Recombinant bacillus subtilis increased in yield of acetylglucosamine

A technology of Bacillus subtilis and glucosamine, which is applied in the field of genetic engineering, can solve problems such as serious environmental pollution, and is not suitable for people with seafood allergies, and achieves the effects of simple construction method, good application prospect and easy use.

Active Publication Date: 2015-04-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, acetyl glucosamine is mainly produced by acid-decomposing chitin in shrimp shells or crab shells. The waste liquid produced by this method is relatively serious for environmental pollution, and the obtained products are easy to cause allergic reactions, so it is not suitable for people with seafood allergies.

Method used

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  • Recombinant bacillus subtilis increased in yield of acetylglucosamine
  • Recombinant bacillus subtilis increased in yield of acetylglucosamine
  • Recombinant bacillus subtilis increased in yield of acetylglucosamine

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1 Knockout of the gene encoding α-acetolactate synthase (alsS) and the gene encoding α-acetolactate decarboxylase (alsD)

[0019] According to the upstream and downstream sequences of the gene encoding α-acetolactate synthase (alsS) and the gene encoding α-acetolactate decarboxylase (alsD) of Bacillus subtilis 168 (ATCC No.27370) published on NCBI, design knockout frame homology arms Amplification primers, left arm upstream and downstream primers are: alsSD-L-F: 5'-CCATGTATAGAGTAGGCCATGCTTCTTTAGC-3' and

[0020] alsSD-L-R:

[0021] 5'-AGGATCCCCGGGTACCGAGCTCCACCCTCACTCCTTATTATGCATTTTAAACGTAAAA-3'; the upper and lower primers of the right arm are: alsSD-R-F:

[0022] 5'-GTCGACCTGCAGGCATGCAAGCAAGAAAAAAAGAAAGCCCCTTTTAGCAGGG-3' and alsSD-R-R:5'-CTACTGCGCTGTCAGAAGCAAAATCAG-3'. The left and right arms contained in the knockout frame were amplified from the Bacillus subtilis 168 genome using the above primers. According to the p7Z6 plasmid sequence published on NCBI ...

Embodiment 2

[0025] The construction of embodiment 2 recombinant Bacillus subtilis

[0026] Transform the constructed knockout frame into Bacillus subtilis BSGN6-P xylA -glmS, through bleomycin resistance plate screening and colony PCR verification, it was confirmed that the gene encoding α-acetolactate synthase (alsS) and the gene encoding α-acetolactate decarboxylase were successfully knocked out, and the recombinant Bacillus subtilis BSGN10 was obtained.

[0027] According to the glucosamine acetylase coding gene (GNA1) in Saccharomyces cerevisiae S288C (ATCC 204508) published on NCBI, the primer GNA1-F was designed:

[0028]5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA1-R: 5'-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3'. The glucosamine acetylase encoding gene (GNA1) was amplified from the Saccharomyces cerevisiae S288C genome using the above primers. The amplified fragment was digested with KpnI and HindIII and then ligated into pP43NMK expression vector. Restrict...

Embodiment 3

[0030] Example 3 Fermentative production of acetylglucosamine

[0031] The seeds cultivated at 37° C. and 200 rpm for 12 hours were transferred to the fermentation medium at an inoculum size of 5%, and cultivated at 37° C. and 200 rpm for 48 hours. After 48 hours of fermentation, the content of acetylglucosamine in the fermentation supernatant reached 38.46g / L, which was 27.1% higher than that of the control bacteria without alsS and alsD knockout (30.25g / L). This was achieved by knocking out the genes encoding α-acetolactate synthase (alsS) and α-acetolactate decarboxylase (alsD), and overexpressing the gene encoding glucosamine acetylase (GNA1) in nagP knockout hosts. Enhanced extracellular production of acetylglucosamine in recombinant Bacillus subtilis.

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Abstract

The invention discloses a recombinant bacillus subtilis increased in yield of acetylglucosamine and belongs to the field of genetic engineering. According to the recombinant bacillus subtilis increased in yield of acetylglucosamine, the recombinant bacillus subtilis BSGN6-PxylA-glmS is taken as an original strain, an alpha-acetolactate synthetase encoding gene and an alpha-acetolacetate decearboxylase encoding gene are knocked out by virtue of homologous recombination, and therefore, the approach by which acetoin and butanediol are generated from 2,3-pyroracemic acid in host bacteria is blocked. In the host bacteria from which the alsS and alsD are knocked out, the glucosamine acetylase encoding gene derived from saccharomyces cerevisiae is excessively expressed, and therefore, the route of synthesis of the acetylglucosamine is enhanced, the yield of the acetylglucosamine in the recombinant bacillus subtilis is increased to 38.46g / L, and a foundation is laid for producing the glucosamine by modifying the bacillus subtilis in the metabolic engineering.

Description

technical field [0001] The invention relates to a recombinant Bacillus subtilis with improved acetylglucosamine production, in particular to a method for improving the production of recombinant Bacillus subtilis acetylglucosamine by knocking out alsS and alsD through homologous recombination, and belongs to the field of genetic engineering. Background technique [0002] Acetyl glucosamine is a kind of monosaccharide in organisms, which widely exists in bacteria, yeast, mold, plants and animals. In the human body, acetylglucosamine is the synthetic precursor of the disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of cartilage and joint tissue functions. Therefore, acetyl glucosamine is widely used as a drug and nutritional dietary supplement to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the field of cosmetics and pharmaceuticals. At present, acetyl glucosamine is mainly produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/75C12P19/26C12R1/125
CPCC12N9/1022C12N9/1029C12N9/88C12P19/26C12Y202/01006C12Y203/01003C12Y401/01005
Inventor 陈坚刘龙堵国成李江华马文龙刘延峰朱妍萩顾洋
Owner JIANGNAN UNIV
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