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

纯化是蛋白质生产的限制步骤。如果蛋白质可以分泌出细胞，纯化就容易得多。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的蛋白质分泌系统的酵母菌株，该系统也能耐受极端的pH和温度水平。CRISPR等基因编辑工具将使原生酵母得到改进。将评估多种蛋白质的生产。该项目还侧重于在高中、社区大学和本科阶段改善代表性不足的少数民族和非传统学生的STEM教育。提高在大学的成功和美国生物技术劳动力的多样性是理想的结果。对酵母菌马氏克鲁维菌的分泌途径进行工程化研究。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。这些筛选应该产生将表型与基因型联系起来的大型数据集。这些数据将用于项目的第二阶段。在第二阶段，基于多重crispr的基因调控将识别协同组合，从而更有效地分泌蛋白质。该研究可能会产生有效的CRISPR gRNA文库，优化的选择策略，对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分泌大幅增加的基因干预文库。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.