Advanced synthetic biology tools for yeast strain development

Background.

The production of recombinant biopharmaceutical proteins is of paramount importance for both public health and a sustainable bio-based economy. Improving production platform, in this case the yeast Komagataella phaffii (syn. Pichia pastoris), is an important tool to make bioprocesses sustainable in the long term and at the same time economically competitive.

Aims and methods.

The aim of this project is to reduce the timelines in strain development for difficult-to-express but therapeutically promising protein formats. For this purpose, technologies that allow for rapid and efficient cell and genome engineering will be further advanced.

Such synthetic biology parts will be identified by systems biology approaches and characterized by flow cytometry using GFP as reporter. Cloning and genetic manipulations will be carried out using state-of the art-methods such as Golden Gate Assembly and CRISPR/Cas9-based homology directed recombination (HDR) established in our laboratory for K. phaffii (Prielhofer et al., 2017, Gassler et al., 2018). The resulting strains will be characterized for growth and productivity in high throughput screening and bioreactor cultivations, e.g. as described in Zahrl et al. 2022.

The work will be carried out in the framework of the Austrian Centre of Biotechnology (acib) in the project “Pichia platform for difficult-to-express protein formats“ (funded by the FFG-COMET program) at the Institute of Microbiology and Microbial Biotechnology (Department of Biotechnology), BOKU University, Vienna.

REFERENCES
Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R. 2019. CRISPR/Cas9-mediated homology-directed genome editing in Pichia pastoris. Methods Mol Biol. 1923:211-225. doi: 10.1007/978-1-4939-9024-5_9.
Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, Sauer M, Mattanovich D, Gasser B, Marx H. 2017. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. BMC Syst Biol. 11(1):123. doi: 10.1186/s12918-017-0492-3.
Zahrl RJ, Prielhofer R, Ata Ö, Baumann K, Mattanovich D, Gasser B. 2022. Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion. Metab Eng. 74:36-48. doi: 10.1016/j.ymben.2022.08.010.