Metabolomics of astaxanthin hyperaccumulation in Haematococcus pluvialis under high light stress

Variation in metabolite profiles of Haematococcus pluvialis(a type of unicellular green algal)under light stress is a key issue of study at the present.To investigate the effect of light intensity on accumulation of astaxanthin in H.pluvialis,a 26-day batch culture experiment of H.pluvialis under the light intensity levels at 73,127,182,236,and 291μmol/(m^(2)·s)was conducted.Therefore,the optimal light intensity and the corresponding metabolic pathways of accumulation in H.pluvialis were determined.Results show that 236μmol/(m^(2)·s)was the optimum light intensity to induce astaxanthin accumulation,at which a maximum content of 9.01 mg/L was achieved on Day 24.A total of 132 metabolites were identified and quantified,of which 38 differential metabolites were highlighted and classified,including 3 fatty acids or intermediates,5 amino acids or derivatives,5 carbohydrates or intermediates,16nucleoside derivatives,and 9 other metabolites using LC-MS/MS technique.Subsequently,16 statistically significant differential metabolic pathways were enriched and annotated based on Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis between the control and the 236μmol/(m^(2)·s)treatment group(P<0.05).In addition,the bioprocesses included cellular basal metabolism and signaling systems,such as carbohydrate metabolism,amino acid metabolism,glycerol and derivatives metabolism,nucleotide and derivative metabolism,and inositol phosphate metabolism were activated and regulated under strong light stress conditions.Moreover,4 hub metabolites containing D-glucose-6-phosphate,L-tyrosine,glycerol-3-phosphate,and L-glutamine were identified,based on which the associated metabolic network was constructed.The study provided a metabolomic view of astaxanthin accumulation in H.pluvialis under strong light stress.

A new approach to promote astaxanthin accumulation via Na_2WO_4 in Haematococcus pluvialis

The optimum concentration of Na_2 WO_4 was explored in relation to the cell density and astaxanthin content in Haematococcus pluvialis. Then, the cellular morphology, nitrate reductase(NR) activity, soluble sugar and protein contents, and chlorophyll ?uorescence were measured, and the transcriptional expression of carotenogenic genes was determined by quantitative real-time PCR. The results showed that 3.0 mmol/L of Na_2 WO_4 was the optimum concentration to induce astaxanthin accumulation, with a maximum content of 49.41±0.13 pg/cell reached on the tenth day. The NR activity decreased signi?cantly and continually after Na_2 WO_4 treatment. The soluble sugar content increased gradually during the experimental period and was eventually signi?cantly higher than that in the control. The soluble protein content increased rapidly,reached a maximum in day 0.5 and day 1 and then decreased. The ef fective photochemical effciency of PSII( F v'/F m') and light saturation( E k) ?rst decreased and then tended to stabilize, and NADP +-glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene expression was correlated with photosynthesis. The transcriptional expression of ipi, psy and bkt was signi?cantly increased compared with that in the control after application of Na_2 WO_4, and the relative expression of ipi reached the highest level on the ?fth day, with a 98.03±1.92-fold increase. Our results describe a new approach to promote the ef fective accumulation of astaxanthin in H. pluvialis by NR inhibitor Na_2 WO_4.

Identification and Functional Characterization of a NovelΔ12 Fatty Acid Desaturase Gene from Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis accumulates large amounts of fatty acids in response to adverse conditions.However,the key fatty acid desaturase genes in H.pluvialis remain unknown.In this study,we cloned and functionally characterized aΔ12 fatty acid desaturase gene,and designated it as HpFAD2.The open reading frame of HpFAD2 consisted of 1137 base pairs and encoded 378 amino acids.The deduced polypeptide showed 70%identity to other endoplasmic reticulumΔ12 fatty acid desaturases,whereas it had only 44%identity to plastidΔ12 fatty acid desaturases.The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulumΔ12 fatty acid desaturases.Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2.Two additional fatty acids(C16:2 and C18:2)were detected in the yeast transformants.The results indicatedΔ12 desaturation activity and substrate preference for C18:1 over C16:1.The transcriptional levels of H.pluvialis HpFAD2 at different growth stages were measured by quantitative polymerase chain reaction(PCR),indicating that the HpFAD2 transcriptional levels were significantly higher in red cells than those in green cells.Our study brings more insight into the fatty acid biosynthetic pathway of H.pluvialis.

