Addgene company, catalog number are 25769.PACYC184 plasmid: NEB company, catalog number E4152S.PAD123 plasmid: Gene, 1999,226 (2): 297- 305.The full name of ONPG are as follows: ortho-nitrophenyl-β-D- galactopyranoside.Bacillus subtilis W168: it is purchased from U.S. Bacillus Genetic Stock Center, article No. 1A308.

Each primer sequence used in embodiment is following (5 ' → 3 '):

WY569:GCGTCGACATAGAACCCAACCGCCTGCTCA；

WY570:AACGATCGACTATCACAGAAGAAACCTGATTACCTCACTACATA；

WY571:TATGTAGTGAGGTAATCAGGTTTCTTCTGTGATAGTCGATCGTT；

WY572:ATTGCGGCCGCCCGAAATAAAATCAGGCAACGT；

WY583:CGTTAATGAAATATCGCCAG；

WY584:TCGAAATCGGCCATAAAGAC.

WY577:CGCGGATCCGAAAGTGTACGAAAGCCAGG；

WY578:GCGCTATCAGGCATTTTTCCTATTAACCCCCCAGTTTCGA；

WY579:TCGAAACTGGGGGGTTAATAGGAAAAATGCCTGATAGCGC；

WY580:ATTGCGGCCGCGTGAAGCGGATCTGGCGATT；

WY587:ATGGCTGTATCCGCTCGCTG；

WY588:ACACCATCGATCAGCAAGGGC.

WY573:CGCGGATCCGGCACGATATTTAAGCTGAC；

WY574:CAACCAGCGACTAACCGCAGAACAAACTCCAGATAAGTGC；

WY575:GCACTTATCTGGAGTTTGTTCTGCGGTTAGTCGCTGGTTG；

WY576:ATTGCGGCCGCGCTGGCAACGCGTCATTTAA；

WY585:GTAACACACACACTTCATCT；

WY586:GATCCCGGATGCTGATTTAG.

WY598:CGCGGATCCATACTGCGATGTGATGGGCC；

WY599:AATACCAGCCCTTGTTCGTGCTCACATCCTCAGGCGATAA；

WY600:TTATCGCCTGAGGATGTGAGCACGAACAAGGGCTGGTATT；

WY601:ATTGCGGCCGCCGTTGCCACTTCAATCCCAC；

WY602:GCTATGCCAACAACGATATG；

WY603:GGTTAATACGCCGGTTGAGC.

WY476:CGCGGATCCGGAACGATTGGTCTGGAAAT；

WY477:GGCTTCAATCAGGTCAAGGATATCCTATCCTCAACGAATTA；

WY478:TAATTCGTTGAGGATAGGATATCCTTGACCTGATTGAAGCC；

WY479:ATTGCGGCCGCCGCGACGGATATTATCAATGAC；

WY497:GCGCCAAAATCCAAAGTAGC；

WY498:ATGTGCGCGCTGGGAAACAT.

WY945:CGCGGATCCTATCTTCGCCGTGACCACTGA；

WY946:ACCGAACATATTACAGGCCAGCGATCCTTTCATTGTGTTGTC；

WY947:GACAACACAATGAAAGGATCGCTGGCCTGTAATATGTTCGGT；

WY948:ATTGCGGCCGCCTCGCGAAGTTCCATCATCCT；

WY949:CCTGTAACGAGCGTAACGACT；

WY950:TATCTTCGCCGTGACCACTGA.

WY914:CCCAAGCTTACAGAGTACACAACATCCATG；

WY1630:CCCAAGCTTCATTAGCACCACCATTACCA；

WY1629:CCCAAGCTTCAGGTAACGGTGCGGGCTGA；

WY1628:CCCAAGCTTCGCGTACAGGAAACACAGAA；

WY1627:CCCAAGCTTGTGCGGGCTTTTTTTTTCGA；

WY913:CCCAAGCTTTCGACCAAAGGTAACGAGGT；

WY1746:CATAGAACCAGAACCAGAACCCAATTGCGCCAGCGGGAAC。

WY1752:CAATTG GGTTCTGGTTCTGGTTCTATGACCATGATTACGGATTCACT；

WY1750:CGCGGATCCACGCGAAATACGGGCAGACA。

The building of embodiment 1, E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT

Using e. coli k12 W3110 as starting strain, (the encoded homoserine succinyl transfer of metA gene is successively knocked out The gene of enzyme), ilvA gene (gene of encoding Thr deaminase), lysA gene (coding diaminapimelate decarboxylase Gene), tdh gene (gene of encoding Thr dehydratase), tdcC gene (gene of encoding Thr absorption and transport albumen) With sstT gene (gene of encoding Thr absorption and transport albumen), chassis engineering bacteria is obtained, E.coli K-12 is named as W3110△metA△ilvA△lysA△tdh△tdcC△sstT。

1, metA gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY569 and WY570 composition PCR amplification is carried out, I-first of DNA fragmentation (metA upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY571 and WY572 composition PCR amplification is carried out, I-second of DNA fragmentation (metA downstream of gene region) is obtained.

(3) it is used as template after mixing I-first of DNA fragmentation and I-second of DNA fragmentation, is drawn using what WY569 and WY572 was formed Object obtains DNA fragmentation I-the third to PCR amplification is carried out.

(4) pKOV plasmid is taken, carries out double digestion with restriction enzyme Sal I and Not I, recycling carrier framework is (about 5.6kb)。

(5) DNA fragmentation I-the third is taken, double digestion is carried out with restriction enzyme Sal I and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid I. According to sequencing result, structure is carried out to recombinant plasmid I and is described as follows: between Sal I and Not the I restriction enzyme site of pKOV plasmid Insert following specific DNA molecular: from upstream to downstream successively as shown in the 245-751 nucleotide of the sequence of sequence table 1 The composition of downstream section shown in 1682-2154 nucleotide of upstream zone and the sequence of sequence table 1.Such as sequence table of metA gene Sequence 1 shown in, 752-1681 nucleotide are open reading frame (metA albumen shown in the sequence 2 of polynucleotide).

(7) recombinant plasmid I is imported into e. coli k12 W3110, obtains the recombinant bacterium of metA gene knockout, is named as E.coli K12 W3110△metA。

The identification method of the recombinant bacterium of metA gene knockout: PCR expansion is carried out using the primer pair that WY583 and WY584 is formed Increase, if obtaining 1375bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On the open reading frame of metA gene be knocked.

2, ilvA gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY577 and WY578 composition PCR amplification is carried out, II-first of DNA fragmentation (ilvA upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY579 and WY580 composition PCR amplification is carried out, II-second of DNA fragmentation (ilvA downstream of gene region) is obtained.

(3) it is used as template after mixing II-first of DNA fragmentation and II-second of DNA fragmentation, is formed using WY577 and WY580 Primer pair carries out PCR amplification, obtains DNA fragmentation II-the third.

(4) pKOV plasmid is taken, carries out double digestion with restriction enzyme Bam HI and Not I, recycling carrier framework is (about 5.6kb)。

(5) DNA fragmentation II-the third is taken, double digestion is carried out with restriction enzyme Bam HI and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid II. According to sequencing result, structure is carried out to recombinant plasmid II and is described as follows: pKOV plasmid Bam HI and Not I restriction enzyme site it Between insert following specific DNA molecular: from upstream to downstream successively as shown in the 140-637 nucleotide of the sequence of sequence table 3 Upstream zone and sequence table the 2183-2712 nucleotide of sequence 3 shown in downstream section composition.IlvA gene such as sequence Shown in the sequence 3 of table, 638-2182 nucleotide are open reading frame (ilvA egg shown in the sequence 4 of polynucleotide It is white).

(7) recombinant plasmid II is imported into E.coli K12 W3110 △ metA, obtains the recombinant bacterium of ilvA gene knockout, ordered Entitled E.coli K-12 W3110 △ metA △ ilvA.

The identification method of the recombinant bacterium of ilvA gene knockout: PCR expansion is carried out using the primer pair that WY587 and WY588 is formed Increase, if obtaining 1344bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On the open reading frame of ilvA gene be knocked.

3, lysA gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY573 and WY574 composition PCR amplification is carried out, III-first of DNA fragmentation (lysA upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY575 and WY576 composition PCR amplification is carried out, III-second of DNA fragmentation (lysA downstream of gene region) is obtained.

(3) it is used as template after mixing III-first of DNA fragmentation and III-second of DNA fragmentation, is formed using WY573 and WY576 Primer pair carries out PCR amplification, obtains DNA fragmentation III-the third.

(5) DNA fragmentation III-the third is taken, double digestion is carried out with restriction enzyme Bam HI and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid III. According to sequencing result, structure is carried out to recombinant plasmid III and is described as follows: pKOV plasmid Bam HI and Not I restriction enzyme site it Between insert following specific DNA molecular: from upstream to downstream successively as shown in the 132-638 nucleotide of the sequence of sequence table 5 Upstream zone and sequence table the 1902-2445 nucleotide of sequence 5 shown in downstream section composition.LysA gene such as sequence Shown in the sequence 5 of table, 639-1901 nucleotide are open reading frame (lysA egg shown in the sequence 6 of polynucleotide It is white).

(7) recombinant plasmid III is imported into E.coli K-12 W3110 △ metA △ ilvA, obtains the weight of lysA gene knockout Group bacterium, is named as E.coli K-12 W3110 △ metA △ ilvA △ lysA.

The identification method of the recombinant bacterium of lysA gene knockout: PCR expansion is carried out using the primer pair that WY585 and WY586 is formed Increase, if obtaining 1302bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On the open reading frame of lysA gene be knocked.

4, tdh gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY598 and WY599 composition PCR amplification is carried out, IV-first of DNA fragmentation (tdh upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY600 and WY601 composition PCR amplification is carried out, IV-second of DNA fragmentation (tdh downstream of gene region) is obtained.

(3) it is used as template after mixing IV-first of DNA fragmentation and IV-second of DNA fragmentation, is formed using WY598 and WY601 Primer pair carries out PCR amplification, obtains DNA fragmentation IV-the third.

(5) DNA fragmentation IV-the third is taken, double digestion is carried out with restriction enzyme Bam HI and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid IV. According to sequencing result, structure is carried out to recombinant plasmid IV and is described as follows: pKOV plasmid Bam HI and Not I restriction enzyme site it Between insert following specific DNA molecular: from upstream to downstream successively as shown in the 227-752 nucleotide of the sequence of sequence table 7 Upstream zone and sequence table the 1779-2271 nucleotide of sequence 7 shown in downstream section composition.Tdh gene such as sequence Shown in the sequence 7 of table, 753-1778 nucleotide are open reading frame (tdh albumen shown in the sequence 8 of polynucleotide).

(7) recombinant plasmid IV is imported into E.coli K-12 W3110 △ metA △ ilvA △ lysA, obtains tdh clpp gene The recombinant bacterium removed is named as E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh.

The identification method of the recombinant bacterium of tdh gene knockout: PCR expansion is carried out using the primer pair that WY602 and WY603 is formed Increase, if obtaining 1434bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On the open reading frame of tdh gene be knocked.

5, tdcC gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY476 and WY477 composition PCR amplification is carried out, V-first of DNA fragmentation (tdcC upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY478 and WY479 composition PCR amplification is carried out, V-second of DNA fragmentation (tdcC downstream of gene region) is obtained.

(3) it is used as template after mixing V-first of DNA fragmentation and V-second of DNA fragmentation, is formed using WY476 and WY479 Primer pair carries out PCR amplification, obtains DNA fragmentation V-the third).

(5) DNA fragmentation V-the third is taken, double digestion is carried out with restriction enzyme Bam HI and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid V. According to sequencing result, structure is carried out to recombinant plasmid V and is described as follows: pKOV plasmid Bam HI and Not I restriction enzyme site it Between insert following specific DNA molecular: from upstream to downstream successively as shown in the 176-700 nucleotide of the sequence of sequence table 9 Upstream zone and sequence table the 1853-2388 nucleotide of sequence 9 shown in downstream section composition.TdcC gene such as sequence Shown in the sequence 9 of table, 701-2032 nucleotide are open reading frame (tdcC egg shown in the sequence 10 of polynucleotide It is white).

(7) recombinant plasmid V is imported into E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh, obtains tdcC The recombinant bacterium of gene knockout is named as E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC.

The identification method of the recombinant bacterium of tdcC gene knockout: PCR expansion is carried out using the primer pair that WY497 and WY498 is formed Increase, if obtaining 1453bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On tdcC gene be knocked such as lower curtate: 701-1852 nucleotide in sequence 9.

6, sstT gene is knocked out

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY945 and WY946 composition PCR amplification is carried out, VI-first of DNA fragmentation (sstT upstream area of gene) is obtained.

(2) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY947 and WY948 composition PCR amplification is carried out, VI-second of DNA fragmentation (sstT downstream of gene region) is obtained.

