Evolutionary engineering of xylose utilizing S. cerevisiae to improve the tolerance against inhibitors present in the pretreated lignocellulosic raw materials

Examensarbete för masterexamen
Master Thesis
Koppram, Rakesh
The development of inhibitor tolerant Saccharomyces cerevisiae is needed for the efficient fermentation of lignocellulosic hydrolysates. In this work, evolutionary engineering was carried out on a xylose utilizing recombinant S. cerevisiae to generate strains tolerant to inhibitors in spruce hydrolysate. A broad range of inhibitors generated in pretreated spruce hydrolysate were chosen from available literature information. Potent inhibitors were selected to represent three inhibitor categories: furans, weak acids and phenolic compounds to make an inhibitor cocktail. Repetitive shake flask experiments were carried out by increasing the concentration of inhibitor cocktail in defined media containing glucose and xylose as carbon sources. The strains were screened for increase in specific growth rate and decrease in length of lag phase. Adaptive evolution was also accompanied by UV induced mutations. Strains developed during the course of adaptive evolution showed 89% increase in specific growth rate and length of lag phase decreased from 48 h to 24 h. The strains were screened on YPD plates containing inhibitor cocktail, YP-hydrolysate and YPX plates for inhibitor tolerance and improved xylose utilization. The two strains RK60-5 and RKU90- 3 were selected and anaerobic fermentations in defined media and spruce hydrolysate were carried out. Both strains displayed higher specific growth rates and glucose consumption rates. In spruce hydrolysate, the maximum ethanol productivity of RK60-5 and RKU90-3 were 0.017 and 0.018 g g-1 h-1 respectively compared to 0.012 g g-1 h-1 of parental strain. However, RK60-5 showed low glycerol yield and no traces of xylitol formation indicating absence of xylose utilization, whereas, RKU90-3 produced considerable levels of xylitol in spruce hydrolysate. Nevertheless, both strains showed high tolerance to inhibitors and the strategy of adaptive evolution proved to be promising. Keywords: Evolutionary engineering, adaptive evolution, inhibitors, spruce hydrolysate, specific growth rate and lag phase.
Energi , Hållbar utveckling , Livsvetenskaper , Biokemisk och bioteknisk processteknik , Energy , Sustainable Development , Life Science , Biochemical process engineering
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