CN110029109B - Sialic acid induced expression element and application - Google Patents

Abstract

The invention discloses a sialic acid induced expression element and application thereof, belonging to the field of genetic engineering. The invention uses pHT plasmid as carrier, in bacillus subtilis, P_ydjOPromoter expression transcription regulation protein NanR, strong promoter P for optimizing and integrating NanR recognition binding sequence_vegThe method realizes the leakage expression of the Bacillus subtilis 168 in an extremely low level in the sialic acid-containing environment, can reduce the leakage expression rate to about 2 percent, and has wide application prospect.

Description

Sialic acid induced expression element and application

Technical Field

The invention relates to a sialic acid induced expression element and application thereof, belonging to the field of genetic engineering.

Background

In the bacillus subtilis, a promoter element capable of inducing and activating expression is widely applied to the field of metabolic engineering. Currently, the commonly used inducible promoter in Bacillus subtilis is xylose inducible promoter P_xylAnd IPTG inducible promoter P_grac. However, xylose inducible promoter P_xylThe inducer xylose can be metabolized and consumed by the bacillus subtilis, and the promoter P is induced by IPTG_gracThe inducer IPTG has a certain toxicity problem to cells. In particular, xylose and IPTG inducible promoters present higher leakage problems. The defects seriously limit the application of the bacillus subtilis in the field of metabolic engineering. Sialic acid is a suitable inducer as a simple carbon source, and is non-toxic to cells and not metabolically consumed by B.subtilis. By genetically engineering the promoter, response elements more suitable for B.subtilis based on sialic acid induction can be obtained. However, how to modify a promoter to reduce its leaky expression is a problem worth intensive study. In addition, Bacillus subtilis lacks the sialic acid native transport pathway and is unable to transport extracellular sialic acid into the cell, which also limits sialic acid toleranceThe application of the expression guide element in the field of bacillus subtilis metabolic engineering.

Disclosure of Invention

To solve the above technical problem, the present invention constructs an inducible expression promoter element responsive to sialic acid, using a constitutive promoter P_ydjOOr a strong promoter P_hbsExpressing the transcription regulatory protein NanR, further by expression in the constitutive promoter P_vegIn the method, a transcription regulatory protein NanR binding sequence TTNANNTGGTATAACAGGTATANAGNTANNNNNTNN is integrated to realize the induced expression of an expression element in the bacillus subtilis BSNanT after sialic acid is added. Further by replacing the NanR promoter P_ydjOIs a strong promoter P_hbsAt P_vegA further addition of one copy of the NanR binding sequence to the promoter results in a very low leakage expression of sialic acid responsive elements without sialic acid addition.

It is a first object of the present invention to provide an inducible expression promoter element responsive to sialic acid; containing (a) or (b);

(a) constitutive promoter P_ydjOTranscription regulatory protein NanR and constitutive promoter P_vegA transcription regulatory protein NanR binding sequence, and the constitutive type P_ydjOThe promoter expresses a transcription regulatory protein NanR; the constitutive promoter P_vegA transcription regulatory protein NanR binding sequence is integrated; the promoter P_ydjOAnd P_vegReverse connection;

(b) constitutive promoter P_hbsTranscription regulatory protein NanR and constitutive promoter P_vegTwo copies of a transcription regulatory protein, a NanR binding sequence, said constitutive P_ydjOThe promoter expresses a transcription regulatory protein NanR; the constitutive promoter P_vegIntegrating a transcription regulatory protein NanR binding sequence; the promoter P_ydjOAnd P_vegAnd connecting in the reverse direction.

In one embodiment of the invention, said constitutive P_ydjOThe nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

In one embodiment of the invention, the transcriptional modulator protein P_hbsThe nucleotide sequence of the promoter is shown as SEQShown in NO. 2.

In one embodiment of the invention, the nucleotide sequence of the NanR binding site sequence is shown in SEQ No. 3.

In one embodiment of the invention, the amino acid sequence of the transcription regulatory protein NanR is shown in SEQ NO. 4.

In one embodiment of the invention, the amino acid sequence of the sialic acid transporter NanT is shown in SEQ No. 5.

In one embodiment of the invention, P incorporates the NanR binding site sequence_vegThe base sequence of the promoter is shown as SEQ NO. 6.

In one embodiment of the invention, P incorporates 2 NanR binding site sequences_vegThe base sequence of the promoter is shown as SEQ NO. 7.

In one embodiment of the invention, the plasmid tool used to construct the promoter for inducible expression of sialic acid is plasmid pHT 01.

In one embodiment of the present invention, said P_vegThe promoter also regulates expression of the marker.

In one embodiment of the invention, the label is a fluorescent protein.

In one embodiment of the invention, the marker is green fluorescent protein, and the amino acid sequence of the marker is shown in SEQ NO.8

In one embodiment of the invention, the inducible expression promoter element is constructed on the pHT01 plasmid.

The second object of the present invention is to provide a method for constructing the expression-inducing element plasmid, comprising the steps of:

1) construction of genomic recombinant integrated NanT fragment:

through fusion PCR, after inserting the sialic acid transport enzyme coding gene NanT of the source escherichia coli into the adeC gene of the bacillus subtilis 168 genome, 1000bp before the termination codon TAA of the adeC gene is used as a left homology arm, 1000bp after the termination codon TAA of the adeC gene is used as a right homology arm, and the zeocin resistance gene and P are₄₃Promoter genes and NanT gene segments, wherein 5 gene segments are fused to obtain a NanT recombinant segment;

2) constructing the transportable sialic acid recombinant bacillus subtilis:

recombining the recombinant fragment obtained in the step 1) to a Bacillus subtilis 168 genome to obtain a recombinant Bacillus subtilis engineering bacterium, which is named as BSNanT;

3) construction of sialic acid response element tool plasmid pHT-ydjO-veg plasmid:

cloning of transcription regulatory protein coding gene NanR gene segment, P_ydjOPromoter Gene fragment, P_vegThe gene fragment and GFP gene fragment, and pHT plasmid fragment, 5 fragments in total were assembled by NEB Gibson Assembly Master Mix Kit to construct the sialic acid response element tool plasmid pHT-ydjO-veg plasmid.

4) Construction of sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-ydjO-veg

And (3) transforming the sialic acid response element tool plasmid pHT-ydjO-veg plasmid in the step 3) into the engineering strain BSNanT in the step 2) to obtain the recombinant bacillus subtilis engineering bacteria, which are named as BSNanT-pHT-ydjO-veg.

