Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris

Ata, Ozge
Prielhofer, Roland
Gasser, Brigitte
Mattanovich, Diethard
Çalık, Pınar
The constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P-GAP), which is one of the benchmark promoters of Pichia pastoris, was analyzed in terms of putative transcription factor binding sites. We constructed a synthetic library with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected transcription factor (TF) genes were overexpressed or deleted to understand their roles on heterologous protein production. Using enhanced green fluorescent protein, an expression strength in a range between 0.35- and 3.10-fold of the wild-type P-GAP was obtained. Another model protein, recombinant human growth hormone was produced under control of selected promoter variants and 1.6- to 2.4-fold higher product titers were reached compared to wild-type P-GAP. In addition, a GAL4-like TF was found to be a crucial factor for the regulation of P-GAP, and its overexpression enhanced the heterologous protein production considerably (up to 2.2-fold compared to the parental strain). The synthetic P-GAP library generated enabled us to investigate the different putative transcription factors which are responsible for the regulation of P-GAP under different growth conditions, ergo recombinant protein production under P-GAP. Biotechnol. Bioeng. 2017;114: 2319-2327. (c) 2017 Wiley Periodicals, Inc.


Transcriptional engineering of GAP promoter for improved recombinant protein production by Pichia pastoris
Ata, Özge; Çalık, Pınar; Mattanovich, Diethard; Department of Biotechnology (2017)
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 bindi...
Transcriptional regulatory proteins in central carbon metabolism ofPichia pastorisandSaccharomyces cerevisiae
Kalender, Ozge; Çalık, Pınar (2020-09-01)
System-wide interactions in living cells and discovery of the diverse roles of transcriptional regulatory proteins that are mediator proteins with catalytic domains and regulatory subunits and transcription factors in the cellular pathways have become crucial for understanding the cellular response to environmental conditions. This review provides information for future metabolic engineering strategies through analyses on the highly interconnected regulatory networks inSaccharomyces cerevisiaeandPichia past...
Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation
Massahi, Aslan; Çalık, Pınar (2016-11-07)
For extracellular recombinant protein production, the efficiency of five endogenous secretion signal peptides (SPs) of Pichia pastoris, SP13 (MLSTILNIFILLLFIQASLQ), SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), SP34 (MRPVLSLLLLLASSVLA), selected based on their D-score which quantifies the signal peptide-ness of a given sequence segment, was investigated using recombinant human growth hormone (rhGH) as the model protein. The expression was conducted under glyceraldehyde-3-p...
Transcriptional regulatory network discovery via multiple method integration: application to e. coli KI2
Sun, Jingjun; Tuncay, Kağan; Haidar, Alaa Abi; Ensman, Lisa; Stanley, Frank; Trelinski, Michael; Ortoleva, Peter (Springer Science and Business Media LLC, 2007-03-30)
Transcriptional regulatory network (TRN) discovery from one method (e. g. microarray analysis, gene ontology, phylogenic similarity) does not seem feasible due to lack of sufficient information, resulting in the construction of spurious or incomplete TRNs. We develop a methodology, TRND, that integrates a preliminary TRN, microarray data, gene ontology and phylogenic similarity to accurately discover TRNs and apply the method to E. coli KI2. The approach can easily be extended to include other methodologies...
Design and construction of double promoter systems and their use in pharmaceutical protein production in P. Pastoris
Demir, İrem; Çalık, Pınar; Department of Chemical Engineering (2019)
Intracellular phenomena such as promoter strength, mRNA secondary structure, translation efficiency and codon preference, 5′-untranslated region processing, and protein turnover, have impacts directly on the expression of heterologous genes. Design of multi-promoter expression systems with constituent strong promoters and engineered promoter variants is a novel metabolic engineering strategy for increasing the promoter strength further, and tuning the expression for recombinant protein (r-protein) productio...
Citation Formats
O. Ata, R. Prielhofer, B. Gasser, D. Mattanovich, and P. Çalık, “Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris,” BIOTECHNOLOGY AND BIOENGINEERING, pp. 2319–2327, 2017, Accessed: 00, 2020. [Online]. Available: