Today’s increasingly complex research and manipulation of biological pathways poses a demand for rapid, controllable gain and loss of biomolecular function. Bioengineering of pathways for industrial processes is hindered due to lack of understanding of non-native proteins in the yeast chassis. Optimizing pathways and adjusting expression of relevant proteins is a tedious task. The 2012 JHU iGEM team set about developing a tool to facilitate optimization and controlling flux of pathways in order to maximize efficiency of manufacture.
Focusing on Optogenetics
Light-inducible proteins are a very modern focus of research, and even more recently a component in engineered systems. We considered PhyB, Cry2, CcaS and the TULIPs system (Strickland, et al. 2012), and any combination of the aforementioned. Finally, we decided on just the TULIPs system because of its relative advantages:
--Immediate response to stimulus and lack thereof, much like an on- and off-switch,
--Required only a single wavelength of light, whereas most others required two (on and off),
--Proven to work in yeast (Strickland, et al. 2012),
--Tunable - small mutations in the light-protein constructs can toggle sensitivity,
--The proteins are small, compared to others. Increases likelihood that proteins can pass through nuclear envelope, or other organelles,
--Does not require addition any exogenous chemicals,
PhyB and CcaS required an exogenous cofactor called phytochromobilin, which does not eliminate the need to add chemicals and contradicts one of our goals. Alternatively, we could have transformed yeast with the genes required to make this chromophore, but we would not like for the success of our overall project to be dependent upon this. Also, a previous and unsuccessful attempt to synthesize PCB in yeast was discouraging (https://2009.igem.org/Team:Harvard/PCB). CcaS also could not be used because we could not find a yeast intracellular signaling pathway that would turn on only the gene we needed; its signaling mechanism also did not allow for precise temporal and spatial control. Cry2 was rather large, and did not have an immediate “off-switch”; it reverts to its inactive form at a late and imprecise time.
Theoretically, the system can be adapted to control loss or gain of just about any function in our model organism. In manufacturing, this allows for light to precisely optimize production throughout an entire bioreactor. For example, if yeast produces a toxic biproduct, but the mechanism to degrade the biproduct is costly, the adapted TULIPs system can be used to turn on the degradation pathway. The pathway’s effect takes place between the precise moment when the blue light is turned on and the precise moment when it is turned off, and can be distributed throughout the bioreactor.
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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