The role of sugar sensing and pathway selection on D-xylose utilization by Saccharomyces cerevisiae
Abstract: Biorefineries have the potential to partially or entirely replace petrochemistry for the production of our daily bulk and fine chemicals. However, this replacement can only be sustainable and cost-effective if the raw material used is cheap, renewable and does not compete with the food and feed industry. One raw material meeting these criteria is lignocellulose from agricultural and forestry waste streams. Lignocellulosic hydrolysates are rich in hexose sugars such as D-glucose and pentose sugars such as D-xylose. The industrial workhorse Saccharomyces cerevisiae readily ferments D glucose into value-added products such as bioethanol and metabolic engineering has enabled the utilization of D-xylose, albeit not with the same efficiency as for D-glucose. The aim of the present thesis has been to investigate two approaches for improving D xylose utilization in S. cerevisiae: introduction of a new pathway for D xylose oxidation and exploration of the signaling response to D xylose, including their engineering. The oxidative Weimberg pathway from the native bacterial host Caulobacter crescentus was introduced into S. cerevisiae. Out of the five enzymes of the pathway, one (XylC) was found to be highly active, leading to the accumulation of toxic D-xylonate, one (XylB) exhibited intermediate activity, two (XylD and XylX) had low activity and one (XylA) was not produced at all in the S. cerevisiae host. The issues observed were resolved by omitting XylC, substituting XylA with KsaD from Corynebacterium glutamicum, deregulating iron homeostasis to increase activity of XylD and introducing a total of 4 copies of the genes encoding the lower pathway (xylD-xylX-ksaD). The resulting strain assimilated 60 % of the available D-xylose through the Weimberg pathway, producing biomass and carbon dioxide, with D xylonate being the main by-product. In parallel, the Weimberg pathway was studied in C. crescentus, showing that this pathway was expressed and active, using both D-xylose and the structurally related pentose sugar L-arabinose. The signaling response towards D xylose was explored using a set of biosensors, coupling the expression of D glucose-responsive promoters to a green fluorescent protein and measuring fluorescence with flow cytometry. D Xylose was found to exert a weak effect on extracellular sensors in wild-type S. cerevisiae but the sugar was sensed in strains capable of D-xylose utilization, although with a response resembling the one observed with low levels of D-glucose. These results indicated that the D-xylose signal is mainly dependent on the intracellular metabolites formed during assimilation. Measurements of a panel of intracellular metabolites identified correlations with the sugar signaling response, independently of whether the metabolites stemmed from D-glucose or D-xylose. The correlation was also maintained in a pgi1Δ strain exhibiting a severe blockage in glycolysis.Engineering of the signaling response within the present thesis included evaluation of deletion mutants found to improve D-xylose utilization. In addition to confirming the increased D-xylose consumption, D-xylose was observed to cause a high D-glucose response within some signaling pathways. Furthermore, the catalytic and regulatory protein hexokinase 2 was engineered for D-xylose tolerance, resulting in a 64 % increase in catalytic activity in the presence of D-xylose, compared to the wild-type enzyme.In conclusion, the present thesis demonstrates novel methods of assaying and interpreting the D-xylose signaling reponse, and describes the first successful introduction of the Weimberg pathway in a eukaryote. Additionally, several potential targets for further engineering of metabolic and signaling pathways are identified and discussed.
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