Changes of pigments and lipids composition in Haematococcus pluvialis vegetative cell as affected by monochromatic red light compared with white light

Much attention has been paid on studies of astaxanthin accumulation process in H aematococcus pluvialis industry. However, growth of H. pluvialis in motile vegetative stage is still the most important and problematic part in the whole cultivation process, such as low growth rate and cell yields. Motile vegetative cells are extremely sensitive to various stresses which make it difficult to maintain the cells of this state to grow. Previous reports showed that motile vegetative cells may have higher biomass yields ifapplied monochromatic red light. However, metabolic responses of these cells are not completely understood, which constraints application of this illumination protocol in industry. The aim of this study was to examine how critical biochemical changes of H. pluvialis motile vegetative cells were af fected by red light when compared with white light. Variation of photosynthetic pigments composition and lipids were mainly studied. By comparing growth process of cultures in red light and white light, prominent variation of pigments composition and lipids changes were observed. The results showed that, even though cell proliferation was the same during exponential growth phase, variation of photosynthetic pigment composition and lipids occurred. The final biomass of cell number was higher in red light group than in white light group. The variations were significant different. Increase or decrease of major photosynthetic membrane lipids to some extent did not influence photosynthesis of the vegetative cells during this phase. However, vegetative cells under polychromatic white light other than monochromatic red light need further metabolic process to adjust its pigment composition and lipids, possibly this is energetically and biochemically unfavorable for motile vegetative cells to growth under white light, a light condition normally not considered as a stress.

Modeling of Biomass Production of Haematococcus pluvialis

Microalgae cultivation is justified by the production of high-value fine chemicals and biofuels, essential to reduce the emissions of gases that cause global warming. This paper presents a study of the growth of microalgae Haematococcus pluvialis considering light conditions from 2000 to 10,000 lux, temperature 22?C and pH in the 6.5-12.5 range. The experiments were performed in 4 liter flat plate photobioreactors using the Rudic culture medium. The biomass growth was measured by counting cells in a Neubauer chamber. Both the light intensity and the pH of the medium influenced the rate of growth of the microalgae. A model with exponential behavior was proposed to describe the production of microalgae biomass over time. A nonlinear autoregressive model based on an Artificial Neural Network was used to predict the dynamic behavior of the pH during the growth of the microalgae at different light intensities. Simulations were carried out to analyze the behavior of biomass production at other light intensities within the range considered.

Sodium acetate can promote the growth and astaxanthin accumulation in the unicellular green alga Haematococcus pluvialis as revealed by a proteomics approach

Haematococcus pluvialis is an ideal natural source of strong antioxidant astaxanthin.Sodium acetate(NaAc)was proven an effective organic carbon source for improving algal growth and astaxanthin production;however,the underlying mechanism remains obscure.To reveal the mechanism of NaAc at the green vegetative stage of H.pluvialis,the physiochemical characteristics and the global protein expression profiles obtained using a tandem mass tag labeling approach were compared between the control(CK)and two NaAc-addition groups.Results show that after NaAc addition,the biomass,nitrate consumption rate,and activities of three carbohydrate metabolism enzymes of H.pluvialis were significantly increased,and the net photosynthetic rate and chlorophyll content decreased.In addition,astaxanthin,total carbohydrates,and total lipids were accumulated,and some red cells appeared in the NaAc5 group.Moreover,317 differentially expressed proteins(DEPs)with the most altered expression patterns were screened out in the CK vs.NaAc5 comparison in our proteomics study.All the DEPs involved in carbohydrate metabolism and lipid metabolism were significantly increased,while most of the photosynthesis-related proteins were depressed in the two NaAc-treated groups.The proteomics results were verified and supported by parallel reaction monitoring approach and physiochemical data.Our findings demonstrate that NaAc promoted the tricarboxylic acid cycle,glyoxylate cycle,and amino acid and lipid synthesis,and inhibited the photo synthe sis-related activities,which consequently speeded up the growth and astaxanthin accumulation in this alga.

