Work

Reprogrammed E. coli for secretion of thermophilic cellulase cocktail for seawater-compatible lignocellulosic bioprocessing

Year: 2026

Type: article

Abstract: Efficient, low-cost cellulase systems are essential for sustainable lignocellulosic biorefining, yet current platforms remain constrained by high production costs, reliance on downstream purification, and dependence on freshwater processing conditions. In this study, we engineered Escherichia coli to co-express and secrete a thermophilic cellulase cocktail consisting of a Bacillus licheniformis processive endoglucanase (H1AD14), a Chaetomium thermophilum cellobiohydrolase (G0SAE6), and a glucose-tolerant engineered β-glucosidase B8CYA8tm (V169C/E173L/I246A B8CYA8) from Halothermothrix orenii . N-terminal tagging with AnsB signal peptide enabled extracellular yields of 0.2–0.4 g/L for each enzyme, bypassing costly purification while retaining activity. The secretion was confirmed via immunoblotting using anti-His antibody, and catalytic activity was validated through enzymatic assays. The absence of E. coli native periplasmic maltose-binding protein (detected with anti-MBP antibody) in the extracellular medium ruled out cell lysis, confirming protein secretion. Using the Golden Gate assembly, we constructed and systematically evaluated synergistic enzyme combinations for hydrolytic performance. By optimizing secretion conditions, including media, inducer concentration, and incubation temperature, the total yield of secreted enzyme cocktail increased from 0.31 g/L to 0.74 g/L. The secreted cocktail effectively hydrolysed diverse lignocellulosic substrates and exhibited 100% activity in seawater, demonstrating potential for freshwater-independent biorefining. By combining secretion engineering, thermostability, and seawater tolerance in a single E. coli platform, this work offers a scalable, purification-free strategy for cellulase production that addresses key economic and environmental barriers to freshwater-independent lignocellulosic biorefining.

Source: Microbial Cell Factories

DOI: 10.1186/s12934-026-03102-x

Authors: Mani Gupta, Aniket Sarkar, Suksham, Debdeep Chatterjee, Supratim Datta

Institution: Indian Institute of Science Education and Research Kolkata

Language: English

Topic: Biofuel production and bioconversion

Subfield: Biomedical Engineering

Field: Engineering

Domain: Physical Sciences

SDGs: Affordable and clean energy, Responsible consumption and production

Open Access status: gold

Estimated APC paid: $3,590