CN-121975641-A - Genetically engineered strain for high-yield secondary metabolite Fumagillin and construction method and application thereof

Abstract

The invention discloses a genetic engineering strain for high-yield secondary metabolite Fumagillin, a construction method and application thereof, wherein the genetic engineering strain is obtained by taking aspergillus fumigatus as chassis bacteria and carrying out hosA gene knockout or hosA catalytic active site mutation. According to the invention, hosA gene knockout strain (delta hosA) and catalytic active site mutant strain (HosA D133A 、HosA H175A 、HosA D210A ) are constructed by means of molecular biology, and the mutant strain is subjected to liquid or solid fermentation under the culture condition of 37 ℃, so that the Fumagillin yield can be remarkably improved. The invention uses histone deacetylase activity change caused by hosA defect to release the inhibition of Fumagillin biosynthesis path and strengthen metabolic flow, provides a high-efficiency and directional production method of high yield Fumagillin, and has important industrial application value.

Inventors

• ZHANG YUANWEI
• ZHOU ZHENGYU
• LU LING
• YUAN CAN

Assignees

• 南京师范大学

Dates

Publication Date

20260505

Application Date

20251230

Claims (10)

1. 1. A genetic engineering strain for high-yield secondary metabolite Fumagillin is characterized in that the genetic engineering strain is obtained by taking aspergillus fumigatus (Aspergillus fumigatus) as chassis bacteria through hosA gene knockout or hosA catalytic active site mutation, and the nucleotide sequence of the hosA gene is shown as SEQ ID NO. 1.
2. 2. The genetically engineered strain for the high yield of secondary metabolites Fumagillin of claim 1, wherein said hosA catalytically active site mutation is a mutation of the hosA gene encoding an amino acid, comprising any one or more of D133A, H175A, D a.
3. 3. The genetically engineered strain of high yield of secondary metabolites Fumagillin of claim 1, wherein the aspergillus fumigatus is wild type or a derivative strain thereof and the aspergillus fumigatus is Aspergillus fumigatus FGSC A1151.
4. 4. The genetically engineered strain for the high yield of secondary metabolites Fumagillin of claim 1, wherein the genetically engineered strain is a hosA gene-deficient strain constructed by homologous recombination techniques to replace the hosA gene coding region with a resistance selection marker or to mutate the hosA catalytically active site.
5. 5. The construction method of the genetically engineered strain for high yield of the secondary metabolite Fumagillin is characterized by comprising the following steps: 1) Designing a knockout vector containing hosA upstream and downstream homology arms or designing and constructing a recombinant replacement vector containing hosA ORF segment with hosA catalytic active site mutation, and taking hygromycin resistance gene (hph) as a screening marker; 2) Converting the linearized hosA knockout fragment or hosA active site mutation substitution fragment into Aspergillus fumigatus protoplast, obtaining positive transformant through PEG mediated transformation, constructing HosA knockout strain delta hosA or catalytic active site mutation strain, namely gene engineering strain of high-yield secondary metabolite Fumagillin.
6. 6. Use of a genetically engineered strain of the high-yield secondary metabolite Fumagillin of claim 1 for the production of high-yield Fumagillin.
7. 7. The use according to claim 6, wherein the genetically engineered strain is subjected to fermentation culture at 30-40 ℃ to induce accumulation of secondary metabolites Fumagillin.
8. 8. The use according to claim 7, wherein the fermentation culture is a liquid shake flask fermentation.
9. 9. Use of the hosA gene of claim 1 to regulate the production of secondary metabolite Fumagillin by aspergillus fumigatus.
10. 10. The use according to claim 9, wherein the aspergillus fumigatus production of the secondary metabolite Fumagillin is enhanced by hosA gene knockout or hosA catalytic active site mutation.

