©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Singlet Oxygen and Peroxyl Radicals Regulate Carotenoid Biosynthesis in Phaffia rhodozyma(*)

(Received for publication, March 13, 1995; and in revised form, May 16, 1995)

William A. Schroeder (§) Eric A. Johnson (¶)

From the Departments of Food Microbiology and Toxicology and of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Carotenoids have recently received considerable interest because of their potential in delaying or preventing degenerative diseases such as arteriosclerosis, cancer, and aging. In this study we show that the active oxygen species singlet oxygen (^1O(2)) and peroxyl radicals differently affect carotenoid composition and biosynthesis in the yeast Phaffia rhodozyma. Photochemical generation of ^1O(2) with rose bengal or alpha-terthienyl induced carotenoid accumulation. In contrast, peroxyl radicals derived from t-butylhydroperoxide (tBOOH) or H(2)O(2) decreased the content of astaxanthin and increased beta-carotene by 4-fold, suggesting end product feedback regulation by astaxanthin or inhibition of biosynthetic enzymes. ^14C labeling of carotenoids during oxidative stress supported the possibility of end product regulation. Carotenoids were bleached by 8 mM tBOOH within 6 h when carotenogenesis was inhibited by thymol. When treated with peroxides, a previously unreported pigment in P. rhodozyma was formed. The carotenoid had a mass of 580 Da and a molecular formula of CHO(3). Chemical derivatizations combined with mass and absorbance spectroscopy tentatively identified the carotenoid as dehydroflexixanthin (3,1`-dihydroxy-2,3,3`,4`-tetradehydro-1`,2`-dihydro-beta,-carotene-4-one). This study provides the first report of induction of astaxanthin biosynthesis by ^1O(2), probable feedback control by astaxanthin, and the oxidative degradation of astaxanthin to novel pigments in P. rhodozyma.


INTRODUCTION

The carotenoid astaxanthin (3,3`-dihydroxy-beta,beta-carotene-4,4`-dione) has attracted considerable interest in recent years because of its potent antioxidant activity and possible role in delaying or preventing degenerative diseases(1, 2) , and also because of the economic value of astaxanthin as a pigment source in salmonid aquaculture(3) . Only a few species of microorganisms produce astaxanthin in nature. Of these limited biological sources, the heterobasidiomycetous yeast Phaffia rhodozyma(4) has attracted the interest of several fermentation companies because it produces astaxanthin as its major carotenoid and attains high levels of biomass (>50 g of dry yeast/liter) in fermentor culture(5) . The fermentation industry has focused on strain development and fermentor control for maximum production of astaxanthin, and limited research has been devoted to the genetics and enzymology of astaxanthin biosynthesis/degradation and of its physiological roles in the yeast. We previously showed that carotenoids protect P. rhodozyma against killing by singlet oxygen (^1O(2))(^1)(6) , and that carotenoids provide a selective advantage to pigmented strains compared to albino mutants. This finding appeared reasonable because the natural habitat of P. rhodozyma is sap fluxes of the birch tree Betula, and the host tree contains an unidentified compound that catalyzes the formation of ^1O(2) on exposure to ultraviolet light(6) . Carotenogenesis may have evolved in P. rhodozyma to ensure survival in face of photogenerated ^1O(2) in the environment.

The yeast P. rhodozyma could serve as an excellent microbial model to study the functions of astaxanthin and other carotenoids in cell aging. Astaxanthin is an extremely potent antioxidant, and its antioxidant activity has been reported to be stronger and to last longer than carotenoids naturally present in vegetables including beta-carotene, lutein, zeaxanthin, beta-cryptoxanthin, and canthaxanthin (7) . Recently, a sexual cycle was described for Phaffia(8) , which should enable biochemical studies to be complemented by genetic analysis of carotenoid function. In earlier reports we demonstrated that carotenoids in P. rhodozyma confer resistance to activated oxygen species. The present study shows that certain oxygen species also regulate carotenoid levels by induction of synthesis and oxidative degradation of pre-existing pools of carotenoids, and through degradation of end products they relieve feedback inhibition of the astaxanthin pathway.


