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\begin{document}

\title{Biotechnological production of the European corn borer sex pheromone in
the yeast Yarrowia lipolytica}



\author[1]{Karolis Petkevicius}%
\author[2]{Eleni Koutsoumpeli}%
\author[2]{Petri Betsi}%
\author[3]{Bao-Jian Ding}%
\author[4]{Kanchana Rueksomtawin Kildegaard}%
\author[4]{Hilbert Jensen}%
\author[4]{Nora Mezo}%
\author[4]{Andrea Mazziotta}%
\author[4]{Anders Gabrielsson}%
\author[4]{Christina Sinkwitz}%
\author[4]{Bettina Lorantfy}%
\author[4]{Carina Holkenbrink}%
\author[3]{Christer Löfstedt}%
\author[5]{Dimitris Raptopoulos}%
\author[2]{Maria Konstantopoulou}%
\author[1]{Irina Borodina}%
\affil[1]{Technical University of Denmark}%
\affil[2]{National Centre for Scientific Research-Demokritos}%
\affil[3]{Lund University}%
\affil[4]{BioPhero ApS}%
\affil[5]{Novagrica Hellas S.A}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of
cereals. Mating disruption with the sex pheromone is a potentially
attractive method for managing this pest. The goal of this study was to
develop a biotechnological method for the production of ECB sex
pheromone. Our approach was to engineer the oleaginous yeast Yarrowia
lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can be
chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the
main pheromone component of the Z-race of O. nubilalis. Fatty acyl-CoA
desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine
different species of Lepidoptera were screened individually and in
combinations. A titer of 29.2\selectlanguage{ngerman}±1.6 mg/L Z11-14:OH was reached in
small-scale cultivation with an optimal combination of a FAD (Lbo\_PPTQ)
from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When
the second copies of FAD and FAR genes were introduced, the titer
improved 2.1-fold. The native FAS1 gene's overexpression led to a
further 1.5-fold titer increase. When the same engineered strain was
cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1±13.4
mg/L of Z11-14:OH was obtained. Fatty alcohols were chemically
acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed
that males of the Z-race of O. nubilalis were responsive to
biologically-derived pheromone blend. Behavioral bioassays in a wind
tunnel revealed attraction of male O. nubilalis at a level similar to
that of the chemically synthesized pheromone used as a control, although
full precopulatory behavior was observed less often. The study paves the
way for the production of ECB pheromone by fermentation.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{1 Introduction}

Crop damage caused by insects is a severe problem in agriculture. Moths
(Lepidoptera) are major insect pests {[}1-3{]}. The European corn borer
(ECB) \emph{Ostrinia nubilalis} is the main pest of maize \emph{Zea
mays} in Europe {[}4{]}. It is estimated that in the absence of
treatment, up to 20\% of the crop may be lost due to the damage
by\emph{O. nubilalis} larvae {[}5{]}. Like other moth species, females
of\emph{O. nubilalis} produce and release a fatty acid-derived sex
pheromone, which attracts conspecific males for mating {[}6{]}. ECB is
polymorphic with respect to its pheromone communication system and two
pheromone races are recognized: the Z-race insects use a 97:3 blend of
(\emph{Z} )-11-tetradecenyl acetate (\emph{Z} 11-14:OAc) to (\emph{E}
)-11-tetradecenyl acetate (\emph{E} 11-14:OAc), while E-race insects use
a 1:99 blend of the same components {[}6,7{]}. On maize in Europe, the
Z-race is most prevalent {[}8{]}.

Mating disruption has been proven an effective and environmentally
friendly solution for crop protection against moths. For mating
disruption, a more or less species-specific pheromone blend is applied
to the fields or orchards to disrupt mating partner detection and, in
this way, decrease the propagation of the pest species {[}9,10{]}.
Currently, pheromones for pest management are produced synthetically
from petrol-derived chemicals {[}11-13{]}. Chemical synthesis typically
comprises multiple steps, uses toxic chemicals and solvents, and
expensive catalysts. Biotechnological production of several insect
pheromone components has already been established in plants and yeasts
{[}14-18{]}. Oleaginous yeast \emph{Yarrowia lipolytica} is particularly
suitable for production of lepidopteran pheromones due to its naturally
high level of fatty acid biosynthesis. Previously, we produced the
precursor of the major pheromone component of \emph{Helicoverpa
armigera} (\emph{Z} 11-16:OH) at 2.5 g/L in \emph{Y. lipolytica} and
proved the effectiveness of yeast-derived insect pheromone in field
trapping experiments {[}16{]}.

In this study, we aimed to produce \emph{Z} 11-14:OAc, the major
pheromone component of the ECB Z-race, using engineered \emph{Y.
lipolytica} as a host and demonstrate the activity of yeast-derived
pheromone on \emph{O. nubilalis} males.

\textbf{2 Materials and methods}

\textbf{2.1 Plasmid construction}

Plasmids were constructed according to Holkenbrink et al., 2018
{[}19{]}. Integration and guide RNA (gRNA) vectors were used to
introduce gene expression constructs into characterized genome sites
of\emph{Y. lipolytica} {[}19{]}.

Primers, synthetic DNA, BioBricks, and plasmids used in this study are
listed in Supplementary Tables S1, S2, S3, and S4, respectively.
BioBricks were amplified by PCR using Phusion U polymerase (Thermo
Fisher Scientific) with the following thermal program: 98\selectlanguage{ngerman}°C for 5 min,
30 cycles of (98°C for 20 s, 54°C for 30 s, 72°C for 30 s/kb), and 72°C
for 4 min. After DNA electrophoresis on 1\% agarose gel, BioBricks were
purified using NucleoSpin\textsuperscript{®} Gel and PCR Clean-up kit
(Macherey-Nagel). The integration vectors were digested with FastDigest
SfaAl (Thermo Fisher Scientific) and nicked with Nb.BsmI (New England
BioLabs). Biobricks with compatible overhangs and nicked integration
vectors were assembled and transformed into \emph{Escherichia
coli}strain DH5\selectlanguage{greek}α \selectlanguage{english}via Uracil-Specific Excision Reaction
(USER\selectlanguage{ngerman}\textsuperscript{®}) cloning. Clones containing the correct
assembly were verified by PCR and Sanger sequencing.

