CN105755033A - General bacillus subtillis combination expression vector for improving expression level of alpha-amylase - Google Patents

Abstract

The invention discloses a universal Bacillus subtilis integrated expression vector capable of increasing the expression level of α-amylase. The present invention relates to a general-purpose Bacillus subtilis integrated expression vector, comprising the following elements: a promoter sequence of Bacillus subtilis, a multiple cloning site sequence for integrating various elements that may be involved in expression regulation, and a replication promoter of Escherichia coli start sequence. The vector of the present invention can not only be stably replicated in Escherichia coli, but also can be integrated into the chromosome of Bacillus subtilis for stable expression. After the carrier of the present invention is integrated into the Bacillus subtilis chromosome, the expression level of alpha-amylase in the Bacillus subtilis can be significantly improved.

Description

A general-purpose Bacillus subtilis integrated expression vector for increasing the expression level of α-amylase

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a general-purpose Bacillus subtilis integrated expression vector, which is particularly suitable for increasing the expression level of α-amylase.

Background technique

Bacillus subtilis ( Bacillus subtilis ) belongs to Gram-positive bacteria and is a non-pathogenic and important industrial microorganism. Its whole genome sequencing work has been completed. Its genetic background and physiological properties are second only to E. coli in understanding. The use of Bacillus subtilis to express foreign proteins has many advantages: (1) high safety, and can be used in industrial production such as food and medicine; (2) can directly release secreted proteins into the medium, which is conducive to separation and purification; (3) has Efficient ability to secrete the target protein, and the eukaryotic-derived recombinant protein secreted by it can still maintain the natural conformation and biological activity in most cases; (4) rapid growth, simple culture conditions, clear understanding of genetic background, and good industrial production Base.

α-amylase (α-amylase, EC3.2.1.1), also known as liquefied amylase, uses glycogen or starch as a substrate, cuts α-1, 4 glycosidic bonds from the inside of the molecule, and the product is dextrin , an oligosaccharide containing more than four glucose residues and a small amount of glucose and maltose. For a long time, α-amylase has been widely used in fields including starch liquefaction, textile desizing, brewing and baking.

The production of α-amylase using the wild-type Bacillus subtilis expression system is usually very low. This is because there are many bottlenecks in the process of protein transport and secretion of α-amylase. On the one hand, when the regulatory elements involved in protein transport in the cell cannot meet the demand α-amylase cannot be transported to the appropriate cell site in time during protein transport and is degraded by intracellular proteases. On the other hand, when the limiting factors involved in protein secretion in cells are lacking, α-amylase is affected Proper folding during secretion causes α-amylase to be degraded by proteases in the cell wall. Therefore, by increasing various restriction regulatory factors involved in protein expression and secretion in Bacillus subtilis, it will be beneficial to increase the expression and secretion of α-amylase.

Contents of the invention

The purpose of the present invention is to provide a general-purpose Bacillus subtilis genome integration expression vector, which integrates various restriction factors that may be involved in the expression and secretion bottleneck of exogenous proteins into the amyE gene of Bacillus subtilis chromosome through homologous recombination In addition, it can greatly increase the expression level of foreign proteins, especially α-amylase.

The plasmid vector of the present invention comprises:

The nucleotide sequence of the grac promoter sequence of Bacillus subtilis is SEQ ID NO:1.

The multiple cloning site MCS for inserting foreign gene fragments has a nucleotide sequence of SEQ ID NO:2.

The nucleotide sequences of the 5' and 3' homology arm sequences used to integrate the amyE site are SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The vector of the present invention also contains the Escherichia coli replicon and the coding sequence of the chloramphenicol resistance gene.

The genetic map of the carrier of the present invention is shown in Figure 2, and its nucleotide sequence is SEQ ID NO:5.

The nucleotide sequence of the overexpression control element PrsA involved in the present invention is SEQ ID NO:6.

The genetic map of the plasmid in Example 3 of the present invention is shown in Figure 3, and its nucleotide sequence is SEQ ID NO:7.

The nucleotide sequences of various regulatory elements SecA, SecDF, SecG, Ffh, SipU, SipV, and SipW in the specific example 4 of the present invention are SEQ ID NOs 8-14.

The integrated vector constructed by the present invention is used to increase the expression level of foreign protein, especially α-amylase, in Bacillus subtilis (such as B. subtilis 168, WB600, WB700, WB800, 1A751, DB104, etc.).

The expression vector constructed by the present invention can be specifically integrated into the amyE gene position of the Bacillus subtilis chromosome by means of homologous recombination, and by overexpressing various restriction factors that may be involved in the bottleneck of exogenous protein expression and secretion in cells, the The transport level of exogenous protein, especially α-amylase in Bacillus subtilis may promote the correct folding of protein, thereby greatly increasing the secretion level of exogenous protein, especially α-amylase in the medium.

Description of drawings:

Fig. 1: The schematic diagram of the construction of the plasmid vector pPgrac-MCS of the present invention;

Fig. 2: the genetic map of plasmid vector pPgrac-MCS of the present invention;

Fig. 3: the genetic map of pPgrac-PrsA in embodiment 3;

Figure 4: Colony PCR verification diagram in Example 3; wherein: M.TIANGENDNA MarkerⅢ; 1. Colony PCR results; 2. Positive control;

Figure 5: SDS-PAGE electrophoresis of the supernatant of the fermentation broth in Example 3: Among them: M.PageRuler ^TM Prestained Protein Ladder; 1.1A751 fermentation broth supernatant; 2.1A751 (amyE::Pgrac-PrsA) fermentation broth supernatant; 3. pMA5-AmyL(1A751) fermentation broth supernatant;

4. The supernatant of pMA5-AmyL (1A751(amyE::Pgrac-PrsA)) fermentation broth.

detailed description

The vector of the present invention uses pDL as the starting plasmid. During the construction process, the grac sequence of the Bacillus subtilis promoter and the multiple cloning site are introduced by means of double enzyme digestion and point mutation, and the original bgaB sequence is replaced. The vector can be efficiently integrated into the amyE position of the Bacillus subtilis chromosome through homologous recombination. The integrated Bacillus subtilis can overexpress various restriction factors that may be involved in the bottleneck of exogenous protein expression and secretion in cells, thereby increasing the secretion level of the exogenous protein in Bacillus subtilis.

The vector construction process and application of the present invention will be described in detail below.

Example 1 Construction of plasmid vector pPgrac-MCS.

The first step is to introduce the Bacillus subtilis Pgrac promoter (including groE promoter, lacO operator and gsiBCD ribosome binding site sequence) between the multiple cloning sites Eco RI and Bam HI of pDL (derived from BGSC, numbered ECE144) ).

Design primers, where the part in black bold font is the restriction site:

Pgrac-F: 5'-CCG GAATTC GAAAAGAATGATGTAAGC -3' (contains Eco RI restriction site),

Pgrac-R: 5'-CGC GGATCC TTCCTCCTTTAATTGG-3' (contains B am HI restriction site).

