Light Intensity Regulates LC-PUFA Incorporation into Lipids of

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Light intensity regulates the LC-PUFA incorporation into lipids of Pavlova lutheri and the final desaturase and elongase activities involved in their biosynthesis. Freddy Guihéneuf, Virginie Mimouni, Gérard Tremblin, and Lionel Ulmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504863u • Publication Date (Web): 15 Jan 2015 Downloaded from http://pubs.acs.org on January 20, 2015

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Journal of Agricultural and Food Chemistry

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Light intensity regulates the LC-PUFA incorporation into lipids of Pavlova lutheri and the

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final desaturase and elongase activities involved in their biosynthesis.

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Freddy Guihéneuf,† Virginie Mimouni,‡ Gérard Tremblin,‡ and Lionel Ulmann,*,‡

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†

Botany and Plant Science, School of Natural Sciences, Ryan Institute, National University of

Ireland Galway, Galway, County of Galway, Ireland

9 10

‡

PRES LUNAM, Université du Maine, Institut Universitaire Mer et Littoral FR-3473 CNRS,

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EA 2160 Mer Molécules Santé, UFR Sciences et Techniques, 72085 Le Mans Cedex, IUT

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Génie Biologique, 53020 Laval Cedex, Pays de la Loire, France

13 14 15 16 17 18 19 20 21 22 23

Corresponding author

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*(L.U.) Phone: + 33 2 43 59 49 59. Fax + 33 2 43 59 49 58. E-mail: Lionel.Ulmann@univ-

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lemans.fr

ACS Paragon Plus Environment

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ABSTRACT

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The microalga Pavlova lutheri is a candidate for the production of omega-3 long-chain

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polyunsaturated fatty acid (LC-PUFA), due to its ability to accumulate both eicosapentaenoic

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and docosahexaenoic acids. Outstanding questions need to be solved to understand the

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complexity of n-3 LC-PUFA synthesis and partitioning into lipids, especially its metabolic

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regulation, and which enzymes and/or abiotic factors control their biosynthesis. In this study,

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the radioactivity of 14C-labeled arachidonic acid incorporated into the total lipids of P. lutheri

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grown under different light intensities and its conversion into labeled LC-PUFA were

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monitored. Our results highlighted for the first time the light-dependent incorporation of LC-

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PUFA into lipids and the light-dependent activity of the final desaturation and elongation

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steps required to synthesize and accumulate n-3 C20/C22 LC-PUFA. The incorporation of

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arachidonic acid into lipids under low light, and the related ∆17-desaturation activity

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measured, explain the variations in fatty acid profile of P. lutheri, especially the accumulation

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of n-3 LC-PUFA such as EPA under low light conditions.

40 41

KEYWORDS

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Pavlova lutheri ; Marine microalga; In-vivo 14C-labeling; n-3 LC-PUFA biosynthesis; LC-

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PUFA incorporation; ∆17-desaturation; Light intensity.

44 45

INTRODUCTION

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Omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) such as eicosapentaenoic

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(EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3) acids have received growing interest

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due to their beneficial health effects.1,2 Currently the main source of these bioactive

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compounds is marine fat fish and fish oils.3 However, due to the constant depletion of fish

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stock caused by overfishing, alternative sources such as microalgae, which may be more


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sustainable via cultivation, are extensively explored to meet the increasing global demand for

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n-3 LC-PUFA.4,5

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Algal-derived n-3 LC-PUFA and their implications for human health have been recently

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reviewed, showing the potential of microalgae as important alternative to fish oil for human

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nutrition, particularly as functional foods playing important roles in early development and

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the prevention and treatment of chronic diseases, notably inflammatory and related

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cardiovascular diseases.6

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Although n-3 LC-PUFA production from autotrophic microalgae is technically possible, the

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metabolic and genetic mechanisms which regulate the major pathways involved in LC-PUFA

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biosynthesis and accumulation are still incompletely described and requires further

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investigations.7,8

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The metabolic plasticity of algae allows them to adapt quickly to changing environmental

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factors.9 It is known that environmental factors (i.e. temperature, salinity, light, nutrients) and

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culture time affect the growth and biochemical composition of microalgae.10-14 In most

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microalgae, environmental conditions promoting n-3 LC-PUFA biosynthesis are usually not

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optimal for growth such as low temperature;15,16 and accumulation of triacylglycerols (TAG)

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occurs mainly during stress, inducing cessation of cell-growth such as nutrient-depletion.17,18

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In addition, LC-PUFA are mainly accumulated in complex polar lipids (i.e., glycolipids and

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phospholipids) constituting the membranes which are intrinsically limited,19 while storage

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lipids are predominantly constructed of saturated (SFA) and monounsaturated (MUFA) fatty

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acids.20,21 Only few species (e.g., Parietochloris incisa, Pavlova lutheri, Nannochloropsis

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oculata, Thalassiosira pseudonana, and Phaeodactylum tricornutum) have the ability to

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accumulate lipids and TAG containing n-3 LC-PUFA.21-24 These species are therefore

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prominent candidates for n-3 LC-PUFA-oil production by autotrophic microalgae.


