Carotenoid Formation Research Articles

Inhibition of the photosynthesis in maize caused by manganese deficiency

The mechanism of the fact that Mn deficiency damages the photosynthesis of plants is not yet fully understood. The main aim of the study was to determine Mn deficiency effects in photophosphorylation and key enzymes of CO 2 assimilation of maize. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mn deficiency and to Mn administered in the Mn-deficient Hoagland’s media. The results showed that Mn deficiency was found to cause extensive declines in plant weight and chlorophyll a content, electron transport and oxygen-evolving rate, photophosphorylation rate, activities of Mg 2+ -ATPase, Ca 2+ -ATPase, Rubisco and Rubisco activase, and mRNA expressions of Rubisco and Rubisco activase of maize, but it only slightly affected chlorophyll b and carotenoid formation. However, Mn addition decreased the inhibition of the photosynthesis in maize caused by Mn deficiency.

Strain-Dependent Carotenoid Productions in Metabolically Engineered Escherichia coli

Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.

Cassava Starch Coating and Citric Acid to Preserve Quality Parameters of Fresh‐Cut “Tommy Atkins” Mango

Combination of citric acid dipping (5 g/L) and cassava starch coating (10 g/L), with and without glycerol (10 g/L), was studied to verify the effectiveness of these treatments to inhibit enzymatic browning, to reduce respiration rate, and to preserve quality parameters of "Tommy Atkins" fresh-cut mangoes during storage at 5 degrees C. Color characteristics (L and C), mechanical properties (stress at failure), weight loss, beta-carotene content, sensory acceptance, and microbial growth of fruits were evaluated during 15 d. The respiration rate of fruit subjected to the treatments was also analyzed. Nontreated fresh-cut mango was used as a control sample. Cassava starch edible coatings and citric acid dipping promoted a decrease in respiration rate of mango slices, with values up to 41% lower than the control fruit. This treatment also promoted better preservation of texture and color characteristics of mangoes and delayed carotenoid formation and browning reactions during storage. Moreover, the treated fruit showed great sensory acceptance by consumers throughout the whole storage period. However, the use of glycerol in the coating formulation was not efficient in the maintenance of quality parameters of fresh-cut mangoes, promoting a higher weight loss of samples, impairing fruit texture characteristics, increasing carotenogenesis, and favoring microbial growth during storage.

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

One amino acid substitution in phytoene desaturase makes Chlorella zofingiensis resistant to norflurazon and enhances the biosynthesis of astaxanthin

A stable Chlorella zofingiensis mutant (E17) produced by chemical mutagen was characterized with respect to growth, astaxanthin biosynthesis, and phytoene desaturation. The mutant E17 could grow well and produce normal levels of colored carotenoids in the presence of 0.25 microM norflurazon, in which the growth of wild type (WT) cells was greatly limited due to inhibited carotenoid formation. Induced by high-light irradiation or glucose, E17 produced 44 or 36% more astaxanthin than WT when cultured in media without norflurazon. A point mutation (C-T) was revealed to occur in the PDS gene of E17, leading to an amino acid change (L516F) in its coding region. The mutated PDS exhibited 31-fold resistance to norflurazon when compared to WT as determined by an in vitro assay. Surprisingly, the mutated PDS exhibited higher efficiency in converting phytoene to zeta-carotene. No difference in PDS transcripts was found between E17 and WT cells cultured either in normal or induced conditions. In contrast, higher transcript levels of beta-carotene ketolase and hydroxylase were found in the E17 cells. Taken together, we conclude that a point mutation in Chlorella PDS gene makes E17 resistant to norflurazon and synthesize higher amounts of carotenoids including astaxanthin.

Effect of High-Oxygen Atmospheres on the Antioxidant Potential of Fresh-Cut Tomatoes

The effect of different initial in-package O2 and CO2 concentrations (2.5 kPa of O2 + 5 kPa of CO2, 10 kPa of O2 + 5 kPa of CO2, and 21, 60, and 80 kPa of O2) on the antioxidant potential of fresh-cut tomatoes was investigated. Changes in individual phenolic compounds, individual carotenoids, vitamin C, and antioxidant capacity as well as in O2, CO2, and ethylene headspace concentrations inside packages were assessed for 21 days at 4 degrees C. High-oxygen and passive atmospheres induced higher production of carotenoids and phenolic compounds. The degradation of the initial content of vitamin C was highly promoted by the presence of oxygen. Lower hydrophilic antioxidant capacity (DPPH assay) was obtained in tomato slices stored under 80 kPa of O2, whereas the antioxidant capacity of the lipophilic fraction was enhanced with oxygen availability inside headspace packages. Therefore, 2.5 kPa of O2 + 5 kPa of CO2 atmospheres not only reduced the formation of carotenoids but also maintained vitamin C in fresh-cut tomatoes.