Use pHT43 as a template, use the above primers to carry out PCR, and mix the components in a sterilized thin-walled centrifuge tube in the following order. The reaction system is:

Upstream primer Pgrac-F (10 μmol/L) 2 μL,

Downstream primer Pgrac-R (10 μmol/L) 2 μL,

DNA template 1 μL,

PrimeSTARMaxPremix (2×) 10 μL,

ddH ₂ O 5 μL,

The total volume is 20 μL.

The amplification conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 5s, 30 cycles; the last cycle was 72°C for 4min.

The PCR product was digested with Eco RI and Bam HI and recovered to obtain the target fragment P1; the vector pDL was digested with Eco RI and Bam HI to recover the target fragment P2. P1 and P2 were ligated overnight at 16°C and transformed into Escherichia coli DH5α to obtain a plasmid named pDL-Pgrac.

In the second step, on the basis of the vector obtained from the above-mentioned plasmid pDL-Pgrac, a multiple cloning site was introduced by way of point mutation to construct a pPgrac-MCS integration vector.

Design primers, where the part in black bold font is the restriction site:

MCS-F: 5'-GCCGCA CTCGAG C GAGCTC CTGCACTGGATG-3' (phosphorylation modification at the 5' end, including Xho I, Sac I restriction sites),

MCS-R: 5'-CGC AAGCTT GTCGA GGTACC TACGTA GGATCC TTCCTC-3' (phosphorylated at the 5' end, including Hind III, Kpn I, Bam HI restriction sites).

Use the plasmid pDL-Pgrac as a template to carry out PCR, and mix the components in a sterilized thin-walled centrifuge tube in the following order. The reaction system is:

Upstream primer MCS-F: (10 μmol/L) 2 μL,

Downstream primer MCS-R: (10 μmol/L) 2 μL,

DNA template 1 μL,

PrimeSTARMaxPremix (2×) 10 μL,

ddH ₂ O 5 μL,

The total volume is 20 μL.

The amplification conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 4min, 30 cycles; the last cycle was 72°C for 4min.

The PCR product was digested with Dpn I at 37°C for 3 hours to digest the template plasmid. The reaction system was as follows:

PCR product 17 μL,

Dpn I (NEB) 1 μL,

10×Cutsmart buffer 2μL,

The total volume is 20 μL.

The target fragment after enzyme digestion was recovered, ligated with T4 ligase overnight at 16°C and transformed into Escherichia coli DH5α to obtain a plasmid, and the correctly sequenced plasmid was named pPgrac-MCS.

Example 2 The plasmid vector pPgrac-PrsA was constructed and integrated into the chromosome amyE of Bacillus subtilis.

The intermolecular chaperone PrsA (nucleotide sequence is SEQ ID NO: 6) can help α-amylase to quickly and correctly fold, reduce the chance of α-amylase being degraded by proteases in the cell wall, and may promote the expression and secretion of α-amylase increase in volume. The carrier of the present invention was used to evaluate the effect of PrsA overexpression on the expression and secretion of α-amylase in Bacillus subtilis.

(1) Construction of plasmid vector pPgrac-PrsA.

Based on the pPgrac-MCS plasmid, the pPgrac-PrsA integration vector was constructed.

Design primers, where the part in black bold font is the restriction site:

PrsA-F: 5'-CGG GGTACC ATGAAGAAAATCGCAATAGCAGCT-3' (including Kpn I restriction site),

PrsA-R: 5'-TGA GAGCTC TTAGCATATTATGTTGCCAACTGT-3' (including Sac I restriction site),

Use the Bacillus subtilis 168 genome as a template to carry out PCR, and mix the components in a sterilized thin-walled centrifuge tube in the following order. The reaction system is:

Upstream primer PrsA-F (10 μmol/L) 2 μL,

Downstream primer PrsA-R (10 μmol/L) 2 μL,

DNA template 1 μL,

PrimeSTARMaxPremix (2×) 10 μL,

ddH ₂ O 5 μL,

The total volume is 20 μL.

The amplification conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 1min, 30 cycles; the last cycle was 72°C for 4min.

The PCR product was digested and recovered with Kpn I and Sac I to obtain the target fragment P3; the vector pDL-MCS was digested with Kpn I and Sac I to recover the target fragment P4. P3 and P4 were ligated overnight at 16°C and transformed into Escherichia coli DH5α to obtain a plasmid named pPgrac-PrsA.

(2) pPgrac-PrsA integrated into Bacillus subtilis chromosome amyE.

Competent cells of Bacillus subtilis 1A751 (preserved in the laboratory) were prepared as follows:

1. Pick a freshly activated 1A751 single colony and insert it into 5mL GMI (10×Spizizen salt ((NH4) ₂ S0 ₄ 2g, K ₂ HPO4 18.3g, KH ₂ PO46g, sodium citrate 1.2g, deionized water to 100mL ) 0.5mL, 2% acid hydrolyzed casein 0.1mL, 5% yeast extract 0.1mL, 40% glucose 0.1mL, 20% magnesium sulfate monohydrate 5μL, 0.5% L-tryptophan 50μL, sterile water to make up to 5mL) culture medium, shake culture at 200rpm at 37℃ for 14-16h;

2. Transfer 500 μL of the above-mentioned bacterial solution to 4.5 mL of GMI medium, and culture at 37°C with shaking at 200 rpm for 4.5 hours;

3. Inoculate 4.25mL GMII with 750μL of the above-mentioned GMI bacterial solution (5mL GMII contains 0.5mL of 10×Spizizen salt, 50μL of 2% acid hydrolyzed casein, 0.1mL of 40% glucose, 40μL of 20% magnesium sulfate monohydrate, and make up to 5mL) in the culture medium, shake culture at 240rpm at 37°C for 1.5h to prepare competent cells.

Take 1 μg of plasmid pPgrac-PrsA and linearize it with Pst I digestion, transform into Bacillus subtilis 1A751, use a plate containing chloramphenicol (25 μg/mL) resistance to screen positive clones, and verify by colony PCR.

Colony PCR primers:

AmyE-PrsA-F:5'-GGAAGGATCCTACGTAGGT-3'

AmyE-PrsA-R:5'-ACTGTCGGAACGAGACTTC-3'

The reaction system is

Upstream primer AmyE-PrsA-F (10 μmol/L) 2 μL,

Downstream primer AmyE-PrsA-R (10 μmol/L) 2 μL,

DNA template 1 μL,

PrimeSTARMaxPremix (2×) 10 μL,

ddH ₂ O 5 μL,

The total volume is 20 μL.

The amplification conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 1min, 30 cycles; the last cycle was 72°C for 4min.

The size of the PCR product band was consistent with the expectation (Figure 4). The colony PCR product was sent for sequencing. The sequencing results showed that Pgrac-PrsA had been integrated into the amyE gene. The constructed strain was named 1A751 (amyE::Pgrac-PrsA) .

Example 3 Fermentative production of α-amylase by Bacillus subtilis 1A751 (amyE::Pgrac-PrsA).

