Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.

中文翻译：

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通过使用CRISPR / Cas9工具在黑曲霉中高表达嗜热毁丝霉高活性且稳定的海藻糖酶及其在乙醇发酵中的应用。

海藻糖酶催化非还原性二糖海藻糖的水解。从6种丝状真菌的海藻糖酶基因中筛选了嗜热毁丝霉的高活性海藻糖酶MthT。由CRISPR / Cas9工具介导的巧妙的多拷贝敲入表达策略和培养基优化被用于提高黑曲霉中MthT的产量，最高可达1698.83 U / mL。由于插入，蛋白质背景显着减弱。重组MthT在pH 5.5和60°C下表现出最佳活性，在酸性条件下（pH 4.5-6.5）在50和60°C之间表现出显着的热稳定性。与使用30％淀粉糖化液的无MthT（34.38％）相比，添加MthT（51.88％）可以显着提高乙醇转化率（乙醇产率/总葡萄糖）。