Researcher(s)
- Helena Lund Hansen, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Wilfred Chen, Chemical Engineering, University of Delaware
- Mark Blenner, Chemical Engineering, University of Delaware
Abstract
Engineering microbes to synthesize pharmaceuticals offers a sustainable alternative to the traditional use of petroleum-based chemical synthesis in the industry. Although this seems promising, an inherent challenge in microbial fermentation is that microbes cannot allocate their resources efficiently between cell growth and product synthesis. To solve this issue, we have engineered a conditional gene expression method that enables microbes to decide between product synthesis and cell growth depending on their environment. We have adapted a CRISPR activation system in which dCas9 targets a specific 20 base pair sequence identified by the single guide RNA (sgRNA). We modified this sgRNA to include an engineered RNA binding protein (RBP) hairpin that contains an MS2 coat protein (MCP) and a metabolite-responsive aptamer. The MCP is attached to a transcriptional activator that can recruit the RNA polymerase to the target gene. The idea with this system is that the RNA polymerase will only be recruited when the cell is in an environment rich in the target metabolite that the aptamer is responsive to, thus creating a conditional transcriptional system. We have previously demonstrated that the system is highly specific to the target metabolite but the 5-fold increase in gene activation remains lower than desired. While characterizing the system, we observed enhanced protein production at lower temperatures likely due to protease recognition of our transcriptional activator as well as unwanted steric clashes leading to incorrect MCP-aptamer complex formation. Finally, to demonstrate the utility of our work, we engineered our system to conditionally regulate the biosynthesis of a valuable natural product, violacein, derived from the target metabolite, tryptophan.