CN108456668A - A kind of ribosome bind site, recombinant expression plasmid, transformant and its application - Google Patents

Abstract

First aspect present invention is related to a kind of ribosome bind site, second aspect is related to including the recombinant expression plasmid of this ribosome bind site, the third aspect is related to including the transformant of the recombinant expression plasmid, the invention further relates to this transformants in fermenting and producing polypeptide, and the further application of the aspect of 1,5 pentanediamine of fermenting and producing.The invention is characterized in that the sequence 5 ' 3 ' of ribosome bind site includes one of sequence as follows：a)、GTCATCAAAA；b)、CGCAAAGATA；c)、AAAGATTTAC.The present invention can reduce the toxic action that 1,5 pentanediamines utilize microbial cell glucose production L lysines during one-step method is using 1,5 pentanediamine of glucose production, improve the yield of 1,5 pentanediamines.

Description

A kind of ribosome bind site, recombinant expression plasmid, transformant and its application

Technical field

First aspect present invention is related to a kind of ribosome bind site, and second aspect is related to including this ribosome binding site The recombinant expression plasmid of point, the third aspect are related to including the transformant of the recombinant expression plasmid, and the invention further relates to this turn Beggar is in fermenting and producing polypeptide, and further fermenting and producing 1, the application of the aspect of 5- pentanediamines.

Background technology

Lysine decarboxylase (L-lysine decarboxylase, abbreviation LDC, EC 4.1.1.18) is widely present in micro- Biology, insect, in animal and higher plant, L-lysine can be sloughed a carboxyl generate 1,5- pentanediamines (cadaverine) and CO2.And the purposes of 1,5- pentanediamine is quite extensive, such as polymerisation synthesizing new nylon can be carried out with binary acid, in work There is very high application value in industry production.Currently, microbial method 1, the production of 5- pentanediamines mainly uses following two methods, micro- Biofermentation produces (abbreviation one-step method) and microbial body exoenzyme catalytic production 1,5- pentanediamines (abbreviation two-step method).

105441497 A of Chinese patent CN are (IPTG/ lactose) inducible promoter-signal peptide-by building structure The expression cassette of lysine decarboxylase gene-terminator so that a large amount of secreting, expressings of lysine decarboxylase gene.First by can be with The Escherichia coli of high-yield L-lysine or Corynebacterium glutamicum strain produce L-lysine using glucose fermentation, then pass through addition The derivants inducing lysine decarboxylase secreting, expressing such as IPTG/ lactose, catalysis L-lysine are converted into cadaverine.It may be implemented in this way 1,5- pentanediamines, i.e. microbial fermentation production L-lysine and coupling catalysed L-lysine decarboxylation are produced using glucose by one step Reaction generates 1,5- pentanediamines.(abbreviation one-step method) but the system leak expression other than needing additional addition IPTG/ lactose Situation is also than more serious, since 1, the 5- pentanediamine concentration that thalline itself is resistant to is limited, if fermentation system mid-early stage expresses 1, the 5- pentanediamines that lysine decarboxylase is converted into are excessive, will cause to poison to thalline, to inhibit thalli growth and profit With process (Qian, the et al., Biotechnol.Bioeng.2011 of glucose production L-lysine；108:93–103). During glucose by one step produces 1,5- pentanediamines, if using constitutive promoter, it can effectively save and ferment The cost of the derivant added in journey, but the expression quantity of lysine decarboxylase must be controlled, otherwise earlier fermentation generate 1,5- Pentanediamine can seriously inhibit the growth of thalline so that fermentation process is interrupted.If the table of regulation and control lysine decarboxylase can be attempted The production quantity of 1,5- pentanediamines is reduced up to amount so that 1, the 5- pentanediamines that are converted into thalli growth and will not utilize Portugal The process that grape sugar produces L-lysine generates depression effect, and if things go on like this, thalline also will produce in incubation to 1,5- penta The tolerance of diamines realizes abundant conversion of the last lysine to 1,5- pentanediamines.But it must assure that remaining lysine simultaneously Cannot be too many, otherwise one side lysine can check the activity that relevant enzyme is synthesized in lysine, on the other hand be given birth in one-step method Produce 1,5- pentanediamines after fermentation, it is necessary to additionally add a large amount of lysine decarboxylase again and convert remaining lysine.

Invention content

In order to screen the mutant strain of different lysine decarboxylase expression quantity, the method pair of nonideal explosives may be used The ribosome bind site (RBS) for influencing gene translation level is mutated, in particular by by the gene cloning of zymoprotein Into a suitable expression vector, then purposive design synthesizes one group of random primer and carries out random mutation to RBS sequences, By high flux screening and method for detecting enzymatic activity is combined, to obtain the mutant plasmid with expected phenotype.

The present invention carries out the sites RBS before lysine decarboxylase gene start codon by the means of nonideal explosives Random mutation, and high flux screening wild-type lysine decarboxylase compared with the method for enzyme activity determination obtains one group of activity is combined to go out The mutant strain now reduced after the RBS sequences for identifying mutation, mutant plasmid is converted into Strains for Lysine Production, Neng Gou One-step method utilizes glucose production 1, reduces toxic action of 1, the 5- pentanediamines to microbial cell during 5- pentanediamines, carries The yield of high 1,5- pentanediamines will have important meaning and help to industrial production.

The first aspect of the present invention purpose is to propose a kind of ribosome bind site, can reduce during the fermentation Expression product and (or) toxic action of the subsequent products to microbial cell, such as can one-step method utilize glucose production 1, Toxic action of 1, the 5- pentanediamines to microbial cell is reduced during 5- pentanediamines, improves the yield of 1,5- pentanediamines.

The sequence 5 ' -3 ' of a kind of ribosome bind site, the ribosome bind site includes that (either) is as follows One of sequence：

a)、GTCATCAAAA；

b)、CGCAAAGATA；

c)、AAAGATTTAC。

The second aspect of the present invention purpose is to propose a kind of recombinant expression plasmid, can reduce table during the fermentation Up to product and (or) toxic action of the subsequent products to microbial cell, such as can one-step method utilize glucose production 1,5- Toxic action of 1, the 5- pentanediamines to microbial cell is reduced during pentanediamine, improves the yield of 1,5- pentanediamines.

A kind of recombinant expression plasmid, including the target gene of recombinant expression plasmid and corresponding ribosome bind site, institute Ribosome bind site is stated as described in any technical solution above, it is preferable that the ribosome bind site is located at initiation codon Sub- upstream -15 arrives -6.

Further, a kind of recombinant expression plasmid, skeleton plasmid are the reproducible skeleton matter in host cell Grain, it is excellent when host cell is in Escherichia coli (Escherichia coli) and hafnia alvei (Hafnia alvei) Select one kind in pUC, pBR322, pACYC plasmid and their plasmid.

Based on the above technical solution further, the target gene of the recombinant expression plasmid is to encode gathering for polypeptide Nucleotide；The polypeptide is enzyme or polypeptide drug；Further, the enzyme is oxidoreducing enzyme, transferase, hydrolysis At least one of enzyme, lyase, isomerase and ligase, the polypeptide drug are in hormone, antibody and growth factor It is at least one；Further, the lyase is decarboxylase, and still further, the decarboxylase is amino acid decarboxylases, is such as relied Propylhomoserin decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase.Again further, it compiles The polynucleotide of code lysine decarboxylase is can to derive from microorganism, and the cell of animal or plant such as derives from Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), Alkaliphilic bacillus (Bacillus Halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), Corynebacterium glutamicum (Corynebacterium glutamicum) or klebsiella oxytoca (Klebsiella Oxytoca lysine decarboxylase gene)；The lysine decarboxylase gene can be from above-mentioned bacterial strains by mutagenesis or Bacterial strain after random mutation or genetic engineering bacterium.It is further preferred that the polynucleotide of coding lysine decarboxylase is cadA The segment of gene, ldcC genes, haldc genes, the segment of cadA genes, the segment of ldcC genes or haldc genes；More into one Preferably, the lysine decarboxylase gene is derived from the induction type lysine decarboxylase gene C adA/ of Escherichia coli to step LdcI(SEQ ID No:1).Lysine decarboxylase can also be that the mutant of the lysine decarboxylase in above-mentioned source is (including natural Mutant and artificial recombination mutant) or active fragment.

The third aspect of the present invention purpose is to propose a kind of transformant, can reduce expression product during the fermentation And (or) toxic action of the subsequent products to microbial cell, for example glucose production 1,5- pentanediamines can be utilized in one-step method During reduce 1,5- pentanediamines to microbial cell utilize glucose production L-lysine toxic action, improve 1,5- penta The yield of diamines.

A kind of transformant, the recombinant expression plasmid of the transformant are the recombination tables as described in any one technical solution above Up to plasmid.

Or a kind of transformant, the genome of the transformant include target gene and corresponding ribosome bind site, The ribosome bind site is it is preferable to which ground, the ribosome bind site are located at upstream from start codon -15 To -6；The target gene is the polynucleotide for encoding polypeptide；The polypeptide is enzyme or polypeptide drug；Further preferably Ground, the enzyme are at least one of oxidoreducing enzyme, transferase, hydrolase, lyase, isomerase and ligase, described Polypeptide drug is at least one of hormone, antibody and growth factor；Further preferably, the lyase is decarboxylation Enzyme, it is further preferred that the decarboxylase is amino acid decarboxylases, such as lysine decarboxylase, tyrosine decarboxylase, arginine Decarboxylase, ornithine decarboxylase or glutamate decarboxylase.

Further, the target gene of the recombinant expression plasmid of the transformant is the poly-nuclear glycosides for encoding lysine decarboxylase Acid, further, the polynucleotide of the coding lysine decarboxylase are derived from Escherichia coli (E.coli), bacillus subtilis Bacterium (B.subtilis), Alkaliphilic bacillus (B.halodurans), streptomyces coelicolor (S.coelicolor), honeycomb Hough Buddhist nun bacterium (H.alvei), corynebacterium glutamicum (C.glutamicum) or klebsiella oxytoca (K.oxytoca) rely Propylhomoserin decarboxylase gene；Further, the polynucleotide for encoding lysine decarboxylase is cadA genes, ldcC genes, haldc bases Cause, the segment of the segment of cadA genes, the segment of ldcC genes or haldc genes；Still further, the cadA genes come Derived from the cadA genes of Escherichia coli (E.coli).

