Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 84-91). Also available for download via the World Wide Web; free to University of Oregon users.

The efficient fermentation of lignocellulosic biomass would enable more economically and environmentally friendly production of biofuels and biochemicals. Yet, Saccharomyces cerevisiae, a platform organism for biofuels and biochemicals production, is unable to convert all of the sugars in lignocellulosic biomass into biofuels and biochemicals mainly due to the lack of a pentose catabolic pathway. Though the advance of genetic engineering enabled S. cerevisiae to utilize pentose sugars, the efficiency of pentose sugar catabolism in S. cerevisiae is still limited. Here, the goal of this research was to confer efficient pentose sugar catabolism to S. cerevisiae by combinatorial and evolutionary engineering. To this end, pentose catabolic pathways were 1) constructed by heterologous expression of pentose catabolic genes, 2) optimized through rational engineering, and 3) further improved through evolutionary engineering. Through these efforts, we reported the highest ethanol yield (0.45 g ethanol / g xylose) and the second highest xylose consumption and ethanol production rates (0.98 g xylose g cell⁻¹ h⁻¹ and 0.44 g ethanol g cell⁻¹ h⁻¹, respectively) in xylose fermentation reported to date. The high performance in xylose fermentation was achieved based on the mutant xylose isomerase (xylA3), which showed 77% increased enzyme activity, engineered through directed evolution. In addition, we have established the first cells capable of growing on arabinose in mimimal medium and demonstrated ethanol production from xylan in minimal medium. The arabinose and xylan catabolic pathways were constructed in S. cerevisiae by expressing novel pentose catabolic genes from a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. In doing so, a complete workflow of bioprospecting to pathway engineering and evolution was detailed as an effective way to transfer a desired phenotype from a non-model organism to a model organism. This study substantially improved the prospect of biofuels and biochemicals production from lignocellulosic biomass by developing efficient pentose utilizing strains, finding new pentose catabolic genes, and suggesting alternative pentose catabolic pathway. Furthermore, the general tools for metabolic engineering demonstrated in this study would also advance microbial strain engineering. / text

Translation is an essential and fundamental process that coverts genetic codes into functional polypeptides by an apparatus called ribosome. In eukaryotic cells, ribosomes are composed of two subunits: the large (60S) subunit and small (40S) subunits. In Saccharomyces cerevisiae, ribosome biogenesis is complex and requires the involvement of over ~170 trans-acting factors. As a growing number of factors were identified related to this essential metabolic pathway, our lab has contributed to functional characterization of the late 60S subunit biogenesis pathway that centers on Nmd3p. This work particularly focuses on characterizing of the nuclear shuttling trans-acting factor Arx1p found in the Nmd3p-60S subunit particle. A working model that describes how Rei1p, another cytosolic trans-acting factor, recycles Arx1p is presented. This work also shows a similar mode of Arx1p recycling by the Hsp40 J-protein, Jjj1p. Furthermore, I have investigated functional interplay between Arx1p and Rpl25p, a 60S ribosomal protein at the polypeptide exit tunnel. These findings further reveal the involvement of Arx1p at the polypeptide exit tunnel in mediating association of other factors with 60S subunits. Beyond its function at the polypeptide exit tunnel, this work also focuses on a function for Arx1p in the export of 60S subunits. In yeast and higher eukaryotes, 60S subunit export depends on the export adaptor Nmd3p via Crm1-dependent pathway. I show that ARX1 interacts with the NES of Nmd3p and nucleoporins. From these results, I propose that Arx1p acts as another export receptor to facilitate 60S subunit export.

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