CN112680433B - Method for producing and secreting protein by using halophilic bacteria - Google Patents

Abstract

The invention discloses a method for producing and secreting protein by using halophilic bacteria. The invention firstly discloses a method for producing and secreting protein by halophilic bacteria, which comprises the steps of fermenting recombinant halophilic bacteria to obtain a fermentation product, and separating the fermentation product to obtain protein; the recombinant halophilic bacteria are obtained by introducing the fusion protein into halophilic bacteria. The invention further discloses a DNA molecule, a fusion gene thereof and a fusion protein expressed by the DNA molecule. The method for producing and secreting protein by using halophilic bacteria and the method for producing PHA by using recombinant halophilic bacteria and taking cheap substrate starch as a carbon source have the characteristics of no need of sterilization, simple equipment, continuous production and the like, can save production cost, is easy to enlarge scale, and has wide application prospect.

Description

Method for producing and secreting protein by using halophilic bacteria

Technical Field

The invention belongs to the field of biotechnology. In particular to a method for producing and secreting protein by using halophilic bacteria.

Background

The development of modern industrial biotechnology is still restricted by the problem of high production cost caused by high energy consumption, high fresh water resource consumption, complex sterile operation and other factors. The advantages of halophilic bacteria as the latest industrially produced chassis bacteria are gradually highlighted. Halophilic bacteria are microorganisms which can grow in a wide salt concentration range, have low requirements on nutrition and are easy to adapt to severe environments.

As the chassis bacteria of the expression system, the halophilic bacteria have at least the following advantages: 1) the seawater is utilized for fermentation, so that the waste of fresh water resources is reduced; 2) the method can adapt to high-salt and high-pH environment, and is not easy to contaminate bacteria in the fermentation process; 3) the open continuous fermentation improves the production efficiency. Therefore, the cost of large-scale industrial production can be effectively reduced (Chen GQ et al.Next Generation Industry Biotechnology Based on Current Opinion in Biotechnology 50(2018) 94-100).

For large scale industrial production, a production process involving expression of intracellular products requires cell separation, lysis and multiple purification steps to separate the product from the cells or fermentation broth. This process is cumbersome, complex and costly, and therefore protein secretion based on halophilic bacteria development is economical, simple and efficient for the production and isolation of downstream products: the fermentation production of the product and the separation of the product are simplified; the complexity of biological production is reduced; improve protein folding and purity, etc. Therefore, the function of conferring protein secretion to halophilic bacteria is very competitive for protein synthesis and saving of production cost of biological products.

Disclosure of Invention

The technical problem to be solved by the invention is how to realize the production and secretion of proteins (enzymes) by halophilic bacteria.

In order to solve the technical problems, the invention provides a DNA molecule.

The DNA molecule of the invention is a signal peptide, and the nucleotide sequence of the signal peptide is shown in the 209-298 th site of the sequence 1.

The application of the DNA molecule in promoting protein secretion is also within the protection scope of the invention.

The invention further provides a fusion gene comprising the DNA molecule.

The fusion gene comprises the DNA molecule and a gene of a coding protein positioned at the downstream of the DNA molecule; the DNA molecule has the function of promoting the secretion of the protein.

The fusion gene also comprises a linker sequence positioned between the DNA molecule and the gene of the coding protein, wherein the linker sequence is the first 15 nucleotide molecules of the gene of the coding protein.

In the fusion gene, the protein is amylase, lipase, cellulase, protease, PhaP, PETase, Lactase or xylanase.

The invention also provides a fusion protein obtained by the fusion gene coding.

The fusion protein comprises a signal peptide with an amino acid sequence shown as 1 st-30 th sites of a sequence 6 and a protein positioned at the C terminal of the signal peptide.

The fusion protein also comprises a linker sequence positioned between the signal peptide and the protein, wherein the linker sequence is the first 5 amino acid residues of the protein.

In the above fusion protein, the protein is amylase, lipase, cellulase, protease, PhaP (polyhydroxyalkanoate (PHA) particle surface binding protein, PETase (Polyethylene terephthalate) degrading enzyme), lacccase (Laccase), or xylanase.

In the above fusion protein, the fusion protein is P1) or P2) or P3):

p1) the fusion protein is recombinant amylase, recombinant lipase, recombinant cellulase, recombinant protease, recombinant PhaP, recombinant PETase, recombinant Lactase or recombinant xylanase;

p2) the protein of the recombinant amylase is amylase, the protein of the recombinant lipase is lipase, the protein of the recombinant cellulase is cellulase, the protein of the recombinant protease is protease, the protein of the recombinant PhaP is PhaP, the protein of the recombinant PETase is PETase, the protein of the recombinant laccrase is laccrase, and the protein of the recombinant xylanase is xylanase;

p3) the recombinant amylase is P11 or P12, the P11 is a protein of which the amino acid sequence is a sequence 6, and the P12 is a protein obtained by modifying the P11 as follows: is a protein obtained by deleting the 31 st to 35 th amino acid residues of the sequence 6 and keeping other amino acid residues of the sequence 6 unchanged;

the recombinant lipase is P21 or P22, the P21 is a protein with an amino acid sequence of sequence 7, and the P22 is a protein obtained by modifying the P21 as follows: is a protein obtained by deleting the 31 st to 35 th amino acid residues of the sequence 7 and keeping other amino acid residues of the sequence 7 unchanged;

the recombinant protease is P31 or P32, the P31 is a protein with an amino acid sequence of sequence 8, and the P32 is a protein obtained by modifying the P31 as follows: is a protein obtained by deleting the 31 st to 35 th amino acid residues of the sequence 8 and keeping other amino acid residues of the sequence 8 unchanged;

the recombinant cellulase is P41 or P42, the P41 is a protein of which the amino acid sequence is a sequence 9, and the P42 is a protein obtained by modifying the P41 as follows: is a protein obtained by deleting the 31 st to 35 th amino acid residues of the sequence 9 and keeping other amino acid residues of the sequence 9 unchanged;

the recombinant PhaP is P51 or P52, the P51 is a protein of which the amino acid sequence is sequence 16, and the P52 is a protein obtained by modifying the P51 as follows: is a protein obtained by deleting amino acid residues 31 to 35 of the sequence 16 and keeping other amino acid residues of the sequence 16 unchanged.

In the above fusion gene, the fusion gene is G1) or G2):

G1) the fusion gene is a gene for coding the recombinant amylase, the recombinant lipase, the recombinant cellulase, the recombinant protease, the recombinant PhaP, the recombinant PETase, the recombinant Lactase or the recombinant xylanase, and is respectively named as a recombinant amylase gene, a recombinant lipase gene, a recombinant cellulase gene, a recombinant protease gene, a recombinant PhaP gene, a recombinant PETase gene, a recombinant Lactase gene or a recombinant xylanase gene;

G2) the recombinant amylase gene is G11 or G12, the G11 is a DNA molecule with a coding sequence of sequence 2, and the G12 is a DNA molecule obtained by modifying the G11 as follows: a DNA molecule obtained by deleting the 91 st to 105 th nucleotides of the sequence 2 and keeping other nucleotides of the sequence 2 unchanged;

the recombinant lipase gene is G21 or G22, G21 is a DNA molecule with a coding sequence of sequence 3, and G22 is a DNA molecule obtained by modifying G21 as follows: a DNA molecule obtained by deleting the 91 st to 105 th nucleotides of the sequence 3 and keeping other nucleotides of the sequence 3 unchanged;

the recombinant protease gene is G31 or G32, the G31 is a DNA molecule with a coding sequence of sequence 4, and the G32 is a DNA molecule obtained by modifying the G31 as follows: a DNA molecule obtained by deleting the 91 st to 105 th nucleotides of the sequence 4 and keeping other nucleotides of the sequence 4 unchanged;

the recombinant cellulase gene is G41 or G42, the G41 is a DNA molecule with a coding sequence of sequence 5, and the G42 is a DNA molecule obtained by modifying the G41 as follows: DNA molecules obtained by deleting the 91 th to 105 th nucleotides of the sequence 5 and keeping other nucleotides of the sequence 5 unchanged;

the recombinant PhaP gene is G51 or G52, the G51 is a DNA molecule with a coding sequence of sequence 15, and the G52 is a DNA molecule obtained by modifying the G51 as follows: and (b) the DNA molecule obtained by deleting the 91 st to 105 th nucleotides of the sequence 15 and keeping other nucleotides of the sequence 15 unchanged.

An expression cassette, a recombinant expression vector or a recombinant bacterium containing the above fusion gene is also within the scope of the present invention.

The recombinant expression vector is obtained by inserting the fusion gene into an expression vector to express the fusion protein. In a specific embodiment of the invention, the expression vector used is pSEVA 321; the recombinant expression vector containing the recombinant amylase gene is pSEVA321-Pporin-SP1-linker-amyL or pSEVA321-Pporin-SP1-amyL or pSEVA321-mmp1-SP1-amyL _bl-opt The recombinant expression vector containing the recombinant lipase gene is pSEVA 321-Pprotin-SP 1-linker-lipase or pSEVA 321-Pprotin-SP 1-lipase, the recombinant expression vector containing the recombinant protease gene is pSEVA 321-Pprotin-SP 1-linker-protease or pSEVA 321-Pprotin-SP 1-protease, the recombinant expression vector containing the recombinant cellulase gene is pSEVA 321-Pprotin-SP 1-linker-cellulose or pSEVA 321-Pprotin-SP 1-cellulose, and the recombinant expression vector containing the recombinant PhaP gene is pSEVA321-mmp1-SP1-linker-phaP or pSEVA321-mmp1-SP 1-cellulose.

In the above-mentioned related biological materials, the recipient bacterium of the recombinant bacterium is halophilic bacterium, specifically Halomonas, more specifically Halomonas bluephagesis TD01(Tan, D.et al (2011) microorganism and linkage of Halomonas by Halomonas TD01. BioResourcer Technol 102(17), 8130-6) or Halomonas bluephagesis TD (Zhao, H.et al (2017) Novel T7-1ike expression system used for Halomonas. MetEng 39, 128-140). The preparation method of the recombinant bacterium comprises the steps of inserting the fusion gene into an expression vector to obtain a recombinant expression vector for expressing the fusion protein, and joining the recombinant expression vector into Halomonas bluephagene TD01 or Halomonas bluephagene TD to obtain the recombinant Halomonas, wherein the recombinant Halomonas can express the corresponding fusion protein.

The invention further provides a method for producing and secreting protein by the halophilic bacteria, which is to ferment the recombinant halophilic bacteria to obtain a fermentation product and separate the protein from the fermentation product.

In the method for producing and secreting proteins by using halophilic bacteria, the fermentation product can be fermentation liquor.

Specifically, according to the difference in protein, methods a1, a2, A3, a4, or a5 are classified:

the method A1 comprises fermenting recombinant halophilic bacteria A1 to obtain a fermentation product, and separating amylase from the fermentation product; the recombinant halophilic bacteria A1 is a recombinant bacterium obtained by introducing the recombinant amylase gene into halophilic bacteria;

the method A2 comprises fermenting recombinant halophilic bacteria A2 to obtain a fermentation product, and separating lipase from the fermentation product; the recombinant halophilic bacteria A2 is a recombinant bacterium obtained by introducing the recombinant lipase gene into halophilic bacteria;

the method A3 comprises fermenting recombinant halophilic bacteria A3 to obtain a fermentation product, and separating protease from the fermentation product; the recombinant halophilic bacteria A3 is a recombinant bacterium obtained by introducing the recombinant protease gene into halophilic bacteria;

the method A4 comprises fermenting recombinant halophilic bacteria A4 to obtain a fermentation product, and separating the fermentation product to obtain cellulose; the recombinant halophilic bacteria A4 are obtained by introducing the recombinant cellulase gene into halophilic bacteria;

the method A5 comprises fermenting recombinant halophilic bacteria A5 to obtain a fermentation product, and separating PhaP from the fermentation product; the recombinant halophilic bacterium A5 is a recombinant bacterium obtained by introducing the recombinant PhaP gene into a halophilic bacterium.

In the above method, the halophilic bacteria is halomonas; specifically, the Halomonas is Halomonas bluephaseensis TD01 or Halomonas bluephaseensis TD.

The invention further provides a method for producing Polyhydroxyalkanoate (PHA) by using the recombinant halophilic bacteria and taking starch as a cheap carbon source as a carbon source.

The method comprises the steps of producing PHA by using the recombinant halophilic bacteria by taking starch as a substrate; the recombinant halophilic bacteria are obtained by introducing the nucleic acid molecule for coding the recombinant amylase into halophilic bacteria.

In the above method, the halophilic bacteria is halomonas; specifically, the Halomonas is Halomonas bluegene TD01, Halomonas bluegene TD delta gabD2-D2(Ye, J.et al. (2018) Engineering of Halomonas bluegene for low cost development of poly (3-hydrobutyl-co-4-hydrobutyl) front glucose Eng 47, 143. 152.) or Halomonas bluegene TD AB (Chen, Y.et al. (2019) chromatography Engineering of the TCA cell bluegene for low cost development of poly (PHBV. v. 32. of polyol) solution for high cost production of sodium chloride of poly (3-8282).