EMPLOYMENT OF ORGANIC ACIDS TO ENHANCE ASTAXANTHIN FORMATION IN HETEROTROPHICCHLORELLA ZOFINGIENSIS

The green alga Chlorella zofingiensis produces significant amounts of the valuable ketocarotenoid astaxanthin under heterotrophic growth conditions. In this study, pyruvate, citrate and malic acid were investigated, for the first time, to promote the formation of secondary carotenoids including astaxanthin in C. zofingiensis in the dark. The addition of pyruvate, citrate and malic acid into the culture medium at a concentration above 10 mM stimulated biosynthesis of astaxanthin and other secondary carotenoids. The addition of 100 mM pyruvate enhanced the yield of astaxanthin from 8.36 to 10.72 mg/L, representing an increase of 28.2%. Citrate and malic acid also had stimulatory effects on the formation of astaxanthin. The results showed that organic acid supplementation was potentially useful for commercial production of astaxanthin by C. zofingiensis in dark heterotrophic culture.

Time‐ and media‐dependent secondary carotenoid accumulation in Haematococcus pluvialis

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.

Conversion of the lycopene monocyclase of Myxococcus xanthus into a bicyclase

Depending on the cyclized hydrocarbon backbone ends, carotenoids can be acyclic, monocyclic, or bicyclic. Lycopene cyclases are the enzymes responsible for catalyzing the formation of cyclic carotenoids from acyclic lycopene. Myxococcus xanthus is a bacterium that accumulates monocyclic carotenoids such as a glycoside ester of myxobacton. We show here that this bacterium possesses a cyclase belonging to the group of the heterodimeric cyclases CrtYc and CrtYd. These two individual proteins are encoded by crtYc and crtYd, which are located in the carotenogenic carA operon of the carB-carA gene cluster, and the presence of both is essential for the cyclization of lycopene. CrtYc and CrtYd from M. xanthus form a heterodimeric cyclase with beta-monocyclic activity, which converts lycopene into monocyclic gamma-carotene, but not into bicyclic beta-carotene like most beta-cyclases. This is an unusual case where two different proteins constitute a lycopene cyclase enzyme with monocyclic activity. We were able to convert this lycopene monocyclase into a lycopene bicyclase enzyme producing beta-carotene, by fusing both proteins with an extra transmembrane domain. The chimeric protein appears to allow a proper membranal disposition of both CrtYc and CrtYd, to perform two cyclization reactions, while a hybrid without the extra transmembrane helix performs only one cyclization.

Manipulation of Phytoene Levels in Tomato Fruit: Effects on Isoprenoids, Plastids, and Intermediary Metabolism

In tomato (Solanum lycopersicum), phytoene synthase-1 (PSY-1) is the key biosynthetic enzyme responsible for the synthesis of fruit carotenoids. To further our understanding of carotenoid formation in tomato fruit, we characterized the effect of constitutive expression of an additional tomato Psy-1 gene product. A quantitative data set defining levels of carotenoid/isoprenoid gene expression, enzyme activities, and metabolites was generated from fruit that showed the greatest perturbation in carotenoid content. Transcriptional upregulation, resulting in increased enzyme activities and metabolites, occurred only in the case of Psy-1, Psy-2, and lycopene cyclase B. For reactions involving 1-deoxy-d-xylulose5-phosphate synthase, geranylgeranyl diphosphate synthase, phytoene desaturase, zeta-carotene desaturase, carotene isomerase, and lycopene beta-cyclase, there were no correlations between gene expression, enzyme activities, and metabolites. Perturbations in carotenoid composition were associated with changes in plastid type and with chromoplast-like structures arising prematurely during fruit development. The levels of >120 known metabolites were determined. Comparison with the wild type illustrated that key metabolites (sucrose, glucose/fructose, and Glu) and sectors of intermediary metabolism (e.g., tricarboxylic [corrected] acid cycle intermediates and fatty acids) in the Psy-1 transgenic mature green fruit resembled changes in metabolism associated with fruit ripening. General fruit developmental and ripening properties, such as ethylene production and fruit firmness, were unaffected. Therefore, it appears that the changes to pigmentation, plastid type, and metabolism associated with Psy-1 overexpression are not connected with the ripening process.

