Exploring biostimulation of plant hormones and nitrate supplement to effectively enhance biomass growth and lutein production with thermo-tolerant Desmodesmus sp. F51.

In this study, the interactive effect of plant hormone-salicylic acid and succinic acid on biomass growth, lutein content, and productivity of Desmodesmus sp. F51 were investigated. The results demonstrated that the synergistic action of salicylic acid and succinic acid could effectively enhance the assimilation of nitrate and significantly improve lutein production. The maximal lutein content 7.01 mg/g and productivity 5.11 mg/L/d could be obtained with a supplement of 100 µM salicylic acid and 2.5 mM succinic acid in batch culture. Furthermore, operation strategy of nitrate fed-batch coupled with supplementation for succinic acid and salicylic acid resulted in further enhancement of lutein content and productivity by 7.50 mg/g and 5.78 mg/L/d, respectively. The performance is better than most of the previously reported values.

Effect of light quality on growth rate, carbohydrate accumulation, fatty acid profile and lutein biosynthesis of Chlorella sp. AE10.

Microalgae are feedstocks for multiple product development based on algal biorefinery concept. The effects of light quality (white, red and blue light emitting diodes) and macro-element starvations on Chlorella sp. AE10 were investigated under 20% CO2 and 850 µmol m-2 d-1. Nitrogen and phosphorus starvations had negative effects on its growth rate. The biomass productivities were decreased from day 1 and the highest one was 1.90 g L-1 d-1 under white light conditions. Phosphorus starvation promoted carbohydrate accumulation under three LED light sources conditions and the highest carbohydrate content was 75.9% using red light. Blue light increased lutein content to 9.58 mg g-1. The content of saturated fatty acids was significantly increased from 37.51% under blue light and full culture medium conditions to 77.44% under blue light and nitrogen starvation conditions. Chlorella sp. AE10 was a good candidate for carbohydrate and lutein productions.

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.

Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1.

The efficiencies of extraction and preservation of lutein from microalgae are critical for the success of its commercialization. In this study, lutein was produced by Chlorella sorokiniana MB-1 via semi-batch mixotrophic cultivation. The microalgal biomass with a lutein content of 5.21mg/g was pretreated by bead-beating and high pressure cell disruption methods, and the lutein content was harvested by a reduced pressure extraction method. The effect of pretreatment, pressure, solvent type, extraction time and temperature on lutein recovery was investigated. Using high pressure pretreatment followed by extraction with tetrahydrofuran (THF) as solvent resulted in high lutein recovery efficiencies of 87.0% (20min) and 99.5% (40min) at 850mbar and 25°C. In contrast, using ethanol as the solvent, 86.2% lutein recovery was achieved under 450mbar, 35°C and 40min extraction. The extracted lutein was stabilized in olive oil or sunflower oil with half-lives of 53.1 and 63.8days, respectively.

Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

Biotechnological production of lutein and its applications.

Lutein is an antioxidant that has gathered increasing attention due to its potential role in preventing or ameliorating age-related macular degeneration. Currently, it is produced from marigold oleoresin, but continuous reports of lutein-producing microalgae pose the question if those microorganisms can become an alternative source. Several microalgae have higher lutein contents than most marigold cultivars and have been shown to yield productivities hundreds of times higher than marigold crops on a per square meter basis. Microalgae and marigold are opposite alternatives in the use of resources such as land and labor and the prevalence of one or the other could change in the future as the lutein demand rises and if labor or land becomes more restricted or expensive in the producing countries. The potential of microalgae as a lutein source is analyzed and compared to marigold. It is suggested that, in the current state of the art, microalgae could compete with marigold even without counting on any of the improvements in microalgal technology that can be expected in the near future.

Outdoor cultivation of microalgae for carotenoid production: current state and perspectives.

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers' demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on beta-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase.

BACKGROUND: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. RESULTS: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. CONCLUSION: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein.

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.

Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.).

To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongly inhibited by iron chelators such as o-phenanthroline or 8-hydroxyquinoline. This suggests the transfer of a reducing equivalent from NADPH to the hydroxylase via ferredoxin and the involvement of an iron activated oxygen insertion process. Based on sequence analysis, the putative histidine clusters involved in the iron coordination were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that carotenoid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both types of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probably for other carotenoid oxygenases as well.

Xanthophyll biosynthesis. Cloning, expression, functional reconstitution, and regulation of beta-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum).

Pepper (Capsicum annuum) beta-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity. The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism. Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could be substituted for ferredoxin. The pepper epoxidase acted specifically on the beta-ring of xanthophylls such as beta-cryptoxanthin, zeaxanthin, and antheraxanthin. The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation. This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium. It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid.

The late steps of carotenoid biosynthesis in plants involve the formation of xanthophylls. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xanthophyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6-epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionality, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full-length cDNA clone encoding a capsanthin capsorubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capsanthin-capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step.