Reconstruction Of Enzyme System And Transcriptional Regulatory Network To Enhance Cellulase Production And Saccharification Ability Of Penicillium Oxalicum

Bioconversion of lignocellulose to liquid fuels such as ethanol and chemicals not only plays an important role in dealing with the energy crisis,but also has many long-term beneficial effects on environmental protection and social development.The large-scale industrial production of cellulosic ethanol is limited by the feedstock,high cost of cellulase and low hydrolysis efficiency.Therefore,it is possible to reduce the cost of cellulase by constructing cellulase high-producers,which is of great significance for promoting the industrial production of cellulosic ethanol.Penicillium oxalicum 114-2 is a cellulase-producing strain isolated by our laboratory.Compared with other cellulase-producing strains,it has higher hemicellulase activities and more diverse enzyme compositions,which is more suitable for the enzymatic hydrolysis of lignocellulose.By far,broad researches about the Penicillium oxalicum,for example,the regulatory network of cellulase expression,the compositions of extracellular cellulase enzyme system and the genomics and proteomics of Penicillium oxalicum,etc.,have been conducted.Based on the researches,in order to further improve expression of extracellular enzyme of Penicillium oxalicum and hydrolysis ability of the enzyme,in this study,high cellulase producing strains are successfully constructed by redesigning the enzyme system and transcriptional regulatory network of Penicillium oxalicum,further,combined with the mutagenic screening,a number of cellulase high-producing-strains with high FPase activity are obtained.The main results of this thesis are as follows:1.High cellulase-producing strains are constructed by redesigning the regulatory pathway of cellulase expression and enzyme system of Penicillium oxalicumUsing RE-7 as starting strain,the recombinant strains TEBS-1 and TEBS-2 are screened by replacing CBH with CBH from Trichoderma reesei,knockouting gene bgl2 and overexpressing EG,BGL1 from Aspergillus niger and swollenin(Swo).Recombinant strain TBgcB-2 is also constructed by replacing CBH,knockouting gene bgl2 and overexpression BGL1 and chimeric transcription factor CXC.The results show that the production of extracellular protein,FPA and the activities of major cellulases such as EG and CBH,are significantly increased when compared to the starting strain,in which,the activity of filter paper of crude enzyme from the TBgcB-2 reaches 9.89±1.04 IU/ml,about 2.15 times of that of starting strain RE-7.The corn stover pretreated by different methods including APCS(ammonium sulfite pretreated corn stover),NPCS(NaOH pretreated corn stover),SPCS(diluted sulfuric acid pretreated corn stover)and LHW(high temperature hot water pretreated corn stover),are hydrolyzed by the crude enzyme from the strain TEBS-2,and found that the conversion of cellulose to glucose at 48 h of enzymatic hydrolysis is 1.5 times,1.67 times,1.48 times and 1.57 times of that of crude enzyme from the strain RE-7,respectively,indicating that the hydrolysis efficiency of crude enzyme from the recombinant strains is greatly improved.Based on above results and previous researches,a strategy of systematic modification of strain is used to enhance cellulase yields of Penicillium oxalicum M12,a uracil deficient strain of Penicillium oxalicum 114-2.A recombinant strain named’CXT’ is obtained by deleting bgl2,creA,cbh1,along with over-expressing bgll from Aspergillus niger,CXC.xlnRA781v and CBH from Trichoderma reesei.It is found that the filter paper activity,extracellular protein concentration and hydrolysis ability of crude enzyme from the CXT are significantly improved.It shows that the modification of the lignocellulose degrading enzyme system and the reconstruction of the regulatory network of Penicillium oxalicum can effectively improve the enzyme activity and protein secretion of the engineering strain.The hydrolysis efficiency of crude enzyme solution of CXT using NPCS,SPCS and LHW as substates are also improved2.Heterologous expression of four hemicellulases in Picha pastoris X-33 and construction of engineering P.oxalicum strains with Xy13A,a-xylosidase and Cax3A high-producing abilityBased on the analysis results of components of hydrolysate and solid residues from enzymatic hydrolysis process several hemicellulase components are expressed in Pichia pastoris,including endo-β-1,4-xylanase from Penicillium oxalicum,α-glucuronidase/β-glucosaminidase,a-xylosidase from Aspergillus niger and α-glucuronidase from Trichoderma reesei.After isolation and purification,enzymatic properties of obtained pure enzyme components are studied.Combined with the researches on hemicellulase in our group,eight kinds of purified hemicellulases are obtained and the effects of the addition of hemicellulases on the conversion of cellulose in the enzymatic hydrolysis of corn stover using CXT crude enzyme are studied.The results show that the addition of β-xylosidase Xy13A,α-xylosidase,and multi-functional enzyme Gax3A with β-glucosidase,arabinofurosidase and xylosidase activity could promote the saccharification of CXT crude enzyme to some extent.The reason may be that the addition of these enzyme components can effectively hydrolyze xylooligosaccharide for reducing the inhibition on cellulase activity.The addition of the multi-functional enzyme Gax3A can also release side chain arabinose group,thus increase the accessibility of cellulase to substrate.In order to further improve the hydrolysis ability of CXT strain,CXT was used as the starting strain,β-xylosidase Xyl3A is overexpressed in strain CXT,then,a-xylosidase and Gax3A are further overexpressed,and the recombinant strain CxylAXG is obtained.Compared with the strain CXT,the conversion of cellulose at 72 h of enzymatic hydrolysis using APCS as substrate reach to 81.3%and increased by 22.8%compared to the control.3.ARTP/ARTP-UV induced mutant breedingTo further improve the enzyme activity and hydrolysis ability of Penicillium oxalicum,all the recombinant strains constructed in this thesis are subjected to ARTP and ARTP-UV mutagenesis.Positive mutant strains with high cellulase activities are preliminary screened by using microcrystalline cellulose plate,then further screened by comparing the filter paper enzyme activity of crude enzymes from the strains.In which,the FPA of the mutant strains CXTA-6 and Cxyl3-9 are increased by 2.04 folds and 2.13 folds,respectively,compared to that of RE-7.