As the global economy begins to shift its focus from conventional fuel sources, the need for effective and cost-efficient fuel alternatives arises. Agricultural waste, which is primarily composed of cellulose, hemicellulose, and lignin, serves as an attractive alternative to these traditional methods. In order to degrade these cellulosic materials into usable components, a cost-effective and efficient method is needed. Bacillus subtilis satiates this requirement, specifically for its ubiquitous usage in the biotechnology industry and efficiency in enzyme secretion, which is an important prerequisite from a microbial host to breakdown large biopolymers. The efficiency of enzymatic secretion in B. subtilis is facilitated in part by signal peptides and their role in enzyme transport across cell membrane. The objective of this project is focused on the development of a B. subtilis based bacterial platform for heterogeneous expression and extracellular export of the hydrolases, namely cellulases for the extracellular depolymerization of cellulose. Towards this goal, various signal peptides and genes were combined, verified, and optimized. Recombinant plasmids were constructed for the expression of two β - glucosidases from Trichoderma resei (Tr) and Neosartorya fischeri (Nf). The secretion of these enzymes were explored with three signal peptides namely YwmC, SacC and AmyE for cellobiose breakdown. The recombinant and wild type strains of B. subtilis WB800N were screened for β - glucosidase activity via p- NPG assay. The highest enzymatic activity was observed in the AmyE- Bg(Tr) sample, with an activity of 6.5 mM/mg/min (5 folds higher than wild type). β - glucosidase plays a key role in the hydrolysis of cellobiose, whereas the endoglucanase attacks the cellulose backbone directly. Therefore, combinations of NprE, YwmC, and Egls signal peptides and endoglucanases from B. subtilis and T. resei are being developed for cellulose depolymerization. The discoveries from this project could be further expanded to develop consolidated bioprocesses for the production of second generation biofuels from lignocellulosic biomass.

Engineering Bacillus subtilis for in situ depolymerization of cellulose

ACS Spring Meeting 2021

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