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Abstract：
BACKGROUND: The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown.
RESULTS: A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose.
CONCLUSIONS: TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.
RESULTS: A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose.
CONCLUSIONS: TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.
摘要：
背景：腐生丝状真菌里氏木霉代表了最多产的纤维素酶生产者之一。通过里氏木霉大量生产木质纤维素分解酶不仅依赖于纤维素酶基因的有效转录,而且依赖于它们在翻译后的有效分泌。然而,有关分泌途径在里氏木霉中高水平生产纤维素酶中的功能作用很少受到关注。RabGTP酶是协调与真核分泌途径相关的各种囊泡运输的关键调节因子。具体来说,Rab7是一种代表性的GTP酶，可调节早期内体向晚期内体的过渡，然后将其融合到液泡以及同型液泡融合。尽管已经报道了内体/液泡途径与分泌途径之间的串扰，Rab7在里氏木霉纤维素酶生产中的功能作用尚不清楚。
结果：在里氏木霉中鉴定并表征了TrRab7。显示TrRab7在里氏木霉营养生长和液泡形态中起重要作用。而敲除Trrab7显著损害了诱导的里氏木霉纤维素酶的产生，关键转录激活因子的过表达，Xyr1，恢复了Trrab7敲低菌株（Ptcu-rab7KD）对葡萄糖的纤维素酶的产生，表明观察到的缺陷纤维素酶生物合成是由受损的纤维素酶基因转录引起的。还发现Trrab7的下调使里氏木霉对包括碳饥饿在内的各种应力更敏感。有趣的是,过表达Snf1，一种被称为能量传感器的丝氨酸/苏氨酸蛋白激酶，部分恢复了Avicel上Ptcu-rab7KD的纤维素酶产量，暗示TrRab7参与对碳饥饿的能量适应，这有助于当里氏木霉从葡萄糖转移到纤维素时成功的纤维素酶基因表达。
结论：TrRab7被证明在里氏木霉的发育和对养分转移导致的碳饥饿的应激反应中起重要作用。这种适应可以允许里氏木霉成功地启动诱导过程,导致有效的纤维素酶生产。本研究为内体/液泡途径在里氏木霉发育和水解酶产生中的功能参与提供了有用的见解。
结果：在里氏木霉中鉴定并表征了TrRab7。显示TrRab7在里氏木霉营养生长和液泡形态中起重要作用。而敲除Trrab7显著损害了诱导的里氏木霉纤维素酶的产生，关键转录激活因子的过表达，Xyr1，恢复了Trrab7敲低菌株（Ptcu-rab7KD）对葡萄糖的纤维素酶的产生，表明观察到的缺陷纤维素酶生物合成是由受损的纤维素酶基因转录引起的。还发现Trrab7的下调使里氏木霉对包括碳饥饿在内的各种应力更敏感。有趣的是,过表达Snf1，一种被称为能量传感器的丝氨酸/苏氨酸蛋白激酶，部分恢复了Avicel上Ptcu-rab7KD的纤维素酶产量，暗示TrRab7参与对碳饥饿的能量适应，这有助于当里氏木霉从葡萄糖转移到纤维素时成功的纤维素酶基因表达。
结论：TrRab7被证明在里氏木霉的发育和对养分转移导致的碳饥饿的应激反应中起重要作用。这种适应可以允许里氏木霉成功地启动诱导过程,导致有效的纤维素酶生产。本研究为内体/液泡途径在里氏木霉发育和水解酶产生中的功能参与提供了有用的见解。
