(609d) Engineering Modular Microbial Communities for Cellulose Utilization and Bioproduct Synthesis

Recent environmental concerns have increased the need to develop systems which can utilize waste materials to make chemicals. Cellulosic biomass, an abundant waste material, can be degraded into sugar monomers to serve as an inexpensive carbon source for microbial systems. Current approaches for biomass utilization rely on pretreatment processes and expensive purification schemes. To address these challenges, microbial communities can be engineered to synergistically perform complex tasks, by dividing the demands among multiple specialized modules, or functional units, to maximize the efficiency of desired product formation. Therefore, we aim to build microbial systems with interchangeable modules, with the first module dedicated to cellulose degradation and the second module specialized for bioproduct synthesis. To engineer synthetic communities for the production of biofuels and pharmaceuticals, we are building and testing mixed cultures of Bacillus megaterium and Escherichia coli strains. We will describe our recent progress engineering these two modules and assembling them for activity.

Complete degradation of cellulose requires a combination of three cellulase activities – an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. We have developed a cellulose degradation module consisting of Bacillus megaterium strains for the secretion of cellulases. A small library of signal peptides (SPs) was selected to tag each cellulase, and thereby used to trigger protein secretion in B. megaterium. Cellulase activity against amorphous cellulose was confirmed through a series of bioassays, and the most active signal peptide constructs were identified for each cellulase. Expression optimization for each strain focused on testing secretion and activity under different expression temperatures, induction time points, expression lengths, and media conditions. Activity of the optimized cellulase secretion strains was characterized individually and in tandem to assess synergistic cellulolytic activity. We have also demonstrated that the secreted cellulase activity can be used to degrade cellulose into glucose monomers to support growth of an E. coli production module. We will describe our progress investigating the effect of different inoculation ratios, expression conditions, and community compositions for the cellulose degradation module to determine optimized cellulase secretion and activity conditions for the system. Further system development has also targeted the second module, with the introduction of a violacein-producing E. coli strain. The cellulose degradation module can be integrated with many different bioproduct synthesis modules to build complex systems for the synthesis of high value products from cellulose.