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Pavlova lutheri, a marine microalga commonly used in aquaculture is a well-known source of

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n-3 LC-PUFA, such as EPA and DHA, reaching up to 53% of its total fatty acid (TFA) under

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specific conditions such as during exponential growth using low light.14 Recently, it has been

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demonstrated that the ability of P. lutheri to accumulate lipid and TAG containing n-3 LC-

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PUFA rely mainly on inorganic carbon availability after nitrate-depletion.24 In this work, the

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authors suggested that bicarbonate supplementation represents a promising strategy to

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stimulate growth and trigger omega-3 enriched oil production in this specie.

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Several metabolic studies using lipid and fatty acid analysis14,21,24-26 or radiolabeled fatty

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acids,22,27,28 as well as molecular studies29-31 have already been performed to investigate the

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effect of growth conditions on lipid and fatty acid composition in microalgae, as well as to

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measure at a transcriptional level the expression of different desaturases and elongases

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involved in n-3 LC-PUFA biosynthesis.29,32,33 As an example, it has been demonstrated that

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the expression of the LC-PUFA front-end desaturase from P. lutheri (PlDES1, ∆4-

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desaturase), which converts 22:5 n-3 and 22:4 n-6 into DHA and 22:5 n-6 respectively, was

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four-fold higher during the mid-exponential growth phase compared to late exponential and

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stationary phases.29 However, no metabolic study attempted to measure directly in vivo the

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effects of environmental factors on desaturase and elongase activities in microalgae. In a

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previous work, it has been provided a method for studying microalgal LC-PUFA biosynthesis

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pathways and desaturase and elongase activities in vivo using externally supplied radiolabeled

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fatty acids as direct substrates.28 Using

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n-6) and arachidonic (ARA, [1-14C] 20:4 n-6) acids as precursors of EPA and DHA, the

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metabolic relationship between the n-6 and n-3 fatty acid series have been demonstrated in P.

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lutheri by ∆17-desaturation of DGLA and ARA into 20:4 n-3 and EPA.

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Herein, using this method, 14C-labeled ARA (14C-ARA) was also used to assess the effect of

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light intensity on the incorporation of LC-PUFA into total lipids, and the regulation of the

14

C-labeled dihomo-γ-linolenic (DGLA, [1-14C] 20:3


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final metabolic steps involved in EPA and DHA synthesis in P. lutheri. Activities of

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conversion of ARA into EPA (∆17-desaturation) and of EPA into DHA (∆5-elongation + ∆4-

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desaturation) were estimated under three different light intensities through the distribution of

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radioactivity into longer and more unsaturated fatty acids using short incubation times (1 and

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3 h). The results of this study represented the first data investigating the influence of abiotic

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factors (i.e., light intensity) on desaturase and elongase activities measured on intact

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microalgal cells “in vivo” using 14C-radiolabeled fatty acids.

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MATERIAL AND METHODS

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Chemicals and radiolabeled fatty acids. Radiolabeled [1-14C] eicosa-5,8,11,14-tetraenoic

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acid ([1-14C] 20:4 n-6;

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purity, were purchased from the Radiochemical Center (GE Healthcare, Amersham,

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Buckinghamshire, UK). All other chemicals were of analytical and HPLC grades and

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purchased from Fisher Scientific Bioblock (Illkirch-Graffenstaden, Bas-Rhin, France).

