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

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...

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Bibliographic Details
Published in:Journal of industrial microbiology & biotechnology 2020, Vol.47 (1), p.133-144
Main Authors: Dong, Liangbo, Lin, Xiaotong, Yu, Dou, Huang, Lianggang, Wang, Bin, Pan, Li
Format: Article
Language:English
Subjects:
Aspergillus niger
Aspergillus niger - genetics
Aspergillus niger - metabolism
Biochemistry
Bioinformatics
Biomedical and Life Sciences
Biotechnology
Biotechnology Methods - Original Paper
CRISPR
CRISPR-Cas Systems
Disaccharides
Enzyme Stability
Ethanol
Ethanol - metabolism
Fermentation
Fungi
Gene expression
Genetic Engineering
Hot Temperature
Inorganic Chemistry
Life Sciences
Microbiology
Myceliophthora thermophila
Optimization
pH effects
Production costs
Saccharification
Sordariales - genetics
Sordariales - metabolism
Starch
Strategy
Thermal stability
Trehalase - genetics
Trehalase - metabolism
Trehalose
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Summary: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.
ISSN:1367-5435
1476-5535
DOI:10.1007/s10295-019-02252-9