5) Construction of optimized sialic acid response element tool plasmid pHT-hbs-veg plasmid

Amplifying strong promoter P by taking Bacillus subtilis 168 genome as template_hbsGene fragment to replace P_ydjOA promoter for increasing the expression level of the transcription regulatory protein NanR; pHT-ydjO-veg plasmid is taken as a template plasmid skeleton gene segment, 2 segments are assembled by a NEB Gibson Assembly Master Mix Kit, and the sialic acid induced expression plasmid pHT-hbs-veg plasmid is constructed.

6) Construction of optimized sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-hbs-veg

And (3) transforming the sialic acid response element tool plasmid pHT-hbs-veg plasmid in the step 5) into the engineering strain BSNanT in the step 2) to obtain the recombinant bacillus subtilis engineering bacteria, which are named as BSNanT-pHT-hbs-veg.

7) Construction of optimized sialic acid inducible expression element plasmid pHT-hbs-veg-2

A sialic acid inducible expression plasmid pHT-hbs-veg-2 plasmid is constructed by adding a NanR binding sequence on a primer by taking the pHT-hbs-veg plasmid as a template.

8) Construction of optimized sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-hbs-veg-2

And (3) transforming the sialic acid response element tool plasmid pHT-hbs-veg-2 plasmid in the step 7) into the engineering strain BSNanT in the step 2) to obtain the recombinant bacillus subtilis engineering bacterium named as BSNanT-pHT-hbs-veg-2.

The third purpose of the invention is to provide a genetically engineered bacterium containing the sialic acid inducible expression element.

In one embodiment of the present invention, the genetically engineered bacterium is a bacillus subtilis host.

The fourth purpose of the invention is to provide the application of the sialic acid inducible expression element plasmid in the field of bacillus subtilis metabolic engineering.

In one embodiment of the invention, the use includes, but is not limited to, activating expression of a downstream gene of interest in the presence of sialic acid.

In one embodiment of the invention, the induction is carried out at 30-37 ℃ and 220rpm in a fermentation medium for 48 h.

Has the advantages that:

(1) the invention integrates a transcription regulatory protein NanR binding site sequence TTNANNTGGTATAACAGGTATANAGNTANNNNNTNN into a constitutive expression promoter P_vegIn (3), the sialic acid-inducible expression promoter element pHT-ydjO-veg was obtained.

(2) The sialic acid inducible expression promoter element provided by the invention has pHT-ydjO-veg activation expression intensity reaching 4000a.u. in bacillus subtilis. The sialic acid inducible expression promoter element has only about 10% of leaky expression level in the absence of sialic acid.

(3) By adding the NanR promoter P_ydjOIs a strong promoter P_hbsObtaining sialic acid inducible expression promoter element pHT-hbs-veg, wherein the activated expression intensity is 3900a.u. Leaky expression level in the absence of sialic acid is from 10% is reduced to 6%.

(4) By passing through at P_vegA NanR binding sequence is further added in the promoter to obtain a sialic acid inducible expression promoter element pHT-hbs-veg-2, and the activation expression intensity of the sialic acid inducible expression promoter element is 3700a.u. The leakage expression level in the absence of sialic acid was further reduced from 6% to 2%. The method lays a foundation for further modifying the sialic acid inducible expression promoter of the bacillus subtilis through genetic engineering. The optimized sialic acid inducible expression promoter provided by the invention is simple in construction method, convenient to use and has good metabolic engineering application prospect.

Detailed Description

Culture conditions of Bacillus subtilis containing sialic acid inducible expression promoter elements:

fermentation medium (g/L): sialic acid 5, tryptone 6, yeast powder 12, ammonium sulfate 6, dipotassium hydrogen phosphate 12.5, potassium dihydrogen phosphate 2.5 and magnesium sulfate 3.

The culture conditions are as follows: culturing at 37 deg.C and 200rpm for 48 h.

The green fluorescent protein and thallus concentration detection method comprises the following steps:

tecan microplate reader: green fluorescent protein detection, excitation wavelength 490nm, emission wavelength 530nm, gain 60. The detection wavelength of the thallus concentration is 600 nm.

Leak expression amount calculation formula (%): x ═ a-b)/c

Wherein a is the fluorescence intensity of the sialic acid-inducible promoter when no sialic acid was added, b is the fluorescence intensity of Bacillus subtilis 168 strain as a control strain, and c is the fluorescence intensity of the sialic acid-inducible promoter when sialic acid was added.

Example 1 construction of a genomic recombinant integration of the NanT fragment

Designing primers L-F: 5'-GTATGTCATGATGCGTGAAGGATCAGTCGCCAAAAACACGC-3' and L-R: 5'-GTTATCCGCTCAAAAAGAGGCACTCCCTAAGGGAGTGCCTCTTTTTATTGCAGTG-3', and amplifying a left arm gene sequence of a recombinant fragment of the NanT transporter shown in SEQ ID No.9 by using a bacillus subtilis 168 genome as a template; design primers S-F: 5'-CCTTAGGGAGTGCCTCTTTTTGAGCGGATAACAATTTCACACAGGAAACAG-3' and S-R:5 ' -CACCTATCATAACGCCAGGGTTTTCCCAGTCACGAC-3', amplifying the spectinomycin-resistant left arm gene sequence of recombinant fragment of NanT transporter SEQ ID NO.10 using P7S6 plasmid as template (disclosed in Zhang, X., Liu, Y., Liu, L., Wang, M., Li, J., Du, G.et al.,2018. modulated pathway engineering of key-carbon-precursor supplied-pathway for improved N-acetyl neuraminic acid production in Bacillus subtilis Biotechnol.Bioeng.115, 2217-31); primers P-F: 5'-GACTGGGAAAACCCTGGCGTTATGATAGGTGGTATGTTTTCGCTTGAACTTTTA-3' and P-R: 5'-GGATATTCTGGGTTGTAGTACTCATGTGTACATTCCTCTCTTACCTATAATGGTACCGC-3' are designed, and a Bacillus subtilis 168-site template is used for amplifying P of a recombinant fragment of the NanT transporter₄₃Promoter gene sequence SEQ ID NO. 11; designing primers N-F: 5'-TAGGTAAGAGAGGAATGTACACATGAGTACTACAACCCAGAATATCCCGTGGTATCGCCATCTCAACCG-3' and N-R: 5'-GGGGTTGACGACCGAGTAGTTCACCTTAACTTTTGGTTTTGACTAAATCGTTTTTGGCGCTGCCAAACGGCACG-3', and amplifying a sialic acid transporter NanT gene sequence of a NanT transporter recombinant fragment shown in SEQ ID NO.12 by using an escherichia coli K12 genome as a template; designing primers R-F: 5'-TTAAGGTGAACTACTCGGTCGTCAACCCCATTAAACCTCCATAATGATCGGCAGGATCATTGGACGGCGCTTTG-3' and R-R: 5'-CCATGTCTGAGCGCCGTGTAGGAGAAAGCATTCATGACATTTTCCG-3', and amplifying a right arm gene sequence SEQ ID NO.13 of a recombinant fragment by using a bacillus subtilis 168 genome as a template; the 5 gene segments (SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13) are constructed into a gene segment for genome integration expression of NanT through a fusion PCR technology, and the gene segment is named as LSPNR.

