Cre/lox system based method for construction of resistance gene free chromosome integrated recombinant bacillus subtilis expressing D-psicose 3-epimerase

A Bacillus subtilis, no resistance gene technology, applied in the field of enzyme genetic engineering, can solve the problems of complex process, difficult separation and purification, and many by-products, etc.

Inactive Publication Date: 2015-09-30
JIANGNAN UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis method has problems such as complicated process, many by-products, difficult separation and purification, and food safety.

Method used

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  • Cre/lox system based method for construction of resistance gene free chromosome integrated recombinant bacillus subtilis expressing D-psicose 3-epimerase
  • Cre/lox system based method for construction of resistance gene free chromosome integrated recombinant bacillus subtilis expressing D-psicose 3-epimerase
  • Cre/lox system based method for construction of resistance gene free chromosome integrated recombinant bacillus subtilis expressing D-psicose 3-epimerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Construction of P43-DPE expression unit

[0024] Introduce the P43 promoter by overlapping PCR, use the primer pair P1 / P2 to amplify the P43 promoter, use the primer pair P3 / P4 to amplify the DPE enzyme gene connection, and design the primers as follows:

[0025] P1: 5’-CC GCTAGC TG ATAGGTGGTA TGTTTTC-3'

[0026] P2: 5’-TAATAAATAC CATGCTTCAT GTGTACATTC CTCTCT-3’

[0027] P3: 5’-AGAGAGGAAT GTACACATGA AGCATGGTAT TTATTA-3’

[0028] P4: 5'- GTC GAC TTAC TTCCATTCAA GCATG -3’

[0029] PCR product 1 was obtained by using P1 and P2 as primers by PCR method. PCR product 2 was obtained by using P3 and P4 as primers.

[0030] Overlap PCR was performed using PCR product 1 and PCR product 2 as templates and P1 and P4 as primers. Its reaction system is as follows:

[0031]

[0032] The reaction program was as follows: pre-denaturation at 98°C for 1 min; 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min; extension at 72°C for 5 min, and cooling t...

Embodiment 2

[0033] Example 2: Construction of integration vector pDGI-7S6-P43-DPE

[0034]Plasmids p7S6 and pDGIEF were digested with SalI and XmaI, respectively, and recovered lox 71- spc - lox 66 and the vector pDGIEF gene fragment were ligated overnight with T4 ligase and transformed into E. coli DH5α competent cells, spread to LB solid medium (containing 100 μg / mL spc ), cultured overnight at 37°C. Pick positive clones, extract plasmids, and perform double enzyme digestion verification. The verified correct plasmid was named pDGI-7S6.

[0035] Plasmids pDGI-7S6 and pP43-DPE were digested with NheI and SalI respectively, and the gene fragments pDGI-7S6 and P43-DPE were recovered, ligated with T4 ligase overnight, and then transformed into E. coli DH5α competent cells, coated with s pc (100 μg / mL) LB plates, cultured overnight at 37°C. Pick positive clones, extract plasmids, perform double enzyme digestion and sequencing verification. The verified and sequenced correct p...

Embodiment 3

[0036] Example 3: Chromosomal Integration

[0037] The recombinant plasmid pDGI-7S6-P43-DPE was linearized with XhoI and then transformed into the host bacteria B. subtilis In 1A751 competent medium, apply to LB solid medium (containing 100 μg / mL spc ), cultured overnight at 37°C, and the transformants of positive clones were selected as B. subtilis 1A751-DPE-spc ( figure 2 ). Making recombinant bacteria B. subtilis 1A751-DPE-spc competent, transform the pTSC plasmid with Cre gene into competent, use the Em (250 μg / mL) LB plate screening, pick positive clones, inoculate with toothpick containing Em (250 μg / mL) and Spc (100 μg / mL) double resistance plate and containing only Em (250 μg / mL) resistance plate, select only with Em resistant without Spc For resistant positive clones, increase the temperature and lose the plasmid pTSC to obtain recombinant bacteria B. subtilis 1A751 / lox -DPE.

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Abstract

Belonging to the technical field of enzyme genetic engineering, the invention relates to a Cre / lox system based method for construction of resistance gene free chromosome integrated recombinant bacillus subtilis expressing D-psicose 3-epimerase. The method includes: taking integration vector pDGIEF as the starting vector, deleting the original spectinomycin (spc) antibiotics resistance gene, and introducing a new antibiotics resistance gene fragment of lox71-spc-lox66 to obtain plasmid pDGI-7S6; fusing a P43 promoter at the upstream of the DPE enzyme gene of ATCC 35704 to construct P43-DPE, constructing the P43-DPE into pDGI-7S6 to obtain pDGI-7S6-P43-DPE; then converting 1A751 competence, under the action of amylase homologous arm, integrating P43-DPE and lox71-spc-lox66 to 1A751 chromosome; utilizing the Cre / lox system to knock out the spc resistance gene, thus obtaining a food grade safe 1A751 / lox-DPE strain producing D-psicose 3-epimerase. The strain is named as Bacillus subtilis SK38.002 with a preservation number of CCTCC NO:M 2015258, has fermentation broth total enzyme activity up to 6U / mL, and has important industrial application value.

Description

technical field [0001] A method for constructing recombinant Bacillus subtilis expressing D-psicose 3-epimerase without resistance gene chromosomal integration based on Cre / lox system, involving a kind of DPE enzyme in Bacillus subtilis food Level expression belongs to the technical field of enzyme gene engineering. Background technique [0002] D-psicose 3-epimerase (DPE enzyme) belongs to the D-tagatose 3-epimerase (DTE for short) family enzyme, which can catalyze the epimerization of various ketose C3 positions , is a good biocatalyst for the production of rare sugars, which can be used alone or coupled with other enzymes to synthesize a variety of carbohydrates, and is widely used in the fields of chemistry, food and pharmaceuticals. Currently, DPE enzyme can catalyze the conversion between D-fructose and D-psicose, and use D-fructose to produce D-psicose. [0003] With the outbreak of a series of chronic diseases caused by excessive high-energy food intake in the worl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P19/24C12P19/02C12R1/125
Inventor 江波沐万孟何伟伟张涛
Owner JIANGNAN UNIV
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