Transform 1A751 and 1A751(amyE::Pgrac-PrsA) with plasmid pMA5-AmyL (AmyL gene is derived from Bacillus licheniformis CICC10181) already in the laboratory carrying the amylase gene of Bacillus licheniformis, and pick one transformant to inoculate Cultivate overnight at 37°C in 2×SR medium (3% peptone, 6% yeast extract, 0.6% K ₂ HPO ₄ ). On the second day, inoculate 30mL of 2×SR culture medium according to the inoculation amount of 1%, ferment at 37°C and 200rpm for 48h, centrifuge the fermented liquid at 12000rpm at 4°C for 10min, and take the supernatant to detect the amylase activity. Two parallel, the results are shown in Table 1.

The detection method of α-amylase activity refers to the national standard method (GBT24401-2009 α-amylase preparation detection method).

Definition of enzyme activity: Under specified conditions, liquefaction of 1 g of soluble starch in 1 hour is 1 unit of enzyme activity, expressed in "U/g (U/mL)". The determination method is as follows: 1. Enzyme solution preparation: prepare enzyme solution with buffer solution, so that the final enzyme concentration is controlled within the range of 65U/mL-70U/mL. 2. Determination: (1) Draw 20mL of soluble starch solution (20g/L) and 5mL of phosphate buffer solution (corresponding to pH6.0) into a test tube. Preheat in a constant temperature water bath at 70°C for 8 minutes. (2) Add 1.00mL of the diluted enzyme solution to be tested, time it immediately, shake it well, and react accurately for 5 minutes. (3) Immediately pipette 1.00 mL of the reaction solution into a test tube pre-filled with 0.5 mL of 0.1 mol/L HCl and 5 mL of dilute iodine solution and shake well. (4) Use the mixture of 0.1mol/L HCl0.5mL and 5mL dilute iodine solution as a blank, and quickly measure the absorbance (A) with a 10mm cuvette at a wavelength of 660nm. According to the absorbance look-up table, obtain the concentration (C) of the test enzyme solution. 3. Calculation: X=C×N×16.67 In the formula: X-sample enzyme activity U/mL (U/g); C-test enzyme solution concentration U/mL; N-sample dilution factor; 16.67-conversion constant. Results obtained are expressed to integers.

Table 1

From the experimental results in Table 1, it can be seen that after the pPgrac-PrsA plasmid was integrated into the chromosome amyE of Bacillus subtilis 1A751, the enzyme activity of α-amylase was significantly improved. Compared with the positive control group (wild type 1A751), the enzyme activity of the test group after shake flask level fermentation can reach 750U/mL, which is 2.42 times higher.

SDS-PAGE electrophoresis detection was performed on the fermentation broth after 48 hours of fermentation, and the detection results are shown in FIG. 4 .

From the results of electrophoresis detection in Figure 4, it can be seen that the expression level of α-amylase contained in the supernatant of the fermentation broth in the test group was significantly higher than that of the positive control group, that is, after the pPgrac-PrsA plasmid was integrated into the Bacillus subtilis 1A751 chromosome amyE, the The secretion of α-amylase was significantly improved.

Example 4 Using the vector of the present invention to evaluate the effect of overexpression of SecA, SecDF, SecG, Ffh, SipU, SipV, SipW, etc. in Bacillus subtilis cells on the expression and secretion of α-amylase.

In addition to the intercellular chaperone PrsA in Example 3, there are many restriction factors that may be involved in the process of protein transport and secretion in cells, including the following categories, respectively (1) SRP (signal peptide recognition particle) Series: Ffh, HBsu, scRNA, etc.; (2) Signal peptidase series: SipS, SipT, SipU, SipV, SipW, etc.; (3) Transporter series: SecA, SecYEG, SecDF, etc.; (4) Molecular chaperone series: GroES , GroEL, DnaK, etc. By constructing various possible regulatory element factors into the Bacillus subtilis integration vector of the present invention, it is integrated into the Bacillus subtilis 1A751 chromosome for overexpression. The existing plasmid pMA5-AmyL (AmyL gene is derived from Bacillus licheniformis CICC10181) was transformed into various constructed Bacillus subtilis strains, and the enzyme activity of α-amylase was detected by fermentation. Part of the regulatory elements (SecA, SecDF, SecG, Ffh, SipU, SipV, SipW, etc., the nucleotide sequence of which is SEQ ID NO: 8-14) can promote the expression and secretion of AmyL in this example to varying degrees.

(A) Construction of plasmid vectors.

Based on the pPgrac-MCS plasmid, the pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Ffh, pPgrac-SipU, pPgrac-SipV, pPgrac-SipW integration vectors were constructed.

Select a suitable multiple cloning site, design primers, and use the Bacillus subtilis 168 genome as a template to perform PCR. Mix the components in a sterilized thin-walled centrifuge tube in the following order. The reaction system is:

Upstream primer (10 μmol/L) 2 μL,

Downstream primer (10μmol/L) 2μL,

DNA template 1 μL,

PrimeSTARMaxPremix (2×) 10 μL,

ddH ₂ O 5 μL,

The total volume is 20 μL.

The amplification conditions were: 98°C for 3min, 1 cycle; 98°C for 10s, 55°C for 15s, 72°C for 2kb/min, 30 cycles; the last cycle was 72°C for 4min.

The PCR product was subjected to double enzyme digestion and recovery to obtain the target fragment P5; the vector pPgrac-MCS was subjected to double enzyme digestion at the corresponding restriction sites to recover the target fragment P6. P5 was ligated with P6 overnight at 16°C and transformed into Escherichia coli DH5α to obtain plasmids, which were named pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Fth, pPgrac-SipU, pPgrac-SipV and pPgrac-SipW.

(2) The plasmid constructed above was integrated into the chromosome amyE of Bacillus subtilis.

According to the preparation method in specific example 3, Bacillus subtilis 1A751 competent cells were prepared. Take 1 μg of the constructed plasmid and select the appropriate enzyme cutting site for single enzyme digestion, transform Bacillus subtilis 1A751, use a plate containing chloramphenicol (25 μg/mL) resistance to screen positive clones, design primers, and colony PCR method authenticating. The verified correct colony PCR products were sent for sequencing, and the verified correct strains were named 1A751 (amyE::Pgrac-SecA), 1A751 (amyE::Pgrac-SecDF), 1A751 (amyE::Pgrac-SecG) , 1A751 ( amyE::Pgrac-Ffh), 1A751 (amyE::Pgrac-SipU), 1A751 (amyE::Pgrac-SipV) and 1A751 (amyE::Pgrac-SipW).

(3) Using the Bacillus subtilis strain constructed in this example to ferment and produce α-amylase.

