Genetic Knockout And Preliminary Exploration Of CRISPR/Cas9 System In High-yield Cellulase Talaromyces Stipitatus Strain

Nowadays cellulose is very abundant carbohydrate on Earth,which is an important component of plants and algal cell walls.Its synthesis and degradation play an important role in the carbon cycle.In addition,with the depletion of non renewable resources such as the coal,petroleum,looking for and using the renewable resources become very popular.One of the major strains used in morden industry for cellulose is filamentous fungi.Talaromyces stipitatus EMM is a self-isolated high-yield cellulase strain by our lab,which was mutated by wild type strain OPC4-1.The protein secretion and enzyme activity stability of EMM have reached a high level,but still can not meet the requirements of industrialization scale.In order to enhance the cellulase production further and make the strain be a stable host strain,it is the effective way to study and regulate the cellulase and hemicellulase production in molecule level.In this thesis,we modify the strain from following aspects:?1?Cellulase is an inducible enzyme,and cellulase of T.stipitatus EMM can be expressed only under the presence of inducer,for example the cellulose?sophorose and lactose,but the expression of cellulase will be repressed by glucose.The creA gene plays a crucial role in the carbon catabolite repression of all the regulatory factors for the cellulase transcription.A creA gene was cloned from T.stipitatus EMM,the results of sequencing and blasting showed the gene cloned from EMM was carbon catabolite repressor gene.And then the plasmid PGcreA1-ApyrF-creA2 was constructed using the molecular biology methodsfor knockout the gene,the plasmid carried two homologous arms and a recycle marker.The knockout gene cassette was transformed into the uracil auxotroph strain EMU6 using the PEG/CaCl₂ protoplasts transformation method.After two times homologous recombination,the creA gene disruption strain was isolated successfully.Morphology analysis showed colony size of?creA21 was smaller and it also grown more spores than control strains.The Fpase showed?creA21 produced more cellulase and xylanase than control strains,and also showed larger clear zones on the selective plates containing 30 g/L glucose and 40 g/L glucose.?2?The secretion of ku protein in organism can promote the occurrence of the NHEJ?nonhomologous end-joining?pathway,thus inhibiting the HR?Homologous Recombination?pathway,so the targeting efficiency is very low.A Ku70 gene was cloned from EMM,the results of sequencing and blasting showed the gene cloned from EMM was the Ku70 gene.And then the plasmid PGApyrF-ku70 was constructed which carrying the two homologous arms and a recycling marker using the molecular biology methods.The knockout gene cassette was transformed into the uracil auxotroph strain EMU6 using the PEG/CaCl₂ protoplasts transformation.After the first homologous recombination,Ku70gene was knocked out,and then second homologous recombination happened under the pressure of5-FOA?5-fluoroorotic acid?for marker recycling.?3?The third generation artificial endonuclease CRISPR/Cas9 system was established preliminarily.The recombinant plasmid PCP-Cas9 was constructed by molecular biology methods which carrying the selective marker hygromycin B,the cellulase promoter Pcbh,the terminator Tcbh and the Cas9 gene?including GFP,Yellow Fluorescent Protein?.Cas9gene was expressed in the T.stipitatus,which lay a foundation for the establishment of CRISPR/Cas9 multi-gene knockout and expression system.