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Construction and application of heterologous expression system of aspergillus nidulans

A technology of Aspergillus nidulans and heterologous expression, applied in the biological field, can solve the problems of slow growth, high difficulty, unclear genetic background, etc.

Active Publication Date: 2018-11-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Aurovertins are a class of anti-tumor compounds isolated for the first time from Dendrosporium, which has defects such as slow growth and unclear genetic background. On this basis, it is difficult to study the biosynthetic pathway of aurovertin, and it is difficult to use heterologous expression Systematic research on fungal natural products is an effective means to discover new aurovertin compounds and analyze their biosynthetic pathways

Method used

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  • Construction and application of heterologous expression system of aspergillus nidulans
  • Construction and application of heterologous expression system of aspergillus nidulans
  • Construction and application of heterologous expression system of aspergillus nidulans

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 Construction of Aspergillus nidulans proteolytic enzyme deletion strain

[0038] Aspergillus nidulans was used as the starting strain for genetic modification. This example uses high-fidelity enzyme PCR to amplify the pyroA auxotrophic marker gene sequence and the Aspergillus nidulans proteolytic enzyme gene dppV, mnn9 and pepA upstream and downstream 1300bp sequences, and then uses the Double-joint PCR method to construct the Aspergillus nidulans proteolytic enzyme respectively Single-gene knockout cassettes for genes dppV, mnn9, and pepA, and gene knockout in Aspergillus nidulans using PEG-mediated protoplast genetic transformation.

[0039] The PEG-mediated protoplast genetic transformation method of Aspergillus nidulans is as follows:

[0040] The used Aspergillus nidulans strain activation culture medium of the present invention is GMM culture medium, and the described solid culture medium of every 1L is prepared as follows: glucose 10g, nitrate mothe...

Embodiment 2

[0052] The construction of embodiment 2 Aspergillus nidulans RsmA gene overexpression strain

[0053] Aspergillus nidulans was used as the starting strain for genetic modification. In this embodiment, first, high-fidelity enzyme PCR is used to amplify the pyrG auxotrophic marker gene, the constitutive strong promoter gpdA promoter sequence, the upstream 1300bp sequence of the RsmA gene and the 1300bp gene fragment of the RsmA gene, and then the double-joint PCR method is used to amplify pyrG auxotrophic marker gene and constitutive strong promoter gpdA promoter construct the overexpression cassette of Aspergillus nidulans RsmA gene, and utilize the protoplast genetic transformation method mediated by PEG to carry out inheritance transformation to Aspergillus nidulans (transformation method is the same as embodiment 1), complete homologous recombination and carry out the overexpression transformation of the RsmA gene in the bacterium, then pick the transformant and extract geno...

Embodiment 3

[0054] Example 3 Construction of laccase-related expression vectors and aurovertin biosynthesis-related expression vectors

[0055] The laccase gene used in this application (as shown in SEQ ID NO: 1) comes from Pycnoporus sanguineus (GenBank: AY510604.1), using Pycnoporus sanguineus genomic DNA as a template, and using high-fidelity enzymes for PCR amplification , respectively cloned into the expression cassette (3476bp) and the laccase gene containing its own promoter sequence of the laccase gene, its terminator sequence, and at the same time using Aspergillus nidulans genomic DNA as a template, using high-fidelity enzymes for PCR amplification, respectively cloned to the gpdA promoter (1513bp) and aflR promoter (660bp) sequences; then use the conventional enzyme-cut ligation method and the Quick-change vector construction technology method to transform Escherichia coli and construct a vector containing the AMA1 autonomous replication sequence (see literature: Aleksenko A, C...

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Abstract

The invention relates to a construction method of a heterologous expression system of aspergillus nidulans. The method includes the following step (1) or step (2): (1) knocking out any one or two of an aspergillus nidulans dppV gene, an mnn9 gene or a pepA gene to construct a proteolytic enzyme knockout mutant strain of the aspergillus nidulans; or (2) overexpressing an RsmA gene in the aspergillus nidulans, and constructing a mutant strain of the aspergillus nidulans with the overexpressed RsmA gene. The proteolytic enzyme knockout mutant strain of the aspergillus nidulans and the mutant strain of the aspergillus nidulans with the overexpressed RsmA gene are helpful for efficient expression production of important industrial proteases. Expression elements of a gpdA promoter and a xylP promoter provided by the invention can regulate the generation direction of different compounds produced when the aspergillus nidulans heterologously expresses natural products of fungi, can be used formodifying or discovering compounds that can be used in medicine, and is of great significance for the research and development of the proteases with important industrial value and potential natural products of fungi.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the construction and application of an Aspergillus nidulans heterologous expression system. Background technique [0002] Filamentous fungi have the characteristics of large expression, high extracellular secretion rate, protein molecular folding and modification system close to higher eukaryotic cells, and exogenous proteins produced with natural activity. Therefore, they are widely used in beverage, cheese and other food industries and in the commercialization of enzymes. It is widely used in production and has become a very attractive expression host system for the production of heterologous recombinant proteins. In basic research, the establishment of a protein expression system is a prerequisite for studying the regulation of enzyme expression in the strain at the molecular level. An important basis for catalytic properties; in applied research, because multi-copy integration i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12P1/02C12R1/66
CPCC12N9/0061C12N15/80C12P1/02C12Y110/03002
Inventor 尹文兵李伟马紫卉
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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