Construction Of Expression System Of Thielavia Terrestris And Expression Of AA9 Family Protein

Lignocellulose is the most abundant renewable biomass resource on the planet.Production of biofuels with lignocellulose as the feedstock is the key to low-carbon sustainable development in the future.Traditional glycoside hydrolases have limited efficiency in degrading lignocellulose due to the tight uncovalent combination among cellulose chains.Achieving methods for efficiently degrading lignocellulose is currently the focus of biofuel production research.Thielavia terrestris is a thermophilic filamentous fungus that normally grows at 40-50°C and has the ability to produce a variety of thermal-stable cellulolytic enzymes.It is a potential industrial strain for cellulase production.Polysaccharide monooxygenases?PMOs?are newly discovered copper-dependent oxidases,which can significantly improve the efficiency of cellulase degradation of lignocellulose,and have broad application prospects.The polysaccharide monooxygenase that acts on lignocellulose in fungi belongs to the AA9?auxiliary activity families 9,AA9?family of proteins,and there are 19 AA9 family protein genes in the genome of the T.terrestris.In this work,a constitutive expression system of T.terrestris was first constructed,and the promoter and the terminator region of glyceraldehyde-3-phosphate dehydrogenase gene?gpd?of T.terrestris were used to construct the expression system.The expression vector pUC-Pgpd-Tgpd was used as the constitutive expression vector and the expression cassette pUC-Pgpd-xyn-Tgpd was constructed using the xylanase gene xyn of T.terrestris to test the efficiency of the expression cassette.The expression cassette was cotransformed into T.terrestris with plasmid pAN7-1.Seven transformants were screened to contain the expression cassette.After fermentation,the relative expression level of the xyn gene in transformed strain was determined to be 39.0 times higher than that of the wild-type strain by realtime fluorescence quantitative PCR.The xylanase activity of the transformant was determined by measuring the xylanase activity of the transformed strain.39.6 U/mL increased by 276.32% relative to the wild-type strain.Two AA9 genes,THITE₁70174 and THITE₁28130 AA9 were selected for homologous expression and activity analysis,and were named as TtAA9 A and TtAA9 B respectively.The AA9 protein expression cassette was constructed using the pUC-Pgpd-Tgpd expression vector and cotransformed into T.terrestris with the pAN7-1 plasmid.Total 9 strains of TtAA9 A transformed strain and 5 strains of TtAA9 B transformed strain were screened to contain the AA9 expression cassette.After fermentation,it was confirmed by SDS-PAGE that there was a clear band at the corresponding protein size in the crude enzyme solution.The crude enzyme solution was added to the cellulase hydrolysis system using rice straw powder and microcrystalline cellulose as substrates,respectively.The results showed that the increase in cellulase activity of TtAA9 A and TtAA9 B was 64.7% and 22.2% in the rice straw substrate.The increase in cellulase activity of TtAA9 A and TtAA9 B in the microcrystalline cellulose substrate was 50.6% and 22.2%.In this work,a constitutive expression system of Thielavia terrestris was successfully constructed,and the expression of two AA9 family proteins was performed through this expression system,and the promotion effect of the expressed product on cellulase activity was verified.This study provides technology basis for improving the cellulase activity with AA9 family proteins.