CN111979257A - Recombinant DNA and application thereof - Google Patents

Abstract

The invention provides a recombinant DNA and application thereof. The recombinant DNA at least comprises a stationary phase specific promoter and a lysine decarboxylase fusion protein gene with a solubility promoting label; wherein the lytic tag is selected from the group consisting of a fluorescent protein, a maltose binding protein, a glutathione transferase, or a combination thereof. The invention provides a new strategy for improving the yield of polyamine produced by fermentation of the recombinant strain.

Description

Recombinant DNA and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to recombinant DNA and application thereof, in particular to application in polyamine production.

Background

1, 5-pentanediamine (also called 1, 5-diaminopentane, cadaverine) is an important pentacarbon compound in the chemical industry, has a wide range of applications, and can be used for producing important chemical raw materials such as polyamide, polyurethane, isocyanate, pyridine, piperidine, and the like. At present, the microbiological method for producing 1, 5-pentanediamine mainly adopts the following two methods: microbial fermentation production and microbial in vitro enzyme catalysis production. Lysine decarboxylase (LDC, EC 4.1.1.18) used in enzyme catalysis production is widely present in microorganisms, insects, animals and higher plants, and can remove one carboxyl group from L-lysine to generate 1, 5-pentanediamine and CO ₂。

In the process of producing 1, 5-pentanediamine by catalyzing lysine by lysine decarboxylase, usually, lysine decarboxylase in a free state or lysine decarboxylase cells are used, or 1, 5-pentanediamine is produced by fermentation by using strains capable of producing lysine and lysine decarboxylase at the same time, but the above production methods cause low recycling efficiency of enzyme or cells, difficult product recovery and high production cost, and are not favorable for industrial production of 1, 5-pentanediamine. Moreover, since the concentration of 1, 5-pentanediamine which is tolerated by the bacterial cells per se is limited, if too much 1, 5-pentanediamine is produced by the conversion of lysine decarboxylase expressed in the early stage of the fermentation system, the bacterial cells are poisoned, and the growth of the bacterial cells and the process of producing L-lysine by using glucose are inhibited (Qian, et al, Biotechnol, Bioeng.2011; 108: 93-103). Therefore, new techniques for obtaining higher yields of 1, 5-pentanediamine are needed.

Disclosure of Invention

The object of the present invention is to provide a recombinant DNA and its use, in particular in the production of polyamines such as 1, 5-pentanediamine.

The invention has the following conception: lysine decarboxylase from thermophilic bacterial strain is screened, and the lysine decarboxylase performs decarboxylation by fusion expression of fluorescent protein and raising temperature; in addition, the use of fluorescent proteins with a color can also unambiguously indicate cells expressing the fusion protein. Furthermore, by means of a promoter in a stationary phase, the expression of lysine decarboxylase can be started only in the stationary phase, so that the yield reduction caused by the poisoning of the host cells by the 1, 5-pentanediamine is greatly reduced, the yield of the 1, 5-pentanediamine produced by fermenting the recombinant strain is obviously improved, the residue of lysine is obviously reduced, and the subsequent process for extracting the 1, 5-pentanediamine is simplified.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a recombinant DNA comprising at least a stationary phase-specific promoter and a lysine decarboxylase fusion protein gene with a lysogenic tag; the fusion protein comprises a dissolving-promoting label and lysine decarboxylase from thermophilic bacteria, and the dissolving-promoting label and the lysine decarboxylase are connected through a Linker;

wherein the lytic tag is selected from the group consisting of a fluorescent protein, a maltose binding protein, a glutathione transferase, or a combination thereof.

In the case of heterologous protein expression in prokaryotes such as E.coli, the maltose binding protein MBP, glutathione transferase GST, and histidine His₇Etc. may promote solubility of the protein and proper folding of the protein. The size of the MBP (maltose binding protein) tag protein is 42kDa, the isoelectric point pI is 5.03, the MBP can increase the solubility of the fusion protein excessively expressed in bacteria, but the larger size of the tag has certain influence on the structure or the function of the protein. GST (glutathione mercaptotransferase) tag protein, the natural size of which is 26KD, and the isoelectric point pI is 6.10.

In the invention, the fluorescent protein is selected from red fluorescent protein, blue-green fluorescent protein, yellow fluorescent protein, orange fluorescent protein or optically highlighted fluorescent protein; preferably at least one of RedStar, tdtomato or mCherry. The inventors have surprisingly found that the above-mentioned fluorescent proteins can be used as a lytic tag in heterologous protein expression, and that their fluorescent properties can also conveniently indicate to the cell that correctly expresses the fusion protein.

In some embodiments, the mCherry is a red fluorescent protein from coral, the best performing one monomeric red fluorescent protein evolved from DsRed (gracewe, et al, Biotechnology Journal,2009,4(6)) with a size of 26KDa and an isoelectric point pI of 5.62.

In some embodiments, the amino acid sequence of mCherry is shown in SEQ ID NO 17 and the nucleotide sequence of the mCherry encoding gene is shown in SEQ ID NO 18, which is codon optimized.

