Xylose utilization in bacteria and yeasts: Novel applications
Abstract
Agricultural biomass is composed of three major components: lignin, cellulose and hemicellulose. The latter constitutes up to 25% of the dry weight of some plant species. Mild acid hydrolysis of agricultural residues yields xylose at 40% of the total sugars. With the threat of quickly diminishing oil reserves and an emphasis on recycling, renewable resource utilization was seen as a significant area of research which could ultimately provide some ecological relief. These developments coincided with the emergence of recombinant DNA techniques and the marriage of the two promised numerous potential applications. Specifically, the conversion of xylose to ethanol and other specialty products by yeasts and bacteria was considered a primary goal. To facilitate this endeavor I have developed useful microbial and biochemical systems, that take advantage of renewable resources and the xylose utilization operon, for industrial and research applications. The E. coli xylulokinase gene was modified and expressed at high levels in yeast for the purpose of improving growth on xylulose and ethanol production. In addition, yeast and E. coli xylulokinase mutants were constructed by gene disruption and transposon mutagenesis, respectively. These mutants will permit an assessment of the role of xylulokinase in xylose and xylulose metabolism. The E. coli xylose isomerase gene (xylA) was used for the development of tools for molecular biology. A direct selection and positive selection cloning systems were constructed which are based on xylose-containing media. The utility of the xylA gene was extended further via the use of the xylose isomerase protein as a heat stable carrier for purifying oligopeptides and through hyperproduction of the enzyme for use in glucose and xylose isomerization. Finally, an examination of cloning strategies for isolating the Lactobacillus brevis xylose isomerase gene, via the use of oligonucleotide probes, was completed.
Degree
Ph.D.
Advisors
Ho, Purdue University.
Subject Area
Food science|Molecular biology|Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.