A modeling and experimental approach to understanding the bottlenecks in xylulose utilization in S. cerevisiae Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ff365877m

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  • The production of fuel ethanol from lignocellulosic biomass has the potential to replace a significant portion of non-renewable transport fuels. Woody feedstocks are composed of cellulose, hemicellulose, and lignin. Glucose, the monomer of cellulose, is readily utilized by wild-type S. cerevisiae, but xylose, which comprises 60% of the sugar in hemicellulose, is not. To make the process economically competitive with conventional fossil fuels, both five and six carbon sugars must be utilized efficiently. One approach to improving xylose utilization is to convert it to the more readily usable xylulose using an extracellular enzyme. Xylulose is taken up by wild-type S. cerevisiae and incorporated into the pentose phosphate pathway. To our knowledge there are no reports that elucidate the kinetics of this pathway, an important hurdle to overcome for strain development. This thesis documents the work carried out to gain a better understanding of the xylulose utilization pathway in S. cerevisiae. The work was comprised of a series of batch fermentations that identified xylulokinase as a limiting enzyme in wild-type strains and transport through the HXT family of hexose transporters as a possible limiting step in xylulokinase enhanced strains. Batch experiments with HXT knockout strains suggest that alternative modes of xylulose transport are possible and may be up regulated in the knockout. An existing genome scale model for S. cerevisiae (iMM904) was used as the basis to develop a dynamic flux balance model. This model was used to verify the batch fermentation findings. The model has strong predictive capacity for xylulose and glucose consumption under anaerobic conditions and sugar levels sufficient to support growth.
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