Metabolic engineering of Pichia pastoris

Background.

Pichia pastoris is one of the most prominent expression systems in biotechnology due to its excellent growth conditions coupled to eukaryotic translation characteristics. Commercial interesting products span from chemicals to biopharmaceutical and industrial relevant macromolecules.

Besides other yeasts, Pichia pastoris is able to grow solely on methanol using a pathway that, until recently, was thought to be only partially localized to peroxisomes (van der Klei, Yurimoto, Sakai, & Veenhuis, 2006). More recent studies suggest that the entire methanol assimilation pathway is localized to peroxisomes contrary to the hypothesis that parts of the assimilation are realized in cytosolic pentose phosphate pathway reactions (Russmayer et al., 2015). These findings together with the growing demand for biomaterials based on non-fossil resources have led to the idea of engineering Pichia pastoris towards assimilation of alternative carbon sources.

Aims and methods.

Major aim of the project is to engineer alternative carbon source assimilation pathways into Pichia pastoris to enable the production of biomaterials based on non-fossil resources. Furthermore, the current knowledge about compartmentalisation of existing Pichia pastoris pathways will be augmented by studying the peroxisomal biology.

Genetic engineering will be performed using state of the art CRISPR/Cas9 and Golden Gate cloning techniques. The strain development strategy will be supported by evolutionary engineering technics. Thermodynamically assisted metabolic modelling approaches will help to assess the feasibility of the planned scenarios. Analytical technics supporting advantageous pathway characteristics will employ 13C labelling of respective substrates coupled to mass spectrometry.

Russmayer, H., Buchetics, M., Gruber, C., Valli, M., Grillitsch, K., Modarres, G., Gasser, B. (2015) Systems-level organization of yeast methylotrophic lifestyle. BMC Biology, 13(1), 80.
van der Klei, I. J., Yurimoto, H., Sakai, Y., & Veenhuis, M. (2006) The significance of peroxisomes in methanol metabolism in methylotrophic yeast. Biochimica et Biophysica Acta - Molecular Cell Research, 1763(12), 1453–1462.