Monday, April 19, 2010
1-15

Effect of glycerol metabolism on xylose fermentation by engineered Saccharomyces cerevisiae

Soo Rin Kim1, Suk Jin HA2, Jin-Ho Choi2, and Yong-Su Jin1. (1) Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 1206 W. Gregory Dr, Urbana, IL 61801, (2) Energy Biosciences Institute, University of Illinois at Urbana-Champaign, 1206 W. Gregory Dr, Urbana, IL 61801

Engineered S. cerevisiae strains constructed through introduction of three genes (XYL1, XYL2 and XYL3) from Pichia stipitis are capable of utilizing xylose as a carbon source. However, ethanol production from xylose is not as efficient as glucose fermentation. Among the putative factors hampering efficient xylose fermentation, it is hypothesized that redox imbalance caused by different preference of cofactors between xylose reductase (XR) and xylitol dehydrogenase (XDH) might elicit higher expression of respiratory enzymes, which results in both xylitol accumulation and lower ethanol yields. In addition to xylitol, glycerol is also produced as a byproduct during xylose fermentation. Because the glycerol producing metabolic pathway also employs different types of cofactors, the activation of glycerol metabolism might serve as a redox balancing pathway which compensates the redox imbalance caused by XR and XDH. Specifically, a metabolic pathway consisting of three metabolic reactions (NADH consuming glyceraldehydes-3-phosphate dehydrogenase, NADPH producing glycerol dehydrogenase and ATP consuming dihydroxyacetone kinase) can convert NADH into NADPH at the expense of one ATP. If these reactions are operational, xylose fermentation become redox neutral. Calculation of elementary flux modes (EFM) supported this hypothesis since EFMs resulting in higher ethanol yields from xylose were only found in metabolic networks including these reactions. In order to confirm this prediction experimentally, GPD1 and GCY1 were individually overexpressed in an engineered S. cerevisiae. The GPD1 overexpressing strain produced less xylitol and more glycerol as compared to the control, which indicates that glycerol production supply enough NAD+ for XDH reaction. However, the GCY1 overexpressing strain showed a reduced xylose uptake rate as compared to the control on the contrary to the prediction. Although drastic improvement of xylose fermentation was not observed as EFM analysis predicted, we confirmed that perturbation of glycerol metabolism highly connected with xylose fermentation.

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