(3) it is used as template after mixing VI-first of DNA fragmentation and VI-second of DNA fragmentation, is formed using WY945 and WY948 Primer pair carries out PCR amplification, obtains DNA fragmentation VI-the third.

(5) DNA fragmentation VI-the third is taken, double digestion is carried out with restriction enzyme Bam HI and Not I, recycles digestion products.

(6) carrier framework that step (4) obtains is connected with the digestion products that step (5) obtains, obtains recombinant plasmid VI. According to sequencing result, structure is carried out to recombinant plasmid VI and is described as follows: pKOV plasmid Bam HI and Not I restriction enzyme site it Between insert following specific DNA molecular: from upstream to downstream successively as shown in the 14-696 nucleotide of the sequence of sequence table 11 Upstream zone and sequence table the 1760-2240 nucleotide of sequence 11 shown in downstream section composition.SstT gene such as sequence Shown in the sequence 11 of list, 701-1945 nucleotide are open reading frame (sstT shown in the sequence 12 of polynucleotide Albumen).

(7) recombinant plasmid VI is imported into E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC, obtained To the recombinant bacterium of sstT gene knockout, it is named as E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ sstT。

The identification method of the recombinant bacterium of sstT gene knockout: PCR expansion is carried out using the primer pair that WY949 and WY950 is formed Increase, if obtaining 1569bp amplified production, is tentatively judged as candidate object bacteria；Further pass through sequence verification strain chromosome On sstT gene be knocked such as lower curtate: 697-1759 nucleotide in sequence 11.

The expression of embodiment 2, attenuator mutant regulation lacZ gene

One, construction recombination plasmid pACYC184-P_PL

1, double chain DNA molecule (promoter P shown in the sequence 13 of composition sequence table_PL)。

2, the double chain DNA molecule prepared using step 1 carries out PCR using the primer pair that WY843 and WY842 is formed as template Amplification, obtains pcr amplification product.

WY843:TGCTCTAGACAATTCCGACGTCTAAGAAA；

WY842:CCCAAGCTTGGTCAGTGCGTCCTGCTGAT。

3, the pcr amplification product for taking step 2 to obtain carries out double digestion with restriction enzyme Xba I and Hind III, returns Receive digestion products.

4, pACYC184 plasmid is taken, double digestion is carried out with restriction enzyme Xba I and Hind III, recycles carrier framework (about 4.1kb).

5, the digestion products of step 3 are connected with the carrier framework of step 4, obtains recombinant plasmid pACYC184-P_PL。

Two, each recombinant plasmid is constructed

1, construction recombination plasmid A

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY914 and WY1746 composition PCR amplification is carried out, pcr amplification product A1 is obtained；Using double chain DNA molecule shown in the sequence 15 of artificial synthesized sequence table as mould Plate carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product A2；By pcr amplification product It is used as template after A1 and pcr amplification product A2 mixing, PCR amplification is carried out using the primer pair that WY914 and WY1750 is formed, is obtained Pcr amplification product A3.

(2) pcr amplification product A3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(3) recombinant plasmid pACYC184-P is taken_PL, with I double digestion of restriction enzyme Hind III and BamH, recycle carrier Skeleton (about 4.0kb).

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid A.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid A: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular A is entered；Specific DNA molecular A is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 172nd to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", (open reading frame is sequence to lacZ gene shown in the sequence 15 of sequence table 1-3075 in 15).Recombinant plasmid A is named as pACYC184-P_PL-thrLA-lacZ914。

2, construction recombination plasmid B

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer of WY1630 and WY1746 composition To PCR amplification is carried out, pcr amplification product B1 is obtained；Double chain DNA molecule shown in sequence 15 with artificial synthesized sequence table is Template carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product B2；PCR amplification is produced It is used as template after object B1 and pcr amplification product B2 mixing, PCR amplification is carried out using the primer pair that WY1630 and WY1750 is formed, Obtain pcr amplification product B3.

(2) pcr amplification product B3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid B.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid B: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular B is entered；Specific DNA molecular B is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 198th to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", lacZ gene shown in the sequence 15 of sequence table.Recombinant plasmid B is named as pACYC184-P_PL-thrLA-lacZ1630。

3, construction recombination plasmid C

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer of WY1629 and WY1746 composition To PCR amplification is carried out, pcr amplification product C1 is obtained；Double chain DNA molecule shown in sequence 15 with artificial synthesized sequence table is Template carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product C2；PCR amplification is produced It is used as template after object C1 and pcr amplification product C2 mixing, PCR amplification is carried out using the primer pair that WY1629 and WY1750 is formed, Obtain pcr amplification product C3.

(2) pcr amplification product C3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid C.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid C: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular C is entered；Specific DNA molecular C is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 236th to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", lacZ gene shown in the sequence 15 of sequence table.Recombinant plasmid C is named as pACYC184-P_PL-thrLA-lacZ1629。

4, construction recombination plasmid D

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer of WY1628 and WY1746 composition To PCR amplification is carried out, pcr amplification product D1 is obtained；Double chain DNA molecule shown in sequence 15 with artificial synthesized sequence table is Template carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product D2；PCR amplification is produced It is used as template after object D1 and pcr amplification product D2 mixing, PCR amplification is carried out using the primer pair that WY1628 and WY1750 is formed, Obtain pcr amplification product D3.

(2) pcr amplification product D3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid D.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid D: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular D is entered；Specific DNA molecular D is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 256th to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", lacZ gene shown in the sequence 15 of sequence table.Recombinant plasmid D is named as pACYC184-P_PL-thrLA-lacZ1628。

5, construction recombination plasmid E

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer of WY1627 and WY1746 composition To PCR amplification is carried out, pcr amplification product E1 is obtained；Double chain DNA molecule shown in sequence 15 with artificial synthesized sequence table is Template carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product E2；PCR amplification is produced It is used as template after object E1 and pcr amplification product E2 mixing, PCR amplification is carried out using the primer pair that WY1627 and WY1750 is formed, Obtain pcr amplification product E3.

(2) pcr amplification product E3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid E.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid E: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular E is entered；Specific DNA molecular E is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 294th to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", lacZ gene shown in the sequence 15 of sequence table.Recombinant plasmid E is named as pACYC184-P_PL-thrLA-lacZ1627。

6, construction recombination plasmid F

(1) using the genomic DNA of e. coli k12 W3110 as template, using the primer pair of WY913 and WY1746 composition PCR amplification is carried out, pcr amplification product F1 is obtained；Using double chain DNA molecule shown in the sequence 15 of artificial synthesized sequence table as mould Plate carries out PCR amplification using the primer pair that WY1752 and WY1750 is formed, obtains pcr amplification product F2；By pcr amplification product It is used as template after F1 and pcr amplification product F2 mixing, PCR amplification is carried out using the primer pair that WY913 and WY1750 is formed, is obtained Pcr amplification product F3.

(2) pcr amplification product F3 is taken, with I double digestion of restriction enzyme Hind III and BamH, recycles digestion products.

(4) digestion products of step (2) are connected with the carrier framework of step (3), obtains recombinant plasmid F.According to sequencing It is described as follows as a result, carrying out structure to recombinant plasmid F: being interleave in Xba I and BamH I restriction enzyme site of plasmid pACYC184 Specific DNA molecular F is entered；Specific DNA molecular F is successively made of following element from upstream to downstream: 13 institute of sequence of sequence table The promoter P shown_PL, the restriction endonuclease recognition sequence of restriction enzyme Hind III, the 310th to 606 nucleosides of sequence 14 of sequence table Acid, catenation sequence " GGTTCTGGTTCTGGTTCT ", lacZ gene shown in the sequence 15 of sequence table.Recombinant plasmid F is named as pACYC184-P_PL-thrLA-lacZ913。

Three, recombinant bacterium is constructed

By pACYC184-P_PL- thrLA-lacZ914 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC914.

By pACYC184-P_PL- thrLA-lacZ1630 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC1630.

By pACYC184-P_PL- thrLA-lacZ1629 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC1629.

By pACYC184-P_PL- thrLA-lacZ1628 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC1628.

By pACYC184-P_PL- thrLA-lacZ1627 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC1627.

By pACYC184-P_PL- thrLA-lacZ913 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as LAC913.

PACYC184 plasmid is imported into E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ SstT obtains recombinant bacterium, is named as LAC control.

Four, betagalactosidase activity measures

Test strain are as follows: LAC914, LAC1630, LAC1629, LAC1628, LAC1627 or LAC913.

1, test strain is seeded to the LB liquid medium of the chloramphenicol containing 34mg/L, 37 DEG C, 220rpm shaken cultivation 12h obtains seed liquor.

2, the seed liquor for taking step 1 to obtain is trained according to the liquid 2YT that 2% inoculum concentration is seeded to the chloramphenicol containing 34mg/L Support base, 37 DEG C, 220rpm culture 6h.

3, after completing step 2,1.5ml is sampled from cultivating system, measures OD_600nmOD value, 1ml is as sample to be tested Detectionofβ-galactosidaseactivity.

The method of detectionofβ-galactosidaseactivity:

(1) 10000 × g of 1ml sample to be tested is centrifuged 5min, collects bacterial sediment, is washed with the PBS buffer solution of pH7.2 Twice, it then is settled to 1ml and abundant suspension thalline with Z-buffer, is placed in spare on ice.Z-buffer:40mM NaH₂PO₄、 60mM Na₂HPO₄、10mM KCl、1mM MgSO₄, 50mM β-mercaptoethanol, pH 7.0.

(2) after completing step (1), 0.05mL is sampled, the ONPG aqueous solution and 0.8mL Z- of 0.2mL 4mg/ml is added Buffer is simultaneously mixed, and reaction initial time is reacted and recorded to 37 DEG C of standings, and 1mL 1M Na is added when system is in faint yellow₂CO₃Water Solution, which terminates, reacts and records the reaction terminating time, measures OD with ultraviolet specrophotometer_420nmValue.

LAC control carries out above-mentioned steps, measures as ultraviolet specrophotometer_OD420nmThe blank control of value.

Beta galactosidase enzyme activity Miller Unit=1000 × OD_420nm/(OD_600nm×t×V)；

T, reaction time (reaction terminating time and the difference for reacting initial time, min)；V, injection volume, 0.05mL.

Beta galactosidase definition living is that each cell decomposes enzyme amount required for 1 μm of olONPG per minute.

The measurement of each bacterial strain three times, is averaged and standard deviation.

It the results are shown in Table 1.Different tests bacterial strain carries out above-mentioned steps, and corresponding beta galactosidase enzyme activity has significance difference Different, the enzyme activity level of LAC1627 is significantly higher than other each bacterial strains.

Table 1

Test strain	Enzyme activity (Miller Unit)
		LAC914	47.72±3.33
LAC1630	26.17±2.71
		LAC1629	31.20±1.17
LAC1628	16.11±1.67
		LAC1627	132.09±4.61
LAC913	22.59±4.23

The expression of embodiment 3, attenuator mutant regulation gfp gene

One, construction recombination plasmid

Construct recombinant plasmid following six: pACYC184-P_PL-thrLA-gfp914、pACYC184-P_PL-thrLA- gfp1630、pACYC184-P_PL-thrLA-gfp1629、pACYC184-P_PL-thrLA-gfp1628、pACYC184-P_PL- ThrLA-gfp1627 and pACYC184-P_PL-thrLA-gfp913。

pACYC184-P_PL- thrLA-gfp914 and pACYC184-P_PLThe difference of-thrLA-lacZ914 is only that: with spy LacZ gene shown in sequence 15 of the different DNA molecular X instead of sequence table.

pACYC184-P_PL- thrLA-gfp1630 and pACYC184-P_PLThe difference of-thrLA-lacZ1630 is only that: being used LacZ gene shown in sequence 15 of the specific DNA molecular X instead of sequence table.

pACYC184-P_PL- thrLA-gfp1629 and pACYC184-P_PLThe difference of-thrLA-lacZ1629 is only that: being used LacZ gene shown in sequence 15 of the specific DNA molecular X instead of sequence table.

pACYC184-P_PL- thrLA-gfp1628 and pACYC184-P_PLThe difference of-thrLA-lacZ1628 is only that: being used LacZ gene shown in sequence 15 of the specific DNA molecular X instead of sequence table.

pACYC184-P_PL- thrLA-gfp1627 and pACYC184-P_PLThe difference of-thrLA-lacZ1627 is only that: being used LacZ gene shown in sequence 15 of the specific DNA molecular X instead of sequence table.

pACYC184-P_PL- thrLA-gfp913 and pACYC184-P_PLThe difference of-thrLA-lacZ913 is only that: with spy LacZ gene shown in sequence 15 of the different DNA molecular X instead of sequence table.

Specific DNA molecular X are as follows: using pAD123 plasmid as template, carried out using the primer pair that WY1751 and WY1748 is formed The 22nd nucleotide of positive number for the pcr amplification product that PCR amplification obtains to the 10th nucleotide reciprocal (wherein has sequence table Gfp gene shown in sequence 16).

