Designing E. coli for the secretion of biomass degrading enzymes

Sarkar, Aniket (2025) Designing E. coli for the secretion of biomass degrading enzymes. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Aniket Sarkar (20MS071)) 20MS071_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

The global transition toward sustainable energy systems has intensified efforts to develop efficient lignocellulosic biorefineries capable of converting plant biomass into biofuels and biochemicals. Central to this process is the enzymatic hydrolysis of cellulose into fermentable sugars. This breakdown requires the synergistic activity of multiple cellulase classes. Conventional approaches rely on separate fermentation steps for enzyme production and biomass saccharification, increasing operational complexity and costs. To address this limitation, we engineered Escherichia coli for the secretion of a cellulase cocktail composed of endoglucanases, exoglucanases, and β-glucosidases. Each enzyme was fused to a secretory tag to facilitate extracellular secretion, enabling direct saccharification. We employed an adapted modular cloning strategy utilizing the Golden Gate and Gibson assembly method to construct the necessary genetic circuits. Furthermore, culture conditions were optimized to enhance the secretion efficiency of the assembled enzyme cocktail. This platform not only simplifies the production process, potentially reducing costs and complexity, but also paves the way for in-depth studies on the synergistic interactions among secreted cellulases for sustainable biofuel and value-added product production.

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