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.