| dc.description.abstract | To achieve a sustainable and economically viable 2G biofuels industry, biorefineries must co-produce high-value, low-volume bioproducts alongside high-volume, low-value cellulosic ethanol. This can be realised with the co-production of the low-calorie sugar substitute, xylitol which has a well-established market, as well as other chemicals. The construction of a xylitol-producing S. cerevisiae strain represents an economically feasible and environmentally friendly approach to xylitol production. Moreover, the exploitation of natural S. cerevisiae strain isolates as bioengineering hosts has the potential to be superior starting points due to their robustness towards process conditions. In this study, the xylitol-producing activities conferred to three natural isolate host strains via conventional and CRISPR-Cas9-mediated δ-integration of three genes encoding a β-xylosidase, β-xylanase and xylose reductase (XR), was evaluated. The effect of over-expressed heterologous protein production on strain robustness and metabolism was also assayed. Our results revealed that the overexpressed XR failed to improve on the xylose reduction ability conferred to our strains, likely by their native GRE3 gene. The exploitation of natural host isolates proved advantageous, given the high heterologous xylanase and xylosidase activities recorded, far-exceeding previously reported studies, which enabled the substrates xylan and xylo-oligosaccharides (XOS) to be used for xylitol production, instead of costly pure xylose. Despite the high levels of heterologous protein production, our engineered natural strains displayed tolerance to acetic acid concentrations higher than 3 g/L but lower than 5 g/L while FIN1-X3 and YI13-X3 displayed tolerance to temperatures as high as 40 °C. Growth analyses revealed that only YI59-X3 displayed somewhat impaired growth, however, no single strain outperformed the other across the recorded assays of this study. The results of this study led us to conclude that the xylose reduction ability of our strains must be enhanced through alternate genetic engineering strategies. Furthermore, the engineering strategies employed for heterologous xylanase and xylosidase activity as well as the use of natural strains as bioengineering hosts, offer considerable potential for use in 2G biorefineries. | en_US |
