High-throughput droplet screening of filamentous fungi on solid substrates.

Droplet-based microfluidics has been frequently used for high-throughput single-cell microbial screening, where individual cells are encapsulated into nanoliter or picoliter-sized droplets and sorted based on measurable phenotypes such as fluorescent proteins or enzyme activity using fluorescence or absorbance-based substrates. However, using droplet microfluidics with filamentous fungi poses a unique challenge: fungal strains tend to escape from droplets as their hyphae grow, causing individual droplets to merge and eventually burst which prevents measuring individual phenotypes. In our work, we demonstrate a strategy to robustly encapsulate and retain spores of filamentous fungi within water-in-oil droplets by incorporating solid substrates into the droplet microenvironment. Using this approach, we show for the first time the encapsulation and growth of two model filamentous fungi species in droplets - Aspergillus oryzae and Trichoderma reesei - with solid substrates supporting spore retention, germination, and early hyphal development without compromising droplet stability. Our goal was to screen for enhanced enzyme production (e.g., α-amylase and cellulase) using fluorescence-based activity assays. To this end, we further characterized our top hits through scale-up fungal fermentation to evaluate improvements in both enzyme production and total protein secretion. Our work overcomes a key technical barrier in applying droplet microfluidics to filamentous fungi that allows for high-throughput screening and discovery of new fungal strains that produce active enzymes and heterologous proteins relevant to industrial applications.