Tuesday, May 3, 2011
Lignocellulosic biomass represents a potential feedstock for microbial conversion into fuels or value-added chemicals. Low concentrations of furfural are formed by the dehydration of pentoses during dilute acid pretreatment of lignocelluloses that significantly inhibit growth and fermentation. Developing biocatalysts that are furfural tolerant is essential for future process simplification. Gene array experiments comparing the parent and a furfural-resistant mutant identified a furfural-inducible operon (bicistronic) that encodes two NADPH-dependent furfural reductases (YqhD and DkgA) under the control of an upstream regulator (YqhC). Both oxidoreductases convert furfural to the less toxic furfuryl alcohol. Plasmid expression of yqhD and dkgA, individually, decreased furfural tolerance. However, deletion of yqhD alone increased tolerance to a greater extent than deletion of both oxidoreductases. A comparison of kinetic parameters revealed that the Km of YqhD for NADPH was unusually low, less than 0.010 mM, while that for DkgA was over twice this value. Both oxidoreductases are proposed to contribute to furfural inhibition by depleting the pools of NADPH and starving biosynthesis. The higher Km DkgA enzyme appears to be beneficial in the absence of YqhD. We have recently identified additional oxidoreductases associated with furfural tolerance. These have been used to develop a general model for detoxification and inhibition that can be used to improve ethanologenic strains of E. coli and to E. coli strains that have been re-engineered for lactate production.