Metabolic engineering of ketocarotenoid biosynthesis in leaves and flowers of tobacco species

Ketocarotenoids and especially astaxanthin are high-valued pigments used as feed additives. Conventionally, they are provided by chemical synthesis. Their biological production is a promising alternative. For the development of a plant production system, Nicotiana glauca, a species with carotenoid-containing yellow pigmented flower petals, was transformed with a cyanobacterial ketolase gene. The resulting plants accumulated 4-ketozeaxantin (adinoxanthin), which is the first ketocarotenoid synthesized in flower petals by genetic modification. Due to the very late flowering in this tobacco species, N. tabacum was used to optimize the yield and ketocarotenoid product pattern by metabolic engineering of the ketolation steps of carotenogenesis. The highly carotenogenic nectary tissue in the flowers represents a model of a flower chromoplast system. By expression of a ketolase gene, it was possible to engineer the biosynthetic pathway towards the formation of 3'-hydroxyechinenone, 3-hydroxyechinenone, 4-ketozeaxanthin, 4-ketozeaxanthin esters, 4-ketolutein and 4-ketolutein esters. Some of these ketocarotenoids were also formed in the leaves of the trangenic plants. In particular, by co-expression of the ketolase gene in combination with a hydroxylase gene under an ubiquitous promoter, the formation of total carotenoids in nectaries increased by more than 2.5-fold. In the nectaries of this type of transformants, more than 50% of the accumulating carotenoids were keto derivatives. In addition, the levels of ketocarotenoid esters were much lower and a higher percentage of the free ketocarotenoids accumulated. These results open new promising perspectives for a successful metabolic engineering of keto-hydroxy carotenoid production in carotenogenic flowers.

Evidence for substantial intramolecular heterogeneity in the stable carbon isotopic composition of phytol in photoautotrophic organisms

The ubiquitous isoprenoid, phytol, was isolated from a range of algae, terrestrial plants and a bacterium, and its two terminal carbon atoms were quantitatively removed by chemical oxidation. The product, 6,10,14-trimethylpentadecan-2-one, was depleted in 13C by 1–4‰ relative to the parent phytol. This difference is significant, and indicates that the pathway for biosynthesis of phytol induces substantial intramolecular stable carbon isotopic fractionation. The nature and magnitude of the fractionation suggest strongly that it is associated both with the biosynthesis of isopentenyl pyrophosphate via the 2-C-methylerythritol-4-phosphate pathway and with the formation of carotenoids and phytol from geranyl-geraniolphosphate. As a result of these large, intramolecular isotopic differences, diagenetic products formed by loss of C, such as pristane, may be naturally depleted in 13C by several permil relative to phytane.

Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants

As a proof of concept, the qualitative and quantitative engineering of carotenoid formation has been achieved in crop plants. Successful reports in tomato, potato, rice, and canola all describe the enhancement of carotenoid with nutritional value, while in model systems such as tobacco and Arabidopsis the engineering of carotenoid to confer abiotic stress has been described. For all the successful applications there have been many examples of unintended/unpredicted phenotypes and results. Typically this has resided from our lack of understanding of carotenoid formation and its regulation. In the present article, we will review advances in carotenoid formation and its regulation to illustrate how metabolic engineering experiments have shed light on regulatory mechanisms.

Genetic engineering of carotenoid formation in tomato

The health promoting and aesthetic qualities of carotenoids have triggered considerable interest in enhancing their levels in crop plants, particularly in fruits and vegetables. One of the main crops of choice is the tomato, since it is readily available in both fresh and processed produce. There are several reports of the use of genetic engineering to increase levels of lycopene and \(\upbeta\)-carotene in ripe fruit. This review will highlight the strategies used to achieve these goals by comparing the use of different genes from plants and microorganisms as well as the choice of promoters.

