Optimal cell factories for membrane protein production

具有重要的社会意义。膜蛋白对于生命、医学和工业生物技术都是必不可少的，但由于(1)丰度低和(2)物理性质难以分离，对膜蛋白的研究仍然很少。然而，20%-30%的蛋白质是膜蛋白，具有支持信号转导、营养输入/输出和细胞间通讯的生物学功能。膜蛋白是有效的药物和疫苗靶点。目前，全球最畅销的十种药物，总销售额为1800亿美元，都是针对膜蛋白的。多种疫苗，包括新冠肺炎、乙肝和百日咳疫苗，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)和抗真菌药物(价值190亿美元)。人工表达膜蛋白在药理学中经常被用来识别药物分子，这些分子可能抑制或增强蛋白质的功能。然而，设计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，减少了新药的识别，并限制了转化潜力。膜蛋白的生产能够在促进人类健康方面取得进展，并为气候变化造成的紧迫社会挑战提供生物启发的解决方案；然而，获得高产量的功能蛋白是取得进展的根本障碍，这一提议解决了这一挑战。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--在某些情况下，需要对数百种不同的实验因素组合进行筛选。这在一定程度上是由于膜蛋白生产的复杂过程，导致细胞生产平台受到多重压力(显着降低细胞生长和生物过程生产率)。细胞膜的脂质成分显著影响蛋白质的功能--可能会使产生的任何蛋白质由于缺乏脂质因素而失去功能。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类型的细胞。这种定制平台的开发限制了功能性膜蛋白的生产能力，从而限制了药物的筛选和开发。我们首先对限制膜蛋白生产的细胞过程进行了详细的系统表征，以应对这些挑战。这将被用来开发一种预测性的计算机辅助设计工具，使设计能够同时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，从而最大化产量和产量。这些系统有可能在不插手的情况下提高产量。我们将设计新的细胞调节系统，能够调节细胞的脂质生成率，使细胞膜的组成和物理性质能够调节，以满足最佳膜蛋白功能所需的条件。我们将开发能够预测最佳膜组成的计算机辅助设计工具。我们将展示我们的新细胞系统生产高价值生物医用膜蛋白的功能，并展示其在工业相关条件下的规模化生产性能。

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.