CN111978407B - Heterologous expression method of lysine decarboxylase from thermophilic bacteria and application thereof - Google Patents

Heterologous expression method of lysine decarboxylase from thermophilic bacteria and application thereof Download PDF

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CN111978407B
CN111978407B CN201910431317.3A CN201910431317A CN111978407B CN 111978407 B CN111978407 B CN 111978407B CN 201910431317 A CN201910431317 A CN 201910431317A CN 111978407 B CN111978407 B CN 111978407B
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CN111978407A (en
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陈玲
周豪宏
雷云凤
刘修才
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CIBT America Inc
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Abstract

The invention relates to a heterologous expression method of lysine decarboxylase from thermophile and application thereof. According to the invention, through screening lysine decarboxylase derived from thermophilic strain, the fusion expression of the lysine decarboxylase derived from thermophilic strain and the lysine decarboxylase derived from thermophilic strain is utilized, and when the lysine decarboxylase derived from thermophilic strain is connected with the fusion promoting label, the lysine decarboxylase derived from thermophilic strain can play a role in lysine decarboxylation; in addition, the use of a lytic tag may also be clearly indicative of cells expressing the fusion protein. The invention provides a new strategy for producing high-yield 1, 5-pentanediamine by fermentation.

Description

Heterologous expression method of lysine decarboxylase from thermophilic bacteria and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a heterologous expression method of lysine decarboxylase from thermophile bacteria and application thereof.
Background
Lysine decarboxylase (L-lysine decarboxylase, abbreviated as LDC, EC 4.1.1.18) is widely found in microorganisms, insects, animals and higher plants, and can strip L-lysine to remove one carboxyl group to produce 1, 5-pentanediamine (cadaverine) and CO 2 . The 1, 5-pentanediamine has quite wide application, for example, the 1, 5-pentanediamine can be polymerized with dibasic acid to synthesize novel nylon, and has high application value in industrial production. At present, the microbial method for producing 1, 5-pentanediamine mainly adopts the following two methods: microbial fermentation production and microbial in-vitro enzyme catalysis production of 1, 5-pentanediamine.
When 1, 5-pentanediamine is produced by microbial fermentation or microbial in-vitro enzyme catalysis, a lysine decarboxylase gene expression cassette is constructed according to a lactose operon, and the Chinese patent application CN 105441497A enables the lysine decarboxylase gene to be secreted and expressed in a large quantity by constructing an expression cassette with a structure of (IPTG/lactose) inducible promoter-signal peptide-lysine decarboxylase gene-terminator. The L-lysine is produced by glucose fermentation through escherichia coli or corynebacterium glutamicum strains capable of producing the L-lysine at first, and then the secretion expression of lysine decarboxylase is induced by adding inducers such as IPTG/lactose and the like to catalyze the conversion of the L-lysine into cadaverine. Thus, the one-step method of producing 1, 5-pentanediamine by glucose can be realized, namely, L-lysine is produced by microbial fermentation and the L-lysine decarboxylation reaction is catalyzed in a coupling way to produce 1, 5-pentanediamine. However, the system has serious leakage expression except the need of adding IPTG/lactose, and because of the limited concentration of 1, 5-pentanediamine tolerated by the thallus, if excessive 1, 5-pentanediamine generated by lysine decarboxylase conversion is expressed in the fermentation system in the early stage, the system can cause toxicity to the thallus, thereby inhibiting the thallus growth and the process of producing L-lysine by utilizing glucose (Qian, et al, biotechnol. Bioeng.2011; 108:93-103), and finally influencing the yield of the catalytically generated 1, 5-pentanediamine.
Disclosure of Invention
The invention aims to provide a heterologous expression method of lysine decarboxylase from thermophile and application thereof.
It is another object of the present invention to provide a process for the fermentative production of 1, 5-pentanediamine in a single step.
The invention is characterized in that: by screening some novel lysine decarboxylases derived from thermophilic strains, the activity of the novel lysine decarboxylases is not or very low at the growth temperature of the lysine production strains (generally 37+/-2 ℃), so that the activity of the novel lysine decarboxylases is not or very low even though the lysine decarboxylases are expressed in the early stage of fermentation, and after the growth of the lysine production strains is finished, the novel lysine decarboxylases are active or have improved activity by changing the temperature, and the catalytically produced L-lysine is converted into 1, 5-pentanediamine.
In order to achieve the purpose of the invention, lysine decarboxylase derived from thermophilic bacterial strain and different protein tags are fused and expressed to help the lysine decarboxylase fold into an active form in a heterologous host cell, and the lysine decarboxylase catalyzes the L-lysine decarboxylation reaction to generate 1, 5-pentanediamine. The recombinant strain can effectively ensure that 1, 5-pentanediamine with lower quantity is generated in the early stage of the process of producing 1, 5-pentanediamine by a glucose one-step method (lysine decarboxylase in the early stage of fermentation has expression but no activity or very low activity), so the toxic effect of the 1, 5-pentanediamine on the growth of thalli is reduced in the early stage of fermentation, and the balance between lysine production and 1, 5-pentanediamine production is maintained; the lysine decarboxylase is then activated by changing the temperature to catalyze the conversion of the produced L-lysine to 1, 5-pentanediamine, preferably at a time point when the lysine-producing strain is grown or the lysine content is no longer increased. The final comprehensive effect can improve the yield of 1, 5-pentanediamine produced by a one-step method, and simultaneously ensure that the residual lysine is as little as possible.
In a first aspect, the invention provides a lysine decarboxylase fusion protein with a pro-lytic tag, the fusion protein comprising a pro-lytic tag and a lysine decarboxylase from thermophilic bacteria, linked by a Linker.
Wherein the pro-lytic tag is selected from fluorescent proteins, maltose binding proteins, glutathione transferase, and the like, or combinations thereof.
When heterologous protein expression is performed in prokaryotes such as E.coli, the maltose binding protein MBP, glutathione transferase GST, and histidine His 7 And the like can promote the solubility of the protein and the correct folding of the protein. MBP (maltose binding protein) tag protein is 42kDa in size and pi=5.03, which increases the solubility of fusion proteins overexpressed in bacteria, but the tag is large and has some effect on the structure or function of the protein. GST (glutathione-sulfhydryl transferase) tag protein, its natural size is 26KD, isoelectric point pi=6.10. The reasons for its application in prokaryotic expression are mainly represented in two aspects, one of which is that it is a highly soluble protein, which can be used to increase the solubility of foreign proteins; secondly, it can be expressed in large quantity in colibacillus, and plays a role in improving the expression quantity.
In the invention, the fluorescent protein is selected from red fluorescent protein, blue-green fluorescent protein, yellow fluorescent protein, orange fluorescent protein or optical highlighting fluorescent protein; preferably RedStar, tdtomato or mCherry. The inventors have unexpectedly found that the above fluorescent proteins can be used as a pro-lytic tag in heterologous protein expression and that their fluorescent properties can also be conveniently indicative of cells that correctly express the fusion protein.
In some embodiments, the mCherry is a red fluorescent protein from coral, the best performing monomeric red fluorescent protein evolved from DsRed (Graewe, et al Biotechnology Journal,2009,4 (6)), size 26 KDa, isoelectric point pi=5.62.
In some embodiments, the amino acid sequence of mCherry is shown in SEQ ID NO. 6 and the nucleotide sequence of the mCherry encoding gene is shown in SEQ ID NO. 5, which is codon optimized.
In a second aspect, the present invention provides a fusion protein using a fluorescent protein as a dissolution-promoting tag, wherein the fusion protein comprises the fluorescent protein and a target protein, which are connected through a Linker.
Further, the present invention provides a fusion protein which is mCherry-Linker-lysine decarboxylase from thermophilic bacteria, or lysine decarboxylase-Linker-mCherry from thermophilic bacteria.
In some embodiments, the lysine decarboxylase is a lysine decarboxylase TeLDC that may be derived from thermophilic bacteria Thermosynechoccus elongatus, having an amino acid sequence as set forth in SEQ ID NO:1, the coding gene is Teldc (GenBank ID BAC09418.1, SEQ ID NO: 2), or a nucleotide sequence identical to SEQ ID NO:1, and having at least 95% sequence identity and equivalent function; the amino acid sequence of the lysine decarboxylase TsLDC from Tepidanaerobacter syntrophicus is shown as SEQ ID NO. 84, the coding gene is Tsldc (GenBank ID GAQ24853.1, SEQ ID NO. 85), or the amino acid sequence has at least 95% sequence identity with SEQ ID NO. 84 and has the same function; can be lysine decarboxylase GkLDC from Geobacillus kaustophilus, the amino acid sequence of which is shown as SEQ ID NO. 86, the coding gene is Gkldc (GenBank ID BAD75350.1, SEQ ID NO. 87), or the amino acid sequence which has at least 95 percent sequence identity with SEQ ID NO. 86 and has the same function; or a lysine decarboxylase TrLDC derived from Thermomicrobium roseum, the amino acid sequence of which is shown as SEQ ID NO. 88, the coding gene is Trldc (GenBank ID ACM05730.1, SEQ ID NO. 89), or an amino acid sequence which has at least 95% sequence identity with SEQ ID NO. 88 and has the same function.
Preferably, the lysine decarboxylase is a lysine decarboxylase TeLDC derived from thermophilic bacteria Thermosynechoccus elongatus, and the amino acid sequence of the lysine decarboxylase is shown as SEQ ID NO:1 is shown in the specification; the coding gene is Teldc, and the nucleotide sequence is shown in SEQ ID NO:2, the nucleotide sequence was optimized according to E.coli codon bias.
In some embodiments of the present invention, in some embodiments,the Linker comprises a flexible Linker composed of a Linker in spiral form or amino acids with low hydrophobicity and low charge effect, and the length of the Linker should be at least 10 amino acids. Preferably, the Linker is a flexible Linker, e.g. (GGGGS) 3 Or (SG) 5-8 The method comprises the steps of carrying out a first treatment on the surface of the More preferably, the Linker is (SG) 5-8 The method comprises the steps of carrying out a first treatment on the surface of the Most preferably, the Linker is SGSGSGSGSG.
In some embodiments, the fusion protein is red fluorescent protein-Linker-TeLDC, red fluorescent protein-Linker-TsLDC, red fluorescent protein-Linker-GkLDC, or red fluorescent protein-Linker-TrLDC.
Preferably, the red fluorescent protein comprises RedStar, tdtomato or mCherry.
In some embodiments, the fusion protein-Linker-lysine decarboxylase from thermophilic bacteria may be the fusion protein MRFP-TeLDC (mCherry-Linker-TeLDC) having the amino acid sequence set forth in SEQ ID NO: shown in figure 7; can be fusion protein TeLDC-MRFP (TeLDC-Linker-mCherry), and the amino acid sequence of the fusion protein is shown as SEQ ID NO: shown as 9; can be fusion protein MRFP-TsLDC (mCherry-Linker-TsLDC), and has an amino acid sequence shown in SEQ ID NO: 92; can be fusion protein MRFP-GkLDC (mCherry-Linker-GkLDC), and has an amino acid sequence shown in SEQ ID NO: 96; it can also be fusion protein MRFP-TrLDC (mCherry-Linker-TrLDC), and the amino acid sequence is shown in SEQ ID NO: shown at 100. Preferably, the fusion protein mCherry-Linker-lysine decarboxylase from thermophilic bacteria is the fusion protein MRFP-TeLDC (SEQ ID NO: 7).
In a third aspect, the invention provides a polynucleotide encoding the fusion protein described above.
Further, the present invention provides a recombinant nucleotide sequence as shown in SEQ ID NO. 8, comprising a nucleotide sequence encoding a fluorescent protein mCherry and a nucleotide sequence encoding a thermophilic bacteria-derived lysine decarboxylase TeLDC in the 5 'to 3' direction.
The present invention provides a recombinant nucleotide sequence as shown in SEQ ID NO. 16, comprising in the 5 'to 3' direction a nucleotide sequence encoding maltose binding protein MBP (SEQ ID NO. 11) (SEQ ID NO. 12) and a nucleotide sequence encoding a thermophilic bacteria-derived lysine decarboxylase TeLDC (SEQ ID NO. 2).
The present invention provides a recombinant nucleotide sequence as shown in SEQ ID No. 18, comprising in the 5 'to 3' direction a nucleotide sequence encoding glutathione transferase GST (SEQ ID No. 13) (SEQ ID No. 14) and a nucleotide sequence encoding thermophilic bacteria-derived lysine decarboxylase TeLDC (SEQ ID No. 2).
In a fourth aspect, the invention provides the use of a fluorescent protein as a pro-lytic tag in the expression of a heterologous protein.
In a fifth aspect, the invention provides biological materials comprising the polynucleotides, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, or engineered bacteria.
In a sixth aspect, the present invention provides an engineering bacterium for producing lysine decarboxylase, which is a strain having an ability to produce L-lysine, and which carries an expression vector for expressing the above fusion protein.
Wherein the original strain of the engineering bacteria is selected from strains in Escherichia, hafnia and other bacteria; preferably, the starting strain is escherichia coli (escherichia coli), hafnia alvei (Hafnia alvei), corynebacterium glutamicum (Corynebacterium glutamicum), bacillus subtilis (Bacillus subtilis) or streptomyces coelicolor (Streptomyces coelicolor), or a strain or genetically engineered bacterium after mutagenesis or random mutation.
The recombinant plasmid may be a pUC or pBR322 plasmid or a derivative thereof, for example pUC18, pUC19, pBR322, pACYC, pET, pSC101 and any derivative thereof.
In a seventh aspect, the present invention provides a method for heterologous expression of lysine decarboxylase derived from thermophilic bacteria, wherein the encoding gene of the fusion protein is constructed into engineering bacteria after codon optimization, and recombinant bacteria are cultured to realize heterologous expression of lysine decarboxylase.
In an eighth aspect, the present invention provides the use of the heterologous expression method described above for the fermentative production of 1, 5-pentanediamine, in particular for the fermentative production of 1, 5-pentanediamine in a one-step process. Constructing the encoding gene of the fusion protein into engineering bacteria with the capacity of producing L-lysine, fermenting and culturing recombinant bacteria and accumulating lysine, and controlling the culture temperature at 20-50 ℃ at the initial stage of fermentation; (b) The rest fermentation stage is controlled at 50-110deg.C to activate lysine decarboxylase, and 1, 5-pentanediamine is produced by conversion.
As used herein, the term "about" when used to modify a value within a temperature range means a reasonable deviation of that value from the value, e.g., within 1 ℃ or 2 ℃ below or above the value recited in the range, and falls within the intended meaning of the value or range.
In some embodiments, step (a) is performed at a temperature of about 25 ℃ to about 45 ℃. In other embodiments, step (a) is performed at a temperature of about 30 ℃ to about 40 ℃. In a further embodiment, step (a) is performed at a temperature of about 35 ℃ to about 39 ℃. In some embodiments, step (b) is performed at a temperature of about 55 ℃ to about 90 ℃. In other embodiments, step (b) is performed at a temperature of about 60 ℃ to about 75 ℃. In a further embodiment, step (b) is performed at a temperature of about 60 ℃ to about 70 ℃.
