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Method for producing glucaric acid by constructing recombinant yeast fermentation

A technology of glucaric acid and yeast, which is applied in the field of metabolic engineering, can solve the problems of inability to produce glucaric acid, etc., and achieve the effect of cheap medium, stable genetics and high expression level

Active Publication Date: 2015-01-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is no report on the fermentation and production of glucaric acid by recombinant yeast. The original yeast strain cannot produce glucaric acid. In view of this, the new recombinant engineering bacteria constructed in the present invention provide new methods and ideas

Method used

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  • Method for producing glucaric acid by constructing recombinant yeast fermentation
  • Method for producing glucaric acid by constructing recombinant yeast fermentation
  • Method for producing glucaric acid by constructing recombinant yeast fermentation

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Construction of recombinant Pichia pastoris GS115 / Udh

[0045] Using the genome of Pseudomonas putida KT2440 as a template, udh-F (sequence shown in SEQ ID NO.7), udh-R (sequence shown in SEQ ID NO.8) as primers to amplify the Udh gene , while using the pGAPZB plasmid as a template, G-F (sequence shown in SEQ ID NO.9), G-R (sequence shown in SEQ ID NO.10) as primers to amplify the GAP promoter. Using the fusion PCR method, the GAP promoter was fused with the Udh gene, and the restriction sites SacI and NotI were respectively introduced into the upstream and downstream of the fusion fragment through primers. The fusion fragment was double digested, connected to the expression vector pPIC9K with corresponding cuts, transformed into E.coliJM109, and the recombinant expression plasmid pPIC9K-GAP-Udh was identified under the premise of ensuring the correct reading frame, and compared by DNA sequencing , the recombinant sequence is correct. The recombinant plasmid...

Embodiment 2

[0046] Example 2 Construction of recombinant bacteria Pichia pastoris GS115 / Udh-MTI

[0047] Using the Pichia pastoris GS115 genome as a template, use primers M-F (sequence shown in SEQ ID NO.11), M-R (sequence shown in SEQ ID NO.12) to amplify the MIOX gene, and primers I-F (sequence shown in SEQ ID NO. 13), I-R (sequence shown in SEQ ID NO.14) amplifies the Ino1 gene. At the same time, using the pGAPZB plasmid as a template, primers T-F (sequence shown in SEQ ID NO.15) and T-R (sequence shown in SEQ ID NO.16) were used to amplify the Pichia pastoris constitutive promoter TEF. The obtained MIOX, TEF, and Ino1 genes were fused in sequence, and two restriction sites XhoI and XbaI were added to both ends of the fusion fragment by designing primers. After double digestion, it was connected to the expression vector pGAPZB with corresponding cuts, and transformed into E.coli TOP10. Under the premise of ensuring the correct reading frame, the recombinant expression plasmid pGAPZB-M...

Embodiment 3

[0052] Example 3 Fermentative production of glucaric acid by recombinant Pichia pastoris

[0053] The recombinant strain Pichia pastoris GS115 / Udh-MTI was fermented. The single clone was inoculated in 25ml of YPD medium (yeast extract 10g / L, peptone 20g / L, glucose 20g / L), and cultured at 30°C and 200rpm for 24h. Inoculate 10% of the inoculum into 50ml (the capacity of the shaker flask is 500ml) fermentation medium for fermentation, and cultivate for 60 hours. The fermentation medium is divided into YPD medium and YPD-MI medium (60 mM inositol is added to YPD medium). 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. figure 2 It is the result of LC-MS detection of glucaric acid in the fermentation broth, wherein A is the standard sample of glucaric acid, and B is the recombinant Pichia pastoris fermentation broth. It can be seen ...

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Abstract

The invention discloses a method for producing glucaric acid by constructing recombinant yeast fermentation and belongs to the metabolic engineering field. A generation path of glucuronic acid is achieved by expressing an inositol-1-phosphate synthase gene (Inol) and an inositol oxygenase gene (MIOX) and meanwhile, aldehyde dehydrogenase (Udh) which is originated from Pseudomonas putida is expressed, so that a synthetic path of glucaric acid is successfully constructed. Recombinant pichia pastoris is used for generating 40mg / L glucaric acid in a YPD fermentation culture medium and 60mg / L glucaric acid in a YPD-MI (60mM inositol is added into the YPD culture medium) fermentation culture medium. The method for producing glucaric acid by constructing recombinant yeast fermentation has a broad development prospect, thereby laying a foundation for biosynthesis of glucaric acid.

Description

technical field [0001] The invention relates to a method for constructing recombinant yeast to ferment and produce glucaric acid, which belongs to the field of metabolic engineering. Background technique [0002] Glucuric acid (glucuric acid, saccharic acid), also known as glucuronic acid, is a sugar acid formed when the aldehyde group and C-6 hydroxyl group of glucose are oxidized to carboxylic acid (such as nitric acid). The melting point is 125-126°C, soluble in water and ethanol, slightly soluble in ether, and is an inhibitor of β-glucuronidase. [0003] Glucaric acid and its derivatives are found in some plants and mammals, especially in some cruciferous vegetables, strawberries and citrus fruits. Glucaric acid has important biological functions and has been identified as "the most valuable biorefinery product" by the U.S. Department of Energy. It can lower cholesterol and be used in the treatment of cancer, and its calcium salt is also a food additive. Glucaric acid...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/58C12R1/84C12R1/865C12R1/73
CPCC12N9/0006C12N9/0069C12N9/90C12P7/58C12Y101/01203C12Y113/99001C12Y505/01004
Inventor 康振陈坚堵国成王淼刘叶
Owner JIANGNAN UNIV
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