Turkish Journal of Biology
http://journals.tubitak.gov.tr/biology/
Research Article
Turk J Biol
(2017) 41: 213-219
© TÜBİTAK
doi:10.3906/biy-1606-10
Converted carotenoid production in Dunaliella salina by using cyclization inhibitors
2-methylimidazole and 3-amino-1,2,4-triazole
Arzu YILDIRIM*, İsmail Hakkı AKGÜN, Meltem CONK DALAY
Department of Bioengineering, Faculty of Engineering, Ege University, İzmir, Turkey
Received: 06.06.2016
Accepted/Published Online: 22.09.2016
Final Version: 20.02.2017
Abstract: Carotenoids are vital for most photosynthetic organisms because of their crucial role in prevention of the damage caused
by excess light or stress conditions like heat or nutrient deprivation. Some of them are also valuable for biotechnology wıth respect to
their colorful feature and antioxidant properties. In this study, a natural β-carotene producer, green microalga Dunaliella salina, was
evaluated for its potential to produce different valuable carotenoids through the inhibition of cyclization reactions in the carotenoid
pathway by the chemical inhibitors 2-methylimidazole (2MI) and 3-amino-1,2,4-triazole (Amitrol). 2MI was shown to be effective on
lutein accumulation in D. salina cultures grown at high temperature, without affecting cell viability. Addition of 2MI to the culture at
1 mM concentration resulted a 1.7-fold increase in lutein content in the cell with the highest amount of 3.45 pg/cell, and an associated
decrease in β-carotene content suggesting that this inhibitor is more effective on lycopene beta-cyclase activity, thus shifting the pathway
from β-carotene arm to the α-carotene direction. The results of this study may give the opportunity to use D. salina for the production
of valuable carotenoids other than β-carotene.
Key words: Dunaliella salina, carotenoids, chemical inhibitors, lutein
1. Introduction
Carotenoids are the group of accessory pigments helping
photosynthesis by absorbing light energy from sunlight at
certain wavelengths. Their colorful feature and antioxidant
properties make them commercially attractive for the
biotechnology industry. As photosynthetic organisms,
microalgae are a valuable natural source for carotenoid
production. One of the well-known species of green
microalgae is Dunaliella salina, showing the ability to
produce high amounts of natural β-carotene in its unicell
in stress conditions like high salt, high temperature, or
nitrogen starvation. The carotenoid pathway leading
to β-carotene production starts from the colorless
carotenoid, phytoene, and continues with the production
of linear lycopene molecule, which is a substrate for the
enzymes lycopene beta- (LCY-b) and lycopene epsilon
cyclases (LCY-e). LCY-b is responsible for the addition of
beta cyclic groups to the two ends of lycopene, forming
β-carotene. On the other hand, the actions of both LCY-e
and LCY-b add one epsilon and one beta ring to the ends
of lycopene, resulting in alpha-carotene, and hydrolysis of
this molecule leads to lutein production (Figure 1).
Most of the pigments in the carotenoid pathway
including xanthophylls have an industrial value, with
*Correspondence: [email protected]
β-carotene, astaxanthin, and lutein having the biggest
share in the market. Therefore, evaluating natural producer
organisms as a source of different pigments is an attractive
field of study.
Molecules such as imidazoles, pyridines, and nicotine
have been shown to inhibit cyclization of the lycopene
molecule, therefore; they have been used to regulate
carotenoid biosynthesis in different organisms. As a
cyclization inhibitor, nicotine was studied to produce
lycopene in both Dunaliella bardawil (Shaish et al.,
1990) and Dunaliella salina (Fazeli et al., 2009); however,
only trace amounts of the final product were observed.
Successful inhibition with nicotine was achieved in
another unicellular green alga, Chlorella regularis, only
under heterotrophic culture conditions (Ishikawa and
Abe, 2004). However, in all cases nicotine severely affected
the cell growth in high concentrations due to its toxicity.
Recently, another lycopene cyclase inhibitor, triethylamine,
was evaluated for lycopene accumulation in D. bardawil
(Liang et al., 2016). Although it was recorded at 0.5 pg/cell
level for this alga, cell growth was negatively affected by
the treatment even at the lowest concentrations (10 ppm).
Feofilova et al. (2006) used a nontoxic azine 2-amino-6methylpyridine (MAP) to block cyclization of lycopene
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Figure 1. A partial illustration of the carotenoid pathway.
in a filamentous fungus, Blakeslea trispora, another
natural producer of β-carotene in the joint cultivation of
its opposite mating types. Pegklidou et al. (2008) used a
slightly toxic inhibitor, 2-methyl imidasole (2MI), for the
same fungus, which resulted in a high lycopene yield in
the fungal culture. Similarly, another lycopene cyclization
inhibitor having slight toxicity, amitrole (3-amino-1,2,4triazole) resulted in the accumulation of lycopene in
leaves of maize with small amounts of β-carotene and
xanthophylls as well in high temperature (Dalla Vecchia
et al., 2001).