Further, the invention provides a method for producing 1, 5-pentanediamine by fermentation, which comprises the steps of constructing a coding gene of the fusion protein into engineering bacteria with L-lysine production capacity, preferably constructing the coding gene into engineering bacteria, preferably escherichia coli after codon optimization, fermenting and culturing recombinant bacteria, controlling the initial fermentation culture temperature at 20-50 ℃, for example 37+/-2 ℃, and carrying out rapid growth and lysine accumulation of bacteria; the rest fermentation stage is controlled at 50-80deg.C, such as 55deg.C+ -2deg.C, to make lysine decarboxylase active or improve activity, and convert lysine to produce 1, 5-pentanediamine. Wherein, preferably, the fusion protein is fluorescent protein-Linker-lysine decarboxylase, more preferably red fluorescent protein-Linker-lysine decarboxylase (TeLDC, tsLDC, gkLDC or TrLDC). In some preferred embodiments of the invention, when the fermentation is cultivated until the lysine content no longer increases, the temperature of the fermentation system is readjusted and controlled to 50-110 ℃. Preferably, the construction can be performed using recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector or engineering bacteria and the like, to engineering bacteria having the ability to produce L-lysine.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
according to the invention, through screening lysine decarboxylase derived from thermophilic strain, the dissolution promoting label is utilized to be fused and expressed with the lysine decarboxylase, when the lysine decarboxylase derived from thermophilic strain is connected with the dissolution promoting label, the lysine decarboxylase can help the lysine decarboxylase to play a role, and compared with lysine decarboxylase CadA derived from escherichia coli, the lysine decarboxylase can play a role simply by increasing the temperature; in addition, the use of a pro-lytic tag may also be clearly indicated for cells expressing the fusion protein, especially fluorescent proteins, which are easier to clearly indicate as pro-lytic tags. The method is applied to the production of 1, 5-pentanediamine, can obviously reduce the cell growth and the cytotoxicity of the 1, 5-pentanediamine generated in the production stage of the L-lysine, and a small amount of 1, 5-pentanediamine can also relieve the feedback inhibition effect of the lysine and improve the yield of the L-lysine; after the temperature is raised, L-lysine can be almost completely converted into 1, 5-pentanediamine, thereby also achieving an increase in the yield of 1, 5-pentanediamine.
In particular, when the red fluorescent protein is used for fusion expression with lysine decarboxylase TeLDC, tsLDC, gkLDC or TrLDC which is screened from thermophilic strains, the 5 'end of the lysine decarboxylase which is derived from thermophilic strains is connected with the 3' end of the red fluorescent protein, so that the lysine decarboxylase can be folded into an active form and exert the function of lysine decarboxylation, compared with the lysine decarboxylase cadA which is derived from escherichia coli, the activity of the lysine decarboxylase CadA is lower at 37+/-2 ℃, the activity of the lysine decarboxylase CadA is higher at 55+/-2 ℃, the lysine decarboxylase CadA can be used for exerting the function simply by increasing the temperature, the yield of 1, 5-pentanediamine is effectively increased when the lysine decarboxylase is applied to the production of 1, 5-pentanediamine, and little L-lysine is remained, so that the industrial difficulty of the subsequent extraction of the 1, 5-pentanediamine is greatly simplified.
Drawings
FIG. 1 shows the SDS-PAGE analysis of intracellular protein supernatants and precipitates after cell lysis in example 2 of the present invention, respectively, for soluble expression of TeLDC.
FIG. 2 shows the soluble expression of MRFP-TeLDC fusion protein by SDS-PAGE of intracellular protein supernatants after cell lysis in example 4 of the present invention.
FIG. 3 shows the detection of soluble expression of TeLDC-MRFP fusion protein by SDS-PAGE of intracellular protein supernatants after cell lysis in example 4 of the present invention.
FIG. 4 shows the detection of soluble expression of MBP-TeLDC fusion proteins by SDS-PAGE of intracellular protein supernatants after cell lysis in example 6 of the present invention.
FIG. 5 shows the soluble expression of GST-TeLDC fusion protein by SDS-PAGE of intracellular protein supernatants after cell lysis in example 8 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Specific steps such as PCR amplification, purification, plasmid extraction, cleavage, ligation of cleavage products, transformation, etc., and condition parameters involved in the following examples were carried out according to the conditions suggested in the specifications of the purchased relevant enzymes and reagents. Wherein the DNA polymerase used for PCR amplification, the restriction enzyme used for enzyme digestion and the ligase used for ligation of enzyme digestion products are all purchased from Takara Bio-engineering (Dalian) Inc. Plasmid extraction kits, DNA gel recovery kits, PCR purification kits were all purchased from corning life sciences (Wu Jiang) inc under the trademark Axygen; primers were purchased from Semer Feishul technology (China) Inc., under the trademark INVITROGEN. The Linker is amino acid with connection function, and different target genes are connected through a nucleotide sequence of the encoded Linker, so that the Linker can be expressed into a single peptide chain in organisms.
The plasmid transformation methods involved in the following examples are as follows: the ligation product was added to 100. Mu.l E.coli BL21 (DE 3) competent cells, and heat shocked at 42℃for 90s after 30min in ice bath. After incubation on ice for 5min 1ml of LB was added. Coated onto corresponding resistant plates.
In some embodiments, the amount of L-lysine and 1, 5-pentanediamine in the medium can be measured by nuclear magnetic resonance methods, and the cellular L-lysine conversion can be calculated by nuclear magnetic resonance methods to measure the amount of 1, 5-pentanediamine produced by lysine decarboxylase catalyzing the conversion of L-lysine.
The primers used in the following examples are as follows:
EXAMPLE 1 cloning of lysine decarboxylase Gene cadA
The lysine decarboxylase (SEQ ID No: 3) encoding gene cadA (SEQ ID No: 4) was amplified from the genome of E.coli MG 1655K 12 (E.coli MG 1655K 12, purchased from Beijing Tian-Zen Biotechnology Co., ltd.) using primers cadA-SacI-F (SEQ ID No: 22) and cadA-XbaI-R (SEQ ID No: 23), and after double cleavage by SacI and XbaI, ligated into the same double digested pUC18 plasmid (purchased from Takara Bio Inc.). By CaCl 2 Competent preparation is carried out by the method, the connection product is transformed into E.coli BL21 (purchased from Bao bioengineering (Dalian) Limited) cells by a heat shock method, ampicillin antibiotics are added into LB culture medium for screening, cloning PCR and sequencing are carried out to verify correctness, and plasmid is extracted to obtain pCIB60 plasmid.
The plasmid pCIB60 is used as a template, and primers cadA-F2 (SEQ ID No: 24) and cadA-R2 (SEQ ID No: 25) are further used for optimizing the sequence of the pCIB60 plasmid upstream of the start codon ATG of the cadA gene, so that the cadA can be successfully translated into protein in E.coli BL21, and the 5' sequence of the cadA gene is replaced by 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3' (SEQ ID No: 20) from 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC-3' (SEQ ID No: 19). After PCR amplification, the PCR product was digested with DpnI restriction enzyme and also transformed into E.coli BL21 by heat shock, and the plasmid pCIB71 was obtained after sequencing and verification.
EXAMPLE 2 cloning and expression validation of lysine decarboxylase Teldc of thermophilic bacterium Thermosynechoccus elongatus
Lysine decarboxylase TeLDC (SEQ ID NO:1,GenBank ID BAC09418.1) from thermophilic strain Thermosynechoccus elongatus synthesizes the gene (SEQ ID NO: 2) by primer splicing after optimizing the full-length codon of the gene, and the codon optimization and gene synthesis method refers to Hoover DM&Lubkowski J Nucleic Acids Research30:10,2002, the spliced Teldc was amplified using TeLDC-SacI-F (SEQ ID No: 26) and TeLDC-XbaI-R (SEQ ID No: 27), double digested with SacI and XbaI, and ligated into the same double digested pCIB71 plasmid. By CaCl 2 Competent cells were prepared by the method, and the ligation products were transformed into E.coli BL21 (purchased from Takara Bio-engineering (Dalian) Co.) competent cells by the heat shock method, screening was performed by adding ampicillin antibiotics to LB medium, cloning PCR and sequencing to verify correct, and plasmids were extracted to obtain pCIB90 plasmids.
The plasmid pCIB90 is used as a template, and primers TeLDC-SacI-F2 (SEQ ID No: 28) and TeLDC-SacI-R2 (SEQ ID No: 29) are further used for optimizing the sequence of the pCIB90 plasmid upstream of the Teldc gene initiation codon ATG, so that the TeLDC can be smoothly translated into protein in E.coli BL21, and the sequence of the Teldc gene initiation codon ATG is replaced by 5'-tgtggaattgtgagctcaataattttgtttaactttaagaaggagaacagct-3' (SEQ ID No: 21) from 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3' (SEQ ID No: 20). After PCR amplification, the PCR product was digested with DpnI restriction enzyme and also transformed into E.coli BL21 by heat shock, and the plasmid pCIB91 was obtained after sequencing and verification.
Plasmids pCIB71 and pCIB91 are transformed into E.coli BL21 cells, respectively, and the cells are spread on LB solid plates containing 100 mug/ml ampicillin for screening to obtain CadA protein expression strain CIB70 and TeLDC expression strain CIB90. After overnight incubation at 37℃3 clones were each picked up and shaken in LB tubes overnight, and equal amounts of bacterial liquid were each taken for SDS-PAGE, i.e.bacterial cells of equal volumes (4 ml) of bacterial liquid were collected, and intracellular proteins were extracted with lysozyme (from Takara Bio-engineering (Dalian) Co., ltd.) and examined for expression of TeLDC and for soluble expression. As a result, as shown in FIG. 1, teLDC was expressed normally and was expressed in a soluble form in a large part, but the expression level of TeLDC was still lower than that of CadA.
EXAMPLE 3 construction of plasmid for fusion expression of TeLDC and Red fluorescent protein
The red fluorescent protein MRFP (i.e., mCherry, SEQ ID No: 6) from coral is synthesized by a method of primer splicing after optimizing the whole length of the gene (mCherry, SEQ ID No: 5), the method of codon optimization and gene synthesis refers to Hoover DM & Lubkowski J, nucleic Acids Research: 10,2002, the spliced MRFP is used as a template, primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and linker-MRFP-R (SEQ ID No: 31) are used for amplification, plasmid pCIB91 is used as a template, primers linker-TeLDC-F (SEQ ID No: 32) and TeLDC-TeLDI-R (SEQ ID No: 27) are used for amplification, the target fragment is cut and recovered by primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and TeLDC-XbaI-R (SEQ ID No: 27) respectively, and the fusion protein PCR fusion of the target fragment and the two-TeLDC-7 (SEQ ID No: 92) is obtained by connecting the primers SacI-MRFP-TeLDC-7 and TeLDC-TeLDI-7' with the fusion of the fusion protein.
The plasmid pCIB91 is used as a template, and primers SacI-TeLDC-MRFP-F (SEQ ID No: 33) and linker-TeLDC-R (SEQ ID No: 34) are used for amplification; the spliced MRFP is used as a template, primers linker-MRFP-F (SEQ ID No: 35) and MRFP-XbaI-R (SEQ ID No: 36) are used for amplification, target fragments are cut and recovered respectively, then fusion PCR is carried out by using the primers SacI-TeLDC-MRFP-F (SEQ ID No: 33) and MRFP-XbaI-R (SEQ ID No: 36), target fragments are purified, double enzyme digestion is carried out by using SacI and XbaI, and the target fragments are connected into a plasmid pCIB91 subjected to the same double enzyme digestion, so that a TeLDC-MRFP fusion protein (SEQ ID No: 9) is obtained to express a plasmid pCIB93, wherein the 3 '-end of a TeLDC encoding gene is connected with the 5' of red fluorescent protein MRFP (SEQ ID No: 10) through a connector sequence.
EXAMPLE 4 construction of strains in which TeLDC and Red fluorescent protein are expressed in fusion
The constructed MRFP-TeLDC fusion protein expression plasmids pCIB92 and TeLDC-MRFP fusion protein expression plasmid pCIB93 are transformed into E.coli BL21 (purchased from Takara Bio-engineering (Dai) Inc.) competent cells, plated on LB plates containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and cultured upside down at 37℃overnight to obtain strains CIB92 and CIB93 in which TeLDC and red fluorescent protein MRFP are fusion expressed. 3 individual clones were individually picked and inoculated into 5ml LB liquid tubes containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and incubated overnight at 37℃at 200 rpm. 4ml of bacterial cells were collected, intracellular proteins were extracted with lysozyme (available from Takara Bio Inc.), and whether fusion proteins MRFP-TeLDC (FIG. 2) and TeLDC-MRFP (FIG. 3) were expressed and soluble were detected by SDS-PAGE. As can be seen from FIGS. 2 and 3, the fusion proteins MFRP-TeLDC and TeLDC-MRFP were both expressed smoothly and were expressed soluble.
EXAMPLE 5 construction of plasmids for fusion expression of TeLDC and maltose binding protein
The first amplification was performed with plasmid Pmal-C5X (available from Getaishan Haiekesai biological Co., ltd.) as a template, primers Plac-MBP-F1 (SEQ ID No: 37) and MBP-R (SEQ ID No: 38), and primers Plac-MBP/GST-F2 (SEQ ID No: 39) and MBP-R (SEQ ID No: 38) were subjected to a second round of amplification to obtain a maltose binding protein MBP encoding gene MBP having a SacI cleavage site at the 5 'end and a ribosome binding site at the 3' end with a partial linker sequence; using plasmid pCIB92 as a template, and using primers TeLDC-F3 (SEQ ID No: 40) and TeLDC-XbaI-R (SEQ ID No: 27) to amplify to obtain a Teldc fragment with a linker sequence at the 5' end; after the target fragments are respectively subjected to gel cutting recovery, fusion PCR amplification is carried out by using a primer Plac-MBP/GST-F2 (SEQ ID No: 39) and a primer TeLDC-XbaI-R (SEQ ID No: 27), the target fragments are recovered by gel cutting, double enzyme cutting is carried out by using SacI and XbaI, and then the target fragments are connected into a plasmid pCIB91 subjected to double enzyme cutting, so that an MBP-TeLDC fusion protein (SEQ ID No: 15) expression plasmid pCIB94 is obtained, wherein the 5 'end of a TeLDC coding gene is connected with the 3' end of a maltose binding protein MRFP (SEQ ID No: 16) through a linker sequence.
EXAMPLE 6 construction of strains in which TeLDC and maltose binding protein are expressed in fusion
The constructed MBP-TeLDC fusion protein expression plasmid pCIB94 was transformed into E.coli BL21 (purchased from Takara Bio Inc.) competent cells, plated on LB plates containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and cultured upside down at 37℃overnight to obtain a strain CIB94 expressed by fusion of TeLDC and maltose binding protein MBP. 3 individual clones were individually picked and inoculated into 5ml LB liquid tubes containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and incubated overnight at 37℃at 200 rpm. 4ml of bacterial cells were collected, and intracellular proteins were extracted with lysozyme (available from Takara Bio Inc.), and whether or not the fusion protein MBP-TeLDC was expressed and soluble was detected by SDS-PAGE (FIG. 4). As can be seen from FIG. 4, the fusion protein MBP-TeLDC was expressed equally well and was expressed in a soluble manner.
EXAMPLE 7 construction of plasmid for fusion expression of TeLDC and glutathione transferase
Taking plasmid pGEX-4T-1 (purchased from GE Healthcare) as a template, performing gel cutting recovery on a target fragment by using primers Plac-GST-F1 (SEQ ID No: 41) and GST-R (SEQ ID No: 42), and performing second round amplification by using primers Plac-MBP/GST-F2 (SEQ ID No: 39) and GST-R (SEQ ID No: 42) to obtain a glutathione transferase GST coding gene GST with a SacI enzyme cutting site at the 5 'end, a ribosome binding site and a partial linker sequence at the 3' end; using plasmid pCIB92 as a template, and using primers TeLDC-F3 (SEQ ID No: 40) and TeLDC-XbaI-R (SEQ ID No: 27) to amplify to obtain a Teldc fragment with a linker sequence at the 5' end; after the target fragments are respectively subjected to gel cutting recovery, fusion PCR amplification is carried out by using a primer Plac-MBP/GST-F2 (SEQ ID No: 39) and a primer TeLDC-XbaI-R (SEQ ID No: 27), the target fragments are recovered by gel cutting, double enzyme cutting is carried out by using SacI and XbaI, and then the target fragments are connected into a plasmid pCIB91 subjected to double enzyme cutting, so that a GST-TeLDC fusion protein (SEQ ID No: 17) expression plasmid pCIB95 is obtained, wherein the 5 'end of a TeLDC coding gene is connected with the 3' end of glutathione transferase GST (SEQ ID No: 18) through a linker sequence.