Example 2 construction of a recombinant Bacillus subtilis that can transport sialic acid

And (3) transforming the recombinant integrated NanT gene segment LSPNR into Bacillus subtilis (Bacillus subtilis)168, and recombining the Bacillus subtilis 168 onto a genome to obtain a recombinant Bacillus subtilis engineering bacterium, which is named as BSNanT.

EXAMPLE 3 construction of sialic acid response element tool plasmid pHT-ydjO-veg plasmid

Primers N-1F:5-AACGACGGCCAGTGAATTCGAGCTCTTATTTCTTTTTGTTGGTGGTCTGACCGAAAG-3 'and N-1R: 5-ACGTAAAAACAAAGGAGGTGAAATGTACACATGGGCCTTATGAACGCATTTGATTCGCA-3' are designed to be used as the Bacillus subtilis budThe genome of bacillus 168 is taken as a template, and a gene segment SEQ ID NO.4 of a transcription regulatory protein coding gene NanR is cloned; primers N-2F:5-GTGTACATTTCACCTCCTTTGTTTTTACGTAATACGATAAATAGGGCCAAAGGT-3 'and N-2R: 5-GTATTACGGAGCACTTCCCATATTATCAAGAAAGCGGGGAATTGTCCTATACC-3' were designed, and the Bacillus subtilis 168 genome was used as a template to clone P_ydjOPromoter gene fragment SEQ ID NO.1, primers N-3F:5-CCCGCTTTCTTGATAATATGGGAAGTGCTCCGTAATACGCTGACAAGAGA-3 'and N-3R: 5-CTTTAAACGATATACCTTTATACCTGTTATACCATTGTACAACACGAGCCCATTTTTGTCAAATAAAATTTAAATT-3' are designed, Bacillus subtilis 168 genome is used as a template, and P is cloned_vegGene segment SEQ ID NO.6, primers N-4F:5-ATAACAGGTATAAAGGTATATCGTTTAAAGGAGGTGAAATGTACACATGGGTAAGGGAGAAGAACTTTTCACT-3 'and N-4R: 5-GCGTGACGTGAATTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAG-3' are designed, a GFP gene segment with a sequence shown as SEQ ID NO.8 is cloned by taking a laboratory-stored plasmid as a template; primers N-5F:5-GAACTATACAAATAATTCACGTCACGCGTCCATGGAGA-3 'and N-5R: 5-GAGCTCGAATTCACTGGCCGTCGTTTTACAAC-3' are designed, a pHT01 framework fragment SEQ ID NO.14 is cloned by taking a plasmid pHT01 as a template, and the 5 fragments in total are assembled by a NEB Gibson Assembly Master Mix Kit to construct a sialic acid response element tool plasmid pHT-ydjO-veg plasmid.

Example 4 construction of sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-ydjO-veg

And transforming the sialic acid response element tool plasmid pHT-ydjO-veg plasmid into the Bacillus subtilis engineering strain BSNanT to obtain the recombinant Bacillus subtilis engineering strain named BSNanT-pHT-ydjO-veg.

Example 5 Bacillus subtilis BSNanT-pHT-ydjO-veg addition of sialic acid to induce fermentation

The strain BSNanT-pHT-ydjO-veg prepared according to the method of example 4 was cultured at 37 ℃ and 200rpm for 48 hours in a fermentation medium containing 5g/L sialic acid. And finally, determining the intensity of the green fluorescent protein of the fermentation liquor to be 4000a.u., and successfully obtaining the sialic acid inducible expression element with strong inducible expression intensity. When sialic acid is not added into the fermentation medium, fermentation is carried out for 48 hours, the green fluorescent protein intensity of the fermentation liquid is 400a.u., and the leakage expression amount is 10%.

EXAMPLE 6 construction of optimized sialic acid response element tool plasmid pHT-hbs-veg plasmid

Primers hbs-F5-TGTGTACATTTCACCTCCTTTGAATGTTTGTCCAACAAAGTGAAGAATTTCTGTATGTA-3 'and hbs-R5-GTATTACGGAGCACTTCCCATAAGGATCAAGGAATAGGATGAAAAAAGGAAAAAAAGGA-3' are designed, a bacillus subtilis 168 genome is used as a template, and a promoter P is cloned_hbsGene segment SEQ ID NO. 2; primers hbs-2F:5-TATGGGAAGTGCTCCGTAATACGCTGA-3 'and hbs-2R: 5-AAAGGAGGTGAAATGTACACATGGGCCT-3' were designed, and plasmid backbone gene fragments shown in SEQ ID NO.15 were cloned using pHT-ydjO-veg plasmid as a template, and assembled by NEB Gibson Assembly Master Mix Kit to construct the sialic acid response element tool plasmid pHT-hbs-veg plasmid.

Example 7 construction of sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-hbs-veg

And (3) transforming the sialic acid response element tool plasmid pHT-hbs-veg plasmid into the Bacillus subtilis engineering strain BSNanT to obtain the recombinant Bacillus subtilis engineering strain named BSNanT-pHT-hbs-veg.

Example 8 fermentation of Bacillus subtilis BSNanT-pHT-hbs-veg with sialic acid addition

The strain BSNanT-pHT-hbs-veg prepared according to the method of example 4 was cultured at 37 ℃ and 200rpm for 48 hours in a fermentation medium containing 5g/L sialic acid. And finally, determining the green fluorescent protein intensity of the fermentation liquor to be 3900a.u., and successfully obtaining the sialic acid inducible expression element with strong inducible expression intensity. When sialic acid is not added into the fermentation medium, fermentation is carried out for 48 hours, the green fluorescent protein intensity of the fermentation liquor is 230a.u., and the leakage expression amount is 6%.

EXAMPLE 9 construction of optimized sialic acid response element tool plasmid pHT-hbs-veg-2

Primers veg 2-F5-TTGATCTGGTATAACAGGTATAAAGGTATACACTTTAAAGGAGGTGAAATGTACACATGGGTAAGG-3 'and veg 2-R5-CTTTATACCTGTTATACCAGATCAAAAACGATATACCTTTATACCTGTTATACCATTGTACAACACGA-3' are designed, pHT-hbs-veg plasmid is used as a template, an amplification product is directly transformed into escherichia coli, and a sialic acid response element tool plasmid pHT-hbs-veg-2 plasmid is constructed.

