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Recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and establishment method and application of recombinant bacillus subtilis

A technology of Bacillus subtilis and fucosyllactose, which is applied in the field of genetic engineering, can solve problems such as limited utilization of lactose, and achieve the effects of simple construction method, easy use and good application prospect.

Active Publication Date: 2019-05-10
BRIGHT DAIRY & FOOD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both pathways require lactose as a substrate, but Bacillus subtilis has limited ability to utilize lactose

Method used

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  • Recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and establishment method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and establishment method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and establishment method and application of recombinant bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Introduce and strengthen the expression of lactose transport enzyme gene lacY

[0025] According to the upstream and downstream sequences of the α-amylase gene amyE of Bacillus subtilis (Bacillus subtilis 168 purchased from the American Type Microorganism Collection, ATCC No.27370), the lactose transport enzyme gene lacY, the P43 promoter and the Bleomyces The sequence of the gene resistance to the drug was constructed by constructing a replacement frame whose sequence is shown in SEQ ID NO.1.

[0026] The constructed replacement frame was electrotransformed into competent cells of Bacillus subtilis 168, the amount of the replacement frame added was 100-300ng, the electroporation conditions were: voltage 2.5kV, electroshock reagent 5ms, recovery at 37°C for 5h, and the final coating concentration was 25μg / mL Bleomycin-resistant LB plates were cultured aerobically at 37°C for 12 hours, and several single clones were selected.

[0027] Due to the presence of the upstream...

Embodiment 2

[0031] Exogenous expression of foreign genes of Bacteroides fragilis and Helicobacter pylori

[0032] According to the sequence of fucokinase and guanyltransferase gene fkp of Bacteroides fragilis (ATCC No.25285) published on NCBI, and the fucoidan of Helicobacter pylori (ATCC No.26695) Glycosyltransferase futC gene sequence, through PCR linear amplification of gene futC, fkp and plasmid pP43NMK and one-step cloning and connection using ClonExpress II One Step Cloning Kit (Vazyme), to construct recombinant plasmid pP43 whose sequence is shown in SEQ ID NO.2 -fkp-futC.

[0033] Use KpnI and PstI endonucleases to double digest the fkp, futC gene sequence and plasmid pP43-mpd, use 50 μl of the system to react at 37°C for 30 minutes, use ligase to perform ligation after purification and recovery, and use 10 μl of the reaction system to react at 16°C for 2 hours. The recombinant plasmid pP43-fkp-futC whose sequence is shown in SEQ ID NO.2 was constructed.

[0034]The constructed ...

Embodiment 3

[0038] Production of 2'-fucosyllactose by fermentation

[0039] The above-mentioned Bacillus subtilis 168L-FF is made into seed liquid, and the formula of seed liquid culture medium is: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L; A single colony is placed in the seed medium and cultivated for 8-10 hours.

[0040] Insert the seed solution into the fermentation medium with an inoculum size of OD value of 0.1. The formula of the fermentation medium is: initial glycerol 20g / L, peptone 6g / L, yeast powder 12g / L, (NH 4 ) SO 4 6g / L, K 2 HPO 4 ·3H 2 O 12.5g / L, KH 2 PO 4 2.5g / L, CaCO 3 5g / L, trace element solution 10ml / L; trace element solution contains: MnSO 4 ·5H 2 O 1.0g / L, CoCl 2 ·6H 2 O0.4g / L, NaMoO 4 2H 2 O 0.2g / L, ZnSO 4 ·7H 2 O 0.2g / L, AlCl 3 ·6H 2 O 0.1g / L, CuCl 2 ·H 2 O0.1g / L, H 3 BO 4 0.05g / L, 5M HCl, cultured at 35°C, 200rpm for 20h.

[0041] At the end of the fermentation, use liquid-mass chromatography to measure the content of 2'-fucosyllactose in...

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Abstract

The invention provides recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and an establishment method and application of recombinant bacillus subtilis. Recombinant bacillus subtilis is obtained in the mode that a lactose transport enzyme gene is subjected to reinforcement expression in a gene group of bacillus subtilis 168, and exogenous fucosyltransferase, fucokinase and gtp-mannose-1-phosphate guanylyltransferase genes are expressed. According to recombinant bacillus subtilis, the lactose transport enzyme gene is subjected to reinforcement expression on the gene group, expression of the lactose transport enzyme is effectively reinforced, the efficiency of transferring exogenous lactose into cytoplasm is improved, the concentration of lactose in cytoplasm is increased, and synthesis of 2'-fucosyllactose is promoted. The establishment method of recombinant bacillus subtilis is simple and convenient to use and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant bacillus subtilis for synthesizing 2'-fucosyllactose and its construction method and application. Background technique [0002] Breast milk contains important nutrients, bioactive substances, factors that stimulate the growth of intestinal flora. Among them, human milk oligosaccharides (Human Milk Oligosaccharides, hMOs) play a key role in many physiological functions, such as promoting the growth of bifidobacteria, inhibiting pathogenic infection, and improving immune response. Cocosylated oligosaccharides (FOSs) in human milk oligosaccharides have gained great interest due to their physiological functions such as their ability to act as receptor analogs for intestinal pathogenic bacteria, to promote immune regulation, and to reduce inflammation. s concern. Because glycosylated oligosaccharides are formed by fucosylation of sugars catalyzed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/12C12R1/125
Inventor 刘龙邓洁莹陈春梅吕雪芹李江华堵国成陈坚
Owner BRIGHT DAIRY & FOOD
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