In some embodiments, the lysine decarboxylase is selected from any one of the following (r) - (r): lysine decarboxylase TelDC derived from thermophilic bacteria Thermosynechococcus elongatus, and the amino acid sequence of the lysine decarboxylase TelDC is shown as SEQ ID NO:1, the coding gene is Teldc (GenBank ID BAC09418.1, SEQ ID NO: 2); ② lysine decarboxylase TsLDC derived from Tepidaaerobacter syntrophicus, the amino acid sequence is shown as SEQ ID NO. 3, and the coding gene is Tsddc (GenBank ID GAQ24853.1, SEQ ID NO. 4); ③ lysine decarboxylase GkLDC from Geobacillus kaustophilus, the amino acid sequence is shown as SEQ ID NO. 5, and the coding gene is Gkldc (GenBank ID BAD75350.1, SEQ ID NO. 6); and the lysine decarboxylase TrLDC derived from Thermomicrobium roseum has the amino acid sequence shown in SEQ ID NO. 7, and the coding gene is Trldc (GenBank ID ACM05730.1, SEQ ID NO. 8). Preferably, the full-length nucleotide sequence is optimized according to the codon preference of Escherichia coli.

In some embodiments, the lysine decarboxylase is identical to SEQ ID NO:1, 3,5 or 7, or at least 80%, at least 85%, at least 90%, at least 95% sequence identity.

In some embodiments, the Linker comprises a Linker in a helical form or a flexible Linker of low hydrophobicity, low charge effect amino acids, which should be at least 10 amino acids in length. Preferably, the Linker is a flexible Linker, such as (GGGGS)₃Or (SG)_5-8(ii) a More preferably, the Linker is (SG)_5-8(ii) a Most preferably, the Linker is SGSGSGSGSG.

In some embodiments, the fusion protein is selected from at least one of fluorescent protein-Linker-TelDC, fluorescent protein-Linker-TsLDC, fluorescent protein-Linker-GkLDC, fluorescent protein-Linker-TrLDC, TeLDC-Linker-fluorescent protein, TsLDC-Linker-fluorescent protein, or TsLDC-Linker-fluorescent protein.

In some embodiments, the fusion protein is mCherry-Linker-TeLDC, TeLDC-Linker-mCherry, mCherry-Linker-TsLDC, mCherry-Linker-GkLDC or mCherry-Linker-TrLDC.

In some embodiments, the fusion protein is any one of the recombinant nucleotide sequences shown in SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, comprising, from the 5 'direction to the 3' direction, a nucleotide sequence encoding a fluorescent protein (SEQ ID NO:5) and a nucleotide sequence encoding a thermophilic lysine decarboxylase.

In some embodiments, the stationary phase specific promoter is selected from any one of pcsiE (SEQ ID NO:20), pbOLA (SEQ ID NO: 21), posmY (SEQ ID NO:22), pkatE (SEQ ID NO:23), P1(SEQ ID NO:24), P2(SEQ ID NO: 25), P3(SEQ ID NO:26), or P4(SEQ ID NO: 27).

In some embodiments, the recombinant DNA comprises at least the following 3 elements: a. a stationary phase specific promoter; b. a red fluorescent protein gene; and c, a lysine decarboxylase gene derived from a thermophilic bacterium; wherein the elements are operatively connected in a-b-c or a-c-b sequence.

In a second aspect, the present invention provides a biological material comprising the above recombinant DNA, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineered bacterium, or the like.

In a third aspect, the present invention provides a recombinant plasmid carrying the above recombinant DNA. Preferably, the issuing plasmid is a pUC or pBR322 plasmid or a derivative plasmid thereof, more preferably pUC18, pUC19, pBR322, pACYC, pET, pSC101 and any derivative plasmid thereof.

In a fourth aspect, the invention provides a genetically engineered bacterium for producing 1, 5-pentanediamine, which is a strain having an ability to produce L-lysine and carries the recombinant DNA, the biological material or the recombinant plasmid.

Wherein the starting strain of the genetic engineering bacteria is selected from the strains in Escherichia (Escherichia) and Hafnia (Hafnia); preferably, the starting strain is escherichia coli (escherichia coli), Hafnia alvei (Hafnia alvei), or a strain or genetically engineered bacterium after mutagenesis or random mutation.

In a fifth aspect, the present invention provides a method for producing 1, 5-pentanediamine, comprising the step of fermenting and culturing the above engineering bacteria to produce 1, 5-pentanediamine.

In a sixth aspect, the invention provides an application of the recombinant DNA in the production of 1, 5-pentanediamine, which comprises (a) constructing the recombinant DNA into an engineering bacterium with the capacity of producing L-lysine, fermenting and culturing the recombinant bacterium and accumulating the lysine, wherein the culture temperature at the initial stage of fermentation is controlled at 20-50 ℃ to carry out the rapid growth of the bacterium and the accumulation of the lysine; (b) the temperature is controlled at 50-110 deg.C in the rest fermentation stage, so that lysine decarboxylase is active, 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 that the value reasonably deviates from the value, e.g., within 1 ℃ or 2 ℃ below or above the value recited within the range is 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 carried out at a temperature of about 30 ℃ to about 40 ℃. In a further embodiment, step (a) is carried out 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 carried out at a temperature of from about 60 ℃ to about 70 ℃.

