CN111394410B

CN111394410B - High-catalytic-activity neuraminic acid synthase and application thereof

Info

Publication number: CN111394410B
Application number: CN202010238268.4A
Authority: CN
Inventors: 刘延峰; 刘龙; 张晓龙; 堵国成; 李江华; 陈坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-02-01
Anticipated expiration: 2040-03-30
Also published as: CN111394410A

Abstract

本发明公开了一种高催化活性神经氨酸合酶及应用，属于遗传工程领域。本发明通过筛选不同来源的N‑乙酰神经氨酸合酶，并以5个不同强度启动子优化N‑乙酰神经氨酸合酶在基因组上的表达水平，将最佳表达强度的NeuB整合到可以合成NeuAc前体物质ManNAc的菌株中，提高了枯草芽孢杆菌的N‑乙酰神经氨酸产量使N‑乙酰神经氨酸在摇瓶中产量达到7.6g/L。The invention discloses a neuraminic acid synthase with high catalytic activity and its application, and belongs to the field of genetic engineering. In the present invention, by screening N-acetylneuraminic acid synthase from different sources, and optimizing the expression level of N-acetylneuraminic acid synthase on the genome with five promoters with different strengths, the NeuB with the best expression strength is integrated into the In the strain synthesizing the NeuAc precursor substance ManNAc, the N-acetylneuraminic acid production of Bacillus subtilis was improved, so that the N-acetylneuraminic acid yield in the shake flask reached 7.6 g/L.

Description

High-catalytic-activity neuraminic acid synthase and application thereof

Technical Field

The invention relates to a high catalytic activity neuraminic acid synthase and application thereof, belonging to the field of genetic engineering.

Background

N-acetylneuraminic acid is a functional monosaccharide and is widely present in microorganisms and mammals. In humans, N-acetylneuraminic acid is involved in a number of physiological processes such as cell recognition, signal transduction, and the like. Therefore, the N-acetylneuraminic acid is widely applied to enhancing the immunity of the infants and promoting the brain development of the infants. At present, N-acetylneuraminic acid is mainly extracted by natural products (eggs, cubilose and the like), and other products are difficult to separate and have higher cost; in addition, the neuraminic acid can be obtained by a whole-cell transformation method, but the substrates of acetylglucosamine and pyruvic acid with higher cost are needed as the substrates, and the production cost of the neuraminic acid is higher due to the lower conversion rate of the substrates.

Bacillus subtilis is a production host widely used as food enzyme preparation and important nutritional chemicals, and the product is certified as "general regulated as safe" (GRAS) level by FDA. Therefore, the efficient de novo synthesis of neuraminic acid by using bacillus subtilis as a host and glucose and other cheap carbon sources as substrates through metabolic engineering is an effective strategy.

The existing N-acetylneuraminic acid metabolic pathway constructed in the hay is mainly a NeuB key enzyme synthetic pathway taking N-acetylglucosamine as a precursor, and the catalytic performance of the existing NeuB enzyme is weak, so that the synthetic efficiency of the neuraminic acid is limited. The existence of this problem severely limits the increase in neuraminic acid production, further limiting its market application.

Disclosure of Invention

In order to solve the problems, the invention improves the yield of the N-acetylneuraminic acid of the bacillus subtilis by screening N-acetylneuraminic acid synthases (NeuB) with different sources and optimizing the expression strength.

The first purpose of the invention is to provide the application of N-acetylneuraminic acid synthase derived from Neisseria meningitidis (Neisseria meningitidis) in improving the yield of N-acetylneuraminic acid of Bacillus subtilis.

In one embodiment, the use is for expressing N-acetylneuraminic acid synthase from Neisseria meningitidis (Neisseria meningitidis) in Bacillus subtilis.

In one embodiment, the N-acetylneuraminic acid synthase is (a) or (b):

(a) protein with amino acid sequence shown as SEQ ID NO. 1;

(b) an enzyme having N-acetylneuraminic acid synthase activity which is substituted, deleted or added with one or more amino acids based on the amino acid sequence of (a).

In one embodiment, the amino acid sequence of the N-acetylneuraminic acid synthase is shown in SEQ ID No. 1.

In one embodiment, the N-acetylneuraminic acid synthase is regulated by a promoter of low strength.

