Abstract

Efficient utilization of both glucose and xylose from lignocellulosic biomass would be economically beneficial for biofuel production. Recombinant Saccharomyces cerevisiae strains with essential genes and metabolic networks for xylose metabolism can ferment xylose; however, the efficiency of xylose fermentation is much lower than that of glucose, the preferred carbon source of yeast. Implications from our previous work suggest that activation of the glucose sensing system may benefit xylose metabolism. Here, we show that deleting cAMP phosphodiesterase genes PDE1 and PDE2 increased PKA activity of strains, and consequently, increased xylose utilization. Compared to the wild type strain, the specific xylose consumption rate (rxylose) of the pde1Δ pde2Δ mutant strains increased by 50%; the specific ethanol-producing rate (rethanol) of the strain increased by 70%. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. Deletion of either RGT2 or SNF3 reduced expression of HXT1 in strains cultured in 1 g L−1 xylose, which suggests that xylose can bind both Snf3 and Rgt2 and slightly alter their conformations. Deletion of SNF3 significantly weakened the expression of HXT2 in the yeast cultured in 40 g L−1 xylose, while deletion of RGT2 did not weaken expression of HXT2, suggesting that S. cerevisiae mainly depends on Snf3 to sense a high concentration of xylose (40 g L−1). Finally, we show that deletion of Rgt1, increased rxylose by 24% from that of the control. Our findings indicate how S. cerevisiae may respond to xylose and this study provides novel targets for further engineering of xylose-fermenting strains.

Highlights

  • Lignocellulosic biomass is considered as an abundant, renewable, and environment-friendly material for biofuels and chemicals production

  • Xylose reductase (XR) and xylitol dehydrogenase (XDH) of Scheffersomyces stipitis or xylose isomerases (XI) of bacteria and fungi have been introduced into S. cerevisiae to build pathways for xylose metabolism

  • In recombinant S. cerevisiae strains, xylose is transported by hexose transporters and metabolized in sequence through the XR-XDH or XI, pentose phosphate, and glycolysis pathways to produce pyruvate, which is converted to ethanol and other products [3,4,5]

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Summary

Introduction

Lignocellulosic biomass is considered as an abundant, renewable, and environment-friendly material for biofuels and chemicals production. In recombinant S. cerevisiae strains, xylose is transported by hexose transporters and metabolized in sequence through the XR-XDH or XI, pentose phosphate, and glycolysis pathways to produce pyruvate, which is converted to ethanol and other products [3,4,5]. Researchers were able to show that extracellular xylose can induce the fluorescent signal of the green fluorescent protein gene (yEGFP3) expressed under the control of the HXT1, 2, and 4 promoters. Their data suggest that the Rgt2/Snf3-Rgt pathway responds to extracellular xylose, the mechanistic details are still not clear [25]. The integration arms of genes were all cloned from the CEN.PK113-5D genome

Cultivation Conditions and Batch Fermentation
Determination of cAMP
Assay of Trehalase Activity
Analysis of Metabolites
Calculation of Physiological Parameters
Results
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