In the above process, the PHA includes, but is not limited to, Polyhydroxybutyrate (PHB), 3-hydroxybutyrate or 4-hydroxybutyrate copolyester (P34HB) or copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

In a specific embodiment of the invention, the halophilic bacteria is Halomonas bluephagenesis TD or Halomonas bluephagenesis TD01, and the PHA is Polyhydroxybutyrate (PHB); the halophilic bacteria are Halomonas bluephagene TD delta gabD2-D2, and the PHA is 3-hydroxybutyric acid or 4-hydroxybutyric acid copolyester (P34 HB); the halophilic bacteria are Halomonas bluephagene TD08AB, and the PHA is 3-hydroxybutyric acid and 3-hydroxyvaleric acid copolyester (PHBV).

The method for producing and secreting protein by using halophilic bacteria and the method for producing PHA by using recombinant halophilic bacteria and taking cheap substrate starch as a carbon source have the characteristics of no need of sterilization, simple equipment, continuous production and the like, can save production cost, is easy to enlarge scale, and has wide application prospect.

Drawings

FIG. 1 is a schematic diagram showing the structure of a GFP expression plasmid carrying a signal peptide SP1 derived from an amylase derived from Bacillus licheniformis, wherein SP1 can be replaced with another type of signal peptide.

FIG. 2 shows the effect of secretion of green fluorescent protein with and without linker; wherein S is _s Represents fluorescence in the supernatant, S _t Indicates total fluorescence, -linker indicates no linker addition and + linker indicates linker addition.

FIG. 3 is a comparison of the activities of different amylases; wherein TD01 is Halomonas TD01 strain, T58: TD-pSEVA321, T47: TD-pSEVA321-mmp1-amyL _TD (derived from TD strain), T88: TD-pSEVA321-mmp1-SP 1' -amyL _b1 (original sequence from Bacillus licheniformis), T89: TD-pSEVA321-mmpl-SP1-amyL _bl-opt (codon-optimized sequence from Bacillus licheniformis).

FIG. 4 shows the secretion of the linker-carrying PhaP protein by Western Blot.

FIG. 5 shows the Western Blot to detect secretion of PhaP protein without linker.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The access to the reagents, the media formulation and the detailed methods used in the following examples of the invention are as follows:

1. and (3) reagent sources:

enzymatic reagents were purchased from ThermoFisher and New England Biolabs (NEB), plasmid extraction kits were purchased from Qiangen (Shanghai, China), synthesis of primers and DNA fragments was accomplished by Scophyta organisms, and synthesis of gene fragments was accomplished by Wuxi Qinglan Biotech Ltd.

2. The formula of the culture medium is as follows:

1) escherichia coli culture medium

LB culture medium: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042), 10g/L NaCl, and the balance water. Adjusting pH to 7.0-7.2, and sterilizing with high pressure steam.

2) Halophilic bacteria culture medium

60LB medium: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042), 60g/L NaCl, and balance water. Adjusting pH to 7.0-7.2, and sterilizing with high pressure steam.

60MMG medium: 60g/L NaCl, 30g/L glucose, 1g/L yeast extract, 2g/L NH ₄ Cl，0.2g/L MgSO ₄ ，9.65g/L Na ₂ HPO ₄ ·12H ₂ O，1.5g/L KH ₂ PO ₄ 10ml/L of trace element solution I and 1ml/L of trace element solution II. Wherein the microelement solution I comprises the following components: 5g/L ferric ammonium citrate, 2g/L CaCl ₂ Formulated with 1M HCl. The composition of the trace element solution II is as follows: 100mg/L ZnSO ₄ ·7H ₂ O，30mg/L MnCl ₂ ·4H ₂ O，300mg/L H ₃ BO ₃ ，200mg/L CoCl ₂ ·6H ₂ O，10mg/L CuSO ₄ ·5H ₂ O，20mg/L NiCl ₂ ·6H ₂ O，30mg/L NaMoO ₄ ·2H ₂ O, formulated with 1M HCl. The final pH of the medium was adjusted to 8-9 with 5M NaOH solution. The above reagents were purchased from the national pharmaceutical group chemical agents corporation.

In the actual culturing process, antibiotics at a concentration of 100. mu.g/mL ampicillin or 25. mu.g/mL chloramphenicol can be added to the above medium to maintain the stability of the plasmid.

The above medium was prepared with deionized water unless otherwise specified.

3.The test method comprises the following steps:

3.1 method of conjugation transformation:

conjugal transformation is an effective method for transferring plasmids into Halomonas TD01(Halomonas bluegene TD01, which is currently stored in the culture collection of institute of microbiology of Chinese academy of sciences, with the collection number of CGMCC 4353; patent number CN 102816729B). In the present invention, the plasmid was transformed into the Halomonas TD01 strain by conjugative transformation from E.coli S17-1 (abbreviated as "E.coli S17-1", from the literature "Simon R, et al. A broad host range ligation organization system for in vivo genetic engineering: transfer microbial mutagenesis in Gram negative bacteria. Bio-Technology 1: 784. sup. 791, 1983").

1)60LB liquid medium for culturing recipient bacterium Halomonas TD01, adding corresponding antibiotic into LB liquid culture medium to culture donor bacterium E.coli S17-1 with plasmid, and culturing to logarithmic phase, OD ₆₀₀ Between 0.6 and 0.8.

2) Each 1mL of the cells was centrifuged at 4 ℃ for 10min at 5,000g in a 1.5mL EP tube, and the cells were collected and washed with LB medium (or 60LB medium).

3) The donor bacteria and the recipient bacteria were mixed at a ratio of 1: 1, and the mixed bacteria were dropped into the center of a 20LB plate, which was kept upright, and cultured at 37 ℃ for 8 hours.

4) To a 1.5mL EP tube was added 50uL of 60LB, and a small amount of lawn scraped from the EP tube was resuspended and spread on a 60LB plate.

5) The plate was inverted in a 37 ℃ incubator until colonies grew.

3.2 measurement of cell Dry weight and PHA content

Collecting thalli after 48 hours of shaking experiments, and detecting the dry weight of cells and the PHA content by the following method:

freeze drying of the thallus: 30ml of bacterial liquid is measured by a measuring cylinder and put into a 50ml centrifuge tube, and the bacteria are collected by centrifugation at 4500rpm for 15 min. The cells were washed twice with a re-suspension of deionized water, centrifuged at 4500rpm for 15min and the supernatant discarded. Freezing the thallus at-80 deg.C for 1 hr, and vacuum freeze drying for more than 12 hr to completely remove water. The weights of the centrifuge tubes before and after sampling were weighed, and the difference was the dry cell weight CDW.

Sample preparation: 30-40 mg of freeze-dried thallus is weighed, accurately weighed and placed in an esterification tube, and 2ml of esterification solution (485 ml of anhydrous methanol is taken, 1g/L of benzoic acid is added, and 15ml of concentrated sulfuric acid is slowly added to prepare about 500ml of esterification solution) and 2ml of chloroform are added. Approximately 20 to 30mg PHA samples were weighed out and treated in the same manner as standard samples. The esterification tube is covered and sealed, and then the reaction is carried out for 4 hours at 100 ℃. After the reaction is finished, cooling the esterification pipe to room temperature, adding 1ml of deionized water, carrying out vortex oscillation until the materials are fully mixed, and standing for layering. After complete separation of the aqueous and organic phases, the lower organic phase was removed for Gas Chromatography (GC) analysis.

Analysis by GC of PHA composition and content: a gas chromatograph model GC-2014 from shimadzu was used. The chromatograph is configured to: an HP-5 type capillary chromatographic column, a hydrogen flame ionization detector FID and an SPL shunt sample inlet; high-purity nitrogen is used as carrier gas, hydrogen is fuel gas, and air is combustion-supporting gas; an AOC-20S autosampler was used, acetone being the wash liquid. The settings of the GC analysis program were: the sample inlet temperature is 240 ℃, the detector temperature is 250 ℃, the initial column temperature is 80 ℃, and the temperature is maintained for 1.5 minutes; ramping up to 140 degrees at a rate of 30 degrees/minute and maintaining for 0 minute; ramping up to 240 degrees at a rate of 40 degrees/minute and maintaining for 2 minutes; the total time was 8 minutes. And quantitatively calculating the PHA composition and content according to peak areas by adopting an internal standard normalization method according to the GC result.

Example 1 screening of Signal peptides derived from different species to obtain Signal peptides with high secretion Capacity

Construction of secretion engineering bacteria

1. Collection of Signal peptide

On Signal peptide database (http:// www.signalpeptide.de /), Bacillus licheniformis, Bacillus subtilis, etc. species were imported to select the "confirm" Signal peptide, giving the following sequence:

SP 1: alpha-amylase: MKQQKRLYARLLTLLFALIFLLPHSAAAAA (from Bacillus licheniformis) (SEQ ID NO: 6, positions 1-30)

SP 2: beta-lactamase: MKLWFSTLKLKKAAAVLLFSCVALAG (from Bacillus licheniformis)

SP 3: chitosanase: MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA (from Bacillus subtilis)

SP 4: beta-lactamase: MKLKTKASIKFGICVGLLCLSITGFTPFFNSTHAEA (from Bacillus subtilis)

SP 5: arabidopsis arabinofuran hydrosole: MRKKCSVCLWILVLLLSCLSGKSAY A (from Bacillus subtilis)

SP6 Endoglucanase: MKRSISIFITCLLITLLTMGGMIASPASA (from Bacillus subtilis)

SP 7: OmpA: MKKTAIAIAVALAGFATVAQA (from E

SP 8: porin: MKKTLLATAIIGALGASAAAQA (from Halomonas bluephagene TD01)

2. Construction of expression vector pSEVA321-Pporin-SP (1-8) -sfGFP

1) Obtaining a desired promoter Pporin

Extracting genomic DNA of Halomonas TD01 as a template, and performing PCR amplification by pfu enzyme with G-Pporin-1TF and G-Pporin-2TR as primers to obtain a PCR product of 150bp shown as 33 th to 182 th sites of a sequence 1, namely the target promoter Pporin.

Wherein, the primer is:

G-Pporin-1TF：5’-atgcctccacaccgctcgtc-3’

G-Pporin-2TR：5’-actcttaaacaaaattatttgtag-3’

and (3) PCR reaction conditions:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 30 cycles; extension was carried out at 72 ℃ for 10 minutes.

PCR amplification System (50. mu.L System):

when the PCR amplification system is prepared, DNA polymerase is added finally.

2) Obtaining of Signal peptide (SP 1-8)

Nucleotide sequence of the signal peptide was synthesized by gene synthesis:

SP1 gene (codon optimized gene):

atgaaacagcagaaacgcctgtatgcccgtcttctgactctgctgttcgccctgattttcctgcttccgcatagcgctgccgctgccgct (sequence 1 at position 209-298)

SP2 gene: atgaaattatggttcagtactttaaaactgaaaaaggctgcagcagtgttgcttttctcttgcgtcgcgcttgcagga

SP3 gene:

cgcaaaaaccgtttcgctcatcatcagggtaaaaaacatggtgaaaacaagtaatgagatcgctgcttttttccaaaaatctgctttttgcatactgattttcat

SP4 gene:

atgaagttgaaaactaaagcgtcaataaaattcggaatatgtgttgggcttttatgtttaagcattactggtttcacaccttttttcaactcaacacatgccgaagca

SP5 gene: atgaggaaaaagtgtagcgtatgtttatggattctagttttattattgagctgcttatctgggaagtctgcgtatgct

SP6 gene: atgaaacggtcaatctctatttttattacgtgtttattgattacgttattgacaatgggcggcatgatggcttcgccggcatcagca

SP7 gene: atgaaaaagacagctatcgcgattgcagtggcactggctggtttcgctaccgtagcgcaggcc

SP8 gene: atgtttaaaaagacgacacttgctctggctgttagtggtttgttgggtgcttcggcagcccaagcg

The lengths are 90, 78, 105, 108, 78, 87, 63, 66bp, respectively. Wherein the nucleotide sequence shown in the 209-298 of the sequence 1 of SP1 and the nucleotide sequence shown in the 209-298 of the sequence 1 of SP1 (namely the nucleotide sequence shown in the 209-298 of the sequence 1) can be replaced by the nucleotide sequence from SP2 to SP8, and the positions corresponding to the sequences are respectively as follows: SP2 is at position 209 and 286, SP3 is at position 209 and 314, SP4 is at position 209 and 317, SP5 is at position 209 and 286, SP6 is at position 209 and 296, SP7 is at position 209 and 271, and SP8 is at position 209 and 274.

