Engineering recombinant microbial communities for cellulose degradation and utilization

Abstract

Recent environmental concerns have intensified the need to develop systems to degrade waste biomass for use as an inexpensive carbon source for microbial chemical production. Current approaches to biomass utilization rely on pretreatment processes that include expensive enzymatic purification steps for the requisite cellulases. To address this challenge, synthetic communities can be engineered to streamline cellulose degradation and utilization, and eliminate the need for costly enzyme purification. Therefore, we aimed to engineer a synthetic microbial system with specialized modules designed for each compartmentalized task, with the first module dedicated to cellulose degradation and the second module specialized for bioproduct synthesis. To construct the cellulose degradation module, EGI1, an endoglucanase, and Cel9AT, a multimodular cellulase, were targeted for secretion from B. megaterium. A small library of signal peptides (SPs) with five amino acid linkers was selected to tag each cellulase for secretion.

Cellulase activity against amorphous cellulose was confirmed, and the most active SP constructs were identified as EGI1 with the LipA SP and Cel9AT with the YngK SP. Both strains were optimized for cellulase expression, and activity of the enzymes was characterized individually and in tandem to demonstrate synergistic cellulolytic activity in coculture. The secreted cellulases were reacted with amorphous cellulose under a range of different conditions, and the resulting glucose supported the growth of the second target module, an E. coli strain to be used for production of violacein, an antimicrobial pharmaceutical product with anti-tumor properties. A series of studies investigated the compatibility of the two modules and the cellulose degradation reaction. The construction of a two-module system for efficient cellulose degradation and utilization demonstrated the value of the modular system design, to allow bacterial strains to be interchanged or linked with other engineered bacterial strains for the efficient production of high value molecules.