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Method for producing glucaric acid by improving yeast fermentation by use of fusion expression

A glucaric acid, fusion expression technology, applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., to reduce competition, reduce degradation, and increase local concentration

Inactive Publication Date: 2015-09-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for better control over how much sugar (glucon) produced from an organism's metabolite called citrate through its own pathway. It involves creating new strains that have improved ability to produce certain sugars such as ribose-5-phosphoriblate-2-semialdehydoxy-4-(methylthymidine)-dihydropyrim iduronium ion). These improvements make it easier than previous methods to achieve higher yields of these compounds while reducing their impact on others involved in the chemical synthesis processes.

Problems solved by technology

Technologies related to producing gluarsulfonic acids with various applications include use in medicine and chemistry industries. These compounds require better ways than current commercial processes due to their unique properties including low cost, ease of manufacturing, safety concerns associated therewith, ability to absorb water vapor when dissolved during respiration, etc., which makes them ideal candidates for medical purposes like treatments on heart disease. Additionally, these materials may find potential benefits in cosmetic industry where they could help improve skin health without causing harmful side effects.

Method used

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  • Method for producing glucaric acid by improving yeast fermentation by use of fusion expression
  • Method for producing glucaric acid by improving yeast fermentation by use of fusion expression
  • Method for producing glucaric acid by improving yeast fermentation by use of fusion expression

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Construction of recombinant Pichia pastoris P.pastoris GS115-(Udh-MIOX)

[0040] Using the pGAPZB plasmid as a template, using G-U-1-F and G-U-1-R as primers to amplify the GAP promoter, using the genome of Pseudomonas putida KT2440 as a template, and using G-U-2-F and U-M-1R as The primers were used to amplify the udh gene, and the pUC57-MIOX plasmid was used as a template, and the miox gene was amplified with U-M-2F and U-M-2R as primers. Using the fusion PCR method, the GAP promoter, udh gene, and miox gene were fused, 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, and transformed into E.coli JM109, and the recombinant expression plasmid pPIC9KGAP-(Udh-MIOX) was identified under the premise of ensuring the correct reading frame. Sequence comparison, the rec...

Embodiment 2

[0041] Example 2 Construction of fusion recombinant Pichia pastoris containing different connecting peptides

[0042] Use the pGAPZB plasmid as a template, use G-U-1-F, G-U-1-R as primers to amplify the GAP promoter, use the genome of Pseudomonas putidaKT2440 as a template, and use G-U-2-F as a forward primer , U-G1-M-1R, U-G2-M-1R, U-G3-M-1R, U-E1-M-1R, U-E2-M-1R, U-E3-M-1R , U-P2-M-1R, U-P4-M-1R, U-P7-M-1R, and U-G2-M-1R were used as reverse primers to amplify the udh gene, and the pUC57-MIOX plasmid was used as a template, U-G1-M-2F, U-G2-M-2F, U-G3-M-2F, U-E1-M-2F, U-E2-M-2F, U-E3-M-2F, U-P2-M-2F, U-P4-M-2F, U-P7-M-2F, U-S3N10-M-2F were used as forward primers, and U-M-2R was used as reverse primer to amplify miox gene. Different connecting peptides were introduced by designing the downstream primers of Udh and the upstream primers of MIOX. Using the fusion PCR method, the GAP promoter, udh gene, and miox gene were fused, and the restriction sites SacI and NotI were res...

Embodiment 3

[0047] Example 3 Different Fusion Modes Recombinant Pichia Pastoris Shake Flask Fermentation Production of Glucaric Acid

[0048] Different recombinant bacteria were cultured in shake flask fermentation. 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 to 50ml (shaking flask capacity is 500ml) fermentation medium YPD-MI medium (add 10g / L inositol in YPD medium) by 10% inoculum amount to ferment, cultivate 96 hours. After the cultivation, take 1ml of the fermentation broth and centrifuge at 8000rpm for 5min, take the supernatant and treat it with Affi-Gel gel to remove most of the impurities in the fermentation broth, filter the treated sample through a 0.22um filter membrane, pass through Product was detected by HPLC. figure 1 It is the case of using different fusion methods to recombine Pichia pastoris to produce glucaric acid, wherein the control strain is a strain ...

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Abstract

The invention discloses a method for producing glucaric acid by improving yeast fermentation by use of fusion expression and belongs to the field of metabolic engineering. According to the method, a myoinositol oxygenase (MIOX) and aldehyde acid dehydrogenase (Udh) are fused and then cloned and expressed in a yeast strain by virtue of direct fusion and addition of different connecting peptides, and therefore, the synthesis way of the glucaric acid is strengthened. By comparison, the yield of the Pichia pastoris GS115-Udh-(GS)1-MIOX is the highest; during shake-flask fermentation culture, 944.67mg/L of glucaric acid is generated in a YPD-MI (adding 10g/L myoinositol to a YPD culture medium) culture medium. During 3L fermentation tank culture, the yield of the glucaric acid can be 6.02g/L. According to the method, the policy of the metabolic engineering is adopted and a microbiological strain is modified to synthesize the target product glucaric acid; and as a result, a firm foundation is laid for efficient production of the glucaric acid by use of the biological method.

Description

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Claims

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Application Information

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Owner JIANGNAN UNIV
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