(288d) Engineering E. Coli for Anaerobic Xylitol Production with Xylose as the Sole Electron Acceptor During Glucose Oxidation

Many studies have focused on the overproduction of fermentation products in genetically modified Escherichia coli. The metabolic pathways exploited typically utilize endogenous metabolic intermediates as electron acceptors for NADH reoxidation to maintain redox balance during anaerobic sugar metabolism. Here we describe E. coli strains which have been engineered to reduce xylose to xylitol as the sole means of regenerating NAD+ during glucose oxidation. The metabolic behavior of these strains is partly fermentative in that energy is derived via substrate level phosphorylation, as well as partly respiratory in that the electron acceptor is external (not derived from glucose). A model strain was tested using a constraint-based stoichiometric network model of E. coli metabolism (SimphenyTM, Genomatica Inc; model adapted from (Reed et al. Genome Biol. 2003)). As expected, growth of the in silico strain is coupled to xylitol production. The model reveals the extent to which the mode of xylose transport and the cofactor specificity of xylose reduction influence cell growth. Experimentally, we constructed the modeled strain by eliminating competing fermentation pathways through corresponding gene deletions (i.e., DldhA, DadhE, Dfrd, DpflB) and preventing xylose metabolism by a xylAB gene deletion. An NADPH-dependent xylose reductase was employed for xylose reduction, thus requiring reducing equivalents to be transferred from NADH to NADP+. Expression of an NADH-insensitive pyruvate dehydrogenase complex mutant (Kim et al. Appl. Environ. Microbiol. 2007) or an NAD+-dependent formate dehydrogenase from Candida boidinii (Berrios-Rivera et al. Metab. Eng 2002) was explored to increase NADH availability and the xylitol yield. This fermentative/respiratory growth-coupled system is being used to study the anaerobic behavior of the E. coli transhydrogenases and xylose transporters.