On the basis of any of the above-described technical solution further, the host strain of the transformant is Escherichia coli (E.coli), bacillus subtilis (B.subtilis), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei) or corynebacterium glutamicum (C.glutamicum), it is further preferred that Escherichia coli (E.coli) or honeycomb breathe out Husband Buddhist nun bacterium (H.alvei).

The fourth aspect of the present invention purpose is to propose a kind of method of fermenting and producing polypeptide, during the fermentation can Reduce expression product and (or) toxic action of the subsequent products to microbial cell, such as can one-step method using glucose life 1,5- pentanediamines are reduced during producing 1,5- pentanediamines to make microbial cell using the murder by poisoning of glucose production L-lysine With the yield of raising 1,5- pentanediamines.

A kind of method of fermenting and producing polypeptide, includes the following steps：

A) transformant of the culture as described in any technical solution above；

B) polypeptide is obtained from the bacterium solution or thalline obtained in step A.

Further, the method for a kind of fermenting and producing polypeptide, is the method for fermenting and producing lysine decarboxylase, including Following steps：

1) transformant of the culture as described in any technical solution above, the purpose base of the recombinant expression plasmid of the transformant Because being the polynucleotide for encoding lysine decarboxylase；

2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1.

The fifth aspect of the present invention purpose is to propose a kind of fermenting and producing 1, the method for 5- pentanediamines, in fermentation process In can reduce expression product and (or) toxic action of the subsequent products to microbial cell, such as can one-step method utilize Portugal Grape sugar reduces 1,5- pentanediamines and utilizes glucose production L-lysine to microbial cell during producing 1,5- pentanediamines Toxic action improves the yield of 1,5- pentanediamines.

A kind of fermenting and producing 1, the method for 5- pentanediamines include the following steps：

The culture transformant that can produce lysine decarboxylase as described above, the host strain of the transformant can also Lysine is produced, the lysine decarboxylase catalysis lysine decarboxylation generates 1,5- pentanediamines.

The sixth aspect of the present invention purpose is to propose a kind of mRNA, with can reduce during the fermentation expression product and (or) toxic action of the subsequent products to microbial cell, for example glucose production 1 can be utilized in one-step method, 5- pentanediamines The toxic action that 1,5- pentanediamines utilize microbial cell glucose production L-lysine is reduced in the process, improves 1,5- penta 2 The yield of amine.

A kind of mRNA, which is characterized in that the sequence of the mRNA includes and ribosome bind site described in claim 1 Corresponding sequence.

Beneficial effects of the present invention：

The present invention is based on the methods of nonideal explosives, and in conjunction with the method for random mutation and high flux screening, later use relies Propylhomoserin conversion ratio and the detection of lysine decarboxylase expression can obtain a series of capable of regulating and controlling lysine decarboxylase difference table Up to the ribosome bind site of intensity, the recombination lysine decarboxylase expression plasmid of these ribosome bind sites is carried, and Obtain a series of mutant strain of lysine decarboxylase difference expressions.Wherein lysine decarboxylase expression declines (this Invention at least declines mutant strain 25%), and lysine decarboxylase recombinant expression plasmid contained therein can be used for subsequently turning Change into Strains for Lysine Production, recombinant bacterial strain can have early period in the process for producing 1,5- pentanediamines using glucose by one step The guarantee of effect generates 1, the 5- pentanediamines of relatively low amount, therefore reduces 1,5- pentanediamines in earlier fermentation and utilize glucose fungus to thalline The toxic effect of body growth maintains production lysine and produces the balance between 1,5- pentanediamines, and last resultant effect can improve The yield of 1, the 5- pentanediamines of Strains for Lysine Production One-step production, while ensureing that remaining lysine is few as possible.The present invention The mutant strain that declines of lysine decarboxylase expression, the substantially binding ability in the sites RBS of ribosomes and mRNA Decline.The present invention can be used not only for reducing the expression quantity of lysine decarboxylase, can be also used for the expression for reducing other polypeptides Amount.

Description of the drawings

Fig. 1 is that (lysine is de- for the SDS-PAGE analyses of wild type and the mutant strain lysine decarboxylase CadA of same OD Carboxylic acid monomer molecule amount about 80kDa)；

Fig. 2 is suppression curve of the various concentration pentanediamine to thalli growth.

Specific implementation mode

Below in conjunction with the accompanying drawings, the present invention will be described in detail.

It uses in the present invention and such as gives a definition：

1. the base sequence used in the present invention, the abbreviation of nucleotide, amino acid (single-letter) are to be based on international theory With applied chemistry federation and international bio federation to the regulation of biological chemical name, European Journal are can refer to The ＂ of the phase of volume 138 the 9th end of the year 1984 of of Biochemistry prepares the finger of the specification containing base sequence and amino acid sequence Lead the abbreviation in mono- texts of ＂, and general abbreviation in biotechnology.

2.RBS sequence representation methods：

Unless otherwise specified, RBS sequences are 5 ' -3 ' expressions, and the sequence that the identification provided in this specification obtains (is shown in Table 2) base before being ATG, wherein RBS are pointed out with black matrix underscore.

The first aspect of the present invention proposes a kind of ribosome bind site, the ribosome bind site, the ribosomes The sequence 5 ' -3 ' of binding site includes one of sequence as follows：

a)、GTCATCAAAA；

b)、CGCAAAGATA；

c)、AAAGATTTAC。

The second aspect of the present invention purpose is to propose a kind of recombinant expression plasmid, the recombinant expression plasmid, including recombination The target gene of expression plasmid and corresponding ribosome bind site, the ribosome bind site any technical solution such as above It is described, it is preferable that the ribosome bind site is located at upstream from start codon -15 and arrives -6.A kind of recombinant expression Plasmid, skeleton plasmid are the reproducible skeleton plasmids in host cell, when host cell is Escherichia coli and Hough Buddhist nun bacterium When middle, preferably pUC, pBR322, pACYC plasmid (being purchased from precious bioengineering (Dalian) Co., Ltd) and their plasmid In one kind.PUC plasmids preferably such as pUC18 and pUC19 plasmids.The target gene of the recombinant expression plasmid is coding polypeptide Polynucleotide；The polypeptide is enzyme or polypeptide drug；Further, the enzyme is oxidoreducing enzyme, transferase, water At least one of enzyme, lyase, isomerase and ligase are solved, the polypeptide drug is in hormone, antibody and growth factor At least one；Further, the lyase is decarboxylase, and still further, the decarboxylase is amino acid decarboxylases, such as Lysine decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase.Again further, The polynucleotide for encoding lysine decarboxylase is to can come from microorganism, the cell of animal or plant, such as derives from Escherichia coli (E.coli), bacillus subtilis (B.subtilis), Alkaliphilic bacillus (B.halodurans), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei), corynebacterium glutamicum (C.glutamicum) or Oaksey support gram The lysine decarboxylase gene of the white bacillus of thunder (K.oxytoca)；The lysine decarboxylase gene can also derive from above-mentioned bacterium Bacterial strain or genetic engineering bacterium of the strain after mutagenesis or random mutation.It is further preferred that coding lysine decarboxylase is poly- Nucleotide is cadA genes, ldcC genes, haldc genes, the segment of cadA genes, the segment of ldcC genes or haldc genes Segment；It is further preferred that the lysine decarboxylase gene is derived from the induction type lysine decarboxylation of Escherichia coli Enzyme gene CadA/LdcI (SEQ ID No:1).Lysine decarboxylase can also be the mutation of the lysine decarboxylase in above-mentioned source Body (including natural mutation and artificial recombination mutant) or active fragment.

The third aspect of the present invention proposes a kind of transformant, and the recombinant expression plasmid of the transformant is as any one of above Recombinant expression plasmid described in technical solution；Or the genome of the transformant includes target gene and corresponding ribosomes knot Site is closed, the ribosome bind site is it is preferable to which ground, the ribosome bind site are located in initiation codon Trip -15 to -6；The target gene of target gene recombinant expression plasmid as described above.Preferably, the transformant Recombinant expression plasmid target gene be encode lysine decarboxylase polynucleotide, encode the poly-nuclear glycosides of lysine decarboxylase Acid is derived from Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), basophilic gemma Bacillus (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum) or klebsiella oxytoca The lysine decarboxylase gene of (Klebsiella oxytoca)；Further, encoding the polynucleotide of lysine decarboxylase is The segment of cadA genes, ldcC genes, haldc genes, the segment of cadA genes, the segment of ldcC genes or haldc genes；More Further, the cadA genes are derived from the cadA genes of Escherichia coli (E.coli).

The host strain of the transformant is Escherichia coli (E.coli), bacillus subtilis (B.subtilis), sky blue Streptomycete (S.coelicolor), hafnia alvei (H.alvei) or corynebacterium glutamicum (C.glutamicum), into one Step is preferably Escherichia coli (E.coli) or hafnia alvei (H.alvei).

The fourth aspect of the present invention has been a kind of method of fermenting and producing polypeptide of proposition, the side of the fermenting and producing polypeptide Method includes the following steps：

A) transformant of the culture as described in any technical solution above, the purpose base of the recombinant expression plasmid of the transformant Because being the polynucleotide for encoding lysine decarboxylase；

B) from step A) in obtained bacterium solution or obtain polypeptide in thalline.

Further, the method for a kind of fermenting and producing polypeptide, is the method for fermenting and producing lysine decarboxylase, including Following steps：

1) transformant of the culture as described in any technical solution above；

2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1).

The fifth aspect of the present invention proposes a kind of fermenting and producing 1, the method for 5- pentanediamines, the fermenting and producing 1,5- penta 2 The method of amine, includes the following steps：

The culture transformant that can produce lysine decarboxylase as described above, the host strain of the transformant can also Lysine is produced, the lysine decarboxylase is catalyzed the lysine decarboxylation and generates 1,5- pentanediamines.