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 col-umn was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus plu-vialis 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 re-sults 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. zofingien-sis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a re-markably 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%).

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.

乙醇-硫酸铵双水相体系萃取虾青素的研究

虾青素具有突出的生理功能和巨大的开发价值。本文研究了乙醇-硫酸铵双水相体系用于雨生红球藻(Haematococcus pluvialis)虾青素粉中虾青素制备的可行性。结果表明:雨生红球藻虾青素粉中含游离虾青素及杂色素。雨生红球藻虾青素粉乙醇提取液与20%或30%硫酸铵溶液形成的双水相可以使部分虾青素形成沉淀集中在上下相之间。当乙醇提取液∶20%硫酸铵(v/v)为1∶1.5时,虾青素沉淀率可达到70%以上,且沉淀中虾青素的纯度较高,提取液中的杂色素基本不沉淀而主要分布在上相。这说明乙醇-硫酸铵双水相体系可用于从雨生红球藻虾青素粉中制备虾青素。

饲料中添加雨生红球藻对七彩神仙鱼生长、体色及抗氧化力的影响

为探讨雨生红球藻（Haematococcus pluvialis）对七彩神仙鱼（Symphysodon haraldi）增色效果以及对机体抗氧化力的影响,分别用添加0（对照组）、3.33、6.66、9.99、13.32和16.65 g/kg雨生红球藻（折算成虾青素添加量分别为0、100、200、300、400和500 mg/kg饲料）的饲料饲喂七彩神仙鱼6周,测定七彩神仙鱼皮肤、鳍条、肌肉和肝脏中的虾青素含量、体表的三刺激值和肝脏总抗氧化能力（T-AOC）和谷胱甘肽（GSH）含量。结果显示：饲料中添加雨生红球藻可以显著提高七彩神仙鱼的增重率,当添加量为9.99 g/kg其增重率最大。七彩神仙鱼皮肤、鳍条、肌肉和肝脏虾青素含量随着雨生红球藻添加量的增加而升高,当雨生红球藻添加量超过6.66 g/kg,各组织中虾青素的含量趋于稳定。随着雨生红球藻添加量增加,皮肤L＊值（亮度）下降、而a＊值（红度）和b＊值（黄度）升高,皮肤中虾青素含量与三刺激值间的相关性均达到高度相关,并与a＊值的相关性最高。随着雨生红球藻添加量的增加,七彩神仙鱼肝脏的T-AOC和GSH的含量也逐渐升高。结果表明,在饲料中添加雨生红球藻可提高七彩神仙鱼的增重率,提高七彩神仙鱼皮肤虾青素含量,改善七彩神仙鱼体色,增强机体的抗氧化能力;雨生红球藻的添加量建议为6.66-9.99 g/kg（虾青素含量为200-300 mg/kg）。

氮磷浓度对雨生红球藻叶绿素荧光参数的影响

在一次性培养过程中,利用水样叶绿素荧光仪(Water-PAM)研究了2个单因子(不同氮浓度、不同磷浓度)对雨生红球藻(Haematococcus pluvialis)叶绿素荧光参数(PSⅡ的最大光能转化效率Fv/Fm、光合电子传递效率ETR)、叶绿素相对含量及细胞密度的影响。单因子方差分析结果表明,氮磷浓度对雨生红球藻的叶绿素荧光参数及生长均有显著的影响(P<0.05)。多重比较结果表明,雨生红球藻进行光合作用和生长最适氮浓度为1760μmol/L;最适磷浓度为18.16μmol/L。相关性分析结果表明:在整个培养周期中,叶绿素相对含量均与细胞密度呈显著的正相关关系。