Description

Genetically engineered strain for high-yield secondary metabolite Fumagillin and construction method and application thereof Technical Field Genetically engineered strain for high-yield secondary metabolite Fumagillin and construction method and application thereof Background Fumagillin is an important fungal secondary metabolite, originally found in aspergillus fumigatus. Fumagillin and derivatives thereof, such as TNP-470 (also known as Fumagillol), are of great interest to the medical community for their unique anti-angiogenic activity. Since it can specifically inhibit methionine aminopeptidase 2 (MetAP 2), fumagillin shows great potential for use in cancer treatment, antiparasitic (microsporidian disease) and the like. Currently Fumagillin is produced mainly by microbial fermentation. However, the yield of Fumagillin of the existing wild type aspergillus fumigatus strain under the traditional fermentation condition is generally low, and the requirements of industrial production and clinical research are difficult to meet, so that the production cost is high. Traditional yield-improving strategies, such as fermentation medium optimization and non-directional mutation breeding, have the defects of byproduct accumulation, huge screening workload, low efficiency, uncertainty of results and the like. Therefore, there is a need for a strategy to enhance Fumagillin production by gene-directed engineering and enhancement of metabolic pathways. In fungal secondary metabolic regulation, epigenetic regulation mechanisms, especially histone modifications, play a key role. Histone deacetylases (Histone Deacetylases, HDACs) are an important class of regulatory factors that inhibit expression of related genes by removing acetyl groups on histones, causing the chromatin structure to shrink. However, there is no technical proposal reported in the literature to realize high yield of Fumagillin by directionally modifying HosA genes and combining specific culture temperature. Disclosure of Invention Aiming at the problems of low yield of fungus secondary metabolite, complicated regulation and control, undefined epigenetic regulation mechanism and the like in the prior art, the invention provides the genetic engineering strain for high-yield secondary metabolite Fumagillin, and the invention eliminates the transcriptional inhibition of Fumagillin biosynthesis gene cluster by deleting or mutating the catalytic active site of histone deacetylase HosA in aspergillus fumigatus in a targeted way to destroy the deacetylation activity, thereby realizing the activation of Fumagillin synthesis path and remarkable improvement of yield. The invention also provides a construction method and application of the genetic engineering strain. In order to achieve the aim, the genetic engineering strain for high-yield secondary metabolite Fumagillin is obtained by taking aspergillus fumigatus FGSC A1151 (Aspergillus fumigatus) as chassis bacteria through hosA gene defect or hosA catalytic active site mutation, and the nucleotide sequence of hosA gene is shown as SEQ ID NO. 1. Wherein the hosA catalytic active site mutation is a mutation of hosA gene coding amino acid, including any one or more of D133A, H175A, D A. Wherein the aspergillus fumigatus is a wild type strain or a derivative strain thereof, and the aspergillus fumigatus is Aspergillus fumigatusFGSC A and 1151. Wherein the genetic engineering strain is hosA gene-defective strain, the construction adopts homologous recombination technology, and hosA gene coding region is replaced by resistance screening mark or hosA conservative catalytic active site mutation. The construction method of the genetically engineered strain for high-yield secondary metabolite Fumagillin comprises the following steps: 1) Designing a knockout vector containing hosA upstream and downstream homology arms or designing and constructing a recombinant replacement vector containing hosA ORF segment with hosA catalytic active site mutation, and taking hygromycin resistance gene (hph) as a screening marker; 3) Converting the linearized hosA knockout fragment or hosA active site mutation substitution fragment into Aspergillus fumigatus protoplast, obtaining positive transformant through PEG mediated transformation, constructing HosA knockout strain delta hosA or catalytic active site mutation strain, namely gene engineering strain of high-yield secondary metabolite Fumagillin. The invention relates to an application of a genetically engineered strain of a high-yield secondary metabolite Fumagillin in the production of high-yield Fumagillin. Wherein the genetically engineered strain is subjected to fermentation culture at 30-40 ℃ to induce accumulation of secondary metabolites Fumagillin. Wherein the fermentation culture is liquid shake flask fermentation. Wherein the hosA gene-deficient aspergillus fumigatus (Aspergillus fumigatus) strain is used in production of Fumagillin under culture conditions of 37 ℃. Wherein