Introduction of theAspergillus fumigatusα-1,2-mannosidase MsdS intoTrichoderma reeseileads to abnormal polarity and improves the ligno-cellulose degradation

Abstract

25-1,2-Mannosidase is an important enzyme essential for N-glycan processing and plays a 26 significant role in the biosynthesis and organization of fungal cell wall. Lacking of α-1,2-27 mannosidase leads to cell wall defect in yeast and filamentous fungi. Trichoderma reesei is 28 known to be non-toxic to human, and its N-glycan on secreted glycoprotein is Man 8 GlcNAc 2 . To 29 evaluate the significance of the N-glycan processing in T. reesei, in this study Aspergillus 30 fumigatus α-1, 2-mannosidase MsdS, an enzyme that cleaves N-linked Man 8 GlcNAc 2 in Golgi to 31 produce Man 6 GlcNAc 2 on secreted glycoprotein, was introduced into T. reesei. The msdS-32 expressing strain Tr-MsdS produced a major glycoform of Man 6 GlcNAc 2 on its secreted 33 glycoproteins, instead of Man 8 GlcNAc 2 in the parent strain. Although the cell wall content of 34 msdS-expressing strain Tr-MsdS was changed, it appeared that the cell wall integrity was not 35 affected. However, phenotypes such as increased conidiation, multiple budding and random 36 branching were observed in strain Tr-MsdS. In addition, expression of MsdS into T. ressei also 37 affected protein secretion and improved the ligno-cellulose degradation of T. reesei. Our results 38 indicate that processing of the N-glycan is species-specific and plays an important role in protein 39 secretion in T. reesei, specially cellulases. Also, our results provide a new strategy to improve 40 cellulases production by interfering the N-glycan processing in T. reesei. 41 42 secretion 44 45 Importance 46For the first time, the N-glycan processing is shown to play an important role in polarized growth 47 and protein secretion in T. reesei. In addition, our results show that alterated N-glycan processing 48 enhances cellulose degradation, which provides a strategy to improve cellulases production in T. 49 reesei. 50 51 52 Trichoderma reesei (syn. Hypocrea jecorina), a mesophilic filamentous fungus identified as 53 GRAS (generally recognized as safe) status by FDA (U.S. Food and Drug Administration) 54 ( Kuhls et al. 1996; Schuster and Schmoll, 2010), is able to produce a wide range of extracellular 55 enzymes in large quantities. T. reesei possesses protein post-translational modification and grows 56 faster than plant, insect or mammalian cells, therefore it has been thought as an attractive 57 expression host. However, the expression of heterologous protein is less efficient than the 58 expression of endogenous proteins in T. reesei and, therefore, efforts have been made to improve 59