雨生红球藻β-胡萝卜素酮化酶基因的cDNA和基因组DNA克隆及序列分析

根据已知的β-胡萝卜素酮化酶(β-carotene ketolase,BKT)的cDNA序列设计引物,利用长距离PCR扩增获得了bkt基因的cDNA完整编码片段及基因组DNA片段,并对这些片断进行了序列测定。cDNA和基因组DNA比对结果表明该基因至少包括5个内含子,它们的剪切位点皆符合GU-AG规律。在比对结果中同时还发现一个19bp的短序列重复,该重复序列在bkt的cDNA和基因组DNA上都发生了多次重复,推测在BKT的表达调控中可能具有一定功能。另外,在序列比对过程中还发现本实验所获得的cDNA和基因组DNA序列与前人报道的cDNA序列之间都存在多处单碱基差异和19bp的短序列差异。

红球藻藻株对光强适应及在工程培养中的应用

于2007～2008年在云南楚雄,利用叶绿素连续荧光分析等技术方法,对比研究了3株雨生红球藻藻株(Haematococcus pluvialis)(H_0、H_2、H_3)不同类型细胞对光强适应能力和光化学机制、生长速率和虾青素累积情况,对比研究了红球藻细胞转化过程的光化学特性与能量分配机制。结果表明,3株雨生红球藻游动细胞、绿色不动细胞和红色不动细胞光合作用对光强需求存在明显差异性,其中绿色游动细胞H_0、H_2和H_3光饱和点分别为750、1000、750 μmol/(m^2·s),绿色不动细胞H_0、H_2和H_3光饱和点分别为750、750、500 μmol/(m^2·s),而红色不动细胞H0、H_2和H_3的光饱和点分别为500、750、500 μmol/(m^2·s)。上述数据意味着红球藻光合作用对光强需求以游动阶段细胞最高,其次为绿色不动细胞,而红色不动细胞对光需求最弱;3株红球藻光合作用对光强需求的顺序为H_2>H_0>H_3。从实际培养效果来看,H_0藻株游动细胞在9月、11月到次年5月生长最好,其中4月、11月比生长速度为藻株H_3的2.19倍和2.17倍,而在6～8月和10月份中,藻株H_2生长比较迅速,但是藻株之间差异性相对较小。针对3株红球藻虾青素累积而言,1～2月、4月、6月、9月、11～12月藻株H0的虾青素含量最高,3月、5月更适宜H_3积累虾青素,其余7～8月和10月H_2藻株积累虾青素更多。综合雨生红球藻细胞生长和虾青素累积二个决定产量的主要因素,作者认为,在秋末、冬、春和夏初等温度和光照相对较低季节,选择藻株H_0规模化培养可获得更好的生产效果,而在光照较强、温度较高和多雨的夏季和秋初季节,应选择藻株H_2培养产量更高。

利用光生物反应器培养雨生红球藻的研究初探

用光生物反应器与光照培养箱两种培养容器培养雨生红球藻(Haematococcus pluvialis),对雨生红球藻在两种容器培养过程中细胞生长、pH值、溶解氧(DO)及虾青素积累情况进行比较,同时比较不同接种密度对光生物反应器培养效果的影响。实验表明,光生物反应器中藻细胞的调整期较短,接种1 d后即进入指数生长阶段,在胁迫阶段则仅需4 d即达到虾青素含量的峰值;将pH值控制在偏碱性条件下(7.75±0.10)有利于藻细胞更好生长;营养培养阶段DO相对饱和度上升至80%,而在胁迫阶段则迅速降低,最低值小于6%;较高的接种密度(2.3×104个/mL)具有较短的营养培养周期(7 d),且因接种密度变化对胁迫周期长短无明显影响(均为4 d),选用较高的接种密度可望降低工业生产成本。

雨生红球藻细胞形态转换和藻落形成差异研究

基于雨生红球藻(Haematococcus pluvialis)培养过程中游动细胞和不动细胞的形态差异与转换,对比研究了两种细胞类型在离心收集与藻落形成效率上的差异。结果表明,雨生红球藻SCCAP K-0084在BYA培养基中培养4~6 d和9~12 d分别以绿色游动细胞与绿色不动细胞为主;早期游动细胞离心收集需要更高的离心力,但500~1 000 g离心5 min可以在不影响细胞存活率的情况下有效收获所有类型细胞;相比于传统直接涂布法,双层琼脂平板法可有效提高平板藻落形成效率,且游动细胞藻落形成效率比不动细胞藻落形成效率更高。TAP培养基由于藻落形成效率较低并不适合于雨生红球藻平板培养与筛选,自养型培养基虽然具有较好的藻落形成效率但是藻落形成速度较慢,相比而言,BYA培养基藻落形成效率较高且生长速度快,更适合用于雨生红球藻细胞的后期平板培养与筛选过程。

