Semi-automated strain engineering and screening in the thermophilic fungus Myceliophthora thermophila for recombinant protein production

Here, Myceliophthora thermophila was engineered into a thermophilic fungal chassis through combinatorial deletion of genes associated with proteolysis, vesicle trafficking, and secretion, resulting in a hyper-secretory strain.

An optimized expression cassette coupled with a laccase reporter enabled activity-based identification of productive transformants. Iterative genome engineering combined withQPix-assisted colony picking enabled parallel recovery and screening of fungal transformants, with 552 colonies recovered after transformation and dilution plating and 96 transformants further evaluated by ABTS-based plate screening. Targeted integration at the MtLacA locus enabled identification of productive recombinants through loss of laccase activity, and transformants frequently carried multicopy integrations.