MATERIALS AND METHODS

Chemicals

Hydrogen peroxide (30% solution, v/v), t-butylhydroperoxide (tBOOH), dimethyl sulfoxide, potassium hydrogen phthalate, rose bengal (RB), mevalonolactone and thymol were from Sigma. Petroleum ether, acetone, acetic anhydride, sodium borohydride, potassium cyanide, and alpha-terthienyl (alpha-T) were from Aldrich. Ethanol (200 proof; USP grade) was a product of Aaper Alcohol and Chemical Co., Shelbyville, KY. Ecolume liquid scintillation mixture was from ICN Radiochemicals (Costa Mesa, CA). (RS)-[2-^14C]Mevalonolactone (55 mCi/mmol) was from Amersham Corp. or from American Radiolabeled Chemicals Inc. (St. Louis, MO). All other chemicals used were of analytical grade or the highest grade commercially available.

Yeast Strains and Growth

P. rhodozyma strains 67-385 and 2A2N were described previously(9, 10) . Yeasts were maintained on slants of yeast extract/malt extract/peptone/glucose medium (YM broth, Difco) with 2% agar (YM agar) and refrigerated at 4 °C. Yeast strains were also stored in 40% glycerol/60% YM broth at -70 °C.

Yeasts were grown as described previously(6, 11) . Briefly, 5 ml of YM broth was inoculated with cells from slants and the yeasts were grown aerobically in roller drum tubes at 20 °C for 2 days until a dry cell weight of 2 mg/ml was obtained. These yeasts were then inoculated at 1% (v/v) into 30 ml of YM broth in 300-ml baffled shake flasks and were grown for 2-5 days depending on the experiment. Yeasts were harvested by centrifugation, and dry cell weight, yeast viability, total carotenoid, and individual carotenoids were analyzed as described previously(11) . To determine total ^14C incorporation into carotenoids, carotenoids were extracted and purified as described previously (11) and quantitated by label incorporation. Additionally, beta-carotene and beta-zeacarotene were isolated by a second TLC run on silica gel using 5% toluene/95% petroleum ether as the mobile phase. After purification, individual bands were scraped and each band reeluted three times with 2 ml of acetone. The pooled acetone extracts were dried inside the glass scintillation vials and dissolved in 10 ml of Ecolume liquid scintillation mixture. The vials were counted in a Beckman model 5801 liquid scintillation counter (Irvine, CA).

Treatment of P. rhodozyma with the Peroxyl Radical Generators H(2)O, tBOOH, and Chemically Generated O

For exposure of P. rhodozyma to peroxyl radicals, cells in YM broth were harvested by centrifugation at 4 °C, washed twice in 0.1 M potassium phthalate buffer (pH 5.0), resuspended in an equal volume of buffer, and exposed to H(2)O(2) or tBOOH. A cell concentration of approximately 5-7 mg ml was used in each experiment. Higher concentrations tended to reduce the reactions of carotenoids, while lower densities tended to cause excessive bleaching.

In certain experiments, cultures were fed with [2-^14C]mevalonolactone (MVA) after 36 h growth in YM broth. The cells were harvested and washed twice in Yeast Nitrogen base (YN; Difco Company, Detroit MI) broth buffered with 0.1 N potassium phthalate (pH 5). Cultures were then resuspended at a cell density of 5-7 mg/ml in buffered YN broth, 30 ml was placed in 300-ml baffled shake flasks, and 300 µl of [2-^14C]MVA (1 M MVA; 10 µCi/ml) was added. The sodium salt of mevalonolactone was prepared immediately prior to use by dissolving 160 mg of mevalonolactone in 600 µl of 2 N NaOH. Two hundred µl of [2-^14C]mevalonolactone (50 µCi/ml) was added and the solution heated to 60 °C for 2 h. The solution was neutralized with HCl and the volume brought to 1 ml.

