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

UNLABELLED: Thermophilic filamentous fungi are widely used for cellulase production at elevated temperatures; however, efficient recombinant protein production in these organisms remains limited because construction and screening of large transformant populations remain difficult. 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 with QPix-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. Through iterative screening and strain improvement, extracellular protein production reached ~1.1 g/L in shake-flask cultures and 5.9 g/L in a preliminary 5-L fermentation after 144 h using glucose as the carbon source. The chassis also supported the secretion of recombinant fungal enzymes and porcine growth factors. Collectively, these results establish a practical semi-automated workflow for recombinant protein production and strain engineering in thermophilic filamentous fungi. IMPORTANCE: Filamentous fungi are important industrial hosts, but strain engineering in these organisms is often constrained by low transformation efficiency, labor-intensive colony isolation, and limited screening throughput. In this study, we established a semi-automated engineering workflow in the thermophilic fungus Myceliophthora thermophila that combines dilution-based colony recovery, QPix-assisted colony isolation, and activity-based screening. More than 500 fungal colonies could be recovered and processed during a single engineering round, allowing rapid evaluation of transformants by ABTS-based plate screening. In addition to enhanced laccase production, the engineered chassis supported the secretion of multiple heterologous proteins, including fungal enzymes and porcine growth factors. These findings demonstrate the potential of thermophilic filamentous fungi as recombinant protein production hosts and provide a practical workflow for improving strain engineering efficiency in filamentous fungal systems.