Figure 3. Controlled expression of the geraniol dehydrogenase and geranial dehydrogenase to reduce by-product formation. a)Geranate titer of GA01 , GA02 , GA03 andGA04 (from left to right) at 24 h (2 g/L isopentenols).b) 3-MC/3-MB concentration of the geranate-producing strains at 24 h. Error bars indicate standard error (n=3).

Testing CdGeDH and CdGaDH with other isoprenoid alcohols

We further examined whether CdGeDH and CdGaDH could oxidize other isoprenoid alcohols into corresponding aldehydes and acids. We selected three natural isoprenoid alcohols, nerol (C10), perillyl alcohol (C10) and farnesol (C15, Figure 4a ) with the expectation that the hydroxyl groups of those compounds would also be oxidized by CdGeDH and CdGaDH into aldehyde or carboxylate group (Figure 4a ). In this case, nerol, perillyl alcohol and farnesol would be oxidized into neral, perillyl aldehyde and farnesal respectively and then would be further oxidized into nerolic acid, perillic acid and farnesoic acid. Nerolic acid is identified as a nassanoff pheromone of the honey bee (Boch & Shearer, 1964). Perillic acid is an antineoplastic agent (Crowell et al., 1992). Farnesoic acid is a key precursor for the biosynthesis of C16 juvenile hormone which plays key roles in both metamorphosis and adult reproductive processes (Liu et al., 2010).
We designed a new strain without the geraniol-producing plasmid (BGA01 ) for the conversion of nerol, perillyl alcohol and farnesol. We cultured BGA01 with 2 g/L of nerol, perillyl alcohol or farnesol with the addition of hexadecane overlay (to reduce alcohol evaporation). Then we monitored substrate consumption and product formation in both aqueous and hexadecane phases with GC/MS. When perillyl alcohol or nerol were supplemented, all added amounts were consumed by BGA01 within 24 h. We could observe clear peaks for their corresponding carboxylic acids (perillic acid and nerolic acid) in both phases by using GC/MS (Figure 4b ). As a control, the parent strain of BGA01 did not form any of these products.Pseudomonas putida DSM 12264 was used to transform limonene into perillic acid via perillyl alcohol (Mirata et al., 2009). The genes responsible for oxidizing perillyl alcohol into perillic acid have been identified (cymB and cymC , encoding cumic alcohol and aldehyde dehydrogenases respectively). It would be useful to compare CymBC and CdGe/GaDH in a future study for improving geranate and/or perillic acid production. To our knowledge, there are no dehydrogenases that can convert nerol into nerolic acid with high product specificity. Therefore, Ge/GaDH should be useful in microbial production of nerolic acid from nerol or simpler building blocks.
We found that farnesol (C15) could only be partially oxidized into its aldehyde, farnesal, by BGA01 . Farnesoic acid was not detected in the reaction mixture and a large amount of farnesol was not utilized. These data provide useful information on the substrate scope of CdGeDH and CdGaDH. Several plant and insect farnesol dehydrogenase and farnesal dehydrogenase have been characterized during the past decade (Bhandari et al., 2010; Mayoral et al., 2009; Satyaveanthan et al., 2021; Seman-Kamarulzaman et al., 2016; Zifruddin et al., 2021). These enzymes could be employed to produce farnesoic acid from farnesol or isopentenols following the workflow established in our study.