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 by 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 light-inducible activation through dimerization of two individually expressed protein halves (split protein), and one for the light-inducible deactivation of protein function by protein relocalization. To test whether we could achieve light-induced alteration of protein function, we first targeted S. cerevisiae cell cycle proteins to generate reversible cell cycle arrest. Cell cycle arrest is an easily measured output as it correlates with bud appearance and size.
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.
The TULIPs system: blue light excitation (<500nm) changes the conformation of LOVpep to allow binding to ePDZ. Figure adapted from Strickland, et al. 2012.
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.
Fluorescence microscopy without blue light excitation of a yeast strain expressing the N and C terminal halves of Whi3 fused to ePDZb/GFP and LOVpep/mCherry, respectively. MCherry fluorescence is visible on the Cy3 channel in b), and GFP fluorescence is visible on the GFP channel in c).
One of the ways in which our system could be applied to pathway control.
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.
Fluorescence microscopy without blue light excitation of yeast expressing Cdc15/ePDZb/GFP and Mid2/LOVpep/mCherry. Mid2 is expected to localize to the plasma membrane, which can be observed from the mCherry fluorescence under the Cy3 filter in b).
Fluorescence microscopy without blue light excitation of yeast expressing Cdc14/ePDZb/GFP and Net1/LOVpep/mCherry. Net1 is expected to localize to the nucleolus, which can be observed from the mCherry fluorescence under the Cy3 filter in b).
One of the ways in which our system could be used to control pathways.
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.
"
