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

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

【背景】烟曲霉α-1,2-甘露糖苷酶Msd S在高尔基体中将N-糖链Man_(8)Glc NAc_(2)加工为成熟分泌糖蛋白的糖型Man_(6)Glc NAc_(2),有研究表明Msd S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Man_(8)Glc NAc_(2),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Msd S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msd S基因的重组质粒并转入里氏木霉中,获得msd S表达菌株Tr-Msd S,分析Tr-Msd S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msd S表达菌株Tr-Msd S中,分泌糖蛋白的主要糖型由出发株的Man_(8)Glc NAc_(2)转变为Man_(6)Glc NAc_(2),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-Msd S菌株产孢、出芽及分枝增多;另外,Msd S的表达还影响蛋白质分泌,在50°C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。

[Background] Aspergillus fumigatus α-1,2-mannosidase MsdS is an enzyme that cleaves N-linked Man_(8) GlcNAc_(2) in Golgi apparatus to produce Man_(6) GlcNAc_(2) 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 Man_(8) GlcNAc_(2) 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 Man_(6) GlcNAc_(2) on its secreted glycoproteins, instead of Man_(8) GlcNAc_(2) 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 °C, 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.