impact of methyl jasmonate on squalene bio synthesis in microalga

8/3/2019 Impact of Methyl Jasmonate on Squalene Bio Synthesis in Microalga

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Short communication

Impact of methyl jasmonate on squalene biosynthesis in microalgaSchizochytrium mangrovei

Cai-Jun Yue a,b, Yue Jiang b,*a College of Life Science and Biotechnology, Heilongjiang August First Land Reclamation University, Daqing 163319, PR Chinab Department of Biology and Kwong Living Trust Food Safety & Analysis Laboratory, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China

1. Introduction

Squalene is an important intermediate in the cholesterol

biosynthetic pathway. It is an essential natural antioxidant to

protect the cells from free radicals and reactive oxygen species. It

plays a major role in releasing oxidative stresses, such as sunlight

exposure [1]. Squalene is also effective in decreasing serum

cholesterol level and has cardioprotective effect on myocardial

infarction in experimental animals [2,3]. Experiments in vitro and

in animal models have shown that squalene can effectively inhibit

colon, lung and skin tumourigenesis in rodents. In some regions,

squalene is a popular folk medicine for chronic liver diseases [4].

Squalene has been regarded as an effective chemopreventive agent

and has the great potential to be used in clinical trial [57].

Thetraditional sources of squaleneare liveroils of deep-seasharks

and whales. Large-scale extraction of squalene from marine animals,however, is nearly impossible as a result of international concernover

the protection of endangered marine animals [8]. Furthermore,

although the application of squalene in cosmetics is in great

quantities, some leading cosmetic companies are phasing out the

use of shark liver oil and other animal-based squalene in their

products in responseto thegrowing pressurefrom theenvironmental

campaign. The potential increasing demand for squalene therefore,

has led to a worldwide interest in searching for other novel and

sustainable sources of this compound. Recently we found the

microalgaSchizochytrium mangrovei, a unicellular thraustochytrid

could be usedas potential producer of squalene[9]. Becausemicrobial

production of squalene using fermentation technology has been

regardedas themostcost-effective strategy forthestable commercial

supply of thisusefulproduct. To furtherincreasethe squalenecontent

of this microalga, in addition to optimize the environmental culture

conditions, the metabolic regulation on the activities of key enzymes

in the squalene biosynthetic pathway is worthy of investigation.

Jasmonates and its methyl ester methyl jasmonate (MJA) are

always considered as potent lipid regulators that modulate various

physiological processes in plants, such as growth, senescence,

reproduction and responses to both mechanical trauma and

pathogenesis [10,11]. Up to now, MJA has been successfully

applied to induce or increase the biosyntheses of many importantsecondary metabolites in plant cell, e.g. ginsenoside and paclitaxel

[1215] that have been widely used by human beings as drugs and

nutraceuticals. However, there has been no such report on the

effect of methyl jasmonate on the activities of the key enzymes in

unicellular microalga for the production of useful metabolite.

Moreover, the information on the effect of methyl jasmonate on

the biosynthesis of essential intracellular intermediate involved in

the primary metabolism is very limited.

In squalene biosynthetic pathway, acetoacetyl-CoA generated

through glycolysis is used to synthesize 3-hydroxy-3-methylglu-

taryl-CoA, which is further converted to farnesyl diphosphate

Process Biochemistry 44 (2009) 923927

A R T I C L E I N F O

Article history:

Received 6 October 2008Received in revised form 19 March 2009

Accepted 26 March 2009

Keywords:

Methyl jasmonate

Squalene synthase

Schizochytrium mangrovei

Squalene

Cholesterol

A B S T R A C T

Squalene is an effective antioxidant and a potential chemopreventive agent. In this work, the effect of

methyl jasmonate (MJA) on squalene biosynthesis in microalga Schizochytrium mangrovei was

investigated. The maximum squalene content (1.17 0.06 mg/g cell dry weight, DW) reached during

the next 3 h after MJA treatment (0.1 mM) at 48 h of cultivation, which was 60% higher than that of control.

The activity of squalene synthase (SS) increased 2-fold over control at this point. The maximum cholesterol

content of 0.45 0.03 mg/g DW was reached at hour 51 when MJA concentration was 0.4 mM, whereas the

squalene content was lower at this point. The observations suggested that the increased squalene content

was resulted from an increased activity of SS. MJA could be used to regulate the key enzymes in squalene

biosynthetic pathway for the increased production of this compound in thraustochytrids. This research also

provided novel information on the stimulation effect of methyl jasmonate on the biosynthesis of essential

intermediate involved in the primary metabolism in microorganism.

2009 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +852 3411 7062; fax: +852 3411 5995.

E-mail address: [email protected] (Y. Jiang).

Contents lists available at ScienceDirect

Process Biochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o

1359-5113/$ see front matter 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2009.03.016
mailto:[email protected]://www.sciencedirect.com/science/journal/13595113http://dx.doi.org/10.1016/j.procbio.2009.03.016http://dx.doi.org/10.1016/j.procbio.2009.03.016http://www.sciencedirect.com/science/journal/13595113mailto:[email protected]


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through several steps. Squalene is then synthesized directly from

farnesyl diphosphate. In cholesterol biosynthetic pathway, squa-lene is the last metabolite preceding sterol ring formation [16].

Within the steps, squalene synthase (EC 2.5.1.21, SS), which

catalyzes a reductive dimerization of two farnesyl diphosphate

(FPP) molecules into squalene, represents a putative branch point

in the isoprenoid biosynthetic pathway diverting carbon flow

specifically to the biosynthesis of sterol and triterpene. It is

therefore considered as a key regulatory point for squalene

metabolism. However, no such information is available on the

regulation of SS for squalene biosynthesis in thraustochytrids.

In this study, the responses of S. mangrovei to the addition of

MJA for squalene biosynthesis were investigated. The content of

squalene, the specific SS activity and cholesterol content in S.

mangrovei under different conditions were analyzed, because

cholesterol is one of the major sterols of thraustochytrids for cellgrowth [17]. Our work aimed to investigate the effectiveness of

MJA on squalene biosynthesis in S. mangrovei in order to propose a

proper strategy to increase the production of squalene by this

microalga. The possibility of using enzyme stimulator on the large

amount accumulation of the indispensable intermediate (i.e.

squalene) whose metabolites are essential for cell growth would

be evaluated. Moreover, the resultfrom this study will also provide

important information on the application of plant elicitorMJA on

the enhanced biosynthesis of primary metabolite in microalgae.

2. Materials and methods

2.1. Heterotrophic growth of S. mangrovei

S. mangrovei used in this study wasisolatedfromdecaying Kandelia candel leaves

from local mangroves in Sai Keng, Hong Kong and maintained on agar slantaccording to the methods described by Fan et al. [18]. The inoculum and culture

were prepared in 250-mL Erlenmeyer flasks (each containing 100 mL of medium)

and incubated at 22 8C in an orbital shaker at 120 rpm in the dark. The medium

compositionwas same asthatdescribed inJianget al.[9]. The inoculum volume was

5% (v/v).

2.2. Methyl jasmonate (MJA) treatment

MJA (0.1 M) was dissolved in dimethyl sulfoxide (DMSO) and sterilized by

passing through 0.22mm polyvinylidenedifluoride (PVDF) syringe filter (Millipore)

before adding to the cell culture. The culture added with same amount of DMSO

without MJAwas used ascontrol.Eachtreatment wasrepeated forthree times.Data

were expressed as mean plus standard deviation of triplicates.

2.3. Determination of cell dry weight (DW)

The cell dry weight was determined according to Fan et al. [18].

2.4. Analyses of squalene and cholesterol contents

The cellular squalene and cholesterol contents were analyzed according to Lu

et al. [19] and Saldanha et al. [20] with some modifications. Freeze-dried cells

(100 mg) were saponified by 4 mL of 10% (w/v) KOH-75% (v/v) ethanol solution at

50 8C for 15 min. The mixture was extracted with 4 mL of hexane for three times.

The hexane layer was collected and evaporated to dryness under N2. The residue

was dissolved in 2.0 mL of acetonitrile for subsequent HPLC analysis. Waters 2695

HPLC (Waters, Milford, MA, USA) equipped with a Waters 2996 photodiode array

detector and a reversed-phase Superspher C18 column (150 4.0 mm i.d. 5mm,

Merck) were used. The separation of squalene and cholesterol was achieved by

usinga mobilephaseof 100% acetonitrileat a constantflow rate of 1.5 mL/minandamobile phase of 100% methanol at a constant flow rate of 1.0 mL/min, respectively.