EXAMPLE 8 construction of strains in which TeLDC and glutathione transferase are expressed in fusion
The constructed GST-TeLDC fusion protein expression plasmid pCIB95 is transformed into E.coli BL21 (purchased from Takara Bio-engineering (Dalian) Co.) competent cells, coated on LB plates containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and cultured upside down at 37℃overnight to obtain a strain CIB95 expressed by fusion of TeLDC and glutathione transferase GST. 3 individual clones were individually picked and inoculated into 5ml LB liquid tubes containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and incubated overnight at 37℃at 200 rpm. 4ml of bacterial cells were collected, and intracellular proteins were extracted with lysozyme (available from Takara Bio Inc.), and whether or not fusion protein GST-TeLDC was expressed and soluble was detected by SDS-PAGE (FIG. 5). As can be seen from FIG. 5, the fusion protein GST-TeLDC was expressed equally well and was expressed in a soluble manner.
Example 9 Activity of lysine decarboxylase TeLDC in different expression forms under different temperature conditions
Bacterial liquid OD of each strain 600 According to the measurement, the bacterial solutions of the strains which do not show obvious difference are respectively taken in equal volumes, the conversion reaction of Lys-HCl is carried out under the conditions of different temperatures (37 ℃,55 ℃,65 ℃ and 75 ℃), 600 mu L of each bacterial solution is taken, 400 mu L of Lys-HCl (L-lysine hydrochloride) and 5 mu L of 20mM PLP (pyridoxal phosphate) are respectively added, and the reaction time is 4 hours. As shown in Table 1, the CIB71 strain expressing CadA can basically convert all Lys-HCl in the system at 37 ℃ or 55 ℃ and the conversion rates are 88.8% and 89.3%, respectively; while the conversion rate is slightly reduced at 65 ℃ and is 61.2%; the conversion of lysine reacted at 75℃was reduced to 6.22%. Compared with the single strain expressing TeLDC, the strain expressing fusion protein TeLDC-MRFP, the strain expressing MBP-TeLDC fusion protein and the strain expressing GST-MRFP fusion protein, the CIB92 strain expressing MRFP-TeLDC fusion protein has obviously improved lysine conversion rate under the reaction condition of 37 ℃,55 ℃,65 ℃ or 75 ℃, wherein the strain is detected at 55 DEG C The highest lysine conversion was measured to be 90.1%, which was only 39.3% at 37℃and reduced compared to 55℃at 65℃for the reaction, 48.7% at 75℃to 3.63%. The MRFP-TeLDC has an optimum temperature of about 55℃as compared with CadA.
TABLE 1 determination of lysine conversion of recombinant cells expressing each lysine decarboxylase
EXAMPLE 10 construction of MRFP-TsLDC, MRFP-TrLDC and MRFP-GkLDC fusion protein expression plasmids
Synthesizing lysine decarboxylase TsLDC from Tepidanaerobacter syntrophicus by using a primer splicing method, wherein the amino acid sequence of the lysine decarboxylase TsLDC is shown as SEQ ID NO. 84, and the coding gene is Tsldc (GenBank ID GAQ24853.1, SEQ ID NO. 85); synthesizing lysine decarboxylase GkLDC from Geobacillus kaustophilus, wherein the amino acid sequence of the lysine decarboxylase GkLDC is shown as SEQ ID NO. 86, and the encoding gene is Gkldc (GenBank ID BAD75350.1, SEQ ID NO. 87); the amino acid sequence of the lysine decarboxylase TrLDC derived from Thermomicrobium roseum is shown as SEQ ID NO. 88, and the coding gene is Trldc (GenBank ID ACM05730.1, SEQ ID NO. 89). Methods of codon optimization and gene synthesis are described in Hoover DM & Lubkowski J Nucleic Acids Research30:10,2002.
The method comprises the steps of using spliced MRFP as a template, using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and linker-MRFP-R (SEQ ID No: 31) for amplification, using spliced genes Tsmdc as a template, using primers linker-TsLDC-F (SEQ ID No: 90) and TsLDC-XbaI-R (SEQ ID No: 91) for amplification, respectively, cutting a target fragment into glue, recovering the glue, using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and TsLDC-XbaI-R (SEQ ID No: 91) for fusion PCR, using SacI and XbaI for double digestion after target fragment purification, and connecting the two digested plasmids into the plasmid pCIB91, and obtaining the MRFP-TeLDC fusion protein (SEQ ID No: 92) for expressing pCIB96, wherein the 5 'end of the TsLDC encoding gene is connected with 3' of red fluorescent protein MRFP (SEQ ID No: 93) through Linker (SGSGSGSGSG).
The method comprises the steps of using spliced MRFP as a template, amplifying by using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and linker-MRFP-R (SEQ ID No: 31), using spliced genes Gkldc as the template, amplifying by using primers linker-GkLDC-F (SEQ ID No: 94) and GkLDC-XbaI-R (SEQ ID No: 95), respectively cutting and recovering a target fragment, performing fusion PCR by using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and GkLDC-XbaI-R (SEQ ID No: 95), purifying the target fragment, and then using SacI and XbaI for double enzyme digestion, and connecting into a plasmid pCIB91 which is cut by the same double enzyme, so as to obtain an MRFP-GkLDC fusion protein (SEQ ID No: 96), and expressing plasmid pCIB97, wherein the 5 'end of the GkLDC encoding gene is connected with red fluorescent protein MRID 3' through Linker (SGSGSGSGSG).
The method comprises the steps of using spliced MRFP as a template, using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and linker-MRFP-R (SEQ ID No: 31) for amplification, using spliced gene Trldc as a template, using primers linker-TrLDC-F (SEQ ID No: 98) and TrLDC-XbaI-R (SEQ ID No: 99) for amplification, respectively performing gel cutting and recovery on a target fragment, using primers SacI-MRFP-TeLDC-F (SEQ ID No: 30) and TrLDC-XbaI-R (SEQ ID No: 99) for fusion PCR, using SacI and XbaI for double digestion after target fragment purification, and connecting the target fragment to plasmid pCIB91 subjected to double digestion to obtain the MRFP-TrLDC fusion protein (SEQ ID No: 100), wherein the 5 'end of the TrLDC encoding gene is connected with 3' of red fluorescent protein MRFP (SEQ ID No: 101).
EXAMPLE 11 construction of MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC fusion protein expression Strain
The constructed MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC fusion protein expression plasmids pCIB96, pCIB97 and pCIB98 are transformed into E.coli BL21 (purchased from Takara Bio Inc.) competent cells, coated on LB plates containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and cultured overnight at 37℃in an inverted manner to obtain strains CIB96, CIB97 and CIB98 expressed by the MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC fusion proteins. 3 individual clones were individually picked and inoculated into 5ml LB liquid tubes containing ampicillin resistance at a final concentration of 100. Mu.g/ml, and incubated overnight at 37℃at 200 rpm.
EXAMPLE 12 detection of lysine decarboxylase Activity of fusion proteins MRFP-TsLDC, MRFP-TrLDC and MRFP-GkLDC under different temperature conditions
Bacterial liquid OD of each strain 600 According to the measurement, the bacterial solutions of the strains which do not show obvious difference are respectively taken in equal volumes, the conversion reaction of Lys-HCl is carried out under the conditions of different temperatures (37 ℃,55 ℃,65 ℃ and 75 ℃), 600 mu L of each bacterial solution is taken, 400 mu L of Lys-HCl (L-lysine hydrochloride) and 5 mu L of 20mM PLP (pyridoxal phosphate) are respectively added, and the reaction time is 4 hours. The results are shown in Table 2, and there was no significant difference in lysine conversion rate at 37℃compared with CIB92 strain expressing MRFP-TeLDC fusion protein; when the reaction is carried out at 55 ℃, the lysine conversion rate of strains CIB96, CIB97 and CIB98 which express fusion proteins MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC is obviously improved, the lysine conversion rate of the CIB96 strain is 83.7%, the lysine conversion rate of the CIB97 strain is 89.9%, the lysine conversion rate of the CIB98 strain is 85.1%, and the optimal temperatures of the strains are all near 55 ℃.
TABLE 2 determination of lysine conversion of recombinant cells expressing each lysine decarboxylase
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EXAMPLE 13 recombinant strains expressing MRFP-TeLDC, MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC fusion proteins for one-step 1, 5-pentanediamine production
The use of the L-lysine-producing E.coli (Escherichia coli) Ela611b strain, which has now been deposited in China center for type culture Collection, address: chinese university of Wuhan, post code 430072, preservation number CCTCC No: m2018736, date of preservation 2018, 11, 1.
Plasmid pCIB71 capable of expressing wild-type lysine decarboxylase cadA and plasmid pCIB92 capable of expressing MRFP-TeLDC fusion protein, plasmid pCIB96 capable of expressing MRFP-TsLDC fusion protein, plasmid pCIB97 capable of expressing MRFP-GkLDC fusion protein and plasmid pCIB98 capable of expressing MRFP-TrLDC fusion protein are respectively transformed into E.coli Ela611b strain capable of producing L-lysine,ela611b-71, ela611b-92, ela611b-96, ela611b-97 and Ela611b-98 strains were obtained, respectively. Three individual clones were individually selected into 5mL liquid medium (containing 4% glucose, 0.1% KH) using the Ela611b strain as a control 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 0.2% yeast extract, 0.01% L-threonine, 0.005% L-isoleucine, 10. Mu.g/mL tetracycline, 100. Mu.g/mL ampicillin), and incubated overnight at 37 ℃. The following day, each strain was then transferred to 100ml of fresh medium containing 30g/L glucose, 0.7% Ca (HCO) 3 ) 2 10. Mu.g/mL tetracycline, 100. Mu.g/mL ampicillin, 0.1% KH 2 PO 4 ,0.1％MgSO 4 ,1.6％(NH 4 ) 2 SO 4 ,0.001％FeSO 4 ,0.001％MnSO 4 Culturing in a medium containing 0.2% yeast extract, 0.01% L-threonine and 0.005% L-isoleucine at 37deg.C for 68 hr, sampling, detecting and calculating the contents of L-lysine and 1, 5-pentanediamine in each medium by nuclear magnetism (Table 2), and reacting at 37deg.C for 4 hr with control strain Ela611b and recombinant strain Ela611 b-71; recombinant strains Ela611b-92, ela611b-96, ela611b-97, ela611b-98 were warmed to 55℃and reacted for 4 hours, and the final L-lysine and 1, 5-pentanediamine content in each medium was detected and calculated using nuclear magnetism (Table 3).
As can be seen from Table 3, after 68 hours of fermentation of the recombinant strain Ela611b-71 expressing CadA, 2.8g/L of L-lysine and 3.6g/L of 1, 5-pentanediamine were detected, and as the fermentation time was prolonged, the amounts of L-lysine and 1, 5-pentanediamine detected in the fermentation broth did not significantly increase, and finally 4.0g/L of 1, 5-pentanediamine was accumulated, and 2.0g/L of L-lysine remained, probably due to the high activity of CadA protein at a temperature of 37℃for lysine growth, the excess of L-lysine synthesized by the strain was catalyzed to 1, 5-pentanediamine earlier, and the accumulation of 1, 5-pentanediamine in the cells was toxic to the cells, which inhibited the metabolism of the cells to some extent, including L-lysine synthesis and conversion of 1, 5-pentanediamine.
While recombinant strain Ela611b-92 expressing thermophilic strain-derived lysine decarboxylase MRFP-TeLDC produces 6.8g/L of L-lysine and 2.0g/L of 1, 5-pentanediamine in 68 hours of fermentation, which indicates that MRFP-TeLDC has lower activity at 37 ℃, weakly converts L-lysine into 1, 5-pentanediamine, can release feedback inhibition of L-lysine to some extent, and simultaneously small amount of 1, 5-pentanediamine does not cause cytotoxicity; when the temperature was raised to 55 ℃, the activity of MRFP-TeLDC was raised, almost all of the remaining L-lysine could be converted to 1, 5-pentanediamine, and finally 5.81g/L of 1, 5-pentanediamine was accumulated, with only 0.04g/L of L-lysine remaining. Likewise, recombinant strain Ela611b-96 expressing thermophilic strain-derived lysine decarboxylase MRFP-TsLDC produced 6.6g/L of L-lysine and 2.2g/L of 1, 5-pentanediamine at 68 hours of fermentation; when the temperature is raised to 55 ℃, the activity of MRFP-TsLDC is raised, the residual L-lysine can be completely converted into 1, 5-pentanediamine, and finally 5.79g/L of 1, 5-pentanediamine is accumulated, and only 0.02g/L of L-lysine remains. The experimental results of recombinant strain Ela611b-97 expressing lysine decarboxylase MRFP-GkLDC derived from thermophilic strain and recombinant strain Ela611b-98 expressing lysine decarboxylase MRFP-TrLDC derived from thermophilic strain are similar, and the recombinant strains all obtain higher 1, 5-pentanediamine yield and almost have no L-lysine residue.
