Fig 1.
An alternative pentose sugar utilization in S. cerevisiae.
The system consists of two exogenous components, XI and KHK, which convert d-xylose to d-xylulose and X1P, respectively. X1P is then metabolized by S. cerevisiae endogenous enzymes—FBA1, GRE2 and/or ADH1—producing glycolaldehyde and DHAP as intermediates and EG and ethanol as final products. XKS1 deletion allows the metabolic flux to be directed to the synthetic pathway via X1P by eliminating a possible route of xylulose utilization via X5P through the PPP. Abbreviations in the figure are: PPP, pentose phosphate pathway; XI, xylose isomerase; KHK, ketohexokinase; FBA1, fructose 1,6-bisphosphate aldolase; GRE2, 3-methylbutanal reductase; ADH1, alcohol dehydrogenase; XKS1, xylulokinase; GRE3, aldose reductase; EG, ethylene glycol; Xylulose-1P, xylulose 1-phosphate; Xylulose-5P/X5P, xylulose 5-phosphate; DHAP, dihydroxyacetone phosphate; GADP, glyceraldehyde 3-phosphate; S7P, sedoheptulose 7-phosphate; F6P, fructose 6-phosphate; E4P, erythrose 4-phosphate.
Fig 2.
Comparisons of fermentation profiles using strain with and without xks1Δ, in the presence and absence of exogenous xylose isomerase (XI) and ketohexokinase (RnKHK) expression.
Concentrations of (A) xylose and (B) ethylene glycol (EG) are shown. NC, negative control. Error bars indicated standard errors, N = 2.
Fig 3.
Effects of overexpression of endogenous enzymes in the alternative xylose utilization pathway.
Strains overexpressing the three endogenous components–FBA1, ADH1, and GRE2—were compared. xks1Δ XI-RnKHK were used as the background strain for all the overexpression comparisons. Concentrations of (A) xylose and (B) ethylene glycol (EG) were shown. OE, overexpression. Error bars indicated standard errors, N = 2.
Fig 4.
Synergistic effects of cellobiose and xylose co-utilization.
(A) Intracellular concentrations of ATP, NAD+ and NADH are shown for fermentations with xylose, cellobiose and its mixture (denoted, X, C and XC respectively) provided to the xks1Δ XI-RnKHK-FBA1-CD strain. (B) xks1Δ XI-RnKHK-CD (denoted in blue) and xks1Δ XI-RnKHK-FBA1-CD (denoted in green) strains were provided with xylose in the presence and absence of cellobiose. Concentrations of ethylene glycol (EG) are shown. X, xylose; C, cellobiose; XC, mixture of xylose and cellobiose; OE, overexpression. Error bars indicated standard errors, N = 5.
Fig 5.
Product titers and relative abundance of metabolites in fermentations with the alternative pathway.
The traditional xylose isomerase pathway (denoted as “PPP”) and the alternative xylose utilization pathway (xks1Δ XI-RnKHK-FBA1-CD denoted as “Bypass”) were compared. (A) Ethanol and ethylene glycol (EG) production. (B) Relative abundance of intracellular ᴅ-xylulose-1-phosphate (X1P), ᴅ-xylulose-5-phosphate (X5P), ᴅ-ribose-5-phosphate (R5P) and ᴅ-sedoheptulose-7-phosphate (S7P), after 4 days of fermentation. Values are normalized to 1 for levels of each compound observed in the Bypass strain. (C) Abundance of metabolites in the Bypass strain provided with xylose (X), a mixture of xylose and cellobiose (XC) and cellobiose only (C) are shown. Samples taken after 4 days of fermentation, and values are normalized to 1 for the levels of each compound observed in the cellobiose-only fermentation. In all three panels, error bars indicated standard errors, N = 5.