It is an object of the invention to the sides by carrying out random mutation to lysine decarboxylase ribosome bind site (RBS) Method builds a series of lysine decarboxylase expression vector of different translation skills, in conjunction with the means of high flux screening and enzyme activity determination Identify one group of RBS that can regulate and control lysine decarboxylase translation.By the lysine decarboxylase expression containing these mutational sites Plasmid is transformed into hafnia alvei strain, can obtain a series of recombinant bacterial strain of different lysine decarboxylase expressions； Lysine decarboxylase expression plasmid containing these mutational sites is transformed into Strains for Lysine Production, grape can utilized A series of recombinant bacterial strain of difference 1,5- pentanediamine yield is obtained when sugar fermentation.

The present invention is first with Escherichia coli (E.coli MG1655K12, purchased from Beijing day bounties Gene Tech. Company Limited) Genome is that template clones lysine decarboxylase CadA encoding genes, and is building up in suitable expression vector, makes it Can in E. coli BL21 or hafnia alvei (H.alvei) successful expression.And then using synthesis with power traction The object and method expanded to plasmid is mutated the RBS sequences at its 5 ' end, and pass through the method for high flux screening and obtain The mutant strain that one group of lysine decarboxylase lysine conversion ratio is raised and lowered.Pass through the RBS of the method identification mutation of sequencing Sequence.These mutant plasmids are further converted into hafnia alvei strain verification since the RBS mutation on plasmid cause Lysine decarboxylase expression change situation；Lysine decarboxylase expression intensity subtracts caused by being mutated again due to RBS Weak plasmid is converted into Strains for Lysine Production, and one-step fermentation is carried out using recombination Strains for Lysine Production, can be notable Improve the yield of the 1,5- pentanediamines of its production.

In some embodiments, for screening since RBS mutation cause the lysine decarboxylase of different expression intensities prominent Selected host cell can be microorganism, plant or zooblast, preferred microorganism cell when variant.In some embodiment party In case, for screening since RBS mutation cause to be selected as host's when the lysine decarboxylase mutant of different expression intensities Microorganism can be Escherichia coli (E.coli), bacillus subtilis (B.subtilis), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei) or corynebacterium glutamicum (C.glutamicum), preferably large intestine bar Bacterium BL21 (E.coli BL21 (DE3)).

In some embodiments, for verifying since RBS mutation lead to the prominent of different lysine decarboxylase expression intensities The host cell for becoming selected when plasmid can be microorganism, plant or zooblast, preferred microorganism cell.In some implementations In scheme, for verifying since RBS mutation cause to be selected as place when the mutant plasmid of different lysine decarboxylase expression intensities Main microorganism can be Escherichia coli (E.coli), bacillus subtilis (B.subtilis), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei) or corynebacterium glutamicum (C.glutamicum), preferably honeycomb breathe out Husband Buddhist nun bacterium (H.alvei).

In some case study on implementation, Strains for Lysine Production is that can produce lysine after being transformed by genetic engineering means Bacterial strain, host strain is Escherichia coli, which is industrial strain suitable for commercial scale.Industrial bacterium Strain can be cultivated in amplification test, including be cultivated in shaking flask and fermentation tank, and the scale of culture can be sufficiently large.

In some embodiments, the temperature for cultivating thalline can be any temperature that can allow thalli growth, appropriate Temperature be 20~40 degree, preferably 30~40 degree, more preferable 35~40 degree.The time of culture can be about 1 day, about 2 days, about 3 It, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days.

Contain polypeptide, peptone, vitamin, trace element and minerals in the culture medium of some case study on implementation, transformant, Such culture medium may include but be not limited to common LB culture mediums (by tryptone, yeast extract and sodium chloride be molten Xie Yushui is made).

In some embodiments, cell lysine conversion ratio is catalyzed lysine using magnetic nuclear resonance method detection lysine The amounts of the 1,5- pentanediamines being converted into calculates.

The PCR amplification mentioned in following embodiment, comparative example, plasmid extraction, digestion, digestion products connection specific step Suddenly, conditional parameter etc. presses the condition progress of the specification suggestion of purchased relevant enzyme and reagent.DNA wherein used in PCR amplification The ligase used in restriction enzyme, digestion products connection used in polymerase, digestion is purchased from precious bioengineering (Dalian) Co., Ltd；Plasmid extraction kit, DNA plastic recovery kits, PCR purification kits are purchased from healthy and free from worry life science (Wujiang) Co., Ltd, trade mark Axygen；Primer is purchased from silent winged scientific and technological (China) Co., Ltd of generation that of match, trade mark INVITROGEN；Have Except illustrating.

Unless otherwise specified, the method for transformation mentioned in following embodiment, comparative example is as follows：By the connection product of 10 μ l or The plasmid of 2 μ l is added into e. coli bl21 (DE3) competent cell of 100 μ l, the heat shock in 42 DEG C after ice bath 20min 90s.The LB culture mediums of 1ml are added after incubation 5min on ice.It is applied in corresponding resistant panel.

OD₆₀₀(OD_562nm) measurement method：The culture medium for not cultivating cell of 3ml is added in the cuvette of 1cm wide, The school light (ABS) is absorbed in UV-8000 ultraviolet-uisible spectrophotometers (Shanghai Yuan Xi Instrument Ltd.) under 600nm (562nm) Zero.Cuvette is cleaned, the culture medium that 2.9ml does not cultivate bacterium is added after drying, is added after the zymotic fluid mixing of 0.1ml in UV- The absorption light value under 600nm (562nm) is tested in 8000 ultraviolet-uisible spectrophotometers.It is to send out that instrument, which shows that numerical value is multiplied by 30, The OD of zymotic fluid₆₀₀(OD_562nm)。

Embodiment 1

1.1, the clone of lysine decarboxylase gene cadA

Using primer cadA--SacI-F and cadA--XbaI-R (primer shown in table 1) by lysine decarboxylase (SEQ ID No:1) encoding gene cadA (SEQ ID No:2) from Escherichia coli MG1655K12 (E.coli MG1655K12, purchased from north Jing Tian bounties Bioisystech Co., Ltd) genome in amplify, after SacI and XbaI double digestions, be connected into same double Connection product is obtained in the pUC18 plasmids (purchased from precious bioengineering (Dalian) Co., Ltd) of digestion.Utilize CaCl₂It is prepared by method The competent cell of E.coli BL21 (purchased from precious bioengineering (Dalian) Co., Ltd), is used in combination heat shock method to turn connection product Change into E.coli BL21 cells, is screened using the ammonia benzyl antibiotic for adding 100 μ g/ml in LB culture mediums, clone PCR After sequence verification (verification cadA sequences are correct), extracting plasmid obtains pCIB60 plasmids.

Using plasmid pCIB60 as template, further utilize cadA-F2 and cadA-R2 to cadA genes in pCIB60 plasmids 5 ' sequences optimize (in order to allow CadA smoothly to state, cadA genes 5 ' end upstream sequences by

5’-ATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC- 3 ' replace with

5’-ATTTCACACAGGAAACAGCT-3’).After PCR amplification, using DpnI restriction enzymes to PCR product into Row digestion, converts also with heat shock method into E.coli BL21, sequence verification (5 ' end upstream sequences of verification cadA genes By 5 '-ATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC- 3 ' replace for 5 '- ATTTCACACAGGAAACAGCT-3 ') after obtain plasmid pCIB71.

Used primer in 1. present invention of table.

The structure in RBS random mutations library and its screening before 1.2 lysine decarboxylase gene C adA coded sequences

Using constructed plasmid pCIB71 in 1.1 as template, random primer cadA-RBS-F and cadA-RBS- are utilized R (table 1) is expanded, and introduces mutation in the process.The prior art is (see Schurr T, Nadir E, Margalit H.Identification and characterization of E.coli ribosomal binding sites by free energy computation..Nucleic Acids Research,1993,21(21):4019-4023) prediction is exhausted - 7 Dao -12 before most of gene start codons (ATG) are ribosome bind site (RBS), and the present invention will be -6 to -15 This section of sequence is considered RBS.

PCR system prepares (50 μ l)：

25μlMax archaeal dna polymerases (2 × Premix, precious bioengineering (Dalian) Co., Ltd)

1 μ l forward primers cadA-RBS-F (10 μM)

1 μ l reverse primers cadA-RBS-R (10 μM)

1 μ l template DNAs (400ng)

22μl ddH₂O to total volume be 50 μ l.

PCR programs：95 DEG C of denaturation 2min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min 30s, 35 are followed Ring；72 DEG C extend eventually, 7min.

After amplification, PCR product is cleaned using Axygen PCR cleanup kits.Restriction enzyme DpnI digestions, after 37 DEG C overnight (16h), digestion products are totally converted to E.coli JM109 (purchased from precious bioengineering (Dalian) Co., Ltd) in, build mutant library.

The plasmid of the recombination lysine decarboxylase mutator in mutant library is extracted, and is converted again to E.coli (while converting in pCIB71 plasmids to E.coli BL21 bacterial strains, obtain bacterial strain CIB71 as a contrast), coating in BL21 bacterial strains Screening primary dcreening operation is carried out in the LB resistant panels containing 100 μ g/ml ampicillins, specifically about 1000 lists of random picking 37 DEG C are cloned in the 96 hole deep-well plates containing liquid LB (+100 μ g/ml ampicillins), and (instrument is purchased from Shanghai generation to 250rpm Flat experimental facilities Co., Ltd, type low capacity total temperature constant temperature culture oscillator free from worldly cares, model SPH-200B) overnight incubation ((16h)).Respectively taken using the volley of rifle fire in the LB culture solutions to a 96 new hole deep-well plates of 600 μ l (while reaction is set and is started not The reacting hole of addition, the after reaction bacterium solution of addition control strain is as blank control), and the lysine salt of 400 μ l is added Acid solution (a concentration of 400g/l) and coenzyme PLP (phosphopyridoxal pyridoxal phosphate, final concentration of 0.1mM), in 37 DEG C, 250rpm (purchase by instrument From Shanghai Shiping Experiment Equipment Co., Ltd., type low capacity total temperature constant temperature culture oscillator free from worldly cares, model SPH-200B) reaction 2hrs, can be with the raising of pH, after reaction in every according to the generation of 1,5- pentanediamines in lysine decarboxylase catalytic process Acid-base indicator (indicator B is added dropwise in a reacting hole：1mg Bromothymol blues are dissolved in 10ml deionized waters；Indicator C： 10mg dimethyl diaminophenazine chlorides are dissolved in 10ml ethyl alcohol；Indicator C and B press 1：1 volume ratio mixes), according to the apparent phase of indicator color picking The mutant strain of raised to the pH of unmutated control strain CIB71 more or few (but opposite blank control still has color change) into Row secondary screening and sequencing identification mutation RBS sequences.

