(449d) Dynamic Regulation of Metabolic Pathways with Toehold-Gated dCas9 Regulators

In metabolic engineering, once a metabolic pathway is expressed, there is an optimization process that aims to increase the production, titer, rate, and yield of the target molecule with a goal to direct the maximum possible amount of metabolic flux toward the pathway of interest. Unlike static regulatory methods, dynamic regulation of metabolic pathways offers the ability to adapt and respond to intracellular changes, as well as changes in the extracellular environment. Because of this, dynamic approaches have greater potential to regulate metabolic pathways when compared to static ones.

A toehold switch is comprised of 2 components: RNA in a hairpin configuration that blocks a Ribosome Binding Site (RBS) and an endogenous piece of RNA capable of opening the hairpin called a trigger. Our labs were able to take the dCas9 system and incorporate the spacer into a modified toehold switch creating a toehold-gated sgRNA (thgRNA) which acts as an orthogonal, transcriptional regulator when activated by an endogenous trigger strand. This system was used to show transcriptional repression of simple and complex metabolic pathways in Escherichia Coli (E. coli). We also looked at a variety of factors including: promoter strength, temperature, and induction time on mCherry fluorescence and Violacein production.

More specifically, first we were able to repress a weak promoter driving the expression of Red Fluorescence Protein (RFP) by about 60% when compared to two control groups: No trigger and a non-binding (scramble) trigger. When looking at the strong T7 promoter, only a small change was observed in the experimental group. We also looked at the effect of the T7 thgRNA at repressing the T7 gene in a strain where RFP was chromosomally integrated. Interestingly, despite the increased strength of the chromosomally integrated strain in comparison to the normal T7 promoter, there was significant repression in the experimental group.

Previously, we introduced the violacein pathway, a five-step metabolic pathway, into a pETM6 vector. The expression of the first three genes, VioA, VioB, and VioE are controlled by the weak mutant T7 promoter G6, the VioD gene is controlled by the 4A6 T7 variant, and the VioC gene is controlled by the 3A2 T7 variant. By incorporating these spacers into thgRNAs, we were able to show repression of individual and multiple genes at once with QRT-PCR being used to measure levels of transcriptional repression. All thgRNAs designed in this experiment were expected to perform best at 30°C and induced with .1 mM IPTG. After we expressed the general T7 thgRNA, the thgRNA that indiscriminately targets all five genes, we found that violacein production decreased by ~75%. Interestingly, when the G6 thgRNA was used, the repression dropped to ~67%; unexpected as the G6 thgRNA was found to be more effective at repressing mCherry. When looking at 3A2 repression, we found very little change in the overall levels of violacein or deoxyviolacein despite QRT-PCR showing a 60% decrease in VioC production. In order to look at 4A6 repression, we chose to use the deoxyviolacein pathway, as proviolacein is significantly harder to produce and measure than prodeoxyviolacein. We found a ~30% reduction in deoxyviolacein expression and a ~70% increase in prodeoxyviolacein expression.

The thgRNAs appear to have a greater effect on the weak T7 promoters, as the weakest promoter (G6) had the most repression while the normal T7 promoter showed no statistical repression at all. We were able to show repression of single and multiple genes in the complex Violacein pathway. Our thgRNAs are able to function as a conditional activation of CRISPR-based systems by using highly predictable toehold-mediated strand displacement reactions. Sequence specific unblocking of the spacer allows for both orthogonality and low cross-talk between thgRNAs. Additionally, these devices do not require the screening of large libraries currently needed to create such specific riboregulators as the CRISPR spacers are so sequence specific.