Deciphering The Role Of Membrane Proteins In The Production Of Cellulases In Penicillium Oxalicum

The cellulase component secreted by Penicillium oxalicum is more abundant and reasonable than Trichoderma reesei,and the p-glucosidase enzyme activity is higher,which has become a research hotspot of biorefinery.Penicillium oxalicum strain HP7-1 is a filamentous fungus that can be used to degrade lignocellulose efficiently from the original forest.The P.oxalicum HP7-1 genome and transcriptome assays have been completed,and the mechanism of cellulase expression regulation has also been revealed.The production of cellulase involves many aspects such as induction of cellulose,regulation of gene expression,synthesis,transport and secretion of cellulase.Transporters play a key role in transporting soluble sugars into cells and forming cellulose-induced signals.For lignocellulose-degrading fungi,the sugar molecule itself is both a carbon source and an important signaling molecule,which is closely related to the induced expression of lignocellulose-degrading enzymes.Therefore,the study of membrane proteins,especially sugar transporters,related to cellulase synthesis is of great significance for further understanding of the degradation and utilization of cellulose.In this study,the PyrG conversion system of Penicillium oxalicum reusable screening marker was first constructed to solve the problem of insufficient screening markers in the transformation system,which facilitated gene knockout and genetic transformation of Penicillium oxalicum.First,the PyrG deletion strain ?ku70APyrG-K(using the kan as a selection marker to knock out the PyrG gene)as the host strain,construct a kan deletion cassette(5kan-T-PyrG-T-3kan),and add the same fragment termination on both sides of the selection marker PyrG.The subsequence TT facilitates subsequent self-shearing.Then,the kan deletion cassette was transformed into ?ku70APyrG-K,and the PyrG label and its self-cleavage were separately screened by using m-met basic medium,PDA medium supplemented with 5'-fluoroorotic acid(5'-FOA)and uracil.Finally,the mutant strain ?ku70?PyrG with the PopyrG gene knockout and no selection marker kan was obtained.The reproducible genetic transformation system with Aku70APyrG as the starting strain and PyrG as the screening marker was successfully constructed.By analyzing the differential transcriptome of P.oxalicum cellulase-producing bacteria or conditions(HP)and low-yielding bacteria or conditions(LP),the transcription levels were significantly different(Fold Change>2.0,P-value and FDR values were ?0.05).3 membrane proteingenes:fibrillar oligotransporter CDT-C(POX06051),glucose/galactose transporter(POX06641),cell wall hydrophobin RodA(POX01764)and a putative aminodeoxylated acid synthase(Salicylate synthase)The gene(POX07269)is a candidate gene.First,four candidate gene knockout mutants ?POX06051,?POX01764,?POX06641,and APOX07269 were constructed,respectively.The amount of secreted proteins of ?POX06051,?POX01764,?POX06641,?POX07269 and?Ku70 under the conditions of Avicel-induced culture of wheat bran was determined,and it was found that there was no significant difference in knockout of POX06051,POX01764,POX06641,POX07269 compared with ?ku70.Under the condition of wheat bran Aicel culture,the?-glucosidase activity of ?POX06051,?POXO1764,?POX06641,?POX07269 and Aku70 increased,and the enzyme activity of mutant strain ?POX01764 increased by 138%compared with ?ku70.The xylanase activity of ?POX06051,?POX01764,?POX06641,?POX07269 decreased compared with ?Ku70,and the xylanase activity of the mutant strain ?POX06051 decreased by 74.5%.Under the conditions of starch culture,the mutant strain APOX06051 cassava amylase activity and soluble amylase activity decreased by 59.7%and 64.5%compared with ?ku70.It is suggested that membrane proteins may be involved in the process of cellulase,hemicellulase and amylase secretion.This study provided theoretical guidance for the transformation of membrane protein to increase the production of penicillium secreted protein.