In some embodiments, the method for producing 1, 5-pentanediamine comprises constructing the recombinant DNA into an engineering bacterium with L-lysine production capability, preferably performing codon optimization on a coding gene, then constructing into the engineering bacterium, preferably Escherichia coli or Hafnia alvei, fermenting and culturing the recombinant bacterium, wherein the culture temperature in the initial stage of fermentation is controlled at 20-50 ℃, for example 37 ℃ +/-2 ℃, so as to perform rapid growth of the bacterium and accumulation of lysine; the remaining fermentation stage controls the temperature to 50-110 deg.C, such as 55 + -2 deg.C, to make lysine decarboxylase active or active, and convert lysine to produce 1, 5-pentanediamine. Wherein, preferably, the fusion protein is fluorescent protein-Linker-lysine decarboxylase (TelDC, TsLDC, GkLDC or TrLDC). Preferably, when the fermentation is continued until the lysine content is no longer increased, the temperature of the fermentation system is raised and controlled to 50-110 ℃. Preferably, the engineered bacterium having the ability to produce L-lysine is constructed using a material such as a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, or an engineered bacterium.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

according to the invention, by screening lysine decarboxylase from thermophilic strains and utilizing fusion expression of a dissolving-promoting label and the dissolving-promoting label, the lysine decarboxylase from thermophilic strains can be helped to play a lysine decarboxylation function when being connected with the dissolving-promoting label, and can be simply enabled to play a function by raising the temperature compared with the lysine decarboxylase CadA from escherichia coli; in addition, the use of a lytic tag may also clearly indicate the cells expressing the fusion protein, especially fluorescent proteins as lytic tags are easier to clearly indicate. The application of the compound can obviously reduce the cell growth and the cytotoxicity of the 1, 5-pentanediamine generated in the L-lysine production stage when being applied to the production of the 1, 5-pentanediamine, and a small amount of the 1, 5-pentanediamine can also remove the feedback inhibition effect of the lysine and improve the yield of the L-lysine; after the temperature is increased, L-lysine can be almost completely converted into 1, 5-pentanediamine, thereby realizing the increase of the yield of the 1, 5-pentanediamine. Meanwhile, a promoter in a stable period is used, and the expression of a downstream lysine decarboxylase gene is started only after the thalli grow to the stable period, so that the yield of 1, 5-pentanediamine produced by fermenting the recombinant strain is comprehensively and remarkably improved.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The specific steps, condition parameters, etc. of PCR amplification, purification, plasmid extraction, enzyme digestion, ligation of enzyme digestion products, transformation, etc. referred to in the following examples were performed according to the conditions suggested by the instructions for the relevant enzymes and reagents purchased. 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) Co., Ltd. The plasmid extraction kit, the DNA gel recovery kit and the PCR purification kit are all purchased from Kangning Life sciences (Wu Jiang) Co., Ltd., trademark Axygen; primers were purchased from Saimer Feishale science and technology (China) Inc. under the trademark INVITROGEN.

The plasmid transformation methods referred to in the following examples are as follows: the ligation product was added to 100. mu.l of E.coli BL21(DE3) competent cells, heat-shocked at 42 ℃ for 90s after ice-cooling for 30 min. After incubation on ice for 5min 1ml of LB was added. Coating on the corresponding resistant plate.

In the present invention, the amounts of L-lysine and 1, 5-pentanediamine in the medium can be detected by a nuclear magnetic resonance method.

The primers used in the following examples are shown in table 1:

TABLE 1 primer information

EXAMPLE 1 cloning of the lysine decarboxylase Gene cadA

The gene cadA (SEQ ID No:31) encoding lysine decarboxylase (SEQ ID No:30) was amplified from the genome of E.coli MG1655K12(E.coli MG1655K12, purchased from Beijing Tiannzze Biotech Co., Ltd.) using primers cadA-SacI-F (SEQ ID No:28) and cadA-XbaI-R (SEQ ID No:29)The plasmid was digested with SacI and XbaI, and ligated into the pUC18 plasmid (obtained from Takara Bio Inc.) which had been digested with SacI and XbaI. By using CaCl₂Preparing competence, transforming the ligation product into Escherichia coli E.coli BL21 (purchased from Takara Bio-engineering Co., Ltd.) cells by a heat shock method, adding ampicillin into an LB culture medium for screening, cloning PCR and sequencing to verify correctness, and extracting plasmids to obtain pCIB60 plasmids.

Plasmid pCIB60 was used as a template, and primers cadA-F2(SEQ ID No:32) and cadA-R2(SEQ ID No:33) were further used to optimize the 5 'sequence of cadA gene in pCIB60 plasmid, so that cadA could be successfully translated into protein in E.coli BL21, and the 5' sequence of cadA gene was replaced with 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3' (SEQ ID No:35) from 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC-3' (SEQ ID No: 34). After PCR amplification, the PCR product was digested with DpnI restriction enzyme, transformed into E.coli BL21 by the same heat shock method, and verified by sequencing to obtain plasmid pCIB 71.

EXAMPLE 2 cloning of lysine decarboxylase Teldc of the thermophilic bacterium Thermosynechococcus elogatus

Lysine decarboxylase TelDC (SEQ ID NO:1, GenBank ID BAC09418.1) from thermophilic strain Thermoynechococcus elongatus, after the full-length codon of the gene is optimized, the gene (SEQ ID NO:2) is synthesized by a primer splicing method, and the method for codon optimization and gene synthesis refers to Hoover DM&Lubkowski J, Nucleic Acids Research 30:10, 2002, amplified the spliced Teldc using TeLDC-SacI-F (SEQ ID No:36) and TeLDC-XbaI-R (SEQ ID No:37), double-digested with SacI and XbaI, and ligated into the same double-digested pCIB71 plasmid. By using CaCl₂Preparing competence, transforming the ligation product into competent cells of Escherichia coli E.coli BL21 (purchased from Boehringer Bio-engineering Co., Ltd.) by a heat shock method, adding ampicillin into an LB culture medium for screening, and extracting plasmids to obtain pCIB90 plasmid after cloning PCR and sequencing verification are correct.

