Research On The Biosynthesis Of Rutamycin And Excavation Of Its Secondary Metabolites

Rutamycins,a kind of 26-membered macrolide,display broad-spectrum antifungal activity.And rutamycins could block the H⁺translocation by binding to the core of F₀ which acted as H⁺channel.So they could inhibit both ATP synthesis and hydrolysis and inhibit the process of oxidative phosphorylation.What's more,rutamycins could also induce the apoptosis of cancer cells,which had an important application prospect in the treatment of cancer and other diseases.By analyzing the chemical structures and the biosynthetic gene cluster of rutamycins,we found that the loading AT domain in the rutamycin PKS recognizes a methylmalonyl-CoA or other types of malonyl-CoA as a starter acyl unit and the subsequent modules extend the acyl chain with 16 extender units,such as malonyl-CoA,methylmalonyl-CoA and ethylmalonyl-CoA.Finally,the acyl chain is hydrolyzed to be macrolides by the TE domain.But the origin of extender units in the rutamycin biosynthesis is not clear.In this dissertation,we tried to understand the biosynthetic pathways of the extender units in the rutamycins producing strain.Firstly,we did a large-scale fermentation of Streptomyces pactum KIB-HL8 to isolate the pure secondary metabolites.And eleven compounds were isolated and purified from the crude extract by silica gel column,Sephadex LH-20 chromatography and semi-preparative HPLC.Their structures,including two new ones,were identified by ¹H NMR,¹³C NMR,2D NMR and MS.One of new compounds is an analogue of rutamycin A.In addition,we found that rutamycin A displayed strong inhibitory activity against Curvularia lunata?Walk?Boed,Fusarium oxysporum f.sp.Vasinfectum,Riziocotinia solani and Fusarium oxysporum.Secondly,we obtained five overlapping cosmids covering the entire rut gene cluster.By individual gene inactivation of rutD and rutE,we found these genes have an effect on the biosynthesis of rutamycins.So we suggested ethylmalonyl-CoA is converted from acety-CoA by the catalysis of thiolase,3-hydroxyacyl-CoA dehydrogenase,ctotonase and crotonyl-CoA reductase.The aims of this study were to find new analogues of rutamycin A and other secondary metabolites from Strepomyces with better antimicrobial activities.Besides,the study of extender units'origin in the rutamycin biosynthesis established foundation to the improvement of the rutamycin-producing strains in genetic engineering.