Fig 1.
Construction of isogenic S. cerevisiae strains expressing a different type of xylose pathways.
(A) Two different xylose pathways. (B) Strain construction using a precise Cas9-based genome integration strategy.
Table 1.
Saccharomyces cerevisiae strains used in this study.
Fig 2.
Effect of PHO13 deletion on xylose fermentation by two xylose-metabolizing strains.
(A) The XYL123 strain expressing the xylose oxidoreductase pathway and (B) the XI-XYL3 strain expressing the xylose isomerase pathway were compared to their corresponding pho13Δ mutants (C and D, respectively). (E, F) Volumetric growth rates (g/L-h) and product yields (g/g) of the xylose fermentations. Fermentations were performed in YP medium containing 40 g/L xylose under oxygen-limited conditions (80 rpm), with a low initial cell density (0.5 g DCW/L). Asterisks denote statistically significant differences (Student's t-test, p < 0.05). n. d.; Not detected.
Table 2.
Fermentation profiles of engineered S. cerevisiae expressing heterologous xylose fermentation pathways.
Table 3.
Fermentation profiles of S. cerevisiae strains expressing the xylose isomerase pathway derived from Orpinomyces sp.
Fig 3.
PHO13 deletion-induced transcriptional and metabolic changes in two xylose-metabolizing strains.
(A) Fold changes in the mRNA levels of the TAL1 gene in the pho13Δ mutants of the XYL123 and the XI-XYL3 strains growing on glucose or xylose. The dashed line is 1, referring to the PHO13 wild type. (B) The intracellular concentrations of sedeheptulose-7-phospahte in the PHO13 wild types (control) and the pho13Δ mutants of the XYL123 and the XI-XYL3 strains growing on xylose. Asterisks denote statistically significant differences (Student's t-test, p < 0.05).
Fig 4.
Adaptive evolution of two xylose-metabolizing strains on xylose.
For adaptive evolution on xylose, growth rates of the XYL123 strains (A) and the XI-XYL3 strains (B) were evaluated under different xylose concentrations. Under growth-liming concentrations of xylose, 40 g/L (C) and 100 g/L (D), the strains were serially subcultured until the described generation numbers.
Fig 5.
Pathway-targeted approaches to improve strains expressing the xylose isomerase pathway.
(A) The target genes to be deleted (gre3Δ, sor1Δ) and the target genes to be overexpressed by integration of a duplicated copy (xylA, XYL3, TAL1). (B) Relative changes in growth rates (g/L-h) on xylose of the engineered strains compared to the XI-XYL3 strain. (C) Comparison of fermentation profiles of the XI-XYL3, (XI)2-XYL3, and δ(XI)-XYL3 strains, and their pho13Δ mutants. All fermentations were performed in YP medium containing 40 g/L xylose under oxygen-limited conditions (80 rpm), with a low initial cell density (0.5 g DCW/L). Different letters represent significant differences across strains within fermentation parameters (Tukey’s test, p < 0.05). n. d.; Not detected.