Engineering and bioprocess optimization of Trichoderma reesei for recombinant production of resilin and spider silk like proteins
Shahin, Gasser (2025)
Diplomityö
2025
School of Engineering Science, Kemiantekniikka
Kaikki oikeudet pidätetään.
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi-fe2025090394291
https://urn.fi/URN:NBN:fi-fe2025090394291
Tiivistelmä
The production of high-performance protein-based biomaterials using microbial hosts offers a promising route toward sustainable alternatives to synthetic polymers. This thesis investigates the heterologous expression of two recombinant structural proteins CBM-resilin-CBM and NT2RepCT using the filamentous fungus Trichoderma reesei. These biomimetic proteins are inspired by insect and spider silks and hold potential for use in soft tissue engineering, surface coatings, and advanced materials.
A major bottleneck in their production is degradation by endogenous fungal proteases. To address this, a targeted CRISPR-Cas9 genome editing approach was applied to delete specific protease genes (e.g., slp2, slp6), previously identified through spiking assays and expression profiling. In parallel, the native transcription factor xyr1 was replaced with a constitutively active xyr1^mut variant, allowing for a shift from lactose- to glucose-based media while maintaining expression from cellulase promoters. This switch supports simplified media formulation, potentially lowering production costs.
Cultivation condition optimization, including pH profiling and media composition, revealed that lower pH (4.0–4.8) enhanced protein stability and minimized degradation. SDS-PAGE and Western blot analyses confirmed improved accumulation of intact CBM-resilin-CBM in protease-deficient and xyr1^mut strains. Initial attempts to express NT2RepCT were unsuccessful, yielding no detectable product in the supernatant, guiding further optimization steps.
This work presents a modular strain engineering strategy combining protease knockout and transcriptional reprogramming to enable stable production of degradation-sensitive proteins in T. reesei. These results lay the foundation for future scale-up of fungal bioprocesses aimed at manufacturing functional biomaterials.
A major bottleneck in their production is degradation by endogenous fungal proteases. To address this, a targeted CRISPR-Cas9 genome editing approach was applied to delete specific protease genes (e.g., slp2, slp6), previously identified through spiking assays and expression profiling. In parallel, the native transcription factor xyr1 was replaced with a constitutively active xyr1^mut variant, allowing for a shift from lactose- to glucose-based media while maintaining expression from cellulase promoters. This switch supports simplified media formulation, potentially lowering production costs.
Cultivation condition optimization, including pH profiling and media composition, revealed that lower pH (4.0–4.8) enhanced protein stability and minimized degradation. SDS-PAGE and Western blot analyses confirmed improved accumulation of intact CBM-resilin-CBM in protease-deficient and xyr1^mut strains. Initial attempts to express NT2RepCT were unsuccessful, yielding no detectable product in the supernatant, guiding further optimization steps.
This work presents a modular strain engineering strategy combining protease knockout and transcriptional reprogramming to enable stable production of degradation-sensitive proteins in T. reesei. These results lay the foundation for future scale-up of fungal bioprocesses aimed at manufacturing functional biomaterials.