The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

The synthesis of astaxanthin esters,independent of the formation of cysts,highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids.     

INHIBITION OF ASTAXANTHIN SYNTHESIS UNDER HIGH IRRADIANCE DOES NOT ABOLISH TRIACYLGLYCEROL ACCUMULATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)1

Under stress conditions, Haematococcus pluvialis Flotow accumulates fatty acid–esterified astaxanthin, in extraplastidial lipid globules. The enhanced accumulation of fatty acids, mainly in triacylglycerols (TAG), among which oleic acid predominates, is linearly correlated with that of astaxanthin. We used inhibitors of either carotenoid or lipid biosynthesis to assess the interrelationship between carotenogenesis and TAG accumulation under high light irradiance as the stress factor. The two carotenogenesis inhibitors used—norflurazon, an inhibitor of phytoene desaturase, and diphenylamine (DPA), an inhibitor of β‐carotene C‐4 oxygenase—suppressed the accumulation of astaxanthin in a concentration‐dependent manner. Concurrently, the accumulation of neutral lipids was significantly less affected. The lipid biosynthesis inhibitor sethoxydim, which inhibits acetyl‐CoA carboxylase, significantly decreased de novo fatty acid synthesis and, in concert, drastically inhibited astaxanthin formation. In the presence of various concentrations of the three inhibitors, the inhibition of astaxanthin was not accompanied by a proportional decrease in oleic acid, which was used as a marker for TAG fatty acids. When astaxanthin synthesis was completely inhibited, the volumetric content of oleic acid was about 60% of the control value when the two carotenogenesis inhibitors (0.05 μM norflurazon or 20 μM DPA) were used and 27% of the control when the lipid‐synthesis inhibitor (50 μM) was used. We suggest therefore that TAG accumulation under high irradiance is not tightly coupled with astaxanthin accumulation, although the correlation between these two processes was demonstrated earlier. Furthermore, we propose that the accumulation of a certain amount of TAG is a prerequisite for the initiation of fatty acid–esterified astaxanthin accumulation in lipid globules.     

Antioxidant activity of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> cells grown in continuous culture as a function of their carotenoid and fatty acid content

The influence of culture conditions on the quality of Haematococcus pluvialis biomass is assessed. Continuously grown cells have been characterised with respect to their astaxanthin, fatty acid content, and antioxidant activity and compared with those of non-growing haematocysts. Moderate limitation of nitrate availability (1.7 mM) under continuous growth conditions favoured the production of reddish palmelloid cells whose extracts possessed antioxidant activity equivalent to that of haematocyst extracts, despite the lower astaxanthin content (0.6%d.wt.), which is compensated by a higher fatty acid level (7.6%d.wt.). Green cells produced under nitrate saturation conditions (>4.7 mM) exhibit only 40% antioxidant activity than palmelloid. In addition, the major fatty acid present in palmelloid cells was oleic acid (40%f.a.), whereas, in both green cells and haematocysts, the main fatty acids were myristic, palmitic, and oleic acid (20–30%f.a. each). Biomass extracts were fractionated and analysed. The antioxidant capacity was a function of both the carotenoid and the fatty acid profiles, the antioxidant capacity of astaxanthin diesters fraction being 60% higher than astaxanthin monoesters fraction and twice than free astaxanthin. In such a way, the evaluation of the quality of H. pluvialis biomass must take into account both variables. When considering the production of H. pluvialis biomass for human consumption, special attention should be paid to the one-step continuous system approach for the generation of cells rich in both astaxanthin and fatty acids, as they have high antioxidant activity but without thick hard cell wall.     

