Method for producing glucaric acid by improving saccharomyces cerevisiae engineered strain fermentation

A technology of glucaric acid and Saccharomyces cerevisiae, which is applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of easy loss, unstable plasmid expression, and unsuitable for industrial production.

Active Publication Date: 2018-06-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the instability and easy loss of plasmid expression in Saccharomyces cerevisiae, this type of genetically engineered bacteria is not suitable for actual industrial production

Method used

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  • Method for producing glucaric acid by improving saccharomyces cerevisiae engineered strain fermentation
  • Method for producing glucaric acid by improving saccharomyces cerevisiae engineered strain fermentation
  • Method for producing glucaric acid by improving saccharomyces cerevisiae engineered strain fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of plasmid pY26-TEF-GPD-MIOX4-UDH

[0043] PUC57-MIOX4-UDH was used as template, and pY26-MIOX4F / pY26-MIOX4R and pY26-UDHF / pY26-UDHR were used as primers to amplify MIOX4 and UDH genes respectively. The recovered product of MIOX4 and the plasmid pY26-TEF-GPD were digested with BglII / NotI respectively to obtain the pY26-MIOX4 plasmid, which was purified and recovered by overnight ligation with T4 ligase to construct the plasmid pY26-TEF-GPD-MIOX4. The pY26-MIOX4-UDH plasmid will be constructed using Gibson Assembly, and then the plasmid pY26-TEF-GPD-UDH plasmid will be digested with EcoRI, and the pY26-TEF-GPD–MIOX4-UDH plasmid will be constructed using Gibson Assembly, and the primers will be veri- pYF and veri-pYR. After colony PCR verification, the plasmid was constructed successfully.

Embodiment 2

[0044] Example 2 Obtaining of integrated fragments DMH-001 and DUH-001 fragments.

[0045] Using the Saccharomyces cerevisiae BY4741 genome as a template, using Delta1F / Delta1R and Delta2F / Delta2R as primers to amplify Delta1 and Delta2 fragments respectively; ID NO.1) is a template, U-GPDF / U-GPDR and M-TEFF / M-TEFR are primers to amplify fragments U-TEF (including promoter GPD, gene UDH, terminator CYC1) and M-TEF respectively TEF (including promoter TEF, gene MIOX4, terminator ADH1); with plasmid pRS313 (preserved in this experiment, its nucleotide sequence shown in SEQ ID NO.2) as a template, primers His1F / His1F and His2F / His2R were respectively amplified Add fragments His1 and His2. The three fragments of Delta1, M-TEF and His1 were subjected to drop-down PCR, using the PCR product as a template and Delta1F / His1R as primers to amplify to obtain DMH-001. His2, U-GPD, and Delta2 were subjected to drop-down PCR, using the PCR product as a template and His2F / Delta2R as primer...

Embodiment 3

[0046] Example 3 High-yield glucaric acid integrated strain Bga-001 obtained

[0047] The above two integrated fragments DMH-001 and DUH-001 were respectively transformed into Saccharomyces cerevisiae BY4741ΔOPI (preserved in our laboratory) by lithium acetate transformation method, coated with SD-His-deficient medium, and the transformants were picked after 2-3 days. In order to integrate successfully recombinant bacteria, insert positive transformants into 10ml YPD liquid medium as seeds, cultivate overnight, transfer 50mlYPD (adding 60mM inositol in the medium) liquid medium with 2% inoculum, shake at 30°C at 250rpm Bed culture 7d. Samples were taken every 12 hours, centrifuged at 13,000 rpm for 5 minutes to obtain the supernatant, and filtered through a 0.22 μm filter membrane. The production of glucaric acid was detected by Hitachi high performance liquid chromatography with a differential detector, the column temperature was 30°C, and the injection volume was 30ul. Thu...

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Abstract

The invention discloses a method for producing glucaric acid by improving saccharomyces cerevisiae engineered strain fermentation, and belongs to the technical field of bioengineering. According to the method disclosed by the invention, Delta sites of multiple copies in a saccharomyces cerevisiae genome are used as exogenous gene module integration sites, so that one-time integration of an exogenous gene module is realized so as to fulfil the aim of expression of the multiple copies. According to the method disclosed by the invention, through an integration expression way, gene engineering bacteria subjected to the integration of genome Delta sites is passaged for 20 times; and under the same fermentation condition, the yield of the glucaric acid is still 3.8g / L, and the method has an important application meaning on industrial production of the glucaric acid.

Description

technical field [0001] The invention relates to a method for improving the fermentative production of glucaric acid by Saccharomyces cerevisiae engineering strains, and belongs to the technical field of bioengineering. Background technique [0002] D-glucaric acid (D-glucaric acid) has been widely studied as a very important organic acid, and it has a wide range of applications in medicine and industry, such as lowering cholesterol, treating diabetes, tumor treatment, etc.[1,2] , or may be used as polymer precursors, including novel nylons and highly branched polyesters [2]. There have been reports on the use of glucaric acid produced from glucose to prepare hydroxylated nylon, which is a biodegradable fiber [2]. In a report written by the Pacific Northwest National Laboratory, the National Renewable Energy Laboratory, and the U.S. Department of Energy, glucaric acid was considered the "top value-added chemical from biomass" [3]. Due to the importance of this organic acid,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/58C12R1/865
CPCC12N9/0006C12N9/0069C12N15/81C12P7/58C12Y101/01203C12Y113/99001
Inventor 邓禹陈娜张晓娟赵运英毛银
Owner JIANGNAN UNIV
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