Customizing amino acid metabolism of Pichia pastoris for recombinant protein production

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems‐level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6‐fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures. Graphical and Lay Summary Amino acids are the building blocks of all proteins. Therefore, the authors hypothesized that the heterologous overexpression of proteins may lead to a limitation in the availability of free amino acids in the cell. Among different metabolic engineering strategies in recombinant strains of the yeast Komagataella phaffii, overexpression of the general amino acid synthesis activator GCN4 led to a marked increase in intracellular amino acid pools and achieved concentrations of a heterologous carboxylesterase.