Investigation of Pathways that Regulate the Expression of Recombinant Proteins in Yeast

在过去的半个世纪里，生物技术的发展使一系列新颖的医疗和工业应用成为可能，从而彻底改变了我们的生活。一个关键的组成部分是我们操纵细胞产生宿主细胞通常不表达的“重组”蛋白质的能力。这些宿主细胞就像工厂一样，可以批量生产大量可用作药物或催化剂的重组蛋白。例如，细胞工厂生产的人胰岛素和促红细胞生成素分别用于治疗糖尿病和贫血。同样，宿主细胞可以产生工业酶，如蛋白酶(分解蛋白质的酶)，这些酶被添加到洗涤剂中。一个主要问题是只有一小群宿主细胞可用，而且许多潜在的新型重组蛋白不能用目前可用的宿主细胞正确表达。这一障碍避免了发现新的生物技术应用。其中一个原因是，由于宿主细胞环境缺乏适当的支持，重组蛋白质并不总是达到它们的3D构象(每个蛋白质为其功能采用特定的形状)。然后这些重组蛋白错误折叠(即采用错误的构象)，从而触发它们在宿主细胞中的降解。我们注意到，酵母细胞中的微小遗传变异可以对我们所研究的模型蛋白质的稳定性产生重大影响。因此，我们建议利用酵母生物多样性和大量酵母突变菌株来筛选潜在的新型宿主细胞，这些细胞表现出增强的表达给定重组蛋白的能力。我们将调查哪些酵母宿主细胞可以产生更高水平的酶家族，这些酶主要用于废水的生物修复、食品加工和生物传感器。这些来源于真菌或植物的许多酶都可以在酵母宿主细胞中表达和分泌。我们已经筛选了一个不同的天然或工业酵母细胞的大型文库，我们已经鉴定了几个来自不同来源的菌株，它们分泌更高水平的被评估的酶。我们将进一步表征这些菌株，以确定为什么会出现这种情况。同时，我们将筛选大量突变细胞库，以确定哪些突变会导致更高的酶表达。利用这些信息，我们将进一步优化宿主细胞进行重组表达。最后，我们将建立一种新的方法来评估潜在的新宿主细胞，看看它们可以在多大程度上用于重组蛋白的表达。值得注意的是，我们将一起表达相关但不同的重组酶的混合池，并确定在给定的评估菌株中哪些是分泌的或不是分泌的。我们的目标之一是建立新的筛选方法来帮助设计新的宿主细胞。同时，我们还希望发现调节细胞环境和蛋白质动态平衡的潜在新的生物学途径。

Developments in biotechnology over the last half century have revolutionized our lives by enabling a broad range of novel medical and industrial applications. One key component has been our ability to manipulate cells to produce "recombinant" proteins that are not normally expressed by the host cells. These host cells, used like factories, can mass-produce a large array of recombinant proteins that can be used as drugs or catalysts. For instance, human insulin and erythropoietin are produced in cell factories to treat diabetes and anemia, respectively. Similarly, host cells can produce industrial enzymes like proteases (enzymes that break proteins), which are added to laundry detergent. A major issue is that only a small regiment of host cells is available, and many potentially novel recombinant proteins cannot be properly expressed using currently available host cells. This hurdle averts the discovery of new biotechnology applications. One reason for this is that recombinant proteins cannot always reach their 3D conformation (each protein adopts a specific shape for its function) due to a lack of appropriate support in the host cellular environment. These recombinant proteins then misfold (i.e. adopt a wrong conformation), which triggers their degradation in host cells. We noticed that minor genetic variations in yeast cells can have dramatic effects in the stability of model proteins that we have studied. Therefore, we propose to take advantage of yeast biodiversity and large collections of yeast mutant strains to screen for potential novel host cells that display enhanced capacity to express a given recombinant protein. We will investigate which yeast host cells can produce higher levels of a family of enzymes that are notably used for bioremediation of waste water, in food processing and biosensors. Many of these enzymes that originate from fungi or plant can be expressed and secreted from yeast host cells. We have screened a large library of diverse natural or industrial yeast cells and we have identified several strains from different origins that secrete higher levels of the assessed enzyme. We will further characterize these strains to determine why that is the case. In parallel, we will screen a large library of mutant cells to determine which mutations cause higher enzyme expression. Using this information, we will bioengineer further optimized host cells for recombinant expression. Finally, we will establish a new method to assess potentially new host cells to see how broadly they can be used for recombinant protein expression. Notably, we will express together a mixed pool of related but different recombinant enzymes and determine which ones are secreted or not in a given assessed strain. One of our goals is to establish new screening methods to help design new host cells. In parallel, we also want to uncover potentially novel biological pathways that regulate the cellular environment and protein homeostasis.