Strategies to Increase the Synthesis of Biorenewable Chemicals Derived from Second-Generation Feedstocks
In response to the exhaustion of carbon feedstock from fossilized sources, dependence on foreign energy supplies, and the threat of climate change, there is an increasing incentive for the production of biofuels and bio-based chemicals. Second-generation biorefineries provide a sustainable approach by relying on lignocellulosic biomass obtained from renewable resources. To increase economic viability and lower the cost of biomass processing there is a promising approach called Consolidated Bioprocessing (CBP). CBP may be accomplished by hydrolysis of lignocellulose’s main components—hemicellulose and cellulose—by displaying a xylanosome and a cellulosome on the surface of the microorganism responsible for synthesis of the desired chemical (e.g. ethanol, medium chain fatty acids, etc.). In the first part of this study, we describe strategies to increase the efficiency of CBP lignocellulosic biomass hydrolysis. First, we developed an expression system and a set of strains that showed enhancements in protein secretion of 5.8-fold to 11.5-fold. Next, we designed and characterized an extracellular glucose sensor that enables selective display of cellulose-degrading enzymes in the presence of the cellulosic fraction of lignocellulose. This is possible by leveraging cellulose-dependent signal amplification. The engineered version of the sensor showed up to 81% higher levels of expression, and a 55%-91% amplification of the signal was observed in the presence of cellobiose. Once saccharification is complete, CBP microorganisms will ideally synthesize chemicals that can functionally substitute those currently generated by the petrochemical industry. Fatty acids with chain lengths between 8 and 12 carbons have a wide variety of industrial applications, including as biofuels and precursors to commodity and fine chemicals. Our laboratory has successfully engineered Saccharomyces cerevisiae to produce hexanoic acid, octanoic acid (C8) and decanoic acid (C10). However, higher levels of production are hindered by the toxic effects of the fatty acids on the cells at increased concentrations. To overcome this limitation, we successfully explored two different strategies in the last part of this study: i) changing the membrane composition to alleviate the effects of C8, and ii) identifying and overexpressing efflux pumps that export C10. This allowed growth improvements of up to 10-fold (C8) and 11-fold (C10).