Princeton University

Jared Toettcher

Cliff Brangwynne

Jose L Avalos

Yongdae Shin

Evan M Zhao

Maxwell Wilson

Agnieszka Gil

Alexander Goglia

Cesar Carrasco-Lopez

A system and method for controlling metabolic enzymes or pathways in cells to produce a chemical above the levels of a wild-type strain is disclosed. The system utilizes cells, including yeasts, bacteria, and molds, having at least two genes capable of being controlled bi-directionally with light, where one gene is turned from off to on when exposed to light and another gene is turned from on to off when exposed to light, the two genes reversing when the light is turned off. Cells may utilize any number of sequences that benefit chemical production, including sequences that: encode for constitutive transcription of light-activated transcription factor fusions; encode for a metabolic enzyme; encode for a repressor; induce expression of metabolic enzymes; and an endogenous or exogenous activator expressed by a constitutive promoter, inducible promoter, or gene circuit. These systems may be coupled to biosensors or protein cascade systems, enabling the monitoring or automation of the fermentation process to optimize production of a desired product. These systems may also allow for optimization and periodic operation of a bioreactor using light pulses.

System and method of optogenetically controlling metabolic pathways for the production of chemicals

Provided herein is an isolated fusion protein comprising a light-responsive domain and a heterologous peptide component. Exposure of the fusion protein to light induces a conformational change in the fusion protein that alters an activity of the fusion protein and the activity is a binding activity selected from an in vitro binding activity, an in vivo extracellular binding activity and an in vivo intracellular binding activity. Also provided are methods of using and identifying the fusion proteins.

Light-responsive fusion proteins for controlling binding to targets

A protein construct including a gene encoding a light-sensitive protein fused to at least one of either a low complexity sequence, an intrinsically disordered protein region (IDR), or a repeating sequence of a linker and another gene encoding a light-sensitive protein. Among the many different possibilities contemplated, the protein construct may also advantageously include cleavage tags. This protein construct may be utilized for a variety of functions, including a method for protein purification, which requires introducing the protein construct into a living cell, and inducing the formation of clusters by irradiating the construct with light. The method may also advantageously include cleaving a target protein from an IDR, and separating the clusters via centrifuge. A kit for practicing in vivo aggregation or liquid-liquid phase separation is also included, the kit including the protein construct and a light source capable of producing a wavelength that the light-sensitive protein will respond to.

Optogenetic tool for rapid and reversible clustering of proteins

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Princeton, NJ, United States of America

Jared Toettcher