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\begin{document}

\title{Biosynthesis of geranate via isopentenol utilization pathway in
Escherichia coli}



\author[1]{Qiuchi Pan}%
\author[1]{Xiaoqiang Ma}%
\author[1]{Hong Liang}%
\author[1]{Yurou Liu}%
\author[1]{Greg Stephanopoulos}%
\author[1]{Kang Zhou}%
\affil[1]{Singapore-MIT Alliance for Research and Technology Centre}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Isoprenoids are a large family of natural products with diverse
structures, which allow them to play diverse and important roles in the
physiology of plants and animals. They also have important commercial
uses as pharmaceuticals, flavouring agents, fragrances, and nutritional
supplements. Recently, metabolic engineering has been intensively
investigated and emerged as the technology of choice for the production
of isoprenoids through microbial fermentation. Isoprenoid biosynthesis
typically originates in plants from acetyl-coA in central carbon
metabolism, however, a recent study reported an alternative pathway, the
Isopentenol Utilization pathway (IUP), that can provide the building
blocks of isoprenoid biosynthesis from affordable C5 substrates. In this
work, we expressed the IUP in \emph{Escherichia coli} to efficiently
convert isopentenols into geranate, a valuable isoprenoid compound. We
first established a geraniol-producing strain in \emph{E. coli} that
uses the IUP. Then, we extended the geraniol synthesis pathway to
produce geranate through two oxidation reactions catalysed by two
alcohol/aldehyde dehydrogenases from \emph{Castellaniella defragrans}.
The geranate titer was further increased by optimizing the expression of
the two dehydrogenases and also parameters of the fermentation process.
The best strain produced 764 mg/L geranate in 24 h from 2 g/L
isopentenols (a mixture of isoprenol and prenol). We also investigated
if the dehydrogenases could accept other isoprenoid alcohols as
substrates.%
\end{abstract}%



\sloppy


\section*{\texorpdfstring{Biosynthesis of geranate via isopentenol
utilization pathway in \emph{Escherichia
coli}}{Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli}}

{\label{biosynthesis-of-geranate-via-isopentenol-utilization-pathway-in-escherichia-coli}}

Qiuchi Pan\textsuperscript{1,2}, Xiaoqiang Ma\textsuperscript{1,2,3},
Hong Liang\textsuperscript{1,2}, Yurou Liu\textsuperscript{1,2}, Gregory
Stephanopoulos\textsuperscript{1,4,*}, Kang Zhou\textsuperscript{1,2,*}

1. Disruptive \& Sustainable Technologies for Agricultural Precision
(DiSTAP), Singapore-MIT Alliance for Research and Technology, Singapore

2. Department of Chemical and Biomolecular Engineering, National
University of Singapore, Singapore

3. Department of Food Science and Technology, School of Agriculture and
Biology, Shanghai Jiao Tong University, Shanghai, China

4. Department of Chemical Engineering, Massachusetts Institute of
Technology, Cambridge, United States

*Corresponding authors: Kang Zhou (kang.zhou@nus.edu.sg) and Gregory
Stephanopoulos (gregstep@mit.edu)

Key words: isopentenol utilization pathway, isoprenoids, geranate,
pathway engineering, metabolic engineering

\section*{ABSTRACT}

{\label{abstract}}

Isoprenoids are a large family of natural products with diverse
structures, which allow them to play diverse and important roles in the
physiology of plants and animals. They also have important commercial
uses as pharmaceuticals, flavouring agents, fragrances, and nutritional
supplements. Recently, metabolic engineering has been intensively
investigated and emerged as the technology of choice for the production
of isoprenoids through microbial fermentation. Isoprenoid biosynthesis
typically originates in plants from acetyl-coA in central carbon
metabolism, however, a recent study reported an alternative pathway, the
Isopentenol Utilization pathway (IUP), that can provide the building
blocks of isoprenoid biosynthesis from affordable C5 substrates. In this
work, we expressed the IUP in \emph{Escherichia coli} to efficiently
convert isopentenols into geranate, a valuable isoprenoid compound. We
first established a geraniol-producing strain in \emph{E. coli} that
uses the IUP. Then, we extended the geraniol synthesis pathway to
produce geranate through two oxidation reactions catalysed by two
alcohol/aldehyde dehydrogenases from \emph{Castellaniella defragrans} .
The geranate titer was further increased by optimizing the expression of
the two dehydrogenases and also parameters of the fermentation process.
The best strain produced 764 mg/L geranate in 24 h from 2 g/L
isopentenols (a mixture of isoprenol and prenol). We also investigated
if the dehydrogenases could accept other isoprenoid alcohols as
substrates.



