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Efficient supply of terpenoid precursors remains a key challenge in metabolic engineering. The two-step isoprenol phosphorylation pathway, which converts isoprenol to isopentenyl diphosphate and dimethylallyl diphosphate via two kinase reactions, offers a streamlined alternative to native biosynthetic routes, yet its efficiency is critically limited by the first phosphorylation step (Kinase-1). Conventional Kinase-1 enzymes exhibit poor catalytic efficiency toward isoprenol, with kcat/Km values approximately four orders of magnitude below those of typical metabolic enzymes. Here, we establish mevalonate kinase (MvaK) as a novel Kinase-1 alternative for the two-step pathway in Escherichia coli. Through bioinformatic screening and lycopene-based functional evaluation, MvaK from Kitasatospora griseola was identified as the most promising candidate among five tested kinases. Two rounds of directed evolution yielded beneficial mutations that improved lycopene production approximately threefold. Molecular docking revealed that poor isoprenol accommodation stems from electrostatic mismatch at residues E33 and H34. Structure-guided rational design on the evolved variant identified E33A/H34L as the best-performing variant among those tested, achieving a lycopene titer of 83.0 ± 1.1 mg/L, representing a 10-fold improvement over wild-type. This work demonstrates that combining bioinformatic mining, directed evolution, and rational design effectively improves enzyme activity toward non-native substrates in terpenoid biosynthesis.
Pan et al. (Mon,) studied this question.