Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Abstracted in Dissertation abstracts, v. 17 (1957) no. 3, p. 584. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 170-173).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1960. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2015 / S. cerevisiae has many properties which have made it the preferred host for the expression and production of a number of recombinant proteins. Xylose is the second most abundant sugar in nature and S. cerevisiae has been engineered to grow on this abundant sugar. Therefore, identifying S. cerevisiae promoters that are strongly induced during growth on xylose will be important in the production of recombinant proteins for the biofuel and other industries. Since xylose is not a native substrate for S. cerevisiae, it is not known how S. cerevisiae promoters will react during growth on xylose. The objective of the study was to evaluate the expression of a reporter gene, the Trichoderma reesei xylanase 2 (XYN2), under the control of five commonly used expression promoters (GPD3, ENO2, PGK1, ADH2 and YG100). Five episomal expression vectors were constructed for this purpose. These vectors were transformed to a recombinant xylose utilizing S. cerevisiae. Xylanase activity assays were used to determine the expression level from each of these promoters. The PGK1 promoter was observed to be the strongest promoter with average activity/OD of 130 nkat/ml/OD on both xylose and glucose. The GPD3 promoter showed the highest average activity/OD of 150 nkat/ml, but xylanase was only produced during growth on glucose. The data presented show that xylose is not a better carbon source than glucose for recombinant protein production in terms of the S. cerevisie promoters evaluated. Further research is required to obtain a yeast strain that grows well on xylose and promoters that show higher level on protein production. Keywords: xylose, promoter, expression, recombinant, S. cerevisiae

Thesis (Ph. D.)--University of Michigan, 1932. / "By Mabel M. Miller and Howard B. Lewis." "Reprinted from the journal of biological chemistry, vol. XCVIII, no. 1 ... October, 1932." Bibliography: p. 140, 149-150.

Thesis (Ph. D.)--University of Michigan, 1932. / "By Mabel M. Miller and Howard B. Lewis." "Reprinted from the journal of biological chemistry, vol. XCVIII, no. 1 ... October, 1932." Bibliography: p. 140, 149-150.

Seventeen species of facultative anaerobic bacteria belonging to three genera (Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol; a sugar alcohol used as a sweetener in the pharmaceutical and food industries. A chromogenic assay of both solid and liquid cultures showed that 10 of the 17 species screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation. The ten bacterial species were studied for the effect of environmental factors, such as temperature, concentration of D-xylose, and aeration, on xylitol production. Under most conditions, Corynebacterium sp. NRRL B 4247 produced the highest amount of xylitol. The xylitol produced by Corynebacterium sp. NRRL B 4247 was confirmed by mass spectrometry. Corynebacterium sp. NRRL B 4247 was studied for the effect of initial D-xylose concentration, glucose, glyceraldehyde, and gluconate, aeration, and growth medium. Corynebacterium sp. NRRL B 4247 produced xylitol only in the presence of xylose, and did not produce xylitol when gluconate or glucose was the substrate. The highest yield of xylitol produced in 24 h (0.57 g/g xylose) was using an initial D-xylose concentration of 75 g/l. Under aerobic conditions the highest xylitol yield was 0.55 g/g while under anaerobic conditions the highest yield was 0.2 g/g. Glyceraldehyde in concentrations greater than 1 g/l inhibited Corynebacterium sp. B 4247 growth and xylitol production. Corynebacterium sp. NRRL B 4247 culture grown in the presence of potassium gluconate (96 g/l) for 48 h and on addition of D-xylose to the media increased accumulation to 10.1 g/l of xylitol after 150 h. Corynebacterium sp. NRRL B 4247 exhibited both NADH and NADPH-dependent xylose reductase activity in cell-free extracts. The NADPH-dependent activity was substrate dependent. The activity was 2.2-fold higher when DL-glyceraldehyde was used as substrate than with D-xylose. In cell-free extracts the difference in xylose reductase and xylitol dehydrogenase activity was highest at 24 h, whereas for cell cultures that were grown in gluconate and xylose, the difference in the reductase and dehydrogenase activities was highest at 12 h after xylose addition. The NAD+ dependent xylitol dehydrogenase activity was low compared to the cells grown without gluconate. The molecular weight of NADPH-dependent xylose reductase protein obtained by gel filtration chromatography was 58 kDa. Initial purification was performed on a DE-52 anion exchange column. Purification using Red Sepharose affinity column resulted in a 58 kDa protein on the SDS PAGE gel and was further purified on a Mono-Q column. The activity stained band on the native gel yielded 58, 49, 39 and 30 kDa bands on the denaturing gel. The peptides of the 58 kDa protein of Corynebacterium sp. B 4247 sequenced by mass spectrometry, identified with E2 and E3 (Bacillus subtilis) components of multi-enzyme system consisting of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and oxo-acid dehydrogenase complex. A 75% match was shown by the peptide "QMSSLVTR" with E-value of 8e-04 to the Saccharomyces cerevisiae protein that was capable of reducing xylose to xylitol. The peptide "LLNDPQLILMEA" had conserved match "LL + DP" over several aldose reductases. The xylose reductase of the yeast Candida tropicalis ATCC 96745 was also purified. The molecular weight of the yeast NADPH-dependent xylose reductase was about 37 kDa on an SDS PAGE / Ph. D.

