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Genetically engineered bacterium for efficiently synthesizing riboflavin and application of genetically engineered bacterium

A technology of genetically engineered bacteria and riboflavin, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve problems such as differences in gene expression levels

Active Publication Date: 2021-07-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that insufficient dissolved oxygen is one of the limiting factors for riboflavin synthesis, and insufficient dissolved oxygen leads to significant differences in the expression levels of genes related to riboflavin synthesis

Method used

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  • Genetically engineered bacterium for efficiently synthesizing riboflavin and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently synthesizing riboflavin and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently synthesizing riboflavin and application of genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Construction of engineering strain RF1-aP

[0050] The gene purR is knocked out from the genome by means of homologous recombination, and the repression of purine metabolism in the cell is released.

[0051] Specific steps are as follows:

[0052] (1) Amplify the upstream homology arm (1000bp) and downstream homology arm (1000bp) of the gene purR according to the primer sequences in Table 1, and separate the PCR product by agarose gel electrophoresis, and cut the gel to recover the target PCR product. Then the homology arm was fused with the knockout Marker by fusion PCR strategy. First, the upstream and downstream fragments were mixed at a volume ratio of 1:1, and an equal volume of PCR enzyme was added to carry out a fusion PCR reaction. The conditions were 98°C for 3min, 98°C for 8s, 61°C for 5s, and 72°C for 2min, and amplified for 13 cycles. The product after the first step reaction is used as a template, and the fusion fragments are amplified using...

Embodiment 2

[0055] Embodiment 2: Construction of engineering strains RF1-aG and RF1-aT

[0056] Place the antisense strands of the genes glnR and tnrA in the strong promoter P 43 Overexpressed under control, the antisense RNA is complementary to the mRNA of the normally expressed genes glnR and tnrA in the cell, thereby preventing the normal translation of GlnR and TnrA and regulating the nitrogen metabolism level in the cell. Inhibition of translation of glnR and tnrA genes by an antisense RNA strategy. Promoter P 43 The antisense RNA of the control genes glnR and tnrA was used to construct engineering strains RF1-aG and RF1-aT.

[0057] Specific steps are as follows:

[0058] 1. Preparation of genetic engineering strain RF1-aG

[0059] (1) Using primer P 43 - AntiglnR-F1 and P 43 - AntiglnR-R1 amplified gene P 43 , primer P 43 -AntiglnR-F2 and P 43 -AntiglnR-R2 amplifies the gene glnR, recovers and purifies the target fragment by agarose gel electrophoresis, then mixes the targ...

Embodiment 3

[0063] Embodiment 3: Construction of engineering strain RF1-gV

[0064] In the process of metabolic modification of industrial microorganisms, VHb is often used to alleviate the dissolved oxygen limitation in the fermentation process and increase the yield of target products. In order to avoid the burden of exogenous gene overexpression on cells and the damage of cells by oxidative stress, a dynamic regulation strategy was used to control the expression of gene vgb.

[0065] Specific steps are as follows:

[0066] (1) with primer P glnR -gfp-F1 and P glnR -gfp-R1 amplifies the promoter sequence of gene glnR and recovers and purifies, then primer P glnR -gfp-F2 and P glnR- The gene gfp sequence amplified by gfp-R2 was recovered and purified, and the above two fragments were mixed at a volume ratio of 1:1, and then PCR enzyme was added for overlapping PCR reaction. The reaction conditions were 98°C for 3min, 98°C for 8s, and 61°C 5s, 2min at 72°C, 13 cycles of amplification...

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Abstract

The invention discloses a genetically engineered bacterium for efficiently synthesizing riboflavin and application of the genetically engineered bacterium, and belongs to the technical field of biological fermentation. The genetically engineered bacterium has the advantages that genes for encoding purR are knocked down, genes for encoding glnR and tnrA are knocked down, hemoglobin from vitreoscilla is overexpressed, dissolved oxygen limitation in the fermentation process is relieved, the energy consumption in the fermentation process is reduced, and the riboflavin yield is increased; and the riboflavin yield of the constructed engineered strain RF1-aPaGaTgV is increased by 50.78% at a shake flask stage, and reaches 2.51 g / L. At the 5-L fermentation level, the highest yield of the riboflavin obtained by adopting the constructed RF1-aPaGaTgV is increased by 45.51% and reaches 10.71 g / L.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for efficiently synthesizing riboflavin and an application thereof, belonging to the technical field of biological fermentation. Background technique [0002] Riboflavin (vitamin B2) is a nutrient element that maintains the normal physiological metabolism of the human body and can only be obtained through diet. In nature, only microorganisms and plant cells have the ability to synthesize riboflavin, while mammalian cells lack the corresponding synthesis pathway. Riboflavin is widely used in our daily life, and can be used as food additives, medicines, health products and cosmetic raw materials. In recent years, the production of riboflavin by microbial fermentation has completely replaced chemical synthesis in industry. The microorganisms that produce riboflavin by fermentation mainly include Bacillus subtilis and Ashbya gossypii. In addition, Candida flareri, E.coli, and Lactobacillus plan...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/75C12P25/00C12R1/125
CPCC07K14/32C07K14/195C12N15/75C12P25/00
Inventor 饶志明尤甲甲杨套伟吴美琪张显徐美娟
Owner JIANGNAN UNIV
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