Enhanced Astaxanthin Production in Escherichia coli via Morphology and Oxidative Stress Engineering.

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.

From Golden Rice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm.

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, ß-carotene ketolase, and ß-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained ß-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.

Carotenoids Production: A Healthy and Profitable Industry.

Carotenoids relevance as natural pigments is mainly due to their uses as colorants, feed supplements, nutraceuticals and for medical, cosmetic, and biotechnological purposes. Since they have putative health beneficial effects, the demand and market of carotenoids are growing significantly. There is a diversity of natural and synthetic carotenoids, but only a few of them are commercially produced, including carotenes (ß-carotene and lycopene) and xanthophylls (astaxanthin, canthaxanthin, lutein, zeaxanthin, and capsanthin). Some biotechnological processes for carotenoids production were established some years ago, but new strains and technologies are being developed nowadays for carotenoids widely in demand. This chapter shows a revision of the main carotenoids from a commercial point of view.

Food Modulation Controls Astaxanthin Accumulation in Eggs of the Sea Urchin Arbacia lixula.

The carotenoid astaxanthin has strong antioxidant properties with beneficial effects for various degenerative diseases. This carotenoid is produced by some microalgae species when cultivated in particular conditions, and, interestingly, it is a predominant carotenoid in aquatic animals throughout a broad range of taxa. Recently, astaxanthin was detected in the eggs of the sea urchin Arbacia lixula in relevant concentrations when this organism was maintained in culture. These results have paved the way for deeper research into astaxanthin production by this species, particularly in regards to how astaxanthin production can be modulated by diet. Results showed that the highest content of astaxanthin in eggs was observed in sea urchins fed on a diet enriched with Spirulina platensis. This result was confirmed by the high antioxidant activity recorded in the egg extracts of these animals. Our results suggest that (i) the sea urchin A. lixula is able to synthesize astaxanthin from precursors obtained from food, and (ii) it is possible to modulate the astaxanthin accumulation in sea urchin eggs by modifying the proportions of different food ingredients provided in their diet. This study demonstrates the large potential of sea urchin cultivation for the eco-sustainable production of healthy supplements for nutraceutical applications.

Safety assessment of astaxanthin derived from engineered Escherichia coli K-12 using a 13-week repeated dose oral toxicity study and a prenatal developmental toxicity study in rats.

Astaxanthin is a natural carotenoid with strong antioxidant activity that has been used for decades as a nutrient/color additive and it has recently been marketed as a health supplement. Astaxanthin can be synthesized in a wide range of microalgae, yeast, and bacteria. As genes directing astaxanthin biosynthesis in various organisms have been cloned, this study assessed the safety of astaxanthin crystal produced by Escherichia coli K-12 harboring plasmids carrying astaxanthin biosynthetic genes. The astaxanthin crystal contains a total carotenoid content of 950 mg/g and an astaxanthin content of 795 mg/g. Subchronic oral toxicity and prenatal developmental toxicity of the astaxanthin in rats were conducted in accordance with the Guidelines of Health Food Safety Assessment promulgated by Food and Drug Administration of Taiwan which is based on OECD guidelines 408 and 414. Both male and female Sprague-Dawley (SD) rats (12 for each gender) receiving the astaxanthin crystal at 1.2, 240.0, or 750.0 mg/kg/day in olive oil via oral gavage for 90 days showed no changes in body weight gains, hematology and serum chemistry values and hepatic enzyme stability, organ integrity and organ weight. Except the higher food consumption observed in rats receiving 750.0 mg/g astaxanthin crystal, administration of the astaxanthin crystal to 25-27 pregnant female rats in each group throughout the period of organogenesis (G6-G15) produced no adverse effects on fetal organogenesis. Based on the results, we propose that the no-observable-adverse-effect level (NOAEL) of the astaxanthin crystal extracted from genetically modified E. coli K-12 is 750.0 mg/kg bw/day.

Carotenoids of Microalgae Used in Food Industry and Medicine.