\textbf{2.2 Strain construction}

All strains constructed in this study are derived from the \emph{Y.
lipolytica} strain ST6629, described in Holkenbrink et al. {[}16{]}.
This strain has modifications related to decreased degradation of fatty
acids/alcohols and an increased pool of fatty acyl-CoAs. \emph{Y.
lipolytica} strain ST4840, which was obtained from Agricultural Research
Service (NRRL, USA), served as a source for genomic DNA (gDNA). gDNA was
extracted using Quick-DNA\selectlanguage{english} Fungal/Bacterial Miniprep Kit (Zymo
Research). The entire list of the strains is provided in Table S5.

Yeast transformations were performed as described in Holkenbrink et al.
{[}19{]} with the modifications described below. \emph{Y.
lipolytica}strains were streaked on Yeast Peptone Dextrose (YPD) plates
(20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, 15 g/L agar) and
grown for 24 h at 28degC. A small patch of biomass was taken with an
inoculation loop and re-streaked on YPD plates containing 0.7 g/L
complete supplement mixture (Formedium). After 24 h incubation at 28degC,
the cells were scraped off, resuspended in 1 mL 0.5 M sterile sucrose
solution, and centrifuged for 5 min at 3,000 g at room temperature. The
supernatant was discarded, the cells resuspended in 0.5 M sucrose
solution, and a volume corresponding to OD\textsubscript{600} 2.6 was
transferred to a sterile tube for transformation. Tubes were centrifuged
for 5 min at 3,000 g at room temperature, and the supernatant was
removed. 500-1,000 ng of gRNA plasmid and integration plasmid, which
previously was linearized with SmiI (Thermo Fisher Scientific) and
gel-purified prior transformation, were added to the pellet. The mixture
was resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L of transformation mix (Supplementary Table S6)
and incubated at 39\selectlanguage{ngerman}°C for 1 h. After the heat shock, the tubes were
centrifuged, the supernatant removed, and the pellet was resuspended in
500 \selectlanguage{greek}µ\selectlanguage{english}L liquid YPD medium. The cells were incubated for 2 h at 28\selectlanguage{ngerman}°C 300
rpm shaking. The pellet was again collected by centrifugation,
resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L of 0.5 M sterile sucrose solution, and plated on
YPD plates containing Hygromycin B (200 mg/L) (Carl Roth) or
Nourseothricin (250 mg/L) (Jena Bioscience) for selection. After 2-3
days of incubation at 28\selectlanguage{ngerman}°C, single colonies appeared, which were tested
for correct integration by PCR using vector-specific primers and primers
complementary to genomic loci close to the integration site
(Supplementary Table S1).

\textbf{2.3 Small scale cultivations}

\emph{Y. lipolytica} strains were inoculated from a YPD agar plate to an
initial OD\textsubscript{600} of 0.2 into 2.5 mL YPG medium (10 g/L
yeast extract, 10 g/L peptone, 40 g/L glycerol) in 24 well-plate
(EnzyScreen). The plate was incubated at 28°C and 300 rpm for 22 hours.
The plate was centrifuged at 3,500 g for 5 min at 20°C, the medium was
removed and the cells were resuspended in 1.25 mL production medium (50
g/L glycerol, 5 g/L yeast extract, 4 g/L
KH\textsubscript{2}PO\textsubscript{4}, 1.5 g/L MgSO\textsubscript{4},
0.2 g/L NaCl, 0.265 g/L CaCl\textsubscript{2}.2H\textsubscript{2}O, 2
mL/L trace elements solution: 4.5 g/L
CaCl\textsubscript{2}.2H\textsubscript{2}O, 4.5 g/L
ZnSO\textsubscript{4}.7H\textsubscript{2}O, 3 g/L
FeSO\textsubscript{4}.7H\textsubscript{2}O, 1 g/L
H\textsubscript{3}BO\textsubscript{3}, 1 g/L
MnCl\textsubscript{2}.4H\textsubscript{2}O, 0.4 g/L
Na\textsubscript{2}MoO\textsubscript{4}.2H\textsubscript{2}O, 0.3 g/L
CoCl\textsubscript{2}.6H\textsubscript{2}O, 0.1 g/L
CuSO\textsubscript{4}.5H\textsubscript{2}O, 0.1 g/L KI, 15 g/L EDTA).
The plate was incubated at 28°C and 300 rpm for 28 h. Each strain was
cultivated in triplicate.

\textbf{2.4 Bioreactor fed-batch cultivations}

Bioreactor fed-batch mode cultivations were carried out in biological
triplicates on initial YPG medium (50 g/L glycerol, 10g/L yeast extract
(Carl Roth), 10 g/L peptone) in controlled stirred bioreactor vessels
with 1.0 L total aerated end working volume (Infors Minifors 2 systems,
Infors AG, Switzerland) at 28°C. All reactors were equipped with pH and
optical dissolved oxygen probes (Hamilton AG, Switzerland) and off-gas
analyzers (BlueSens GmbH, Germany). pH was controlled at 4.5±0.1 with
automated addition of a 4 M solution of NaOH. The dissolved oxygen
control was set at a minimum threshold of 20\% with a cascade control by
gradually increasing the stirrer speed of two six-blade Rushton turbines
and the aeration rate using Eve fermentation control software (Infors
AG, Switzerland). The initial aeration rate was set to 1 L/min, with
stirring at 400 rpm. The CO\textsubscript{2} (\%) and
O\textsubscript{2}(\%) in the off-gas were monitored continuously during
the fed-batch cultivations. Bioreactor cultures of strain \emph{Y.
lipolytica} ST9253 were inoculated from shake flask precultures in the
exponential growth phase (250 mL baffled shake flasks, 40 mL cultivation
volume, YPG 40 g/L glycerol, 10 g/L yeast extract, 10 g/L peptone) to a
starter fermentation volume of 620 mL. After the depletion of the
initially supplied glycerol carbon source, the cultures were fed with a
nutrient-rich feed solution (700 g/L glycerol, 10 g/L yeast extract, 10
g/L (NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4}). The
composition of the feed solution was set to facilitate the production of
the target compounds after an initial biomass buildup phase. Off-line
samples were taken regularly to analyze residual glycerol concentration
(Megazyme Inc. assay kit), optical density at 600 nm (Genesys
photometer, Thermo Fischer Scientific), cell dry weight, and fatty
alcohol concentrations.

