Engineering a responsive, heterologous mevalonate pathway in Escherichia coli using microarrays

Isoprenoids are a highly diverse class of natural compounds that have received wide spread commercial use as flavors, fragrances, and pharmaceuticals. However, their supply is often limited due to their low abundance in their natural sources and difficult routes of chemical synthesis. To alleviate this limitation, the Keasling Lab has engineered Escherichia coli to produce large quantities of the ultimate precursors, the isoprenyl diphosphates, by introducing the mevalonate pathway from Saccharomyces cerevisiae. Coupled with engineered terpene synthases and tailoring enzymes from their native sources, we now have the ability to make these molecules from simple sugars in a controlled environment. The engineered heterologous pathway however bypassed the native regulation of E. coli's isoprenoid pathway and the unregulated expression of the pathway resulted in growth inhibition due to the accumulation of the isoprenyl diphosphates. Microarray analysis of the inhibited cells identified the PhoPQ regulon as a concerted response to the toxicity. The PhoPQ regulon is E. coli's regulatory two component system to respond to magnesium concentrations in the environment. Further analysis of the growth conditions revealed that the toxicity occurred when the magnesium and glucose concentrations in the media were low, validating the microarray results and leading to the hypothesis that the accumulation of the negatively charged diphosphate molecules disrupted the divalent cation's normal role in the cell. Enzyme assays with luciferase, which uses ATP and magnesium as cofactors, confirmed that farnesyl diphosphate was inhibitory at an enzyme level. Fluorescent sensors were designed using promoters of these genes upregulated and downregulated in response to farnesyl diphosphate accumulation. The sensors were tested for their dose dependent response to farnesyl diphoshpate accumulation and these promoters were then used to regulate the heterologous pathway. In the absence of other methods to regulate the pathway in response to the intermediates' accumulation, we have used standard inducible promoters that give a static output in response to an exogenous inducer and have no connection to the flux through the pathway. However, at industrial scale production the use of these exogenous inducers is not feasible. Using the responsive promoters return dynamic regulation to the pathway and obviates the need for an exogenous inducer. When the responsive promoters were cloned 5’ of the isoprenyl diphsophates' downstream enzyme and the pathway was still regulated by the inducible promoters, production was less than the inducible promoter regulating the downstream enzyme. However, when the entire pathway was regulated by the responsive promoters, production doubled relative to the inducible pathway. In addition, the responsive pathway reduced acetate accumulation, allowed faster glucose consumption, and increased cell growth