Highly robust production strains and platform processes are critical to achieve high protein yields through cultivation for extended induction time. In a given expression system, the cell yield, protein productivity and quality of post-transcriptional modifications are all influenced by cultivation processes.
Here, systems approaches including transcriptomics, proteomics and metabolomics are applied to understand changes in gene/protein expression and metabolite profiles under different process conditions. Metabolomic analyses comparing more or less robust Pichia strains producing the identical antibody sequence showed significantly different accumulation patterns of metabolites in carbohydrate, lipid, nucleotide and amino acid pathways during the methanol induction phase. Media supplementation based on the analyses of metabolite depletion increased induction time while maintaining robustness of strain, which resulted in 20% higher productivity of antibody. Moreover, based on this combined with pathway integrated transcriptome analysis, we genetically re-engineered the methanol assimilation pathway by deleting one of the two genes encoding dihydroxyacetone synthase (DAS), which is responsible for the first step in the incorporation of formaldehyde into central carbon metabolism. In microreactor-scale fermentation, antibody productivity in these strains was improved by up to two-fold and further evaluation is underway in larger-scale bioreactors.
This integrative approach has led to significant enhancements of the glycoengineered Pichia expression platform.