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Short Communication A novel source of dihomo-g-linolenic acid: Possibilities and limitations of DGLA production in the high-density cultures of the D5 desaturase-mutant microalga incisa

Said Abu-Ghosh1, Dipasmita Pal-Nath1, Dana Markovitch1, Alexei Solovchenko2, Shoshana Didi- Cohen1, Isabel Portugal1, Inna Khozin-Goldberg1, Zvi Cohen1 and Sammy Boussiba1

1 Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of Drylands, the Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion, Israel 2 Department of Bioengineering, Faculty of Biology, Moscow State University, Moscow, Russia

The D5 desaturasemutant (P127)ofthe microalga Lobosphaeraincisa isa promising organismforlarge-scale production of the valuable LC-PUFA dihomo-g-linolenic acid (DGLA, 20:3 n-6). We examined the potential of P127 for DGLA production under nitrogen (N) starvation conditions, triggering the deposition of DGLA in triacylglycerols, and developed a strategy for optimization of the DGLA productivity in high- density cultures. Towards this end, the effects of initial biomass concentration (1, 2, and 4 g L1) and PAR irradiance (170 and 400 mmol m2 s1) on DGLA and total fatty acid (TFA) production were studied. The highest DGLA and TFA percentages (10 and 38% of dry weight, respectively) were displayed by the cultures initiated at 1 g L1 and grown under a moderate irradiance. Higher irradiances and lower starting biomass content facilitated oleic acid accumulation at the expense of DGLA. Maximum volumetric productivities of TFA and DGLA were recorded in the cultures started at 2 g L1 biomass and grown under 400 mmol PAR m2 s1. We show that a sufficiently high starting culture density should be combined with a mild light stress to facilitate the production of biomass enriched in DGLA-containing triacylglycerols.

Practical applications: The D5 desaturase mutant of L. incisa, P127, is a rare green source of DGLA, a LC-PUFA with valuable pharmaceutical and neutraceutical properties. We report on the optimization of DGLA production by the nitrogen-starving indoor cultures of P127. Stresses, such as N starvation and/or high irradiances, promote accumulation of lipids by microalgal cells. On the other hand, excessively severe stress is deteriorative for the target LC-PUFA accumulation. An important outcome of the present work is establishing physiological constrains of DGLA productivity and finding an approach to balance starting cell density vs. irradiance in order to maximize DGLA yields. Findings of the present work lay a foundation of the efficient production of DGLA using upscaled cultures of P127.

Keywords: High-density culture / LC-PUFA / Microalgal biotechnology / Molecular species / Parietochloris incisa / Triacylglycerols Received: September 3, 2014 / Revised: November 25, 2014 / Accepted: December 5, 2014 DOI: 10.1002/ejlt.201400430

Additional supporting information may be found in the online version of this article at the publisher’s :web-site.

Correspondence: Inna Khozin-Goldberg, J. Blaustein Institutes for fatty acid; OA, oleic acid; PAR, photosynthetically active radiation; (T)FA, Desert Research, Ben-Gurion University of the Negev, Sede Boqer (total) fatty acids; TAG, triacylglycerol Campus, Midreshet Ben-Gurion 84990, Israel E-mail: [email protected] Fax: þ972-8-6596742

Abbreviations: AA, arachidonic acid; DGLA, dihomo-g-linolenic acid; Present address of Said Abu-Ghosh, The Mina and Everard Goodman FAME, Fatty acid methyl esters; LC-PUFA, long-chain polyunsaturated Faculty of Life Sciences, Bar Ilan University, Ramat Gan, 52900, Israel.

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1 Introduction production of the LC-PUFA. In the present work, we examined DGLA production by N-depleted P127 cultures There is increasing market demand for the long-chain initiated at different cell densities, under moderate and high polyunsaturated fatty acids (LC-PUFAs) of omega-3 and PAR levels, to obtain an insight into the relationships between omega-6 families due to their diverse health-beneficial effects DGLA productivity and the cultivation conditions necessary [1, 2]. The omega-6 LC-PUFA dihomo-g-linolenic acid for the subsequent upscaling. (DGLA, 20:3 n-6) is the immediate precursor of arachidonic acid (AA, 20:4 n-6). In the n-6 LC-PUFA biosynthesis pathway, these LC-PUFA give rise to distinctive biologically 2 Materials and methods active eicosanoids such as DGLA-derived prostaglandin E1