WY1751:TTG GGTTCTGGTTCTGGTTCT ATGAGTAAAGGAGAAGAACTTTTCACT；

WY1748:CGCGGATCCCTTGCATGCCTGCAGGAGAT。

Two, recombinant bacterium is constructed

By pACYC184-P_PL- thrLA-gfp914 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP914.

By pACYC184-P_PL- thrLA-gfp1630 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP1630.

By pACYC184-P_PL- thrLA-gfp1629 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP1629.

By pACYC184-P_PL- thrLA-gfp1628 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP1628.

By pACYC184-P_PL- thrLA-gfp1627 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP1627.

By pACYC184-P_PL- thrLA-gfp913 imports E.coli K-12 W3110 △ metA △ ilvA △ lysA △ Tdh △ tdcC △ sstT, obtains recombinant bacterium, is named as GFP913.

PACYC184 plasmid is imported into E.coli K-12 W3110 △ metA △ ilvA △ lysA △ tdh △ tdcC △ SstT obtains recombinant bacterium, is named as GFP control.

Three, expression of the flow cytometry analysis GFP in cell colony

Test strain are as follows: GFP914, GFP1630, GFP1629, GFP1628, GFP1627, GFP913 or GFP control are (empty White control).

1, test strain is seeded to the LB liquid medium of the chloramphenicol containing 34mg/L, 37 DEG C, 220rpm shaken cultivation 2h, Thalline were collected by centrifugation.

2, the thallus for taking step 1 to obtain is suspended with the PBS buffer solution of pH7.2, obtains OD_600nmThe bacteria suspension that value is 0.5.

3, the bacteria suspension for taking step 2 to obtain is counted with stream type cell analyzer (FACSCalibur type, U.S. company BD) 50000 cells analyze experimental result using FlowJ software.

The corresponding the result is shown in Figure 1 of each test strain and table 2 (average value of 50000 cells).In Fig. 1, flora fluorescence Distribution curve is respectively GFP1627, GFP913, GFP914, GFP1630, GFP1629, GFP1628 and GFP control from right to left. The fluorescence level of GFP1627 improves 30 to 1280 times than other bacterial strains.

Table 2

Embodiment 4 prepares alanine

One, the building of recombinant plasmid

1, double chain DNA molecule shown in the sequence 13 of composition sequence table.

2, the double chain DNA molecule synthesized using step 1 carries out PCR using the primer pair that WY843 and WY1760 is formed as template Amplification, obtains pcr amplification product.

3, the pcr amplification product for taking step 2 to obtain carries out double digestion with restriction enzyme Xba I and BamH I, recycling Digestion products.

4, pACYC184 plasmid is taken, double digestion is carried out with restriction enzyme Xba I and BamH I, recycles carrier framework (about 3.8kb).

5, the digestion products of step 3 are connected with the carrier framework of step 4, obtains recombinant plasmid pACYC184-P_PL2。

6, using the genomic DNA of bacillus subtilis W168 as template, using the primer pair of WY1785 and WY1778 composition PCR amplification is carried out, pcr amplification product is obtained.

7, using the genomic DNA of bacillus subtilis W168 as template, using the primer pair of WY1786 and WY1778 composition PCR amplification is carried out, pcr amplification product is obtained.

8, with I double digestion recombinant plasmid pACYC184-P of restriction enzyme BamH I and Sph_PL2, recycle carrier framework (about 4.2kb).

9, the pcr amplification product for taking step 6 to obtain recycles digestion with I double digestion of restriction enzyme BamH I and Sph Product.

10, the digestion products of step 9 are connected with the carrier framework of step 8, obtains recombinant plasmid, is named as pACYC184-P_PL-ald_WT.According to sequencing result, to pACYC184-P_PL-ald_WTIt carries out structure to be described as follows: by pACYC184 Specific DNA molecular I is inserted between I restriction enzyme site of plasmid Xba I and Sph；Specific DNA molecular I from upstream to downstream successively by Following element composition: promoter P shown in the sequence 13 of sequence table_PL, the restriction endonuclease recognition sequence of restriction enzyme BamH I, sequence Double chain DNA molecule shown in the sequence 17 of list.

11, the pcr amplification product for taking step 7 to obtain recycles enzyme with I double digestion of restriction enzyme BamH I and Sph Cut product.

12, the digestion products of step 11 are connected with the carrier framework of step 8, obtains recombinant plasmid, is named as pACYC184-P_PL-ald_5UTRthrA.According to sequencing result, to pACYC184-P_PL-ald_5UTRthrAIt carries out structure to be described as follows: will Specific DNA molecular II is inserted between I restriction enzyme site of pACYC184 plasmid Xba I and Sph；Specific DNA molecular II from upstream extremely Downstream is successively made of following element: promoter P shown in the sequence 13 of sequence table_PL, the digestion of restriction enzyme BamH I Identify sequence, double chain DNA molecule shown in the sequence 18 of sequence table.

WY843:TGCTCTAGACAATTCCGACGTCTAAGAAA；

WY1760:CGCGGATCCGGTCAGTGCGTCCTGCTGAT；

WY1785:CGCGGATCCCACATATACAGGAGGAGACAGA；

WY1786:

CGCGGATCCGTGCGGGCTTTTTTTTTCGACCAAAGGTAACGAGGTAACAACCATGATCATAGGGGTTCCTAAAGA；

WY1778:ACATGCATGCGTCATAATTCGTGAAATGGTCTCT。

Two, the building of alanine engineering bacteria

By pACYC184-P_PL-ald_WTE. coli k12 W3110 is imported, recombinant bacterium is obtained, is named as E.coli K-12 W3110/pACYC184-P_PL-ald_WT。

By pACYC184-P_PL-ald_5UTRthrAE. coli k12 W3110 is imported, recombinant bacterium is obtained, is named as E.coli K-12 W3110/pACYC184-P_PL-ald_5UTRthrA。

PACYC184 plasmid is imported into e. coli k12 W3110, recombinant bacterium is obtained, is named as E.coli K-12 W3110/pACYC184。

Three, the shake flask fermentation of alanine engineering bacteria

Test strain are as follows: E.coli K-12 W3110/pACYC184-P_PL-ald_WT、E.coli K-12 W3110/ pACYC184-P_PL-ald_5UTRthrAOr E.coli K-12 W3110/pACYC184.

1, test strain is taken, streak inoculation is in the solid LB media plate of the chloramphenicol containing 34mg/L, 37 DEG C of stationary cultures 12 hours.

2, after completing step 1, the lawn on picking plate is seeded in 3mL LB liquid medium, 37 DEG C, 220rpm vibration Culture 12h is swung, seed liquor is obtained.

3, after completing step 2, seed liquor is seeded in 30mL fermentation medium according to 3% inoculum concentration, 37 DEG C, 220rpm shake culture.

Fermentation medium: glucose 20.0g/L, yeast powder 2.0g/L, peptone 4g/L, ammonium sulfate 6.0g/L, di(2-ethylhexyl)phosphate Hydrogen potassium 2.0g/L, epsom salt 1.0g/L, glycine betaine 1.0g/L, calcium carbonate 15.0g/L, micro-mixed liquor 1mL/L are remaining Amount is water.

In incubation, it is set to maintain 6.8-7.0 with the pH value that ammonium hydroxide adjusts reaction system.

It is primary every 3-4h sampling in incubation, glucose content is detected using bio-sensing analyzer SBA-40D, When the glucose content in system is lower than 5g/L, adds glucose and the concentration of glucose in system is made to reach 10g/L.

It is sampled after cultivating 12h and 48h, 12000g is centrifuged 2 minutes, takes supernatant, detects l-Alanine concentration.

After cultivating 12h and 48h, the l-Alanine concentration in fermentation supernatant is shown in that Fig. 2 and table 3 (repeat being averaged for test three times Value ± standard deviation).

Table 3

After culture 12 hours, using the engineering for 5 ' the non-translational region Expression elements regulation ald gene expression that the present invention screens Bacterium E.coli K-12 W3110/pACYC184-P_PL-ald_5UTRthrAL-Alanine is prepared than control strain E.coli K-12 W3110/pACYC184-P_PL-ald_WTImprove 98.6%.After culture 48 hours, using 5 ' the non-translational region tables of the invention screened Up to the engineering bacteria E.coli K-12 W3110/pACYC184-P of element regulation ald gene expression_PL-ald_5UTRthrAPrepare L- third Propylhomoserin is than control strain E.coli K-12 W3110/pACYC184-P_PL-ald_WT40.8% is improved, offer of the present invention is provided The applications of 5 ' non-translational region Expression elements can significantly improve the fermentation yield of alanine.

The detection method of l-Alanine content in fermentation liquid: high-efficient liquid phase technique, in bibliography (amino acid and biology money Source, 2000,22,59-60) it is optimized on the basis of amino acid detection method in, (2,4-dinitrofluorobenzene that the specific method is as follows (FDBN) column front derivation high-efficient liquid phase technique):

It takes 10 μ L supernatants in 2mL centrifuge tube, 200 μ L 0.5M NaHCO is added₃1% (volume of aqueous solution and 100 μ L Than) FDBN- acetonitrile solution, dark place heated at constant temperature 60min, then cools to room temperature in 60 DEG C of water-baths, and 700 μ L are added 0.04mol/L KH₂PO₄Aqueous solution (pH=7.2 ± 0.05 adjusts pH with 40g/L KOH aqueous solution) simultaneously shakes up, and stands 15min, Then it filters and collects filtrate.Filtrate is used for loading, and sample volume is 15 μ L.

Chromatographic column used is C18 column (ZORBAX Eclipse XDB-C18,4.6*150mm, Agilent, USA)；Column temperature: 40℃；Ultraviolet detection wavelength: 360nm；Mobile phase A is 0.04mol/L KH₂PO₄(40g/ is used in pH=7.2 ± 0.05 to aqueous solution 100mL KOH aqueous solution adjusts pH), Mobile phase B is 55% (volume ratio) acetonitrile solution, and mobile phase total flow is 1mL/min.

Elution process: the volume parts that elution initial time (0min) mobile phase A accounts for mobile phase total flow are 86%, flow The volume parts that phase B accounts for mobile phase total flow are 14%；Elution process is divided into 4 stages, mobile phase A and flowing in each stage The volume parts that phase D accounts for mobile phase total flow are linear change；1st stage (be total to since initial time and carry out 2min) terminates When mobile phase A account for that the volume parts of mobile phase total flow are 88%, Mobile phase B accounts for the volume parts of mobile phase total flow and is 12%, mobile phase A accounts for the body of mobile phase total flow at the end of the 2nd stage (since the 1st finish time in stage be total to carry out 2min) Product number is 86%, to account for the volume parts of mobile phase total flow be 14% to Mobile phase B, and the 3rd stage is (from the 2nd finish time in stage Start total to carry out 6min) at the end of mobile phase A account for that the volume parts of mobile phase total flow are 70%, that Mobile phase B accounts for mobile phase is total The volume parts of flow are 30%, and mobile phase A accounts at the end of the 4th stage (be total to since the 3rd finish time in stage and carry out 10min) The volume parts of mobile phase total flow are 30%, to account for the volume parts of mobile phase total flow be 70% to Mobile phase B.

Standard curve is made by standard items of commercially available l-Alanine, calculates the l-Alanine concentration of sample.

Finally, it should be noted that obviously, the above embodiment is merely an example for clearly illustrating the present invention, and simultaneously The non-restriction to embodiment.For those of ordinary skill in the art, it can also do on the basis of the above description Other various forms of variations or variation out.There is no necessity and possibility to exhaust all the enbodiments.And thus drawn The obvious changes or variations that Shen goes out are still in the protection scope of this invention.

