CN111411066A - Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof - Google Patents
Double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof Download PDFInfo
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Abstract
The invention discloses a double-way compound neuraminic acid-producing bacillus subtilis and a construction method thereof, belonging to the field of genetic engineering.A N-acetylneuraminic acid synthase and an N-acetylneuraminic acid aldolase are simultaneously integrated on a genome to be expressed, and 3 promoters with different strengths are selected to optimize the expression quantity of NeuB and NanA genes, so that the final yield reaches 8.3 g/L in a shake flask, and a foundation is laid for further improving the yield of the bacillus subtilis N-acetylneuraminic acid.
Description
Technical Field
The invention relates to a dual-way composite neuraminic acid-producing bacillus subtilis and a construction method 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 current N-acetylneuraminic acid metabolic pathway constructed in the bacillus subtilis is mainly a NeuB key enzyme synthetic pathway taking N-acetylglucosamine as a precursor, and because the intracellular phosphoenolpyruvate (precursor of the NeuB for synthesizing neuraminic acid) of the bacillus subtilis is low in concentration, the synthesis 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 application further introduces N-acetylneuraminic acid aldolase (NanA) on the basis of a way of strengthening NeuB as a characteristic enzyme, pyruvate is used as a precursor substance, and 3 promoters with different strengths are selected to optimize the expression levels of NeuB and NanA so as to obtain the strain with improved N-acetylneuraminic acid yield.
The first purpose of the invention is to provide a recombinant bacillus subtilis, which integrates and expresses N-acetylneuraminic acid synthase (NeuB) and N-acetylneuraminic acid aldolase (NanA) through promoters with different strengths, and the nucleotide sequence of the promoter is shown in any one of SEQ ID NO. 3-5.
In one embodiment, the amino acid sequence of N-acetylneuraminic acid synthase (NeuB) is shown in SEQ ID No. 1.
In one embodiment, the amino acid sequence of N-acetylneuraminic acid aldolase (NanA) is shown in SEQ ID No. 7.
In one embodiment, the nucleotide sequence of the P1 promoter is set forth in SEQ ID NO. 9.
In one embodiment, the nucleotide sequence of the P2 promoter is set forth in SEQ ID No. 10.
In one embodiment, the nucleotide sequence of the P3 promoter is set forth in SEQ ID NO. 11.
In one embodiment, the recombinant Bacillus subtilis further overexpresses glucosamine-6-phosphate-N-acetyltransferase (Gna1) and N-acetylglucosamine isomerase (Age).
In one embodiment, the amino acid sequence of the glucosamine-6-phosphate-N-acetyltransferase (Gna1) is set forth in SEQ ID No. 3; the amino acid sequence of the N-acetylglucosamine isomerase (Age) is shown as SEQ ID NO. 5.
In one embodiment, the recombinant Bacillus subtilis expresses glucosamine-6-phosphate-N-acetyltransferase from the P1 promoter and N-acetylglucosamine isomerase from the P2 promoter.
In one embodiment, the recombinant Bacillus subtilis expresses glucosamine-6-phosphate-N-acetyltransferase from the P1 promoter, N-acetylglucosamine isomerase from the P2 promoter, and N-acetylneuraminic acid synthase and N-acetylneuraminic acid aldolase from the P1 promoter.
In one embodiment, the Bacillus subtilis is Bacillus subtilis BSGN 6-comK; the construction method of the Bacillus subtilis BSGN6-comK is disclosed in a paper 'modulated path engineering of key-preferred-paths for improved N-acetyl amino acid production in Bacillus subtilis'.
The second purpose of the invention is to provide a construction method of the recombinant bacillus subtilis, which is to respectively construct recombinant integrated fragments of glucosamine-6-phosphate-N-acetyltransferase gene, N-acetylglucosamine isomerase gene, N-acetylneuraminic acid synthase gene and N-acetylneuraminic acid aldolase gene, and then transform one or more recombinant integrated fragments onto the bacillus subtilis genome; the recombinant integration segment is formed by fusing and connecting a gene left arm, a promoter segment, a gene segment and a gene right arm in sequence.
In one embodiment of the present invention, the nucleotide sequence of the promoter fragment is selected from SEQ ID NO. 9-11.
