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.

Physiological and Metabolomics Analyses Reveal the Roles of Fulvic Acid in Enhancing the Production of Astaxanthin and Lipids in Haematococcus pluvialis under Abiotic Stress Conditions.

In this study, it was found that fulvic acid (FA) enhanced the contents of astaxanthin and lipids in Haematococcus pluvialis under high light and nitrogen starvation conditions by 2- and 1.2-fold, respectively. Meanwhile, the carbohydrate and chlorophyll contents were decreased by FA induction, whereas the levels of reactive oxygen species (ROS) and glutathione (GSH) as well as the expression of astaxanthin and lipid biosynthetic genes were increased. To further explore the interrelation between FA and the biosynthesis of astaxanthin and lipids, a metabolomics analysis of H. pluvialis by combined FA and abiotic stress exposure was conducted by using LC-MS/MS. The contents of some cytoprotective metabolites and signal molecules, including d-maltose, succinate, malic acid, melatonin (MT), and some amino acids, were increased under FA induction and abiotic stress conditions. These metabolites are intermediates in the TCA cycle and Calvin cycle, providing more precursors for the synthesis of astaxanthin and lipids. Moreover, the signal molecules might contribute to enhancing the abiotic stress tolerance. This study provided new insights into the regulatory mechanism of FA on astaxanthin and lipid accumulation in H. pluvialis.

Arabidopsis thaliana Plants Engineered To Produce Astaxanthin Show Enhanced Oxidative Stress Tolerance and Bacterial Pathogen Resistance.

Carotenoids play key roles in photosynthesis and photoprotection. Few multicellular plants produce the ketocarotenoid astaxanthin, a strong antioxidant; however, Arabidopsis thaliana lines overexpressing the Chlamydomonas reinhardtii ß-carotene ketolase (CrBKT) accumulated high amounts of astaxanthin in the leaves. In this study, we investigated the changed regulation of key metabolic pathways and the tolerance of the engineered plants to biotic and abiotic stresses resulting from the heterologous expression of CrBKT. Transcriptome analysis identified 1633 and 1722 genes that were differentially expressed in the leaves and siliques, respectively, of CrBKT-overexpressing plants (line CR5) as compared to wild-type Arabidopsis. These genes were enriched in the carotenoid biosynthetic pathways, and plant hormone biosynthesis and signaling pathways. In particular, metabolic profiling showed that, as compared to the wild-type leaves and siliques, overexpression of CrBKT increased the levels of most amino acids, but decreased the contents of sugars and carbohydrates. Furthermore, CR5 plants had lower sensitivity to abscisic acid (ABA) and increased tolerance to oxidative stress. CR5 plants also exhibited enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study provides insight into the regulation of carotenoids and the related pathways, which may be involved in plant response to oxidative stress and pathogen infection.

Ethanol induced jasmonate pathway promotes astaxanthin hyperaccumulation in Haematococcus pluvialis.

Haematococcus pluvialis is a main biological resource for the antioxidant astaxanthin production, however, potential modulators and molecular mechanisms underpinning astaxanthin accumulation remain largely obscured. We discovered that provision of ethanol (0.4%) significantly triggered the cellular astaxanthin content up to 3.85% on the 4th day of treatment. Amongst, 95% of the accumulated astaxanthin was esterified, particularly enriched with monoesters. Ultrastructural analysis revealed that ethanol altered cell wall structure and physiological properties. Antioxidant analyses revealed that astaxanthin accumulation offset the ethanol induced oxidative stress. Ethanol treatment reduced carbohydrates while increased lipids and jasmonic acid production. Transcriptomic analysis uncovered that ethanol orchestrated the expression of crucial genes involved in carotenogenesis, e.g. PSY, BKT and CRTR-b were significantly upregulated. Moreover, methyl jasmonic acid synthesis was induced and played a major role in regulating the carotenogenic genes. The findings uncovered the novel viewpoint in the intricate transcriptional regulatory mechanisms of astaxanthin biosynthesis.