Cultures were also exposed to chemically generated ^1O(2) produced by photoactivation of RB or alpha-T. RB was prepared and used as described previously(6) . Illumination of RB or alpha-T with 550 or 366 nm light, respectively, produces ^1O(2) by photocatalyzed conversion of ground state triplet oxygen (^3O(2)) to ^1O(2)(12, 13) . For alpha-T treatment, alpha-T was dissolved in ethanol as a 1000-fold stock solution. The culture inoculum (10%, v/v) was exposed to alpha-T and 366 nm light for 5 min as described previously(6) . Inoculum samples were plated before and after treatment with alpha-T and UV light to determine the degree of inactivation of P. rhodozyma by the treatment. Cells were then cultured in the dark and sampled over 4 days. After culturing, yeasts were harvested by centrifugation and dry cell weight and carotenoids analyzed.

Identification of Unknown Carotenoid Formed by Exposure of the Yeast to tBOOH or H(2)O

A previously unreported carotenoid was detected in minor quantities in cells exposed to tBOOH or H(2)O(2). Repeated incubations of several batches of cells with H(2)O(2) were carried out to obtain sufficient pigment for analysis. The carotenoid was initially purified in two chromatographic runs on activated silica plates (Silica Gel 60, 20 20 cm, 0.25 mm thickness, E. Merck, Darmstadt, Germany) using a solvent system of 20% acetone/80% petroleum ether. It was further purified by two chromatographic runs on activated silica plates using 40% acetone/60% petroleum ether as the solvent system. The unknown was collected over several weeks and stored at -70 °C under N(2) in the dark. Acetylation, silylation, and NaBH(4) reduction were performed according to Davies(14) . Mass spectrometry data were collected in the electron impact mode on an MA-50TC ultrahigh resolution mass spectrometer (Kratos Ltd.) and data collected on a Kratos DS-55 data acquisition system. About 100 µg of the purified carotenoid was used for mass spectrometry. Although unlikely, the possibility that this carotenoid could be an artifact of isolation cannot be entirely discounted since sufficient pigment was not available to perform NMR and CD spectroscopy.

Statistical Validation of Results

All experiments in this report were performed in duplicate or triplicate and replicated at least once. The error estimates presented in the text and figures for yeast mass and carotenoid content represent 1 standard deviation from the mean.


RESULTS

Induction of Carotenogenesis by ^1O

In a previous study we showed that astaxanthin protects P. rhodozyma from killing by ^1O(2) generated by photoactivated RB or alpha-T(6) . In the present study we investigated the influence of exposure to ^1O(2) and ROO on carotenoid formation, degradation, and composition. Wild-type P. rhodozyma cultures were grown in flasks containing YM broth and increasing concentrations of RB. The flasks were continuously illuminated with 550 nm light, which generates ^1O(2)(12) . Continual incubation in the presence of 4-6 µM RB increased total pigmentation by 40% after 24 and 48 h (Fig. 1A). Pigmentation usually increases with slow growth rates; however, the RB-associated pigment increase was not growth rate-associated since the dry cell weight after 24 h was actually slightly higher in the 4 and 6 µM RB-treated cultures compared to the untreated culture. The relatively small incremental increase in carotenoid level may have been due to limitations in the quanta of light reacting with RB in the turbid yeast suspension. No significant change in the relative levels of individual carotenoids was found by TLC analysis (data not shown). Higher RB concentrations of 8 and 10 µM decreased pigmentation to approximately the basal level of the control, which was caused by killing of a proportion of the yeast population as indicated by lower cell densities in the higher RB levels.


Figure 1: Carotenoid content in P. rhodozyma strain 67-385 exposed to photochemically generated ^1O(2). A, RB + 550 nm light; B, alpha-T + 366 nm light.



We were interested if brief exposure of the inoculum to ^1O(2) would predispose the cells to produce increased levels of carotenoid during subsequent dark incubation possibly by gene activation as proposed by Hodgson and Murillo (15) for Myxococcus. Pigmentation was increased in P. rhodozyma when the inoculum was treated for 5 min with alpha-T activated by 366 nm light, followed by culturing for up to 4 days in the dark (Fig. 1B). At alpha-T concentrations greater than 10 µM, the pigment levels decreased, which was associated with cell killing. At concentrations of alpha-T > 10 µM, cell survival after treatment in the population was < 50%, while treatments with lower concentrations all maintained at least 65% viability. Xanthophyll synthesis increased maximally during stationary phase, the stage in the growth cycle in which oxygen uptake is unaffected by CN (see next section). These results are intriguing because they show that brief exposure of the cells in the inoculum is sufficient to increase the levels of pigmentation during subsequent dark fermentation. The most likely explanation for the increased levels is that carotenoid gene expression was increased by the relatively brief pulse of ^1O(2) and expression continued throughout the incubation.