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Culture and radiolabeling conditions. Pavlova lutheri (CCAP 931/6) were batch cultivated

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on artificial seawater as previously described in 500 ml Erlenmeyer flasks using a working

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volume of 300 ml under controlled temperature (15±1 °C) and a light-dark cycle of 14 h light

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using three different light intensities (20, 100 or 340 µmol photons m-2 s-1).28 Cultures in mid-

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exponential growth phase (day 14) and mid-light period were incubated with 0.3 µM

14

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ARA ammonium salt during 1 and 3 h. The availability and/or accessibility of the

14

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radiolabeled fatty acid substrates have been shown to be non-limiting under such incubation

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times for P. lutheri grown under intermediate conditions (15±1 °C, 100 µmol photons m-2 s-

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14

C-ARA), specific activity 2.07 GBq mmol-1, 99.3% radiochemical

CC-

1 28

). To avoid any changes of growth conditions (i.e. decrease in light availability per cell), no

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concentration of the cells has been performed prior incubation with 14C-ARA. The final cell

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density on day 14 was determined with a Malassez improved bright-line hemocytometer, after


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immobilizing the cells with Lugol 5%. The cell density was estimated to be 2.3, 3.1 and

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4.4×106 cell ml-1 at the incubation time, for the three light intensities (20, 100 or 340 µmol

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photons m-2 s-1, respectively). After incubation, algal cells were gently harvested by

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centrifuging at low-speed (1200 g, 10 min), re-suspended and washed three times with fresh

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medium. The pellets obtained were then frozen, and stored at -70 °C prior to lipid extraction

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and analysis. Three replicate cultures were grown for each incubation time.

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Lipid radiolabeling determination. The total lipids were extracted using a modified version

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as previously described14,34 and fatty acid methyl esters (FAME) were obtained as described35

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for gas chromatography analysis. The distribution of radioactivity between the different

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labeled fatty acids was determined by the reversed-phase HPLC method,36 using a P1500 high

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pressure pump (Thermo Separation Products, Les Ulis, Essonne, France), equipped with a 410

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differential refractometer (Waters, Milford, MA, USA) and a Lichrocart column (Lichrospher

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100 RP-18, 5 mm, 250mm × 4mm internal diameter, Merck, Darmstadt, Germany). The

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samples were eluted using acetonitrile/water solvent system (95:5 v/v) with a flow rate of 1

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ml min-1, and the FAME were collected at the detector outlet. Figure 1 displays a

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chromatogram of a mixture of FAME separated using the reversed-phase HPLC method

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described. 14C-radioactivity incorporated in the total lipid fraction was highly recovered into

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the FAME (percentage of

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identified using pure standards (Sigma-Aldrich, St. Quentin Fallavier, Isère, France). The

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radioactivity measured in the total lipid fraction and each individual FAME were expressed in

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DPM (disintegrations per minute) after correction of the quenching by ESR (External

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Standard Ratios) method using a Wallac 1410 Liquid Scintillation Counter (LSC, ECG

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Instrument, Perkin-Elmer, Waltham, MA, USA) and liquid scintillation cocktail (ACS, GE

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Healthcare, Amersham, Buckinghamshire, UK).

14

C recovered into the FAME > 95%). Each individual peak was


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14

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Fatty acid analysis by GC-FID. Before incubation with

C-ARA, cultures grown under

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similar conditions were used to assess the effect of light intensity on P. lutheri fatty acid

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composition. The total lipids were extracted and FAME were prepared as previously

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described and analyzed with a FOCUS gas chromatography apparatus (Thermo Electron

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Corporation, Les Ulis, Essonne, France) equipped with a flame ionization detector (GC-FID),

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and a fused-silica capillary column (CP Sil-88 25 m×0.25 mm id capillary column, Varian,

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Les Ulis, Essonne, France). The GC-FID conditions were similar to those used previously.28

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Pure standards (Sigma-Aldrich, St. Quentin Fallavier, Isère, France) were used to identify the

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fatty acids by comparing the peak retention times of the samples and standards.

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Parameters calculation and expression. All results and parameters were determined after

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counting of the radioactivity in DPM. Radioactivity incorporated into the total lipids (Table 1)

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was expressed in percentage of the radioactivity incorporated into the total lipids in relation to

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the total radioactivity added in the medium (calculated and expressed both in DPM per

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million cells). According to the moles of 14C-ARA available per million cells in the medium

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and the percentage of radioactivity incorporated into the total lipids, results were expressed in

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pmol per million cells, and in fmol per minute and per million cells. Distribution of the

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radioactivity in each FAME (Figure 4) were expressed in percentage of the total DPM

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radioactivity incorporated into labeled FAME. Enzymatic conversion rates (Table 2) were

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expressed in percentage of conversion of substrate into longer chain and more unsaturated

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fatty acids as described by the following equations (1) and (2).