In the present study, D. salina was treated with the
chemical inhibitors 2MI and amitrole as cyclization
blocking agents. They were shown to be successful at
stopping cyclization reactions in different organisms
but had not been studied for D. salina. It was aimed to
convert the carotenoid pathway to different products,
and explore the potential of these two inhibitors for the
production of different valuable pigments from this
alga. Our experiments with 2MI resulted in a significant
214
shift to lutein from β-carotene in the culture with a 1.7fold increased accumulation with respect to the control
without disturbing cell viability. The results show that 2MI
is a useful inhibiting agent and could further be used in
studies for conversion of the carotenoid pathway.
2. Materials and methods
2.1. Dunaliella salina strain and stock preparation
The Dunaliella salina (EGEMACC-24) strain was obtained
from Ege University Microalgae Culture Collection,
Turkey. A 1-L stock culture was grown in a 2-L flask on an
incubator shaker at 100 rpm under 50 µmol photons/m2 s
of illumination with cool white fluorescent lamps at 12:12
photoperiod at 24 °C, and grown for 1 week up to 1 × 106
cell/mL, and then diluted 1:10 into a 250-mL flask for each
experiment.
2.2. Media and experimental conditions
Dunaliella medium (Mojaat et al., 2008) containing 1 mL
of 1000X trace element solution consisting of 185 mM
YILDIRIM et al. / Turk J Biol
H3BO3, 2 mM FeCl3, 7 mM MnCl2, 1 mM ZnCl2, 1 mM
CoCl2, 1 mM CuCl2, and 5 mM Na2EDTA was used as
growth medium at pH 7.5. Next 50 mL of cultures were
grown in 250-mL flasks on an incubator shaker at 100 rpm
under 50 µmol photons/m2 s of illumination with cool
white fluorescent lamps at 12:12 photoperiod between 28
and 35 °C for 21 days.
2.3. Cyclization inhibitors and culture growth
Amitrole and 2-methylimidazole (2MI) were supplied
by Sigma-Aldrich (St. Louis, MO, USA). Both inhibitors
were added to the cultures at final concentrations of 1, 2,
and 3 mM on day 1. The effect of the inhibitors on cell
growth was observed by cell count using a Neubauer
hemocytometer. Specific growth rate of the cultures was
calculated according to the following formula (Andersen,
2005):
µ = ln(X2) – ln(X1)/t2 – t1,
where µ is specific growth rate, X2 is the cell number/mL
on day 21 of growth, X1 is the 4th day 4, t2 and t1 represent
the time (day) of the growth.
2.4. Carotenoid extraction and U-HPLC analysis
Chlorophyll and total carotenoid extractions were
performed with acetone/water (80:20 v/v) quantified by
spectrophotometric calculations (Ultraspec 1100 pro)
according to Lichtenthaler and Buschmann (2001). In
order to characterize the carotenoid profile U-HPLC
analysis was performed in control and inhibitor-treated
cultures. Total pigments were extracted from 40 mL of
cultures and centrifuged at 2000 rpm for 5 min. Pellets
were resuspended in 4 mL of acetone (100%) and vortexed
for 5 min at maximum speed and centrifuged again for
5000 rpm for 5 min. Solvent phase-carrying dissolved
carotenoids were transferred to glass vials and dried in
a SpeedVac Concentrator vacuum evaporator (Thermo
Scientific) at 35 °C. The residue was dissolved in 1 mL
of acetone for the analysis. Standard molecules for lutein
and β-carotene were purchased from ChromaDex Corp.
U-HPLC analysis was performed according to Yildirim et
al. (2014).
2.5. Statistical analysis
Three independent data sets for each experimental
condition were evaluated for lutein and β-carotene
accumulation. The mean values from each inhibitor were
compared to the control group by t-tests analysis using
Microsoft Excel, and P-values below 0.005 (P < 0.005)
accepted as statistically significant.
