Genetically engineered bacterium for producing 4-hydroxyisoleucine and application of genetically engineered bacterium

A technology of hydroxyisoleucine and isoleucine, which is applied in the field of metabolic engineering, can solve the problems of long fermentation period, low yield of 4-hydroxyisoleucine and high production cost, and achieves low production cost, easy control, high yield effect

Active Publication Date: 2021-03-12
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the above needs, the precursor L-isoleucine and (or) α-ketoglutarate, low yield of 4-hydroxyisoleucine, For bottleneck problems such as long fermentation period and high pro

Method used

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  • Genetically engineered bacterium for producing 4-hydroxyisoleucine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 4-hydroxyisoleucine and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing 4-hydroxyisoleucine and application of genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Construction of 4-hydroxyisoleucine synthesis pathway in TUIE03

[0033] (1) amplification of ido gene and

[0034] Using the ido gene fragment shown in SEQ ID NO.1 as a template, primers ido-1 and ido-2 were designed to amplify the ido gene fragment, and the PCR product was subjected to agarose gel electrophoresis and recovered with a gel recovery kit.

[0035] (2) The construction of recombinant plasmid pSTV28-ido is

[0036] Plasmid pSTV28 and gene fragment ido were digested with restriction endonucleases EcoR I and Hind III respectively. Then, the linearized pSTV28 plasmid and ido fragment were recovered and purified using a gel recovery kit, and ligated with T4 ligase. The ligation product was transformed into E.coli DH5α competent cells, evenly spread on the LB solid medium plate containing 0.05mmol / L chloramphenicol after recovery, and cultured overnight at 37°C. The next day, a single colony on the plate was selected for colony PCR verification using...

Embodiment 2

[0039] Example 2: Construction of sucA weakened bacteria HIL02

[0040] (1) Construction of overlapping fragments

[0041] Using the Escherichia coli W3110 genome as a template, primers sucA-1 / sucA-2 and sucA-3 / sucA-4 were used to amplify the upstream and downstream homology arms of the sucA promoter, in which the primers contained the weak promoter BBA_J23114 sequence, and the PCR product After recovery, the fusion fragment U comprising the upper and lower homology arms of the sucA promoter and the promoter BBA_J23114 was obtained by overlapping PCR PsucA -BBA_J23114-D PsucA .

[0042] (2) Construction of pGRB-sucA plasmid

[0043] Design and synthesize gRNA 20bp forward and reverse sequences RB-1 and RB-2 according to the sucA promoter sequence. After annealing, the two are connected to the plasmid using the recombination kit ClonExpress II One Step Cloning Kit (Nanjing Nuoweizan Medical Technology Co., Ltd.) pGRB, the recombinant plasmid pGRB-sucA was obtained by transf...

Embodiment 3

[0046] Embodiment 3: Construction of 4-hydroxyisoleucine producing bacteria HIL03

[0047] The recombinant plasmid pSTV28-ido was extracted and transformed into strain HIL02 by electrotransformation. The strain HIL03 was obtained by chloramphenicol resistance plate screening and colony PCR identification.

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Abstract

The invention relates to a genetically engineered bacterium for producing 4-hydroxyisoleucine and application of the genetically engineered bacterium, and belongs to the field of metabolic engineering. According to the strain, TUIE03 is taken as a host, and expression of an alpha-ketoglutarate dehydrogenase E1 subunit encoding gene sucA is weakened while an isoleucine dioxygenase encoding gene idois overexpressed. After the strain is fermented for 24-48h, the yield of the 4-hydroxyisoleucine reaches 32.5-62.4 g/L, and the L-isoleucine is not detected in the whole fermentation process, which indicates that the L-isoleucine is completely converted. The fermentation process adopted by the method is simple, precursors L-isoleucine and alpha-ketoglutaric acid do not need to be added, and the method is easy to control, low in production cost and beneficial to popularization and application of industrial production.

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 metabolic engineering. Background technique: [0002] 4-Hydroxyisoleucine is a hydroxylated compound of L-isoleucine. 4-Hydroxyisoleucine is the main active substance in fenugreek seeds, a traditional hypoglycemic drug. 4-Hydroxyisoleucine can promote insulin secretion, improve insulin resistance, lower blood sugar, and lower blood lipids. It is useful in the treatment of diabetes Good application prospects. The main production methods of 4-hydroxyisoleucine are extraction, chemical synthesis, enzyme catalysis and microbial fermentation. At present, industrial production mainly adopts the extraction method of fenugreek seeds. However, 6.7kg of fenugreek seeds are needed to extract 1.0g of 4-HIL, which leads to low yield, high cost of raw materials and complicated process. Chemical synthesis and enzymati...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/113C12P13/06C12R1/19
CPCC12N9/0008C12N9/0069C12P13/06C12Y102/04002C12Y113/11052
Inventor 张成林韩世宝郑颖楠林蓓蓓徐皓然李洋陈宁
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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