Isoprenoids comprise a large class of chemicals of significant interest due to their diverse properties. Biological production of isoprenoids is considered to be the most efficient way for their large-scale production. Isoprenoid biosynthesis has thus far been dependent on pathways inextricably linked to glucose metabolism. These pathways suffer from inherent limitations due to their length, complex regulation, and extensive cofactor requirements. Here, we present a synthetic isoprenoid pathway that aims to overcome these limitations. This isopentenol utilization pathway (IUP) can produce isopentenyl diphosphate or dimethylallyl diphosphate, the main precursors to isoprenoid synthesis, through sequential phosphorylation of isopentenol isomers isoprenol or prenol. After identifying suitable enzymes and constructing the pathway, we attempted to probe the limits of the IUP for producing various isoprenoid downstream products. The IUP flux exceeded the capacity of almost all downstream pathways tested and was competitive with the highest isoprenoid fluxes reported.

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Metabolic engineering is the practice of using directed genetic manipulations to rewire cellular metabolism primarily with the aim to transform the organism into a single-celled chemical factory. Using biological processes, we can produce more complex chemicals in a more sustainable way. This is particularly important for chemicals which are hard to synthesize using traditional chemistry. However, cells have evolved for growth and must be engineered to produce a single chemical at commercially viable levels. This review focuses on the strategies used to rewire cellular metabolism to produce chemicals using isoprenoid production in Escherichia coli as an example that illustrates many of the challenges faced in metabolic engineering.

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far. K E Y W O R D Sbiocatalysis, enzyme kinetics, isoprenoids, metabolic control analysis, taxadiene

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