Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei
Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei
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直接时空追踪活体里氏木霉中纤维素酶的定位和分泌
DOI：
10.1186/s13068-019-1538-0
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发表时间：
2019-08-20
影响因子：
6.3
通讯作者：
Lin, Fengming
中科院分区：
文献类型：
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作者：
Li, Chengcheng;Pang, Ai-Ping;Lin, Fengming
背景：丝状真菌在细胞外分泌纤维素酶和半纤维素酶等水解酶，是自然界中重要的植物生物质清道夫和酶工业的主力。与真菌产纤维素酶机制的广泛研究不同，对纤维素酶在真菌中的时空分布和分泌规律的研究较少，阻碍了对真菌产纤维素酶分子机制的深入了解。reeseiRUT-C30，分别表达红色荧光蛋白DsRed标记的β-葡糖苷酶（BGL）、纤维二糖水解酶（CBH）和内切葡聚糖酶（CMC）形式。在这些菌株的协助下，我们发现所有三种纤维素酶组分BGL，CBH和CMC扩散到整个真菌菌丝体中，主要积累在菌丝顶端。这些酶定位于内质网、高尔基体、液泡和细胞膜/壁，而不定位于细胞间隔，并大量分泌到培养基中。此外，CBH和CMC的主要分泌比BGL更早开始。布雷菲德菌素A（Brefeldin A，BFA）完全阻断了里氏木霉纤维素酶的表达和分泌。分别表达BGL、CBH和CMC的DsRed融合版本的T. reesei RBGL、RCBH和RCMC中，首次以动态方式直接观察了纤维素酶在T. reesei中的分布和分泌，初步绘制了T.为揭示里氏木霉纤维素酶的分泌途径提供了依据。结果表明，纤维素酶的分泌主要通过传统的ER-高尔基体分泌途径进行，并可能通过非传统的蛋白质分泌途径进行辅助。
Background:Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production.Result:RecombinantTrichoderma reeseistrains RBGL, RCBH, and RCMC were successfully constructed fromT. reeseiRUT-C30, expressing red fluorescent protein DsRed-tagged versions of β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion inT. reesei.Conclusion:Based on recombinantT. reeseiRBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production inT. reeseiwere first visualized directly in a dynamic way, preliminarily mapping the location and secretion ofT. reeseicellulase and providing evidence for revealing the secretion pathways of cellulase inT. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER-Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.