3) Obtaining of target Gene sfGFP

The gene of IGEM No. BBa _ I746916 is used as a template, G-gfp-3 TF and G-gfp-4 TR are used as primers, and PCR amplification is carried out by pfu enzyme to obtain a 717bp PCR product shown in the 299-th 1015 site of the sequence 1, namely the target gene sfGFP, the position of the target gene sfGFP in the sequence 1 is exemplified by the plasmid containing SP1 in figure 1, and the position of the plasmid containing other signal peptides is slightly changed according to the size and the position of the nucleotide length of the signal peptide.

Wherein, the primer is: G-sfGFP-3 TF: 5'-atgcgtaaaggcgaagagct-3'

G-sfGFP-4 TR：5’-tcatttgtacagttcatccataccatgc-3’

The PCR reaction conditions and the PCR amplification system are the same as the step 1).

4) Amplification of vector backbone fragments

A pfu enzyme-based PCR amplification using a plasmid pSEVA321(Silva-Rocha, R., de Lorenzo, V., 2013.The Standard European Vector Architecture (SEVA): a coherent plant for The analysis and purification of complex prokarstic phenols. nucleic Acids Res.41, 666. cndot. 675.) as a template and 321-F and 321-R as primers gave a total of 3580bp PCR products shown in sequence 1, 1016. cndot. 4563 and 1-32, i.e., Vector backbone fragments whose positions in sequence 1 are exemplified by SP1 in FIG. 1, and whose positions in plasmids containing other signal peptides vary slightly depending on The size and position of The nucleotide length of The signal peptide.

Wherein, the primer is: 321-F: 5'-gtcgtgactgggaaaaccctggcg-3'

321-R：5’-tcctgtgtgaaattgttatccgct-3’

The PCR reaction conditions and the PCR amplification system are the same as the step 1).

Connecting the PCR products of 1), 2), 3) and 4) by a Gibson assembly homologous recombination method to obtain a connecting product, transforming the connecting product into E.coli S17-1 to obtain a transformant, sequencing, selecting a correctly sequenced product, storing the correctly sequenced product, culturing the correctly sequenced product by using an LB culture medium overnight, and then upgrading the plasmid.

Wherein, the reaction conditions of the Gibson assembly homologous recombination method are as follows:

the PCR products were mixed, made up to 10. mu.l with water and then treated at 50 ℃ for 1 hour.

Gibson assembly homologous recombination system (10uL system):

performing PCR amplification by pfu enzyme using verification primers F24 and R24 to obtain a glue running strip, selecting positive transformants with the size of 1031bp, extracting plasmids of the positive transformants, and sequencing to obtain plasmids containing PCR products obtained in the steps 1), 2), 3) and 4), wherein eight plasmids, namely pSEVA321-Pporin-SP (1-8) -sfGFP, are obtained in the step 2) and are respectively recorded as pSEVA321-Pporin-SPl-sfGFP, pSEVA321-Pporin-SP2-sfGFP, pSEVA321-Pporin-SP3-sfGFP, pSEVA321-Pporin-SP4-sfGFP, pSEVA321-Pporin-SP5-sfGFP, pSEVA321-Pporin-SP6-sfGFP, pSEVA 321-SP 3535 7-sfGFP, and pSEVA-SP-8-sfGFP; the nucleotide sequence of the pSEVA321-Pporin-SP1-sfGFP is shown as a sequence 1, the structural schematic diagram of the plasmid is shown as a figure 1, and the nucleotide sequence of other plasmids replaces the sequence of the signal peptide SP1 with other corresponding signal peptides;

wherein, the verification primer is: f24: 5'-agcggataacaatttcacacagga-3'

R24：5’-cgccagggttttcccagtcacgac-3’

3. Construction of recombinant bacterium s17-1-pSEVA321-PPorin-SP (1-8) -sfGFP and Halomonas TD01-pSEVA321-PPorin-SP (1-8) -sfGFP

The expression vector pSEVA321-Pporin-SP (1-8) -sfGFP prepared in the step 2 is transferred into E.coli S17-1 by an electrotransfer method to obtain a recombinant strain S17-1-pSEVA321-Pporin-SP (1-8) -sfGFP. The halomonas TD01 is used as a recipient strain, E.coli S17-1-pSEVA321-Pporin-SP (1-8) -sfGFP is used as a donor strain to carry out conjugation transformation to obtain 8 halomonas TD01 containing different signal peptide plasmids, which are respectively named as TD01-pSEVA321-Pporin-SP1-sfGFP, TD01-pSEVA321-Pporin-SP2-sfGFP, TD01-pSEVA321-Pporin-SP3-sfiGFP, TD01-pSEVA321-Pporin-SP4-sfGFP, TD01-pSEVA321-Pporin-SP5-sfGFP, TD01-pSEVA321-Pporin-SP6-sfGFP, TD01-pSEVA 321-SP 7-sfGFP, TD 01-pS321-Pporin-SP 53-sP-SP 8, and the monoclonal LB plate with resistance on a single-strain screen.

4. Screening signal peptide with strong secretion capacity by green fluorescent protein

The 8 halomonas TD01 strains (i.e., TD01-pSEVA 321-Pporrin-SP 1-sfGFP, TD01-pSEVA 321-Pporrin-SP 2-sfGFP, TD01-pSEVA 321-Pporrin-SP 3-sfGFP, TD01-pSEVA 321-Pporrin-SP 4-sfGFP, TD01-pSEVA 321-Pporrin-SP 5-sfGFP, TD01-pSEVA 321-Pporrin-SP 6-sfGFP, TD 01-EVA 321-Pporrin-SP 7-sfGFP, TD01-pSEVA 321-Pporrin-SP 8-sfGFP) which are respectively joined in step 3, are respectively cultured in a deep well plate, after 12h, transferred to 1mL of new culture medium in an amount of 1h, cultured for 12h, the intensity of supernatant is measured by a photometric meter, the supernatant is separated by 600 rpm, the intensity of supernatant is measured, the supernatant is increased, the supernatant is 4500rpm is measured, the secretion capacity is stronger.

As shown in Table 1, a signal peptide SP1 with strong secretion ability and large total protein expression amount was selected as one of the elements for the subsequent construction of a secretion platform of Halomonas.

TABLE 1 sfGFP secretion of different signal peptides

Example 2 optimization of protein secretion System protein secretion Effect of Halomonas with and without linker was compared by Green fluorescence

On the basis of example 1, the first 15 nucleotide sequences of the target gene sfGFP (i.e., the nucleotide sequence shown at position 299-313 of the sequence 1 and encoding the first 5 amino acid residues of GFP) were inserted between the SP1 of the pSEVA 321-Pporrin-SP 1-sfGFP and the target gene GFP of the plasmid shown in FIG. 1, and the plasmid was designated as pSEVA 321-Pporrin-SP 1-linker-sfGFP (linker-added plasmid), while pSEVA 321-Pporrin-SP 1-sfGFP was a non-linker-added plasmid.

The pSEVA321-Pporin-SP1-linker-sfGFP (plasmid with linker) and the pSEVA321-Pporin-SP1-sfGFP (plasmid without linker) are transferred into E.coli S17-1 by an electrotransfer method to obtain recombinant bacteria S17-1-pSEVA321-Pporin-SP1-linker-sfGFP and S17-1-pSEVA321-Pporin-SP1-sfGFP respectively. Halomonas TD01 was used as a recipient strain, recombinant strain s17-1 was used as a donor strain, and subjected to conjugation transformation to obtain 2 species of Halomonas TD01 (i.e., recombinant strains TD01-pSEVA321-Pporin-SP1-sfGFP and TD01-pSEVA321-Pporin-SP1-linker-sfGFP), and single clones were screened on Cm-resistant 60LB (LB containing 60g/L NaCl) plates.

Recombinant bacteria TD01-pSEVA321-Pporin-SP1-sfGFP and TD01-pSEVA321-Pporin-SP1-linker-sfGFP are respectively cultured in a deep-well plate, after 12 hours of culture, the recombinant bacteria are transferred into a new 1mL culture medium according to the transfer amount of 1 percent, after 12 hours of culture, OD600 is measured by a spectrophotometer, the centrifugation is carried out for 20 minutes at 4500rpm, supernate and sediment are separated, the sfGFP intensity of the supernate is measured by a microplate reader, and the higher the sfGFP intensity is, the stronger the secretion capacity is. The detection result is shown in fig. 2, and it can be seen from the figure that sfGFP in total or in supernatant is significantly improved after the linker is added, which indicates that the insertion of a sequence (i.e., linker) identical to the first 15 nucleotides of the target gene between the signal peptide and the target gene can significantly increase the protein secretion capacity.

Example 3 secretion of proteins by Halomonas secretion platform

1. Secretion of amylase, lipase, protease and cellulase is realized through a halomonas secretion platform

1) Obtaining of Amylase encoding genes amyL and linker-amyL of target genes

The method comprises the steps of searching an amylase amyL gene of Bacillus licheniformis on NCBI, using amyL-F1 and amyL-R as primers after codon optimization to perform PCR amplification by pfu enzyme to obtain a PCR product shown as 106 th and 1554 th positions of a sequence 2, namely the amylase coding gene amyL of a target gene, and using amyL-F2 and amyL-R as primers to perform PCR amplification by pfu enzyme to obtain a PCR product shown as 91 th to 1554 th positions of the sequence 2, namely linker-amyL of the target gene.

Wherein, the primer is: amyL-F1: 5'-aatttaaatggcaccctgatgcagtacttc-3'

amyL-F2：5’-aatttaaatggcaccaatttaaatggcaccctgatgcagtacttc-3’

amyL-R：5’-ttagcgctgcacgtaaatgc-3’

2) Obtaining target gene lipase encoding gene lipase and linker-lipase

Synthesizing a lipase gene by taking the lipase gene of Bacillus licheniformis as a template, and carrying out PCR amplification by taking lip-F1 and lip-R as primers and pfu enzyme to obtain a PCR product shown in 106 th-1545 th position of a sequence 3, namely the lipase of a target gene lipase coding gene. And carrying out PCR amplification by using lip-F2 and lip-R as primers and pfu enzyme to obtain a PCR product shown as 91-1554 th site of the sequence 3, namely the target gene linker-lip.

Wherein, the primer is: lip-F1: 5'-ttgagtaagaagattaagaagtcc-3'

lip-F2：5’-ttgagtaagaagattttgagtaagaagattaagaagtcc-3’

lip-R：5’-ctaaagtttaagccaatttgcca-3’

3) Obtaining target gene protease coding gene protease and linker-protease

Synthesizing a protease gene by taking the protease gene of Bacillus licheniformis as a template, and carrying out PCR amplification by taking pro-F1 and pro-R as primers and pfu enzyme to obtain a PCR product shown as 106 th-651 th position of a sequence 4, namely a target gene protease coding gene protease. Using pro-F2 and pro-R as primers, and carrying out PCR amplification by pfu enzyme to obtain a PCR product shown as 91-651 th site of the sequence 4, namely the target gene linker-protease.

Wherein, the primer is: pro-F1: 5'-atgtcatcttttcacgcaacgacgat-3'

pro-F2：5’-atgtcatcttttcatgtcatcttttcacgcaacgacgat-3’

pro-R：5’-ctattcaagttcttctacaatgat-3’

4) Obtaining target gene cellulase coding genes cellulose and linker-cellulose

After codon optimization is carried out on the cellulose gene of Bacillus licheniformis, the codon is taken as a template, cellulose-F and cellulose-R are taken as primers, pfu enzyme is used for PCR amplification, and a PCR product shown as 106 th and 1104 th bits of a sequence 5, namely the target gene cellulose coding gene cellulose, is obtained. Using cellula-F2 and cellula-R as primers, and using pfu enzyme to perform PCR amplification to obtain a PCR product shown as 91 th to 1104 th sites of the sequence 5, namely the target gene linker-cellula.

Wherein, the primer is: cell-F1: 5'-tttagtgccgcgaatgttttctcttac-3'

cellulase-F2：5’-tttagtgccgcgaattttagtgccgcgaatgttttctcttac-3’

cellulase-R：5’-atgaaacggtcaatttctg-3’

5) Amplification of vector backbones

PCR amplification was performed with pfu enzyme using pSEVA321-Pporin-SP1-sfGFP as a template and 321-S-F and 321-S-R as primers to obtain a 3846bp PCR product, i.e., a vector backbone, as shown in SEQ ID NO. 10.

Wherein, the primer is: 321-S-F: 5'-gtcgtgactgggaaaaccctggcg-3'

321-S-R：5’-agcggcagcggcagcgct-3’

And (3) respectively connecting the PCR products obtained in the steps 1), 2), 3) and 4) with the PCR product obtained in the step 5) by a Gibson assembly homologous recombination method to obtain a connecting product, and transforming the connecting product into E.coli S17-1 to obtain a transformant.

