Heterologous-expression Of ?-glucosidases And Application Of CRISPR-Cas9 System In Penicillium Oxalicum

As the rate of fossil fuel consumption continues to increase and the environmental and resource crises increase,there is an urgent need for alternative energy sources such as bioethanol,biodiesel and biohydrogen.Plant-derived biomass is the most abundant renewable resource on the planet.The economic transformation of biomass raw materials to fuels and chemicals can effectively alleviate the energy and environmental crisis.Filamentous fungi can efficiently express lignocellulase and degrade plant biomass efficiently,which plays an essential role in the natural carbon utilization cycle.At present,the low hydrolysis efficiency of the filamentous fungal lignocellase system leads to the high cost of enzyme use,which is the main bottleneck in the economic conversion of lignocellulosic biomass.The filamentous fungus lignocellulose degrading enzyme system is composed of various hydrolases,mainly including exo-cellulase,endo-cellulase and ?-glucosidase.?-glucosidase is one of the important components in the cellulase system.Its main function is to efficiently convert cello-oligosaccharide or cellobiose into glucose,which plays an important role in the degradation of cellulose.However,the content of glucosidase accounts for a relatively low proportion of the whole enzyme system,making it one of the main factors limiting the efficient conversion of biomass by cellulase.Therefore,targeting high-yield lignocellulase-producing Penicillium oxalicum strain,reconstituting the expression regulation of cellulase and constructing of high-performance strains producing ?-glucosidase are of great significance for the further efficient use of cellulase in biomass resources.The main results of the thesis are as follows: 1.Over-expression of bgl1 from Aspergillus niger in Penicillium oxalicumIn the previous study,a cellulase-producing P.oxalicum strain PT3-1 was constructed,and its cellulase expression level was significantly higher than that of the original strain,but the expression level of ?-glucosidase was relatively low.To solve this problem,heterologously expressing of other filamentous fungal-derived ?-glucosidases was performed.Previous studies have found that ?-glucosidase derived from A.niger has higher specific activity and higher glucose tolerance,which will help to improve the degradation efficiency of cellulose.In this study,three overexpression cassettes of A.niger-derived ?-glucosidase were constructed,using the bgl2 promoter,cbh1 promoter and PDE₀2864 gene promoter of P.oxalicum as promoters for the heterologous expression cassette of A.niger beta-glucosidase gene.The above three expression cassettes were expressed in high-yield cellulase strain PT3-1,and a highyield ?-glucosidase strain C3-1 was obtained.The ?-glucosidase activity of the C3-1 mutant strain reached 172.6 U/mL after 120 h of fermentation,which was 244-fold and 156-fold higher than that of the wild strain?WT?114-2 and the starting strain PT 3-1,respectively.Besides,the filter paper assay of strain C3-1 was 7-fold and 1.5-fold higher compared to the parent strain and WT,resulting in levels of up to 3.2 U/mL.A strain of high-filtration paper enzyme strain CD46-1 was obtained.The highest activity of filter paper at 144 h after fermentation was 3.43 U/mL,which was 7.5 times and 1.6 times higher than that of wild strain 114-2 and the original strain PT 3-1,respectively.Extracellular proteins also increased significantly.2.Optimization of ?-rec/six Recombination System to Construct High-yield ?-Glucosidase StrainIn the previous study,a recycling marker system by employing the ?-rec/six sitespecific recombination technology was established in P.oxalicum.However,after removal of the screening marker by this system,the ?-serine recombinase gene remains on the chromosome,which may adversely affect other sites on the chromosome.Through literature research,the ?-rec/six recombination system was optimized,and a recombination system without traces of Rec recombinase in the transformant genome was constructed.When the screening marker was removed by this system,the ?-serine recombinase gene was also excised,avoiding the adverse effects caused by the sustained expression of the ?-serine recombinase gene.This system can be used for the construction of subsequent ?-glucosidase high-yield strains.3.Application of CRISPR-Cas9 System in P.oxalicumThe CRISPR-Cas9 system is a powerful gene editing method that has been widely used in filamentous fungi.However,the application of this gene editing system has not been reported in P.oxalicum.In this study,we expressed the plasmid carrying the Cas9 protein gene in Penicillium oxalicum.A variety of RNA polymerase type III?Pol III?promoters for in vivo transcription of sgRNA were screened,and a variety of different sgRNA expression cassettes were constructed.The application of the CRISPR-Cas9 gene editing system Penicillium oxalicum was initially explored.