ABSTRACT
WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.
Subject(s)
Eukaryota , Gene Expression Regulation, Plant , Phylogeny , Plant Proteins/metabolism , Plants/metabolism , Stress, Physiological/genetics , Transcription Factors/metabolismABSTRACT
The unicellular green alga Haematococcus pluvialis is the best source of natural astaxanthin (AST) in the world due to its high content under stress conditions. Although high light (HL) can effectively induce AST biosynthesis, the specific mechanisms of light signal perception and transduction are unclear. In the current study, we used transcriptomic data of normal (N), high white light (W), and high blue light (B) to study the mechanisms of light inducing AST accumulation from the point of photoreceptors. The original data of 4.0 G, 3.8 G, and 3.6 G for N, W, and B were obtained, respectively, by the Illumina Hi-seq 2000 sequencing technology. Totally, 51 954 unigenes (at least 200 bp in length) were generated, of which, 20 537 unigenes were annotated into at least one database (NR, NT, KO, SwissProt, Pfam, GO, or KOG). There were 1 255 DEGs in the W vs N, 1 494 DEGs in the B vs N, and 1 008 DEGs in the both W vs N and B vs N. KEGG enrichment analysis revealed that photosynthesis, oxidative phosphorylation, carotenoid biosynthesis, fatty acids biosynthesis, DNA replication, nitrogen metabolism, and carbon metabolism were the significantly enriched pathways. Moreover, a large number of genes encoding photoreceptors and predicted interacting proteins were predicted in Haematococcus transcriptome data. These genes showed significant differences at transcriptional expression levels. In addition, 15 related DEGs were selected and tested by qRT-PCR and the results were significantly correlated with the transcriptome data. The above results indicate that the signal transduction pathway of "light signal - photoreceptors - interaction proteins - (interaction proteins - transcription factor/transcriptional regulator) - gene expression - AST accumulation" might play important roles in the regulation process, and provide reference for further understanding the transcriptional regulation mechanisms of AST accumulation under HL stress.
Subject(s)
Chlorophyta/genetics , Gene Expression Profiling , Signal Transduction/genetics , Transcriptome/genetics , XanthophyllsABSTRACT
Astaxanthin is widely applied as a nutraceutical, pharmaceutical, and aquaculture feed additive because of its high antioxidant activity. Haematococcus pluvialis is a microalgal species that can largely accumulate astaxanthin under adverse environmental conditions. Here we review the research progress of astaxanthin biosynthesis in H. pluvialis, including the induction and regulation of massive astaxanthin, the relationship between astaxanthin synthesis, photosynthesis and lipid metabolism.
Subject(s)
Chlorophyceae , Chlorophyta , Microalgae , XanthophyllsABSTRACT
Resumen En la actualidad la astaxantina de origen natural es uno de los pigmentos carotenoides con importantes aplicaciones en la industria alimenticia, farmacéutica y cosmética, debido a sus grandes propiedades dentro de las que se destaca su gran poder antioxidante, efecto preventivo del cáncer, incremento de la respuesta inmune, inhibición de los radicales libres entre muchas otras. Haematococcus pluvialis es una microalga verde de agua dulce y es una de las fuentes naturales con mayor producción de astaxantina ya que es capaz de acumular hasta un 3% de astaxantina en peso seco. El objetivo del presente trabajo fue determinar el medio de cultivo y las condiciones óptimas para el crecimiento y la producción de astaxantina a partir de Haematococcus pluvialis. La influencia de diferentes factores como el pH, temperatura, agitación, aireación CO2 e iluminación favorecen el crecimiento celular, al darle un ambiente óptimo a la microalga. Para determinar las condiciones nutricionales óptimas, se evaluó el efecto de diferentes medios de cultivo (BBM, OHM, RM) en Birreactores de 500mL con 350mL de medio y 1x104cel/ml de inóculo en fase exponencial, las condiciones de cultivo empleadas fueron: pH. 6.7 a 7, CO2 al 5%, fotoperiodo de 16 horas luz 8 oscuridad, irradianza 70μE/m2s; Los resultados mostraron que el mayor crecimiento o producción celular se obtuvo en el medio RM con 7,5 x 105 cel/ml en el día 36, y la mayor producción de astaxantina se obtuvo en el medio RM con una concentración de 8.3 μg/ml en el tratamiento 4.