The laboratory already has plasmid pMA5-AmyL (AmyL gene is derived from Bacillus licheniformis CICC10181) to transform 1A751 and other constructed integrated strains respectively, pick one transformant and inoculate them in 2×SR medium (3% peptone, 6% yeast extract, 0.6% K ₂ HPO4), and cultured overnight at 37°C. On the second day, inoculate 30 mL of 2×SR culture medium according to the inoculum amount of 1%, ferment at 37°C and 200 rpm for 48 hours, centrifuge the fermentation liquid at 12,000 rpm at 4°C for 10 minutes, and take the supernatant to detect the amylase activity. The results are shown in Table 2.

The detection method of α-amylase enzyme activity refers to: GBT24401-2009 detection method of α-amylase preparation.

For the specific method, refer to the method in Example 3.

Table 2

From the experimental results in Table 2, it can be known that after plasmids such as pPgrac-SecA, pPgrac-SecDF, pPgrac-SecG, pPgrac-Fth, pPgrac-SipU, pPgrac-SipV, pPgrac-SipW were integrated into the chromosome amyE of Bacillus subtilis 1A751, the α- The secretion of amylase has a certain promoting effect. Compared with the positive control group (wild-type 1A751), the enzyme activity after shake flask horizontal fermentation could reach (287-345) U/mL, which was increased by 0.2-0.5 times respectively.

Therefore, the Bacillus subtilis integrated vector constructed by the present invention has certain versatility, and various restriction factors that may be involved in protein transport, expression and secretion can be inserted into the The vector is thus integrated into the chromosome amyE of Bacillus subtilis, and through the overexpression of various regulatory elements, the expression and secretion level of foreign protein, especially α-amylase, is promoted.

SEQUENCELISTING

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

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atcctgcatcatgaatatccttcatattgtgttttaacgtattgaacgaccaattccatg1560

