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Method for producing glucaric acid by constructing recombinant saccharomyces cerevisiae and fermenting

A technology for recombining Saccharomyces cerevisiae and Saccharomyces cerevisiae, applied in the field of bioengineering, can solve problems such as MIOX activity decline and MIOX instability, and achieve high conversion rate, easy separation, and suitable for large-scale fermentation

Active Publication Date: 2017-09-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is the instability of MIOX, during the stable phase, the activity of MIOX will decline rapidly even in the absence of glucaric acid
However, there is no relevant report on the preparation of glucaric acid by yeast metabolic engineering.

Method used

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  • Method for producing glucaric acid by constructing recombinant saccharomyces cerevisiae and fermenting
  • Method for producing glucaric acid by constructing recombinant saccharomyces cerevisiae and fermenting
  • Method for producing glucaric acid by constructing recombinant saccharomyces cerevisiae and fermenting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Obtainment of BY4741opilΔSaccharomyces cerevisiae strain.

[0029] Using the pUG6 plasmid as a template, the primers PUG 6F and PUG 6R amplify the knockout frame (including the loxp-kan-loxp component), and the knockout frame has 50 bp bases in the upstream and downstream of the OPI1 gene as homology arms. Transform Saccharomyces cerevisiae BY4741 competent with lithium acetate chemical transformation method, spread the G418 resistance plate, and grow a single colony after 2-3 days. Use primers OPI-1 and OPI-4 to verify the correct bands by colony PCR, culture the correct strain in shake flasks to the logarithmic phase, and extract the genome for verification. This strain was used as a competent strain to transform the pSH65 plasmid, and galactose induced Cre to excise Kan. After verifying that the excision was correct, it was continuously passaged in YPD liquid medium to lose the plasmid pSH65, and the strain BY4741opi1Δ that knocked out the OPI1 gene was obt...

Embodiment 2

[0031] Example 2 Construction of recombinant strain BY4741opi1Δ-INO1-INM1

[0032] Select the constitutive plasmid pY26-GPD-TEF plasmid, use SmaI to single-digest the plasmid, use Gibson Assembly to assemble and construct pY26-INO1 plasmid; then use EcoRI to single-enzyme plasmid pY26-INO1, use Gibson Assembly to construct pY26-INO1- INM1 plasmid. Positive clones were verified using verification primers pY26F and pY26R, and the results were judged by the size of the electrophoresis band.

[0033] Plasmids Pmri-21 and pY26-INO1-INM1 were digested with BglII and SalI respectively, the Pmri21 plasmid and INO1-GPD-TEF-INM1 fragments were recovered by gel electrophoresis, and the corresponding cuts were connected with T4 ligase to obtain the recombinant plasmid Pmri-GPD- TEF-INO1-INM1, use BlpI single-digestion plasmid to linearize it, transform Saccharomyces cerevisiae BY4741opi1Δ, spread G418 resistance plate, and grow a single colony after 2-3 days. Colony PCR with primers INO...

Embodiment 3

[0034] Example 3 Construction of recombinant bacterial group BY4741opi1Δ-INO1-INM / MIOX-UDH

[0035] Fragment MIOX4 is from Arabidopsis thaliana, codon-optimized, as shown in SEQ ID NO:3, and inserted into pUC57 plasmid restriction site BglII / NotI to obtain pUC57-MIOX4. Fragment UDH is from Pseudomonas putida. After codon optimization, as shown in SEQ ID NO:4, it was inserted into EcoRI and SacII of pUC57-MIOX4 to obtain pUC57-MIOX4-UDH. Using pUC57-MIOX4-UDH as a template, using pY26-MIOX4F / pY26-MIOX4R and pY26-UDHF / pY26-UDHR as primers to amplify the MIOX4 and UDH genes respectively, purified by PCR product purification kit, and cut pY26 with SmaI -GPD-TEF plasmid, the fragments were recovered by the gel kit, and the pY26-UDH plasmid was assembled by GibsonAssembly; then the plasmid pY26-UDH plasmid was digested with EcoRI, and the pY26-UDH–MIOX4 plasmid was assembled by GibsonAssembly, and the primers were verified as pY26F and pY26R. Transform Saccharomyces cerevisiae BY4...

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Abstract

The invention discloses a method for producing glucaric acid by constructing recombinant saccharomyces cerevisiae and fermenting and belongs to the technical field of bioengineering. The method comprises the following steps: knocking out an OPI gene of saccharomyces cerevisiae BY4741; introducing myo inositol-1-phosphate synthase and inositol monophosphatase from the saccharomyces cerevisiae, inositol from arabidopsis thaliana and uronic acid dehydrogenase from pseudomonas syringae into the saccharomyces cerevisiae through constitutive plasmid pY26-GPD-TEF, and realizing approach construction of the saccharomyces cerevisiae from glucose to the glucaric acid. According to the method disclosed by the invention, by use of a constructed metabolic pathway, non-selectivity, high energy consumption property and high cost performance of a chemical oxidation method are avoided; the low-priced glucose is directly transformed into D-glucaric acid with high added value by fermentation culture of engineering bacteria; the method has the advantages of mild reaction conditions, high transformation rate and fewer byproducts; production cost can be greatly reduced.

Description

technical field [0001] The invention relates to a method for constructing recombinant Saccharomyces cerevisiae to ferment and produce glucaric acid, which 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. It has a wide range of applications in medicine and industry, such as lowering cholesterol, treating diabetes, tumor treatment, etc., or may be used as Polymer precursors, including novel nylons and highly branched polyesters. There have been reports on the use of glucaric acid produced from glucose to prepare hydroxylated nylon, which is a biodegradable fiber. In a report written by Pacific Northwest National Laboratory, National Renewable Energy Laboratory, and the U.S. Department of Energy, glucaric acid was considered a "top value-added chemical from biomass". Due to the importance of this organic acid, it is getting more and more attention from ac...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P7/58C12R1/865
CPCC12N9/0006C12N9/0069C12N9/16C12N9/90C12N15/81C12P7/58C12Y101/01203C12Y113/99001C12Y301/03072
Inventor 邓禹陈娜张晓娟毛银
Owner JIANGNAN UNIV
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