1.3RBS mutant libraries the selection result and mutational site sequencing identification

Primary dcreening operation is carried out to the CadA fallibility PCR mutant libraries of structure, have in 1000 monoclonals 51 in 96 hole deep holes The judgement that can not grown in plate is false positive clones；It is apparent that 15 clone's color changes are screened in remaining 949 clones altogether It is better than the mutant strain of control strain, screens 14 monoclonal color changes significantly lower than unmutated control strain CIB71's Mutant strain.Secondary screening (secondary screening method and primary dcreening operation are same) twice is carried out to this 29 clones.Therefrom it is determined that 3 clones are apparent after secondary screening There is the lysine decarboxylase activity less than control.There are 3 plants of monoclonals to be apparently higher than the lysine decarboxylase activity of control.

This 6 clones are further seeded to 5ml LB liquid mediums (containing 100 μ g/ml ampicillins) to train It supports, 37 DEG C, (instrument is purchased from Shanghai Shiping Experiment Equipment Co., Ltd. to 250rpm, and type low capacity total temperature constant temperature incubation free from worldly cares is shaken Swing device, model SPH-200B) overnight (16h).Sequencing identification is carried out to the sequence before lysine decarboxylase gene after extracting plasmid RBS sequences are mutated, sequencing result is as shown in table 2 below.

Table 2：The 6 plant mutant body mutational sites identification screened in CadA fallibility mutant libraries.

1.4 lysine decarboxylase CadA mutain lysine conversion ratios measure

Plasmid and control pCIB71 plasmids that extracted in 1.3 8 are cloned are converted respectively to honeycomb Hough Buddhist nun (H.alvei) it in competent cell, is coated in the LB resistant panels containing 100 μ g/ml ampicillins and is screened.Into One step distinguishes each 5 monoclonals of picking, is cultivated, 37 using 5ml LB liquid mediums (containing 100 μ g/ml ampicillins) DEG C, 250rpm (instrument be purchased from Shanghai Shiping Experiment Equipment Co., Ltd., type low capacity total temperature constant temperature culture oscillator free from worldly cares, Model SPH-200B) overnight (16h).It takes respectively in the LB culture solutions to EP pipes of 600 μ l respectively cloned, and relying for 400 μ l is added Propylhomoserin hydrochloric acid solution (a concentration of 400g/l) and coenzyme PLP (phosphopyridoxal pyridoxal phosphate, final concentration of 0.1mM), natural pH is (about 6.7), in 37 DEG C, (instrument is purchased from Shanghai Shiping Experiment Equipment Co., Ltd., type low capacity total temperature constant temperature training free from worldly cares to 250rpm Support oscillator, model SPH-200B) reaction 2hrs, after reaction to reaction solution carry out 12000rpm centrifugation 3min (room temperature from Scheming, brand and model Beckman coulter microfuge 16centrifuge, similarly hereinafter), on the reaction solution for taking 500 μ l Clearly in nuclear magnetic tube, and 100 μ l D are added₂O：DMSO=30：1 (mass ratio) is used as internal standard, is reacted using magnetic resonance detection The amount of remaining lysine in system (calculates 1,5- penta with the amount of this 1,5- pentanediamine for calculating generation and lysine conversion ratio The mole of diamines divided by remaining lysine and 1, the integral molar quantity of 5- pentanediamines), and then calculate relying relatively for opposite CIB71 Propylhomoserin conversion ratio, the results are shown in Table 3.

3. nuclear-magnetism of table detects lysine conversion ratio and OD of 3 plants of CadA mutant strains compared with wild-type strain₆₀₀。

As shown in table 3, in the identical reaction time, containing mutation RBS plasmids pCIB71-RBS1, pCIB71-RBS2 and The lysine conversion ratio of the mutant strain of pCIB71-RBS3 respectively be compare CIB71 bacterial strains 38%, 58% and 75%, and this three Plant mutant strain grows no significant difference compared with the control.Thus analysis is since RBS mutation cause lysine decarboxylase to express water It is flat to decline, it is converted so as to cause mutant strain CIB71-RBS-1, CIB71-RBS-2 and CIB71-RBS-3 cell effect lysine Rate declines.And this five plant mutants bacterial strain of CIB71-RBS-4, CIB71-RBS5, CIB71-RBS-6 lysine conversion compared with the control Rate is all significantly increased, and is the 189%, 168% and 177% of control respectively；Wherein CIB71-RBS-4, CIB71-RBS-5, The growth of this three plant mutants strain of CIB71-RBS-6 is compared with the control without significant changes, in order to further verify these mutant strain phases It is lysine decarboxylase gene expression amount caused by the simple mutation because of RBS to increasing or decreasing for control lysine conversion ratio Raising, rather than be that the conversion ratio caused by thalli growth velocity variations increases or decreases, further pass through protein expression Horizontal analysis is determined.

The extraction and SDS-PAGE electrophoretic analysis of 1.5 mutation lysine decarboxylase CadA

Utilize CIB71, CIB71-RBS-1, CIB71-RBS-2, CIB71-RBS-3, CIB71-RBS-4, CIB71- in 1.4 The bacterium solution of RBS-5, CIB71-RBS-6 mutant strain, and the OD of this several plants of bacterium is adjusted to by phase according to the OD of the bacterial strain measured in 1.4 Same level takes the bacterium solution of the wild type and mutant strain of the same OD of same volume (4ml), 12000rpm to centrifuge 10min, abandon Clearly.The intracellular protein of each bacterial strain is extracted as follows：

1) (the Buffer 1 (pH 8) of buffer 1 of 1ml are used:50mM Tris-HCl pH 8；25mM NaCl；2mM EDTA thalline (hereinafter referred to as sample)) is resuspended；

2) 1hr is frozen for -20 DEG C；

3) 37 DEG C, 15min melts sample；

4) add 5 μ l lysozymes (purchased from precious bioengineering (Dalian) Co., Ltd), and continue to be incubated 3hrs at 37 DEG C, during which Reverse mixing is several times；

5) 1hr is frozen for -20 DEG C；

6) 37 DEG C, 15min melts sample；

7) the DNase I (purchased from precious bioengineering (Dalian) Co., Ltd) of 1 μ l are added, continue to be incubated 1hrs at 37 DEG C；

8) 12000rpm centrifuges 10min, takes in supernatant to a new EP pipe, it is ensured that supernatant does not contain cell fragment；

9) supernatant is stored in 4 DEG C of refrigerators, spare；Precipitation is suspended with the buffer 1 of 1ml, spare.

Each 64 μ l of the intracellular protein supernatant of each bacterial strain are taken respectively, and 5 × SDS-PAGE sample-loading buffers that 16 μ l are added (are matched Side：250mM Tris-HCl (pH6.8), 10% (w/v) SDS, 0.5% (w/v) bromophenol blue, 50% (v/v) glycerine, 5% (v/v) Beta -mercaptoethanol；) the denatured by boiling 10min in boiling water bath, after of short duration cooling, 12000rpm centrifuge 2min, take 10 μ l supernatants into Row SDS-PAGE analyses, as a result as shown in Fig. 1.It can be seen that mutant strain CIB71-RBS-1, CIB71-RBS-2 and CIB71- The trend that lysine decarboxylase CadA expression quantity in RBS-3 supernatants decreased significantly compared with compareing CIB71, from protein expression The RBS that mutation is demonstrated in level makes the translation rate of lysine decarboxylase occur being remarkably decreased；And CIB71-RBS-4, The lysine decarboxylase of CIB71-RBS-5 and CIB71-RBS-6 mutant strains expression significantly improves compared with the control, also from egg The RBS that these three mutation are demonstrated on white level makes the translation rate of lysine decarboxylase occur significantly rising.

Comparative example 1

The inhibition that the 1,5- pentanediamines of various concentration grow microorganism

The plasmid pCIB71 that lysine decarboxylase CadA will be expressed is converted into Escherichia coli CIB103-2 bacterial strains It arrives

CIB103-71 bacterial strains.The specific construction method of wherein CIB103-2 bacterial strains is as follows：

1. building tetracycline efflux pump (tetracycline effluxpump) expression vector skeleton：

Utilize primer psyn-1 and psyn-2 synthetic promoter sequence (SEQ ID NO:10), wherein primer psyn-1 packets open Promoter sequences and one section of sequence homologous with pUC18, primer psyn-2 include one section of sequence homologous with pUC18, the two PCR Primer is used to expand a part of sequence of the promoter sequence and pUC18 carriers of synthesis, restricted including multiple cloning sites Restriction endonuclease EcoRI and ScaI are used for the DNA fragmentation that digest amplification comes out, and further connect structure in pUC18 and obtain pUC10.

2. building tetracycline efflux pump expression vector：

Utilize primer tetA-F and tetA-R tetracycline efflux pump TetA (SEQ ID NO:11) encoding gene (tetA, SEQ ID NO:12) it, expands and obtains from E. coli cloning vector pBR322, using being connected to after SacI and XbaI double digestions PCIB20 is obtained in pCIB10 plasmids.

