Optimization of the Isopentenol Utilization Pathway for Isoprenoid Synthesis in Escherichia coli .

Engineering microbes to produce isoprenoids can be limited by the competition between product formation and cell growth because biomass and isoprenoids are naturally derived from central metabolism. Recently, a two-step synthetic pathway was developed to partially decouple isoprenoid formation from central carbon metabolism. The pathway used exogenously added isopentenols as substrates. In the present study, we systematically optimized this isopentenol utilization pathway in Escherichia coli by comparing enzyme variants from different species, tuning enzyme expression levels, and using a two-stage process. Under the optimal conditions found in this study, ∼300 mg/L lycopene was synthesized from 2 g/L isopentenol in 24 h. The strain could be easily modified to synthesize two other isoprenoid molecules efficiently (248 mg/L β-carotene or 364 mg/L R-(-)-linalool produced from 2 g/L isopentenol). This study lays a solid foundation for producing agri-food isoprenoids at high titer/productivity from cost-effective feedstocks.