In one embodiment of the invention, the genetically engineered bacterium is a Bacillus subtilis BSGN6-comK as a host.
The third purpose of the invention is to provide a method for improving the yield of the neuraminic acid in the bacillus subtilis, which is to strengthen the expression of N-acetylneuraminic acid synthase (NeuB) and N-acetylneuraminic acid aldolase (NanA) through a strong promoter.
In one embodiment, the method further enhances the expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase.
In one embodiment, the nucleotide sequence of the strong promoter is shown in SEQ ID NO.10 or SEQ ID NO. 11.
The fourth purpose of the invention is to provide the application of the recombinant bacillus subtilis in the production of neuraminic acid and derivatives thereof.
In one embodiment, the recombinant Bacillus subtilis is inoculated in L B culture medium, cultured for 12-18 h to obtain a seed solution with OD of 6-10, and then inoculated into a fermentation culture medium for fermentation at an inoculation amount of 1-10%.
In one embodiment, the recombinant Bacillus subtilis is cultured under the condition of 30-37 ℃ for 16-72 hours.
Has the advantages that:
(1) the invention reduces the protein synthesis pressure of key enzymes on cells by optimizing the expression levels of N-acetylneuraminic acid synthase (NeuB) and N-acetylneuraminic acid aldolase (NanA) on genomes through 3 promoters (P1, P2 and P3) with different strengths, and further integrates the N-acetylneuraminic acid synthase (NeuB) and the N-acetylneuraminic acid aldolase (NanA) with optimized expression levels into the same recombinant bacillus subtilis, so that the yield of the bacillus subtilis N-acetylneuraminic acid is increased to be more than 9.5 g/L from 2.75 g/L;
(2) the invention provides a multi-path construction strategy, and the construction method is simple, convenient to use and has good metabolic engineering application prospect.
Detailed Description
The amino acid sequence of the 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 amino acid sequence of glucosamine-6-phosphate-N-acetyltransferase (Gna1) is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4;
the amino acid sequence of the N-acetylglucosamine isomerase (Age) is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6;
the amino acid sequence of the N-acetylneuraminic acid aldolase (NanA) is shown as SEQ ID NO.7, and the nucleotide sequence is shown as SEQ ID NO. 8;
the nucleotide sequence of the promoter P1 is shown as SEQ ID NO. 9; the nucleotide sequence of the promoter P2 is shown as SEQ ID NO. 10; the nucleotide sequence of the promoter P3 is shown as SEQ ID NO. 11;
the amino acid sequence of the Escherichia coli-derived NeuB enzyme is shown as SEQ ID NO.12, and the nucleotide sequence is shown as SEQ ID NO. 13;
the amino acid sequence of the NeuB enzyme derived from Moritella viscosa is shown as SEQ ID NO.14, and the nucleotide sequence is shown as SEQ ID NO. 15.
The fermentation medium (g/L) comprises glucose 60, tryptone 6, yeast powder 12, ammonium sulfate 6, dipotassium hydrogen phosphate 12.5, potassium dihydrogen phosphate 2.5 and magnesium sulfate 3.
The detection method of N-acetylneuraminic acid comprises Agilent liquid chromatography, wherein a chromatographic column is Aminex HPX-87H column (300 × 7.8.8 mM), an absorption peak is detected by ultraviolet 210nm, a mobile phase is 10mM sulfuric acid, the flow rate is 0.5m L/min, and the time of the N-acetylneuraminic acid appearing is about 9.8 minutes.
EXAMPLE 1 construction of a genomic recombinant integration Gna1 fragment
Taking a bacillus subtilis 168 genome as a template, designing primers Gna 1-L-F: 5'-CGTGATATCGTCATTCAGTCTCTTGAACGCCA-3' and Gna 1-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCATGTTCTTTTTAGTTAGACGATTTTAATACAAGCCTCGCCA-3', and amplifying, recombining and integrating a Gna1 left-arm gene fragment;
synthesizing fragments of promoters P1, P2 and P3 shown as SEQ ID NO. 9-11;
synthesizing a gene segment which is shown as SEQ ID NO.4 and codes Gna 1;
a right-arm gene fragment of Gna1 is amplified, recombined and integrated by taking a Bacillus subtilis 168 genome as a template and primers Gna 1-R-L: 5'-ATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCCCAGTCATAAAATAGTTTTCCTAATAAGACCTGG-3' and Gna 1-R-R: 5'-cctacttaagctgctaccacttgtga-3'.
