With corn and food prices increasing new feedstocks must be found for the ethanol plants currently in use. Cellulosic agricultural wastes contain mostly glucose but also a considerable amount of xylose, a five carbon sugar that current industrial S. cerevisiae strains cannot ferment. The work here describes the design and testing of an artificial chromosome containing the genes for xylose isomerase (XI, from Piromeyces sp. E2) and xylulokinase (XKS, from Yersinia pestis) two enzymes that should allow for xylose fermentation. The artificial chromosome construct was confirmed by PCR analysis and DNA sequencing. Western blot analysis demonstrated that the SUMO-XI-XKS polyprotein is cleaved into separate active proteins by the internal proteases of the yeast host yielding XI and XKS proteins at a roughly equimolar ratio. The recombinant strain containing the artificial chromosome consumed 7 times more xylose in a minimal xylose medium and 4 times more xylose in whole corn hydrolysate than the control yeast strain. Surprisingly, the recombinant containing yeast grew faster on glucose (full use of 70 g/L in 48 hours versus 96+ hours for the control yeast). This finding suggests that the addition of the XI and XKS genes to the host S. cerevisiae strain is impacting other metabolic pathways in addition to providing a xylose utilization pathway.
|Advisor:||Fry, Michelle R., Hughes, Stephen R.|
|Commitee:||McQuade, Kristi L., Taylor, Max A.|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Illinois|
|Source:||MAI 52/06M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Genetics, Biochemistry|
|Keywords:||Xylose isomerase, Xylose utilizing yeast, Xylulokinase, Yeast artificial chromosome|
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