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∆17 des = [ Σ (DPMEPA+DHA) / Σ (DPMARA+EPA+DHA) ] × 100

(1)

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∆5 elo + ∆4 des = [ DPMDHA / Σ (DPMEPA+DHA) ] × 100

(2)

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Enzymatic conversion rates expressed in fmol of substrate converted per minute and per

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million cells were determined using the percentage of conversion (∆17 des or ∆5 elo + ∆4


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des) previously calculated and the radioactivity incorporated into the total lipids and

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expressed in the same unit.

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Statistical analyses. One-way analysis of variance (ANOVA) was used to examine the effect

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of light intensity (n=3). The Student-Newman-Keuls (SNK) multiple comparison test was

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used to test the differences between groups. Differences were considered significant at a

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probability level of 0.05. All statistics were performed with Statgraphics (version Centurion

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XV) software (StatPoint Technologies, Warenton, VA, USA).

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RESULTS AND DISCUSSION

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Effect of light intensity on total fatty acid composition. Among the abiotic parameters (e.g.,

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light, temperature, CO2 disponibility, pH, and nutrients), light outstands as a key

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environmental factor controlling growth and chemical composition of photosynthetic cells. In

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microalgae, variations in light regime impose numerous adaptation mechanisms involving

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changes in pigment content, biochemical composition, and the capacities of the

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photosynthetic apparatus.37-39 In particular, changes in lipid content and fatty acid profile are

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observed in response to changes in the light intensity.14,40,41 In our work, P. lutheri showed a

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decrease in the proportions of LC-PUFA, in particular stearidonic acid (18:4 n-3) and EPA,

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associated with an increased in SFA, especially palmitic acid (16:0), with increasing light

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intensity (Figure 2). This results are similar to those published for P. lutheri grown under

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identical conditions.14 This previous study showed that the highest PUFA levels, such as those

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of EPA, were predominantly found in the galactolipid fraction when the cells were grown at

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low light; and it has been suggested that accumulation of LC-PUFA in the galactolipids may

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facilitate thylakoid membrane fluidity, and therefore the velocity of electron flow involved in

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photosynthesis during light acclimatization. It is also important to notice that ARA accounted

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for less than 2% of TFA in P. lutheri suggesting its intermediate metabolic function for longer


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and more unsaturated LC-PUFA synthesis. While EPA and DHA accumulate and reach up to

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29% under low light and 18% under high light of TFA, respectively.

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Incorporation of

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pathway. According to the results reported on table 1, the incorporation of 14C-radioactivity

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issued from

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incubation. The percentage of

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radioactivity initially present in the culture medium. After 1 h of incubation and under low

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light intensity (20 µmol photons m-2 s-1), the percentage of 14C incorporated into the total lipid

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fraction is 2.5 time higher (50% of the radioactivity initially present in the culture medium)

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than the percentage obtained at 100 and 340 µmol photons m-2 s-1 (19 and 22%, respectively).

208

Similarly, the amount of 14C incorporated and expressed in pmol per million cells is highest

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when the cells are incubated under low light. The average of substrate incorporated per

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minute and per million cells decreased with increasing light intensity. After 3 h of incubation,

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the percentage of

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(approximately 52% of the radioactivity initially present in the culture medium), and the

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amount of 14C incorporated per minute decreased with increasing light intensity. It has been

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previously demonstrated using [1-14C] 16:0 and [1-14C] 20:3n-6 as radiolabeled fatty acid

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substrates that 70-80% of the radioactivity incorporated into the cells were recovered in the

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total lipids of P. lutheri whatever the incubation time applied (3, 10 and 24 h).28 We may

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assume similar results with 14C-ARA in this study, and suggest that the difference may be due

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to incomplete extraction of the total lipids or losses during this process. The

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incorporated in P. lutheri cells was left in the medium suggesting its non-limitation over 1 or

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3 h of incubation as previously showed.28

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According to these results, Figure 3 illustrates a suggest way of incorporation of

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into complex lipids of P. lutheri using a schematic representation of lipid biosynthesis in

14

C-ARA through a potential light-dependent lipid biosynthesis

14

C-ARA into total lipids is dependent of the light intensity and the time of 14

C incorporated in the total lipid fraction reaches 52% of the

14

C incorporated were highest at 20 and 100 µmol photons m-2 s-1


14

C-ARA non-

14

C-ARA

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microalgae. Currently two locations for de novo lipid biosynthesis have been suggested in

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microalgae, and more specifically in Chlamydomonas reinhardtii, being the plastid

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prokaryotic pathway and the endoplasmic reticulum (ER) eukaryotic pathway.42 The

226

radiolabeled

227

diffusion, esterified to CoA by the long chain acyl-CoA synthases (LACS), and therefore

228

associated into the acyl-CoA pool. The LACS gene may be light-dependant and could

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therefore explained the fast and strong 14C-ARA incorporation into total lipids observed in P.