Sequence table

<160> 19

<210> 1

<211> 2305

<212> DNA

<213>Escherichia coli

<400> 1

cgttaatgaa atatcgccag ttccacatcc atgcgcaatc agcggtactc agtgatagtg 60

cggtcatggc aatgcttaag cagaaataat cgtgtcacca ttggtgggta ctaaacctga 120

agttcagccc accgggatga gaaaaaatcg cctacgcccc cacatacgcc agattcagca 180

acggatacgg tttccccaaa tcgtccacct cagagcgtcc cgtaacctta aaacccacct 240

tcttatagaa cccaaccgcc tgctcatttt gctcattaac gttggttgtc agttccggtg 300

ccatcgagag cgcatgctcc accagcaccc gacctacgcc gcagccgcgc acatcaggat 360

cgataaacag cgcatccata tgctgcccac ttagcaacat aaatccaacc ggctgatccc 420

gctcattaac cgcgacccac aacggcgctt ccggcaggaa ggaacgaact aggtcctcca 480

gctcggtccg atactctgct gatagaaaat cgtgagtggc atcgacagaa cgacaccaaa 540

tcgcaacgag ttcctcccct tcctcatgcc gtgagcggcg aatactaata accattttct 600

ctccttttag tcattcttat attctaacgt agtcttttcc ttgaaacttt ctcaccttca 660

acatgcaggc tcgacattgg caaattttct ggttatcttc agctatctgg atgtctaaac 720

gtataagcgt atgtagtgag gtaatcaggt tatgccgatt cgtgtgccgg acgagctacc 780

cgccgtcaat ttcttgcgtg aagaaaacgt ctttgtgatg acaacttctc gtgcgtctgg 840

tcaggaaatt cgtccactta aggttctgat ccttaacctg atgccgaaga agattgaaac 900

tgaaaatcag tttctgcgcc tgctttcaaa ctcacctttg caggtcgata ttcagctgtt 960

gcgcatcgat tcccgtgaat cgcgcaacac gcccgcagag catctgaaca acttctactg 1020

taactttgaa gatattcagg atcagaactt tgacggtttg attgtaactg gtgcgccgct 1080

gggcctggtg gagtttaatg atgtcgctta ctggccgcag atcaaacagg tgctggagtg 1140

gtcgaaagat cacgtcacct cgacgctgtt tgtctgctgg gcggtacagg ccgcgctcaa 1200

tatcctctac ggcattccta agcaaactcg caccgaaaaa ctctctggcg tttacgagca 1260

tcatattctc catcctcatg cgcttctgac gcgtggcttt gatgattcat tcctggcacc 1320

gcattcgcgc tatgctgact ttccggcagc gttgattcgt gattacaccg atctggaaat 1380

tctggcagag acggaagaag gggatgcata tctgtttgcc agtaaagata agcgcattgc 1440

ctttgtgacg ggccatcccg aatatgatgc gcaaacgctg gcgcaggaat ttttccgcga 1500

tgtggaagcc ggactagacc cggatgtacc gtataactat ttcccgcaca atgatccgca 1560

aaatacaccg cgagcgagct ggcgtagtca cggtaattta ctgtttacca actggctcaa 1620

ctattacgtc taccagatca cgccatacga tctacggcac atgaatccaa cgctggatta 1680

atcttctgtg atagtcgatc gttaagcgat tcagcacctt acctcaggca ccttcgggtg 1740

ccttttttat ttccgaaacg tacctcagca ggtgaataaa ttttattcat attgttatca 1800

acaagttatc aagtattttt aattaaaatg gaaattgttt ttgattttgc attttaaatg 1860

agtagtctta gttgtgctga acgaaaagag cacaacgatc cttcgttcac agtggggaag 1920

ttttcggatc catgacgagg agctgcacga tgactgaaca ggcaacaaca accgatgaac 1980

tggctttcac aaggccgtat ggcgagcagg agaagcaaat tcttactgcc gaagcggtag 2040

aatttctgac tgagctggtg acgcatttta cgccacaacg caataaactt ctggcagcgc 2100

gcattcagca gcagcaagat attgataacg gaacgttgcc tgattttatt tcggaaacag 2160

cttccattcg cgatgctgat tggaaaattc gcgggattcc tgcggactta gaagaccgcc 2220

gcgtagagat aactggcccg gtagagcgca agatggtgat caacgcgctc aacgccaatg 2280

tgaaagtctt tatggccgat ttcga 2305

<210> 2

<211> 309

<212> PRT

<213>Escherichia coli

<400> 2

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

1 5 10 15

Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu

20 25 30

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

35 40 45

Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln

50 55 60

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

65 70 75 80

Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln

85 90 95

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

100 105 110

Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu

115 120 125

Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu

195 200 205

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

210 215 220

Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala

225 230 235 240

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

245 250 255

Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr

260 265 270

Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp

275 280 285

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

290 295 300

Asn Pro Thr Leu Asp

305

<210> 3

<211> 2889

<212> DNA

<213>Escherichia coli

<400> 3

atggctgtat ccgctcgctg gaacacgcct acagcaaaga cggcggcctg gcggtgctct 60

acggtaactt tgcggaaaac ggctgcatcg tgaaaacggc aggcgtcgat gacagcatcc 120

tcaaattcac cggcccggcg aaagtgtacg aaagccagga cgatgcggta gaagcgattc 180

tcggcggtaa agttgtcgcc ggagatgtgg tagtaattcg ctatgaaggc ccgaaaggcg 240

gtccggggat gcaggaaatg ctctacccaa ccagcttcct gaaatcaatg ggtctcggca 300

aagcctgtgc gctgatcacc gacggtcgtt tctctggtgg cacctctggt ctttccatcg 360

gccacgtctc accggaagcg gcaagcggcg gcagcattgg cctgattgaa gatggtgacc 420

tgatcgctat cgacatcccg aaccgtggca ttcagttaca ggtaagcgat gccgaactgg 480

cggcgcgtcg tgaagcgcag gacgctcgag gtgacaaagc ctggacgccg aaaaatcgtg 540

aacgtcaggt ctcctttgcc ctgcgtgctt atgccagcct ggcaaccagc gccgacaaag 600

gcgcggtgcg cgataaatcg aaactggggg gttaataatg gctgactcgc aacccctgtc 660

cggtgctccg gaaggtgccg aatatttaag agcagtgctg cgcgcgccgg tttacgaggc 720

ggcgcaggtt acgccgctac aaaaaatgga aaaactgtcg tcgcgtcttg ataacgtcat 780

tctggtgaag cgcgaagatc gccagccagt gcacagcttt aagctgcgcg gcgcatacgc 840

catgatggcg ggcctgacgg aagaacagaa agcgcacggc gtgatcactg cttctgcggg 900

taaccacgcg cagggcgtcg cgttttcttc tgcgcggtta ggcgtgaagg ccctgatcgt 960

tatgccaacc gccaccgccg acatcaaagt cgacgcggtg cgcggcttcg gcggcgaagt 1020

gctgctccac ggcgcgaact ttgatgaagc gaaagccaaa gcgatcgaac tgtcacagca 1080

gcaggggttc acctgggtgc cgccgttcga ccatccgatg gtgattgccg ggcaaggcac 1140

gctggcgctg gaactgctcc agcaggacgc ccatctcgac cgcgtatttg tgccagtcgg 1200

cggcggcggt ctggctgctg gcgtggcggt gctgatcaaa caactgatgc cgcaaatcaa 1260

agtgatcgcc gtagaagcgg aagactccgc ctgcctgaaa gcagcgctgg atgcgggtca 1320

tccggttgat ctgccgcgcg tagggctatt tgctgaaggc gtagcggtaa aacgcatcgg 1380

tgacgaaacc ttccgtttat gccaggagta tctcgacgac atcatcaccg tcgatagcga 1440

tgcgatctgt gcggcgatga aggatttatt cgaagatgtg cgcgcggtgg cggaaccctc 1500

tggcgcgctg gcgctggcgg gaatgaaaaa atatatcgcc ctgcacaaca ttcgcggcga 1560

acggctggcg catattcttt ccggtgccaa cgtgaacttc cacggcctgc gctacgtctc 1620

agaacgctgc gaactgggcg aacagcgtga agcgttgttg gcggtgacca ttccggaaga 1680

aaaaggcagc ttcctcaaat tctgccaact gcttggcggg cgttcggtca ccgagttcaa 1740

ctaccgtttt gccgatgcca aaaacgcctg catctttgtc ggtgtgcgcc tgagccgcgg 1800

cctcgaagag cgcaaagaaa ttttgcagat gctcaacgac ggcggctaca gcgtggttga 1860

tctctccgac gacgaaatgg cgaagctaca cgtgcgctat atggtcggcg gacgtccatc 1920

gcatccgttg caggaacgcc tctacagctt cgaattcccg gaatcaccgg gcgcgctgct 1980

gcgcttcctc aacacgctgg gtacgtactg gaacatttct ttgttccact atcgcagcca 2040

tggcaccgac tacgggcgcg tactggcggc gttcgaactt ggcgaccatg aaccggattt 2100

cgaaacccgg ctgaatgagc tgggctacga ttgccacgac gaaaccaata acccggcgtt 2160

caggttcttt ttggcgggtt agggaaaaat gcctgatagc gcttcgctta tcaggcctac 2220

ccgcgcgaca acgtcatttg tggttcggca aaatcttcca gaatgcctca attagcggct 2280

catgtagccg ctttttctgc gcacacacgc ccagctcaaa cggcgttttc tcatcgctgc 2340

gctctaaaat catcacgcgg ttacgcaccg gttcggggct gttttccagc accacttccg 2400

gcaacaatgc cacgccacag ccgagtgcca ccatcgatac catcgcttca tgcccgccaa 2460

ccgtggcgta aatcatcggg ttactgattt tattgcgtcg aaaccacagt tcaatgcggc 2520

ggcgtaccgg cccctgatcg gccataataa acggcaccgt tgaccagtcc ggcttctcta 2580

ccgacacctg attacgcacc gggcagggca gcgcgggggc aatcagcact actgccagat 2640

tctccagcat cgaaaacgcc actgcgccgg gcaaggtttc cggtttaccc gcaatcgcca 2700

gatccgcttc accagtgacc accttttcca tcgcatctgc cgcatcacca gtagtaagtt 2760

taatctccac cgacgggtgt tccgcgcgga agcgatccag aatcggcggc agatggctgt 2820

aggcagcggt caccgagcag aagatatgta attcgccaga gagcgacggc ccttgctgat 2880

cgatggtgt 2889

<210> 4

<211> 514

<212> PRT

<213>Escherichia coli

<400> 4

Met Ala Asp Ser Gln Pro Leu Ser Gly Ala Pro Glu Gly Ala Glu Tyr

1 5 10 15

Leu Arg Ala Val Leu Arg Ala Pro Val Tyr Glu Ala Ala Gln Val Thr

20 25 30

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

35 40 45

Leu Val Lys Arg Glu Asp Arg Gln Pro Val His Ser Phe Lys Leu Arg

50 55 60

Gly Ala Tyr Ala Met Met Ala Gly Leu Thr Glu Glu Gln Lys Ala His

65 70 75 80

Gly Val Ile Thr Ala Ser Ala Gly Asn His Ala Gln Gly Val Ala Phe

85 90 95

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

100 105 110

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

115 120 125

Leu Leu His Gly Ala Asn Phe Asp Glu Ala Lys Ala Lys Ala Ile Glu

130 135 140

Leu Ser Gln Gln Gln Gly Phe Thr Trp Val Pro Pro Phe Asp His Pro

145 150 155 160

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

165 170 175

Asp Ala His Leu Asp Arg Val Phe Val Pro Val Gly Gly Gly Gly Leu

180 185 190

Ala Ala Gly Val Ala Val Leu Ile Lys Gln Leu Met Pro Gln Ile Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Met Lys Asp Leu Phe Glu Asp Val Arg Ala Val Ala Glu Pro Ser

