Quorum-Sensing Linked RNAi for Dynamic Pathway Control in Saccharomyces Cerevisiae

Many metabolic engineering strategies involve the deletion or over-expression of genes which subsequently reduce or eliminate biomass formation. However, in order to achieve high compound titers a production strain must be able to grow to a high population density by accumulating biomass. This means that the most productive genetic modifications cannot be implemented unless population-growth and production phases are separated. To achieve this separation it is necessary to dynamically control the expression of relevant genes so that a population can grow free of metabolic burden to a high density before switching to production mode. We enabled dynamic regulation by using a synthetic quorum sensing circuit in S. cerevisiae which utilises pheromone mediated cell-to-cell communication to autonomously trigger gene expression at a high population density. Dynamic silencing of gene expression was enabled by combining the quorum sensing circuit with an RNA interference (RNAi) module which targets mRNA for degradation according to base-pair complementarity of expressed RNA hairpins. As a demonstration we used the quorum sensing-RNAi circuit to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (pHBA), a valuable aromatic chemical used in liquid crystal polymers. Quorum sensing triggered a production phase with the expression of a chorismate lyase for pHBA formation (UBiC gene), feedback resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (ARO4 gene), and transketolase (TKL1 gene) for increased precursor supply. This dynamic regulation also allowed the implementation of engineering strategies identified by elementary flux mode analysis as highly productive but inhibitory of biomass formation, after a population growth phase. To demonstrate this capacity we conditionally silenced the expression of ARO7, TRP3, and ZWF1 genes which improve pHBA yield, but severely limit growth if deleted. We also silenced a pyruvate kinase gene (CDC19) which when deleted with traditional methods is not only inhibitory to growth, but is lethal. This approach facilitated the attainment of the most highly productive flux modes after the population had grown to a high density such that lethal gene silencing did not limit biomass formation and therefore titer. Using simple shake-flask fermentations, this approach resulted in the highest recorded pHBA titer in yeast of 166 mg/L.