TABLE 3 detection of levels of L-lysine and 1, 5-pentanediamine in strains capable of simultaneously expressing lysine-synthesizing protein and lysine decarboxylase
Wherein n.d. indicates no detection.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> CIBT American Co., ltd, shanghai Kaiser Biotechnology research and development center Co., ltd
<120> method for heterologous expression of lysine decarboxylase derived from thermophilic bacteria and use thereof
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1 5 10 15
Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu Ala Gln Pro Thr Gly Val
20 25 30
Leu Ala Glu Ala Gln Ala Val Val Ala Ala Thr Val Gly Ser Asp Arg
35 40 45
Ala Trp Phe Leu Val Asn Gly Ala Thr Gly Gly Leu Leu Ala Ala Leu
50 55 60
Leu Ala Thr Val Gly Pro Gly Asp Arg Val Leu Val Gly Arg Asn Val
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His Arg Ser Val Ile Ala Gly Leu Val Leu Ala Gly Ala Lys Pro Val
85 90 95
Tyr Leu Gly Val Gly Val Asp Pro Gln Trp Gly Leu Pro Trp Pro Val
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Thr Arg Asp Val Val Ala Ala Gly Leu Ala Ala Tyr Pro Asp Thr Lys
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Ala Val Val Leu Val Ser Pro Thr Tyr Glu Gly Leu Cys Ser Pro Leu
130 135 140
Leu Glu Ile Ala Gln Cys Val His Asn His Gly Val Pro Leu Ile Val
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Asp Glu Ala His Gly Ser His Phe Ala Tyr His Pro Ala Phe Pro Val
165 170 175
Thr Ala Leu Ala Ala Gly Ala Asp Val Val Val Gln Ser Trp His Lys
180 185 190
Thr Leu Gly Thr Leu Thr Gln Thr Ala Val Leu His Leu Lys Gly Glu
195 200 205
Arg Val Ser Ala Glu Arg Leu Ser Gln Ala Leu Asn Leu Val Gln Thr
210 215 220
Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala Leu Glu Gly Ala Gly Val
225 230 235 240
Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr Gly Arg Leu Leu Gln Trp
245 250 255
Val Lys Thr Phe Glu Trp Pro Leu Pro Arg Trp Gln Pro Pro Gly Ile
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Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly Thr Trp Pro Ile Gly Leu
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catcgtagcg tgattgcggg cttggtactg gctggcgcaa aaccggtgta tcttggcgtc 300
ggcgtcgatc cacaatgggg tctgccgtgg cccgtgaccc gggacgttgt cgcggcaggc 360
ttggctgcgt accccgacac caaggcggtc gtacttgtaa gtcctaccta tgaaggcctg 420
tgctcgccgc tgttagaaat cgcgcagtgc gtgcataatc atggcgtacc gctgattgtc 480
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gctggggctg acgtcgtcgt tcagtcatgg cacaaaacgt tgggcacgct gacccaaacg 600
gcggtgctgc atctgaaagg cgaacgcgtg tcggcagagc ggctgagcca ggcgttgaat 660
ctggtgcaga cctcgagccc gaactattgg cttctggccg cacttgaagg tgccggggtc 720
cagatggcgc agcagggcga acagatttat ggccggctgc tgcagtgggt aaaaacattt 780
gagtggcctt tgccgcggtg gcagcctcca ggaatccccc aagatcctct gcgtttgacc 840
ctggggacgt ggccgattgg tttaaccgga tttgcactgg atgaactttt acaacctcag 900
ataattgcgg aatttccaag cgggcgtagc ctgacctttt gtctgggtct gggcacaaca 960
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tcgccgcgtg aagcgtactt ttgcccgcag cgtagcatac cgcttcgtgc agctcttaat 1140
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Ile Val Tyr Pro Asn Asp Arg Asp Asp Leu Leu Lys Leu Ile Glu Asn
35 40 45
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
85 90 95
Arg Leu Gln Ile Ser Phe Phe Glu Tyr Ala Leu Gly Ala Ala Glu Asp
100 105 110
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
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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
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Lys Ser Asp Ile Ser Ile Ser Val Ser Glu Leu Gly Ser Leu Leu Asp
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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
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Lys Ile Val Gly Met Tyr Ser Ala Pro Ala Gly Ser Thr Ile Leu Ile
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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
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Gly Gly Ile Pro Gln Ser Glu Phe Gln His Ala Thr Ile Ala Lys Arg
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Thr Leu Asp Val Lys Ser Ile His Phe Asp Ser Ala Trp Val Pro Tyr
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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
420 425 430
Ile Lys Phe Arg Lys Glu Ile Lys Arg Leu Arg Thr Glu Ser Asp Gly
435 440 445
Trp Phe Phe Asp Val Trp Gln Pro Asp His Ile Asp Thr Thr Glu Cys
450 455 460
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
500 505 510
Ile Val Ala Lys Tyr Leu Asp Glu His Gly Ile Val Val Glu Lys Thr
515 520 525
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
625 630 635 640
Val Gly Arg Ile Asn Ala Asn Met Ile Leu Pro Tyr Pro Pro Gly Val
645 650 655
Pro Leu Val Met Pro Gly Glu Met Ile Thr Glu Glu Ser Arg Pro Val
660 665 670
Leu Glu Phe Leu Gln Met Leu Cys Glu Ile Gly Ala His Tyr Pro Gly
675 680 685
Phe Glu Thr Asp Ile His Gly Ala Tyr Arg Gln Ala Asp Gly Arg Tyr
690 695 700
Thr Val Lys Val Leu Lys Glu Glu Ser Lys Lys
705 710 715
<210> 4
<211> 2148
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 4
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
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> 5
<211> 711
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaata a 711
<210> 6
<211> 236
<212> PRT
<213> mushroom coral (mushroom coral)
<400> 6
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 7
<211> 683
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Glu Pro Leu Leu Arg Ala Leu Trp Gly
245 250 255
Thr Ala Leu Glu Gln Asp Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu
260 265 270
Ala Gln Pro Thr Gly Val Leu Ala Glu Ala Gln Ala Val Val Ala Ala
275 280 285
Thr Val Gly Ser Asp Arg Ala Trp Phe Leu Val Asn Gly Ala Thr Gly
290 295 300
Gly Leu Leu Ala Ala Leu Leu Ala Thr Val Gly Pro Gly Asp Arg Val
305 310 315 320
Leu Val Gly Arg Asn Val His Arg Ser Val Ile Ala Gly Leu Val Leu
325 330 335
Ala Gly Ala Lys Pro Val Tyr Leu Gly Val Gly Val Asp Pro Gln Trp
340 345 350
Gly Leu Pro Trp Pro Val Thr Arg Asp Val Val Ala Ala Gly Leu Ala
355 360 365
Ala Tyr Pro Asp Thr Lys Ala Val Val Leu Val Ser Pro Thr Tyr Glu
370 375 380
Gly Leu Cys Ser Pro Leu Leu Glu Ile Ala Gln Cys Val His Asn His
385 390 395 400
Gly Val Pro Leu Ile Val Asp Glu Ala His Gly Ser His Phe Ala Tyr
405 410 415
His Pro Ala Phe Pro Val Thr Ala Leu Ala Ala Gly Ala Asp Val Val
420 425 430
Val Gln Ser Trp His Lys Thr Leu Gly Thr Leu Thr Gln Thr Ala Val
435 440 445
Leu His Leu Lys Gly Glu Arg Val Ser Ala Glu Arg Leu Ser Gln Ala
450 455 460
Leu Asn Leu Val Gln Thr Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala
465 470 475 480
Leu Glu Gly Ala Gly Val Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr
485 490 495
Gly Arg Leu Leu Gln Trp Val Lys Thr Phe Glu Trp Pro Leu Pro Arg
500 505 510
Trp Gln Pro Pro Gly Ile Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly
515 520 525
Thr Trp Pro Ile Gly Leu Thr Gly Phe Ala Leu Asp Glu Leu Leu Gln
530 535 540
Pro Gln Ile Ile Ala Glu Phe Pro Ser Gly Arg Ser Leu Thr Phe Cys
545 550 555 560
Leu Gly Leu Gly Thr Thr Gln Thr Met Leu Glu Thr Leu Ala Asp Arg
565 570 575
Leu Lys Ser Val Tyr Thr Glu Tyr Cys His Asn Ala Pro Leu Pro Pro
580 585 590
Leu Ala Ile Pro Ser Ile Pro Ser Cys Gln Glu Pro Ala Leu Ser Pro
595 600 605
Arg Glu Ala Tyr Phe Cys Pro Gln Arg Ser Ile Pro Leu Arg Ala Ala
610 615 620
Leu Asn Glu Ile Ser Ala Glu Thr Ile Ala Pro Tyr Pro Pro Gly Ile
625 630 635 640
Pro Thr Val Ile Ala Gly Glu Arg Phe Thr Glu Ser Val Ile Ala Thr
645 650 655
Leu Gln Thr Leu Gln Glu Leu Gly Ala Glu Met Val Gly Ala Ser Asp
660 665 670
Pro Thr Leu Gln Thr Leu Arg Ile Cys Lys Val
675 680
<210> 8
<211> 2052
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat ggaaccatta cttcgcgcac tgtgggggac cgcgctggaa 780
caggacctta gcgaacttcc gggtcttgac aatttagcgc aaccaaccgg cgtgttagcc 840
gaagcgcaag ctgtggtcgc tgcgacggtc ggctctgatc gtgcgtggtt tctggtgaac 900
ggcgctactg gcggcctgct tgcggcttta cttgcgaccg taggtcccgg cgaccgggtg 960
ctggttggcc gtaatgtgca tcgtagcgtg attgcgggct tggtactggc tggcgcaaaa 1020
ccggtgtatc ttggcgtcgg cgtcgatcca caatggggtc tgccgtggcc cgtgacccgg 1080
gacgttgtcg cggcaggctt ggctgcgtac cccgacacca aggcggtcgt acttgtaagt 1140
cctacctatg aaggcctgtg ctcgccgctg ttagaaatcg cgcagtgcgt gcataatcat 1200
ggcgtaccgc tgattgtcga cgaagcacat ggcagtcatt tcgcgtatca tccggcattt 1260
cctgtgaccg cgttagctgc tggggctgac gtcgtcgttc agtcatggca caaaacgttg 1320
ggcacgctga cccaaacggc ggtgctgcat ctgaaaggcg aacgcgtgtc ggcagagcgg 1380
ctgagccagg cgttgaatct ggtgcagacc tcgagcccga actattggct tctggccgca 1440
cttgaaggtg ccggggtcca gatggcgcag cagggcgaac agatttatgg ccggctgctg 1500
cagtgggtaa aaacatttga gtggcctttg ccgcggtggc agcctccagg aatcccccaa 1560
gatcctctgc gtttgaccct ggggacgtgg ccgattggtt taaccggatt tgcactggat 1620
gaacttttac aacctcagat aattgcggaa tttccaagcg ggcgtagcct gaccttttgt 1680
ctgggtctgg gcacaacaca gactatgctg gagacgcttg cagatcgcct gaagagcgtc 1740
tataccgaat attgccataa tgcgcccttg cctccgttgg cgataccgtc tattccgagc 1800
tgtcaggaac ccgcgctttc gccgcgtgaa gcgtactttt gcccgcagcg tagcataccg 1860
cttcgtgcag ctcttaatga aatctcggct gaaaccattg ccccgtaccc tcccggcata 1920
cctaccgtga tcgctgggga gcgctttacc gaaagtgtta ttgcgactct gcaaacgctg 1980
caggaattag gtgcggaaat ggtaggggca agcgatccga ccttacaaac cctgcggata 2040
tgtaaagtgt aa 2052
<210> 9
<211> 683
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Met Glu Pro Leu Leu Arg Ala Leu Trp Gly Thr Ala Leu Glu Gln Asp
1 5 10 15
Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu Ala Gln Pro Thr Gly Val
20 25 30
Leu Ala Glu Ala Gln Ala Val Val Ala Ala Thr Val Gly Ser Asp Arg
35 40 45
Ala Trp Phe Leu Val Asn Gly Ala Thr Gly Gly Leu Leu Ala Ala Leu
50 55 60
Leu Ala Thr Val Gly Pro Gly Asp Arg Val Leu Val Gly Arg Asn Val
65 70 75 80
His Arg Ser Val Ile Ala Gly Leu Val Leu Ala Gly Ala Lys Pro Val
85 90 95
Tyr Leu Gly Val Gly Val Asp Pro Gln Trp Gly Leu Pro Trp Pro Val
100 105 110
Thr Arg Asp Val Val Ala Ala Gly Leu Ala Ala Tyr Pro Asp Thr Lys
115 120 125
Ala Val Val Leu Val Ser Pro Thr Tyr Glu Gly Leu Cys Ser Pro Leu
130 135 140
Leu Glu Ile Ala Gln Cys Val His Asn His Gly Val Pro Leu Ile Val
145 150 155 160
Asp Glu Ala His Gly Ser His Phe Ala Tyr His Pro Ala Phe Pro Val
165 170 175
Thr Ala Leu Ala Ala Gly Ala Asp Val Val Val Gln Ser Trp His Lys
180 185 190
Thr Leu Gly Thr Leu Thr Gln Thr Ala Val Leu His Leu Lys Gly Glu
195 200 205
Arg Val Ser Ala Glu Arg Leu Ser Gln Ala Leu Asn Leu Val Gln Thr
210 215 220
Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala Leu Glu Gly Ala Gly Val
225 230 235 240
Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr Gly Arg Leu Leu Gln Trp
245 250 255
Val Lys Thr Phe Glu Trp Pro Leu Pro Arg Trp Gln Pro Pro Gly Ile
260 265 270
Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly Thr Trp Pro Ile Gly Leu
275 280 285
Thr Gly Phe Ala Leu Asp Glu Leu Leu Gln Pro Gln Ile Ile Ala Glu
290 295 300
Phe Pro Ser Gly Arg Ser Leu Thr Phe Cys Leu Gly Leu Gly Thr Thr
305 310 315 320
Gln Thr Met Leu Glu Thr Leu Ala Asp Arg Leu Lys Ser Val Tyr Thr
325 330 335
Glu Tyr Cys His Asn Ala Pro Leu Pro Pro Leu Ala Ile Pro Ser Ile
340 345 350
Pro Ser Cys Gln Glu Pro Ala Leu Ser Pro Arg Glu Ala Tyr Phe Cys
355 360 365
Pro Gln Arg Ser Ile Pro Leu Arg Ala Ala Leu Asn Glu Ile Ser Ala
370 375 380
Glu Thr Ile Ala Pro Tyr Pro Pro Gly Ile Pro Thr Val Ile Ala Gly
385 390 395 400
Glu Arg Phe Thr Glu Ser Val Ile Ala Thr Leu Gln Thr Leu Gln Glu
405 410 415
Leu Gly Ala Glu Met Val Gly Ala Ser Asp Pro Thr Leu Gln Thr Leu
420 425 430
Arg Ile Cys Lys Val Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Met
435 440 445
Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met
450 455 460
Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu
465 470 475 480
Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala
485 490 495
Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile
500 505 510
Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro
515 520 525
Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys
530 535 540
Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr
545 550 555 560
Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu
565 570 575
Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr
580 585 590
Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala
595 600 605
Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His
610 615 620
Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln
625 630 635 640
Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His
645 650 655
Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg
660 665 670
His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys
675 680
<210> 10
<211> 2052
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggaaccat tacttcgcgc actgtggggg accgcgctgg aacaggacct tagcgaactt 60
ccgggtcttg acaatttagc gcaaccaacc ggcgtgttag ccgaagcgca agctgtggtc 120
gctgcgacgg tcggctctga tcgtgcgtgg tttctggtga acggcgctac tggcggcctg 180
cttgcggctt tacttgcgac cgtaggtccc ggcgaccggg tgctggttgg ccgtaatgtg 240
catcgtagcg tgattgcggg cttggtactg gctggcgcaa aaccggtgta tcttggcgtc 300
ggcgtcgatc cacaatgggg tctgccgtgg cccgtgaccc gggacgttgt cgcggcaggc 360
ttggctgcgt accccgacac caaggcggtc gtacttgtaa gtcctaccta tgaaggcctg 420
tgctcgccgc tgttagaaat cgcgcagtgc gtgcataatc atggcgtacc gctgattgtc 480
gacgaagcac atggcagtca tttcgcgtat catccggcat ttcctgtgac cgcgttagct 540
gctggggctg acgtcgtcgt tcagtcatgg cacaaaacgt tgggcacgct gacccaaacg 600
gcggtgctgc atctgaaagg cgaacgcgtg tcggcagagc ggctgagcca ggcgttgaat 660
ctggtgcaga cctcgagccc gaactattgg cttctggccg cacttgaagg tgccggggtc 720
cagatggcgc agcagggcga acagatttat ggccggctgc tgcagtgggt aaaaacattt 780
gagtggcctt tgccgcggtg gcagcctcca ggaatccccc aagatcctct gcgtttgacc 840
ctggggacgt ggccgattgg tttaaccgga tttgcactgg atgaactttt acaacctcag 900
ataattgcgg aatttccaag cgggcgtagc ctgacctttt gtctgggtct gggcacaaca 960
cagactatgc tggagacgct tgcagatcgc ctgaagagcg tctataccga atattgccat 1020
aatgcgccct tgcctccgtt ggcgataccg tctattccga gctgtcagga acccgcgctt 1080
tcgccgcgtg aagcgtactt ttgcccgcag cgtagcatac cgcttcgtgc agctcttaat 1140
gaaatctcgg ctgaaaccat tgccccgtac cctcccggca tacctaccgt gatcgctggg 1200
gagcgcttta ccgaaagtgt tattgcgact ctgcaaacgc tgcaggaatt aggtgcggaa 1260
atggtagggg caagcgatcc gaccttacaa accctgcgga tatgtaaagt gtctggttct 1320
ggttctggtt ctggttctgg tatggtgtct aaaggcgagg aagataatat ggcgattatc 1380
aaagaattta tgcgttttaa agtgcatatg gaaggcagcg tgaatgggca tgagtttgaa 1440
attgaaggcg aaggagaagg ccgtccgtat gaaggcaccc agaccgctaa actgaaagtg 1500
accaaaggcg gaccactgcc gtttgcgtgg gacattctga gcccgcagtt tatgtatggc 1560
agcaaagcgt atgtgaaaca tccggcggat attccggatt atctgaaact gagctttccg 1620
gagggcttca aatgggaacg tgtgatgaat tttgaagatg gcggcgtggt gaccgtgacc 1680
caggatagca gcctgcaaga cggcgaattc atttacaagg tgaagctgcg tggcaccaac 1740
tttcccagcg atggcccggt gatgcagaaa aagaccatgg gctgggaggc gagcagcgaa 1800
cgtatgtacc cggaggatgg cgcgctgaag ggcgaaatta agcagcgtct gaagttaaaa 1860
gatggtgggc actatgatgc ggaagtgaaa accacctata aagcgaaaaa accggtgcag 1920
ttaccaggcg cttataatgt gaacattaag ctggatatta ccagccataa tgaagattat 1980
accattgtgg aacagtatga gcgtgcggag ggacggcata gcacgggcgg aatggatgaa 2040
ctgtataaat aa 2052
<210> 11
<211> 408
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
370 375 380
Glu Gly Arg Ile Ser His Met Ser Met Gly Gly Arg Asp Ile Val Asp
385 390 395 400
Gly Ser Glu Phe Pro Ala Gly Asn
405
<210> 12
<211> 1227
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60
ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120
ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180
atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240
accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300
aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360
gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420
aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480
ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540
gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600
aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660
ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720
gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780
ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840
ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900
ggtgccgtag cgctgaagtc ttacgaggaa gagttggtga aagatccgcg tattgccgcc 960
actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020
tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080
gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140
aacctcggga tcgagggaag gatttcacat atgtccatgg gcggccgcga tatcgtcgac 1200
ggatccgaat tccctgcagg taattaa 1227
<210> 13
<211> 226
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
210 215 220
Gly Ser
225
<210> 14
<211> 681
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatccta a 681
<210> 15
<211> 855
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
370 375 380
Glu Gly Arg Ile Ser His Met Ser Met Gly Gly Arg Asp Ile Val Asp
385 390 395 400
Gly Ser Glu Phe Pro Ala Gly Asn Ser Gly Ser Gly Ser Gly Ser Gly
405 410 415
Ser Gly Met Glu Pro Leu Leu Arg Ala Leu Trp Gly Thr Ala Leu Glu
420 425 430
Gln Asp Leu Ser Glu Leu Pro Gly Leu Asp Asn Leu Ala Gln Pro Thr
435 440 445
Gly Val Leu Ala Glu Ala Gln Ala Val Val Ala Ala Thr Val Gly Ser
450 455 460
Asp Arg Ala Trp Phe Leu Val Asn Gly Ala Thr Gly Gly Leu Leu Ala
465 470 475 480
Ala Leu Leu Ala Thr Val Gly Pro Gly Asp Arg Val Leu Val Gly Arg
485 490 495
Asn Val His Arg Ser Val Ile Ala Gly Leu Val Leu Ala Gly Ala Lys
500 505 510
Pro Val Tyr Leu Gly Val Gly Val Asp Pro Gln Trp Gly Leu Pro Trp
515 520 525
Pro Val Thr Arg Asp Val Val Ala Ala Gly Leu Ala Ala Tyr Pro Asp
530 535 540
Thr Lys Ala Val Val Leu Val Ser Pro Thr Tyr Glu Gly Leu Cys Ser
545 550 555 560
Pro Leu Leu Glu Ile Ala Gln Cys Val His Asn His Gly Val Pro Leu
565 570 575
Ile Val Asp Glu Ala His Gly Ser His Phe Ala Tyr His Pro Ala Phe
580 585 590
Pro Val Thr Ala Leu Ala Ala Gly Ala Asp Val Val Val Gln Ser Trp
595 600 605
His Lys Thr Leu Gly Thr Leu Thr Gln Thr Ala Val Leu His Leu Lys
610 615 620
Gly Glu Arg Val Ser Ala Glu Arg Leu Ser Gln Ala Leu Asn Leu Val
625 630 635 640
Gln Thr Ser Ser Pro Asn Tyr Trp Leu Leu Ala Ala Leu Glu Gly Ala
645 650 655
Gly Val Gln Met Ala Gln Gln Gly Glu Gln Ile Tyr Gly Arg Leu Leu
660 665 670
Gln Trp Val Lys Thr Phe Glu Trp Pro Leu Pro Arg Trp Gln Pro Pro
675 680 685
Gly Ile Pro Gln Asp Pro Leu Arg Leu Thr Leu Gly Thr Trp Pro Ile
690 695 700
Gly Leu Thr Gly Phe Ala Leu Asp Glu Leu Leu Gln Pro Gln Ile Ile
705 710 715 720
Ala Glu Phe Pro Ser Gly Arg Ser Leu Thr Phe Cys Leu Gly Leu Gly
725 730 735
Thr Thr Gln Thr Met Leu Glu Thr Leu Ala Asp Arg Leu Lys Ser Val
740 745 750
Tyr Thr Glu Tyr Cys His Asn Ala Pro Leu Pro Pro Leu Ala Ile Pro
755 760 765
Ser Ile Pro Ser Cys Gln Glu Pro Ala Leu Ser Pro Arg Glu Ala Tyr
770 775 780
Phe Cys Pro Gln Arg Ser Ile Pro Leu Arg Ala Ala Leu Asn Glu Ile
785 790 795 800
Ser Ala Glu Thr Ile Ala Pro Tyr Pro Pro Gly Ile Pro Thr Val Ile
805 810 815
Ala Gly Glu Arg Phe Thr Glu Ser Val Ile Ala Thr Leu Gln Thr Leu
820 825 830
Gln Glu Leu Gly Ala Glu Met Val Gly Ala Ser Asp Pro Thr Leu Gln
835 840 845
Thr Leu Arg Ile Cys Lys Val
850 855
<210> 16
<211> 2568
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60
ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120
ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180
atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240
accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300
aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360
gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420
aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480
ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540
gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600
aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660
ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720
gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780
ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840
ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900
ggtgccgtag cgctgaagtc ttacgaggaa gagttggtga aagatccgcg tattgccgcc 960
actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020
tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080
gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140
aacctcggga tcgagggaag gatttcacat atgtccatgg gcggccgcga tatcgtcgac 1200
ggatccgaat tccctgcagg taattctggt tctggttctg gttctggttc tggtatggaa 1260
ccattacttc gcgcactgtg ggggaccgcg ctggaacagg accttagcga acttccgggt 1320
cttgacaatt tagcgcaacc aaccggcgtg ttagccgaag cgcaagctgt ggtcgctgcg 1380
acggtcggct ctgatcgtgc gtggtttctg gtgaacggcg ctactggcgg cctgcttgcg 1440
gctttacttg cgaccgtagg tcccggcgac cgggtgctgg ttggccgtaa tgtgcatcgt 1500
agcgtgattg cgggcttggt actggctggc gcaaaaccgg tgtatcttgg cgtcggcgtc 1560
gatccacaat ggggtctgcc gtggcccgtg acccgggacg ttgtcgcggc aggcttggct 1620
gcgtaccccg acaccaaggc ggtcgtactt gtaagtccta cctatgaagg cctgtgctcg 1680
ccgctgttag aaatcgcgca gtgcgtgcat aatcatggcg taccgctgat tgtcgacgaa 1740
gcacatggca gtcatttcgc gtatcatccg gcatttcctg tgaccgcgtt agctgctggg 1800
gctgacgtcg tcgttcagtc atggcacaaa acgttgggca cgctgaccca aacggcggtg 1860
ctgcatctga aaggcgaacg cgtgtcggca gagcggctga gccaggcgtt gaatctggtg 1920
cagacctcga gcccgaacta ttggcttctg gccgcacttg aaggtgccgg ggtccagatg 1980
gcgcagcagg gcgaacagat ttatggccgg ctgctgcagt gggtaaaaac atttgagtgg 2040
cctttgccgc ggtggcagcc tccaggaatc ccccaagatc ctctgcgttt gaccctgggg 2100
acgtggccga ttggtttaac cggatttgca ctggatgaac ttttacaacc tcagataatt 2160
gcggaatttc caagcgggcg tagcctgacc ttttgtctgg gtctgggcac aacacagact 2220
atgctggaga cgcttgcaga tcgcctgaag agcgtctata ccgaatattg ccataatgcg 2280
cccttgcctc cgttggcgat accgtctatt ccgagctgtc aggaacccgc gctttcgccg 2340
cgtgaagcgt acttttgccc gcagcgtagc ataccgcttc gtgcagctct taatgaaatc 2400
tcggctgaaa ccattgcccc gtaccctccc ggcataccta ccgtgatcgc tggggagcgc 2460
tttaccgaaa gtgttattgc gactctgcaa acgctgcagg aattaggtgc ggaaatggta 2520
ggggcaagcg atccgacctt acaaaccctg cggatatgta aagtgtaa 2568
<210> 17
<211> 673
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
210 215 220
Gly Ser Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Met Glu Pro Leu
225 230 235 240
Leu Arg Ala Leu Trp Gly Thr Ala Leu Glu Gln Asp Leu Ser Glu Leu
245 250 255
Pro Gly Leu Asp Asn Leu Ala Gln Pro Thr Gly Val Leu Ala Glu Ala
260 265 270
Gln Ala Val Val Ala Ala Thr Val Gly Ser Asp Arg Ala Trp Phe Leu
275 280 285
Val Asn Gly Ala Thr Gly Gly Leu Leu Ala Ala Leu Leu Ala Thr Val
290 295 300
Gly Pro Gly Asp Arg Val Leu Val Gly Arg Asn Val His Arg Ser Val
305 310 315 320
Ile Ala Gly Leu Val Leu Ala Gly Ala Lys Pro Val Tyr Leu Gly Val
325 330 335
Gly Val Asp Pro Gln Trp Gly Leu Pro Trp Pro Val Thr Arg Asp Val
340 345 350
Val Ala Ala Gly Leu Ala Ala Tyr Pro Asp Thr Lys Ala Val Val Leu
355 360 365
Val Ser Pro Thr Tyr Glu Gly Leu Cys Ser Pro Leu Leu Glu Ile Ala
370 375 380
Gln Cys Val His Asn His Gly Val Pro Leu Ile Val Asp Glu Ala His
385 390 395 400
Gly Ser His Phe Ala Tyr His Pro Ala Phe Pro Val Thr Ala Leu Ala
405 410 415
Ala Gly Ala Asp Val Val Val Gln Ser Trp His Lys Thr Leu Gly Thr
420 425 430
Leu Thr Gln Thr Ala Val Leu His Leu Lys Gly Glu Arg Val Ser Ala
435 440 445
Glu Arg Leu Ser Gln Ala Leu Asn Leu Val Gln Thr Ser Ser Pro Asn
450 455 460
Tyr Trp Leu Leu Ala Ala Leu Glu Gly Ala Gly Val Gln Met Ala Gln
465 470 475 480
Gln Gly Glu Gln Ile Tyr Gly Arg Leu Leu Gln Trp Val Lys Thr Phe
485 490 495
Glu Trp Pro Leu Pro Arg Trp Gln Pro Pro Gly Ile Pro Gln Asp Pro
500 505 510
Leu Arg Leu Thr Leu Gly Thr Trp Pro Ile Gly Leu Thr Gly Phe Ala
515 520 525
Leu Asp Glu Leu Leu Gln Pro Gln Ile Ile Ala Glu Phe Pro Ser Gly
530 535 540
Arg Ser Leu Thr Phe Cys Leu Gly Leu Gly Thr Thr Gln Thr Met Leu
545 550 555 560
Glu Thr Leu Ala Asp Arg Leu Lys Ser Val Tyr Thr Glu Tyr Cys His
565 570 575
Asn Ala Pro Leu Pro Pro Leu Ala Ile Pro Ser Ile Pro Ser Cys Gln
580 585 590
Glu Pro Ala Leu Ser Pro Arg Glu Ala Tyr Phe Cys Pro Gln Arg Ser
595 600 605
Ile Pro Leu Arg Ala Ala Leu Asn Glu Ile Ser Ala Glu Thr Ile Ala
610 615 620
Pro Tyr Pro Pro Gly Ile Pro Thr Val Ile Ala Gly Glu Arg Phe Thr
625 630 635 640
Glu Ser Val Ile Ala Thr Leu Gln Thr Leu Gln Glu Leu Gly Ala Glu
645 650 655
Met Val Gly Ala Ser Asp Pro Thr Leu Gln Thr Leu Arg Ile Cys Lys
660 665 670
Val
<210> 18
<211> 2022
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatcctc tggttctggt tctggttctg gttctggtat ggaaccatta 720
cttcgcgcac tgtgggggac cgcgctggaa caggacctta gcgaacttcc gggtcttgac 780
aatttagcgc aaccaaccgg cgtgttagcc gaagcgcaag ctgtggtcgc tgcgacggtc 840
ggctctgatc gtgcgtggtt tctggtgaac ggcgctactg gcggcctgct tgcggcttta 900
cttgcgaccg taggtcccgg cgaccgggtg ctggttggcc gtaatgtgca tcgtagcgtg 960
attgcgggct tggtactggc tggcgcaaaa ccggtgtatc ttggcgtcgg cgtcgatcca 1020
caatggggtc tgccgtggcc cgtgacccgg gacgttgtcg cggcaggctt ggctgcgtac 1080