In one embodiment, the promoter comprises a nucleotide sequence set forth in any one of SEQ ID nos. 3 to 7.

The second purpose of the invention is to provide a genetically engineered bacterium which has the synthetic capacity of N-acetyl-D-mannosamine (NeuAc) and expresses N-acetylneuraminic acid synthase shown in SEQ ID NO. 1.

In one embodiment, the N-acetylneuraminic acid synthase is integrated into Bacillus subtilis, which can synthesize N-acetyl-D-mannosamine (Mannac).

In one embodiment, the genetically engineered bacteria are host Bacillus subtilis BSGN 6-comK.

In one embodiment, the genetically engineered bacteria also potentiate the expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase.

In one embodiment, the amino acid sequence of the glucosamine-6-phosphate-N-acetyltransferase (Gna1) is set forth in SEQ ID No. 9.

In one embodiment, the amino acid sequence of the N-acetylglucosamine isomerase (Age) is shown in SEQ ID NO. 11.

In one embodiment, the glucosamine-6-phosphate-N-acetyltransferase is expressed under the control of a promoter as set forth in SEQ ID NO. 3.

In one embodiment, the N-acetylglucosamine isomerase is expressed under the control of a promoter represented by SEQ ID NO. 8.

The third purpose of the invention is to provide the application of the bacillus subtilis engineering bacteria in the production of N-acetylneuraminic acid or derivatives thereof.

In one embodiment, the recombinant bacillus subtilis is inoculated in an LB culture medium, cultured for 12-18 h to obtain a seed solution, and then inoculated into a fermentation culture medium for fermentation in an inoculation amount of 1-10% by volume.

In one embodiment, the bacillus subtilis engineering bacteria are fermented for 16-72 hours at 30-37 ℃.

In one embodiment, the derivative product includes, but is not limited to, the antiviral drug zanamivir or oseltamivir.

Has the advantages that:

(1) according to the invention, through screening NeuB enzymes from different sources, the NeuB derived from Neisseria meningitidis (Neisseria meningitidis) has higher catalytic activity compared with sources such as escherichia coli and the like at the same low expression level, and the yield of NeuAc can be improved to 7.6g/L from 0.1 g/L.

(2) According to the invention, the expression level of N-acetylneuraminic acid synthase (NeuB) on a genome is optimized by 5 promoters (P1-P5) with different intensities, and the NeuB with the optimal expression intensity is integrated into a strain capable of synthesizing N-acetylneuraminic acid (NeuAc) precursor N-acetyl-D-aminommannose (Mannac), so that the yield of the neuraminic acid is increased, and the yield of the neuraminic acid after 72h of shake flask horizontal fermentation is increased from 3.7g/L to 7.6 g/L.

Detailed Description

The amino acid sequence of N-acetylneuraminic acid synthase (NeuB) enzyme derived from Neisseria meningitis is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2;

the nucleotide sequence of the promoter P1 is shown as SEQ ID NO. 3; the nucleotide sequence of the promoter P2 is shown as SEQ ID NO. 4; the nucleotide sequence of the promoter P3 is shown as SEQ ID NO. 5; the nucleotide sequence of the promoter P4 is shown as SEQ ID NO. 6; the nucleotide sequence of the P5 promoter is shown as SEQ ID NO.7, and the nucleotide sequence of the P10 promoter is shown as SEQ ID NO. 8;

the amino acid sequence of glucosamine-6-phosphate-N-acetyltransferase (Gna1) is shown as SEQ ID NO.9, and the nucleotide sequence is shown as SEQ ID NO. 10;

the amino acid sequence of the N-acetylglucosamine-isomerase (Age) is shown as SEQ ID NO.11, and the nucleotide sequence is shown as SEQ ID NO. 12;

the amino acid sequence of the Escherichia coli-derived NeuB enzyme is shown as SEQ ID NO.13, and the nucleotide sequence is shown as SEQ ID NO. 14;

the amino acid sequence of the NeuB enzyme derived from Moritella viscosa is shown as SEQ ID NO.15, and the nucleotide sequence is shown as SEQ ID NO. 16.

Seed medium (g/L): tryptone 10, yeast extract 5, NaCl 10.

Fermentation medium (g/L): 60 parts of glucose, 6 parts of tryptone, 12 parts of yeast powder, 6 parts of ammonium sulfate, 12.5 parts of dipotassium phosphate, 2.5 parts of potassium dihydrogen phosphate and 3 parts of magnesium sulfate.

