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Coenzyme self-sufficient Escherichia coli as well as construction method and application thereof

A technology of Escherichia coli and coenzyme, which is applied in the application field of catalyzing and synthesizing glufosinate-ammonium, can solve problems such as insufficient concentration of coenzyme, and achieve the effects of shortening the reaction process, slowing down the short half-life and improving the space-time yield.

Pending Publication Date: 2022-01-07
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology can be used for studying enzyme networks involved with biosynthesis or other biological functions such as regulation of energy production from photosynthetic organisms. It allows researchers to study how these systems work together more effectively than before they were developed due to their ability to produce certain compounds that have important benefits on living cells.

Problems solved by technology

Technologies described in this patented technical solution involve developing new ways to produce various types of molecules called lysinsulokines, particularly those found naturally within plants. These molecular structures act like natural plant growth promoter systems through their ability to control both levels of production and concentrations of free radicals generated during photosynthetic processings without causing unwanted side effects on certain components inside living microbes.

Method used

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  • Coenzyme self-sufficient Escherichia coli as well as construction method and application thereof
  • Coenzyme self-sufficient Escherichia coli as well as construction method and application thereof
  • Coenzyme self-sufficient Escherichia coli as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1: Knockout of Coenzyme Decomposition Gene

[0049] Single or double knockout of mudC, mazG and nadR on Escherichia coli BL21 (DE3), wherein the gene knockout plasmid used in the present invention is pCasRed, pCRISPR-gDNA (sgRNA) and homology arm (donor) into Escherichia On coli BL21(DE3), Cas9 / sgRNA induces a double-strand break at the target gene locus, and the recombinase Red integrates the donor into the target gene to achieve gene knockout, which is verified by sequencing. mudC sgRNA, mudCdonor, mazG sgRNA, mazG donor, nadR sgRNA, and nadR donor are respectively shown in SEQ ID NO 16 to SEQ ID NO 21 in the sequence table. The changes in intracellular coenzyme concentration of the strains obtained by different knockout combinations are shown in Table 1.

[0050] Table 1: Coenzyme changes after gene knockout

[0051] strain NADP(H) coenzyme concentration (μmol / g) E.coli BL21(DE3) 21.4 E.coli BL21(DE3)ΔmudC 24.7 E.coli BL21(DE3)Δ...

Embodiment 2

[0053] Example 2: Construction of Escherichia coli comprising NADH kinase

[0054] In this example, five NADH kinase sequences were selected from the NCBI database through literature reports, which were derived from Corynebacterium glutamicum (CgNadK) (accession number NC_003450.3), Escherichia coli (EcNadK) (accession number NC_000913.3), Methanocaldococcus jannaschii (MjNadK) (accession number NC_000009.12), Entamoeba histolytica (EhNadK) (accession number NW_001914860.1), Saccharomycescerevisiae (ScNadK) (accession number NC_001148.4), and the whole gene synthesis after codon optimization, nucleoside The acid sequences are respectively shown in SEQ ID NO.1-NO.5.

[0055] MjNadK, EcNadK, CgNadK, EhNadK, ScNadK were respectively cloned into the first multiple cloning site of plasmid pCDFDuet-1 by one-step cloning method:

[0056] Design carrier linearization primer 1 and primer 2, with the beginning and end of the linearized vector each 10-15 bp as homologous sequences, design...

Embodiment 3

[0089] Example 3: Construction of co-expression Escherichia coli

[0090]The recombinant Escherichia coli E. coli BL21(DE3) / pETDuet-1-PPTGDHE3-GDH containing glutamate dehydrogenase and glucose dehydrogenase has been constructed in the previous work of our laboratory (patent publication number CN109609475A). E.coli BL21(DE3) / pETDuet-1-PPTGDHE3-GDH, E.coli BL21(DE3)MN / pCDFDuet-1-MjNadK, E.coli BL21(DE3)MN / pCDFDuet-1-EcNadK, E.coli BL21(DE3)MN / pCDFDuet-1-CgNadK, E.coli BL21(DE3)MN / pCDFDuet-1-EhNadK, E.coli BL21(DE3)MN / pCDFDuet-1-ScNadK, E.coli BL21(DE3)MN / pCDFDuet-1-nadE, E.coli BL21(DE3)MN / pCDFDuet-1-nadD, E.coli BL21(DE3)MN / pCDFDuet-1-pncB were respectively inoculated into LB liquid medium, cultured for 12 hours and then extracted Plasmids, pETDuet-1-PPTGDHE3-GDH, pCDFDuet-1-MjNadK, pCDFDuet-1-EcNadK, pCDFDuet-1-CgNadK, pCDFDuet-1-EhNadK, pCDFDuet-1-ScNadK, pCDFDuet-1-nadE pCDFDuet- 1-nadD, pCDFDuet-1-pncB, measure the nucleic acid concentration, take 200ng pETDuet-1-PPTGDH...

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Abstract

The invention provides coenzyme self-sufficient Escherichia coli, a construction method of the coenzyme self-sufficient Escherichia coli and application of the coenzyme self-sufficient Escherichia coli in synthesis of L-glufosinate-ammonium. According to the invention, NADH kinase and a key enzyme of a coenzyme synthetic pathway are expressed in Escherichia coli, an enzyme for decomposing the coenzyme is knocked out, and in a cell culture process, by adding a combined metabolic intermediate, a concentration of intracellular NADP (H) is increased by at least 50%, and the catalytic activity of glutamate dehydrogenase is increased by 2 times. In a process of asymmetric reductive amination of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid, the NADH kinase continues to convert intracellular NAD (H) into the NADP (H), the lowering of the activity of the glutamate dehydrogenase caused by a too short half-life period of the NADP (H) is slowed down, an asymmetric reductive amination reaction process is shortened by at least 5h, and the space time yield of the reaction is remarkably increased.

Description

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Claims

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Application Information

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Owner ZHEJIANG UNIV OF TECH
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