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

酵母中重组蛋白表达调控途径的研究

• 批准号: RGPIN-2022-03787
• 负责人: Mayor, Thibault
• 金额: $ 4.88万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760918
• 关键词: Investigation; Pathways; Regulate; Expression; Recombinant

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.

生物技术在过去的半个世纪的发展已经彻底改变了我们的生活，使广泛的新颖的医疗和工业应用。一个关键的组成部分是我们能够操纵细胞产生“重组”蛋白质，这些蛋白质通常不会被宿主细胞表达。这些宿主细胞就像工厂一样，可以大量生产大量重组蛋白质，这些蛋白质可以用作药物或催化剂。例如，人类胰岛素和促红细胞生成素是在细胞工厂中生产的，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），这些酶被添加到洗衣粉中。一个主要的问题是，只有一小群宿主细胞是可用的，许多潜在的新的重组蛋白不能正确地表达使用目前可用的宿主细胞。这一障碍阻碍了新的生物技术应用的发现。其中一个原因是，由于宿主细胞环境缺乏适当的支持，重组蛋白质并不总能达到其3D构象（每个蛋白质都采用特定的形状来发挥其功能）。这些重组蛋白然后错误折叠（即采用错误的构象），这引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能对我们研究的模型蛋白质的稳定性产生巨大影响。因此，我们建议利用酵母的生物多样性和酵母突变株的大量收集来筛选潜在的新宿主细胞，这些宿主细胞表现出增强的表达给定重组蛋白的能力。我们将研究哪些酵母宿主细胞可以产生更高水平的酶家族，这些酶家族特别用于废水的生物修复，食品加工和生物传感器。许多来源于真菌或植物的酶可以从酵母宿主细胞中表达和分泌。我们已经筛选了大量不同的天然或工业酵母细胞库，并且我们已经鉴定了来自不同来源的分泌更高水平的评估酶的几种菌株。我们将进一步描述这些菌株，以确定为什么会出现这种情况。同时，我们将筛选一个大型突变细胞库，以确定哪些突变导致更高的酶表达。利用这些信息，我们将生物工程进一步优化重组表达的宿主细胞。最后，我们将建立一种新的方法来评估潜在的新宿主细胞，以了解它们可用于重组蛋白表达的范围有多广。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，并确定哪些酶在给定的评估菌株中分泌或不分泌。我们的目标之一是建立新的筛选方法，以帮助设计新的宿主细胞。与此同时，我们还希望发现调节细胞环境和蛋白质稳态的潜在新生物途径。