EXAMPLE 10 construction of sialic acid inducible expression element Bacillus subtilis BSNanT-pHT-hbs-veg-2

And (3) transforming the sialic acid response element tool plasmid pHT-hbs-veg-2 plasmid into the Bacillus subtilis engineering strain BSNanT to obtain the recombinant Bacillus subtilis engineering strain named BSNanT-pHT-hbs-veg-2.

Example 11 Bacillus subtilis BSNanT-pHT-hbs-veg-2 addition of sialic acid to induce fermentation

The strain BSNanT-pHT-hbs-veg-2 prepared according to the method of example 4 was cultured at 37 ℃ and 200rpm for 48 hours in a fermentation medium containing 5g/L sialic acid. Finally, the intensity of the green fluorescent protein of the fermentation liquor is measured to be 3700a.u., and the sialic acid inducible expression element with strong inducible expression intensity is successfully obtained. When sialic acid is not added into the fermentation medium, the fermentation is carried out for 48 hours, the green fluorescent protein intensity of the fermentation liquor is 75a.u., and the leakage expression amount is 2%.

The sialic acid concentration is adjusted, and other culture conditions are not changed, so that the fluorescence intensity is 3500-3700 a.u. when the sialic acid content in the fermentation medium is 1-10 g/L, and the intensity is not changed greatly.

Comparative example 1:

constructing an expression element library according to the same strategy as in examples 1 to 8, and selecting P_asdPromoter, P_gltXPromoter, P_yvyDPromoter, P_csbAPromoter, P_phrFPromoter and P_yceCPromoters (disclosed in the papers Yang, S., Du, G., Chen, J., Kang, Z.,2017. in Characterisation and application of endogenous phase-dependent promoters in Bacillus subtilis. appl. Microbiol. Biotechnol.101, 4151-61.) A total of 6 promoters integrated the NanR binding sequence, showing that P_asdPromoter, P_gltXPromoter, P_yvyDPromoter, P_csbAPromoter, P_phrFPromoter and P_yceCThe promoters, 6 promoters in total, were not activated by sialic acid for expression.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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gaggagttct gagaattggt atgccttata agtccaatta acagttgaaa acctgcatag 120

