真菌烯化吲哚生物碱的生物合成及Perforalactone CD 促溶酶体生成研究

	真菌烯化吲哚生物碱的生物合成及Perforalactone CD 促溶酶体生成研究; Study of Fungal Prenylated Indole Alkaloids Biosynthesis and the Abilities to Induce Lysosomal Biosynthesis Activity of Perforalactone CD
	刘帅
导师	邸迎彤
摘要	The fungal prenylated indole alkaloids are mainly derived from the Penicillium and Aspergillus. These indole alkaloids have diverse biological activities of medicinal value such as antitumor and antibacterial. Due to its structural diversity and wide range of biological activities, prenylated indole alkaloids bearing bicyclo[2.2.2]diazaoctane core has been a hot topic of research in natural product chemistry. The biosynthetic pathway and the related enzymatic catalytic mechanism still leave much to be desired. The biosynthetic pathway was elucidated by bioinformatic analysis, in vivo gene knockout, heterologous expression of gene clusters, in vivo feeding of precursor compounds, in vitro enzyme catalysis, co-localization of enzymes, docking, site-directed mutagenesis and DFT computational. Detailed elucidation of the enzymatic catalytic mechanism of the pyran ring, 3S-spiro-ψ-indoxyl，spiro-2R-oxindole，3R-hydroxyindolenine and bicyclo[2.2.2]diazaoctane ring system in nature. This study provides a firm theoretical and material basis for further research discoveries on such compounds with medicinal value. (1) Identification of the spe gene cluster. First, the genome of Aspergillus sp. KM017 was sequenced, and then antiSMASH analysis identified a gene cluster spe associated with prenylated indole alkaloids biosynthesis. Bioinformatic analysis showed that the spe gene cluster contained a total of seven genes, including non-ribosomal polypeptide synthase (NRPS) SpeA, prenyltransferase SpeB/D, P450 monooxygenase SpeC/G, FAD-dependent oxidoreductase SpeE, FAD-dependent monooxygenase SpeF. RNA-seq showed that all seven genes were expressed between 3 and 9 days. We isolated the compound 7 accumulated in the mutant strain ΔspeC. It was demonstrated that gene cluster spe and DPIA biosynthesis are related. (2) SpeABCD is involved in the biosynthesis of 9. We heterologously expressed the four enzymes SpeABCD by Aspergillus nidulans A1145. An-SpeAB, An-SpeABC, and An-SpeABCD produced the compounds 7-9. So speACBD is involved in the biosynthesis of 9. (3) FAD oxidoreductase SpeE catalyzed 6π-electrocyclization to form pyran ring. An-SpeABCDE strain can produce 10 and the mutant ΔspeE can accumulate the intermediate 9. The determination of 10 absolute configurations is by X-ray. Feeding experiments and in vitro assays confirmed that SpeE catalyzed 9 to form pyran ring in compound 10. Culture solution reaction demonstrated that SpeE with a signal peptide is a secreted protein. DFT calculation revealed that SpeE, a FAD oxidoreductase with a BBE structural domain, can catalyze the dehydrogenation of C6-OH in 9 to form the oQM intermediate which then form the pyran ring in 10 via 6π-electrocyclization. (4) Cytochrome P450 monooxygenase SpeG and FAD monooxygenase SpeF synergistically catalyzed to form prenylated indole alkaloids bearing bicyclo[2.2.2]diazaoctane core. As the knockout mutant strain ΔspeG can accumulate the intermediates 11-12, it can be hypothesized
	2021-11
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74630
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	刘帅. 真菌烯化吲哚生物碱的生物合成及Perforalactone CD 促溶酶体生成研究, Study of Fungal Prenylated Indole Alkaloids Biosynthesis and the Abilities to Induce Lysosomal Biosynthesis Activity of Perforalactone CD[D],2021.

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