A kind of Aspergillus producing monacolin j and its construction method and application

A technology for producing monacolin and constructing a method is applied in the field of biopharmaceuticals, which can solve the problems of large difference in yield, easy degradation and accumulation of lovastatin, and achieve the effects of enhancing fermentation stability, simplifying production process and reducing environmental pollution.

Active Publication Date: 2022-07-29
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, the synthesis regulation mechanism of secondary metabolites is very complex, blocking the synthesis pathway may have a significant impact on the entire synthesis pathway, and other upstream intermediates cannot effectively accumulate while the final product disappears
Moreover, some intermediate products are unstable and easy to degrade, or the downstream route of intermediate products is not unique, and may be intermediate products of multiple synthetic pathways, so knocking out a downstream gene may not necessarily achieve the accumulation of intermediate products
[0005] The synthesis of secondary metabolites is usually significantly disturbed by environmental factors, and small differences in some unknown environmental factors may have a greater impact on the synthesis of secondary metabolites
Lovastatin is a polyketide secondary metabolite. In the industrial production process, the yield of lovastatin varies greatly between batches, which not only increases the difficulty of production process control but also affects production efficiency.

Method used

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  • A kind of Aspergillus producing monacolin j and its construction method and application
  • A kind of Aspergillus producing monacolin j and its construction method and application
  • A kind of Aspergillus producing monacolin j and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1, Aspergillus genetic transformation

[0075] 1. Preparation of protoplasts

[0076] The spores of Aspergillus HZ01 were inoculated into 50 mL of IPM liquid medium so that the spore concentration was about 10 7 cells / mL, cultured at 200rmp and 32°C for 12-18h. The growing mycelia were collected by filtration through a sterile monolayer of 500-mesh nylon cloth and washed with sterile 0.6M MgSO. 4 The solution was rinsed three times, dried and placed in a sterile 50ml conical flask. An appropriate amount of enzymolysis solution was added according to the weight of mycelium (10ml of enzymolysis solution was added per 1g of mycelium), and treated at 30°C and 60rpm for 1-3h. The mixed solution after the enzymolysis was first filtered with 8 layers of lens-wiping paper, and the filtrate was collected. Protoplasts were collected by centrifugation at 4°C, 4000 rpm, washed once with pre-cooled 1.0 M sorbitol solution, and then pre-cooled with STC (STC composition: 1....

Embodiment 2

[0079] Example 2. Complete knockout of lovF gene

[0080] Knock out the lovF gene to complete the knockout of all copies. All primer positions and gene knockout strategies are as follows figure 1 shown.

[0081] 1. Knock out the first copy of lovF

[0082] (1) Construction of lovF gene targeting element lovF-KO1

[0083] According to Aspergillus terreus NIH2624 genome information (GenBank: AAJN00000000.1) and Aspergillus terreus HZ01 lovastatin synthesis gene cluster information, the following primers were designed and synthesized:

[0084] U1-lovF-F: 5'-gctcccatagctatggttggc-3' (SEQ ID No. 1);

[0085] U1-lovF-R: 5'-GTTCAATCACCATCTCCCTTAgagtcttcaagacgatcgcagc-3' (SEQ ID No. 2);

[0086] D1-lovF-F: 5'-CGTATTTCTCCGCCTGTGTGatctgcgagtggctggtcgatc-3' (SEQ ID No. 3);

[0087] D1-lovF-R: 5'-gcatctcagaacgggatgctg-3' (SEQ ID No. 4).

[0088] Using Aspergillus HZ01 genomic DNA as a template, pfu DNA polymerase (Fermentas, catalog number: EP0501) was used for PCR amplification, an...

Embodiment 3

[0144] Example 3. Construction of LovE strains overexpressing specific transcriptional regulators

[0145] 1. Design and synthesize the following primers:

[0146] Uku80-F: 5'-agcacaaacatattgatcagc-3' (SEQ ID No. 31);

[0147] pyrGAn-R: 5'-GGATCCTCCCAGAGTGTAAgcatcaaatcgtcgtaccgca-3' (SEQ ID No. 32);

[0148] TtrpC-F3: 5'-aagcttgagatccacttaacgttactgaaatcatc-3' (SEQ ID No. 33);

[0149] Dku80-R: 5'-gaaggcgaaaagtagtctcgtg-3' (SEQ ID No. 34).

[0150] Using plasmid pXH-106 as a template, PCR amplification was performed with primer pairs Uku80-F / pyrGAn-R and TtrpC-F3 / Dku80-R to obtain the upstream homology arm Uku80-pyrGAn and the downstream homology arm TtrpC-Dku80.

[0151] 2. Design and synthesize the following primers:

[0152] lovE-F: 5'-ACAACTCATCAATCATCACatggctgcagatcaaggtat-3' (SEQ ID No. 35);

[0153] lovE-R: 5'-GTTAAGTGGATCTCAAGCTTcatggaggaatattgttga-3' (SEQ ID No. 36).

[0154] Using Aspergillus HZ01 genomic DNA as a template, the lovE gene fragment was obtained by...

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Abstract

The invention discloses a monacolin J-producing Aspergillus and its construction method and application. The construction method of a monacolin J-producing Aspergillus disclosed in the invention includes the step of completely blocking the expression of polyketide synthase LovF in the Aspergillus. The invention achieves the accumulation of monacolin J by completely knocking out the lovF gene to block the synthesis of lovastatin, and then using a constitutive promoter to overexpress the transcriptional regulator lovE on this basis, so that the monacolin J of the constructed strain is constructed. The yield of Lin J was further significantly improved, and the fermentation stability of the strain was enhanced. The strain of the present invention can be used to directly ferment and produce monacolin J, thereby simplifying the production process of simvastatin, reducing costs, improving production efficiency and reducing environmental pollution.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a monacolin J-producing Aspergillus and a construction method and application thereof; furthermore, the invention relates to the production of monacolin J. Background technique [0002] Cardiovascular and cerebrovascular diseases are a serious threat to human health, and their morbidity and mortality ranks first in many countries and regions. Hyperlipidemia (hypercholesterolemia) is an important cause of cardiovascular and cerebrovascular diseases. Therefore, preventing hyperlipidemia, regulating blood lipids and reducing blood lipids have become the keys to preventing and treating cardiovascular and cerebrovascular diseases. Based on the above reasons, cholesterol-lowering drugs have great market prospects and have been at the forefront of the world's best-selling drugs for many years. [0003] Simvastatin (Zocor) is an important hypolipidemic drug developed by Merck, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N15/90C12N1/15C12P7/42C12R1/66
CPCC12N15/80C12N15/902C12N9/1029C12P7/42C12N15/90C12R2001/66C12N1/145
Inventor 吕雪峰黄雪年杨勇郑玲辉滕云
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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