Optimal cell factories for membrane protein production

SOCIETAL IMPORTANCE. Membrane proteins are essential for life, medicine and industrial biotechnology and yet remain understudied due to their (1) low abundance and (2) physical properties which makes isolation difficult. However, 20-30% of all proteins are membrane proteins with biological functions underpinning signal transduction, nutrient import/export, and cell-to-cell communication. Membrane proteins are potent drug and vaccine targets. Currently, the top ten selling medicines worldwide, and in total US$ 180 billion worth of drug sales, target membrane proteins. Multiple vaccines, including those for COVID-19, Hepatitis B and pertussis, target virus surface or bacterial membrane proteins. Non-pharmaceutical markets include biopesticides (worth US$ 3 billion) and antifungals (worth US$ 19 billion). Artificial expression of membrane proteins is frequently used in pharmacology to identify drug molecules, which may inhibit or enhance protein function. However, engineering high levels of functional membrane protein production remains challenging, limiting the efficiency of drug screening, reducing identification of new drugs and limiting transformative potential. Production of membrane proteins enables progress in promoting human health and provides bio-inspired solutions to pressing societal challenges caused by climate change; however, obtaining high yields of functional protein constitutes a fundamental roadblock to progress which this proposal addresses.

SCIENTIFIC CHALLENGE. Despite the importance of membrane proteins in these key industries, their production remains challenging with production of each protein requiring trial and error methods - in some cases requiring screening of 100s of different combinations of experimental factors. This is in part due to the complex process of membrane protein production which results in multiple stresses on cellular production platforms (significantly reducing cell growth and bioprocess productivity). The cell membrane's lipid composition significantly impacts protein function - potentially rendering any proteins produced non-functional due to lack of lipid factors. Therefore, scientists must test multiple different cell types in an expensive and laborious process. This bespoke platform development limits production capacity of functional membrane proteins and, in doing so limits drug screening and development.

PROJECT OUTLINE. We address these challenges by first carrying out a detailed systematic characterization of the cellular processes which limit membrane protein production. This will be used to develop a predictive computer aided design tool to enable designs which facilitate both production and cell growth. We will use these tools to design and implement autonomous feedback control strategies that enable living cells to self-regulate protein production in response to stress and therefore maximise production and yield. These systems have the potential to enable increased yields in a 'hands off manner'. We will engineer new cellular regulatory systems capable of tuning the cell's lipid production rate so that the cell membranes' composition and physical properties can be tuned to meet those needed for optimal membrane protein function. We will develop computer aided design tools which enable the prediction of optimal membrane composition. We will demonstrate the function of our new cellular systems to produce high value biomedical membrane proteins and demonstrate their production performance at scale in industrially relevant conditions.

Heterologous expression of membrane proteins underpins significant global activities in the discovery of medicines, biopesticides, antifungals and the delivery of vaccines to humans and animals; however, obtaining high yields of functional protein remains a significant challenge. This application establishes new technologies for efficient biomanufacture of medically and commercially relevant membrane proteins. Our research programme couples whole cell systems modelling frameworks and lipid-protein molecular dynamics simulations, with experimental strain engineering to develop new Escherichia coli strains capable of autonomously tuning protein production and with variable lipid composition to overcome key roadblocks for the efficient production of membrane proteins. Our proof-of-concept study establishes a new team with recognised expertise in synergistic technologies and industrial engagement which will be a springboard for industrial impact and extension of the platform approach to new organisms.