Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Development of Saccharomyces cerevisiae as a recombinant antibody factory

The yeast Saccharomyces cerevisiae has been widely used as an expression host for the manufacturing of products like biofuels, small molecules, and of recombinant proteins. To increase the yields of economically interesting proteins, the secretory pathway has been engineered extensively, however, secretion titers have often remained low. One example for these short-comings are full-length IgG antibodies, which are currently mostly produced in CHO cells, although microbial and plant based production platforms are emerging. We believe that S.cerevisiae has the potential to become an industrially relevant antibody factory. In this thesis, using targeted and random screening approaches, we aimed to identify genetic factors that can beused to create strains with an increased IgG secretion efficiency. First, we focused on genes that are regulated by the unfolded protein response and encode proteins affecting the ER folding environment. We enlarged the functional folding space in the ER through deletion of the OPI1 gene, a modification that increased IgG titers by up to 4.8-fold. Out of a screen of folding catalysts and molecular chaperones, the over expression of the peptidyl-prolyl isomerase Cpr5p provided the most beneficial effect, increasing IgG titers by up to3.26-fold. Finally, by combining the OPI1 deletion with CPR5 overexpression IgG secretion was increased over tenfold when compared with the wild type background. In contrast, in a set of strains with deletions of genes encoding proteins of the ER associated degradation pathway only deletion of HTM1 increased titers by 1.15-fold. Development of a clearance assay allowedus to distinguish differences in cellular IgG clearance among the ERAD deletion strains. As targets for rational strain engineering are limited, we developed a high throughput method for screening a transposon mediated yeast deletion library and identified genes that influence IgG secretion. With this approach, we were able to identify the genes VPS30 and TAR1 that after deletion improved IgG secretion by up to 2.5-fold and up 1.13-fold, respectively, thus validating the applicability of the method. Finally, we aimed to gain insight into the changes that recombinant antibody production inflicts on selected intracellular metabolites. Metabolic footprints of strains expressing a scFv, a scFv-Fc fusion, and a full-length IgG were found to besignificantly different based on a semi-quantitative metabolomics method. The most apparent changes were found in metabolites involved in amino acid and redox metabolism. In conclusion, we identified genes at several places along the secretory pathway that can beused to improve IgG secretion in S. cerevisiae

Following nature's roadmap

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. A subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high-throughput ELISA platform was selected. Co-expression of the mammalian chaperone BiP, the co-chaperone GRP170, or the peptidyl-prolyl isomerase FKBP2, with the antibody improved specific product yields up to two-fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, the authors demonstrate that speeding up peptidyl-prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. The findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast-based antibody factory.Peer reviewe