Study On The Functions Of Regulatory Proteins Involved In The Hyphae Morphology And Protein Secretion Of Filamentous Fungi

In the application of industrial enzyme production technology,cellulase and hemicellulase are used to degrade lignocellulose and other renewable resources to transform into various low-carbon and environmentally friendly biofuels and various chemical products.It is of great significance to slow down the consumption of non-renewable resources,environmental protection,and sustainable resource and economic development.Filamentous fungi represented by Trichoderma reesei and Penicillium oxalicum are the main producers of lignocellulose degrading enzymes.Studies have shown that extracellular proteins are mainly secreted from the apex of the hyphae.The morphology of filamentous fungi affects the production of extracellular proteins and some strains with altered hyphae morphology show higher enzyme production capacity.Mycelial growth,secretory vesicle transport and protein secretion are closely related,suggesting that they are key factors for industrial filamentous fungi to produce extracellular proteins.CrzA(calcineurin-responsive zinc finger 1)is an important transcriptional regulator involved in the process of fungal cell wall remodeling.As an important downstream response factor of calcineurin(CN)in cells,CrzA plays an important role in regulating the formation of fungal hyphae.Exocyst complex is an important tethering factor in the tethering process between vesicles and plasma membrane.It is essential for the constitutive secretion and polarizing exocytosis of cells,and affect the polar growth of hyphae and the apical secretion of extracellular proteins.The research of this paper is mainly divided into two parts:(1)The regulation mechanism of CrzA in the mycelial morphology and protein secretion of P.oxalicum was studied,and the interaction between CrzA and other proteins was explored.The possible mechanism by which CrzA regulates transcription and therefore affects the synthesis of extracellular cellulase was proposed.(2)The subunits of the Exocyst complex were identified in T.reesei which has strong protein secretion ability.And the functions of the four more important subunits of Sec3,Sec5,Sec15 and Exo70 in the hyphae morphology and protein secretion process of T.reesei were preliminary explored.The research contents and results are as follows:1.Study on the functional of CrzA in P.oxalicum.Taking P.oxalicum 114-2 as the starting strain,the effects of CrzA on mycelial morphology,the transcription of cellulase-encoding genes,and cellulase synthesis were studied.The results show that CrzA plays an important role in the colony extension,conidia formation and extracellular protein secretion of P.oxalicum.The normal expression of CrzA is the key factor of the growth of the hyphae,the normal transcription of cellulase-encoding genes and the normal secretion of extracellular proteins.The deletion of CrzA severely damages the hyphae morphology of P.oxalicum,which is mainly reflected in the decrease in colony diameter,damage to hyphae extension,increase in hyphae branching,sharp reduction in spore yield,and no loosening of mycelium during liquid fermentation,instead,it presents a denser form of mycelium pellets.The deletion of CrzA significantly down-regulates the expression levels of conidia pigment synthesis genes and spore wall synthesis-related genes of P.oxalicum,and also has a regulatory effect on the expression levels of genes related to the sporulation process.The transcription levels of BrlA,FlbA,FIbB,FlbC,and StuA coding genes in ?crzA are significantly reduced.The above five proteins have been proved to be early regulatory transcription factors that control the conidia formation in P.oxalicum,which explains the reason for the reduction of conidia in ?crzA mutant.Compared with the starting strain 114-2,the ?crzA strain's ability to produce cellulase and the total amount of extracellular protein secreted are greatly reduced.The results of expression profile analysis showed that the absence of CrzA can broadly down-regulate the expression levels of cellulase-encoding genes,indicating that CrzA can cause a decrease in cellulase synthesis by affecting the expression of cellulase-encoding genes.Using tandem affinity purification technology has captured 6 proteins,including Cyc8,that interact with CrzA protein,combined with the results of transcriptome studies,three possible ways for P.oxalicum CrzA to regulate cellulase gene expression are proposed:(1)CrzA directly binds to the promoter region of certain cellulase-encoding genes,and then recruits Tupl-Cyc8 complex,to activate the expression of cellulase-encoding genes.When CrzA is absent,the expression of cellulase-encoding genes is inhibited;(2)After CrzA binds to the promoter of Xyr1 cellulase-encoding gene,it activates the expression of gene xyr1.When CrzA is absent,the expression of gene xyr1 is down-regulateed and indirectly inhibits the expression of cellulase-encoding gene;(3)When CrzA is absent,the transcriptional factor CreA recruits more Tupl-Cyc8 complex,and inhibits the expression of cellulase-encoding genes.2.Preliminary study on the function of Exocyst complex in T.reesei.Eight subunits(Sec3?Sec5?Sec6?Sec8?Sec10?Sec15?Exo70 and Exo84)of the Exocyst complex in T.reesei which has strong protein secretion ability have been identified,and confirmed that they are all necessary proteins for the growth of the strain,their deletion caused the death of fungus.The four more important subunits,Sec3 and Exo70 which directly interact with the plasma membrane,Sec15 which directly interacts with vesicles,and the central protein Sec5 that mediate the interaction between the remaining subunits,were selected,and the copper ion response promoter replacement strategy was used to explore their functions in T.reesei.The results of the study show that the four subunits of Sec3,Sec5,Sec15,and Exo70 are closely related to the process of T.reesei hypha apex extension and hyphae branching,reflecting the important role of the Exocyst complex in the growth of the hypha apex.The colony of the mutant strain Ptucl-sec5 almost no longer extends in the agar medium containing 80?M Cu2+,the hyphal growth unit length is reduce by 81.5%compared with the original strain,and the hyphae branches are significantly increased.It indicates that Sec5,as the central protein that mediates the interaction between the other subunits of the Exocyst complex,plays an extremely important role in the polar extension and branching of the mycelium.Under different carbon source induction conditions,the mutant strains Ptcul-sec5,Ptcul-sec15 and Ptcu1-exo 70 show different protein secretion capabilities.When the expression level of Sec5 is down-regulated,Ptcul-sec5 mutant strains shows lower protein secretion ability in pure cellulose induction medium containing copper ions,indicating that Sec5 plays a positive regulatory role in this process.However,Ptcu1-sec15 and Ptcu1-exo 70 don't show significant differences under the same conditions.In addition,when Ptuc1-sec5,Ptcu1-sec15 and Ptcu1-exo70 are cultured in the cellulase induction medium containing 2%corn steep liquor and 2%microcrystalline cellulose,none of them show significantly different extracellular protein secretion ability from the starting strain.It indicates the complexity of regulation of the important subunits of Sec5,Sec 15,and Exo70 when they response to different exogenous signals.