ACCUMULATION OF OLEIC ACID IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) UNDER NITROGEN STARVATION OR HIGH LIGHT IS CORRELATED WITH THAT OF ASTAXANTHIN ESTERS1

The chlorophyte Haematococcus pluvialis accumulates large quantities of astaxanthin under stress conditions. Under either nitrogen starvation or high light, the production of each picogram of astaxanthin was accompanied by that of 5 or 3–4 pg of fatty acids, respectively. In both cases, the newly formed fatty acids, consisting mostly of oleic (up to 34% of fatty acids in comparison with 13% in the control), palmitic, and linoleic acids, were deposited mostly in triacylglycerols. Furthermore, the enhanced accumulation of oleic acid was linearily correlated with that of astaxanthin. Astaxanthin, which is mostly monoesterified, is deposited in globules made of triacylglycerols. We suggest that the production of oleic acid‐rich triacylglycerols on the one hand and the esterification of astaxanthin on the other hand enable the oil globules to maintain the high content of astaxanthin esters.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%). Supported by the Frontier Research Grant of the SCSIO, the Hundred Talents program of Chinese Academy of Sciences, and National Natural Sciences of China projects (Grant No. 40776087)     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

Astaxanthin biosynthesis enhanced by reactive oxygen species in the green algaHaematococcus pluvialis

The unicellular green algaHaematococcus pluvialis has recently attracted great interest due to its large amounts of ketocarotenoid astaxanthin, 3,3′-dihydroxy-β,β-carotene-4,4′-dione, widely used commercially as a source of pigment for aquaculture. In the life cycle ofH. pluvialis, astaxanthin biosynthesis is associated with a remarkable morphological change from green motile vegetative cells into red immotile cyst cells as the resting stage. In recent years we have studied this morphological process from two aspects: defining conditions governing astaxanthin biosynthesis and questioning the possible function of astaxanthin in protecting algal cells against environmental stress. Astaxanthin accumulation in cysts was induced by a variety of environmental conditions of oxidative stress caused by reactive oxygen species, intense light, drought, high salinity, and high temperature. In the adaptation to stress, abscisic acid induced by reactive oxygen species, would function as a hormone in algal morphogenesis from vegetative to cyst cells. Furthermore, measurements of bothin vitro andin vivo antioxidative activities of astaxanthin clearly demonstrated that tolerance to excessive reactive oxygen species is greater in astaxanthin-rich cysts than in astaxanthin-poor cysts or astaxanthin-less vegetative genesis and carotenogenesis, and the accumulated astaxanthin in cysts can function as a protective agent against oxidative stress damage. In this study, the physiological roles of astaxanthin in stress response and cell protection are reviewed.     

Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis

During light induction for astaxanthin formation in Haematococcus pluvialis, we substituted photoautotrophic induction for heterotrophic induction using acetate, both to prevent contamination by heterotrophs due to addition of organic carbon and to enhance carbon assimilation in the induced cells. Strong photoautotrophic induction was performed by N-deprivation of photoautotrophically grown Haematococcus cells followed by supplementation with bicarbonate (HCO₃^–) or CO₂. Bicarbonate-induced cells contained more astaxanthin than acetate-induced cells, and even further enhancement of astaxanthin accumulation was achieved by continuous CO₂ supply. The maximum astaxanthin content (77.2 mg g^–1 biomass, 3.4-fold higher than with heterotrophic induction) was obtained under conditions of 5% CO₂, yielding astaxanthin concentration and productivity of 175.7 mg l^–1 and 6.25 mg l^–1 day^–1, respectively. The results indicate that photoautotrophic induction is more effective than heterotrophic induction for astaxanthin synthesis in H. pluvialis.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe²⁺ led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557–583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of β-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of β-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).     

Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-starvation and high light conditions

Haematococcus pluvialis gave the highest astaxanthin accumulation rate (2.7 mg l^–1 day^–1) and total astaxanthin content ( 22.7 mg g^–1 biomass). Astaxanthin accumulation in Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Chorella zofingiensis and Scenedesmus vacuolatus was, respectively, 19.2, 14.3, 10.9, 6.8 and 2.7 mg astaxanthin g^–1 biomass, respectively.     

