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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)
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国家自然科学基金重点项目(31630016);广西八桂学者项目(2016A24)


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

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Sharma Ghimire Prakriti,欧阳浩淼,赵光亚,解明明,周慧,杨静华,金城. 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)[J]. 微生物学通报, 2021, 48(10): 3432-3448

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  • 收稿日期:2021-01-27
  • 录用日期:2021-03-12
  • 在线发布日期: 2021-10-12
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