期刊
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
卷 47, 期 1, 页码 133-144出版社
OXFORD UNIV PRESS
DOI: 10.1007/s10295-019-02252-9
关键词
Trehalase; Aspergillus niger; CRISPR; Cas9; Multi-copy; Ethanol fermentation
资金
- National Natural Science Foundation of China [31871736]
- Research and Development Plan in Key Areas of Guangdong Province [2018B020205002]
- Guangdong Provincial Key Laboratory of Advanced Biofermentation Technology Enterprise in Flavoring Food [2017B030302002]
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 degrees C, and exhibited prominent thermal stability between 50 and 60 degrees 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.
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