Biosynthesis method of glucuronic acid and glucuric acid

A technology of glucuronic acid and aldol dehydrogenase, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effect of efficient production

Active Publication Date: 2015-01-28
江苏弘康未来营养科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the inositol oxygenase gene (MIOX) derived from Pichia pastoris is rarely reported

Method used

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  • Biosynthesis method of glucuronic acid and glucuric acid
  • Biosynthesis method of glucuronic acid and glucuric acid
  • Biosynthesis method of glucuronic acid and glucuric acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The construction of embodiment 1 recombinant escherichia coli

[0027] Using the genomes of Pichia pastoris GS115 and Pseudomonas putida KT2440 as templates respectively, MIOX and Udh were obtained, and the primer for amplifying the MIOX gene was F1 (the nucleotide sequence is shown in SEQ ID NO.5 ), R1 (nucleotide sequence as shown in SEQ ID NO.6), the primers for amplifying the Udh gene are F2 (nucleotide sequence as shown in SEQ ID NO.7), R2 (nucleotide sequence as shown in SEQ ID NO.7) shown in NO.8). The primers are as follows (the underlined part is the primer restriction site):

[0028] MIOX

[0029] F1: CGC GGATCC GTAAAAGGAGAAAAAAAATGTCAGTACAAAAAAAGCACGAAG

[0030] R1: CCG GAATTC TCAAAACTTTACCAGCTTTTGAGG

[0031] Udh

[0032] F2: GGAATTC CATATG ACCACTACCCCCCTTCAATC

[0033] R2: CCG CTCGAG TTAGTTGAACGGGCCGGCCACGGC

[0034] Digest the fragment containing the MIOX gene with the corresponding restriction endonuclease, and connect it with the expressio...

Embodiment 2

[0036]Embodiment 2 Recombinant Escherichia coli fermentation produces glucuronic acid and glucaric acid

[0037] Pick a single colony from the LB plate containing Kana antibiotic resistance and inoculate it into a Erlenmeyer flask for culture. The liquid volume is 25mL / 250mL, and cultured at 37°C and 200r / min for 10-14h. Transfer the cultivated seed culture solution to a 500mL Erlenmeyer flask containing 50mL of inositol-containing fermentation medium according to the inoculum amount of 1%, and cultivate at 37°C and 200rmin-1. The bacterium grew to OD600=0.6, and was induced by adding 0.1 mM IPTG, and at the same time adjusted the temperature to 30° C., and continued to cultivate for 48 h. After the cultivation, 1ml of the fermentation broth was centrifuged at 8000rpm for 10min, and the supernatant was filtered through a 0.22um filter membrane, and the product was detected by LC-MS. Such as Figure 1-4 as shown, figure 1 , figure 2 They are the LC-MS detection charts of g...

Embodiment 3

[0038] Embodiment 3 recombinant escherichia coli fermentation produces the method for glucuronic acid or glucaric acid

[0039] 1. Strains: E.coli BL21(DE3) / pRSFD-MIOX (for the synthesis of glucuronic acid) and E.coli BL21(DE3) / pRSFD-MIOX-Udh (for the synthesis of glucuronic acid)

[0040] 2. Culture medium:

[0041] Seed medium: LB medium (peptone 10g / L, sodium chloride 10g / L yeast powder 5g / L)

[0042] Fermentation medium: LB-MI medium (60 mM inositol was added to LB medium).

[0043] If necessary, add Kan50 mg / L of Kanna antibiotics to the culture medium.

[0044] 3. Training conditions:

[0045] (1) Seed culture: Pick a single colony from the LB plate containing Kana antibiotic resistance and inoculate it into a Erlenmeyer flask for culture. The liquid volume is 25mL / 250mL, and cultured at 37°C and 200r / min for 10-14h.

[0046] (2) Shake flask fermentation culture: Transfer the cultivated seed culture solution to a 500mL Erlenmeyer flask containing 50mL fermentation me...

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Abstract

The invention discloses a biosynthesis method of glucuronic acid and glucuric acid, belonging to the field of metabolic engineering. The biosynthesis method is characterized by expressing myo inositol oxygenase (MIOX) from pichia pastoris in Escherichia coli to convert myo inositol into glucuronic acid, and expressing aldehyde acid dehydrogenase (Udh) from pseudomonas putida to convert produced glucuronic acid into glucuric acid, thereby successfully establishing a synthesis path from myo inositol to glucuric acid. Recombinant Escherichia coli established by the method has very great application prospect, and a new idea is provided for generating glucuronic acid and glucuric acid through conversion by the biological method.

Description

technical field [0001] The invention relates to a method for biosynthesizing glucuronic acid and glucaric acid, belonging to the field of metabolic engineering. Background technique [0002] Glucuronic acid, the full name of D-(+)-glucuronic acid (D-glucuronic acid), glucuronic acid widely exists in animals and plants, and has important biological functions. It can combine toxic substances containing hydroxyl, amino, carboxyl, sulfhydryl and other groups, enhance the water solubility of toxic substances, make them quickly excreted from the kidneys, and play a detoxification role. At the same time, glucuronic acid can combine with the active group-phenolic hydroxyl group on the conjugated molecule, reducing the biological effects of related hormones and drugs. Its derivative glucuronolactone is also a kind of health care medicine, which is beneficial to the detoxification of the liver, and can prevent and treat epidemic hepatitis, liver cirrhosis, food and drug poisoning, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N1/21C12P19/02C12P7/44C12R1/19
CPCC12N9/0069C12P7/58C12P19/02C12Y113/99001
Inventor 康振陈坚堵国成王淼刘叶
Owner 江苏弘康未来营养科技有限公司
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