Construction Of Cellulase Over-producing Strain Of Penicillium Oxalicum By Red/ET And ?-Rec/six Recombination Technology

Lignocellulosic biomass is the most important renewable resource for biorefineries.However,the characters of difficult biodegradation for lignocellulose hinder the efficient use of these renewable resources.Filamentous fungi have efficient expression of cellulase properties,and can efficiently express cellulase and degrade lignocellulose.Penicillium oxalicum is a filamentous fungus with high yield of cellulase isolated from the soil.Compared to Trichoderma reesei,P.oxalicum can express much more cellulase components.The degradation of biomass utilization process need large amounts of cellulase,and it is necessary to further improve the cellulase expression level of the existing industrial strains through rational transformation,Multi-target genetic manipulation of P.oxalicum is the key to rational construction of strains.Therefore,how to break through the genetic screening marker of P.oxalicum and establish the technical method for the operation of its continuous and multi-gene fragment is the key problem that needs to be solved urgently.The main results of this paper are as follows:1.Cellulase gene cluster was constructed by Red/ET recombination systemRed/ET recombination is a DNA engineering technique based on the Red operator(Red?/Redp/Redy)and the Rac phage RecE/RecT recombination system of ? phage.The genetic manipulations of insert,knock,mutation and modifications for large DNA molecules could be simple and fast through Red/ET recombination technology.At present,the amplification for long fragment used for transformation was limited by PCR.The construction of polycellulase gene expression cluster by Red/ET recombination system could effectively improve the efficiency of single transformation,that is,one-time transformation could achieve the expression of various cellulase genes,at the same time to improve the efficiency of a variety of cellulase activities.In this work,a 15 kb cellulase gene expression cassette was designed.The expression cassette was transformed into P.oxalicum 114-2,and the highest activity of FPA was more than 50 times higher than that of the original strain effect.The successful application of this technology provides a rapid and effective method for the construction of high-yield cellulase strains of Penicillium oxalicus.2.Construct ?-Rec/six screening marker recovery systemIn the process of constructing high-yield cellulase strains of P.oxalicum,the knockout and overexpression of genes are often dependent on the screening of marker genes.However,the limited kind of marker genes limits the genetic manipulation of high-yield cellulase strains.The use of marker genes,especially antibiotic resistance genes,often has an adverse effect on environmental and industrial applications.Therefore,how to break through the genetic screening marker of P.oxalicum and establish a method for continuous gene manipulation is a key issue that needs to be solved.The ?-Rec/six screening marker recovery system is based on the p-Rec recombinase in bacteria that recognizes and acts on two repeating six sequences that specifically cleave fragments between two repeating six sequences.Using this,the use of pyrG gene in Aspergillus nidulans was used to reuse the screening marker pyrG in P.oxalicum deficient strain M12,which broke the long-standing screening marker limitation.3.Construction of high-yield endo-cellulase and xylanase strains using Red/ET and ?-Rec/six recombination systemOn the basis of previous work,we have established a technical method for the treatment continuous and polygene fragments of P.oxalicum.We combined Red/ET and the ?-Rec/six recombination system to construct high-yield endo-cellulase and xylanase strains.In this study,three expression genes were designed,including two endo-cellulase gene clusters EEC and EXDC.The two expression cassettes were transformed into the screening strain to reuse strain 7-1-1 to obtain REEC and REX strains;also includes a xylanase gene cluster XXC,used to transform the REX strain to obtain XXC strain.The resulting transformants have achieved the reuse of screening markers,and enzyme activity has also been a certain increase,the FPA activity of REX strain was improved by 1.5 times,and the activity of Xylanase was increased by 13 times.The FPA activity of XXC strain was increased by 5 times and xylanase activity increased by 2 times.The results of the above experiments show that the combination of Red/ET recombination system and ?-Rec/six self-deletion system can be used to construct high-yield cellulase and hemicellulase strains,but only the recombinant enzyme Without reaching the desired level,future work needs to continue to explore strategies to improve the expression of recombinant enzymes.