Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biomass Conversion

Background:

• Cellulases are key enzymes for biomass hydrolysis.
• Enhancing cellulase activity and stability is crucial for efficient biofuel production.
• Talaromyces emersonii Cel7A (TeCel7A) is a thermophilic enzyme with potential for industrial applications.

Purpose of the Study:

• To improve the performance of TeCel7A by fusing it with various carbohydrate-binding modules (CBMs).
• To investigate the effect of CBMs on cellulose binding, thermal stability, and hydrolytic activity.
• To evaluate the efficacy of engineered enzymes in biomass hydrolysis.

Main Methods:

• Construction and expression of chimeric cellobiohydrolases (TeCel7A fused with bacterial and fungal CBMs).
• Purification and characterization of fusion proteins, including cellulose binding assays and thermal stability measurements (T m).
• Enzymatic activity assays on microcrystalline cellulose and lignocellulosic feedstock at various temperatures.

Main Results:

• Fusion proteins exhibited enhanced cellulose binding and high thermal stability (T m: 72–77 °C).
• A bacterial CBM3 fusion (from CipA) significantly boosted activity on microcrystalline cellulose.
• The best CBM3 fusion enzyme outperformed Trichoderma reesei Cel7A at moderate and high temperatures (up to 65 °C) and showed potency in lignocellulosic biomass hydrolysis at 70 °C.

Conclusions:

• Fusion with specific CBMs, particularly bacterial CBM3, is an effective strategy to enhance cellulase performance.
• Engineered TeCel7A variants demonstrate potential for efficient and high-temperature biomass conversion.
• This work contributes to the development of robust enzymes for the biofuel and biochemical industries.