\section*{INTRODUCTION}

{\label{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, \emph{Colletotrichum
graminicola} and \emph{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 \emph{Stephanitis
pyrioides} and \emph{Aedes} \emph{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 \emph{C. defragrans} cells during its growth
on\selectlanguage{greek}\emph{β} -\selectlanguage{english}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 \emph{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 \emph{E.
coli}strain 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.



\section*{METHODS}

{\label{methods}}

\emph{\textbf{Chemicals}}

All chemicals were purchased from Sigma-Aldrich unless otherwise
specifically mentioned. Synthetic DNA and oligos were synthesized by
Integrated DNA Technologies.

\emph{\textbf{Plasmids and strains construction}}

All plasmids constructed in the current study were conducted by using
Guanin/Thymine standard (Ma et al., 2019). Constructed plasmids were
verified by Sanger sequencing (service provider: Bio Basic Asia Pacific
Pte Ltd, Singapore). Each of the constructed plasmids was introduced
into \emph{E. coli} \selectlanguage{greek}\textbf{MG1655\_DE3 (\emph{ΔrecA, ΔendA}, DE3)}\selectlanguage{english} via
standard electroporation protocol (Ma et al., 2019). Successfully
constructed strains were isolated on Luria Bertani (LB) agar (BD, DIFCO)
plate containing proper antibiotics. All plasmids and strains used in
this study are summarized in \textbf{Table 1} .

\textbf{Table 1} . Plasmids and strains used in this study.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Strains & Plasmids\tabularnewline
\midrule
\endhead
\textbf{GE01} & pIG\_01
(P\textsubscript{BAD}\_EcthiM\_MvIPK\_Ecidi\_P\textsubscript{T7}\_ObgeS\_Aggpps\_Amp\textsuperscript{R}\_pMB1)\tabularnewline
\textbf{GA01} & pIG\_01\tabularnewline
& pGA\_01
(P\textsubscript{cymO}\_CdGeDH\_CdGaDH\_Spect\textsuperscript{R}\_pAC)\tabularnewline
\textbf{GA02} & pIG\_01\tabularnewline
& pGA\_02
(P\textsubscript{cymO}\_CdGeDH\_CdGaDH\_Spect\textsuperscript{R}\_pSC101)\tabularnewline
\textbf{GA03} & pIG\_01\tabularnewline
& pGA\_03
(P\textsubscript{T7}\_CdGeDH\_CdGaDH\_Spect\textsuperscript{R}\_pSC101)\tabularnewline
\textbf{GA04} & pIG\_01\tabularnewline
& pGA\_04
(P\textsubscript{thrC3}\_CdGeDH\_CdGaDH\_Spect\textsuperscript{R}\_pSC101)\tabularnewline
\textbf{BGA01} & pGA\_01\tabularnewline
\bottomrule
\end{longtable}

\emph{E. coli} \selectlanguage{greek}\textbf{MG1655\emph{\_ΔrecA\_ΔendA\_}DE3}\selectlanguage{english} was used as the
parent strain. Spect\textsuperscript{R}: spectinomycin resistance.
Amp\textsuperscript{R}: ampicillin resistance.

