A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae

Authors: HOHENSCHUH, WILLIAM - Oregon State University; Hector, Ronald - Ron; MURTHY, GANTI - Oregon State University

Submitted to: Bioresource Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2015
Publication Date: 2/15/2015
Publication URL: http://handle.nal.usda.gov/10113/62054
Citation: Hohenschuh, W., Hector, R.E., Murthy, G.S. 2015. A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae. Bioresource Technology. 188:153-160.

Interpretive Summary: Economically viable production of lignocellulosic ethanol requires efficient conversion of feedstock sugars to ethanol. The yeast commonly used for industrial ethanol production, Saccharomyces cerevisiae, cannot use xylose, the main five-carbon sugar in biomass, but it can metabolize xylulose, an enzymatically-derived isomer. Xylulose metabolism is slow relative to glucose and requires further development. Strain development to improve utilization requires an understanding of the metabolic bottlenecks. Here, the kinetics of xylulose transport into the cell and phosphorylation of xylulose were investigated. Xylulose phosphorylation by the enzyme xylulokinase was identified as a limiting step in wild type S. cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through non-specific glucose transporters (HXT family). A genome-scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain the limitation of xylulose utilization in S. cerevisiae. This information will be valuable to further engineer S. cerevisiae strains with improved utilization of biomass-derived sugars for the production of renewable fuels and chemicals.

Technical Abstract: Economically viable production of lignocellulosic ethanol requires efficient conversion of feedstock sugars to ethanol. Saccharomyces cerevisiae cannot ferment xylose, the main five-carbon sugars in biomass, but can ferment xylulose, an enzymatically derived isomer. Xylulose fermentation is slow relative to glucose and requires further development. Strain development to improve utilization requires an understanding of the utilization bottlenecks. Here, batch fermentations were used to probe the kinetics of xylulose transport and phosphorylation. Xylulose phosphorylation by xylulokinase was identified as limiting in wild type S. cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through the HXT family of non-specific glucose transporters. A genome scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain xylulose utilization limitation in S. cerevisiae.