Understanding and improving microbial biofuel tolerance as a result of efflux pump expression through genetic engineering and mathematical modeling

• Main Author: Turner, William James
• Format: Others
• Language: en
• Published: ScholarWorks @ UVM 2014
• Subjects: Biofuel; Efflux pumps; Pinene; Tolerance; Engineering; Microbiology
• Online Access: http://scholarworks.uvm.edu/graddis/322; http://scholarworks.uvm.edu/cgi/viewcontent.cgi?article=1321&context=graddis

Recent advances in synthetic biology have enabled the construction of non-native metabolic pathways for production of next-generation biofuels in microbes. One such biofuel is the jet-fuel precursor α-pinene, which can be processed into high-energy pinene dimers. However, accumulation of toxic biofuels in the growth medium limits the possible fuel yield. Overexpression of transporter proteins such as efflux pumps can increase tolerance to biofuels by pumping them out of the cell, thus improving fuel yields. However, too many efflux pumps can compromise the cell as well, creating a trade-off between biofuel toxicity and pump toxicity. In this work we improve the conditions of this trade-off in order to increase pinene tolerance in E. coli. We do so by constructing strains incorporating multiple efflux pumps from a variety of organisms and then testing them for tolerance in growth assay experiments. Previous research has suggested that certain combinations of efflux pumps can confer additional tolerance compared to the individual pumps themselves. However, the functional form of the combination of the tolerance provided by each pump and the toxicity due to their simultaneous activity is unknown. Using differential equations, we developed a growth model incorporating the trade-offs between toxicity of α-pinene and efflux pump activity to describe the dynamics of bacterial growth under these conditions. By analyzing biofuel toxicity and the effects of each efflux pump independently through a series of experiments and mathematical models, we propose a functional form for their combined effect on growth rate. We model the mean exponential growth rate as a function of pump induction and biofuel concentration and compare these results to experimental data. We also apply this technique to modeling toxicity of ionic liquids, a class of corrosive salts that has emerged as and effective chemical for pretreatment of biofuel production feedstock. We compare a model for a variety of ionic liquid responsive efflux pump controllers to that of an IPTG inducible controller and show agreement with experimental data, supporting the model's utility to test control schemes before conducting experiments. The overall goal of this project is to use modeling to guide design of tolerance mechanisms to improve overall biofuel yield.