3. the structure of three albumen LysC, DapA and LysA reading frames on lysine synthetic pathway：

In genome of E.coli encode Bio-synthetic pathway of lysine on three albumen gene, lysC, dapA and lysA：Aspartokinase (LysC or AKIII, encoded by lysC), dihydrodipicolinate synthase (DapA or DHDPS, by DapA is encoded), diaminopimelate decarboxylase (LysA is encoded by lysA), in these three gene clonings to plasmid vector, thus three A albumen LysC (SEQ ID NO:13),DapA(SEQ ID NO:15),LysA(SEQ ID NO:17) it may be implemented in large intestine Overexpression in bacillus.First with primer lysC-F and lysC-R by gene lysC (SEQ ID NO:14) from Escherichia coli It amplifies and in the genome of E.coli MG1655K12, same double enzymes are connected to later with SacI and BamHI are double digested Plasmid pCIB7 is obtained in the plasmid pUC18 cut.Using primer dapA-F and dapA-R by gene dapA (SEQ ID NO:16) from It amplifies and in the genome of E. coli MG1655K12, be connected to later with BamHI and XbaI are double digested Plasmid pCIB8 is obtained in the plasmid pCIB7 of same double digestion.Using primer lysA-F and lysA-R by gene lysA (SEQ ID NO:18) amplified from the genome of E. coli MG1655K12 come, with XbaI and SalI it is double digested after It is connected in the plasmid pCIB8 of same double digestion and obtains plasmid pCIB9.Further utilize lysC-F and lysA-R by these three Gene is amplified from pCIB9 together to be come, and double digested using SacI and SalI, is connected to the plasmid of same double digestion PCIB32 is obtained in pCIB10.

4. aspartokinase enzyme coding gene mutational vector is built：

In order to improve the yield of lysine, further to aspartokinase gene (LysC or AKIII, by lysC, SEQ ID NO:18 codings) it is mutated, specifically, using primer 318-F, 318-R, 323-F, 323-R expand to obtain LysC-1 (LysC-M318I, G323D, SEQ.ID NO:21) encoding gene lysC-1 (SEQ ID NO:20), and by replacing pCIB32 In wild type lysC, obtain plasmid pCIB43.LysC-1 after mutation can further weaken the feedback inhibition of lysine.

5. building the coexpression vector of other three kinds of albumen Asd, DapB and AspC in lysine synthesis access：

Further, the other three gene asd, dapB and aspC in access is synthesized to lysine to be overexpressed.These Gene is separately encoded following three albumen：Aspartate semialdehyde dehydrogenase (Asd (SEQ ID NO:21), encoded by asd), two Pyridinium hydroxide dicarboxylic acids reductase (DapB or DHDPR (SEQ ID NO:23), encoded by dapB), aspartate transaminase (AspC (SEQ ID NO:25) it, is encoded by aspC).Using primer asd-F and asd-R by asd (SEQ ID NO:22) from Escherichia coli It amplifies and in MG1655K12 genomes, after SacI and BamHI double digestions, be connected to structure in pUC18 and obtain pCIB12；Using primer dapB-F and dapB-R by dapB (SEQ ID NO:24) from Escherichia coli MG1655K12 genomes It amplifies and, after BamHI and XbaI double digestions, be connected to structure in pCIB12 and obtain pCIB13；Utilize primer aspC-F With aspC-R by aspC (SEQ ID NO:26) amplify and from Escherichia coli MG1655K12 genomes, using XbaI and After SalI double digestions, it is connected to structure in pCIB13 and obtains pCIB31；Using primer tetA-F3 and tetA-R3, with plasmid PCIB20 is template, and amplification obtains tetA encoding genes, builds to obtain using pCIB31 is connected into after XhoI and SphI double digestions pCIB59。

6. building six gene co-expressing plasmids in lysine synthesis access：

LysC-1, dapA, and lysA are amplified to come together from pCIB43 using primer LAL-F and LAL-R, After ApaI and KpnI double digestions, it is connect with the plasmid pCIB59 of same double digestion, obtains pCIB103-2 plasmids.

7. building CIB103-2 bacterial strains：

The pCIB103-2 plasmids built are converted into e. coli bl21, Escherichia coli CIB103-2 bacterial strains are obtained.

Respectively picking CIB103-71 bacterial strains monoclonal to containing various concentration (0,5,10,15,20,25,30,35, The culture medium of 1,5- pentanediamines 40g/L) (contains 4% in 5mL fluid nutrient mediums<Mass percent, following components are same>Grape Sugar, 0.1%KH₂PO₄, 0.1%MgSO₄, 1.6% (NH₄)₂SO₄, 0.001%FeSO₄, 0.001%MnSO₄, the extraction of 0.2% yeast Object (is purchased from England OXOID LTD.), 0.05%L- methionine, 0.01%L- threonines, 0.005%L- isoleucines, 10 μ G/mL tetracyclines) in, in 37 DEG C, (instrument is purchased from Shanghai Shiping Experiment Equipment Co., Ltd. to 250rpm, and type low capacity free from worldly cares is entirely warm Spend constant temperature culture oscillator, model SPH-200B) overnight incubation (for 24 hours), it is dense using each 1,5- pentanediamines of spectrophotometric determination The OD of degree condition hypothallus_562nm.As a result as shown in Fig. 2, it can be seen that when 1,5- pentanediamines concentration gradually increases from 0 to 20g/L The OD of added-time, thalline drastically decline；After 1,5- pentanediamine concentration is more than 20g/L, it is nearly no detectable thalli growth.

Embodiment 2

Strains for Lysine Production One-step production 1,5- pentanediamines

By wild plasmid pCIB71 and random mutation screen containing lysine acid decarboxylase expression quantity can be caused The mutant plasmid (pCIB71-RBS1, pCIB71-RBS2, pCIB71-RBS3) of the RBS sequences of reduction is converted respectively to can give birth to In the Escherichia coli CIB103-2 for producing lysine, CIB103-71, CIB103-71-RBS1, CIB103-71-RBS2 are respectively obtained With CIB103-71-RBS3 bacterial strains.As a contrast with CIB103-2 bacterial strains, respectively in picking monoclonal to 5mL fluid nutrient mediums (contain 4%<Mass percent, following components are same>Glucose, 0.1%KH₂PO₄, 0.1%MgSO₄, 1.6% (NH₄)₂SO₄, 0.001%FeSO₄, 0.001%MnSO₄, 0.2% yeast extract (purchased from England OXOID LTD.), 0.05%L- egg ammonia Acid, 0.01%L- threonines, 0.005%L- isoleucines, 10 μ g/mL tetracyclines, 100 μ g/mL ampicillins), in 37 DEG C, (instrument is purchased from Shanghai Shiping Experiment Equipment Co., Ltd., type low capacity total temperature constant temperature culture oscillator free from worldly cares, model to 250rpm SPH-200B) overnight incubation (for 24 hours).Second day, each bacterial strain be switched to again 100ml it is fresh contain 30g/L glucose, 0.7% (mass percent) Ca (HCO₃)₂, in 37 DEG C in the culture medium of 10 μ g/mL tetracyclines and 100 μ g/mL ampicillins, (instrument is purchased from Shanghai Shiping Experiment Equipment Co., Ltd., standard type large capacity constant-temperature shaking incubator, model SPH- to 170rpm 211B) continue culture 72 hours, the lysine in each culture medium and 1, the content (table of 5- pentanediamines are detected and calculated using nuclear-magnetism 4).As shown in table 4, with mutant strain CIB103-71-RBS3, the expression of CIB103-71-RBS2 and CIB103-71-RBS1 bacterial strains The amount of lysine decarboxylase continuously decrease, the yield of 1,5- pentanediamine gradually rises, and also means that more lysines hairs Conversion has been given birth to, lysine has thus been may be decreased relevant enzyme is synthesized to lysine and checked, the yield of lysine also can be into one Step increases and is converted into 1,5- pentanediamines, is embodied in the L-lysine and 1 of measurement, the total amount of 5- pentanediamines gradually rises.

Table 4. detects the bacterial strain lysine and 1,5- penta 2 that can express lysine synthetic proteins and lysine decarboxylase simultaneously The level of amine.

SEQUENCE LISTING

<110>Shanghai Cathay Biological Technology Research Center Co., Ltd

Kai Sai biological industrys Co., Ltd

<120>A kind of ribosome bind site, recombinant expression plasmid, transformant and its application

<130> PA16011-2

<160> 56

<170> PatentIn version 3.5

<210> 1

<211> 715

<212> PRT

<213> Escherichia coli

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

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Glu Pro Ile Arg Glu Leu His Arg Ala Leu Glu Arg Leu Asn Phe Gln

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Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn

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

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Glu Leu Cys Glu Glu Ile Ser Lys Met Asn Glu Asn Leu Pro Leu Tyr

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

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

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Ile Ala Asn Lys Ile Lys Gln Thr Thr Asp Glu Tyr Ile Asn Thr Ile

115 120 125

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

130 135 140

Tyr Thr Phe Cys Thr Pro Gly His Met Gly Gly Thr Ala Phe Gln Lys

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

165 170 175

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

180 185 190

His Ser Gly Pro His Lys Glu Ala Glu Gln Tyr Ile Ala Arg Val Phe

195 200 205

Asn Ala Asp Arg Ser Tyr Met Val Thr Asn Gly Thr Ser Thr Ala Asn

210 215 220

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

225 230 235 240

Asp Arg Asn Cys His Lys Ser Leu Thr His Leu Met Met Met Ser Asp

245 250 255

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

260 265 270

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

275 280 285

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

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

305 310 315 320

Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr

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Thr Asn Phe Ser Pro Ile Tyr Glu Gly Lys Cys Gly Met Ser Gly Gly

340 345 350

Arg Val Glu Gly Lys Val Ile Tyr Glu Thr Gln Ser Thr His Lys Leu

355 360 365

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

370 375 380

Asn Glu Glu Thr Phe Asn Glu Ala Tyr Met Met His Thr Thr Thr Ser

385 390 395 400

Pro His Tyr Gly Ile Val Ala Ser Thr Glu Thr Ala Ala Ala Met Met

405 410 415

Lys Gly Asn Ala Gly Lys Arg Leu Ile Asn Gly Ser Ile Glu Arg Ala

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

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Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys

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Trp Pro Leu Arg Ser Asp Ser Thr Trp His Gly Phe Lys Asn Ile Asp

465 470 475 480

Asn Glu His Met Tyr Leu Asp Pro Ile Lys Val Thr Leu Leu Thr Pro

485 490 495

Gly Met Glu Lys Asp Gly Thr Met Ser Asp Phe Gly Ile Pro Ala Ser

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Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr

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

530 535 540

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

545 550 555 560

Asp Leu Asn Leu Arg Val Lys Asn Met Leu Pro Ser Leu Tyr Arg Glu

565 570 575

Asp Pro Glu Phe Tyr Glu Asn Met Arg Ile Gln Glu Leu Ala Gln Asn

580 585 590

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

595 600 605

Ala Phe Glu Val Leu Pro Thr Met Val Met Thr Pro Tyr Ala Ala Phe

610 615 620

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

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Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val

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

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Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly

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Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr

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gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300

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actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420

cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480

agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540

tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600

gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660

tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720

gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780

tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840

cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900

gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960

acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020

ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080

gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140

aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200

ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260

ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320

cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380

acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440

aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500

gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560

catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620

atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680

gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740

tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800

aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860

tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920

gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980

ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040

gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100

gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA-RBS

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ggataacaat ttcacacagg aaacagct 28

<210> 4

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<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-1

<400> 4

ggataacaat ttcgtcatca aaacagct 28

<210> 5

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-2

<400> 5

ggataacaat ttccgcaaag atacagct 28

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-3

<400> 6

ggataacaat ttcaaagatt taccagct 28

<210> 7

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-4

<400> 7

ggataacaat ttcgagagga ggacagct 28

<210> 8

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-5

<400> 8

ggataacaat ttcaagggaa attcagct 28

<210> 9

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> cadA RBS-6

<400> 9

ggataacaat ttcttaaaga gggcagct 28

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Promoter

<400> 10

agtttattct tgacatgtag tgagggggct ggtataat 38

<210> 11

<211> 396

<212> PRT

<213> Artificial Sequence

<220>

<223> TetA

<400> 11

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

1 5 10 15

Asp Ala Val Gly Ile Gly Leu Val Met Pro Val Leu Pro Gly Leu Leu

20 25 30

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

35 40 45

Leu Ala Leu Tyr Ala Leu Met Gln Phe Leu Cys Ala Pro Val Leu Gly

50 55 60

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

65 70 75 80

Leu Gly Ala Thr Ile Asp Tyr Ala Ile Met Ala Thr Thr Pro Val Leu

85 90 95

Trp Ile Leu Tyr Ala Gly Arg Ile Val Ala Gly Ile Thr Gly Ala Thr

100 105 110

Gly Ala Val Ala Gly Ala Tyr Ile Ala Asp Ile Thr Asp Gly Glu Asp

115 120 125

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

130 135 140

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

145 150 155 160

Ala Pro Phe Leu Ala Ala Ala Val Leu Asn Gly Leu Asn Leu Leu Leu

165 170 175

Gly Cys Phe Leu Met Gln Glu Ser His Lys Gly Glu Arg Arg Pro Met

180 185 190

Pro Leu Arg Ala Phe Asn Pro Val Ser Ser Phe Arg Trp Ala Arg Gly

195 200 205

Met Thr Ile Val Ala Ala Leu Met Thr Val Phe Phe Ile Met Gln Leu

210 215 220

Val Gly Gln Val Pro Ala Ala Leu Trp Val Ile Phe Gly Glu Asp Arg

225 230 235 240

Phe Arg Trp Ser Ala Thr Met Ile Gly Leu Ser Leu Ala Val Phe Gly

245 250 255

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

260 265 270

Arg Phe Gly Glu Lys Gln Ala Ile Ile Ala Gly Met Ala Ala Asp Ala

275 280 285

Leu Gly Tyr Val Leu Leu Ala Phe Ala Thr Arg Gly Trp Met Ala Phe

290 295 300

Pro Ile Met Ile Leu Leu Ala Ser Gly Gly Ile Gly Met Pro Ala Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Leu Ala Trp Ile Val Gly Ala Ala Leu Tyr Leu Val Cys Leu Pro Ala

370 375 380

Leu Arg Arg Gly Ala Trp Ser Arg Ala Thr Ser Thr

385 390 395

<210> 12

<211> 1191

<212> DNA

<213> Artificial Sequence

<220>

<223> tetA

<400> 12

atgaaatcta acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc 60

ataggcttgg ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc 120

atcgccagtc actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca 180

cccgttctcg gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta 240

cttggagcca ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac 300

gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc 360

gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc 420

ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat 480

gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta 540

atgcaggagt cgcataaggg agagcgtcga ccgatgccct tgagagcctt caacccagtc 600

agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt 660

atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc 720

tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc 780

ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt 840

atcgccggca tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc 900

tggatggcct tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg 960

caggccatgc tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc 1020

gcggctctta ccagcctaac ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc 1080

gcctcggcga gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc 1140

tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg a 1191

<210> 13

<211> 449

<212> PRT

<213> Escherichia coli

<400> 13

Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr

85 90 95

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

100 105 110

Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu

115 120 125

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

130 135 140

Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala

145 150 155 160

Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu

165 170 175

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

180 185 190

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

195 200 205

Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro

210 215 220

Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile

225 230 235 240

Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala

245 250 255

Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile

260 265 270

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

275 280 285

Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu

290 295 300

Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His

305 310 315 320

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

325 330 335

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

340 345 350

Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln

355 360 365

Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu

370 375 380

Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys

385 390 395 400

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

405 410 415

Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro

420 425 430

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

435 440 445

Glu

<210> 14

<211> 1350

<212> DNA

<213> Escherichia coli

<400> 14

atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60

aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120

tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180

ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240

ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300

gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca cggcgagctg 360

atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420

gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480

gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540

acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600

agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660

accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720

gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780

ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840

gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900

gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960

tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020

ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080

actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140

gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200

ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260

ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320

caaaaactgc atagtaattt gtttgagtaa 1350

<210> 15

<211> 292

<212> PRT

<213> Escherichia coli

<400> 15

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

1 5 10 15

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

20 25 30

Ala Ser Gly Thr Ser Ala Ile Val Ser Val Gly Thr Thr Gly Glu Ser

35 40 45

Ala Thr Leu Asn His Asp Glu His Ala Asp Val Val Met Met Thr Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ile Val Gly Cys Leu Thr Val Thr Pro Tyr Tyr Asn Arg Pro Ser Gln

