<i>De Novo</i> Biosynthesis of Methyl Isobutyl Ketone by Engineered <i>Escherichia coli</i>

Methyl isobutyl ketone (MIBK) serves as a critical high-grade organic solvent and chemical intermediate in biofuel extraction, rubber antioxidants, and polymer synthesis. Current industrial production relies on petroleum-dependent processes involving high-pressure acetone condensation, and to date, there has been no previous report on the biosynthesis of MIBK directly from glucose. In this work, we designed a novel biosynthetic pathway comprising three modules: (i) conversion of isovalerate (IVA) to isovaleryl-CoA by <i>Megasphaera elsdenii</i> CoA transferase (MePCT); (ii) C5+C2 carbon chain elongation via <i>Cupriavidus necator</i> thiolase (CnBktB)-catalyzed reverse β-oxidation; and (iii) decarboxylation to MIBK by <i>Solanum habrochaites</i> methylketone synthase 1 (MKS1). This pathway achieved 103.61 ± 0.52 mg/L MIBK from IVA with 33.1% substrate conversion efficiency. Integration with an endogenous IVA production system enabled <i>de novo</i> synthesis from glucose, yielding 27.31 ± 0.57 mg/L MIBK. To address IVA-induced toxicity and enhance efficiency, we developed a toxicity-driven directed evolution platform and identified the CnBktB mutant M158H, which exhibited a 65.7% reduction in <i>K</i>_m (from 173.4 ± 22.9 to 59.5 ± 11.8 μM) and a 55.7% increase in <i>K</i>_cat (from 1.524 to 2.373 s^-1) toward isovaleryl-CoA. The mutant showed enhanced growth under high IVA stress and further increased the yield of glucose from MIBK synthesis to 55.23 ± 2.01 mg/L. This work establishes a complete microbial platform for MIBK biosynthesis and opens a new approach to green solvent production.