Cell culture and media

Seed cultures were started in 14 mL round-bottom falcon tubes, which
were incubated at 37 \selectlanguage{ngerman}°C/250 rpm overnight. Cell density was measured by
Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) at
the wavelength of 600 nm. The overnight grown seed culture was used to
inoculate (1.5\%, v/v) 25 mL of K3 medium (Zhou et al., 2015) in 250 mL
shake flasks. Subsequent cell culture processes were conducted at 30
°C/250 rpm. The K3 medium composition was as follows (working
concentration): 10 g/L glucose, 10 g/L tryptone, 5 g/L yeast extract,
13.3 g/L KH\textsubscript{2}PO\textsubscript{4}, 4 g/L
(NH\textsubscript{4})\textsubscript{2}HPO\textsubscript{4}, 0.0084 g/L
EDTA, 0.0025 g/L CoCl\textsubscript{2}, 0.015 g/L MnCl\textsubscript{2},
0.0015 g/L CuCl\textsubscript{2}, 0.003 g/L
H\textsubscript{3}BO\textsubscript{3}, 0.0025 g/L
Na\textsubscript{2}MoO\textsubscript{4}, 0.008 g/L Zn
(CH\textsubscript{3}COO)\textsubscript{2}, 0.06 g/L Fe(III) citrate and
1.3 g/L MgSO\textsubscript{4}. Antibiotics (100 \selectlanguage{greek}µ\selectlanguage{english}g/mL ampicillin and/or
50 \selectlanguage{greek}µ\selectlanguage{english}g/mL spectinomycin) were added according to \textbf{Table 1} , and
pH was adjusted to 7 using 400 g/L sodium hydroxide solution. Inducers
were added when cell density (OD\textsubscript{600}) reached
\textasciitilde{}0.5 to trigger expression. 0.01 mM IPTG, 4 g/L
L-arabinose and 0.03 mM cumic acid were added at this stage when gene
expression was controlled by P\textsubscript{T7},
P\textsubscript{BAD}and P\textsubscript{cymO}, respectively. Cells were
collected 6 hours after induction.

The grown cells were centrifuged at 4,000 g for 8 min. Cell pellets were
resuspended in 5 mL of fresh K3 medium to a final OD of 20. In the fresh
K3 medium, 20 g/L L-arabinose, 10 g/L tryptone and 5 g/L yeast extract
were used as carbon sources. 0.05 mM IPTG or 0.03 mM cumic acid was used
in the second gene expression stage using P\textsubscript{T7} or
P\textsubscript{cymO}. One millilitre of re-suspended cells was
transferred to a 14 mL round-bottom falcon tube. A total of 2 g/L
isoprenol and prenol at a molar ratio of 3:1 was supplemented as
substrate. A 15\% v/v hexadecane layer was used under same conditions
(as specified in \textbf{Results and Discussion} ). The culture was
incubated at 30 \selectlanguage{ngerman}°C/250 rpm for 24 h. For the biotransformation of nerol,
perillyl alcohol and farnesol, 2 g/L of nerol, perillyl alcohol and
farnesol were added instead of isoprenol and prenol.

\emph{\textbf{Extraction and quantification of geraniol and geranate in
organic phase}}

Two hundred millilitres of cell culture (including organic layer) were
collected and transferred to a 1.7 mL centrifuge tube (Axygen) followed
by centrifugation at 10,000 g for 10 min. Two microliters of the
hexadecane phase was taken and diluted with 98 \selectlanguage{greek}µ\selectlanguage{english}L ethyl acetate (EA).
One microliter of diluted sample was analyzed by gas chromatography-mass
spectrometry (GC/MS, 5977B, Agilent Technologies) to quantify geraniol
and geranate in the hexadecane phase. HP-5MS capillary column (30 m \selectlanguage{ngerman}×
0.25 mm, 0.25 \selectlanguage{greek}µ\selectlanguage{english}m film thickness, Agilent Technologies) was used, with
helium as the carrier gas. The following oven temperature program was
carried out: 50 \selectlanguage{ngerman}°C for 1 min, 50--100 °C at a rate of 5 °C/min, 100 °C
for 1 min, 100--200 °C at a rate of 50 °C/min, 200 °C for 1 min,
200--300 °C at a rate of 50 °C/min and 300 °C for 1 min. The reported
geraniol and geranate titer is the sum of the products in the hexadecane
phase and aqueous phase when two phases were used.

Extraction and quantification of isopentenols and geraniol in aqueous
phase Two hundred millilitres of cell culture (including organic layer)
were collected and transferred to a 1.7 mL centrifuge tube (Axygen)
followed by centrifugation at 10,000 g for 10 min. One hundred
microliters of supernatant was mixed with one hundred microliters of EA
and the mixture was vortexed for 30 min. The solution was then
centrifuged at 10,000 g for 10 min. The EA phase (1 \selectlanguage{greek}μ\selectlanguage{english}L) was analyzed by
GC/MS to quantify geraniol and isopentenols in the aqueous phase. The
same GC/MS method was used as described above.