275 280 285

Gly Ala Leu Ala Leu Ala Gly Met Lys Lys Tyr Ile Ala Leu His Asn

290 295 300

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

305 310 315 320

Phe His Gly Leu Arg Tyr Val Ser Glu Arg Cys Glu Leu Gly Glu Gln

325 330 335

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

340 345 350

Leu Lys Phe Cys Gln Leu Leu Gly Gly Arg Ser Val Thr Glu Phe Asn

355 360 365

Tyr Arg Phe Ala Asp Ala Lys Asn Ala Cys Ile Phe Val Gly Val Arg

370 375 380

Leu Ser Arg Gly Leu Glu Glu Arg Lys Glu Ile Leu Gln Met Leu Asn

385 390 395 400

Asp Gly Gly Tyr Ser Val Val Asp Leu Ser Asp Asp Glu Met Ala Lys

405 410 415

Leu His Val Arg Tyr Met Val Gly Gly Arg Pro Ser His Pro Leu Gln

420 425 430

Glu Arg Leu Tyr Ser Phe Glu Phe Pro Glu Ser Pro Gly Ala Leu Leu

435 440 445

Arg Phe Leu Asn Thr Leu Gly Thr Tyr Trp Asn Ile Ser Leu Phe His

450 455 460

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

465 470 475 480

Leu Gly Asp His Glu Pro Asp Phe Glu Thr Arg Leu Asn Glu Leu Gly

485 490 495

Tyr Asp Cys His Asp Glu Thr Asn Asn Pro Ala Phe Arg Phe Phe Leu

500 505 510

Ala Gly

<210> 5

<211> 2565

<212> DNA

<213>Escherichia coli

<400> 5

gtaacacaca cacttcatct aaagagagta attcggtacg ttctgttccc gcaggcgtat 60

ggagcgtttc agtgagtcct aaatcatgac gctgggccga gagccactct tcaagtagcg 120

gtgattcctg gggcacgata tttaagctga catcgggata acgtgccaga aagggttgca 180

ggagctgcgg taaaaaagat tgcgaaaaga ccggcaggca ggcaatagac agttctccct 240

ggcgaaactc gcgcagactt tctgcggcgc tgacaatgcg atccagtccg taccaggatc 300

gttgcacttc ttcaaacaga cgcagtcctt gcacggtagg atgtaatcgc ccacgtacgc 360

gctcaaacaa tttcagcccg atcaccttct caaagcgcgc aagttcgcgg ctgacggttg 420

gctgtgaggt gtgtagcagg tgtgccgcct cagtcaggct tccggcggtc attaccgcat 480

gaaaaatttc aatatgacgt aagttaacgg cggccattag cgctctctcg caatccggta 540

atccatatca tttttgcata gactcgacat aaatcgatat tttttattct ttttatgatg 600

tggcgtaatc ataaaaaagc acttatctgg agtttgttat gccacattca ctgttcagca 660

ccgataccga tctcaccgcc gaaaatctgc tgcgtttgcc cgctgaattt ggctgcccgg 720

tgtgggtcta cgatgcgcaa attattcgtc ggcagattgc agcgctgaaa cagtttgatg 780

tggtgcgctt tgcacagaaa gcctgttcca atattcatat tttgcgctta atgcgtgagc 840

agggcgtgaa agtggattcc gtctcgttag gcgaaataga gcgtgcgttg gcggcgggtt 900

acaatccgca aacgcacccc gatgatattg tttttacggc agatgttatc gatcaggcga 960

cgcttgaacg cgtcagtgaa ttgcaaattc cggtgaatgc gggttctgtt gatatgctcg 1020

accaactggg ccaggtttcg ccagggcatc gggtatggct gcgcgttaat ccggggtttg 1080

gtcacggaca tagccaaaaa accaataccg gtggcgaaaa cagcaagcac ggtatctggt 1140

acaccgatct gcccgccgca ctggacgtga tacaacgtca tcatctgcag ctggtcggca 1200

ttcacatgca cattggttct ggcgttgatt atgcccatct ggaacaggtg tgtggtgcta 1260

tggtgcgtca ggtcatcgaa ttcggtcagg atttacaggc tatttctgcg ggcggtgggc 1320

tttctgttcc ttatcaacag ggtgaagagg cggttgatac cgaacattat tatggtctgt 1380

ggaatgccgc gcgtgagcaa atcgcccgcc atttgggcca ccctgtgaaa ctggaaattg 1440

aaccgggtcg cttcctggta gcgcagtctg gcgtattaat tactcaggtg cggagcgtca 1500

aacaaatggg gagccgccac tttgtgctgg ttgatgccgg gttcaacgat ctgatgcgcc 1560

cggcaatgta cggtagttac caccatatca gtgccctggc agctgatggt cgttctctgg 1620

aacacgcgcc aacggtggaa accgtcgtcg ccggaccgtt atgtgaatcg ggcgatgtct 1680

ttacccagca ggaaggggga aatgttgaaa cccgcgcctt gccggaagtg aaggcaggtg 1740

attatctggt actgcatgat acaggggcat atggcgcatc aatgtcatcc aactacaata 1800

gccgtccgct gttaccagaa gttctgtttg ataatggtca ggcgcggttg attcgccgtc 1860

gccagaccat cgaagaatta ctggcgctgg aattgcttta actgcggtta gtcgctggtt 1920

gcatgatgac ttgcctccag cgacggagtt gacactgaat gacgacgtac cagcgtcgga 1980

ctaaagacat tagtgatttc cgggagaggg cgattatccg ccagcgccaa agccagttcg 2040

gcagcctggg tcgccatcgt cacgattggg taacgcacgg tggtcaggcg cggacgcaca 2100

tagcgtgaca ccagcacatc atcaaagcca attaacgaaa tctcacccgg tacatcaata 2160

ccattatcat tgagaacgcc catcgcaccc gccgccattg aatcgttata acaggctacc 2220

gcagtgaaat ttcttcctcg tcccaaaagc tcggtcattg cctgttcgcc gccgctttcg 2280

tctggttcgc caaatgtcac cagccggtca ttggccgcaa taccactttc agcaagggca 2340

tcgtaatacc cttgcagacg atcttcggcg tcagaaatag agtggttaga gcacagataa 2400

ccaatgcggg tatgaccttg ctgaattaaa tgacgcgttg ccagccaggc accgtaacga 2460

tcgtccagag caatacaacg gttttcaaag ccaggcagga tacggttgat cagcaccata 2520

ccgggcattt gtttcattaa tgaggctaaa tcagcatccg ggatc 2565

<210> 6

<211> 420

<212> PRT

<213>Escherichia coli

<400> 6

Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn

1 5 10 15

Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp

20 25 30

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

35 40 45

Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu

50 55 60

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

65 70 75 80

Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp

85 90 95

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

100 105 110

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

115 120 125

Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn

130 135 140

Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu

145 150 155 160

Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp

165 170 175

Val Ile Gln Arg His His Leu Gln Leu Val Gly Ile His Met His Ile

180 185 190

Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met

195 200 205

Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala

210 215 220

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

225 230 235 240

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

245 250 255

Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe

260 265 270

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

275 280 285

Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp

290 295 300

Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser

370 375 380

Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly

385 390 395 400

Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu Leu Leu Ala

405 410 415

Leu Glu Leu Leu

420

<210> 7

<211> 2460

<212> DNA

<213>Escherichia coli

<400> 7

gctatgccaa caacgatatg caggagctgg aagcacgtct gaaagaagcg cgtgaagccg 60

gtgcgcgtca tgtgctgatc gccaccgatg gtgtgttctc aatggacggc gtgattgcca 120

acctgaaggg cgtttgcgat ctggcagata aatatgatgc cctggtgatg gtagacgact 180

cccacgcggt cggttttgtc ggtgaaaatg gtcgtggttc ccatgaatac tgcgatgtga 240

tgggccgggt cgatattatc accggtacgc ttggtaaagc gctgggcggg gcttctggtg 300

gttataccgc ggcgcgcaaa gaagtggttg agtggctgcg ccagcgttct cgtccgtacc 360

tgttctccaa ctcgctggca ccggccattg ttgccgcgtc catcaaagta ctggagatgg 420

tcgaagcggg cagcgaactg cgtgaccgtc tgtgggcgaa cgcgcgtcag ttccgtgagc 480

aaatgtcggc ggcgggcttt accctggcgg gagccgatca cgccattatt ccggtcatgc 540

ttggtgatgc ggtagtggcg cagaaatttg cccgtgagct gcaaaaagag ggcatttacg 600

ttaccggttt cttctatccg gtcgttccga aaggtcaggc gcgtattcgt acccagatgt 660

ctgcggcgca tacccctgag caaattacgc gtgcagtaga agcatttacg cgtattggta 720

aacaactggg cgttatcgcc tgaggatgtg agatgaaagc gttatccaaa ctgaaagcgg 780

aagagggcat ctggatgacc gacgttcctg taccggaact cgggcataac gatctgctga 840

ttaaaatccg taaaacagcc atctgcggga ctgacgttca catctataac tgggatgagt 900

ggtcgcaaaa aaccatcccg gtgccgatgg tcgtgggcca tgaatatgtc ggtgaagtgg 960

taggtattgg tcaggaagtg aaaggcttca agatcggcga tcgcgtttct ggcgaaggcc 1020

atatcacctg tggtcattgc cgcaactgtc gtggtggtcg tacccatttg tgccgcaaca 1080

cgataggcgt tggtgttaat cgcccgggct gctttgccga atatctggtg atcccggcat 1140

tcaacgcctt caaaatcccc gacaatattt ccgatgactt agccgcaatt tttgatccct 1200

tcggtaacgc cgtgcatacc gcgctgtcgt ttgatctggt gggcgaagat gtgctggttt 1260

ctggtgcagg cccgattggt attatggcag cggcggtggc gaaacacgtt ggtgcacgca 1320

atgtggtgat cactgatgtt aacgaatacc gccttgagct ggcgcgtaaa atgggtatca 1380

cccgtgcggt taacgtcgcc aaagaaaatc tcaatgacgt gatggcggag ttaggcatga 1440

ccgaaggttt tgatgtcggt ctggaaatgt ccggtgcgcc gccagcgttt cgtaccatgc 1500

ttgacaccat gaatcacggc ggccgtattg cgatgctggg tattccgccg tctgatatgt 1560

ctatcgactg gaccaaagtg atctttaaag gcttgttcat taaaggtatt tacggtcgtg 1620

agatgtttga aacctggtac aagatggcgg cgctgattca gtctggcctc gatctttcgc 1680

cgatcattac ccatcgtttc tctatcgatg atttccagaa gggctttgac gctatgcgtt 1740

cgggccagtc cgggaaagtt attctgagct gggattaaca cgaacaaggg ctggtattcc 1800

agccctttta tctgaggata atctgttaaa tatgtaaaat cctgtcagtg taataaagag 1860

ttcgtaattg tgctgatctc ttatatagct gctctcatta tctctctacc ctgaagtgac 1920

tctctcacct gtaaaaataa tatctcacag gcttaatagt ttcttaatac aaagcctgta 1980

aaacgtcagg ataacttcag aggtcgtcgg taatttatga tgaacagcac caataaactt 2040

agtgttatta ttccgttata taatgcgggc gatgatttcc gcacttgtat ggaatcttta 2100

attacgcaaa cctggactgc tctggaaatc attattatta acgatggttc aacggataat 2160

tctgttgaaa tagcaaagta ttacgcagaa aactatccgc acgttcgttt gttgcatcag 2220

gcgaatgctg gcgcatcggt ggcgcgtaat cgtgggattg aagtggcaac gggcaaatat 2280

gtcgcttttg tcgatgctga cgatgaagtc tatcccacca tgtacgaaac gctgatgacc 2340

atggcgttag aggacgacct cgacgtggcg cagtgcaacg ctgactggtg ttttcgtgaa 2400

acgggagaaa cctggcaatc catccccacc gatcgccttc gctcaaccgg cgtattaacc 2460

<210> 8

<211> 341

<212> PRT

<213>Escherichia coli

<400> 8

Met Lys Ala Leu Ser Lys Leu Lys Ala Glu Glu Gly Ile Trp Met Thr

1 5 10 15

Asp Val Pro Val Pro Glu Leu Gly His Asn Asp Leu Leu Ile Lys Ile

20 25 30

Arg Lys Thr Ala Ile Cys Gly Thr Asp Val His Ile Tyr Asn Trp Asp

35 40 45

Glu Trp Ser Gln Lys Thr Ile Pro Val Pro Met Val Val Gly His Glu

50 55 60

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

65 70 75 80

Ile Gly Asp Arg Val Ser Gly Glu Gly His Ile Thr Cys Gly His Cys

85 90 95

Arg Asn Cys Arg Gly Gly Arg Thr His Leu Cys Arg Asn Thr Ile Gly

100 105 110

Val Gly Val Asn Arg Pro Gly Cys Phe Ala Glu Tyr Leu Val Ile Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ile Met Ala Ala Ala Val Ala Lys His Val Gly Ala Arg Asn Val Val