3. Results
3.1. Effect of chemical inhibition on cell viability
Chemical inhibitors in 1, 2, and 3 mM concentrations
were applied to D. salina cultures in order to obtain the
optimum concentrations for culture growth and carotenoid
accumulation. 2MI at 2 and 3 mM concentrations (2MI2 and 2MI-3) was highly toxic to the cells, so that the
cultures were almost completely bleached after day 10 of
the experiment. On the other hand, 1 mM of the same
inhibitor (2MI-1) resulted in a slight decrease in cell
number (2.9 × 106 cell/mL) with respect to the control
(3.3 × 106 cell/mL) (Figure 2). All three concentrations of
amitrole (A-1, A-2, and A-3) were tolerable for the culture
growth, with 3 mM (A-3) being the most inhibitive (Figure
3). Specific growth rates for 2MI-1, A-1, and A-2 had no
harmful effect on culture growth, and were slightly lower
than the control, being 0.112, 0.109, and 0.096, respectively
(Table).
3.2. Effect of chemical inhibitors on chlorophyll and
carotenoid content
Since 2MI-2, 2MI-3, and A-3 caused inhibition on culture
growth, total carotenoid and chlorophyll contents were
affected by the chemical application likewise. However,
total carotenoid contents for 2MI-1, A-1, and A-2 were
almost constant compared to the control (Figure 4).
Although 2MI-1 resulted in a slight decrease in chlorophyll
accumulation, it was not significantly different from the
control culture. Similarly, A-1- and A-2-treated cultures
had no negative effect on chlorophyll or carotenoid
accumulation; hence cultures were able to preserve cell
viability during production.
3.3. Characterization of the carotenoid profile
The efficiency of inhibitors at the stated concentrations was
identified by U-HPLC analysis. Lycopene accumulation
Figure 2. Cell growth for the control (C) and 2-methylimidazoletreated cultures at 1 mM (2MI-1), 2 mM (2MI-2), and 3mM
(2MI-3) concentrations.
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Table. Growth rates of control and inhibited cultures.
Figure 3. Cell counts based on amitrole treatment at 1 mM (A-1),
2 mM (A-2), and 3 mM (A-3) concentrations versus the control
(C).
was not observed in the 2MI- or amitrole-treated cultures
(Figure 5). The amount of lutein with 2MI-1 significantly
increased from 6.682 mg/L to 10.038 mg/L, and per cell
lutein content rose from 2.025 pg/cell to 3.455 pg/cell,
giving the highest level of lutein accumulation (1.7-fold)
with respect to the control. However, cultures with amitrole
were inefficient in converting the pathway through lutein
accumulation (Figure 6). All 2MI-1, A-1, and A-3 cultures
resulted in a reduction in β-carotene content almost at the
same ratio (1.6-fold) with the increase observed for lutein
with 2MI-1 (Figure 7).
4. Discussion
β-Carotene and lutein are two important pigments mainly
protecting the cell from reactive singlet oxygen and triplet
chlorophyll formations caused by extreme light access
that occurs during photosynthesis (Telfer, 2002; Jahnz and
Holzwarth, 2012).
Dunaliella salina is a well-known natural source
for β-carotene (Johnson and Schroeder, 1995; Pulz and
Gross, 2004; Ye et al., 2008), and the alga produces this
pigment as a response to the extreme stress conditions
that occur during summer (Del Campo, 2007). Light
stress is the major promoter and high temperature is one
of the conditions that triggers carotenoid biosynthesis
in photosynthetic organisms, and it is also stated to be
effective on carotenoid production, mainly for lutein and
216
Name and concentration of the
chemical inhibitor
Specific growth rate (µ)
2MI-1
0.112
A-1
0.109
A-2
0.096
A-3
0.038
Control
0.132
β-carotene synthesis in Dunaliella sp. (Guedes et al., 2011).
In the present study, high temperature was chosen as
a stress condition for the accumulation of carotenoids in
Dunaliella salina. Culture conditions were effective on
carotenoid production as observed in the control culture.
However, both of the inhibitors resulted in a decrease in
β-carotene accumulation (Figure 7). Although the cyclic
group addition to the linear lycopene molecule was
the target activity for chemical inhibition, no lycopene
accumulation was observed in any of the inhibited cultures.
It has also been shown in tomato (Shaish et al., 1990) and
Arabidopsis (Cunningham et al., 1996) that temperatures
above 30 °C may inhibit lycopene accumulation but not
β-carotene production in the cell. This was explained by an
alternative pathway, where neurosporene, an intermediate
molecule in lycopene synthesis, is the precursor for cyclic
carotenoid formation instead of lycopene (Shaish et al.,
1990; Cunningham, 1996) (Figure 1). Based on the studies
with D. bardawil with different cyclization inhibitors
(nicotine, CPTA, and MPTA) Shaish et al. (1990) suggested
that both pathways are active for β-carotene biosynthesis
in this alga.
