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

纯化是蛋白质生产中的限制性步骤。如果蛋白质可以分泌出细胞，纯化就容易得多，也简单得多。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，该系统也能耐受极端pH和温度水平。基因编辑工具，如CRISPR，将能够改善天然酵母。将对多种蛋白质的生产进行评价。该项目还侧重于改善高中、社区学院和本科阶段代表性不足的少数民族和非传统学生的STEM教育。提高大学的成功率和美国生物技术劳动力的多样性是期望的结果。全基因组CRISPR-Cas9敲除、CRISPRi和CRISPRa文库将用于鉴定影响蛋白质产生和分泌的关键基因。这些筛选应该生成将表型与基因型联系起来的大型数据集。这些数据将用于该项目的第二阶段。在第二阶段，基于多重CRISPR的基因调控将识别协同组合，从而导致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的蛋白质分泌大幅增加的基因干预文库。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.