(PGE1), which is assumed to reduce inflammation, modulate 2.1 Strain and cultivation conditions vascular reactivity and to play role in cholesterol homeostasis [3–5]. DGLA offers potential for the treatment of inflam- The D5 desaturase mutant P127 was obtained by chemical matory disorders including atopic eczema, psoriasis, asthma, mutagenesis of the wild-type L. incisa strain, deposited in the atherosclerosis and arthritis [6] as well as in suppressing Culture Collection of at Goettingen University under ID cancerogenesis [7–9]. Therefore, sustainable sources of of SAG 2468. The mutant strain was cultivated in one-liter DGLA are very much sought after. glass columns (internal diameter 6 cm) in a temperature- Microalgae are gaining increasing attention as a renew- regulated water bath at 25°C and constantly bubbled with air: able source of value-added LC-PUFA [2, 10, 11]. However, CO2 (98 : 2, v/v). The pre-cultures were maintained at in contrast to n-3 LC-PUFA common in microalgae, n-6 LC- logarithmic phase by daily dilution with fresh BG-11 PUFA are rarely accumulated in appreciable amounts [10]. medium[20] to the chlorophyll (Chl)content of10–15 mg L1 Thus, DGLA as the metabolic precursor for AA normally at 170 mmol PAR m2 s1. To induce N starvation, the cells constitutes only a small percentage in the fatty acid profile of from pre-cultureswere resuspended in N-freeBG-11 medium. cellular lipids in any native species. In search for a natural The initial biomass concentrations were adjusted to 1 (low source of DGLA, the screening of the D5 desaturase mutants density, LD), 2 (medium density, MD), or 4 g L1 (high of the green microalga Lobosphaera (formerly Parietochloris) density, HD) and corresponding Chl of 30, 60, or 120 mg L1. incisa [12], the oleaginous freshwater species (Trebouxio- The cultures were grown under a PAR flux of 170 mmol m2 phyceae, ) known as a richest photosynthetic s1 (designated as moderate light) or 400 mmol m2 s1 (high source of AA [13], was carried out. Under nitrogen (N) light)continuouslyprovidedbywhitefluorescentlamps.These starvation conditions, ca. 20% of cell dry weight (DW) and particular irradiances were chosen as, respectively, incapable about 60% of the total fatty acids (TFA) in L. incisa is and capable of the induction of high-light stress response in constituted by AA, over 90% of which is associated with N-starved P127 cultures initiated at 1 g L1 DW [21]. The triacylglycerols (TAG) [12, 14]. A D5 desaturase mutant of culture growth was monitored via Chl and DW content [15]. L. incisa, designated P127, is unable to convert DGLA to AA. Under N starvation, P127 accumulates DGLA in an amount 2.2 Fatty acid analysis up to 30% of the TFA, also in the TAG [12, 15], making this algal strain a promising source of DGLA. Fatty acid content and composition of P127 cell lipids were Many microalgal species respond to environmental determined by gas chromatography of fatty acid methyl esters stresses, especially to high irradiances and N starvation, (FAME) [22]. The FAME were identified by co-chroma- with increased production of TAG [16] albeit accompanied tography with authentic standards. The data shown represent by slowdown of culture growth. Accordingly, a major mean values with a range of less than 5% for major peaks biotechnological challenge is to find cultivation conditions (over 10% fatty acids) and 10% for minor peaks of the that facilitate sustained productivity of TAG-enriched samples, each analyzed in duplicate. biomass under variable stress conditions [16]. Moreover, excessive irradiances are deleterious for the biosynthesis of 2.3 Analysis of molecular species of TAG TAG-associated LC-PUFA, including AA and DGLA in L. incisa wild type and P127, respectively [17]. Total lipids were extracted from the lyophilized samples The goal of enhancing biomass yield and productivity calls (50 mg) of P127 biomass and separated into classes as for increased culture cell densities, which, in turn, require previously described [13]. higher irradiance to drive a more efficient light utilization [18] TAG molecular species were resolved by reverse-phase although the effects of cell density, especially in the domain HPLC equipped with a LiChrospher RP-18 column >1gL1 remain sofarmuch less elucidatedthan theirradiance (100 mm length, 5 mM, Merck), and either spectrophoto- effects per se [19]. Therefore, it is essential to find a proper metric detector (Lamda-Max 481, Waters) or evaporative balance between the initial cell density and the irradiance in the light scattering detector (ELSD IIA, Varex, Burtonsville, N-starved cultures of L. incisa to achieve a sustained MD, USA). The elution of the TAG species was achieved

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com 762 S. Abu-Ghosh et al. Eur. J. Lipid Sci. Technol. 2015, 117, 760–766 with the system comprised by acetonitrile (solvent A) and acetonitrile: ethanol : hexane (40:20:20, v/v/v, lower phase) (solvent B) at a flow rate of 1.5 mL min1. For the first 25 min, the TAG were eluted isocratically by 30% A:70% B mixture following by linear gradient elution changed to 0% A:100% B for another 30 min with detection at 205 nm. The fractions corresponding to individual peaks were collected and identified by GC-FID. ELSD detection was used for quantification of the TAG molecular species (evaporation temperature of 122°C). Sodiated TAG molecular species were also quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [23].