A gene fragment of Gna1 left arm, a gene fragment of promoter (P1, P2 or P3) and Gna1 and a gene fragment of Gna1 right arm are subjected to fusion PCR to obtain a recombinant integrated Gna1 gene fragment, and the recombinant integrated Gna1 gene fragment is named as Gna1-1, Gna1-2 and Gna1-3 (corresponding to the promoters P1, P2 and P3) according to the difference of the promoters.
Example 2 construction of a genomic recombinant integration Age fragment
Using 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 fragments;
synthesizing fragments of promoters P1, P2 and P3 shown as SEQ ID NO. 9-11;
synthesizing a gene segment for coding Age shown as SEQ ID NO. 6;
and (3) amplifying, recombining and integrating an Age right arm gene fragment by taking a bacillus subtilis 168 genome as a template and primers Age-R-L: 5'-ATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCCCAGTCATAAAATAGTTTTCCTAATAAGACCTGG-3' and Age-R-R: 5'-ATAACCAACGCAGCAAGTGGCAACCT-3'.
The gene sequence of the left arm of the Age fragment, the promoter fragment (P1, P2 or P3 respectively), the Age gene fragment and the gene sequence of the right arm of the Age fragment are subjected to fusion PCR to obtain a recombinant integrated Age gene fragment, and the recombinant integrated Age gene fragment is named Age-1, Age-2 and Age-3 (corresponding to the promoters P1, P2 and P3 respectively) according to the difference of the promoters.
Example 3 construction of a genomic recombinant integration NeuB fragment
Using a bacillus subtilis 168 genome as a template, designing primers NeuB-L-F: 5'-CGGTGTCTGTATATCACAAAAATAGTGAGCAGGGTAACGA-3' and NeuB-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCCACCTATTTTGTTACAGCGTGTGCCACTTTTATGCA-3', and amplifying, recombining and integrating a NeuB left arm gene fragment;
synthesizing fragments of promoters P1, P2 and P3 shown as SEQ ID NO. 9-11;
synthesizing a gene segment which is shown as SEQ ID NO.2 and codes NeuB;
the genome of the bacillus subtilis 168 is taken as a template, and NeuB-R-L: 5'-TAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCGCTTGAACAGCTTTTTTTGAATACCTTGTCCAGCT-3' and Age-R-R: 5'-GCGTCATCGCAGTTTTTGCACCTGACT-3' are used for amplifying, recombining and integrating NeuB right arm gene fragments.
Constructing a recombinant integrated NeuB gene segment by the amplified NeuB left arm gene segment, promoter segment (P1, P2 or P3), NeuB gene segment and NeuB right arm segment through a fusion PCR technology, and respectively naming the NeuB gene segment as NeuB-1, NeuB-2 and NeuB-3 (respectively corresponding to promoters P1, P2 and P3) according to the difference of the promoters.
Example 4 construction of a genomic recombinant integration NanA fragment
Using a bacillus subtilis 168 genome as a template, designing primers NanA-L-F: 5'-GATGTTGCAGTCACAGTTAGTTGATTAGAGTTAGCAGCA-3' and NanA-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCTTTTTACGGCCCTGTGCCACAACTTACT-3', and amplifying, recombining and integrating a NanA left arm gene fragment;
synthesizing fragments of promoters P1, P2 and P3 shown as SEQ ID NO. 9-11;
synthesizing a NanA fragment shown as SEQ ID NO. 8;
a gene segment of the right arm of NanA is amplified, recombined and integrated by taking a bacillus subtilis 168 genome as a template and primers NanA-R-L: 5'-GAATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCGAATAAGTCCAAGACGGAAAGCCTGCGGA-3' and NanA-R-R: 5'-GGATATTAACATGTACGCAATACTGCTGCTGT-3'.
A NanA left arm gene fragment, a promoter fragment (P1, P2 or P3), a NanA gene fragment and a NanA fragment right arm gene sequence are respectively subjected to fusion PCR to construct a recombinant integrated NanA gene fragment, and the recombinant integrated NanA gene fragment is named as NanA-1, NanA-2 and NanA-3 (respectively corresponding to a promoter P1, a promoter P2 and a promoter P3) according to different promoters.