雨生红球藻铁型超氧化物酶基因的克隆与序列分析

利用已报道的FeSOD保守区域,设计简并引物,通过简并PCR的方法获得了雨生红球藻(Haematococcus pluvialis)FeSOD(HpFeSOD)cDNA的部分序列,然后采用RACE方法分别克隆到5′端和3′端。拼接后得到HpFeSOD cDNA全长,该基因全长为1 138 bp,ORF为684 bp,编码227个氨基酸。把已经得到的HpFeSOD序列推导成氨基酸序列与一些已知物种的FeSOD氨基酸序列相比较,雨生红球藻FeSOD与杜氏藻、衣藻、团藻、石莼、颤藻的同源性为89%、83%、78%、70%和63%。分子系统学分析表明,雨生红球藻FeSOD与杜氏藻FeSOD聚在一起,介于真菌与高等植物之间并且真核微藻FeSOD与高等植物的同源性更高,推测其在进化上与高等植物的亲源关系更近。

Effects of dietary astaxanthin enrichment on enhancing the colour and growth of red tilapia,Oreochromis sp.

The aim of this research was to examine the effects of dietary inclusion of astaxanthin from Haematococcus pluvialis in commercial fish feed on the colour and growth of red tilapia,Oreochromis sp..A 6-week feeding trial was conducted at concentration of astaxanthin at 0%(T1),4%(T2),8%(T3)and 12%(T4)in feed formulation to examine the growth performance,colour pigmentation on skin,texture analysis and carotenoid contents.Results showed an increment of certain growth parameter in fish fed with astaxanthin enriched diet.Increased in weight gain(WG),protein efficiency ratio(PER)was obtained in fish fed with astaxanthin enriched compared with control.All treatments with astaxanthin-enriched provided higher redness(a*)to skin than negative control that directly influence the colour of the fish.It can be concluded that inclusion of astaxanthin from green algae into commercial fish feed can be used as an alternative natural carotenoid source in Oreochromis sp.diets to ensure good pigmentation,hence improving the skin colour for more appealing and subsequently increase the market value of the fish.

雨生红球藻蓝光受体向光素(PHOT)互作蛋白鉴定及烟草异源表达的生理效应

向光素(PHOT)是植物和绿藻特有的蓝光受体,鉴定PHOT的互作蛋白及探究其功能为阐明PHOT介导的信号途径与生理功能奠定基础。本研究采用酵母双杂交技术从雨生红球藻cDNA文库中筛选HaePHOT的候选互作蛋白,共获得23个阳性克隆,其中4个克隆注释为DnaJ。利用RNA-Seq数据,对雨生红球藻中HaePHOT与DnaJ的表达模式进行相关性分析,发现二者呈很强的正相关;进一步分析HaePHOT或DnaJ与虾青素合成及积累的关键基因(PSY、PDS、LYCB、BKT、BCH、CYP97B、KAS、FAD)表达量的相关性,发现HaePHOT及DnaJ分别与LYCB、BKT、BCH、CYP97B呈正相关,而与PSY、PDS、KAS、FAD呈负相关。瞬时转化烟草结果显示,在正常光(白光)下,野生烟草和过表达HaePHOT的烟草植株叶绿素a、叶绿素b和类胡萝卜素差异不显著;但蓝光下差异显著,且转基因烟草色素含量较高。qRT-PCR结果显示叶绿素a加氧酶(CAO)、原叶绿素酸酯氧化还原酶(POR)、八氢番茄红素合酶(PSY)、八氢番茄红素脱氢酶(PDS)和番茄红素β-环化酶(LYCB)编码基因在蓝光照射及转基因烟草中的表达显著提高,均与叶绿素和类胡萝卜素含量的变化趋势一致。这些发现为后续解析HaePHOT及其互作蛋白介导的调控机制提供了理论基础。