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Recombinant bacillus subtilis for producing 3-fucosyllactose as well as construction method and application of recombinant bacillus subtilis

A technology of Bacillus subtilis and fucosyllactose, which is applied in the field of recombinant strain construction of Bacillus subtilis to reduce the cost of separation and purification

Active Publication Date: 2022-05-06
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no technology or report on the synthesis of 3-FL using Bacillus subtilis

Method used

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  • Recombinant bacillus subtilis for producing 3-fucosyllactose as well as construction method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing 3-fucosyllactose as well as construction method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing 3-fucosyllactose as well as construction method and application of recombinant bacillus subtilis

Examples

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Embodiment 1

[0043] Embodiment 1: Construction of recombinant Bacillus subtilis 1643FL

[0044] Bacillus subtilis 1643FL integrates two DNA sequences on the genome of Bacillus subtilis 6051a, that is, integrates manA-manB-manC cassette (SEQ ID NO: 1) at the manA site, and after eliminating the erythromycin resistance gene, in The xylA-xylB site integrates the gmd-wcaG-futA cassette (SEQ ID NO:2), see figure 1 , is a schematic diagram of the construction of recombinant Bacillus subtilis 1643FL. The specific construction method of Bacillus subtilis 1643FL is as follows:

[0045] Firstly, a linear DNA fragment manA-manB-manC was prepared, which was obtained by fusion PCR of 6 fragments, which were U-manA, ermC, t7m, manB, manC and D-manA. U-manA and D-manA were amplified using the Bacillus subtilis ATCC 6061a genome as a template; manB and manC were amplified using Escherichia coli BL21 (DE3) as a template; ermC; t7m was amplified using Escherichia coli expression plasmid pET28a as a templ...

Embodiment 2

[0049] Example 2: Construction of recombinant Bacillus subtilis 1643FL-fcoA2

[0050] Recombinant Bacillus subtilis 1643FL-fcoA2 is based on Bacillus subtilis 1643FL, and lacY (SEQ ID NO: 3) encoding Escherichia coli BL21 (DE3) lactose permease is integrated into the fcoA2 site of 1643FL (attached figure 2 ), the specific operation is as follows: first construct fcoA2-PxylA-lacY, which consists of 5 fragments (U-fcoA2, Cmg, PxylA, lacYg and D-fcoA2). U-fcoA2 and D-fcoA2 were amplified from the genome of Bacillus subtilis ATCC 6061a; lacYg was amplified from Escherichia coli BL21 (DE3); PxylA and Cmg were amplified from artificially synthesized fragments. Wherein, the primer sequence of PCR amplification is as follows: the F-terminal primer sequence of amplifying U-fcoA2 (as shown in SEQ ID NO:29); The R-terminal primer sequence of amplifying U-fcoA2 (as shown in SEQ ID NO:30 Shown); Amplify the F-terminal primer sequence of Cmg (as shown in SEQ ID NO:31); Amplify the R-termi...

Embodiment 3

[0052] Example 3: Construction of recombinant Bacillus subtilis 1643FL-fcoA1

[0053] The recombinant Bacillus subtilis 1643FL-fcoA2 integrates lactose permease lacY into Bacillus subtilis 1643FL, and integrates a DNA fragment (SEQ ID NO: 4) into the fcoA1 site of 1643FL. The first is to prepare linear DNA fcoA1-PxylA-lacY, which is obtained by fusion PCR of 5 fragments, which are U-fcoA1, Cmy, PxylA, lacYy and D-fcoA1 respectively. Among them, U-fcoA1 and D-fcoA1 were amplified using the genome of Bacillus subtilis ATCC 6061a as a template; lacYy was amplified using Escherichia coli BL21 (DE3) as a template; PxylA and Cmy were amplified using artificially synthesized DNA fragments as a template. The primer sequences for PCR amplification are as follows. Amplify the F-terminal primer sequence of U-fcoA1 (as shown in SEQ ID NO:39); amplify the R-terminal primer sequence of U-fcoA1 (as shown in SEQ ID NO:40); amplify the F-terminal primer sequence of Cmy (As shown in SEQ ID NO...

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Abstract

The invention discloses recombinant bacillus subtilis for producing 3-fucosyllactose as well as a construction method and application of the recombinant bacillus subtilis. The recombinant bacillus subtilis is a recombinant bacillus subtilis 1643FL, a recombinant bacillus subtilis 1643FL-fcoA2, a recombinant bacillus subtilis 1643FL-fcoA1, a recombinant bacillus subtilis 1643FL-fcoA2, a recombinant bacillus subtilis According to the recombinant bacillus subtilis 1643FL, fusion gene segments for coding fructose 6-phosphate isomerase, phosphomannose mutase and mannose-1-phosphate guanosine transferase are integrated at a manA site of the bacillus subtilis, and fusion gene segments for coding GDP-mannose dehydratase, GDP-L-fucose synthetase, alpha-1, 2, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3, 4-trimethyl-1, 3-trimethyl-1, 3-trimethyl-1, 3-trimethyl-1, 3-trimethyl-1, 3-trimethyl-1, 3-trimethyl-1 3, obtaining a fusion gene segment of fucosyltransferase; the recombinant bacillus subtilis 1643FL-fcoA2 is obtained by integrating a gene segment for coding lactose osmose at a fcoA2 site of the recombinant bacillus subtilis 1643FL, and the recombinant bacillus subtilis 1643FL-fcoA2 is a recombinant bacillus subtilis 1643FL-fcoA2. The recombinant bacillus subtilis 1643FL-fcoA1 is obtained by integrating a gene segment for coding lactose osmose at a fcoA1 site of the recombinant bacillus subtilis 1643FL. The recombinant bacillus subtilis 1643FL-fcoA1 is a recombinant bacillus subtilis 1643FL-fcoA1. The recombinant bacillus subtilis disclosed by the invention can be used for producing the 3-fucosyllactose at high yield by utilizing a relatively cheap carbon source.

Description

technical field [0001] The invention belongs to the technical field of construction of recombinant strains of Bacillus subtilis, and in particular relates to a recombinant Bacillus subtilis for producing 3-fucosyllactose and a construction method and application thereof. Background technique [0002] The content of human milk oligosaccharides (HMOs) in human milk is as high as 15g / L, which is the third most abundant component after lactose and lipids in human milk. Among them, fucosylated oligosaccharides including 2'-FL (2'-fucosyllactose) and 3-FL (3-fucosyllactose) are unique to breast milk. 3-FL can inhibit the attachment of pathogenic bacteria on epithelial cells, thereby preventing infectious diseases. At the same time, adding 3-FL to formula milk powder can reduce the symptoms of diarrhea in infants. Therefore, 3-FL has both medical and nutritional value. [0003] Like 2'-FL, 3-FL can be isolated from human milk, or synthesized by chemical means or enzymatic methods...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/00C12R1/125
CPCC12N15/75C12P19/00C12N9/90C12N9/1241C12N9/88C12N9/0006C12N9/1051C12Y503/01C12Y504/02008C12Y207/07013C12Y402/01008C12Y101/01271
Inventor 孙俊松纪明华谢雨康陈艾史吉平
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI