Ultrahigh-throughput screening of Trichoderma reesei strains capable of carbon catabolite repression release and cellulase hyperproduction using a microfluidic droplet platform.

Trichoderma reesei is the most well-known cellulase producer in the biorefinery industry. Its cellulase biosynthesis is repressed by glucose via carbon catabolite repression (CCR), making CCR-releasing strains with cellulase hyperproduction desirable. Here, we employed a microfluidic droplet platform to culture and screen T. reesei mutants capable of CCR release and cellulase overproduction from extensive mutagenesis libraries. With 3 mutagenesis rounds, about 6.20 × 103 droplets were sorted from a population of 1.51 × 106 droplets in a period of 4.4 h; 76 recovery mutants were screened on flask fermentation, and 2 glucose uptake retarded mutants, MG-9-3 and MG-9-3-30, were eventually isolated. We also generated a hypercellulase producer, M-5, with CCR release via a single mutagenesis round. The hyphal morphology and molecular mechanisms in the mutants were analyzed. This versatile approach combined with a comprehensive understanding of CCR release mechanisms will provide innovative and effective strategies for low-cost cellulase production.

A novel high-throughput approach for transforming filamentous fungi employing a droplet-based microfluidic platform.

Droplet-based microfluidic technology is a powerful tool for single-cell cultivation and rapid isolation of bacteria, yeasts and algae. However, it has been of limited use for studies of filamentous fungi due to the fast growth of their branched hyphae. The long regeneration time for fungal protoplasts and low-throughput screening methods are inherent problems for current genetic transformation techniques. Therefore, we have developed a novel droplet-based method for the filamentous fungus Trichoderma reesei expressing green fluorescent protein (GFP) as a marker. This approach presented several outstanding advantages over the traditional transformation method, including a 7-fold reduction in time for T. reesei protoplast regeneration, an 8-fold increase in regeneration frequency, and a screening speed of up to 8,000 droplets min-1. In this study, we encapsulated and incubated the gfp-transformed T. reesei protoplasts in droplets for 24 h, screened the droplets in a high-throughput assay, and eventually collected a transformant library with over 96 % of the candidates transformed with the marker gene. This versatile approach should make fungi more amenable to genetic manipulation and encourage strain improvements for industrial applications.