3 Results and discussion

3.1 The combination of starting cell density and irradiance determines the kinetics of the culture growth parameters

In the present study, we focused on finding an optimal for the accumulation of DGLA balance of starting cell density and irradiance to which the culture is exposed during its cultivation under N starvation. It is conceivable that the amount of light effectively reaching each cell in the microalgal Figure 1. Changes in chlorophyll (a,c) and biomass (b,d) content of suspension is influenced by the incident irradiance on the the P127 cultures initiated at (1)low,(2) moderate and (4)highcell surface of the cultivation vessel as well as by the cell density density (1, 2, and 4 mg DW L1, respectively) grown under either and their pigment content. This problem is even more moderate (170 mmol m 2 s 1)orhigh(400mmol m 2 s 1)PAR important in the case of N-starving cultures which are prone irradiance undernitrogen starvation. Chl, chlorophyll; DW, dry weight. to photooxidative stress deteriorative for biomass and LC- PUFA productivity [17]. On the other hand, high-density The highest biomass accumulation rate (and the highest final microalgal cultures are deemed to be more robust and biomass content) was displayed by the MD and HD cultures efficient in the production of fine chemicals and biodie- whereas the LD cultures rapidly (within 3 6 days) reached sel [18]. With these considerations in mind, we determined stationary phase. Obviously, the culture started at the lowest the effects of different combinations of these parameters on density suffered from the high light-stress exacerbated by N biomass productivity and the dynamics of Chl content in the depletion. This suggestion is supported by the largest extent N-starving cultures, demonstrating DGLA deposition in of the decline in Chl (from 30 to ca. 8 mg L1) recorded in TAG (Fig. 1). the LD culture (Fig. 1C). By contrast, the combination of In the cultures grown at the different irradiances, Chl a higher starting cell density (2 g DW L1) and higher content increased transiently and then declined at a rate irradiance turned to be optimal from the standpoint of inversely dependent on the irradiance (Fig. 1A,C) likely biomass accumulation. Notably, a further increase in starting manifesting a different degree of combined high light- and N cell density did not result in a corresponding increase in starvation stresses. biomass accumulation rate. The slow-down of the HD Under the moderate irradiance (170 mmol photons m2 s1), culture growth could be caused by self-inhibition, as by an low starting density (LD) cultures featured the highest insufficient gas exchange and O2 build-up; these limitations biomass accumulation rate whereas in those started at could be mitigated, to a certain extent, by an increase in the moderate (MD) or high density (HD) it was lower (Fig. 1B). mixing and aeration rates and medium replenishment [19]. Taking into account high volumetric Chl content of these cultures, one can suggest that the latter cultures are to a 3.2 Interactive effects of cell density and irradiance certain degree light-limited. On the other hand, the HD and N starvation on the accumulation of TFA and cultures eventually attained the highest biomass content DGLA (Fig. 1B, curve 4). An increase in the irradiance to 400 mmol quanta m2 s1 Since actual LC-PUFA productivity of algal cultures influenced profoundly the growth kinetics of P127 (Fig. 1D). depends, apart from biomass yield, on fatty acid (FA)

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com Eur. J. Lipid Sci. Technol. 2015, 117, 760–766 Optimization of DGLA production by P127 in dense culture 763 enrichment of the biomass as well as on the proportion the target LC-PUFA in the FA profile, we followed the changes in these parameters under our experimental conditions (Figs. 2 and 3,, Supplementary Tables S1 and S2). The extent and the kinetics of FA production by P127, as biomass production, were affected by both starting cell density and irradiance (Figs. 2A,C). Under moderate irradiance, the pattern of DGLA enrichment in biomass essentially followed the trends recorded for TFA percentages (Figs. 2B,D). DGLA enrichment was impaired by high irradiance, excepting the HD culture. The TFA and DGLA productivity and accumulation rates were generally higher in the cultures grown under the high irradiance (cf. panels A, B and C, D in Fig. 3). These cultures accumulated a high FA amount within seven days of cultivation whereas in the cultures grown at the moderate irradiance this process was remarkably slower. Comparison of volumetric TFA productivity reciprocally affected by biomass yield and FA enrichment (Fig. 3A,C) clearly showed that the moderate irradiance was insufficient for induction of high TFA production in the HD cultures (curve 4 in Figs. 2A and 3B). On the other hand, the FA