The neuraminic acid detection method comprises the following steps: agilent liquid chromatography: the chromatographic column is Aminex HPX-87H column (300X 7.8mM), the absorption peak is detected by ultraviolet 210nm, the mobile phase is 10mM sulfuric acid, and the flow rate is 0.5 mL/min. The neuraminic acid peak time was 9.8 minutes.

EXAMPLE 1 construction of a genomic recombinant integration Gna1 fragment

Using a bacillus subtilis 168 genome as a template, designing primers Gna1-L-F: 5'-CGTGATATCGTCATTCAGTCTCTTGAACGCCA-3' and Gna1-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCATGTTCTTTTTAGTTAGACGATTTTAATACAAGCCTCGCCA-3', and amplifying, recombining and integrating a Gna1 left-arm gene fragment;

synthesizing a promoter P1 segment shown as SEQ ID NO. 3; synthesizing a gene segment which is shown as SEQ ID NO.10 and codes Gna 1;

taking a Bacillus subtilis 168 genome as a template, and performing amplification by using a primer Gna 1-R-L: 5'-ATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCCCAGTCATAAAATAGTTTTCCTAATAAGACCTGG-3' and Gna 1-R-R: 5'-CCTACTTAAGCTGCTACCACTTGTGA-3', amplifying, recombining and integrating Gna1 right arm gene segments.

The gene fragment of Gna1 left arm, the gene fragment of P1 promoter, Gna1 and the gene fragment of Gna1 right arm are fused by PCR technology to construct a recombinant integrated Gna1 gene fragment, which is named Gna 1-1.

Example 2 construction of a genomic recombinant integration Age fragment

Taking a bacillus subtilis 168 genome as a template, designing primers Age-L-F: 5'-CGTGATATCGTCATTCAGTCTCTTGAACGCCA-3' and Age-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCATGTTCTTTTTAGTTAGACGATTTTAATACAAGCCTCGCCA-3', and amplifying, recombining and integrating Age left arm gene segments;

synthesizing a promoter fragment of a promoter P10 shown as SEQ ID NO. 8;

synthesizing a gene segment which is shown as SEQ ID NO.12 and codes Age;

taking a hay genome as a template, and carrying out PCR by using a primer Age-R-L: 5'-ATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCCCAGTCATAAAATAGTTTTCCTAATAAGACCTGG-3' and Age-R-R: 5'-ATAACCAACGCAGCAAGTGGCAACCT-3', amplifying and recombining an Age right arm gene segment.

The Age left arm gene fragment, the P10 promoter fragment (shown in SEQ ID NO. 8), the Age gene fragment and the Age right arm gene fragment are constructed into a recombinant integrated Age gene fragment by a fusion PCR technology, and the recombinant integrated Age gene fragment is named as Age-10.

Example 3 construction of a genomic recombinant integration NemNeuB fragment

Using a bacillus subtilis 168 genome as a template, designing primers NemNeuB-L-F: 5'-CGGTGTCTGTATATCACAAAAATAGTGAGCAGGGTAACGA-3' and NemNeuB-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCCACCTATTTTGTTACAGCGTGTGCCACTTTTATGCA-3', and amplifying, recombining and integrating a NemNeuB left arm gene fragment;

respectively synthesizing fragments of promoters P1-P5 shown as SEQ ID NO. 3-7;

synthesizing a gene fragment of the N-acetylneuraminic acid synthase gene NeuB shown as SEQ ID NO. 2;

taking a bacillus subtilis 168 genome as a template, and carrying out amplification by using a primer NemNeuB-R-L: 5'-TAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCGCTTGAACAGCTTTTTTTGAATACCTTGTCCAGCT-3' and NemNeuB-R-R: 5'-GCGTCATCGCAGTTTTTGCACCTGACT-3', amplifying, recombining and integrating the NemNeuB right arm gene fragment.

The gene fragment of the left arm of NemNeuB, the promoter fragment (shown as SEQ ID NO. 3-7 respectively), the gene fragment of the NemNeuB and the gene fragment of the right arm of NemNeuB are constructed into a recombinant and integrated NemNeuB gene fragment by a fusion PCR technology, and the gene fragment is named as NemNeuB-1-NemNeuB-5 according to different promoters.

