Biofuels Production projects

Designer Secretion Pumps for Fuel-Producing Bacteria

The microbial conversion of sugars to biofuels is a promising new technology, but the fuels produced are often toxic to the microbial producers. One promising solution is to alter the pumps that microbes use to secrete antibiotics and other toxic chemicals to secrete the fuel product out of the cell instead. Understanding the natural diversity of such pumps and how to modify them for the efflux of desired chemicals will be critical for successful microbial biofuel production. This group is characterizing and genetically optimizing protein-based efflux pumps to secrete two important classes of biofuels from gram-negative bacteria.

project Highlights

2012 Highlights

With this project, our goal was to develop pumps that secrete biofuels from Escherichia coli that are producing these fuels. This potentially would increase growth rates, as the fuels are toxic, and increase the amount of fuel produced. In 2012, we used directed evolution to further improve upon the pumps that we engineered in 2011. We identified two new versions of the AcrB pump that is native to E. coli, each of which give rise to higher growth rates in the presence of alcohols such as butanol, pentanol, and heptanol. We also have experimental evidence that this higher growth is due to the activity of the pump and is specific for alcohols – there is a decrease in tolerance to the natural antibiotic substrates. Finally, we demonstrated that some pumps increase titer and productivity of butanol-producing E. coli. While this is exciting, we also observed that making too many copies of the pump is detrimental to cell growth, so we designed a genetic circuit to keep the level of the pump at reasonable levels. This circuit is still being optimized but will soon be combined with our engineered pumps in the butanol-production strain.

2011 Highlights

Tullman-Ercek’s group developed two pumps that conferred higher growth rates in the presence of toxic levels of butanol in laboratory strains of Escherichia coli. They also combined pumps with butanol-production strains from the Chang lab and demonstrated slight butanol production increases in E. coli in the presence of pumps. Researchers also determined that the two pumps also confer higher growth rates in the presence of other alcohols, but do not increase tolerance to the antibiotic substrate of the wild type pump.

2010 Highlights

Tullman-Ercek’s group designed a system for and demonstrated functional expression of heterologous pump systems in E. coli, determined selection conditions for pump optimization, and designed diversified libraries of two efflux pumps.

2009 Highlights

Tullman-Ercek’s group selected five candidate tripartite efflux pump systems for characterization and engineering. Members of the group have designed the directed evolution experiments that will be used to create mutated versions of the efflux pumps. Mutant pumps that confer higher tolerance levels to the fuels of interest will be uncovered using a competition-based growth selection scheme. Successfully engineered pumps are expected to increase the yield of toxic biofuels in E. coli and any other gram-negative host organism compatible with efflux pump expression and assembly.

 


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