cccgacacca aggcggtcgt acttgtaagt cctacctatg aaggcctgtg ctcgccgctg 1140
ttagaaatcg cgcagtgcgt gcataatcat ggcgtaccgc tgattgtcga cgaagcacat 1200
ggcagtcatt tcgcgtatca tccggcattt cctgtgaccg cgttagctgc tggggctgac 1260
gtcgtcgttc agtcatggca caaaacgttg ggcacgctga cccaaacggc ggtgctgcat 1320
ctgaaaggcg aacgcgtgtc ggcagagcgg ctgagccagg cgttgaatct ggtgcagacc 1380
tcgagcccga actattggct tctggccgca cttgaaggtg ccggggtcca gatggcgcag 1440
cagggcgaac agatttatgg ccggctgctg cagtgggtaa aaacatttga gtggcctttg 1500
ccgcggtggc agcctccagg aatcccccaa gatcctctgc gtttgaccct ggggacgtgg 1560
ccgattggtt taaccggatt tgcactggat gaacttttac aacctcagat aattgcggaa 1620
tttccaagcg ggcgtagcct gaccttttgt ctgggtctgg gcacaacaca gactatgctg 1680
gagacgcttg cagatcgcct gaagagcgtc tataccgaat attgccataa tgcgcccttg 1740
cctccgttgg cgataccgtc tattccgagc tgtcaggaac ccgcgctttc gccgcgtgaa 1800
gcgtactttt gcccgcagcg tagcataccg cttcgtgcag ctcttaatga aatctcggct 1860
gaaaccattg ccccgtaccc tcccggcata cctaccgtga tcgctgggga gcgctttacc 1920
gaaagtgtta ttgcgactct gcaaacgctg caggaattag gtgcggaaat ggtaggggca 1980
agcgatccga ccttacaaac cctgcggata tgtaaagtgt aa 2022
<210> 19
<211> 69
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa 60
ttcgagctc 69
<210> 20
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
tgtggaattg tgagcggata acaatttcac acaggaaaca gctgagctc 49
<210> 21
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tgtggaattg tgagctcaat aattttgttt aactttaaga aggagaacag ct 52
<210> 22
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tccgagctca tgaacgttat tgcaatattg 30
<210> 23
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gcctctagac cacttccctt gtacgagc 28
<210> 24
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
agctgtttcc tgtgtgaaat 20
<210> 26
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggcgagctca tggaaccatt acttcgcgc 29
<210> 27
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
ggctctagat tacactttac atatccgcag gg 32
<210> 28
<211> 55
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
aataattttg tttaacttta agaaggagaa cagctatgga accattactt cgcgc 55
<210> 29
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gttctccttc ttaaagttaa acaaaattat tgagctcaca attccacaca acatacgag 59
<210> 30
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ggcgagctca ataattttgt ttaactttaa gaaggagaac agctatggtg tctaaaggc 59
<210> 31
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
accagaacca gaaccagaac cagaaccaga tttatacagt tcatccattc cgcc 54
<210> 32
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
tctggttctg gttctggttc tggttctggt atggaaccat tacttcgcgc actgtgg 57
<210> 33
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ggcgagctca ataattttgt ttaactttaa gaaggagaac agctatggaa ccattac 57
<210> 34
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
accagaacca gaaccagaac cagaaccaga cactttacat atccgcaggg 50
<210> 35
<211> 59
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
tctggttctg gttctggttc tggttctggt atggtgtcta aaggcgagga agataatat 59
<210> 36
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ggctctagat tatttataca gttcatccat tccgcccg 38
<210> 37
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
aattttgttt aactttaaga aggagaacag ctatgaaaat cgaagaaggt aaactggt 58
<210> 38
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
ccagaaccag aaccagaacc agaattacct gcagggaatt cggat 45
<210> 39
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gcttccggct cgtatgttgg agctcaataa ttttgtttaa ctttaagaag gagaacag 58
<210> 40
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
tctggttctg gttctggttc tgg 23
<210> 41
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ttgtttaact ttaagaagga gaacagctat gtcccctata ctaggttatt ggaaaatt 58
<210> 42
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ccagaaccag aaccagaacc agaggatcca cgcggaacca gat 43
<210> 43
<211> 66
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ggcgaattca gtttattctt gacatgtagt gagggggctg gtataatgag ctcggtaccc 60
ggggat 66
<210> 44
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
ggcagtactc aaccaagtca ttctgagaat agtg 34
<210> 45
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
ggcgagctca cacaggaaac agaccatgaa atctaacaat gcgctcatc 49
<210> 46
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
ggctctagat caacgacagg agcacgatc 29
<210> 47
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
ggcgagctca cacaggaaac agaccatgtc tgaaattgtt gtctcc 46
<210> 48
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
ggcggatcct tactcaaaca aattactatg cag 33
<210> 49
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
ggcggatcca cacaggaaac agaccatgtt cacgggaagt attgtc 46
<210> 50
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
ggctctagat tacagcaaac cggcatgc 28
<210> 51
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
ggctctagaa cacaggaaac agaccatgcc acattcactg ttcagc 46
<210> 52
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
ggcgtcgact taaagcaatt ccagcgccag 30
<210> 53
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
ggcctcgaga gtttattctt gacatgtagt gagg 34
<210> 54
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
ggcgcatgct caacgacagg agcacgatc 29
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
cagcctgaat atactgcatt ctc 23
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
gagaatgcag tatattcagg ctg 23
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gcattctcgc gatttcctcg 20
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
cgaggaaatc gcgagaatgc 20
<210> 59
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ggcgagctca cacaggaaac agaccatgaa aaatgttggt tttatcgg 48
<210> 60
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
ggcggatcct tacgccagtt gacgaagc 28
<210> 61
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ggcggatcca cacaggaaac agaccatgca tgatgcaaac atccg 45
<210> 62
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
ggctctagat tacaaattat tgagatcaag tacatctc 38
<210> 63
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
ggctctagaa cacaggaaac agaccatgtt tgagaacatt accgcc 46
<210> 64
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
ggcgcatgcg acctcgaggt agtcgactta cagcactgcc acaatcg 47
<210> 65
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
ggcggtacca gtttattctt gacatgtagt gagg 34
<210> 66
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
ggcgggccct taaagcaatt ccagcgcca 29
<210> 67
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
agtttattct tgacatgtag tgagggggct ggtataat 38
<210> 68
<211> 1191
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 68
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> 69
<211> 396
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 69
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> 70
<211> 1350
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 70
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> 71
<211> 449
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 71
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> 72
<211> 879
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 72
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> 73
<211> 292
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 73
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> 74
<211> 1263
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 74
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> 75
<211> 420
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 75
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> 76
<211> 1349
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 76
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> 77
<211> 449
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 77
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> 78
<211> 1104
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 78
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> 79
<211> 350
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 79
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> 80
<211> 822
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 80
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> 81
<211> 273
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 81
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> 82
<211> 1191
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 82
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> 83
<211> 396
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 83
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> 84
<211> 482
<212> PRT
<213> Tepidanaerobacter syntrophicus
<400> 84
Met Glu Lys Gln Glu Ile Asn Lys Phe Ser Lys Thr Pro Leu Ile Gln
1 5 10 15
Ala Leu Lys Glu Tyr Glu Lys Lys Asp Ser Leu Arg Phe His Met Pro
20 25 30
Gly His Lys Gly Arg Cys Pro Lys Gly Val Phe Cys Asp Ile Lys Glu
35 40 45
Asn Leu Phe Gly Trp Asp Val Thr Glu Ile Pro Gly Leu Asp Asp Phe
50 55 60
Ala Gln Pro Glu Gly Pro Ile Lys Glu Ala Gln Glu Lys Leu Ser Ala
65 70 75 80
Leu Tyr Gly Ala Asp Thr Ser Tyr Phe Leu Val Asn Gly Ala Thr Ser
85 90 95
Gly Ile Ile Ser Met Met Ala Gly Ala Leu Ser Glu Lys Asp Lys Ile
100 105 110
Leu Ile Pro Arg Thr Ser His Lys Ser Val Leu Ser Gly Leu Ile Leu
115 120 125
Thr Gly Ala Ser Ala Ala Tyr Ile Met Pro Glu Arg Cys Glu Glu Leu
130 135 140
Gly Val Tyr Ala Gln Val Glu Pro Cys Ala Ile Thr Asn Lys Leu Ile
145 150 155 160
Glu Asn Pro Asp Ile Lys Ala Ile Leu Val Thr Asn Pro Val Tyr Gln
165 170 175
Gly Phe Cys Pro Asp Ile Ala Arg Val Ala Glu Ile Ala Lys Glu Arg
180 185 190
Gly Thr Thr Leu Leu Ala Asp Glu Ala Gln Gly Pro His Phe Gly Phe
195 200 205
Ser Lys Lys Val Pro Gln Ser Ala Gly Lys Phe Ala Asp Ala Trp Val
210 215 220
Gln Ser Pro His Lys Met Leu Thr Ser Leu Thr Gln Ser Ala Trp Leu
225 230 235 240
His Ile Lys Gly Asn Arg Ile Asp Lys Glu Arg Leu Glu Asp Phe Leu
245 250 255
His Ile Val Thr Thr Ser Ser Pro Ser Tyr Ile Leu Met Ala Ser Leu
260 265 270
Asp Gly Thr Arg Glu Leu Ile Glu Glu Asn Gly Asn Ser Tyr Ile Glu
275 280 285
Lys Ala Val Glu Leu Ala Gln Lys Ala Arg Tyr Glu Ile Asn Asn Ser
290 295 300
Thr Val Phe Tyr Ala Pro Gly Gln Glu Ile Leu Gly Lys Tyr Gly Ile
305 310 315 320
Ser Ser Gln Asp Pro Leu His Leu Met Val Asn Val Ser Cys Ala Gly
325 330 335
Tyr Thr Gly Tyr Asp Ile Glu Lys Ala Leu Arg Glu Asp Phe Ser Ile
340 345 350
Tyr Ala Glu Tyr Ala Asp Leu Cys Asn Val Tyr Phe Leu Ile Thr Phe
355 360 365
Ser Asn Thr Leu Glu Asp Ile Lys Gly Leu Leu Ala Val Leu Ser His
370 375 380
Phe Lys Pro Leu Lys Asn Lys Val Lys Pro Cys Phe Trp Ile Lys Asp
385 390 395 400
Leu Pro Lys Val Ala Leu Glu Pro Lys Lys Ala Phe Lys Leu Pro Ala
405 410 415
Lys Ser Val Pro Phe Lys Asp Ser Ala Gly Ser Val Ser Lys Arg Pro
420 425 430
Leu Val Pro Tyr Pro Pro Gly Ala Pro Leu Val Met Pro Gly Glu Ile
435 440 445
Ile Glu Lys Glu His Ile Glu Met Ile Asn Glu Ile Leu Asn Ser Gly
450 455 460
Gly Tyr Cys Gln Gly Val Thr Ser Glu Lys Phe Ile Gln Val Val Thr
465 470 475 480
Asp Phe
<210> 85
<211> 1449
<212> DNA
<213> Tepidanaerobacter syntrophicus
<400> 85
atggagaagc aagagattaa caagttctct aagaccccgc tcatccaagc gctgaaagaa 60
tacgagaaaa aggattctct gcgtttccac atgccaggtc acaaaggccg ttgtccaaaa 120
ggtgtttttt gcgatattaa ggagaacctg ttcggttggg atgttaccga aatcccgggt 180
ctggatgact tcgctcaacc ggaaggtccg atcaaggaag cacaggagaa actgtctgcg 240
ctgtacggtg ccgacacctc ctatttcctc gttaatggtg caacctctgg tatcatttct 300
atgatggcgg gtgctctgtc cgaaaaggac aaaatcctga tcccgcgtac cagccataag 360
agcgtactct ctggtctgat tctcactggc gcctctgcgg cgtacatcat gccggagcgt 420
tgcgaagagc tgggtgttta cgcacaggtg gaaccttgtg ccatcaccaa caaactgatc 480
gagaacccgg atatcaaagc gattctggtt accaacccag tgtaccaggg tttctgcccg 540
gacatcgcgc gtgttgcgga aatcgcgaaa gaacgcggta ccaccctgct cgcagacgaa 600
gcgcaaggcc cacatttcgg cttttccaag aaagttccgc agtctgcggg taagttcgcg 660
gatgcgtggg ttcagtcccc tcacaaaatg ctgacgagcc tgacccaatc tgcgtggctg 720
cacatcaagg gcaatcgtat cgacaaggaa cgtctggaag actttctcca catcgttacc 780
acctcttctc cgtcttacat cctcatggcg tctctggacg gtacccgcga gctgattgaa 840
gaaaacggta actcctacat tgaaaaggcg gttgaactgg ctcagaaagc gcgttatgaa 900
atcaacaact ctactgtttt ctacgcgcca ggccaggaga ttctcggtaa atacggtatt 960
tcttctcagg acccgctgca tctgatggtt aatgtttctt gcgcgggtta cacgggctac 1020
gacatcgaaa aagccctgcg tgaggacttt tctatctacg ccgaatacgc ggacctgtgt 1080
aacgtttact tcctcattac gtttagcaat accctggagg acattaaagg tctcctcgcg 1140
gttctgtctc acttcaaacc gctcaaaaac aaagttaaac cgtgcttctg gatcaaagac 1200
ctgccgaaag ttgcgctgga gccaaagaag gcgttcaaac tgccggcgaa atctgtgcct 1260