Example 4 construction of Bacillus subtilis with Gna1 Gene recombinantly integrated

The recombinant integrated Gna1-1 gene fragment was transformed into Bacillus subtilis BSGN6-comK (the construction method of the strain is disclosed in the article "modulated path engineering of key carbon-precursor supplied-path for amplified N-acetyl amino acid production in Bacillus subtilis") genome, and the obtained recombinant Bacillus subtilis was named as BS-Gna 1.

Example 5 construction of Bacillus subtilis with recombinant integration of Age Gene

The gene fragment of the recombinant integrated Age-10 was transformed into the genome of recombinant Bacillus subtilis BS-Gna1 constructed in example 4, and the obtained recombinant Bacillus subtilis was named BSG-Age-10.

Example 6 construction of Bacillus subtilis recombinantly integrating NemNeuB Gene

The gene fragments recombined and integrated with NemNeuB-1 to NemNeuB-5 are respectively transformed to the Bacillus subtilis BSG-Age-10 genome constructed in the embodiment 5, and the obtained recombined Bacillus subtilis is named as BSGA-NemNeuB-1 to BSGA-NemNeuB-5.

Respectively inoculating the recombinant bacillus subtilis BSGA-NemNeuB-1-BSGA-NemNeuB-5 into an LB culture medium, culturing for 12-18 h to obtain a seed solution with OD about 8, transferring the seed solution into a fermentation culture medium by an inoculation amount of 1% by volume, culturing for 72h at 37 ℃ and 200rpm, and determining the yield of NeuAc in the fermentation broth as follows: 7.6g/L, 3.4g/L, 3.1g/L, 2.8g/L, 3.7 g/L.

Comparative example 1: expression effects of N-acetylneuraminic acid synthases of different origins

The N-acetylneuraminic acid synthase NemNeuB in example 3 is replaced by NeuB enzyme (the nucleotide sequence is shown in SEQ ID NO. 14) derived from Escherichia coli, a NemNeuB left arm gene fragment, a P1 promoter fragment (shown in SEQ ID NO. 3), a NeuB gene fragment and a NemNeuB right arm gene fragment are fused in sequence according to the same method, and then the recombinant Bacillus subtilis is constructed according to the method in example 6, and the result shows that the NeuAc yield of the constructed recombinant Bacillus subtilis is only 0.1g/L under the same culture conditions in example 6.

Comparative example 2: effect of N-acetylneuraminic acid synthases of different origins on expression Effect

The yields of neuraminic acid after fermentation for 16 to 72 hours under the same culture conditions as in example 6 are shown in table 1, wherein the NeuB genes derived from e.coli K1 and Moritella viscosa are expressed in bacillus subtilis BSG-Age-10, respectively, according to the same strategy as in example 6.

TABLE 1 recombinant Bacillus subtilis neuraminic acid production expressing NeuB from different sources

Comparative example 3: effect of different promoters on expression Effect of different enzymes

According to the same strategy as that in example 2, P10 promoters are respectively replaced by P1-P5, recombinant integrated Age gene fragments are constructed and respectively named Age-1, Age-2, Age-3, Age-4 and Age-5 and are respectively integrated on the genome of bacillus subtilis BS-Gna1, and the obtained recombinant bacillus subtilis is respectively named BSG-Age-1-BSG-Age-5.

The recombinant integrated NemNeuB-1 gene fragments were transformed into recombinant Bacillus subtilis BSG-Age-1-BSG-Age-5, respectively, according to the same strategy as in example 6, and fermented under the same conditions as in example 6, and the results showed that the NeuAc yields of the recombinant Bacillus subtilis BSG-Age-1-BSG-Age-5 fermented for 72 hours were 1.5g/L, 2.2g/L, 2.5g/L, 3.5g/L, and 3.2g/L, respectively.

