Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof

A technology of genetically engineered strains and epimerase, which is applied in the direction of genetic engineering, isomerase, and microbial-based methods, can solve the problems of increasing the complexity of product purification and increasing the cost of protein production, so as to reduce production costs , high-efficiency secretion and expression, and the effect of simplifying the protein purification process

Active Publication Date: 2016-05-25
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, the DPEase enzymes in the literature and patents are mainly expressed in E. coli. The outer membrane of E. coli contains lipopolysaccharide, which is an endotoxin that can cause fever in humans and mammals, and the removal of endotoxin in the final product This obviously increases the complexity of product purification; in addition, in Escherichia coli, protein synthesis usually occurs in the cell, in order to obtain a large amount of target protein, it is necessary to destroy the cell to release the protein in the cell. The additional process will inevitably increase the production cost of the protein

Method used

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  • Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof
  • Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof
  • Genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0026] Example 1D- Obtaining of Psicose 3-epimerase Gene Fragment

[0027] According to the sequence of the DPEase coding gene rdpe derived from Ruminococcussp. Subsequent expression vector construction. The synthesized gene was connected to the pUC57 vector and named pUC57-RDPE.

Embodiment 2D

[0028] Example 2D-constitutive expression of psicose 3-epimerase

[0029] Plasmids pUC57-RDPE and pMA5 were subjected to NdeI and BamHI double enzyme digestion treatment respectively; the double enzyme digestion reaction conditions were 37°C water bath for 3h. Gel-recover the double-digested products to obtain the rdpe fragment with NdeI and BamHI restriction sites at both ends and the linearized pMA5 vector, and connect the two with T4 ligase in a refrigerator at 4°C overnight; The product was transformed into DH5α colonic competent cells, resistance screening was performed with ampicillin (100 μg / mL), positive clones were screened by colony PCR, and plasmids were extracted for sequencing verification. For plasmid construction results, see figure 1 , the constructed recombinant expression plasmid containing the rdpe gene was named pMA5-RDPE.

[0030] The constitutive expression plasmid pMA5-RDPE was transformed into 1A751 competent cells by Spizizen transformation method, s...

Embodiment 3D

[0032] Example 3D-Inducible expression of psicose 3-epimerase

[0033] xylose promoter P xylA Sequence, maltose promoter P glv sequence and sucrose promoter P sacB The sequences are shown in SEQIDNO.2, SEQIDNO.3 and SEQIDNO.4 respectively, and primers xylA-F / xylA-R, glv-F / glv-R and sacB-F / sacB-R were designed. Using xylA-F / xylA-R as primers and plasmid pHCMC04 as template, PCR was performed to obtain P xylA Fragment; using glv-F / glv-R and sacB-F / sacB-R as primers, and using the Bacillus subtilis 1A751 genome as a template to obtain P glv and P sacB fragment. Primers pMA5-RDPE-F / pMA5-RDPE-R were designed, and pMA5-RDPE was used as a template for PCR to obtain a linearized pMA5-RDPE vector fragment. The primer sequences described above are as follows:

[0034] XylA-F:5'-CAGCCTCGCAGAGCACACACTTTATGGGCGCGCCTTTCCCAGTCACGACGTTGTAAAAC-3'

[0035] XylA-R:5'-CAATAAGCGTAATAAATACCATATTTCATATGATTTCCCCCCTTTGATTTAAGTGATTTCC-3'

[0036] Glv-F:5'-CAGCCTCGCAGAGCACACACTTTATGGGCGCGCCCGCG...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a genetic engineering strain capable of effectively secreting D-psicose 3-epimerase and a construction method thereof. A D-psicose 3-epimerase gene rdpe from rumen bacterium Ruminococcus sp. 5_1_39B_FAA is obtained firstly, recombinant plasmid pMA5-RDPE construction and bacillus subtilis conversion are conducted, and then constitutive and secretive expression of RDPE in bacillus subtilis is achieved. By comparing three glucose-induced promoters, the optimal inducible promoter P[xylA] is obtained, and the RDPE secretion level is increased remarkably. By knocking out the xylose utilization gene xylAB (xylA and xylB), the xylose metabolism pathway of bacillus subtilis is blocked, the secretion amount of RDPE is further increased, and the optimal induced concentration of the inducer xylose is reduced to 0.5% from 4.0%. Finally, the engineered strain 1A751SD-XR is evaluated in a 7.5 L fermentation tank, and the RDPE secretion level can be as high as 95 U / mL and 2.6 g / L.

Description

technical field [0001] The invention belongs to the field of industrial biotechnology, and in particular relates to a Bacillus subtilis genetically engineered strain that efficiently secretes and expresses D-psicose 3-epimerase and a construction method thereof, and uses the genetically engineered strain to ferment and produce D-psicose 3-epimerase. - Use of psicose 3-epimerase. Background technique [0002] Rare sugars are a class of monosaccharides and their derivatives that exist in nature but in very small amounts. With the gradual understanding of these sugars, the research on functional rare sugars centered on psicose and tagatose has developed rapidly and attracted the attention of many researchers. D-psicose (D-Psicose) is a rare sugar, which is the epimer of D-fructose at the C3 position. D-Psicose is present in extremely small amounts in some commercial sugars and agricultural products and is difficult to synthesize chemically. For example, D-psicose is found in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/61C12N15/75C12N9/90C12R1/125
Inventor 张大伟陈景奇付刚朱玥明孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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