Zeaxanthin Production Research Articles

A synoptic survey of musty/muddy odor metabolites and microcystin toxin occurrence and concentration in southeastern USA channel catfish ( Ictalurus punctatus Ralfinesque) production ponds

A synoptic profile was conducted in July–August 2000 to assess the occurrence of off-flavor compounds beta-cyclocitral, 2-methylisoborneol (MIB), and geosmin, and the cyanobacterial toxin microcystin in catfish production ponds. A total of 485 ponds were sampled during a 1-week period; sample collections were weighted to reflect state area in production. Over 47% of ponds had detectable toxin concentrations, although less than 10% of ponds exceeded 1 ng/ml microcystin. MIB and/or geosmin off-flavors occurred in 25% of the samples. Beta-cyclocitral was detected in over 98% of ponds, and was strongly correlated with presence of myxoxanthin ( r=0.94) and zeaxanthin ( r=0.82), both indicative of cyanobacterial production of this compound. Off-flavor compounds (geosmin and MIB) were correlated to a lesser degree with zeaxanthin production ( r=0.78), whereas aphanizyphyl was better correlated with MIB and geosmin ( r=0.89 and 0.56, respectively), again suggesting cyanobacterial production of these compounds.

A mutant of the green alga Dunaliella salina constitutively accumulates zeaxanthin under all growth conditions.

A novel mutant (zea1) of the halotolerant unicellular green alga Dunaliella salina is impaired in the zeaxanthin epoxidation reaction, thereby lacking a number of the beta-branch xanthophylls. HPLC analysis revealed that the zea1 mutant lacks neoxanthin (N), violaxanthin (V) and antheraxanthin (A) but constitutively accumulates zeaxanthin (Z). Under low-light physiological growth conditions, the zea1 (6 mg Z per g dry weight or 8 x 10(-16) mol Z/cell) had a substantially higher Z content than the wild type (0.2 mg Z per g dry weight or 0.5 x 10(-16) mol Z/cell). Lack of N, V, and A did not affect photosynthesis or growth of the zea1 strain. Biochemical analyses suggested that Z constitutively and quantitatively substitutes for N, V, and A in the zea1 strain. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin, a high-value bioproduct.

Effects of desiccation on photosynthesis pigments and the ELIP-like dsp 22 protein complexes in the resurrection plant Craterostigma plantagineum

Desiccation and abscisic acid treatment lead to major changes in thylakoid membranes of the desiccation-tolerant plant Craterostigma plantagineum. The chlorophyll contents and proteins of the light harvesting complexes decrease during desiccation, although some chlorophyll is retained in the dehydrated state. The xanthophyll cycle pigment zeaxanthin, however, increased. Under these conditions, a 22 kDa ELIP-like desiccation-induced protein (dsp 22) accumulated in the thylakoid membranes. Fractionation of pigment–protein complexes of stressed plants revealed that the dsp 22 protein co-localized with the carotenoid zeaxanthin. Inhibition of zeaxanthin production had a negative effect on the accumulation of the dsp 22 protein. It is suggested that dsp 22 contributes to the protection against photoinhibition caused by dehydration.

Application of a Complete Factorial Design for the Production of Zeaxanthin by Flavobacterium sp.

Utilizing a four-liter fermentor and applying a complete factorial design 2(3), the combined effects of agitation speed, aeration rate, and corn steep liquor concentration on zeaxanthin production by Flavobacterium sp. were studied. Maximum growth and production of total carotenoids and zeaxanthin were obtained at 600 rpm, 2 vvm and 4.6% corn steep liquor. Under these conditions, zeaxanthin represented 86% of the total carotenoids produced. Lower values of the variables studied resulted in lower growth, volumetric production of zeaxanthin and total carotenoids, and favored the formation of other carotenoids such as beta-carotene and canthaxanthin. The positive effects on growth and total carotenoids and zeaxanthin formation were in a large extent due to the interaction of agitation/corn steep liquor. However, aeration also had a positive effect on growth.

Application of a complete factorial design for the production of zeaxanthin by Flavobacterium sp.

Utilizing a four-liter fermentor and applying a complete factorial design 2 3, the combined effects of agitation speed, aeration rate, and corn steep liquor concentration on zeaxanthin production by Flavobacterium sp. were studied. Maximum growth and production of total carotenoids and zeaxanthin were obtained at 600 rpm, 2 vvm and 4.6% corn steep liquor. Under these conditions, zeaxanthin represented 86% of the total carotenoids produced. Lower values of the variables studied resulted in lower growth, volumetric production of zeaxanthin and total carotenoids, and favored the formation of other carotenoids such as β-carotene and canthaxanthin. The positive effects on growth and total carotenoids and zeaxanthin formation were in a large extent due to the interaction of agitation/corn steep liquor. However, aeration also had a positive effect on growth.

Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803.

The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis in Synechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and the Synechocystis beta-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP and crtB, respectively) were introduced into Synechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, and crtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the beta-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the beta-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtO gene, encoding beta-carotene ketolase, which converts beta-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

Light energy dissipation in Quercus ilex resprouts after fire

Holm oak (Quercus ilex) plants that have resprouted after fire have higher photosynthetic capacity than control plants in intact vegetation. In this study, branches detached from forest plants were fed with dithiothreitol (DTT) in the laboratory to inhibit zeaxanthin production and thus reduce the dissipation of light energy as heat. This allowed us to test the hypothesis that plants with greater photosynthetic capacity, and therefore greater photo-chemical sink strength, would suffer a lower reduction in photochemical efficiency under stressful conditions. Greater rates of photochemistry in resprouts, which exhibited increased photosynthesis (A), leaf conductance (g), quantum yield of PSII (ΔF/Fm′) and photochemical quenching (qP), were related to lower non-radiative dissipation of excess energy as indicated by 1 – (Fv′/Fm′). However, the fraction of energy remaining of that used in photo-chemistry or dissipated thermally in the PSII antennae was similar in resprouts and controls and was not affected by DTT, especially under high irradiance conditions. Zeaxanthin involvement in PSII protection operated in resprouts and controls since DTT induced the same kind of response (NPQ decrease) but was lower in resprouts. These chloro-phyll fluorescence results suggest the participation of some additional mechanism for energy dissipation. Light capture characteristics of the photosynthetic apparatus did not differ between resprouts and controls, and leaf age did not play a determining role in the differences observed.

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.

Biosynthesis of carotenoids in bittermelon at high temperature

Detached mature green bittermelon fruit were ripened at 25 and 35° to determine the influence of temperature on the biosynthesis of carotenoids in the fruit tissues. Carotenoid biosynthesis was inhibited in the seed aril at 35°. The production of α-carotene, α-cryptoxanthin, β-cryptoxanthin, zeaxanthin and violaxanthin in the pericarp was not significantly affected at high temperature while β-carotene and the total carotenoids increased during the later stages of ripening at 35°. Carotenogenesis in the aril and in the pericarp responded differently to high temperature probably because the chromoplasts in the tissues metamorphose from different plastid types.

Influence of carbon and nitrogen sources on Flavobacteriumgrowth and zeaxanthin biosynthesis.

Growth and production of zeaxanthin by Flavobacterium sp were studied using different carbon and nitrogen sources in a chemically defined medium. The best growth was supported by sucrose, but glucose yielded similar carotenoid concentrations. Both asparagine and glutamine stimulated growth and pigment formation. Carotenoid production and glucose consumption increased as a function of asparagine concentration. In the presence of asparagine, high glucose concentrations decreased pigment production without affecting biomass formation. In the absence of glucose, asparagine could not support growth and zeaxanthin production. When compared to the effect of 55 mM glucose, 10 mM oxaloacetate increased growth and carotenoid production. Pyruvate and other intermediates of the citric acid cycle showed a similar stimulatory effect. The intermediates of glycolysis: glucose 6-phosphate and fructose 1,6-diphosphate did not support growth. These results suggest that Flavobacterium sp utilizes asparagine primarily as a nitrogen source for growth and production of zeaxanthin.

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on s-carotene and zeaxanthin production which reached 1.6 mg g−1 dry wt.

The transiently pubescent young leaves of plane (Platanus orientalis) are deficient in photodissipative capacity

We have analyzed reflectance changes and carotenoid composition of young and mature leaves of Platanus orientalis L. in order to test the hypothesis that the transient occurrence of highly absorptive and reflective leaf hairs of young leaves (M. Ntefidou and Y. Manetas 1996, Aust. J. Plant Physiol. 23: 535–538) may be correlated to a weakly developed photodissipative capacity in the chloroplast. Compared to mature leaves, young leaves showed negligible reflectance changes at 530 nm upon sudden illumination, possibly indicating a limited production of zeaxanthin. In addition, actual pigment analysis confirmed lower pools of xanthophyll cycle components and reduced capacity for violaxanthin photoconversion in young leaves. Accordingly, the epoxidation state at saturating photon fluence rates was particularly high. A notable feature of xanthophyll cycle interconversions in young leaves was the inability to drive the system to complete de‐epoxidation, as antheraxanthin in the light was always higher than zeaxanthin. Among the rest of the carotenoids, the levels of β‐carotene were particularly low. Moreover, most of the photosynthetic pigments were considerably bleached when young leaves were exposed to high light. The above results strongly suggest that young leaves possess a limited photodissipative capacity and therefore, the presence of leaf hairs affords protection against excess light. When the leaf has matured and presumably the concentrations of photoprotective compounds are adequate, the loss of hairs is not of consequence. In fact, their presence on mature leaves may reduce the photosynthetically active radiation to non‐saturating levels for photosynthesis.

Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids.

Carotenoids are of great commercial interest and attempts are made to produce different carotenoids in transgenic bacteria and yeasts. Development of appropriate systems and optimization of carotenoid yield involves transformation with several new genes on suitable plasmids. Therefore, the non-carotenogenic bacterium Escherichia coli JM101 was transformed in our study with several genes that mediated the biosynthetic production of the carotenoid zeaxanthin in this host. Selection of plasmids for the introduction of five essential genes for zeaxanthin formation showed that a pACYC-derived plasmid was the best. Multiplasmid transformation generally decreased production of zeaxanthin. By cotransformation with different plasmids, limitations in the biosynthetic pathway were found at the level of geranylgeranyl-pyrophosphate synthase and beta-carotene hydroxylase. In our study a maximum zeaxanthin content of 289 micrograms/g dry weight was obtained. This involved the construction of a plasmid that mediate high-level expression of beta-carotene hydroxylase. The level of expression was demonstrated on protein gels and solubilization by the mild detergent Brij 78 revealed that a significant portion of the expressed enzyme is located in the E. coli membranes where it can exert its catalytic function. Based on the results obtained, new strategies for vector construction and strain selection were proposed which could increase the present concentrations drastically. Optimal growth conditions of the transformed E. coli strains for carotenoid formation were found at a temperature of 28 degrees C and a cultivation period of 2 days.

Cloning, sequencing and expressing the carotenoid biosynthesis genes, lycopene cyclase and phytoene desaturase, from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% ( Erwinia herbicola), 37.4% ( Erwinia uredovora) and 22.9% ( Synechococcus sp.), and those between phytoene desaturase genes are 50.3% ( E. herbicola), 54.7% ( E. uredovora) and 39.6% ( Rhodobacter capsulatus).

Operation of the Xanthophyll Cycle in Non-Stressed and Stressed Cells of Dunaliella salina Teod. in Response to Diurnal Changes in Incident Irradiation: A Correlation with Intracellular β-Carotene Content

Summary Changes in carotenoid composition were monitored on a diurnal basis in cells of Dunaliella salina Teod. grown outdoors, under natural irradiance in mass culture. Stressed cells contained approximately four times more, β-carotene than non-stressed cells. The net change in β-carotene content however, was similar for both treatments and correlated closely with changes in photon flux density during the diurnal cycle. Xanthophyll levels were higher in stressed cells suggesting that excess light was not the only factor involved in stimulating carotenoid accumulation, although xanthophyll content increased in nonstressed cells to a level similar to that of stressed cells, with increasing photon flux density. Light-response curves of zeaxanthin and β-carotene formation displayed a biphasic nature in stressed cells. In nonstressed cells however, zeaxanthin formation was rapid and the descending light-response curve showed little change in net zeaxanthin levels. High β-carotene-containing cells had substantial amounts of zeaxanthin and levels of this carotenoid remained higher in stressed cells throughout the diurnal cycle. Changes in intracellular β-carotene occurred coincident with operation of the xanthophyll cycle during the diurnal period. Non-stressed and stressed cells showed similar de-epoxidation of the xanthophyll cycle at peak photon flux denisty. Although non-stressed cells formed more β-carotene and zeaxanthin in response to increasing irradiance, stressed cells formed β-carotene and zeaxanthin more rapidly i.e. at lower photon flux density. Zeaxanthin formation in stressed cells occurred as a consequence of violaxanthin de-epoxidation. By comparasion, zeaxanthin production in non-stressed cells occurred by two probable mechanisms: 1, by rapid de-epoxidation of violaxanthin and 2, a slow but continuous de novo synthesis which increased the total zeaxanthin pool.

The carotenoids of Flavobacterium strain R1560

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3'R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, "zeta-carotene" (7,8,7',8'-tetrahydro-psi, psi-carotene plus 7,8,11,12-tetrahydro-psi, psi-carotene), neurosporene, lycopene, beta-zeacarotene, gamma-carotene, beta-carotene, beta-cryptoxanthin, rubixanthin, 3-hydroxy-beta-zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of "half-molecule" reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.