Bacillus subtilis for improving yield of 2'-fucosyllactose and construction method and application of bacillus subtilis

A technology of Bacillus subtilis and fucosyllactose, which is applied in the field of genetic engineering, can solve the problem of low 2'-FL yield and achieve the effect of increasing the initial translation rate

Active Publication Date: 2021-11-12
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of low yield of 2'-FL produced by microbial fermentation, the present invention provides a method for improving 2'-FL fermentation yield through metabolic engineering technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Reconstitution of Bacillus subtilis WCY1 and WCY2

[0034] (a) Artificially synthesized gene fragment P srfa -fkp, using the Bacillus subtilis 168 genome as a template, using primers amye-U-F, amye-U-R to amplify the gene amye-U, using primers amye-D-F, amye-D-R to amplify the gene amye-D, using the plasmid F19- 1 (SEQ ID NO.5) was used as a template, and F19-amye-F and F19-amye-R were used to amplify to obtain fragment F19-1H.

[0035] (b) the four fragments amye-U, amye-D, F19-1H and P obtained in step (a) srfa - The fkp gene fragments were cloned in one step according to the equimolar ratio configuration, and the connection conditions were: 50°C, 60min. After heat-shocking the ligated reaction system into Escherichia coli JM109 competent, use primers amye-U-F and amye-D-R colony PCR to verify and sequence. A single colony with correct sequencing was inserted into 2mL LB liquid medium and cultured at 37°C for 16 hours, and then the plasmid F19-fkp was ext...

Embodiment 2

[0055] Example 2 Reconstitution of Bacillus subtilis WCY3 and WCY4

[0056] (a) Artificially synthesized gene fragment P 43 -glcp, using the Bacillus subtilis 168 genome as a template, using primers apre-U-F, apre-U-R to amplify the gene apre-U, using primers apre-D-F, apre-D-R to amplify the gene apre-D, using the plasmid F19- 3 (SEQ ID NO.7) was used as a template, and F19-apre-F and F19-apre-R were used to amplify to obtain fragment F19-3H.

[0057] (b) the four fragments apre-U, apre-D, F19-3H and P obtained in step (a) 43 -glcp gene fragments were cloned in one step according to the equimolar ratio configuration, and the connection conditions were: 50°C, 60min. After heat-shocking the ligated reaction system into Escherichia coli JM109 competent, the primers apre-U-F and apre-D-R colony PCR were used to verify and sequenced. A single colony with correct sequencing was inserted into 2mL LB liquid medium and cultured at 37°C for 16 hours, and then the plasmid F19-glcp wa...

Embodiment 3

[0073] Example 3 Reconstitution of Bacillus subtilis WCY5

[0074] (a) Artificially synthesized gene fragment P 43 -lac12, using the Bacillus subtilis 168 genome as a template, using primers ganA-U-F, ganA-U-R to amplify the gene ganA-U, using primers ganA-D-F, ganA-D-R to amplify the gene ganA-D, using the plasmid F19- 4 (SEQ ID NO.8) was used as a template, and fragment F19-4H was amplified by using F19-ganA-F and F19-ganA-R.

[0075] (b) the four fragments ganA-U, ganA-D, F19-4H and P obtained in step (a) 43 The -lac12 gene fragments were cloned in one step according to the equimolar ratio configuration, and the connection conditions were: 50°C, 60min. After heat-shocking the ligated reaction system into Escherichia coli JM109 competent, use primers ganA-U-F and ganA-D-R colony PCR to verify and sequence. A single colony with correct sequencing was inserted into 2mL LB liquid medium and cultured at 37°C for 16 hours, and then the plasmid F19-lac12 was extracted using the...

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PUM

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Abstract

The invention discloses bacillus subtilis for improving the yield of 2-fucosyllactose and a construction method and application of the bacillus subtilis. Bacillus subtilis 168 serves as an original strain, a constitutive promoter Psrfa is adopted to express an exogenous gene bifunctional enzyme L-fucokinase fkp on a genome, a constitutive promoter P43 is adopted to express exogenous genes alpha-1,2-fucosyltransferase futC, lactose transporter lac12 and endogenous gene monosaccharide protease glcp on the gene respectively, and ribosome binding site RBS sequences of fkp, futC, lac12 and glcp are respectively optimized to obtain the bacillus subtilis for improving the yield of the 2'-fucosyllactose. By optimizing the RBS sequence, 2'-FL metabolic pathway key gene is systematically optimized, the initial translation rate of the gene is improved, the final 2'-FL fermentation yield reaches 1.0 g/L, the operation is simple and convenient, and the improvement effect is remarkable.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bacillus subtilis for improving the production of 2'-fucosyllactose, a construction method and application thereof. Background technique [0002] 2'-fucosyllactose (2'-FL) is an oligosaccharide composed of three monosaccharide molecules, and its content in breast milk is about 2.4g / L, which is the most abundant in breast milk of oligosaccharides. 2'-FL has good nutritional value and physiological activity. Experiments have proved that it can inhibit the adhesion of pathogenic bacteria to specific receptors on the surface of epithelial cells and reduce the risk of pathogenic infection in infants, thereby promoting the growth of beneficial microorganisms in the intestinal tract as a soluble prebiotic . At present, both the European Union and FDA have approved that 2’-FL can be added to infant formula milk powder, and the market prospect is broad. Both c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/00C12R1/125
CPCC12N15/75C12N9/1051C12N9/50C12P19/00
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰于文文
Owner BRIGHT DAIRY & FOOD
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