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lutheri grown under low light. TAG and polar lipids are then assembled using a complex

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process involving diverse enzymatic activities as presented in Figure 3. By consequence, 14C-

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ARA or longer and more unsaturated

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(see the section on LC-PUFA biosynthesis by alternative desaturation and elongation steps),

234

such

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successive acylation steps of the Kennedy pathway.

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production of TAG may also derive from membrane lipids in the acyl-CoA-independent

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pathway using transacylases; or by means of specific lipase activity on membrane lipids

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which liberates fatty acyl groups for TAG synthesis.42 A similar process using similar

239

enzymes could also be involved in the formation of complex lipids, and in the remodelling of

240

fatty acids between membranes. After incubation with 14C-ARA, the high percentage of 14C-

241

radioactivity incorporated into total lipids of P. lutheri grown under low light could therefore

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also be explain by an overexpression of all or certain genes encoding acyltransferases or other

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proteins involved in the process. Especially, enzymes with strong substrate specificities for

244

lipid membranes containing LC-PUFA such as EPA and DHA. The ability of P. lutheri to

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partition both DHA and EPA to TAG, compared to other microalgal species raises the

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question of how this process is regulated.24 It may involve acyltransferases with different

247

substrate specificities. This could also partially explain the strong

14

14

C-ARA used in this study may be incorporated into P. lutheri cells by

C-EPA and

14

C-radiolabeled fatty acids subsequently synthetized

14

C-DHA, may be incorporated into TAG or complex lipids throughout 43,44

Alternatively, fatty acyl donors for


14

C-ARA (or other

10

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synthetized

C-LC-PUFA) incorporation into lipids under low light, and potentially

249

partitioned to TAG or complex lipids. Therefore, high level of expression of specific

250

enzymes, such as acyltransferases, especially under low light conditions, could promote the

251

partitioning of LC-PUFA into complex lipids and could explained the high level of LC-PUFA

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(e.g., EPA) observed in P. lutheri grown under low light intensity, and more specifically

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linked to polar lipids (i.e., glycolipids and phospholipids) constituting the membranes.14,26

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Finally, a strong ∆17-desaturation activity under low light could also explain the high

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percentage of 14C-ARA incorporated and needed as substrate for the synthesis of longer and

256

more unsaturated fatty acids such as EPA and DHA as described in the following section.

257 258

Effect of light intensity on the final desaturase and elongase steps involved in n-3 LC-

259

PUFA biosynthesis. In P. lutheri, the major radiolabeled fatty acids obtained from 14C-ARA

260

were

261

percentage of 14C-radioactivity incorporated into EPA and DHA (respectively 38-40% and 2-

262

3% of the total fatty acids labeled) were observed with the level of light. However, after 3 h of

263

incubation, significant variations in the distribution of 14C-radioactivity incorporated into the

264

fatty acids were observed with changes in light intensity. The percentage of

265

significantly higher when the cells were incubated under 100 µmol photons m-2 s-1 (61% of

266

the total fatty acids labeled), and the percentage of 14C-DHA decreased under high light (340

267

µmol photons m-2 s-1). Therefore, in addition to the high percentage of 14C-ARA incorporated

268

in the total lipids, the light intensity seems to affect the last desaturation and elongation

269

activities involved in EPA and DHA synthesis in P. lutheri. These results are in accordance

270

with several other authors, and can contribute to explain how the levels of EPA and DHA may

271

vary with light intensity in microalgae.14,40,45 Whatever the light intensity, the results also

14

C-EPA and 14C-DHA (figure 4A and 4B). After 1 h of incubation, no changes in the


14

C-EPA were

11


14

C-ARA decreased while those of

Page 12 of 28

14

show that the proportion of

273

increased with the time of incubation (data not showed). 28

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The effect of light intensity on the percentage of conversion of ARA into longer and more

275

unsaturated fatty acids, and the related conversion rates (expressed in fmoles of substrate

276

converted per minute and per million cells) are reported in table 2. The highest percentage of

277

conversion of ARA to EPA was obtained under 100 µmol photons m-2 s-1 after 3 h of

278

incubation. Under these conditions, 68% of

279

converted to EPA and DHA. The results also show that the highest percentage of conversion

