Next Generation Pichia Toolbox Development: New high throughput screening strategies for the selection of the high producing P. pastoris clones
SUPERVISOR: Diethard MATTANOVICH
PROJECT ASSIGNED TO: Helena GODON
The yeast Pichia pastoris is established as a powerful eukaryotic host for recombinant protein production. Its popularity can be attributed to its ability to rapidly grow to high cell densities, perform post-translational modifications, secrete high amounts of recombinant products and to the wide array of tools and techniques available for cell engineering. However, it has been observed that the efficiency of production and secretion also varies according to the protein target, highlighting the importance of strain engineering and continuous improvement.
Certain proteins, requiring complex and numerous post-translational modifications are not inclined to be produced easily. It has been shown that the co-expression of enzymes and chaperones can improve secretion and productivity. One of the objectives pursued in this project will be to develop production strains and tailored processes fit for production of complex proteins with the workhorse Pichia pastoris. The genes corresponding to the proteins of interest will be cloned into Lonza’s proprietary expression system. Synthetic biology tools will be applied to test libraries of helper factors to identify the best combinations suitable for individual proteins.
In the last steps of cell engineering, fast and efficient screening of large number of clones is necessary to identify the best performers. At the moment, the available screening workflow is laborious, time-consuming as well as limited to a low number of clones (100-400) that can be screened. Hence, the probability of identifying so-called super-producers is very low. To remedy this issue, another objective pursued in this project will be to establish an optimized high throughput screening workflow, enabling the screening of up to millions of clones and raising the chances of singling out the best producing clones. The leading technology for rapid clone separation is FACS. This technique requires coupling a fluorescence read-out to the individual producing clone. Two approaches are proposed to address this. The first approach is to develop an intracellular sensor readout where the productivity of the clones will be indirectly related to the expression of a fluorescent marker protein. The fluorescent marker serves as the signal for FACS. The second approach consists in developing single cell encapsulation combined with a transient tagging technique using stop codon readthrough.
The objectives of this doctoral study are to generate a deeper knowledge on complex protein production and take part in the development of a next generation toolbox for Pichia pastoris with the final aim of streamlining the workflow from strain generation and optimization to screening for the highest producing clones.