BACKGROUND: Since the industrial revolution, the consumption of processed food increased dramatically. During processing, food material loses many of its natural properties. OBJECTIVE: The simple restoration of the original properties of the processed food as well as fortification require food supplementation with compounds prepared chemically or of natural origin. The observations that natural food additives are safer and better accepted by consumers than synthetic ones have strongly increased the demand for natural compounds. Because some of them have only a low abundance or are even rare, their market price can be very high. This is the case for most carotenoids of natural origin to which this review is dedicated. The increasing demand for food additives of natural origin contributes to an accelerated depletion of traditional natural resources already threatened by intensive agriculture and pollution. To overcome these difficulties and satisfy the demand, alternative sources for natural carotenoids have to be found. In this context, photosynthetic microalgae present a very high potential because they contain carotenoids and are able to produce particular carotenoids under stress. Their potential also resides in the fact that only ten thousands of microalgal strains have been described while hundred thousands of species are predicted to exist. Carotenoids have been known for ages for their antioxidant and coloring properties, and a large body of evidence has been accumulated about their health potential. CONCLUSION: This review summarizes both the medicinal and food industry applications of microalgae with emphasis on the former. In addition, traditional and alternative microalgal sources used for industrial carotenoid extraction, the chemical and physical properties, the biosynthesis and the localization of carotenoids in algae are also briefly discussed.

Engineered maize as a source of astaxanthin: processing and application as fish feed.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Carotenoids exclusively synthesized in red pepper (capsanthin and capsorubin) protect human dermal fibroblasts against UVB induced DNA damage.

Photoprotection by dietary carotenoids has been linked to their antioxidant properties, in particular quenching of singlet molecular oxygen and scavenging of peroxyl radicals. Here, we compared the DNA-protection and antioxidant effects of selected carotenoids exclusively synthesized in red pepper (capsanthin and capsorubin) to the xanthophyll lutein. Preincubation of human dermal fibroblasts (hdf) with capsanthin and capsorubin significantly counteracted UVB induced cytotoxicity at doses between 0 and 300 mJ cm(-2). Pretreatment of hdf with capsanthin, capsorubin or lutein (1 µM) significantly decreased the formation of DNA strand breaks following irradiation with UVB light. All carotenoids studied decreased caspase-3 cleavage (a marker for UVB-induced apoptosis), however, caspase dependent PARP-1 cleavage was not affected suggesting that the remaining caspase activity is sufficient to promote UVB-induced apoptosis. It is conceivable that carotenoids selectively interfere with cellular responses activated by UVB-mediated damage. Our findings indicate that capsanthin and capsorubin exhibit similar properties to lutein and could be used as a dietary supplement to improve natural photoprotection.

Gradient domestication of Haematococcus pluvialis mutant with 15% CO2 to promote biomass growth and astaxanthin yield.

In order to increase biomass yield and reduce culture cost of Haematococcus pluvialis with flue gas from coal-fired power plants, a screened mutant by nuclear irradiation was gradually domesticated with 15% CO2 to promote biomass dry weight and astaxanthin yield. The biomass yield of mutant after 10 generations of 15% CO2 domestication increased to 1.3 times as that with air. With the optimization of nitrogen and phosphorus concentration, the biomass dry weight was further increased by 62%. The astaxanthin yield induced with 15% CO2 and high light of 135 µmol photons m(-2) s(-1) increased to 87.4mg/L, which was 6 times higher than that induced with high light in air.

Effect of red cyst cell inoculation and iron(II) supplementation on autotrophic astaxanthin production by Haematococcus pluvialis under outdoor summer conditions.

The negative effect of heat stress on the autotrophic astaxanthin production by Haematococcus pluvialis has been observed during outdoor culture in summer. Under the summer conditions, the proliferation of vegetative cells was highly halted in the green stage and the inducibility in the biosynthesis of astaxanthin was partly hindered in the red stage. Herein, under outdoor summer conditions in which variations of the diurnal temperature occur, heat-stress-driven inefficient vegetative growth of H. pluvialis was highly improved by inoculating the red cyst cells; thereby, maintaining relatively moderate intracellular carotenoid levels in the green stage. Subsequently, a remarkably enhanced astaxanthin titer was successfully obtained by supplementing 50 µM iron(II) to induce the heat stress-driven Haber-Weiss reaction in the red stage. As a result, the productivity of astaxanthin in the cells cultured under summer temperature conditions (23.4-33.5 °C) using the two methods of red cell (cyst) inoculation and the iron(Fe(2+)) supplementation was increased by 147% up to 5.53 mg/L day compared with that of the cells cultured under spring temperature conditions (17.5-27.3 °C). Our technical solutions will definitely improve the annual natural astaxanthin productivity in H. pluvialis in locations confronted by hot summer weather, particularly in large-scale closed photobioreactor systems.

Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids.

To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4' ß-oxygenase (crtW) and 3, 3' ß-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future.

Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt.

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis ß-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed ß-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from ß-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Proteomic and metabolomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous using different carbon sources.

BACKGROUND: Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable). RESULTS: A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis. Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions. CONCLUSIONS: The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Astaxanthin: sources, extraction, stability, biological activities and its commercial applications--a review.

There is currently much interest in biological active compounds derived from natural resources, especially compounds that can efficiently act on molecular targets, which are involved in various diseases. Astaxanthin (3,3'-dihydroxy-ß, ß'-carotene-4,4'-dione) is a xanthophyll carotenoid, contained in Haematococcus pluvialis, Chlorella zofingiensis, Chlorococcum, and Phaffia rhodozyma. It accumulates up to 3.8% on the dry weight basis in H. pluvialis. Our recent published data on astaxanthin extraction, analysis, stability studies, and its biological activities results were added to this review paper. Based on our results and current literature, astaxanthin showed potential biological activity in in vitro and in vivo models. These studies emphasize the influence of astaxanthin and its beneficial effects on the metabolism in animals and humans. Bioavailability of astaxanthin in animals was enhanced after feeding Haematococcus biomass as a source of astaxanthin. Astaxanthin, used as a nutritional supplement, antioxidant and anticancer agent, prevents diabetes, cardiovascular diseases, and neurodegenerative disorders, and also stimulates immunization. Astaxanthin products are used for commercial applications in the dosage forms as tablets, capsules, syrups, oils, soft gels, creams, biomass and granulated powders. Astaxanthin patent applications are available in food, feed and nutraceutical applications. The current review provides up-to-date information on astaxanthin sources, extraction, analysis, stability, biological activities, health benefits and special attention paid to its commercial applications.

Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene ß-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

Metabolic engineering of Mucor circinelloides for zeaxanthin production.

Mucor circinelloides is a ß-carotene producing zygomycete amenable to metabolic engineering using molecular tools. The crtS gene of the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous encodes the enzymatic activities ß-carotene hydroxylase and ketolase, allowing this yeast to produce the xanthophyll called astaxanthin. Here we describe the fermentation of X. dendrorhous in astaxanthin producing conditions to purify mRNA for the cloning of the cDNA from the crtS gene by RT-PCR. Further construction of an expression plasmid and transformation of M. circinelloides protoplasts allow the heterologous expression of the crtS cDNA in M. circinelloides to obtain ß-cryptoxanthin and zeaxanthin overproducing transformants. These two xanthophylls are hydroxylated compounds from ß-carotene. These results show that the crtS gene is involved in the conversion of ß-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching.

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.

Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants.

ß-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting ß-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85%), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.

Spirulina is an effective dietary source of zeaxanthin to humans.

Zeaxanthin is a predominant xanthophyll in human eyes and may reduce the risk of cataracts and age-related macular degeneration. Spirulina is an algal food that contains a high concentration of zeaxanthin. In order to determine the zeaxanthin bioavailability of spirulina for dietary supplementation in humans, spirulina was grown in nutrient solution with ²H2O for carotenoid labelling. Single servings of ²H-labelled spirulina (4.0-5.0 g) containing 2.6-3.7 mg zeaxanthin were consumed by fourteen healthy male volunteers (four Americans and ten Chinese) with 12 g dietary fat. Blood samples were collected over a 45 d period. The serum concentrations of total zeaxanthin were measured using HPLC, and the enrichment of labelled zeaxanthin was determined using LC-atmospheric pressure chemical ionisation-MS (LC-APCI-MS). The results showed that intrinsically labelled spirulina zeaxanthin in the circulation was detected at levels as low as 10 % of the total zeaxanthin for up to 45 d after intake of the algae. A single dose of spirulina can increase mean serum zeaxanthin concentration in humans from 0.06 to 0.15 µmol/l, as shown in our study involving American and Chinese volunteers. The average 15 d area under the serum zeaxanthin response curve to the single dose of spirulina was 293 nmol × d/µmol (range 254-335) in American subjects, and 197 nmol × d/µmol (range 154-285) in Chinese subjects. It is concluded that the relative bioavailability of spirulina zeaxanthin can be studied with high sensitivity and specificity using ²H labelling and LC-APCI-MS methodology. Spirulina can serve as a rich source of dietary zeaxanthin in humans.