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

J Ind Microbiol Biotechnol. 2020 Jan;47(1):133-144. doi: 10.1007/s10295-019-02252-9. Epub 2019 Nov 30.


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.

Keywords: Aspergillus niger; CRISPR/Cas9; Ethanol fermentation; Multi-copy; Trehalase.

MeSH terms

  • Aspergillus niger / genetics
  • Aspergillus niger / metabolism*
  • CRISPR-Cas Systems
  • Enzyme Stability
  • Ethanol / metabolism*
  • Fermentation
  • Hot Temperature
  • Sordariales / genetics
  • Sordariales / metabolism*
  • Trehalase / genetics
  • Trehalase / metabolism*


  • Ethanol
  • Trehalase