The 5 ' sequence of the Teldc gene in the pCIB90 plasmid was further optimized by using the primers TelDC-SacI-F2(SEQ ID No:38) and TelDC-SacI-R2(SEQ ID No:39) with the plasmid pCIB90 as a template, so that the TelDC could be successfully translated into protein in E.coli BL21, and the sequence upstream of the start codon ATG of the Teldc gene was replaced with 5'-tgtggaattgtgagctcATCGATAAGCTTGATATCGAATTCTTAACTTTAAGAAGGAATATACAT-3' (SEQ ID No:41) from 5'-tgtggaattgtgagcggataacaATTTCACACAGGAAACAGCTGAGCTC-3' (SEQ ID No: 40). After PCR amplification, the PCR product was digested with DpnI restriction enzyme, transformed into E.coli BL21 by the same heat shock method, and verified by sequencing to obtain plasmid pCIB 91.

Example 3 construction of a plasmid for fusion expression of Red fluorescent protein and lysine decarboxylase derived from thermophilic bacteria

The following methods for codon optimization and gene synthesis are referred to Hoover DM & Lubkowski J, Nucleic Acids Research 30:10, 2002.

(1) After codon optimization of red fluorescent protein MRFP (mCherry, SEQ ID No:17) from coral, the gene (mCherry, SEQ ID No:18) was synthesized by primer splicing.

(2) Construction of red fluorescent egg (MRFP) -TelDC fusion expression plasmid: spliced MRFP is taken as a template, primers SacI-MRFP-TelDC-F (SEQ ID No:42) and linker-MRFP-R (SEQ ID No:43) are utilized for amplification, using plasmid pCIB91 as a template, using primers linker-TelDC-F (SEQ ID No:44) and TelDC-XbaI-R (SEQ ID No:37) to amplify, cutting and recovering target fragments, fusion PCR was performed using primers SacI-MRFP-TeldC-F (SEQ ID No:42) and TeldC-XbaI-R (SEQ ID No:37), and after purification of the target fragment, the double digestion is carried out by SacI and XbaI, and the double digestion is connected into a plasmid pCIB91 which is also subjected to double digestion, the expression plasmid pCIB92 of the MRFP-TelDC fusion protein (SEQ ID No:9) is obtained, wherein the 5 'end of the TelDC coding gene is connected with the 3' end of the red fluorescent protein MRFP (SEQ ID No:10) through a linker (SGSGSGSGSG).

(3) Construction of Red fluorescent protein (MRFP) -TsLDC fusion expression plasmid: a lysine decarboxylase TsLDC derived from Tepidaaerobacter syntrophicus is synthesized by a primer splicing method, the amino acid sequence of the lysine decarboxylase TsLDC is shown as SEQ ID NO. 3, and the coding gene is Tsdc (GenBank ID GAQ24853.1, SEQ ID NO. 4). Spliced MRFP is taken as a template, primers SacI-MRFP-TelDC-F (SEQ ID No:42) and linker-MRFP-R (SEQ ID No:43) are utilized for amplification, the spliced gene Tsddc is taken as a template, primers of linker-TsdDC-F (SEQ ID No:45) and TsdDC-XbaI-R (SEQ ID No:46) are utilized for amplification, after respectively cutting and recovering the target fragments, fusion PCR was performed using primers SacI-MRFP-TeldC-F (SEQ ID No:42) and TsLDC-XbaI-R (SEQ ID No:46), and after purification of the target fragment, the double digestion is carried out by SacI and XbaI, and the double digestion is connected into a plasmid pCIB91 which is also subjected to double digestion, obtaining an expression plasmid pCIB96 of MRFP-TelDC fusion protein (SEQ ID No:11), wherein the 5 'end of the TsLDC coding gene is connected with the 3' end of the red fluorescent protein MRFP through a linker (SGSGSGSGSG) (SEQ ID No: 12).

(4) Constructing a red fluorescent protein (MRFP) -TrLDC fusion expression plasmid: the lysine decarboxylase GkLDC derived from Geobacillus kaustophilus is synthesized by primer splicing, the amino acid sequence of the lysine decarboxylase GkLDC is shown as SEQ ID NO. 5, and the coding gene is Gkldc (GenBank ID BAD75350.1, SEQ ID NO. 6). Spliced MRFP is taken as a template, primers SacI-MRFP-TelDC-F (SEQ ID No:42) and linker-MRFP-R (SEQ ID No:43) are utilized for amplification, amplifying by using the spliced gene Gkldc as a template and using primers linker-GklDC-F (SEQ ID No:47) and GklDC-XbaI-R (SEQ ID No:48), cutting and recovering target fragments, fusion PCR was performed using primers SacI-MRFP-TeldC-F (SEQ ID No:42) and GkLDC-XbaI-R (SEQ ID No:48), and after purification of the target fragment, the double digestion is carried out by SacI and XbaI, and the double digestion is connected into a plasmid pCIB91 which is also subjected to double digestion, obtaining the expression plasmid pCIB97 of MRFP-GkLDC fusion protein (SEQ ID No:13), wherein the 5 'end of the GkLDC coding gene is connected with the 3' end of the red fluorescent protein MRFP (SEQ ID No: 14) through a linker (SGSGSGSGSG).

