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Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain

A technology for glucaric acid and Saccharomyces cerevisiae is applied in the field of improving the efficiency of synthesizing glucaric acid by engineering strains of Saccharomyces cerevisiae, which can solve the problems of unbalanced flow of host cells, lack of natural metabolism regulation mechanism of host cells in heterologous proteins, etc., and achieve stable expression. Effect

Active Publication Date: 2020-04-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When pathways constructed by metabolic engineering strategies contain heterologous enzymes, there is an imbalance in the host cell flux because the heterologous protein lacks the host cell's natural metabolic regulation mechanism

Method used

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  • Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain
  • Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain
  • Method for improving efficiency of synthesizing glucaric acid by saccharomyces cerevisiae engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Obtaining of MIOX4-linker-udh integrated fragment

[0029] Using the genome of Bga-001 (disclosed in a patent application with publication number CN108220176A: a method for improving the fermentative production of glucaric acid by Saccharomyces cerevisiae engineering strains) as a template, first use MIOX4-LF / MIOX4-LR as primers to amplify PTEF1-MIOX4 fragment; then amplified with UDH-F (0-12) and UDH-R without linker, and contains the following 12 different linkers: GSG, GSGGGGS, GSGEAAAK, GSGEAAAKEAAAK, SGMGSSSN, PTPTP, PTPTPTPTP, PTPTPTPTPTPTPTP , EAAAAK, EAAAAKEAAAAAK, EAAAAKEAAAAKEAAAAK, and UDH-TCYC1 fragments of SSSNNNNNNNNNN, and then use MIOX4-LF and UDH-R to perform fusion PCR on the above two fragments to obtain PTEF1-MIOX4-UDH-TCYC1 DNA fragments connected by 12 different linkers ( figure 1 and figure 2 ).

[0030]Then with the genome of Bga-001 as a template, the HIS3 fragment shown in SEQID NO.5 (containing the upstream homologous sequence o...

Embodiment 2

[0031] Embodiment 2: Detection of MIOX4-linker-udh integrated bacterial strain

[0032] Extract the genome of the yeast transformant obtained by the above method, use MD-Check-(opi1)F / MD-Check-(opi1)R as primers to amplify the extracted genome, and verify whether the MIOX-UDH1 fusion protein is integrated into the BY4741opi1Δ strain genome, image 3 It can be explained that there are MIOX and UDH fusion proteins on the genome of the verified strains, and finally the correct integrated strains were obtained, which were named Bga-L1-Bga-L13 respectively.

Embodiment 3

[0033] Example 3: Fermentative synthesis of glucaric acid by fusion expression engineered bacteria

[0034] 1. Preparation of seed solution: Streak the bacteria preserved in glycerol on the plate, pick a single colony and inoculate it in 10 mL of YPD medium, culture at 30°C, 250 rpm, and shaker for 24 hours to obtain the seed solution.

[0035] 2. Fermentation conditions: Inoculate the seed liquid into a 250mL Erlenmeyer flask containing 50mL liquid medium according to the inoculum amount of 5%, the glucose concentration in the fermentation medium is 20g / L, and add 10.8g / L inositol . Cultivate for 240 hours at 30° C. with a shaker rotation speed of 250 rpm.

[0036] 3. Product detection: Take 1 mL of fermentation broth, centrifuge at high speed for 10 minutes, retain the supernatant and filter it with a 0.22 μm filter membrane, and the filtrate is the sample. It can be detected by liquid chromatography-mass spectrometry (LC-MS), or detected by high performance liquid chromat...

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Abstract

The invention, which belongs to the technical field of bioengineering, discloses a method for improving the efficiency of synthesizing glucaric acid by a saccharomyces cerevisiae engineering strain. An opi1 deletion strain under the background of saccharomyces cerevisiae BY4741 is used as an original strain, myoinositol oxygenase MIOX4 derived from Arabidopsis thaliana and uronic acid dehydrogenase udh derived from pseudomonas syringae are linked through different linkers; and then integration to genome of a saccharomyces cerevisiae opi1 deletion strain is performed. After fusion expression, under the same fermentation condition, the maximum yield of the Bga-L12 engineering bacteria reaches 1.45 g / L, and the yield of the Bga-L12 engineering bacteria is increased by about 5 times by being compared with that of saccharomyces cerevisiae engineering bacteria which are not subjected to MIOX4 and udh fusion expression. And a foundation is laid for further metabolic transformation of the glucaric acid engineering bacteria.

Description

technical field [0001] The invention relates to a method for improving the efficiency of glucaric acid synthesis by engineering strains of Saccharomyces cerevisiae, belonging to the technical field of bioengineering. Background technique [0002] Glucaric acid is a dibasic acid formed by oxidizing the aldehyde group and C6 hydroxyl group of glucose to carboxyl group. Glucaric acid exists in nature, such as cruciferous vegetables (cauliflower, etc.), fruits (such as citrus, etc.) and mammalian metabolites are found in glucaric acid. Glucaric acid and its derivatives have important applications in medicine. Since glucaric acid and its lactones can effectively inhibit the activity of β-glucuronidase, they become good anticancer substances. Moreover, glucaric acid also has the effect of lowering cholesterol; glucaric acid-1,4-lactone has antioxidant effect and can inhibit diabetes. Studies have found that glucaric acid can form polyamide through polycondensation reaction, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/58C12R1/865
CPCC12N9/0006C12N9/0069C12P7/58C12Y101/01203C12Y113/99001
Inventor 邓禹赵运英李杰李国辉毛银周胜虎
Owner JIANGNAN UNIV
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