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   . 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.
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.
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