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Multipath composite neuraminic acid producing bacillus subtilis and application thereof

A technology of Bacillus subtilis and acetylneuraminic acid, which is applied in the field of genetic engineering, can solve problems such as cell pressure, unfavorable cell growth and product synthesis, and limit market application, so as to reduce the pressure of protein synthesis and improve the application prospect of metabolic engineering.

Active Publication Date: 2020-07-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for better understanding about how bacterial cells produce neurologically active substances called neuraminic acids during their growth process. It also includes various techniques like gene manipulation or chemical modification to improve these processes such as increasing specificity towards certain types of molecules while minimizing unwanted side reactions. Overall, this innovative approach helps create new compounds with improved properties useful applications in biotechnology.

Problems solved by technology

This patents describes how nervein produced during certain stages of life has become increasingly popular due to their ability to stimulate neural cells while also being able to protect them against harmful substances like bacteria or viruses. However, current methods require expensive materials and produce low yields per gram of material compared to more affordable options.

Method used

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  • Multipath composite neuraminic acid producing bacillus subtilis and application thereof
  • Multipath composite neuraminic acid producing bacillus subtilis and application thereof
  • Multipath composite neuraminic acid producing bacillus subtilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of Genome Recombination Integrated NeuC Fragment

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

[0041] 5'-GCGAACAGGCATCCTATACACTGGGACAA-3' and

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

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

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

[0045] Using the Bacillus subtilis 168 genome as a template, with primer NeuC-R-L:

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

[0047] 5'-CTAACACAATCCATTTTGAAGATGCCTTTTTGCA-3', amplified and recombined NeuC right arm gene fragment;

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

Embodiment 2

[0049] Example 2 Construction of Genome Recombination Integration NanE Fragment

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

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

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

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

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

[0055] 5'-CAACGATTGCGTTTAATGTCAGCATCAGCCATACA-3', amplifies and integrates the NanE right arm gene fragment.

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

Embodiment 3

[0057] Example 3 Construction of Genome Recombination and Integration of Gna1 Fragment

[0058] Using the Bacillus subtilis 168 genome as a template, design primers Gna1-L-F: 5'-CGTGATATCGTCATTCAGTCTCTTGAACGCCA-3' and Gna1-L-R: 5'-CGCAATAACGCAGGCGTTCTGTGACATTAACTTATTTCATGTTTCTTTTTAGTTAGACGATTTTAATACAAGCCTCGCCA-3' to amplify the recombinant integrated Gna1 left arm gene fragment;

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

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

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

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

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Abstract

The invention discloses multipath composite neuraminic acid producing bacillus subtilis and an application thereof, and belongs to the field of genetic engineering. The expression levels of key enzymes, namely UDP-N-acetylglucosamine-2-epimerase, N-acetylglucosamine-6-phosphate isomerase, glucosamine-6-phosphoric acid-N-acetyltransferase, N-acetylglucosamine isomerase and N-acetylneuraminate synthase on genomes in three neuraminic acid synthesis paths are sequentially optimized by 10 promoters with different intensities, so that the protein synthesis pressure of enzyme expression on cells is reduced; and three neuraminic acids are further integrated into the same engineering bacillus subtilis, so that the bacillus subtilis with the increased N-acetylneuraminic acid yield is obtained, the neuraminic acid yield under the shake flask level reaches 10.4 g/L, and a foundation is laid for further increasing the NeuAc yield of the bacillus subtilis.

Description

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Claims

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Application Information

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Owner JIANGNAN UNIV
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