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