180 185 190

Ile Thr Asp Val Asn Glu Tyr Arg Leu Glu Leu Ala Arg Lys Met Gly

195 200 205

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

210 215 220

Ala Glu Leu Gly Met Thr Glu Gly Phe Asp Val Gly Leu Glu Met Ser

225 230 235 240

Gly Ala Pro Pro Ala Phe Arg Thr Met Leu Asp Thr Met Asn His Gly

245 250 255

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

260 265 270

Trp Thr Lys Val Ile Phe Lys Gly Leu Phe Ile Lys Gly Ile Tyr Gly

275 280 285

Arg Glu Met Phe Glu Thr Trp Tyr Lys Met Ala Ala Leu Ile Gln Ser

290 295 300

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

305 310 315 320

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

325 330 335

Ile Leu Ser Trp Asp

340

<210> 9

<211> 2732

<212> DNA

<213>Escherichia coli

<400> 9

gatgccaaaa ggtgcgccaa aatccaaagt agcggcaacg tgcgactact ccgcagaagt 60

cgttctgcat ggtgataact tcaacgacac tatcgctaaa gtgagcgaaa ttgtcgaaat 120

ggaaggccgt atttttatcc caccttacga tgatccgaaa gtgattgctg gccagggaac 180

gattggtctg gaaattatgg aagatctcta tgatgtcgat aacgtgattg tgccaattgg 240

tggtggcggt ttaattgctg gtattgcggt ggcaattaaa tctattaacc cgaccattcg 300

tgttattggc gtacagtctg aaaacgttca cggcatggcg gcttctttcc actccggaga 360

aataaccacg caccgaacta ccggcaccct ggcggatggt tgtgatgtct cccgcccggg 420

taatttaact tacgaaatcg ttcgtgaatt agtcgatgac atcgtgctgg tcagcgaaga 480

cgaaatcaga aacagtatga ttgccttaat tcagcgcaat aaagtcgtca ccgaaggcgc 540

aggcgctctg gcatgtgctg cattattaag cggtaaatta gaccaatata ttcaaaacag 600

aaaaaccgtc agtattattt ccggcggcaa tatcgatctt tctcgcgtct ctcaaatcac 660

cggtttcgtt gacgcttaat taattcgttg aggataggat atgagtactt cagatagcat 720

tgtatccagc cagacaaaac aatcgtcctg gcgtaaatca gataccacat ggacgttagg 780

cttgtttggt acggcaatcg gcgccggggt gctgttcttc cctatccgcg caggttttgg 840

cggactgatc ccgattcttc tgatgttggt attggcatac cccatcgcgt tttattgcca 900

ccgggcgctg gcgcgtctgt gtctttctgg ctctaaccct tccggcaaca ttacggaaac 960

ggtggaagag cattttggta aaactggcgg cgtggttatc acgttcctgt acttcttcgc 1020

gatttgccca ctgctgtgga tttatggcgt tactattacc aataccttta tgacgttctg 1080

ggaaaaccag ctcggctttg caccgctgaa tcgcggcttt gtggcgctgt tcctgttgct 1140

gctgatggct ttcgtcatct ggtttggtaa ggatctgatg gttaaagtga tgagctacct 1200

ggtatggccg tttatcgcca gcctggtgct gatttctttg tcgctgatcc cttactggaa 1260

ctctgcagtt atcgaccagg ttgacctcgg ttcgctgtcg ttaaccggtc atgacggtat 1320

cctgatcact gtctggctgg ggatttccat catggttttc tcctttaact tctcgccaat 1380

cgtctcttcc ttcgtggttt ctaagcgtga agagtatgag aaagacttcg gtcgcgactt 1440

caccgaacgt aaatgttccc aaatcatttc tcgtgccagc atgctgatgg ttgcagtggt 1500

gatgttcttt gcctttagct gcctgtttac tctgtctccg gccaacatgg cggaagccaa 1560

agcgcagaat attccagtgc tttcttatct ggctaaccac tttgcgtcca tgaccggtac 1620

caaaacaacg ttcgcgatta cactggaata tgcggcttcc atcatcgcac tcgtggctat 1680

cttcaaatct ttcttcggtc actatctggg aacgctggaa ggtctgaatg gcctggtcct 1740

gaagtttggt tataaaggcg acaaaactaa agtgtcgctg ggtaaactga acactatcag 1800

catgatcttc atcatgggct ccacctgggt tgttgcctac gccaacccga acatccttga 1860

cctgattgaa gccatgggcg caccgattat cgcatccctg ctgtgcctgt tgccgatgta 1920

tgccatccgt aaagcgccgt ctctggcgaa ataccgtggt cgtctggata acgtgtttgt 1980

taccgtgatt ggtctgctga ccatcctgaa catcgtatac aaactgtttt aatccgtaac 2040

tcaggatgag aaaagagatg aatgaatttc cggttgtttt ggttattaac tgtggttcgt 2100

cttcgattaa gttttccgtg ctcgatgcca gcgactgtga agtattaatg tcaggtattg 2160

ccgacggtat taactcggaa aatgcattct tatccgtaaa tgggggagag ccagcaccgc 2220

tggctcacca cagctacgaa ggtgcattga aggcaattgc atttgaactg gaaaaacgga 2280

atttaaatga cagtgtggcc ttaattggcc accgcatcgc tcacggcggc agtattttta 2340

ccgagtccgc cattattacc gatgaagtca ttgataatat ccgtcgcgtt tctccactgg 2400

cacccctgca taattacgcc aatttaagtg gtattgaatc ggcgcagcaa ttatttccgg 2460

gcgtaactca ggtggcggta tttgatacca gtttccacca gacgatggct ccggaagctt 2520

atttatacgg cctgccgtgg aaatattatg aagagttagg tgtacgccgt tatggtttcc 2580

acggcacgtc gcaccgctat gtttcccagc gcgcacattc gctgctgaat ctggcggaag 2640

atgactccgg cctggttgtg gcgcatcttg gcaatggcgc gtcaatctgc gcggttcgca 2700

acggtcagag tgttgatacc tcaatgggaa tg 2732

<210> 10

<211> 443

<212> PRT

<213>Escherichia coli

<400> 10

Met Ser Thr Ser Asp Ser Ile Val Ser Ser Gln Thr Lys Gln Ser Ser

1 5 10 15

Trp Arg Lys Ser Asp Thr Thr Trp Thr Leu Gly Leu Phe Gly Thr Ala

20 25 30

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

35 40 45

Leu Ile Pro Ile Leu Leu Met Leu Val Leu Ala Tyr Pro Ile Ala Phe

50 55 60

Tyr Cys His Arg Ala Leu Ala Arg Leu Cys Leu Ser Gly Ser Asn Pro

65 70 75 80

Ser Gly Asn Ile Thr Glu Thr Val Glu Glu His Phe Gly Lys Thr Gly

85 90 95

Gly Val Val Ile Thr Phe Leu Tyr Phe Phe Ala Ile Cys Pro Leu Leu

100 105 110

Trp Ile Tyr Gly Val Thr Ile Thr Asn Thr Phe Met Thr Phe Trp Glu

115 120 125

Asn Gln Leu Gly Phe Ala Pro Leu Asn Arg Gly Phe Val Ala Leu Phe

130 135 140

Leu Leu Leu Leu Met Ala Phe Val Ile Trp Phe Gly Lys Asp Leu Met

145 150 155 160

Val Lys Val Met Ser Tyr Leu Val Trp Pro Phe Ile Ala Ser Leu Val

165 170 175

Leu Ile Ser Leu Ser Leu Ile Pro Tyr Trp Asn Ser Ala Val Ile Asp

180 185 190

Gln Val Asp Leu Gly Ser Leu Ser Leu Thr Gly His Asp Gly Ile Leu

195 200 205

Ile Thr Val Trp Leu Gly Ile Ser Ile Met Val Phe Ser Phe Asn Phe

210 215 220

Ser Pro Ile Val Ser Ser Phe Val Val Ser Lys Arg Glu Glu Tyr Glu

225 230 235 240

Lys Asp Phe Gly Arg Asp Phe Thr Glu Arg Lys Cys Ser Gln Ile Ile

245 250 255

Ser Arg Ala Ser Met Leu Met Val Ala Val Val Met Phe Phe Ala Phe

260 265 270

Ser Cys Leu Phe Thr Leu Ser Pro Ala Asn Met Ala Glu Ala Lys Ala

275 280 285

Gln Asn Ile Pro Val Leu Ser Tyr Leu Ala Asn His Phe Ala Ser Met

290 295 300

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

305 310 315 320

Ile Ile Ala Leu Val Ala Ile Phe Lys Ser Phe Phe Gly His Tyr Leu

325 330 335

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

340 345 350

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

355 360 365

Ile Phe Ile Met Gly Ser Thr Trp Val Val Ala Tyr Ala Asn Pro Asn

370 375 380

Ile Leu Asp Leu Ile Glu Ala Met Gly Ala Pro Ile Ile Ala Ser Leu

385 390 395 400

Leu Cys Leu Leu Pro Met Tyr Ala Ile Arg Lys Ala Pro Ser Leu Ala

405 410 415

Lys Tyr Arg Gly Arg Leu Asp Asn Val Phe Val Thr Val Ile Gly Leu

420 425 430

Leu Thr Ile Leu Asn Ile Val Tyr Lys Leu Phe

435 440

<210> 11

<211> 2645

<212> DNA

<213>Escherichia coli

<400> 11

atagcattcc ggctatcttc gccgtgacca ctgacccgtt cattgtgctg acctcaaacc 60

tgtttgcgat cctcggcctg cgtgcgatgt atttcctgct ggcgggcgta gcagagcgtt 120

tctcgatgct caaatatggc ctggcggtga ttctggtgtt tatcggtatc aagatgctga 180

ttgtcgactt ctaccatatt ccaatcgccg tctcgctggg cgtggtgttt ggcattctgg 240

tgatgacgtt tattatcaac gcctgggtga attatcggca tgataagcag cggggtggat 300

aatttttaat ctgcctaagc cgtgtaccct gtcattaaca tgagcaccgt tttctccctc 360

tcccttccag ggagagggtc ggggtgaggg taatttttcg caccgatgct ggcctgttcc 420

cctcacccta accctctccc caaacggggc gaggggactg accgagtcct tttttgatgt 480

tgtcatcagt ctggaagccg cacgttggct ttatttttat gtcaaagaaa tgtaaccatt 540

aagtttcaaa atatgacctc tctttaaaat ccagcatttt tcgcttcccg aagctgtaac 600

tttccttata ctcgaccttg caaacacttt gttacatcct gaaagatgcg tcgacagaac 660

gcaccaggga tgtgcgacaa cacaatgaaa ggatcgaaaa atgactacgc aacgttcacc 720

ggggctattc cggcgtctgg ctcatggcag cctggtaaaa caaatcctgg tcggccttgt 780

tctggggatt cttctggcat ggatctcaaa acccgcggcg gaagctgttg gtctgttagg 840

tactttgttc gtcggcgcac tgaaagccgt tgcccccatc ctggtgttga tgctggtgat 900

ggcatctatt gctaaccacc agcacgggca gaaaaccaat atccgcccta ttttgttcct 960

ctatctactg ggcaccttct ctgctgctct ggccgcagta gtcttcagct ttgccttccc 1020

ttctaccctg catttatcca gtagcgcggg tgatatttcg ccgccgtcag gcattgtcga 1080

agtgatgcgc gggctggtaa tgagcatggt ttccaacccc atcgacgcgc tgctgaaagg 1140

taactacatc gggattctgg tgtgggcgat cggcctcggc ttcgcactgc gtcacggtaa 1200

cgagaccacc aaaaacctgg ttaacgatat gtcgaatgcc gttaccttta tggtgaaact 1260

ggtcattcgc ttcgcaccga ttggtatttt tgggctggtt tcttctaccc tggcaaccac 1320

cggtttctcc acactgtggg gctacgcgca actgctggtc gtgctggttg gctgtatgtt 1380

actggtggcg ctggtggtta acccattgct ggtgtggtgg aaaattcgtc gtaacccgtt 1440

cccgctggtg ctgctgtgcc tgcgcgaaag cggtgtgtat gccttcttca cccgcagctc 1500

tgcagcgaac attccggtga atatggcgct gtgtgaaaag ctgaatctgg atcgcgatac 1560

ctattccgtt tctattccgc tgggagccac catcaatatg gcgggcgcag caatcactat 1620

taccgtgttg acgctggctg cggttaatac gctgggtatt ccggtcgatc tgcccacggc 1680

gctgctgttg agcgtggtgg cttctctgtg tgcctgtggc gcatccggcg tggcgggggg 1740

gtctctgctg ctgatcccac tggcctgtaa tatgttcggt atttcgaacg atatcgccat 1800

gcaggtggtt gccgtcggct ttatcatcgg cgtattgcag gactcttgcg aaaccgcgct 1860

gaactcttca actgacgtgc tgttcactgc ggcagcttgc caggcagaag acgatcgtct 1920

ggcaaatagc gccctgcgta attaattgtt taaccccttt cgtctacggc ggaaggggtt 1980

ttctcaactt taaacggatc aattcccctt ttctgcatcc gccagaaacg aatgatattc 2040

aggccattca taagcagaaa actaccctca atcatcgtgc cgcctatcga ccccgcccag 2100

aagttgtgaa tcacccagca acacgttgaa aaccacatta cgcagcgcat ggtcagccct 2160

ttacagcaga atagcgccca ggtactgaca atcgtgccga taaccggcaa tagttcgaca 2220

ggatgatgga acttcgcgag gccaattccg ccagtcagca caataaaaat cgccattacc 2280

cataagctgc gcgtgcgtaa ggtaatcaat gtacgaatgg cattaaggat ggcactggca 2340

ccagcgggat aggtgcccag aagaaaaaaa tgtacgccaa taacggcgct atagaccgaa 2400

agctgctttt tgaagcgacg ttcgtcacga ttgaaaaatg ttgtgatacc aatcagaaag 2460

gcgatgacac ccacgccctg ggccagccaa tacgcggtca tgataaatcc ttagcaggta 2520

tggaaaagca aacggcgctt cacattatga aacgccgttt tttattaaca actcatttcg 2580

actttatagc gttacgccgc ttttgaagat cgccagttcg cggaagtcgt tacgctcgtt 2640

acagg 2645

<210> 12

<211> 414

<212> PRT

<213>Escherichia coli

<400> 12

Met Thr Thr Gln Arg Ser Pro Gly Leu Phe Arg Arg Leu Ala His Gly

1 5 10 15

Ser Leu Val Lys Gln Ile Leu Val Gly Leu Val Leu Gly Ile Leu Leu

20 25 30

Ala Trp Ile Ser Lys Pro Ala Ala Glu Ala Val Gly Leu Leu Gly Thr

35 40 45

Leu Phe Val Gly Ala Leu Lys Ala Val Ala Pro Ile Leu Val Leu Met

50 55 60

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

65 70 75 80

Ile Arg Pro Ile Leu Phe Leu Tyr Leu Leu Gly Thr Phe Ser Ala Ala

85 90 95

Leu Ala Ala Val Val Phe Ser Phe Ala Phe Pro Ser Thr Leu His Leu

100 105 110

Ser Ser Ser Ala Gly Asp Ile Ser Pro Pro Ser Gly Ile Val Glu Val

115 120 125

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

130 135 140

Leu Lys Gly Asn Tyr Ile Gly Ile Leu Val Trp Ala Ile Gly Leu Gly

145 150 155 160

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

165 170 175

Met Ser Asn Ala Val Thr Phe Met Val Lys Leu Val Ile Arg Phe Ala

180 185 190

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

195 200 205

Phe Ser Thr Leu Trp Gly Tyr Ala Gln Leu Leu Val Val Leu Val Gly

210 215 220

Cys Met Leu Leu Val Ala Leu Val Val Asn Pro Leu Leu Val Trp Trp

225 230 235 240

Lys Ile Arg Arg Asn Pro Phe Pro Leu Val Leu Leu Cys Leu Arg Glu

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Cys Ala Cys Gly Ala Ser Gly Val Ala Gly Gly Ser Leu Leu Leu Ile