Glyceryl trinitrate blocks staphyloxanthin and biofilm formation in Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an important nosocomial bacterium that is responsible for a number of infections that may be fatal. The treatment of such infections is limited by emergence of antibiotic resistance. Targeting virulence of Staphylococcus aureus may provide an alternative option to antibiotic that may bypass the emergence of resistant strains due to lack of stress on viability. OBJECTIVES: Investigation of the ability of glyceryl trinitrate (GTN) to inhibit staphyloxanthin, biofilm and tolerance to oxidative stress. METHODS: The disk sensitivity method was used to detect the methicillin resistance of Staphylococcus aureus. The effect of sub-inhibitory concentration of GTN on biofilm formation, staphyloxanthin production and tolerance to oxidative stress was evaluated. Molecular docking study was used to investigate the ability of GTN to bind to dehydrosqualene synthase enzyme. RESULTS: GTN showed a significant inhibition of biofilm, staphyloxanthin and tolerance to oxidative stress. In the molecular docking study, it was found that GTN could bind to dehydrosqualene synthase enzyme by hydrogen bonding, electrostatic interaction and pi-cation interaction. CONCLUSION: The present study revealed the ability of GTN to serve as a potential anti-virulence candidate for attenuation of S. aureus pathogenicity.

Light Elicits Astaxanthin Biosynthesis and Accumulation in the Fermented Ultrahigh-Density Chlorella zofinginesis.

The growth and astaxanthin production of Chlorella zofingiensis were examined under both heterotrophic and photoautotrophic conditions, and it was found that, in comparison to the photoautotrophic mode, the heterotrophic mode led to high algal densities but attenuated intracellular astaxanthin accumulation. Following the heterotrophy-photoautotrophy transition, a considerable increase in the astaxanthin content was observed, accompanied by the upregulation of key carotenogenic genes, including phytoene synthase (PSY), ß-carotenoid hydroxylase (CHYb), ß-carotenoid ketolase 1 (BKT1), and ß-carotenoid ketolase 2 (BKT2). In contrast, the astaxanthin content and carotenogenic genes underwent an opposite change following the photoautotrophy-heterotrophy transition, suggesting the key role of light in stimulating astaxanthin biosynthesis. To improve the astaxanthin production by C. zofingiensis, a novel heterotrophy-photoinduction culture strategy without dilution was developed and evaluated. The astaxanthin content and productivity reached 2.7 mg g-1 of dry weight and 9.9 mg L-1 day-1, respectively, which were 4.0- and 2.5-fold higher than that obtained under the heterotrophic condition.

Nonnatural biosynthetic pathway for 2-hydroxylated xanthophylls with C50-carotenoid backbone.

Carotenoids are structurally diverse pigments with various important biological functions. There has been a large interest in the search for novel carotenoid structures, since only a slight structural changes can result in a drastic difference in their biological functions. Carotenoid-modifying enzymes show remarkable substrate promiscuity, allowing rapid access to a vast set of novel carotenoids by combinatorial biosynthesis. We previously constructed a nonnatural carotenoid biosynthetic pathway in Escherichia coli that can produce C50 carotenoids having a longer chain than their natural C40 counterparts. In this study, a carotenoid 2,2'-hydroxylase (crtG) from Brevundimonas sp. SD212 was coexpressed together with our laboratory-engineered C50-zeaxanthin and C50-astaxanthin biosynthetic pathways. We identified six novel nonnatural C50-xanthophylls, namely, C50-nostoxanthin, C50-caloxanthin, C50-adonixanthin, C50-4-ketonostoxanthin, C50-2-hydroxyastaxanthin, and C50-2,2'-dihydroxyastaxanthin.

Strategy and regulatory mechanisms of glutamate feeding to enhance astaxanthin yield in Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous is an excellent industrial source for production of natural astaxanthin, but the yield of astaxanthin is relative low due to the contradiction between biomass weight and astaxanthin accumulation. Glutamate, a metabolite connecting nitrogen and carbon metabolisms, is probably a promising entry point to interfere cellular metabolisms. Thus, the effect of glutamate on cell growth and astaxanthin accumulation in X. dendrorhous was investigated. Results showed that glutamate feeding facilitated glucose consumption and further led to the increment of astaxanthin content with little influence of cell growth. A comparative proteomics study was applied to decipher the regulatory mechanisms of enhanced astaxanthin biosynthesis in X. dendrorhous as a response to the glutamate feeding. The expressions of proteins with the highest degree of fold change were involved in carbohydrate, amino acids, and carotenogenesis metabolisms as well as redox and stress-associated metabolisms. In addition, a possible regulatory model of enhanced astaxanthin accumulation in response to glutamate feeding in X. dendrorhous is also proposed.

Accumulation of Astaxanthin Was Improved by the Nonmotile Cells of Haematococcus pluvialis.