Performing PCR amplification with pfu enzyme using verification primers F24 and R24 to obtain gel-running bands, selecting positive transformants with bands of 2077bp, 2102bp, 1754bp, 1769bp, 860bp, 875bp, 1313bp and 1328bp, extracting plasmids of the positive transformants, sequencing to obtain plasmids containing PCR products obtained in steps 1), 2), 3) and 4), wherein the plasmids are respectively marked as pSEVA321-Pporin-SP1-amyL, pSEVA321-Pporin-SP1-linker-amyL, pSEVA321-Pporin-SP 1-lipase, pSEVA321-Pporin-SP1-linker-lipase, pSEVA321-Pporin-SP1-linker, pSEVA 321-SP 1-linker-protease, pSEVA321-Pporin-SP 1-linker-SP 1-protease, and pSEVA 321-SP-1-linker-SP 1-SP-SP-protease, and the map thereof can be referred to the figure, except that GFP was replaced with the gene for the enzyme corresponding to the above-mentioned sequence.

Wherein, the verification primer is: f24: 5'-agcggataacaatttcacacagga-3'

R24：5’-cgccagggttttcccagtcacgac-3’

7) Construction of recombinant bacterium s17-1 and recombinant halomonas TD01

Transferring the plasmids pSEVA321-Pporin-SP1-amyL, pSEVA321-Pporin-SP1-linker-amyL, pSEVA321-Pporin-SP 1-lipase, pSEVA321-Pporin-SP1-linker-lipase, pSEVA321-Pporin-SP1-protease, pSEVA321-Pporin-SP1-linker-protease, pSEVA321-Pporin-SP 1-cellulose, and pSEVA321-Pporin-SP 1-linker-cellulose into E.coli S17-1 by an electrotransformation method respectively to obtain 8 recombinant bacteria S17-1. Halomonas TD01 is taken as a recipient bacterium, the recombinant bacterium S17-1 is respectively taken as a donor bacterium to carry out conjugation transformation to obtain Halomonas TD01 (namely recombinant bacterium TD01-pSEVA321-Pporin-SP1-amyL, TD01-pSEVA321-Pporin-SP 1-line-amyL, TD01-pSEVA321-Pporin-SP 8-lipase, TD01-pSEVA321-Pporin-SP1-linker-lipase, TD01-pSEVA321-Pporin-SP 6-protease, pSTD 01-Pporin-SP 27-SP 1-SP 1-linker-lipase, TD 01-pSEVA-Pporin-SP 6-protease, pSTD 01-SP 01-Pporin-SP 27-SP 3884-SP, TD-SP 1 and TD-SP 1) which contain genes coding amylase amyL, amylase, lipase-amyL, lipase-amyL, lipase-SP 1-SP 1-SP 9-SP 23, single colonies were screened on Cm-resistant 60LB (LB containing 60 g/LNaCl) plates.

8) The secretion of amylase, lipase, cellulase and protease is verified by enzyme activity experiments

The aforementioned halomonas TD01 strains (i.e., recombinant strains TD01-pSEVA 321-Pporrin-SP 1-amyL, TD01-pSEVA 321-Pporrin-SP 1-linker-amyL, TD01-pSEVA321-Pproin-SP1-lipase, TD01-pSEVA321-Pproin-SP1-linker-lipase, TD01-pSEVA 321-Pporrin-SP 1-protease, TD01-pSEVA 321-Pporrin-SP 1-linker-protease, TD 01-EVA 321-Pprorin-SP 1-cellulose, TD01-pSEVA 321-Pprorin-SP 1-linker-lipase) respectively, which are respectively joined with plasmids containing amylase, lipase, protease and cellulase genes, are cultured in 20mL, and then transferred to a new medium with a transfer speed of 1% after 12h culture, the transfer speed is measured by 20 rpm, 20mL of 20min, and then transferred to a new medium with a transfer speed of 20 rpm, the supernatant and the pellet were separated. The supernatant separated as above was subjected to the following enzyme activity test. The method comprises the following specific steps:

a. amylase Activity assay

The amylase activity test is based on the dinitrosalicylic acid method and comprises a two-part solution, a reaction solution and a stop solution. Wherein the reaction solution is prepared by dissolving 0.5% soluble starch in 20Mm sodium acetate solution; the stop solution was a mixed solution of 0.4M sodium hydroxide, 22mM dinitrosalicylic acid (3, 5-dinitrosalicylic acid), 1.1M KCl (-) -tartrate tetrahydrate, all of the above-mentioned reagents being purchased from sigma. The experimental procedure was to mix 200ul of the reaction solution with 50ul of crude enzyme obtained by concentrating the supernatant obtained after culturing recombinant bacteria TD01-pSEVA 321-Porin-SP 1-amyL and TD01-pSEVA 321-Porin-SP 1-linker-amyL, incubate at 37 ℃ for 5min, add 200ul of the stop solution, heat at 100 ℃ for 5min, and measure the absorbance at 542 nm. Enzyme activity is defined as the number of moles of glucose released per minute per unit volume, corresponding to the amount of glucose released by amylase.

The present method references "Ohdan, K.et al, characteristics of two forms of alpha-and structural imaging. Appl.environ.Microbiol.65, 4652-4658(1999), Gujin Zhang et al, excellular amplification of recombinant proteins from the carrier protein Yebf in Escherichia coli, natural biotechnology" (2005) "

b. Lipase Activity assay

Lipase is measured by lipase kit (CAS No. MAK046, Sigma-Aldrich) at 542nm for absorbance of supernatants isolated after incubation of recombinant bacteria TD01-pSEVA 321-Pprein-SP 1-lipase and TD01-pSEVA 321-Pprorin-SP 1-linker-lipase, respectively, one unit of lipase is defined as the amount of enzyme that will produce 1.0 mole of glycerol from triglycerides per minute at 37 ℃.

c. Cellulase Activity test

Cellulase activity assays were performed using materials, equipment and procedures specified in the Sigma non-specific cellulase activity assay protocol. In this assay, 50mM sodium acetate trihydrate buffer was adjusted to pH and the reaction was maintained at pH 5.0. The substrate source was 5% (w/v) Sigmacell Microcrystal Type 20 from Sigma, a product from Sigma, at 37 ℃ for 120 minutes with continuous shaking at 200rpm, and the light absorption of supernatants isolated after incubation of recombinant bacteria TD01-pSEVA321-Pporin-SP 1-cellulose and TD01-pSEVA321-Pporin-SP 1-linker-cellulose, respectively, was tested at 340nm and glucose concentration was determined using glucose (HK) assay reagent as indicator. 1 unit of cellulase released 1.0. mu. mol glucose from cellulose within 1 hour at pH 5.0 at 37 ℃ (incubation time 2 hours).

d. Protease activity assay

Protease activity was performed using the materials (see, in particular, the document Cupp-Enyard, C. (2008) Sigma's non-specific protease activity assay-case as a substrate J. Vis. exp. 19: 899.) equipment and procedures described in the Sigma non-specific protease activity assay protocol. In this reaction, potassium phosphate buffer (pH 7.5) and casein as a substrate were used. When the protease digests casein, the amino acid tyrosine is released along with other amino acids and peptide fragments. Folin and Ciocalteus phenol or Folin reagents react mainly with free tyrosine to generate blue chromophores, and supernatants isolated after incubation of recombinant bacteria TD01-pSEVA 321-Porin-SP 1-protease and TD01-pSEVA 321-Porin-SP 1-linker-protease were measured spectrophotometrically at an absorbance value of 660nm using an Epoch BioTek 96 well plate reader spectrophotometer. The protease activity of the samples was determined in units, defined as micromolar amounts of tyrosine equivalents released from casein per minute at 37 ℃.

According to the enzyme activity experiments, the results are shown in a table 2, the enzyme activities of various enzymes are detected in the separated supernatant, and the results show that the halomonas TD can secrete amylase, lipase, protease and cellulase to the outside of cells under the guidance of signal peptide, and the enzyme activity of the carried linker is better than that of the non-carrying linker.

TABLE 2 enzymatic Activity of different enzymes

Example 4 comparison of the Activity of different amylases

1) Gene of interest amyL _TD Obtained by

Taking amylase gene of Halomonas bluephasegenetics TD01 as a template, taking amyTD-F and amyTD-R as primers, and carrying out PCR amplification by using pfu enzyme to obtain a PCR product of 1770bp shown in sequence 11, namely the target gene amyL _TD 。

Wherein, the primer is: amyTD-F: 5'-atgaaacaacaaaaacggctttacgc-3', respectively;

amyTD-R：5’-ctatctttgaacataaattgaaaccgaccc-3’

2) gene of interest amyL _b1 Obtained by

PCR amplification was performed with pfu enzyme using the amylase gene (synthesis) of Bacillus licheniformis as a template and amybl-F and amybl-R as primers to obtain a 1539bp PCR product shown in SEQ ID No. 12, i.e., the objective gene SP 1' -amyL _bl Wherein the 1 st to 90 th positions of the sequence 12 are nucleotide sequences of signal peptide without codon optimization (indicated by ' SP1 '), and the 91 st to 1539 th positions of the sequence 12 are nucleotide sequences of amylase without codon optimization (indicated by ' amyL _bl "means").

Wherein, the primer is: amybl-F: 5'-atgaaacagcagaaacgcctgtatgcccgtcttct-3', respectively;

amybl-R：5’-ttagcgctgcacgtaaatgctc-3’

3) gene of interest amyL _bl-opt Obtained by

Taking the amylase gene (synthesized) of Bacillus licheniformis after codon optimization as a template, taking amybl-opt-F and amybl-opt-R as primers, and carrying out PCR amplification by using pfu enzyme to obtain a PCR product of 1539bp shown in a sequence 13, namely the target gene SP1-amyL _bl-opt Wherein, the 1 st to 90 th sites of the sequence 13 are the nucleotide sequence of a signal peptide SP1 subjected to codon optimization, and the sequence is the same as the sequence shown in the 1 st to 90 th sites of the sequence 2; the nucleotide sequence of amylase for codon optimization at positions 91-1539 of the sequence 12 (denoted as "amyL _bl-opt "represents)" which is identical to the sequence shown in the 106 th and 1554 th positions of the sequence 2.

Wherein, the primer is: amybl-opt-F: 5'-atgaaacagcagaaacgcctgtatgcccgtcttctg-3'

amybl-opt-R：5’-ttagcgctgcacgtaaatgctca-3’

4) Obtaining of inducible promoters

The selection of inducible promoters is documented in the following literature: zhao, h.et al, (2017) Novel T7-like expression systems used for halonas. metabeng 39, 128-140. Wherein, the sequence of the inducible promoter is shown as sequence 14.

5) Amplification of vector backbones

PCR amplification is carried out by taking pSEVA321 as a template and 321-S-F and 321-S-R as primers and pfu enzyme to obtain a PCR product, namely a vector skeleton, of 3580bp in total, as shown in 1016, 4563 and 1-32 of a sequence 1.

Wherein, the primer is: 321-S-F: 5'-gtcgtgactgggaaaaccctggc-3'

321-S-R：5’-tcctgtgtgaaattgttatccgct-3’

Respectively connecting the PCR product obtained in the steps 1), 2) and 3) and the vector skeleton obtained in the step 4) and 5) by a Gibson assembly homologous recombination method to obtain connecting products, and transforming the connecting products into E.coli S17-1 to obtain transformants respectively.

Carrying out PCR amplification by using verification primers F24 and R24 and pfu enzyme to obtain a glue running strip, selecting positive transformants with the strips of 2007bp, 1776bp and 1776bp respectively, extracting plasmids of the positive transformants, sending the plasmids to a sequencing unit, and obtaining a result that the plasmids respectively contain the target gene amyL _TD ，SP1’-amyL _bl ，SP1-amyL _bl-opt Plasmid fragment, designated pSEVA321-mmp1-amyL _TD ，pSEVA321-mmp1-SP1’-amyL _bl ，pSEVA321-mmp1-SP1-amyL _bl-opt 。

Wherein, the verification primer is: f24: 5'-agcggataacaatttcacacagga-3'

R24：5’-cgccagggttttcccagtcacgac-3’

The obtained plasmid pSEVA321-mmp1-amyL _TD ，pSEVA321-mmp1-SP1’-amyL _bl 、pSEVA321-mmp1-SP1-amyL _bl-opt And the empty vector pSEVA321 is transferred into E.coli S17-1 by an electrotransformation method respectively to obtain 4 recombinant bacteria S17-1. Halomonas TD (the source of the strain is shown in the following documents: ZHao et al. novel T7-like expression system used for Halomonas. Metabolic Engineering (2017): 39: 128-14) is used as a receptor strain, and a recombinant strain S17-1 is respectively used as a donor strain to carry out joint transformation to obtain a recombinant strain TD-pSEVA321-mmp1-amyL _TD 、TD-pSEVA321-mmp1-SP1’-amyL _bl 、TD-pSEVA321-mmp1-SP1-amyL _b1-opt And the recombinant strain TD-pSEVA321, and single colonies were screened on Cm-resistant 60LB (LB containing 60g/L NaCl) plates.

