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Steroid side chain modification gene and its application

A technology of steroidal compounds and compounds, applied in the field of biosynthesis of steroidal compounds, can solve problems such as limiting the biosynthesis of green glomycin compounds

Active Publication Date: 2020-05-01
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the study of the biosynthetic mechanism of green glomycin compounds, there are still many key issues that have not been resolved, especially the reaction mechanism and key genes of the side chain break modification at the C17 position, which greatly limits the potential of green glomycin. biosynthesis of compounds

Method used

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  • Steroid side chain modification gene and its application
  • Steroid side chain modification gene and its application
  • Steroid side chain modification gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] The acquisition of embodiment 1 candidate gene

[0158] After whole-genome sequencing of Nodulisporium Sp. (65-12-7-1), which is rich in demethoxyviridin, the related functional genes were predicted by gene prediction software, and A gene cluster related to the synthesis of demethoxygreen glomycin was obtained ( figure 1 b), found 3 new genes VidF (polypeptide sequence as shown in SEQ ID NO: 1, nucleic acid sequence as shown in SEQ ID NO: 4), VidP (polypeptide sequence as shown in SEQ ID NO: 2, nucleic acid sequence as shown in SEQ ID NO: 2) ID NO: 5) and VidH (the polypeptide sequence is shown in SEQ ID NO: 3, the nucleic acid sequence is shown in SEQ ID NO: 6) as the final candidate research genes.

Embodiment 2

[0159] Example 2 gene knockout experiments proved that these three genes are closely related to the break and modification of the C17 side chain

[0160] The CRISPR / Cas9 knockout system was used to knock out the selected three genes (vidF, vidP, vidH) one by one, and the previously constructed Nodulisporium sp. (No.65-12-7-1) JN001 stably expressing the Cas9 protein Three stable knockout strains were constructed from strains (Zheng, Y.-M. et al. Development of a versatile and conventional technique forgene disruption in filamentous fungi based on CRISPR-Cas9 technology. Sci. Rep. 7, 9250, 2017) ΔvidF-JN1001, ΔvidP-JN1001 and ΔvidH-JN1001.

[0161] It can be seen from the results that ( figure 2 ), in the JN1001 strain of the control group, demethoxygreen glomycin (compound 1) was highly expressed, but in the knockout strains of the three genes, no demethoxygreen glomycin could be produced. Among them, in ΔvidF-JN1001, compound 7 or 6 was enriched, while in ΔvidP-JN1001, onl...

Embodiment 3

[0162] Example 3 VidF, VidP and VidH catalyze the cleavage and modification of the C17 side chain

[0163] In order to determine the functions of the three genes, recombinant VidF was purified in Escherichia coli ( Figure 4 ), carried out in vitro enzyme catalysis test, the results showed that VidF can catalyze the Bayer-Villiger oxidative rearrangement reaction (Baeyer-Villiger reaction) at the C17-C20 position of compound 7 (3-Dihydrovirone), thus obtaining compound 5 (Nodulisporiviridin N) ( Figure 5 ). Since VidP and VidH cannot be expressed in Escherichia coli, VidP and VidH were respectively introduced into Aspergillus oryzae A. Compound 5 (the catalytic product of VidF) is used as a substrate to produce compound 4 (Nodulisporiviridin M) with a hydroxyl group at the C17 position; VipH can further use compound 4 as a substrate to catalyze the conversion of the hydroxyl group at the C17 position to =O, Thereby obtaining compound 9 (Nodulisporiviridin K) ( Image 6 ),...

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Abstract

The invention belongs to the field of genetic engineering and biosynthesis, particularly relates to biosynthesis of steroid compounds and provides genes which play key roles in side chain breaking insynthesis of viridin compounds and are named VidF, VidP and VidH respectively and coding polypeptides of the genes. Polypeptide sequences are shown in SEQ ID NO:1-3. Nucleotide sequences are shown inSEQ ID NO:4-6.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biosynthesis, in particular to the biosynthesis of steroid compounds. Background technique [0002] Since the discovery of viridin in 1945 (Brian, P.W. & McGowan., J.C.Viridin: a highlyfungistatic substance produced by Trichoderma viride. Nature 156, 144-145 (1945)), a large number of studies have shown that this type of compound has a spectrum of antifungal effects, Anti-inflammatory and anti-bacterial activities, in particular, have also been found to be nanomolar PI3K-based enzyme inhibitors, of which wortmannin has been developed as a commercial PI3K inhibitor and is widely used. Due to the important role of the signaling molecule PI3K in tumors, green glomycin compounds as PI3K inhibitors have been found to have important anti-tumor activities, among which PX-886, a semi-synthetic analogue of wortmannin, has recently entered the II phase clinical research. Due to their special structu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N9/04C12N9/16C12N15/53C12N15/55C12P33/20C12P33/00
CPCC12N9/0006C12N9/0071C12N9/16C12P33/005C12P33/20
Inventor 高昊胡丹陈国栋姚新生秦盛莹王高乾吕建明李韶阳
Owner JINAN UNIVERSITY
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