Chemoenzymatic construction of a programmable synthetic endoplasmic reticulum

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A fundamental goal in synthetic biology is to build artificial cells that have many of the basic functions of natural cells, as well as specific functions that are useful in medicine and biotechnology. This project focuses on building a synthetic subcellular compartment or organelle known as the endoplasmic reticulum, since this organelle would provide artificial cells with essential functions such as the ability to synthesize proteins, process proteins and synthesize lipids. Broader outcomes of this proposal include graduate student training and educating the public about designer cell organelles and their importance through outreach lectures to high school students and the general public. Bioethical consequences related to generating a synthetic organelle will also be considered. To generate a synthetic ER de novo, the chemoenzymatic synthesis of non-canonical phospholipids will be combined with recombinant proteins that influence membrane curvature and fusion. The proposed program to generate and study a synthetic ER will follow these three closely coupled objectives: (1) chemical and enzymatic steps will be developed to generate a library of non-canonical phospholipid membranes similar to the lipid membranes of living cells. (2) the minimal lipid composition requirements necessary for generating an ER-like reticulated membrane architecture will be explored. The interactions of these lipids with membrane-bound proteins that play a critical role in membrane curvature stabilization and fusion respectively in yeast, will be monitored. Furthermore, different combinations of non-canonical lipids will be tested to explore how biomimetic phospholipids affect membrane properties; and (3) the differences in properties between natural and synthetic spherical, tubular, and reticular membrane geometries will be investigated. In particular, the kinetics of internalized reactions, reversibility of the steady-state morphology, mixing of lipids in reticular membranes, diffusion through the lumen of the synthetic ER, and network connectivity will be characterized. To equip the synthetic ER with functionality, a simple post-translational network by localizing two fluorescently labeled glycosyltransferases will be developed within a synthetic reticular network for post-translational modification of a protein substrate. The proposed studies will establish the groundwork for developing artificial reticulated organelles that mimic the ER and facilitate future programmable functions such as post-translational modification, biomolecular trafficking, sensing, and protein turnover.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.