Characterization of transcriptional regulatory proteins

Background.

Yeasts are attractive hosts capable to synthesize high amounts of recombinant proteins for biopharmaceutical or industrial purposes. Among them, the methylotrophic yeast Pichia pastoris plays a major role. However, many complex secretory proteins still cannot be produced at the desired high levels during industrial production processes. Although impressive increases in productivity have been reached by engineering of protein folding and secretion for some products (reviewed by Delic, et al., 2014a and Idiris, et al., 2010), there is still continuing effort needed on this topic. Besides the overexpression or knockout of individual proteins involved in the secretory process, concerted engineering of the cellular response by modulation of transcriptional regulatory proteins has proven to be a promising approach to increase productivity in P. pastoris (Delic, et al., 2014b; Gasser, et al., 2006; Guerfal, et al., 2010; Ruth, et al., 2014).

Gene expression is regulated at various levels, including epigenetic and transcriptional control of gene expression in response to external signals. The endowment of a given organism with transcriptional regulatory proteins, which regulate distinct gene sets in response to different signals, has a strong influence on its regulatory behavior and phenotype. Up to now, a large number of transcriptional regulators present in P. pastoris are of uncharacterized function, meaning that their application to targeted cell engineering is limited.

Aims and methods.

To shed light onto regulatory targets of yet uncharacterized transcriptional regulators in P. pastoris, this project aims to characterize their necessity in different stress conditions and their impact on cellular organization, particularly focusing on production relevant conditions and organelles, as well as to identify the transcription factors binding sites and regulons of selected regulators.

The function of transcriptional regulators will be assessed by generation of the respective gene knock-outs and overexpressions, and arraying of these strains on media representing different growth and stress conditions. In addition to growth ability in the presence or absence of stressors and different nutrient sources, morphology of the strains on their macroscopic and microscopic level will be embraced.

For some selected transcriptional regulators, the regulons and the respective binding sites on the DNA will be identified using next-generation sequencing approaches (RNA-Seq and ChIP-Seq, respectively).

Delic, M., Gongrich, R., Mattanovich, D. and Gasser, B. (2014a). Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins. Antioxid Redox Signal 21, 414-37.
Delic, M., Graf, A. B., Koellensperger, G., Haberhauer-Troyer, C., Hann, S., Mattanovich, D. and Gasser, B. (2014b). Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion. Microbial Cell 1, 376 - 386.
Gasser, B., Maurer, M., Gach, J., Kunert, R. and Mattanovich, D. (2006). Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnol Bioeng 94, 353-61.
Guerfal, M., Ryckaert, S., Jacobs, P. P., Ameloot, P., Van Craenenbroeck, K., Derycke, R. and Callewaert, N. (2010). The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 9, 49.
Idiris, A., Tohda, H., Kumagai, H. and Takegawa, K. (2010). Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86, 403-17. Ruth, C., Buchetics, M., Vidimce, V., Kotz, D., Naschberger, S., Mattanovich, D., Pichler, H. and Gasser, B. (2014). Pichia pastoris Aft1--a novel transcription factor, enhancing recombinant protein secretion. Microb Cell Fact 13, 120.