Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

Abstract

Xylose is the second most abundant sugar in lignocellulose, but it cannot be used as carbon source by budding yeast Saccharomyces cerevisiae. Rational promoter elements engineering approaches were taken for efficient xylose fermentation in budding yeast. Among promoters surveyed, HXT7 exhibited the best performance. The HXT7 promoter is suppressed in the presence of glucose and derepressed by xylose, making it a promising candidate to drive xylose metabolism. However, simple ectopic expression of both key xylose metabolic genes XYL1 and XYL2 by the HXT7 promoter resulted in massive accumulation of the xylose metabolic byproduct xylitol. Through the HXT7-driven expression of a reported redox variant, XYL1-K270R, along with optimized expression of XYL2 and the downstream pentose phosphate pathway genes, a balanced xylose metabolism toward ethanol formation was achieved. Fermented in a culture medium containing 50 g/L xylose as the sole carbon source, xylose is nearly consumed, with less than 3 g/L xylitol, and more than 16 g/L ethanol production. Hence, the combination of an inducible promoter and redox balance of the xylose utilization pathway is an attractive approach to optimizing fuel production from lignocellulose.

Keywords: Saccharomyces cerevisiae; expression; promoter; xylitol; xylose.

FIGURE 1

Fermentation results with E6, E7, E8, and E9 engineered strains in a YPX medium containing 50 g/L xylose. Samples were taken at different time points, and fermentation data were obtained through HPLC analysis. Time-dependent (A) xylose consumption; (B) ethanol production; and (C) xylitol accumulation. Error bars represent standard deviations from three independent experiments.

FIGURE 2

Effects of XYL1 expression driven by different promoters on the xylose fermentation. (A–D) Fermentation results of strains E9T1 (A), E9F1 (B), E9H1 (C), and E9 (D) in a YPX medium with 50 g/L xylose are shown. (E) Comparison of relative transcription levels of XYL1 and XYL2 for strains E9, E9F1, E9T1, and E9H1. (F,G) Fermentation results of D9H1 (F) and D9 (G) in the YPX medium with 50 g/L xylose. Error bars represent standard deviations from three independent experiments.

FIGURE 3

Glucose and xylose metabolic pathways along with key genes and enzymes involved. Abbreviations of related genes: TAL1, Transaldolase; TKL1, Transketolase; RPE1, Ribulose 5-phosphate epimerase; RKI1, Ribose 5-phosphate isomerase; PYK1, Pyruvate kinase; G-6-P, Glucose-6-phosphate; DHAP, Dihydroxyacetone phosphate; Ga-3P, 3-phosphoglyceraldehyde; PEP, Phosphoenolpyruvate; and PYR, Pyruvate.

FIGURE 4

Improvement of xylose fermentation efficiency by altered expression of XYL2 and key genes in PPP. (A,B) Fermentation results of E9H1B8 (A) and E9H1H2B8 (B) in a YPX medium containing 50 g/L xylose. (C) Comparison of relative transcription levels of XYL1 and XYL2 in strains E9H1B8 and E9H1H2B8. (D) The fermentation performance of D9H1H2B8 was evaluated in a YPX medium containing 50 g/L xylose. Error bars represent standard deviations from three independent experiments.

FIGURE 5

Expression levels of XYL1 and XYL2 driven by the HXT7 promoter under different fermentation conditions. (A) Comparison of transcription levels of XYL1 and XYL2 in strains E9, E9H1B8, and E9H1H2B8 in a YPX medium containing 20 g/L glucose to logarithmic growth phase. (B,C) Comparison of transcription levels of XYL1 (B) and XYL2 (C) for the indicated strains in a YPX medium containing 10 g/L xylose and 10 g/L glucose to logarithmic growth phase. Error bars represent standard deviations from three independent experiments.