(485n) Engineering Tolerance and Determining Toxicity of Furfural On E. Coli for Improved Lignocellulosic Biomass Utilization

Lignocellulosic biomass provides a renewable feedstock for microbial conversion to biofuels and commodity chemicals. Pre-treated hydrolysate from corn stover contains complex mixtures of microbial inhibitors that are known to act synergistically, especially when the sugar degradation product furfural is present, limiting efficient growth and conversion. Our goal is to identify genetic elements capable of conferring increased tolerance toward hydrolysate and to isolate the inhibitors responsible specifically, enabling elucidation of their toxic modes of action. In this work, furfural has been studied independently as well as a defined hydrolysate designed to represent all the modes of inhibitory toxicity, thus enabling isolation of genetic effects. A genomic library selection was performed using E. coli K12 DNA on solid minimal media supplemented with up to 5 g/l furfural, from which twenty randomly selected colonies were screened from the surviving populations. All sequenced clones belong to one of four specific genomic regions. During growth in liquid batch cultures at 1 g/l furfural, representative clones from these four regions exhibit a > 200% increase in specific growth. A defined hydrolysate has been created, based on the known composition of ammonium pre-treated hydrolysate, thus defining the necessary components for toxicity of this hydrolysate at 50% (the concentration at which this hydrolysate results in 100% cell death). The presence and concentration of constitutive components has been manipulated for parallel genomic analysis, the results of which are discussed.