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Engineering strain for efficient biosynthesis of glucaric acid (GA) and application of engineering strain

A technology of glucaric acid and engineering strains, which is applied in the field of engineering strains for efficient biosynthesis of glucaric acid, can solve the problems of difficulty in obtaining high-yield glucaric acid, long metabolic pathways, and low glucaric acid yield, etc., and achieves the concentration of the product. The conversion rate of the reaction is improved, the conversion route is short, and the production cost is low.

Inactive Publication Date: 2020-09-11
UNIV OF JINAN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above have successfully obtained glucaric acid, but in the construction of recombinant bacteria, inositol oxygenase MIOX is used, which is the rate-limiting enzyme for the production of glucaric acid, which leads to the accumulation of inositol and the low production of glucaric acid , and the metabolic pathway is too long, all of which require two to three steps of oxidation-reduction reactions, making it difficult to obtain high-yield glucaric acid. Therefore, how to simplify the synthesis process of glucaric acid and increase its yield has become an important issue in the industrial production process of glucaric acid. important research direction

Method used

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  • Engineering strain for efficient biosynthesis of glucaric acid (GA) and application of engineering strain
  • Engineering strain for efficient biosynthesis of glucaric acid (GA) and application of engineering strain
  • Engineering strain for efficient biosynthesis of glucaric acid (GA) and application of engineering strain

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Embodiment 1

[0031] Embodiment 1: Construction of recombinant escherichia coli

[0032] Udh genomes derived from Pseudomonas synxantha strain KENGFT3 (Pseudomonas synxantha strain KENGFT3) and Pseudomonas fragilis P121 (Pseudomonas fragi strain P121) were respectively used as templates to amplify glucuronate dehydrogenase (Udh) primers F1, R1, F2, R2, primers are as follows:

[0033] F1: 5'-ATGCGTCCGGCGTAGA-3'

[0034] R1: 5'-GATTATGCGGCCGTGTACAA-3'

[0035] F2: 5'-TTGTACACGGCCGCATAATC-3'

[0036] R2: 5'GCTAGTTATTGCTCAGCGG3'

[0037] Introduce the Udh gene into the pETDuet-1 vector, connect it through the restriction site, construct the recombinant plasmid pETDuet-2×Udh, and transform the expression vector into the host strain E.coli BL21(DE3); spread / streak on the plate , select the positive clones to extract the plasmid, and verify it by enzyme digestion and electrophoresis. The verification is correct and sent to Shanghai Shining Molecular Biotechnology Co., Ltd. for sequencing. The...

Embodiment 2

[0038] Embodiment 2: Recombinant escherichia coli produces glucaric acid

[0039] Pick a single colony from the LB plate containing ampicillin antibiotic resistance, inoculate it into a 25mL test tube containing 5mL medium, and cultivate it at 37°C and 220rpm for 12h; transfer the cultured seed solution to In a 250mL Erlenmeyer flask with 50mL LB medium, culture at 37°C and 220rpm, when OD 600 When the value was 1.0, 0.1mM TPTG was added for induction, and cultured at 32°C and 220rpm for 18h; after the cultivation, 1mL of fermentation broth was taken and centrifuged at 12000rpm for 5min. Take 0.9 mL supernatant, add 50 mg Affi-Gel for treatment, and filter the treated liquid through a 0.22 μm filter membrane for mass spectrometry and liquid phase analysis.

[0040] Such as Figure 1-4 as shown, figure 1 , image 3 They are the mass spectrogram of the glucaric acid standard and the liquid chromatogram of the glucaric acid standard respectively, and the [M-1] of the glucaric...

Embodiment 3

[0041] Embodiment 3: the method that recombinant escherichia coli prepares glucaric acid

[0042] Recombinant Escherichia coli was inoculated on the LB solid medium, and the medium composition was (g / L): tryptone 10, yeast extract 5, sodium chloride 10, agar 15, pH 7.0, and the concentration of ampicillin finally reached 100 μg / L. mL, incubate at 37°C for 16h; pick a single colony and inoculate it on LB slant medium, and incubate at 37°C for 24h; take a loop and inoculate in a 25mL test tube containing 5mL LB liquid medium, at 36.5°C, 220rpm , shaker culture 12h;

[0043] Inoculate the bacterial liquid in the test tube into a 250mL shake flask containing 50mL LB medium at an inoculum amount of 1%, the concentration of ampicillin finally reaches 100μg / mL, the pH is 6.9, and culture on a shaking table at 36°C and 220rpm 2-3h, to OD 600 When the value reaches 0.6, add IPTG to make the final concentration reach 0.4mM, and culture on a shaker at 32°C and 220rpm for 24h;

[0044]...

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Abstract

The invention provides an engineering strain for efficient biosynthesis of glucaric acid (GA) and an application of the engineering strain. The method successfully constructs a synthesis path from glucuronic acid to GA by expressing Udh derived from Pseudomonas synxantha strain KENGFT3 and Pseudomonas fragi strain P121 in Escherichia coli; recombinant Escherichia coli is subjected to cell transparent treatment, and when the cell concentration is 20 g / L, the content of GA reaches 975.5 mg / L+ / -0.15. The recombinant strain and a transformation process provided by the present invention have greatadvantages in the preparation of the GA, and have the potential for industrial application.

Description

technical field [0001] The invention relates to the technical field of metabolic engineering, in particular to an engineering strain for efficiently biosynthesizing glucaric acid and its application. Background technique [0002] Glucaric acid (Glucaric Acid, GA) is a glucose derivative containing 4 chiral carbon atoms, usually in the form of chiral compound D-glucaric acid, which will spontaneously oxidize in aqueous solution, usually in the form of lactone exist. [0003] Glucaric acid naturally exists in fruits such as apples and citrus and cruciferous vegetables such as broccoli and cabbage, and is also secreted in a small number of mammals and humans (Chen N, Wang J, Zhao Y, et al. Microbial CellFactories (2018) 17:67-80). Studies have shown that glucuronic acid and its derivatives, gluconic acid 1,4-lactone (DSL) can reduce the content of cholesterol in the human body and regulate the content of certain hormones in the body, which helps to improve the autoimmune mech...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/70C12N1/38C12P7/58C12R1/19
CPCC12N1/38C12N9/0006C12N15/70C12P7/58C12Y101/01203
Inventor 叶春江牛林林董启圣赵晓畅
Owner UNIV OF JINAN
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