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Escherichia coli recombinant bacterium for synthesizing inositol by efficiently utilizing glucose as well as construction method and application of escherichia coli recombinant bacterium

A technology of Escherichia coli and recombinant bacteria, applied in the biological field, can solve the problems of low utilization rate of raw materials, environmental pollution, high cost of substrates and catalysts, etc.

Pending Publication Date: 2021-11-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has disadvantages such as harsh reaction conditions, low utilization rate of raw materials, many by-products, strict requirements on equipment, and environmental pollution.
However, the chemical synthesis method also has defects such as high substrate and catalyst costs, low yield, difficult separation and purification, and poor product performance.

Method used

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  • Escherichia coli recombinant bacterium for synthesizing inositol by efficiently utilizing glucose as well as construction method and application of escherichia coli recombinant bacterium
  • Escherichia coli recombinant bacterium for synthesizing inositol by efficiently utilizing glucose as well as construction method and application of escherichia coli recombinant bacterium
  • Escherichia coli recombinant bacterium for synthesizing inositol by efficiently utilizing glucose as well as construction method and application of escherichia coli recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1, Construction of the recombinant vector pINS co-expressing TbIPS and EcIMP

[0077] 1. Obtaining the pIPS gene fragment

[0078] Using the artificially synthesized sequence containing sequence 1 (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) as a template, PCR amplification was performed using primers P1 and P2 to obtain the PCR product, which is the coding gene sequence of inositol-3-phosphate synthase TbIPS (shown in sequence 1 in the sequence listing). The primer sequences are as follows:

[0079] P1: 5'-GCGCGGCAGCCTCGAGATGCCGGCAGTGCGTACGAA-3';

[0080] P2: 5'-CCGAGCTCACCACTAGTTTAGCTGCCTACGCCACGC-3'.

[0081] The PCR product was detected by 1% agarose gel electrophoresis, the size of the PCR product was about 1600bp, consistent with the target fragment, and named pIPS. The pIPS gene fragment was gel recovered.

[0082] 2. Construction of pBAD / HisB-TbIPS recombinant vector

[0083] After the carrier pBAD / HisB was double-digested with XhoI a...

Embodiment 2

[0091] Embodiment 2, the construction of escherichia coli mutant INS01, INS02 and INS03

[0092] Escherichia coli mutants INS01, INS02 and INS03 were specifically constructed by using the P1 phage-mediated transfection method. Taking INS01 as an example, the specific construction steps are as follows:

[0093] 1. Obtaining the P1 transfection solution of the donor bacteria

[0094] The donor bacterium BW25113Δpgi::Kan was inoculated in LB medium (the solvent was water, the solute and its concentration were as follows: 10mM MgCl 2 , 5mM CaCl 2 and 0.1g / 100mL glucose), cultivate for about 1 hour, add wild-type P1 phage, and cultivate for 1-3 hours. Add a few drops of chloroform and shake for a few minutes, centrifuge to get the supernatant to obtain phage P1virΔpgi.

[0095] 2. Using P1 phage transduction technology to construct E. coli knockout strain INS01::Kan.

[0096] Take 1.5mL of overnight cultured SG104 (recipient bacteria), centrifuge at 6000rpm for 2 minutes, then u...

Embodiment 3

[0104] Embodiment 3, the construction of Escherichia coli recombinant bacteria MI01, MI02, MI03, MI04, MI05

[0105] The expression vector pINS constructed in Example 1 was transformed into Escherichia coli BW25113 and Escherichia coli mutants SG104, INS01, INS02 and INS03 by chemical transformation, and positive clones were screened on an LB plate containing streptomycin (50 μg / mL), to obtain corresponding recombinant strains. Among them, the recombinant strain obtained by transforming Escherichia coli BW25113 with the expression vector pINS was named MI01; the recombinant strain obtained by transforming the Escherichia coli mutant SG104 with the expression vector pINS was named MI02; the recombinant strain obtained by transforming the Escherichia coli mutant INS01 with the expression vector pINS Named as MI03; the recombinant strain obtained by transforming the E. coli mutant INS02 with the expression vector pINS was named MI04; the recombinant strain obtained by transformin...

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Abstract

The invention discloses an escherichia coli recombinant bacterium for synthesizing inositol by efficiently utilizing glucose as well as a construction method and application of the escherichia coli recombinant bacterium. The construction method of the escherichia coli recombinant bacterium for producing the inositol, provided by the invention, comprises the steps: introducing an inositol-3-phosphate synthetase gene and an inositol monophosphate enzyme gene into host bacteria to obtain the escherichia coli recombinant bacteria for producing the inositol, wherein the host bacteria are wild escherichia coli or mutant escherichia coli; and the mutant escherichia coli is obtained by knocking out a glucose-6-phosphate isomerase gene or a glucophosphomutase gene or a glucose-6-phosphate dehydrogenase gene in an SG104 genome of the mutant escherichia coli. Glucose can be efficiently utilized to synthesize the inositol by utilizing the escherichia coli recombinant strain for producing the inositol, and the yield of the inositol can be up to 375 mM (67.5 g / L). For inositol production, huge economic benefits can be generated, and the method has great popularization and application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an Escherichia coli recombinant bacterium efficiently utilizing glucose to synthesize inositol and a construction method thereof, and the application of the strain in producing inositol. Background technique [0002] Inositol, also known as cyclohexyl alcohol, is a saturated cyclic polyol. Inositol is an essential substance for various cell life activities and widely exists in plants, animals and microorganisms. Inositol is a water-soluble group B vitamin, which is widely used in food, medicine, cosmetics, feed and other industries. (1) As a nutritional agent for metabolic activities in animals and humans, inositol has been added to many foods, such as functional drinks, infant milk powder, etc.; (2) Inositol participates in the metabolism of the body, and related experiments have proved that inositol It can be used to treat various diseases such as diabetes, fatty live...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/61C12N15/55C12N15/54C12N15/53C12N1/21C12P7/02C12R1/19
CPCC12N15/70C12N15/52C12N9/90C12N9/92C12N9/16C12N9/1205C12N9/0006C12P7/02C12Y505/01004C12Y301/03C12Y503/01009C12Y207/0101C12Y101/01049
Inventor 胡美荣王雷尤然张莎莎陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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