Abstract Currently astaxanthin of natural origin is one of the carotenoids with important applications in the food, pharmaceutical and cosmetics, due to their large properties within its powerful antioxidant, cancer preventive effect, increase the immune response, inhibiting free radicals among many others. Haematococcus pluvialis is a freshwater green microalgae and is one of the largest natural sources of astaxanthin production as it is able to accumulate up to 3% astaxanthin by dry weight. The aim of this study was to determine the culture medium and the optimal conditions for growth and production of astaxanthin from Haematococcus pluvialis. The influence of different factors such as pH, temperature, agitation, aeration and lighting, CO2, promote cell growth, by giving an optimal environment to microalgae. To determine the optimal nutritional conditions for cell growth the effect of different culture media (BBM, OHM, RM) in bioreactors of 500 ml with 350mL of medium and 1x104 cells/ml inoculum was evaluated in exponential phase, the culture conditions employed they were: pH. 6.7 to 7, 5% CO2, 16 hours photoperiod Light 8 dark, irradiance 70 μE / m2s; The results showed that the highest growth and cell reproduction was obtained in the middle RM with 7.5 x 105 cells /ml on day 36, and increased production of astaxanthin was obtained in the middle RM with a concentration of 8.3 ug / ml in the treatment 4.
Subject(s)
Humans , Microalgae , Food Industry , Culture Media , Cell EnlargementABSTRACT
Astaxanthin, a red carotenoid, has been known to possess excellent antioxidant activity and various biological activities, thereby attracting attention as a functional food material.The safety of astaxanthin administered orally has been demonstrated in human clinical studies for about ten years.In this review, we summarized the clinical studies related to safety, as well as studies on genotoxicity, and acute and subchronic toxicity, with a focus on AstaREAL, an astaxanthin product derived from <i>Haematococcus pluvialis</i> which has been reported in numerous human clinical studies to be safe and to have multiple health benefits.Furthermore, based on the latest research, we reviewed the effect of astaxanthin on drug-metabolizing enzymes involved in drug interactions, and concluded that the safety of <i>H. pluvialis</i>-derived astaxanthin, AstaREAL has been widely confirmed.
ABSTRACT
The aim of this work was to investigate the growth performance of Diaphanosoma birgei fed with two Chlorophyceae algae, Ankistrodesmus gracilis and Haematococcus pluvialis using monoalgal diets and simpler mixed diets. D. birgei was daily fed on four treatments: 1) 100% Ankistrodesmus gracilis (Ag); 2) 100% Haematoccocus pluvialis (Hp); 3) 25% A. gracilis + 75% H. pluvialis (Ag-25+Hp-75) and 4) 75% A. gracilis + 25% H. pluvialis (Ag-75+Hp-25). The fecundity curve of D. birgei showed that the mixed feed Ag-25+Hp-75 and temperature 24±2°C triggered fast fecundity at approximately two days. The fecundity was low when based only on H. pluvialis (Hp), albeit with greater longevity (19 days) and a higher number of broods (8). D. birgei fed on Ag and Ag-75+Hp-25 diets in this experiment sustained higher growth rate and higher lipid content in these treatments. The present study showed that A. gracilis diet and mixed microalgae diets tested were able to support the egg production and development of D. birgei.
O objetivo deste estudo foi avaliar o crescimento de uma espécie de Cladocera Diaphanosoma birgei alimentada com dois tipos de microalgas Chlorophyceae, Ankistrodesmus gracilis e Haematococcus pluvialis cultivadas em monocultura e cultura mista. Quatro dietas foram utilizadas: 1) 100% Ankistrodesmus gracilis (Ag); 2) 100% Haematoccocus pluvialis (Hp); 3) cultura mista com 25% A. gracilis + 75% H. pluvialis (Ag-25+Hp-75) e 4) cultura mista com 75% A. gracilis + 25% H. pluvialis (Ag-75+Hp-25). A curva de fecundidade de D. birgei mostrou que a dieta mista Ag-25+Hp-75 e temperatura de 24±2°C promoveu rápida fecundidade em aproximadamente dois dias. A fecundidade foi baixa quando D. birgei foi alimentada somente com H. pluvialis (Hp), porém foi observada maior longevidade (19 dias) e consequentemente, maior número de descendentes (8). As dietas Ag and Ag-75+Hp-25 apresentaram efeito direto na taxa de crescimento de D. birgei com elevados teores de lipídios nestes tratamentos. O presente estudo mostrou que as dietas contendo somente A. gracilis e as dietas mistas de microalgas foram capazes de manter níveis adequados na produção de ovos e no desenvolvimento de D. birgei.