Extraction and quantification of geranate, 3-methyl-crotonate and
3-methyl-3-butanoate in aqueous phase

One hundred microliters of cell culture were collected and centrifuged
at 10,000 g for 10 min. Four hundred microliters of methanol was mixed
with the obtained supernatant and vortexed for 1 h. The mixture was then
centrifuged at 10,000 g for 10 min to remove the precipitated enzymes
and the supernatant was then analysed. An Ultra Performance Liquid
Chromatography (UPLC, Waters ACQUITY) linked with a Time-of-flight Mass
Spectrometry (TOFMS, Bruker micrOTOF II) was used to quantify geranate,
3-methyl-crotonate (3-MC) and 3-methyl-3-butanoate (3-MB). The column
used was Poroshell 120 EC-C18 column (2.1 \selectlanguage{ngerman}× 50 mm, 2.7 \selectlanguage{greek}μ\selectlanguage{english}m, Agilent
Technologies). Two mobile phases were used: A) 0.1 \% trifluoroacetic
acid in water; B) acetonitrile. The gradient was as follows: 0--2 min,
95\% A; 2-2.5 min, 95\%-90\% A; 2.5-3.5 min, 90\% A, 3.5-5.5 min,
90\%-60\% A, 5.5-9 min, 60\% A; 9-10 min, 60\%-95\% A; 10-11 min, 95\%
A. The flow rate was 0.25 mL/min. Electrospray ionization was used and
the mass spectrometry was operated to scan 50--800 m/z in positive mode
with 2,500 V end plate voltage and 3,800 V capillary voltage. Nebulizer
gas was provided in 1 bar, drying gas temperature was 9 mL/min, and dry
gas temperature was 200 \selectlanguage{ngerman}°C. Sample injection volume was 10 \selectlanguage{greek}μ\selectlanguage{english}L.

\section*{RESULTS AND DISCUSSION}

{\label{results-and-discussion}}

\subsection*{\texorpdfstring{Engineering \emph{E. coli} to produce
geraniol from
isopentenols}{Engineering E. coli to produce geraniol from isopentenols}}

{\label{engineering-e.-coli-to-produce-geraniol-from-isopentenols}}

We first engineered the IUP in \emph{E. coli} strains for the
heterologous production of geraniol (\textbf{Figure 1} ). In prior work
we had found that using \emph{E. coli} hydroxyethylthiazole kinase
(EcthiM) (Clomburg et al., 2019) and \emph{Methanococcus
vannielii}isopentenyl 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 \emph{EcthiM} and
\emph{MvIPK}in this study too for the biosynthesis of IPP and DMAPP as
precursors for geraniol synthesis. We also overexpressed \emph{E. coli}
\emph{idi}to better balance the ratio of DMAPP to IPP, and geranyl
diphosphate synthase (Aggpps) from \emph{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
\emph{Ocimum basilicum} was overexpressed for converting the
intermediate GPP into geraniol (Iijima et al., 2004). Genes involved in
IUP (\emph{EcthiM, MvIPK} and \emph{Ecidi} ) were co-expressed in one
operon under the control of an inducible promoter
(P\textsubscript{BAD}). Downstream genes for geraniol production
(\emph{Aggpps} and \emph{Obges} ) were co-expressed in another operon
driven by another inducible promoter (P\textsubscript{T7}). These two
operons were placed in one plasmid (\textbf{Figure 2a} ). The resulting
geraniol-producing strain was named\textbf{GE01} . Strain and plasmid
information are included in

\textbf{Table 1.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} The metabolic pathway to synthesize geraniol and
geranate from isoprenol/prenol. The host organism used in this study
was\emph{E. coli} . IP: Isopentenyl monophosphate; IPP: Isopentenyl
diphosphate; DMAP: Dimethylallyl monophosphate; DMAPP: Isomer
dimethylallyl diphosphate; GPP: Geranyl diphosphate; 3-MC:
3-Methyl-crotonate; 3-MB: 3-Methyl-3-butanoate. EcthiM: \emph{E.
coli}hydroxyethylthiazole kinase; MvIPK: \emph{Methanococcus
vannielii}isopentenyl phosphate kinase; Ecidi: \emph{E. coli} IPP
isomerase; Aggpps: \emph{Abies grandis} geranyl diphosphate synthase;
Obges:\emph{Ocimum basilicum} geraniol synthase; CdGeDH:
\emph{Castellaniella defragrans} geraniol dehydrogenase; CdGaDH:
\emph{Castellaniella defragrans} geranial dehydrogenase.

