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Inositol oxidase mutant, as well as coding gene and application thereof

A technology of inositol oxidase and coding gene, which is applied in the field of inositol oxidase mutant and its coding gene and application, and can solve the problem that GA production cannot be further improved

Active Publication Date: 2018-05-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have found that when the GA synthesis pathway takes Escherichia coli as a host, when the GA production is higher than 5g / L, due to the presence of pH-mediated acid toxicity accompanying the production process (Moon, T.S., et al., Use of modular,synthetic scaffolds for improvedproduction of glucaric acid in engineered E.coli.Metabolic Engineering,2010.12(3):p.298-305.), GA production cannot be further improved, which makes it necessary to replace the host

Method used

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  • Inositol oxidase mutant, as well as coding gene and application thereof
  • Inositol oxidase mutant, as well as coding gene and application thereof
  • Inositol oxidase mutant, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 The acquisition of mutant inositol oxidase

[0034] Using the plasmid pACYCDuet-MIOX-Udh as a template, using error-prone PCR technology (outsourced to Nanjing GenScript Company), the random mutation library of MIOX was obtained, and then the library was electrotransformed (2500V, 6ms) into Escherichia coli BL21 (DE3), in Cultured on LB plates containing chloramphenicol (34 μg / mL) for 12 hours, and positive transformants were screened. Using GA biosensor (Rogers, J.K. and G.M. Church, Genetically encoded sensors enable real-time observation of metabolite production. Proceedings of the National Academy of Sciences of the United States of America, 2016.113(9): p.2388-2393.) to participate The strains with higher GA production were screened out by the one-pot dual-bacteria method, and the GA production was measured by LC / MS. The three strains with the highest GA production were selected for sequencing. Compared with the wild-type inositol oxidase (NP_064361.2)...

Embodiment 2

[0040] Embodiment 2 Contains the construction of the host cell of recombinant plasmid vector

[0041] According to the two amino acid residue mutation sites detected by sequencing in Example 1, using the wild-type inositol oxidase coding gene (Genebank: NM_019977) as a template, the 244th nucleotide was mutated from G to T by site-directed mutagenesis , The 518th nucleotide is mutated from G to A, and the SUMO-MIOX (D82Y / S173N) gene sequence of the double-site mutation is obtained, and it is recombined into the NdeI and the MCS2 of the plasmid vector pACYCDuet-1 and pACYCDuet-MIOX-Udh Between the XhoI restriction sites, the recombinant plasmid vector pACYCDuet-MIOX(D82Y / S173N) was obtained (see figure 1 ) and pACYCDuet-MIOX(D82Y / S173N)-Udh, the plasmid vector pACYCDuet-MIOX and the above-mentioned original plasmid vector and recombinant plasmid vector were respectively transformed into Escherichia coli BL21 (DE3) competent cells (outsourced to Nanjing GenScript Company), Esch...

Embodiment 3

[0044] Enzyme digestion and electrophoresis of the recombinant plasmid vector of embodiment 3

[0045] The Escherichia coli BL21 (DE3) bacteria liquid containing pACYCDuet-1 and pACYCDuet-MIOX (D82Y / S173N) prepared in Example 2 were respectively inoculated in the chloramphenicol concentration of 34 μg / mL at a volume ratio of 1:500. In LB liquid medium, activate overnight at 37°C and 225rpm for 12h, use a plasmid extraction kit Plasmid Mini Kit I (purchased from OMEGA Bio-tek) and follow the instructions of the plasmid extraction kit to extract the plasmid in the bacterial liquid.

[0046] The empty plasmid pACYCDuet-1 and recombinant plasmid pACYCDuet-MIOX (D82Y / S173N) extracted above were digested with NdeI and XhoI (purchased from Thermo Scientific), and subjected to agarose gel electrophoresis. The electrophoresis results are shown in figure 2 .

[0047] Among them, the double digestion 20μL reaction system: plasmid, 200ng; endonuclease XhoI, 1μL; endonuclease NdeI, 1μL; ...

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Abstract

The invention relates to an inositol oxidase mutant, as well as a coding gene and application thereof, belonging to the technical field of gene engineering. According to the inositol oxidase mutant, plasmids pACYCDuet-MIOX-Udh containing wild inositol oxidase coding genes are adopted as a template, an error PCR technology is used to obtain an inositol oxidase MIOX random mutation library; a high GA yield inositol oxidase mutant can be obtained by high throughput screening of biosensor, and the inositol oxidase mutant has relatively high catalytic activity and can be applied to the fields of enzyme engineering, gene engineering, metabolic engineering and the like.

Description

technical field [0001] The invention relates to an inositol oxidase mutant, its coding gene and its application, and belongs to the technical field of genetic engineering. Background technique [0002] Glucaric acid (Glucaric acid, GA) is a six-carbon monosaccharide derivative, which is widely found in vegetables, fruits and other plants, as well as a few mammals. Glucaric acid is also used in a wide variety of applications, from dietary supplements such as calcium D-gluconate, to cholesterol-lowering and cancer-fighting drug research. GA is classified by the U.S. Department of Energy as one of the 30 compounds with the highest value return, not only because of its own use, but also because of its many higher value derivatives, such as nylon, plastic and food Additives (Werpy, T., and G. Petersen. 2004. Top. value added chemicals from biomass, volume 1—results of screening for potential candidates from sugars and synthesis gas. U.S. De-partment of Energy, Washington, DC. ht...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/81C12N1/21C12N1/19C12P19/02C12P7/58C12R1/21C12R1/865
CPCC12N9/0069C12P7/58C12P19/02C12Y113/99001
Inventor 生举正王凤山郑爽
Owner SHANDONG UNIV
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