Study On The ?-L-arabinofuranosidases From Penicillium Oxalicum And The Construction Of Hemicellulase High-producing Strains

Enzymatic hydrolysis of polysaccharides in plant cell wall is of importance in several industrial processes.Arabinofuranosyl residues on side chains of hemicellulose and pectin are major components in many plant cell walls.?-L-arabinofuranosidases can remove L-arabinoses from these polysaccharides and their addition can effectively improve the conversion efficiency of xylan to xylose and promote the overall biomass to soluble sugar.Their important applications range from corn starch and animal feed processing to biofuel production,etc.In addition,hemicelluloses in plant cell walls form a complex structure.Arabinofuranosyl residues linked to xylan backbone can be esterified with ferulic or p-coumaric acids and then cross-linked with other arabinoxylan chains or lignin to form a network structure that greatly impact the full degradation of the lignocellulose substrates.So,the full degradation of lignocellulosic biomass often requires synergy of a series of hemicellulases.The further optimization of hemicellulase components on the basis of the high-yield cellulases of existing strains is conducive to improving the hydrolysis efficiency of hemicellulose-rich biomass and reducing the costs of biomass conversion.Penicillium oxalicum is an industrially important filamentous fungus that produces lignocellulose degrading enzymes,and its genome contains diverse hemicellulase genes.Finding new good-performance arabinofuranosidases and constructing hemicellulase high-yield genetically engineered strains will benefit the optimization of lignocellulose-degrading enzyme systems,utilization of hemicellulose,and further promotion of the enzymatic hydrolysis process of complex polysaccharides.In this thesis,we mainly studied the ?-L-arabinofuranosidases from P.oxalicum and constructed several hemicellulase high-producing strains.The main research contents and results are as follows:1.Effect of transcription factor AraR mutant on ?-L-arabinofuranosidase system of Penicillium oxalicumA constitutively active mutant of AraR was constructed by introducing the A731V mutation based on the sequence homology between transcription factors AraR and XlnR of P.oxalicum.The mutant AraRA731V driven by a constitutive promoter was overexpressed in P.oxalicum CXC,a strain exhibiting cellulose-independent cellulase production,and the resulting strain was named CXc-gAraRA731V.The AraRA731V-overexpressing strain can synthesize ?-L-arabino1Uranosidases in carbon starvation medium and the level of ?-L-arabinofuranosidase activity was comparable to that of wild-type strain cultivated in the cellulose inducing medium.Compared with CXC,the CXC-gAraRA731V strain showed a 54-fold increase in?-L-arabinofuranosidase production,an activity of 28.7 U mL-1 was achieved at 120 h under an inducing condition,which was generally higher than those reported in the literature.Transcription levels of 11 ?-L-arabinofuranosidase and/or ?-xylosidase genes were significantly upregulated in CXC-gAraRA731V relative to CXC.In addition,CXC-gAraRA731V also showed a 7.4-fold increase in ?-galactosidase production relative to CXC and the transcription level of aga27A encoding ?-galactosidase was upregulated by 17.4-fold in CXC-gAraRA731V relative to CXC.In the degradation of natural substrate gum arabic,the crude enzyme from CXC-gAraRA731V produced 0.98g L-1 of reducing sugar,which was 4.5 times higher than that of CXC with the same protein dosage.2.Cloning and expression of ?-L-arabinofuranosidases from Penicillium oxalicum To construct a P.oxalicum protein expression system,cbh1 of P.oxalicum was overexpressed under the control of constitutive promoter PubiC or PpgmA in M12,an uracil auxotrophic strain as the host for protein expression here,with pyrG as the selection marker gene.The recombinant strains cultivated in glucose medium exhibited high expression levels of the target protein CBH1,and the crude enzymes of the recombinant strains showed cellobiohydrolase activity.Seven?-L-arabinofuranosidases predicted in the annotation of P.oxalicum genome were successfully expressed using the above new protein expression system,and the hydrolytic activity of the crude enzymes on the synthetic substrate pNPA was determined.Only Abf51 A?PDE 06546?and Abf54A?PDE₀9988?exhibited obvious pNPAase activity,and other proteins showed no hydrolysis activity on pNPA.3.Construction of hemicellulase high-producing strains of Penicillium oxalicumDeletion of carbon catabolite repressor CreA was combined with the overexpression of AraRA731V in CXC strain and the resulted strain gAraRA731V-AcreA showed hyperproduction of ?-L-arabinofuranosidase.The ?-L-arabinofuranosidase activity of gAraRA731V-?creA reached 64.48 U/mL,which is the highest level in filamentous fungi reported so far.In addition,the deletion of CreA also increased the main cellulases and hemicellulases production to different degrees,and the extracellular crude enzymes of gAraRA731V-?creA had better performance in the degradation of corn fiber than the starting strain CXC-gAraRA731V.Via combining manipulations of the main ?-glucosidase gene bgl2 and the genes encoding hemicellulase components,high-producing strains of ?-xylosidase and ferulic acid esterase were successfully constructed respectively.For transformant RX6 strain,bgl2 was knocked out together with the overexpression of xyl3A that encodes the main?-xylosidase in P.oxalicum.The cellulase activity of crude enzymes from RX6 was obviously higher than that of the starting strain RE-6,and ?-xylosidase activity reached 22.18 U/mL,which was 38.2 times that of RE-6.For transformant RX2 strain,xyl3A was overexpressed driven by the bgl2 promoter,and its ?-xylosidase activity reached 47.76 U/mL,which was 82.3 times higher than that of the starting strain RE-6,Deletion of bgl2 was combined with the overexpression of fae1A in RE-6 and the resulted transformants RF2 and RF3 showed doubled production of ferulic acid esterase compared to RE-6,with an activity of 6.25 U mL-1 and 6.38 U mL-1,respectively.Of course,the deletion of bgl2 enhanced cellulolytic enzyme production to varying degrees in RF2 and RF3.