\textbf{2.5 Extraction and derivatization of lipids}

1 mL of cultivation broth was sampled into 4 mL glass vials and
centrifuged at 3,500 g for 5 min at 20°C. The supernatant was removed,
and 1 mL of 1 M HCl in anhydrous methanol was added to the pellet.
Samples were vortexed for 20 s and incubated at 70°C for 2 h for
methanolysis reaction to proceed. Samples were cooled down to room
temperature, 1 mL of 1 M NaOH in methanol, 500 \selectlanguage{greek}µ\selectlanguage{english}L of saturated NaCl
solution in water, 1 mL of hexane, and 10 \selectlanguage{greek}µ\selectlanguage{english}L of internal standard (2 g/L
of methyl nonadecanoate (19:Me) in hexane) were added sequentially.
Vials were vortexed for 10 s, centrifuged at 3,500 g for 5 min at 20\selectlanguage{ngerman}°C,
and the upper organic layer was taken for analysis.

\textbf{2.6 Extraction of fatty alcohols from small-scale samples}

In small-scale screening experiments, 1 mL of cell culture was sampled
into 4 mL glass vials and centrifuged at 3,500 g, 5 min, 21°C. The
supernatant was removed, and 1 mL of ethyl acetate:ethanol mixture
(85:15, v/v) was added to the pellet together with 10 \selectlanguage{greek}µ\selectlanguage{english}L of internal
standard (2 g/L of methyl nonadecanoate (19:Me) in ethyl acetate). Vials
were vortexed for 20 s and incubated for 1 h, followed by 5 min
vortexing. 300 \selectlanguage{greek}µ\selectlanguage{english}L of water was added, samples vortexed for 10 s,
centrifuged at 3,500 g, 5 min, 21\selectlanguage{ngerman}°C, and the upper organic layer was
taken for analysis.

Samples from bioreactors were processed as follows. 100 \selectlanguage{greek}µ\selectlanguage{english}L of cell
culture was taken, and the total broth was processed in the same way as
for small-scale samples except that the first centrifugation step was
omitted.

\textbf{2.7 Extraction and work-up of fatty alcohols from bioreactor
cultivation for acetylation reaction}

The 2.4 L of broth from the bioreactor cultivation was centrifuged at
3,000 g for 5 min at room temperature, and the supernatant was separated
from the pellet. Fatty alcohols were extracted from the supernatant by
adding 2.2 L of ethyl acetate and incubating on a multi-vortexer for 1
h. Organic phase was decanted. To extract fatty alcohols from the
pellet, 100 mL of ethyl acetate was added to the pellet, and the mix was
shaken on a multi-vortexer for 6 h . The organic phase was decanted, 100
mL of fresh solvent was added to the biomass pellet, and the mixing
repeated for 1 h. The organic phase was decanted. All ethyl acetate
fractions from the supernatant and pellet extraction were combined and
dried with anhydrous sodium sulfate. The solvent was removed in a rotary
evaporator.

The extracted crude fatty alcohol mixture was passed through a silica
gel column, which was prepared as described in {[}20{]}. The silica was
washed with hexane and then subsequently with a gradient of hexane/ethyl
acetate at the proportion of 95:5 to 40:60 (\% v/v). Purest fractions
based on thin-layer chromatography were collected, and the solvent was
evaporated in a rotary evaporator.

\textbf{2.8 Acetylation of fatty alcohols}

The purified fatty alcohol mixture was acetylated by using a 1.2 molar
equivalent of acetic anhydride and 0.1 equivalent of sodium acetate
anhydrous (equivalents were based on 14:OH). The reaction mixture was
allowed to stir for 1 h at 80\selectlanguage{ngerman}°C in a water bath and then transferred to
a separatory funnel where it was diluted with ethyl acetate and washed
with a saturated solution of sodium bicarbonate. The organic phase was
dried with anhydrous sodium sulfate, and the solvent evaporated in
rotary evaporator.

\textbf{2.9 Gas chromatography-mass spectrometry (GS-MS) analysis}

Analysis was carried out on an Agilent GC 7820A coupled to a MS 5977B,
equipped with a split/spitless injector and a DB-Fatwax UI column (30 m
x 250 \selectlanguage{greek}μ\selectlanguage{english}m x 0.25 \selectlanguage{greek}μ\selectlanguage{english}m). The operation parameters were: 1 \selectlanguage{greek}µ\selectlanguage{english}L injection,
split ratio 20:1 for FAMEs and alcohol samples and 10:1 for acetates,
injector temperature 220 \selectlanguage{ngerman}°C, constant flow 1 mL/min of helium, oven ramp
80°C for 1 min, 20 °C/min to 150°C, then 1°C/min to 200°C, then 20°C/min
to 230°C. MS was scanning between \emph{m/z} 30 and 350. For compound
identification, reference standards were purchased from Pherobank, and
retention times together with mass spectra were compared to the sample
of interest. Quantification was made based on 6 points calibration
curves. Visual inspection of chromatograms was performed using
MassHunter Qualitative Analysis Navigator B.08.00 (Agilent), while
quantification was performed using MS Quantitative Analysis
(Quant-My-Way) (Agilent).

\textbf{2.10 Electrophysiological responses of male \emph{Ostrinia
nubilalis}}

Antennal responses of male ECB adults to biologically-derived pheromone
blend (BioPhe) were evaluated by electroantennography (EAG) using a
commercially available electroantennographic system (Ockenfels Syntech
GmbH, Buchenbach, Germany). The antenna of a virgin two-to-three-day old
male adult was excised from the head using micro-scissors and attached
between two stainless-steel wire electrodes with the assistance of
electrically conductive gel (G008, Fiab, Italy). The base of the antenna
was connected to the indifferent electrode, whilst the distal end was
connected to the recording electrode after cutting off a few segments of
the tip. The signal was amplified 10X by a Universal AC/DC pre-amplifier
probe connected to the recording electrode, and the analog signal was
amplified and detected with a data acquisition controller (IDAC-4,
Ockenfels Syntech GmbH).

