N-糖链加工影响里氏木霉形态发生并提高木质纤维素降解能力（英文）

Altered N-glycan processing in Trichoderma reesei affects the morphogenesis and improves the degradation of lignocellulose

摘要:

[背景] 烟曲霉α-1，2-甘露糖苷酶MsdS在高尔基体中将N-糖链Man₈GlcNAc₂加工为成熟分泌糖蛋白的糖型Man₆GlcNAc₂，有研究表明MsdS与烟曲霉的形态发生、细胞壁合成及蛋白质分泌密切相关；与烟曲霉不同的是，里氏木霉的成熟分泌糖蛋白上的N-糖链结构为Man₈GlcNAc₂，细胞却能正常生长，说明丝状真菌N-糖链的加工具有物种特异性，但其生物学意义不明。[目的] 为研究N-糖链加工对里氏木霉细胞生长及蛋白质分泌的影响，本研究将烟曲霉MsdS转入里氏木霉中以改变其成熟分泌糖蛋白的糖型。[方法] 构建带有烟曲霉msdS基因的重组质粒并转入里氏木霉中，获得msdS表达菌株Tr-MsdS，分析Tr-MsdS菌株的生长表型、N-糖组、蛋白质分泌途径和纤维素酶活性的变化。[结果] 在里氏木霉msdS表达菌株Tr-MsdS中，分泌糖蛋白的主要糖型由出发株的Man₈GlcNAc₂转变为Man₆GlcNAc₂，细胞壁组分发生变化，但细胞壁完整性未受影响；与出发株相比，Tr-MsdS菌株产孢、出芽及分枝增多；另外，MsdS的表达还影响蛋白质分泌，在50℃时降解纤维素和β-葡聚糖的能力分别提高9.9%和32.2%。[结论] 研究结果表明，N-糖链的加工可影响里氏木霉蛋白质，尤其是纤维素酶的分泌，干扰N-糖链加工可能是提高里氏木酶纤维素酶产量的新策略。

Abstract:

[Background] Aspergillus fumigatus α-1,2-mannosidase MsdS is an enzyme that cleaves N-linked Man₈GlcNAc₂ in Golgi apparatus to produce Man₆GlcNAc₂ on mature secreted glycoproteins. MsdS has been shown to play a significant role in morphogenesis, cell wall synthesis and protein secretion in A. fumi gatus. Unlike A. fumigatus, Trichoderma reesei produces Man₈GlcNAc₂ on its mature secreted glycoproteins and grows normally. These observations suggest a species-specific N-glycan processing in filamentous fungi, however, its biological significance keeps unclear. [Objective] To evaluate the effects of the N-glycan processing on cell growth and protein secretion in T. reesei, A. fumigatus MsdS was introduced into T. reesei to change the glycoform on mature secreted proteins. [Methods] The recombinant plasmid haboring the msdS gene was constructed and transformed into T. reesei to obtain the msdS-expressing strain Tr-MsdS. The phenotypes, N-glycome, protein secretory pathway and cellulase activity were analysed. [Results] The msdS-expressing strain Tr-MsdS produced a major glycoform of Man₆GlcNAc₂ on its secreted glycoproteins, instead of Man₈GlcNAc₂ in the parent strain. Although the cell wall content of msdS-expressing strain Tr-MsdS was changed, it appeared that the cell wall integrity was not affected. However, phenotypes such as increased conidiation, multiple budding and random branching were observed in strain Tr-MsdS. In addition, expression of MsdS in T. ressei also affected protein secretion and increased the acivities of cellulose and β-mannan degradation by 9.9% and 32.2% at 50℃, respectively. [Conclusion] Our results indicate that the N-glycan processing plays an important role in protein secretion in T. reesei, especially cellulases. Also, our results provide a new strategy to improve cellulases production by interfering the N-glycan processing in T. reesei.