Expression of a Heterologous Xylose Transporter in a Saccharomyces cerevisiae Strain Engineered to Utilize Xylose Improves Aerobic Xylose Co-consumption

Authors: Hector, Ronald - Ron; Qureshi, Nasib; Hughes, Stephen; Cotta, Michael

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2008
Publication Date: 9/1/2008
Citation: Hector, R.E., Qureshi, N., Hughes, S.R., Cotta, M.A. 2008. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption. Applied Microbiology and Biotechnology. 80(4):675-684.

Interpretive Summary: Lignocellulosic substrates used for bioconversion to fuel ethanol contain a mixture of five and six carbon sugars. Saccharomyces cerevisiae, the yeast commonly used for industrial fermentations, cannot utilize the five carbon sugars in these substrates, thus limiting the amount of ethanol that can be made from lignocellulosic feedstocks. Yeast strains have been engineered to ferment xylose, the most abundant five carbon sugar in these feedstocks. However, they consume xylose at sub-optimal rates for industrial use. One limitation in these strains is the lack of a xylose-specific transport system for getting xylose inside the cell. In this investigation, a xylose transporter was expressed in S. cerevisiae cells that were also engineered to utilize xylose. The effect of a xylose transporter on xylose co-consumption with glucose was analyzed. We determined that the presence of a xylose transport system to allow better uptake of xylose into the cell improved xylose utilization. Up to 2-fold more xylose was co-consumed with glucose and ethanol concentration, yield, and specific productivity were increased. The results of this investigation show that further increases in fermentation of xylose from biomass feedstocks can be attained by improving the uptake of xylose. Understanding the limitations of xylose utilization will help generate strains more suited for industrial conversion of biomass to fuel ethanol.

Technical Abstract: Strains of Saccharomyces cerevisiae have been engineered to utilize xylose by expression of the genes for xylose reductase and xylitol dehydrogenase, or xylose isomerase. These strains are still limited in their ability to efficiently use xylose. Unlike native xylose assimilating yeasts such as Pichia stipitis, S. cerevisiae does not contain xylose-specific transport systems. The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption. We expressed two heterologous transporters from Arabidopsis thaliana in S. cerevisiae strains that were previously engineered to utilize xylose. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Our results from aerobic cultivation with glucose/xylose mixtures indicate that expression of the transporters increased xylose consumption rates (prior to glucose depletion) by up to 100% compared to the control strain. Increased xylose consumption in the presence of glucose also correlated with increased ethanol concentration, yield, and productivity. During the xylose/glucose co-consumption phase, the best performing strains had up to a 14% increase in maximum ethanol concentration, 9% increase in ethanol yield, and 46% increase in specific ethanol productivity. It was concluded that in these strains xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.