Oxygen Utilization in P. rhodozyma Cultures

In addition to formation of ^1O(2) by light-activated reactions, ^1O(2) and other active oxygen species can be generated in dark biological systems by oxygen-utilizing respiratory systems(16, 17, 18) . These active oxygen species primarily derive from spillover of oxidative metabolism and also from the activities of alternative pathways of oxygen metabolism mediated by P-450 cytochromes, flavin oxygenases, and peroxisomal metabolism(18) . Consequently, we determined utilization of O(2) in wild-type P. rhodozyma cells as a function of culture age (Fig. 2). Although total O(2) uptake remained relatively constant throughout growth and in stationary phase, there was a nearly complete shift from CN-sensitive to CN-insensitive respiration in the stationary phase, supporting the importance of alternative pathways of O(2) utilization in astaxanthin biosynthesis. Carotenoids, particularly the oxygenated xanthophylls, are synthesized at the maximum rate during the stationary phase (19) or when respiration is blocked by antimycin(10, 20) .


Figure 2: Oxygen uptake by P. rhodozyma strain 67-385 and sensitivity to 1.3 mM KCN as a function of culture age.



Alterations in Relative Levels of Carotenoids by Exposure to tBOOH

We considered that ^1O(2) may be chemically synthesized in the cell through the reaction of two peroxyl radicals (16) (Reaction 1).

Reaction 1

We therefore treated the yeast with the peroxyl-radical generator tBOOH. Unexpectedly, treatment with tBOOH caused substantial changes in the carotenoid composition of the yeast ( Table 1and Fig. 3). Total carotenoid levels stayed relatively constant during exposure to 8 mM tBOOH for 6 h. During this incubation period, there was a sharp decrease in astaxanthin concentration and a 4-fold increase in the concentration of beta-carotene (Fig. 3). Examination of the individual carotenoids in the cells by TLC also showed the presence of a previously unreported carotenoid in P. rhodozyma. In addition to increase in beta-carotene and decrease in astaxanthin, some other pigments were degraded or increased. HDCO and torulene decreased in content, while early intermediates in the pathway such as -carotene and echinenone increased slightly. beta-Zeacarotene was also detected in small quantities, suggesting asymmetric cyclization of the linear precursors.




Figure 3: beta-Carotene and astaxanthin levels in P. rhodozyma strain 67-385 as a function of exposure time to 8 mM tBOOH.



Destruction of carotenoids also occurred but to a lesser extent when cells were treated with H(2)O(2) (Table 2). Since both tBOOH and carotenoids are hydrophobic, whereas H(2)O(2) is hydrophilic, lower H(2)O(2) reactivity could have been caused by poor interaction of the reactants in the cell or by limited diffusion of H(2)O(2) through the cytoplasmic membrane. We showed earlier that P. rhodozyma contains a low level of catalase compared to Saccharomyces cerevisiae(11) . Since catalase decomposes H(2)O(2), whereas tBOOH is a suicide substrate for catalase(21) , it is also possible that H(2)O(2) was partly eliminated from the cell by catalase thereby decreasing its reactivity.



Peroxyl radicals appear to react directly with carotenoids causing degradation by free radical mechanisms(7, 22, 23, 24) . To attempt to determine if peroxyl radicals were reacting with carotenoids, an inhibitor of carotenogenesis (thymol) was added 3 h prior to treatment with tBOOH (Table 3). In the presence of thymol, significant bleaching of the cells and degradation of carotenoids occurred, while incubation with thymol alone did not bleach the carotenoids (data not shown). Cells treated with tBOOH without thymol partly replenished their complement of carotenoids by de novo synthesis during the 6-h exposure.



