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- Title
Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.
- Authors
Li, Fangya; Liu, Qian; Li, Xiaolin; Zhang, Chenyang; Li, Jingen; Sun, Wenliang; Liu, Dandan; Xiao, Dongguang; Tian, Chaoguang
- Abstract
Objective: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass. Results: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0–9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT. Conclusions: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.
- Subjects
THERMOPHILIC fungi; ENZYMES; FUNGAL enzymes; BIOMASS chemicals; AMYLASES; INDUSTRIAL capacity; CRISPRS
- Publication
Biotechnology Letters, 2020, Vol 42, Issue 7, p1181
- ISSN
0141-5492
- Publication type
Article
- DOI
10.1007/s10529-020-02882-5