catgaagaatggttccgcttttgatcgacggtgctgtaagctcattcgatttgttcgccg1620

tttcagcactcgcagccgccggtcctgccagaaccaaatgaaacagcaataaaaatccag1680

cgaataacggcagtaaagaggttttgaatcgttttgcaaacattcttgacactccttatt1740

tgattttttgaagacttacttcggagtcaaaaatccctcttacttcattcttccgcttcc1800

tcctttcaaaccgatgtgaagactggagaattttgttaattcttgaagacgaaagggcct1860

cgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcagg1920

tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattc1980

aaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaag2040

gaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttg2100

ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtt2160

gggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttt2220

tcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggt2280

attatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaa2340

tgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag2400

agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac2460

aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac2520

tcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacac2580

cacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttac2640

tctagcttcccggcaacaattaatatagactggatggaggcggataaagttgcaggaccact2700

tctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg2760

tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagt2820

tatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat2880

aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttta2940

gattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataa3000

tctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtaga3060

aaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac3120

aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt3180

tccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagcc3240

gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaat3300

cctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaag3360

acgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc3420

cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaag3480

cgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac3540

aggagagcgcacgaggggagcttccagggggaaacgcctggtatctttatagtcctgtcgg3600

gtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcct3660

atggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgc3720

tcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttga3780

gtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagga3840

agcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg3900

catatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacact3960

ccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgac4020

gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc4080

gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcgg4140

taaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtcc4200

agctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgtta4260

agggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatg4320

ggggtaatgataccgatgaacgagagaggatgctcacgatacgggttactgatgatgaac4380

atgcccggttatggaacgttgtgagggtaaacaactggcggtatggatgcggcgggacc4440

agagaaaatcactgttcatgggggtaatgataccgatgaaacgagagaggatgctcacga4500

tacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactgg4560

cggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgtta4620

atacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaaca4680

taatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaaga4740

ccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgct4800

cgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcc4860

tcaacgacaggagcacgatcatgcgcacccgtggccaggacccaacgctgcccgagatgc4920

gccgcgtgcggctgctggagatggcggacgcgatggatatgttctgccaagggttggttt4980

gcgcattcacagttctccgcaagaattgattggctccaattcttggagtggtgaatccgt5040

tagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacg5100

caacgcgggggaggcagacaaggtatagggcggcgcctacaatccatgccaacccgttcca5160

tgtgctcgccgaggcggcataaatcgccgtgacgatcagcggtccaatgatcgaagttag5220

gctggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcct5280

ggacagcatggcctgcaacgcgggcatcccgatgccgccggaagcgagaagaatcataat5340

ggggaaggccatccagcctcgcgtcgcgactaagaaaatgccgtcaaatccgctcgccat5400

gacttcactaacgatgcctttgaaaatcttcaagttcttttctactaattcaaggcgtgt5460

ctcaccaggtttttggtttgctccggcgcaaatgcagacaatatcagcatccttgcaggg5520

tatgtttctctttgatgtctttttgtttgtgaagtatttcacatttatattgtgcaacac5580

ttcacaaacttttgcaagagaaaagttttgtctgatttatgaacaaaaaagaaaccatca5640

ttgatggtttctttcggtaagtcccgtctagccttgccctcaatggggaagagaaccgct5700

taagcccgagtcatttatataaaccatttagcacgtaatcaaagccaggctgattctgacc5760

gggcacttgggcgctgccattattaaaaatcacttttgcgttggttgtatccgtgtccgc5820

aggcagcgtcagcgtgtaaattccgtctgcatttttagtcattggttttccaggccaaga5880

tccggtcaattcaattactcggctcccatcatgtttatagatataagcatttacctggct5940

ccaatgattcggattttgatagccgatggttttggccgacgctggatctcttttaacaaa6000

actgtatttctcggtcctcgttacaccatcactgttcgttccttttaacatgatggtgta6060

tgttttgccaaatggatctccttttccgattgtgaattgatctccatccttaaacgccgt6120

ctctggtccattattgatttgataaacggcttttgttgtattcgcatctgcacgcaaggt6180

aatcgtcagttgatcattgaaagaatgtgttacacctgttttgtaattctcaaggaaaac6240

atgaggcgcttttgcaatatcatcaggataaagcacagctacagacctggcattgatcgt6300

gcctgtcagtttaccatcgttcacttgaaatgaacccgctccagctttatgtcatacct6360

gccatcaggcaattttgttgccgtattgatagagacagaggatgaacctgcatttgccag6420

cacaacgccatgtgagccgcgctgattcataaatatctggttgtttccattcgggttcga6480

gagttcctcaggctgtccagccatcacattgtgaaatctattgaccgcagtgatagcctg6540

atcttcaaataaagcactcccgcgatcgcctatttggcttttccccgggaacctcacacc6600

atttccgcctccctcaggtctggaaaagaaaagaggcgtactgcctgaacgagaagctat6660

caccgcccagcctaaacggatatcatcatcgctcatccatgtcgacgctctcccttatgc6720

gactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgca6780

aggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccacc6840

atacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcg6900

gtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacg6960

atgcgtccggcgtagaggatctggagctgtaatataaaaaccttcttcaactaacggggc7020

aggttagtgacattagaaaaccgactgtaaaaagtacagtcggcattatctcatattata7080

aaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcat7140

gataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattac7200

ctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattga7260

tattaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcatttt7320

caggtataggtgttttgggaaacaatttccccgaaccatttatatttctctacatcagaaa7380

ggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatg7440

ttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaact7500

ctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcacta7560

agaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacac7620

taatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaata7680

tctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcct7740

aaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaa7800

tccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactt7860

tatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacatt7920

aaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttct7980

ttcttatcttgataataagggtaactattgccgatgataagctgtcaaacatgagaattc8040

gaaaagaatgatgtaagcgtgaaaaattttttatcttatcacttgaaattggaagggaga8100

ttctttattataagaattgtggaattgtgagcggataacaattcccaattaaaggaggaa8160

ggatcctacgtaggtacctcgacaagcttgcggccgcactcgagcgagct8210

<210>6

<211>879

<212> DNA

<213> Bacillus subtilis

<400>6