The decrease in concentrations of astaxanthin and certain other carotenoids in the presence of thymol and tBOOH suggests that carotenoids react directly with peroxyl radical. These data suggest that peroxyl radical degrades astaxanthin, in turn relieving feedback inhibition of carotenoid biosynthesis. However, an alternate explanation for the increased beta-carotene levels coincidental to the decrease in astaxanthin content is that peroxyl radical inhibits or eliminates the activity of the oxygenation enzymes that converts carotenes to xanthophylls. In order to distinguish between these two possibilities, we fed 36-h-old cultures with [2-^14C] MVA in the presence or absence of 8 mM tBOOH and incubated the cells for an additional 36 h. Under oxidative stress, total MVA incorporation into carotenoids increased 6-fold. Incorporation into beta-carotene and beta-zeacarotene increased 4-fold, whereas incorporation into astaxanthin increased over 2-fold. If peroxyl radical inhibited the beta-carotene oxygenases, reduced incorporation into the xanthophylls would be expected. However, if peroxyl radical degradation of astaxanthin relieved feedback inhibition, then MVA incorporation would be expected to increase as the biosynthetic pathway becomes more active (Table 4). Similar results were seen after 12 h of exposure to labeled MVA (data not shown). These results support the hypothesis of feedback regulation by astaxanthin in P. rhodozyma.



Formation of a Previously Unidentified Carotenoid in P. rhodozyma Formed during Treatment with H(2)Oor tBOOH

A previously unreported carotenoid was detected in cells treated with H(2)O(2) or tBOOH. In order to generate detectable levels of the pigment, it was necessary to expose cultures to >4 mM H(2)O(2). However, levels of production of the pigment were essentially constant (10 µg/g) at greater H(2)O(2) concentrations and for exposure times of 15 min to 9 h (data not shown). However, the physiological state of the cell critically affected the production of this pigment when cultures were under oxidative stress. Log phase cultures were necessary for pigment production. Stationary phase or heat-killed cultures were incapable of production. Furthermore, cycloheximide (10 mM) and chloramphenicol (1 mM) did not affect pigment production, suggesting that the required enzymatic activity is not induced by H(2)O(2) exposure.

The unknown pigment was purified and subjected to a number of derivitization procedures (acetylation, silylation, and NaBH(4) reduction). The compound and the resulting derivatives were subjected to measurements of polarity, as judged by silica gel TLC mobility, visible light spectra, and low resolution electron impact mass spectroscopy. From these analyses, it was determined that the carotenoid's molecular formula was CHO(3). This xanthophyll contains a chromophore of 14 conjugated double bonds. It has one cyclized beta ring containing a ketone at the 4-position and a hydroxyl at the 3-position. The other end group is in the configuration containing a hydroxyl group at the 1`-position. Taken together, these data indicate that the pigment is identical or very similar to dehydroflexixanthin (3,1`-dihydroxy-2,3,3`,4`-tetradehydro-1`,2`-dihydro-beta,-carotene-4-one). This is only a tentative structure. Because of the small amounts isolated (<100 µg), it was not possible to perform NMR and CD spectroscopic analyses to unequivocally identify the carotenoid.


DISCUSSION

Active oxygen species including O, H(2)O(2), OH, and ^1O(2) have been suggested as biological reactants that delay or prevent degenerative diseases such as cancer, arteriosclerosis, cataracts, and increase longevity in eukaryotes(18, 27, 28) . Oxidative damage to DNA, proteins, and other cellular components accumulated with age and decreased the life span in model eukaryotes such as Drosophila. The source of these oxidant by-products in cells is probably excessive oxidative metabolism through respiration, oxidative bursts of phagocytic cells, peroxisomes, cytochrome P-450 systems, and flavin-containing oxygenases(18) . Metabolism in P. rhodozyma associated with astaxanthin biosynthesis is extremely oxygen-demanding. A mathematical model indicates that 21 mol of O(2) are required per mol of astaxanthin synthesized(29) . We have often noticed in flask and fermentor cultures of P. rhodozyma that carotenoid levels, particularly the xanthophylls, accumulate at a maximum rate in older cultures when growth has terminated and O(2) uptake is resistant to CN or to antimycin. Likewise, carotenoid concentrations increase when the yeast is grown on non-fermentable carbon sources or in the presence of antimycin(10, 20, 30) , conditions that may require oxygen utilizing pathways other than mitochondrial respiration.

