Pichia pastoris genetics for strain development

Background.

The yeast Pichia pastoris is established as a highly productive protein production system. However, the performance of different strains varies, pointing at genetically encoded differences in the protein synthesis and secretion pathway. Due to the complexity of the entire protein production machinery, these differences cannot easily be identified by direct comparison of genome sequences.

Based on ribosomal DNA sequencing P. pastoris has been moved into a new genus, Komagataella (Yamada et al. 1995. Biosci Biotechnol Biochem. 59, 439-444), which to date consists of seven species (Naumov et al. 2018. Antonie Van Leeuwenhoek, doi: 10.1007/s10482-018-1028-6). With this increasing understanding of genetic diversity among the Komagataella strains the utilization of diversity for strain breeding comes to reality.

Most yeasts can undergo a sexual cycle including mating, meiosis, and spore formation, leading to a recombination of the genomes of the mating partners. While this cycle is well understood for a few model yeasts, there is still a lot of open questions for many yeasts of biotechnological interest, such as Pichia pastoris. We have recently published the first stable mating-type strains and an in-depth characterization of mating regulation (Heistinger et al. 2018. Mol Cell Biol. 38:2 e00398-17). This opens huge opportunities to recombine natural genomes, engineer complex genetic traits and evolve production strains.

Project plan.

The aim of this project is the characterization of genetic traits determining the protein production potential of Pichia pastoris and related species. The method spectrum will include crossing of yeast strains, analysis of the offspring with classical genetic methods, next-generation sequencing and analysis of quantitative trait loci by QTL mapping as well as state of the art molecular biology methods like Golden Gate cloning and CRISPR/Cas9. The strain family created in the course of this development will be tested for productivity with several model proteins in comparison to the parental strains, including testing in lab scale fermentation.