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

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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.