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Elucidating Mechanisms of Acetate Tolerance in E. Coli Using Scales

Nicholas R. Sandoval and Dr. Ryan T. Gill. Department of Chemical and Biological Engineering, University of Colorado, ECCH 111, Campus Box 424, Boulder, CO 80309

Creating biofuels from hemicellulosic and cellulosic biomass is an important part in transitioning away from the petroleum-based transportation fuel economy. Acetate is a major toxic side product of the requisite pretreatment steps of the feedstock to the microorganisms producing biofuels. Conferring tolerance of acetate upon the microorganism will increase the viability of the biofuels economy.

The SCalar Analysis of Library Enrichments (SCALEs) method, created previously by the Gill lab at the University of Colorado, combines a traditional genomic library selection with DNA microarray technology providing genotypic data for selected clones. The method employs multiple libraries of different, but defined, insert sizes to allow greater resolution of beneficial genomic regions. The objective is to find mechanisms of acetate inhibition on E. coli growth. These mechanisms can be elucidated using SCALEs. Sections of the genome that confer acetate tolerance, when over-expressed, will be found in a SCALEs selection.

We report here two selections using an E. coli K12 library with a 1.75 g/L or a 2.5 g/L acetate culture titrated to a pH of ~7.0 with potassium hydroxide. The cultures were monitored over a three day period with serial transfers to ensure cultures were not in stationary phase. Top selected clones show an increase in growth rate between 60% to over 100% against control.

Microarray studies were performed and analyzed using the SCALEs method. Regions of the genome were found that conferred tolerance the most. Validation growth rate studies were done via subcloning. These top performing regions contained genes that were important in amino acid transport and metabolism and cell envelope biogenesis. SCALEs data underwent a pathway analysis and those pathways most enriched included amino acid metabolism pathways and central metabolism pathways.