Synthesis Regulation And Fermentaton Optimization Of Chaetoglobosin A

Biological pesticide is one solution to solve the current problems such as pollution,poison and drug tolerance caused by chemical pesticides.Natural products with the advantages of chemical(easy to store and transport)and living biological(environment harmlessness)pesticides received widespread attention.Chaetomium globosum is an important source of bioactive natural products,but its main antifungal substance is still not clear.Culture environment suitable for the synthesis of the active substances and other influential factors in vivo or vitro also have no definite research results.Therefore,the primary purpose of this study is to determine the main antifungal substance secreted by C.globosum and its properties.Then,use means of genetic engineering and fermentation engineering to find genes or nutrients which can promote the synthesis of active substances,aiming at the improvement of yield or productivity.In order to determine main extracellular antifungal active substances of C.globosum,isolation of pathogens was the first job.From the surface of potatoes infected by dry rot,two strains of fusarium were isolated,and the one with growth vigor was chosen for further research.Through the inhibition effects of C.globosum and its fermentative supernatant on pathogens,it was confirmed that there were antifungal active substances outside the cell,and these substances were fat soluble with acetone to be optimal extract solvent.Thin layer chromatography method was used to separate the compounds,and the antifungal effect of bands showed the main active substance in the crude extract.Mass Spectrometry and Nuclear Magnetic Resonance identified the active substance at nature culture condition to be chaetoglobosin A.According to the results of stability tests,the optimal storage condition of chaetoglobosin A was determined as low temperature(<25 oC),dry(humidity<75%)and dark environment.Antifungal activities tests showed that chaetoglobosin A could inhibit the mycelia growth and spores germination of Fusarium sporotrichioides with the IC50 and MIC value to be 4.34 and 10.50 μg/m L,respectively.Experiment in vitro showed that chaetoglobosin A could prevent potato dry rot efficiently,and this substance had potential application value for biological pesticide.In order to further determine the status of main antifungal substance of chaetoglobosin A,the cell walls of F.sporotrichioides were used to induce C.globosum to obtain an antifungal β-1,3-glucanase,of which best catalytic condition was at 45 oC and p H 5.0.The enzyme was inhibited by various heavy metal ions but activated by Ba2+,and hydrolyze β-1,3-glucan characteristically to be glucose one by one.Its antifungal activities depended on a much higher concentration(100 μg/m L)than that of chaetoglobosin A.After ruling out the possibility of induced substance,chaetoglobosin A was finally identified as the major antifungal substance secreted by C.globosum.For more higher yield or productivity of chaetoglobosin A,Cger gene,which related to the extension of C chain in polyketide synthesis,was firstly overexpressed in C.globosum.The results showed that overexpression led to the change of expression sequence and the improvement of productivity despite the constant of final yield.Secondly,in order to find the gene inhibiting the synthesis of chaetoglobosin A,a randomly inserted T-DNA was used to mutated the genome of C.globosum.After the selection of transformant with higher yields of chaetoglobosin A,the aimed gene was found by the inverse PCR.Although the production improvement brought by insertion mutation disappeared after several passages under the effect of compensation mechanism,a histone acetylation enzyme gene Cghd I with inhibiting effect on synthesis of chaetoglobosin A was found in this study.In addition to genetic engineering,fermentation engineering was another way to improve the yield or productivity of chaetoglobosin A.Firstly,through the analysis of the structure and synthetic process of chaetoglobosin A,a series of possible precursor or raw materials were added into the medium with sufficient nutrition and the yields of chaetoglobosin A in and after fermentation were detected.Based on the data,it was found that tryptophan,n-propanol and n-butanol could not only greatly improve the productivity of chaetoglobosin A,bu also increase the final yields slightly.Then,for the decrease of cost on chaetoglobosin A fermentation,a fermentation-purification process was established by using cornstalks as main substrate.Through extraction,deacidifying and degreasing on fermented products,contents of effective component in crude extract increased from 4.80% to 19.17%,and this process had no negative effect on the activity of chaetoglobosin A according to the active detection.Finally,in order to further improve the initial contents of chaetoglobosin A in fermented products,the culture condition was optimized,and the optimum value of cornstalks was 11.66 g,ammonium chloride 0.67 g and temperature 25.45 oC.The main extracellularly antifungal substance and its properties were determined in this study The related gene and metabolism regulation were researched and a low-cost cornstalk fermentation-purification process was established.All of these provided theories for the research on biological pesticides of natural products and were the basis of the application.