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Application of glycosyltransferase gene

A technology of glycosyltransferase and gene, which is applied in the field of microbial gene function and application, can solve the problems of poor strain stability, high extraction and purification costs, and low EPS synthesis, and achieve the effect of improving screening efficiency

Inactive Publication Date: 2021-01-29
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, under normal circumstances, the amount of EPS synthesis is generally not high, and the strain stability is poor, and factors such as high extraction and purification costs restrict the large-scale application of lactic acid bacteria EPS in industrial production. The transformation of the combination of optimal culture conditions can effectively increase the amount of EPS synthesis and reduce costs, and make the industrial production of EPS possible.

Method used

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  • Application of glycosyltransferase gene
  • Application of glycosyltransferase gene
  • Application of glycosyltransferase gene

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Experimental program
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Effect test

Embodiment 1

[0029]Example 1: Glycosyltransferase gene (orf 1595、cps 4I) Knockout homology arm clone

[0030]1. PCR amplification of the upstream and downstream homology arms

[0031]Use TAKARA bacterial genomic DNA extraction kit (Bao Biological Engineering Co., Ltd.) to extract the food-borne Lactobacillus plantarum YM 4-3 genome, and the specific operation is carried out according to the kit instructions:

[0032]Targeting genesorf 1595, using the extracted genome as a template, usingorf 1595-up-F (5’-CCGGAATTC GATGGATCTAAAGCGGTGTT-3’ underline is the restriction siteEcoR Ⅰ),orf 1595-up-R (5’- CGCTGAATGAATTGCCTTTGTGCCGCATGTATTTGTAAGG-3’) andorf 1595-down-F (5’- CCTTACAAATACATGCGGCACAAAGGCAATTCATTCAGCG-3’),orf 1595-down-R(5’-CCCAAGCTT GTGGCAAGCCGTCAACGA-3’, underlined is the restriction siteHind Ⅲ) Amplify to get upstream and downstream homology armsorf 1595-up (992bp),orf 1595-down (1156bp);

[0033]Targeting genescps 4I, using the extracted genome as a template, using primer pairs respectivelycps 4I-up-...

Embodiment 2

[0040]Example 2:orf 1595、cps 4I gene single knockout vector construction

[0041]Use restriction enzymesEcoR Ⅰ.Hind Ⅲ andEcoR Ⅰ.Hind Ⅲ Simultaneous restriction digestion of the correctly sequenced gene knockout fragment and temperature-sensitive plasmid pFED760;orf The 1595 restriction digestion system is:EcoR Ⅰ, 2µL;Hind Ⅲ, 2µL; 1× H buffer, 4µL; gene knockout fragment or pFED760, 10~16µL; add sterile deionized water to 20µL.cps The 4I digestion system is:EcoR Ⅰ, 1 µL;Hind Ⅲ, 1 µL; 1× M buffer, 4 µL; gene knockout fragment or pFED760, 30 µL; add sterile deionized water to 40 µL. After digestion at 37°C for 4 hours, the digested product was recovered. According to the target gene: vector = 4:1~2:1 (molar ratio), after loading the sample, add T4 DNA ligase and ligate at 16°C for 12-16 hours; use heat shock transformation method The ligation product was introduced into E. coli DH5α competent cells, and then spread on the erythromycin-LB solid plate; after overnight culture at 28°C, the...

Embodiment 3

[0042]Example 3:orf 1595 orcps Construction of 4I gene knockout strain

[0043]1,orf 1595 orcps Introduction of 4I gene knockout vector into competent cells of Lactobacillus plantarum

[0044]Prepare Lactobacillus plantarum competent cells according to the method reported by Fei Yongtao (2015, South China University of Technology master's thesis); add 10μL gene knockout vector pFED760-Δ to 90~100μL Lactobacillus plantarum competent respectivelyorf 1595 or pFED760-Δcps 4I, mix gently, transfer to the pre-cooled electroporation cup after ice bath for 5 minutes, and perform the electroshock according to the parameters of 1.25kv / cm, 200Ω; after the electroporation is completed, quickly add 900μL of fresh MRS culture medium to the electroporation cup, and use the pipette tip After gently pipetting and mixing, transfer the mixed solution to a sterile 1.5mL centrifuge tube and incubate at 28°C for 2.5~3 hours to recover the cells. After culture, the bacterial solution was centrifuged at 8000 rpm...

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Abstract

The invention discloses an application of a glycosyltransferase gene, namely an application of the glycosyltransferase gene in improving the synthesis of lactobacillus plantarum exopolysaccharides; wherein the glycosyltransferase gene is a gene orf1595 or a gene cps4I, the nucleotide sequence of the gene orf1595 is as shown in SEQ ID NO: 1, and the nucleotide sequence of the gene cps4I is as shownin SEQ ID NO: 2; an orf1595 or cps4I gene and a temperature-sensitive plasmid are recombined to construct a knockout vector, the knockout vector is introduced into a food-borne lactobacillus plantarum competent cell, and then a gene knockout strain is constructed through homologous recombination; a phenol sulfuric acid method is adopted to determine the exopolysaccharide production capacity of the strain, it is found that the exopolysaccharide production capacity of the knocked-out strain is lower than that of a wild strain, orf1595 or cps4I genes play a key role in exopolysaccharide synthesis, and the method has huge potential in the fields of exopolysaccharide biosynthesis research and application.

Description

Technical field[0001]The invention belongs to the function and application field of microbial genes, and specifically relates to glycosyltransferase genesorf 1595、cps Application of 4I in enhancing the biosynthesis of extracellular polysaccharide of Lactobacillus plantarum YM 4-3.Background technique[0002]Lactic Acid Bacteria (Lactic Acid Bacteria) is a general term for a class of microorganisms that are generally regarded as safe (GRAS) that ferment glucose or use lactose to produce lactic acid. It not only exists in inorganic environments, but also common in human and animal intestines. It is traditionally related to fermented foods and is closely related to human culture and welfare. Historically, lactic acid bacteria have been The positive contributions to quality and safety are well known.[0003]Lactobacillus exopolysaccharide (EPS) is an important secondary metabolite produced by lactic acid bacteria, which has attracted widespread attention and recognition in recent years. Mic...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/75C12N15/66C12N15/65C12N1/21C12P19/04C12R1/25
CPCC12N9/1048C12N15/75C12N15/66C12N15/65C12P19/04
Inventor 杨恩熊永刚柳陈坚罗义勇
Owner KUNMING UNIV OF SCI & TECH
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