Test stimulus was the biologically-derived pheromone blend BioPhe
containing 7.2\% \emph{Z} 11-14:OAc, according to GC-MS analysis.
Commercially available \emph{Z} 11-14:OAc, the major sex pheromone of
the\emph{O. nubilalis} Z-race, was used as a reference compound. The
BioPhe blend, as well as commercially available pheromone compounds,
were diluted in pentane. For each stimulus, a 10 \selectlanguage{greek}µ\selectlanguage{english}L aliquot of the
solution was pipetted to a piece of filter paper (7 x 30 mm, Whatman no.
1), and the solvent was allowed to evaporate before being inserted into
a Pasteur pipette for odor delivery. Control stimulus consisted of a
clean Pasteur pipette with a filter paper impregnated with solvent
(pentane). Stimuli were provided as 0.3~s air puffs into a continuous
flow of filtered and humidified air. The airflow, at 25~
cm\textsuperscript{3}/s rate, tube diameter 1 cm, was generated by an
air stimulus controller (CS-55, Syntech, The Netherlands).

The reference stimulus, consisting of a filter paper carrying 100 ng
of\emph{Z} 11-14:OAc, was provided at regular intervals during each
recording session. The EAG response to each reference stimulus was
defined as 100\%, and all responses to test stimuli between adjacent
references were normalized in \% relative to the references. At least 1
min for weak and moderate stimuli and 5 min for strong stimuli were
allowed between successive stimulations to let the antenna recover. The
BioPhe blend was tested at a 100 ng dose (containing \textasciitilde{}7
ng \emph{Z} 11-14:OAc), as well as at a 1 \selectlanguage{greek}μ\selectlanguage{english}g dose (\emph{i.e} ., 70
ng\emph{Z} 11-14:OAc), in order to reach an amount of major sex
pheromone comparable to the 100 ng dose of the \emph{Z} 11-14:OAc
stimulus.

The minor sex pheromone, \emph{E} 11-14:OAc, was also tested, as well as
both isomers at a 97:3 Z:E ratio, based on the ratio released from the
female moth gland as reported in the literature for ECB Z-race
{[}7,21{]} . In addition, considering the high amount of tetradecyl
acetate (14:OAc) in the BioPhe blend (\textasciitilde{}50\%), the EAG
response to 50 ng of this compound was also evaluated (for consistency
reasons 50 ng is approx. the amount of 14:OAc contained in the 100 ng
BioPhe test dose). All aforementioned stimuli were tested on a total of
20 antennal preparations.

\textbf{2.11 Behavioral bioassays}

Behavioral studies were performed in a dark room under red light (2.5-3
lux) at 21\selectlanguage{ngerman}±2°C and 70±5\% relative humidity. Male moths were tested in a
wind tunnel as described in {[}22{]}. For odor delivery, a piece of
triangular-shaped filter paper (2.5 cm base, 4 cm height) was pipetted
with appropriate volume of the test solution and, following solvent
evaporation, was hung from the suction hook in the tunnel.

Pheromone standards and BioPhe samples were dissolved in pentane. The
major and minor sex pheromone components \emph{Z} 11-14:OAc and\emph{E}
11-14:OAc, respectively, were mixed at a 97:3 ratio and were used as a
positive control at a 50 \selectlanguage{greek}μ\selectlanguage{english}g dose, which, through preliminary testing,
was concluded to be the minimum amount eliciting positive courtship
behavior, i.\emph{e.,} source-oriented flight pattern, abdominal hair
pencils display and abdomen curling upon contact. Additionally, to
evaluate potential antagonistic effects of BioPhe blend contaminants on
the males' responses, 14:OAc (most prominent component
\textasciitilde{}50\% of BioPhe blend) and \emph{Z} 9-11:OAc
(\textasciitilde{}9\% of BioPhe blend) have been tested.

A dose of 700\selectlanguage{greek}μ\selectlanguage{english}g BioPhe was used in the bioassays (containing
50\selectlanguage{greek}μ\selectlanguage{english}g\emph{Z} 11-14:OAc and 350\selectlanguage{greek}μ\selectlanguage{english}g 14:OAc) and a dose of 350\selectlanguage{greek}μ\selectlanguage{english}g was added
to 50\selectlanguage{greek}μ\selectlanguage{english}g pheromone standard (97:3) to emulate abundance and ratio in the
BioPhe. Also a dose of 50\selectlanguage{greek}μ\selectlanguage{english}g pheromone standard (97:3) with the addition
of 4.5\selectlanguage{greek}μ\selectlanguage{english}g of \emph{Z} 9-16:OAc (9\% present in the BioPhe blend) was
tested, as to our knowledge the role of \emph{Z} 9-16:OAc has not been
investigated as antagonist to male response. Other components of BioPhe,
such as \emph{Z} 9-14:OAc have already been reported in the literature
as antagonist to male response {[}23,24{]}. Behavioral tests were
conducted between the 3\textsuperscript{rd} and 5\textsuperscript{th}
hour in the scotophase {[}24,25{]}. Two hours before testing, 2-5 days
old male moths were transferred in individual 400 mL clear plastic cups
covered with perforated lids and left in the conditions of the dark room
to acclimate. For each test, the odor source was attached to the tunnel
ceiling, and after 30 seconds, an individual male was released in the
tunnel, and its behavior was recorded for 10 min.

The following sequences of behavior were recorded: close approach (less
than 10 cm) and contact with the source (landing). In addition, the
approach and landing steps were further analyzed into four grades, and
each grade was assigned its corresponding value. For the landing step,
grades were discriminated as follows: grade 1) brief contact with the
source (no landing), grade 2) landing on source for 1-2 sec, grade 3)
landing on source for more than 2 seconds, and grade 4) landing on
source accompanied by hairpencils display and/or abdomen curling
(copulation attempt). Similarly, for the approach step were
discriminated as: grade 1) close approach to source for 1-2 sec, grade
2) close approach with zig-zag patterned flight for \textgreater{} 2
sec, grade 3) close approach with zig-zag patterned flight and
hairpencils display for 1-2 sec, grade 4) close approach with zig-zag
patterned flight and hairpencils display for \textgreater{} 2 sec.

Between treatments, the flight tunnel inner walls and suction hook were
wiped with acetone and left to aerate for 10 min before the next
treatment. For each treatment, 30 males were tested. Males were used
once and were discarded after testing.

\textbf{2.12 Statistical analyses}

The electrophysiological and behavioral data were subjected to analysis
of variance (ANOVA) (SAS Institute, 2000). The means of
electrophysiological data and the response grades were separated using
the Tukey (honestly significant difference, HSD) test at P = 0.05.