gagagctatg cgggtttttt attttacata atgatacata atttaccgaa acttgcggaa 180

cataattgag gaatcataga attttgtcaa aataatttta ttgacaacgt cttattaacg 240

ttgatataat ttaaatttta tttgacaaaa atgggctcgt gttgtacaat ggtataacag 300

gtataaaggt atatcgtttt tgatctggta taacaggtat aaaggtatac acttt 355

<210> 8

<211> 238

<212> PRT

<213> Artificial sequence

<400> 8

Met Gly Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

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

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

50 55 60

Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg

65 70 75 80

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

85 90 95

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

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

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

225 230 235

<210> 9

<211> 1068

<212> DNA

<213> Artificial sequence

<400> 9

gtatgtcatg atgcgtgaag gatcagtcgc caaaaacacg ctcaatgtgc tgccggcggt 60

gaatgaaaag aacgcacgcc ggttcttttt ctgtacggat gataagcatg tggatgattt 120

attgtcagag ggaagtgtaa accatcaggt gaaaatggcg attcaagccg gacttaatcc 180

gtttttagcc tatcagctag gaagcctcaa tgcagccgaa tgctacggat tagatacaaa 240

gggagcgatt gccccgggtt ttgacgctga tttgcttttt gtatctgatc tggaaaatgt 300

cactgtcaca atgacgatgg taaaagggca gactgttgct gaagacagca aagcggtcta 360

tcaggatcat gcttcaactg cagcaccaga tcaggcactg cttgattctg ttaagcttgc 420

tgctcctctt aacaaacagg attttcatat gccaatcgat tcagagcagc agatcaatgt 480

cattcaaatc ataccaaatc agcttgaaac acgattagta caagttccgg ctcctgttgc 540

ccgcgaattt gagcctgaca ctgagcttga tttgttaaag attgcagttg tcgagcggca 600

taaaggatta aaagaaaccg gacttggtgt tgtgaaaggt tttggattca agagcggagc 660

gattgccaca accatttcac acgactccca taatattatt gccgtcggaa cgaatgatga 720

ggatatcgcg gcggcagtta ataagctgca ggaaattggc ggaggattaa caattataaa 780

aaatggggaa gagctccatt cagtaccgct gccgattgca gggttattat ccgaccaatc 840

tgcagagcaa gtgaatcaaa gcttgctgac gcttcatgat aaattgtcgt taatcggttt 900

cacaggcgga tttaatccat ttttgacatt gtcgttttta gcgttgcctg tcattcctga 960

tattaaaatg acgactacgg gattattcga tgtaaaatca tttcaacaca tatcactgca 1020

ataaaaagag gcactccctt agggagtgcc tctttttgag cggataac 1068

<210> 10

<211> 1267

<212> DNA

<213> Artificial sequence

<400> 10

gagcggataa caatttcaca caggaaacag ctatgaccat gattacgaat tcgagctcgg 60

tacccgggga tcctctagag attgtaccgt tcgtatagca tacattatac gaagttatcg 120

attttcgttc gtgaatacat gttataataa ctataactaa taacgtaacg tgactggcaa 180

gagatatttt taaaacaatg aataggttta cacttacttt agttttatgg aaatgaaaga 240

tcatatcata tataatctag aataaaatta actaaaataa ttattatcta gataaaaaat 300

ttagaagcca atgaaatcta taaataaact aaattaagtt tatttaatta acaactatgg 360

atataaaata ggtactaatc aaaatagtga ggaggatata tttgaataca tacgaacaag 420

ttaataaagt gaaaaaaata cttcggaaac atttaaaaaa taaccttatt ggtacttaca 480

tgtttggatc aggagttgag agtggactaa aaccaaatag tgatcttgac tttttagtcg 540

tcgtatctga accattgaca gatcaaagta aagaaatact tatacaaaaa attagaccta 600

tttcaaaaaa aataggagat aaaagcaact tacgatatat tgaattaaca attattattc 660

agcaagaaat ggtaccgtgg aatcatcctc ccaaacaaga atttatttat ggagaatggt 720

tacaagagct ttatgaacaa ggatacattc ctcagaagga attaaattca gatttaacca 780

taatgcttta ccaagcaaaa cgaaaaaata aaagaatata cggaaattat gacttagagg 840

aattactacc tgatattcca ttttctgatg tgagaagagc cattatggat tcgtcagagg 900

aattaataga taattatcag gatgatgaaa ccaactctat attaacttta tgccgtatga 960

ttttaactat ggacacgggt aaaatcatac caaaagatat tgcgggaaat gcagtggctg 1020

aatcttctcc attagaacat agggagagaa ttttgttagc agttcgtagt tatcttggag 1080

agaatattga atggactaat gaaaatgtaa atttaactat aaactattta aataacagat 1140

taaaaaaatt ataaataact tcgtatagca tacattatac gaacggtaga atcgtcgacc 1200

tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 1260

ggcgtta 1267

<210> 11

<211> 300

<212> DNA

<213> Artificial sequence

<400> 11

tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60

taataactga caaacatcac cctcttgcta aagcggccaa ggacgccgcc gccggggctg 120

tttgcgttct tgccgtgatt tcgtgtacca ttggtttact tatttttttg ccaaggctgt 180

aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240

gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300

<210> 12

<211> 1491

<212> DNA

<213> Artificial sequence

<400> 12

atgagtacta caacccagaa tatcccgtgg tatcgccatc tcaaccgtgc acaatggcgc 60

gcattttccg ctgcctggtt gggatatctg cttgacggtt ttgatttcgt tttaatcgcc 120

ctggtactca ccgaagtaca aggtgaattc gggctgacga cggtgcaggc ggcaagtctg 180

atctctgcag cctttatctc tcgctggttc ggcggcctga tgctcggcgc tatgggtgac 240

cgctacgggc gtcgtctggc aatggtcacc agcatcgttc tcttctcggc cgggacgctg 300

gcctgcggct ttgcgccagg ctacatcacc atgtttatcg ctcgtctggt catcggcatg 360

gggatggcgg gtgaatacgg ttccagcgcc acctatgtca ttgaaagctg gccaaaacat 420

ctgcgtaaca aagccagtgg ttttttgatt tcaggcttct ctgtgggggc cgtcgttgcc 480

gctcaggtct atagcctggt ggttccggtc tggggctggc gtgcgctgtt ctttatcggc 540

attttgccaa tcatctttgc tctctggctg cgtaaaaaca tcccggaagc ggaagactgg 600

aaagagaaac acgcaggtaa agcaccagta cgcacaatgg tggatattct ctaccgtggt 660

gaacatcgca ttgccaatat cgtaatgaca ctggcggcgg ctactgcgct gtggttctgc 720

ttcgccggta acctgcaaaa tgccgcgatc gtcgctgttc ttgggctgtt atgcgccgca 780

atctttatca gctttatggt gcagagtgca ggcaaacgct ggccaacggg cgtaatgctg 840

atggtggtcg tgttgtttgc tttcctctac tcatggccga ttcaggcgct gctgccaacg 900

tatctgaaaa ccgatctggc ttataacccg catactgtag ccaatgtgct gttctttagt 960

ggctttggcg cggcggtggg atgctgcgta ggtggcttcc tcggtgactg gctgggaacc 1020

cgcaaagcgt acgtttgtag cctgctggcc tcgcagctgc tgattattcc ggtatttgcg 1080

attggcggcg caaacgtctg ggtgctcggt ctgttactgt tcttccagca aatgcttgga 1140

caagggatcg ccgggatctt accaaaactg attggcggtt atttcgatac cgaccagcgt 1200

