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Method for improving yield of bacillus subtilis acetylglucosamine

A technology of Bacillus subtilis and glucosamine, applied in the field of metabolic engineering, can solve problems such as no reported phosphatase, and achieve the effects of improving cell growth, eliminating toxic effects, and reducing excessive accumulation

Pending Publication Date: 2020-11-06
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no phosphatase with the same function has been reported from Bacillus subtilis

Method used

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  • Method for improving yield of bacillus subtilis acetylglucosamine
  • Method for improving yield of bacillus subtilis acetylglucosamine
  • Method for improving yield of bacillus subtilis acetylglucosamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Construction of phosphatase YwpJ knockout box

[0026] According to the Bacillus subtilis (Bacillus subtilis 168, purchased from the American Type Microorganism Collection, ATCC No.27370) published on NCBI, according to the published upstream and downstream sequences of the phosphatase gene ywpJ, the upstream and downstream homology arm amplification primers were designed,

[0027] The upstream homology arm amplification primers are (SEQ ID NO.2\3):

[0028] ywpJ-U-F:TACGCTCCTGCAAGTGTCAAGG,

[0029] ywpJ-U-R: GCTATACGAACGGTAGAATCTC GTTTGTCAGTCTTTCCTTCATCCTGCTCT;

[0030] The downstream homology arm amplification primers are (SEQ ID NO.4\5):

[0031] ywpJ-D-F: TATACGAACGGTAGCGCACATA TGATGAAACATTTGTTGTAAGT,

[0032]ywpJ-D-R: TATTTAAAGGAAGAGCAAACCGAGC.

[0033] Bleomycin resistance gene amplification primers are (SEQ ID NO.6\7):

[0034] zeo-F: ATGAAGGAAAGGACTGACAA ACGAGATTTCTACCGTTCGTATAGC

[0035] zeo-R: CAAATGTTTCATCATATGTGCG CTACCGTTCGTATAAT...

Embodiment 2

[0037] Example 2: Construction of glucosamine 6 phosphate deaminase gene nagBB knockout box

[0038] According to the published upstream and downstream sequences of the glucosamine 6-phosphate deaminase gene nagBB (Gene ID: 938425), the upstream and downstream homology arm amplification primers were designed,

[0039] The upstream homology arm amplification primers are (SEQ ID NO.8\9):

[0040] nagBB-U-F:TTAACAATTCAAACAGCGAGCCG,

[0041] nagBB-U-R: GCTATACGAACGGTAGAATCTC ACTTACAGAGTTCTTCGTAGTGCT;

[0042] The downstream homology arm amplification primers are (SEQ ID NO.10\11):

[0043] nagBB-D-F: GCATACATTATACGAACGGTAG CGCCGATTATAAAAGCAGCTCAAAA,

[0044] nagBB-D-R: GCGTATAAAAAATATGATGCAGGCC.

[0045] Bleomycin resistance gene amplification primers are (SEQ ID NO.12\13):

[0046] zeo-F: AGCACTACGAAGAACTCTGTAAGT GAGATTCTACCGTTCGTATAGC

[0047] zeo-R: TTTTGAGCTGCTTTATAATCGGCG CTACCGTTCGTATAATGTATGC

[0048] Using fusion PCR technology, the upstream homology arm of ...

Embodiment 3

[0049] Example 3: Construction of murQ knockout box

[0050] According to the published upstream and downstream sequences of the acetylmuramic acid 6-phosphate synthase gene murQ (Gene ID: 938882), the upstream and downstream homology arm amplification primers were designed,

[0051] The upstream homology arm amplification primers are (SEQ ID NO.14\15):

[0052] murQ-U-F: AAAAAATCACCGGCAGCGGAGTAA,

[0053] murQ-U-R: GCTATACGAACGGTAGAATCTC TTAATGGTTCTGACATGAGGATGCC;

[0054] The downstream homology arm amplification primers are (SEQ ID NO.16\17):

[0055] murQ-D-F: GCATACATTATACGAACGGTAG CATTACCATCCATGATAAGGAGAGA,

[0056] murQ-D-R: AATTGTGTGAGGCTGATCGTTGT.

[0057] Bleomycin resistance gene amplification primers are (SEQ ID NO.18\19):

[0058] zeo-F: GGCATCCTCATGTCAGAACCATTAA GAGATTCTACCGTTCGTATAGC

[0059] zeo-R: TCTCTCCTTATCATGGATGGTAATG CTACCGTTCGTATAATGTATGC

[0060] Using fusion PCR technology, the upstream homology arm of murQ, the bleomycin resistance gen...

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Abstract

The invention discloses a method for improving yield of bacillus subtilis acetylglucosamine. According to the invention, knockout verification is carried out to obtain a result that key phosphatase YwpJ can catalyze GlcNAc-6-P to take a dephosphorylation effect to generate GlcNAc; by overexpressing the phosphatase YwpJ, excessive accumulation of intracellular sugar phosphates is reduced, a toxic effect of the excessively accumulated sugar phosphates on cells is eliminated, and the sugar phosphates are effectively secreted out of the cells, so that cell growth is improved; and GlcNAc syntheticprecursor degradation pathway key genes murQ and nagBB are further knocked out, degradation of a precusor substance is blocked, waste of carbon flow is reduced, more carbon flow is enabled to be transformed into products, a GlcNAc yield of a final recombinant strain FMIP34 reaches 26.1g / L and is increased by 61.1%, and a transformation rate of the GlcNAc reaches 0.483g per gram of glucose.

Description

technical field [0001] The invention relates to a method for increasing the yield of Bacillus subtilis acetylglucosamine, which belongs to the technical field of metabolic 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. Bacillus subtilis is widely used as a production host for food enzyme preparations and important nutritional chemicals, and its products are certified by the FDA as "generally regarded as safe" (GRAS) safety level. Therefore, using metabolic engineering to construct recombinant Bacillus subtilis is an effective way to produce food-safe acetylglucosamine. [0003] The previously constructed recombinant Bacillus subtilis SFMI (Δnag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/55C12N15/60C12N1/21C12P19/26C12R1/125
CPCC12N9/16C12N9/78C12N9/88C12N15/75C12P19/26C12Y305/99006C12Y402/01126
Inventor 刘龙卢健行刘长峰陈坚堵国成李江华吕雪芹牛腾飞
Owner JIANGNAN UNIV
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