Example 5 construction of recombinant Bacillus subtilis with Gna1 Gene recombinantly integrated
The recombinant integrated Gna1-1 gene fragment constructed in example 1 was transformed into the genome of Bacillus subtilis BSGN6-comK (the construction method is disclosed in the article "modulated path engineering of key carbon-precursor supplied-path for amplified N-acetyl amino acid production in Bacillus subtilis"), and the obtained recombinant Bacillus subtilis was named as BS-Gna 1.
Example 6 construction of recombinant Bacillus subtilis with Age Gene integrated by genome recombination
The gene fragment of the recombinant integrated Age-2 constructed in example 2 was transformed into the genome of the recombinant Bacillus subtilis BS-Gna1 constructed in example 5, and the obtained recombinant Bacillus subtilis was named BSG-Age-2.
Example 7 construction of recombinant Bacillus subtilis having NeuB Gene recombinantly integrated in genome
The gene fragments of recombinant and integrated NeuB-1, NeuB-2 and NeuB-3 constructed in example 3 were transformed into the genome of recombinant Bacillus subtilis BSG-Age-2 constructed in example 6, and the obtained recombinant Bacillus subtilis was named BSGA-NeuB-1, BSGA-NeuB-2 and BSGA-NeuB-3, respectively.
Respectively inoculating the recombinant bacillus subtilis BSGA-NeuB-1, the recombinant bacillus subtilis BSGA-NeuB-2 and the recombinant bacillus subtilis BSGA-NeuB-3 into L B culture medium for culturing for 12-18 hours to obtain seed liquid with OD of about 6, then respectively inoculating the seed liquid into a fermentation culture medium according to the volume by 1 percent of the inoculation amount, culturing for 72 hours at 37 ℃ and 200rpm, and measuring the yield of NeuAc in the fermentation liquid to be 7.6 g/L, 1.9 g/L and 1.7 g/L respectively.
Example 8 construction of Bacillus subtilis by genomic recombination and integration of the NanA Gene
The gene fragments of recombinant integrated NanA-1, NanA-2 and NanA-3 constructed in example 4 were transformed onto the genome of recombinant Bacillus subtilis BSGA-NeuB-1 constructed in example 7, and the obtained recombinant Bacillus subtilis were named BSGAN-NanA-1, BSGAN-NanA-2 and BSGAN-NanA-3, respectively.
Respectively inoculating the recombinant bacillus subtilis BSGAN-NanA-1, the recombinant bacillus subtilis BSGAN-NanA-2 and the recombinant bacillus subtilis BSGAN-NanA-3 into L B culture medium for culturing for 12-18 hours to obtain seed liquid with OD of about 6, then respectively inoculating the seed liquid into a fermentation culture medium according to 1% of inoculation amount, culturing for 72 hours in the fermentation culture medium at 37 ℃ and 200rpm, and finally determining that the yield of NeuAc detected in the fermentation liquid is respectively 9.5 g/L, 8.1 g/L and 7.7 g/L, thereby successfully obtaining the engineering strain capable of improving the yield of NeuAc, and the yield of the BSGAN-NanA-1 engineering strain is highest and reaches 9.5 g/L.
Comparative example 1: NeuAc synthesis via only the Age-NeuB pathway
According to the same strategy of the embodiment 6-7, only an Age-NeuB approach is strengthened to synthesize NeuAc without regulation and control of a promoter, the constructed recombinant strain is cultured in a fermentation medium for 72 hours at 37 ℃ and 200rpm, and the highest yield of NeuAc can only reach 2.75 g/L.
Comparative example 2: effect of N-acetylneuraminic acid synthases of different origins on expression Effect
Coli K1 and Moritella viscosa-derived NeuB genes were expressed in Bacillus subtilis BSGN6-comK by the same strategy as in example 5, respectively, and the expression of the genes was regulated by different promoters, respectively, the neuraminic acid yields after fermentation for 72h of the constructed recombinant Bacillus subtilis were as shown in Table 1, and were only 4.5 g/L at the maximum, under the same culture conditions as in example 10.