gcagcgggcc tgggctttac ctacaacgtt ggcgcattgg gcggtgcact ggccccaatc 1260

atcggcgcgt tgatcgctca acgtctggat ctgggtactg cgctggcatc gctctcgttc 1320

agtctgacgt tcgtggtgat cctgctgatt gggctggata tgccttctcg cgttcagcgt 1380

tggttgcgcc cggaagcgtt gcgtactcat gacgctatcg acggtaaacc attcagcggt 1440

gccgtgccgt ttggcagcgc caaaaacgat ttagtcaaaa ccaaaagtta a 1491

<210> 13

<211> 1081

<212> DNA

<213> Artificial sequence

<400> 13

ggtgaactac tcggtcgtca accccattaa acctccataa tgatcggcag gatcattgga 60

cggcgctttg ttttttcgta caagaaaggt gcaagcgtgt ctgtaatttc gtttttgatt 120

tcagaccatt gagtcgtttt tcgttccatt actttttgta agtgatttga aatgagctct 180

tgagcgtcgt tgatcaagtc accagattct ctcatgtaca caaatcctct ggaaatcaaa 240

tcaggacccg ctgaaatctt gaagtcgtcc atgtcaatgc tgacaacaac gatgacaagt 300

ccttcttcag agagaattct gcgatcacga agtacgatat tgccaatatc accgataccg 360

cttccgtcaa tgtacactga accggacggg atttttcctg caactgaagc ctcatcgcct 420

ttaagtgcta atacttcacc attatccatg ataaagcagt tttcctctgg gatgccacaa 480

tctgttgcaa gtttgacatg catcttttgc attctgtact caccgtgaat cggcatgaag 540

aatttaggct tgattaaacg aagcatcagc ttctgttctt cctgtccgcc gtgaccggat 600

gtatggatat cgttaagagg gccgtgaata acctcagcac ccgcacgata cagctggttg 660

attgttcgac tcacgctgat tgtgttgcct gggatagggg atgaagaaaa tacgactgta 720

tccccaggat tgattgaaat ttggcggtgt gtgccgtttg caattcttga taacgccgcc 780

attggttccc cttggcttcc tgtacataaa attgttactt tattagcagg catacgattg 840

atttcgttat gctcaataaa cgtattttta ggacagttaa tataaccgag tgtctgtcca 900

atttcgatag ccgattccat actgcgtcca aatacggcaa cttttcttcc attttgtaca 960

gcagcttcaa ttacctgctg caagcggtga atattcgacg caaatgtggc gaagataatt 1020

cggccgtcca ccttgcggaa aatgtcatga atgctttctc ctacacggcg ctcagacatg 1080

g 1081

<210> 14

<211> 6420

<212> DNA

<213> Artificial sequence

<400> 14

gaactataca aataattcac gtcacgcgtc catggagaga actatacaaa taattcacgt 60

cacgcgtcca tggagatctt tgtctgcaac tgaaaagttt ataccttacc tggaacaaat 120

ggttgaaaca tacgaggcta atatcggctt attaggaata gtccctgtac taataaaatc 180

aggtggatca gttgatcagt atattttgga cgaagctcgg aaagaatttg gagatgactt 240

gcttaattcc acaattaaat taagggaaag aataaagcga tttgatgttc aaggaatcac 300

ggaagaagat actcatgata aagaagctct aaaactattc aataacctta caatggaatt 360

gatcgaaagg gtggaaggtt aatggtacga aaattagggg atctacctag aaagccacaa 420

ggcgataggt caagcttaaa gaacccttac atggatctta cagattctga aagtaaagaa 480

acaacagagg ttaaacaaac agaaccaaaa agaaaaaaag cattgttgaa aacaatgaaa 540

gttgatgttt caatccataa taagattaaa tcgctgcacg aaattctggc agcatccgaa 600

gggaattcat attacttaga ggatactatt gagagagcta ttgataagat ggttgagaca 660

ttacctgaga gccaaaaaac tttttatgaa tatgaattaa aaaaaagaac caacaaaggc 720

tgagacagac tccaaacgag tctgtttttt taaaaaaaat attaggagca ttgaatatat 780

attagagaat taagaaagac atgggaataa aaatatttta aatccagtaa aaatatgata 840

agattatttc agaatatgaa gaactctgtt tgtttttgat gaaaaaacaa acaaaaaaaa 900

tccacctaac ggaatctcaa tttaactaac agcggccaaa ctgagaagtt aaatttgaga 960

aggggaaaag gcggatttat acttgtattt aactatctcc attttaacat tttattaaac 1020

cccatacaag tgaaaatcct cttttacact gttcctttag gtgatcgcgg agggacatta 1080

tgagtgaagt aaacctaaaa ggaaatacag atgaattagt gtattatcga cagcaaacca 1140

ctggaaataa aatcgccagg aagagaatca aaaaagggaa agaagaagtt tattatgttg 1200

ctgaaacgga agagaagata tggacagaag agcaaataaa aaacttttct ttagacaaat 1260

ttggtacgca tataccttac atagaaggtc attatacaat cttaaataat tacttctttg 1320

atttttgggg ctatttttta ggtgctgaag gaattgcgct ctatgctcac ctaactcgtt 1380

atgcatacgg cagcaaagac ttttgctttc ctagtctaca aacaatcgct aaaaaaatgg 1440

acaagactcc tgttacagtt agaggctact tgaaactgct tgaaaggtac ggttttattt 1500

ggaaggtaaa cgtccgtaat aaaaccaagg ataacacaga ggaatccccg atttttaaga 1560

ttagacgtaa ggttcctttg ctttcagaag aacttttaaa tggaaaccct aatattgaaa 1620

ttccagatga cgaggaagca catgtaaaga aggctttaaa aaaggaaaaa gagggtcttc 1680

caaaggtttt gaaaaaagag cacgatgaat ttgttaaaaa aatgatggat gagtcagaaa 1740

caattaatat tccagaggcc ttacaatatg acacaatgta tgaagatata ctcagtaaag 1800

gagaaattcg aaaagaaatc aaaaaacaaa tacctaatcc tacaacatct tttgagagta 1860

tatcaatgac aactgaagag gaaaaagtcg acagtacttt aaaaagcgaa atgcaaaatc 1920

gtgtctctaa gccttctttt gatacctggt ttaaaaacac taagatcaaa attgaaaata 1980

aaaattgttt attacttgta ccgagtgaat ttgcatttga atggattaag aaaagatatt 2040

tagaaacaat taaaacagtc cttgaagaag ctggatatgt tttcgaaaaa atcgaactaa 2100

gaaaagtgca ataaactgct gaagtatttc agcagttttt tttatttaga aatagtgaaa 2160

aaaatataat cagggaggta tcaatattta atgagtactg atttaaattt atttagactg 2220

gaattaataa ttaacacgta gactaattaa aatttaatga gggataaaga ggatacaaaa 2280

atattaattt caatccctat taaattttaa caaggggggg attaaaattt aattagaggt 2340

ttatccacaa gaaaagaccc taataaaatt tttactaggg ttataacact gattaatttc 2400

ttaatggggg agggattaaa atttaatgac aaagaaaaca atcttttaag aaaagctttt 2460

aaaagataat aataaaaaga gctttgcgat taagcaaaac tctttacttt ttcattgaca 2520

ttatcaaatt catcgatttc aaattgttgt tgtatcataa agttaattct gttttgcaca 2580

accttttcag gaatataaaa cacatctgag gcttgtttta taaactcagg gtcgctaaag 2640

tcaatgtaac gtagcatatg atatggtata gcttccaccc aagttagcct ttctgcttct 2700

tctgaatgtt tttcatatac ttccatgggt atctctaaat gattttcctc atgtagcaag 2760

gtatgagcaa aaagtttatg gaattgatag ttcctctctt tttcttcaac ttttttatct 2820

aaaacaaaca ctttaacatc tgagtcaatg taagcataag atgtttttcc agtcataatt 2880

tcaatcccaa atcttttaga cagaaattct ggacgtaaat cttttggtga aagaattttt 2940

ttatgtagca atatatccga tacagcacct tctaaaagcg ttggtgaata gggcatttta 3000

cctatctcct ctcattttgt ggaataaaaa tagtcatatt cgtccatcta cctatcctat 3060

tatcgaacag ttgaactttt taatcaagga tcagtccttt ttttcattat tcttaaactg 3120

tgctcttaac tttaacaact cgatttgttt ttccagatct cgagggtaac tagcctcgcc 3180

gatcccgcaa gaggcccggc agtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 3240

tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 3300

ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 3360

ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 3420

gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 3480

caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 3540

ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 3600

cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 3660

gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 3720

taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 3780

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 3840

agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 3900

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 3960

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4020

tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 4080

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 4140

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 4200

agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 4260

gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 4320

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 4380

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 4440

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 4500

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 4560

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4620

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 4680

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 4740

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 4800

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 4860

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 4920

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 4980

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 5040

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 5100

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca tgcttaagtt 5160

attggtatga ctggttttaa gcgcaaaaaa agttgctttt tcgtacctat taatgtatcg 5220

ttttagaaaa ccgactgtaa aaagtacagt cggcattatc tcatattata aaagccagtc 5280

attaggccta tctgacaatt cctgaataga gttcataaac aatcctgcat gataaccatc 5340

acaaacagaa tgatgtacct gtaaagatag cggtaaatat attgaattac ctttattaat 5400

gaattttcct gctgtaataa tgggtagaag gtaattacta ttattattga tatttaagtt 5460

aaacccagta aatgaagtcc atggaataat agaaagagaa aaagcatttt caggtatagg 5520

tgttttggga aacaatttcc ccgaaccatt atatttctct acatcagaaa ggtataaatc 5580

ataaaactct ttgaagtcat tctttacagg agtccaaata ccagagaatg ttttagatac 5640

accatcaaaa attgtataaa gtggctctaa cttatcccaa taacctaact ctccgtcgct 5700

attgtaacca gttctaaaag ctgtatttga gtttatcacc cttgtcacta agaaaataaa 5760

tgcagggtaa aatttatatc cttcttgttt tatgtttcgg tataaaacac taatatcaat 5820

ttctgtggtt atactaaaag tcgtttgttg gttcaaataa tgattaaata tctcttttct 5880

cttccaattg tctaaatcaa ttttattaaa gttcatttga tatgcctcct aaatttttat 5940

ctaaagtgaa tttaggaggc ttacttgtct gctttcttca ttagaatcaa tcctttttta 6000

aaagtcaata ttactgtaac ataaatatat attttaaaaa tatcccactt tatccaattt 6060

tcgtttgttg aactaatggg tgctttagtt gaagaataaa agaccacatt aaaaaatgtg 6120

gtcttttgtg tttttttaaa ggatttgagc gtagcgaaaa atccttttct ttcttatctt 6180

gataataagg gtaactattg ccgatcgtcc attccgacag catcgccagt cactatggcg 6240

tgctgctagc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 6300

gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 6360

gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc 6420

<210> 15

<211> 8261

<212> DNA

<213> Artificial sequence

<400> 15

cccgctttct tgataatatg ggaagtgctc cgtaatacgc tgacaagaga gaaagggctt 60

ggaggtattg aaacaagagg agttctgaga attggtatgc cttataagtc caattaacag 120

ttgaaaacct gcataggaga gctatgcggg ttttttattt tacataatga tacataattt 180

accgaaactt gcggaacata attgaggaat catagaattt tgtcaaaata attttattga 240

caacgtctta ttaacgttga tataatttaa attttatttg acaaaaatgg gctcgtgttg 300

tacaatggta taacaggtat aaaggtatat cgtttaaagg aggtgaaatg tacacatggg 360

taagggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt 420

taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact 480

tacccttaaa tttatttgca ctactggaaa gcttcctgtt ccttggccaa cacttgtcac 540

tactcttact tatggtgttc aatgcttttc aagataccca gatcatatga agcggcacga 600

cttcttcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct tcttcaagga 660

cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc tcgtcaacag 720

aatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc acaagttgga 780

atacaactac aactcccaca acgtatacat catggcagac aaacaaaaga atggaatcaa 840

agttaacttc aaaattagac acaacattga agatggaagc gttcaactag cagaccatta 900

tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc 960

cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga 1020

gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaat aattcacgtc 1080

acgcgtccat ggagatcttt gtctgcaact gaaaagttta taccttacct ggaacaaatg 1140

gttgaaacat acgaggctaa tatcggctta ttaggaatag tccctgtact aataaaatca 1200

ggtggatcag ttgatcagta tattttggac gaagctcgga aagaatttgg agatgacttg 1260

cttaattcca caattaaatt aagggaaaga ataaagcgat ttgatgttca aggaatcacg 1320

gaagaagata ctcatgataa agaagctcta aaactattca ataaccttac aatggaattg 1380

atcgaaaggg tggaaggtta atggtacgaa aattagggga tctacctaga aagccacaag 1440

gcgataggtc aagcttaaag aacccttaca tggatcttac agattctgaa agtaaagaaa 1500

caacagaggt taaacaaaca gaaccaaaaa gaaaaaaagc attgttgaaa acaatgaaag 1560

ttgatgtttc aatccataat aagattaaat cgctgcacga aattctggca gcatccgaag 1620

ggaattcata ttacttagag gatactattg agagagctat tgataagatg gttgagacat 1680

tacctgagag ccaaaaaact ttttatgaat atgaattaaa aaaaagaacc aacaaaggct 1740

gagacagact ccaaacgagt ctgttttttt aaaaaaaata ttaggagcat tgaatatata 1800

ttagagaatt aagaaagaca tgggaataaa aatattttaa atccagtaaa aatatgataa 1860

gattatttca gaatatgaag aactctgttt gtttttgatg aaaaaacaaa caaaaaaaat 1920

ccacctaacg gaatctcaat ttaactaaca gcggccaaac tgagaagtta aatttgagaa 1980

ggggaaaagg cggatttata cttgtattta actatctcca ttttaacatt ttattaaacc 2040

ccatacaagt gaaaatcctc ttttacactg ttcctttagg tgatcgcgga gggacattat 2100

gagtgaagta aacctaaaag gaaatacaga tgaattagtg tattatcgac agcaaaccac 2160

tggaaataaa atcgccagga agagaatcaa aaaagggaaa gaagaagttt attatgttgc 2220

tgaaacggaa gagaagatat ggacagaaga gcaaataaaa aacttttctt tagacaaatt 2280

tggtacgcat ataccttaca tagaaggtca ttatacaatc ttaaataatt acttctttga 2340

tttttggggc tattttttag gtgctgaagg aattgcgctc tatgctcacc taactcgtta 2400

tgcatacggc agcaaagact tttgctttcc tagtctacaa acaatcgcta aaaaaatgga 2460

caagactcct gttacagtta gaggctactt gaaactgctt gaaaggtacg gttttatttg 2520

gaaggtaaac gtccgtaata aaaccaagga taacacagag gaatccccga tttttaagat 2580

tagacgtaag gttcctttgc tttcagaaga acttttaaat ggaaacccta atattgaaat 2640

tccagatgac gaggaagcac atgtaaagaa ggctttaaaa aaggaaaaag agggtcttcc 2700

aaaggttttg aaaaaagagc acgatgaatt tgttaaaaaa atgatggatg agtcagaaac 2760

aattaatatt ccagaggcct tacaatatga cacaatgtat gaagatatac tcagtaaagg 2820

agaaattcga aaagaaatca aaaaacaaat acctaatcct acaacatctt ttgagagtat 2880

atcaatgaca actgaagagg aaaaagtcga cagtacttta aaaagcgaaa tgcaaaatcg 2940

tgtctctaag ccttcttttg atacctggtt taaaaacact aagatcaaaa ttgaaaataa 3000

aaattgttta ttacttgtac cgagtgaatt tgcatttgaa tggattaaga aaagatattt 3060

agaaacaatt aaaacagtcc ttgaagaagc tggatatgtt ttcgaaaaaa tcgaactaag 3120

aaaagtgcaa taaactgctg aagtatttca gcagtttttt ttatttagaa atagtgaaaa 3180

aaatataatc agggaggtat caatatttaa tgagtactga tttaaattta tttagactgg 3240

