Investigation On Preliminarily Catalytic Mechanism Of A Terpenoid Synthase From Leucosceptrum Canum And Metabolites Of Three Microorganisms

This dissertation is composed of four chapters.Chapter 1 describes the catalytic mechanism of a diterpenoid/sesterterpenoid synthase from Leucosceptrum canum.Chapter 2 reports the metabolites and their antibacterial activity of an engineered Escherichia coli Lhy.Chapter 3 expounds the chemical constituents and their bioactivity of two medicinal fungi Shiraia bambusicola and Hypocrella bambusae.Chapter 4 mostly states about the metabolites and their biological activities of H.bambusae and drug-delivery of hypocrellins.Chapter 1 Investigation on preliminarily catalytic mechanism of a terpenoid synthase from Leucosceptrum canumSesterterpenoids,which consist of five isoprene structural units,are derived from geranylfarnesyl diphosphate(GFPP)which could be cyclized by sesterterpenoid synthases to generate various sesterterpenoids.In previous research,we cloned and identified a novel bifunctional diterpenoid/sesterterpenoid synthase LcTPS25 from the glandular trichomes of L.canum.Its active center and regulation mechanism controlling two kinds of terpenoid products are worthy of investigation.Based on conventional molecular biology techniques,11 mutant enzymes of LcTPS25 were obtained by site-directed mutagenesis.Enzyme activity using in vivo analysis showed that seven mutant enzymes including LcTPS25-277Y/W,-277Y/A,-415I/S,-429W/G,-447T/A,-505N/A and-509V/F resulted in the significant decrease of both sesterterpenoids and diterpenoid synthase activities,among which LcTPS25-277Y/W produced neither diterpenoid nor sesterterpenoids.The LcTPS25-411L/A resulted in little impact in both sesterterpenoid and diterpene synthase activity.Nevertheless,LcTPS25-516F/A showed significantly increase of sesterterpenoid synthase activity.In addition,two mutant enzymes LcTPS25-419M/G and-512R/A also led to the increase of sesterterpenoid synthase activity.Besides,the function of LcTPS25 in planta was also studied through transient co-expression of LcTPS25 and GFPP synthase in Nicotiana benthamiana.Two transient expression vectors of LcTPS25 and GFPP synthase were constructed,and then co-transformed into N.benthamiana using Agrobacterium-mediated approach.It’s found that enzyme active products in N.benthamiana were identical with that in E.coli,which suggested that the function of LcTPS25 might not be affected by expressing host.This study has uncovered the preliminarily catalytic mechanism of LcTPS25,which lays the foundation for further investigation on catalytic mechanism of plant sesterterpenoid synthase,and provides scientific basis for biosynthesis and metabolic engineering of sesterterpenoids.Chapter 2 Metabolites and their antibacterial activity of an engineered Escherichia coli LhyNematodes are pathogenic microorganisms,and plant parasitic nematodes have caused great harm to crops all over the world.Thermolides,a kind of metabolites derived from bacterial type hybrid polyketone synthase-non ribosomal peptide synthase,showed significantly nematicidal activity.An engineered E.coli Lhy,containing a polyketone synthase-non-ribosomal peptide synthase cluster,an alpha/beta hydrolase fold,a glutathione S-transferase,a P450,a major facilitator superfamily and an enoyl reductase was constructed previously.The metabolites of the ethyl acetate extraction from the fermentation broth of engineered E.coli Lhy were separated and purified by silica column chromatography,RP18 column chromatography and semi-preparation high performance liquid chromatography(HPLC).Fifteen compounds were isolated and characterized by spectroscopic techniques.Seven of them are acyl substitute of chloramphenicol(glhy9-glhyl5).Bioassay revealed that the antibacterial activities of glhy10-glhyl5 were lower than that of chloramphenicol,suggesting bacterial could convert chloramphenicol into low toxic derivative by acylation,which provides the basis for further studies on the detoxification mechanism of chloramphenicol in bacterial.Chapter 3 Chemical constituents of Shiraia bambusicola and Hypocrella bambusae and their cytotoxic activity.Shiraia bambusicola and Hypocrella bambusae are Chinese traditional medical fungi using to treat with gastrosia and rheumatic arthritis.The major chemical components of these two fungi are the same,but the differences between them are rarely reported.Fourteen and eight compounds were isolated by various column chromatographies and characterized by spectroscopic techniques from their methanolic extraction,respectively.Six common compounds are hypocrellin A(gSbl),hypocrellin B(gSb2),hypocrellin C(gSb3),ergosterol peroxide(gSb6),3,6,8-trihydroxy-1-methylanthone(gHb7)and 3,8-dihydroxy-6-methoxy-l-methylanthone(gHb8).Furthermore,HPLC analysis showed that hypocrellin A-C are the same three major constituents of them.In addition,compounds ergosta-7,22E-dien-3β,5α,6β-triol(gSb8)and ergosta-7,22E-diene-2β,3α,9a-triol(gSb1O)from S.bambusicola,and compounds hypocrellin D(gHb4),(+)-grifulvin(gHb5),gHb7 and gHb8 from H.bambusae were reported for the first time.The major distrinct constituents are gSb4 and gHb5.Bioassy studied showed that gSb4 exhibited potent cytotoxic activity against human lung adenocarcinoma,human carcinoma and human breast cancer cells.gSbl also showed significant inhibitory activity against three cell lines,but the cytotoxic activity of gHb5 was relatively weak.This study suggested S.bambusicola might have better application prospect than H.bambusae in antitumor field,which provides reference value for application and utilization of these two Chinese traditional medicines.Chapter 4 Chemical constituents and their biological activity in Hypocrella bambusaeThis review summarized the chemical constituents of H.bambusae and the research advance of their biological activities reported until 2019 and drug-deliveries of hypocrellins.