The squalene and cholesterol were monitored at wavelength of 195 and 205 nm,

respectively. The peaks of squalene and cholesterol were identified based on their

retentiontime and UV spectra against those obtained fromsqualeneand cholesterol

standards, respectively (Sigma Chemical Co., USA). The quantification was done

according to the external calibration graphs obtained from the peak areas vs.

different concentrations of squalene and cholesterol standards, respectively. All

samples and standards were filtered through 0.45 mm membrane filters before

injection.

2.5. Preparation and assay of squalene synthase (SS)

The assay was performed as described by Okada et al. [21] with some

modifications. Briefly, the cell pellets harvested from100 mL brothwere suspended

in 45 mL of solution A consisting of 50 mM TrisHCl (pH 7.2), 5 mM 2-

mercaptoethanol and 20 mM MgCl2. The suspension was centrifuged (4000 g)

at 4 8C for 10 min. The cell pellet (1 g) was frozen by liquid nitrogen andhomogenized in ice bath with 5 mL of extraction buffer consisting of 250 mM Tris

HCl buffer (pH 7.2), 250 mM sucrose, 5 mM 2-mercaptoethanol, 20 mM MgCl2 and

1 mM PMSF. The homogenate was centrifuged at 5000 g for 10 min at 4 8C. The

supernatant was collected and filtered through three layers of gauze. The filtrate

was used as enzyme extract for subsequent SS assay. The reaction mixture (0.2 mL)

consisted 0.15 mL of solution B (250 mM TrisHCl buffer (pH 7.2), 10 mM NADPH,

10 mM MgCl2, 2.5 mM 2-mercaptoethanol, 0.23 mM farnesyl diphosphate) and

0.05 mL of enzyme extract. The one without the addition of farnesyl diphosphate

was used as blank. The reaction was kept at 35 8C for 30 min and terminated by

adding 0.4 mL n-hexane followed by mixing and centrifugation at 15,000g for

3 min. The hexane was collected and the residue reaction mixture was extracted

with 0.4 mL hexane twice. The hexane layers were combined and evaporated to

dryness under N2. The residue was re-dissolved in 0.04 mL of acetonitrile for

squalene analysis as indicated above. The amount of squalene converted from

farnesyl diphosphate catalyzed by SS was obtained by subtracting the squalene

content of the blank from that of the reaction mixture with the addition of farnesyl

diphosphate.The specific activity of SS wasexpressed as nmol squalene/mg protein(Pr)/h. The protein content was measured by Bradford method [22] with bovine

serum albumin (Sigma Co., USA) as standard.

3. Results and discussion

3.1. Influence of MJA on the growth and squalene biosynthesis in S.

mangrovei

As no effect of DMSO (ranging from 0.05 to 0.4 mL in every

100 mL culture broth) on the growth, squalene and cholesterol

contents as well as the activity of SS in S. mangrovei was observed

(data not shown), DMSO was selected as the solvent to dissolve

MJA in this study. Effects of MJA on the growth and squalene

biosynthesis of S. mangrovei were investigated by adding MJAranging from 0 to 0.4 mM after 48-h cultivation (Tables 1 and 2).

For cell growth, the cell dry weight (DW) at hours 60 and 72 were

apparently lower than that of control when the concentration of

MJA was 0.4 mM. But no obvious difference was observed when

the concentrationof MJAranged from 0.05 to 0.2 mM (Table 1). The

squalene content was greatly increased after low concentration

MJA treatment but decreased when MJA concentration was high

(Table 2). The maximal squalene content of 1.17 0.06 mg/g DW

wasreached,whichwas 1.6-fold of that of control, duringthe next 3 h

after MJA (0.1 mM) treatment at cultivation hour of 48.

During MJA treatment, squalene content in S. mangrovei

increased rapidly (60% increment compared to the control) after

the treatment of 0.1 mM MJA at 48 h. But the incrementcould only

be maintained for a short period of time. This might be due to the

Fig. 1. Responses of squalene synthase (SS) of S. mangrovei to MJA addition at

cultivation hour of 48.Symbols:(^) 0 mM MJA;(~) 0.1 mM MJA;(&) 0.4 mMMJA.

