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

ABSTRACT 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. Graphical Abstract Graphical Abstract A repeated high-throughput mutagenesis workflow for strain improvements of Trichoderma reesei using the droplet-based microfluidic platform