Collaborative Research: Data-driven engineering of the yeast Kluyveromyces marxianus for enhanced protein secretion

合作研究：马克斯克鲁维酵母的数据驱动工程，以增强蛋白质分泌

• 批准号: 2323983
• 负责人: Nancy Da Silva
• 金额: $ 35万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-02-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2323983&HistoricalAwards=false
• 关键词: Collaborative; Research; Data; driven; engineering

Purification is the limiting step in protein production. If the protein can be secreted out of the cell, purification is much easier and simpler. Certain yeasts are proficient at protein secretion. The objective of this project is to develop a yeast strain with a robust protein secretion system that is also tolerant to extreme pH and temperature levels. Gene editing tools such as CRISPR will enable the improvement of a native yeast. Production of a broad range of proteins will be evaluated. This project also focuses on improving STEM education for underrepresented minorities and non-traditional students at the high school, community college, and undergraduate levels. Improving success in college and the diversity of the US biotechnology workforce are desired outcomes.The secretory pathway of the yeast Kluyveromyces marxianus will be engineered. Genome-wide CRISPR-Cas9 knockout, CRISPRi, and CRISPRa libraries will be used to identify critical genes influencing protein production and secretion. These screens should generate large datasets that link phenotype-to-genotype. This data will be used to inform a second phase of the project. In the second phase, multiplexed CRISPR-based gene-regulation will identify synergistic combinations leading to more efficient protein secretion. The research could result in effective CRISPR gRNA libraries, optimized selection strategies, fundamental knowledge of the secretory pathway in this emerging industrial yeast, and a library of gene interventions leading to substantially higher protein secretion for a range of proteins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

纯化是蛋白质生产的限制步骤。如果蛋白质可以分泌到细胞外，纯化就容易得多、简单得多。某些酵母擅长蛋白质分泌。该项目的目标是开发一种具有强大蛋白质分泌系统的酵母菌株，该菌株还能够耐受极端的 pH 和温度水平。 CRISPR 等基因编辑工具将能够改进天然酵母。将评估多种蛋白质的生产。该项目还致力于改善高中、社区学院和本科生中代表性不足的少数族裔和非传统学生的 STEM 教育。提高大学成功率和美国生物技术劳动力的多样性是理想的结果。马克斯克鲁维酵母的分泌途径将被改造。全基因组 CRISPR-Cas9 敲除、CRISPRi 和 CRISPRa 文库将用于识别影响蛋白质产生和分泌的关键基因。这些屏幕应该生成将表型与基因型联系起来的大型数据集。该数据将用于为该项目的第二阶段提供信息。在第二阶段，基于 CRISPR 的多重基因调控将识别协同组合，从而实现更有效的蛋白质分泌。该研究可能会产生有效的 CRISPR gRNA 文库、优化的选择策略、这种新兴工业酵母分泌途径的基础知识，以及导致一系列蛋白质显着更高的蛋白质分泌的基因干预文库。该奖项反映了 NSF 的法定使命通过使用基金会的智力优点和更广泛的影响审查标准进行评估，并被认为值得支持。