Figure 3. Volumetric contents of (a,c) total fatty acids and (b,d) DGLA in the P127 cultures, initiated at different cell densities and grown under either (a,b) moderate or (c,d) high PAR irradiance. For curve designations, see Fig. 1.

production of LD culture was impaired under the high irradiance (Fig. 3C, curve 1). This is compatible with previously established deteriorative effect of excessive irradiances on LC-PUFA production in L. incisa [16, 17, 24]. The combination of a sufficiently high starting cell density (in our case, equivalent to 2 g L1 DW) with the high irradiance was most favorable for TFA and DGLA accumulation in P127 cultures concurrently with most efficient biomass production. Previous observations in our laboratory have shown that the wild-type L. incisa is capable of committing cell division by multiple fission of mother cells at the onset of N starvation, in particular at high initial cell densities, giving rise to a substantial increase in biomass [25]. A similar phenomenon was observed in the present work with the P127 mutant strain. High biomass productivity of P127 under conditions of external N deficiency argues for a highly tuned and efficient mobilization and cellular redistribution of the Figure 2. Total fatty acid (a,c) and (b,d) DGLA enrichment of endogenous N reserves under optimized conditions. We biomass (expressed as percentage of DW) in the P127 cultures, speculate that the cultivation conditions that were identified initiated at different cell densities, and grown under either (a,b) in the present work allowed for a gradual depletion of moderate or (c,d) high irradiance under nitrogen starvation. For nitrogenous compounds, continuing carbon assimilation, curve designations, see Fig. 1. resulting in substantial biomass increase under N starvation.

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The dynamic nature of metabolic changes in microalgae N-replete cells DGLA accumulated mainly in polar lipids under N depletion and existence of N sparing mechanisms (Fig. S1). Importantly, both DGLA and oleic acid (18:1 supports this assumption [26], however further studies in n-9, OA) constituted the major acyl groups of TAG in L. incisa are obviously required. P127 (Fig. S2). A substantial increase in the relative Notably, the highest rate of TFA and DGLA abundance of OA-enriched TAG was recorded in the accumulation (0–7 days) was inversely related to the N-starving P127 cells (Table S1). initial biomass concentration. This was also the case for OA is a key precursor for LC-PUFA biosynthesis in the biomass enrichment with AA in wild-type L. incisa P127; furthermore an increase in the monounsaturated which was the highest under lower irradiances (250 mmol OA over PUFA on the background of TAG accumulation photons m2 s1)[27]. is a common manifestation of stresses induced by high light and N starvation in green microalgae [see e.g. 3.3 TAG molecular species profiling and 15,16]. A similarly tight relationship between OA/DGLA irradiance-dependent changes of fatty and carotenoid-to-chlorophyll ratio (not shown), another acid profile in N-starving P127 cells stress marker in L. incisa [15], supports this suggestion. We have previously hypothesized that the augmented 18:1 Different combinations of cultivation parameters affect the content in the TAG of P127, as compared to the wild biosynthesis of structural vs. storage lipids. Therefore, to type, where AA can rich up to 60% of TAG, is associated optimize cultivation of microalgae for the yield of a value- with the reduced expression of the genes of the entire LC- added LC-PUFA, it is crucial to analyze the lipid class PUFA biosynthesis pathway, particularly under N starva- predominantly harboring the LC-PUFA of interest. The tion conditions [12]. Hence, a high proportion of OA in alterations in the FA profile in wild-type L. incisa and P127 the mutant manifests a bottleneck in DGLA production, under N-starvation largely manifest the changes in FA and deserves special attention in the context of cultivation composition of TAG comprising more than 95% of total condition optimization. cell lipids (and almost all neutral lipids, NL) [24, 28]. To A conspicuous increase was documented in the dissect the interactive effects of the stresses, we compared proportion of OA in the total FA profile (Tables S2 and FA profiles and contents of NL (Table 1) and polar lipids S3). The magnitude of this effect was proportional to from the LD cultures and from the cells grown in the N- the irradiance and inversely related with the initial cell supplemented medium under otherwise similar conditions density (Fig. S3B). Therefore, it is not surprising that (Fig. S1). The LD cultures were chosen for this OA percentage was closely related with TFA content comparison as, arguably, the most stressed under the (r2 ¼ 0.99; Fig. S3A) driven primarily by enhanced TAG conditions employed. N-starvation triggered a sharp biosynthesis in the N-starving cells [16, 10]. In view of these increase in NL, which increased to 30% DW (Table 1). considerations, OA/DGLA ratio may be employed as a After 3 days of cultivation, ca. 90% of total acyl groups convenient stress marker during optimization of P127 and 95% of DGLA were accumulated in NL, whereas in cultivation conditions.

