Who are your proposed end users? How do you envision others using your project? How would you implement your project in the real world?
One of the application prospects of our project is to use engineered yeasts as biosensor to detect specific substances. We position our end users as environmental monitoring agencies, who can use our engineered yeasts to detect some contaminants in the environment. We plan to select proteins that fail to be detected with existing methods as our targets. Through modifying G protein-coupled receptors, cells can respond to specific signals and activate the intracellular reporter pathway, thus realizing the detection of contaminants in the environment. Especially for some substances that cannot be efficiently detected through chemical methods.
Another application scenario is the construction of cell factory. Ste5ΔN-CTM mutant can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes1. Fermentation engineers can use such characteristic to regulate specific reactions through appropriate induction conditions. For example, chassis can be induced at various stages to avoid the toxic effect of the accumulated intermediates. Additionally, the activation of yeast mating-specific signaling pathways arrests the cell cycle in G1 phase, allowing more carbon sources to be applied to product synthesis2. Therefore, using pheromone-responsive promoters to induce the synthesis of non-growth-coupled metabolites is worth considering. Compared to other signaling transduction processes, mating-specific signal pathways are relatively independent of the asexual life history of yeast. Thus, using this pathway for gene expression regulation, to a certain extent, enables to reduce the crosstalk between signal pathways.
Besides, the promoter engineering of pheromone-responsive promoters in Saccharomyces cerevisiae also provides a library of promoters, which facilitates the precise control of gene expression for metabolic optimization. And through the construction of pgal1-Ste5ΔN-CTM cassette in yeast, we managed to use galactose instead of pheromone to activate the yeast mating specific signaling pathway. Considering the high price of pheromones, this may greatly reduce costs for related research.
What are the safety aspects you would need to consider?
The use of biosensors to detect proteins must be carried out in a closed bench. Once the engineered yeasts leaking into the environment, they may cause some harm to the environment. Therefore, a leak-proof device will be designed to improve the safety of our project before it being put into practical application.
What other challenges would you need to consider?
In our project, using galactose as the inducer changes the carbon source which may affect cell growth.
The G protein coupled receptor in the pheromone signal pathway can be modified to detect some special proteins. But the antigen-antibody reaction has been widely used in protein detection. And the construction of highly specific antibodies has mature schemes, which are simpler than the modification of G protein coupled receptors.
The escape of engineered yeasts from the laboratory will destroy the ecological balance of the natural yeast community. We need to make further considerations for environmental safety through the waste classification mechanism and recycling.
 Lamson, R. E., Takahashi, S., Winters, M. J., and Pryciak, P. M. (2006) Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity, Current Biology 16, 618-623.
 Heiman, M. G., and Walter, P. (2000) Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating, The Journal of cell biology 151, 719-730.