Team:Johns Hopkins-Wetware/Project

Synthetic biology is helping to solve problems such as malnutrition and disease through the production of compounds like vitamins and medications. Using microorganisms to produce these compounds can provide a most cost-effective solution and thus make them more readily available. This year, the Johns Hopkins wetware team presents tools to control cellular processes in yeast, which can be applied to the optimization of non-native biosynthetic pathways used in cell-based manufacturing of compounds. Our two projects are an ethanol level self-regulation system, and a system for optogenetic control of protein function.

In industrial fermentation, the buildup of toxic intermediates and byproducts keeps productivity from reaching its full potential. In yeast, ethanol toxicity is the major chemical stress. To reduce ethanol stress, we constructed an ethanol-level self-regulation system consisting of the human cytochrome p450 CYP2E1 driven by a library of ethanol-induced promoters. CYP2E1 catalyzes the conversion of ethanol to acetaldehyde and then to acetate. When the ethanol level exceeds the optimal level, expression of CYP2E1 is triggered, which breaks down the excess ethanol. Using this tool, we have demonstrated a way to decrease ethanol concentration under fermentation conditions without negatively impacting cell growth.

The ability to inducibly control protein function in vivo can be used to regulate flux through a biosynthetic pathway, minimizing stress on the host cell and maximizing production of a desired compound. Here we use the ePDZ/LOVpep light-induced dimerization system to demonstrate the activation and deactivation of proteins in S. cerevisaie. The use of light as a control mechanism has the advantages of being fast-acting, reversible, and amenable to automation in industrial applications. As proof-of-concept, we have applied our system in two ways: (i) testing light-induced activation of protein function by restoring a complete protein from two non-functional halves; and (ii) demonstrating deactivation of protein function through protein localization. In the context of pathway engineering, the tunability of the ePDZ/LOVpep system is a major advantage as enzyme function can be modulated.