atgaagaaaatcgcaatagcagctatcactgctacaagcatcctcgctctcagtgcttgc60

agcagcggcgacaaagaagttatcgcaaaaacagacgcaggcgatgtcacaaaaggcgag120

ctttacacaaacatgaagaaaacagctggcgcaagcgtactgacacagctagtgcaagaa180

aaagtattggacaagaagtataaagtttcggataaagaaattgacaacaagctgaaagaa240

tacaaaacgcagcttggcgatcaatatactgccctcgaaaagcaatatggcaaagattac300

ctgaaagaacaagtaaaatatgaattgctgacacaaaaagcggctaaagataacatcaaa360

gtaacagacgccgatatcaaagagtactgggaaggcttaaaaggcaaaatccgtgcaagc420

cacatccttgttgctgataaaaagacagctgaagaagtagagaaaaagctgaaaaaaggc480

gagaagtttgaagaccttgcgaaagaatactcaacagacagctctgcttcaaaaggcggg540

gatcttggctggttcgcaaaagaaggccaaatggacgaaacattcagcaaagctgcattc600

aaattaaaaacaggtgaagtcagtgatcctgtcaaaacgcaatacggctaccatatcatt660

aaaaagacagaagaacgcggcaaatatgatgatatgaaaaaagaactgaaatctgaagtg720

cttgaacaaaaattaaatgacaacgcagctgttcaggaagctgttcaaaaagtcatgaag780

aaggctgacatcgaagtaaaagataaagatctgaaagacacatttaatacatcttcaaca840

agcaacagcacttcttcatcttcaagcaattctaaataa879

<210>7

<211>9101

<212> DNA

<213> Artificial sequence

<400>7

catgaagaaaatcgcaatagcagctatcactgctacaagcatcctcgctctcagtgcttg60

cagcagcggcgacaaagaagttatcgcaaaaacagacgcaggcgatgtcacaaaaggcga120

gctttacacaaacatgaagaaaacagctggcgcaagcgtactgacacagctagtgcaaga180

aaaagtattggacaagaagtataaagtttcggataaagaaattgacaacaagctgaaaga240

atacaaaacgcagcttggcgatcaatatactgccctcgaaaagcaatatggcaaagatta300

cctgaaagaacaagtaaaatatgaattgctgacacaaaaagcggctaaagataacatcaa360

agtaacagacgccgatatcaaagagtactgggaaggcttaaaaggcaaaatccgtgcaag420

ccacatccttgttgctgataaaaagacagctgaagaagtagagaaaaagctgaaaaaagg480

cgagaagtttgaagaccttgcgaaagaatactcaacagacagctctgcttcaaaaggcgg540

ggatcttggctggttcgcaaaagaaggccaaatggacgaaacattcagcaaagctgcatt600

caaattaaaaacaggtgaagtcagtgatcctgtcaaaacgcaatacggctaccatatcat660

taaaaagacagaagaacgcggcaaatatgatgatatgaaaaaagaactgaaatctgaagt720

gcttgaacaaaaattaaatgacaacgcagctgttcaggaagctgttcaaaaagtcatgaa780

gaaggctgacatcgaagtaaaagataaagatctgaaagacacatttaatacatcttcaac840

aagcaacagcacttcttcatcttcaagcaattctaaataaagaagtctcgttccgacagt900

tggcaacataatatgctaagagctcctgcactggatggtggcgctggatggtaagccgct960

ggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcc1020

tgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaacc1080

gaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggc1140

ggaaaacctcagtgtgacgctccccgccgcgtccccacgccatcccgcatctgaccaccag1200

cgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcagg1260

ctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatca1320

gttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccc1380

taacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgtt1440

gcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggca1500

gcatcagggggaaaaccttattatcagccggaaaacctaccggattgatggtagtggtca1560

aatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattgg1620

cctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgca1680

agaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtc1740

agacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcga1800

attgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacag1860

tcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatg1920

gctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagt1980

atcggcggaattacagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaa2040

ataataataaccgggcaggccatgtctgcccgtatttcgcgtaaggaaatccattatgta2100

ctatttaattctgcgtgacatcccatcgatcagaccagtttttaatttgtgtgtttccat2160

gtgtccagtttggaatactcttaacctcattggaaatcgcggcataatcactggtggtat2220

gattgatgaccgcgtcaacaatgacctttatgccatattcttcagcggctgcaacacattt2280

ctttaaattcttgttcagtacctaagtaacggttgccaatttgatacgatgtcggctgat2340

acagccagtaccagttcgacatgcttttatctccttgattcccttcctttacttggttaa2400

tcggagatgtctgaatggctgtatatcctgcatcatgaatatccttcatattgtgtttta2460

acgtattgaacgaccaattccatgcatgaagaatggttccgcttttgatcgacggtgctg2520

taagctcattcgatttgttcgccgtttcagcactcgcagccgccggtcctgccagaacca2580

aatgaaacagcaataaaaatccagcgaataacggcagtaaagaggttttgaatcgttttg2640

caaacattcttgacactccttatttgattttttgaagacttacttcggagtcaaaaatcc2700

ctcttacttcattcttccgcttcctcctttcaaaccgatgtgaagactggagaattttgt2760

taattcttgaagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgat2820

aataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctat2880

ttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata2940

aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccct3000

tattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaa3060

agtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaa3120

cagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttt3180

taaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcgg3240

tcgccgcataacactattctcagaatgacttggttgagtactcaccagtcacagaaaagca3300

tcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataa3360

cactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttt3420

gcacaacatggggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagc3480

cataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaa3540

actattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga3600

ggcggataaagttgcaggaccttctgcgctcggcccttccggctggctggtttattgc3660

tgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccaga3720

tggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatga3780

acgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcaga3840

ccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggat3900

ctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgtt3960

ccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct4020

gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgcc4080

ggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacc4140

aaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc4200

gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtc4260

gtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctg4320

aacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata4380

cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta4440

tccggtaagcggcagggtcggaacaggagagcgcacgaggggagcttccagggggaaacgc4500

ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtg4560

atgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggtt4620

cctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgt4680

ggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccga4740

gcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttac4800

gcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgc4860

cgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccc4920

cgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgct4980

tacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatca5040

ccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacag5100

atgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctgg5160

cttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctcc5220

gtgtaagggggatttctgttcatgggggtaatgataccgatgaacgagagaggatgctca5280

cgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaac5340

tggcggtatggatgcggcgggaccagagaaaatcactgttcatggggggtaatgataccga5400

tgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactgg5460

aacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactc5520

agggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagc5580

atcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagac5640

tttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgc5700

agcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggc5760

aacccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggcc5820