(5) Constructing a red fluorescent protein (MRFP) -GkLDC fusion expression plasmid: the lysine decarboxylase TrLDC derived from Thermomicrobium roseum is synthesized by primer splicing, the amino acid sequence of the TrLDC is shown as SEQ ID NO. 7, and the coding gene is Trldc (GenBank ID ACM05730.1, SEQ ID NO. 8). Spliced MRFP is taken as a template, primers SacI-MRFP-TelDC-F (SEQ ID No:42) and linker-MRFP-R (SEQ ID No:43) are utilized for amplification, amplifying by using a spliced gene Trldc as a template and primers of linker-TrLDC-F (SEQ ID No:49) and TrLDC-XbaI-R (SEQ ID No:50), cutting and recovering target fragments, fusion PCR was performed using primers SacI-MRFP-TeldC-F (SEQ ID No:42) and TrLDC-XbaI-R (SEQ ID No:50), and after purification of the target fragment, the double digestion is carried out by SacI and XbaI, and the double digestion is connected into a plasmid pCIB91 which is also subjected to double digestion, obtaining an expression plasmid pCIB98 of MRFP-TrLDC fusion protein (SEQ ID No:15), wherein the 5 'end of the TrLDC coding gene is connected with the 3' end of the red fluorescent protein MRFP (SEQ ID No:16) through a linker (SGSGSGSGSG).

Example 4 construction of a Strain in which lysine decarboxylase derived from thermophilic bacteria and Red fluorescent protein are fusion-expressed

The constructed MRFP-TeLDC, MRFP-TsLDC, MRFP-gkdc and MRFP-TrLDC fusion protein expression plasmids pCIB92, pCIB96, pCIB97 and pCIB98 were transformed into e.coli BL21 (purchased from baozoite bioengineering (gangreng) limited) competent cells, plated on LB plates containing ampicillin resistance at a final concentration of 100 μ g/ml, and cultured overnight at 37 ℃ in an inverted manner, respectively, to obtain MRFP-TeLDC, MRFP-TsLDC, MRFP-gkc and MRFP-TrLDC fusion protein expression strains CIB92, CIB96, CIB97 and CIB 98. Each 3 individual colonies were picked and inoculated into 5ml LB liquid tubes containing ampicillin resistance at a final concentration of 100. mu.g/ml, and cultured overnight at 37 ℃ and 200 rpm.

Example 5 detection of lysine decarboxylase Activity of fusion proteins MRFP-TeLDC, MRFP-TsLDC, MRFP-TrLDC and MRFP-GkLDC under different temperature conditions

Bacterial liquid OD of each strain₆₀₀No obvious difference is shown by determination, bacterial solutions of the strains with the same volume are respectively taken, Lys-HCl conversion reaction 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 into the bacterial solutions with the concentration of 400g/L, and the reaction time is 4 h. As shown in Table 2, compared with CIB92 strain expressing MRFP-TelDC fusion protein, the lysine conversion rate was not significantly different when reacting at 37 ℃; when the reaction is carried out at 55 ℃, the lysine conversion rates of strains CIB96, CIB97 and CIB98 for expressing fusion proteins MRFP-TsLDC, MRFP-GkLDC and MRFP-TrLDC are obviously improved, and the lysine conversion rates of CIB96 strainsThe lysine conversion was 83.7%, that of the CIB97 strain was 89.9%, and that of the CIB98 strain was 85.1%, and their optimum temperatures were around 55 ℃.

TABLE 2 determination of lysine conversion in recombinant cells expressing the respective lysine decarboxylase

Example 6 construction of strains in which the stationary phase promoter induces expression of lysine decarboxylase

Using the genome of Escherichia coli MG1655K12(E.coli MG1655K12, available from Beijing Tiannzze biotechnology Co., Ltd.) as a template, primers pcsiE-F (SEQ ID No: 51)/pcsiE-R (SEQ ID No: 52), pbolA-F (SEQ ID No: 53)/pbolA-R (SEQ ID No: 54), posmY-F (SEQ ID No: 55)/posmY-R (SEQ ID No: 56), pkatE-F (SEQ ID No: 57)/pkatE-R (SEQ ID No: 58) were amplified to obtain the stationary phase promoter pcsiE (SEQ ID No: 20), pbolA (SEQ ID No: 21), posmY (SEQ ID No: 22), pkatE (SEQ ID No: 23), and after double digestion with KpnI and ClaI, respectively, they were ligated to the same double digested plasmid pCIB92, IB96, pCIB97, pCIB98, pCIB92-101, pCIB92-102, pCIB92-103, pCIB92-104, pCIB96-101, pCIB96-102, pCIB96-103, pCIB96-104, pCIB97-101, pCIB97-102, pCIB97-103, pCIB97-104, pCIB98-101, pCIB98-102, pCIB98-103 and pCIB98-104 were obtained.

Double-stranded DNA sequences (5 '-3') of 4 promoters (P, P, P and P) as listed in Table 3 were synthesized using a gene sequence synthesis method commonly used in the art, and ligated to cleavage sites of KpnI and ClaI at the 5 'and 3' ends of the sequences, respectively, and then ligated to plasmids pCIB, pCIB, pCIB and pCIB double-cleaved with KpnI and ClaI, respectively, to obtain plasmids (Table 3), pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P, pCIB-P and pCIB-P containing the 4 promoters.

