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Application of dihydropteroate synthase gene folP

A technology of dihydropteroate synthase and gene, applied in genetic engineering, plant genetic improvement, and microbial-based methods, etc., can solve problems such as less research, lack of systematic research on folic acid synthesis genes, and new functions to be discovered , to achieve the effect of improving the screening efficiency

Active Publication Date: 2019-05-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to increase the production of natural folic acid and meet human needs, much attention has been paid to studying the key genes of folic acid biosynthesis in the above-mentioned pathways or changing the metabolism of folic acid in plants and microorganisms through genetic engineering. ground research, some new functions are yet to be discovered
[0006] At present, in the research on the regulation mechanism of microbial folic acid biosynthesis pathway, only the branch of p-aminobenzoic acid metabolism has been studied in depth, while the other branch of pterin metabolism has been less studied

Method used

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  • Application of dihydropteroate synthase gene folP
  • Application of dihydropteroate synthase gene folP
  • Application of dihydropteroate synthase gene folP

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Dihydropteroate Synthase Gene folP knockout homology arm clone

[0034] 1. PCR amplification of upstream and downstream homology arms

[0035] The genome of food-borne Lactobacillus plantarum YM 4-3 was extracted using the Bacterial Genomic DNA Extraction Kit (Betech Biotechnology Co., Ltd., China), and the specific operation was performed according to the kit instructions. folP The 990 bp sequence upstream of the start codon ATG in the gene coding region is used as the upstream homology arm, and the 992 bp sequence downstream of the stop codon TAG is used as the length of the downstream homology arm;

[0036] Using the extracted genome as a template, the primer pair up-PF (5'-GG ACTAGT GGCAAGTGTGAAG-3', underlined Speech I restriction site) + up-PR (5’-ATCGTCAAAATCATCCGGACTACTCCACCGATCCTTCC-3’) and down-PF (5’-GGAAGGATCGGTGGAGTAGTCCGGATGATTTTGACGAT-3’) + down-PR (5’-T

[0037] G GAATTC CATTATCACGCTTATCTTG-3', underlined Eco RI restriction site) t...

Embodiment 2

[0044] Example 2: folP Gene knockout vector construction

[0045] restriction endonuclease Speech I and Eco RI performed synchronous enzyme digestion on the sequenced correct gene knockout fragment and the temperature-sensitive plasmid pFED760 respectively. The enzyme digestion system is: Speech I, 1 µL; Eco RI, 1 µL; 1×H buffer, 2 µL; gene knockout fragment or pFED760, 10-16 µL; add sterilized deionized water to 20 µL, digest at 37°C for 4 h; : Carrier = 4:1~2:1 (molar ratio) After adding the sample, add T4 DNA ligase and ligate at 16°C for 12~16 h. The ligation product was introduced into Escherichia coli DH5α competent cells by the heat shock transformation method, and then spread on the erythromycin-LB solid plate. After culturing overnight at 28°C, extract plasmids from 10-15 single colony cells and use Speech I and Eco RI digestion verification to obtain a positive plasmid, named pFED760- folP .

Embodiment 3

[0046] Example 3: folP Gene knockout strain construction

[0047] 1, folP Gene knockout vector introduced into Lactobacillus plantarum competent cells

[0048] Lactobacillus plantarum competent cells were prepared according to the method reported by Fei Yongtao (2015, master's degree thesis of South China University of Technology). Add 10 μL gene knockout vector pFED760- folP , mixed gently, put in an ice bath for 5 min, then transferred to a pre-cooled electric shock cup, and electric shock was performed according to the parameters of 12.5 kv / cm, 200 Ω. After the electric shock was completed, quickly add 900 μL of fresh MRS culture solution to the electro-cup, blow and mix gently with a pipette tip, then transfer the mixture to a sterile 1.5 mL centrifuge tube, and incubate at 28°C for 2.5-3 h to make the cells Resuscitate; after cultivation, centrifuge the bacterial solution at 8,000-10,000 rpm for 3 minutes, discard 900 μL of the supernatant, resuspend the bacterial cel...

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Abstract

The invention discloses application of a dihydropteroate synthase gene folP, namely the application of the dihydropteroate synthase gene folP in improvement of folic acid biosynthesis of microbial strains or in increase of biomass of the microbial strains. A knockout vector is created by recombination of the gene folP and a temperature-sensitive plasmid pFED760, and is introduced into a competentcell of food-borne lactobacillus plantarum, and accordingly, an folP gene knockout strain is formed by homologous recombination. Results show that the folP gene is related to the cell morphology and the growth state of the strains; the folic acid production ability of the delta folP strains is found to be lower than that of wild-type strains through liquid chromatography-mass spectrometry, so thatthe folP gene plays a key role in folic acid synthesis; the folP gene has great potential in research and application of the folic acid biosynthesis.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular to dihydropteroate synthase gene folP In improving Lactobacillus plantarum ( Lactobacillus plantarum ) application in folic acid biosynthesis. Background technique [0002] Folic acid, chemically called pteroylglutamic acid (pteroylglutamic acid), is a kind of pterin, p-aminobenzoic acid ( p -aminobenzoic acid, p ABA) and one or more glutamic acid combined water-soluble B vitamins, namely vitamin B9. Chemically synthesized folic acid contains only one glutamic acid tail, while natural folate consists of multiple glutamic acid tails. [0003] Folic acid has important physiological functions, and people cannot survive without folic acid. According to reports, folic acid is involved in physiological metabolic processes such as DNA and RNA biosynthesis, DNA repair, amino acid metabolism and hemoglobin synthesis. In addition, because folic acid can provide a large amou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/74C12N1/21C12P17/18C12R1/25
Inventor 罗义勇柳陈坚龙云
Owner KUNMING UNIV OF SCI & TECH
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