Historically, heterologous protein production has been challenging using the hyper-cellulolytic fungus, Trichoderma reesei. T. reesei is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for identification of successful transformants. In this work, we have applied the 2A-peptide multi-gene expression system to co-express four enzymes, which include three cellulases, a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1); as well as the enhanced Green Fluorescent Protein, (eGFP), used here as a marker for monitoring expression levels. We designed a new chassis vector, pTrEno-4X-2A for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each of the cellulases was assessed using pNP glycosides and the chromogenic substrate, AZCL-HE-cellulose, which confirmed the functionality of the enzymes. Expression and activity of these enzymes was proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. Although all three cellulase proteins were successfully expressed, their expression levels varied significantly. Specifically, up to an 18-fold difference was observed between the first and the third gene within the 2A-peptide construct, based on protein quantitation. The availability of this new screening tool is expected to greatly impact multi-enzyme applications, such as production of complex commercial enzyme formulations and the study of metabolic pathway enzymes, especially those destined for cell free applications.

中文翻译：

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用于生产里氏木霉复合酶制剂的多重蛋白表达系统

从历史上看，使用高纤维素分解真菌里氏木霉生产异源蛋白质一直具有挑战性。里氏木霉以转化效率低、同源重组频率低和用于识别成功转化体的边际筛选系统而著称。在这项工作中，我们应用 2A 肽多基因表达系统共表达四种酶，包括三种纤维素酶、一种纤维二糖水解酶 (CBH1)、一种内切葡聚糖酶 (EG1) 和一种 β-D-葡萄糖苷酶 (BGL1) ; 以及增强型绿色荧光蛋白 (eGFP)，此处用作监测表达水平的标记。我们为这项工作设计了一个新的底盘载体 pTrEno-4X-2A。这些纤维素酶的表达通过实时定量逆转录 PCR 和免疫印迹分析得到证实。使用 pNP 糖苷和显色底物 AZCL-HE-纤维素评估每种纤维素酶的活性，这证实了酶的功能。这些酶的表达和活性与 eGFP 荧光水平成正比，从而验证了该筛选技术的可靠性。尽管所有三种纤维素酶蛋白均已成功表达，但它们的表达水平差异很大。具体而言，基于蛋白质定量，在 2A 肽构建体中的第一个和第三个基因之间观察到高达 18 倍的差异。这种新筛选工具的可用性预计将极大地影响多酶应用，例如复杂商业酶制剂的生产和代谢途径酶的研究，尤其是那些用于无细胞应用的酶。