\textbf{3 Results}

\textbf{3.1 Screening of fatty acyl-CoA desaturases}

In \emph{O. nubilalis} , a [?]11 desaturase acts on myristoyl-CoA
(14:CoA), which in turn is generated by \selectlanguage{greek}β-\selectlanguage{english}oxidation of the abundant
fatty acid synthesis product hexadecanoyl-CoA (16:CoA), and generates a
mixture of\emph{E} /\emph{Z} 11-14:CoA {[}6,26{]}. In \emph{Y.
lipolytica} , myristic acid is a minor component of the total fatty acid
pool, comprising only about 0.25\% of the total fatty acids {[}27{]}. To
increase the supply of 14:CoA in \emph{Y. lipolytica} , we substituted
isoleucine 1220 of the fatty acid synthase ketoacyl synthase domain to
phenylalanine (Fas2p\textsuperscript{I1220F}). This mutation was
proposed to hinder the binding of longer acyl-CoAs in the active site
tunnel of the FAS complex and was reported to result in the production
of shorter chain acyl-CoAs {[}27{]}. The mutation has already been
proven to be effective in the production of \emph{Z} 9-14:OH, where it
increased the titer 15-fold {[}16{]}. In this study, we introduced the
FAS mutation into\emph{Y. lipolytica} strain ST6629, which was
previously developed for decreased degradation of fatty alcohols and
decreased storage lipid synthesis {[}16{]}. The resulting ST7982 showed
8.4-fold increase in myristic acid production compared to the parental
strain (Supplementary Figure S1).

Next, seven [?]11 desaturases with previously documented\emph{E} /\emph{Z}
11-14:CoA activity were tested in strain ST7982 (Figure 1, Figure S2).
These were the desaturase Lbo\_PPTQ from the grapevine moth
\emph{Lobesia botrana} {[}28{]}, Onu11 from the European corn
borer\emph{O. nubilalis} {[}26{]}, EpoE11 from the light brown apple
moth\emph{Epiphyas postvittana} {[}29{]}, CroZ11 from the oblique banded
leafroller moth \emph{Choristoneura rosaceana} {[}30{]}, CpaE11 from the
spotted fireworm moth \emph{Choristoneura parallela} {[}31{]}, Hzead11
from the corn earworm \emph{H. zea} {[}32{]}, and Msextad11 from the
tobacco hornworm \emph{Manduca sexta} {[}33{]}. The resulting strains
were cultivated, their lipids methanolized into FAMEs, and analyzed on
GC-MS. Lbo\_PPTQ expression resulted in the highest content of\emph{Z}
11-14:acid. The \emph{Z} /\emph{E} isomer ratio was 9:2. For the strain
expressing the OnuE11 desaturase, no new compounds could be detected,
indicating that this desaturase is most likely not well expressed or
active in \emph{Y. lipolytica} . Likewise, no activity was detected for
the desaturases, EpoE11 and CpaE11, which were previously reported to
produce \emph{E} 11-14:acid in another yeast,\emph{Saccharomyces
cerevisiae} , albeit in minuscule amounts {[}29,31{]}. The strain
expressing CroZ11 produced a mixture of\emph{E} /\emph{Z} 11-14:acid
with an excess of the Z isomer; however, the amounts of both isomers
were lower compared to the strain expressing Lbo\_PPTQ. The desaturase
Hzead11 appeared to be a promiscuous [?]11 desaturase with a high
preference towards 16:acid (Figure 1). Apart from\emph{Z} 11-16:acid, we
also detected \emph{E} /\emph{Z} 11-14:acid and some other unsaturated
fatty acids, which remain to be identified. (Figure S2). The Msextad11
desaturase-expressing strain produced\emph{E} /\emph{Z} 11-14:acid in
ratio 1:1, where the amount of the E isomer was the highest among the
tested desaturases.

In summary, Lbo\_PPTQ resulted in the highest content and purity
of\emph{Z} 11-14:acid and was therefore chosen for establishing the
biosynthetic pathway towards ECB pheromone in \emph{Y. lipolytica} .

\textbf{3.2 Screening of fatty acyl-CoA reductases}

We further screened four fatty acyl-CoA reductases (FARs) to identify
the reductase with the highest activity and selectivity towards\emph{Z}
11-14:acid. The four reductases were: HarFAR from cotton bollworm
\emph{H. armigera} {[}34{]}, SlitpgFARII from African cotton leafworm
\emph{Spodoptera littoralis} {[}35{]}, and two reductases from\emph{O.
nubilalis} , OnuFAR\_E and OnuFAR\_Z {[}36{]}. First, we expressed the
four reductases individually in the strain ST7982. The resulting strains
were cultivated as follows: first, the cells were grown for biomass
propagation for 22 h, followed by a production phase in media
supplemented with equal amounts of \emph{E} and\emph{Z} 11-14:Me (500
mg/L of each isomer) lasting for 28 hours. Fatty alcohols were extracted
from the broth and quantified on GC-MS. HarFAR and SlitpgFARII
expressing strains produced around 40 and 14 mg/L of\emph{E} 11-14:OH
and \emph{Z} 11-14:OH from the corresponding methyl esters,
respectively, and did not exhibit a preference towards any isomer.
OnuFAR\_E produced very low amounts (\textless{}1 mg/L) of\emph{E}
/\emph{Z} 11-14:OH, which were below the quantification limit. However,
manual inspection of the chromatograms showed that this reductase had a
bias towards \emph{E} isomer, as expected. No fatty alcohols were
detected in the strain expressing OnuFAR\_Z (Figure 2, Figure S3).

Next, for \emph{de novo} production of \emph{Z} 11-14:OH, we expressed
the four reductases in the strain ST9992, which contained
Fas2p\textsuperscript{I1220F} mutation and expressed desaturase
Lbo\_PPTQ. Interestingly, while HarFAR resulted in 2.8-fold higher
product titer than SlitpgFARII in the feeding assay above, in this
experiment with \emph{de novo} production, a higher \emph{Z} 11-14:OH
titer was obtained in strain with SlitpgFARII, 37.7\selectlanguage{ngerman}±2.6 mg/L versus
29.2±1.6 mg/L for HarFAR strain.

However, the strain expressing SlitpgFARII additionally produced
402.5±17.3 mg/L of 14:OH and 54.7± 4.0 mg/L of \emph{Z} 9-16:OH, which
is 2.2 and 1.8-fold higher compared to HarFAR, respectively (Figure 2B).
Very high activity towards tetradecanoate of SlitpgFARII was also
observed by Antony et al. {[}35{]}, who reported that among a wide
variety of saturated, mono-, and di-unsaturated fatty acyl substrates,
tetradecanoate was converted into alcohol most efficiently.

