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Gene mining method combining functional sequence and structural simulation, NADH preference type glufosinate-ammonium dehydrogenase mutant and application

A technology of glufosinate-ammonium and dehydrogenase, which is applied in genomics, instrumentation, proteomics, etc., can solve the problem of low activity of asymmetric amination reduction

Pending Publication Date: 2021-06-04
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to solve the problem that the existing glufosinate-ammonium dehydrogenase has low activity for the asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphono)-butyric acid, and provides a NADH-preferred glufosinate-ammonium Phosphine dehydrogenase mutants and using the NADH-preferring glufosinate-ammonium dehydrogenase mutant gene recombinant bacteria and their crude enzyme solution as biocatalysts for chiral biosynthesis of L-glufosinate-ammonium

Method used

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  • Gene mining method combining functional sequence and structural simulation, NADH preference type glufosinate-ammonium dehydrogenase mutant and application
  • Gene mining method combining functional sequence and structural simulation, NADH preference type glufosinate-ammonium dehydrogenase mutant and application
  • Gene mining method combining functional sequence and structural simulation, NADH preference type glufosinate-ammonium dehydrogenase mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Step 1: Analyze the characteristic sequences that NADH-type glutamate dehydrogenase should possess: ① protein size: the length of the candidate protein (300-500 amino acids), ② two necessary characteristic sequences of glufosinate-ammonium dehydrogenase: the first The segment is GGGKGG, where the X position represents any amino acid, and the second segment is one of VVTG, FVTG, VLTG, VFTG, FITG, FFTG, VVFG, FVFTG, VLFG, VFFG, FLFG, FFFG, ③The characteristic sequence of NADH binding : GXRVXXG.

[0048] Step 2: Search the gene bank: use the above characteristic sequences to perform iterative PSI-BLAST search and cluster analysis on the NCBI microbial genome resources and then the NCBINR sequence database (containing about 100 million protein genes), and obtain 15 clusters, 15 The degrees of aggregation of the clusters are 0.82, 0.76, 0.71, 0.66, 0.65, 0.58, 0.43, 0.42, 0.40, 0.39, 0.38, 0.34, 0.33, 0.32, 0.30 (arranged from high to low).

[0049] Step 3: Select represent...

Embodiment 2

[0050] Example 2: Construction and screening of glufosinate-ammonium dehydrogenase mutant library

[0051] The codon-optimized LcGDH amino acid sequence of Example 1 (the codon-optimized nucleotide sequence is shown in SEQ ID No.1), and the LcGDH gene obtained by gene synthesis by Hangzhou Qingke Biotechnology Co., Ltd. was cloned into On the NcoI of MCS1 (Multiple Cloning Site 1) of the plasmid pETDuet, construct the recombinant expression vector pETDuet-LcGDH, retain the His-Tag gene of the plasmid itself, transform it into E. coli BL21 (DE3), and send it to Hangzhou Qingke Biotechnology Co., Ltd. synthesized wild-type glufosinate-ammonium dehydrogenase engineering bacteria E.coli BL21(DE3) / pETDuet-LcGDH.

[0052] The glucose dehydrogenase gene EsGDH was cloned from Exiguobacterium sibiricum ZJBML01011 (the nucleotide sequence is shown in GenBank accession number KM817194.1), and constructed into the recombinant expression vector pETDuet-LcGDH by Vazyme's OneStep Cloning Kit...

Embodiment 3

[0060] Embodiment 3: Induced expression of glufosinate-ammonium dehydrogenase mutant engineering bacteria

[0061] The wild-type glufosinate-ammonium dehydrogenase and glucose dehydrogenase in Example 2 are set out to co-express strain E.coliBL21(DE3) / pETDuet-1-LcGDH-EsGDH and the glufosinate-ammonium dehydrogenase mutant with glucose dehydrogenase Enzyme co-expression strains:

[0062] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G)-EsGDH,

[0063] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V345A-M91A)-EsGDH,

[0064] E.coli BL21(DE3) / pETDuet-1-LcGDH(A144G-V375F-M91A)-EsGDH,

[0065] Inoculate them into LB liquid medium containing a final concentration of 50 μg / mL ampicillin, culture at 37°C for 8 hours, and inoculate them into fresh LB liquid culture medium containing a final concentration of 50 μg / mL ampicillin at an inoculum volume concentration of 2%. culture medium at 37°C and 180 rpm for 2 hours, and then add 0.1mM IPTG to the culture medium at a final concentration of 18°C ​​fo...

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Abstract

The invention discloses a gene mining method combining a functional sequence and structural simulation, an NADH preference type glufosinate-ammonium dehydrogenase mutant and an application. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence of NADH type glutamate dehydrogenase; (2) searching a gene pool according to the characteristic sequence; (3) performing clustering analysis and protein structure simulation on the searched genes; and (4) selecting a gene with high gene polymerization degree and a protein structure similar to that of known glufosinate-ammonium dehydrogenase as a candidate gene. The method comprises the following steps: carrying out gene mining to obtain wild glufosinate-ammonium dehydrogenase which is derived from Lysinibacillus composti and has an amino acid sequence shown as SEQ ID No.2, carrying out mutation screening to obtain an NADH preference type glufosinate-ammonium dehydrogenase mutant, and selecting a mutation site from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; and (3) A144G. The mutant enzyme can be subjected to catalytic reaction by using cheap coenzyme NAD.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene mining method combining functional sequence and structure simulation, NADH-preferred glufosinate-ammonium dehydrogenase mutants and applications. Background technique [0002] Glufosinate-ammonium (phosphinothricin, also known as glufosinate, referred to as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono]-butyric acid, which is the second most resistant herbicide in genetically modified crops in the world. Developed and produced by Sturt (now owned by Bayer after several mergers), it is also known as glufosinate ammonium salt, Basta, Buster, etc. It is a phosphonic acid herbicide, and the non-selective (killing) contact herbicide is glutamine Synthetic enzyme inhibitors. [0003] Glufosinate-ammonium has two optical isomers, L-glufosinate-ammonium and D-glufosinate-ammonium. But only the L-type has physiological activity, and is easy to decompose in the soil...

Claims

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Application Information

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IPC IPC(8): G16B20/50G16B35/00G16B50/30
CPCG16B20/50G16B50/30G16B35/00
Inventor 薛亚平程峰张嘉敏邹树平徐建妙郑裕国
Owner ZHEJIANG UNIV OF TECH
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