Studies On Co-regulation Mechanism Of Sporulation And Cellulase Expression In Penicillium Oxalicum

Lignocellulosic biomass is the largest reserves of renewable resource on the earth. With the intensification of fossil fuels shortage and environment pollution, effective use of these low-cost resource to produce biofuel and biochemical is an efficient way to protect environment and maintain sustainable economic development. To achieving these goals, lignocellulosic materials are hydrolyzed to fermentable sugars by fungal enzymes is one of the most widely accepted biorefinery schemes.The filamentous fungus Penicillium oxalicum can secrete balanced extracellular lignocellulolytic enzyme system and the JU-A10-T obtained by multiple rounds of mutagenesis has been used for industrial-scale cellulase production for16years. Thus, deep investigation of P.oxalicum cellulase regulation mechanisms would be important for making full use of lignocellulose and the other renewable resources, as well as promoting economic and social sustainable development. However, the conidiation ability greatly decreased in the mutant strains. Therefore, the combination of filamentous fungus conidia and cellulase production will provide a theoretical basis for further high yield strain improvement.The main results of the thesis are as follows:1. Study of the global regulator LaeA in regulation of conidia and cellulase production.LaeA is a highly conserved global regulator of filamentous fungi, involved in regulating primary metabolism, secondary metabolism and asexual development.After deletion of LaeA, the number of conidia was sharply dropped when incubated in glucose, lactose, glycel or MCC (microcrystalline cellulose) as the sole carbon source medium, indicating that LaeA plays an important role on regulating the asexual development. Furthermore, double knockout strain AlaeAAcreA conidiation was almostlly suppressed, suggesting that CreA is not only a global regulator of cellulase production but also a key regulator of conidiation. In addition, when strains were cultivated in CW (cellulose+wheat bran) medium, the cellulase activity of AlaeA was up-regulated in the early stage of the fermentation but declined rapidly in the late stage. Morever, expression levels of the major extracellular lignocellulolytic enzyme genes were completely consistent with this. Furthermore, the cellulase activity of double knockout strain AlaeAAcreA, overexpression strains ΔlaeA-PgpdA::clrB and AlaeA-PgpdA::xlnR were consistent with ΔlaeA but higher in the early stage of fermentation. Besides, the cellulase activity of"WT-PgpdA::laeA and A10T-PgpdA::laeA were declined slightly.2. Studies on the mediation between LaeA and BrlA in regulation of conidia and cellulase production.The transcription levels of brlA was almost silent by determining the expression levels of the major asexual development genes of AlaeA. After deletion of BrlA, the mutant blocks asexual sporulation compared with the wild-type strain. The occurrence of higher branching frequency was found in ΔbrlA mutant strain.On the third day of fermentation, ΔbrlA indicated an89.1%increase in the cellulase activity compared with the wild-tpe strain. Morever, expression levels of the major extracellular lignocellulolytic enzyme genes were completely consistent with this. Putative BrlA-binding sites were found in almost all the major extracellular lignocellulolytic enzyme promoters. BrlA is assumed a negative regulator of cellulase expression. Without affecting cellulase activity, overexpression BrlA in JU-A10-T leads to3.4times rising of the conidia. Comprehensive analysis shows that the LaeA regulate conidia and cellulase production partially tthrough mediating BrlA. In order to obtain direct experimental evidence, we successfully obtained LaeA protein-protein interactions and BrlA protein-protein interactions respectively using TAP pull-down techniques. And this will provide a solid foundation for analysis of its functions.