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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

  • Biotechnology Methods - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

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.

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Funding

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).

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Authors and Affiliations

  1. Room 315 Building B6, School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu town, Guangzhou, 510006, Guangdong, China

    Liangbo Dong, Xiaotong Lin, Dou Yu, Lianggang Huang, Bin Wang & Li Pan

  2. Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, Guangzhou, 510006, China

    Bin Wang & Li Pan

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  1. Liangbo Dong
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  2. Xiaotong Lin
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  3. Dou Yu
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  4. Lianggang Huang
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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.

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Correspondence to Li Pan.

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Dong, L., Lin, X., Yu, D. et al. 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 47, 133–144 (2020). https://doi.org/10.1007/s10295-019-02252-9

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