We chose HarFAR for further strain engineering due to its high activity
and better selectivity towards \emph{Z} 11-14:CoA than SlitpgFARII.

\textbf{3.3. Metabolic engineering for improved production
of\emph{Z}11-14:OH}

After selecting Lbo\_PPTQ and HarFAR as the biosynthetic enzymes
for\emph{Z} 11-14:OH production, we proceeded with further metabolic
engineering strategies to increase the product titer (Figure 3A). First,
we introduced two gene copies of HarFAR into the high myristate
producing strain ST7982 to enable biosynthesis of fatty alcohols. The
resulting strain ST8225 produced 318.3±52.4 mg/L total fatty alcohols,
among which 14:OH constituted 67.5\% and was the most abundant. In order
to produce \emph{Z} 11-14:OH, we expressed Lbo\_PPTQ in ST8225 leading
to ST8373, which yielded \emph{2} 1.5±2.2 mg/L of the target compound.
Compared to the parental strain, this strain made 46.7\% more of the
total fatty alcohols. Then we introduced a second copy of Lbo\_PPTQ and
this led to a further 3-fold improvement of \emph{Z} 11-14:OH titer.

We anticipated that further improvement could be achieved by
overexpressing native \emph{FAS1} from \emph{Y. lipolytica} . Previous
studies in \emph{S. cerevisiae} showed that the activity of FAS complex
is enhanced when the copy number of \emph{FAS1} gene is increased
{[}37{]}. Indeed, overexpression of \emph{FAS1} (strain ST9253) resulted
in 60.7\% improvement in the total fatty alcohol titer and increased
production of \emph{Z} 11-14:OH by 54.7\%, resulting in 93.9±11.7 mg/L
of\emph{Z} 11-14:OH in small-scale cultivation.

\textbf{3.4 Production of \emph{Z}11-14:OAc by fed-batch fermentation
and alcohol acetylation}

The engineered strain ST9253 (MATa \emph{ku70} \selectlanguage{english}[?] \emph{Cas9} \emph{hfd4}
\selectlanguage{english}[?]\emph{hfd1} \selectlanguage{english}[?] \emph{pex10} \selectlanguage{english}[?] \emph{fao1} \selectlanguage{english}[?] \emph{hfd2} \selectlanguage{english}[?] \emph{hfd3} \selectlanguage{english}[?]
GPAT\_100bpPr Fas2p\textsuperscript{I1220F}
2x\emph{Lbo\_PPTQ}2x\emph{HarFAR} \selectlanguage{english}|\emph{FAS1} ) was cultivated in
controlled bioreactors in fed-batch mode. The initial batch phase served
as a biomass propagation phase. After the depletion of the initial
carbon source supplied in the batch phase, the composition of the
fed-batch feed solution was set to facilitate the production of fatty
alcohols. In the first 42 h of fermentation, the accumulation of biomass
was targeted, and stationary phase was reached after 49 h (Figure3B).
The start of the fed-batch facilitated a shift towards nitrogen-limited
conditions and thereby transition from the biomass build-up phase to
production of fatty alcohols.

In the fatty alcohol production phase of the fermentation process, we
observed a constant increase in specific yield. The specific production
yield of \emph{Z} 11-14:OH peaked at the very end of fermentation and
reached 0.007 g product/g DW. At this time point, the titer of\emph{Z}
11-14:OH was 188.1+-13.4 mg/L, while the cell dry weight reached 28.2+-0.7
g/L (Figure 3B). The titer of 14:OH reached 1,350.1+-188.1 mg/L. It was
the most abundant fatty alcohol in the fermentation broth. Tight control
of parameters such as pH, dissolved oxygen and glycerol levels in the
bioreactor cultivations led to a 2-fold improvement in the target
compound titer compared to the small-scale cultivations.

After 120 h of fed-batch fermentation, fatty alcohols were extracted
with organic solvent and purified on a silica column. The resulting
fatty alcohol mixture containing approximately 250 mg of\emph{Z}
11-14:OH was acetylated with acetic acid anhydride. It resulted in full
conversion, where 320.1+-13.4 mg of \emph{Z} 11-14:OAc was obtained, and
no traces of alcohols were left at the end of the reaction (Figure 4).
The resulting product is referred to as BioPhe, for biologically-derived
pheromone.

\textbf{3.5 Electrophysiological responses of male \emph{Ostrinia
nubilalis}}

We studied electrophysiological responses of male \emph{O.
nubilalis}insect antennas to pure chemical pheromone components, BioPhe
product, and several blends. As expected, EAG responses of ECB Z-race
male moths were higher for 100 ng \emph{Z} 11-14:OAc than for \emph{E}
11-14:OAc (Figure 5A). Also, the 97:3 (\emph{Z} 11-14:OAc/\emph{E}
11-14:OAc) mixture at 100 ng dose elicited the same magnitude of
response as the major pheromone component (\emph{Z} 11-14:OAc). The
biologically-derived pheromone BioPhe at 100 ng elicited a significantly
lower response, as expected, because \emph{Z} 11-14:OAc is present just
at 7.2\% \emph{ergo}and only about 7 ng of \emph{Z} 11-14:OAc were
directed to the antenna. When the quantity of BioPhe was increased
ten-fold, the EAG response was also increased. In this treatment, the
amount of \emph{Z} 11-14:OAc present in the biologically-derived mixture
was of the same order of magnitude as the reference (\emph{i.e} .,
\textasciitilde{}70 ng). However, it should be noted, that some of the
other BioPhe components may also contribute to the augmented response
recorded. In this context, we tested the effect of 14:OAc, given the
fact that it comprises about 50\% of BioPhe. At 50 ng dose, analogous to
that contained in 100 ng BioPhe, it was clearly detected by the antenna
but to a much lesser degree than the reference compound.

\textbf{3.6 Behavioral bioassays}

Male ECB moths responded with approach and landing to 50 \selectlanguage{greek}μ\selectlanguage{english}g of the
pheromone blend corresponding to the \emph{Z} -race (Figure 5B). As
BioPhe contains just 7.2\% \emph{Z} 11-14:OAc, a 700 \selectlanguage{greek}μ\selectlanguage{english}g stimulus of
BioPhe was tested in lab bioassays (containing approx. 50 \selectlanguage{greek}μ\selectlanguage{english}g\emph{Z}
11-14:OAc). Significantly fewer source-oriented approaches and fewer
landings were observed compared to the response elicited by the optimal
Z-race pheromone, indicating the presence of compound(s) interfering
with the insects' anticipated behavior.