We adopted a two-stage fermentation protocol for isoprenoid production
(Ma et al., 2022). In brief, the first stage is for cell growth and gene
expression. The engineered strains were cultured in a medium containing
glucose, tryptone and yeast extract with the goal to accumulate cell
biomass. Six hours after induction, cells were collected for use in the
second stage fermentation. In this stage, cells were re-suspended in
fresh media (OD\textsubscript{600} = 20) containing 2 g/L isopentenols
(1.5 g/L isoprenol and 0.5 g/L prenol). 15\% (v/v) of hexadecane was
added as organic overlay to continuously extract the produced geraniol.
The reported geraniol titer is the sum of the geraniol in the aqueous
and organic phases. \textbf{GE01} consumed 1.5 g/L isopentenols
(\textbf{Figure 2e} ) and produced 750 mg/L geraniol within 24 h in the
second stage (\textbf{Figure 2c} ). The geraniol-producing strain and
two-stage fermentation protocol were used in the subsequent experiments.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} \textbf{a)} Illustration of the plasmid used in
geraniol production in \textbf{GE01} (\emph{pIG\_01} ).
\textbf{b)}Illustration of the plasmid used in converting geraniol into
geranate in\textbf{GA01} (\emph{pGA\_01} ). Production of \textbf{c)}
geraniol with\textbf{GE01} and \textbf{d)} geranate with \textbf{GA01}
with and without hexadecane within 24 h. No geranate was detected in the
culture of \textbf{GE01} and no geraniol was detected in the culture
of\textbf{GA01. e)} Isopentenol consumption by \textbf{GE01}
or\textbf{GA01} within 24 h with hexadecane. \textbf{f)} Formation of
by-products (3-MC and 3-MB) by \textbf{GE01} or \textbf{GA01} within 24
h. n.d., not detected. \textbf{g)} Extracted ion chromatogram (EIC) of
3-MC (m/z=101.06) and geranic acid (m/z=169.12) standards. 3-MC was
eluted at 4.8 min and geranic acid was eluted at 9.2 min. \textbf{h} )
mass spectra of 3-MC and geranic acid. Error bars indicate standard
error (n=3).

\subsection*{\texorpdfstring{Pathway construction for conversion of
geraniol into geranate in \emph{E.
coli}}{Pathway construction for conversion of geraniol into geranate in E. coli}}

{\label{pathway-construction-for-conversion-of-geraniol-into-geranate-in-e.-coli}}

To convert geraniol into geranate, we co-expressed \emph{CdGeDH}
and\emph{CdGaDH} from \emph{C. defragrans} in one operon in a separate
plasmid (\emph{pGA01} ) under the control of a strong inducible promoter
P\textsubscript{cymO} (\textbf{Figure 2b} ). We named the geranate
production strain \textbf{GA01} . After the fermentation, Liquid
Chromatography Mass Spectrometry (LC/MS) was used to quantify geranate
in the aqueous phase (\textbf{Figure 2g, h} ); Gas Chromatography Mass
Spectrometry (GC/MS) was used to quantify geranic acid in the organic
overlay. The fermentation produced 298 mg/L geranate with
\textbf{GA01}(\textbf{Figure 2d} ). Geraniol and geranial were not
detected in the culture of \textbf{GA01} , indicating that CdGeDH and
CdGaDH have high catalytic efficiency with geraniol and geranial,
respectively.

