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Mycoleptodiscin type indole sesquiterpene compound as well as biosynthesis method and application thereof

A compound and microorganism technology, applied in the fields of genetic engineering and biosynthesis, can solve the problems of unclear biosynthesis pathway, restrict the biosynthesis of mycoleptodicin compounds, etc., and achieve the effects of high stereoselectivity, low environmental pollution, and simple process.

Pending Publication Date: 2022-04-15
THE FIRST AFFILIATED HOSPITAL OF JINAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no bibliographical report about the biosynthesis of mycoleptodiscin in the prior art, and its indole heteroterpenoids petromindole and 17-hydroxyeujindole, which have terpene and indole ring C-3 and C-4 positions, are connected simultaneously. The biosynthetic pathway is also unclear, which greatly limits the biosynthesis of mycoleptodiscin-like compounds

Method used

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  • Mycoleptodiscin type indole sesquiterpene compound as well as biosynthesis method and application thereof
  • Mycoleptodiscin type indole sesquiterpene compound as well as biosynthesis method and application thereof
  • Mycoleptodiscin type indole sesquiterpene compound as well as biosynthesis method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: the screening of bacterial strain

[0065]Mycoleptodiscus sp.JUN2018AT0063Y is a filamentous fungus collected from the leaves of a calamus plant in Baiyun Mountain, Guangzhou. It is white when cultured on PDA medium, and the pigment gradually accumulates and turns gray-black in the later stage. Mycoleptodiscus sp.JUN2018AT0063Y is easily inactivated when stored on PDA medium for long-term storage at 4°C. Vacuum freeze-drying or ultra-low temperature freezing can be used for long-term storage. The strain was deposited on December 06, 2021 at the General Microbiology Center of China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCCNo.23889.

[0066] Mycoleptodiscus sp.JUN2018AT0063Y uses a variety of common laboratory fermentation media for small-scale fermentation to analyze metabolites. The results show ...

Embodiment 2

[0067] Embodiment 2: the acquisition of candidate genes

[0068] Mycoleptodiscus sp.JUN2018AT0063Y was stored on potato agar (PDA) medium at room temperature, and a small amount of mycelium was inoculated into potato liquid (PDB) medium at 28°C with a rotation speed of 200 rpm and shaken for 3 days. The mycelia were collected by filtration, ground with liquid nitrogen, and the total DNA was extracted by the phenol-chloroform method. Sequencing was performed using the Illumina HiSeq 2500 sequencing platform. Sequence analysis was completed using the software SPAdes version 3.5.0 (http: / / cab.spbu.ru / software / spades / ), and then assembled through multiple Kmer values, and finally the best results were obtained by combining the assembly results of each Kmer value. Then, GapFiller version 1.11 was used to fill in GAPs on the spliced ​​contig, and finally PrInSeS-Gversion 1.0.0 was used for sequence correction to correct editing errors and small fragment insertions and deletions dur...

Embodiment 3

[0070] Embodiment 3: Construct gene mutant strain on the basis of original bacterial strain

[0071] The three selected genes (MycA, MycB, MycC) were knocked out one by one using the CRISPR / Cas9 knockout system. The specific method is as follows:

[0072] (1) Protoplast preparation and Cas9 expression strain construction

[0073] 1) Inoculate wild-type Mycoleptodiscus sp.JNU2018AT0063Y into 10 mL of DPY medium (dextrin 2%, polypeptone 1%, yeast extract powder 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.5%, natural pH), 28 ℃, 200rpm shaking culture for 2 days.

[0074] 2) Inoculate the seed solution into 100mL of new DPY medium at 28°C and culture at 200rpm for 24 hours.

[0075] 3) Use the sterilized syringe filter to filter, press dry the bacteria, take out the bacteria with a sterilized medicine spoon, put them into a new 50mL centrifuge tube, add 10mL of PBS solution containing 0.7M NaCl Medium (pH 6.0, containing 0.5% snailase and 0.5% driselase). ...

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Abstract

The invention belongs to the field of genetic engineering and biosynthesis, and particularly relates to biosynthesis of an indole sesquiterpene compound. The invention provides a bacterial strain for producing mycoleptodiscin A and a bacterial strain for producing mycoleptodiscin B. The bacterial strain for producing the mycoleptodiscin A and the bacterial strain for producing the mycoleptodiscin B are provided. The invention provides key action genes for forming a 6-6-6-6-5 five-ring structural skeleton by cyclizing a sesquiterpene part and connecting the sesquiterpene part with a C-4 site of an indole ring in biosynthesis of a mycoleptodiscin compound, and the key action genes are respectively named as MycA, MycB and MycC as well as encoding polypeptides of the key action genes. The invention also provides an application of the polypeptide in the biosynthesis of the mycoleptodiscin compound. According to the present invention, the mycoleptodiscin compound obtained through the biosynthesis method has the cytotoxic activity and the antibacterial activity similar to the cytotoxic activity and the antibacterial activity of the positive control or the mycoleptodiscin A or the mycoleptodiscin B;

Description

technical field [0001] The invention belongs to the field of genetic engineering and biosynthesis, in particular to the biosynthesis and application of Mycoleptodiscin type indole sesquiterpene compounds. Background technique [0002] Mycoleptodiscin A / B was first isolated from the tropical plant Desmotes in comparabilis Mycoleptodiscus sp.F0194 by Cubilla-Rios et al. in 2013 (Cubilla-Rios, L. et al., Mycoleptodiscin A and B, cytotoxic alkaloids from the endophytic fungus Mycoleptodiscus sp.F0194 . J. Nat. Prod. 2013, 76, (4), 741-744.). In the evaluation of biological activity, mycoleptodiscin B showed significant growth inhibitory activity on tumor cells H460, A2058, G522-T1 and PC-3 with IC500.6-0.78μM, while mycoleptodiscin A was not evaluated for biological activity because of its low biomass . Later, researchers reported that Mycoleptodiscin B showed significant growth inhibitory activity of Bacillus subtilis (MIC 0.5 μg / mL) and Staphylococcus aureus (MIC 1 μg / mL) (D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P17/10C12N9/10C12N15/54C12N9/02C12N9/00C12N15/53C12N15/52C12N15/81C12N15/80C12N1/19C12N1/15C07D209/08C07D209/12C07D209/56A61P35/00A61P31/04A61P31/10C12R1/645C12R1/69C12R1/865
Inventor 秦盛莹唐小龙胡丹高昊
Owner THE FIRST AFFILIATED HOSPITAL OF JINAN UNIV