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Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli

A technology of Escherichia coli and construction method, applied in the field of production of riboflavin, strains of Escherichia coli and construction, can solve the problems such as the production of riboflavin by Escherichia coli, and achieve the effect of improving yield and yield

Active Publication Date: 2014-06-18
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] After searching, there is currently no report on the production of riboflavin using metabolically engineered Escherichia coli

Method used

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  • Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli
  • Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli
  • Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Design the ribosome binding site RBS1 shown in SEQ ID NO.37, the ribosome binding site RBS2 shown in SEQ ID NO.38, the ribosome binding site RBS3 shown in SEQ ID NO.39, and Ribosome binding site RBS4 shown in SEQ ID NO.40 and ribosome binding site RBS5 shown in SEQ ID NO.41.

Embodiment 2

[0036] Using amplification primers FuECribA-F (SEQ ID NO.1) / FuECribA-R (SEQ ID NO.2) to use the genome of Escherichia coli MG1655 (purchased from CGSC) as a template to artificially design the ribA gene on the riboflavin synthetic metabolic pathway The ribosome binding site RBS1 and the coding sequence were amplified to obtain Fragment 1 (see figure 2 ).

[0037] Using amplification primers FuECribB-F (SEQ ID NO.3) / FuECribB-R (SEQ ID NO.4) to use the genome of Escherichia coli MG1655 (purchased from CGSC) as a template to artificially design the ribB gene on the riboflavin synthetic metabolic pathway The ribosome binding site RBS2 and the coding sequence were amplified to obtain Fragment 2 (see figure 2 ).

[0038]Using amplification primers FuECribD-F (SEQ ID NO.5) / FuECribD-R (SEQ ID NO.6) to use the genome of Escherichia coli MG1655 (purchased from CGSC) as a template to artificially design the ribD gene on the riboflavin synthetic metabolic pathway The ribosome binding...

Embodiment 3

[0042] Using amplification primers FuECribA-F (SEQ ID NO.1) / FuECribC-R (SEQ ID NO.10), using fragment 1, fragment 2, fragment 3, fragment 4 and fragment 5 obtained in Example 2 as templates, by Overlap-extension polymerase chain reaction spliced ​​these five genes in the order of RBS1-ribA-RBS2-ribB-RBS3-ribD-RBS4-ribE-RBS5-ribC and named the operon EC10 (see Figure 4 ), cloned into plasmid pTrc99a at the EcoRI / HindIII site (see image 3 , purchased from Addgene) after digestion, enzyme ligation, transformation, verification and other operations, the plasmid p20C-EC10 was obtained (see Figure 5 ), and the plasmid was transformed into Escherichia coli Escherichia coli MG1655 to obtain the RF01S strain.

[0043] Among them, the formula of LB liquid medium is: 10g / L peptone, 5g / L yeast extract, 10g / L NaCl, adjust the pH to 7.0, and sterilize under 0.1Mpa pressure for 20min. The formula of LB solid medium is: add 15g / L agar powder to LB liquid medium with adjusted pH value, an...

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Abstract

The invention discloses an Escherichia coli for producing riboflavin and constructing method and use of Escherichia coli. The preparation method comprises the following steps: designing ribosome bind sites RBS1, RBS2, RBS3, RBS4 and RBS5, respectively connecting the ribosome bind sites with genes ribA, ribB, ribD, ribE and ribC into fragments, splicing the fragments in order and enzyme-cutting and linking to a plasmid pTrc99a to obtain plasmid p20C-EC10, transferring into the Escherichia coli to obtain RF01S strain, transforming related gene of glycolytic pathway and ED pathway of the RF01S strain so as to improve the output of the riboflavin, and transforming the consumption pathway of the riboflavin to decrease the metabolism velocity of the riboflavin so as to further improve the riboflavin accumulation, inserting a Ptrc strong promoter into genome of the strain to obtain the Escherichia coli for producing the riboflavin. The constructed Escherichia coli strain has clear genetic background, and the glucose can be used as substrate to produce the riboflavin, and the shake-flask fermenting result is more than 1g / L, and a foundation is provided for improving the riboflavin output and yield.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to a riboflavin-producing Escherichia coli strain, a construction method and application. Background technique [0002] Vitamin B2, also known as riboflavin, is a yellow natural water-soluble B vitamin. Vitamin B2, as one of the 13 essential vitamins, is slightly soluble in water. Riboflavin is an important prosthetic group of many enzyme systems in the body - the constituents of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). As a coenzyme of flavoprotein, it participates in the metabolism of hydrogen transfer, plays an important role in the metabolic process of respiration and biological oxidation, is an essential vitamin for life activities, and is a necessary nutrient for maintaining normal substance metabolism in humans and animals. . In addition, riboflavin has been widely used in feed, food, medicine and other fields a...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P25/00C12R1/19
Inventor 陈涛林振泉徐志博王智文赵学明
Owner TIANJIN UNIV
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