Astaxanthin Biosynthetic Genes Research Articles

Comparative transcriptome analysis unveils mechanisms underlying the promoting effect of potassium iodide on astaxanthin accumulation in Haematococcus pluvialis under high light stress

Astaxanthin produced by Haematococcus pluvialis is an economically valuable ketocarotenoid pigment that benefits the health of human being. Astaxanthin can be accumulated in H. pluvialis cells up to 3% under high light stress. In this study, by adding 1 mM potassium iodide (KI) into the culture media of H. pluvialis cells under high light condition (HK), 26%–35% more astaxanthin than that of high light alone (HL) was obtained. As compared with HL that all cells turned into astaxanthin-rich non-motile ones at day 5 and day 10, about 30%–35% cells in HK remained flagella, which were motile without thickened secondary cell wall. Notably, these astaxanthin-rich motile cells contained more astaxanthin than astaxanthin-containing non-motile cells. The maximal photochemical efficiency (Fv/Fm) was higher in HK while the actual photochemical efficiency (Fv’/Fm') was lower in HK as compared with those in HL. Transcriptomes of cells in HK and HL groups were significantly different, especially at day 5 and day 10, which were consistent with above-mentioned morphological differences between HK and HL cells. KI promoted astaxanthin accumulation by up-regulating the transcription levels of astaxanthin biosynthetic genes and by simultaneously down-regulating the transcription levels of genes involved in other pathways that compete for precursors of astaxanthin biosynthesis. Transcriptomic data also revealed significant changes between HK and HL cells in genes involved in cell wall biosynthesis (57), flagella biosynthesis and cell movement (620), ROS homeostasis (42), and transcriptional factors (42). The possible mechanisms underlying KI's promoting role in H. pluvialis cells under high light were discussed based on morphological and transcriptomic data. In summary, this study provides a new method to increase astaxanthin accumulation in H. pluvialis, and its output expands current understanding of survival strategies and astaxanthin accumulation of H. pluvialis under high light stress.

Single-Stage Astaxanthin Production Enhances the Nonmevalonate Pathway and Photosynthetic Central Metabolism in Synechococcus sp. PCC 7002.

The natural pigment astaxanthin is widely used in aquaculture, pharmaceutical, nutraceutical, and cosmetic industries due to superior antioxidant properties. The green alga Haematococcus pluvialis is currently used for commercial production of astaxanthin pigment. However, slow growing H.pluvialis requires a complex two-stage stress-induced process with high light intensity leading to increased contamination risks. In contrast, the fast-growing euryhaline cyanobacterium Synechococcus sp. PCC 7002 (Synechococcus 7002) is able to reach high density under stress-free phototrophic conditions, and is therefore a promising metabolic engineering platform for astaxanthin production. In the present study, genes encoding β-carotene hydroxylase and β-carotene ketolase, from the marine bacterium Brevundimonas sp. SD212, are integrated into the endogenous plasmid of Synechococcus 7002, and then expressed to biosynthesize astaxanthin. Although Synechococcus 7002 does not inherently produce astaxanthin, the recombinant ZW strain yields 3 mg/g dry cell weight astaxanthin from CO2 as the sole carbon source, with significantly higher astaxanthin content than previous cyanobacteria reports. Synechococcus 7002 astaxanthin productivity reached 3.35 mg/L/day after just 2 days in a continuous autotrophic process, which is comparable to the best H.pluvialis astaxanthin productivities when factoring in growth times. Metabolomics analysis reveals increases in fractions of hexose-, pentose-, and triose phosphates along with intermediates involved in the nonmevalonate pathway. Dynamic metabolomics analysis of 13C labeled metabolites clearly indicates flux enhancements in the Calvin cycle and glycolysis resulting from the overexpression of astaxanthin biosynthetic genes. This study suggests that cyanobacteria may enhance central metabolism as well as the nonmevalonate pathway in an attempt to replenish depleted pigments such as β-carotene and zeaxanthin.

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 (86.54mgL-1day-1) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5mgg-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.

Genetic transformation of the astaxanthin biosynthetic genes bkt and crtR-B into apple tree to increase photooxidation resistance

Apples may suffer sunburn during growing season because of photooxidation damage. Astaxanthin have a stronger antioxidant activity against photooxidation compared to its precursors zeaxanthin and β-carotene. Here, we transferred β-carotene ketolase (bkt) and β-carotene hydroxylase (crtR-B), two genes encoding key enzymes for astaxanthin biosynthesis, into “Brookfield Gala” apple plants using pCAMBIA1302-bkt-crtR-B via transformation mediated by Agrobacterium tumefaciens EHA105. We aimed to confer a stronger antioxidant ability against sunburn to apple plants by regulating metabolic pathways of carotenoids. RT-PCR and qPCR analysis confirmed that bkt and crtR-B genes were expressed simultaneously in transgenic plants. Southern blotting analysis confirmed that the transgene was stably integrated and determined transgene copy number. Transgenic plants synthesized novel ketocarotenoids, resulting in an accumulation of astaxanthin up to 12.06 μg/g and of canthaxanthin up to 6.38 μg/g per leaf fresh weight. The light antioxidant ability of transgenic plantlets was stronger than that of non-transgenic ones in high light conditions. We speculate that the transgenic apples accumulating astaxanthin are resistant to photooxidation and can therefore prevent sunburn.