aggacccaacgctgcccgagatgcgccgcgtgcggctgctggagatggcggacgcgatgg5880

atatgttctgccaagggttggtttgcgcattcacagttctccgcaagaattgattggctc5940

caattcttggagtggtgaatccgttagcgaggtgccgccggcttccatcaggtcgaggt6000

ggcccggctccatgcaccgcgacgcaacgcggggaggcagacaaggtataggggcggcgcc6060

tacaatccatgccaacccgttccatgtgctcgccgaggcggcataaatcgccgtgacgat6120

cagcggtccaatgatcgaagttaggctggtaagagccgcgagcgatccttgaagctgtcc6180

ctgatggtcgtcatctacctgcctggacagcatggcctgcaacgcggtcatcccgatgcc6240

gccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgactaagaa6300

aatgccgtcaaatccgctcgccatgacttcactaacgatgcctttgaaaatcttcaagtt6360

cttttctactaattcaaggcgtgtctcaccagggtttttggtttgctccggcgcaaatgca6420

gacaatatcagcatccttgcagggtatgtttctctttgatgtctttttgtttgtgaagta6480

tttcacatttatattgtgcaacacttcacaaacttttgcaagagaaaagttttgtctgat6540

ttatgaacaaaaaagaaaccatcattgatggtttctttcggtaagtcccgtctagccttg6600

ccctcaatggggaagagaaccgcttaagcccgagtcatttatataaaccatttagcacgta6660

atcaaagccaggctgattctgaccgggcacttgggcgctgccattattaaaaatcacttt6720

tgcgttggttgtatccgtgtccgcaggcagcgtcagcgtgtaaattccgtctgcattttt6780

agtcattggttttccaggccaagatccggtcaattcaattactcggctcccatcatgttt6840

atagatataagcatttacctggctccaatgattcggattttgatagccgatggttttggc6900

cgacgctggatctcttttaacaaaactgtatttctcggtcctcgttacaccatcactgtt6960

cgttccttttaacatgatggtgtatgttttgccaaatggatctccttttccgattgtgaa7020

ttgatctccatccttaaacgccgtctctggtccattattgatttgataaacggcttttgt7080

tgtattcgcatctgcacgcaaggtaatcgtcagttgatcattgaaagaatgtgttacacc7140

tgttttgtaattctcaaggaaaacatgaggcgcttttgcaatatcatcaggataaagcac7200

agctacagacctggcattgatcgtgcctgtcagtttaccatcgttcacttgaaatgaacc7260

cgctccagctttatgtgtcatacctgccatcaggcaattttgttgccgtattgatagagac7320

agaggatgaacctgcatttgccagcacaacgccatgtgagccgcgctgattcataaatat7380

ctggttgtttccattcgggttcgagagttcctcaggctgtccagccatcacattgtgaaa7440

tctattgaccgcagtgatagcctgatcttcaaataaagcactcccgcgatcgcctatttg7500

gcttttccccgggaacctcacaccatttccgcctccctcaggtctggaaaagaaaagagg7560

cgtactgcctgaacgagaagctatcaccgcccagcctaaacggatatcatcatcgctcat7620

ccatgtcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtagtagttg7680

aggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagt7740

cccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaag7800

tggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacct7860

gtggcgccggtgatgccggccacgatgcgtccggcgtagaggatctggagctgtaatata7920

aaaaccttcttcaactaacggggcaggttagtgacattagaaaaccgactgtaaaaagta7980

cagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaa8040

tagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaag8100

atagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggta8160

gaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaa8220

taatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaac8280

cattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattcttta8340

caggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggct8400

ctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtat8460

ttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttctt8520

gttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgttt8580

gttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttat8640

taaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttactt8700

gtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaat8760

atatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgcttt8820

agttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggattt8880

gagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatg8940

ataagctgtcaaacatgagaattcgaaaagaatgatgtaagcgtgaaaaattttttatct9000

tatcacttgaaattggaagggagattctttattataagaattgtggaattgtgagcggat9060

aacaattcccaattaaaggaggaaggatcctacgtaggtac9101

<210>8

<211>2526

<212> DNA

<213> Bacillus subtilis

<400>8

atgcttggaattttaaataaaatgtttgatccaacaaaacgtacgctgaatagatacgaa60

aaaattgctaacgatattgatgcgattcgcggagactatgaaaatctctctgacgacgca120

ttgaaacataaaacaattgaatttaaagagcgtcttgaaaaaggggcgacaacggatgat180

cttcttgttgaagctttcgctgttgttcgagaagcttcacgccgcgtaacaggcatgttt240

ccgtttaaagtccagctcatggggggcgtggcgcttcatgacggaaatatagcggaaatg300

aaaacaggggaagggaaaacattaacgtctaccctgcctgtttattaaatgcgttaacc360

ggtaaaggcgtacacgtcgtgactgtcaacgaatacttggcaagccgtgacgctgagcaa420

atggggaaaattttcgagtttctcggtttgactgtcggtttgaatttaaactcaatgtca480

aaagacgaaaaacgggaagcttatgccgctgatattacttactccacaaacaacgagctt540

ggcttcgactatttgcgtgacaatatggttctttaaagagcagatggttcagcgcccg600

cttcattttgcggtaatagatgaagttgactctattttaattgatgaagcaagaacaccg660

cttatcatttctggacaagctgcaaaatccactaagctgtacgtacaggcaaatgctttt720

gtccgcacgttaaaagcggagaaggattacacgtacgatatcaaaacaaaagctgtacag780

cttactgaagaaggaatgacgaaggcggaaaaagcattcggcatcgataacctctttgat840

gtgaagcatgtcgcgctcaaccaccacatatcaaccaggccttaaaagctcacgttgcgatg900

caaaaggacgttgactatgtagtggaagacggacaggttgttattgttgattccttcacg960

ggacgtctgatgaaaggccgccgctacagtgaggggcttcaccaagcgattgaagcaaag1020

gaagggcttgagattcaaaacgaaagcatgaccttggcgacgattacgttccaaaactac1080

ttccgaatgtacgaaaaacttgccggtatgacgggtacagctaagacagaggaagaagaa1140

ttccgcaacatctacaacatgcaggttgtcacgatccctaccaacaggcctgttgtccgt1200

gatgaccgcccggatttaatttaccgcacgatggaaggaaagtttaaggcagttgcggag1260

gatgtcgcacagcgttacatgacgggacagcctgttctagtcggtacggttgccgttgaa1320

acatctgaattgatttctaagctgcttaaaaacaaaggaattccgcatcaagtgttaaat1380

gccaaaaaccatgaacgtgaagcgcagatcattgaagaggccggccaaaaaggcgcagtt1440

acgattgcgactaacatggcggggcgcggaacggacattaagcttggcgaaggtgtaaaa1500

gagcttggcgggctcgctgtagtcggaacagaacgacatgaatcacgccggattgacaat1560

cagcttcgaggtcgttccggacgtcaggggagaccccggggattactcaattttatctttct1620

atggaagatgaattgatgcgcagattcggagctgagcggacaatggcgatgcttgaccgc1680

ttcggcatggacgactctactccaatccaaagcaaaatggtatctcgcgcggttgaatcg1740

tctcaaaaacgcgtcgaaggcaataacttcgattcgcgtaaacagcttctgcaatatgat1800

gatgttctccgccagcagcgtgaggtcatttataagcagcgctttgaagtcattgactct1860

gaaaacctgcgtgaaatcgttgaaaatatgatcaagtcttctctcgaacgcgcaattgca1920

gcctatacgccaagagaagagcttcctgaggagtggaagcttgacggtctagttgatctt1980

atcaacacaacttatcttgatgaaggtgcacttgagaagagcgatatcttcggcaaagaa2040

ccggatgaaatgcttgagctcatttatggatcgcatcatcacaaaatataatgagaaggaa2100

gagcaattcggcaaagagcaaatgcgcgaattcgaaaaagttatcgttcttcgtgccgtt2160

gattctaaatggatggatcatattgatgcgatggatcagctccgccaagggattcacctt2220

cgtgcttacgcgcagacgaacccgcttcgtgagtatcaaatggaaggttttgcgatgttt2280

gagcatatgattgaatcaattgaggacgaagtcgcaaaatttgtgatgaaagctgagatt2340

gaaaacaatctggagcgtgaagaggttgtacaaggtcaaacaacagctcatcagccgcaa2400

gaaggcgacgataacaaaaaagcaaagaaagcaccggttcgcaaagtggttgatatcgga2460

cgaaatgccccatgccactgcggaagcgggaaaaaatataaaaattgctgcggccgtact2520

gaatag2526

<210>9

<211>2214

<212> DNA

<213> Bacillus subtilis

<400>9

atgaaaaaaggacgcttgattgcgtttttccttttcgttctattgatcggcacgggcttg60

ggctactttacgaagcctgccgctaacaatattacgttaggattggatttgcaaggcgga120

tttgaggtgctgtatgatgtacagcctgtaaaaaaaggtgacaaaatcacaaaagacgtt180

ctggtcagcacagtagaggcactgaaccgccgggccaatgttctcggtgtcagcgaaccg240

aacatccaaattgaagggaataaccggattcgcgttcagctcgctggcgtgacaaaccaa300

aacagagcgcgtgaaattttggcgactgaagcgcagctttctttcagagatgcaaatgat360

aaggaactgttaaacggtgctgatctagtcgaaaacggcgctaaacaaacttatgatagc420

acaacaaatgagccaattgtcacgattaagctgaaagacgctgataaatttggtgaagtg480

accaagaaggtcatgaaaatggcgccaaacaaccagcttgtcatttggttggattatgat540

aaaggtgattcctttaagaaagaagttcaaaaagagcatcctaaatttgtatccgctcca600

aatgtaagtcaggaactaaatacaactgatgtaaaaattgaaggtcatttcacagctcaa660

gaagcgaaagatttagccagcattttaaacgcaggcgcacttcctgtgaaactgactgaa720