100 105 110

Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu His Thr Asp Leu

115 120 125

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

130 135 140

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

145 150 155 160

Lys Glu Ala Thr Gly Asn Leu Thr Arg Val Asn Gln Ile Lys Glu Leu

165 170 175

Val Ser Asp Asp Phe Val Leu Leu Ser Gly Asp Asp Ala Ser Ala Leu

180 185 190

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

195 200 205

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

210 215 220

His Phe Ala Glu Ala Arg Val Ile Asn Gln Arg Leu Met Pro Leu His

225 230 235 240

Asn Lys Leu Phe Val Glu Pro Asn Pro Ile Pro Val Lys Trp Ala Cys

245 250 255

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

260 265 270

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

275 280 285

Ala Gly Leu Leu

290

<210> 16

<211> 879

<212> DNA

<213> Escherichia coli

<400> 16

atgttcacgg gaagtattgt cgcgattgtt actccgatgg atgaaaaagg taatgtctgt 60

cgggctagct tgaaaaaact gattgattat catgtcgcca gcggtacttc ggcgatcgtt 120

tctgttggca ccactggcga gtccgctacc ttaaatcatg acgaacatgc tgatgtggtg 180

atgatgacgc tggatctggc tgatgggcgc attccggtaa ttgccgggac cggcgctaac 240

gctactgcgg aagccattag cctgacgcag cgcttcaatg acagtggtat cgtcggctgc 300

ctgacggtaa ccccttacta caatcgtccg tcgcaagaag gtttgtatca gcatttcaaa 360

gccatcgctg agcatactga cctgccgcaa attctgtata atgtgccgtc ccgtactggc 420

tgcgatctgc tcccggaaac ggtgggccgt ctggcgaaag taaaaaatat tatcggaatc 480

aaagaggcaa cagggaactt aacgcgtgta aaccagatca aagagctggt ttcagatgat 540

tttgttctgc tgagcggcga tgatgcgagc gcgctggact tcatgcaatt gggcggtcat 600

ggggttattt ccgttacggc taacgtcgca gcgcgtgata tggcccagat gtgcaaactg 660

gcagcagaag ggcattttgc cgaggcacgc gttattaatc agcgtctgat gccattacac 720

aacaaactat ttgtcgaacc caatccaatc ccggtgaaat gggcatgtaa ggaactgggt 780

cttgtggcga ccgatacgct gcgcctgcca atgacaccaa tcaccgacag tggtcgtgag 840

acggtcagag cggcgcttaa gcatgccggt ttgctgtaa 879

<210> 17

<211> 420

<212> PRT

<213> Escherichia coli

<400> 17

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> 18

<211> 1263

<212> DNA

<213> Escherichia coli

<400> 18

atgccacatt cactgttcag caccgatacc gatctcaccg ccgaaaatct gctgcgtttg 60

cccgctgaat ttggctgccc ggtgtgggtc tacgatgcgc aaattattcg tcggcagatt 120

gcagcgctga aacagtttga tgtggtgcgc tttgcacaga aagcctgttc caatattcat 180

attttgcgct taatgcgtga gcagggcgtg aaagtggatt ccgtctcgtt aggcgaaata 240

gagcgtgcgt tggcggcggg ttacaatccg caaacgcacc ccgatgatat tgtttttacg 300

gcagatgtta tcgatcaggc gacgcttgaa cgcgtcagtg aattgcaaat tccggtgaat 360

gcgggttctg ttgatatgct cgaccaactg ggccaggttt cgccagggca tcgggtatgg 420

ctgcgcgtta atccggggtt tggtcacgga catagccaaa aaaccaatac cggtggcgaa 480

aacagcaagc acggtatctg gtacaccgat ctgcccgccg cactggacgt gatacaacgt 540

catcatctgc agctggtcgg cattcacatg cacattggtt ctggcgttga ttatgcccat 600

ctggaacagg tgtgtggtgc tatggtgcgt caggtcatcg aattcggtca ggatttacag 660

gctatttctg cgggcggtgg gctttctgtt ccttatcaac agggtgaaga ggcggttgat 720

accgaacatt attatggtct gtggaatgcc gcgcgtgagc aaatcgcccg ccatttgggc 780

caccctgtga aactggaaat tgaaccgggt cgcttcctgg tagcgcagtc tggcgtatta 840

attactcagg tgcggagcgt caaacaaatg gggagccgcc actttgtgct ggttgatgcc 900

gggttcaacg atctgatgcg cccggcaatg tacggtagtt accaccatat cagtgccctg 960

gcagctgatg gtcgttctct ggaacacgcg ccaacggtgg aaaccgtcgt cgccggaccg 1020

ttatgtgaat cgggcgatgt ctttacccag caggaagggg gaaatgttga aacccgcgcc 1080

ttgccggaag tgaaggcagg tgattatctg gtactgcatg atacaggggc atatggcgca 1140

tcaatgtcat ccaactacaa tagccgtccg ctgttaccag aagttctgtt tgataatggt 1200

caggcgcggt tgattcgccg tcgccagacc atcgaagaat tactggcgct ggaattgctt 1260

taa 1263

<210> 19

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<223> LysC-1

<400> 19

Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr

85 90 95

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

100 105 110

Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu

115 120 125

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

130 135 140

Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala

145 150 155 160

Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu

165 170 175

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

180 185 190

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

195 200 205

Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro

210 215 220

Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile

225 230 235 240

Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala

245 250 255

Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile

260 265 270

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

275 280 285

Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu

290 295 300

Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Ile Leu His

305 310 315 320

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

325 330 335

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

340 345 350

Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln

355 360 365

Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu

370 375 380

Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys

385 390 395 400

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

405 410 415

Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro

420 425 430

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

435 440 445

Glu

<210> 20

<211> 1349

<212> DNA

<213> Artificial Sequence

<220>

<223> lysC-1

<400> 20

tgtctgaaat tgttgtctcc aaatttggcg gtaccagcgt agctgatttt gacgccatga 60

accgcagcgc tgatattgtg ctttctgatg ccaacgtgcg tttagttgtc ctctcggctt 120

ctgctggtat cactaatctg ctggtcgctt tagctgaagg actggaacct ggcgagcgat 180

tcgaaaaact cgacgctatc cgcaacatcc agtttgccat tctggaacgt ctgcgttacc 240

cgaacgttat ccgtgaagag attgaacgtc tgctggagaa cattactgtt ctggcagaag 300

cggcggcgct ggcaacgtct ccggcgctga cagatgagct ggtcagccac ggcgagctga 360

tgtcgaccct gctgtttgtt gagatcctgc gcgaacgcga tgttcaggca cagtggtttg 420

atgtacgtaa agtgatgcgt accaacgacc gatttggtcg tgcagagcca gatatagccg 480

cgctggcgga actggccgcg ctgcagctgc tcccacgtct caatgaaggc ttagtgatca 540

cccagggatt tatcggtagc gaaaataaag gtcgtacaac gacgcttggc cgtggaggca 600

gcgattatac ggcagccttg ctggcggagg ctttacacgc atctcgtgtt gatatctgga 660

ccgacgtccc gggcatctac accaccgatc cacgcgtagt ttccgcagca aaacgcattg 720

atgaaatcgc gtttgccgaa gcggcagaga tggcaacttt tggtgcaaaa gtactgcatc 780

cggcaacgtt gctacccgca gtacgcagcg atatcccggt ctttgtcggc tccagcaaag 840

acccacgcgc aggtggtacg ctgatgtgca ataaaactga aaatccgccg ctgttccgcg 900

ctctggcgct tcgtcgcaat cagactctgc tcactttgca cagcctgaat atactgcatt 960

ctcgcgattt cctcgcggaa gttttcggca tcctcgcgcg gcataatatt tcggtagact 1020

taatcaccac gtcagaagtg agcgtggcat taacccttga taccaccggt tcaacctcca 1080

ctggcgatac gttgctgacg caatctctgc tgatggagct ttccgcactg tgtcgggtgg 1140

aggtggaaga aggtctggcg ctggtcgcgt tgattggcaa tgacctgcca aaagcctgcg 1200

gcgttggcaa agaggtattc ggcgtactgg aaccgttcaa cattcgcatg atttgttatg 1260

gcgcatccag ccataacctg tgcttcctgg tgcccggcga agatgccgag caggtggtgc 1320

aaaaactgca tagtaatttg tttgagtaa 1349

<210> 21

<211> 350

<212> PRT

<213> Escherichia coli

<400> 21

Met Lys Asn Val Gly Phe Ile Gly Trp Arg Gly Met Val Gly Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

