Date

2018

Author

Gündüz Ergün, Burcu

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The aim of the Ph.D. thesis was designing novel engineered promoter variants (NEPVs) to obtain strong ethanol regulated promoters for improved recombinant protein (r-protein) production, and to clarify the role of certain transcription factors (TFs) on the regulation of P. pastoris PADH2 and PAOX1. Promoter architecture of PADH2 was redesigned by nucleosome optimization and modification of transcription factor binding sites and, the induction mechanism of PAOX1 was changed to ethanol as sole carbon and energy source. Engineered PADH2 allowed a 476% increase in enhanced green fluorescent protein (eGFP) synthesis compared to that of PADH2-wt. NEPVs of PAOX1 spanning an activity range between 74% and 130% of the PAOX1-wt in fermentations with ethanol; and allow maximum 197% increase in eGFP synthesis in fermentations with methanol. Subsequent measurements of the eGFP transcript levels confirmed stronger transcriptional capacities of the NEPVs. The second model protein extracellular human serum albumin (HSA) production was also studied with the promising NEPVs. Average extracellular HSA yield per gram of wet-cell-weight (YP/X) was 0.26, 0.38, 1.13, and 0.48 mg/g with PADH2-wt, PADH2-NucOpt, PADH2-OptCat, and PAOX1-mod, respectively under ethanol induction. In methanol-based defined medium with PAOX1-wt and PAOX1-mod YP/X was calculated as 0.85 and 1.34 mg/g, respectively. In order to clarify the role of the TFs in the regulation of PADH2 and PAOX1, and to create novel r-protein expression systems, overexpression and knock-out P. pastoris strains of Adr1(Mxr1), Aca1, Cat8-1, and Cat8-2 were designed and constructed. Cat8-2 overexpression created a derepressible r-protein production system under limited glucose condition with PADH2 reached 37% of the expression reached with ethanol induction. A very potent activation by derepression was observed with PAOX1-mod in adr1Δ strain with 206% of the expression reached with methanol-induced PAOX1-wt with limited glucose. Adr1 was found to be the main regulator of PAOX1 but not PADH2. Cat8-1 and Cat8-2 were both the activators of PADH2 and PAOX1-v; but in fact Cat8-1 is more effective. The cat8-1cat8-2Δ strain lost its ethanol utilization ability. The strongest expression potential by PADH2-wt and PAOX1-mod was obtained in adr1Δ strains under methanol induction, although the cells lost their ability to grow. It is conclusively demonstrated that the NEPVs and transcriptionally engineered r-protein expression platforms are promising candidates for industrial r-protein production processes.

Subject Keywords

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

URI

http://etd.lib.metu.edu.tr/upload/12622786/index.pdf
https://hdl.handle.net/11511/27812

Collections

Graduate School of Natural and Applied Sciences, Thesis