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: C/N ratio and light intensity

We investigated the effect of carbon/nitrogen (C/N) ratio on astaxanthin synthesis in Haematococcus pluvialis during photoautotrophic induction by continuous input of both CO₂–air mixture and intense light. When H. pluvialis was induced by constant irradiance induction at 200 μmol photon m⁻² s⁻¹, there was a positive correlation with astaxanthin content and C/N ratio, which was similar to the case for heterotrophic induction. Lower C/N ratios did not retard Haematococcus encystment, but did increase culture biomass, resulting in a decrease in astaxanthin production because of light limitation. However, induction using variable irradiance showed that reduction of astaxanthin production at low C/N ratios was successfully overcome by simply increasing the light intensity from 200 to 300 μmol photon m⁻² s⁻¹ to overcome the light limitation. This resulted in a greatly enhanced astaxanthin synthesis in proportion to cell density in cultures with low C/N ratios. Our results indicate that light intensity is more critical than C/N ratio in astaxanthin production by H. pluvialis during photoautotrophic induction.     

Proteomic analysis of molecular response to oxidative stress by the green alga<Emphasis Type="Italic"> Haematococcus pluvialis</Emphasis> (Chlorophyceae)

Rapidly growing, green motile flagellates of Haematococcus pluvialis can transform into enlarged red resting cysts (aplanospores) under oxidative stress conditions. However, it is not known what initial molecular defense mechanisms occur in response to oxidative stress, and may ultimately lead to cellular transformation. In this study, global-expression profiling of cellular proteins in response to stress was analyzed by two-dimensional gel electrophoresis, image analysis, and peptide mass fingerprinting. Oxidative stress was induced in cultures of green flagellates by addition of acetate and Fe²⁺, and exposure to excess light intensity. Overall, 70 proteins were identified with altered expression patterns following stress induction. Some key proteins involved in photosynthesis and nitrogen assimilation were down-regulated, whereas some mitochondrial respiratory proteins were transiently up-regulated after the onset of stress. Most of the identified proteins, particularly those from the families of superoxide dismutase, catalase, and peroxidase, were transiently up-regulated, but reverted to down-regulation during the 6 days of stress. On the other hand, cellular accumulation of the antioxidant astaxanthin occurred well after initiation of oxidative stress and reached its maximum cellular level after six or more days of stress. It appears that the early stress response involves multiple enzymatic defense processes that play a critical role upon onset of stress and also during the early transition of green vegetative cells to red cysts. As cyst development continues, the intensive, enzyme-mediated initial responses were largely replaced in mature red cysts by accumulation of the molecular antioxidant astaxanthin. This study provides the first direct evidence for a massive, and concerted up-regulation of multiple antioxidative defense mechanisms, both spatially and temporarily, to protect H. pluvialis cells against oxidative stress.Abbreviations 2-DE Two-dimensional gel electrophoresis - IPI Isopentenyl-diphosphate -isomerase - HSP Heat-shock protein - MALDI–TOF Matrix-assisted laser desorption/ionization–time of flight - ROS Reactive oxygen species - SOD Superoxide dismutase     

Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress

Under stressful environments, many green algae such as Haematococcus pluvialis accumulate secondary ketocarotenoids such as canthaxanthin and astaxanthin. The carotenogenesis, responsible for natural phenomena such as red snows, generally accompanies larger metabolic changes as well as morphological modifications, i.e., the conversion of the green flagellated macrozoids into large red cysts. Astaxanthin accumulation constitutes a convenient way to store energy and carbon, which will be used for further synthesis under less stressful conditions. Besides this, the presence of high amount of astaxanthin enhances the cell resistance to oxidative stress generated by unfavorable environmental conditions including excess light, UV-B irradiation, and nutrition stress and, therefore, confers a higher survival capacity to the cells. This better resistance results from the quenching of oxygen atoms for the synthesis itself as well as from the antioxidant properties of the astaxanthin molecules. Therefore, astaxanthin synthesis corresponds to a multifunctional response to stress. In this contribution, the various biochemical, genetic, and molecular data related to the biosynthesis of ketocarotenoids by Haematococcus pluvialis and other taxa are reviewed and compared. A tentative regulatory model of the biochemical network driving astaxanthin production is proposed.     

Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.