| The filamentous fungus Trichoderma reesei is capable of synthesizing and secreting large quantities of cellulase and hemicellulase enzymes,making it widely utilized in various industrial production fields.With advantages such as efficient protein synthesis,high safety(GRAS certified),efficient protein modification capabilities,and the ability to utilize inexpensive and complex raw materials,T.reesei has been developed as a host cell for the expression and secretion of heterologous proteins.However,unexpected conditions are often encountered during heterologous protein production,where the expression and secretion levels are significantly lower than those of endogenous(hemi)cellulases.Currently,strategies used to improve heterologous protein synthesis and secretion levels primarily focus on enhancing transcriptional levels,and there is limited identification and characterization of secretionpromoting elements,which restricts the application and development of T.reesei as a host cell.Therefore,this study investigates two aspects:(1)screening highly efficient endogenous secreted proteins in T.reesei to identify short fusion expression elements that can promote the expression and secretion of heterologous proteins,and(2)modifying commonly used CBHI signal peptides in T.reesei to obtain signal peptide forms that significantly promote the expression and secretion of heterologous proteins.The main research results obtained in this study are as follows:1.XYNII,a novel fusion expression element that was screened from various endogenous enzymes,has been identified to significantly enhance the expression and secretion of heterologous proteins.Based on existing literature and previous research,the N-terminal CBM domains of four highly secreted and relatively short endogenous proteins from Trichoderma reesei,CBHⅡ,EGⅡ,SWO,and the full-length sequence of XYNⅡ,were selected as candidate fusion elements and fused to the N-terminus of the target heterologous protein,LZ8.Results showed that XYNⅡ exhibited the best ability to promote the secretion of the target heterologous protein.To further verify this,XYN Ⅱ was used as a fusion element to guide the expression and secretion of two heterologous Xylanases,and the results demonstrated that XYN II significantly enhanced their expression and secretion levels,surpassing the currently known short fusion element,CBH Ⅱ-CBM.By introducing a KEX2 cleavage site between XYN Ⅱ and the target heterologous protein,and co-expressing with KEX2,effective separation between XYN Ⅱ and the target heterologous protein was achieved.These results indicate that XYN Ⅱ as a new fusion element can promote the efficient synthesis and secretion of heterologous proteins in T.reesei.On the other hand,Xylanase plays a significant role in the efficient degradation of lignocellulose.Compared to cellulases,the Xylanase level in T.reesei is relatively low,thus,the construction of a Xylanase overproducing strain is of great significance.In this study,when using XYNII as a fusion element to guide the expression of the heterologous Xylanase,Xyn10A,it was found that the fusion expression of XYNII and Xyn10A significantly increased the extracellular Xylanase activity in T.reesei,reaching up to 270U/mL,thus generating a highproducing Xylanase strain with great application potential.2.By increasing the hydrophobicity of the signal peptide of the CBHI cellulase from Trichoderma reesei through mutations,a mutant form was obtained that significantly enhanced the expression and secretion of the heterologous xylanase XynST11.Short and cleavable signal peptides are preferred for heterologous protein secretion in production systems.In Trichoderma reesei,the cellulase CBHI is the component with the highest extracellular secretion level,and its signal peptide is often used to guide the expression and secretion of heterologous proteins.In this study,we first modified the CBHI signal peptide by introducing two leucine residues between the signal peptide N-region and H-region to increase hydrophobicity.It was found that the mutant signal peptide significantly enhanced the expression and secretion of the heterologous xylanase XynST11,and was superior to the CBHII signal peptide,which also has high hydrophobicity.Further experiments showed that the positively charged amino acids in the signal peptide N-region and C-region play important roles in maintaining signal peptide function.In addition,it was found that there may be some adaptability between the CBHI signal peptide and its downstream sequence in guiding protein translocation and secretion.When the guiding sequence was extended downstream by 35 amino acids based on the CBHI signal peptide,it was able to promote the expression and secretion of the heterologous protein XynST11. |
