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High-yield riboflavin Escherichia coli engineering strain, and construction and fermentation method thereof

A technology of Escherichia coli and engineering strains, applied in the field of bioengineering technology and application and fermentation engineering, to achieve the effects of short production cycle, increased output and energy saving

Active Publication Date: 2016-04-13
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] After searching, there is no report on the use of metabolic engineering to simultaneously transform the pfkA, edd and eda genes of Escherichia coli, and to produce riboflavin by fermentation

Method used

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  • High-yield riboflavin Escherichia coli engineering strain, and construction and fermentation method thereof
  • High-yield riboflavin Escherichia coli engineering strain, and construction and fermentation method thereof
  • High-yield riboflavin Escherichia coli engineering strain, and construction and fermentation method thereof

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Embodiment 1

[0053] With the Cat-F primer shown in SEQIDNo.6 and the Cat-R shown in SEQIDNo.7 as a primer, with pCas9 as a template, the chloramphenicol acetyltransferase (Cat) gene is amplified by PCR, and then assembled by CEPC. The Cat gene and the pSC101 replicon from the pTKRed plasmid were assembled into a new plasmid pRB01, such as figure 2 shown.

Embodiment 2

[0055] The rib-F primer shown in SEQIDNo.4 and the rib-R shown in SEQIDNo.5 were used as primers, and p20C_EC10 was used as a template to obtain the fragment rib-int shown in SEQIDNo.1 by PCR amplification. The pRB01 plasmid was cut with two restriction enzymes, ScaI and HindIII, and the digested product was purified, and rib-int was ligated into a new plasmid pLR01 by T4 DNA ligase, such as figure 1 shown.

Embodiment 3

[0057] Pick E.coliMG1655 to LB liquid medium, culture at 37°C, 200rpm, when the cell concentration is between 0.4-0.6 (indicated by the absorbance value at 600nm), it will be made competent. Then, by electroporation, the pLR01 plasmid was introduced into E.coliMG1655, and positive transformants were selected by chloramphenicol, and the obtained strain was named EC-rib01.

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Abstract

The invention discloses a high-yield riboflavin Escherichia coli engineering strain, and a construction and fermentation method thereof. The construction method comprises the following steps: (1) obtaining a rib-int segment; (2) constructing a pLR 01 plasmid; (3) constructing an EC-rib 01 strain; and (4) constructing an EC-rib 02 strain, and knocking out the pfkA gene in the glycolytic pathway of the strain EC-rib 01 genome and the edd and eda genes in the ED pathway, thereby obtaining the strain EC-rib 02. The riboflavin yield of the high-yield riboflavin Escherichia coli engineering strain EC-rib 02 reaches 10.47 g / L or above, which is the maximum riboflavin yield for the existing Escherichia coli. The production cycle of the EC-rib 02 strain is 60-80 hours, which is shorter than the existing common riboflavin engineering strain production cycle, thereby saving the energy.

Description

technical field [0001] The invention belongs to the fields of bioengineering technology and application and fermentation engineering, and in particular relates to an Escherichia coli strain for producing riboflavin, a construction method and a fermentation technology. Background technique [0002] Riboflavin, also known as vitamin B 2 , is a yellow natural water-soluble B vitamin. Its molecular formula is C 17 h 20 o 6 N 4 , with a molecular weight of 376, is one of the 13 essential vitamins for the human body. Riboflavin is an important prosthetic group of many enzyme systems in the body - a precursor for the synthesis 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 respiration and biological oxidation, is an indispensable vitamin in life activities, and is a necessary nutrient to maintain the normal substance metabolism of human and ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P25/00C12R1/19
CPCC07K14/245C12N9/1033C12N9/12C12P25/00C12Y203/01028C12Y207/01011
Inventor 陈涛刘双康培王智文赵学明
Owner TIANJIN UNIV
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