Our Solution

Since natural gut microbes lack the enzymes needed to efficiently break down plant cell walls, the ability of BSFL to digest and absorb nutrients from plant-based food waste is limited. To address this issue, our project fills the gap by engineering a microbial system capable of producing and secreting cellulolytic enzymes that can break down the fibrous fraction of food waste prior to BSFL introduction. This pretreatment approach could shorten composting times while providing a sustainable solution to food waste accumulation.

Bacillus subtilis JA18

We selected Bacillus subtilis JA18 as the gene source for our endoglucanase due to its demonstrated enzymatic robustness: the recombinant endo-β-1,4-glucanase—expressed in E. coli—exhibits optimal activity at pH 5.8 and 60 °C, while maintaining remarkable stability across a broad pH range (4.0–12.0) and up to 60 °C (Liu et al., 2006). These thermal properties match the elevated temperatures (40–60 °C, occasionally approaching 70 °C in thermophilic phases) observed during food waste degradation and composting (Voběrková et al., 2020). Instead of using JA18 as our chassis organism, we cloned and expressed the endoglucanase gene directly in E. coli BL21, combining the stability and efficiency of the JA18 enzyme with the customizability of the E. coli expression system.

Endo-β-1,4-glucanase gene from Bacillus subtilis JA18 plays a key role because it produces an enzyme that cuts apart the internal bonds of cellulose. By doing this, it breaks down the plant cell wall structure into smaller sugars. This process makes fibrous material much easier to degrade, allowing microbes to use the nutrients more efficiently and helping BSFL digest food waste more effectively. Unlike exoglucanases, which cut from the ends of chains, endoglucanase works internally. It quickly shortens polymers and increases the surface area of the substrate, speeding up the overall breakdown process (Liu et al., 2006). When expressed in E. coli, this gene allows our engineered system to release active cellulase into the environment. It directly targets the fibrous part of food waste. In this way, the endoglucanase enhances microbial digestion while transforming the structural complexity of plant biomass, converting a natural limitation into a controllable and efficient pretreatment process.

To enable secretion of the recombinant endoglucanase, we fused the JA18 endo-β-1,4-glucanase gene with YebF, a well-characterized periplasmic protein, which we expressed in E. coli using our designed vector that can be exported into the extracellular medium via the Sec pathway. This strategy allows the cellulase to bypass cell lysis and directly accumulate outside the bacterial cell, where it can interact with the fibrous fraction of food waste. The fusion exploits the natural ability of YebF to carry heterologous proteins across the inner membrane, ensuring that the enzyme reaches the extracellular environment in an active form (Zhang et al., 2006). By using YebF-mediated secretion, we address one of the major limitations of intracellular enzyme expression—the need for costly downstream purification or artificial cell disruption—thereby making our system more efficient and practical for food waste pretreatment.