Compound as well as synthetic gene cluster and application thereof

A compound and gene cluster technology applied in the field of microbial medicine

Active Publication Date: 2021-04-02
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention activates recessive genes through heterologous expression to obtain 4 novel sesquiterpeno...

Method used

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  • Compound as well as synthetic gene cluster and application thereof
  • Compound as well as synthetic gene cluster and application thereof
  • Compound as well as synthetic gene cluster and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] [Example 1] Construction of Aspergillus oryzae transformant Ao-AuS1

[0060] Inoculate the spores of the strain 094102 in the fungus II liquid medium, culture at 28°C, 220rpm for 2 days, collect about 20mg of mycelium and add it to a 2mL nucleic acid extraction tube with a small amount of glass beads at the bottom (high temperature sterilization is required to avoid exogenous genes interference). Add 500 μL of sterile water, shake in the nucleic acid extractor for 1 min, then add 500 μL of phenol: chloroform: isoamyl alcohol solution, vortex and mix well, then centrifuge at 12,000 rpm for 10 min, and take the upper aqueous layer as a template for polymerase chain reaction (PCR).

[0061] Referring to the sequence of SAC11189, the primer pair 11189F and 11189R were designed to amplify the gene Au11189. MCLAB 2×High-Fidelity Master Mixon was used to carry out PCR reactions separately, each target gene reaction system was 50 μL, and the components were as follows: 25 μL o...

Embodiment 2

[0064] [Example 2] Aspergillus oryzae transformant Ao-AuS1 fermentation and metabolite detection

[0065] The Aspergillus oryzae transformants obtained in Example 1 were inoculated in 100 mL of DPY liquid medium, cultured at 28° C. and 220 rpm for 7 days, and the mycelium was collected with gauze. Add an equal volume of acetone to the mycelia and soak overnight, ultrasonically crush for 1 hour, repeat three times, and filter through a Buchner funnel to obtain an acetone-water layer. After concentrating under reduced pressure to remove acetone, the aqueous solution was extracted three times with an equal volume of n-hexane; all n-hexane layers were combined and concentrated under reduced pressure to obtain a crude extract. Use 1 mL of n-hexane to redissolve the crude extract and filter for GC-MS detection ( Figure 4-5 ).

[0066] Conclusion: In the gene cluster SAC11189, terpene synthase Au11189 catalyzes the production of four sesquiterpene compounds 1-4 (m / z 340), which ca...

Embodiment 3

[0067] [Example 3] Separation and structure confirmation of precursor compound 1-4

[0068] The Aspergillus oryzae transformant Ao-AuS1 obtained in Example 1 was inoculated in 28L DPY liquid medium, cultured at 28°C and 220rpm for 7 days, the mycelium was collected, and treated according to the method described in Example 2 to obtain a crude extract. Redissolve about 20 g of the obtained crude extract in a small amount of n-hexane, use Qingdao Haiyang 100-200 mesh silica gel to mix the sample, and fill the silica gel column with 200-300 mesh silica gel. Use n-hexane for elution, collect the eluate with glass test tubes, each tube is about 8 mL, and perform GC-MS detection to determine the components. Combine the eluent containing the target sesquiterpene compound, concentrate under reduced pressure and redissolve with 1 mL of acetone, filter and carry out HPLC semi-preparative separation and purification ( Figure 7 ).

[0069] The structures of precursor compounds 1-4 were ...

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Abstract

The invention provides a compound as well as a synthetic gene cluster and an application thereof. The compound is prepared from a precursor compound aspergidienes A-D and a sesterterpene compound aspergiols A-D, and the aspergiols A-D has the anti-tumor activity, and the sesterterpene compound aspergiols A-D has the anti-tumor activity. The biosynthetic gene cluster SAC11189 composed of a terpenesynthase gene Au11189 and a cytochrome P450 enzyme gene Au11188 is obtained in aspergillus fungi 094102 through genome mining. An aspergillus oryzae expression system is used for expressing each genein a gene cluster to obtain four sesquiterpene compounds aspergiols A-D with novel structures. In-vitro cytotoxic activity test results show that the assergils A-D shows different degrees of inhibitory activity on a plurality of tumor cells, and a possible candidate compound is provided for the development of novel anti-tumor drugs.

Description

technical field [0001] The invention belongs to the field of microbial medicine, and specifically relates to a class of compounds, their synthetic gene clusters and applications. Background technique [0002] Cancer poses a huge threat to human life and health. According to the statistics of the National Cancer Center in 2019, the death of malignant tumors accounted for 23.91% of all deaths among residents, and the morbidity and mortality showed an increasing trend year by year. The severe prevention and control situation made The development and development of novel anticancer drugs has become more urgent. [0003] Natural products are an important source of antineoplastic drugs, and about half of antineoplastic drugs are directly or indirectly derived from natural products, including terpenes, alkaloids, polyphenols, etc. (Newman, D.J, & Cragg, G.M. Natural products as sources of new drugs from 1981to 2014. Journal of Natural Products, 2016, 79, 629-661.). Sesquiterpene ...

Claims

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Application Information

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IPC IPC(8): C07C29/74C07C29/76C07C29/78C07C29/86C07C35/44C07C7/00C07C7/10C07C7/12C07C7/14C07C13/66A61K31/045A61K31/015A61P35/00C12N1/15C12N15/80C12N15/53C12N15/52C12P15/00C12R1/69
CPCC07C29/74C07C29/76C07C29/78C07C29/86C07C35/44C07C7/005C07C7/10C07C7/12C07C7/14C07C13/66A61K31/045A61K31/015A61P35/00C12N15/52C12N9/0077C12N9/00C12N15/80C12P15/00C07B2200/07C07C2603/40Y02A50/30
Inventor 洪葵郭晶晶邓子新
Owner WUHAN UNIV
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