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
The recombinant integrated Age-1 and Age-3 constructed in example 2 were transformed into the recombinant Bacillus subtilis BS-Gna1 genome constructed in example 5 according to the same strategy as in example 6, and the neuraminic acid yields of the obtained strains were 0.5 g/L and 3.1 g/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
<110> university of south of the Yangtze river
<120> double-way composite neuraminic acid-producing bacillus subtilis and construction method thereof
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Leu Asp Gln Glu Ala Phe Glu Lys Arg Phe Glu Ala Met Arg Thr Ser
50 55 60
Val Pro Asn Tyr His Ile Val Val Ile Glu Asp Ser Asn Ser Gln Lys
65 70 75 80
Val Val Ala Ser Ala Ser Leu Val Val Glu Met Lys Phe Ile His Gly
85 90 95
Ala Gly Ser Arg Gly Arg Val Glu Asp Val Val Val Asp Thr Glu Met
100 105 110
Arg Arg Gln Lys Leu Gly Ala Val Leu Leu Lys Thr Leu Val Ser Leu
115 120 125
Gly Lys Ser Leu Gly Val Tyr Lys Ile Ser Leu Glu Cys Val Pro Glu
130 135 140
Leu Leu Pro Phe Tyr Ser Gln Phe Gly Phe Gln Asp Asp Cys Asn Phe
145 150 155 160
Met Thr Gln Arg Phe
165
<210>4
<211>498
<212>DNA
<213> Artificial sequence
<400>4
atgagccata tcttcgacgc atctgtactg gctccacata ttcctagtaa ccttcctgat 60
aatttcaagg tgagaccact ggcaaaggat gatttttcga agggatatgt cgacctgctg 120
tcacaattga cgtcagttgg aaaccttgac caagaagcat ttgagaaacg atttgaggcg 180
atgagaacaa gcgtaccgaa ttatcacatc gtagtaattg aggattccaa cagccagaaa 240
gtggtggcgt ctgctagttt ggttgttgaa atgaaattca ttcatggggc cggatcaagg 300
ggtcgtgttg aagatgttgt cgtcgataca gaaatgcgcc ggcaaaaatt aggtgccgtg 360
cttttaaaaa ctttggtgtc acttggcaaa tctttaggcg tctacaaaat aagcctcgaa 420
tgcgtcccgg aattactccc gttctattcc caatttggct ttcaggatga ctgtaatttt 480
atgacccagc gcttttaa 498
<210>5
<211>388
<212>PRT
<213> Artificial sequence
<400>5
Met Gly Lys Asn Leu Gln Ala Leu Ala Gln Leu Tyr Lys Asn Ala Leu
1 5 10 15
Leu Asn Asp Val Leu Pro Phe Trp Glu Asn His Ser Leu Asp Ser Glu
20 25 30
Gly Gly Tyr Phe Thr Cys Leu Asp Arg Gln Gly Lys Val Tyr Asp Thr
35 40 45
Asp Lys Phe Ile Trp Leu Gln Asn Arg Gln Val Trp Thr Phe Ser Met
50 5560
Leu Cys Asn Gln Leu Glu Lys Arg Glu Asn Trp Leu Lys Ile Ala Arg
65 70 75 80
Asn Gly Ala Lys Phe Leu Ala Gln His Gly Arg Asp Asp Glu Gly Asn
85 90 95
Trp Tyr Phe Ala Leu Thr Arg Gly Gly Glu Pro Leu Val Gln Pro Tyr
100 105 110
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
<210>6
<211>1167
<212>DNA
<213> Artificial sequence
<400>6
atgggcaaaa acttacaagc tctggcccag ctttataaaa atgccctgct taacgatgtg 60
cttccgtttt gggaaaatca ttcattagat agcgaaggcg gatattttac atgcctggat 120
agacagggca aagtctacga tacagataaa tttatctggc ttcaaaaccg ccaggtttgg 180
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>7
<211>297
<212>PRT
<213> Artificial sequence
<400>7
Met Ala Thr Asn Leu Arg Gly Val Met Ala Ala Leu Leu Thr Pro Phe
1 5 10 15
Asp Gln Gln Gln Ala Leu Asp Lys Ala Ser Leu Arg Arg Leu Val Gln
20 25 30
Phe Asn Ile Gln Gln Gly Ile Asp Gly Leu Tyr Val Gly Gly Ser Thr
35 40 45
Gly Glu Ala Phe Val Gln Ser Leu Ser Glu Arg Glu Gln Val Leu Glu
50 55 60
Ile Val Ala Glu Glu Ala Lys Gly Lys Ile Lys Leu Ile Ala His Val
65 70 75 80
Gly Cys Val Ser Thr Ala Glu Ser Gln Gln Leu Ala Ala Ser Ala Lys
85 90 95
Arg Tyr Gly Phe Asp Ala Val Ser Ala Val Thr Pro Phe Tyr Tyr Pro
100 105 110
Phe Ser Phe Glu Glu His Cys Asp His Tyr Arg Ala Ile Ile Asp Ser