While geraniol was partitioned more favourably into the organic phase of
the \textbf{GE01} culture (\textbf{Figure 2c} ), most of the geranate
accumulated in the aqueous phase of the \textbf{GA01} culture
(\textbf{Figure 2d} ). This was expected because geraniol (LogP = 3.56)
(Griffin et al., 1999) is more hydrophobic than geranate (LogP = 2.8)
(Ko et al., 2021). Hexadecane is frequently used to prevent loss of
volatile products (such as geraniol). Using hexadecane, however,
complicates downstream product purification (it is challenging to
separate geraniol from hexadecane). When we removed the hexadecane from
the \textbf{GE01} culture, the geraniol titer was reduced to 8\% of the
control (with hexadecane, \textbf{Figure 2c} ). We, however, found that
a similar geranate titer (268 mg/L) was achieved with or without
hexadecane (\textbf{Figure 2d} ). Hexadecane was not used in the rest of
this study to simplify the fermentation process.

Next, we determined the consumption of isopentenols in the cultures, and
found that \textbf{GA01} consumed more isopentenols than
\textbf{GE01}(\textbf{Figure 2e} ). Since the titer of geranate in the
\textbf{GA01}culture was much lower than that of geraniol in the
\textbf{GE01}culture (\textbf{Figure 2c,d} ), this was an indication
that by-products were formed along with geranate by the
geranate-producing \textbf{GA01} . We hypothesized that the hydroxyl
group of isoprenol and prenol may also be oxidized by CdGeDH and CdGaDH
to form corresponding carboxylates (3-methyl-crotonate {[}3-MC{]} and
3-methyl-3-butanoate {[}3-MB{]},\textbf{Figure 1} ), as these oxidation
reactions would compete with the downstream reaction of geranate
biosynthesis. We confirmed this hypothesis by analysing the aqueous
phase of \textbf{GE01} and\textbf{GA01} cultures with LC/MS
(\textbf{Figure 2f} ). As shown in\textbf{Figure 2f} , 773 mg/L of
by-products (3-MC and 3-MB) were produced by \textbf{GA01} while no
by-products were found in the culture of \textbf{GE01} (\textbf{Figure
2f} ).

\subsection*{Tuning expression of CdGeDH and CdGaDH to reduce by-product
formation}

{\label{tuning-expression-of-cdgedh-and-cdgadh-to-reduce-by-product-formation}}

It is possible that the expression level of CdGeDH and CdGaDH was too
high in \textbf{GA01} , rapidly oxidizing a large amount of isopentenols
into the by-products and competing with EcthiM (the first kinase in IUP)
for substrate. This suggests a reduction in the expression level of
CdGeDH and CdGaDH so that more isopentenols could enter the IUP for
production of geranate. To test this hypothesis, we constructed a new
plasmid (\emph{pGA02} ) with a low copy number replication origin
(pSC101; copy number: \textasciitilde{}5) to weaken the expression of
CdGeDH and CdGaDH (\emph{pGA01} used pAC replication origin; copy
number: 10-15). We replaced \emph{pGA01} with \emph{pGA02}
in\textbf{GA01} , creating a new strain (\textbf{GA02} ). The geranate
produced by \textbf{GA02} (764 mg/L) was almost three times that
produced by \textbf{GA01} (\textbf{Figure 3a} ), and the concentration
of the by-products (3-MC and 3-MB) was substantially reduced (from 865
mg/L to 436 mg/L) (\textbf{Figure 3b} ). Then we replaced the
P\textsubscript{cymO} promoter in \textbf{GA02} with
P\textsubscript{T7}or P\textsubscript{thrC3} (Anilionyte et al., 2018),
creating \emph{E. coli} \textbf{GA03} and \textbf{GA04} , respectively.
These two strains produced a similar amount of geranate as \textbf{GA02}
(\textbf{Figure 3a} ), but with significantly reduced amount of
by-products (68 mg/L and 190 mg/L 3-MC/3-MB by \textbf{GA03} and
\textbf{GA04} , respectively,\textbf{Figure 3b} ). A low IPTG
concentration (0.05 mM) was used in this study, so P\textsubscript{T7}
could be weaker than P\textsubscript{thrC3} and P\textsubscript{cymO}.

