Engineering K. Phaffii for methane utilisation

Our project, Pheast, endeavored to design and test an engineered cell-factory to use methane as a feedstock, using the methylotrophic yeast, Komagataella phaffii GS115 (K. phaffii). Currently, there is a big incentive for developing efficient gene editing tools such as CRISPR/Cas9-based platforms for engineering K. phaffii), as this yeast is a popular production chassis for industrial enzymes and biotherapeutics, due to its recombinant protein production abilities and low secretion of endogenous proteins[1] [2] [3] [4]. This project consisted of several milestones, and iterations through the engineering cycle to reach our goal of developing an efficient toolbox for the genetic engineering of K. phaffii. For an efficient use of such a toolbox it is necessary to do both deletions and insertions of genes of interest. Our toolbox does both.

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To validate the use of our CRISPR/Cas9 toolbox for genomic integration and gene deletion K. phaffii we went through two iterations of the engineering cycle. At the end of the second cycle, and after several rounds of troubleshooting, we experimentally validated our designed CRISPR based toolbox.


[1] Yang, Z., & Blenner, M. (2020). Genome editing systems across yeast species. Current Opinion in Biotechnology, 66, 255-266.
[2] Need for Crispr-based tools in k phaffi 2021: Cai, P., Duan, X., Wu, X., Gao, L., Ye, M., & Zhou, Y. J. (2021). Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Research, 49(13), 7791–7805.
[3] Gao, J., Jiang, L., & Lian, J. (2021). Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products. Synthetic and systems biotechnology, 6(2), 110-119.
[4] Karbalaei, M., Rezaee, S. A., & Farsiani, H. (2020). Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 235(9), 5867–5881.
[5] Nødvig, C., Nielsen, J., Kogle, M., & Mortensen, U. (2015). A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLOS ONE, 10(7). doi: 10.1371/journal. Pone.0133085
[6] Nødvig, C., Hoof, J., Kogle, M., Jarczynska, Z., Lehmbeck, J., Klitgaard, D., & Mortensen, U. (2018). Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genetics And Biology, 115, 78-79. doi: 10.1016/j.fgb.2018.01.004
[7] Garcia Vanegas, K., Lehka, B. J., & Mortensen, U. H. (2017). SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae. Microbial Cell Factories, 16(25), 25.