Engineering an organelle with selective small molecule permeability for compartmentalizing multi-enzyme pathways

面包酵母可以作为一种廉价的、自我复制的工厂，用于生产许多有价值的化学品。为了实现这一点，来自植物和其他来源的基因被转移到酵母中。酵母可以在大规模发酵罐中廉价生长。然而，这些途径在酵母中通常不起作用。这个项目将设计一个细胞器，它的行为就像一个专门的容器，可以被设计成满足各种途径的需要。这种合成细胞器也将是有价值的，当部分途径，是有毒的细胞。我们将组建一个由来自不同背景的五名本科生组成的团队，并将这些学生培养为合成生物学的未来领导者。他们将获得分子细胞生物学和工程方面的专业知识。该团队将在支持性环境中从同行指导中获益。他们还将接受其他资深科学家的指导。该研究项目的中心目标是将过氧化物酶体重新编程为合成细胞器，以满足各种代谢途径的需求，为代谢工程提供可推广和灵活的区室化策略。过氧化物酶体为这种重编程提供了一个有希望的起点，因为它在许多酵母物种（包括酿酒酵母）中不需要生存力。通过添加短的C-末端信号肽序列，可以将杂合蛋白有效地导入过氧化物酶体。然而，许多代谢途径的区室化的关键限制是过氧化物酶体对小于约700道尔顿的分子的天然渗透性。这种渗漏的原因被假设为是由于过氧化物酶体膜蛋白具有允许这些小分子扩散穿过该膜屏障的孔。许多期望的应用将需要降低泄漏程度。因此，细胞器生物发生不需要的过氧化物酶体膜蛋白将被敲除，以构建具有降低的小分子渗透性的设计过氧化物酶体。过氧化物酶体渗透性可以使用实验室内开发的酶螯合测定法在体内测量。有毒蛋白质和多酶途径将被测试，以探测这些工程过氧化物酶体的能力，以支持在单一蛋白质和途径水平的区室化。这一奖项反映了NSF的法定使命，并已被认为是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的支持。

Baker's yeast can be used as an inexpensive, self-replicating factory for the environment-friendly production of many valuable chemicals. To carry this out genes from plants and other sources are transferred into yeast. Yeast can grow cheaply in large-scale fermenters. However, these pathways often do not work well in yeast. This project will engineer an organelle which behaves as a specialized container that can be engineered to meet the needs of a variety of pathways. This synthetic organelle will also be valuable when parts of the pathway, are toxic to the cell. We will assemble a team of five undergraduate students from varied backgrounds and train these students as future leaders in synthetic biology. They will gain expertise in molecular cellular biology and engineering. This team will gain from peer-to-peer mentoring within a supportive environment. They will also be mentored by other senior scientists. The central aim of this research project is to reprogram the peroxisome as a synthetic organelle to meet the needs of a variety of metabolic pathways, providing a generalizable and flexible compartmentalization strategy for metabolic engineering. The peroxisome provides a promising starting point for this reprogramming as it is not required for viability in many yeast species, including Saccharomyces cerevisiae. Heterologous proteins can be efficiently imported into the peroxisome via the addition of a short C-terminal signal peptide sequence. A critical limitation for compartmentalization of many metabolic pathways, however, is the natural permeability of peroxisomes to molecules smaller than approximately 700 Daltons. The cause of this leakiness is hypothesized to result from peroxisome membrane proteins with orifices allowing these small molecules to diffuse across this membrane barrier. Many desired applications will require the degree of leakiness to be reduced. Accordingly, peroxisome membrane proteins not required for organelle biogenesis will be knocked-out to build a designer peroxisome with reduced small molecule permeability. Peroxisome permeability can be measured in vivo using enzyme sequestration assays developed within the lab. A toxic protein and multi-enzyme pathway will be tested to probe the ability of these engineered peroxisomes to support compartmentalization at both the single protein and pathway levels.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.