The isopentenol utilization pathway: enzyme engineering, metabolic integration, and applications in microbial...

Terpenoids are among the most structurally diverse and commercially important natural products, with applications in pharmaceuticals, flavors, fragrances, and biofuels. Controllable supply of the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) remains a central limitation for microbial terpenoid production. The native mevalonate (MVA) and methylerythritol phosphate (MEP) pathways are deeply integrated with sterol homeostasis, central carbon metabolism and redox balance. Their distributed regulation, cofactor demands and scale-dependent bottlenecks complicate further intensification. The isopentenol utilization pathway (IUP) offers an orthogonal alternative: a compact, ATP-only, alcohol-fed bypass converting exogenous isoprenol and prenol to IPP and DMAPP in two kinase steps plus isomerization. This review treats the IUP as a configurable precursor module rather than a single pathway variant. Enzyme-level sections cover entry-kinase and isopentenyl phosphate kinase structure, kinetics, and engineering, including the kinetic imbalance that places most flux control in the first phosphorylation step in canonical C5 configurations, together with the feeding, energetic and downstream conditions under which control shifts elsewhere. Host-level sections examine expression hierarchies, construct design, host choice and compartmentalization, and the interaction between IUP flux, toxicity management, and native precursor pathways. Cell-free cascades and techno-economic analysis then identify the regimes, and the quantitative margins, within which an alcohol-fed bypass outperforms further MVA or MEP optimization.