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步骤。如果蛋白质可以分泌出细胞外，纯化就容易得多，也更简单。某些酵母菌精通蛋白质分泌。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，该系统还能耐受极端的pH和温度水平。CRISPR等基因编辑工具将使本地酵母的改良成为可能。将对广泛的蛋白质生产进行评估。该项目还侧重于改善高中、社区大学和本科阶段代表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。将对马尔克斯克鲁维酵母的分泌途径进行工程设计。全基因组的CRISPR-Cas9基因敲除、CRISPRi和CRISPRa文库将用于识别影响蛋白质生产和分泌的关键基因。这些筛查应该生成将表型与基因型联系起来的大型数据集。这些数据将用于为该项目的第二阶段提供信息。在第二阶段，基于多路CRISPR的基因调控将识别导致更有效的蛋白质分泌的协同组合。这项研究可以产生有效的CRISPR gRNA文库，优化的选择策略，这种新兴工业酵母中分泌途径的基础知识，以及导致一系列蛋白质分泌显著增加的基因干预文库。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.