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A kind of high riboflavin-producing Escherichia coli engineering strain and its construction and fermentation method

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 saving energy, increasing production, and shortening the production cycle

Active Publication Date: 2018-11-09
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of high riboflavin-producing Escherichia coli engineering strain and its construction and fermentation method
  • A kind of high riboflavin-producing Escherichia coli engineering strain and its construction and fermentation method
  • A kind of high riboflavin-producing Escherichia coli engineering strain and its construction and fermentation method

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Experimental program
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Embodiment 1

[0053] With the Cat-F primer shown in SEQ ID No.6 and the Cat-R shown in SEQ ID No.7 as a primer, with pCas9 as a template, the chloramphenicol acetyltransferase (Cat) gene was amplified by PCR, and then passed CEPC Assembly technology, the Cat gene and the pSC101 replicon from the pTKRed plasmid are assembled into a new plasmid pRB 01, such as figure 2 shown.

Embodiment 2

[0055] Using the rib-F primer shown in SEQ ID No.4 and the rib-R shown in SEQ ID No.5 as primers, using p20C_EC10 as a template, the fragment rib shown in SEQ ID No.1 was obtained by PCR amplification -int. The pRB 01 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 pLR 01 by T4 DNA ligase, such as figure 1 shown.

Embodiment 3

[0057] Pick E.coli MG1655 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 electrotransformation, the pLR 01 plasmid was introduced into E.coliMG1655, and positive transformants were screened out by chloramphenicol, and the obtained strain was named EC-rib 01.

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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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Patent Type & Authority Patents(China)
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