In the ethanol self-regulation project, we limited the concentration of a toxic metabolite in order to reduce stress on the yeast, with the goal of allowing the yeast to divert more cellular resources towards manufacturing a desired compound. For industrial fermentation, this would be a convenient method with which to increase yield, since the yeast controls the ethanol level itself, without any outside input. However, this strategy for regulating chemical concentration is dependent on the availability of both a promoter that is induced by the chemical, and an enzyme that can degrade that chemical. Because these may not always be readily available, we decided to build an optogenetic protein control system that could be applied to any pathway in order to optimize the flux through that pathway.
This project consists of two modules: One for the light-inducible activation of protein function by restoring a split protein, and one for the light-inducible deactivation of protein function by protein relocalization. To test whether we could achieve light-induced protein activation and deactivation, we targeted proteins related to the cell cycle in order to generate reversible cell cycle arrest.
The basis of our optogenetic system is the TUnable, Light-controlled Interacting Protein tags (TULIPs) system developed in the Glotzer lab at the University of Chicago. TULIPs consists of two proteins: LOVpep, a light-oxygen-voltage domain from an Avena sativa phototropin, and ePDZ, an engineered PDZ domain. Upon excitation with blue light, LOVpep undergoes a conformational change which allows binding to ePDZ. The response time of the system is fast: dimerization following blue light stimulation occurs on the order of seconds, and dissociation after turning off blue light occurs within minutes. In addition, the kinetics of the binding interaction are tunable by mutagenesis of both the LOVpep and ePDZ domains.
To test the protein activation system, we selected endogenous yeast cell cycle checkpoint proteins whose overexpression is known to arrest the cell cycle with the idea that blue light stimulation should activate the function of these proteins and cause the cell cycle to arrest.
We designed and constructed two 'acceptor vectors' which can be used to easily express the N or C-terminal halves of a protein as a fusion with LOVpep or ePDZ and a fluorescent tag for localization tracking. We have co-transformed a total of 9 unique split protein expression vector pairs into yeast and have confirmed expression through fluorescent microscopy. Currently, we are beginning the first light-induction experiments to see if cell-cycle arrest is achieved.
We envision that optogenetic protein activation could be used to rapidly and reversibly change the flow through a biosynthetic pathway. For example, one could activate a protein to break down a toxic intermediate at a certain level, balancing the flow through the pathway and the stress on the cell.
To use the cell cycle to test light-inducible loss of function, we selected two sets of proteins endogenous to yeast: one set of 'cell-cycle' proteins essential for the progression of the cell cycle, and one set of 'tether' proteins with specific cellular localization patterns. By fusing ePDZ to a cell-cycle protein and LOVpep to a tether protein, we expect that upon blue light stimulation, binding between ePDZ and LOVpep will relocalize the cell cycle protein to the tether protein's location, such that the cell-cycle protein cannot perform its function and the cell cycle will be arrested.
We have constructed 7 yeast strains with unique combinations of cell-cycle and tether genes tagged with LOVpep/ePDZ and a fluorescent protein for tracking cellular localization. We have confirmed expression of both fusion proteins and observed the correct sub-cellular localization of our tether proteins through fluorescent microscopy. Currently, we are beginning light-induction experiments to see if protein re-localization is observed and if cell-cycle arrest occurs.
Here we have presented this optogenetic system as a tool to improve the manufacture of important chemical compounds. In an industrial setting, a fermentation vat could be programmed to optimize flow through a biosynthetic pathway simply by turning a blue light on or off. However, this system has many other important applications. This system allows quick, reversible, spatio-temporal control of protein function, and is orthogonal in yeast.
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