Porteomics Analysis Of Rhizopus Chinensis With Different Mycelial Morphology In Submerged Culture

Filamentous fungi are important strains in industrial fermentation. Mycelial morphologyis closely related with the production and metabolism of the desired product. Rhizopus chinensisCCTCC M201021can form dispersed, pellet and clump mycelia in submerged fermentation,and the fermentation of membrane-bound lipase was significantly affected by mycelialmorphology. In this study, haploid strains were isolated and chosen as experimental strains,proteomics method such as two-dimensional electrophoresis was used to analyze the differentlyexpressed protein between two typical mycelial morphology. The mechanism how the fungalmorphology would affect lipase fermentation in submerged fermentation was discussed. Theresults are of great value in both associated metabolic mechanisms of filamentous fungi andregulation of lipase fermentation. The main results are as follows:(1) Conidia of R.chinensis were isolated, and30haploid strains were obtained. Insubmerged fermentation of lipase. mycelial morphology of each strain was corresponding tolipase activity, that is, the clump mycelia exhibits higher lipase activity compared to dispersedmycelia. Similar results were obtained by controlling mycelial morphology by differentinoculums. The results showed that genotype might be not the main factor that affects therelationship between mycelial morphology and lipase production.(2) Two-dimensional electrophoresis system of R. chinensis was established and optimizedafter comparison of main operating conditions, and was as follows: disrupting cell with thecombining of liquid nitrogen and mechanical agitation, precipitating proteins with TCA/acetone,passively rehydrating proteins with24cm pH4~7linear strips. The sample loading quantityfor eachstripwas1200μg. Finally,2-Dgel map withno obvious verticaland horizontal stripes,low background, high resolution and good reproducibility was obtained. Approximately800protein spots were isolated from one2-DE protein map, and the mach rate was85%.(3) Differential proteomics analysis was carried out between two typical mycelialmorphology of R. chinensis: dispersed and clump mycelia. A total of80protein spots belongingto66proteins were identified:48up-regulated spots,20down-regulated spots and12uniquelyexpressed spots. No desirable results were obtained by proteomics of membrane proteins,however, the identification of membrane proteins with the method that combines SDS-PAGEand MALDI-TOF/TOF showed lipase was the major membrane protein in R. chinensis.(3) The66differentially expressed proteins were classified with KEGG. The resultsshowed that differentially expressed proteins covers almost all aspects of cell life, illustratingthat the effect of mycelial morphology was extremely complicated. The fact that most proteinsrelated to protein folding, cell growth, carbon and lipid metabolism were upregulated,indicating that these processes might be enhanced in clump mycelia. The strengthen of processes related to protein folding, prenylation, cytoskeleton formation probably contributedto the expression and localization of membrane-bound lipase, while the difference betweensugar and energy metabolism might be the result of differences in oxygen metabolic...