State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao 266237, P. R. China.
J Ind Microbiol Biotechnol. 2023 Feb 17;50(1). doi: 10.1093/jimb/kuad002.
The laccases from white-rot fungi exhibit high redox potential in treating phenolic compounds. However, their application in commercial purposes has been limited because of the relatively low productivity of the native hosts. Here, the laccase A-encoding gene lacA of Trametes sp. AH28-2 was overexpressed under the control of the strong promoter of cbh1 (Pcbh1), the gene encoding the endogenous cellobiohydrolase 1 (CBH1), in the industrial workhorse fungus Trichoderma reesei. Firstly, the lacA expression cassette was randomly integrated into the T. reesei chromosome by genetic transformation. The lacA gene was successfully transcribed, but the laccase couldn't be detected in the liquid fermentation condition. Meanwhile, it was found that the endoplasmic reticulum-associated degradation (ERAD) was strongly activated, indicating that the expression of LacA probably triggered intense endoplasmic reticulum (ER) stress. Subsequently, the lacA expression cassette was added with the downstream region of cbh1 (Tcbh1) to construct the new expression cassette lacA::Δcbh1, which could replace the cbh1 locus in the genome via homologous recombination. After genetic transformation, the lacA gene was integrated into the cbh1 locus and transcribed. And the unfolded protein response (UPR) and ERAD were only slightly induced, for which the loss of endogenous cellulase CBH1 released the pressure of secretion. Finally, the maximum laccase activity of 168.3 U/l was obtained in the fermentation broth. These results demonstrated that the reduction of secretion pressure by deletion of endogenous protein-encoding genes would be an efficient strategy for the secretion of heterologous target proteins in industrial fungi.
ONE-SENTENCE SUMMARY: The reduction of the secretion pressure by deletion of the endogenous cbh1 gene can contribute to heterologous expression of the laccase (LacA) from Trametes sp. AH28-2 in Trichoderma reesei.
白腐真菌中的漆酶在处理酚类化合物时表现出较高的氧化还原电位。然而，由于其天然宿主的生产力相对较低，其在商业用途中的应用受到限制。在这里，通过控制内源性纤维二糖水解酶 1 (CBH1) 的基因 cbh1 (Pcbh1) 的强启动子，在工业用菌里氏木霉中过表达了来自 Trametes sp. AH28-2 的漆酶 A 编码基因 lacA。首先，通过遗传转化将 lacA 表达盒随机整合到 T. reesei 染色体中。lacA 基因成功转录，但在液体发酵条件下无法检测到漆酶。同时，发现内质网相关降解 (ERAD) 被强烈激活，表明 LacA 的表达可能引发强烈的内质网 (ER) 应激。随后，将 lacA 表达盒与 cbh1 的下游区域 (Tcbh1) 连接起来构建新的表达盒 lacA::Δcbh1，该表达盒可以通过同源重组取代基因组中的 cbh1 基因座。遗传转化后，lacA 基因整合到 cbh1 基因座并转录。未折叠蛋白反应 (UPR) 和 ERAD 仅被轻度诱导，因为内源性纤维素酶 CBH1 的缺失释放了分泌压力。最终，发酵液中获得了 168.3 U/l 的最大漆酶活性。这些结果表明，通过删除内源性蛋白编码基因来降低分泌压力将是在工业真菌中分泌异源靶蛋白的有效策略。
