Understanding the bug that makes our drugs: a multi-OMICs approach to engineer Escherichia coli for improved production of recombinant periplasmic proteins in the biopharmaceutical industry.

Around 35% of therapeutic proteins produced in E. coli (the main bacterial expression system) are antibody fragments that are secreted to the periplasm [1]. However, optimizing expression requires trial and error and issues such as secretion system overloading and periplasmic inclusion body formation can occur. In this thesis, proteomics and transcriptomics were used to analyse strains expressing a model therapeutic protein (an anti-TNF scFv) in the cytoplasm and periplasm under industrially relevant fermentation conditions. This data was then used to engineer strains and media that were tested for improved expression and secretion of periplasmic recombinant proteins. ScFv secretion affected expression of multiple genes and proteins involved in stress (heat shock, envelope stress, iron stress and oxidative stress), cellular components (proteases and chaperones, the Sec secretion system and flagella) and metabolic pathways (oxidative phosphorylation, fatty acid, amino acid and ribosome synthesis). High levels of scFv expression (0.125 mM IPTG) that overloaded the secretion system also affected expression of the CpxR envelope stress response, pilus genes, osmotic stress, and membrane proteins. Data from this study was used to develop and test strains altering expression of 18 genes and media components. Of these, 3 alterations to expression of genes involved in SecY and membrane protein degradation, improved scFv expression and secretion under shake flask conditions when inducing high levels of scFv expression (0.125 mM IPTG) that overloaded the secretion system. Initial data indicated that 2 of these strains improved yields of secreted scFv 134% and 156% (12 hours post-induction) under industrially relevant fermentations conditions and improved expression of different recombinant periplasmic proteins (HGH, sfGFP and Hel4). Overall, this project has improved our understanding of cellular responses to recombinant protein secretion and has led to the development of novel methods to improve biologics production.