A strategy for boosting astaxanthin accumulation in green microalga Haematococcus pluvialis by using combined diethyl aminoethyl hexanoate and high light

Haematococcus pluvialis is one of the best natural sources of the high-value antioxidant astaxanthin. The use of biotic or abiotic elicitors is an effective strategy to improve astaxanthin production. In this study, a strategy combining diethyl aminoethyl hexanoate (DA-6) and high light was used to promote the accumulation of astaxanthin in H. pluvialis LUGU. The effect of DA-6 on the astaxanthin content, reactive oxygen species (ROS), and transcriptional expression of six astaxanthin biosynthetic genes in H. pluvialis LUGU was investigated. The results showed that treatment with 0.1 mM DA-6 increased the astaxanthin content to 30.95 mg g−1, which was 2.01-fold higher than that of the control (15.43 mg g−1). Moreover, adding exogenous DA-6 significantly accelerated the transcription of chy, pds, and ptox2, which are responsible for substrate conversion and electron transport during the process of astaxanthin formation under conditions of high light. The level of reactive oxygen species (ROS) and the upregulation of astaxanthin biosynthesis-related genes increased astaxanthin accumulation under DA-6 high light conditions. This work is beneficial in developing an efficient strategy for the hyperproduction of astaxanthin.

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.

Astaxanthin biosynthetic pathway: Molecular phylogenies and evolutionary behaviour of Crt genes in eubacteria

Astaxanthin (3, 3′-dihydroxy-β,β-carotene-4,4′-dione) is a high-value ketocarotenoid synthesized by several species of microalgae, plants, bacteria, and fungi and widely used as a potent antioxidant for human health and also a coloring agent. About six genes (CrtI, CrtL, CrtO, CrtW, CrtR and CrtZ) involve in the biosynthesis of astaxanthin. Phylogenetic analysis of astaxanthin biosynthetic pathway genes and their evolutionary rate variations has been studied among major phylogenetic groups of organisms such as plant, algae, and bacteria. The maximum likelihood (ML) algorithm was used in protein coding DNA sequences to infer the evolutionary relationship for the Crt genes among different eukaryotic and prokaryotic lineages. The phylogenetic analysis suggests that evolutionary pattern of Crt genes in eukaryotic and prokaryotic is characterized by even such as lateral gene transfer and gene duplication events. The specific amino acids present in the respective enzymes are the reason for their functional divergence. The dN values indicate that astaxanthin biosynthetic genes are more conserved in plant and algae than in any other bacterial phyla. This study provides the knowledge of individual Crt enzymes in different organisms that helps to increase the production of astaxanthin which is a biologically significant apocarotenoid that has various uses ranging from human health to cosmetic purposes.

Enhanced astaxanthin production from a novel strain of Haematococcus pluvialis using fulvic acid

Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. Morphological and genomic characterization of a novel strain of H. pluvialis LUGU, which was isolated from Lugu Lake in Yunnan Plateau, was described in this study. The algae can accumulate astaxanthin under abiotic stress. Thus, the influences of fulvic acid (FA) on biomass and astaxanthin were investigated, and transcriptional levels of three astaxanthin biosynthetic genes in H. pluvialis, namely, phytoene desaturase gene (PDS), lycopene cyclase gene (LCY), and β-carotene hydroxylase gene (CHY), were measured. The astaxanthin content increased by 86.89% and 9.78% in 5 and 10mgL−1 FA-treated algal cells, respectively. The transcriptional levels of PDS and CHY genes were also enhanced under FA induction. Results suggested that high level of astaxanthin accumulation may correlate astaxanthin biosynthetic genes with FA induction. This research demonstrated a useful strategy to efficiently produce astaxanthin by using H. pluvialis.

Enhanced production of astaxanthin in Paracoccus sp. strain N-81106 by using random mutagenesis and genetic engineering

Astaxanthin is the most powerful antioxidant of all carotenoids and has great commercial value for use in the aquacultural, pharmaceutical, and food industries. The marine bacterium Paracoccus sp. strain N-81106 is one of the natural producers of astaxanthin, but its astaxanthin productivity is too low for economically feasible industrial production. We tried to improve the productivity of astaxanthin by this strain by a combination of (i) random mutagenesis and (ii) gene cloning/overexpression of astaxanthin-biosynthetic genes. In the first step, we isolated astaxanthin-overproducing mutants after random mutagenesis of strain N-81106. The mutants isolated, NG5 and NG9, showed greater production of astaxanthin than did the wild-type strain N-81106. The yield of astaxanthin in the mutants was about 17 times that in strain N-81106. In the second step, we cloned the astaxanthin-biosynthesis genes of strain N-81106 in Escherichia coli by using a broad-host-range vector, and we then mobilized the cloned genes from E. coli to strains N-81106, NG5, and NG9. The recombinant NG5 produced astaxanthin at 58mg/l—56 times the production of the original strain. The yield was further increased by fed-batch fermentation to reach 480mg/l culture fluid.

Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions

Haematococcus pluvialis, a green alga, accumulates carotenoids, predominantly astaxanthin, when exposed to stress conditions. In the present work, changes in the pigment profile and expression of carotenogenic genes under various nutrient stress conditions and their regulation were studied. Nutrient stress and higher light intensity in combination with NaCl/sodium acetate (SA) enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g(-1) of dry biomass, respectively. Expression of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHY) were up-regulated under all the stress conditions studied. However, the extent of expression of carotenogenic genes varied with stress conditions. Nutrient stress and high light intensity induced expression of astaxanthin biosynthetic genes, BKT and CHY, transiently. Enhanced expression of these genes was observed with SA and NaCl/SA, while expression was delayed with NaCl. The maximum content of astaxanthin recorded in cells grown in medium with SA and NaCl/SA correlated with the expression profile of the astaxanthin biosynthetic genes. Studies using various inhibitors indicated that general carotenogenesis and secondary carotenoid induction were regulated at both the transcriptional and the cytoplasmic translational levels. The induction of general carotenoid synthesis genes was independent of cytoplasmic protein synthesis while BKT gene expression was dependent on de novo protein synthesis.

In Vitro Characterization of Astaxanthin Biosynthetic Enzymes

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase.