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Genetic engineering bacterium highly producing D-allulose and application of genetic engineering bacterium

A technology of genetically engineered bacteria and psicose, applied in the field of bioengineering, can solve the problems of low yield, many by-products, difficult separation and purification, etc., and achieve the effect of reducing production costs

Active Publication Date: 2020-05-29
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The methods for preparing D-psicose mainly include chemical synthesis and biotransformation. The chemical synthesis is mainly synthesized through reactions such as catalytic hydrogenation, addition reaction, and Ferrier rearrangement, but there are reaction steps in these chemical synthesis methods. disadvantages such as many, harsh reaction conditions, low yield, many by-products and difficult separation and purification

Method used

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  • Genetic engineering bacterium highly producing D-allulose and application of genetic engineering bacterium
  • Genetic engineering bacterium highly producing D-allulose and application of genetic engineering bacterium
  • Genetic engineering bacterium highly producing D-allulose and application of genetic engineering bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1: DTEase gene target fragment amplification

[0030] By PCR method to DTEase-pMD TM The 18-T vector is used as a template, and DTE-F and DTE-R are used as primers to amplify and obtain the target fragment product of DTEase gene;

[0031] The primer sequences are as follows:

[0032] DTE-F: 5'-TGCACTGCAGATGTTGATATCCCCCTTTCGAG-3'(Pst I),

[0033] DTE-R: 5'-GGAAGCTTTCAGGCGTTCCGTGCGGC-3' (Hind III).

[0034] The PCR reaction system is as follows:

[0035]

[0036] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; 35 cycles of 98°C for 10 s, 56°C for 15 s, and 72°C for 1 min; extension at 72°C for 5 min. Finally, the target fragment of the DTEase gene (as shown in SEQ ID NO.1) was obtained through amplification.

Embodiment 2

[0037] Example 2: Cloning vector DTEase- -Build of T1

[0038] The target fragment of the DTEase gene obtained by amplification and -T1 (Beijing Quanshijin Biotechnology Co., Ltd.) performs the ligation reaction according to the following system:

[0039]

[0040] The reaction process is as follows: Gently mix the reaction mixture, react in a PCR instrument at a temperature of 25°C for 10 minutes, and place in an ice bath for 2 to 3 minutes after the reaction.

[0041]Add the ligation product to 100 μL Trans1-T1 competent cells (add the ligation product when the competent cells are just thawed), flick and mix well, and keep in an ice bath for about 30 min; heat shock in a water bath at 42°C for 30 s, and immediately place on ice for 2 min; add 250 μL of SOC medium equilibrated to room temperature, and incubate at 200 rpm at 37°C for 1 h; take 200 μL of the bacterial solution and spread it on LB solid medium (containing 50 μg / mL Kan), and incubate overnight at 37°C. Pic...

Embodiment 3

[0045] Embodiment 3: Construction of expression vector DTEase-pP43NMK

[0046] The plasmid DTEase- -T1 and pP43NMK were digested with Pst I and Hind III respectively, and the gene fragments were recovered from the gel, and then transformed into E.coli DH5α competent cell (the specific method is the same as in Example 2 -T1 transformation process), spread on LB plates containing Amp (50 μg / mL), and culture overnight at 37°C. Pick positive clones, use DTE-F and DTE-R as primers to screen positive clones by bacterial liquid PCR, extract plasmids, double enzyme digestion (Pst I and Hind III) and sequence verification. The recombinant plasmid verified and sequenced correctly was named as DTEase-pP43NMK ( figure 1 ).

[0047] The ligation reaction system of the linearized gene fragment kit is as follows (target fragment: linearized vector = 1:100 or more):

[0048]

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Abstract

The invention discloses a genetic engineering bacterium highly producing D-allulose and application of the genetic engineering bacterium. A food-grade strain bacillus subtilis is used as a host bacterium, D-tagatose 3-epimerase (DTEase) is efficiently expressed through gene recombination, and a cell factory for producing D-allulose by directly using D-fructose is formed. The genetic engineering bacterium highly producing the D-allulose is obtained by screening, through optimization of a fermentation process, the yield of the D-allulose reaches 4.56 g / L, and the conversion rate of a substrate is 56.26%, and the production efficiency of the D-allulose is 0.19 g / L*h.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetic engineering bacterium with high production of D-psicose and its application. Background technique [0002] Rare sugar, the International Sugar Society (ISRS) defines rare sugar as "a class of monosaccharides and their derivatives that exist in nature but are very low in content". Although the content in nature is very small, these substances play a great role in diet, health care, medicine and other fields. D-allulose is an epimer at the C-3 position of D-fructose, and is an important rare sugar. D-tagatose 3-epimerase (abbreviated as DTEase) can catalyze the epimerization of various ketose C3 positions, and is a biocatalyst for the effective production of D-psicose. The application of genetically engineered bacteria fermentation technology can catalyze the isomerization between D-fructose and D-psicose, and directly use the substrate D-fructose to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/24C12P19/02C12R1/125
CPCC12N9/90C12P19/02C12P19/24C12Y501/03
Inventor 张宇张俊吕鹏梅梁翠谊徐惠娟王忠铭
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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