Employment of reactive oxygen species to enhance astaxanthin formation in Chlorella zofingiensis in heterotrophic culture

The advantages of using heterotrophic cultivation for commercial production of the high-value ketocarotenoid astaxanthin by microalgae have been emphasized in recent years [Chen F. High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol 1996;14:421–5; Chen F, Chen H, Gong X. Mixotrophic and heterotrophic growth of Haematococcus lacustris and rheological behaviour of the cell suspensions. Bioresour Technol 1997;62:19–24; Ip PF, Chen F. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochem 2005;40:733–8]. In a heterotrophic culture where light is not provided, the employment of oxidative stress is essential for promoting the formation of secondary carotenoids including astaxanthin. In the present study, oxidative treatment imposed by chemically generated reactive oxygen species (ROS) including the hydroxyl radicals ( OH) and singlet oxygen ( 1O 2) for inducing carotenogenesis and astaxanthin production by C. zofingiensis in heterotrophic culture was investigated. OH stimulated secondary carotenoids accumulation significantly, whereas 1O 2 did not. The addition of 0.1 mM hydrogen peroxide to the culture leading to the generation of OH enhanced the yield of astaxanthin from 9.9 mg l −1 to 12.58 mg l −1. These results indicate that the application of a suitable type and dosage of ROS is potentially useful for mass production of astaxanthin by C. zofingiensis in heterotrophic culture.

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Directed evolution of farnesyl diphosphate (FPP, C 15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C 20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme. The 12 evolved clones having enhanced C 20 GGPP production have characteristic mutations in the conserved regions of prenyl diphosphate synthases (designated regions I through VII). Some of these mutations (I76T, Y79S, Y79H, C75Y, H83Y, and H83Q) are found near or before the conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl synthases. Molecular modeling suggested a mechanism for chain length determination for these mutations including substitutions at the 1st and 9th amino acids upstream of the FARM that have not been reported previously. In addition, a mutation on a helix adjacent to the FARM within the substrate-binding pocket (D115G) suggests a novel mechanism for chain length determination. One mutant IspA clone carries a mutation of C155G at the 2nd amino acid upstream of conserved region IV (GQxxDL), which was recently found to be an important region controlling the chain elongation of a Type III GGPP synthase. One IspA clone carries mutations (T234A and T249I) near the conserved second aspartate rich motif (SARM). As a verification of the in vivo activity of the mutant clones (represented as C 40 carotenoid formation), we confirmed the product distribution of wild-type and mutant IspA using an in vitro assay.

Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark

Dark-heterotrophic growth of microalgae eliminates the requirement for light and therefore, facilitates large-scale cultivation of microalgae, especially in denser cultures [Trends Biotechnol. 14 (1996) 421]. In this study, the heterotrophic production potential of the secondary carotenoid astaxanthin by the green microalga Chlorella zofingiensis was investigated. The alga showed excellent growth on glucose-supplemented media in batch culture. The maximum specific growth rate and astaxanthin yield were 0.031 h −1 and 10.3 mg l −1, respectively obtained at glucose concentrations of 20 and 50 g l −1, which were the highest ever reported in heterotrophic algal cultures. In the absence of light, formation of secondary carotenoids was mostly dependent on the initial carbon and nitrogen balance in the medium; enhanced biosynthesis of secondary carotenoids including astaxanthin was found in the medium with a high C/N ratio (i.e. C/N ratio=180). The light-independent astaxanthin-producing ability of C. zofingiensis suggested that the alga might be potentially employed for commercial production of astaxanthin on a large-scale.

GENETICALLY MODIFIED TOMATO LINES WITH ELEVATED AND ALTERED CAROTENOID COMPOSITION

Transgenic tomato lines have been produced with increased and altered carotenoid compositions. The biosynthetic steps in carotenoid formation that have been subjected to genetic engineering include phytoene synthesis and desaturation as well as lycopene cyclisation. Homozygous (T 3 ) lines containing an additional phytoene synthase from the bacterium Erwinia uredovora, expressed in a fruit-specific manner show increases in phytoene, lycopene and β-carotene levels of 2.4, 1.8 and 2.2-fold respectively (Fraser et al., 2002). Homozygous (up to T 6 ) lines containing a constitutively-expressed phytoene desaturase from E. uredovora show an elevated (up to 4-fold) level of β-carotene and other xanthophylls (Romer et al. 2000). However, the total carotenoid content is reduced 2-fold. In contrast transgenic lines (up to T 3 ) expressing lycopene β-cyclase show increased β-carotene levels with no reduction in total carotene content. These genetically modified varieties have been subjected to detailed molecular and biochemical analysis at the gene, protein and enzyme-activity level as well as metabolic control analysis. Evidence for regulatory mechanisms such as feedback inhibition and metabolite channelling has been revealed.

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate.

Two strains were selected--the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/l min, 220 rpm), a temperature of 30 degrees C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high beta-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.

A novel type of lycopene epsilon-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4.

Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.