5) Comparison of enzyme activities of different strains

The above-mentioned genes are respectively combined with target gene amyL _TD ，amyL _bl ，amyL _bl-opt Halomonas TD strain (recombinant strain TD-pSEVA321-mmp 1-amyL) _TD 、TD-pSEVA321-mmp1-SP1’-amyL _bl 、TD-pSEVA321-mmp1-SP1-amyL _bl-op ) The halomonas TD strain (namely the recombinant strain TD-pSEVA321) and the control group halomonas TD strain which are jointed with the empty vector are respectively cultured in 20mL, after 12h of culture, the strains are transferred into a new 20mL culture medium according to the transfer amount of 1 percent for 12h of culture, OD600 is measured by a spectrophotometer, centrifugation is carried out at 4500rpm for 20min, and supernatant and sediment are separated. Subjecting the separated supernatant to the following amylaseAnd (4) testing enzyme activity. The method comprises the following specific steps:

the amylase activity test is based on the dinitrosalicylic acid method and comprises a two-part solution, a reaction solution and a stop solution. Wherein the reaction solution is prepared by dissolving 0.5% soluble starch in 20Mm sodium acetate solution; the stop solution was a mixed solution of 0.4M sodium hydroxide, 22mM dinitrosalicylic acid (3, 5-dinitrosalicylic acid), 1.1M KCl (-) -tartrate tetrahydrate, all of the above-mentioned reagents being purchased from sigma. The experimental procedure was to mix 200ul of the reaction solution with 50ul of the crude enzyme after concentration of the separated supernatant, incubate at 37 ℃ for 5min, add 200ul of the stop solution, heat at 100 ℃ for 5min, and measure the absorbance at 542nm of the separated supernatant after culturing the above-mentioned strain. Enzyme activity is defined as the number of moles of glucose released per minute per unit volume, corresponding to the amount of glucose released by amylase.

The present method is described in "Ohdan, K.et al (1999) Characteristics of two fbrms of alpha-amylases and structural engineering. apple Environ Microbiol 65(10), 4652-8.Zhang, G.et al (2006) excellar accumulation of recombinant proteins fused to the carrier protein Yebf in Escherichia coli. Nat Biotechnol 24(1), 100-4.

The results are shown in FIG. 3, and indicate that the recombinant bacterium TD-pSEVA321-mmp1-amyL _TD The recombinant strain TD-pSEVA321-mmp1-SP1 '-amyL has substantially no extracellular amylase activity in the supernatant isolated after the culture of the recombinant strain TD-pSEVA321 (shown as "T58") and the control Pseudomonas halophila TD strain (shown as "TD" in the figure), while the recombinant strain TD-pSEVA321-mmp1-SP 1' -amyL has substantially no extracellular amylase activity in the supernatant _bl (T88 in the figure) after the culture, the amylase activity in the separated liquid has a certain amylase activity, and the amylase activity has a certain amylase activity with the recombinant bacterium TD-pSEVA321-mmp1-SP 1' -amyL _bl Compared with the recombinant strain TD-pSEVA321-mmp1-SP1-amyL _bl-opt (T89 in the figure) shows higher amylase activity in the supernatant separated after the culture, which indicates that the halomonas TD strain can not utilize starch, and in addition, the signal peptide SP1 and the amylase of the invention can obviously provide the activity of the amylase after being subjected to codon optimization.

Example 5 Halomonas secretion platform for achieving secretion of Phap protein

1) Acquisition of target genes phaP and linker-phaP

Taking halomonas TD01 as a template, taking phaP-F1 and phaP-R as primers, and carrying out PCR amplification by pfu enzyme to obtain a PCR product of 351bp shown in the 106-rd and 456-th positions of the sequence 15, namely the target gene phaP. The primers phaP-F2 and phaP-R are used for carrying out PCR amplification by pfu enzyme to obtain a PCR product of 366bp shown in 91-456 bit of the sequence 15, namely the target gene linker-phaP.

Wherein, the primer is: phaP-F1: 5'-atgaatatggacgtgatcaagagcttta-3'

phaP-F2：5’-atgaatatggacgtg atgaatatggacgtgatcaagagcttta-3’

phaP-R：5’-ttaggccttgcccgtgctcttcttg-3’

2) Amplification of vector backbones

Using pSEVA321-mmp1-amyL _bl-opt Using 321-S-F and 321-S-R as primers, and pfu enzyme to perform PCR amplification to obtain 3769bp PCR product, i.e. vector skeleton, shown as sequence 17.

Wherein, the primer is: 321-S-F: 5'-gtcgtgactgggaaaaccctggcg-3', respectively;

321-S-R：5’-ctagtatttctcctctttctctagtaactctta-3’

and (2) respectively connecting the PCR products obtained in the step 1) with the PCR products obtained in the step 2) by a Gibson assembly homologous recombination method to obtain connecting products, and transforming the connecting products into E.coli S17-1 to obtain transformants.

Carrying out PCR amplification by using verification primers F24 and R24 and pfu enzyme to obtain a glue running strip, selecting positive transformants with the size of 588bp and 603bp, extracting plasmids of the positive transformants, and sequencing to obtain plasmids containing a target gene phaP fragment and a target gene linker-phaP, wherein the plasmids are respectively marked as pSEVA321-mmp1-SP1-phaP and pSEVA321-mmp1-SP 1-linker-phaP.

Wherein, the verification primer is: f24: 5'-agcggataacaatttcacacagga-3'

R24：5’-cgccagggttttcccagtcacgac-3’

The obtained plasmids pSEVA321-mmp1-SP1-phaP and pSEVA321-mmp1-SP1-linker-phaP are respectively transferred into E.coli S17-1 by an electrotransformation method to obtain 2 recombinant bacteria S17-1. Halomonas TD01 was used as a recipient strain, and recombinant strain S17-1 was used as a donor strain to perform joint transformation, to obtain recombinant strain TD01(pSEVA321-mmp1-SP1-phaP) and recombinant strain TD01(pSEVA321-mmp1-SP1-linker-phaP), and single colonies were screened on a Cm-resistant 60LB (LB containing 60g/L NaCl) plate.

3) Verification of phaP secretion by Western-Blot

The aforementioned Halomonas TD strain (i.e., recombinant bacteria TD-pSEVA321-mmp1-SP1-phaP and TD-pSEVA321-mmp1-SP1-linker-phaP) and the control Halomonas TD strain, each of which contains linker-phaP and phaP genes, were cultured in 20mL of medium, cultured for 12 hours, transferred to a new 20mL of medium in an amount of 1% of the transferred medium, cultured for 12 hours, centrifuged at 4500rpm for 20min, and the supernatant and the precipitate were separated. To analyze phaP expression, the pellet is dissolved in

Master Mix lysine Solution (Norway root, Mass.) and processed according to the instructions. Soluble fraction and insoluble fraction of cells were prepared after centrifugation at 16000 Xg at 4 ℃. SDS-PAGE analysis was performed using 12% Bis-Tris gels (Life Technologies, NP0342Box, USA) mounted in Mini Gel Tank (Life Technologies). The amount of soluble protein was determined using BCA protein assay kit (Thermo Scientific). Gel lanes were scanned with the Fluro-ChemTM FC3 system (ProteinSample). For large scale extraction of proteins, cell pellets were suspended in a volume of PBS and then disrupted with a nano homogenizer (ATS Engineering INC, canada). Cell lysates were concentrated 50-fold using an Amicon Ultra-15 centrifugal filter unit (10kDa MWCO, merchandib, ma).

As shown in fig. 4 and 5, it was found that the phaP protein with linker could be secreted to the outside of the cell, but much of the phaP protein remained in the cell. The phaP without the linker is hardly secreted to the outside of the cell, and the protein expressed in the cell is very little, which indicates that the addition of the linker promotes the secretion of the signal peptide to the protein phaP.

Example 6 conversion of an inexpensive carbon Source to PHA, modeled on an Amylase

Constructing a protein secretion platform to prove that the signal peptide from amylase can secrete different proteins, the engineering bacteria can secrete industrial enzymes and convert substrates into various products;

1. recombinant bacterium TD-pSEVA321-mmp1-SP1-amyL _bl-opt Shake flask experiment for PHB Synthesis (prepared in example 4)

In order to test the recombinant bacteria TD-pSEVA321-mmp1-SP1-amyL _bl-opt The ability to synthesize PHB was determined using the reference bacterium Halomonas TD and the recombinant bacterium TD-pSEVA321-mmp1-SP1-amyL _bl-opt (shaking flask experiment is carried out; recombinant bacteria TD-pSEVA321-mmp1-SP1-amyL _b1-opt For example, for producing PHB, a recombinant bacterium TD-pSEVA321-mmp1-SP1-amyL was selected _bl-opt The single colony of (2) was cultured in 20mL of LB60 (LB containing 60g/L NaCl) medium at 37 ℃ overnight at 200rpm, and was inoculated once and cultured for 12 hours; then inoculating the culture solution into 50mL MM culture medium (pH 8.5-9.0, NaCl concentration 60g/L), with the concentration of 20g/L glucose and 20g/L starch respectively according to the inoculation amount of 5% (v/v), culturing for 48 hours in a shaking flask at 37 ℃ and 200rpm to obtain a bacterial solution, centrifuging, washing and drying by ice to obtain the cell dry weight of the sample, and detecting the content percentage (%) of PHB in the cell dry weight by gas chromatography. Three sets of parallels were set for each condition. The cell dry weight and PHB content are shown in Table 3. As can be seen from table 3: recombinant bacterium TD-pSEVA321-mmp1-SP1-amyL _bl-opt Can replace glucose as a carbon source to produce PHB, and the yield of the PHB is the highest in a starch culture medium of 20 g/L.

TABLE 3 comparison of the addition of different carbon sources

2. Recombinant bacterium TD delta gabD2-D2-pSEVA321-mmp1-SP1-amyL _bl-opt Shake flask experiment for synthesis of P34HB

Taking the Halomonas bluephagene TD Δ gabD2-D2 as an example, which is a salt capable of synthesizing PHBV, the strain is described in the following documents: ye, J.et al. (2018) Engineeringof Halomonas bluegene for low cost production of poly (3-hydroxybutyric-co-4-hydroxybutyric) from glucose Eng 47, 143-152. in this strain, P3HB4HB can be produced using glucose as a sole carbon source. Over-expressing plasmid pSEVA321-mmp1-SP1-amyL on the basis of the strain _b1-opt (prepared in example 4), recombinant bacterium TD. DELTA. gabD2-D2-pSEVA321-mmp1-SP1-amyL was obtained _bl-opt So that the starch has the capability of degrading starch and takes the starch as a carbon source.

Firstly, selecting recombinant bacteria TD delta gabD2-D2-pSEVA321-mmp1-SP1-amyL _bl-opt The single colonies of (2) were cultured overnight at 37 ℃ and 200rpm in 20mL of LB60 (LB containing 60g/L NaCl) medium; then respectively inoculating the cells into 50mL MM culture medium (pH 8.5-9.0 and NaCl concentration 60g/L) according to the inoculation amount (v/v) of 5 percent, fixing the glucose concentration to 20g/L, setting the starch concentration to 20g/L, setting the culture temperature to 37 ℃, setting the rotation speed to 200rpm, shaking the bottles for 48 hours to obtain bacterial liquid, obtaining the dry cell weight of the sample after centrifugation, washing and ice drying, and then detecting the content percent (%) of PHB in the dry cell weight by gas chromatography. Three sets of parallels were set for each condition. The dry cell weight, PHBV content and 3HV ratio are shown in Table 4, from which it can be seen that: halomonas TD delta gabD2-D2-pSEVA321-mmp1-SP1-amyL _bl-opt The production of P34HB can be carried out using starch.

TABLE 4 TD. delta. gabD2-D2-pSEVA321-mmp1-SP1-amyL _bl-om Synthesis of P34HB at different starch concentrations

3. Recombinant bacterium TD08AB-pSEVA321-mmp1-SP1-amyL _bl-opt Shake flask experiment for synthesizing PHBV

The Halomonas bluephagene TD08AB, which is a salt capable of synthesizing PHBV, is described in the following documents: yin, J.et al (2015) Effects of chromosomal gene copy number and location on polyhydroxyakanoate synthesis by Escherichia coli and Halomonas sp.appl Microbioltechnol 99(13), 5523-34. At this pointOverexpression plasmid pSEVA321-mmp1-SP1-amyL based on strain _bl-optl (prepared in example 4), recombinant bacterium TD08AB-pSEVA321-mmp1-SP1-amyL was obtained _bl-opt 。

Firstly, selecting recombinant bacteria TD08AB-pSEVA321-mmp1-SP1-amyL _bl-opt The single colonies of (2) were cultured overnight at 37 ℃ and 200rpm in 20mL of LB60 (LB containing 60g/L NaCl) medium; then, 50mL of MM medium (pH 8.5-9.0, NaCl concentration 60g/L) was inoculated with 5% inoculum size (v/v), glucose concentration was fixed at 30g/L, and acetic acid was set at 4 concentration gradients: 0g/L, 2g/L, 4g/L and 6 g/L. Culturing at 37 deg.C and 200rpm, shaking for 48 hr to obtain bacterial liquid, centrifuging, washing with water, and freeze-drying to obtain dry cell weight, and detecting PHB content (%) by gas chromatography. Three sets of parallels were set for each condition. The cell dry weight, PHBV content and 3HV ratio are shown in Table 5. As can be seen from table 5: halomonas TD08AB-pSEVA321-mmp1-SP1-amyL _bl-opt The production of P34HB can be carried out using starch.