ttcaaagatt ctgctggtag cgtttctaaa cgcccgctgg ttccgtatcc gccaggtgcg 1320
ccactcgtga tgccgggtga gatcattgag aaagagcaca tcgagatgat taatgaaatt 1380
ctcaactctg gcggctactg ccagggtgtt acgtctgaaa agttcattca ggttgtaacc 1440
gatttctaa 1449
<210> 86
<211> 490
<212> PRT
<213> Geobacillus kaustophilus
<400> 86
Met Ser Gln Leu Glu Thr Pro Leu Phe Thr Gly Leu Leu Glu His Met
1 5 10 15
Lys Lys Asn Pro Val Gln Phe His Ile Pro Gly His Lys Lys Gly Ala
20 25 30
Gly Met Asp Pro Glu Phe Arg Ala Phe Ile Gly Asp Asn Ala Leu Ala
35 40 45
Ile Asp Leu Ile Asn Ile Ser Pro Leu Asp Asp Leu His His Pro Lys
50 55 60
Gly Met Ile Lys Arg Ala Gln Glu Leu Ala Ala Glu Ala Phe Gly Ala
65 70 75 80
Asp Tyr Thr Phe Phe Ser Val Gln Gly Thr Ser Gly Ala Ile Met Thr
85 90 95
Met Val Met Ser Val Ala Gly Pro Gly Asp Lys Ile Ile Val Pro Arg
100 105 110
Asn Val His Lys Ser Val Met Ser Ala Ile Val Phe Ser Gly Ala Thr
115 120 125
Pro Ile Phe Ile His Pro Glu Ile Asp Lys Glu Leu Gly Ile Ser His
130 135 140
Gly Ile Thr Pro Gln Ala Val Glu Lys Ala Leu Arg Gln His Pro Asp
145 150 155 160
Ala Lys Gly Val Leu Val Ile Asn Pro Thr Tyr Phe Gly Ile Ala Gly
165 170 175
Asp Leu Lys Lys Ile Val Asp Ile Ala His Ser Tyr Asn Val Pro Val
180 185 190
Leu Val Asp Glu Ala His Gly Val His Ile His Phe His Glu Asp Leu
195 200 205
Pro Leu Ser Ala Met Gln Ala Gly Ala Asp Met Ala Ala Thr Ser Val
210 215 220
His Lys Leu Gly Gly Ser Leu Thr Gln Ser Ser Ile Leu Asn Val Arg
225 230 235 240
Glu Gly Leu Val Ser Ala Lys His Val Gln Ala Ile Leu Ser Met Leu
245 250 255
Thr Thr Thr Ser Thr Ser Tyr Leu Leu Leu Ala Ser Leu Asp Val Ala
260 265 270
Arg Lys Gln Leu Ala Thr Lys Gly Arg Glu Leu Ile Asp Lys Ala Ile
275 280 285
Arg Leu Ala Asp Trp Thr Arg Arg Gln Ile Asn Glu Ile Pro Tyr Leu
290 295 300
Tyr Cys Val Gly Glu Glu Ile Leu Gly Thr Glu Ala Thr Tyr Asp Tyr
305 310 315 320
Asp Pro Thr Lys Leu Ile Ile Ser Val Lys Glu Leu Gly Leu Thr Gly
325 330 335
His Asp Val Glu Arg Trp Leu Arg Glu Thr Tyr Asn Ile Glu Val Glu
340 345 350
Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile Ile Thr Pro Gly Asp Thr
355 360 365
Glu Arg Glu Ala Ser Leu Leu Val Glu Ala Leu Arg Arg Leu Ser Lys
370 375 380
Gln Phe Ser His Gln Ala Glu Lys Gly Ile Lys Pro Lys Val Leu Leu
385 390 395 400
Pro Asp Ile Pro Ala Leu Ala Leu Thr Pro Arg Asp Ala Phe Tyr Ala
405 410 415
Glu Thr Glu Val Val Pro Phe His Glu Ser Ala Gly Arg Ile Ile Ala
420 425 430
Glu Phe Val Met Val Tyr Pro Pro Gly Ile Pro Ile Phe Ile Pro Gly
435 440 445
Glu Ile Ile Thr Glu Glu Asn Leu Lys Tyr Ile Glu Thr Asn Leu Ala
450 455 460
Ala Gly Leu Pro Val Gln Gly Pro Glu Asp Asp Thr Leu Gln Thr Leu
465 470 475 480
Arg Val Ile Lys Glu Tyr Lys Pro Ile Arg
485 490
<210> 87
<211> 1473
<212> DNA
<213> Geobacillus kaustophilus
<400> 87
atgtctcagc tcgagacccc tctgttcacc ggtctgctcg aacacatgaa gaaaaacccg 60
gtccagtttc acattccagg tcacaagaaa ggtgctggta tggaccctga gttccgtgcg 120
tttatcggtg ataacgcgct cgcgatcgac ctgatcaaca tctcccctct cgacgacctc 180
caccacccga aaggcatgat caaacgtgcg caggaactgg ctgcggaagc gtttggcgcg 240
gactacacgt tcttcagcgt tcaaggcacc agcggtgcca tcatgacgat ggtaatgtct 300
gttgcgggtc cgggcgataa gatcatcgtc cctcgtaacg ttcacaaatc tgttatgtct 360
gccatcgttt tctctggcgc gacccctatt ttcatccacc cggaaatcga taaggagctg 420
ggtattagcc acggtattac cccgcaggcc gtggagaaag ccctgcgtca acaccctgat 480
gctaaaggcg ttctggtaat caacccgact tatttcggta tcgcgggtga cctcaaaaag 540
atcgttgaca tcgcgcactc ttataatgtg ccggtcctgg tagatgaagc gcacggtgtt 600
catattcact tccacgagga cctcccactc agcgcaatgc aggcgggtgc ggatatggcg 660
gcgacgtccg tgcacaagct gggcggtagc ctgactcagt cttccattct gaacgtacgc 720
gaaggtctgg tttctgctaa acacgtgcaa gcgattctct ctatgctgac caccacttct 780
acctcttatc tgctgctggc ttccctggac gtagcgcgta aacagctggc aaccaaaggt 840
cgtgaactca tcgacaaagc catccgcctc gcggattgga cccgtcgcca gattaacgag 900
atcccgtacc tctactgcgt gggtgaagag atcctgggta ccgaagcaac ctacgactac 960
gatccgacta aactgatcat cagcgtaaaa gaactcggtc tcactggcca tgacgttgag 1020
cgttggctcc gtgaaaccta caatatcgaa gttgaactgt ctgacctcta taacatcctc 1080
tgcatcatca ccccgggtga tactgagcgc gaagcgtctc tcctggtgga agcactgcgc 1140
cgtctgtcta aacaattctc ccatcaggcc gaaaagggta tcaaacctaa ggttctcctg 1200
ccggatattc ctgccctcgc cctgacgcct cgtgacgcgt tctatgcgga aaccgaagtc 1260
gttccgttcc atgagtccgc cggtcgtatc atcgcggagt ttgtaatggt ttacccaccg 1320
ggcatcccaa tcttcatccc tggcgagatt atcactgagg aaaacctgaa atacatcgaa 1380
accaacctgg cggctggcct cccggttcag ggcccagaag acgacacgct gcagaccctc 1440
cgtgtcatta aagaatacaa accaattcgt taa 1473
<210> 88
<211> 495
<212> PRT
<213> Thermomicrobium roseum
<400> 88
Met Ser Glu Glu Gln Gln Arg Ala Pro Tyr Leu Glu Gln Trp Leu Ala
1 5 10 15
Tyr Val Asp Glu Cys Val Ile Pro Phe Thr Thr Pro Gly His Lys Gln
20 25 30
Gly Arg Gly Ala Pro Pro Glu Phe Val Ala Ala Phe Gly Glu Arg Ala
35 40 45
Leu Ala Leu Asp Ile Pro His Asp Gly Gly Thr Phe Asp Ala His Leu
50 55 60
Glu His Asp Pro Leu Val Ala Ala Glu Arg Leu Ala Ala Ala Leu Trp
65 70 75 80
Gly Ala Arg Asp Ala Val Phe Leu Val Asn Gly Ser Thr Thr Gly Asn
85 90 95
Leu Ala Ala Leu Leu Thr Leu Gly Arg Pro Gly Gln Pro Ile Val Val
100 105 110
Thr Arg Ala Met His Lys Ser Leu Leu Ala Gly Leu Val Leu Ser Gly
115 120 125
Ala Arg Pro Val Tyr Val Val Pro Ala Val His Pro Glu Ser Gly Ile
130 135 140
Leu Leu Asp Leu Pro Pro Glu Ser Val Ala Gln Ala Leu Ala Ala Trp
145 150 155 160
Pro Asp Ala Thr Ala Val Ala Leu Val Ser Pro Thr Tyr Thr Gly Val
165 170 175
Thr Ser Asp Thr Ala Glu Leu Ala Ala Leu Cys His Ala His Gly Val
180 185 190
Pro Leu Phe Val Asp Glu Ala Trp Gly Pro His Leu Pro Phe His Pro
195 200 205
Ala Leu Pro Ala Ala Ala Ile Pro Ser Gly Ala Asp Leu Ala Val Thr
210 215 220
Ser Leu His Lys Leu Ala Gly Ser Leu Thr Gln Thr Ala Leu Leu Leu
225 230 235 240
Met Ala Gly Asn Leu Val Asp Gln Ala Gln Leu Arg Ala Ala Thr Ala
245 250 255
Met Val Gln Thr Thr Ser Pro Ala Ala Phe Leu Tyr Ala Ser Leu Asp
260 265 270
Ala Ala Arg Arg Arg Leu Ala Leu Glu Gly Glu Gln Leu Leu Ala Arg
275 280 285
Thr Leu Glu Leu Ala Glu His Ala Arg Arg Glu Leu Ala Ala Ile Pro
290 295 300
Gly Leu Glu Val Val Gly Pro Glu Ile Val Ala Gly Arg Pro Gly Ala
305 310 315 320
Gly Phe Asp Arg Thr Arg Leu Val Val Asp Val Gln Gly Phe Gly Leu
325 330 335
Thr Gly Leu Glu Val Lys Arg Ile Leu Arg Arg Asp Phe Arg Ile Ala
340 345 350
Ala Glu Met Ala Asp Leu Val Ser Val Val Phe Leu Ile Thr Ile Gly
355 360 365
Asp Thr Pro Glu Thr Ile Ala Ala Leu Val Ala Ala Phe Arg Ala Leu
370 375 380
Ala Ala Asp Arg Thr Arg Pro Asp Cys Ala Ala Gly Arg Arg Ala Val
385 390 395 400
Arg Ala Leu Leu Arg Ser Thr Gly Pro Ile Val Ala Gly Ala Pro Gln
405 410 415
Ala Met Thr Pro Arg Glu Ala Phe Phe Ala Pro Ala Glu Arg Val Pro
420 425 430
Leu Ala Asp Ala Val Gly Arg Val Ala Ala Glu Pro Val Thr Pro Tyr
435 440 445
Pro Pro Gly Ile Pro Val Leu Ala Pro Gly Glu Val Val Arg Pro Glu
450 455 460
Val Val Glu Phe Leu Gln Ala Gly Arg Ala Ala Gly Met Arg Phe Asn
465 470 475 480
Gly Ala Ser Asp Pro Thr Leu Ala Thr Leu Arg Val Val Arg Ala
485 490 495
<210> 89
<211> 1488
<212> DNA
<213> Thermomicrobium roseum
<400> 89
atgtctgaag aacagcaacg tgctccgtac ctggagcaat ggctggcgta cgttgacgag 60
tgcgttatcc cgtttaccac tccgggtcac aaacaaggtc gcggtgcgcc accggagttc 120
gttgcggcgt tcggtgaacg tgcgctcgct ctggacattc cgcatgacgg tggcaccttt 180
gacgcgcatc tggaacatga cccgctcgtt gccgccgaac gtctggctgc cgcactgtgg 240
ggtgcacgcg atgcggtgtt tctggttaac ggttccacca ctggtaacct ggcggctctg 300
ctcactctcg gtcgcccagg tcagccgatt gttgttactc gtgccatgca taagagcctg 360
ctggcaggtc tggtcctgag cggtgctcgc cctgtctacg ttgtaccggc cgtacaccca 420
gaatccggta tcctcctcga tctccctccg gaatctgttg cgcaggcgct ggccgcgtgg 480
cctgatgcga cggctgtagc tctggtgtcc ccgacctaca ctggcgttac ctctgacact 540
gctgaactgg cagccctctg tcacgctcat ggtgttccac tgtttgttga tgaagcgtgg 600
ggtccgcacc tcccgttcca tccagcactc ccagcagcag ctattccgtc tggtgccgat 660
ctggcggtta cttctctgca caaactggcg ggttccctca cccaaaccgc tctcctcctg 720
atggcaggca acctcgtaga ccaagcccag ctgcgtgcag ccacggcaat ggtgcaaacc 780
accagccctg cagccttcct gtacgcgtcc ctggatgctg cccgtcgccg tctcgcgctc 840
gaaggtgaac agctcctcgc acgtactctc gagctggctg agcacgctcg ccgtgaactc 900
gccgccatcc cgggtctgga ggtggtcggt ccagaaattg ttgcgggtcg tccgggtgcc 960
ggcttcgatc gtactcgcct cgttgttgac gttcagggtt tcggtctgac tggcctcgaa 1020
gtaaagcgta tcctgcgtcg tgacttccgt attgcagctg aaatggcaga tctcgtctct 1080
gttgttttcc tcatcaccat cggtgacacc ccagagacca tcgctgccct ggtagcagct 1140
ttccgtgcac tcgctgctga ccgtacccgt ccagactgtg ctgccggtcg tcgtgcagta 1200
cgcgccctcc tccgttctac cggtccgatc gtcgcgggtg ctcctcaggc gatgaccccg 1260
cgtgaagctt tcttcgctcc agctgagcgc gttccgctcg cggatgccgt cggtcgtgtt 1320
gcagccgagc cggttacccc atatccgcct ggtattccgg tactggcccc aggtgaagtg 1380
gttcgcccgg aggtagttga attcctccag gcaggccgtg ccgctggtat gcgtttcaat 1440
ggcgcgtctg acccgactct ggcgaccctc cgtgtcgttc gtgcctaa 1488
<210> 90
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
tctggttctg gttctggttc tggttctggt atggagaagc aagagattaa caagttc 57
<210> 91
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
ggctctagat tagaaatcgg ttacaacctg aatg 34
<210> 92
<211> 728
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 92
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Glu Lys Gln Glu Ile Asn Lys Phe Ser
245 250 255
Lys Thr Pro Leu Ile Gln Ala Leu Lys Glu Tyr Glu Lys Lys Asp Ser
260 265 270
Leu Arg Phe His Met Pro Gly His Lys Gly Arg Cys Pro Lys Gly Val
275 280 285
Phe Cys Asp Ile Lys Glu Asn Leu Phe Gly Trp Asp Val Thr Glu Ile
290 295 300
Pro Gly Leu Asp Asp Phe Ala Gln Pro Glu Gly Pro Ile Lys Glu Ala
305 310 315 320
Gln Glu Lys Leu Ser Ala Leu Tyr Gly Ala Asp Thr Ser Tyr Phe Leu
325 330 335
Val Asn Gly Ala Thr Ser Gly Ile Ile Ser Met Met Ala Gly Ala Leu
340 345 350
Ser Glu Lys Asp Lys Ile Leu Ile Pro Arg Thr Ser His Lys Ser Val
355 360 365
Leu Ser Gly Leu Ile Leu Thr Gly Ala Ser Ala Ala Tyr Ile Met Pro
370 375 380
Glu Arg Cys Glu Glu Leu Gly Val Tyr Ala Gln Val Glu Pro Cys Ala
385 390 395 400
Ile Thr Asn Lys Leu Ile Glu Asn Pro Asp Ile Lys Ala Ile Leu Val
405 410 415
Thr Asn Pro Val Tyr Gln Gly Phe Cys Pro Asp Ile Ala Arg Val Ala
420 425 430
Glu Ile Ala Lys Glu Arg Gly Thr Thr Leu Leu Ala Asp Glu Ala Gln
435 440 445
Gly Pro His Phe Gly Phe Ser Lys Lys Val Pro Gln Ser Ala Gly Lys
450 455 460
Phe Ala Asp Ala Trp Val Gln Ser Pro His Lys Met Leu Thr Ser Leu
465 470 475 480
Thr Gln Ser Ala Trp Leu His Ile Lys Gly Asn Arg Ile Asp Lys Glu
485 490 495
Arg Leu Glu Asp Phe Leu His Ile Val Thr Thr Ser Ser Pro Ser Tyr
500 505 510
Ile Leu Met Ala Ser Leu Asp Gly Thr Arg Glu Leu Ile Glu Glu Asn
515 520 525
Gly Asn Ser Tyr Ile Glu Lys Ala Val Glu Leu Ala Gln Lys Ala Arg
530 535 540
Tyr Glu Ile Asn Asn Ser Thr Val Phe Tyr Ala Pro Gly Gln Glu Ile
545 550 555 560
Leu Gly Lys Tyr Gly Ile Ser Ser Gln Asp Pro Leu His Leu Met Val
565 570 575
Asn Val Ser Cys Ala Gly Tyr Thr Gly Tyr Asp Ile Glu Lys Ala Leu
580 585 590
Arg Glu Asp Phe Ser Ile Tyr Ala Glu Tyr Ala Asp Leu Cys Asn Val
595 600 605
Tyr Phe Leu Ile Thr Phe Ser Asn Thr Leu Glu Asp Ile Lys Gly Leu
610 615 620
Leu Ala Val Leu Ser His Phe Lys Pro Leu Lys Asn Lys Val Lys Pro
625 630 635 640
Cys Phe Trp Ile Lys Asp Leu Pro Lys Val Ala Leu Glu Pro Lys Lys
645 650 655
Ala Phe Lys Leu Pro Ala Lys Ser Val Pro Phe Lys Asp Ser Ala Gly
660 665 670
Ser Val Ser Lys Arg Pro Leu Val Pro Tyr Pro Pro Gly Ala Pro Leu
675 680 685
Val Met Pro Gly Glu Ile Ile Glu Lys Glu His Ile Glu Met Ile Asn
690 695 700
Glu Ile Leu Asn Ser Gly Gly Tyr Cys Gln Gly Val Thr Ser Glu Lys
705 710 715 720
Phe Ile Gln Val Val Thr Asp Phe
725
<210> 93
<211> 2187
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat ggagaagcaa gagattaaca agttctctaa gaccccgctc 780
atccaagcgc tgaaagaata cgagaaaaag gattctctgc gtttccacat gccaggtcac 840
aaaggccgtt gtccaaaagg tgttttttgc gatattaagg agaacctgtt cggttgggat 900
gttaccgaaa tcccgggtct ggatgacttc gctcaaccgg aaggtccgat caaggaagca 960
caggagaaac tgtctgcgct gtacggtgcc gacacctcct atttcctcgt taatggtgca 1020
acctctggta tcatttctat gatggcgggt gctctgtccg aaaaggacaa aatcctgatc 1080
ccgcgtacca gccataagag cgtactctct ggtctgattc tcactggcgc ctctgcggcg 1140
tacatcatgc cggagcgttg cgaagagctg ggtgtttacg cacaggtgga accttgtgcc 1200
atcaccaaca aactgatcga gaacccggat atcaaagcga ttctggttac caacccagtg 1260
taccagggtt tctgcccgga catcgcgcgt gttgcggaaa tcgcgaaaga acgcggtacc 1320
accctgctcg cagacgaagc gcaaggccca catttcggct tttccaagaa agttccgcag 1380
tctgcgggta agttcgcgga tgcgtgggtt cagtcccctc acaaaatgct gacgagcctg 1440
acccaatctg cgtggctgca catcaagggc aatcgtatcg acaaggaacg tctggaagac 1500