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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Ser Arg Ala Ala Ala Leu Arg Leu Gln Arg Met Asp Ile Pro Ala Tyr

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Lys Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys Leu Val

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Ala Ser Phe Gly Lys Pro Ile Ile Leu Ser Thr Gly Met Asn Ser Ile

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Glu Ser Ile Lys Lys Ser Val Glu Ile Ile Arg Glu Ala Gly Val Pro

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Tyr Ala Leu Leu His Cys Thr Asn Ile Tyr Pro Thr Pro Tyr Glu Asp

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Val Arg Leu Gly Gly Met Asn Asp Leu Ser Glu Ala Phe Pro Asp Ala

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Ile Ile Gly Leu Ser Asp His Thr Leu Asp Asn Tyr Ala Cys Leu Gly

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Ala Val Ala Leu Gly Gly Ser Ile Leu Glu Arg His Phe Thr Asp Arg

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Met Asp Arg Pro Gly Pro Asp Ile Val Cys Ser Met Asn Pro Asp Thr

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Phe Lys Glu Leu Lys Gln Gly Ala His Ala Leu Lys Leu Ala Arg Gly

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Gly Lys Lys Asp Thr Ile Ile Ala Gly Glu Lys Pro Thr Lys Asp Phe

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Val Val Ala Ser Ala Ser Leu Val Val Glu Met Lys Phe Ile His Gly

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Ala Gly Ser Arg Gly Arg Val Glu Asp Val Val Val Asp Thr Glu Met

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Arg Arg Gln Lys Leu Gly Ala Val Leu Leu Lys Thr Leu Val Ser Leu

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Gly Lys Ser Leu Gly Val Tyr Lys Ile Ser Leu Glu Cys Val Pro Glu

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Met Thr Gln Arg Phe

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Leu Cys Asn Gln Leu Glu Lys Arg Glu Asn Trp Leu Lys Ile Ala Arg

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Asn Gly Ala Lys Phe Leu Ala Gln His Gly Arg Asp Asp Glu Gly Asn

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Trp Tyr Phe Ala Leu Thr Arg Gly Gly Glu Pro Leu Val Gln Pro Tyr

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Asn Ile Phe Ser Asp Cys Phe Ala Ala Met Ala Phe Ser Gln Tyr Ala

115 120 125

Leu Ala Ser Gly Glu Glu Trp Ala Lys Asp Val Ala Met Gln Ala Tyr

130 135 140

Asn Asn Val Leu Arg Arg Lys Asp Asn Pro Lys Gly Lys Tyr Thr Lys

145 150 155 160

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

165 170 175

Leu Ala Asn Leu Thr Leu Glu Met Glu Trp Leu Leu Pro Gln Glu Thr

180 185 190

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

195 200 205

Leu Asp Gln Glu Gln Gly Leu Met Tyr Glu Asn Val Ala Pro Asp Gly

210 215 220

Ser His Ile Asp Cys Phe Glu Gly Arg Leu Ile Asn Pro Gly His Gly

225 230 235 240

Ile Glu Ala Met Trp Phe Ile Met Asp Ile Ala Arg Arg Lys Asn Asp

245 250 255

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

260 265 270

Phe Ala Trp Asp Asn Glu Tyr Gly Gly Leu Tyr Tyr Phe Met Asp Ala

275 280 285

Ala Gly His Pro Pro Gln Gln Leu Glu Trp Asp Gln Lys Leu Trp Trp

290 295 300

Val His Leu Glu Ser Leu Val Ala Leu Ala Met Gly Tyr Arg Leu Thr

305 310 315 320

Gly Arg Asp Ala Cys Trp Ala Trp Tyr Gln Lys Met His Asp Tyr Ser

325 330 335

Trp Gln His Phe Ala Asp Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu

340 345 350

Asn Arg Arg Gly Glu Val Leu Leu Asn Leu Lys Gly Gly Lys Trp Lys

355 360 365

Gly Cys Phe His Val Pro Arg Ala Met Tyr Leu Cys Trp Gln Gln Phe

370 375 380

Glu Ala Leu Ser

385

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<213> Artificial sequence

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acattttcta tgctttgtaa ccagctggaa aaaagagaaa actggctgaa aatcgctcgc 240