Several studies have implied that carotenoids have an important role as antioxidants in P. rhodozyma(6, 9, 11) . We demonstrated earlier that astaxanthin provides a defense against killing by ^1O(2)(6) , and this defense is probably important in the yeast's natural habitat. It appears that ^1O(2) may be important in contributing to death of some fungal populations in certain habitats. This is particularly true of organisms exposed to light, such as in the phylloplane and in fluxes of trees, and that some biochemical pathways such as carotenoid biosynthesis may have evolved in part to protect against damaging actions of ^1O(2).

Although well studied in photosynthetic systems, the roles of ^1O(2) in cell metabolism and longevity in nonphotosynthetic microorganisms and mammalian cells have not been investigated to the same extent as other oxygen species such as H(2)O(2), peroxyl radicals, and O. Singlet oxygen is known to occur as a result of respiratory burst in mammalian cells, by transfer of energy from light in plants, and through lipid peroxidation. Other mechanisms of dark ^1O(2) formation in chemical and biological systems have recently been demonstrated, including generation through the Haber-Weiss reaction (17) and on acidification of aqueous hypochlorite(31) . Krinsky (16) hypothesized several years ago that O, H(2)O(2), and peroxyl radicals may spontaneously form ^1O(2) in the cell.

Our data suggest that ^1O(2) may induce carotenoid synthesis in P. rhodozyma by gene activation. A brief pulse of exposure in the inoculum yielded a yeast population that produced more astaxanthin in a dark fermentation. Hence, continual pressure by ^1O(2) was not necessary to stimulate carotenogenesis. Hodgson and Murillo (26) presented an interesting model of regulation of carotenogenesis in Myxococcus xanthus, in which ^1O(2) was produced at the cell membrane by reaction of blue light and O(2) with protoporphyrin IX induced carotenogenesis. Induction of carotenogenesis by blue light in P. rhodozyma has been reported previously (20, 32) . Induction by light may involve an ^1O(2) intermediate. The direct relation of ^1O(2) to photoregulation of carotenoid synthesis in other organisms has not been studied to our knowledge. However, photoregulation has been shown to affect the production of carotenogenic enzymes in certain organisms. Fontes et al. (33) showed that the carR gene, encoding phytoene dehydrogenase, was induced by light. Light also induced the formation of hydroxymethylglutaryl-CoA reductases in the carotenogenic yeast Rhodotorula minuta(34) . We detected no significant change in the relative amounts of the various carotenoids after ^1O(2) exposure, indicating that ^1O(2) induction in P. rhodozyma occurs early in the biosynthetic pathway and possibly affects regulatory genes or proteins.

We also noted that peroxyl radicals generated by tBOOH dramatically affected carotenoid levels and composition in P. rhodozyma. Peroxyl radical can react directly with carotenoids as well as many other molecules in cells, and may form ^1O(2) under specific conditions. Peroxyl radicals, because of their relatively long life in the cell, their ability to diffuse, and their selectivity, are potentially more harmful than other types of radicals (7, 35) . Certain carotenoids are very effective quenchers of peroxyl radicals(7, 23) . Our data support the hypothesis that carotenoids react in vivo with peroxyl radicals. Exposure of P. rhodozyma to tBOOH or H(2)O(2) markedly altered the relative concentrations of the carotenoid composition. In particular, astaxanthin was destroyed and the concentration of carotenes, particularly beta-carotene, were increased. Since the carotene pool was increased as the astaxanthin concentration diminished, it is possible that astaxanthin regulates carotenoid biosynthesis by feedback inhibition of carotene synthesis early in the pathway. The addition of the biosynthesis inhibitor thymol together with tBOOH resulted in rapid destruction of the carotenoids within minutes, suggesting that there is a rapid turnover of carotenoids in P. rhodozyma. These results imply that astaxanthin biosynthesis involves a kinetically active and yet highly regulated and dynamic system involving induction (by ^1O(2)), degradation of xanthophylls by peroxyl radicals, and rapid replenishment of carotenoid pools when the xanthophylls are destroyed.