TABLE 3 stationary phase promoter and lysine decarboxylase combinations and expression plasmids thereof

Example 7 comparison of lysine and 1, 5-Pentanediamine production by strains with different promoters inducing expression of lysine decarboxylase

In this example, Escherichia coli Ela6116(Escherichia coli Ela6116) strain capable of producing L-lysine, which is now deposited in China center for type culture Collection, addresses: wuhan, Wuhan university, zip code 430072, preservation number CCTCC No: m2018736, date of deposit 2018, 11 months and 1 day.

The plasmids pCIB71, pCIB92, pCIB96, pCIB97, pCIB98 and the 32 plasmids listed in Table 3 were transformed into the E.coli Ela611b strain, respectively, to obtain the corresponding 37 recombinant strains. Using Escherichia coli Ela611b strain as a control, three monoclonals were picked up in 5mL of liquid medium (containing 4% glucose and 0.1% KH)₂PO₄,0.1％MgSO₄,1.6％ (NH₄)₂SO₄,0.001％FeSO₄,0.001％MnSO₄0.2% yeast extract, 0.01% L-threonine, 0.005% L-isoleucine, 10. mu.g/mL tetracycline, 100. mu.g/mL ampicillin) at 37 ℃ overnight. The next day, each strain was transferred to 100ml fresh medium containing 30g/L glucose, 0.7% Ca (HCO)₃)₂10. mu.g/mL tetracycline and 100. mu.g/mL ampicillin, 0.1% KH₂PO₄，0.1％MgSO₄,1.6％(NH₄)₂SO₄,0.001％FeSO₄,0.001％MnSO₄0.2% yeast extract, 0.01% L-threonine, 0.005% L-isoleucine at 37 ℃ for a further 68 h. After the culture is finished, sampling, detecting and calculating the content of the L-lysine and the 1, 5-pentanediamine in each culture medium by utilizing nuclear magnetic resonance (Table 4), and then continuing the reaction for 4 hours at 37 ℃ by using a control strain Ela611b and a recombinant strain Ela611 b-71; the other recombinant strains were heated to 55 ℃ and reacted for 4 hours, and the final contents of L-lysine and 1, 5-pentanediamine in each culture medium were calculated by nuclear magnetic detection (Table 4).

As can be seen from Table 4, the CadA-expressing recombinant strain Ela611b-71 fermented for 68h detected 2.8 g/L-lysine and 3.6 g/L1, 5-pentanediamine, and as the fermentation time increased, the amount of L-lysine and 1, 5-pentanediamine detected in the fermentation broth did not increase significantly, and 4.0 g/L1, 5-pentanediamine and 2.0 g/L-lysine remained, which is probably due to the high activity of CadA protein at 37 ℃ of lysine growth temperature, the excessive L-lysine synthesis of the strain was catalyzed into 1, 5-pentanediamine earlier, and the intracellular accumulation of 1, 5-pentanediamine generated toxicity to the cell, and inhibited the cell metabolism to some extent, including L-lysine synthesis and 1, and (3) converting 5-pentanediamine.

In addition, as can be seen from Table 4, the recombinant strains Ela611b-92, Ela611b-96, Ela611b-97 and Ela611b-98, which constitutively express lysine decarboxylase derived from thermophilic strains, produce 5.9-6.8g/L of L-lysine and 1.8-2.2g/L of 1, 5-pentanediamine in 68h of fermentation, which indicates that the lysine decarboxylase derived from thermophilic bacteria has lower activity at 37 ℃, weakly converts the L-lysine into the 1, 5-pentanediamine, can release the feedback inhibition of the L-lysine to a certain extent, and a small amount of the 1, 5-pentanediamine does not cause cytotoxicity; when the temperature is increased to 55 ℃, the activity of lysine decarboxylase is increased, the residual L-lysine can be completely converted into 1, 5-pentanediamine, 5.1-5.8g/L of 1, 5-pentanediamine is finally accumulated, and only 0.02-0.09g/L of L-lysine is remained.

And the promoter in the stationary phase is used, the transcription of the downstream gene is started theoretically only after the thalli enter the stationary phase, so that the accumulation of the downstream gene in the lysine production process can be further reduced, and tests show that the L-lysine level of each recombinant strain (strain numbers 7-38) using the promoter in the stationary phase is further improved and the yield of the 1, 5-pentanediamine generated by conversion is reduced compared with 4 recombinant strains (strain numbers 3-6) using a constitutive promoter in fermentation for 68 hours. When the temperature is increased to 55 ℃, the activity of the fusion expression protein of the red protein and the decarboxylase from the thermophilic bacteria is increased, the residual L-lysine is continuously converted into 1, 5-pentanediamine almost completely, and finally 1, 5-pentanediamine with the concentration of more than 5g/L is accumulated, and almost no L-lysine remains.

TABLE 4 detection of the levels of L-lysine and 1, 5-pentanediamine of strains capable of simultaneously expressing lysine-synthesizing protein and lysine decarboxylase

Note: n.d. indicates no detection.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Shanghai Kaiser Biotechnology research and development center, Inc

CIBT USA

<120> a recombinant DNA and use thereof

<130> KHP181116615.0

<160> 58

<170> SIPOSequenceListing 1.0

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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

435

<210> 2

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<213> Thermomyces (Thermosynechococcus elongatus)

<400> 2

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 gtaa 1314

<210> 3

<211> 482

<212> PRT

<213> thermophilic bacterium (Tepidana aerobacter syntrophicus)

<400> 3

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

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<212> DNA

<213> thermophilic bacterium (Tepidana aerobacter syntrophicus)

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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> 5

<211> 490

<212> PRT

<213> thermophilic bacterium (Geobacillus kaustophilus)

