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Recombinant bacillus subtilis and application

A technology of Bacillus subtilis and Bacillus subtilis is applied in the field of recombinant microorganism genetic engineering and can solve the problems of large carbon flux and the like

Inactive Publication Date: 2018-09-04
SHANDONG RUNDE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, phosphoenolpyruvate can also be converted to pyruvate by pyruvate kinase, resulting in a greater carbon flux to the Krebs cycle

Method used

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  • Recombinant bacillus subtilis and application
  • Recombinant bacillus subtilis and application
  • Recombinant bacillus subtilis and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] KHG / KDPG aldolase gene kdgA

[0023] According to the upstream and downstream sequences of the pyruvate kinase encoding gene pckA of Bacillus subtilis (Bacillus subtilis 168 purchased from the American Type Microorganism Collection, ATCC NO.27370) published on NCBI, and the sequence of the bleomycin resistance gene, the sequence was constructed Knockout box as shown in SEQ ID NO.1.

[0024] The specific construction process is as follows: the starting strain is based on B. subtilis 168, the genotype is modified as follows: ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔptaΔglckΔpckA::lox72, and the promoter P xylA ,P 43 Control the recombinant expression of glmS and GNA1. The KHG / KDPG aldolase gene kdgA of the recombinant Bacillus subtilis BSGNK-PxylA-glmS-P43-GNA1 is knocked out by constructing a KHG / KDPG aldolase coding gene knockout frame, and the knockout frame is knocked out by homologous recombination. The bleomycin resistance gene zeo replaced the KHG / KDPG aldolase gene kdgA i...

Embodiment 2

[0028] Determination method of acetylglucosamine:

[0029] High performance liquid chromatography (HPLC) detection method: Agilent 1200, RID detector, NH Column (250 * 4.6mm, 5 μ m), mobile phase: 70% acetonitrile, flow rate 0.75mL / min, column temperature 30 ℃, injection volume is 10 μL.

[0030] Seed medium (g / L): tryptone 10, yeast powder 5, NaCl10.

[0031] The fermentation medium is a compound medium; the compound medium is calculated in g / L, containing: glucose 60, peptone 6, yeast powder 12, (NH 4 ) SO 4 6,K 2 HPO 4 ·3H 2 O 12.5, KH 2 PO 4 2.5, CaCO 3 5. Trace element 10ml / L; trace element solution contains in g / L: MnSO 4 ·5H 2 O 1.0, C O cl 2 ·6H 2 O 0.4, NaMoO 4 2H 2 O 0.2, ZnSO 4 ·7H 2 O 0.2, AlCl 3 ·6H 2 O 0.1, CuCl 2 ·H 2 O 0.1, H 3 BO 4 0.05 with 5M HCl.

[0032]Culture conditions: Transfer the seeds cultivated at 37°C and 220rpm for 12h to the fermentation medium with an inoculation amount of 5%, add the inducer xylose 5g / L after 2h inocula...

Embodiment 3

[0034] Fermentation of Acetyl Glucosamine

[0035] The seeds cultivated at 37° C. and 220 rpm for 12 hours were transferred to the fermentation medium at an inoculum size of 5%, and cultivated at 30-37° C. and 200-220 rpm for 48-52 hours. After 36 hours of fermentation, the content of acetylglucosamine in the fermentation supernatant reached 11.13g / L, which was 67.8% higher than that of the strain before the knockout (see attached Figure 1-2 Shown), realized the improvement of the extracellular production of acetylglucosamine in the recombinant Bacillus subtilis.

[0036] Table 1 Cell growth and acetylglucosamine synthesis of BSGNK and control bacteria BSGNKA1

[0037]

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Abstract

The invention belongs to the technical field of recombinant microbial genetic engineering, particularly relates to a construction method of a recombinant bacillus subtilis strain, and further relatesto application of the recombinant bacillus subtilis strain, in particular to the application thereof to synthesis of acety-lglucosamine. The construction method of a recombinant bacillus subtilis comprises the steps as follows: using bacillus subtilis BSGNK-PxylA-glmS-P43-GNA1 as a starting strain, knocking out KHG / KDPG aldolase gene kdgA, and obtaining the recombinant bacillus subtilis strain. The recombinant bacillus subtilis, provided by the invention, can efficiently utilize glucose to synthesize the acety-lglucosamine, and the fermentation yield of shake flask can reach 7.34 g / L, which is11.84% higher than that before the knockout, thereby laying a foundation for further transforming the bacillus subtilis to produce glucosamine through metabolic engineering.

Description

technical field [0001] The invention belongs to the technical field of recombinant microorganism genetic engineering, and specifically relates to a method for constructing a recombinant Bacillus subtilis strain, and also relates to the application of the above-mentioned recombinant Bacillus subtilis strain, in particular to its application in the synthesis of acetylglucosamine. 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 prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC12N9/88C12P19/26C12Y401/02014C12Y401/0202C12Y401/03016
Inventor 卢伟刘长峰张弘治马善丽卢健行
Owner SHANDONG RUNDE BIOTECH CO LTD
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