C.-J. Yue, Y. Jiang/ Process Biochemistry 44 (2009) 923927924


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indispensability of squalene and its metabolites are essential for

cell growth. If we further increased the treatment concentration of

MJA, however, the growth inhibition was observed. If MJA was

added at cultivation time of 24 and 72 h, respectively, the effects of

MJA on squalene biosynthesis and cell growth ofS. mangrovei were

similar to those obtained from MJA treatment at 48 h (data not

shown). MJA was an effective elicitor in plant cell to induce the

increased accumulation of terpenoid, phytoecdysteroid, etc.,

through terpene synthesis and mevalonic pathway [2325]. The

results from this study suggested that MJA did have effects on the

growth and squalene synthesis of S. mangrovei and the influence

might be MJA-dose dependent. However, the influence of MJA onsqualene biosynthesis in microalga has not yet been investigated

although theinhibitory effectof MJA on cell growth was previously

observed in mycorrhizal fungi [26] and plant cells [20]. To further

understand how MJA affected squalene biosynthesis,the activity of

squalene synthase and cholesterol content were investigated upon

the addition of MJA. Because SS directly catalyzes the conversionof

farnesyl diphosphate to squalene which can be further converted

to cholesterol.

3.2. Influence of MJA on cholesterol content and activity of squalene

synthase (SS)

The effects of different concentrations of MJA on cholesterol

content in S. mangrovei are shown in Table 3. At cultivation time of

51 h, thecholesterol contentwas increasedwith an increase of MJA

level from 0 to 0.4 mM. The highest cholesterol content

(0.45 0.03 mg/g DW) was reached when MJA concentration was

0.4 mM, whereas the squalene content was the lowest in the same

culture.

Fig. 1 shows the activities of SS in cells treated with MJA (from

0.1 to 0.4 mM) 48 h after cultivation. When the concentration of

MJA was 0.1 mM, the specific activity of SS reached the highest

(3.62 0.36 nmol/mg Pr/h) when culture was 51 h old, which was 2-

fold than that of control. At the same time, squalene content reached

the highest as shown in Table 2. In squalene biosynthetic pathway,

squalene synthase catalyzes the condensation of two molecules offarnesyl diphosphate for the generation of presqualene diphosphate

which subsequently converts to squalene. This is the first committed

step and the specific reaction in cholesterol biosynthesis [27]. It was

pointed out that MJA treatment could induce the activation of the

transcripts of SS and increase the activity of SS in plant cells [28]. Our

result was consistent with previous reports. However, the specific SS

activity ofS. mangroveitreated with 0.4 mM MJAwas also increasedat

hour 51 although the increment was not comparable to those cells

treated with 0.1 mM MJA. These results indicated that higher content

of squalene wasclosely related to higheractivityof SS whereas higher

activity of SS would not definitely lead to higher content of squalene.

This might be due to the activation of downstream steps in

cholesterol biosynthetic pathway by high concentration of MJA as

shown in Fig. 2. In animal cells, it was indicated that the increased SS

Table 2

Effects of different MJA concentrations on the squalene content of S. mangrovei.

MJAa concentration (mM) Squalene content (mg/g DW)

24 (h) 48 (h) 51 (h) 54 (h) 60 (h) 72(h) 96(h)

0 0.45 0.02 0.68 0.03 0.74 0.03a 0.78 0.04a 0.82 0.06a 0.67 0.04 0.12 0.01

0.05 0.45 0.02 0.68 0.03 0.87 0.04b 0.75 0.05a 0.76 0.05a 0.66 0.03 0.12 0.01

0.1 0.45 0.02 0.68 0.03 1.17 0.06c 0.90 0.05bc 0.94 0.06b 0.69 0.05 0.13 0.01

0.2 0.45 0.02 0.68 0.03 0.84 0.05b 0.83 0.04b 0.79 0.04a 0.65 0.04 0.13 0.01

0.4 0.45 0.02 0.68 0.03 0.69 0.03 0.76 0.05a 0.77 0.03a 0.64 0.04 0.11 0.01

Meanswith the sameletters(ac) notedin a single columnare not significantly differentaccordingto Tukeys Honestly SignificantDifferencemultiple-comparisontest withafamily error rate of 0.05.

a Addition of MJA at cultivation hour of 48.

Table 3

Influences of different MJA concentrations on the cholesterol content of S. mangrovei.