aatggagcca aatttctggc acaacatggc agagatgatg aaggaaactg gtattttgct 300

ttaacacgcg gcggagaacc gctggttcaa ccgtataata tttttagcga ttgctttgca 360

gcgatggcct tttctcagta tgcattagcg tcaggagaag aatgggcaaa agatgttgct 420

atgcaagcct ataataacgt gctgagacgc aaagataacc cgaaaggcaa atacacaaaa 480

acatatccgg gaacaagacc gatgaaagct ttagccgttc cgatgattct ggcgaacctg 540

acacttgaaa tggaatggtt actgccgcaa gaaacactgg aaaatgtgct tgctgccaca 600

gtccaggaag ttatgggcga ttttcttgat caagaacagg gattaatgta tgaaaacgtc 660

gctccggatg gctcacatat cgattgcttt gaaggacgcc tgattaatcc gggccatgga 720

atcgaagcga tgtggtttat tatggatatc gctagacgca aaaacgatag caaaacaatc 780

aaccaggcgg ttgatgttgt gttaaatatc ctgaactttg cttgggataa cgaatacggc 840

ggactttact actttatgga tgcagcgggc catccgccgc aacagctgga atgggatcaa 900

aaactttggt gggtgcatct tgaaagctta gtcgcactgg cgatgggcta tagattaaca 960

ggacgcgatg catgttgggc gtggtatcaa aaaatgcatg attattcttg gcagcatttt 1020

gcagatccgg aatatggcga atggtttgga tatcttaaca gacgcggcga agtgcttctg 1080

aacctgaaag gcggaaaatg gaaaggatgc tttcatgtcc cgagagccat gtatctgtgt 1140

tggcaacagt ttgaagcact ttcataa 1167

<210> 13

<211> 346

<212> PRT

<213> Escherichia coli

<400> 13

Met Ser Asn Ile Tyr Ile Val Ala Glu Ile Gly Cys Asn His Asn Gly

1 5 10 15

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

20 25 30

Val Asn Ala Val Lys Phe Gln Thr Phe Lys Ala Asp Lys Leu Ile Ser

35 40 45

Ala Ile Ala Pro Lys Ala Glu Tyr Gln Ile Lys Asn Thr Gly Glu Leu

50 55 60

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

65 70 75 80

Tyr Leu His Leu Met Glu Tyr Ala Val Ser Leu Asn Leu Asp Val Phe

85 90 95

Ser Thr Pro Phe Asp Glu Asp Ser Ile Asp Phe Leu Ala Ser Leu Lys

100 105 110

Gln Lys Ile Trp Lys Ile Pro Ser Gly Glu Leu Leu Asn Leu Pro Tyr

115 120 125

Leu Glu Lys Ile Ala Lys Leu Pro Ile Pro Asp Lys Lys Ile Ile Ile

130 135 140

Ser Thr Gly Met Ala Thr Ile Asp Glu Ile Lys Gln Ser Val Ser Ile

145 150 155 160

Phe Ile Asn Asn Lys Val Pro Val Gly Asn Ile Thr Ile Leu His Cys

165 170 175

Asn Thr Glu Tyr Pro Thr Pro Phe Glu Asp Val Asn Leu Asn Ala Ile

180 185 190

Asn Asp Leu Lys Lys His Phe Pro Lys Asn Asn Ile Gly Phe Ser Asp

195 200 205

His Ser Ser Gly Phe Tyr Ala Ala Ile Ala Ala Val Pro Tyr Gly Ile

210 215 220

Thr Phe Ile Glu Lys His Phe Thr Leu Asp Lys Ser Met Ser Gly Pro

225 230 235 240

Asp His Leu Ala Ser Ile Glu Pro Asp Glu Leu Lys His Leu Cys Ile

245 250 255

Gly Val Arg Cys Val Glu Lys Ser Leu Gly Ser Asn Ser Lys Val Val

260 265 270

Thr Ala Ser Glu Arg Lys Asn Lys Ile Val Ala Arg Lys Ser Ile Ile

275 280 285

Ala Lys Thr Glu Ile Lys Lys Gly Glu Val Phe Ser Glu Lys Asn Ile

290 295 300

Thr Thr Lys Arg Pro Gly Asn Gly Ile Ser Pro Met Glu Trp Tyr Asn

305 310 315 320

Leu Leu Gly Lys Ile Ala Glu Gln Asp Phe Ile Pro Asp Glu Leu Ile

325 330 335

Ile His Ser Glu Phe Lys Asn Gln Gly Glu

340 345

<210> 14

<211> 1041

<212> DNA

<213> Artificial sequence

<400> 14

atgtctaaca tctacatcgt ggcagaaatc ggctgcaatc ataacggatc agtcgatatc 60

gcgagagaaa tgattttaaa agctaaagaa gccggcgtga acgctgtcaa atttcaaaca 120

tttaaagccg ataaactgat cagcgcaatt gcgccgaaag cagaatacca aatcaaaaac 180

