Enteric bacterial catalysts for fuel ethanol production

The technology is available to produce fuel ethanol from renewable lignocel lulosic biomass. The current challenge is to assemble the various process o ptions into a commercial venture and begin the task of incremental improvem ent. Current process designs for lignocellulose are far more complex than g rain to ethanol processes. This complexity results in part from the complex ity of the substrate and the biological limitations of the catalyst. Our wo rk at the University of Florida has focused primarily on the genetic engine ering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate d ecarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrate d into the chromosome of Escherichia coli B for use with hemicellulose-deri ved syrups. The resulting strain, KO11, produces ethanol efficiently from a ll hexose and pentose sugars present in the polymers of hemicellulose. By u sing the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccha rification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and se crete high levels of endoglucanase has also been added to strain P2, furthe r reducing the requirement for fungal cellulase. The general approach for t he genetic engineering of new biocatalysts using the PET operon has been mo st successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Man y opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for th e efficient fermentation of both hemicellulosic and cellulosic substrates.