Genetic Modification Of Talaromyces Pinophilus EMU For The Enhancement Of Cellulase Production And Construction Of Heterologous Expression System Using CRISPR/Cas9 Technology

Talaromyces pinophilus EMU is a uracil auxotrophic strain with high cellulase yield.In order to further increase the production of endogenous proteins and make the strain a cell factory for heterologous protein expression,strain EMU was genetically modified as follows.Firstly,to increase the probability of homologous recombination in the process of gene knockout or insertion,a ku70 mutant strain EMU-△ku70 was obtained using CRISPR/Cas9 system.Afterwards,two morphologically related genes,tps2 and mpg1,were mutated and their effects on protein production were studied.The mutation of the tps2 encoding trehalose-6-phosphate phosphatase seriously affected the growth of hyphae and spore formation,and destroyed the integrity of the cell wall.The cellulase activity of two tps2 mutant strains △ tps④and △ tps⑥were 16.66±0.79 IU/mL and 16.64±0.68 IU/mL,respectively,and showed higher activity than the EMU strain(12.13±0.81 IU/mL).The concentration of extracellular protein in △ tps④ and △ tps⑥(11.43 ± 0.48 mg/mL,11.52± 0.34 mg/mL)was significantly higher than that of EMU strain(8.09 ± 0.57 mg/mL),suggesting that cell wall defects caused by tps2 mutation can increase protein secretion.The Mannose-1-phosphate guanylyltransferase is involved in the maintenance of cell wall integrity and protein glycosylation.The three mutants of mpg1 show different morphologies and enzyme production capabilities.The morphology of △mpgl-③ changed significantly,the FPA was as high as 15.21±0.85 IU/mL,and its enzyme production capacity was increased by about 41.2%compared with the EMU strain.Furthermore,filamentous fungal protein is easily degraded by proteases,resulting in low levels of heterologous proteins.Therefore,extracellular aspartate endopeptidase and tripeptidyl peptidase were mutated in EMU-△ku70,and two Eap mutants and the strain EMU-△ku70-△Eap⑦-△TppA③with double mutation of Eap and TppA were obtained.The TppA mutant strain showed lower protease activity(34.6±3.4 mU/mL),which was 85.3%of the EMU strain,and the protein yield and cellulase activity were 22.6%and 19.2%higher than the EMU strain,respectively.For heterologous protein expression,the main protein product(CBH1)of EMU strain was knocked out to reduce the background of its self-secreted protein,and strain EMU-△ku70-△Eap⑦-△TppA③-△cbh231(CEA231)was obtained.We used Aspergillus nidulans tef1 exogenous strong constitutive promoter and EMU endogenous inducible cbh1 promoter to express the heterologous protein Elaeis guineensis lipase.EGlipase recombinant strain SEA⑨ driven by the tef1 promoter was screened by PEG-mediated transformation.EGlipase driven by the cbh1 promoter was expressed in strain CEA231.RT-PCR verified that the EGlipase gene was transcribed into mRNA,but SDS-PAGE analysis showed that SEA⑨and CEA231 did not produce new suspected EGlipase.We believe that the EGlipase is derived from higher plants and has problems with low protein expression lever or mRNA secondary structure.