Subject(s)
Animals , Aquaculture , Animal Feed/analysis , Chlorophyta , Cladocera/growth & development , Cladocera/physiology , Fertility , Longevity , Nutritive Value , Time FactorsABSTRACT
The fresh water unicellular alga Haematococcus pluvialis is a promising natural source of astaxanthin. The present study investigated the transcriptional expression of carotenoid genes for astaxanthin accumulation in H. pluvialis using real-time fluorescence quantitative PCR (qRT-PCR). With treatments of 20 and 40 mg/L of gibberllin A3 (GA3), five genes ipi-1, ipi-2, psy, pds and bkt2 were up-regulated with different expression profiles. GA20 (20 mg/L of GA3) treatment had a greater effect on transcriptional expression of bkt2 than on ipi-1 ipi-2, psy and pds (>4-fold up-regulation). However, GA40 (40 mg/L of GA3) induced more transcriptional expression of ipi-2, psy and bkt2 than both ipi-1 and pds. The expression of lyc, crtR-B and crtO for astaxanthin biosynthesis was not affected by GA3 in H. piuvialis. In the presence of GA3, astaxanthin biosynthesis genes of ipi-1, pds and bkt2 were up-regulated at transcriptional level, psy at post-transcriptional level, whereas ipi-2 was up-regulated at both levels. The study could potentially lead to a scale application of exogenous GA3 in astaxanthin production with H. pluvialis just like GAs perform in increasing crops production and it would provide new insight about the multifunctional roles of carotenogenesis in response to GA3.
Subject(s)
Carotenoids/genetics , Dose-Response Relationship, Drug , Fresh Water , Gene Expression Regulation, Plant/drug effects , Gibberellins/pharmacology , Plant Growth Regulators/pharmacology , Transcription, Genetic/drug effects , Volvocida/drug effects , Volvocida/genetics , Volvocida/metabolism , Xanthophylls/metabolismABSTRACT
Las microalgas son fuente de un gran número de compuestos bioactivos de interés industrial, como los carotenoides que se utilizan como colorantes naturales en alimentación animal y humana, así como en la industria farmacéutica, cosmética y en la acuicultura. Además se han propuesto como agentes efectivos en la prevención de una variedad de enfermedades, debido a su capacidad antioxidante, inmunoregulaora, anti-inflamatoria y anti-cancerígena. El ketocarotenoide astaxantina es el más importante desde el punto de vista biotecnológico. Hoy la mayor cantidad de astaxantina es producida por síntesis química y es vendida a un precio de US $2500/kg. El alto precio y el incremento en la demanda para este compuesto, especialmente de origen natural, en las diferentes industrias, hace que sea de interés la producción astaxantina a partir de microalgas como el Haematococcus pluvialis, que acumula cantidades importantes (más del 4%/g de peso seco) y de mejor calidad que las obtenidas por otras fuentes como levaduras y plantas. La acumulación del pigmento en H. pluvialis ocurre durante la transformación de la microalga desde el estado vegetativo (fase verde) a aplanospora (fase roja) cuando cesa su crecimiento en la fase estacionaria. Los tipos de estrés que inducen a la acumulación de astaxantina son temperatura, intensidad lumínica, ciclos de luz/oscuridad, concentración de nutrientes, pH, especies reactivas de oxígeno, sales y presencia de inhibidores de procesos metabólicos a diferente nivel. Es importante resaltar que esta microalga es de difícil cultivo; así como en la obtención del pigmento en cantidades de interés, debido a su ciclo celular complejo. De igual forma, un mayor entendimiento de las bases moleculares de la relación -condiciones de estrés-inducción- acumulación de astaxantina en H. pluvialis, podría ser útil para aumentar la productividad de astaxantina.
