Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing. We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer. This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

中文翻译：

---

在没有纤维素酶基因诱导剂的情况下，转录因子 ACE3 的修饰增强了里氏木霉的蛋白质生产。

里氏木霉是最著名的纤维素分解生物之一，它产生大量用于降解木质纤维素物质的一整套细胞外纤维素酶和半纤维素酶。因此，里氏木霉是一种重要的生物技术宿主，它在商业上用于酶生产，包括天然和外来来源。在里氏木霉中生产酶的许多策略依赖于 cbh1 和其他纤维素酶基因启动子以实现高水平表达，并且这些启动子需要通过槐糖、乳糖或其他诱导剂诱导，以在制造过程中实现高生产率。我们描述了一种通过在里氏木霉中过表达转录因子 ACE3 来产生高水平分泌蛋白的方法。我们改进了 ace3 基因结构并确定了特定的 ACE3 变体，这些变体能够以葡萄糖为唯一碳源（即，在没有诱导剂的情况下）产生分泌的纤维素酶和半纤维素酶。这些特定的 ACE3 变体在 N 端包含全长 Zn2Cys6 双核簇结构域，在 C 端包含定义长度的截断。当在真菌细胞中以中等水平表达时，ACE3 变体可诱导葡萄糖上纤维素酶和半纤维素酶的高水平表达（即，在没有诱导剂的情况下），并进一步提高乳糖或葡萄糖/槐糖上的表达（即，在诱导剂存在的情况下）。最后，我们证明了该方法适用于工业菌株和发酵条件，在诱导剂不存在和存在的情况下均能提高蛋白质产量。这项研究表明，ACE3 变体的过表达能够在没有诱导剂的情况下实现高水平的蛋白质生产，并在存在诱导剂的情况下促进蛋白质生产。这是提高蛋白质生产力和降低制造成本的有效方法。