Construction Of Genetic Transformation System Of Aspergillus Flavipes And Study On The Function Of LaeA

This thesis consists of three parts: the first part is the construction of genetic transformation system for Aspergillus flavipes.The second part is the targeted knockout of genes regulated by Aspergillus flavipes.The third part is the metabolic analysis of Lae A mutant and the isolation and identification of differential signals.The fourth part is the summary and prospect.The fifth part is the summary of the research progress on the regulation of fungal secondary metabolites.Microorganisms produce a large number of special metabolites with various structures,which have a wide range of biological activities.However,the fact that many specialized metabolite biosynthesis gene clusters(BGCs)are not expressed in laboratory culture hinders the realization of this potential.In this paper,we discussed the strategy of silent BGC expression in fungi,and the methods of separation and structure characterization of metabolites.The research object of this paper is Aspergillus flavipes.Firstly,we explored the gene knockout method of Aspergillus flavipes.This part of work is very basic and important for the study of the whole biosynthesis of Aspergillus flavipes.Secondly,we studied the influence of five regulatory genes on the metabolites of Aspergillus flavipes.It was found that the mutant of Lae A gene knocked out by Aspergillus flavipes produced a new compound flavipemycin A and three known compounds with increased yield.We used the reverse silica gel column,dextran gel Sephadex LH-20 and semi preparation column to separate these 4 compounds,and used NMR,IR,UV and CD for structural characterization.At the end of this paper,we also reviewed the progress of the research on the regulatory genes of fungal secondary metabolites.We briefly summarized the main concepts of the genomic driven discovery of microbial natural products,and discussed the conditions for the development of multi validity and pathway specific methods to induce the production of non or low expression metabolites in conventional laboratories.