PKS/TPS Hybrid Natural Products From Two Mutant Strains Of Arthrobotrys Oligospora And Their Bioactivities

This dissertation consists of four chapters.Chapter 1 elaborated the investigation on specific PKS/TPS hybrid metabolites of the mutant strain ΔAOL_s00215g274 from Arthrobotrys oligospora and their bioactivities.Chapter 2 discribed the investigation on specific PKS/TPS hybrid metabolites of the mutant strain ΔAOL_s00215g278 from Arthrobotrys oligospora and their bioactivities.Chapter 3 discussed the biosynthesis pathways of arthrosporols in Arthrobotrys oligospora.Chapter 4 reviewed the advances in epoxyquinone natural products.In our previous study,a series of new sesquiterpenyl epoxy-cyclohexenoids(SECs)named arthrobotrisins A-C and arthrosporols A-C were isolated from Arthrobotrys oligospora YMF1.3170.These compounds were found to show not only inhibitory activity against the formation of conidiophores,but also regulatory ability toward the formation of two-dimensional fungal networks,thus have been considered as potential agents for biological control of nematodes.Therefore,our lab has carried out research on the biosynthesis pathways of arthrosporols.In view of the novel hybrid carbon skeleton of this family of natural products consisting of an epoxycyclohexenol coupled with a rare monocyclic sesquiterpenol substructure,key biosynthetic enzymes such as polyketide synthase(PKS)and terpene synthase(TPS)should be involved in their biosynthesis.Ten candidate genes which were presumably involved in the biosynthesis of arthrosporols were targeted,and the corresponding mutant strains were obtained by knockout of the ten genes.In this dissertation,the specific PKS/TPS hybrid metabolites of two mutant stains(ΔAOL_s00215g274 andΔAOL_s00215g278)were traced and isolated with HPLC and multiple column chromatographic techniques,and were identified by spectroscopic methods including 1D and 2D NMR,MS,UV and IR.Altogether,27 compounds including 22 new ones were obtained and characterized,among which were a number of key biosynthetic intermediates and their metabolites,and an unusual 13-membered macrolide with a new carbon skeleton.Biological assays showed that some of these PKS/TPS hybrid metabolites exhibited cytotoxic,anti-inflammatory,immunosuppressive activities and nematode paralysis effect.The results would help unravel the biosynthetic pathways of these signal small molecules from Arthrobotrys oligospora,and provide basis for further exploration of the biological functions of this unique family of natural products.Chapter 1 Specific PKS/TPS metabolites of the mutant strainΔ AOL_s00215g274 of Arthrobotrys oligospora and their bioactivitiesIt was reported by Holm et al that AOL_s00215g274 might be an important gene encoding the enzyme involved in the oxirane formation of SECs,therefore the specific PKS/TPS metabolites of the mutant strainΔ AOL_s00215g274 of Arthrobotrys oligospora was investigated.In total 11 compounds(274-1~274-11),including 11 new ones(274-1~274-11),were isolated and identified.Among them were 8 new sesquiterpenyl epoxy-cyclohexenoids(SECs)(274-2,274-4,274-5,274-6,274-8,274-9,274-10 and 274-11),a 13-membered macrolide possessing a new carbon skeleton(274-7),2 oxirane-opened SECs(274-1 and 274-3).All these compounds are either intermediates or metabolites in the biosynthetic pathway of arthrosporols.The discovery of a series of oxirane-containing SECs from mutant strainΔ AOL_s00215g274 suggested that AOL_s00215g274 should not be involved in the oxirane formation step.Biological assays indicated that 274-2 and 274-4 were significantly cytotoxic against human lung adenocarcinoma cell line NCI-H1975,human hepatoma cell line Hep G2 and human breast cancer cell line MCF-7,IC50 about 10 μM.Compound 274-5 exhibited moderate cytotoxicity against NCI-H1975,Hep G2 and MCF-7 cells.Compound 274-6 displayed weak cytotoxicity against Hep G2 and MCF-7 cells,and moderate immunosuppressive activity on the release of IL-2 at 20 μM.Chapter 2 Specific PKS/TPS metabolites of the mutant strainΔ AOL_s00215g278 of Arthrobotrys oligospora and their bioactivitiesAOL_s00215g278 was presumably another important gene involved in the oxirane formation of SECs,therefore the specific PKS/TPS metabolites of the mutant strainΔ AOL_s00215g278 of Arthrobotrys oligospora was also investigated.Fourteen compounds(278-1~278-14)were isolated and identified,including 2 known compounds 278-1 and 278-2 that were the anticipated key intermediates in the biosynthetic pathway of arthrosporols,and 11 new PKS/TPS hybrid metabolites(278-3~278-6,278-8~278-14).Among the new compounds were 4 derivatives of farnesylated phenol(278-3~278-6)and four rhamnosides(278-11~278-14),and two pairs of SEC diastereomers(278-7~278-10).Both the number and content of SECs were greatly decreased in mutant strainΔ AOL_s00215g278,suggesting that AOL_s00215g278 was likely involved in the oxirane formation of SECs.Compound 278-1 exhibited moderate cytotoxicity against NCI-H1975 and Hep G2 cells,IC50 about 50 μM.,and 278-2 and 278-11 showed similar cytotoxicity against NCI-H1975,Hep G2 and MCF-7 cells,including 278-2 displayed the more cytotoxicity,IC50 at 12 μM.Compound 278-7、278-9(40 μM),278-10(1 μM)and 278-11(20 μM)had moderate immunosuppressive activity on the release of IL-2,and 278-7 displayed weak anti-inflammatory activity on the release of IL-6 at 40 μM.Chapter 3 Biosynthesis pathways of arthrosporols in Arthrobotrys oligosporaBased on our previous work that proved the first three steps of the biosynthesis of arthrosporols,and the discovery of two important PKS/TPS hybrid intermedidates(278-1 and 278-2)as well as a series of new SECs from the two mutant strains of Arthrobotrys oligospora in this study,a biosynthetic pathway for arthrosporols was proposed.It seems that one molecule of acetyl Co A and three molecules of malonyl Co A were condensed by a polyketide synthase to form m-cresol,which was transformed into 6-methylsalicylic acid by a decarboxylase and then into toluquinol by a hydroxylase.Subsequently,toluquinol was converted into farnesyl hydroquinone(278-2)by a farnesyl transferase.Very likely 278-2 was spontaneously oxidated to produce farnesyl quinone(278-1).Farnesyl epoxy-quinones were then formed by oxidases,which yielded diversified SECs under various post-modification enzymes especially reductases and oxidases.Finally,SECs with a monocyclic sesquiterpenol like arthrosporols were produced by some sesquiterpene synthase-like enzymes.Chapter 4 Advances in epoxyquinone natural productsEpoxyquinone natural products are a special class of compounds composed of quinone or hydroquinone and oxirane substructures.This classs of natural products widely distributed around the nature and displayed extensive biological functions.In this chapter,advances in representative epoxyquinone natural products and their bioactivities in the past thirty years were reviewed.Hopefully it will be helpful for further in-depth investigation and utilization of these unique natural products.