Engineering cellular stress for enhanced extracellular bioproduction

The production of recombinant protein is powerful technique widely employed in biology research labs around the world and for the industrial-scale production of therapeutic proteins and industrial enzymes. Within the pharmaceutical sector, protein-based biopharmaceuticals constitute 8 out of the 10-bestselling products, and the global protein-based biopharmaceutical market had sales worth $120b in 2017. A major component of the manufacturing cost of these products is associated with extraction of the product from the host production cells, and isolation and purification procedures. This has a major impact upon the affordability and equitable global access to biologic-medicines once off patent, as the high manufacturing cost limits possible price reductions when compared to small-molecule generic medicines. As such, adoption of new methods to facilitate the isolation of high purity and quality product, would offer great benefit in reducing the cost and challenges, and hence benefit society.

Proteins destined for secretion out of host cells are directed toward the secretion pathways by the presence of specific signal peptides. However, in a recent study we demonstrated that under conditions of stress bacterial cells, which are commonly used for recombinant protein production, undergo an excretion phenomenon whereby proteins are translocated into the extracellular environment, even without a signal peptide. Confirming the presence of alternative translocation/excretion pathways and understanding their function and regulation are thus important for fundamental microbiology and biotechnology. In this study we will seek to understand and harness this excretion phenomenon for the biotechnological production of protein-based biopharmaceuticals. The potential to more effectively produce these medicines, used in the treatment of diabetes or certain cancers, could lower the cost of the therapy and so reduce the burden upon healthcare providers, e.g. the NHS. Within this study will seek to understand the ability of E. coli cells to excrete and produce biopharmaceuticals with enhanced productivity. This research project will generate technology and knowledge that will help maintain the UK's competitive edge, and will produce highly trained and skilled research personnel.

The apparent mislocalization or excretion of cytoplasmic proteins is a commonly observed phenomenon in both bacteria and eukaryotes. However, reports on the mechanistic basis and the cellular function of this so-called "nonclassical protein secretion" are limited. We recently reported that protein overexpression in recombinant cells and antibiotic-induced translation stress in wild-type Escherichia coli cells both lead to excretion of cytoplasmic protein (ECP) [Morra et al. mBio 2018]. Condition-specific metabolomic and proteomic analyses, combined with genetic knockouts, indicated a role for both the large mechanosensitive channel (mscL) and the alternative ribosome rescue factor A (arfA) in ECP. Collectively, the findings indicated that MscL-dependent protein excretion is positively regulated in response to both osmotic stress and arfA-mediated translational stress. The identified cellular stresses cause bacterial cells to excrete both recombinant and endogenous cytoplasmic proteins, with no loss in cell viability. The productivity of this excretion has been shown to be comparable to classical secYEG-mediated secretion in E. coli; this provides a novel cellular export paradigm for potential industrial-scale protein production.

In this study we will seek to exploit the recently identified ability of bacterial cells undergo excretion of cytoplasmic proteins in a biotechnological context for the production of high-value human therapeutic proteins. Firstly, the stress response mechanism and regulation of excretion will be temporally characterised using promoter activity and multiplexed transcriptomic analyses. Secondly, we will develop a genetic toolbox to permit user-controlled regulation and activation of excretion. Finally, we will seek to implement genetic engineered strains in a continuous production mode, using advanced fermentation process design and optimisation tools for the extracellular production of therapeutic proteins from bacterial cells.

WHO WILL BENEFIT: Pharmaceutical, biotech and contract manufacturing organisations producing human therapeutic proteins on the hundred gram to kilogram scale could use the knowledge and technology being developed here to produce biopharmaceuticals in E. coli with less cost and time. Drug discovery companies would also benefit from novel excretion/translocation technologies that permit the supply of drug targets. Additionally, many chemical companies that employ biocatalysts, such as Novozymes, DSM, Lonza, BASF and Dr. Reddy's, could also use these new tools for the extracellular production of enzymes. Finally, there are many companies, such as ThermoFisher, Qiagen, Promega, Merck Millipore, Bio-Rad, GE healthcare, New England BioLabs, and Sigma-Aldrich, who both produce technical enzymes and sell commercial expression systems. Any number of these companies could benefit through licensing agreements to use new systems based on the technologies developed in this project. More efficient production of biopharmaceuticals could also reduce cost, limiting burden on healthcare providers, e.g. the NHS. In addition, cost reduction would also permit equitable global access to therapeutic including in ODA-countries. Finally, this research project will also generate technology and knowledge that will help maintain the UK's competitive edge and will produce highly trained and skilled research personnel.

HOW WILL THEY BENEFIT: We will actively seek to communicate our findings to the wider community, through scientific meetings and scholarly publications (by continuing to publish in top journals such as: mBio, NAR, JACS, PNAS, and Angew Chemie). We will seek to disseminate the findings to academic and industrial R&D communities through domain specific international meetings, e.g. Gordon Conference Series and PepTalk. We will seek to work closely with personnel involved in exploitation and commercial impact both inside the university and outside, for example via the Biotechnology KTN. With support from the university TTO (UMIP), any new IP generated will be filed prior to publication/disclosure, allowing potential commercialization/ exploitation projects to be explored in the future.