A previous study successfully engineered \emph{Acinetobacter sp. Tol
5}to transform geraniol into geranate (Usami et al., 2020). Their
geranate titer was similar to what we achieved in this study. Our
process, however, used a cheaper substrate (isopentenols instead of
geraniol) and was much faster (24 h instead of 144 h).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Controlled expression of the geraniol dehydrogenase
and geranial dehydrogenase to reduce by-product formation.
\textbf{a)}Geranate titer of \textbf{GA01} , \textbf{GA02} ,
\textbf{GA03} and\textbf{GA04} (from left to right) at 24 h (2 g/L
isopentenols).\textbf{b)} 3-MC/3-MB concentration of the
geranate-producing strains at 24 h. Error bars indicate standard error
(n=3).

\subsection*{Testing CdGeDH and CdGaDH with other isoprenoid
alcohols}

{\label{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, \textbf{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 (\textbf{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
(\textbf{BGA01} ) for the conversion of nerol, perillyl alcohol and
farnesol. We cultured \textbf{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 \textbf{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 (\textbf{Figure 4b} ). As a control, the
parent strain of BGA01 did not form any of these
products.\emph{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 (\emph{cymB} and \emph{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 \textbf{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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} . \textbf{a)} Oxidation of perillyl alcohol, nerol and
farnesol to aldehyde and carboxylate forms. \textbf{b)} GC/MS extracted
ion chromatograms (EIC) of aqueous phase sample of biotransformation of
(i) perillyl alcohol, (ii) nerol and (iii) farnesol with
\textbf{BGA01}(lower row) and its parent strain (upper row).
\textbf{BGA01} harbored the plasmid overexpressing CdGeDH and CdGaDH
under the control of P\textsubscript{T7}. Peaks numbered on each EIC are
identified as 1, perillyl alcohol; 2, perillic acid; 3, nerol; 4,
nerolic acid; 5, geranic acid; 6 \& 7, farnesol; 8, farnesal.

\section*{CONCLUSION}

{\label{conclusion}}

In this study, we successfully constructed geranate-producing \emph{E.
coli} strains via IUP. We applied a two-stage fermentation protocol to
increase the product titer. We found a competing pathway for geranate
production, and optimized the expression level of the oxidoreductases to
reduce by-product formation. The best strains produced 764 mg/L of
geranate from 2 g/L isopentenols within 24 h, among the highest geranate
titers reported in the literature. To our knowledge, this is the first
study demonstrating the engineering of \emph{E. coli} as microbial cell
factory for \emph{de novo} production of geranate using simple and cheap
isopentenol substrates. The optimization approaches followed in this
study may be applied to similarly reduce by-product formation in the
production of other value-added isoprenoid compounds. We suggest that
follow up studies improve the substrate specificity of CdGeDH and CdGaDH
via protein engineering, and thus provide a basis host cell for further
increasing the geranate titer through genetic and process engineering. A
potential challenge might be the toxicity of geranate to \emph{E.
coli}at higher concentrations. This issue could be addressed by using a
more robust host (e.g., \emph{Pseudomonas} species), evolving the
engineered\emph{E. coli} in the presence of geranate, and/or
sequestering geranate on charged resin or in a solvent.

\subsection*{ACKNOWLEDGMENTS}

{\label{acknowledgments}}

This work was financially supported by National Research Foundation
Singapore through a Singapore-MIT Alliance for Research and Technology
project (funding identifier: R-279-000-587-592) and an Intra-CREATE
project (funding identifier: A-0001440-00-00).

\subsection*{AUTHOR CONTRIBUTIONS}

{\label{author-contributions}}

Qiuchi Pan and Kang Zhou conceived and designed the research. Xiaoqiang
Ma contributed key materials and analysis tools. Qiuchi Pan, Xiaoqiang
Ma, Yurou Liu, Hong Liang performed the analysis. Qiuchi Pan wrote the
original draft of the manuscript. Qiuchi Pan, Xiaoqiang Ma, Kang Zhou
and Gregory Stephanopoulos edited the manuscript.

\subsection*{CONFLICT OF INTERESTS}

{\label{conflict-of-interests}}

The authors declare that there are no conflict of interests.

\subsection*{DATA AVAILABILITY
STATEMENT}

{\label{data-availability-statement}}

All of the data used to support the claims of this study have been
presented in the form of figures and/or tables, which are available in
this manuscript.

\par\null

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