Designing a New Entry Point into Isoprenoid Metabolism by Exploiting Fructose-6-Phosphate Aldolase Side Reactivity of <i>Escherichia coli</i>

The 2C-methyl-d-erythritol-4-phosphate (MEP) pathway in <i>Escherichia coli</i> has been highlighted for its potential to provide access to myriad isoprenoid chemicals of industrial and therapeutic relevance and discover antibiotic targets to treat microbial human pathogens. Here, we describe a metabolic engineering strategy for the <i>de novo</i> construction of a biosynthetic pathway that produces 1-dexoxy-d-xylulose-5-phosphate (DXP), the precursor metabolite of the MEP pathway, from the simple and renewable starting materials d-arabinose and hydroxyacetone. Unlike most metabolic engineering efforts in which cell metabolism is reprogrammed with enzymes that are highly specific to their desired reaction, we highlight the promiscuous activity of the native <i>E. coli</i> fructose-6-phosphate aldolase as central to the metabolic rerouting of carbon to DXP. We use mass spectrometric isotopomer analysis of intracellular metabolites to show that the engineered pathway is able to support <i>in vivo</i> DXP biosynthesis in <i>E. coli</i>. The engineered DXP synthesis is further able to rescue cells that were chemically inhibited in their ability to produce DXP and to increase terpene titers in strains harboring the non-native lycopene pathway. In addition to providing an alternative metabolic pathway to produce isoprenoids, the results here highlight the potential role of pathway evolution to circumvent metabolic inhibitors in the development of microbial antibiotic resistance