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Genetically engineered bacterium for synthesizing D-allulose, and construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and psicose, applied in the fields of biotechnology and genetic engineering, can solve problems such as limitations and achieve high-efficiency expression

Active Publication Date: 2020-12-15
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Bacillus subtilis can use D-fructose as a carbon source to satisfy its own growth, which also limits its ability to produce D-psicose as an engineered bacterium to a certain extent

Method used

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  • Genetically engineered bacterium for synthesizing D-allulose, and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for synthesizing D-allulose, and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for synthesizing D-allulose, and construction method and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: Construction of recombinant Bacillus subtilis

[0040] 1.1 Using Bacillus subtilis WB600 as the starting strain, the fructokinase coding gene gmuE was knocked out to obtain the mutant strain WB600 (ΔgmuE).

[0041] Using Bacillus subtilis WB600 as the starting strain, the gene gmuE encoding fructokinase was knocked out. Using the Bacillus subtilis WB600 genome as a template, two pairs of primers ΔgmuE-A-F / R and ΔgmuE-B-F / R (as shown in Table 1) were used to amplify the upstream and downstream homology arms of the target gene, and the two PCR products obtained were of the same size. is 500bp. Perform overlapping PCR on the two fragments to obtain a linear target fragment with a size of 1000 bp, purify and recover the PCR product, and store it at -20°C for future use. The purified PCR amplified product was double-digested with BamHI and Not I, and then ligated with the pKSV7 plasmid after the same double-digestion, and T 4 DNA ligase was ligated at 16°C ...

Embodiment 2

[0078] Example 2: Recombinant genetically engineered bacteria are used to synthesize D-psicose

[0079] Single colonies of Bacillus subtilis WB600, recombinant Bacillus subtilis WB600 / AgDAE-pMA5 and WB600(ΔgmuEΔfruAΔlevG) / AgDAE-pMA5 were picked and inoculated in 5 mL liquid LB medium containing 50 μg / mL kanamycin sulfate, at 37 °C, 220r / min shaking culture overnight. The overnight culture was then inoculated in 50 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate at an inoculum size of 2%. 37°C, 220r / min shaking culture for 24-48h, for constitutive expression. Centrifuge the cells after constitutive expression, collect the precipitate, wash the cells twice with 0.8% normal saline, then resuspend the cells with 1×PBS buffer (pH 7.4), add 500g of / L fructose was used as a substrate to catalyze the reaction at 60°C. After 20 hours of reaction, the transformed supernatant was collected and analyzed by HPLC-ELSD method. The results are shown in Table 2. Finally, the re...

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Abstract

The invention discloses a genetically engineered bacterium for synthesizing D-allulose, and a construction method and application of the genetically engineered bacterium. The invention provides a bacillus subtilis genetically engineered bacterium capable of synthesizing the D-allulose by D-fructose, and a construction method of the bacillus subtilis genetically engineered bacterium. Particularly,bacillus subtilis WB600 is taken as a host bacterium, a fructokinase gene on the genome of the host bacterium and the genes of PTS system fructose specific translocator components IIABC and IID are knocked out to obtain a bacillus subtilis mutant, and a D-allulose-3-epimerase encoding gene from Arthrobacter globiformis M30 is subjected to free expression in the host bacterium to obtain recombinantbacillus subtilis. By use of the genetically engineered bacterium disclosed by the invention, the substrate D-allulose can be fully utilized, the yield of biosynthesis D-allulose is greatly improvedso as to be favorable for industrial production of the D-allulose, and the genetically engineered bacterium has a wide application prospect.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for synthesizing D-psicose and its construction method and application, belonging to the technical fields of biotechnology and genetic engineering. Background technique [0002] D-psicose is an important member of the rare sugar family and a new type of low-energy sweetener. Due to its high sweetness and low energy, D-psicose is considered to be an ideal sweetener and an effective substitute for sucrose. It can help manufacturers reduce sucrose in formulations for the development of low-calorie food and beverages. At the same time, D-psicose can undergo Maillard reaction, which helps to improve food quality and can perfectly replace sugar. In addition, D-psicose also has unique physiological functions, and has great potential application value in the field of clinical medicine. For example, in the treatment of diseases such as obesity, diabetes, hypertension, hyperlipidemia, and atheroscler...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P19/24C12P19/02C12R1/125
CPCC12N9/1205C12N9/90C07K14/32C12N15/75C12P19/24C12P19/02C12Y207/01004C12Y503/01
Inventor 秦慧民路福平毛淑红李超
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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