Microalgae are a source of a large number of bioactive compounds of industrial importance, such as carotenoids used as natural colorants in food and feed, as well as in pharmaceuticals, cosmetics and aquaculture. They also have been studied as effective compounds for the prevention of different diseases due to their antioxidant, immunoregulatory, anti-inflammatory and anticarcinogenic properties. In biotechnology applications astaxanthin is the most important ketocarotenoide. Currently most astaxanthin is produced by chemical synthesis and sold at U.S. $ 2500/kg. The high price and increasing demand of this compound in different industries, especially of natural origin creates an interest in the astaxanthin production from microalgae as Haematococcus pluvialis that accumulate significant amounts (more than 4%/g dry weight) and better quality what is obtained from sources such as yeast and plants. The pigment accumulation in H. pluvialis occurs during the transformation of microalgae from the vegetative state (green phase) to aplanospora (red phase) when growth ends in the stationary phase. The types of stress that induce astaxanthin accumulation are temperature, light intensity, cycles of light / dark, nutrient concentration, pH, reactive oxygen species, salts and presence of metabolic processes inhibitors at different levels. Is important to take in account that this microalgae is hard to grow and obtain the pigment in amounts of interest could be complicated due to complex cell cycle. Similarly, a better understanding of the molecular basis of the relationship, stress-inducing conditions, astaxanthin accumulation in H. pluvialis, might be helpful for increasing productivity of astaxanthin.
Subject(s)
Humans , Stress, Psychological , Plants , Carotenoids , GeneticsABSTRACT
The fresh-water green unicellular alga Haematococcus pluvialis is known to accumulate astaxanthin under stress conditions. In the present study, transcriptional expression of eight genes involved in astaxanthin biosynthesis exposed to EBR (25 and 50 mg/L) was analyzed using qRT-PCR. The results demonstrated that both 25 and 50 mg/L EBR could increase astaxanthin productivity and the eight carotenogenic genes were up-regulated by EBR with different expression profiles. Moreover, EBR25 induction had a greater influence on the transcriptional expression of ipi-1, ipi-2, crtR-B, lyc and crtO (> 5- fold up-regulation) than on psy, pds, bkt; EBR50 treatment had a greater effect on the transcriptional expression of ipi-2, pds, lyc, crtR-B, bkt and crtO than on ipi-1 and psy. Furthermore, astaxanthin biosynthesis under EBR was up-regulated mainly by ipi1־ and psy at the post-transcriptional level, pds, lyc, crtR-B, bkt and crtO at the transcriptional level and ipi-2 at both levels.
Subject(s)
Brassinosteroids/pharmacology , Carotenoids/biosynthesis , Chlorophyta/genetics , Plant Growth Regulators/pharmacology , RNA, Messenger/metabolism , Steroids, Heterocyclic/pharmacology , Analysis of Variance , Carotenoids/genetics , Chlorophyta/cytology , Real-Time Polymerase Chain Reaction , RNA, Messenger/genetics , Transcription, Genetic , Xanthophylls/biosynthesisABSTRACT
The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, i.e. astaxanthin up to 4%dry weight under stress conditions. Phytoene synthase is considered to be the first rate limiting enzyme in carotenoid biosynthesis pathway in H. pluvialis. The cDNA and genomic genes of phytoene synthase, i.e. psy from H.pluvialis were cloned and characterized.Result showed that psy had one open reading frame of 1 200 bp encoding a putative polypeptide of 400 amino acids which was interrupted by four introns. Phylogenetic analysis revealed that psy from green algae formed a monophyletic clade, and its closer relationship was higher plants. By using genomic walking approach, an approximate 1 kb 5′ flanking region ofpsy gene was cloned and a number of putative cis-regulatory elements were revealed. Fusing a 297 bp internal sequence (-297 to -1 bp from the translation initiation codon ofpsy) with the reporter gene, i.e. lacZ before attemptedintroducing the construct into the green alga via particle bombardment resulted in lacZ transient expression.
ABSTRACT
International research advances and current situation of industrialization in the last decade on the biological character and inducing conditions of natural astaxanthin accumulation in Haematococcus pluvialis cells are reviewed in present paper. Current situation of mass cultivation of Haematococcus pluvialis for industrial production of natural astaxanthin by use of photobioreactors is described. The industrialization prospect of astaxanthin in China is also proposed.