TABLE 5 TD08AB-pSEVA321-mmp1-SP1-amyL _bl-opt PHBV synthesis at different starch concentrations

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> Qinghua university

<120> a method for producing and secreting protein using halophilic bacteria

<130> GNCFY196095

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 4563

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttaattaaag cggataacaa tttcacacag gaatgcctcc acaccgctcg tcacatcctg 60

ttgcgttcac tggaatccca gtataagatt tgacctgcga gcaagctgtc accggatgtg 120

ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt ttgtttaaga 180

gttactagag aaagaggaga aatactagat gaaacagcag aaacgcctgt atgcccgtct 240

tctgactctg ctgttcgccc tgattttcct gcttccgcat agcgctgccg ctgccgctat 300

gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt ctggtggaac tggatggtga 360

tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa 420

actgacgctg aagttcatct gtactactgg taaactgccg gtaccttggc cgactctggt 480

aacgacgctg acttatggtg ttcagtgctt tgctcgttat ccggaccata tgaagcagca 540

tgacttcttc aagtccgcca tgccggaagg ctatgtgcag gaacgcacga tttcctttaa 600

ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa 660

ccgcattgag ctgaaaggca ttgactttaa agaagacggc aatatcctgg gccataagct 720

ggaatacaat tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcat 780

taaagcgaat tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca 840

ctaccagcaa aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct 900

gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct 960

ggagttcgta accgcagcgg gcatcacgca tggtatggat gaactgtaca aatgagtcgt 1020

gactgggaaa accctggcga ctagtcttgg actcctgttg atagatccag taatgacctc 1080

agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga 1140

gaatccaggg gtccccaata attacgattt aaattggcga aaatgagacg ttgatcggca 1200

cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga 1260

gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac 1320

caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc 1380

tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa 1440

gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc 1500

tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca 1560

cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata 1620

ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga 1680

aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc 1740

ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc 1800

cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat 1860

tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca 1920

acagtactgc gatgagtggc agggcggggc gtaatttgac ttttgtcctt ttccgctgca 1980

taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga 2040

tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag 2100

ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct 2160

tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc gtaggccggc 2220

cgcgatgcag gtggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt 2280

gcaatgcacc aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc 2340

ttcgcaggct tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca 2400

catgaggcgg aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc 2460

gaacatccgt cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta 2520

gtggtcgcca gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt 2580

tttcttgcca cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc 2640

aacgcggtcg gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc 2700

cttcgtgtaa taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt 2760

gaaggtgatc ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac 2820

cagttcgtca tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt 2880

gacgtggaaa atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt 2940

gaacagggca gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc 3000

aatatcgaac aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc 3060

ggcctgcttg gcttcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt 3120

ggtcgtcata gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg 3180

ttcgagacga cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag 3240

ttgcacgctg tcgcgctcga tcttggccgt agcttgctgg actatcgagc cgacggactg 3300

gaaggtttcg cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc 3360

ggaaaacccc gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat 3420

tcaccctcct tgcgggattg ccccggaatt aattccccgg atcgatccgt cgatcttgat 3480

cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc 3540

ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc 3600

gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt 3660

gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc 3720

tgcggactgg ctttctacgt ggctgccatt tttggggtga ggccgttcgc ggccgagggg 3780

cgcagcccct ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg 3840

ggcacccccc ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag 3900

gtttataaat attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg 3960

gcggaaaccc ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag 4020

gtgcgcccct catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct 4080

gtcagtagtc gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca 4140

catcatctgt gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca 4200

gctccacgtc gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga 4260

gtcggcccct caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc 4320

gaggtatcca caacgccggc ggccctacat ggctctgctg tagtgagtgg gttgcgctcc 4380

ggcagcggtc ctgatccccc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 4440

tacggcgcgc ccagctgtct agggcggcgg atttgtccta ctcaggagag cgttcaccga 4500

caaacaacag ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg 4560

cct 4563

<210> 2

<211> 1554

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct aatttaaatg gcaccaattt aaatggcacc 120

ctgatgcagt acttcgaatg gtacatgccg aacgacggcc aacactggaa acgcctgcaa 180

aacgactctg cctatctggc cgaacatggc attaccgccg tttggatccc gccggcctat 240

aaaggtacca gccaggccga tgttggttat ggtgcctacg acctgtacga cctgggcgaa 300

ttccaccaga aaggcaccgt gcgcaccaaa tacggcacca aaggcgaact tcagagcgcc 360

atcaaatctc tgcacagccg cgacatcaac gtttacggcg acgtggtgat caaccacaaa 420

ggcggtgccg atgccactga agatgttacc gccgtggaag ttgaccctgc cgatcgcaat 480

cgtgtgatta gcggcgaaca cctgatcaaa gcctggaccc acttccattt tccgggtcgc 540

ggtagcactt acagcgactt caaatggcac tggtaccact ttgacggcac cgactgggat 600

gaaagccgca aactgaaccg catctacaaa ttccagggca aagcctggga ttgggaagtg 660

agcaacgaaa acggcaacta cgactacctg atgtacgccg acatcgacta tgatcacccg 720

gatgtggccg ccgaaattaa acgttggggc acctggtatg ccaacgaact gcagctggac 780

ggttttcgtc tggatgccgt gaaacacatc aaattcagct tcctgcgcga ctgggttaat 840

cacgtgcgcg aaaaaaccgg caaagaaatg ttcaccgtgg ccgaatactg gcagaatgac 900

ctgggtgccc tggaaaacta cctgaacaag accaacttca accacagcgt gttcgatgtg 960

ccgctgcact atcaattcca cgccgcctct actcaaggcg gtggttacga catgcgcaaa 1020

ctgctgaacg gcaccgtggt tagcaaacac ccgctgaaaa gcgtgacctt cgtggacaac 1080

cacgacactc aaccgggtca gagcctggaa agcactgtgc agacctggtt caaaccgctg 1140

gcctacgcct ttattctgac ccgcgaaagc ggttatccgc aggtgttcta cggtgacatg 1200

tacggcacca aaggcgatag ccagcgtgaa atcccggccc tgaaacacaa aatcgaaccg 1260

atcctgaaag cccgcaaaca gtacgcctat ggcgcccagc acgactattt tgaccaccac 1320

gacatcgttg gctggactcg cgaaggcgat agcagcgttg ccaattctgg tctggccgcc 1380

ctgattactg atggtccggg cggtgccaaa cgtatgtatg tgggccgtca aaacgccggt 1440

gaaacctggc atgacatcac tggcaaccgt agcgaaccgg tggtgatcaa ctctgaaggc 1500

tggggcgaat ttcatgtgaa tggcggcagc gtgagcattt acgtgcagcg ctaa 1554

<210> 3

<211> 1545

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct ttgagtaaga agattttgag taagaagatt 120

aagaagtcca atcttacgga tgagacttat tatcgtataa gccaacgttc ttacaactac 180

gattatctca gaaaaaaatt aaaaaacaaa gagtacatac ggataaattc atctgtttcc 240

ggagccacct actggtatgt tgataaaatt aaaacagacg aagacaccgg gttagatgcg 300

gccgttctat cccaggccga aaataaaaac ggcaagtggg tgaaatccga ccaccccaaa 360

aacgtcgttg tagcttttgc gggaactgat ccggggaagg acccgttaag tgacgtagag 420

caagcagaca ttaatcatat cgtcttagga aacgatccga aagataaaac acaatatgtc 480

gtcaagaaag atgcgaaaga tatgtctaag acgttcggta gatatattgg ttcgatggaa 540

caaactgcca tgcttgagtc cggagattat aaattaatta caaaaacatc gcaaatcgat 600

caagccgatc aacttgtgcg ggaagttaaa caaaagtata aaggcacctc gacagttatt 660

tcaaccaccg gacactcgct cggcggcgca gaggctgaat acagcgcggt caacaatgat 720

atctatgccg tcgcgtttaa tagcccctca atcgttcacc tgcattctga tgaaaaacag 780

aaagaaatta ataacggaga ttataactct tatgtaaaat ctatcattaa tccggatgat 840

atggtcggtg ccggttggtg ggacgaattc gatcgccaca atgggactac catctacaca 900

aaagaccctt ctattgcaac ggctaatcgc gaagaaagac ttgacggcaa taaactccaa 960

caggttggaa gaaaccttct gtatttcgct aatacactaa tctttcaaaa tccggataca 1020

catgggataa ataagtcgaa tttctctttc gacgaaaacg gcaacgttca aaatatagag 1080

ggcgatgaac tcgtttatga caaaaattta aaagcgatgc tcccaccgga agttgcctcc 1140

ggaagtggcg caattaaagt aacacccgaa gttgccaagc agctcgcgca aaaagtaaat 1200

gcgatcatcg atgacttgcg gacgatgaaa cgggaagccg aaaacgctta tcaggaacac 1260

gacgccgaga tcaacgacct gaagcacgat acctaccgtc aggtcggcca cggtttatac 1320

gacaagctga cactcgagga tgtaaacaat acgctgaaaa acctggcgca gtcgtttgac 1380

aaaaaaggca atccgctatt ttacgatgtt catgccgaaa aagcgtacat cgcctcatta 1440

aaaaatacga tttcagattt agaagacatc agcgggtacc tggcgcagat tgcaaaagat 1500

tttaaatcga aagataaaat gctggcaaat tggcttaaac tttag 1545

<210> 4

<211> 651

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct atgtcatctt ttcacatgtc atcttttcac 120

gcaacgacga tattcgccgt ccagcacaac ggaaaaagcg cgatggcggg agacggccag 180

gttacgttcg gtcaggctgt cgtcatgaag cataccgcta gaaaggtaag aaagctcttc 240

aacgggaaag tcattgccgg ctttgccggt tctgttgcgg acgctttcac gctctttgaa 300

atgtttgaag cgaagcttga agagtacaac ggcaatttac agcgtgcggc agtcgagctt 360

gcaaaagagt ggcgaagcga taaagtcctc agaaagcttg aggcgatgct gatcgtcatg 420

aacgctgaca gcatgctgct cgtttccgga acaggcgagg tgattgaacc tgacgacggc 480

attttggcga tcggctcagg cggaaactat gccctggcag ccggaagggc gttgaaacgt 540

catgcaggcg gccagcttga cgcaagagcg attgcgaggg cctcacttga aaccgcgggc 600

gaaatctgtg tgtatacgaa cgatcagatc attgtagaag aacttgaata g 651

<210> 5

<211> 1104

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct tttagtgccg cgaattttag tgccgcgaat 120

gttttctctt acgaatgttc ctgaagcagt taaatctgta agcggccagc cgcctgtttt 180

agatgctcct ggctttaacg ctgaggatga ttcctgctta tcagaaagat tccagttcac 240

ccagctgatt ttcttgctgt cgagataatt cagccattcc cgcgactggc caaggaatac 300

accgccattt ccagacgcat cgcttgttcc ccattccgtc acgaaaatcg gcgctccttt 360

gctgagtgcg tagtttgctt tatcccgtaa aaattggcca tgtgtgccgg cataaaaatg 420

aagcgcgtac atgacgtttg catcttttat ctggtcatcg gcagcatcat tcacatcctg 480

gctccatgta ccggttccga caatgatgat gttgtctgga tcatttttgc ggataacgga 540

aatcacttct tccgcatacg gtttaatatc acgcttccag ttcacgtcac cgtttggttc 600

gtttgcaatt tcataaatga cgtttggcgt gtttccgtaa agacttgaca tctccttgaa 660

aaattctttt gccttctctt tattttggtt tgggttgccg tcatttaaaa tatgccagtc 720

aatgatgaca tatatcccaa gttcttttgc cgcttcaacc gcttctttta ctttattttt 780

cacggacggg ttgtcaatat aaccgccatc tgccgtatac atcgccgcgc ggaaaacggt 840

tatgccccaa tcgtctctca gccattttaa gctgtcttta ttgacgaaat cgccatacca 900

ttgcaatcca tgtgaactga tccctttcag ttgtaccgct ttgccatctc ggtttacaag 960

ttgtgtacct tttatgctaa gctgaccatt cttggctact ggcgtttttg ttcctgctgc 1020

tgatgccggc gaaggcagca agccgcccat tgtcaatacc gcaatcaata aacacgtaat 1080

aaaaacagaa attgaccgtt tcat 1104

<210> 6

<211> 517

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

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

20 25 30

Asn Gly Thr Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr

35 40 45

Met Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala

50 55 60

Tyr Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ile Asn Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp

