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Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

This work describes the characterization of the CAN1 gene, thought to encode the arginine permease of yeast. I have identified the RNA transcript of this gene and obtained the DNA sequence, which specifies a highly hydrophobic protein with multiple potential membrane-spanning domains. I use a gene fusion approach to identify sequences within the CAN1 protein that can translocate adjacent sequences across the E.R. membrane in vitro and extend these observations by finding that the topology of E.R. insertion is conserved up to the plasma membrane in vivo. Using a series of CAN1 gene fusions to the secreted yeast killer toxin, I find that the pathway of membrane protein export to the cell surface need not be functionally distinct from that of secreted proteins. Finally, I describe a mutation in the CAN1 gene that leads to altered rates of lysine uptake and results in growth inhibition and rapid plasmid loss in the presence of lysine.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 105-116).

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: John T. Koh, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1982. / Typescript (photocopy).

Thesis (Ph.D.) -- University of Adelaide, Department of Genetics, 1977.