280

of EPA to DHA (∆5-elongation + ∆4-desaturation) are obtained under 20 and 100 µmol

281

photons m-2 s-1 after 3 h of incubation (10-11% of the

282

DHA). Meanwhile, whatever the incubation time, the desaturation and elongation conversion

283

rates measured, especially for the ∆17-desaturase, were highest under low light. These rates

284

were also higher after 1 h of incubation, compared to those obtained after 3 h of incubation

285

except under 100 µmol photons m-2 s-1. The highest conversion rates were consequently

286

obtained under low light (20 µmol photons m-2 s-1) after 1 h of incubation (∆-17 desaturation:

287

486, ∆5-elongation + ∆4-desaturation: 96, fmol min-1 106 cell-1). Similarly to the results

288

obtained during incubation of P. lutheri with [1-14C] 16:0 or [1-14C] 20:3 n-6,28 conversion

289

rates are closely linked to the amount of substrate incorporated. Through photosynthetic

290

mechanisms, the regulatory role played by light regarding gene expression coding for

291

desaturases and elongases could explain the variations of the enzymatic conversion rates

292

observed. Previous study showed that the levels of mRNA transcripts coding for ∆12-, ∆15-,

293

and ∆6-desaturases (DesA, DesB, and DesD, respectively) of the cyanobacteria Synechocystis

294

PCC6803 were 10 times higher for cells grown under light conditions, compared to those

295

grown in the dark.46 The desaturation of fatty acids as an adaptive response to shifts in light

296

intensity in five microalga species was also proposed.37 In this study, the authors suggested

14

C-EPA and

14

272

C-DHA

C-ARA incorporated in the total lipids was

14

C-radioactivity were converted to


12

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297

that the optimisation of photosynthetic process resulted in some increase of the relative

298

content of the most unsaturated fatty acids such as EPA in various unicellular microalgae,

299

which exhibited some similarity in their mechanisms of response to changes in light

300

conditions. Moreover, in their study, the fatty acid desaturation was shown to correlate with

301

the photosynthetic activity, in particular the activity of PSI (photosystem I).37

302

In this work, as previously described,28 the ∆17-desaturation activity involved in the

303

conversion of ARA to EPA was confirmed, as well as the metabolic relationship between the

304

n-6 and n-3 fatty acid series (Figure 5). In addition, microalgal desaturases and elongases are

305

either acyl-CoA dependent, or glycerolipid-linked (e.g. phospho-linked) dependent. As

306

examples, acyl-CoA ∆6-desaturases were isolated from microalgae Ostreococcus tauri and

307

Micromonas pusilla;47,48,51 while two membrane bound desaturases with ∆6- and ∆5-

308

desaturase activities were isolated from the freshwater microalga Parietochloris incisa.33 As

309

highlighted in Figure 5, the radiolabeled 14C-ARA incorporated in the total lipids of P. lutheri

310

may therefore have been converted to 14C-EPA and 14C-DHA before or after to be esterified

311

to complex lipids by the acyl-CoA dependent or the glycerolipid-linked dependent LC-PUFA

312

biosynthesis pathways, respectively. The strong incorporation of

313

under low light, and the strong related ∆17-desaturation activity measured, enables us to

314

assume it to be an adaptive response which provides alterations to lipid-protein interactions in

315

the membrane that may be important for the self-assembly of active chlorophyll-protein

316

complexes for the photosynthetic apparatus. The consequent accumulation of n-3 LC-PUFA

317

such as EPA in P. lutheri under low light condition could therefore facilitate thylakoid

318

membrane fluidity, and the velocity of electron flow involved in photosynthesis during low

319

light.14,49

320

In conclusion, the present results have confirmed the metabolic relationship between n-6 and

321

n-3 fatty acid series and measured the effect of light intensity on the ∆17-desaturation activity.


14

C-ARA into total lipids

13


Page 14 of 28

322

Our findings also suggested a light-dependent incorporation of LC-PUFA into structured

323

lipids through different enzymes (i.e., acyltransferases) involved in the biosynthesis of lipids

324

(TAG and membrane lipids); as well as highlight the light-dependent activities of the final

325

desaturation and elongation steps required for n-3 C20/C22 LC-PUFA formation in P. lutheri.

326

Therefore, the strong incorporation of 14C-ARA under low light in association with the strong

327

∆17-desaturation activity could facilitate thylakoid membrane fluidity by LC-PUFA

328

accumulation in the structural lipids (i.e., glycolipids and phospholipids), and therefore

329

provides better lipid-protein interactions in the membrane enhancing the velocity of electron

330

flow involved in photosynthesis during light adaptation.