In the BioPhe sample 14:OAc comprises almost 50\% of the total amount.
To emulate a BioPhe sample, 350 \selectlanguage{greek}μ\selectlanguage{english}g 14:OAc were added to the 50 \selectlanguage{greek}μ\selectlanguage{english}g
pheromone blend (97:3). In this case, males scored significantly lower
in both approach and landing. This is an indication that 14:OAc at this
dose interferes with the precopulatory behavior of \emph{O. nubilalis.}

Similarly, when 4.5 \selectlanguage{greek}μ\selectlanguage{english}g of \emph{Z} 9-16:OAc was added to the 50 \selectlanguage{greek}μ\selectlanguage{english}g
pheromone blend (97:3), significantly fewer landings were recorded, but
the frequency of approaches did not differ significantly from what was
observed in response to the native pheromone .

\textbf{4 Discussion}

We have evaluated the performance of seven FADs and four FARs, which are
involved in insect sex pheromone biosynthesis, to establish the
production of \emph{Z} 11-14:OH in \emph{Y. lipolytica} . Among FADs,
Lbo\_PPTQ showed the highest production of (\emph{Z}
)--11--tetradecenoate and was selected for further strain engineering.
Several desaturases, namely, Onu11, EpoE11, and CpaE11, were not active.
The desaturase Onu11 was previously assayed in other recombinant hosts,
such as \emph{S. cerevisiae} and \emph{Nicotiana benthamiana} with
divergent results. In \emph{S. cerevisiae} , it generated \emph{E}
/\emph{Z} 11-14:acid in ratio 4:5 and also produced\emph{Z} 11-16:acid
{[}26{]}, while no activity of this enzyme was observed in \emph{N.
benthamiana} {[}14{]}\emph{.} The other two desaturases that were
non-functional in our study, EpoE11 and CpaE11, were reported to act on
14:acid and produce \emph{E} 11-14:acid in\emph{S. cerevisiae} , but the
amounts were very low {[}29,31{]}. One possible explanation is that the
non-functional desaturases were poorly expressed or misfolded. A recent
study by Bu\selectlanguage{polish}č\selectlanguage{english}ek et al. showed that different moth desaturases could have
different expression levels {[}38{]}. Codon-optimization could also have
been suboptimal {[}39{]}.

After examining FADs, we screened several FARs for increased specificity
towards fatty acids with C14 chain length. Previously, HarFAR was shown
to prefer (\emph{Z} )--9--tetradecenoate over (\emph{Z}
)--11--hexadecenoate and (\emph{Z} )--9--hexadecenoate {[}34{]}. We
hypothesized that this reductase should also be able to act on (\emph{E}
/\emph{Z} )-11- tetradecenoate, which is structurally similar to
(\emph{Z} )-9-tetradecenoate. Indeed, a feeding experiment and
co-expression with Lbo\_PPTQ confirmed that HarFAR could produce\emph{E}
/\emph{Z} 11-14:OH from corresponding acids. (\emph{E} /\emph{Z} )-11-
tetradecenoate was also converted into alcohol by \emph{Y. lipolytica}
expressing SlitpgFARII. This FAR previously produced the highest amounts
of \emph{Z} 11-14:OH among the four tested reductases from
\emph{Spodoptera} spp. {[}35{]}. OnuFAR\_E and OnuFAR\_Z were shown to
be selective for E and Z isomers of 11-tetradecenoate, respectively
{[}36{]}, however, in our study, OnuFAR\_E showed little and OnuFAR\_Z
no activity. The same results were obtained when these enzymes were
screened in the plant \emph{N. benthamiana} {[}14{]}. One possible
explanation for the lack of functionality of these enzymes in yeast and
plant expression hosts could be incorrect folding of the reductases in
the ER membrane, which has a different lipid composition in yeasts and
plants than in insects {[}40-43{]}.

In order to improve the production of \emph{Z} 11-14:OH, we employed
metabolic engineering strategies, such as integration of multiple copies
of genes and enhancement of precursor supply by overexpression
of\emph{FAS1} subunit of \emph{Y. lipolytica} . In small-scale
cultivations of the engineered yeast strain, we achieved 93.9 \selectlanguage{ngerman}±11.7 mg/L
of\emph{Z} 11-14:OH. On the way of building the production strain
(ST9253), we observed that introduction of desaturase Lbo\_PPTQ not only
contributed to the biosynthesis of \emph{Z} 11-14:OH, but it also
improved the total fatty alcohol titer. It was previously shown that
overexpression of the native \emph{OLE1} desaturase increased fatty acid
biosynthesis in general, likely because the fatty acid synthase complex
FAS is less inhibited by unsaturated fatty acyl-CoAs than by saturated
fatty acyl-CoAs {[}44-46{]}.

For collection of pheromone for EAG and behavioral tests, the engineered
strain was fermented in controlled 1 L bioreactors in fed-batch mode. We
used glycerol as the carbon source with a high carbon-to-nitrogen ratio
to favor fatty alcohol production {[}47-52{]}. We obtained 188.1±13.4
mg/L of \emph{Z} 11-14:OH. The strain also produced large amounts (over
1.3 g/L) of the saturated by-product 14:OH, indicating a significant
limitation of the desaturation step. This may be improved by further
strain engineering and fermentation optimization in the future. To our
knowledge, this is the first study showing the production of\emph{Z}
11-14:OH in a microbial host. Previously this pheromone precursor was
recombinantly synthesized in plant \emph{N. benthamiana}yielding 14 \selectlanguage{greek}µ\selectlanguage{english}g
from 1 g leaf tissue {[}14{]}. While \emph{Y. lipolytica}has been
engineered for the production of fatty alcohols in multiple other
studies, the common products are naturally unsaturated or saturated
fatty alcohols {[}48,49,53-56{]}.

To convert the fermented alcohol into acetate, which is the active sex
pheromone component of \emph{O. nubilalis} , a chemical acetylation step
was performed, which resulted in full conversion and yielded 320\selectlanguage{ngerman}±13.4 mg
of \emph{Z} 11-14:OAc. Interestingly, based on the current knowledge
about insect pheromone biosynthesis, this reaction should be catalyzed
by acyltransferases in insects {[}6,57{]}. However, until now, no
enzymes from moths catalyzing this reaction have been found even though
some gene candidates have been proposed and tested {[}58{]}.