acaggagaat tagaatctca gctggaaatg acgaaaaaac tggaaatgaa atacgatgat 240

taccttcatc tgatggaata cgcagtcagc ctgaatcttg atgtttttag cacaccgttt 300

gatgaagatt ctattgattt tctggcgtca ctgaaacaaa aaatctggaa aattccgtca 360

ggcgaactgc ttaaccttcc gtacctggaa aaaatcgcta aacttccgat cccggataag 420

aaaattatca ttagcacagg catggccaca atcgatgaaa tcaaacagtc tgtctcaatc 480

tttatcaata acaaagtccc ggttggaaac atcacaatcc tgcattgtaa cacagaatat 540

ccgacaccgt ttgaagatgt taaccttaac gctatcaacg atctgaaaaa acattttccg 600

aaaaacaaca tcggcttttc tgatcattca agcggatttt atgcagcgat tgctgccgtt 660

ccgtatggca tcacatttat cgaaaaacat tttacactgg ataaaagcat gtctggaccg 720

gatcatcttg cttcaatcga accggatgaa ctgaaacatc tttgcattgg cgttagatgt 780

gtggaaaaat cactgggatc aaatagcaaa gttgtgacag ccagcgaaag aaaaaacaaa 840

atcgttgcac gcaaatctat catcgcgaaa acagaaatca aaaaaggaga agtgttttca 900

gagaaaaata tcacaacaaa aagaccgggc aacggaatta gcccgatgga atggtataat 960

ttactgggca aaatcgcgga acaagatttt atcccggatg aacttatcat ccatagcgaa 1020

tttaaaaacc agggagaata a 1041

<210> 15

<211> 347

<212> PRT

<213> Moritella viscosa

<400> 15

Met Thr Asn Pro Val Phe Glu Ile Ser Gly Arg Lys Val Gly Leu Asp

1 5 10 15

Tyr Ala Pro Leu Val Ile Ala Glu Ile Gly Ile Asn His Glu Gly Ser

20 25 30

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

35 40 45

Glu Ile Ile Lys His Gln Thr His Val Ile Glu Asp Glu Met Ser Ser

50 55 60

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

65 70 75 80

Ile Met Asp Arg Cys Ser Leu Asn Glu Glu Asp Glu Ile Lys Leu Lys

85 90 95

Lys Tyr Ile Glu Ser Lys Gly Ala Ile Phe Ile Ser Thr Pro Phe Ser

100 105 110

Arg Ala Ala Ala Leu Arg Leu Glu Arg Met Gly Val Ser Ala Tyr Lys

115 120 125

Ile Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Leu Asp Leu Ile Ala

130 135 140

Ser Tyr Gly Lys Pro Val Ile Leu Ser Thr Gly Met Asn Asp Ile Pro

145 150 155 160

Ser Ile Arg Lys Ser Val Glu Ile Phe Arg Lys Tyr Lys Thr Pro Leu

165 170 175

Cys Leu Leu His Thr Thr Asn Leu Tyr Pro Thr Pro Asp His Leu Ile

180 185 190

Arg Ile Gly Ala Met Glu Glu Met Gln Arg Glu Phe Ser Asp Val Val

195 200 205

Val Gly Leu Ser Asp His Ser Ile Asp Asn Leu Ala Cys Leu Gly Ala

210 215 220

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

225 230 235 240

Ala Arg Ser Gly Pro Asp Ile Cys Cys Ser Met Asp Gly Ala Glu Cys

245 250 255

Ala Glu Leu Ile Ser Gln Ser Lys Arg Met Ala Gln Met Arg Gly Gly

260 265 270

Ser Lys Gly Ala Val Lys Glu Glu Gln Val Thr Ile Asp Phe Ala Tyr

275 280 285

Ala Ser Val Val Thr Ile Lys Glu Ile Lys Ala Gly Glu Ala Phe Thr

290 295 300

Lys Asp Asn Leu Trp Val Lys Arg Pro Gly Thr Gly Asp Phe Leu Ala

305 310 315 320

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

325 330 335

Phe Asp Val Gln Leu Lys Lys Glu Phe Ile Lys

340 345

<210> 16

<211> 1044

<212> DNA

<213> Artificial sequence

<400> 16

atgacaaatc cggtctttga aatttctggc agaaaagttg gacttgatta tgccccgtta 60

gtgatcgcag aaattggcat caaccatgaa ggatcactga aaacagcctt tgaaatggtg 120

gatgcagcga ttgaaggcgg agcagaaatc atcaaacatc aaacacatgt cattgaagat 180

gaaatgtcaa gcgaagcaaa gaaagttatc ccgggcaatg ctgatgtgag catctacgaa 240

atcatggata gatgctctct gaacgaagaa gatgaaatca aactgaaaaa atacatcgaa 300

