Komagataella phaffii strain:NRRL Y-11430 Genome sequencing. Komagataella phaffii strain:NRRL Y-11430

GlycoFi has reported the complete humanization of the N-glycosylation pathway of the yeast Pichia pastoris. This glycoengineered Pichia platform combines a widely used and effective protein expression host with the ability to confer human glycans in a highly specified manner to any secreted protein produced in this yeast. While an attractive alternative to traditional mammalian expression hosts, this technology also introduces a vision for tailoring glycosylation of protein therapeutics for purpose. However, the genetic engineering leveraged to generate these strains can result in compromised viability and process compatibility, which complicates process development and scale up. Moreover, the iterative nature of the engineering is encumbered by the potential for introduction of undesirable mutations. To better enable this type of complex genetic and process engineering whole genome profiling methods were applied to: 1) identify key gene expression differences between wild type P. pastoris and glycoengineered Pichia strains; 2) determine key drivers of growth and viability differences in glycoengineered strains to aid in development of genetic or process improvements and 3) enable the choice and optimization of preferred engineered lineages by identifying ancillary genetic lesions (either potentially beneficial or deleterious). These experiments revealed significant transcriptional changes in nearly 75% (FDR < 0.01) of the genes in the currently annotated P. pastoris genome, indicating a massive overhaul of cellular physiology that has occurred because of the re-engineering of the secretory pathway in these strains. However, despite the extent of the transcriptional changes, several important biological gene expression patterns could be observed that may help to explain underlying phenotypic differences between the strains. These changes indicate pathway level cellular reprogramming which may have resulted either directly or indirectly from the genetic engineering and offer the potential to better define and control these responses. Wild-type P. pastoris generally displayed increased responses to oxidative stress and decreased translation-related gene expression, strategies which may be consistent with prolonged survival during methanol growth, whereas the glycoengineered strains showed increased expression in secretory pathway, cell wall, and lipid biosynthesis-related genes. Prominent among these genes were those involved in cell wall composition and structure, amino acid metabolism, and fatty acid oxidation. Next generation sequencing of the same strains as well as other parallel lineage strains revealed several interesting single nucleotide polymorphisms that may be responsible for driving some of the transcriptomic changes observed and also provides critical genomic data that can allow for strain lineage selection and optimization. Synthesis of these profiling methods, combined with an improving genomic annotation and a wealth of genetic tools, provides a more fundamental understanding of the impact of N-glycosylation pathway engineering and should lead to the identification of factors affecting strain and process robustness as well as product titer and quality.