The yeast-derived pheromone blend caused a response of \emph{O.
nubilalis} males in electroantennogram experiments similar to what could
be expected based on the responses to synthetic pheromone compounds.
Furthermore, the blend was attractive to insects in behavioral bioassays
in a wind tunnel to the same level as the chemically synthesized
pheromone blend. However, full precopulatory behavior was observed less
often. In order to induce a complete the ECB precopulatory behavior, a
higher purity may be required, as certain by-products present in the
blend may hinder the complete expression of the precopulatory behavior.
Notably, reduced precopulatory behavior is not necessarily a hindrance
and may even be a benefit for mating disruption. Therefore, activity
studies in the field are warranted.

In summary, we have successfully employed yeast \emph{Y. lipolytica} for
production of \emph{O. nubilalis} sex pheromone precursor\emph{Z}
11-14:OH and showed that the resulting yeast-derived pheromone was
biologically active in modulating the behavior of \emph{O. nubilalis}
males.

\textbf{Acknowledgements}

KP acknowledges the funding from Innovationsfonden under grant agreement
number 8053-00179B. This project has received funding from the European
Union's Horizon 2020 research and innovation programme under grant
agreement No. 760798 (OLEFINE) and from the Bio-Based Industries Joint
Undertaking (BBI JU) under the European Union's Horizon 2020 research
and innovation programme under grant agreement No: 886662 (PHERA). IB
and CL acknowledge the financial support from the Novo Nordisk
Foundation under grant agreements No. NNF15OC0016592 and No.
NNF10CC1016517. CL acknowledges funding from the Swedish Foundation for
Strategic Research (No. RBP 14--0037, Oil Crops for the Future). The
authors thank the Agricultural Research Service (NRRL, USA) for the
yeast strain. We thank Blanka Toth for assistance with monitoring and
sampling during fed-batch fermentation.

\textbf{Conflict of interest}

IB, CH, CL, BJD are co-inventors on patent applications WO2016207339,
WO2018109167, and WO2018109163. KP, KRK, HJ, NM, AM, AG, CS, BL, CH, IB
have financial interest in BioPhero ApS. DR has financial interest in
Novagrica SA.

\textbf{Data Availability Statement}

Not applicable

\textbf{Author Contributions}

The concept of the study was developed by KP, IB, MK, DR, and CL. KP,
IB, MK, DR, and CL acquired the funding for the research. The
investigation was carried out by KP, EK, PCB, KRK, HJ, NM, AM, AG, CS,
BL, and CH. B-J D and CL provided Lbo\_PPTQ sequence. IB, MK, DR, and CL
supervised the work. KP, CH, IB, CL, and MK drafted the manuscript. All
authors participated in reviewing and editing the manuscript.

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\par\null

\textbf{Figures and legends}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Characterization of [?]11 desaturases.
\textbf{A.}Production of \emph{E} /\emph{Z} 11-14:acid and
\textbf{B.}\emph{Z} 11-16:acid by \emph{Y. lipolytica} containing
different moth fatty acyl-CoA desaturases. Quantification of fatty acids
was done after derivatization into methyl esters. Results are obtained
from three biological replicates. Error bars indicate standard
deviations.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Characterization of reductases. \textbf{A.} Profiles
of \emph{E} /\emph{Z} 11-14:OH produced by \emph{Y. lipolytica}
containing different reductases when cultivation media was supplemented
with a mixture containing equal amounts (500 mg/L) of\emph{E} /\emph{Z}
11-14:Me. \textbf{B.} Fatty alcohol profiles of\emph{Y. lipolytica}
strains expressing Lbo\_PPTQ together with different reductases. The
control strain has no reductase introduced. Results are obtained from
three biological replicates. Error bars indicate standard deviations.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Production of \emph{Z} 11-14:OH in small scale
cultivations and in 1 L fed-batch fermentation. \textbf{A.} Metabolic
engineering of \emph{Y. lipolytica} for improved production of\emph{Z}
11-14:OH. High myristate producing strain ST7982, containing mutation in
\selectlanguage{greek}α-\selectlanguage{english}subunit of the fatty acyl synthase complex
(Fas2p\textsuperscript{I1220F}) was used as background strain. Results
are obtained from three biological replicates. \textbf{B.} Fed-batch
fermentation results of ST9253. Data obtained for optical density (OD),
cell dry weight (DW), glycerol, and \emph{Z} 11-14:OH concentration are
represented as averages from three bioreactors and standard deviations
are calculated which are shown as error bars.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} GC-MS chromatogram representing fatty alcohol sample
before (above) and after (below) acetylation. Numbers in red represent
the amount of each fatty acetate (mg\selectlanguage{ngerman}±SD) in final sample. Measurements
were performed in technical triplicates. Error bars indicate standard
deviations.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Performance of biologically-derived \emph{O.
nubilalis} sex pheromone. \textbf{A.} Electrophysiological responses of
male \emph{O. nubilalis} antennae to biologically-derived pheromone
blend (BioPhe), standard compounds (\emph{Z} 11-14:OAc, \emph{E}
11-14:OAc and 14:OAc), and mixtures of the standard compounds. Response
to 100 ng of \emph{Z} 11-14:OAc was defined as 100\%, and all responses
normalized in \% relative to this reference. Means followed by the same
letter are not significantly different (P\textgreater{} 0.5, Tukey's
studentized range {[}HSD{]} test, F=51.650, df=5, P=0.000) \textbf{B} .
Behavioral responses of \emph{O. nubilalis} males to yeast-derived
pheromone blend (BioPhe), standard compounds (\emph{Z} 11-14:OAc,
\emph{E} 11-14:OAc, 14:OAc) and mixtures of the standard compounds. Phe
97:3 represents pheromone composition originally found in \emph{O.
nubilalis} Z-race and is generated from standard compounds \emph{Z}
11-14:OAc,\emph{E} 11-14:OAc in ratio 97:3. Means followed by the same
letter are not significantly different (P\textgreater{} 0.05, Tukey's
studentized range {[}HSD{]} test). Approach, F=4.579 df=3, P=0.005,
Landing, F=5.899, df=3, P=0.001)

\selectlanguage{english}
\FloatBarrier
\end{document}