331 332

ABBREVIATIONS USED

333

LC-PUFA, long-chain polyunsaturated fatty acids; EPA, eicosapentaenoic acid; DHA,

334

docosahexaenoic acid; TAG, triacylglycerols; SFA, saturated fatty acids; MUFA,

335

monounsaturated fatty acids; TFA, total fatty acids; DGLA, dihomo-γ-linolenic acid; ARA,

336

arachidonic acid; HPLC, high performance liquid chromatography; FAME, fatty acid methyl

337

esters; ESR, external standard ratios; GC-FID, gas chromatography-flame ionization detector;

338

ANOVA, analysis of variance; ER, endoplasmic reticulum; LACS, long chain acyl-CoA

339

synthases.

340 341

ACKNOWLEDGMENTS

342

This work was financially supported by the French Ministère de l’Enseignement Supérieur et

343

de la Recherche (MESR) and by the FP7-KBBE European Collaborative Project GIAVAP.

344 345 346

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Page 15 of 28


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two novel microalgal enzymes involved in the conversion of the ω3-fatty acid,

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docosahexaenoic acid biosynthesis. Phytochemistry 2007, 68 (6), 785-796.

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photoacclimation of Haslea ostrearia (Bacillariophyta): effect of irradiance on growth rates,

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(41) Tzovenis, I.; De Pauw, N.; Sorgeloos, P. Optimisation of T-ISO biomass production rich

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in essential fatty acids: I. Effect of different light regimes on growth and biomass production.

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Aquaculture 2003, 216 (1-4), 203-222.

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(44) Khozin-Goldberg, I.; Cohen, Z. Unraveling algal lipid metabolism: Recent advances in

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gene identification. Biochimie 2011, 93 (1), 91-100.

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(45) Meireles, L. A.; Guedes, A. C.; Malcata, F. X. Lipid class composition of the microalga

473

Pavlova lutheri: eicosapentaenoic and docosahexaenoic acid. J. Agric. Food Chem. 2003, 51

474

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475

(46) Kis, M.; Zsiros, O.; Farkas, T.; Wada, H.; Nagy, F.; Gombos, Z. Light-induced

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expression of fatty acid desaturase genes. Proc. Natl. Acad. Sci. USA 1998, 95, 4209-4214.

477

(47) Domergue, F.; Abbadi, A.; Zähringer, U.; Moreau, H.; Heinz, E. In vivo characterization

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of the first acyl-CoA ∆6-desaturase from a member of the plant kingdom, the microalga

479

Ostreococcus tauri. Biochem. J. 2005, 389 (2), 483-490.

480

(48) Petrie, J. R.; Shrestha, P.; Mansour, M. P.; Nichols, P. D.; Liu, Q.; Singh, S. P. Metabolic

481

engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA

482

∆6-desaturase with ω3-preference from the marine microalga Micromonas pusilla. Metab.

483

Eng. 2010, 12 (3), 233-240.

484

(49) Guihéneuf, F.; Ulmann, L.; Tremblin, G.; Mimouni, V. Light-dependent utilization of

485

two radiolabelled carbon sources, sodium bicarbonate and sodium acetate, and relationships

486

with long chain polyunsaturated fatty acids synthesis in the microalga Pavlova lutheri

487

(Haptophyta). Eur. J. Phycol. 2011, 46 (2), 143-152.


20

Page 21 of 28


488

Table 1. Effect of light intensity on the radioactivity incorporated into the total lipids of P.

489

lutheri grown under 20, 100 and 340 µmol photons m-2 s-1 and a temperature of 15 °C after 1

490

and 3 h of incubation with 0.3 µM 14C-ARA (37296 DPM per ml) as substrate†

Light intensity µmol photons m-2 s-1

491

3h

1h

Incubation time 20

100

340

20

100

340

% radioactivity

50.4 ± 2.5 c

18.7 ± 0.8 a

21.9 ± 2.0 a

51.7 ± 0.7 c

52.3 ± 1.3 c

27.6 ± 2.9 b

pmol × 106 cell-1

66.4 ± 0.9 c

17.4 ± 1.0 a

15.1 ± 2.3 a

66.2 ± 1.9 c

52.7 ± 8.3 b

19.2 ± 2.5 a

fmol min-1 × 106 cell-1

1107 ± 14 d

290 ± 16 b

252 ± 39 a

368 ± 11 c

293 ± 46 b

107 ± 14 a

†

Results are expressed as percentage of incorporated radioactivity, as pmol of incorporated

492

substrate per million cells, and fmol of incorporated substrate per minute per million cells.