aattaataat taacacgtag actaattaaa atttaatgag ggataaagag gatacaaaaa 3300

tattaatttc aatccctatt aaattttaac aaggggggga ttaaaattta attagaggtt 3360

tatccacaag aaaagaccct aataaaattt ttactagggt tataacactg attaatttct 3420

taatggggga gggattaaaa tttaatgaca aagaaaacaa tcttttaaga aaagctttta 3480

aaagataata ataaaaagag ctttgcgatt aagcaaaact ctttactttt tcattgacat 3540

tatcaaattc atcgatttca aattgttgtt gtatcataaa gttaattctg ttttgcacaa 3600

ccttttcagg aatataaaac acatctgagg cttgttttat aaactcaggg tcgctaaagt 3660

caatgtaacg tagcatatga tatggtatag cttccaccca agttagcctt tctgcttctt 3720

ctgaatgttt ttcatatact tccatgggta tctctaaatg attttcctca tgtagcaagg 3780

tatgagcaaa aagtttatgg aattgatagt tcctctcttt ttcttcaact tttttatcta 3840

aaacaaacac tttaacatct gagtcaatgt aagcataaga tgtttttcca gtcataattt 3900

caatcccaaa tcttttagac agaaattctg gacgtaaatc ttttggtgaa agaatttttt 3960

tatgtagcaa tatatccgat acagcacctt ctaaaagcgt tggtgaatag ggcattttac 4020

ctatctcctc tcattttgtg gaataaaaat agtcatattc gtccatctac ctatcctatt 4080

atcgaacagt tgaacttttt aatcaaggat cagtcctttt tttcattatt cttaaactgt 4140

gctcttaact ttaacaactc gatttgtttt tccagatctc gagggtaact agcctcgccg 4200

atcccgcaag aggcccggca gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 4260

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4320

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4380

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4440

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4500

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4560

tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 4620

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 4680

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 4740

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 4800

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4860

gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4920

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4980

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5040

gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5100

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5160

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5220

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5280

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5340

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5400

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5460

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5520

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5580

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5640

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5700

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5760

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5820

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5880

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5940

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6000

ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 6060

gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 6120

gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcat gcttaagtta 6180

ttggtatgac tggttttaag cgcaaaaaaa gttgcttttt cgtacctatt aatgtatcgt 6240

tttagaaaac cgactgtaaa aagtacagtc ggcattatct catattataa aagccagtca 6300

ttaggcctat ctgacaattc ctgaatagag ttcataaaca atcctgcatg ataaccatca 6360

caaacagaat gatgtacctg taaagatagc ggtaaatata ttgaattacc tttattaatg 6420

aattttcctg ctgtaataat gggtagaagg taattactat tattattgat atttaagtta 6480

aacccagtaa atgaagtcca tggaataata gaaagagaaa aagcattttc aggtataggt 6540

gttttgggaa acaatttccc cgaaccatta tatttctcta catcagaaag gtataaatca 6600

taaaactctt tgaagtcatt ctttacagga gtccaaatac cagagaatgt tttagataca 6660

ccatcaaaaa ttgtataaag tggctctaac ttatcccaat aacctaactc tccgtcgcta 6720

ttgtaaccag ttctaaaagc tgtatttgag tttatcaccc ttgtcactaa gaaaataaat 6780

gcagggtaaa atttatatcc ttcttgtttt atgtttcggt ataaaacact aatatcaatt 6840

tctgtggtta tactaaaagt cgtttgttgg ttcaaataat gattaaatat ctcttttctc 6900

ttccaattgt ctaaatcaat tttattaaag ttcatttgat atgcctccta aatttttatc 6960

taaagtgaat ttaggaggct tacttgtctg ctttcttcat tagaatcaat ccttttttaa 7020

aagtcaatat tactgtaaca taaatatata ttttaaaaat atcccacttt atccaatttt 7080

cgtttgttga actaatgggt gctttagttg aagaataaaa gaccacatta aaaaatgtgg 7140

tcttttgtgt ttttttaaag gatttgagcg tagcgaaaaa tccttttctt tcttatcttg 7200

ataataaggg taactattgc cgatcgtcca ttccgacagc atcgccagtc actatggcgt 7260

gctgctagcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 7320

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 7380

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctct 7440

tatttctttt tgttggtggt ctgaccgaaa gcgtgccagg tagcagagac gctgttgaga 7500

tgcgattgca acgcacgatc ggcttcgtca ggatcatgac ggcggatcgc atcaacgatc 7560

gcaatatgct gttgataact aacgttgtta tgttcgtgca gtgcctgatc ggtaaccgtt 7620

gggcgtgcgg caataagcca gtcgagcagg gcaacgtgga tcgccatgaa gattgggtta 7680

ccggggatct ccgccagcac gcggtggaaa tcaacgtctg aacgaatgaa tgccgcgttg 7740

ttatccagcg actgactgtt gatttccagt gcttttgcca gcaaatcgat ttgctcatcg 7800

gtggcatgtt cagccgcata gcgcaccaga ctggattcaa agaacagacg taattgttcg 7860

aaatgggcaa tcccaccggg atgagaaagg aaatctttcg ccatgccgga aagctcaccg 7920

atgatagtgt ccgcagaagg acgcgagacg cgagcgcgtt cgccgttgtt tatttgcacc 7980

agacctttgc gttttaacgc tgccagcgct tcacgcaccg aaggacgccc gacgttaaag 8040

aacgccatca gttcgcgttc agacggtaat tgttcacctt cgccaaattc acgacggcgg 8100

atcatctgtt ccagctcttc ttccaccatt tcggagagtt ttttacgcgc cagcgggcgg 8160

ctacgcaagt tgcgaccaat tgcaggtgaa gaatcttcgg tttgcgaatc aaatgcgttc 8220

ataaggccca tgtgtacatt tcacctcctt tgtttttacg t 8261

Claims (9)

1. An inducible expression element responsive to sialic acid; wherein the expression element comprises: constitutive promoter P _hbs A transcription regulatory protein NanR, a constitutive promoter P integrated with one or two transcription regulatory protein NanR binding sequences _veg Said constitutive form P _hbs The promoter expresses a transcription regulatory protein NanR; the promoter P _hbs And P _veg The transcription direction is opposite; the nucleotide sequence of the NanR binding sequence is shown in SEQ NO. 3.

2. Inducible expression element according to claim 1 wherein the constitutive promoter P _veg 2 copies of the transcription regulatory protein, the NanR binding sequence, were integrated.

3. The inducible expression element of claim 2 further comprising a sialic acid transporter, NanT, having the amino acid sequence shown in SEQ No. 5.

4. The inducible expression element of any one of claims 1 to 3 further comprising a marker; the marker is expressed by promoter P _veg Regulating expression.

5. A plasmid containing the expression-inducing element according to any one of claims 1 to 4.

6. A genetically engineered bacterium comprising the inducible expression element according to any one of claims 1 to 3, wherein Bacillus subtilis is used as a host.

7. The use of the inducible expression element of any one of claims 1 to 4 in the field of Bacillus subtilis metabolic engineering.

8. Use according to claim 7, wherein expression of the gene of interest is activated in a culture environment in the presence of sialic acid.

9. The use according to claim 8, wherein the range is 30 to 37^oAnd C, culturing at 180-250 rpm for at least 24 hours.