(256a) A Novel Two Step Pathway for Isoprenoid Synthesis

Isoprenoids are a large class of more than 50,000 natural compounds, found in plants and many living organisms. They are of significant interest due to their varied applications in fields such as medicine, flavors, fragrances and cosmetics. The field of isoprenoid biosynthesis has thus far been dependent on the methyl-erythritol (MEP) pathway or the mevalonate (MVA) pathway, both of which are linked to glucose metabolism and suffer from limitations due to their length, their complex regulation and their extensive cofactor requirements.

Here, we present a novel synthetic isoprenoid pathway, which we term the Isopentenol Utilization Pathway (IUP), that aims to overcome these limitations. This pathway is able to produce isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP); the main precursors to isoprenoid synthesis, through the sequential phosphorylation of the isopentenol isomers isoprenol or prenol.

We first conducted in-vitro enzymatic assays to identify suitable enzymes for each step of the pathway. After establishing the viability of the IUP pathway as the sole isoprenoid pathway, we then attempted to probe the limits of the pathway for the production of various isoprenoid downstream products, including taxadiene and lycopene. Furthermore, in our efforts to estimate flux through the pathway and identify bottlenecks, we conducted pulse-chase experiments and measured metabolic intermediate profiles. After minimal optimization of the downstream operon, we were able to achieve through the IUP an IPP flux competitive with some of the best reported in literature.