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.