The current commercial production of natural astaxanthin is mainly carried out using Haematococcus pluvialis vegetative cells in the "two-stage" batch mode. The motile vegetative cells are more sensitive to stress than nonmotile vegetative cells, thereby affecting the overall astaxanthin productivity in H. pluvialis cultures. In this study, we compared the differences between motile cells and nonmotile cells in astaxanthin productivity, morphological changes, the mortality rate, and the diameter of the formed cysts. The experimental design was achieved by two different types H. pluvialis cell under continuous light of 80 µmol photons m-2 s-1 for a 9-day induction period. The highest astaxanthin concentration of 48.42 ± 3.13 mg L-1 was obtained in the nonmotile cell cultures with the highest the productivity of 5.04 ± 0.15 mg L-1 day-1, which was significantly higher than that in the motile cell cultures. The microscopic examination of cell morphological showed a large number of photooxidative damaged cells occurring in the motile cell cultures, resulting in higher cell mortality rate (22.2 ± 3.97%) than nonmotile cell cultures (9.6 ± 0.63%). In addition, the analysis results of cell diameter statistics indicated that nonmotile cells were more conducive to the formation of large astaxanthin-rich cysts than motile cells. In conclusion, the works presented here suggest that the accumulation of astaxanthin was significantly improved by nonmotile cells of H. pluvialis, which provided a possibility of optimizing the existing H. pluvialis cultivation strategy for the industrial production.

Role of media composition in biomass and astaxanthin production of Haematococcus pluvialis under two-stage cultivation.

In the present study, the effects of four different culture media on the growth, astaxanthin production and morphology of Haematococcus pluvialis LUGU were studied under two-step cultivation. The interactions between astaxanthin synthesis and secondary messengers, reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) were also investigated. In the first green vegetative cell stage, maximal biomass productivity (86.54 mg L-1 day-1) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5 mg g-1) occurred in BG-11 medium, which was higher than in any other media. The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any other media, whereas the ROS content was lower. Biochemical and physiological analyses suggested that the ROS, MAPK and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H. pluvialis under different culture media conditions. This study proposes a two-step cultivation strategy to efficiently produce astaxanthin using microalgae.

Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles.

The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 µmol/m2/s. Further increase in light intensity to 750 µmol/m2/s enhanced the biomass productivity to 1821.5 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.

Directed Coevolution of ß-Carotene Ketolase and Hydroxylase and Its Application in Temperature-Regulated Biosynthesis of Astaxanthin.

Because it is an outstanding antioxidant with wide applications, biotechnological production of astaxanthin has attracted increasing research interest. However, the astaxanthin titer achieved to date is still rather low, attributed to the poor efficiency of ß-carotene ketolation and hydroxylation, as well as the adverse effect of astaxanthin accumulation on cell growth. To address these problems, we constructed an efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein engineering and dynamic metabolic regulation. First, superior mutants of ß-carotene ketolase and ß-carotene hydroxylase were obtained by directed coevolution to accelerate the conversion of ß-carotene to astaxanthin. Subsequently, the Gal4M9-based temperature-responsive regulation system was introduced to separate astaxanthin production from cell growth. Finally, 235 mg/L of (3 S,3' S)-astaxanthin was produced by two-stage, high-density fermentation. This study demonstrates the power of combining directed coevolution and temperature-responsive regulation in astaxanthin biosynthesis and may provide methodological reference for biotechnological production of other value-added chemicals.

Illustrating and Enhancing the Biosynthesis of Astaxanthin and Docosahexaenoic Acid in Aurantiochytrium sp. SK4.

The marine thraustochytrids are a promising source of docosahexaenoic acid (DHA) and the ketocarotenoid astaxanthin. In this study, the biosynthetic pathways of these two important metabolites in Aurantiochytrium sp. SK4 was illustrated by the analyses of the genome, transcriptome, key enzymes, and pathway products. Two sets of genes were involved in two pathways for the biosynthesis of fatty acids. The absence of Δ-15 desaturase genes and the presence of docosapentaenoic acid (DPA), up to 12% of total fatty acids suggest that Aurantiochytrium sp. SK4 may synthesize DHA mainly via a polyketide synthase (PKS) pathway. Three enzymes, namely geranyl diphosphate synthase (GPPS), farnysyl diphosphate synthase (FPPS), and geranylgeranyle diphosphate synthase (GGPPS) were found to be involved in the formation of GGPP that was subsequently catalyzed to ß-carotene by a trifunctional CrtIBY enzyme. ß-Carotene might be ketolated and then hydroxylated into astaxanthin based on the carotenoid profiles. The formation of GGPP was proposed to be the limiting steps for carotenoid production. Overexpression of the Archaeoglobus GPS together with the Escherichia coli isopentenyl pyrophosphate isomerase, and Vitreoscilla hemoglobin resulted in not only 1.85- and 5.02-fold increases of total carotenoids and astaxanthin, but also 2.40- and 2.74-fold increases of total fatty acids and DHA. This study provides insights into the biosynthesis of carotenoids and fatty acids in Aurantiochytrium.