Incubation of yeasts with tBOOH and H(2)O(2) also resulted in the production of a new carotenoid, tentatively identified as dehydroflexixanthin, which may actually be a carotenoid breakdown product. The formation of this product was rapid, occurring in less than 15 min, and did not require new protein synthesis. However, it was produced only in live (unheated) cells, implying degradation entails an in vivo cellular response to the presence of peroxyl radicals and is catalyzed by specific enzymes.

The actual biosynthetic pathway to astaxanthin in P. rhodozyma is enigmatic. An (9) proposed a primary and an alternate biosynthetic pathway for astaxanthin. This was based on the detection of carotenoids that do not logically fit into the pathway proposed initially by Andrewes et al.(26) . However, this proposal did not take into account the possibility that some of the carotenoids detected in P. rhodozyma may be enzymatically derived oxidative products formed in reactions with radicals. The actual astaxanthin biosynthetic pathway in P. rhodozyma may indeed be the one originally proposed by Andrewes et al.(26) ; however, at the time he also could not account for the presence of HDCO (3-hydroxy-3`,4`-didehydro-beta,-carotene-4-one) in substantial cellular quantities. This pigment may also be an enzymatic degradation product of another xanthophyll. This hypothesis is currently being investigated in our laboratory. The occurrence of asymmetric pigments may be due to the activity of a bifunctional enzyme complex that catalyzes reactions on both ends of the fairly symmetrical carotene precursors. Under stress, catalysis is disrupted and asymmetric products are produced which may be further modified by oxidative and degradative reactions.

In summary, our results suggest that induction by ^1O(2), end product inhibition by astaxanthin, and the activity of degradative pathways in concert control the levels of carotenoids in P. rhodozyma. The distinct differences in carotenoid metabolism mediated by ^1O(2) and peroxyl radicals can be explained by their differing interactions with carotenoids. Singlet oxygen is detoxified by carotenoids through changes in energy state but without disruption of covalent structure of the carotenoids. In contrast, carotenoids covalently react with peroxyl radicals and then are further degraded, possibly by reactions involving cytochrome P-450 or flavin-oxygenases as occurs with other terpenoids and steroids. The astaxanthin pathway in P. rhodozyma appears to involve a dynamic system in which carotenoids are in a constant state of synthesis and degradation. Both the synthetic and degradative systems appear to respond to oxidative stress very rapidly. Exposure to ^1O(2) can alter the total levels of carotenoid intermediates in minutes to hours, and distinct degradative products are also formed rapidly in response to peroxyl radicals. P. rhodozyma appears to have evolved a sophisticated system with the ability to rapidly alter its carotenogenic capacity depending on both environmental conditions as well as the physiological state of the cell.


FOOTNOTES

*
This work was supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI, and by Gist-brocades, Delft, Netherlands. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Science Foundation fellowship.

To whom correspondence should be addressed: Dept. of Food Microbiology and Toxicology, 1925 Willow Dr., University of Wisconsin, Madison, WI 53706. Tel.: 608-263-7944; Fax: 608-263-1114; eajohnso{at}facstaff.wisc.edu.

^1
The abbreviations used are: ^1O(2), singlet oxygen; O, superoxide radical; ROO, peroxyl radical; tBOOH, t-butylhydroperoxide; RB, rose bengal; alpha-T, alpha-terthienyl; YN, Yeast Nitrogen Base; YM, yeast malt broth; MVA, mevalonic acid lactone; HDCO, 3-hydroxy-3,4`-didehydro-beta,-caroten-4-one; dehydroflexixanthin, 3,1`-dihydroxy-2,3,3`,4`-tetradehydro-1`,2`-dihydro-beta,-carotene-4-one.


ACKNOWLEDGEMENTS

We thank Tricia C. Maleniak for expert technical assistance and Roland Rowell for the mass spectrometry work. We also thank Katharina Schiedt of F. Hoffmann-La Roche Ltd. for helpful discussions.


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