Method for constructing engineering strain for producing flavin mononucleotide, and applications of engineering strain

A single nucleotide and engineering strain technology, applied in the field of bioengineering, can solve problems such as difficult to realize microbial fermentation production of flavin mononucleotide, achieve the effect of realizing fermentation output value, eliminating by-products, and reducing production costs

Inactive Publication Date: 2018-07-27
BEIHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the conventional genetic engineering idea of ​​"excessive accumulation and secretion

Method used

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  • Method for constructing engineering strain for producing flavin mononucleotide, and applications of engineering strain
  • Method for constructing engineering strain for producing flavin mononucleotide, and applications of engineering strain
  • Method for constructing engineering strain for producing flavin mononucleotide, and applications of engineering strain

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Embodiment 1

[0034] 1. Construction of plasmids for riboflavin synthesis pathway:

[0035] The codon-optimized riboflavin synthesis proteins RibA, RibD, RibE and RibH of Bacillus subtilis were introduced into Escherichia coli to construct recombinant Escherichia coli that over-produced riboflavin.

[0036] like figure 2 As shown in middle a, each exogenous gene is designed and synthesized into the form of biological bricks. EcoRI, XbaI restriction site and ribosome binding site (ribosomebinding site, RBS for short) are sequentially designed upstream of the coding sequence of each gene. Wherein, the RBS sequence is: AAAGAGGA GAAA. The ribosome binding site is 6 bp away from the ATG site of the coding sequence. SpeI and PstI restriction sites are designed downstream of the coding sequence of each gene. Order and synthesize the genes ribA, ribD, ribE, and ribH (sequences shown in SEQ ID No.1-4) in the form of bio-bricks from a gene synthesis service company, and separately link them into...

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Abstract

The invention discloses a method for constructing an engineering strain for producing flavin mononucleotide, and applications of the engineering strain. According to the method, by using a synthetic biology technology, an intracellular flavinpool component and endometrial secretion are regulated in newly-constructed or existing riboflavin fermenting bacteria, and an inside-cell-membrane-interstitium enzyme catalytic step is novelly added, such that the high-purity flavin mononucleotide is produced through fermentation with microorganisms; and the method specifically comprises: (1) introducinga bifunctional enzyme riboflavin kinase/flavin adenine dinucleotide producing enzyme into riboflavin fermentation bacteria to convert the intracellular flavinpool component from riboflavin to flavin adenine dinucleotide, (2) introducing a flavin adenine dinucleotide internal membrane transporter, and transporting the intracellular flavin adenine dinucleotide into cell membrane interstitium, and (3) introducing 5'-nucleotidase to lyse and convert the flavin adenine dinucleotide in the cell membrane interstitium into the flavin mononucleotide. According to the present invention, under the shaking fermentation condition, the fermentation purity of the flavin mononucleotide (accounting for the total flavin compounds) can be up to 92.4%.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for constructing an engineering bacterium producing flavin mononucleotide and its application. Background technique [0002] Flavin mononucleotide (FMN for short) is an expensive compound with a wide range of uses. In organisms, flavoprotein mononucleotide, as a coenzyme of flavoprotein, participates in the redox reaction of cells and plays an important role in basic metabolism. Many microorganisms and plants can synthesize flavin compounds by themselves, while higher animals can only ingest them from food. When they are deficient, they can lead to hair loss, skin inflammation, vision impairment and other diseases. Therefore, as a water-soluble vitamin, flavin mononucleotide is often used as clinical medicine, food and feed additive, food coloring, etc. [0003] In addition, flavin mononucleotide has a redox active center and is an important electron transfer ca...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12P19/32
CPCC12N15/63C12N2800/101C12N2800/22C12P19/32
Inventor 王华杨昀郭林
Owner BEIHANG UNIV
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