130 135 140

Ala Thr Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn

145 150 155 160

Arg Val Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His

165 170 175

Phe Pro Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr

180 185 190

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

195 200 205

Tyr Lys Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn

210 215 220

Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro

225 230 235 240

Asp Val Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu

245 250 255

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

260 265 270

Ser Phe Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys

275 280 285

Glu Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu

290 295 300

Glu Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val

305 310 315 320

Pro Leu His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr

325 330 335

Asp Met Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu

340 345 350

Lys Ser Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Lys Ile Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala

420 425 430

Gln His Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu

435 440 445

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

450 455 460

Gly Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly

465 470 475 480

Glu Thr Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile

485 490 495

Asn Ser Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser

500 505 510

Ile Tyr Val Gln Arg

515

<210> 7

<211> 514

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Leu Ser

20 25 30

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

35 40 45

Thr Tyr Tyr Arg Ile Ser Gln Arg Ser Tyr Asn Tyr Asp Tyr Leu Arg

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Val Lys Ser Asp His Pro Lys Asn Val Val Val Ala Phe Ala Gly

115 120 125

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

130 135 140

Asn His Ile Val Leu Gly Asn Asp Pro Lys Asp Lys Thr Gln Tyr Val

145 150 155 160

Val Lys Lys Asp Ala Lys Asp Met Ser Lys Thr Phe Gly Arg Tyr Ile

165 170 175

Gly Ser Met Glu Gln Thr Ala Met Leu Glu Ser Gly Asp Tyr Lys Leu

180 185 190

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

195 200 205

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

210 215 220

His Ser Leu Gly Gly Ala Glu Ala Glu Tyr Ser Ala Val Asn Asn Asp

225 230 235 240

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

245 250 255

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

260 265 270

Lys Ser Ile Ile Asn Pro Asp Asp Met Val Gly Ala Gly Trp Trp Asp

275 280 285

Glu Phe Asp Arg His Asn Gly Thr Thr Ile Tyr Thr Lys Asp Pro Ser

290 295 300

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

305 310 315 320

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

325 330 335

Asn Pro Asp Thr His Gly Ile Asn Lys Ser Asn Phe Ser Phe Asp Glu

340 345 350

Asn Gly Asn Val Gln Asn Ile Glu Gly Asp Glu Leu Val Tyr Asp Lys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Pro Leu Phe Tyr Asp Val His Ala Glu Lys Ala Tyr Ile Ala Ser Leu

465 470 475 480

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

485 490 495

Ile Ala Lys Asp Phe Lys Ser Lys Asp Lys Met Leu Ala Asn Trp Leu

500 505 510

Lys Leu

<210> 8

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Met Ser

20 25 30

Ser Phe His Met Ser Ser Phe His Ala Thr Thr Ile Phe Ala Val Gln

35 40 45

His Asn Gly Lys Ser Ala Met Ala Gly Asp Gly Gln Val Thr Phe Gly

50 55 60

Gln Ala Val Val Met Lys His Thr Ala Arg Lys Val Arg Lys Leu Phe

65 70 75 80

Asn Gly Lys Val Ile Ala Gly Phe Ala Gly Ser Val Ala Asp Ala Phe

85 90 95

Thr Leu Phe Glu Met Phe Glu Ala Lys Leu Glu Glu Tyr Asn Gly Asn

100 105 110

Leu Gln Arg Ala Ala Val Glu Leu Ala Lys Glu Trp Arg Ser Asp Lys

115 120 125

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

130 135 140

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

145 150 155 160

Ile Leu Ala Ile Gly Ser Gly Gly Asn Tyr Ala Leu Ala Ala Gly Arg

165 170 175

Ala Leu Lys Arg His Ala Gly Gly Gln Leu Asp Ala Arg Ala Ile Ala

180 185 190

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

195 200 205

Gln Ile Ile Val Glu Glu Leu Glu

210 215

<210> 9

<211> 368

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Met Lys

20 25 30

Arg Ser Ile Met Lys Arg Ser Ile Ser Ile Phe Ile Thr Cys Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Gly Thr Gln Leu Val Asn Gln Asn Gly Lys Pro Val Gln Leu Lys Gly

85 90 95

Ile Ser Ser His Gly Leu Gln Trp Phe Gly Asp Tyr Val Asn Lys Asp

100 105 110

Thr Leu Lys Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala

115 120 125

Ala Met Tyr Thr Ala Asp Gly Gly Tyr Ile Glu Asn Pro Ser Val Lys

130 135 140

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

145 150 155 160

Val Ile Ile Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn

165 170 175

Lys Glu Lys Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly

180 185 190

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

195 200 205

Val Asn Trp Lys Arg Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser

210 215 220

Val Ile Arg Lys Asn Asp Pro Asp Asn Ile Ile Ile Thr Gly Thr Gly

225 230 235 240

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

245 250 255

Ala Asn Val Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln

260 265 270

Phe Leu Arg Asp Lys Ala Asp Tyr Ala Leu Ser Lys Gly Ala Pro Ile

275 280 285

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

290 295 300

Tyr Leu Asp Gln Ser Arg Glu Trp Leu Asn Tyr Leu Asp Ser Lys Lys

305 310 315 320

Ile Ser Trp Val Asn Trp Asn Leu Ser Asp Lys Gln Glu Ser Ser Ser

325 330 335

Ala Leu Lys Pro Gly Ala Ser Lys Thr Gly Gly Trp Pro Leu Ser Asp

340 345 350

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

355 360 365

<210> 10

<211> 3846

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttaattaaag cggataacaa tttcacacag gaatgcctcc acaccgctcg tcacatcctg 60

ttgcgttcac tggaatccca gtataagatt tgacctgcga gcaagctgtc accggatgtg 120

ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt ttgtttaaga 180

gttactagag aaagaggaga aatactagat gaaacagcag aaacgcctgt atgcccgtct 240

tctgactctg ctgttcgccc tgattttcct gcttccgcat agcgctgccg ctgccgctgt 300

cgtgactggg aaaaccctgg cgactagtct tggactcctg ttgatagatc cagtaatgac 360

ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg 420

tgagaatcca ggggtcccca ataattacga tttaaattgg cgaaaatgag acgttgatcg 480

gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt 540

tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 600

taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 660

tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 720

aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 780

tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 840

tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 900

ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 960

tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 1020

tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 1080

ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 1140

gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 1200

acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt gacttttgtc cttttccgct 1260

gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca 1320

cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt 1380

cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc 1440

ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgtaggcc 1500

ggccgcgatg caggtggctg ctgaaccccc agccggaact gaccccacaa ggccctagcg 1560

tttgcaatgc accaggtcat cattgaccca ggcgtgttcc accaggccgc tgcctcgcaa 1620

ctcttcgcag gcttcgccga cctgctcgcg ccacttcttc acgcgggtgg aatccgatcc 1680

gcacatgagg cggaaggttt ccagcttgag cgggtacggc tcccggtgcg agctgaaata 1740

gtcgaacatc cgtcgggccg tcggcgacag cttgcggtac ttctcccata tgaatttcgt 1800

gtagtggtcg ccagcaaaca gcacgacgat ttcctcgtcg atcaggacct ggcaacggga 1860

cgttttcttg ccacggtcca ggacgcggaa gcggtgcagc agcgacaccg attccaggtg 1920

cccaacgcgg tcggacgtga agcccatcgc cgtcgcctgt aggcgcgaca ggcattcctc 1980

ggccttcgtg taataccggc cattgatcga ccagcccagg tcctggcaaa gctcgtagaa 2040

cgtgaaggtg atcggctcgc cgataggggt gcgcttcgcg tactccaaca cctgctgcca 2100

caccagttcg tcatcgtcgg cccgcagctc gacgccggtg taggtgatct tcacgtcctt 2160

gttgacgtgg aaaatgacct tgttttgcag cgcctcgcgc gggattttct tgttgcgcgt 2220

ggtgaacagg gcagagcggg ccgtgtcgtt tggcatcgct cgcatcgtgt ccggccacgg 2280

cgcaatatcg aacaaggaaa gctgcatttc cttgatctgc tgcttcgtgt gtttcagcaa 2340

cgcggcctgc ttggcttcgc tgacctgttt tgccaggtcc tcgccggcgg tttttcgctt 2400

cttggtcgtc atagttcctc gcgtgtcgat ggtcatcgac ttcgccaaac ctgccgcctc 2460

ctgttcgaga cgacgcgaac gctccacggc ggccgatggc gcgggcaggg cagggggagc 2520

cagttgcacg ctgtcgcgct cgatcttggc cgtagcttgc tggactatcg agccgacgga 2580

ctggaaggtt tcgcggggcg cacgcatgac ggtgcggctt gcgatggttt cggcatcctc 2640

ggcggaaaac cccgcgtcga tcagttcttg cctgtatgcc ttccggtcaa acgtccgatt 2700

cattcaccct ccttgcggga ttgccccgga attaattccc cggatcgatc cgtcgatctt 2760

gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc 2820

ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa 2880

accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg 2940

cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt 3000

ttctgcggac tggctttcta cgtggctgcc atttttgggg tgaggccgtt cgcggccgag 3060

gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg ttcgagaagg 3120

gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct ggttaaaaac 3180

aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt ggccgaaaaa 3240

cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct caaatgtcaa 3300

taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc gcgcccctca 3360

tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc ccaggcttgt 3420

ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt gcgaggctgg 3480

ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg ccgcgccggg 3540

tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg ccaagttttc 3600

cgcgaggtat ccacaacgcc ggcggcccta catggctctg ctgtagtgag tgggttgcgc 3660

tccggcagcg gtcctgatcc cccgcagaaa aaaaggatct caagaagatc ctttgatctt 3720

ttctacggcg cgcccagctg tctagggcgg cggatttgtc ctactcagga gagcgttcac 3780

cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg 3840

atgcct 3846

<210> 11

<211> 1770

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgaccccat ttgagcagac ggtccatcaa ttcctttcgc acctctacgg cccccgcgcc 60

agcgagatac agcgccgcat tggtcaacac attgagcact ttcgccgtgc gtcacaggcg 120

ccagccacaa cacaaaatat ggcggctgaa agcggtaacg caccaacatg gagcgaaaaa 180

gatcagtggg tcattagtta cggtgattcc attgtggcag aggaaacgcc cccacttgct 240

gtgcttaatg agtttcttca acgctatttg ggcgagcgca ttagcggcgt gcatgtactg 300

cctttttttc cttggagtag cgatgatggc ttctcagtca ttcattaccg tgaggttaat 360

ccagagctag gtgattggga gcacattcgc gagctagcca gccactatga cgtcatggcc 420

gacttagtgc ttaatcacgt atctcgtgag tcactgtggt ttgtagatta tctaaccggc 480

agcctgccag gtcgtgatta ctttattgaa gcagacccag aaaccgatgt ttctgacgtt 540

attcggccac gcagcagccc attgctagtg cccatttcca cgcgccgagg cactcgccat 600

gtatgggcga cattttcaga agatcagatt gatttaaact ttgaaaatcc tgatgtattg 660

ctagagtttg ttggcatttt gctgttttac cttcaacagg gcgtgcgcat cattcgcctg 720

gatgcgatag cgtttttatg gaagcgcctg ggaacatctt gcatccacct ccctgaaacc 780

catacggttg tccgtttact gcgcgccatt gtggatgaaa tatcgcctgg cacgctgata 840

attaccgaga ccaatgtccc tcacgcagaa aacatcagtt actttggcct agaacggtta 900

gccaatgggg cgcctgacga agcccatatg gtctatcaat ttgccctacc accactgcta 960

ttgcatacac tcacccgcgg tgaagccact acccttcaaa catggctttc cagcctgccg 1020

atactgccca gacactgcac ctatttaaac ttcacagcga gccatgatgg tattggtgtg 1080

cgtcctttag aagggctgct cccggaccat gaacgagatg cgctgctaga gctgatgcat 1140

cgctttggtg gctttgtcag tatgcgcagc aatcccgatg gtagtgactc gccctatgag 1200

atcaacatta cctggtttga ggccatgcgc ggcacgcgta gaggaccaga tccctggcaa 1260

attgcccgct tcctttgcag ccaggccatt atgctcacgc ttcagggcat tcccgccctc 1320

tatattcata cactgaccgg cacccttaac gatgttgaag gggtggaacg cagcggccgt 1380

ttacgttcaa ttaatcgccg ccgttggcag cgtgacgaat tagccctgtt acttgaaagc 1440

ccttcaacac caacccacga tgtttttcat gcccttagcc gcctgttgga gctacggcgg 1500

gtagaaccct gctttcaccc caacgccaca cagcgcgtac tgcccacacc tcctgaattg 1560

ttagccattg agcgaggacc actcagtgac ggccgccgcc tgcttgccct gtttaatgtc 1620

accgacacgc tgctgccatt agatagtgtg ggagaagcac ttgagcaggc gcttagccaa 1680

catgtttggc gtgctttaga catgcaaccc cccggagagg aaaccgcgct accgccctat 1740

gcaataagat ggatggtggc agacacatga 1770

<210> 12

<211> 1539

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgaaacaac aaaaacggct ttacgcccga ttgctgacgc tgttatttgc gctcatcttc 60

