Optimal cell factories for membrane protein production

社会的重要性。膜蛋白对生命、医学和工业生物技术至关重要，但由于其(1)丰度低和(2)物理性质难以分离，膜蛋白的研究仍处于不足状态。然而，所有蛋白质中有20-30%是膜蛋白，具有支持信号转导、营养输入/输出和细胞间通信的生物学功能。膜蛋白是有效的药物和疫苗靶点。目前，全球十大畅销药物，总销售额达1800亿美元，都是针对膜蛋白的。多种疫苗，包括COVID-19、乙型肝炎和百日咳疫苗，针对病毒表面或细菌膜蛋白。非药品市场包括生物农药（价值30亿美元）和抗真菌剂（价值190亿美元）。人工表达膜蛋白在药理学中经常被用来鉴定药物分子，这可能会抑制或增强蛋白质的功能。然而，工程高水平的功能性膜蛋白生产仍然具有挑战性，限制了药物筛选的效率，减少了新药的鉴定，限制了转化潜力。膜蛋白的生产促进了人类健康的进步，并为气候变化引起的紧迫社会挑战提供了生物灵感的解决方案；然而，获得高产量的功能性蛋白质构成了本提案所解决的进展的根本障碍。科学的挑战。尽管膜蛋白在这些关键行业中很重要，但它们的生产仍然具有挑战性，每种蛋白质的生产都需要反复试验的方法，在某些情况下需要筛选100种不同的实验因素组合。这部分是由于膜蛋白生产的复杂过程导致细胞生产平台的多重压力（显着降低细胞生长和生物过程生产力）。细胞膜的脂质成分显著影响蛋白质的功能——由于缺乏脂质因子，可能导致任何产生的蛋白质失去功能。因此，科学家必须在一个昂贵而费力的过程中测试多种不同的细胞类型。这种定制平台的开发限制了功能性膜蛋白的生产能力，从而限制了药物筛选和开发。项目大纲。我们通过首先对限制膜蛋白生产的细胞过程进行详细的系统表征来解决这些挑战。这将用于开发一种预测性计算机辅助设计工具，使设计能够促进生产和细胞生长。我们将使用这些工具来设计和实施自主反馈控制策略，使活细胞能够自我调节蛋白质生产以应对压力，从而最大限度地提高产量和产量。这些系统有可能以“不干预的方式”提高产量。我们将设计新的细胞调节系统，能够调节细胞的脂质生产速率，使细胞膜的组成和物理性质可以调整，以满足最佳膜蛋白功能所需的那些。我们将开发计算机辅助设计工具，以预测最佳的膜组成。我们将展示我们的新细胞系统在生产高价值生物医学膜蛋白方面的功能，并展示它们在工业相关条件下的大规模生产性能。

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.