<400> 5

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> 6

<211> 1473

<212> DNA

<213> thermophilic bacterium (Geobacillus kaustophilus)

<400> 6

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> 7

<211> 495

<212> PRT

<213> Thermomicrobium roseum

<400> 7

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> 8

<211> 1488

<212> DNA

<213> Thermomicrobium roseum

<400> 8

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> 9

<211> 683

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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> 10

<211> 2052

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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> 11

<211> 728

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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> 12

<211> 2187

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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> 13

<211> 736

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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> 14

<211> 2211

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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> 15

<211> 741

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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> 16

<211> 2226

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

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

<210> 17

<211> 711

<212> DNA

<213> Mushroom coral (mushroom coral)

<400> 17

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> 18

<211> 236

<212> PRT

<213> Mushroom coral (mushroom coral)

<400> 18

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> 19

<211> 276

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 60

gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 120

cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 180

cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 240

ctttatgctt ccggctcgta tgttgtgtgg aattgt 276

<210> 20

<211> 235

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 20

tgctttttcc gatcgtcacg gcgatgttta tcgcgaacag atggtggact ttatccttag 60

cgcgttgaat ccgcagaact aacccatgat cgctagcacg ataatcattc acaaaaccac 120

cttaagacat gctaatccac tggtcagaac agtttaagat gagaaaaatt ctgtgacgct 180

tgccaacatt tctgatgatt agcattccct tcgccatttc cttgagcaaa cttta 235

<210> 21

<211> 238

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 21

tgtttggtaa aaattcccgc catcataaca ttgccaacgg cgaggggaag tgggtaaggc 60

atgtaaattc atcatgttga cgaaataatc gcccctggta aaagaaacac tgatgcgagg 120

cctgtgtttc aatctttaaa tcagtaaact tcatacgctt gacggaaaaa ccaggacgaa 180

acctaaatat ttgttgttaa gctgcaatgg aaacggtaaa agcggctagt atttaaag 238

<210> 22

<211> 233

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 22

ctcgcttaca tcgctaccag catggtcaac ctgcgcctgg cacaggaacg ttatccggac 60

gttcagttcc accagacccg cgagcattaa ttcttgcctc cagggcgcgg tagccgctgc 120

gccctgtcaa tttcccttcc ttattagccg cttacggaat gttcttaaaa cattcacttt 180

tgcttatgtt ttcgctgata tcccgagcgg tttcaaaatt gtgatctata ttt 233

<210> 23

<211> 237

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 23

gcagaaatga ctctcccatc agtacaaacg caacatattt gccacgcagc atccagacat 60

cacgaaacga atccatcttt atcgcatgtt ctggcggcgc gggttccgtg cgtgggacat 120

agctaataat ctggcggttt tgctggcgga gcggtttctt cattactggc ttcactaaac 180

gcatattaaa aatcagaaaa actgtagttt agccgattta gcccctgtac gtcccgc 237

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tcccgccaaa tccctaaaat tgttctatac tgtattg 37

<210> 25

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tcccgccaaa ttattaaaat tgttctatac tgtattg 37

<210> 26

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgcgttcccg cctttagggg caattgttct atactgtatt g 41

<210> 27

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tcccgccaaa tctgcaaaat tgttctatac tgtattg 37

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tccgagctca tgaacgttat tgcaatattg 30

<210> 29

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gcctctagac cacttccctt gtacgagc 28

<210> 30

<211> 715

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 30

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

1 5 10 15

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

20 25 30

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

50 55 60

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

65 70 75 80

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

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> 31

<211> 2148

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 31

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> 32

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

atttcacaca ggaaacagct atgaacgtta ttgcaatatt gaat 44

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

agctgtttcc tgtgtgaaat 20

<210> 34

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa 60

ttcgagctc 69

<210> 35

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

tgtggaattg tgagcggata acaatttcac acaggaaaca gctgagctc 49

<210> 36

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ggcgagctca tggaaccatt acttcgcgc 29

<210> 37

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ggctctagat tacactttac atatccgcag gg 32

<210> 38

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

cttgatatcg aattcttaac tttaagaagg aatatacata tggaaccatt acttcgcgc 59

<210> 39

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gttaagaatt cgatatcaag cttatcgatg agctcacaat tccacacaac atacgag 57

<210> 40

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

tgtggaattg tgagcggata acaatttcac acaggaaaca gctgagctc 49

<210> 41

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

tgtggaattg tgagctcatc gataagcttg atatcgaatt cttaacttta agaaggaata 60

tacat 65

<210> 42

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ggcgagctca tcgataagct tgatatcgaa ttcttaactt taagaaggaa tatacatatg 60

gtgtctaaag gc 72

<210> 43

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

accagaacca gaaccagaac cagaaccaga tttatacagt tcatccattc cgcc 54

<210> 44

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

tctggttctg gttctggttc tggttctggt atggaaccat tacttcgcgc actgtgg 57

<210> 45

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

tctggttctg gttctggttc tggttctggt atggagaagc aagagattaa caagttc 57

<210> 46

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

ggctctagat tagaaatcgg ttacaacctg aatg 34

<210> 47

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

tctggttctg gttctggttc tggttctggt atgtctcagc tcgagacccc tctgttc 57

<210> 48

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ggctctagat taacgaattg gtttgtattc tttaatg 37

