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Bacillus subtilis for producing 2 '-fucosyllactose and application thereof

A technology of Bacillus subtilis and fucosyllactose, applied in the field of genetic engineering, can solve the problems of inability to meet industrial production and high cost of L-fucose, and achieve the effect of strong application value and industrial production potential

Pending Publication Date: 2022-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production of 2’-fucosyllactose by the remedial route still has a high cost of producing raw material L-fucose, which cannot meet the purpose of industrial production

Method used

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  • Bacillus subtilis for producing 2 '-fucosyllactose and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of recombinant Bacillus subtilis genetically engineered bacteria producing 2'-fucosyllactose

[0042] The plasmid pHT-XCR6 was transformed into Bacillus subtilis competent cells, positive clones containing the target plasmid were obtained through chloramphenicol resistance screening, and positive single clones were prepared into Bacillus subtilis competent cells containing plasmid pHT-XCR6.

[0043] According to the genome information of Bacillus subtilis 168 (Bacillus subtilis 168 purchased from the American Type Microorganism Collection Center, ATCC No.27370) published on the National Center for Biotechnology Information (NCBI), the upper and lower gene sequences of the integrated manA gene were determined, and the strong constitutive The promoter P43 and the manA gene sequence of the Bacillus subtilis 168 strain were used to construct the manA integrated expression cassette. The mannose-6-phosphate isomerase gene, manA, was integrated into the Bacillus s...

Embodiment 2

[0058] Construction of recombinant Bacillus subtilis genetically engineered bacteria producing 2'-fucosyllactose

[0059] The plasmid pHT-XCR6 was transformed into Bacillus subtilis competent cells, positive clones containing the target plasmid were obtained through chloramphenicol resistance screening, and positive single clones were prepared into Bacillus subtilis competent cells containing plasmid pHT-XCR6.

[0060] According to the genome information of Bacillus subtilis 168 published on NCBI, the upstream and downstream gene sequences of the integrated manA gene were determined, and the manA integrated expression cassette was constructed with the strong constitutive promoter P9 and the manA gene sequence of the Bacillus subtilis 168 strain. The mannose-6-phosphate isomerase gene, manA, was integrated into the Bacillus subtilis genome and expressed using the strong constitutive promoter P9.

[0061] The constructed manA integrated expression cassette was connected to the p...

Embodiment 3

[0075] Construction of recombinant Bacillus subtilis genetically engineered bacteria producing 2'-fucosyllactose

[0076] The plasmid pHT-XCR6 was transformed into Bacillus subtilis competent cells, positive clones containing the target plasmid were obtained through chloramphenicol resistance screening, and positive single clones were prepared into Bacillus subtilis competent cells containing plasmid pHT-XCR6.

[0077] According to the genome information of Bacillus subtilis 168 published on NCBI, the upstream and downstream gene sequences of the integrated manA gene were determined, and the manA integrated expression cassette was constructed with the strong constitutive promoter P43 and the manA gene sequence of the Bacillus subtilis 168 strain. The mannose-6-phosphate isomerase gene, manA, was integrated into the Bacillus subtilis genome and expressed using the strong constitutive promoter P43.

[0078] The constructed manA integrated expression cassette was connected to the...

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Abstract

The invention provides recombinant bacillus subtilis for producing 2 '-fucosyllactose and application of the recombinant bacillus subtilis, and belongs to the technical field of genetic engineering. According to the recombinant bacillus subtilis, bacillus subtilis is used as an original strain, and a mannose-6-phosphate isomerase gene manA, a phosphomannose mutase gene manB, a mannose-1-phosphoguanosine transferase gene manC, a GDP-mannose dehydratase gene gmd, a GDP-fucose synthase gene wcaG and alpha-1, 2-dihydroxy-4-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1-(2-hydroxyphenyl)-1 The recombinant bacillus subtilis is obtained by integrating a 1, 2-fucosyl transferase gene futC onto a bacillus subtilis genome and knocking out beta-galactosidase genes yesZ and ganA. The invention also provides application of the recombinant bacillus subtilis in fermentation synthesis of the 2 '-fucosyllactose, the yield of the 2'-fucosyllactose in fermentation in a 5L fermentation tank reaches 70.08 g / L, and the recombinant bacillus subtilis has relatively high application value and industrial production potential.

Description

technical field [0001] The technical field of genetic engineering of the present invention particularly refers to a recombinant Bacillus subtilis producing 2'-fucosyllactose and its application. Background technique [0002] Human milk oligosaccharides (HMOs) are a general term for a class of non-single-structure, non-digestible carbohydrates in human milk, which play an important role in the health, growth and development of infants, and have been used as functional components of infant formula. Element. 2'-Fucosyllactose (2'-FL) is the most abundant breast milk oligosaccharide, accounting for about 1 / 3 of the total breast milk oligosaccharides, and is currently the most potential breast milk oligosaccharides. 2'-Fucosyllactose has multiple functions such as regulating intestinal flora, resisting the adhesion of pathogenic bacteria, regulating immunity, and promoting the development and repair of the nervous system. It has been approved by many countries or regions as an ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/61C12N15/60C12N15/54C12N15/53C12N15/56C12N1/21C12P19/12C12P19/14C12P19/24C12R1/125
CPCC12N15/75C12N1/20C12N9/90C12N9/1241C12N9/88C12N9/0006C12N9/2402C12N9/2471C12N15/52C12Y503/01008C12Y504/02008C12Y207/07013C12Y402/01047C12Y101/01271C12Y302/01063C12P19/12C12P19/14C12P19/24
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰张权威
Owner JIANGNAN UNIV
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