ttgctgcctc attctgcagc agcggcggca aatcttaatg ggacgctgat gcagtatttt 120

gaatggtaca tgcccaatga cggccaacat tggaagcgtt tgcaaaacga ctcggcatat 180

ttggctgaac acggtattac tgccgtctgg attcccccgg catataaggg aacgagccaa 240

gcggatgtgg gctacggtgc ttacgacctt tatgatttag gggagtttca tcaaaaaggg 300

acggttcgga caaagtacgg cacaaaagga gagctgcaat ctgcgatcaa aagtcttcat 360

tcccgcgaca ttaacgttta cggggatgtg gtcatcaacc acaaaggcgg cgctgatgcg 420

accgaagatg taaccgcggt tgaagtcgat cccgctgacc gcaaccgcgt aatttcagga 480

gaacacctaa ttaaagcctg gacacatttt cattttccgg ggcgcggcag cacatacagc 540

gattttaaat ggcattggta ccattttgac ggaaccgatt gggacgagtc ccgaaagctg 600

aaccgcatct ataagtttca aggaaaggct tgggattggg aagtttccaa tgaaaacggc 660

aactatgatt atttgatgta tgccgacatc gattatgacc atcctgatgt cgcagcagaa 720

attaagagat ggggcacttg gtatgccaat gaactgcaat tggacggttt ccgtcttgat 780

gctgtcaaac acattaaatt ttcttttttg cgggattggg ttaatcatgt cagggaaaaa 840

acggggaagg aaatgtttac ggtagctgaa tattggcaga atgacttggg cgcgctggaa 900

aactatttga acaaaacaaa ttttaatcat tcagtgtttg acgtgccgct tcattatcag 960

ttccatgctg catcgacaca gggaggcggc tatgatatga ggaaattgct gaacggtacg 1020

gtcgtttcca agcatccgtt gaaatcggtt acatttgtcg ataaccatga tacacagccg 1080

gggcaatcgc ttgagtcgac tgtccaaaca tggtttaagc cgcttgctta cgcttttatt 1140

ctcacaaggg aatctggata ccctcaggtt ttctacgggg atatgtacgg gacgaaagga 1200

gactcccagc gcgaaattcc tgccttgaaa cacaaaattg aaccgatctt aaaagcgaga 1260

aaacagtatg cgtacggagc acagcatgat tatttcgacc accatgacat tgtcggctgg 1320

acaagggaag gcgacagctc ggttgcaaat tcaggtttgg cggcattaat aacagacgga 1380

cccggtgggg caaagcgaat gtatgtcggc cggcaaaacg ccggtgagac atggcatgac 1440

attaccggaa accgttcgga gccggttgtc atcaattcgg aaggctgggg agagtttcac 1500

gtaaacggcg ggtcggtttc aatttatgtt caaagatag 1539

<210> 13

<211> 1539

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct aatttaaatg gcaccctgat gcagtacttc 120

gaatggtaca tgccgaacga cggccaacac tggaaacgcc tgcaaaacga ctctgcctat 180

ctggccgaac atggcattac cgccgtttgg atcccgccgg cctataaagg taccagccag 240

gccgatgttg gttatggtgc ctacgacctg tacgacctgg gcgaattcca ccagaaaggc 300

accgtgcgca ccaaatacgg caccaaaggc gaacttcaga gcgccatcaa atctctgcac 360

agccgcgaca tcaacgttta cggcgacgtg gtgatcaacc acaaaggcgg tgccgatgcc 420

actgaagatg ttaccgccgt ggaagttgac cctgccgatc gcaatcgtgt gattagcggc 480

gaacacctga tcaaagcctg gacccacttc cattttccgg gtcgcggtag cacttacagc 540

gacttcaaat ggcactggta ccactttgac ggcaccgact gggatgaaag ccgcaaactg 600

aaccgcatct acaaattcca gggcaaagcc tgggattggg aagtgagcaa cgaaaacggc 660

aactacgact acctgatgta cgccgacatc gactatgatc acccggatgt ggccgccgaa 720

attaaacgtt ggggcacctg gtatgccaac gaactgcagc tggacggttt tcgtctggat 780

gccgtgaaac acatcaaatt cagcttcctg cgcgactggg ttaatcacgt gcgcgaaaaa 840

accggcaaag aaatgttcac cgtggccgaa tactggcaga atgacctggg tgccctggaa 900

aactacctga acaagaccaa cttcaaccac agcgtgttcg atgtgccgct gcactatcaa 960

ttccacgccg cctctactca aggcggtggt tacgacatgc gcaaactgct gaacggcacc 1020

gtggttagca aacacccgct gaaaagcgtg accttcgtgg acaaccacga cactcaaccg 1080

ggtcagagcc tggaaagcac tgtgcagacc tggttcaaac cgctggccta cgcctttatt 1140

ctgacccgcg aaagcggtta tccgcaggtg ttctacggtg acatgtacgg caccaaaggc 1200

gatagccagc gtgaaatccc ggccctgaaa cacaaaatcg aaccgatcct gaaagcccgc 1260

aaacagtacg cctatggcgc ccagcacgac tattttgacc accacgacat cgttggctgg 1320

actcgcgaag gcgatagcag cgttgccaat tctggtctgg ccgccctgat tactgatggt 1380

ccgggcggtg ccaaacgtat gtatgtgggc cgtcaaaacg ccggtgaaac ctggcatgac 1440

atcactggca accgtagcga accggtggtg atcaactctg aaggctgggg cgaatttcat 1500

gtgaatggcg gcagcgtgag catttacgtg cagcgctaa 1539

<210> 14

<211> 189

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgcctccac accgctcgtc acatcctgcc catgagttaa ttatatttgt ggcattatag 60

ggagaattgt gagcgctcac aattagctgt caccggatgt gctttccggt ctgatgagtc 120

cgtgaggacg aaacagcctc tacaaataat tttgtttaag agttactaga gaaagaggag 180

aaatactag 189

<210> 15

<211> 456

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgaaacagc agaaacgcct gtatgcccgt cttctgactc tgctgttcgc cctgattttc 60

ctgcttccgc atagcgctgc cgctgccgct atgaatatgg acgtgatgaa tatggacgtg 120

atcaagagct ttaccgagca gatgcaaggc ttcgccgccc ccctcacccg ctacaaccaa 180

ctgctggcca gcaacatcga gcagctgacc cggttgcagc tggcctccgc caacgcctac 240

gccgaactgg gcctcaacca gttgcaggcc gtgagcaagg tgcaggacac ccagagtctg 300

gctgccctcg gcacagtgca gctggagacc gccagccagc tctcccgcca gatgctggac 360

gacatccaga agctgagcgc cctcggccag cagttcaagg aagagctgga tgtcctgacc 420

gcggacggca tcaagaagag cacgggcaag gcctaa 456

<210> 16

<211> 151

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe

1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Met Asn

20 25 30

Met Asp Val Met Asn Met Asp Val Ile Lys Ser Phe Thr Glu Gln Met

35 40 45

Gln Gly Phe Ala Ala Pro Leu Thr Arg Tyr Asn Gln Leu Leu Ala Ser

50 55 60

Asn Ile Glu Gln Leu Thr Arg Leu Gln Leu Ala Ser Ala Asn Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Lys Lys Ser Thr Gly Lys Ala

150

<210> 17

<211> 3769

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttaattaaag cggataacaa tttcacacag gaatgcctcc acaccgctcg tcacatcctg 60

cccatgagtt aattatattt gtggcattat agggagaatt gtgagcgctc acaattagct 120

gtcaccggat gtgctttccg gtctgatgag tccgtgagga cgaaacagcc tctacaaata 180

attttgttta agagttacta gagaaagagg agaaatacta ggtcgtgact gggaaaaccc 240

tggcgactag tcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg 300

atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaggggtcc 360

ccaataatta cgatttaaat tggcgaaaat gagacgttga tcggcacgta agaggttcca 420

actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt 480

caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat 540

cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata 600

accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca 660

agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc 720

gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg 780

ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc 840

ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt 900

tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca 960

ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg 1020

gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg 1080

ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg 1140

agtggcaggg cggggcgtaa tttgactttt gtccttttcc gctgcataac cctgcttcgg 1200

ggtcattata gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg 1260

ccaaagggtt cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg 1320

tgaagtaggc ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc 1380

ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag gccggccgcg atgcaggtgg 1440

ctgctgaacc cccagccgga actgacccca caaggcccta gcgtttgcaa tgcaccaggt 1500

catcattgac ccaggcgtgt tccaccaggc cgctgcctcg caactcttcg caggcttcgc 1560

cgacctgctc gcgccacttc ttcacgcggg tggaatccga tccgcacatg aggcggaagg 1620

tttccagctt gagcgggtac ggctcccggt gcgagctgaa atagtcgaac atccgtcggg 1680

ccgtcggcga cagcttgcgg tacttctccc atatgaattt cgtgtagtgg tcgccagcaa 1740

acagcacgac gatttcctcg tcgatcagga cctggcaacg ggacgttttc ttgccacggt 1800

ccaggacgcg gaagcggtgc agcagcgaca ccgattccag gtgcccaacg cggtcggacg 1860

tgaagcccat cgccgtcgcc tgtaggcgcg acaggcattc ctcggccttc gtgtaatacc 1920

ggccattgat cgaccagccc aggtcctggc aaagctcgta gaacgtgaag gtgatcggct 1980

cgccgatagg ggtgcgcttc gcgtactcca acacctgctg ccacaccagt tcgtcatcgt 2040

cggcccgcag ctcgacgccg gtgtaggtga tcttcacgtc cttgttgacg tggaaaatga 2100

ccttgttttg cagcgcctcg cgcgggattt tcttgttgcg cgtggtgaac agggcagagc 2160

gggccgtgtc gtttggcatc gctcgcatcg tgtccggcca cggcgcaata tcgaacaagg 2220

aaagctgcat ttccttgatc tgctgcttcg tgtgtttcag caacgcggcc tgcttggctt 2280

cgctgacctg ttttgccagg tcctcgccgg cggtttttcg cttcttggtc gtcatagttc 2340

ctcgcgtgtc gatggtcatc gacttcgcca aacctgccgc ctcctgttcg agacgacgcg 2400

aacgctccac ggcggccgat ggcgcgggca gggcaggggg agccagttgc acgctgtcgc 2460

gctcgatctt ggccgtagct tgctggacta tcgagccgac ggactggaag gtttcgcggg 2520

gcgcacgcat gacggtgcgg cttgcgatgg tttcggcatc ctcggcggaa aaccccgcgt 2580

cgatcagttc ttgcctgtat gccttccggt caaacgtccg attcattcac cctccttgcg 2640

ggattgcccc ggaattaatt ccccggatcg atccgtcgat cttgatcccc tgcgccatca 2700

gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga 2760

gggcgcccca gctggcaatt ccggttcgct tgctgtccat aaaaccgccc agtctagcta 2820

tcgccatgta agcccactgc aagctacctg ctttctcttt gcgcttgcgt tttcccttgt 2880

ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg gactggcttt 2940

ctacgtggct gccatttttg gggtgaggcc gttcgcggcc gaggggcgca gcccctgggg 3000

ggatgggagg cccgcgttag cgggccggga gggttcgaga agggggggca ccccccttcg 3060

gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa aacaaggttt ataaatattg 3120

gtttaaaagc aggttaaaag acaggttagc ggtggccgaa aaacgggcgg aaacccttgc 3180

aaatgctgga ttttctgcct gtggacagcc cctcaaatgt caataggtgc gcccctcatc 3240

tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc 3300

ccctcaagtg tcaataccgc agggcactta tccccaggct tgtccacatc atctgtggga 3360

aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc tggccagctc cacgtcgccg 3420

gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag 3480

tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt ttccgcgagg tatccacaac 3540

gccggcggcc ctacatggct ctgctgtagt gagtgggttg cgctccggca gcggtcctga 3600

tcccccgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gcgcgcccag 3660

ctgtctaggg cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa 3720

aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcct 3769

Claims (3)

1. A method for producing and secreting amylase by using halophilic bacteria, which is characterized by comprising the following steps: the method comprises the steps of fermenting recombinant halophilic bacteria to obtain a fermentation product, and separating amylase from the fermentation product; the recombinant halophilic bacteria are obtained by introducing recombinant amylase genes into halophilic bacteria;

the halophilic bacteria are halophilic bacteriaHalomonas bluephagenesis；

The coding sequence of the recombinant amylase gene is shown as a sequence 2.

2. A recombinant amylase gene characterized by: the coding sequence is shown as sequence 2.

3. A method for producing PHA using halophilic bacteria, comprising: the method comprises the steps of producing PHA by using the recombinant halophilic bacteria by taking starch as a substrate; a recombinant bacterium obtained by introducing the recombinant amylase gene described in claim 2 into a halophilic bacterium; the halophilic bacteria are halomonasHalomonas bluephagenesis。

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