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Genetic engineering bacterium for producing 4-hydroxyisoleucine and application thereof

A technology of hydroxyisoleucine and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of low 4-HIL yield and the like, and achieve the effect of improving enzyme activity

Active Publication Date: 2019-06-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But relatively speaking, the production of 4-HIL is still relatively low

Method used

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  • Genetic engineering bacterium for producing 4-hydroxyisoleucine and application thereof
  • Genetic engineering bacterium for producing 4-hydroxyisoleucine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of a bacterial strain expressing Bacillus thuringiensis derived Btido

[0024] The isoleucine dioxygenase encoding gene Btido shown in SEQ ID NO.1 was synthesized by chemical total synthesis. The construct contains Btido and is driven by the strong promoter P tacM The recombinant expression vector pJYW-5-Btido that controls its expression, wherein the expression vector pJYW-5 has been disclosed in the patent with publication number CN103834679B. The recombinant plasmid was transformed into the L-isoleucine producing strain SN01 (published in the paper of ApplMicrobiol Biotechnol, 2015, 99(9):3851-3863) to obtain the recombinant engineering strain SN02.

[0025] Press bacterial strain SN02 according to final OD 562 The inoculum size of 1.8 is inoculated in the fermentation medium, wherein, the fermentation medium composition is: glucose 140g / L, (NH 4 ) 2 SO 4 20g / L, corn syrup 10g / L, KH 2 PO 4 1g / L, MgSO 4 0.5g / L, FeSO 4 0.5g / L, CaCO 3...

Embodiment 2

[0027]Example 2 Constructing the bacterial strain expressing Bwido derived from Bacillus westerii

[0028] The isoleucine dioxygenase encoding gene Bwido shown in SEQ ID NO.2 was synthesized by chemical total synthesis. The construct contained Bwido and was driven by the strong promoter P tacM The recombinant expression vector pJYW-5-Bwido, which controls its expression, transforms the recombinant plasmid into Corynebacterium glutamicum SN01 to obtain the recombinant engineering strain SZ01.

[0029] Fermentation was carried out under the same fermentation conditions as in Example 1, and the results showed that under the same fermentation time, the output of 4-HIL synthesized by SZ01 fermentation was 38.42mM (5.65g / L), the total amount of 4-HIL and L-isoleucine is 78.45mM.

Embodiment 3

[0030] Example 3 Construction of a strain that co-expresses Btido and mqo and knocks out aceA

[0031] The Btido shown in SEQ ID NO.1 and the mqo shown in SEQ ID NO.3 were connected to the plasmid pJYW-5 to obtain the recombinant plasmid pJYW-5-Btido-mqo. The upstream homology arm fragment of aceA, the kanamycin resistance gene with loxP site, and the downstream homology arm fragment of aceA were sequentially connected to the vector pBlueScript to construct the knockout plasmid pBS-aceA. The knockout plasmid pBS-aceA was transformed into Corynebacterium glutamicum SN01 to knock out the aceA gene. Then, the plasmid pDTW109 was transformed into the aceA-knockout Corynebacterium glutamicum SN01, and the kanamycin resistance gene fragment remaining on the genome was removed. The gene knockout method and the pDTW109 plasmid sequence have been disclosed in the patent publication number CN103409446A. The recombinant plasmid pJYW-5-Btido-mqo was transformed into aceA knockout Coryne...

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Abstract

The invention discloses a genetic engineering bacterium for producing 4-hydroxyisoleucine and application thereof, and belongs to the field of genetic engineering. Two isoleucine dioxygenase encodinggenes Btido and Bwido in different species are co-expressed in an L-isoleucine producing bacterium to enhance the enzymatic activity of isoleucine dioxygenase and promote the L-isoleucine accumulatedby the bacterium to have faster hydroxylation reaction to produce more 4-hydroxyisoleucine. The yield of the 4-HIL is increased to 91.56mM, and the conversion rate of the L-isoleucine to the 4-HIL is86%. By means of fermentation medium optimization, the yield of 4-HIL of the genetic engineering bacterium is increased to 111.11 mM at the shake flask level, and the conversion rate of the L-isoleucine to the 4-HIL reaches 98%. The bacterium proves the feasibility and the advantages of co-expression of double genes, and lays a good foundation for industrial application.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for producing 4-hydroxyisoleucine and an application thereof, belonging to the field of genetic engineering. Background technique [0002] Diabetes is a metabolic disorder with a high prevalence rate. At present, more than 400 million people suffer from diabetes, and this number is still increasing. (2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) is a natural non-protein amino acid that can promote insulin secretion, reduce the resistance of peripheral tissues such as liver, muscle, and fat to insulin, and regulate blood lipids It has a good application prospect in the treatment of diabetes and its complications. At present, the main preparation method of 4-HIL is to extract from fenugreek seeds, but the yield is very low and it is difficult to meet the market demand. So since 2002, people have been looking for a chemical-enzymatic method to synthesize 4-HIL, but they have not gone further because th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
Inventor 史锋张舒萍李永富
Owner JIANGNAN UNIV
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