MJAa concentration (mM) Cholesterol content (mg/g DW)

24 (h) 48 (h) 51 (h) 54 (h) 60 (h) 72(h) 96(h)

0 0.54 0.02 0.34 0.02 0.33 0.02a 0.33 0.03a 0.34 0.02 0.37 0.03 0.51 0.03

0.05 0.54 0.02 0.34 0.02 0.35 0.02ab 0.33 0.02a 0.32 0.02 0.35 0.02 0.51 0.04

0.1 0.54 0.02 0.34 0.02 0.36 0.02bc 0.35 0.02ab 0.33 0.02 0.34 0.03 0.52 0.02

0.2 0.54 0.02 0.34 0.02 0.39 0.03c 0.37 0.02bc 0.34 0.02 0.36 0.02 0.51 0.04

0.4 0.54 0.02 0.34 0.02 0.45 0.03cd 0.37 0.02bc 0.35 0.04 0.37 0.03 0.51 0.03

Meanswith thesame letters (ad) noted in a singlecolumn arenot significantlydifferent accordingto Tukeys Honestly Significant Difference multiple-comparison test with

a family error rate of 0.05.a

Addition of MJA at cultivation hour of 48.

Table 1

Effects of different MJA concentrations on the growth of S. mangrovei.

MJAa concentration (mM) Cell dry weight (g/L)

24 (h) 48 (h) 51 (h) 54 (h) 60 (h) 72(h) 96(h)

0 1.97 0.09 5.67 0.26 6.02 0.28 6.12 0.34 7.64 0.36a 9.60 0.44a 11.4 0.6

0.05 1.97 0.09 5.67 0.26 5.85 0.28 6.12 0.29 7.50 0.45ab 9.33 0.41ab 11.2 0.5

0.1 1.97 0.09 5.67 0.26 5.82 0.25 6.35 0.35 7.60 0.30a 9.22 0.47ab 11.4 0.6

0.2 1.97 0.09 5.67 0.26 5.83 0.35 6.40 0.34 7.40 0.34ab 9.10 0.44ab 11.3 0.7

0.4 1.97 0.09 5.67 0.26 5.79 0.27 6.32 0.32 7.11 0.33b 9.04 0.42b 11.0 0.6

Means with the same letters (a and b) noted in a single column are not significantly different according to Tukeys Honestly Significant Difference multiple-comparison test

with a family error rate of 0.05.a Addition of MJA at cultivation hour of 48.

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activity in liver of male mice led to an increase of both squalene and

cholesterol syntheses [29]. MJA could not only stimulate mRNA level

and the activity of oxidosqualene cyclase which catalyzes the

conversion of oxidosqualene to lanosterol in Glycyrrhiza glabra

[30,31], but also the mRNA level of squalene epoxidase which

catalyzes the second committed step in cholesterol biosynthesis

converting squalene to oxidosqualene in Panax ginseng [32]. On the

other hand, squalene synthetase represents an ideal site for selective

inhibition of cholesterol without interfering with the production of

key nonsterol compounds that play important roles in the regulationof cellular growth and metabolism. It has been demonstrated clearly

that zaragozic acid A (ZGA-A) could effectively inhibit SS activity

through competitive inhibition followed by mechanism-based

irreversible inactivation [33]. In our study, with the addition of

ZGA-A at concentrations of 0.1 and 1.8mM when the cell was

cultivated for 36 h, the squalene contents decreased drastically about

52.5% and 70% compared to the control, respectively (data not

shown). It was postulated that the biosynthesis of squalene could be

enhanced by regulating the activity of squalene synthase through the

adding of MJA in S. mangrovei. MJA could be used to stimulate the

accumulation of not only the secondary metabolite in plant cell but

also the primary metabolite in microorganism through its activity on

the key enzymes. The result also showed that the production of

essential intermediate involved in the primary metabolism in

microalgae could be enhanced by the application of certain plant

elicitor.

In this study we found the MJA treated cells became yellow in

the later stage of cultivation. We hypothesized that MJA might

affect the further steps in isoprenoid pathway, such as carotenoids

synthesis [34]. The other works will be done to further study the

co-effects of SS stimulatorsand the inhibitors of other keyenzymes

in cholesterol biosynthetic pathway on the accumulation of

squalene in S. magrovei.

Acknowledgment

This research was supported by the RGC (the Research Grants

Council of Hong Kong SAR).

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impact of methyl jasmonate on squalene bio synthesis in microalga

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