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Gene-deficient type Amycolatopsis sp. capable of highly producing vanillin, construction method and application thereof

A technique of Amycolatopsis and genetic defects, applied in the field of genetic engineering, can solve the problems of no research on secondary metabolism and less research on transcription factors

Active Publication Date: 2021-12-14
SHAANXI HEALTHFUL BIOLOGICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the ROK family regulatory genes are widely distributed in prokaryotes, but there are few studies on the transcription factors of this family in Streptomyces, and there is no research on the secondary metabolism of ROK family regulatory genes in Amycolatopsis.

Method used

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  • Gene-deficient type Amycolatopsis sp. capable of highly producing vanillin, construction method and application thereof
  • Gene-deficient type Amycolatopsis sp. capable of highly producing vanillin, construction method and application thereof
  • Gene-deficient type Amycolatopsis sp. capable of highly producing vanillin, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Construction of ROK-TR1 gene deletion, gene complementation, and gene overexpression strains

[0065] On the NCBI website, the sequence of the transcription regulator ROK-TR1 of the ROK family in Amycolatopsis sp. ATCC 39116 strain was compared according to the gene sequence of Amycolatopsis sp.ATCC 39116. The gene sequence is shown in SEQ ID NO:1 , the encoded protein accession number is WP_020420567.1.

[0066] 1. Construction of Amycolatopsis bacterium with deletion of ROK-TR1 gene

[0067] Using TR1-U-FOR / TR1-U-REV and TR1-D-FOR / TR1-D-REV as primers and Amycolatopsis genome as template, PCR amplifies the upstream and downstream of ROK-TR1 gene about 1.1kb each homology arm. At the same time, the above two TR1-UP and TR1-DOWN fragments were connected to the HindIII / EcoRI site of the pKC1139 plasmid through the Assembly kit to complete the construction of the plasmid pKC1139-TR1. The pKC1139-TR1 plasmid map is as follows figure 1 shown.

[0068] The pKC...

Embodiment 2

[0074] Example 2: Construction of ROK-TR2 gene deletion, gene complementation, and gene overexpression strains

[0075] On the NCBI website, the sequence of the transcription regulator ROK-TR2 of the ROK family in wild-type Amycolatopsis sp. ATCC 39116 was compared according to the gene sequence of the Amycolatopsis sp.ATCC 39116 strain. The encoded protein accession number is WP_020421841 .1.

[0076] 1. Construction of Amycolatopsis with ROK-TR2 gene deletion

[0077] Using TR2-U-FOR / TR2-U-REV and TR2-D-FOR / TR2-D-REV as primers, Amycolatopsis genome as template, PCR amplifies the upstream and downstream of ROK-TR2 gene about 1.1kb each homology arm. At the same time, the above two TR2-UP and TR2-DOWN fragments were connected to the HindIII / EcoRI site of the pKC1139 plasmid through the Assembly kit to complete the construction of the plasmid pKC1139-TR2. The pKC1139-TR2 plasmid map is as follows figure 2 shown.

[0078] The pKC1139-TR2 plasmid was transformed into Amycol...

Embodiment 3

[0084] Example 3: Construction of ROK-TR3 gene deletion, gene complementation, and gene overexpression strains

[0085] On the NCBI website, the sequence of the transcription regulator ROK-TR3 of the ROK family in Amycolatopsis sp. ATCC 39116 strain was compared according to the gene sequence of Amycolatopsis sp. .

[0086] 1. Construction of Amycolatopsis bacterium with deletion of ROK-TR3 gene

[0087] Using TR3-U-FOR / TR3-U-REV and TR3-D-FOR / TR3-D-REV as primers, and the Amycolatopsis genome as a template, PCR amplifies the upstream and downstream of ROK-TR3 gene about 1.1kb each homology arm. At the same time, the above two TR3-UP and TR3-DOWN fragments were connected to the HindIII / EcoRI site of the pKC1139 plasmid through the Assembly kit to complete the construction of the plasmid pKC1139-TR3. The pKC1139-TR3 plasmid map is as follows image 3 shown.

[0088] The pKC1139-TR3 plasmid was transformed into Amycolatopsis HM-141 by the method of conjugative transfer exper...

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Abstract

The invention provides an application of deletion of ROK family transcription regulation genes ROK-TR1, ROK-TR2 or ROK-TR3 in improving vanillin production of Amycolatopsis sp., and especially relates to a gene-deficient type Amycolatopsis sp. capable of highly producing vanillin. The Amycolatopsis sp. is deleted with the transcription regulation gene ROK-TR1 in the ROK family of the Amycolatopsis sp. The invention also provides a construction method of the Amycolatopsis sp. and an application of the Amycolatopsis sp. in producing vanillin. After the ROK-TR1 gene is knocked out, the vanillin yield of the Amycolatopsis sp. is increased by 33% compared with that of an original strain, the expression in the transcription regulation genes in the three ROK families is the most prominent, when the ROK-TR1 gene is overexpressed, the vanillin yield is reduced by 34% compared with that of the original strain, and the technical support is provided for further increasing the vanillin fermentation yield in industrial production.

Description

【Technical field】 [0001] The invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a gene-defective amycolatopsis, and also relates to the application of the amycolatopsis in the production of vanillin. 【Background technique】 [0002] Vanillin, also known as vanillin (3-methoxy-4-hydroxybenzaldehyde), is one of the most widely used seasonings in the world and is widely used in food, medicine, cosmetics, agriculture and other fields. Vanillin has the reputation of "the king of food spices". It has vanilla bean aroma and rich milky aroma, and can play a role in enhancing fragrance and flavor in food; in medicine, vanillin is a synthetic compound An important raw material for medicines; vanillin can be used as a flavoring agent in cosmetics, perfumes and other fields; it can also be used as a ripening agent and yield-increasing agent for crops in agricultural production. Due to its wide range of applications, the ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/74C12N15/90C12P7/24C12R1/01
CPCC12N15/74C12N15/902C07K14/195C12P7/24C12N1/20
Inventor 孟永宏强珊郭建琦牛永洁杨璐
Owner SHAANXI HEALTHFUL BIOLOGICAL ENG
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