Aspergillus for producing monacolin J, and construction method and application thereof

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

Active Publication Date: 2020-03-03
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Aspergillus for producing monacolin J, and construction method and application thereof
  • Aspergillus for producing monacolin J, and construction method and application thereof
  • Aspergillus for producing monacolin J, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1, Aspergillus genetic transformation

[0075] 1. Protoplast preparation

[0076] The spores of Aspergillus HZ01 were inoculated into 50mL IPM liquid medium, so that the spore concentration was about 10 7 cells / mL, cultured at 200rmp, 32°C for 12-18h. Collect the growing mycelium by filtering with a sterile single layer of 500-mesh nylon cloth, and filter with sterile 0.6M MgSO 4 The solution was rinsed three times, pressed dry and placed in a sterile 50ml Erlenmeyer flask, adding an appropriate amount of enzymatic solution according to the weight of mycelium (add 10ml of enzymatic solution per 1g of mycelium), and treated at 30°C and 60rpm for 1-3h. Filter the mixture after the above enzymolysis with 8 layers of lens-cleaning paper, and collect the filtrate. Collect protoplasts by centrifugation at 4°C and 4000rpm, wash once with pre-cooled 1.0M sorbitol solution, and then wash with pre-cooled STC (STC composition: 1.0M sorbitol, 50mM Tris-HCl (pH 8.0), 5...

Embodiment 2

[0079] Example 2, complete knockout of the lovF gene

[0080] Knockout the lovF gene to complete the knockout of all copies, all primer positions and gene knockout strategy procedures are as follows figure 1 shown.

[0081] 1. Knockout of the first lovF copy

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

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

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

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

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

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

[0088] Genomic DNA of Aspergillus HZ01 was used as a template, and pfu DNA polymerase (Fermentas, catalog number: EP0501) was used ...

Embodiment 3

[0144] Embodiment 3, the construction of overexpression-specific transcription regulator LovE bacterial strain

[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 the plasmid pXH-106 as a template, PCR amplification was performed with primer pairs Uku80-F / pyrGAn-R and TtrpC-F3 / Dku80-R respectively 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'-ACAACTCATCAATCATCATCAC atggctgcagatcaaggtat-3' (SEQ ID No. 35);

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

[0154] Aspergillus HZ01 genome DNA was used as a template, and ...

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Abstract

The invention discloses an aspergillus for producing monacolin J and a construction method and application thereof. The construction method of the monacolin J-producing aspergillus comprises the stepof completely blocking the expression of polyketide synthase LovF in aspergillus. According to the invention, the accumulation of monacolin J is realized by completely knocking out the lovF gene and blocking the synthesis of lovastatin; and on this basis, the transcription regulation factor lovE is overexpressed by a constitutive promoter, so that the monacolin J yield of the constructed strain isfurther significantly improved, and the fermentation stability of the strain is enhanced. Monacolin J can be directly fermented by the strain of the invention, so that the production process of simvastatin is simplified, the cost is reduced, the production efficiency is improved, and the environmental pollution is reduced.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a monacolin J-producing Aspergillus and its construction method and application; further, the invention relates to the production of monacolin J. Background technique [0002] Cardiovascular and cerebrovascular diseases seriously threaten human health, and its morbidity and mortality rank first in many countries and regions. Hyperlipidemia (hypercholesterolemia) is an important cause of cardiovascular and cerebrovascular diseases, so preventing hyperlipidemia, regulating blood lipids and reducing blood lipids has become the key 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, namely Zocor, is an important blood lipid-lowering drug developed by Merck, which can effe...

Claims

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Application Information

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Patent Type & Authority Applications(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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