Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system

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 - gfp OE- amyR Δ alp-1 Δ res-1 Δ cre-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.