College of Life Science, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, China.
Microb Cell Fact. 2012 Jun 18;11:84. doi: 10.1186/1475-2859-11-84.
The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II.
The transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively.
This work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.
真菌里氏木霉是表达同源或异源基因的重要工具，通常使用诱导型 cbh1 启动子。然而，在某些情况下，组成型表达比诱导型表达更可取，因为诱导型表达会导致产生不需要的纤维素酶成分。在这项工作中，筛选了里氏木霉的组成型启动子，并成功地用于高水平同源表达木聚糖酶 II。
通过定量实时 RT-PCR 分析了里氏木霉中参与葡萄糖代谢的 13 个关键基因的转录谱。结果表明，在高葡萄糖条件下，pdc（编码丙酮酸脱羧酶）和 eno（编码烯醇酶）基因的 mRNA 水平远高于其他基因。使用 pdc 和 eno 基因的启动子，构建了同源表达木聚糖酶 II 的重组里氏木霉菌株，在高葡萄糖浓度的培养基中，培养上清液分别产生 9266IU/ml 和 8866IU/ml 的木聚糖酶活性。木聚糖酶 II 的产率分别为 1.61g/L（使用 pdc 启动子）和 1.52g/L（使用 eno 启动子），分别约占里氏木霉分泌的总蛋白的 83%和 82%。
本工作通过 RT-qPCR 在里氏木霉中筛选组成型启动子，并使用这些启动子获得了迄今为止重组木聚糖酶 II 的最高表达。
