High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation

  • Liangbo Dong
  • Xiaotong Lin
  • Dou Yu
  • Lianggang Huang
  • Bin Wang
  • Li PanEmail author
Biotechnology Methods - Original Paper


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.


Trehalase Aspergillus niger CRISPR/Cas9 Multi-copy Ethanol fermentation 


Author contributions

LD, XL and DY performed the experiments. LD, LH and LP designed the study, analyzed the data and wrote the paper. BW interpreted and polished the writing. All authors discussed the results and implications and commented on the manuscript at all stages. All authors have read and approved the final manuscript.


This work was supported by the National Natural Science Foundation of China (Grant number 31871736), the Research and Development Plan in Key Areas of Guangdong Province (Grant number 2018B020205002) and the Guangdong Provincial Key Laboratory of Advanced Biofermentation Technology Enterprise in Flavoring & Food (Grant number 2017B030302002).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

10295_2019_2252_MOESM1_ESM.docx (626 kb)
Supplementary material 1 (DOCX 626 kb)


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Room 315 Building B6, School of Biology and Biological EngineeringSouth China University of Technology, Guangzhou Higher Education Mega CenterGuangzhouChina
  2. 2.Guangdong Provincial Key Laboratory of Fermentation and Enzyme EngineeringGuangzhouChina

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