Maximizing Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis
Dhinakar Kompala
University of Colorado, Boulder

Zymomonas mobilis has been metabolically engineered to broaden their substrate utilization range to include D-xylose and L-arabinose at the National Renewable Energy Laboratory in Golden, CO. Both chromosomally-integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose D-xylose have been created by incorporating 4 genes: 2 genes xylA and xylB  encoding xylose utilization metabolic enzymes, xylose isomerase and xylulokinase and 2 genes  talB andtktA  encoding pentose phosphate pathway enzymes, transaldolase and transketolase.  While the proof-of principle that the metabolically engineered Z. mobilis strains are able to ferment bother glucose and xylose to ethanol has been previously established, our current research undertakes detailed quantitiative investigations on the enhanced metabolic network to maximize the ethanol production from glucose and xylose by these strains.

Two different xylose-fermenting Z. mobilis strains were grown on glucose-xylose mixtures in computer-controlled fermentors to analyze the extracellular metabolite concentrations as well as the activities of several intracellular enzymes from the xylose and glucose consumption pathways.  Dynamic profiles of these enzymes show dramatic increases in the activities of the two xylose utilizing enzymes immediately after the depletion of the preferred sugar, glucose.  We are now addressing the regulatory mechanisms underlying these reproducible increases.  First, the issues of regulation at the protein synthesis level versus the enzyme activity level is being resolved through quantification of the key intracellular protein concentrations through proteomic analysis using 2 D gel electrophoresis techniques.   In parallel, we are characterizing the intracellular concentrations of the key metabolites along the network, namely the phosphorylated carbohydrates through NMR spectroscopy.  Subsequently, the issues of transporter limitations as well as the gene expression regulation and dosage effects will be addressed in the next year.

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