340 345 350

Pro Leu Ala Cys Asn Met Phe Gly Ile Ser Asn Asp Ile Ala Met Gln

355 360 365

Val Val Ala Val Gly Phe Ile Ile Gly Val Leu Gln Asp Ser Cys Glu

370 375 380

Thr Ala Leu Asn Ser Ser Thr Asp Val Leu Phe Thr Ala Ala Ala Cys

385 390 395 400

Gln Ala Glu Asp Asp Arg Leu Ala Asn Ser Ala Leu Arg Asn

405 410

<210> 13

<211> 162

<212> DNA

<213>artificial sequence

<400> 13

caattccgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 60

cacgaggccc tttcgtcttc acctcgagtc cctatcagtg atagagattg acatccctat 120

cagtgataga gatactgagc acatcagcag gacgcactga cc 162

<210> 14

<211> 5020

<212> DNA

<213>artificial sequence

<400> 14

agcttttcat tctgactgca acgggcaata tgtctctgtg tggattaaaa aaagagtgtc 60

tgatagcagc ttctgaactg gttacctgcc gtgagtaaat taaaatttta ttgacttagg 120

tcactaaata ctttaaccaa tataggcata gcgcacagac agataaaaat tacagagtac 180

acaacatcca tgaaacgcat tagcaccacc attaccacca ccatcaccat taccacaggt 240

aacggtgcgg gctgacgcgt acaggaaaca cagaaaaaag cccgcacctg acagtgcggg 300

cttttttttt cgaccaaagg taacgaggta acaaccatgc gagtgttgaa gttcggcggt 360

acatcagtgg caaatgcaga acgttttctg cgtgttgccg atattctgga aagcaatgcc 420

aggcaggggc aggtggccac cgtcctctct gcccccgcca aaatcaccaa ccacctggtg 480

gcgatgattg aaaaaaccat tagcggccag gatgctttac ccaatatcag cgatgccgaa 540

cgtatttttg ccgaactttt gacgggactc gccgccgccc agccggggtt cccgctggcg 600

caattgaaaa ctttcgtcga tcaggaattt gcccaaataa aacatgtcct gcatggcatt 660

agtttgttgg ggcagtgccc ggatagcatc aacgctgcgc tgatttgccg tggcgagaaa 720

atgtcgatcg ccattatggc cggcgtatta gaagcgcgcg gtcacaacgt tactgttatc 780

gatccggtcg aaaaactgct ggcagtgggg cattacctcg aatctaccgt cgatattgct 840

gagtccaccc gccgtattgc ggcaagccgc attccggctg atcacatggt gctgatggca 900

ggtttcaccg ccggtaatga aaaaggcgaa ctggtggtgc ttggacgcaa cggttccgac 960

tactctgctg cggtgctggc tgcctgttta cgcgccgatt gttgcgagat ttggacggac 1020

gttgacgggg tctatacctg cgacccgcgt caggtgcccg atgcgaggtt gttgaagtcg 1080

atgtcctacc aggaagcgat ggagctttcc tacttcggcg ctaaagttct tcacccccgc 1140

accattaccc ccatcgccca gttccagatc ccttgcctga ttaaaaatac cggaaatcct 1200

caagcaccag gtacgctcat tggtgccagc cgtgatgaag acgaattacc ggtcaagggc 1260

atttccaatc tgaataacat ggcaatgttc agcgtttctg gtccggggat gaaagggatg 1320

gtcggcatgg cggcgcgcgt ctttgcagcg atgtcacgcg cccgtatttc cgtggtgctg 1380

attacgcaat catcttccga atacagcatc agtttctgcg ttccacaaag cgactgtgtg 1440

cgagctgaac gggcaatgca ggaagagttc tacctggaac tgaaagaagg cttactggag 1500

ccgctggcag tgacggaacg gctggccatt atctcggtgg taggtgatgg tatgcgcacc 1560

ttgcgtggga tctcggcgaa attctttgcc gcactggccc gcgccaatat caacattgtc 1620

gccattgctc agggatcttc tgaacgctca atctctgtcg tggtaaataa cgatgatgcg 1680

accactggcg tgcgcgttac tcatcagatg ctgttcaata ccgatcaggt tatcgaagtg 1740

tttgtgattg gcgtcggtgg cgttggcggt gcgctgctgg agcaactgaa gcgtcagcaa 1800

agctggctga agaataaaca tatcgactta cgtgtctgcg gtgttgccaa ctcgaaggct 1860

ctgctcacca atgtacatgg ccttaatctg gaaaactggc aggaagaact ggcgcaagcc 1920

aaagagccgt ttaatctcgg gcgcttaatt cgcctcgtga aagaatatca tctgctgaac 1980

ccggtcattg ttgactgcac ttccagccag gcagtggcgg atcaatatgc cgacttcctg 2040

cgcgaaggtt tccacgttgt cacgccgaac aaaaaggcca acacctcgtc gatggattac 2100

taccatcagt tgcgttatgc ggcggaaaaa tcgcggcgta aattcctcta tgacaccaac 2160

gttggggctg gattaccggt tattgagaac ctgcaaaatc tgctcaatgc aggtgatgaa 2220

ttgatgaagt tctccggcat tctttctggt tcgctttctt atatcttcgg caagttagac 2280

gaaggcatga gtttctccga ggcgaccacg ctggcgcggg aaatgggtta taccgaaccg 2340

gacccgcgag atgatctttc tggtatggat gtggcgcgta aactattgat tctcgctcgt 2400

gaaacgggac gtgaactgga gctggcggat attgaaattg aacctgtgct gcccgcagag 2460

tttaacgccg agggtgatgt tgccgctttt atggcgaatc tgtcacaact cgacgatctc 2520

tttgccgcgc gcgtggcgaa ggcccgtgat gaaggaaaag ttttgcgcta tgttggcaat 2580

attgatgaag atggcgtctg ccgcgtgaag attgccgaag tggatggtaa tgatccgctg 2640

ttcaaagtga aaaatggcga aaacgccctg gccttctata gccactatta tcagccgctg 2700

ccgttggtac tgcgcggata tggtgcgggc aatgacgtta cagctgccgg tgtctttgct 2760

gatctgctac gtaccctctc atggaagtta ggagtctgac atggttaaag tttatgcccc 2820

ggcttccagt gccaatatga gcgtcgggtt tgatgtgctc ggggcggcgg tgacacctgt 2880

tgatggtgca ttgctcggag atgtagtcac ggttgaggcg gcagagacat tcagtctcaa 2940

caacctcgga cgctttgccg ataagctgcc gtcagaacca cgggaaaata tcgtttatca 3000

gtgctgggag cgtttttgcc aggaactggg taagcaaatt ccagtggcga tgaccctgga 3060

aaagaatatg ccgatcggtt cgggcttagg ctccagtgcc tgttcggtgg tcgcggcgct 3120

gatggcgatg aatgaacact gcggcaagcc gcttaatgac actcgtttgc tggctttgat 3180

gggcgagctg gaaggccgta tctccggcag cattcattac gacaacgtgg caccgtgttt 3240

tctcggtggt atgcagttga tgatcgaaga aaacgacatc atcagccagc aagtgccagg 3300

gtttgatgag tggctgtggg tgctggcgta tccggggatt aaagtctcga cggcagaagc 3360

cagggctatt ttaccggcgc agtatcgccg ccaggattgc attgcgcacg ggcgacatct 3420

ggcaggcttc attcacgcct gctattcccg tcagcctgag cttgccgcga agctgatgaa 3480

agatgttatc gctgaaccct accgtgaacg gttactgcca ggcttccggc aggcgcggca 3540

ggcggtcgcg gaaatcggcg cggtagcgag cggtatctcc ggctccggcc cgaccttgtt 3600

cgctctgtgt gacaagccgg aaaccgccca gcgcgttgcc gactggttgg gtaagaacta 3660

cctgcaaaat caggaaggtt ttgttcatat ttgccggctg gatacggcgg gcgcacgagt 3720

actggaaaac taaatgaaac tctacaatct gaaagatcac aacgagcagg tcagctttgc 3780

gcaagccgta acccaggggt tgggcaaaaa tcaggggctg ttttttccgc acgacctgcc 3840

ggaattcagc ctgactgaaa ttgatgagat gctgaagctg gattttgtca cccgcagtgc 3900

gaagatcctc tcggcgttta ttggtgatga aatcccacag gaaatcctgg aagagcgcgt 3960

gcgcgcggcg tttgccttcc cggctccggt cgccaatgtt gaaagcgatg tcggttgtct 4020

ggaattgttc cacgggccaa cgctggcatt taaagatttc ggcggtcgct ttatggcaca 4080

aatgctgacc catattgcgg gtgataagcc agtgaccatt ctgaccgcga cctccggtga 4140

taccggagcg gcagtggctc atgctttcta cggtttaccg aatgtgaaag tggttatcct 4200

ctatccacga ggcaaaatca gtccactgca agaaaaactg ttctgtacat tgggcggcaa 4260

tatcgaaact gttgccatcg acggcgattt cgatgcctgt caggcgctgg tgaagcaggc 4320

gtttgatgat gaagaactga aagtggcgct agggttaaac tcggctaact cgattaacat 4380

cagccgtttg ctggcgcaga tttgctacta ctttgaagct gttgcgcagc tgccgcagga 4440

gacgcgcaac cagctggttg tctcggtgcc aagcggaaac ttcggcgatt tgacggcggg 4500

tctgctggcg aagtcactcg gtctgccggt gaaacgtttt attgctgcga ccaacgtgaa 4560

cgataccgtg ccacgtttcc tgcacgacgg tcagtggtca cccaaagcga ctcaggcgac 4620

gttatccaac gcgatggacg tgagtcagcc gaacaactgg ccgcgtgtgg aagagttgtt 4680

ccgccgcaaa atctggcaac tgaaagagct gggttatgca gccgtggatg atgaaaccac 4740

gcaacagaca atgcgtgagt taaaagaact gggctacact tcggagccgc acgctgccgt 4800

agcttatcgt gcgctgcgtg atcagttgaa tccaggcgaa tatggcttgt tcctcggcac 4860

cgcgcatccg gcgaaattta aagagagcgt ggaagcgatt ctcggtgaaa cgttggatct 4920

gccaaaagag ctggcagaac gtgctgattt acccttgctt tcacataatc tgcccgccga 4980

ttttgctgcg ttgcgtaaat tgatgatgaa tcatcagtaa 5020

<210> 15

<211> 3113

<212> DNA

<213>artificial sequence

<400> 15

atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 60

ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc 120

gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc 180

tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct 240

gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc 300

tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg 360

acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg 420

cgaattattt ttgatggcgt taactcggcg tttcatctgt ggtgcaacgg gcgctgggtc 480

ggttacggcc aggacagtcg tttgccgtct gaatttgacc tgagcgcatt tttacgcgcc 540

ggagaaaacc gcctcgcggt gatggtgctg cgctggagtg acggcagtta tctggaagat 600

caggatatgt ggcggatgag cggcattttc cgtgacgtct cgttgctgca taaaccgact 660

acacaaatca gcgatttcca tgttgccact cgctttaatg atgatttcag ccgcgctgta 720

ctggaggctg aagttcagat gtgcggcgag ttgcgtgact acctacgggt aacagtttct 780

ttatggcagg gtgaaacgca ggtcgccagc ggcaccgcgc ctttcggcgg tgaaattatc 840

gatgagcgtg gtggttatgc cgatcgcgtc acactacgtc tgaacgtcga aaacccgaaa 900

ctgtggagcg ccgaaatccc gaatctctat cgtgcggtgg ttgaactgca caccgccgac 960

ggcacgctga ttgaagcaga agcctgcgat gtcggtttcc gcgaggtgcg gattgaaaat 1020

ggtctgctgc tgctgaacgg caagccgttg ctgattcgag gcgttaaccg tcacgagcat 1080

catcctctgc atggtcaggt catggatgag cagacgatgg tgcaggatat cctgctgatg 1140

aagcagaaca actttaacgc cgtgcgctgt tcgcattatc cgaaccatcc gctgtggtac 1200

acgctgtgcg accgctacgg cctgtatgtg gtggatgaag ccaatattga aacccacggc 1260

atggtgccaa tgaatcgtct gaccgatgat ccgcgctggc taccggcgat gagcgaacgc 1320

gtaacgcgaa tggtgcagcg cgatcgtaat cacccgagtg tgatcatctg gtcgctgggg 1380

aatgaatcag gccacggcgc taatcacgac gcgctgtatc gctggatcaa atctgtcgat 1440

ccttcccgcc cggtgcagta tgaaggcggc ggagccgaca ccacggccac cgatattatt 1500

tgcccgatgt acgcgcgcgt ggatgaagac cagcccttcc cggctgtgcc gaaatggtcc 1560

atcaaaaaat ggctttcgct acctggagag acgcgcccgc tgatcctttg cgaatacgcc 1620

cacgcgatgg gtaacagtct tggcggtttc gctaaatact ggcaggcgtt tcgtcagtat 1680

ccccgtttac agggcggctt cgtctgggac tgggtggatc agtcgctgat taaatatgat 1740

gaaaacggca acccgtggtc ggcttacggc ggtgattttg gcgatacgcc gaacgatcgc 1800

cagttctgta tgaacggtct ggtctttgcc gaccgcacgc cgcatccagc gctgacggaa 1860