tttctccaca tcgttaccac ctcttctccg tcttacatcc tcatggcgtc tctggacggt 1560
acccgcgagc tgattgaaga aaacggtaac tcctacattg aaaaggcggt tgaactggct 1620
cagaaagcgc gttatgaaat caacaactct actgttttct acgcgccagg ccaggagatt 1680
ctcggtaaat acggtatttc ttctcaggac ccgctgcatc tgatggttaa tgtttcttgc 1740
gcgggttaca cgggctacga catcgaaaaa gccctgcgtg aggacttttc tatctacgcc 1800
gaatacgcgg acctgtgtaa cgtttacttc ctcattacgt ttagcaatac cctggaggac 1860
attaaaggtc tcctcgcggt tctgtctcac ttcaaaccgc tcaaaaacaa agttaaaccg 1920
tgcttctgga tcaaagacct gccgaaagtt gcgctggagc caaagaaggc gttcaaactg 1980
ccggcgaaat ctgtgccttt caaagattct gctggtagcg tttctaaacg cccgctggtt 2040
ccgtatccgc caggtgcgcc actcgtgatg ccgggtgaga tcattgagaa agagcacatc 2100
gagatgatta atgaaattct caactctggc ggctactgcc agggtgttac gtctgaaaag 2160
ttcattcagg ttgtaaccga tttctaa 2187
<210> 94
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
tctggttctg gttctggttc tggttctggt atgtctcagc tcgagacccc tctgttc 57
<210> 95
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
ggctctagat taacgaattg gtttgtattc tttaatg 37
<210> 96
<211> 736
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 96
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Ser Gln Leu Glu Thr Pro Leu Phe Thr
245 250 255
Gly Leu Leu Glu His Met Lys Lys Asn Pro Val Gln Phe His Ile Pro
260 265 270
Gly His Lys Lys Gly Ala Gly Met Asp Pro Glu Phe Arg Ala Phe Ile
275 280 285
Gly Asp Asn Ala Leu Ala Ile Asp Leu Ile Asn Ile Ser Pro Leu Asp
290 295 300
Asp Leu His His Pro Lys Gly Met Ile Lys Arg Ala Gln Glu Leu Ala
305 310 315 320
Ala Glu Ala Phe Gly Ala Asp Tyr Thr Phe Phe Ser Val Gln Gly Thr
325 330 335
Ser Gly Ala Ile Met Thr Met Val Met Ser Val Ala Gly Pro Gly Asp
340 345 350
Lys Ile Ile Val Pro Arg Asn Val His Lys Ser Val Met Ser Ala Ile
355 360 365
Val Phe Ser Gly Ala Thr Pro Ile Phe Ile His Pro Glu Ile Asp Lys
370 375 380
Glu Leu Gly Ile Ser His Gly Ile Thr Pro Gln Ala Val Glu Lys Ala
385 390 395 400
Leu Arg Gln His Pro Asp Ala Lys Gly Val Leu Val Ile Asn Pro Thr
405 410 415
Tyr Phe Gly Ile Ala Gly Asp Leu Lys Lys Ile Val Asp Ile Ala His
420 425 430
Ser Tyr Asn Val Pro Val Leu Val Asp Glu Ala His Gly Val His Ile
435 440 445
His Phe His Glu Asp Leu Pro Leu Ser Ala Met Gln Ala Gly Ala Asp
450 455 460
Met Ala Ala Thr Ser Val His Lys Leu Gly Gly Ser Leu Thr Gln Ser
465 470 475 480
Ser Ile Leu Asn Val Arg Glu Gly Leu Val Ser Ala Lys His Val Gln
485 490 495
Ala Ile Leu Ser Met Leu Thr Thr Thr Ser Thr Ser Tyr Leu Leu Leu
500 505 510
Ala Ser Leu Asp Val Ala Arg Lys Gln Leu Ala Thr Lys Gly Arg Glu
515 520 525
Leu Ile Asp Lys Ala Ile Arg Leu Ala Asp Trp Thr Arg Arg Gln Ile
530 535 540
Asn Glu Ile Pro Tyr Leu Tyr Cys Val Gly Glu Glu Ile Leu Gly Thr
545 550 555 560
Glu Ala Thr Tyr Asp Tyr Asp Pro Thr Lys Leu Ile Ile Ser Val Lys
565 570 575
Glu Leu Gly Leu Thr Gly His Asp Val Glu Arg Trp Leu Arg Glu Thr
580 585 590
Tyr Asn Ile Glu Val Glu Leu Ser Asp Leu Tyr Asn Ile Leu Cys Ile
595 600 605
Ile Thr Pro Gly Asp Thr Glu Arg Glu Ala Ser Leu Leu Val Glu Ala
610 615 620
Leu Arg Arg Leu Ser Lys Gln Phe Ser His Gln Ala Glu Lys Gly Ile
625 630 635 640
Lys Pro Lys Val Leu Leu Pro Asp Ile Pro Ala Leu Ala Leu Thr Pro
645 650 655
Arg Asp Ala Phe Tyr Ala Glu Thr Glu Val Val Pro Phe His Glu Ser
660 665 670
Ala Gly Arg Ile Ile Ala Glu Phe Val Met Val Tyr Pro Pro Gly Ile
675 680 685
Pro Ile Phe Ile Pro Gly Glu Ile Ile Thr Glu Glu Asn Leu Lys Tyr
690 695 700
Ile Glu Thr Asn Leu Ala Ala Gly Leu Pro Val Gln Gly Pro Glu Asp
705 710 715 720
Asp Thr Leu Gln Thr Leu Arg Val Ile Lys Glu Tyr Lys Pro Ile Arg
725 730 735
<210> 97
<211> 2211
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat gtctcagctc gagacccctc tgttcaccgg tctgctcgaa 780
cacatgaaga aaaacccggt ccagtttcac attccaggtc acaagaaagg tgctggtatg 840
gaccctgagt tccgtgcgtt tatcggtgat aacgcgctcg cgatcgacct gatcaacatc 900
tcccctctcg acgacctcca ccacccgaaa ggcatgatca aacgtgcgca ggaactggct 960
gcggaagcgt ttggcgcgga ctacacgttc ttcagcgttc aaggcaccag cggtgccatc 1020
atgacgatgg taatgtctgt tgcgggtccg ggcgataaga tcatcgtccc tcgtaacgtt 1080
cacaaatctg ttatgtctgc catcgttttc tctggcgcga cccctatttt catccacccg 1140
gaaatcgata aggagctggg tattagccac ggtattaccc cgcaggccgt ggagaaagcc 1200
ctgcgtcaac accctgatgc taaaggcgtt ctggtaatca acccgactta tttcggtatc 1260
gcgggtgacc tcaaaaagat cgttgacatc gcgcactctt ataatgtgcc ggtcctggta 1320
gatgaagcgc acggtgttca tattcacttc cacgaggacc tcccactcag cgcaatgcag 1380
gcgggtgcgg atatggcggc gacgtccgtg cacaagctgg gcggtagcct gactcagtct 1440
tccattctga acgtacgcga aggtctggtt tctgctaaac acgtgcaagc gattctctct 1500
atgctgacca ccacttctac ctcttatctg ctgctggctt ccctggacgt agcgcgtaaa 1560
cagctggcaa ccaaaggtcg tgaactcatc gacaaagcca tccgcctcgc ggattggacc 1620
cgtcgccaga ttaacgagat cccgtacctc tactgcgtgg gtgaagagat cctgggtacc 1680
gaagcaacct acgactacga tccgactaaa ctgatcatca gcgtaaaaga actcggtctc 1740
actggccatg acgttgagcg ttggctccgt gaaacctaca atatcgaagt tgaactgtct 1800
gacctctata acatcctctg catcatcacc ccgggtgata ctgagcgcga agcgtctctc 1860
ctggtggaag cactgcgccg tctgtctaaa caattctccc atcaggccga aaagggtatc 1920
aaacctaagg ttctcctgcc ggatattcct gccctcgccc tgacgcctcg tgacgcgttc 1980
tatgcggaaa ccgaagtcgt tccgttccat gagtccgccg gtcgtatcat cgcggagttt 2040
gtaatggttt acccaccggg catcccaatc ttcatccctg gcgagattat cactgaggaa 2100
aacctgaaat acatcgaaac caacctggcg gctggcctcc cggttcaggg cccagaagac 2160
gacacgctgc agaccctccg tgtcattaaa gaatacaaac caattcgtta a 2211
<210> 98
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
tctggttctg gttctggttc tggttctggt atgtctgaag aacagcaacg tgctccg 57
<210> 99
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
ggctctagat taggcacgaa cgacacggag gg 32
<210> 100
<211> 741
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 100
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ser Gly Ser Gly
225 230 235 240
Ser Gly Ser Gly Ser Gly Met Ser Glu Glu Gln Gln Arg Ala Pro Tyr
245 250 255
Leu Glu Gln Trp Leu Ala Tyr Val Asp Glu Cys Val Ile Pro Phe Thr
260 265 270
Thr Pro Gly His Lys Gln Gly Arg Gly Ala Pro Pro Glu Phe Val Ala
275 280 285
Ala Phe Gly Glu Arg Ala Leu Ala Leu Asp Ile Pro His Asp Gly Gly
290 295 300
Thr Phe Asp Ala His Leu Glu His Asp Pro Leu Val Ala Ala Glu Arg
305 310 315 320
Leu Ala Ala Ala Leu Trp Gly Ala Arg Asp Ala Val Phe Leu Val Asn
325 330 335
Gly Ser Thr Thr Gly Asn Leu Ala Ala Leu Leu Thr Leu Gly Arg Pro
340 345 350
Gly Gln Pro Ile Val Val Thr Arg Ala Met His Lys Ser Leu Leu Ala
355 360 365
Gly Leu Val Leu Ser Gly Ala Arg Pro Val Tyr Val Val Pro Ala Val
370 375 380
His Pro Glu Ser Gly Ile Leu Leu Asp Leu Pro Pro Glu Ser Val Ala
385 390 395 400
Gln Ala Leu Ala Ala Trp Pro Asp Ala Thr Ala Val Ala Leu Val Ser
405 410 415
Pro Thr Tyr Thr Gly Val Thr Ser Asp Thr Ala Glu Leu Ala Ala Leu
420 425 430
Cys His Ala His Gly Val Pro Leu Phe Val Asp Glu Ala Trp Gly Pro
435 440 445
His Leu Pro Phe His Pro Ala Leu Pro Ala Ala Ala Ile Pro Ser Gly
450 455 460
Ala Asp Leu Ala Val Thr Ser Leu His Lys Leu Ala Gly Ser Leu Thr
465 470 475 480
Gln Thr Ala Leu Leu Leu Met Ala Gly Asn Leu Val Asp Gln Ala Gln
485 490 495
Leu Arg Ala Ala Thr Ala Met Val Gln Thr Thr Ser Pro Ala Ala Phe
500 505 510
Leu Tyr Ala Ser Leu Asp Ala Ala Arg Arg Arg Leu Ala Leu Glu Gly
515 520 525
Glu Gln Leu Leu Ala Arg Thr Leu Glu Leu Ala Glu His Ala Arg Arg
530 535 540
Glu Leu Ala Ala Ile Pro Gly Leu Glu Val Val Gly Pro Glu Ile Val
545 550 555 560
Ala Gly Arg Pro Gly Ala Gly Phe Asp Arg Thr Arg Leu Val Val Asp
565 570 575
Val Gln Gly Phe Gly Leu Thr Gly Leu Glu Val Lys Arg Ile Leu Arg
580 585 590
Arg Asp Phe Arg Ile Ala Ala Glu Met Ala Asp Leu Val Ser Val Val
595 600 605
Phe Leu Ile Thr Ile Gly Asp Thr Pro Glu Thr Ile Ala Ala Leu Val
610 615 620
Ala Ala Phe Arg Ala Leu Ala Ala Asp Arg Thr Arg Pro Asp Cys Ala
625 630 635 640
Ala Gly Arg Arg Ala Val Arg Ala Leu Leu Arg Ser Thr Gly Pro Ile
645 650 655
Val Ala Gly Ala Pro Gln Ala Met Thr Pro Arg Glu Ala Phe Phe Ala
660 665 670
Pro Ala Glu Arg Val Pro Leu Ala Asp Ala Val Gly Arg Val Ala Ala
675 680 685
Glu Pro Val Thr Pro Tyr Pro Pro Gly Ile Pro Val Leu Ala Pro Gly
690 695 700
Glu Val Val Arg Pro Glu Val Val Glu Phe Leu Gln Ala Gly Arg Ala
705 710 715 720
Ala Gly Met Arg Phe Asn Gly Ala Ser Asp Pro Thr Leu Ala Thr Leu
725 730 735
Arg Val Val Arg Ala
740
<210> 101
<211> 2226
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
atggtgtcta aaggcgagga agataatatg gcgattatca aagaatttat gcgttttaaa 60
gtgcatatgg aaggcagcgt gaatgggcat gagtttgaaa ttgaaggcga aggagaaggc 120
cgtccgtatg aaggcaccca gaccgctaaa ctgaaagtga ccaaaggcgg accactgccg 180
tttgcgtggg acattctgag cccgcagttt atgtatggca gcaaagcgta tgtgaaacat 240
ccggcggata ttccggatta tctgaaactg agctttccgg agggcttcaa atgggaacgt 300
gtgatgaatt ttgaagatgg cggcgtggtg accgtgaccc aggatagcag cctgcaagac 360
ggcgaattca tttacaaggt gaagctgcgt ggcaccaact ttcccagcga tggcccggtg 420
atgcagaaaa agaccatggg ctgggaggcg agcagcgaac gtatgtaccc ggaggatggc 480
gcgctgaagg gcgaaattaa gcagcgtctg aagttaaaag atggtgggca ctatgatgcg 540
gaagtgaaaa ccacctataa agcgaaaaaa ccggtgcagt taccaggcgc ttataatgtg 600
aacattaagc tggatattac cagccataat gaagattata ccattgtgga acagtatgag 660
cgtgcggagg gacggcatag cacgggcgga atggatgaac tgtataaatc tggttctggt 720
tctggttctg gttctggtat gtctgaagaa cagcaacgtg ctccgtacct ggagcaatgg 780
ctggcgtacg ttgacgagtg cgttatcccg tttaccactc cgggtcacaa acaaggtcgc 840
ggtgcgccac cggagttcgt tgcggcgttc ggtgaacgtg cgctcgctct ggacattccg 900
catgacggtg gcacctttga cgcgcatctg gaacatgacc cgctcgttgc cgccgaacgt 960
ctggctgccg cactgtgggg tgcacgcgat gcggtgtttc tggttaacgg ttccaccact 1020
ggtaacctgg cggctctgct cactctcggt cgcccaggtc agccgattgt tgttactcgt 1080
gccatgcata agagcctgct ggcaggtctg gtcctgagcg gtgctcgccc tgtctacgtt 1140
gtaccggccg tacacccaga atccggtatc ctcctcgatc tccctccgga atctgttgcg 1200
caggcgctgg ccgcgtggcc tgatgcgacg gctgtagctc tggtgtcccc gacctacact 1260
ggcgttacct ctgacactgc tgaactggca gccctctgtc acgctcatgg tgttccactg 1320
tttgttgatg aagcgtgggg tccgcacctc ccgttccatc cagcactccc agcagcagct 1380
attccgtctg gtgccgatct ggcggttact tctctgcaca aactggcggg ttccctcacc 1440
caaaccgctc tcctcctgat ggcaggcaac ctcgtagacc aagcccagct gcgtgcagcc 1500
acggcaatgg tgcaaaccac cagccctgca gccttcctgt acgcgtccct ggatgctgcc 1560
cgtcgccgtc tcgcgctcga aggtgaacag ctcctcgcac gtactctcga gctggctgag 1620
cacgctcgcc gtgaactcgc cgccatcccg ggtctggagg tggtcggtcc agaaattgtt 1680
gcgggtcgtc cgggtgccgg cttcgatcgt actcgcctcg ttgttgacgt tcagggtttc 1740
ggtctgactg gcctcgaagt aaagcgtatc ctgcgtcgtg acttccgtat tgcagctgaa 1800
atggcagatc tcgtctctgt tgttttcctc atcaccatcg gtgacacccc agagaccatc 1860
gctgccctgg tagcagcttt ccgtgcactc gctgctgacc gtacccgtcc agactgtgct 1920
gccggtcgtc gtgcagtacg cgccctcctc cgttctaccg gtccgatcgt cgcgggtgct 1980
cctcaggcga tgaccccgcg tgaagctttc ttcgctccag ctgagcgcgt tccgctcgcg 2040
gatgccgtcg gtcgtgttgc agccgagccg gttaccccat atccgcctgg tattccggta 2100
ctggccccag gtgaagtggt tcgcccggag gtagttgaat tcctccaggc aggccgtgcc 2160
gctggtatgc gtttcaatgg cgcgtctgac ccgactctgg cgaccctccg tgtcgttcgt 2220
gcctaa 2226

Claims (3)

1. A method for producing 1, 5-pentanediamine by fermentation is characterized in that (a) a coding gene of fusion protein is constructed into engineering bacteria with L-lysine production capacity, recombinant bacteria are fermented and cultured, lysine accumulation is carried out, and the initial fermentation culture temperature is controlled at 20-45 ℃; (b) The rest fermentation stage is controlled at 55-65deg.C to make lysine decarboxylase active, and 1, 5-pentanediamine is produced by conversion;
wherein, the fusion protein is selected from amino acid sequences shown in SEQ ID NO:7, wherein the amino acid sequence of the fusion protein MRFP-TeLDC is shown as SEQ ID NO:92, the amino acid sequence of the fusion protein MRFP-TsLDC is shown as SEQ ID NO:96, or a fusion protein MRFP-GkLDC with an amino acid sequence shown as SEQ ID NO:100, a fusion protein MRFP-TrLDC shown in the specification;
the original strain of the engineering bacteria is escherichia coliEscherichia. coli）。
2. The method according to claim 1, wherein the construction is performed using recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or engineering bacteria material when constructing an engineering bacteria having an ability to produce L-lysine.
3. The method according to claim 1, wherein the temperature of the fermentation system is readjusted and controlled to 55-65 ℃ when the fermentation is incubated until the lysine content no longer increases.
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