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A kind of multi-path compound neuraminic acid-producing Bacillus subtilis and its application

A technology of Bacillus subtilis and acetylneuraminic acid, which is applied in the field of genetic engineering, can solve the problems of cell pressure, unfavorable cell growth, and product synthesis and improvement, and achieve the effect of reducing protein synthesis pressure and improving the application prospect of metabolic engineering

Active Publication Date: 2022-08-09
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

[0004] The N-acetylneuraminic acid metabolic pathway currently constructed in subtilis is mainly through the NeuC key enzyme synthesis pathway with UDP-N-acetylglucosamine as the precursor, the Age key enzyme synthesis pathway with N-acetylglucosamine as the precursor, and N -NanE key enzyme synthesis pathway with acetylglucosamine-6 ​​phosphate as the precursor, because the pathway involves many exogenous enzyme proteins, introducing multiple enzyme proteins at the same time will easily lead to cell stress, which is not conducive to cell growth and product synthesis, so it is difficult to achieve multiple The strategy of pathway compounding, the existence of this problem severely limits the improvement of neuraminic acid production, further limiting its market application

Method used

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  • A kind of multi-path compound neuraminic acid-producing Bacillus subtilis and its application
  • A kind of multi-path compound neuraminic acid-producing Bacillus subtilis and its application
  • A kind of multi-path compound neuraminic acid-producing Bacillus subtilis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of genome recombination and integration of NeuC fragments

[0040] Using the Bacillus subtilis 168 genome as a template, the primer NeuC-L-F was designed:

[0041] 5'-GCGAACAGGCATCCTATACACTGGGACAA-3' and

[0042] NeuC-L-R: 5'-ACCGAGCTCGAATTCTTATTAGACGGAGTCTTTTTTGCTTTTGCCAATCAGACGTGTAA-3', amplify and integrate NeuC left arm gene fragment;

[0043] Synthesize fragments of promoters P1-P10 shown in SEQ ID NO. 6-15;

[0044] Synthesize the NeuC gene sequence shown in SEQ ID NO.16;

[0045] Using the genome of Bacillus subtilis 168 as a template and primers NeuC-R-L:

[0046] 5'-ACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCGACGGAGTCTTTTTTTATTTCGTTTTTAAGAAGTAGG-3' and NeuC-R-R:

[0047] 5'-CTAACACAATCCATTTTGAAGATGCCTTTTTGCA-3', amplify and integrate the NeuC right arm gene fragment;

[0048] The amplified NeuC left arm gene fragment, promoter fragment (respectively shown in SEQ ID NO. 1 to 10), NeuC gene fragment and NeuC right arm fragmen...

Embodiment 2

[0049] Example 2 Construction of genome recombination and integration of NanE fragments

[0050] Using the Bacillus subtilis 168 genome as a template, the primers NanE-L-F: 5'-GTGTTCGTAGTCTCTCGGGAGAGTCATTCCATGA-3' and NanE-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCGCGTTTAAGAGAACAGGCCTTGGTTTGTGACA-3' were designed to amplify and integrate the left arm gene fragment of NanE;

[0051] Synthesize fragments of promoters P1-P10 shown in SEQ ID NO. 6-15;

[0052] Synthesize the NanE gene fragment shown in SEQ ID NO.17;

[0053] Using the genome of Bacillus subtilis 168 as a template and primers NanE-R-L:

[0054] 5'-GAATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCATGACTGTCAGTTCTTTCAGCCGCT-3' and NanE-R-R:

[0055] 5'-CAACGATTGCGTTTAATGTCAGCATCAGCCCATACA-3', amplified and recombined the NanE right arm gene fragment.

[0056] The amplified NanE left arm gene fragment, promoter fragment (respectively shown in SEQ ID NO. 6 to 15), NanE gene fragment and NanE right arm fr...

Embodiment 3

[0057] Example 3 Construction of genome recombination and integration of Gna1 fragments

[0058] Using the Bacillus subtilis 168 genome as a template, primers Gna1-L-F: 5'-CGTGATATCGTCATTCAGTCTCTTGAACGCCA-3' and Gna1-L-R: 5'-CGCAATAACGCAGGCGTTTCTGTGACATTAACTTATTTCATGTTCTTTTTAGTTAGACGATTTTAATACAAGCCTCGCCA-3' were designed to amplify and integrate the left arm gene fragment of Gna1;

[0059] Synthesize fragments of promoters P1-P10 shown in SEQ ID NO. 6-15;

[0060] Synthesize the gene fragment encoding Gna1 as shown in SEQ ID NO.18;

[0061] Using the genome of Bacillus subtilis 168 as a template, the primers Gna1-R-L: 5'-ATAACTTGTCAGACTGCCGGGAAATCCCGGCAGTCTTTTTTCCATTAAAACACGGCCCAGTCATAAAATAGTTTTCCTAATAAGACCTGG-3' and Gna1-R-R: 5'-CCTACTTAAGCTGCTACCACTTGTGA-3' were used to amplify and integrate the right arm gene fragment of Gna1.

[0062] The amplified Gna1 left arm gene fragment, the promoter fragment (respectively shown in SEQ ID NO. 6-15), the Gna1 gene fragment and the Gn...

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Abstract

The invention discloses a multi-path compound neuraminic acid-producing Bacillus subtilis and an application thereof, belonging to the field of genetic engineering. The present invention sequentially optimizes the key enzymes UDP-N-acetylglucosamine-2-epimerase and N-acetylglucosamine-6-phosphate isomerase in the three neuraminic acid synthesis pathways through 10 promoters with different strengths, and the expression levels of glucosamine-6-phosphate-N-acetyltransferase, N-acetylglucosamine isomerase and N-acetylneuraminic acid synthase on the genome, reducing the protein synthesis caused by the expression of the enzymes on cells pressure, and further integrated the three neuraminic acids into the same strain of Bacillus subtilis engineering bacteria to obtain Bacillus subtilis with improved N-acetylneuraminic acid production, and its yield at the shake flask level reached 10.4g / L, It lays a foundation for further improving the production of Bacillus subtilis NeuAc.

Description

technical field [0001] The invention relates to a multi-path compound neuraminic acid-producing Bacillus subtilis and its application, and belongs to the field of genetic engineering. Background technique [0002] N-acetylneuraminic acid is a functional monosaccharide widely found in microorganisms and mammals. In the human body, N-acetylneuraminic acid is involved in many physiological processes such as cell recognition and signal transduction. Therefore, N-acetylneuraminic acid is widely used to enhance infant immunity and promote infant brain development. At present, N-acetylneuraminic acid mainly adopts the natural product extraction method (egg, bird's nest, etc.), and other products are difficult to separate and the cost is high; in addition, it can be obtained by the method of whole cell transformation, but it requires high-cost substrates Acetyl-glucosamine and pyruvate are substrates, resulting in higher cost. [0003] Bacillus subtilis is widely used as a host f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/60C12P19/26C12R1/125
CPCC12N9/88C12Y205/01056C12P19/26
Inventor 刘延峰刘龙张晓龙堵国成李江华陈坚
Owner JIANGNAN UNIV
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