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A genetically engineered strain for efficiently secreting d-psicose 3-epimerase, its construction method and its application

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 protein production costs and increasing the complexity of product purification, so as to reduce production costs , high-efficiency secretion and expression, and the effect of simplifying the protein purification process

Active Publication Date: 2019-09-03
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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  • A genetically engineered strain for efficiently secreting d-psicose 3-epimerase, its construction method and its application
  • A genetically engineered strain for efficiently secreting d-psicose 3-epimerase, its construction method and its application
  • A genetically engineered strain for efficiently secreting d-psicose 3-epimerase, its construction method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Obtaining of D-psicose 3-epimerase gene fragment

[0027] According to the source of rumen bacteria ( Ruminococcus sp. 5_1_39B_FAA) DPEase encoding gene rdpe The whole gene synthesis is carried out, and the 5' and 3' ends of the nucleotide sequence of the gene are respectively introduced Nde I and Bam HI restriction enzyme cutting site to facilitate the subsequent construction of expression vectors. The synthesized gene was connected to the pUC57 vector and named pUC57-RDPE.

Embodiment 2

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

[0029] Plasmids pUC57-RDPE and pMA5 were carried out separately Nde I and Bam HI double enzyme digestion treatment; the reaction condition of double enzyme digestion is 37°C water bath for 3 h. The double-digested products were recovered by gel respectively to obtain Nde I and Bam HI restriction site rdpe The fragment and the linearized pMA5 vector were ligated with T4 ligase overnight in a refrigerator at 4°C; the ligated product was transformed into DH5α colon competent cells, screened for resistance with ampicillin (100 μg / mL), and colonies passed Positive clones were screened by PCR, and plasmids were extracted for sequencing verification. For plasmid construction results, see figure 1 , the constructed contains the rdpe The recombinant expression plasmid of the gene was named pMA5-RDPE.

[0030] The constitutive expression plasmid pMA5-RDPE was transformed into 1A751 competent cells by Spiz...

Embodiment 3

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

[0033] xylose promoter P xylA Sequence, maltose promoter P glv sequence and sucrose promoter P sacB The sequences are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.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'-CAGCCTCGCAGAGCACACACTTTATGGGCGC...

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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 Patents(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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