Table 1. Fatty acid composition of neutral lipids (NL) of P127 cultivated in complete BG11 (þN) and nitrogen-depleted (-N) media under irradiance of 170 mmol PAR m 2 s 1 and at initial cell density of 1 g L 1 (LD)

Fatty acid composition (% of total fatty acids)

16:0 16:1a 16:2 16:3 18:0 18:1 18:1 18:2 18:3 18:3 20:3 Time NL % DGLA % Medium (days) DW DW n-6 n-3 n-9 n-7 n-6 n-6 n-3 n-6

þN 3 1.1 0.3 8.4 7.3 2.1 1.8 3.3 13.9 2.4 12.2 1.3 3.7 30.2 N 14.6 4.2 8.7 0.5 tr 0.2 2.3 36.3 2.8 13.5 1.6 0.8 2.8 þN 7 10.2 3.5 8.1 1.8 tr tr 4.8 28.7 1.4 13.4 1.8 0.6 33.9 N 28.3 8.7 7.9 0.6 tr tr 2.2 38.2 2.4 12.7 1.2 0.5 30.7 þN 14 20.6 6.8 8.2 0.9 tr tr 4.1 33.2 1.3 13.6 1.7 0.5 32.7 N 33.2 10.3 7.4 0.5 tr tr 1.9 38.0 3.2 12.9 1.1 0.5 31.1 aTotal of 16:1 isomers. 20:1, 20:2 n-6, 20:4 n-3 and 22:0 were also present at less than 1%. tr - traces; The data represent a mean of two analytical repeats of the pooled biomass samples collected in the representative experiment.

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4 Conclusions linolenic acid in breast cancer cells. Breast Cancer Res. Treat. 2002, 72,203–219. doi: 10.1023/A1014968415759 Both light-limitation and excessive irradiation impaired [8] Watkins, G., Martin, T. A., Bryce, R., Mansel, R. E., Jiang, biomass, TFA and DGLA productivities of N-starving W. G., Gamma-linolenic acid regulates the expression and fi secretion of SPARC in human cancer cells. Prostaglandins P127 cultures. By contrast, at suf ciently high starting cell – 1 Leukot Essent Fatty Acids 2005, 72, 273 278. doi: 10.1016/j. density (2 g L under our conditions) the increased plefa.2004.12.004 irradiance was conductive for DGLA production by P127. [9] Tabolacci, C., Lentini, A., Provenzano, B., Gismondi, A. An added benefit of high-density cultures is an increase in et al., Similar antineoplastic effects of nimesulide, a selective biomass productivity meaning a shorter cultivation cycle in COX-2 inhibitor, and prostaglandin E1 on B16-F10 murine the case of the bi-phasic cultivation approach. Admittedly, melanoma cells. Melanoma Res. 2010, 20, 273–279. doi: 10.1097/Cmr.0b013e328339d8ac the parameters found in this work may not hold for every combination of cultivation conditions. Therefore, for each [10] Khozin-Goldberg, I., Iskandarov, U., Cohen, Z., LC-PUFA from photosynthetic microalgae: occurrence, biosynthesis, cultivation system, the optimal conditions with regard to the and prospects in biotechnology. Appl. Microbiol. Biotechnol. effective degree of stress applied to the culture should be 2011, 1–11. doi: 10.1007/s00253–011-3441-x evaluated. This process might be streamlined by the use of [11] Skjånes, K., Rebours, C., Lindblad, P., Potential for green the characteristic stress markers, such as OA/DGLA ratio, microalgae to produce hydrogen, pharmaceuticals and with important biotechnological applications. other high value products in a combined process. Crit. Rev. Biotechnol. 2013, 33, 172–215. doi: 10.3109/ 07388551.2012.681625 The authors acknowledge the financial support of PTT Global [12] Iskandarov, U., Khozin-Goldberg, I., Cohen, Z., Selection Chemical Public Company Limited (Thailand). S.AG, D.M. of a DGLA-producing mutant of the microalga Parietochloris incisa: I. Identification of mutation site and expression of and D.P. acknowledge the support from the Albert Katz School of VLC-PUFA biosynthesis genes. Appl. Microbiol. Biotechnol. Desert Research; D.P. and I.P. acknowledge the support from the 2011, 90, 249–256. 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