tcaaaaggcg ctatctttat ctcaacaccg tttagccgcg ctgccgcact gagacttgaa 360

cgcatgggag ttagcgccta taaaattggc tctggagaat gcaataacta tccgctgctt 420

gatcttattg cgtcttatgg caaaccggtc atcttatcaa caggaatgaa tgatattccg 480

tctatcagaa aatcagttga aatctttcgc aaatacaaaa caccgctttg tttactgcat 540

acaacaaacc tgtatccgac accggatcat cttattagaa tcggcgcaat ggaagaaatg 600

caacgcgaat ttagcgatgt tgtggtcgga ctgagcgatc attctatcga taacctggct 660

tgtctgggag ctgtggctgc tggagcttct gtcctggaaa gacattttac agataacaaa 720

gctcgctcag gcccggatat ttgctgtagc atggatggag cggaatgtgc tgaacttatc 780

tctcaatcaa aaagaatggc ccagatgcgc ggcggatcaa aaggcgcagt caaagaagaa 840

caggttacaa ttgattttgc ctatgcaagc gttgtgacaa ttaaagaaat caaagccgga 900

gaagcattta caaaagataa tctgtgggtt aaacgcccgg gcacaggaga ttttcttgcg 960

gatgattatg aaatgctttt aggcaagaaa gcaagccaaa acattgattt tgatgtgcag 1020

ctgaagaaag aatttatcaa ataa 1044

Claims

1. the application of N-acetylneuraminic acid synthase derived from Neisseria meningitidis ( Neisseria meningitidis ) in improving the yield of Bacillus subtilis N-acetylneuraminic acid, characterized in that the N-acetylneuraminic acid synthase The amino acid sequence of the enzyme is shown in SEQ ID NO.1.

2. a Bacillus subtilis, is characterized in that, has the synthetic ability of N-acetyl-D-mannosamine, and expresses the N-acetylneuraminic acid synthase of Neisseria meningitidis ( Neisseria meningitidis ) source; Also strengthens Expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase; the N-acetylneuraminic acid synthase has the amino acid sequence shown in SEQ ID NO.1; the The amino acid sequence of glucosamine-6-phosphate-N-acetyltransferase is shown in SEQ ID NO.9; the amino acid sequence of the N-acetylglucosamine isomerase is shown in SEQ ID NO.11.

3 . The Bacillus subtilis according to claim 2 , wherein the expression of the N-acetylneuraminic acid synthase is regulated by a low-strength promoter. 4 .

4. Bacillus subtilis according to claim 3, is characterized in that, the nucleotide sequence of described promoter is as shown in any of SEQID NO.3～7.

5. The Bacillus subtilis according to any one of claims 2 to 4, wherein the gene encoding the N-acetylneuraminic acid synthase is integrated into the subtilis that can synthesize N-acetyl-D-mannosamine Bacillus genome.

6. The Bacillus subtilis according to any one of claims 2 to 4, wherein the expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase is also enhanced.

7. Bacillus subtilis according to claim 6, is characterized in that, regulates glucosamine-6-phosphate-N-acetyltransferase expression by the promoter shown in SEQ ID NO.3, or by SEQ ID NO.3. The promoter shown in 8 regulates N-acetylglucosamine isomerase expression.

8. the application of the arbitrary described Bacillus subtilis of claim 2～7 in producing N-acetylneuraminic acid or its derivative product.

9. A method for producing neuraminic acid, characterized in that the fermentation of Bacillus subtilis described in any one of claims 2 to 7 is applied.

10. method according to claim 9, is characterized in that, described fermentation is carried out under 30～37 ^℃ .