Acidic cultivation of Haematococcus pluvialis for improved astaxanthin production in the presence of a lethal fungus.

An acidic cultivation strategy was developed to prevent contamination of a lethal fungus Paraphysoderma sedebokerensis in Haematococcus pluvialis culture for astaxanthin production. Instead of generally used neutral pH, an acidic condition (pH 4) was applied to the cultivation, resulting in a significant inhibition of the fungal contamination. This could be ascribed to the acidity-associated denaturation of a surface protein of P. sedebokerensis, which plays an important role in recognition of H. pluvialis. Stress relief strategies including stepwise light irradiation and naturally occurring nitrogen deficiency were employed in the induction stage to minimize the reduction of astaxanthin production caused by acidic pH. Accordingly, an astaxanthin titer of 84.8 mg L-1 was obtained, which is 141-fold of that from the completely contaminated culture and double of that without the stress relief methods. This strategy provides a persistent contamination control method that can be used for practical astaxanthin production by H. pluvialis.

Stepwise pathway engineering to the biosynthesis of zeaxanthin, astaxanthin and capsanthin in rice endosperm.

Carotenoid pigments are valuable components of the human diet. A notable example is ß-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond ß-carotene, we specifically used a Capsicum ß-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus ß-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart ß-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 µg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 µg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 µg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on ß-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension.

Genome and Transcriptome Sequencing of the Astaxanthin-Producing Green Microalga, Haematococcus pluvialis.

Haematococcus pluvialis is a freshwater species of Chlorophyta, family Haematococcaceae. It is well known for its capacity to synthesize high amounts of astaxanthin, which is a strong antioxidant that has been utilized in aquaculture and cosmetics. To improve astaxanthin yield and to establish genetic resources for H. pluvialis, we performed whole-genome sequencing, assembly, and annotation of this green microalga. A total of 83.1 Gb of raw reads were sequenced. After filtering the raw reads, we subsequently generated a draft assembly with a genome size of 669.0 Mb, a scaffold N50 of 288.6 kb, and predicted 18,545 genes. We also established a robust phylogenetic tree from 14 representative algae species. With additional transcriptome data, we revealed some novel potential genes that are involved in the synthesis, accumulation, and regulation of astaxanthin production. In addition, we generated an isoform-level reference transcriptome set of 18,483 transcripts with high confidence. Alternative splicing analysis demonstrated that intron retention is the most frequent mode. In summary, we report the first draft genome of H. pluvialis. These genomic resources along with transcriptomic data provide a solid foundation for the discovery of the genetic basis for theoretical and commercial astaxanthin enrichment.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Enhancing Astaxanthin Accumulation in Haematococcus pluvialis by Coupled Light Intensity and Nitrogen Starvation in Column Photobioreactors.

Natural astaxanthin mainly derives from a microalgae producer, Haematococcus pluvialis. The induction of nitrogen starvation and high light intensity is particularly significant for boosting astaxanthin production. However, the different responses to light intensity and nitrogen starvation needed to be analyzed for biomass growth and astaxanthin accumulation. The results showed that the highest level of astaxanthin production was achieved in nitrogen starvation, and was 1.64 times higher than the control group at 11 days. With regard to the optimization of light intensity utilization, it was at 200 µmo/m²/s under nitrogen starvation that the highest astaxanthin productivity per light intensity was achieved. In addition, both high light intensity and a nitrogen source had significant effects on multiple indicators. For example, high light intensity had a greater significant effect than a nitrogen source on biomass dry weight, astaxanthin yield and astaxanthin productivity; in contrast, nitrogen starvation was more beneficial for enhancing astaxanthin content per dry weight biomass. The data indicate that high light intensity synergizes with nitrogen starvation to stimulate the biosynthesis of astaxanthin.

Photosystem-II D1 protein mutants of Chlamydomonas reinhardtii in relation to metabolic rewiring and remodelling of H-bond network at QB site.

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency - HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, ß-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the QB site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.

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.