Figure 1. The metabolic pathway to synthesize geraniol and geranate from isoprenol/prenol. The host organism used in this study wasE. coli . IP: Isopentenyl monophosphate; IPP: Isopentenyl diphosphate; DMAP: Dimethylallyl monophosphate; DMAPP: Isomer dimethylallyl diphosphate; GPP: Geranyl diphosphate; 3-MC: 3-Methyl-crotonate; 3-MB: 3-Methyl-3-butanoate. EcthiM: E. colihydroxyethylthiazole kinase; MvIPK: Methanococcus vannieliiisopentenyl phosphate kinase; Ecidi: E. coli IPP isomerase; Aggpps: Abies grandis geranyl diphosphate synthase; Obges:Ocimum basilicum geraniol synthase; CdGeDH: Castellaniella defragrans geraniol dehydrogenase; CdGaDH: Castellaniella defragrans geranial dehydrogenase.
We adopted a two-stage fermentation protocol for isoprenoid production (Ma et al., 2022). In brief, the first stage is for cell growth and gene expression. The engineered strains were cultured in a medium containing glucose, tryptone and yeast extract with the goal to accumulate cell biomass. Six hours after induction, cells were collected for use in the second stage fermentation. In this stage, cells were re-suspended in fresh media (OD600 = 20) containing 2 g/L isopentenols (1.5 g/L isoprenol and 0.5 g/L prenol). 15% (v/v) of hexadecane was added as organic overlay to continuously extract the produced geraniol. The reported geraniol titer is the sum of the geraniol in the aqueous and organic phases. GE01 consumed 1.5 g/L isopentenols (Figure 2e ) and produced 750 mg/L geraniol within 24 h in the second stage (Figure 2c ). The geraniol-producing strain and two-stage fermentation protocol were used in the subsequent experiments.