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 by 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 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 secretion of heterologous target proteins in industrial fungi.

中文翻译：

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通过替换里氏木霉中的主要纤维素酶 CBH1 来提高异源栓菌漆酶 (LacA) 的产量

来自白腐真菌的漆酶在处理酚类化合物时表现出高氧化还原电位。然而，由于本地主机的生产力相对较低，它们在商业用途上的应用受到了限制。在这里，栓菌属的漆酶 A 编码基因 lacA。AH28-2 在 cbh1 (Pcbh1) 强启动子的控制下过表达，cbh1 是编码内源性纤维二糖水解酶 1 (CBH1) 的基因，在工业主力真菌里氏木霉中。首先，通过遗传转化将 lacA 表达盒随机整合到里氏木霉染色体中。lacA基因转录成功，但液体发酵条件下检测不到漆酶。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，在lacA表达盒中加入cbh1下游区域（Tcbh1），构建新的表达盒lacA::Δcbh1，通过同源重组替代基因组中的cbh1位点。遗传转化后，lacA 基因被整合到 cbh1 位点并被转录。并且未折叠蛋白反应（UPR）和ERAD仅被轻微诱导，为此内源性纤维素酶CBH1的缺失释放了分泌压力。最后，在发酵液中获得了 168.3 U/L 的最大漆酶活性。