115 120 125
Ala Asp Gly Leu Pro Met Val Val Tyr Asn Ile Pro Ala Leu Ser Gly
130 135 140
Val Lys Leu Thr Leu Asp Gln Ile Asn Thr Leu Val Thr Leu Pro Gly
145 150 155 160
Val Gly Ala Leu Lys Gln Thr Ser Gly Asp Leu Tyr Gln Met Glu Gln
165 170 175
Ile Arg Arg Glu His Pro Asp Leu Val Leu Tyr Asn Gly Tyr Asp Glu
180 185 190
Ile Phe Ala Ser Gly Leu Leu Ala Gly Ala Asp Gly Gly Ile Gly Ser
195 200 205
Thr Tyr Asn Ile Met Gly Trp Arg Tyr Gln Gly Ile Val Lys Ala Leu
210 215 220
Lys Glu Gly Asp Ile Gln Thr Ala Gln Lys Leu Gln Thr Glu Cys Asn
225 230 235 240
Lys Val Ile Asp Leu Leu Ile Lys Thr Gly Val Phe Arg Gly Leu Lys
245 250255
Thr Val Leu His Tyr Met Asp Val Val Ser Val Pro Leu Cys Arg Lys
260 265 270
Pro Phe Gly Pro Val Asp Glu Lys Tyr Leu Pro Glu Leu Lys Ala Leu
275 280 285
Ala Gln Gln Leu Met Gln Glu Arg Gly
290 295
<210>8
<211>894
<212>DNA
<213> Artificial sequence
<400>8
atggcaacga atttacgtgg cgtaatggct gcactcctga ctccttttga ccaacaacaa 60
gcactggata aagcgagtct gcgtcgcctg gttcagttca atattcagca gggcatcgac 120
ggtttatacg tgggtggttc gaccggcgag gcctttgtac aaagcctttc cgagcgtgaa 180
caggtactgg aaatcgtcgc cgaagaggcg aaaggtaaga ttaaactcat cgcccacgtc 240
ggttgcgtca gcaccgccga aagccaacaa cttgcggcat cggctaaacg ttatggcttc 300
gatgccgtct ccgccgtcac gccgttctac tatcctttca gctttgaaga acactgcgat 360
cactatcggg caattattga ttcggcggat ggtttgccga tggtggtgta caacattcca 420
gccctgagtg gggtaaaact gaccctggat cagatcaaca cacttgttac attgcctggc 480
gtaggtgcgc tgaaacagac ctctggcgat ctctatcaga tggagcagat ccgtcgtgaa 540
catcctgatc ttgtgctcta taacggttac gacgaaatct tcgcctctgg tctgctggcg 600
ggcgctgatg gtggtatcgg cagtacctac aacatcatgg gctggcgcta tcaggggatc 660
gttaaggcgc tgaaagaagg cgatatccag accgcgcaga aactgcaaac tgaatgcaat 720
aaagtcattg atttactgat caaaacgggc gtattccgcg gcctgaaaac tgtcctccat 780
tatatggatg tcgtttctgt gccgctgtgc cgcaaaccgt ttggaccggt agatgaaaaa 840
tatctgccag aactgaaggc gctggcccag cagttgatgc aagagcgcgg gtga 894
<210>9
<211>116
<212>DNA
<213> Artificial sequence
<400>9
tcatagacct gaaaaggtct ttttttgtac tcttaataat aaaaagaaga tgaaacttgt 60
ttaaggattg aacgtagtag ataataatat taaaactgag aaaggaggtg ataaaa 116
<210>10
<211>97
<212>DNA
<213> Artificial sequence
<400>10
ttgaggaatc atagaatttt gacttaaaaa tttcagttgc ttaatcctac aattcttgat 60
ataatattct catagtttga aaaaggaggt gataaaa 97
<210>11
<211>116
<212>DNA
<213> Artificial sequence
<400>11
attttgtcaa aataatttta ttgacaacgt cttattaacg ttgatataat ttaaatttta 60
tttgacaaaa atgggctcgt gttgtacaat aaatgtagtg aaaggaggtg ataaaa 116
<210>12
<211>346
<212>PRT
<213> Artificial sequence
<400>12
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
130135 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
290295 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>13
<211>1041
<212>DNA
<213> Artificial sequence
<400>13
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>14
<211>347
<212>PRT
<213> Artificial sequence
<400>14
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>15
<211>1044
<212>DNA
<213> Artificial sequence
<400>15
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 (10)
1. The recombinant bacillus subtilis is characterized in that N-acetylneuraminic acid synthase and N-acetylneuraminic acid aldolase are integrated and expressed through promoters with different strengths, and the nucleotide sequence of the promoter is selected from SEQ ID NO. 9-11.