<210> 49

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

tctggttctg gttctggttc tggttctggt atgtctgaag aacagcaacg tgctccg 57

<210> 50

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

ggctctagat taggcacgaa cgacacggag gg 32

<210> 51

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

ggggtacctg ctttttccga tcgtcacg 28

<210> 52

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

ccatcgatta aagtttgctc aaggaaatgg c 31

<210> 53

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

ggggtacctg tttggtaaaa attcccg 27

<210> 54

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

ccatcgatct ttaaatacta gccgctttta c 31

<210> 55

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

ggggtaccct cgcttacatc gctaccagc 29

<210> 56

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ccatcgataa atatagatca caattttgaa 30

<210> 57

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

ggggtaccgc agaaatgact ctcccatc 28

<210> 58

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

ggatcgatgc gggacgtaca ggggc 25

Claims (10)

1. Recombinant DNA, characterized in that the recombinant DNA comprises at least a stationary phase specific promoter and a lysine decarboxylase fusion protein gene with a lysotropic tag; the fusion protein comprises a solubilizing label and lysine decarboxylase from thermophilic bacteria, and the solubilizing label and the lysine decarboxylase are connected through a Linker;

wherein the lytic tag is selected from the group consisting of a fluorescent protein, a maltose binding protein, a glutathione transferase, or a combination thereof.

2. The recombinant DNA according to claim 1, wherein the fluorescent protein is selected from the group consisting of a red fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, and an optically highlighted fluorescent protein; preferably at least one of RedStar, tdtomato or mCherry; and/or the presence of a gas in the gas,

the lysine decarboxylase is selected from any one of the following ((r) - (r)): lysine decarboxylase TelDC derived from thermophilic bacteria Thermosynechococcus elongatus, and the amino acid sequence of the lysine decarboxylase TelDC is shown as SEQ ID NO: 1 is shown in the specification; ② lysine decarboxylase TsLDC derived from Tepidanerobacter syntrophicus, the amino acid sequence is shown as SEQ ID NO. 3; ③ lysine decarboxylase GkLDC derived from Geobacillus kaustophilus, the amino acid sequence of which is shown as SEQ ID NO. 5; tetra-lysine decarboxylase TrLDC derived from Thermomicrobium roseum, and the amino acid sequence of the TrLDC is shown as SEQ ID NO. 7;

Preferably, the lysine decarboxylase is identical to SEQ ID NO: 1,3,5 or 7, or at least 80%, at least 85%, at least 90%, at least 95% sequence identity; more preferably, the fusion protein is selected from at least one of fluorescent protein-Linker-TelDC, fluorescent protein-Linker-TsLDC, fluorescent protein-Linker-GkLDC, fluorescent protein-Linker-TrLDC, TeLDC-Linker-fluorescent protein, TsLDC-Linker-fluorescent protein or TsLDC-Linker-fluorescent protein.

3. The recombinant DNA according to claim 1, wherein the Linker comprises a Linker in a helical form or a flexible Linker composed of amino acids with low hydrophobicity and low charge effect, wherein the length of the Linker is at least 10 amino acids;

preferably, the Linker is a flexible Linker, more preferably (GGGGS)₃Or (SG)_5-8。

4. The recombinant DNA of claim 1, wherein the stationary phase specific promoter is selected from any one of pcsiE, pbOLA, posmY, pkatE, P1, P2, P3 or P4, and the nucleotide sequences thereof are shown in SEQ ID NO. 20-27, respectively.

5. The recombinant DNA according to any one of claims 1 to 4, wherein the recombinant DNA comprises at least the following 3 elements: a. a stationary phase specific promoter; b. a red fluorescent protein gene; and c, a lysine decarboxylase gene derived from a thermophilic bacterium; wherein the elements are operably linked in the order a-b-c or a-c-b.

6. A biological material comprising the recombinant DNA according to any one of claims 1 to 5, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineered bacterium.

7. A recombinant plasmid carrying the recombinant DNA according to any one of claims 1 to 5; preferably, the starting plasmid is a pUC or pBR322 plasmid or a derivative thereof, more preferably pUC18, pUC19, pBR322, pACYC, pET, pSC101 and any derivative thereof.

8. A genetically engineered bacterium producing 1, 5-pentanediamine, wherein the genetically engineered bacterium carries the recombinant DNA of any one of claims 1 to 5, the biomaterial of claim 6, or the recombinant plasmid of claim 7;

wherein the starting strain of the genetic engineering bacteria is selected from the strains in Escherichia (Escherichia) and Hafnia (Hafnia); preferably, the starting strain is escherichia coli (e.coli), bacillus subtilis (b.subtilis), streptomyces coelicolor (s.coelicolor), hafnia alvei (h.alvei), corynebacterium glutamicum (c.glutamicum), or a strain or genetically engineered bacterium after mutagenesis or random mutation.

A method for producing 1, 5-pentanediamine, which comprises the step of fermentatively culturing the genetically engineered bacterium of claim 8 to produce 1, 5-pentanediamine.

10. Use of the recombinant DNA according to any one of claims 1 to 5 for the production of 1, 5-pentanediamine, wherein (a) the recombinant DNA according to any one of claims 1 to 5 is constructed into an engineered bacterium having an ability to produce L-lysine, the recombinant bacterium is cultured by fermentation and the accumulation of lysine is carried out, the culture temperature at the initial stage of fermentation is controlled to be 20 to 50 ℃; (b) the temperature is controlled at 50-110 deg.C in the rest fermentation stage, so that lysine decarboxylase is active, and 1, 5-pentanediamine is produced by conversion.