Between the two chemical inhibitors used in this
study, 2MI (imidazole) and amitrole (triazole) both
belong to the azole group, and, in general, imidazoles
and triazoles differ from each other with respect to the
mechanism of inhibition of the cytochrome P450 enzyme,
by whether the molecule binds to the heme iron atom of
ferric cytochrome P450 from the N3 position or the heme
group from the N4 position, respectively (Riviere and
Papich, 2009). Cytochrome P450 is an important enzyme
that functions as a catalyzer for O2 cleavage and binding
in many biochemical reactions, and also playing an
important role in photosynthesis as an electron receiver in
plants (Lassen et al., 2014). Amitrole has been previously
shown to cause severe damage to the chloroplast of wheat
leaves (Dalla Vecchia et al., 2001) and barley (Agnolucci et
al., 1996) in cold temperature (20 °C); hence it negatively
affects photosynthesis and growth. On the other hand,
high temperature (30 °C) resulted in an increase in
carotenoid biosynthesis along with chlorophylls in both
YILDIRIM et al. / Turk J Biol
Figure 4. Comparison of total chlorophylls (CHLs) and carotenoids (CARs) accumulated during
inhibitor treatment in high temperature conditions.
Figure 5. Carotenoid profiles showing lutein and β-carotene accumulations in the control (left column upper row), 2MI-1 (left
column lower row), A-1 (right column upper row), and A-2 (right column lower row).
plants, which corresponds to our study. Dalla Vecchia et
al. (2001) explained that effect with possible temperaturesensitive steps in the carotenoid pathway; therefore, it can
be concluded that the temperature of choice may act as
identifier for the direction of carotenoid biosynthesis.
As a chemical inhibitor for cyclization of lycopene,
nicotine was used in several studies previously; however, it
negatively affected cell growth due to its highly toxic nature
(McDermott et al., 1973; Fazeli et al., 2009; Doddaiah et
al., 2013). Lycopene accumulation in an algal culture by
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Figure 6. Lutein concentrations obtained from U-HPLC analysis.
Figure 7. β-Carotene concentrations obtained from U-HPLC analysis.
nicotine inhibition was only achieved in a heterotrophically
grown Chlorella regularis Y-21, a high carotenoid producer
mutant (Ishikawa and Abe, 2004). Our previous attempts
to grow D. salina in the presence of nicotine resulted in
complete bleaching of the cells (data not shown). Therefore,
a less toxic inhibitor, 2-methylimidazole, was chosen to
convert the carotenoid pathway along with amitrole. In
our study, inhibition of carotenoid biosynthesis in D. salina
culture with 2MI between the temperatures 28 and 35 °C
showed similar effects observed by Doddaiah et al. (2013)
for Dunaliella bardawil using glyphosate and DCMU as
inhibiting agents under high light stress. Both chemicals
resulted in increased levels of lutein accumulation and a
decrease in β-carotene. We observed the same effect with
2MI-1, indicating that this inhibitor might be more effective
on lycopene beta cyclase than lycopene alpha cyclase, and
it directs the pathway to the α-carotene branch followed by
lutein synthesis.
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Lutein is a high value pigment with a known effect on
vision and eye-related diseases (Abdel-Aal et al., 2013).
Many algal species including Chlorella sp. (Del Campo et
al., 2000; Jeon et al., 2014), Muriellopsis sp., and Tetracystis
sp. (Del Campo et al., 2000) were evaluated for their
potential on lutein production. The highest accumulation
of lutein was achieved under high light conditions at 28 °C
with Muriellopsis sp., and high temperatures were stated to
stimulate carotenogenesis in this alga as well.
We observed a shift of the carotenoid pathway towards
lutein accumulation without any negative effect on culture
growth with 1 mM concentration of 2MI. Compared
to Dunaliella salina, both Chlorella and Muriellopsis
have higher growth rates, and both species have more
advantages with respect to overall culture yield. However,
our result for per cell lutein accumulation with 2MI1 was almost 7 times higher (3.445 pg/cell) than the
results obtained with Muriellopsis sp. (0.51 pg/cell) by
YILDIRIM et al. / Turk J Biol
Del Campo et al. (2000). In order to evaluate and use D.
salina efficiently for lutein production, a two-phase system
focusing on biomass production in the first stage and
lutein accumulation in the second would be a valuable
approach, as previously successfully applied for β-carotene
production in D. bardawil by Ben-Amotz et al. (1995) and
Hejazi et al. (2004).
In the present study, our results showed that 2MI-1
is promising and can be used as a tool for conversion of
carotenoid biosynthesis in D. salina without disturbing cell
viability. The results of this study may give the opportunity
to use D. salina as a host for the production of valuable
carotenoids other than β-carotene, such as lutein.
Acknowledgment
This work was supported by the projects from the Turkish
General Directorate of Agricultural Research and Policy
(TAGEM/10/ARGE/06).
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