Plant biomass serves as a carbon and energy source for Streptomyces spp. which secrete degradative enzymes capable of breaking down the complex plant biomass into simple saccharides. Hemicellulose is a major component of plants and is composed of five and six carbon sugars, such as xylose and glucose. Enzymatic degradation of hemicellulose to obtain desired sugars has been a cornerstone of many industries, as well as the subject of worldwide research for additional sources of efficient enzymes for substrate conversion. In this study, environmentally-derived Streptomyces isolates were screened for their ability to hydrolyze oat-spelt and birchwood xylan in agar-based high throughput activity screens. Of the isolates tested, eight displayed high levels of substrate-degrading activity and were chosen for further characterization which included 16S rRNA gene analysis, microscopic analysis from both liquid and agar grown cultures, xylanase-specific activity, lignin peroxidase production and indole acetic acid production.Qualitative assessment of extracellular lactone signalling for all eight isolates was also performed. Putative lactone signalling was observed for Streptomyces isolates JLS1-C4, JLS1-A6, JLS2-D6 and KT1-B1 which exhibited xylanase-specific activities of 0.622 μmol/min/mg, 0.0243 μmol/min/mg, 0.721 μmol/min/mg, and 0.706 μmol/min/mg respectively. Streptomyces isolates JLS1-F12 and JLS1-C12 did not exhibit lactone signalling but did exhibit xylanase-specific activities of 0.125 μmol/min/mg and 0.0688 μmol/min/mg respectively. No xylanase-specific activity was detected for isolates JLS2- C7 and KT1-B8; however lactone signalling was observed for isolate KT1-B8. Streptomyces isolate JLS1-A6 degraded birchwood xylan optimally at pH 4 and 28°C with a maximal xylanase activity of 1.56 x10-3 μmol/min/mg. / UOIT

Zugl.: Düsseldorf, Universiẗat, Diss., 1991.

No description available.

Xylose is a pentose sugar that forms a substantial proportion of the monosaccharides released from lignocellulosic biomass after hydrolysis. Therefore, the economic and commercial viability of biofuel production from lignocellulosic material via microbial fermentation relies upon maximising the metabolism of monosaccharides like xylose. As such, second generation biofuels are becoming a focus of biofuel innovation because of the depleting fossil fuel reserves and increasing levels of carbon emissions. Even so the majority of current biofuel production uses glucose as a carbon source from corn, wheat or sugar cane. This conflicts with food production and has prompted the food versus fuel debate. The introduction of xylose metabolising pathways into current biofuel production microorganisms like yeast, which cannot utilize xylose, would allow xylose use from lignocellulosic biomass. The xylose-reductase (XR) pathway from fungi utilise the xylose reductase, xylose dehydrogenase and xylulokinase genes, whereas the xylose isomerase (XI) pathway from bacteria consists of xylose isomerase and xylulokinase. In this project plasmid constructs containing the two pathways were successfully introduced into yeast. The genes were further integrated into specific chromosomal sites for comparison. Depending on the type of media used, some xylose uptake and ethanol production could be demonstrated for some of these strains, but overall levels of xylose use did not reach a level likely to impact upon commercial biofuel production. As a result, several strategies were investigated with a view to increasing xylose metabolism and ethanol production from the strains. Alterations were made to the cassette design for the xylose enzyme genes, such as gene promoter replacement or removal of the epitope tag. A pentose specific transporter, GXF1, from Candida tropicalis was also introduced. However, none of these strategies improved xylose use. A further approach, which led to minor increases in xylose metabolism, was deletion of the PHO13 gene, which is thought to impact upon expression of pentose phosphate genes. One further goal of this work was to investigate whether xylose metabolism could be connected to butanol production, as butanol has superior properties as a biofuel in yeast. Unfortunately, butanol was not detected from heterologous butanol producing strains bearing the plasmid based XI pathway, presumably because the growth and health of these strains was quite poor. Overall this project has demonstrated that S. cerevisiae is able to metabolise hemicellulosic xylose to ethanol using heterologous pathways, however, the very low levels generated mean that a great deal of genetic and metabolic engineering would be required for optimisation of biofuel production for commercial viability.