Heterotrophic fermentation is a widely used means in the biotechnological, pharmaceutical and food industries for the large-scale production of desired products. However, two problems are often encountered during its application: the limitation of dissolved oxygen level in high cell density cultures, and the high cost of the carbon substrates for supporting growth. In this study these issues are solved through the expression of the hemoglobin gene from Vitreoscilla (VHb), which is known to be able to facilitate intracellular oxygen delivery, and the use of low-cost waste carbon sources for fermentation, respectively. Aurantiochytrium sp. MP4 (non-pigmented) and SK4 (pigmented) were chosen for the present study because of members of this genus has been consideredas potential producers of biodiesel as well asastaxanthin in recent years. VHb gene was successfully expressed in Aurantiochytrium sp. MP4 and SK4, and three transformants of MP4 (TMP4-VHb1, TMP4-VHb10 and TMP4-VHb24) and one transformant of SK4 (TSK4-VHb2) were obtained, respectively. TMP4-VHb24 andTSK4-VHb2 were selected for further study. It was found that VHb expression led to higher maximum biomass under microaerobic conditions and improved oxygen utilization in high cell density fermentation. Moreover, under aerobic conditions, there was an increase of total cellular fatty acid content by 10% and 44%forTMP4-VHb24 and TSK4-VHb2,respectively, and the pigmented strain TSK4-VHb2 produced 9-fold higher astaxanthin content than the control (i.e., SK4), showing that heterologous expression of VHb as a promising approach to improving growth or secondary metabolite biosynthesis in the fermentation process. The mechanism underlying the effects of VHb expression was further investigated using TMP4-VHb24. Expression of VHb led to a higher adenylate energy charge and hence higher metabolically available energy in the cells, especially at the late exponential phase. In addition, VHb expression promoted the rate of consumption of NADH during the period from the late exponential phase to the stationary phase, in which NADH carried electrons from the oxidation of carbon substrates in energy pathways for ATP synthesis. VHb also increased the resistance of the host cells to nitrosative stress, but not to oxidative stress. In terms of utilization of cheap carbon substrates, TMP4-VHb24 and TSK4-VHb2were found to grow well on the waste carbon sources including cane molasses and crude glycerol. Under such conditions, however, the contents of fatty acids and astaxanthin were generally lowered compared to the use of glucose-based media. Therefore, the growth and product synthesis in TMP4-VHb24 was further optimized using the response surface methodology, and it was found to be effective. In conclusion, the expression of VHb in Aurantiochytrium sp. MP4 and SK4 was effective in relieving limitation on the growth due to low oxygen availability at high cell density cultivation, while increasing the cellular astaxanthin and fatty acid biosyntheses under aerobic conditions. Also, the VHb-expressing strains were able to utilize low-cost waste carbon sources, and the response surface methodology could be employed to optimize fermentation effectively in order to lower the cost of fermentation. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Abstract Sheep fat contains relatively high levels of saturated fatty acids while poly-unsaturated fatty acids (PUFA) are toxic to cellulolytic bacteria and are also saturated in the rumen. Stabilization of residual oil in sunflower oilcake by conversion into calcium salts would be advantageous. Alpha lipoic acid acts as an anti-oxidant to ameliorate the effects of oxidative stress caused by high dietary levels of PUFA. Residual oil (14%) in mechanically extracted (expeller) sunflower oilcake (SFOC) was saponified in situ and compared in a complete feedlot diet (100 g crude protein and 31 g extractable fat/kg feed) with commercially extracted oilcake containing 2.4% residual oil (control diet with 29 g fat and 123 g crude protein kg feed) fed to four groups of 10 SA Mutton Merino weaner lambs (ca. 23 kg) for nine weeks. Both diets were fed either with, or without a weekly oral dosing of 500 mg α-lipoic acid. Fatty acid composition was determined on back fat samples while thiobarbituric acid-reactive substances (TBARS) were determined on samples of m. longissimus dorsi stored for zero and six months and displayed for six days. The back fat of the lambs on the saponified expeller SFOC diets contained significantly higher levels of saturated fatty acids and lower levels of mono-unsaturated fatty acids. PUFA were unaffected. High TBARS levels (>1.0) were found after six months storage. Non significant increases in TBARS were observed in the groups that received lipoic acid.

Not available / text

published_or_final_version / Chemistry / Master / Master of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

Fatty acid transport across the plasma membrane is an important site of regulation in skeletal muscle lipid metabolism, and is governed by a number of fatty acid transport proteins including, CD36, FABPpm, and FATP1 and 4. While each transporter is capable of independently stimulating fatty acid transport, less is known about their specific functions under various metabolic conditions, although CD36 appears to be key. The purpose of this thesis was to examine skeletal muscle fatty acid metabolism in several rodent models where CD36 has been altered, particularly via whole body deletion, by muscle specific overexpression, or in the face of permanent redistribution of CD36 to the plasma membrane. Using these models, this thesis sought to answer the following questions: 1) Is caffeine-stimulated fatty acid oxidation CD36-dependent? 2) Does CD36 function in tandem with FABPpm, and does this enhance fatty acid uptake at the plasma membrane and/or influence the metabolic fate of incoming fatty acids? 3) Is intramuscular lipid distribution altered in a rodent model of obesity, in which CD36-mediated fatty acid uptake is increased? Specific novel findings include the following: 1) Caffeine-stimulated calcium release can elicit the translocation of a number of fatty acid transporters in skeletal muscle, but CD36 is essential for caffeine-induced increases in fatty acid uptake and oxidation. 2) In spite of difficulties associated with protein co-overexpression, it appears that simultaneous overexpression of CD36 and FABPpm enhances fatty acid transport across the plasma membrane, and that these transporters may collaborate to increase insulin-induced fatty acid esterification and AICAR-induced oxidation. 3) Finally, in the obese Zucker rat model, augmented CD36-dependent fatty acid transport into muscle in combination with elevated lipid supply, results in lipid accretion within the IMF region of muscle, an effect that could not be explained by compartment-specific changes in selected glycerolipid synthesizing enzymes. Taken together, these studies emphasize the importance of CD36 in the regulation of plasmalemmal fatty acid transport, and further elucidate the metabolic implication of CD36 alteration on overall skeletal muscle metabolism.

No description available.

No description available.

The hypothesis of this study was that dietary n-3 fatty acids protect against phenytoin (P) teratogenicity by inhibiting embryonic prostaglandin H synthase bioactivation of P and/or by delaying embryonic development. Female caesarian-derived (CD-1) mice were fed a safflower (SAFF)- or a cod liver/linseed oil (CLO/LO)-based diet for three weeks prior to impregnation and throughout pregnancy. The CLO/LO diet, compared to the SAFF diet, reduced malformations and fetal growth retardation due to P. Open eye defect was the only anomaly induced by P in CLO/LO fetuses while P produced cleft palates only in SAFF fetuses. Since the period of maximal susceptibility to open eye defect occurs before palatal closure, this result suggests that the CLO/LO diet delayed development relative to the SAFF diet. In Exp 2, dietary n-6/n-3 fatty acid ratios were reflected in maternal hepatic phospholipids. Lipid peroxidation (LPO) was induced in maternal hepatic tissues by the SAFF diet while LPO was induced by P only in CLO/LO embryos.