gcaaaacacc agcagcagtt tttccagttc cgtttatccg ggcaaaccat cgaagtgacc 1920

agcgaatacc tgttccgtca tagcgataac gagctcctgc actggatggt ggcgctggat 1980

ggtaagccgc tggcaagcgg tgaagtgcct ctggatgtcg ctccacaagg taaacagttg 2040

attgaactgc ctgaactacc gcagccggag agcgccgggc aactctggct cacagtacgc 2100

gtagtgcaac cgaacgcgac cgcatggtca gaagccgggc acatcagcgc ctggcagcag 2160

tggcgtctgg cggaaaacct cagtgtgacg ctccccgccg cgtcccacgc catcccgcat 2220

ctgaccacca gcgaaatgga tttttgcatc gagctgggta ataagcgttg gcaatttaac 2280

cgccagtcag gctttctttc acagatgtgg attggcgata aaaaacaact gctgacgccg 2340

ctgcgcgatc agttcacccg tgcaccgctg gataacgaca ttggcgtaag tgaagcgacc 2400

cgcattgacc ctaacgcctg ggtcgaacgc tggaaggcgg cgggccatta ccaggccgaa 2460

gcagcgttgt tgcagtgcac ggcagataca cttgctgatg cggtgctgat tacgaccgct 2520

cacgcgtggc agcatcaggg gaaaacctta tttatcagcc ggaaaaccta ccggattgat 2580

ggtagtggtc aaatggcgat taccgttgat gttgaagtgg cgagcgatac accgcatccg 2640

gcgcggattg gcctgaactg ccagctggcg caggtagcag agcgggtaaa ctggctcgga 2700

ttagggccgc aagaaaacta tcccgaccgc cttactgccg cctgttttga ccgctgggat 2760

ctgccattgt cagacatgta taccccgtac gtcttcccga gcgaaaacgg tctgcgctgc 2820

gggacgcgcg aattgaatta tggcccacac cagtggcgcg gcgacttcca gttcaacatc 2880

agccgctaca gtcaacagca actgatggaa accagccatc gccatctgct gcacgcggaa 2940

gaaggcacat ggctgaatat cgacggtttc catatgggga ttggtggcga cgactcctgg 3000

agcccgtcag tatcggcgga attccagctg agcgccggtc gctaccatta ccagttggtc 3060

tggtgtcaaa aataataata accgggcagg ccatgtctgc ccgtatttcg cgt 3113

<210> 16

<211> 717

<212> DNA

<213>artificial sequence

<400> 16

atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60

gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120

aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180

gtcactactt tcgggtatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag 240

catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300

aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360

aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420

ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480

atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540

cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600

ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660

cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717

<210> 17

<211> 1242

<212> DNA

<213>artificial sequence

<400> 17

cacatataca ggaggagaca gatatgatca taggggttcc taaagagata aaaaacaatg 60

aaaaccgtgt cgcattaaca cccgggggcg tttctcagct catttcaaac ggccaccggg 120

tgctggttga aacaggcgcg ggccttggaa gcggatttga aaatgaagcc tatgagtcag 180

caggagcgga aatcattgct gatccgaagc aggtctggga cgccgaaatg gtcatgaaag 240

taaaagaacc gctgccggaa gaatatgttt attttcgcaa aggacttgtg ctgtttacgt 300

accttcattt agcagctgag cctgagcttg cacaggcctt gaaggataaa ggagtaactg 360

ccatcgcata tgaaacggtc agtgaaggcc ggacattgcc tcttctgacg ccaatgtcag 420

aggttgcggg cagaatggca gcgcaaatcg gcgctcaatt cttagaaaag cctaaaggcg 480

gaaaaggcat tctgcttgcc ggggtgcctg gcgtttcccg cggaaaagta acaattatcg 540

gaggaggcgt tgtcgggaca aacgcggcga aaatggctgt cggcctcggt gcagatgtga 600

cgatcattga cttaaacgca gaccgcttgc gccagcttga tgacatcttc ggccatcaga 660

ttaaaacgtt aatttctaat ccggtcaata ttgctgatgc tgtggcggaa gcggatctcc 720

tcatttgcgc ggtattaatt ccgggtgcta aagctccgac tcttgtcact gaggaaatgg 780

taaaacaaat gaaacccggt tcagttattg ttgatgtagc gatcgaccaa ggcggcatcg 840

tcgaaactgt cgaccatatc acaacacatg atcagccaac atatgaaaaa cacggggttg 900

tgcattatgc tgtagcgaac atgccaggcg cagtccctcg tacatcaaca atcgccctga 960

ctaacgttac tgttccatac gcgctgcaaa tcgcgaacaa aggggcagta aaagcgctcg 1020

cagacaatac ggcactgaga gcgggtttaa acaccgcaaa cggacacgtg acctatgaag 1080

ctgtagcaag agatctaggc tatgagtatg ttcctgccga gaaagcttta caggatgaat 1140

catctgtggc gggtgcttaa ttcacaataa gcttgcagaa agatttctgc aggctttttt 1200

attttttaaa aggaaaaaag agaccatttc acgaattatg ac 1242

<210> 18

<211> 1262

<212> DNA

<213>artificial sequence

<400> 18

gtgcgggctt tttttttcga ccaaaggtaa cgaggtaaca accatgatca taggggttcc 60

taaagagata aaaaacaatg aaaaccgtgt cgcattaaca cccgggggcg tttctcagct 120

catttcaaac ggccaccggg tgctggttga aacaggcgcg ggccttggaa gcggatttga 180

aaatgaagcc tatgagtcag caggagcgga aatcattgct gatccgaagc aggtctggga 240

cgccgaaatg gtcatgaaag taaaagaacc gctgccggaa gaatatgttt attttcgcaa 300

aggacttgtg ctgtttacgt accttcattt agcagctgag cctgagcttg cacaggcctt 360

gaaggataaa ggagtaactg ccatcgcata tgaaacggtc agtgaaggcc ggacattgcc 420

tcttctgacg ccaatgtcag aggttgcggg cagaatggca gcgcaaatcg gcgctcaatt 480

cttagaaaag cctaaaggcg gaaaaggcat tctgcttgcc ggggtgcctg gcgtttcccg 540

cggaaaagta acaattatcg gaggaggcgt tgtcgggaca aacgcggcga aaatggctgt 600

cggcctcggt gcagatgtga cgatcattga cttaaacgca gaccgcttgc gccagcttga 660

tgacatcttc ggccatcaga ttaaaacgtt aatttctaat ccggtcaata ttgctgatgc 720

tgtggcggaa gcggatctcc tcatttgcgc ggtattaatt ccgggtgcta aagctccgac 780

tcttgtcact gaggaaatgg taaaacaaat gaaacccggt tcagttattg ttgatgtagc 840

gatcgaccaa ggcggcatcg tcgaaactgt cgaccatatc acaacacatg atcagccaac 900

atatgaaaaa cacggggttg tgcattatgc tgtagcgaac atgccaggcg cagtccctcg 960

tacatcaaca atcgccctga ctaacgttac tgttccatac gcgctgcaaa tcgcgaacaa 1020

aggggcagta aaagcgctcg cagacaatac ggcactgaga gcgggtttaa acaccgcaaa 1080

cggacacgtg acctatgaag ctgtagcaag agatctaggc tatgagtatg ttcctgccga 1140

gaaagcttta caggatgaat catctgtggc gggtgcttaa ttcacaataa gcttgcagaa 1200

agatttctgc aggctttttt attttttaaa aggaaaaaag agaccatttc acgaattatg 1260

ac 1262

<210> 19

<211> 378

<212> PRT

<213>artificial sequence

<400> 19

Met Ile Ile Gly Val Pro Lys Glu Ile Lys Asn Asn Glu Asn Arg Val

1 5 10 15

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

20 25 30

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

35 40 45

Ala Tyr Glu Ser Ala Gly Ala Glu Ile Ile Ala Asp Pro Lys Gln Val

50 55 60

Trp Asp Ala Glu Met Val Met Lys Val Lys Glu Pro Leu Pro Glu Glu

65 70 75 80

Tyr Val Tyr Phe Arg Lys Gly Leu Val Leu Phe Thr Tyr Leu His Leu

85 90 95

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

100 105 110

Ala Ile Ala Tyr Glu Thr Val Ser Glu Gly Arg Thr Leu Pro Leu Leu

115 120 125

Thr Pro Met Ser Glu Val Ala Gly Arg Met Ala Ala Gln Ile Gly Ala

130 135 140

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

145 150 155 160

Val Pro Gly Val Ser Arg Gly Lys Val Thr Ile Ile Gly Gly Gly Val

165 170 175

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

180 185 190

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

195 200 205

Phe Gly His Gln Ile Lys Thr Leu Ile Ser Asn Pro Val Asn Ile Ala

210 215 220

Asp Ala Val Ala Glu Ala Asp Leu Leu Ile Cys Ala Val Leu Ile Pro

225 230 235 240

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

245 250 255

Lys Pro Gly Ser Val Ile Val Asp Val Ala Ile Asp Gln Gly Gly Ile

260 265 270

Val Glu Thr Val Asp His Ile Thr Thr His Asp Gln Pro Thr Tyr Glu

275 280 285

Lys His Gly Val Val His Tyr Ala Val Ala Asn Met Pro Gly Ala Val

290 295 300

Pro Arg Thr Ser Thr Ile Ala Leu Thr Asn Val Thr Val Pro Tyr Ala

305 310 315 320

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

325 330 335

Ala Leu Arg Ala Gly Leu Asn Thr Ala Asn Gly His Val Thr Tyr Glu

340 345 350

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

355 360 365

Leu Gln Asp Glu Ser Ser Val Ala Gly Ala

370 375

Claims

1.DNA molecule first, as shown in the 294th to 606 nucleotide of sequence 14 of sequence table.

2. DNA molecular first described in claim 1 is promoting the application in destination gene expression as controlling element.

3. application as claimed in claim 2, it is characterised in that: the starting that the DNA molecular first is located at the target gene is close Between numeral and the promoter of the target gene.

4.DNA molecule second, successively includes following element from upstream to downstream: DNA molecular first, mesh described in promoter, claim 1 Gene.

5.DNA molecule third successively includes following element from upstream to downstream: DNA molecular first described in claim 1, the third ammonia of coding The gene of acidohydrogenase.

6.DNA molecule fourth, successively includes following element from upstream to downstream: DNA molecular third described in promoter, claim 5.

7. the recombinant plasmid second containing DNA molecular second described in claim 4 or the weight containing DNA molecular third described in claim 5 Group plasmid third or the recombinant plasmid fourth containing DNA molecular fourth described in claim 6.

8. the recombinant bacterium second containing DNA molecular second described in claim 4 or the recombination containing DNA molecular third described in claim 5 Bacterium third or recombinant bacterium fourth containing DNA molecular fourth described in claim 6.

9. application of the recombinant bacterium third or recombinant bacterium fourth described in claim 8 in production l-Alanine.