INTRODUCTION

The isoprenoid family comprises more than 65,000 compounds (Berthelot et al., 2012) that have found many useful applications in the manufacturing of drugs, fragrances, food additives, colorants, and rubber and advanced biofuels (Gershenzon & Dudareva, 2007). Currently, these compounds are produced for commercial use by extraction from plants (Daletos et al., 2020). In order to satisfy increasing market demand and reduce production cost, microbial production of isoprenoids is investigated (Chang & Keasling, 2006). Metabolic engineering has enabled the construction of strains with attractive properties for isoprenoid production in microbial hosts (Li et al., 2020; Schempp et al., 2018).
Nearly all isoprenoids are synthesized from two precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are biosynthesized in nature by two distinct metabolic pathways, the methylerythritol phosphate (MEP) pathway, which is present in most bacteria and plastids of plant cells, and the mevalonate (MVA) pathway, which functions in most eukaryotes, archaea, and certain bacteria. IPP and DMAPP are subsequently condensed to generate geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP). These linear diphosphate intermediates can be further functionalized into various structures and the diversity of these reactions is responsible for the synthesis of many diverse isoprenoid compounds (Daletos et al., 2020). The IPP and DMAPP precursors of the two native pathways are derived from central metabolism, requiring many reaction steps for the conversion of a typical carbon substrate like glucose to an isoprenoid molecule. These pathways also compete with cell biosynthesis for building blocks and, as such, are subject to native host regulation.
To overcome the above problems, a novel pathway (IUP) was recently proposed whereby the two key precursors, IPP and DMAPP, are synthesized from isopentenols by only two consecutive phosphorylation steps (Chatzivasileiou et al., 2019). IUP comprises only two enzymes that sequentially phosphorylate isoprenol (or prenol) into isopentenyl phosphate (or dimethylallyl phosphate), and then into IPP (or DMAPP). DMAPP and IPP are isomerized into each other by IPP isomerase (IDI) to balance their ratio. Compared with the MVA and MEP pathways, IUP does not require any building blocks from central metabolism and is less energetically demanding (Liu et al., 2020; Luo et al., 2020; Ward et al., 2019).
Geranate is a valuable C10 isoprenoid compound with broad industrial applications. Geranate can be used as a perfuming agent in cosmetics (Mi et al., 2014) and has also been identified as a superior antifungal agent against two main phytopathogens of corn, Colletotrichum graminicola and Fusarium graminearum . As such, it was recently produced in a transgenic maize plant to control fungal disease outbreak (Yang et al., 2011). Geranate also has potential as insecticide since it possesses excellent insecticidal activity against Stephanitis pyrioides and Aedes aegypti as well as high biting deterrent activity (Ali et al., 2013). Moreover, geranate is known to be a tyrosinase inhibitor and inhibits melanin synthesis (Wang & Hebert, 2006) in applications of skin depigmentation. However, the production of geranate by engineered microbes has not been systematically pursued.
Geranate could be obtained from the oxidation of geraniol, a commercially important fragrance molecule (Chen & Viljoen, 2010). Geraniol in turn can be synthesized from GPP and has been produced from isopentenols and other simple substrates through microbial fermentation. Geraniol is formed in C. defragrans cells during its growth onβ -myrcene via hydration and isomerization (Brodkorb et al., 2010). Geranate is also identified as an intermediate in this culture, and the relevant proteins (geraniol dehydrogenase [CdGeDH] and geranial dehydrogenase [CdGaDH]) in C. defragrans were purified and sequenced (Luddeke et al., 2012).
In this work, we first established a geraniol production pathway which produces 750 mg/L geraniol from 2 g/L isopentenols in 24 h. Then we extended the geraniol production pathway to produce geranate through two oxidation reactions catalysed by CdGeDH and CdGaDH. After optimizing the expression level of CdGeDH and CdGaDH, the engineered E. colistrain could produce up to 764 mg/L geranate from 2 g/L isopentenols within 24 h. We also confirmed that producing geranate did not require an organic overlay because of its high water-solubility. The use of organic overlays complicates production and downstream processing and increases the product purification cost. As such, processes that do not require such organic overlays for the isoprenoid production are advantageous.
Additionally, we found that CdGeDH and CdGaDH can oxidize various C5 to C15 isoprenoid alcohols. The geranate-producing strain developed in this study provides a promising basal strain for developing manufacturing processes for the production of geranate and its derivatives.