493

Values are expressed as the mean ± SD (n=3). Means assigned different manuscript letters

494

were statistically different (p < 0.05), and results are reported in ascending order: a < b < c.

495


21


Page 22 of 28

496

Table 2. Enzymatic conversion rate determinations involved in n-3 LC-PUFA synthesis in P.

497

lutheri grown under 20, 100 and 340 µmol photons m-2 s-1 and a temperature of 15 °C after

498

incubation with 0.3 µM 14C-ARA (37296 DPM per ml) during 1 and 3 h†

Light intensity µmol photons m-2 s-1

3h

1h

Incubation time 20

100

340

20

100

340

43.9 ± 0.2 a

41.1 ± 4.1 a

40.9 ± 2.4 a

56.6 ± 0.8 b

67.6 ± 1.9 c

52.7 ± 3.0 b

8.7 ± 0.2 c

6.5 ± 0.2 b

4.6 ± 0.1 a

10.5 ± 0.8 d

9.8 ± 0.7 cd

6.3 ± 0.8 b

% conversion ARA into EPA (∆17 des) EPA into DHA (∆5 elo + ∆4 des) Conversion rate (fmoles min-1 106 cell-1) ARA into EPA (∆17 des) EPA into DHA (∆5 elo + ∆4 des) 499

486.4 ± 8.4 d 119.2 ± 18.4 b 102.7 ± 9.8 b 95.7 ± 0.4 f

18.8 ± 1.6 c

11.5 ± 1.9 b

206.9 ± 2.5 c 200.1 ± 32.8 c

55.9 ± 8.4 a

38.7 ± 3.2 e

6.6 ± 1.2 a

28.4 ± 4.0 d

†

Results are expressed as the percentage of conversion of the substrate into longer chain and

500

more unsaturated fatty acids, and in fmol of substrate converted per minute and per million

501

cells. Values are expressed as the mean ± SD (n=3). Means assigned different manuscript

502

letters were statistically different (p < 0.05), and results are reported in ascending order: a < b

503

< c.


22

Page 23 of 28


Refractometer response

4 67 2 1 3 5

8 9 10

504 505

Figure 1. Chromatogram of Mix FAME standards separated using the reversed-phase HPLC

506

method. 1, 18:4 n-3; 2, 20:5 n-3 (EPA); 3: 22:6 n-3 (DHA); 4, 18:3 n-3; 5, 20:4 n-6 (ARA); 6,

507

18:2 n-6; 7, 20:3 n-6; 8, 18:1 n-9; 9, 16:0; 10, 18:0.

508


23


Page 24 of 28

Fatty acid composition (% TFA)

40

c

30

b a b

20

b a

b

a

a b

10

a

0

509 510

Figure 2. Total lipid fatty acid composition (% TFA) of P. lutheri grown under 20, 100 and

511

340 µmol photons m-2 s-1 (white, grey and black bars, respectively), a temperature of 15 °C, a

512

light-dark cycle of 14 h light, and harvested in mid-exponential growth phase (day 14) and

513

mid-light period. Results are expressed as the mean ± SD (n=3). Means assigned different

514

manuscript letters were statistically different (p < 0.05), and results are reported in ascending

515

order: a < b < c.

516


24

Page 25 of 28


517

518 519 520

Figure 3. Schematic representation of lipid biosynthesis in microalgae and suggested ways (in

521

red) of incorporation of

522

adapted from previous studies.42

14

C-ARA into complex lipids of P. lutheri. Figure modified and

523


25


Page 26 of 28

% Radioactivity

524 80

80 A

60

60

80 B b b

b

40

40

20

20

a

60 a

40

a

20 b

c

0

b

a

a

0

0

525 526 527

Figure 4. Effect of light intensity on the distribution of radioactivity in the main individual

528

fatty acid fractions of P. lutheri grown under 20, 100 and 340 µmol photons m-2 s-1 (white,

529

grey and black bars, respectively) and a temperature of 15 °C after incubation with 14C-ARA

530

during 1 h (A) or 3 h (B). Results are expressed as percentage of total radiolabeled fatty acids;

531

mean ± SD (n=3). Means assigned different manuscript letters were statistically different (p

Light Intensity Regulates LC-PUFA Incorporation into Lipids of

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