Abstract
Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.
Highlights
Global climate change has accelerated efforts to find eco-friendly alternatives for fossil fuels
When fermenting 40 g/L xylose under oxygen-limited conditions with a low initial cell density (0.5 g DCW/L), the resulting strains showed different phenotypes; while the XYL123 strain consumed over 90% xylose and produced ethanol within 72 h (Fig 2A), the xylose isomerase (XI)-XYL3 strain consumed 10% xylose in the same time period and no ethanol was detected (Fig 2B and Table 2)
The result suggested that the expression level of the xylose isomerase gene (xylA) gene is one of the most critical factor for efficient xylose consumption, and the δ(XI)-XYL3 strain may have not reached to an optimal level of the xylA expression
Summary
Global climate change has accelerated efforts to find eco-friendly alternatives for fossil fuels. In engineered strains of S. cerevisiae expressing the xylose oxidoreductase pathway, the rate of xylose consumption and ethanol productivity are relatively high, but xylitol, glycerol, and acetate are accumulated as byproducts [3,5]. It was further confirmed that pho13Δ leads to transcriptional and metabolic shifts toward efficient xylose fermentation [17,18] It has not been clearly understood how strains expressing the isomerase pathway can be improved, there have been several attempts of genome sequencing of the evolved strains expressing the isomerase pathway [19,20,21,22,23,24]. Deletion of the PHO13 gene, adaptive evolution, the upregulation of the PP pathway, and some other strategies were performed to identify the most critical and simple factor to improve the strain expressing the xylose isomerase pathway
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