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A kind of glufosinate-ammonium dehydrogenase mutant and its application in the production of l-glufosinate-ammonium

A glufosinate-ammonium and dehydrogenase technology, applied to the glufosinate-ammonium dehydrogenase mutant and its application in the production of L-glufosinate-ammonium, can solve the problem of low activity of asymmetric amination reduction

Active Publication Date: 2020-12-25
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of glufosinate-ammonium dehydrogenation in view of the problem that the existing glufosinate-ammonium dehydrogenase has low activity to 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid asymmetric amination reduction Enzyme mutants and using the glufosinate-ammonium dehydrogenase mutant gene recombinant bacteria and their crude enzyme solution as biocatalysts for L-glufosinate-ammonium chiral biosynthesis, the activity of the catalyst increased by 12 times, the initial substrate D,L-glufosinate concentration increased 8 times

Method used

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  • A kind of glufosinate-ammonium dehydrogenase mutant and its application in the production of l-glufosinate-ammonium
  • A kind of glufosinate-ammonium dehydrogenase mutant and its application in the production of l-glufosinate-ammonium
  • A kind of glufosinate-ammonium dehydrogenase mutant and its application in the production of l-glufosinate-ammonium

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Construction and screening of glufosinate-ammonium dehydrogenase mutant library

[0039] The glufosinate-ammonium dehydrogenase gene dygdh cloned from Pseudomonas monteilii (Pseudomonas monteilii) WP_060477601.1 1 (The nucleotide sequence is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2), the glufosinate-ammonium dehydrogenase gene dygdh obtained from Pseudomonas japonicaWP_042124798.1 clone 2 (The nucleotide sequence is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ ID No.4), the glufosinate-ammonium dehydrogenase gene dygdh obtained from Thiopseudomonas denitrificansWP_101496154 clone 3 (The nucleotide sequence is shown in SEQ ID No.5, and the amino acid sequence is shown in SEQ ID No.6), and the expression vector pETDuet-dygdh was constructed respectively 1 , pETDuet-dygdh 2 , pETDuet-dygdh 3 , keep the His-Tag gene of the vector itself, transform it into Escherichia coli E.coli BL21(DE3), and obtain the pa...

Embodiment 2

[0055] Example 2: Induced expression of glufosinate-ammonium dehydrogenase parent engineering bacteria, glufosinate-ammonium dehydrogenase mutant engineering bacteria, formate dehydrogenase engineering bacteria, D-amino acid oxidase engineering bacteria, alcohol dehydrogenase engineering bacteria

[0056] 1. Construction of engineering bacteria

[0057] Construction of D-amino acid oxidase engineering bacteria E.coli BL21(DE3) / pET24b-daao:

[0058] The D-amino acid oxidase gene sequence (GenBank: NO.XM_016418953.1, nucleotide sequence: SEQ ID No.9) derived from yeast Rhodotorula gracilis was introduced into the vector pET-24b(+) to construct the recombinant The expression vector pET-24b-daao uses E.coli BL21(DE3) as the host bacterium to construct the engineering bacterium E.coli BL21(DE3) / pET24b-daao.

[0059] Formate dehydrogenase engineering bacteria construction:

[0060] The formate dehydrogenase gene sequence (GenBank: No.WP_013726924.1, nucleotide sequence: SEQ ID No....

Embodiment 3

[0068] Example 3: Mutant library screening

[0069] The glufosinate-ammonium dehydrogenase parent and glucose dehydrogenase prepared by the method in Example 2 co-express wet thallus or glufosinate-ammonium dehydrogenase mutants and glucose dehydrogenase co-expression wet thallus as a catalyst, with intermediate The product 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid is used as the substrate, glucose is used as the coenzyme regeneration substrate, ammonium sulfate is added, no exogenous NADPH or NADP+ is added, and the endogenous NADPH of the bacteria is used. Use pH 7.4, 100mM phosphate buffer as the reaction medium to form a 10mL reaction system, the amount of catalyst is 25g / L based on the final concentration of wet bacteria, the final concentration of substrate is 300mM, the final concentration of glucose is 450mM, and the final concentration of ammonium sulfate is 750mM. React at 600 rpm for 5 minutes, take 100 μL of the reaction solution, add 5 μL of hydrochloric...

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Abstract

The invention discloses a glufosinate-ammonium dehydrogenase mutant, and application of the glufosinate-ammonium dehydrogenase mutant in producing ammonium 2-amino-4(hydroxymethylphosphinyl)-L-butanoate through multienzyme coupling. The glufosinate-ammonium dehydrogenase mutant is obtained through carrying out single mutation at a 375th site of amino acid as shown in SEQ ID No. 2, SEQ ID No. 4 andSEQ ID No. 6. The specific enzyme activity of the glufosinate-ammonium dehydrogenase mutant DyGDH-V3755 prepared through the invention is improved by 11 to 12 times compared with parent glufosinate-ammonium dehydrogenase, and the maximum feeding of the substrate ammonium 2-amino-4(hydroxymethylphosphinyl)-D / L-butanoate reaches to 800mM, so that the glufosinate-ammonium dehydrogenase mutant has abetter industrial application prospect.

Description

technical field [0001] The present invention relates to the construction of glufosinate-ammonium dehydrogenase DyGDH mutant, the development of glufosinate-ammonium dehydrogenase recombinant bacteria and enzymes in 2-amino-4-(hydroxymethylphosphinyl)-L-ammonium butyrate (commonly known as L -glufosinate-ammonium) chiral biosynthesis. Background technique [0002] Glufosinate-ammonium is the second most resistant herbicide in genetically modified crops in the world. It was developed and produced by Hearst (now owned by Bayer). It is a phosphoric acid herbicide and a glutamine synthetase inhibitor. It is non-selective ( Natural) contact herbicides. [0003] The activity of glufosinate-ammonium is between that of glyphosate and paraquat, and has the advantages of high activity, low toxicity, easy degradation, and environmental friendliness; in addition, it can also be used to screen transgenic crops resistant to glufosinate-ammonium, so it is widely used , are generally favor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12P13/04
CPCC12N9/0016C12P13/04C12Y104/03003Y02P20/584
Inventor 薛亚平程峰李恒郑裕国
Owner ZHEJIANG UNIV OF TECH
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