Project description:We used RNA sequencing to analyse the differences in gene expression between the homothallic K. phaffii CBS2612 wild type and heterothallic a- and α-type strains during growth in rich medium and under mating conditions. This data was used to investigate mating-type specific gene expression and allowed the identification of the K. phaffii a-factor genes.
Project description:The yeast Komagataella phaffii is a promising alternative host for manufacturing of therapeutic proteins. Deletion of unneeded endogenous proteins could increase the secreted titer of recombinant proteins by redirecting cellular resources. Genetic engineering in non-model hosts is hampered by limited annotation of genes, especially essential genes. In this study, we identified the set of endogenous secreted proteins in K. phaffii and attempted to disrupt these genes. We designed, transformed, and sequenced a pooled CRISPR-Cas9 knockout library to determine which genes are essential. With this knowledge, we rapidly disrupted up to 9 consecutive genes in K. phaffii. Engineered strains exhibited a ~20x increase in the production of human serum albumin and a 2x increase in the production of a monoclonal antibody. The pooled CRISPR-Cas9 library and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant therapeutic proteins and enzymes.
Project description:Investigation of whole genome gene expression level changes in Pichia stipitis CBS 6054 grown aerobically in xylose, compared to the same strain grown aerobically in glucose. A six array study using total RNA recovered from three separate cultures of Pichia stipitis CBS 6054 grown in glucose and three separate cultures of Pichia stipitis CBS 6054 grown in xylose. Each array measures the expression level of 374,100 probes (average probe length 53.6 +/- 4.1 nt) tiled across the Pichia stipitis CBS 6054 genome with a median spacing distance of 33 nt. During data processing, probes are filtered to include only those probes corresponding to annotated protein-coding genes.
