Date

2017

Author

Ata, Özge

Metadata

Show full item record

Item Usage Stats

335
views

121
downloads

Cite This

The objective of this PhD thesis is to enhance the expression strength of glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) for improved recombinant protein (r-protein) production through modifying the transcription factors (TF) that regulate the functioning of PGAP by transcriptional engineering. PGAP was analyzed in terms of putative TF binding sites. A synthetic library was constructed with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected TFs genes were overexpressed or deleted to understand their roles on r-protein production. Using enhanced green fluorescent protein (eGFP) as the first model protein, an expression strength in a range between 0.35- and 3.10-fold of the wild-type PGAP was obtained in Pichia pastoris. Another model protein, recombinant human growth hormone (rhGH) was produced under control of selected promoter variants, i.e. P9 (Gal4-like binding site was duplicated) and P10 (Rfx1 binding site was deleted), and 2.4- to 1.6-fold higher product titers were reached compared to wild-type PGAP in 24 deep well plates, respectively. Finally, combining two approaches resulted in 2.2-fold increase in wet cell weight specific rhGH yield with P9 that was coupled with overexpression of a Gal4-like TF compared to PGAP in fed-batch bioreactor cultivation. In addition to its role on enhancing r-protein production under PGAP, the effect of overexpression of Gal4-like TF on central carbon metabolism of P. pastoris was investigated. The specific glucose consumption rate and ethanol production rates were increased by 1.7- and 8-fold in P. pastoris Gal4-like transcription factor overexpression mutants compared to wild-type P. pastoris, respectively. 13C labelled metabolic flux and RNASeq analyses results exhibited that overexpression of Gal4-like TF caused downregulation in pentose phosphate pathway and increased the fluxes through lower glycolysis pathway significantly. This higher and imbalanced glycolytic flux caused an overflow metabolism which triggered the fermentative pathway. Taken together, it was revealed that overexpression of Gal4-like transcription factor resulted in a switch from Crabtree negative to Crabtree positive behavior.

Subject Keywords

Pichia pastoris., Recombinant proteins., Genetic transcription., Promoters (Genetics).

URI

http://etd.lib.metu.edu.tr/upload/12621611/index.pdf
https://hdl.handle.net/11511/26962

Collections

Graduate School of Natural and Applied Sciences, Thesis