Method for biotransformation of trichothecene

A technology of trichothecenes, biotransformation, applied in the directions of biochemical equipment and methods, chemical instruments and methods, and botany equipment and methods

Pending Publication Date: 2022-04-01
帝斯曼奥地利有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As preventative measures have not been successful in stopping mycotoxin contamination, there is a need to find a way to provide materials that detoxify and decontaminate as well as provide methods to decontaminate contaminated substances

Method used

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  • Method for biotransformation of trichothecene
  • Method for biotransformation of trichothecene
  • Method for biotransformation of trichothecene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0178] Construction of the expression host:

[0179] In order to be able to detect the activity of plant GST in the standard colorimetric method, a suitable Escherichia coli host strain was constructed, in which the standard substrate (chloro-2,4-dinitrobenzene (CDNB)) of the colorimetric method showed activity. The source GST gene (gstA) is inactivated. The gstA gene in the T7 polymerase-based E. coli expression host "T7 Express" was disrupted as follows: first, plasmid pDK46 (with a temperature-sensitive origin of replication, and from strain BW25113 / pKD46 ( http: / / cgsc2.biology.yale.edu / Strain.php? ID=68099 ) The obtained arabinose-induced phage λ recombination system (γβexo)) was introduced into the expression strain T7 Express. The PCR product obtained from the gstA mutant strain JW1627-1 (http: / / cgsc2.biology.yale.edu / Strain.php?ID=107667) from a systematic knockout pool of E. coli was then used to transform arabinose-induced competence cell. The PCR product obta...

Embodiment 2

[0187] Cloning and expression of GST candidates:

[0188] Candidates were cloned into the E. coli expression vector pCA02 encoding an N-terminal 6x His-tag and a maltose-binding domain. This vector carries the ColE1 origin, an ampicillin marker, a copy of the lac repressor and the T7 promoter and lac operator sequences.

[0189] The empty vector pCA02 was used as a negative control.

[0190] Cloning of TrGST-C02:

[0191] The wheat glutathione S-transferase TRIAE_CS42_1DL_TGACv1_062916_AA0221650 was amplified from the genomic DNA of Chinese spring wheat with primers TrGST-C02_upstr_fw and TrGST-C02_3'UTR_rv, and cloned into pMiniT( PCR cloning kit). Exon 1 was amplified with primers TrGST-C02_GA-E1-fw and TrGST-C02_GA-E1-rv and exon 2 was amplified from pMiniT clones with primers TrGST-C02_GA-E2-fw and TrGST-C02_GA-E2-rv . Fusion PCR was performed with primers TrGST-C02_GA-E1-fw and TrGST-C02_GA-E2-rv. The PCR product was digested with NdeI / EcoRI and ligated into pCA0...

Embodiment 3

[0269] It was shown that TrGST-C02 and TrGST-C12 can also open the epoxides of A-type and macrocyclic (D-type) trichothecenes. The following compounds lack the conjugated C8-ketocharacteristic of trichothecenes type B (required for Michael adduct formation), so adduct formation is due to epoxide ring opening. The calculations for the expected masses of the adducts with the respective compounds are given in the table below.

[0270] table 5

[0271] name Mode C H N O S neutral mass [M+H]+ T-2 C24H34O9 24 34 0 9 466.2203 467.2276 GSH C10H17N3O6S 10 17 3 6 1 307.0838 308.0911 T-2-GSH 34 51 3 15 1 773.3041 774.3114

[0272] Table 6

[0273] name Mode C H N O S neutral mass [M+H]+ Trichoderma C17H24O4 17 24 4 292.1675 293.1747 GSH C10H17N3O6S 10 17 3 6 1 307.0838 308.0911 TCM-GSH 27 41 3 10 1 599.2513 600.2585

[0274] Table 7

[0275] ...

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Abstract

The present invention relates to a novel method for detoxification of trichothecene contaminated materials. More specifically, the present invention relates to a method of bioconverting trichothecene by contacting a material contaminated with trichothecene with an exogenous non-animal glutathione-S-transferase (GST) having substrate specificity for the epoxy ring of trichothecene. The invention also relates to recombinant GST as well as to transgenic plants and animals expressing said GST.

Description

technical field [0001] The present invention relates to novel methods for detoxifying trichothecene-contaminated materials. More specifically, the present invention relates to a method for the biotransformation of trichothecenes by combining a trichothecene-contaminated material with an exosome specific for the epoxy ring of the trichothecenes. Source non-animal glutathione-S-transferase (GST) exposure was performed. The invention also relates to recombinant GSTs and transgenic plants and animals expressing said GSTs. Background technique [0002] Fungi produce a large number of metabolites that are not essential for life but may provide fungi with an ecological advantage in certain environments. Such metabolites are called secondary metabolites. Fungal secondary metabolites include plant growth regulators (e.g., gibberellins), pharmaceutically acceptable compounds (e.g., penicillins, lovastatins), pigments (e.g., carotenoids), and mycotoxins (e.g., trichothecenes, equin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82A23L5/20A23K10/14A23K10/30
CPCC12N9/1088A23L5/25A23K10/14C12N15/8242A23K10/30C07K14/415
Inventor M·锡勒G·亚当M·多普勒R·舒马赫尔K·库格勒K·F·X·马耶尔G·维森贝格尔H·米克迈尔W·施维格尔M·霍弗尔H·伯斯迈尔B·施泰纳
Owner 帝斯曼奥地利有限公司
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