RESULTS AND DISCUSSION
Engineering E. coli to produce
geraniol from
isopentenols
We first engineered the IUP in E. coli strains for the
heterologous production of geraniol (Figure 1 ). In prior work
we had found that using E. coli hydroxyethylthiazole kinase
(EcthiM) (Clomburg et al., 2019) and Methanococcus vannieliiisopentenyl phosphate kinase (MvIPK) (Couillaud et al., 2019) yielded
higher lycopene titers relatively to other combinations of IUP kinases
tested (Ma et al., 2022). Thus, we used EcthiM and MvIPKin this study too for the biosynthesis of IPP and DMAPP as precursors
for geraniol synthesis. We also overexpressed E. coli idito better balance the ratio of DMAPP to IPP, and geranyl diphosphate
synthase (Aggpps) from Abies grandis to catalyse the condensation
of DMAPP and IPP into GPP (Burke & Croteau, 2002). The gene of a
truncated geraniol synthase (Obges) from sweet basil Ocimum
basilicum was overexpressed for converting the intermediate GPP into
geraniol (Iijima et al., 2004). Genes involved in IUP (EcthiM,
MvIPK and Ecidi ) were co-expressed in one operon under the
control of an inducible promoter (PBAD). Downstream
genes for geraniol production (Aggpps and Obges ) were
co-expressed in another operon driven by another inducible promoter
(PT7). These two operons were placed in one plasmid
(Figure 2a ). The resulting geraniol-producing strain was namedGE01 . Strain and plasmid information are included in