Gly Thr Thr Gly Thr Leu Gln Asp Ala Phe Asp Leu Glu Ala Leu Lys

50 55 60

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

65 70 75 80

Ile Tyr Pro Lys Leu Arg Glu Ser Gly Trp Gln Gly Tyr Trp Ile Asp

85 90 95

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

100 105 110

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

115 120 125

Thr Phe Val Gly Gly Asn Cys Thr Val Ser Leu Met Leu Met Ser Leu

130 135 140

Gly Gly Leu Phe Ala Asn Asp Leu Val Asp Trp Val Ser Val Ala Thr

145 150 155 160

Tyr Gln Ala Ala Ser Gly Gly Gly Ala Arg His Met Arg Glu Leu Leu

165 170 175

Thr Gln Met Gly His Leu Tyr Gly His Val Ala Asp Glu Leu Ala Thr

180 185 190

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

195 200 205

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

210 215 220

Ser Leu Ile Pro Trp Ile Asp Lys Gln Leu Asp Asn Gly Gln Ser Arg

225 230 235 240

Glu Glu Trp Lys Gly Gln Ala Glu Thr Asn Lys Ile Leu Asn Thr Ser

245 250 255

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

260 265 270

Cys His Ser Gln Ala Phe Thr Ile Lys Leu Lys Lys Asp Val Ser Ile

275 280 285

Pro Thr Val Glu Glu Leu Leu Ala Ala His Asn Pro Trp Ala Lys Val

290 295 300

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

305 310 315 320

Val Thr Gly Thr Leu Thr Thr Pro Val Gly Arg Leu Arg Lys Leu Asn

325 330 335

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

340 345 350

<210> 22

<211> 1104

<212> DNA

<213> Escherichia coli

<400> 22

atgaaaaatg ttggttttat cggctggcgc ggtatggtcg gctccgttct catgcaacgc 60

atggttgaag agcgcgactt cgacgccatt cgccctgtct tcttttctac ttctcagctt 120

ggccaggctg cgccgtcttt tggcggaacc actggcacac ttcaggatgc ctttgatctg 180

gaggcgctaa aggccctcga tatcattgtg acctgtcagg gcggcgatta taccaacgaa 240

atctatccaa agcttcgtga aagcggatgg caaggttact ggattgacgc agcatcgtct 300

ctgcgcatga aagatgacgc catcatcatt cttgaccccg tcaatcagga cgtcattacc 360

gacggattaa ataatggcat caggactttt gttggcggta actgtaccgt aagcctgatg 420

ttgatgtcgt tgggtggttt attcgccaat gatcttgttg attgggtgtc cgttgcaacc 480

taccaggccg cttccggcgg tggtgcgcga catatgcgtg agttattaac ccagatgggc 540

catctgtatg gccatgtggc agatgaactc gcgaccccgt cctctgctat tctcgatatc 600

gaacgcaaag tcacaacctt aacccgtagc ggtgagctgc cggtggataa ctttggcgtg 660

ccgctggcgg gtagcctgat tccgtggatc gacaaacagc tcgataacgg tcagagccgc 720

gaagagtgga aagggcaggc ggaaaccaac aagatcctca acacatcttc cgtaattccg 780

gtagatggtt tatgtgtgcg tgtcggggca ttgcgctgcc acagccaggc attcactatt 840

aaattgaaaa aagatgtgtc tattccgacc gtggaagaac tgctggctgc gcacaatccg 900

tgggcgaaag tcgttccgaa cgatcgggaa atcactatgc gtgagctaac cccagctgcc 960

gttaccggca cgctgaccac gccggtaggc cgcctgcgta agctgaatat gggaccagag 1020

ttcctgtcag cctttaccgt gggcgaccag ctgctgtggg gggccgcgga gccgctgcgt 1080

cggatgcttc gtcaactggc gtaa 1104

<210> 23

<211> 273

<212> PRT

<213> Escherichia coli

<400> 23

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

1 5 10 15

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

20 25 30

Leu Gly Ala Ala Leu Glu Arg Glu Gly Ser Ser Leu Leu Gly Ser Asp

35 40 45

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

50 55 60

Ser Leu Asp Ala Val Lys Asp Asp Phe Asp Val Phe Ile Asp Phe Thr

65 70 75 80

Arg Pro Glu Gly Thr Leu Asn His Leu Ala Phe Cys Arg Gln His Gly

85 90 95

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

100 105 110

Ala Ile Arg Asp Ala Ala Ala Asp Ile Ala Ile Val Phe Ala Ala Asn

115 120 125

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

130 135 140

Lys Val Met Gly Asp Tyr Thr Asp Ile Glu Ile Ile Glu Ala His His

145 150 155 160

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

165 170 175

Ala Ile Ala His Ala Leu Asp Lys Asp Leu Lys Asp Cys Ala Val Tyr

180 185 190

Ser Arg Glu Gly His Thr Gly Glu Arg Val Pro Gly Thr Ile Gly Phe

195 200 205

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

210 215 220

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

225 230 235 240

Met Thr Phe Ala Asn Gly Ala Val Arg Ser Ala Leu Trp Leu Ser Gly

245 250 255

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

260 265 270

Leu

<210> 24

<211> 822

<212> DNA

<213> Escherichia coli

<400> 24

atgcatgatg caaacatccg cgttgccatc gcgggagccg gggggcgtat gggccgccag 60

ttgattcagg cggcgctggc attagagggc gtgcagttgg gcgctgcgct ggagcgtgaa 120

ggatcttctt tactgggcag cgacgccggt gagctggccg gagccgggaa aacaggcgtt 180

accgtgcaaa gcagcctcga tgcggtaaaa gatgattttg atgtgtttat cgattttacc 240

cgtccggaag gtacgctgaa ccatctcgct ttttgtcgcc agcatggcaa agggatggtg 300

atcggcacta cggggtttga cgaagccggt aaacaagcaa ttcgtgacgc cgctgccgat 360

attgcgattg tctttgctgc caattttagc gttggcgtta acgtcatgct taagctgctg 420

gagaaagcag ccaaagtgat gggtgactac accgatatcg aaattattga agcacatcat 480

agacataaag ttgatgcgcc gtcaggcacc gcactggcaa tgggagaggc gatcgcccac 540

gcccttgata aagatctgaa agattgcgcg gtctacagtc gtgaaggcca caccggtgaa 600

cgtgtgcctg gcaccattgg ttttgccacc gtgcgtgcag gtgacatcgt tggtgaacat 660

accgcgatgt ttgccgatat tggcgagcgt ctggagatca cccataaggc gtccagccgt 720

atgacatttg ctaacggcgc ggtaagatcg gctttgtggt tgagtggtaa ggaaagcggt 780

ctttttgata tgcgagatgt acttgatctc aataatttgt aa 822

<210> 25

<211> 396

<212> PRT

<213> Escherichia coli

<400> 25

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

1 5 10 15

Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly

20 25 30

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

35 40 45

Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn

50 55 60

Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu

65 70 75 80

Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg

85 90 95

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

100 105 110

Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp

210 215 220

Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val

225 230 235 240

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

245 250 255

Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe

260 265 270

Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala

275 280 285

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

290 295 300

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

305 310 315 320

Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg

325 330 335

Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly

340 345 350

Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr

355 360 365

Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn

370 375 380

Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu

385 390 395

<210> 26

<211> 1191

<212> DNA

<213> Escherichia coli

<400> 26

atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60

cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120

ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180

accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240

ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300

ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360

aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420

ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480

gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540

tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600

tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660

ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720

gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780

gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840

gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900

gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960

atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020

atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080

ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140

acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-SacI-F

<400> 27

tccgagctca tgaacgttat tgcaatattg 30

<210> 28

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-XbaI-R

<400> 28

gcctctagac cacttccctt gtacgagc 28

<210> 29

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-F2

<400> 29

atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-R2

<400> 30

agctgtttcc tgtgtgaaat 20

<210> 31

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-RBS-F

<220>

<221> misc_feature

<222> (15)..(24)

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

<400> 31

cggataacaa tttcnnnnnn nnnncagcta tgaacgttat tgcaatattg aatcacatg 59

<210> 32

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer cadA-RBS-R

<400> 32

gaaattgtta tccgctcaca attccacaca ac 32

<210> 33

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer psyn-1

<400> 33

ggcgaattca gtttattctt gacatgtagt gagggggctg gtataatgag ctcggtaccc 60

ggggat 66

<210> 34

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer psyn-2

<400> 34

ggcagtactc aaccaagtca ttctgagaat agtg 34

<210> 35

<211> 49

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer tetA-F

<400> 35

ggcgagctca cacaggaaac agaccatgaa atctaacaat gcgctcatc 49

<210> 36

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer tetA-R

<400> 36

ggctctagat caacgacagg agcacgatc 29

<210> 37

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer lysC-F

<400> 37

ggcgagctca cacaggaaac agaccatgtc tgaaattgtt gtctcc 46

<210> 38

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer lysC-R

<400> 38

ggcggatcct tactcaaaca aattactatg cag 33

<210> 39

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer dapA-F

<400> 39

ggcggatcca cacaggaaac agaccatgtt cacgggaagt attgtc 46

<210> 40

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer dapA-R

<400> 40

ggctctagat tacagcaaac cggcatgc 28

<210> 41

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer lysA-F

<400> 41

ggctctagaa cacaggaaac agaccatgcc acattcactg ttcagc 46

<210> 42

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer lysA-R

<400> 42

ggcgtcgact taaagcaatt ccagcgccag 30

<210> 43

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer tetA-F3

<400> 43

ggcctcgaga gtttattctt gacatgtagt gagg 34

<210> 44

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer tetA-R3

<400> 44

ggcgcatgct caacgacagg agcacgatc 29

<210> 45

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 318-F

<400> 45

cagcctgaat atactgcatt ctc 23

<210> 46

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 318-R

<400> 46

gagaatgcag tatattcagg ctg 23

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 323-F

<400> 47

gcattctcgc gatttcctcg 20

<210> 48

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer 323-R

<400> 48

cgaggaaatc gcgagaatgc 20

<210> 49

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer asd-F

<400> 49

ggcgagctca cacaggaaac agaccatgaa aaatgttggt tttatcgg 48

<210> 50

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer asd-R

<400> 50

ggcggatcct tacgccagtt gacgaagc 28

<210> 51

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer dapB-F

<400> 51

ggcggatcca cacaggaaac agaccatgca tgatgcaaac atccg 45

<210> 52

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer dapB-R

<400> 52

ggctctagat tacaaattat tgagatcaag tacatctc 38

<210> 53

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer aspC-F

<400> 53

ggctctagaa cacaggaaac agaccatgtt tgagaacatt accgcc 46

<210> 54

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer aspC-R

<400> 54

ggcgcatgcg acctcgaggt agtcgactta cagcactgcc acaatcg 47

<210> 55

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LAL-F

<400> 55

ggcggtacca gtttattctt gacatgtagt gagg 34

<210> 56

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LAL-R

<400> 56

ggcgggccct taaagcaatt ccagcgcca 29

Claims (12)

1. a kind of ribosome bind site, which is characterized in that the sequence 5 ' -3 ' of the ribosome bind site include/is as follows Shown in one of sequence：

a)、GTCATCAAAA；

b)、CGCAAAGATA；

c)、AAAGATTTAC。

2. a kind of recombinant expression plasmid, including the target gene of recombinant expression plasmid and corresponding ribosome bind site, special Sign is that the ribosome bind site is as described in any technical solution of claim 1, it is preferable that the ribosome binding site Point is located at upstream from start codon -15 and arrives -6.

3. a kind of recombinant expression plasmid as claimed in claim 2, which is characterized in that the skeleton plasmid of the recombinant expression plasmid It is one kind in pUC, pBR322, pACYC plasmid and their plasmid.

4. such as a kind of recombinant expression plasmid of claim 2-3 any one of them, which is characterized in that the recombinant expression plasmid Target gene is the polynucleotide for encoding polypeptide；The polypeptide is enzyme or polypeptide drug；It is further preferred that the enzyme It is at least one of oxidoreducing enzyme, transferase, hydrolase, lyase, isomerase and ligase, the polypeptide drug is At least one of hormone, antibody and growth factor；Further preferably, the lyase is decarboxylase, further excellent Selection of land, the decarboxylase are amino acid decarboxylases, such as lysine decarboxylase, tyrosine decarboxylase, arginine decarboxylase, ornithine Decarboxylase or glutamate decarboxylase.

5. a kind of recombinant expression plasmid according to claim 4, which is characterized in that the recombinant expression plasmid of the transformant Target gene be encode lysine decarboxylase polynucleotide；Preferably, the polynucleotide of the coding lysine decarboxylase It is derived from Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), basophilic gemma bar Bacterium (Bacillus halodurans), streptomyces coelicolor (Streptomyces coelicolor), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum) or klebsiella oxytoca The lysine decarboxylase gene of (Klebsiella oxytoca)；It is further preferred that the poly-nuclear glycosides of coding lysine decarboxylase Acid is the piece of cadA genes, ldcC genes, haldc genes, the segment of cadA genes, the segment of ldcC genes or haldc genes Section；It is further preferred that the cadA genes are derived from the cadA genes of Escherichia coli (E.coli).

6. a kind of transformant, which is characterized in that the genome of the transformant includes that target gene and corresponding ribosomes combine Site, the ribosome bind site are as described in claim 1, it is preferable that the ribosome bind site is located at initiation codon Sub- upstream -15 arrives -6；The target gene is the polynucleotide for encoding polypeptide；The polypeptide is enzyme or polypeptide drug； It is further preferred that the enzyme be in oxidoreducing enzyme, transferase, hydrolase, lyase, isomerase and ligase at least One kind, the polypeptide drug are at least one of hormone, antibody and growth factor；Further preferably, described to split conjunction Enzyme is decarboxylase, it is further preferred that the decarboxylase is amino acid decarboxylases, such as lysine decarboxylase, tyrosine decarboxylation Enzyme, arginine decarboxylase, ornithine decarboxylase or glutamate decarboxylase；

Alternatively, the recombinant expression plasmid of the transformant is such as claim 2-5 any one of them recombinant expression plasmids.

7. transformant as claimed in claim 6, which is characterized in that the recombinant expression plasmid of the transformant is such as claim Recombinant expression plasmid described in any one of 5 technical solutions.

8. the host strain of transformant as claimed in claims 6 or 7, the transformant is Escherichia coli (E.coli), withered grass bud Spore bacillus (B.subtilis), streptomyces coelicolor (S.coelicolor), hafnia alvei (H.alvei) or glutamic acid rod Shape bacillus (C.glutamicum) is further preferred that Escherichia coli (E.coli) or hafnia alvei (H.alvei).

9. a kind of method of fermenting and producing polypeptide, which is characterized in that include the following steps：

A claim 6-8 any one of them transformants) are cultivated；

B) polypeptide is obtained from the bacterium solution or thalline obtained in step A.

10. a kind of method of fermenting and producing polypeptide as claimed in claim 9, which is characterized in that be that fermenting and producing lysine is de- The method of carboxylic acid, includes the following steps：

1) culture such as claim 7-8 any one of them transformants；

2) lysine decarboxylase is obtained in the bacterium solution or thalline that are obtained from step 1.

11. a kind of fermenting and producing 1, the method for 5- pentanediamines, which is characterized in that include the following steps：

Culture such as claim 7-8 any one of them transformants, the host strain of the transformant can produce lysine, described to rely Propylhomoserin decarboxylation enzymatic lysine decarboxylation generates 1,5- pentanediamines.

12. a kind of mRNA, which is characterized in that the sequence of the mRNA includes and ribosome bind site described in claim 1 Corresponding sequence.