Recent environmental concerns have increased the need to develop systems that can convert biological waste materials to chemicals. Cellulosic biomass can be degraded into sugar monomers and serve as an inexpensive carbon source for microbial chemical production systems. Current approaches for biomass utilization rely on pretreatment processes that include expensive enzymatic degradation steps. Streamlining the cellulose degradation and utilization processes into one system would eliminate the need for costly enzyme purification. To address this challenge, microbial communities can be engineered to synergistically perform complex tasks, dividing the demands among specialized units to improve the efficiency of 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 engineered to express and secrete cellulases. A small library of signal peptides (SPs) was selected to tag each cellulase for secretion from B. megaterium. Cellulase activity against amorphous cellulose was confirmed, and the signal peptide constructs that led to highest levels of secretion and activity were identified for each cellulase. The optimized cellulase secretion strains were characterized in monoculture and coculture, demonstrating synergistic cellulolytic activity. We have further verified that the secreted cellulases can support growth of an E. coli production module via sugars released from cellulose degradation. We will discuss the effect of inoculation ratio, expression conditions, and community compositions on the assembly of a coculture system utilizing cellulose to produce violacein, an antimicrobial pharmaceutical product with anti-tumor properties. The cellulose degradation strains will be further integrated with alternate bioproduct synthesis modules to build new cellulose-based systems.

Engineering modular microbial communities for cellulose utilization and bioproduct synthesis

255th National Meeting (2018)

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