2. The recombinant Bacillus subtilis of claim 1, wherein the N-acetylneuraminic acid synthase is derived from Neisseria meningitidis (Neisseria meningitidis).
3. The recombinant Bacillus subtilis of claim 1 or 2, wherein the expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase is further enhanced.
4. The recombinant Bacillus subtilis of claim 3, wherein the expression of glucosamine-6-phosphate-N-acetyltransferase is regulated by the promoter shown in SEQ ID No.9, and the N-acetylglucosamine isomerase is expressed by the promoter shown in SEQ ID No. 10.
5. The recombinant Bacillus subtilis according to claim 3, wherein glucosamine-6-phosphate-N-acetyltransferase, N-acetylneuraminic acid synthase and N-acetylneuraminic acid aldolase in the recombinant Bacillus subtilis are each expressed under the control of a promoter represented by SEQ ID No.9, and N-acetylglucosamine isomerase is expressed under the control of a promoter represented by SEQ ID No. 10.
6. The recombinant Bacillus subtilis of any one of claims 1 to 5, wherein the Bacillus subtilis is Bacillus subtilis BSGN 6-comK.
7. A method for constructing the recombinant Bacillus subtilis of any one of claims 1 to 6, wherein recombinant integration fragments of the glucosamine-6-phosphate-N-acetyltransferase gene, the N-acetylglucosamine isomerase gene, the N-acetylneuraminic acid synthase gene and the N-acetylneuraminic acid aldolase gene are constructed, respectively, and then one or more of the recombinant integration fragments are transformed into the Bacillus subtilis genome; the recombinant integration segment is formed by fusing and connecting a gene left arm, a promoter segment, a gene segment and a gene right arm in sequence; the nucleotide sequence of the promoter fragment is selected from SEQ ID NO. 9-11.
8. A method for increasing the yield of neuraminic acid in Bacillus subtilis is characterized in that the expression of N-acetylneuraminic acid synthase and N-acetylneuraminic acid aldolase is enhanced through a strong promoter, and the expression of glucosamine-6-phosphate-N-acetyltransferase and N-acetylglucosamine isomerase is enhanced; the nucleotide sequence of the strong promoter is shown in any one of SEQ ID NO. 9-11.
9. Use of the recombinant Bacillus subtilis of any one of claims 1 to 6 for the production of N-acetylneuraminic acid and derivatives thereof.
10. A method for producing neuraminic acid is characterized in that the recombinant bacillus subtilis of any one of claims 1 to 6 is inoculated into L B culture medium, cultured for 12 to 18 hours to obtain seed liquid, and then transferred into a fermentation culture medium by the inoculation amount of 1 to 10% for fermentation for 16 to 72 hours at the temperature of 30 to 37 ℃.
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| CN111394292A (en) * | 2020-03-30 | 2020-07-10 | 江南大学 | Multi-way composite neuraminic acid-producing bacillus subtilis and application thereof |
| WO2024036508A1 (en) * | 2022-08-17 | 2024-02-22 | 深圳大学 | Fusion protease, fusion expression vector, engineered bacterium, and method for producing n-acetylneuraminic acid |
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