aagtattcgacatcagtaggcgcgcaattcggccagcaggctctccatgatacggtgttt780

gccggtattgtcggtatcgcaattattttcttatttatgcttttctattaccgtctgccg840

ggattaatcgcggtgattacgctgtctgtttatatctacattacactccagatctttgac900

tggatgaatgccgtactcacgcttccgggaattgccgctctcattttaggtgtcgggatg960

gctgttgacgccaacattattacctatgagcggattaaagaagagctcaagctaggaaag1020

tcagtccgctctgccttccgttcaggaaacagacggtcatttgcgacgatttttgacgcg1080

aatattacaaccattattgcagcggttgtgctctttatctttgggacaagctctgttaaa1140

gggtttgcgacaatgctgatcctatcgattttgacaagctttatcactgccgttttctta1200

tcgagatttctcctcgctctccttgtggaaagcagatggcttgatcggaaaaaaggctgg1260

tttggtgtcaataagaaacatatcatggatattcaggatacggatgaaaatacagagccg1320

catacgccattccaaaaatgggatttcacgagcaaacgcaaatacttctttattttctcc1380

agtgcggtcacggttgccgggattattatcctgcttgtgttcaggctgaatcttggcatt1440

gactttgcaagcggtgcacggattgaagtgcaaagcgaccataagctgacgacagagcaa1500

gttgagaaggattttgaatctctgggtatggaccctgatactgtagttctgtcaggcgaa1560

aagagcaatatcggtgttgcccgttttgtcggggtgccagataaagaaaccattgcaaaa1620

gtaaaaacgtattttaaagacaaatacggatctgatccaaatgtcagcacagtttcaccg1680

acagtcggtaaggagctggcgagaaatgcgctgtacgcagttgctatagcttctattggc1740

atcatttattacgtttcaatccgattcgaatacaaaatggcgattgctgccatcgcctca1800

ttgctatatgacgcattctttatcgtcacgttcttcagtatttacaaggcttgaggtagat1860

gttacattcatcgcggccatcttgacgataatcgggtattccattaacgatacaatcgtt1920

acatttgacagggtccgcgagcatatgaaaaagcgtaagccgaaaacctttgccgatctg1980

aaccatattgtaaacctgagcctgcagcaaacctttacacgttcaattaacactgtatta2040

accgttgtgattgttgttgtgacattgctgatctttggagcatcttctattactaacttc2100

tcaattgctttatggtcgggctgttaacaggcgtttatattcttctctatacattgccgca2160

caaatttggcttgcatggaaaggaagagaactgaaaaaagattcggcgcaataa2214

<210>10

<211>231

<212> DNA

<213> Bacillus subtilis

<400>10

atgcacgcagttttgattaccttattggttatcgtcagcattgcacttattattgtcgtt60

ttgcttcaatccagtaaaagtgccggattatctggtgcgatttcaggcggagcggagcag120

ctcttcgggaaacaaaaagcaagaggtcttgattaattttgcaccgcattacggtagtg180

ctggcagtcttgtttttcgtgttaacgattgcgcttgcttatatcctatag231

<210>11

<211>1341

<212> DNA

<213> Bacillus subtilis

<400>11

atggcatttgaaggattagccgaccgactgcagcagacgatttctaaaatccgcggaaaa60

gggaaagtcagcgaacaagatgtaaaagagatgatgcgtgaggtccgtcttgcgctgctt120

gaggctgacgttaactttaaagtagtcaaggattttgtcaaaaaagtaagtgaacgcgct180

gtaggccaagacgtcatgaaaagtctgacgcccggccagcaggtcattaaagttgttcaa240

gaggaactgactgagctgatgggcggcgaagagagcaaaatcgccgtcgcaaaaaggccg300

ccgactgttattatgatggtcggtctccaaggtgccggtaaaacgacaacaagcggtaag360

cttgcgaatctgctgcgcaaaaagcataatcgcaaaccgatgctggttgctgccgatatt420

taccgcccagccgcaattaagcagctggaaacactcggcaaacagcttgatatgcctgtt480

ttctctcttggcgatcaggtcagtcctgtagaaatagctaaacaggctattgagaaagcc540

aaggaagaacattatgactacgtcattttggatacggcagggcgcttgcatatcgaccat600

gaactgatggatgaactgaccaacgtcaaagaaatcgcgaatccggaagaaattttcctg660

gttgtcgattcaatgaccggtcaggacgctgtgaatgttgccaaaagctttaatgaacag720

ctcggtttaaccggtgttgtgttgactaagctggatggagacacacgcggcggggctgcg780

ctttctattcgcgctgtcacaaacacgccaattaagtttgcaggtttgggcgaaaagctt840

gatgcgttagagccgttccatcctgaacgcatggcatcaaggattctcggcatgggcgac900

gtgctgacattgattgaaaaagcacaggccagcgttgatgaagacaaagccaaagagctg960

gaacaaaaaatgagaacgatgagcttcacattggacgattttctggagcagctcgggcaa1020

gtcagaaacatggggccgcttgatgagcttctgcaaatgatgccgggtgcaggtaaaatg1080

aagggcctgaaaaacatccaagttgatgaaaaacagctgaatcatgtggaagcaatcatc1140

aaatcaatgactgttcttgaaaaagaacagccggatattatcaatgccagccggcggaag1200

cggattgcaaaaggaagcgggacatccgtacaggaagtcaaccgtctgcttaagcagttt1260

gatgaaatgaaaaaaatgatgaagcagatgacaaacatgtcaaaaggcaagaaaaaaggg1320

tttaagctaccttttatgtaa1341

<210>12

<211>564

<212> DNA

<213> Bacillus subtilis

<400>12

ttgaatgcaaaaacaatcacgttaaagaaaaaaagaaaaatcaaaacgatcgttgtactc60

agtatcatttatgatcgcagctctcatttttacgatcagattggtgttttacaagcctttt120

cttattgaaggatcatcaatggccccaacgcttaaagactcagaaagaattctggttgat180

aaagcagtcaaatggactggcgggtttcacagaggagacatcatagtcattcatgacaaa240

aagagcggccgctcatttgtcaaacgtttaatcggtttgcctggtgacagcattaaaatg300

aaaaatgatcagctatacataaatgataaaaaggtggaagaaccatacttaaaggaatat360

aaacaggaggtcaaagagtcgggtgtaaccttaacaggtgacttcgaagttgaggttcct420

tccggtaaatattttgtgatgggagataaccgtctcaattcactggatagcagaaacgga480

atgggcatgccttctgaggacgatatcatcggtactgaatctctcgtcttttatccattc540

ggtgagatgagacaggcaaaataa564

<210>13

<211>507

<212> DNA

<213> Bacillus subtilis

<400>13

atgaaaaaacggttttggtttcttgccggtgtagtgtccgttgttctcgccattcaggtt60

aaaaatgctgtctttattgattacaaggtagaaggcgtcagtatgaacccgaccttccag120

gaaggaaacgaattgttggtcaataaattttcgcatcgatttaaaaccatccatcgtttt180

gacatcgtcctttttaaaggccctgatcataaagtgctgattaaacgggtaatcggcttg240

cccggtgaaacgatcaaatataaagatgatcagctgtatgtgaacggaaagcaggttgct300

gagccatttttgaagcatttgaaatctgtttctgccggcagccatgtaacgggtgatttt360

tctttgaaagatgtgacgggaacaagcaaggtgccgaaaggaaaatattttgtcgttgga420

gataatcgcatatacagcttcgacagccggcattttggtccgataagagaaaaaaatatt480

gtcggtgtgatttctgatgccgaataa507

<210>14

<211>573

<212> DNA

<213> Bacillus subtilis

<400>14

atgaagctgatcagtaatattttatacgtgatcatctttactcttattattgtgctgaca60

cttgtcgtgatttcaacacgttcatccggggggagagccggcagtgtttgggtatacgctg120

aaatcagttctgtcaggttcgatggagccggaggttcaatacaggttccttaatattggtc180

aaagaaatcactgatgtgaaagagctccaaaaaggtgacgttattacattttgcaggat240

gcaaatacggcggtcacccacagaattgttgacataacaaagcaaggagaccatttgtta300

tttaaaacaaaaggtgataataatgcagcagctgattcagcgcctgtatcggacgaaaat360

gttcgcgcgcaatacacaggttttcagcttccatatgccggctatatgcttcattttgcc420

agccagccgattggaacggctgtattattgattgttcccggcgtgatgctgttagtttac480

gcttttgtgacgatcagcagcgccattagagaaattgaaagaaagacaaaagccttggaa540

acagatacaaaggacagcaccatgtctacttaa573

Claims (6)

1. A Bacillus subtilis integrated expression vector for improving the expression level of α-amylase, characterized in that its vector sequence is sequence 5. 2. a Bacillus subtilis integrated expression vector as claimed in claim 1, is characterized in that: comprise the grac promoter sequence of Bacillus subtilis, be used for integrating the polyclonal that may relate to expression control relevant element in various Bacillus subtilis The site sequence is used to integrate the gene 5' and 3' homology arm sequences of the amyE site, and also includes the Escherichia coli replicon and the coding sequence of the chloramphenicol resistance gene. 3. the Bacillus subtilis integrated expression vector according to claim 2, is characterized in that the grac promoter sequence of Bacillus subtilis, is used for inserting the multiple cloning site sequence of various expression regulatory element fragments, is used for integrating amyE site The 5' and 3' homology arm sequences of the gene, the nucleotide sequences of which are sequence 1, sequence 2, sequence 3 and sequence 4, respectively. 4. The Bacillus subtilis integrated expression vector according to claim 1, characterized in that its integrated host cell is Bacillus subtilis. 5. A genetic engineering strain expressing α-amylase according to claim 4, specifically Bacillus subtilis with a cell wall chaperone PrsA gene (GenebankAccessionNumberX57271.1) integrated with a grac promoter at the amyE site of the genome Host cells can obviously promote the expression and secretion of α-amylase. 6. The Bacillus subtilis genetically engineered strain that integrates other restriction factors (SecA, SecDF, SecG, Ffh, SipU, SipV, SipW) related to exogenous protein expression and secretion bottlenecks according to claim 4, to α-amyloid The expression and secretion of enzymes can be promoted in different degrees.